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Enlightening the Path to Protein Engineering: Chemoselective Turn-On Probes for High-Throughput Screening of Enzymatic Activity
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Cite this: Chem. Rev. 2023, 123, 6, 2832–2901
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https://doi.org/10.1021/acs.chemrev.2c00304
Published February 28, 2023

Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Many successful stories in enzyme engineering are based on the creation of randomized diversity in large mutant libraries, containing millions to billions of enzyme variants. Methods that enabled their evaluation with high throughput are dominated by spectroscopic techniques due to their high speed and sensitivity. A large proportion of studies relies on fluorogenic substrates that mimic the chemical properties of the target or coupled enzymatic assays with an optical read-out that assesses the desired catalytic efficiency indirectly. The most reliable hits, however, are achieved by screening for conversions of the starting material to the desired product. For this purpose, functional group assays offer a general approach to achieve a fast, optical read-out. They use the chemoselectivity, differences in electronic and steric properties of various functional groups, to reduce the number of false-positive results and the analytical noise stemming from enzymatic background activities. This review summarizes the developments and use of functional group probes for chemoselective derivatizations, with a clear focus on screening for enzymatic activity in protein engineering.

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1. Introduction

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Protein engineering involves the design and creation of proteins with specific, desired functionalities. (1) For applications in synthetic chemistry, these efforts generally involve changing the substrate specificity, increasing the enzymatic activity, inverting and/or improving stereoselectivity, and increasing the stability of the biocatalysts to solvent, pH, temperature, proteolytic agents, etc. (2) All modifications aim to complement or even completely replace reactions that use hazardous or environmentally unfavorable reagents or conditions. The demands of synthetic chemistry, however, do not necessarily align with nature’s repertoire of enzymatically catalyzed reactions. (3) Consequently, tools for the improvement of existing and the development of new enzymatic catalysts are needed.
In recent years, it has become possible to use rational structure-based and de novo design to create new active enzymes. These new methods have been demonstrated to work in improving catalytic performance, broadening substrate range, increasing or inverting existing stereoselectivity, and in creating “new-to-nature” functionality (4) to obtain products with high value for industrial applications. (5) In contrast to specific design, methods that test all (or almost all members) of a given population (“screening”) are typically used when there is no prior knowledge of a protein structure or when rational design seems to only deliver incremental improvements. (6−8)
These screening methods are often subsumed under the term “directed evolution,” pioneered in the 1990s and awarded with the 2018 Nobel Prize in Chemistry. (9) Random mutagenesis (by error-prone PCR or gene shuffling) enables the researcher to explore uncharted territory of chemical reactions by unguided substitutions of the genetic code, thus causing exchanges of single amino acids or regions of an enzyme. Goldsmith and Tawfik found a correlation between increases in activity and the number of mutations in the protein; large improvements (>103-fold activity of the wild type) required an average of five mutations per 10-fold improvement in catalytic efficiency. (10) A specific example: To cover the whole sequence space of a protein with 300 amino acids by using random mutations at five random positions, 1.9.7 × 1012 variants would have to be assessed. (11) Such large numbers of experiments practically exclude the use of chromatographic methods (e.g., GC, HPLC) and can only be managed by spectroscopic techniques, using chromo- or fluorogenic reporters, with short analysis times, low detection limits, and high sensitivity (Figure 1). (6)
Many studies rely on chromo- or fluorogenic substrates that mimic the true target or on enzyme-coupled chromo- or fluorogenic assays for the detection of a wide range of catalytic activities. (12) The major drawback of these methods is that they generate indirect experimental evidence of the desired activity but no analytical signal from the relevant transformation. The most reliable hits are achieved by detecting the target product.
Functional group assays (FGA) use the functionality of a target molecule as an analytical handle. They thereby reduce the number of false-positive results, and the analytical noise from enzymatic background activities. (13,14) In this review, we summarize the development and use of functional group probes for the chemoselective detection of a broad range of target molecules in physiological media (e.g., aqueous buffered solutions, cell culture media). We approach the topic primarily in the context of protein engineering, focusing on the applicability of those tools to the development of new biocatalysts. With this summary, we wish to target two fields that are heavily involved in this area of research: biochemistry and synthetic organic chemistry. We aim to equip the biochemist with a toolset to pursue the discovery of valuable biocatalysts, and we hope to motivate organic chemists to identify and tackle underdeveloped areas in the field of assay development. The review is structured from a chemist’s point of view, ordered by functional groups typically encountered in molecules of interest in academic and industrial research and development: amines, alcohols, aldehydes, ketones, phenols, epoxides, diols, thiols, etc. This classification is intended to enable easy access to information and offer chemical solutions to challenges in screening for enzymatic activity. Compatible buffer ranges, spectral regions of the respective assay product, and the commercial availability of the presented probes are additionally highlighted.

2. Assays in High-Throughput Screening

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High-throughput screening is essentially a technical method in which every detail matters. If a good photometric assay is available, a library of enzymes based on a single site-saturation mutagenesis (e.g., using the NNK codon) can be easily screened for activity in a single 96-well microtiter plate. A combinatorial library of two residues (using NNK codons, as before) requires about 4000 clones (for statistical reasons), or 10 384-well plates. (15) When there are even more amino acids in the enzyme that need to be mutated and there is no better way of designing variants than combinatorics, the number of variants to be screened rapidly exceeds the (human) capacity for handling plates and performing manipulations, with or without automation. Miniaturization can offer a significant improvement to throughput, with certain implications on the applied downstream analytics used for the high-throughput screening (HTS; 104–105 variants to analyze), and the development of novel (ultra) high-throughput (uHTS; 106–108 variants) screening methods becomes critical.
There are various scalable methods to obtain experimental information about structure–property relationships in enzymes. Generally, the methods with the highest throughput are in vitro binding assays. (16) The data they produce is sometimes rich enough to complete even complicated scientific tasks, such as solving a protein structure from a deep mutational scan. They are not suitable when the goal is to optimize a biocatalyst for conditions relevant in an industrial process.
Most approaches rely on the spectroscopic detection of target or reporter molecules from absorbance or fluorescence measurements in the visual range, or close to it. Whenever a chromatographic separation is included before detection, throughput drops greatly. Fluorescence-activated cell sorting (FACS) can be used if (i) the reaction is run in cells, (ii) the reporter or product is fluorescent, and (iii) it does not permeate between cells. Recent developments in microfluidic fluorescence-activated droplet sorting (FADS) circumvent the issue of cell permeability by compartmentalizing cells in picoliter-sized water-in-oil droplets but in turn require analytes to be hydrophilic to prevent cross-talk between droplets; applications are currently limited to very polar fluorophores. (17,18) Only few metabolites of interest have useful absorbance or fluorescence, and thus artificial fluorogenic substrates are needed. These artificial probes are often custom-designed and synthesized for a single application only.
High-throughput screening is still in its infancy and largely an academic pursuit where new approaches are being developed for linking enzymatic activity to a spectroscopic signal (Figure 2). While UV absorbance-based assays can detect a broad range of compounds, their sensitivity is usually lower than fluorescence-based ones. Compounds in crude cell lysates might interfere with the absorptive detection of the analyte of choice. Therefore, ratiometric “turn-on” probes, linking product formation to a proportional increase of a ratio of fluorescence signals, constitute the majority in the vast landscape of reporters (Figure 2). (13,19,20) The increase in fluorescence intensity can result from various molecular implementations: (i) the inherent fluorescence of the desired product, formed by the enzymatic reaction (Figure 2a), (ii) a coupled and strongly correlated process that produces a fluorescent molecule, e.g., a cofactor recycling system (Figure 2b), (iii) enhanced transcription of fluorescent biosensors (Figure 2c), (iv) the concomitant formation of fluorescent metabolites (Figure 2d), (v) by induction of Förster resonance energy transfer (FRET) or suppression of photoinduced electron transfer (PET) processes (Figure 2e), or (vi) a tandem reaction producing a fluorescent molecule in a cascade, downstream of the targeted reaction (Figure 2g).
Not all the modes to generate a fluorescent signal listed above are equally applicable to any problem at hand. Given that many relevant products are not or only weakly fluorescent, direct monitoring of their formation is often not feasible (Figure 2a). Not all enzymatic transformations offer possibilities for indirect read-outs, e.g., from cofactor recycling (Figure 2b,d). Many protein engineering efforts thus use analytical tools based on fluorogenic substrates that mimic the chemical properties of the target compound to some degree while still being easy to synthesize. (12) Screening for enzymatic activity with such mimics, however, bears the risk of identifying biocatalysts that are better at producing the mimic and not necessarily the target product. An aphorism capturing this issue, nicknamed as the First Law of Directed Evolution, that is often quoted, says: “You get what you screen for.” (21,22) Assays based on mimics might be useful for proof-of-concept studies or the detection of new catalytic activity; they are not powerful in the optimization of biocatalysts.
Functional group probes (Figure 2f) use chemoselectivity, a result of steric and electronic properties of a functional group, as an analytical handle. This simplification, reducing the analytical complexity to a distinct interconversion of one functional group to another, often enables a clear discrimination of the desired enzymatic activity, even in the presence of substrates and interfering molecules from cellular background. Functional group probes offer an attractive option to reduce the number of false-positive results and the analytical noise from enzymatic or cellular background activities. (13,14) Over the last decades, many functional group sensors have been developed, often benefiting from research in bioconjugation chemistry and vice versa. Among these examples, many have proven to be applicable in protein engineering, when HTS was used to test large libraries of enzyme variants.

3. About High Throughput

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In any screening campaign, every manipulation decreases throughput. For instance, if one has to start with plating out cells after transformation, an agar-plate assay will likely have a higher throughput than one based on microtiter plates; (23) or, transferring lysate from a deep-well plate (for cell growth) to an optical plate (for reading) halves throughput by doubling the number of plates to be handled. As a rule of thumb, throughput doubles when the analysis is performed in the same plate as the cultivation of the microorganism producing the enzyme. That also requires the assay reagents to not damage the DNA, which encodes the sequence of a potential hit.

Figure 1

Figure 1. Change of analytical approach depending on screening scale and mutant library size: Increase in library size urges a shift from chromatographic methods to spectroscopic detection for (ultra) high-throughput screenings: (u)HTS. TLC: thin-layer chromatography; GC: gas chromatography; HPLC: high-performance liquid chromatography; (μ)MS: (microscale) mass spectrometry. Created with BioRender.com.

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Assays where reagents for detection at the end can be added from the beginning of the reaction (e.g., in a single master mix with the substrate) are better than assays with multiple components that need to be added in a certain order. (24) There are limitations to such straightforward optimization of protocols. For instance, some reagents react with substrates or enzymes, inactivating them, or are unstable in water in general (e.g., fluorescamine 18 (25)) or at certain pH (e.g., DTNB 232 (26)). They can only be added at a later stage, when the chosen reaction time has passed or even just before analysis. The duration should be chosen in consideration of the dynamic range of the detection method without extra dilution (or concentration). Variations between wells on microtiter plates (in growth rates and/or expression levels, even for the same variant) are often large and significant, resulting in cases where relative specific activities cannot be estimated by merely quantifying the product. Continuous kinetic measurements are unsuitable because every moment one sample stays in the reader longer than necessary corresponds to other variants not being screened. Normalization to the actual enzyme content (e.g., using tagged-protein complementation assays like the split-GFP (27) or split-luciferase assays (28)) is possible, but it can be expensive and time-consuming─it is therefore rarely done.
The power of HTS lies in producing information by interrogating many variants, not scrutinizing few. For the purpose of discovery, end-point measurements often provide information as good as kinetic measurements. For this reason, HTS is typically stronger in the discovery of new activities or the early rounds of improvement than in improving enzymes with substantial activity. This trend is a conundrum because initial improvements in activity are often simpler to achieve than further optimization: quantitative performance of the assay has to become more granular, more sensitive, and more reliable at later stages than in the beginning. Driven by this increasing challenge, HTS methods are used for tasks at which they do not excel by design, and work-arounds are introduced (e.g., use of lower substrate concentrations, subsequent dilution steps). The dynamic range of assay systems thus needs to match the aim of the study (discovery or optimization).
The requirements for HTS-capable assay development are high. Ideally, an assay makes as few mechanical and fluidic manipulations as possible, use stable compounds, is sensitive, selective, simple, and has a large dynamic range. Any assay that can run at room temperature is simpler than one requiring heating or refrigeration. High temperatures, extreme pH, corrosive, or toxic chemicals and solvents (especially, volatiles and halogenated ones) should be avoided, as they can make assays incompatible with common equipment (plastic multiwell microtiter plates and their readers). It is critical that assays work under aqueous conditions because crude cell lysates are prepared using aqueous buffers. Therefore, this review focuses mainly on probes used to analyze aqueous samples.

4. Sensitivity and Selectivity

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The best functional group probes are those where fluorescence solely depends on the concentration of the analyte. Such a simple relation can be typically observed in systems with only one enzyme and substrate present in the reaction. Many assays, however, require additional components (e.g., more than one substrate or enzyme, cofactors, activators or inhibitors, stabilizers, surfactants, cosolvents, detection reagents, etc.), making the selective detection of the main product challenging.

Figure 2

Figure 2. Overview of several ratiometric absorption or fluorescence turn-on strategies applied in directed enzyme evolution in biological systems: (a) direct detection of activity by functional group transformations or cleavage reaction of the target substrate, (b) indirect detection of conversion via dye cofactor recycling with a concomitant increase in signal, (c) triggered enhanced transcription of fluorescent biosensors, (d) formation of the dye in a downstream reaction upon cleavage of a metabolic reporter, detection via suitable recognition domain or subsequent reaction with a secondary chromo-/fluorogenic substrate, (e) induction of Förster resonance energy transfer (FRET) or suppression of photoinduced electron transfer (PET), (f) functional group transformation is detected via subsequent reaction with functional group selective assay component, (g) tandem reaction cascade of a model-substrate (mimic) unmasks chromo-/fluorophore.

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Crude cell lysates, which are the preferred formulation of enzymes used in HTS, compromise the detection because they introduce many intracellular metabolites in high concentrations, often much higher than the concentration of the product. For instance, in Escherichia coli, metabolites present in the mM range include several amino acids (glutamate, at 96 mM, aspartate, valine, glutamine, alanine, and citrulline), several nucleotide phosphates or thiols (e.g., glutathione, at 17 mM, or coenzyme A, at 1.4 mM). (29) The high concentrations of these and other metabolites complicates the detection of low enzyme activities with rather nonspecific colorimetric probes. For example, the derivatization of primary amines using o-phthaladehyde (OPA, 22) in the presence of 2-mercaptoethanol (ME) (24,30) may be influenced by endogenous thiols because evolution of the signal is dependent on the thiol coupling partner.
Buffers too can obfuscate the results of an assay when they contain constituents that might interfere with the chemoselectivity of the probe. For instance, Good’s buffers contain amines, which may therefore compete with the detection of an amino functionality in the product.
Functional group assays work best when the functional group is being created from another in the reaction (i.e., a functional group interconversion, FGI). For example, carboxylate reductases produce aldehydes from carboxylic acids, amidases produce amines from amides, and thioesterases produce thiols from thioesters. In such straightforward cases, probes only need to discriminate one functional group from another and not between two of the same type. Hence, many probes are useful for FGI screening. (31)
In many other reactions (e.g., aminotransferases, isomerases, etc.), functional groups are not interconverted but moved around or between molecules, making chemoselectivity essential and challenging. Probes that cannot differentiate between substrate and product bearing the same functional group (e.g., by forming conjugates with different spectral properties from each) would require separation prior to detection (e.g., extraction or chromatography). Because the substrate is usually present in high concentration to saturate the enzyme (typically, ∼ 100-fold the KM value), specificity for the product by ways of vastly different reaction rate constants is important (because with similar constants, specifically detecting a product at low concentration, is noisy against the background from the substrate).
Examples for the power of chemoselective probes are (i) o-diacetylbenzene (DAB, 26), which can discriminate amines in amino acids from primary amines, used in HTS of amino acid decarboxylases, (32,33) (ii) β-aryloxy fluorescein aldehyde 38 reported by Leslie et al., (34) which undergoes accelerated β-elimination by secondary amines (34) and is valuable for detecting reactions that convert primary to secondary amines (e.g., methyltransferases).
Manipulating the reaction conditions can also be used to change selectivity of an assay by changing the reactivity of the target. For example, fluorescamine 18, a probe for amines, can under acidic conditions be used for the selective detection of aromatic amines in the presence of aliphatic amines because the pKa values of these types of amines are sufficiently different. (35)
Because the selectivity profile of most reported assays is not extensively characterized (even for the small number of commercially available probes), it is often unclear to protein engineers which reagent might be the most suitable to detect the product of interest and many need to be tested. Additionally, many studies on assay development add the analyte in large excess over the detection probe. While this approach is advantageous to show the optical performance of the assay, it does not guarantee its practicability for screenings. It would add great value if research groups developing new assays characterized the selectivity of a new probe in commonly used reaction media toward various derivatives of the same substance or reactivity class (e.g., nucleophiles, electrophiles, oxidants) and (cellular) interfering molecules.
The goal of this review is to present protein engineers, biochemists, and chemists with an overview of available options for the detection of compounds of interest. For synthetic chemists, we provide a summary of known starting points for the development of even more selective probes.

5. Principles for Designing Functional Group Probes

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The detection of organic functional groups is widespread in chemistry. Thin-layer chromatography, (36) which is taught in beginner chemistry laboratory courses, often uses chemoselective, chromogenic stains based on reactions with functional groups. Historically, the discovery and development of staining agents improved the characterization of new compounds via derivatization and enabled reliable and fast reaction analytics. Many selective reagents enjoyed great popularity, including Ehrlich’s reagent 1, (37) 2,4-dinitrophenylhydrazine (DNPH) 109, (38) phosphomolybdic acid, (39) Fe(III)Cl3/hydroxylamine, (40) Hanessian’s cerium stain, (41) Simon’s reagent, (42) among others, and are still used for illicit drug screenings (Scheme 1).

Scheme 1

Scheme 1. Representative Reagents Typically Used in Thin-Layer Chromatography (TLC) Staining Solutions
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Only a few of these classic probes have been applied in protein engineering, so far, likely because of the drawbacks that many of these agents entail: they are largely incompatible with biological systems, have poor selectivity or sensitivity, and inadequate optical properties. Nevertheless, some emerged as the structural or conceptual basis for many probes that followed.
Designing chemoselective probes has several requirements to enable their general application: (14) (i) Most importantly, the probe must react selectively with the target analyte in the presence of substrates and other background (from cells or the medium). Chemoselectivity has to be sufficiently high to promote the preferential reaction with the desired analyte even in the presence of molecules with similar functional groups. (ii) Probes should react fast under actual screening conditions. A HTS assay should not be limited in throughput by the reaction rate of the probe. (iii) If cells need to be kept alive (e.g., for cofactor recycling), probes have to be nontoxic.
Two options commonly serve as the starting point for initial development: (i) the discovery of a suitable, highly selective chromo- or fluorogenic reaction and subsequent refinement of the chemical probe’s spectral properties, or (ii) the incorporation of a chemical sensing mechanism into an already established chromophore which causes a desired optical read-out.
Detecting targets at low concentration with a short lifetime brings particularly stringent requirements for selectivity and reactivity. Probes that meet these requirements can enable special methods, such as single-cell droplet assays, or HTS campaigns that search for the proverbial “needle in the haystack.”
In addition to defining the chemical properties of a probe, the choice of the chromogenic platform is also essential. Knowledge about the genesis of and control over the optical signal is necessary for the rational design of good probes. Spectroscopic properties of organic molecules at visible and near-visible wavelengths are closely associated with their electronic (delocalized) structures. (43) Extension of a conjugated π–electron system upon reaction is a common element in designing chromogenic probes, which generally leads to a bathochromic shift in the absorption maximum. It does not simultaneously guarantee the emission of a fluorescent signal, which is only observed for a small fraction of absorbers due to the action of prominent quenching mechanisms, such as internal conversion (IC) or intersystem crossing (ISC). (44) Fluorescence is favored over absorption as the signal-generating mode due to a better signal-to-noise ratio and thus lower detection limits. Generally, it is easier to see a strong signal against a weak background than to look for weakening of a strong background.
Most turn-on fluorescent probes are based on a change of the electronic structure of the chromogenic dye, (45,46) triggered by changes to the size of a delocalized electron system (e.g., degree of conjugation of π–electron systems) or to the distribution of electrons in the system (by the introduction, manipulation, or removal of electron-donating or withdrawing groups). The resulting changes in absorption coefficients, quantum yields, and/or Stokes shifts can be measured and, if chemoselective, related to changes of the analytical target. Other mechanisms of fluorescent assays to generate a signal include: suppression of a photoinduced electron transfer (PET), (47) enabling a fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores, (48) modulating an intramolecular charge transfer (ICT), (49) or enabling an excited-state intramolecular proton transfer (ESIPT). (50) Recent developments, such as aggregation-induced emission (AIE), (51) twisted intramolecular charge transfer (TICT), (52) electronic energy transfer (EET), (53) or C═N isomerization, have not yet found widespread application in the design of reaction-based turn-on probes.
There are excellent reviews on general design principles of fluorescent chemosensors, with a focus on chemoselective bioimaging of endogenous metabolites, (14) fluorescent probes in redox biology applications, (54) imaging of reactive oxygen, nitrogen, or sulfur species (ROS, NOS, RSS), (55,56) anions and cations, (57) screening for defined enzymatic functionalities, (13,58) or the high-throughput aspect of selected methods. (7) Here, we will elaborate on reaction-based probes in the context of protein engineering. We outline the rational design of most known functional-group probes; some of them have not (yet) been used in protein engineering.

6. Chromo- and Fluorogenic Probes for the Detection of Amines and Amino Acids

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Amines are a highly valuable class of compounds used in the chemical, pharmaceutical, and agrochemical industries. They are frequently used as intermediates for a vast number of bioactive compounds, especially in their optically pure form. (59) Efficient methods for their enantioselective preparation typically require sophisticated catalyst design or the use of transition metals. (60,61) Hence, chemists have turned their attention to enzymes, which have since been established as an excellent approach for the preparation and use of amines as building blocks. (364)

Figure 3

Figure 3. (top) Summary of general properties of amines, reactivity trends toward amine-selective probes, and expected cross-reactivity. (bottom) Compatible pH range and buffers in reported amine-selective assays. Bold numbers indicate the most commonly used pH values.

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To assess the progress of transformations that move amino groups intermolecularly, a probe with a significant difference in reactivity between substrate and product is required. Large differences often result from an increase of substitution and steric bulk. Primary amines and ammonia have the highest reactivity and thus can be efficiently discriminated from secondary amines and most amino acids. Tertiary amines are mostly unreactive toward conjugation due to the decreased steric accessibility. (663)
Amidases have the biggest potential to benefit from these chemical probes because they release amino compounds as products. The strong nucleophilicity of this functional group creates possibilities for electrophilic reactions in addition or substitution reactions. Cross-reactivity in biocatalytic studies can be expected with other nucleophiles present (e.g., thiols, cyanides, and, if not targeted themselves, amino acids). The rate of those side reactions depends on the actual substrate, the conjugation partner, pH, temperature, and buffer composition.
Amines are generally mildly basic, and reactions with aliphatic amines are thus confined to a pH range of approximately 8–10 to avoid deactivation by protonation. Aromatic amines can be selectively targeted in neutral or slightly acidic regimes because their pKa values are generally lower than those of aliphatic analogues (4.5 vs 9). (664)
Amine-containing buffers (e.g., Tris; tris(hydroxymethyl)aminomethane) must be avoided when using amine-reactive probes because the rate of the reaction with the buffer would greatly exceed that with amino groups in the product (Figure 3).
Commonly used enzymes to yield amine-containing compounds, include amidases, (62) lipases, (63,64) transaminases (TA), (65) reductive aminases (RA), (66,67) imine reductases (IRED), (68) monoamine oxidases (MAO), (69) and lyases. (70) Several studies have described the potential of these catalysts (Scheme 2). In a seminal study, Savile et al. applied substrate walking, modeling, and a mutational approach to establish a highly potent transaminase for the synthesis of the antidiabetic compound sitagliptin 3 (Scheme 2a). (71) Their best variant included 27 mutations and converted the ketone 2 to the chiral amine 3 at 200 g/L substrate loading in 50% DMSO, with a 92% assay yield and full enantioselectivity. Challenged with the sterically substantially hindered bicyclic amine 5, Weiß et al. applied protein engineering for the optimization of a fold class I ((S)-selective amine transaminase). (72) Saturation mutagenesis and two rounds of directed evolution facilitated the kinetic resolution of the racemic substrate 4 in excellent yield and full stereocontrol (Scheme 2b). Interestingly, reactions performed with the prochiral ketone as starting material determined the mutant to be unsuitable for direct asymmetric synthesis. Machine-directed evolution was applied by Novartis researchers as a potent enzyme engineering strategy by employing a moderately stereoselective imine reductase as the model system. (73) One screening cycle already yielded a library of highly active mutants with a dramatically shifted activity distribution compared to that of the traditional directed evolution methodology. Using their most selective and active mutant, the authors synthesized the H4 receptor antagonist ZPL389 7 in 72% yield with >99% ee (Scheme 2c). Smart rational engineering of amidase AJ270 from Rhodococcus erythropolis by the group of Wang enabled the desymmetrization of heterocyclic meso-dicarboxamides by generating only 10 variants. (74) Using their biocatalytic platform, they enabled access to both antipodes of O,N-heterocyclic and carbocyclic dicarboxamides 9 in excellent yields and full stereocontrol (Scheme 2d). Another protein engineering example for amidases was delivered by the group of McLeish. (75) A comparison of several members of the enzyme family inferred that residues involved in substrate recognition were not conserved. Substitution of position G202, crucial to substrate binding, to slightly bulkier alanine or valine residues, prompted a dramatic decrease in the kcat/KM value of mandelamide 10a with concomitant increase in lactamide 10b activity, effectively transforming the biocatalyst into an aliphatic amidase (Scheme 2e). Given the scope of this review, however, only a subset of the stated biocatalysts can be efficiently targeted using the presented methodology.

Scheme 2

Scheme 2. Representative Selection of Enzymatic Transformations for the Production of Amine-Containing Products
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6.1. Nucleophilic Aromatic Substitution (SNAr)

SNAr reactions of para-substituted electron-poor aromatic systems enable the emergence of an optical signal (Figure 4). The change in absorbance (or fluorescence) is commonly explained by the formation of electronic “push-pull” structures: The incorporation of electron-donating groups (EDGs) by reaction with the amine and the pre-existing electron-withdrawing groups (EWGs) in the para position enable ICT processes. (76)

Figure 4

Figure 4. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on nucleophilic aromatic substitution (SNAr) with aromatic molecules containing electron-withdrawing groups (EWG). Abs: wavelength typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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The most common leaving groups are halides (Cl–, F–) and sulfonates (SO3–); fluorides are most reactive. Historically, this concept was used by Sanger for the detection of amines in insulin (Sanger’s reagent: 2,4-dinitrofluorobenzene, DNFB). (78) Nitrated aryl systems have high absorption coefficients and led to the development of nitrobenzoxadiazole (NBD) reagents. Ghosh et al. were the first to report the use of NBD-Cl 12 as a fluorogenic nucleofuge and noticed different optical signatures depending on the reaction partner. Whereas primary and secondary aliphatic amines formed highly fluorescent products, reactions with O, S, and N-Ar nucleophiles only gave weak fluorescence. Tertiary amines were found to be unreactive. (79) Watabe et al. switched to the fluoro-derivative of NBD, which is 50–100-fold more reactive than NBD-Cl, for the detection of hydroxyproline and proline in blood plasma. (80) Recent studies used NBD-Cl 12 for the detection of l-ornithine (81) and NBD-F 13 for the derivatization of Alendronate. (82) In a recent study, Annenkov et al. showed that NBD-Cl can be a viable derivatizing agent even for tertiary amines at room temperature, albeit in dioxane. (77) The substituted products are believed to undergo subsequent Hofmann elimination or substitution by the expelled chloride ion and form a mixture of highly fluorescent NBD adducts. Application in aqueous systems was not investigated.
NBD-fluorophores, with excitation maxima between 460 and 490 nm, are often incompatible with other green-fluorescent reporters, such as the broadly used green fluorescent protein (GFP) with an excitation maximum at 488 nm. To circumvent this overlap and thus improve their usefulness in in vivo studies, Benson et al. substituted the oxygen in the ring with a quaternary carbon (SCOTfluors 14a–14e, Figure 5). These probes showed tunable emissions in the near-infrared (NIR) spectrum under physiological conditions and were applied for in vivo cell imaging. (83)

Figure 5

Figure 5. Structures and spectral properties of SCOTfluors 14a–e. aValues determined in EtOH. bQY (quantum yield) in dioxane using acridine orange, fluorescein, rhodamine 101, and Cy5 as standards. Reproduced with permission from ref (83). Copyright 2019 Wiley-VCH under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/.

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Another historical example of an SNAr reagent is 2,4,6-trinitrobenzenesulfonic acid (TNBS 15), which was first described by Okuyama and Satake in 1960. (84) Formation of aniline derivatives upon reactions with aliphatic amines can be traced by observing intense orange bands. Even though it is explosive, TNBS has still found broad use in protein modification of terminal amino acids because it selectively reacts with primary amines. (85−88) Recently, Olivero et al. used the reagent in combination with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA, 24) (cf. section 6.3) for the quantification of proteins bound to nanoparticles. (89) With the emergence of new fluorogenic scaffolds, Kim et al. incorporated the amino-responsive function into the BODIPY fluorophore. (90) Upon reaction with lysine residues, the excitation wavelength of the BODIPY derivative 16 shifts from 525 to 409 nm and could be used for fluorescent labeling under mild conditions (37 °C, 2 h) (Figure 6).

Figure 6

Figure 6. Labeling scheme of lysine residues in protein with 16. (a) Normalized absorbance spectra (dashed-lines) and fluorescence emission spectra (solid-lines) of 16 (1 μM) and lysozyme labeled with 16 (5 μg/mL lysozyme, 2 h incubation at 37 °C), measured in HEPES buffer (10 mM, pH 7.4, 0.1% dimethyl sulfoxide). The emission spectra were obtained with excitation at 485 and 375 nm, respectively. The inset photo was taken under UV light (365 nm). (b) A plot of time-dependent (0–4 h) fluorescence intensity at 409 nm. The excitation wavelength was 375 nm. Inset box: lysozyme activity calculated using an assay kit at 2 h incubation point. Means and standard deviations were calculated from triplicate measurements. Reproduced with permission from ref (90). Copyright 2017 Korean Chemical Society; Wiley-VCH.

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6.2. Michael Addition (Type) Reactions

As functional group probes, Michael acceptors make use of the nucleophilicity of amines in neutral and basic aqueous solutions. Most probes of this type (Figure 7) undergo a Michael addition and subsequent β-elimination: upon 1,4-conjugate addition of the amine, the leaving group is expelled, forming a stable enamine.

Figure 7

Figure 7. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on Michael addition type reactions. Abs: wavelength typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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A prominent example is epicocconone 17 (Figure 8), a yellow dye isolated from the fungus Epicoccum nigrum. (91) Elucidation of the structure enabled its application for the detection and staining of primary amines. (92) The conjugate that forms upon reaction with lysine has a high molar absorptivity (104 M–1cm–1), strong orange to red fluorescence (λem = 610 nm), and a large Stokes shift (100 nm). The stability of the enamine depends on the pH and is highest at 2.4. (92) The derivatization is reversible at neutral pH, which enabled the analysis of proteins by staining gels after electrophoresis. (93) Substitution of the heptatriene chain in 17, especially with a p-chloropyridyl functionality, increases fluorescence yields without impacting the performance in staining. (94)

Figure 8

Figure 8. Epicocconone 17 is the active ingredient in Deep Purple Total Protein Stain and is responsible for the apparent noncovalent staining of proteins in polyacrylamide gels and electroblots. The reaction of 17 with amines has shown that 17 reacts reversibly with primary amines to produce a highly fluorescent enamine that is readily hydrolyzed by base or strong acid. Such conditions are used in postelectrophoretic analysis, such as peptide mass fingerprinting or Edman degradation. Reproduced with permission from ref (92). Copyright 2005 American Chemical Society.

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Fluorescamine 18, an intrinsically nonfluorescent reagent, is another commonly used probe for the selective derivatization of primary amines. (25) Although secondary amines and alcohols react with the probe, they do not form fluorescent products. Furthermore, the reaction with alcohols is reversible and thus tolerated in amine screenings. (95) Unfortunately, due to the poor hydrolytic stability of the reagent, an excess must always be used to achieve reliable derivatization, (96,97) but because the hydrolyzed product of 18 is not fluorescent, this assay enjoys high popularity. The maximum in reactivity and fluorescence appears at pH 9, but assays performed at pH 6–8 still provide acceptable results with aliphatic amines.
Acetic acid can be used as a reaction medium to quantify aromatic amines, as shown by Rinde and Troll. (35) Aromatic amines remain unprotonated even in this acidic milieu, making selective detection in the presence of aliphatic amines possible. Two recent studies applied this reagent in HTS: Murugayah et al. used 18 to detect activity of N-acyl-l-homoserine lactone (AHL) acylase, (98) and Ashby et al. used it to detect the interaction between proteins and nanoparticles (changes in the structure of the studied proteins caused a change in fluorescence, which made conformational changes upon interactions with nanoparticles detectable). (99)
1,2-Naphthoquinone-4-sulfonate (NQS), also called Folin’s reagent 19, is one of the most commonly utilized chromogenic reagents for the determination of illicit drugs and pharmaceuticals containing aliphatic or aromatic amines. (100) The reaction of NQS with amines forms an orange dye, which is stable over a broad pH range (2–11). The rate of the reaction is independent of the pH but impacted by the amine substrate. (101) Most assays are complete in less than 20 min (at pH 9–10). (102) Besides amines, it is also possible to detect sulfamides and tetrazoles.
Ylidenemalononitriles (compounds 20a–c) are another useful group of compounds for amine assays, as shown by Longstreet et al. (103) They are weakly fluorescent, but upon reaction with (some) primary amines form cyclic amidines with a 900-fold increase in emission intensity. Several derivatives were synthesized (Figure 9, 20a–20c), spanning the spectral excitation (λex = 390–510 nm) and emission windows (λem = 500–555 nm). Their use was demonstrated in the derivatization of a human transferrin glycoprotein at pH 7, albeit in 50% DMSO.

Figure 9

Figure 9. Normalized emission spectra of ylidenemalononitrile enamines 20a–c in CH2Cl2 at room temperature. Insets show photographs of 1 mM solutions of 20a–c in CH2Cl2 irradiated at 365 nm. Reproduced with permission from ref (103). Copyright 2014 American Chemical Society.

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A recent example of a red-emitting probe, PMB 21, was reported by Sathiskumar and Easwaramoorthi. (104) In this compound, phenothiazine and 1,3-dimethylbarbituric acid are connected via a methylene bridge. Fluorogenic properties of the probe are observed upon reaction with several primary aliphatic and electron-rich aniline systems. The described mechanism suggests that addition of the amine leads to the cleavage of the barbituric acid moiety and the formation of phenothiazine-conjugated imines. Higher substituted amines and deactivated aromatic rings did not give any signal. The strong dependence of the fluorescence emission on the reaction partner can be explained by large differences in ICT processes in the products. Compound 21 was used for the detection of 3-amino-l-tyrosine in buffered medium.

6.3. Isoindole Formation

Another approach for the detection of amines is entrapping them in an isoindole structure (Figure 10). In a double-condensation reaction, o-phthalaldehydes, such as compound 22, and amines form highly reactive o-quinoid structures. (24,30) Initial attempts to form isoindolines used reducing agents, e.g., 2-mercaptoethanol (ME) or bisulfite, (105) to “stabilize” these products. Analysis of the highly fluorescent conjugates, however, revealed that 1-substituted isoindoles are formed, their emission depending on the structures of the parent dialdehyde and trapping nucleophiles. Practical trapping agents include aliphatic thiols (e.g. ME, cysteine) sulfites, or cyanide. In consequence, this multicomponent system can also be used for the detection of thiols or cyanides. Especially the electron-withdrawing CN-substituent is believed to stabilize the resulting conjugates. (106) The addition sequence of the reagents is crucial for the intensity of the fluorescence emission. (24) For instance, addition of the thiol before the amine was beneficial (but not necessary) for strong fluorescence. Amino acid conjugates gave a strong fluorescent signal in the range of pH 7–12. The reactions, however, were typically performed above pH 8 to enable a broad spectrum of amines to be detected. With thiols as nucleophiles, a substantial amount of thiolate (pKa ∼ 11) is required. (30) Generally, sterically accessible amines react fastest with the isoindole probes, reducing the derivatization time to several minutes for simple primary amines. (111,112) The fluorescence emission, however, depends on multiple factors, including pH, the trapping nucleophile, the detectable amine, and even temperature. Ammonia generates significant background emission: its conjugates are 100–1000-fold more emissive than those of amines. (107)

Figure 10

Figure 10. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on isoindole formation with o-phthalaldehyde derivatives and o-diacetylbenzene. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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Enlarging the π–electron system, e.g., with naphthalene as in NDA 23, or analogues, (108,109) is generally accompanied by a large red-shift and increase in sensitivity. A systematic study by de Montigny et al. (106) revealed a strong dependence of the Stokes shift and the quantum yield of the conjugates on the trapping nucleophile. While reactions with 22 were ideally performed with ME, 23 was shown to be practically nonfluorescent with this thiol donor. Cyanide can be used with both dialdehydes, but premixed solutions of 23 with cyanide were prone to decomposition; they had to be prepared separately. Reactions with amino acids were shown to proceed fastest between pH 8.5 and 10, with a maximum at ∼9.5 (23/CN detecting alanine).
CBQCA 24 (89,665) and 5-furoylquinoline-3-carboxaldehyde (FQ, 25) (666−668) are additional analogues for the same class. FQ is several orders of magnitude more sensitive than its parent compound OPA 22, or fluorescamine 18, in the quantification of bovine serum albumin (BSA), but long incubation periods are necessary (90 min for FQ, compared to 30 min for 22). Compound 24 has been used to quantify proteins bound to nanoparticles in HTS by the group of De Crescenzo. (89) Hapuarachchi and Aspinwall used a combination of 22 and SAMSA-F, a thiol-bearing fluorescein derivative for the labeling of primary amines and amino acids. (110) Although their method required the subsequent separation of the tagged amines from the unreacted probe by capillary electrophoresis, it showed how mechanistic understanding can be used in design. Recently, Medici et al. showed that the ketone analogue of 22, o-diacetylbenzene (DAB, 26), can efficiently differentiate between primary amines and their corresponding amino acids, thus enabling HTS of amino acid decarboxylases. (32) As with 22, ME was used as the trapping nucleophile. Given that the resulting isoindole does not condense to a fully aromatic product, a study by Choi et al. attempted to elucidate the structure of the actual fluorophore. (33)
The presented probes have been used for detection of amines, amino acids, and even ammonia as nitrogen sources in cellular systems. Examples using compound 22 include screening for activity of: (i) catalase, measured via the H2O2 triggered depletion of GSH, (113) (ii) nitrilase, by detecting ammonia, (114) (iii) aminotransferase, (115) by ME-catalyzed formation of fluorescent pyrazino-diisoindole-diones 27 (Scheme 3). Unexpectedly, ME was not incorporated into the product but was nevertheless needed as a catalyst. The fluorophore exhibited a blue-shifted excitation maximum at 410 nm, only observed with vicinal diamines; other diamines did not show measurable fluorescence upon excitation at this wavelength.

Scheme 3

Scheme 3. Reaction of 22 with Vicinal Diamine Resulting in the Formation of Pyrazino-diisoindole-dione 27
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6.4. Amide Formation via Activated Ester Cleavage

Activated esters are the synthetic chemist’s mimic of nature’s highly reactive thioester or acyl phosphates used for selective amidations. In recent years, many probes were developed by incorporating metastable leaving groups, such as mixed anhydrides, NHS esters (N-hydroxysuccinimide), (116) pentafluorophenyl (PFP)-, (117) or thiophenols, (118) into established fluorescent molecules (e.g., fluorescein, (119) coumarin, (120) BODIPY, (121)etc.; Figure 11). (122) The majority of such designs modify the molecular structure of the dye in a region distant from the fluorophore core, which typically does not significantly change the spectral properties of the dye and thus requires the separation of the conjugate from any unreacted probe after the reaction. (123−126) Conversely, the incorporation of the reactive handle directly into the fluorophore modulates the fluorescent emission and led to the development of reactive turn-on probes.

Figure 11

Figure 11. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on amide formation using activated esters. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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Typically, the turn-on effect is achieved by liberating fluorogenic carboxy or hydroxy moieties from their initial state, when they are trapped as reactive anhydrides, carbonates, or labile esters. Two examples for ratiometric turn-on probes: (i) 6-(dimethylamino)naphtho[2,3-c]furan-1,3-dione (ANH, 28) (127) and derivatives thereof (128) as well as isatoic anhydride analogues. (129,130) ANH has great potential as an environment- and solvent-sensitive turn-on fluorophore, but it is only weakly fluorescent in water. An increase in fluorescence was only observed upon conjugation to apolar protein domains. To tackle the challenge of poor aqueous solubility of a majority of apolar turn-on-based (NHS-) probes, the group of Ogle derivatized fluorogenic isatoic anhydride 29 with a quaternary ammonium handle. (130) This permanently charged group substantially increased the solubility in water, no longer requiring cosolvents for conjugation, e.g., with BSA. They furthermore demonstrated the efficiency of their probe using a rapid-injection NMR experiment: a 10-fold excess of n-butylamine completely reacted with their probe in less than 5 s.
All reagents of this type, however, were unstable toward hydrolysis, with half-lives of 16 h (pH 7) and 2 h (pH 8.4). This reactivity is generally expected of any activated ester, as hydrolysis under basic conditions always competes with the desired reaction.
Similar to the well-established methods for amine conjugation using cleavable moieties, the group of Kim adapted their meso-carboxyBODIPY 30 probe and achieved a remarkable turn-on effect upon reaction of its activated acid, (131) initially with the NHS- 30a and PFP 30b esters (Figure 12) and later also with thioester 30c. (132) Using their PFP derivative 30b, they showed a 3000-fold increase in fluorescence upon reaction with MeNH2 under physiological conditions in less than 5 min. A similar rate was observed for simple primary aliphatic mono- and diamines. An increase in steric bulk caused a substantial decrease in reactivity and required reaction times of 60 min to reach similar turn-on factors (e.g., 900-fold with cyclohexylamine). Tertiary and aromatic amines were unreactive. Compound 30a produced a strong fluorescent signal upon conjugation to lysine or arginine after 5 min under physiological conditions. The other canonical amino acids only gave a weak signal, likely due to a steric effect. Compound 30b was not tested with amino acids.

Figure 12

Figure 12. (top) Synthesis of fluorogenic meso-active-ester-BODIPYs 30a/30b and their conversion to fluorescent meso-amide-BODIPY products upon reaction with amines. (bottom) Absorption (a) and emission (b) spectra of 30b (10 μM) upon treatment with MeNH2 (20 μM) in CH3CN as a function of time (0–5 min) at 25 °C. λex = 470 nm. (inset) Relative fluorescence intensity at 546 nm as a function of incubation time, in the absence (red) and the presence (green) of MeNH2. (c) Relative fluorescence intensity at 546 nm as a function of [MeNH2] (0–50 μM). (inset) Calibration curve of fluorescence intensity at 546 nm vs [MeNH2] (0–6 μM). (d) Photographs of 30b (20 μM) upon addition of MeNH2 (left to right: 0, 3, 5, 10, 20 μM) under ambient light (left) and 365 nm UV irradiation (right). Incubation time = 5 min. Reproduced with permission from ref (131). Copyright 2020 American Chemical Society.

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Figure 13

Figure 13. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on pyridinium salt formation with pyrylium salts. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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NHS-derivative 30a has remarkable hydrolytic stability at pH 7.4, with a half-life of 2.5 d at 25 °C. Interestingly, changing its reactive moiety to a thioester caused a large decrease in reactivity: It would only react with cysteine, subsequently undergoing a reaction cascade known as native chemical ligation (NCL; intermolecular conversion of a labile thioester to a stable amide) and eventually showing turn-on behavior. As with the 8-SMe–BODIPY 16 (cf. section 6.1), only amino or amide-conjugates gave the desired increase in fluorescence; esters or thiol ethers remained nonemitters. The importance of the correct placement of amino groups on the BODIPY core was highlighted in a paper by Esnal et al. (133) Other dyes bearing such reactive groups could potentially give a similar turn-on behavior upon cleavage of the fluorescence-concealing group, but they have yet to be explored (Figure 14). (134)

6.5. Pyrylium Salt Formation

A fascinating approach for the selective detection of amines builds on initial reports by Katritzky (135) from 1984. The reaction of his, from then on eponymous, pyrylium salts with amines, forming stable pyridinium salts, was taken up by Wetzl et al. (136) Twenty years later, their group modified the pyrylium moieties with color-forming p-anilino substituents (31 and 32), which resulted in a new class of chromogenic labels, the so-called “Chameleon” labels (Figure 13). The class, although not limited to pyrylium salts, generally shows strong bathochromic shifts upon conjugation. Due to their straightforward synthesis from the corresponding aldehydes and methyl pyrylium salts, they have already been applied in HTS as chromogenic reagents for the detection of biogenic amines (Figure 14). (137,138)

Figure 14

Figure 14. Image of a Py-1 31 sensing microplate reacted with various concentrations of histamine (the numbers indicate the concentrations of histamine in μg mL–1). Reproduced with permission from ref (137). Copyright 2011 The Royal Society of Chemistry.

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Figure 15

Figure 15. Colors under daylight and the colors of fluorescence in solution of pH 7.0 under a UV lamp (365 nm) of label 31 and of some other Py labels when conjugated to serum albumin at 37 °C. Note that label 33 does not react but remains blue (at any concentration). Reproduced with permission from ref (141). Copyright 2011 The Royal Society of Chemistry.

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Although Py-1 31 is the most widely used derivative, (138−140) many other spectral bands are addressable by modification of the aromatic core as exemplified by the benzothiazole derivative 33 (Figure 15). The reactivity of those salts could furthermore be fine-tuned by modifying the substituents of the pyrylium moiety. (141) Primary amines and amino acids (or proteins containing terminal amino groups) could be efficiently labeled at physiological conditions in less than 30 min. Anilines required reactions to be run at 50 °C and in organic solvents. Reactions with secondary amines or ammonia did not form colored products and thus did not interfere with the assay. These probes were stable in acidic solution (pH < 5) but decomposed quickly even at slightly basic pH. The hydrolysis products, however, also did not interfere with the optical read-out. (142)

Figure 16

Figure 16. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on the formation of thioureas or 2-aminothiazoles from isothiocyanates. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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6.6. Thiourea or 2-Aminothiazole Formation

Isothiocyanates (ITCs) have long known to be reactive toward a great number of nucleophiles and have been used for the synthesis of stable thiocarbamates and thioureas (Figure 16). (143,144) Fluorescein isothiocyanate (FITC) is used for protein labeling, relying on the strong nucleophilicity of amines (compared to alcohols or water). It has disadvantages similar to the active esters of fluorescein discussed above: relatively small changes in the electronic structures upon conjugation require separation of the tagged amines from the unreacted label. (145,146) Thus, only a few examples of efficient use as turn-on probes have been reported.

Figure 17

Figure 17. (top) Fluorescence spectra of unbound 9-ITPPo 35 (blue) and its covalent adduct ATAZPo (red) with BSA (a) and gold nanoclusters (AuNCs) (b). (bottom) Absorbance of a mixture of 35 (15 μM) and n-butylamine (3 mM) in toluene: Structures, time curves of concentration, and spectral profiles of the reactant, the intermediate, and the product (from left to right). Reproduced with permission from ref (148). Copyright 2015 The Royal Society of Chemistry.

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The group of Belfield, experienced in the synthesis of fluorene-based dyes, reported the synthesis and simple modification of isothiocyanate dyes, such as compound 34, that exhibited a small hypsochromic shift upon conjugation with an amine. (147) Using their probe, they performed the exemplary derivatization of reelin, a large extracellular matrix glycoprotein, at physiological pH. Although in these experiments the unreacted fluorophore was removed after the reaction, they could show a large increase in quantum yield upon thiourea formation, in DMSO.
Recently, Planas et al. used another prominent scaffold, porphycene, as a fluorogenic sensor. (148) They modified it (9ITPPo 35; Figure 17) to give a 70 nm Stokes shift to the near-IR upon conjugation, enabling spectral discrimination from the unreacted probe. Isolation of the reaction products of 35 with several primary, secondary, and even aromatic amines revealed that the underlying cause for the color change was a cyclization of the initially formed thiourea into a 2-aminothiazole fragment. Although synthetic methods for this type of cyclization are known, (149,150) the spontaneous cyclization, particularly for these scaffolds, had previously not been reported. (151) For a proof of concept, derivatizations of BSA, and amino-functionalized gold nanoclusters were performed in carbonate buffer at pH 9.2 and in EtOH. Although the first step (formation of thiourea) was completed in less than 15 min, the fluorescence continued to shift significantly due to the rate-limiting cyclization, which required prolonged reaction times. Biocompatible auxiliaries to the cyclization might thus lead to the development of new turn-on methods based on these ITC scaffolds.

Figure 18

Figure 18. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on hemiaminal, aminal, or Schiff’s base formation. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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6.7. Hemiaminal, Aminal, or Schiff’s Base Formation

Another approach for the detection of amines is based on the disruption of electron-withdrawing properties of carbonyl, imine, or imino groups (Figure 18). When amines add to such functional groups and form stable hemiaminals, they tend to induce a hypsochromic shift of conjugated electron systems. (282) In some cases, further elimination (of water, alcohols, or amines) to imines or iminium salts is favored, and these transformations have also been shown to result in changed spectral features.

Figure 19

Figure 19. Amine-sensing panels of 37 (10 μmol L−1) in water: (1) 37; (2) butylamine; (3) tert-butylamine; (4) benzylamine; (5) cyclohexylamine; (6) ethylene diamine; (7) 1,3-diaminopropane; (8) cadaverine; (9) morpholine; (10) ephedrine; (11) 4-aminopyridine; (12) ethanolamine. Reproduced with permission form ref (154). Copyright 2012 Wiley-VCH.

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The first turn-on probes based on the formation of Schiff’s bases were reported by the group of Glass in 2003: (152) They used coumarin aldehydes, such as compound 36, for the detection of amino acids and primary amines. By mimicking chelation-based metal sensors, based on the protonation of the formed imine (Scheme 4), they achieved a 26-fold increase in fluorescence at pH 7.4 (buffered) with glycine as the target. Water-soluble analogues containing aliphatic tertiary amine groups as well as carboxylic acid functionalities were also synthesized but not yet spectroscopically characterized. Alkylation of the 4-position of the coumarin was found to be necessary for a large change in absorption: the imine adopts a trans-configuration due to steric repulsion.

Scheme 4

Scheme 4. Sensing Mechanism of 36: Chelation of the Formed Iminium Species by the Coumarin Moiety Triggers a Turn-on Response (152)
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A second example emerged from the experience with cross-shaped (“cruciform”) fluorophores by the group of Bunz. (153) Based on initial designs of such dyes, Kumpf and Bunz synthesized the water-soluble, aldehyde-appended distyrylbenzene 37, which showed strong turn-on behavior in aqueous systems. (154) Their reaction with a broad set of (biogenic) primary and secondary amines as well as amino acids at μM concentrations yielded different Stokes shifts upon the formation of hemiaminal or imine structures, with emissions in the range from 450 to 520 nm. Among the tested amino acids, only cysteine, arginine, and lysine gave an increase in fluorescence. The signal strength was strongly dependent on the amine. Whereas morpholine exhibited merely a 10-fold turn-on factor, the reaction with benzylamine or ethylenediamine caused a ∼200–300-fold increase in fluorescence. Compound 37 and its aminal and imine products were stable in neutral aqueous solution.
A recent example of the use of Schiff’s base formation was reported by Leslie et al. (Scheme 5). (34) This study, aiming to optimize their previously reported chemodosimeter for the detection of ozone, revealed that the intermediately formed β-aryloxy fluorescein aldehyde 38 undergoes β-elimination, which is rate-determining and catalyzed by amines. The addition of pyrrolidine increased the rate 30-fold and drove the reaction to completion in less than 3 min; the uncatalyzed reaction took 1.5 h. From a selection of cyclic secondary amines, they identified azetidine as the best catalyst. Anilines were excluded from the reaction screening due to their known reactivity with air and ozone. The authors speculated that the efficiency of the catalyst was primarily dependent on steric effects and the pKa of the amine. The rate enhancement was greatest at pH 6 to 7, and the elimination product of 38 showed the highest fluorescence between pH 5 to 9. However, due to the focus of the study on detecting ozone, use of 38 as an amine-selective probe was not investigated.

Scheme 5

Scheme 5. Ozone Sensing Mechanisms Employing 38. (34)
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Another reagent relying on the nucleophilic attack of the amine onto an electrophilic center was developed by Wu et al. (669) Their synthetically readily accessible iminium dye BI-CA-ID 39 effectively recognized primary amines upon reaction by ratiometric discoloration of the dye solution and emergence of a blue-shifted fluorescence emission with a peak at 382 nm. The reaction with primary and secondary amines was reported to be instantaneous. Completely discoloring of sample solutions at 40 μM in less than 1 min (containing 800 μM amine) in 50% ACN (acetonitrile)/water mixtures was observed. Even tertiary amines were reported to be viable for the assay, when allowing reaction times of up to 10 min. Aromatic amines were reported to be completely unreactive toward the probe.
Trifluoroacetyl probes (e.g., 40 and 41) are another class of compounds not yet proven to be applicable in aqueous systems but with frequent occurrence in literature. Although we generally limit this review to reactive probes for aqueous solutions, these compounds deserve an honorable mention. Initial reports by Mohr already exemplified the usefulness of these dyes as potent fluorogenic sensors for amines in organic solvents. (155−157) At the core, stilbene or azobenzene motifs are responsible for the spectral features of the dyes; they have since then been modified into even larger delocalized structures. (158) Their kinetics and thermodynamics have been studied, (159) they have been turned into star-shaped, tripodal fluorophores with variable reactive trifluoroacetyl reactive sites, (160) and incorporated into a BINOL-scaffold for the detection of diamines. (161−163) A demonstration of their viability in aqueous systems is yet to be published.

7. Chromo- and Fluorogenic Probes for Alcohols

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Alcohols are crucial building blocks for the manufacturing of agrochemicals, flavor and fragrance compounds, and pharmaceutical drugs, especially chiral alcohols in their optically pure form. Typical methods to prepare them enantioselectively typically require the use of expensive, complicated chiral catalysts or transition metals, which can be toxic. (164−166) Biocatalytic approaches are considered an effective and green alternative due to their mild reaction conditions and remarkable enantioselectivity. A recent review by Chen and de Souza highlighted the recent developments in the field. (167) Although a large variety of biocatalysts is available for the formation of chiral alcohols, only few are used in combination with functional group probes. One possible explanation is the relatively low reactivity of alcohols, which impedes selective targeting, especially in an environment where many competitive, strong nucleophiles are present. Below, we highlight a few pertinent examples, chosen from the many success stories of applications and optimization of such enzymes.

Figure 20

Figure 20. Summary of general properties of alcohols and expected cross-reactivity (bottom).

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Nature offers a broad repertoire of catalysts for the synthesis of chiral alcohols (Table 1). (168) Using directed evolution, Zhang et al. achieved the reduction of 1-(4′-chlorophenyl)-1-(pyridine-2′-yl)-methyl ketone (CPMK, 42; Scheme 6a). (670) They identified a crucial mutation (S273A) among a library of 2000 clones using the colorimetric DNPH 109 assay to quantify unconverted starting material. In an attempt to identify new enzymes based on sequence similarities, the group of Pohl discovered a new (R)-selective HNL from Arabidopsis thaliana. (170) This enzyme converted a wide range of benzaldehydes, and aliphatic aldehydes and ketones, to the corresponding cyanohydrins and was established as an alternative to other known (R)-selective HNLs. For instance, the enzyme formed the cyanohydrin of 2-hexanone 44 with 95% ee (48% conversion; Scheme 6b).
Table 1. List of Enzyme Classes for the Enantioselective Synthesis of Chiral Alcohols
catalystactivity
alcohol dehydrogenases (ADH) (171) and ketoreductases (KRED) (172)asymmetric reduction of carbonyl compounds
α-ketoacid carboligases (173) or aldolases (174)decarboxylative or regular aldol reactions
hydroxynitrile lyases (HNL) (175)addition of cyanide to aldehydes forming cyanohydrins
(Michael) hydratases (176)addition of water to unactivated or α,β-unsaturated compounds
epoxide hydrolases (177)enantioselective hydrolysis of epoxides affording vicinal diols
oxidases (178)oxidative functionalization of C–H bonds
haloacid dehalogenases, (179) haloalkane dehalogenases (180) or halohydrin dehalogenases (181)hydrolysis of 2-haloalkanoic acids, halogenated aliphatics, or vicinal haloalcohols
lipases (182) or esterases (183)(dynamic) kinetic resolution of racemic mixtures

Scheme 6

Scheme 6. Representative Selection of Enzymatic Transformations for the Production of Alcohol-Containing Products
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Chen et al. tackled the challenging enzymatic Michael addition of water motivated by initial reports for the formation of (S)-3-hydroxy-3-methylfuranone 47 from 3-methylfuran-2(5H)-one 46 using Rhodococcus rhodochrous ATCC 17895. (184) They compared six closely related Rhodococcus strains and found similar activities for the transformation with all six bacteria. Optimization of the process parameters of their initially studied strain enabled the formation of 47 in 82% isolated yield and 95% ee on gram scale (Scheme 6c).
Lehwald et al. reported the first asymmetric intermolecular carboligation of an aldehyde and a ketone using a ThDP-dependent flavoenzyme from Yersinia pseudotuberculosis (Scheme 6d). (185) Their catalytic system overcame the known hurdle of low electrophilicity and increased steric bulk of ketones (e.g., 48), which typically hinders aldol reactions with these substrates. The carboligase is promiscuous regarding the substrate, accepting a broad range of cyclic and open-chain ketones, diketones, and α- or β-ketoesters.
An unusual example for the synthesis of chiral alcohols was reported by Hammer et al. (186) They exploited the highly Brønsted-acidic active site of a squalene hopene cyclase (SHC) from Alicyclobacillus acidocaldarius and enabled a stereoselective Prins reaction of (R)-citronellal 50. A single point mutation in the active site (I261A) facilitated the stereospecific cyclization to (−)-iso-isopulegol 51, among the four possible products (Scheme 6e).
Enzymatic late-stage modifications are a flourishing trend in total synthesis. Biocatalysts are uncontested in their selectivity, especially in transformations of highly decorated compounds. The group of Stoltz used an enzymatic approach for the late-stage oxidation of 52 at the C-7 position and screened a small library of cytochrome P450 catalysts. (187) A double mutant was shown to selectively oxidize the intermediate 52 to the corresponding alcohol 53, which was subsequently treated with Dess–Martin periodinane to form the desired natural product Nigelladine A 54 (Scheme 6f).
Many protein engineering efforts rely on indirect detection methods using coupled enzymatic reactions, summarized below. The most widely applied protocol oxidizes the produced alcohols with appropriately chosen alcohol dehydrogenases (ADHs) or alcohol oxidases (AOs), generating NADH, which can be quantified via the reduction of MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide 55, forming purple-colored formazan 56 (λabs,max = 550 nm) (Scheme 7). (188−190)

Scheme 7

Scheme 7. MTT Assay: Detection of NAD(P)H or “Reducing Equivalents” via the Reduction of MTT 55 to the Purple Formazan 56
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The oxidized products can also be directly detected using carbonyl-selective assays (cf. sections 9 and 10). Chromogenic Purpald 113 has already been used for this purpose by detecting formaldehyde (FA), for example, in the directed evolution of a terpene synthase (191) and in engineering a CYP153A35 oxidase to enhance its ω-hydroxylation activity toward palmitic acid. (192)
Luciferase can be used to further oxidize aldehydes (produced via ADH catalysis from the target alcohols) to quantify alcohol production by the emergence of a blue-green luminescence (λem,max = 490 nm). (193,194)
The drawbacks and limitations of these enzymatically coupled detection methods have motivated the development of alcohol-sensitive probes despite the known issues with selectivity in relevant reaction environments. These probes rely either on an approach similar to that described for detecting amines (nucleophilic attack on electrophilic carbonyl species, cf. section 6.7) or on coordinating metals. Unfortunately, these methods have been found broadly incompatible with aqueous media or poorly sensitive, so far impeding their application in protein engineering. The competition with other nucleophilic species, biogenic amines, but mostly water, creates too much noise in detection. Separation of the analytes from competing nucleophiles (e.g., extraction into an organic solvent) might ultimately be necessary to overcome these limitations (Figure 20).

7.1. Hemiacetal Formation

The reversible formation of hemiacetals from aldehydes or trifluoroacetyl groups can be used to detect alcohols, as the addition of the alcohol changes the spectral properties of these probes (Figure 21). This mechanism is identical for amino and carboxylate probes and will create background signals in the presence of these substance classes (cf. sections 6.7 and 11.3).

Figure 21

Figure 21. Summary of chromo- or fluorogenic turn-on probes for the selective detection of alcohols based on hemiacetal formation. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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The group of Cosa applied this method with their meso-BODIPY scaffolds 57, which become fluorescent upon nucleophilic addition of primary alcohols (Figure 22). (195,196) Conversely, amines formed nonfluorescent imines. Incorporation of electron-withdrawing substituents (e.g., CN) onto the BODIPY core and the use of catalytic amounts of p-toluenesulfonic acid strongly improved the reaction rate. Still, the reaction with water was 10 times faster, and assays had to be performed in dry organic media.

Figure 22

Figure 22. (a) Fluorescence–time trajectories following the reaction of 4.5 μM of compound 57 (R′ = Cl, R′′ = H) and 1.09 M methanol (MeOH), ethanol (EtOH), butanol (BuOH), ethylene glycol (EtyGly), water (H2O), or ethanedithiol at 21 °C in ACN supplemented with 0.333 mM of p-TsOH. (b) Apparent rate constants for various nucleophiles tested were obtained from fitting the intensity–time trajectories in (a). Reproduced with permission from ref (195). Copyright 2017 American Chemical Society.

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Sawminathan and Iyer designed the polarity-sensitive fluorescent dye 58, based on an imidazole core functionalized with a conjugated push–pull system. (197) Addition of MeOH and EtOH triggered a hypsochromic shift of the fluorescence maximum, enabling the ratiometric detection of these alcohols in ACN. Compound 58 was applied for the determination of MeOH in biodiesel and used to detect vapors with paper-based test strips (Figure 23).

Figure 23

Figure 23. Photographs showing the color changes of sensor 58 before and after the addition of methanol vapor. The images were taken under UV irradiation (365 nm). Reproduced with permission from ref (197). Copyright 2021 Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

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Mohr et al. extensively studied solvatochromic trifluoroacetyl stilbenes (e.g., compound 59), (156,198,199) which are used for the detection of amines (cf. section 6.7) and found a hypsochromic shift of absorption upon hemiacetal formation in many apolar and polar organic solvents. The reaction with EtOH could be either followed by an increase in absorbance at 360 nm or as fluorogenic turn-off probe (λex,max = 450 nm, λem,max = 570 nm) in buffered media. The fluorescent effect was only observed at concentrations of EtOH above 10 v/v%.
Takano et al. developed the trifluoroacetyl compound BC-1 60, synthesized from a lyophilized bacteriochlorin derivative. (200) This alcohol-sensitive probe fluoresces upon excitation either at 431 or 731 nm, circumventing potential optical interference from the sample. The probe was adsorbed on PVC (because the study was focused on developing alcohol-responsive membranes), giving a 2.5-fold increase in fluorescence with 25 v/v% EtOH in buffered solutions at pH 4–8.

7.2. Coordination to Metals and Chelation

Alcohols can be detected using reactions where they act as ligands, are coordinated to metals, or are chelated by conformationally rigid organic structures (Figure 24). Zhao et al. developed the probe ZR1 61, which tautomerizes upon coordination to an alcohol, (201) causing an increase in ICT and concomitant increase in purple color and fluorescence (at 605 nm). The reaction is selective for MeOH; other alcohols did not react. This selectivity was demonstrated by titrating an ethanolic solution of the probe with methanol. The presence of water was problematic: concentrations as low as 15% eradicated the signal, making an application in aqueous environments impossible.

Figure 24

Figure 24. Summary of chromo- or fluorogenic turn-on probes for the selective detection of alcohols based on the coordination to chelation centers. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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A probe developed by Roy et al. uses an aluminum species for the selective detection of MeOH. (202) Displacement of coordinating ethoxide on PPY 62 by MeOH caused a change in geometry from the tetrahedral to the octahedral complex, suppressing intramolecular PET and causing an approximately 7-fold increase in fluorescence at 405 nm. A small percentage of water (5 v/v%) was necessary to observe the emission. Higher concentrations than 5% were detrimental, leading to complete annihilation of the signal at 75% and above.
The group of Wang developed a so-called donor−π-system–acceptor (D−π–A) probe TTO 63, using click chemistry to form the triazole as a coplanar π–electron bridge between the sensing units. (203) This approach enabled the facile synthesis of several structural analogues. The emissive properties of the probe were strongly dependent on the solvent and were shown to be selective for MeOH; there was only weak fluorescence with EtOH and none with any higher alcohols. Similar to 62, an increase in water concentration was detrimental to the observed signal intensity.

7.3. Ring Opening of 3-Aminorhodanine

A new strategy for turn-on probes responsive to MeOH was reported by Kumar et al. They synthesized Schiff’s bases of 3-aminorhodanine with aldehyde-bearing fluorophores, which undergo ring-opening methanolysis at the imide C–N bond (Figure 25). The probes are typically nonemissive due to inhibited isomerization of the C═N bond and additionally active PET from the lone pair of the aldimine N atom to the fluorophore. The initially studied coumarin probe RS 64 was proven to be unreactive toward water and alcohols, including EtOH, nPrOH, and nBuOH; other nucleophiles were not tested. (204)

Figure 25

Figure 25. Summary of chromo- or fluorogenic turn-on probes for the selective detection of alcohols based on the ring-opening of 3-aminorhodanine derivatives. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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The function of the probe was demonstrated in aqueous and ethanolic solutions after 15 min reaction times, with a limit of detection (LOD) of 0.042 wt % MeOH in water. Later, the authors reported a hydroxynaphthalene analogue NRSB 65, (205) which had similar functionality and reactivity trends, except for EtOH (also detected). Incubation with increasing MeOH concentrations (0–10 wt %) in ACN demonstrated a potential for colorimetric detection with the naked eye (LOD: 0.46 wt %; Figure 26).

Figure 26

Figure 26. (a) The visible color change of 65 (5 mM) in different solvents: (i) hexane, (ii) toluene, (iii) cyclohexane, (iv) ethyl acetate, (v) chloroform, (vi) dichloromethane, (vii) tetrahydrofuran, (viii) dioxane, (ix) ACN, (x) MeOH, (xi) EtOH, (xii) iPrOH, and (xiii) nBuOH. (b) The absorption spectra of 65 in different solvents, (c) time-dependent absorption spectra (0–30 min) of 5 mM 65 in MeOH; inset: plot of optical density at 470 nm vs time. (d,e) Absorption spectra of 65 (5 mM) at different ratios of ACN:EtOH (0–10%) and ACN:MeOH (0–10%), respectively. Reproduced with permission from ref (205). Copyright 2019 Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

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The conjugate of 65 with MeOH was only weakly fluorescent. The addition of Al3+ to the ring-opened product greatly increased the emission due to chelation-enhanced fluorescence (CHEF) by inhibiting cis–trans isomerization as well as PET. Hence, both MeOH and Al3+ are necessary for a strong turn-on response.

8. Chromo- And Fluorogenic Probes for Diols

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Diols are found in many relevant compounds, including pharmaceuticals (206,207) and natural products. (208,209) They are used as precursors (206,207,210−213) in the synthesis of sulfites, sulfates, epoxides, and carbonyl compounds. (214,215) Cyclohexadiene diols have been frequently used as starting material for carbohydrates, alkaloids, prostaglandins, and terpenes. (216) The reactivity of diols is similar to that of alcohols: They can react as nucleophiles forming ethers, or esters with a carbonyl donor (Figure 27). Furthermore, they can be oxidized to carbonyl compounds or undergo dehydration or condensation reactions used in polyester synthesis. (215,217,218)

Figure 27

Figure 27. (top) Summary of general properties of diol sensors and expected cross-reactivity. (bottom) Compatible pH range and buffers in reported diol-selective assays. Bold numbers indicate the most commonly used pH values.

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Common enzymatic approaches to generate vicinal diols use epoxide hydrolases, (219) dicarbonyl reductases, (207) or dioxygenases. (220,221) Zhao et al. reported the discovery of several microbial epoxide hydrolases (EH) that enabled the diastereoselective synthesis of (R,R)-diol 67 or (S,S)-diol 69via desymmetrization of various aliphatic and aromatic meso-epoxides 66 and 68 (Scheme 8a,b). (222) Makarova et al. designed four different synthetic routes for ent-oxycodone 72. In all approaches, the initial synthetic step employed a toluene dioxygenase (TDO) to obtain a diene-diol 71 from a whole-cell fermentation with E. coli (Scheme 8c). (223)

Scheme 8

Scheme 8. Representative Selection of Enzymatic Transformations for the Production of Diol-Containing Products
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Enzymatic procedures yielding 1,3-diols include the enzymatic reduction of 1,3-diketones with Pichia farinosa IAM 4682, (224) aldol additions with aldolases, (225) or the reduction of hydroxy ketones with ketoreductases. (226) Wu et al. employed a diketoreductase from Acinetobacter baylyi for the stereoselective double reduction of ethyl 3,5-diketo-6-benzyloxy hexanoate 73 to ethyl 3R,5S-dihydroxy-6-benzyloxy hexanoate 74, which is used in the synthesis of statin drugs (Scheme 8d). They optimized the whole-cell catalytic system coexpressing diketoreductase and glucose dehydrogenase to work without added cofactors at a high substrate concentration (15-fold increase from base level). (227) Deoxyribose 5-phosphate aldolase (DERA) from Shewanella halifaxensis was used to synthesize the 1,3-diol 76a, an intermediate in the biocatalytic synthesis of islatravir 76b (Scheme 8e). In a nine-enzyme cascade, the product could be obtained in a single aqueous solution without isolation steps in 51% yield. (228)
Numerous diol-sensing assays employ boronic acid-based probes and make use of the formation of fluorescent cyclic boronate esters as a read-out. Works in this area have been reviewed extensively. (209,229−233) Other approaches are based on indirect detection, such as converting diols to aldehydes and their subsequent detection with aldehyde-sensing assays. It remains challenging to detect diols selectively. Particularly, the discrimination between carbohydrates with minor differences in reactivity and stereochemistry is problematic. (234)

8.1. Esterification of Boronic Acids

One approach to detect 1,2- or 1,3-cis-diols is to use boronic acid derivatives: They react reversibly in aqueous solution, forming cyclic boronates (Figure 28). (235) Since initial reports by Czarnik et al. and Shinkai et al., various boronic acid-based fluorescent probes have been developed. (236,237) In 1992, Yoon and Czarnik were the first to describe the use of anthranylboronic acid as a fluorescent probe for polyols in water. (237) In general, fluorescent boronic acid-based probes consist of a fluorophore as a signal unit and a boronic acid as a recognition site. (238) Probes with various fluorophores, including naphthalene, anthracene, quinoline, coumarin, cyanine, and BODIPY, have been developed. (229,230) Upon binding of the diol, a change in fluorescence based on processes, like PET, ICT, or FRET, can be observed. (209,230,233,239)

Figure 28

Figure 28. Summary of fluorogenic turn-on probes for the selective detection of diols based on the formation of fluorescent boronate esters. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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The interaction between boronic acids and diols is pH-dependent. Boronic acids (neutral) are trigonal planar, whereas the boronates (anionic) are tetrahedral; their esters behave analogously. Usually, the pKa values of the boronate esters are lower than those of boronic acids. (233)
In general, equilibrium constants of ester formation with neutral boronic acids are lower than with boronates. Therefore, common hypotheses are that (i) diol binding is favored at a higher pH value when mainly anionic boronate is present and (ii) that boronic acids with low pKa values tend to have high affinities for diols, and (iii) that a pH value higher than the pKa of the boronic acid is favorable for diol binding. There are reports contradicting these statements, indicating that not only the pKa of the boronic acid but also the pKa of the diol needs to be considered. (233,240)
Steric hindrance, buffer composition, and the solvent play a crucial role as well. Generally, diols with a small distance between their oxygen atoms bind to boronic acids with higher affinity, quantified by a small (and constrained) dihedral angle in the diol and consequently an O–B–O bond angle that favors sp3 hybridization of the tetrahedral molecule.
Boronic acids are Lewis acids and might interact with buffer components in that way, particularly if the buffer contains Lewis bases, e.g., phosphate or chloride, influencing how the probe reacts with water or diols. (233,241,242,240) Cross-reactivities might arise with functional groups such as amino alcohols, cyanides, alcohols, fluorides, α-hydroxy acids, or α-amino acids. (239,241)
Many boronic acid-based probes have been developed for various structurally different saccharides. Gao et al. developed the naphthalene- and ICT-based probe 4-(dimethylamino)naphthalene-1-boronic acid (4-DMANBA, 77) to detect saccharides in phosphate buffer at pH 7.4. The amino group acted as a donor and the boron atom as an acceptor in the ICT process. A 41-fold increase in emission intensity at 445 nm was observed upon adding fructose, which was the largest reported increase with an ICT-based probe at that time. Besides fructose, also galactose, glucose, sorbitol, and tagatose showed fluorescent responses. Moreover, the water-soluble probe could be readily synthesized in one step. (243) The authors observed that a change in the substitution pattern strongly influenced the fluorescence properties. The derivative 6-DMANBA 79 proved to be a turn-off fluorescent probe, whereas 5-DMANBA 78 is a ratiometric probe showing large changes in fluorescence intensity at 513 and 433 nm upon binding to diols. (244,245)
Zhang et al. described several water-soluble naphthalene-based probes for saccharides and observed that 5-(monomethylamino)-naphthalene-1-boronic acid (5-MMANBA, 80) and 5-aminonaphthalene-1-boronic acid (5-ANBA, 81) showed an even higher increase in emission intensity (71-fold and 66-fold, respectively) upon the addition of fructose than previously reported structural analogues. (246) From their studies of carbohydrates on the surfaces of cells, Yang et al. identified quinoline fluorophores as particularly stable and water-soluble analogues. For example, 8-quinolineboronic acid (8-QBA, 82) is a useful probe for the detection of carbohydrates in aqueous solutions at physiological pH. At a pH < 5, background fluorescence was observed. Addition of fructose led to a 47-fold fluorescence increase at pH 7.5. A fluorescence enhancement was also observed with galactose, tagatose, and arabinose. (247) The structural analogue 5-QBA 83 exhibited a similar enhancement of fluorescence. Its binding constants were higher than 82, and the probe could be applied from pH 3.5 and higher. (248)
Cheng et al. developed various fluorescent isoquinolinylboronic acid-based probes (IQBA) 84–86, some with opposite changes in fluorescence intensity depending on whether fructose or glucose was added. Weak binding of these probes to cis-cyclohexanediol and binding of some probes with methyl α-d-glucopyranose as a model glycoside was observed. (249)
Elfeky reported a probe, 4-(5-dimethylamino-naphthalene-1-sulfonamido)-3-(4-iodophenyl)butanoic acid (DNSBA 87), which, when combined with a diol quencher, showed fluorescence recovery in the presence of various saccharides and nucleosides at pH 8.2. In contrast, the binding of the hydroxylated acids citric acid, tartaric acid, and glucuronic acid prompted a decrease in fluorescence intensity. (238)
Near-infrared (NIR) probes have several advantages over emitters in the visible range, particularly no interference from the autofluorescence of biomolecules. Samaniego Lopez et al. developed a water-soluble NIR turn-on probe 88, which consisted of a tricarbocyanine, a piperazine unit as a linker, and a boronic acid moiety. They observed that the selectivity was pH-dependent: at pH 7.4, the probe had a high affinity for fructose and sorbitol but not for galactose, glucose, or mannose. Increasing the pH from neutral to basic increased the fluorescence. The probe also showed a fluorescent response upon binding to mucin, human serum albumin, and asialofetuin. (250)
Wang et al. reported the fluorescent, rhodamine B-based probe ATP-Red 1 89, which rapidly and selectively reported intracellular ATP levels using a multisite-binding strategy. In the presence of ATP, the boronic acid formed an ester with the ribose moiety of ATP, and π–π stacking between the xanthene and the adenine moieties and electrostatic interactions of the amino group with the phosphate groups took place. (251)
Huang et al. introduced a concept to improve selectivity, based on ensembles of boronic acid receptors (90). Interactions of π–electron systems and the pyridinium cation and interactions between the diols and the boronic acids led to the formation of conjugates with d-fructose, (unordered 1:1 stoichiometry) and with d-glucose (ordered 2:1 stoichiometry). The ordered complex gave strong fluorescence at 510 nm, but the unordered was only weakly fluorescent at 380 and 400 nm. Binding of the probe to d-glucose was approximately four times stronger than with d-fructose. To improve this ratio, Huang et al. added phenylboronic acid, which binds to fructose 40 times stronger than to glucose, to improve the selectivity for glucose. (252)
Another concept for carbohydrate detection with boronic acid derivatives uses the combination of a cationic viologen compound linked to a boronic acid moiety and a fluorescent anionic dye. The fluorescence of the dye is quenched by coordination to the viologen compound and restored in the presence of saccharides. Vilozny et al. applied a system composed of the boronic acid-appended viologens 3,3′-o-BBV 91/4,4′-o-BBV 92 and commercially available, fluorescent 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS, 93) for enzymatic assays of sucrose phosphorylase (E.C. 2.4.1.7) and phosphoglucomutase (PGM, E.C. 5.4.2.2). (253) The binding of the enzymatic products, fructose and glucose-6-phosphate, to the boronic acid moieties led to the recovery of fluorescence, which allowed the successful HTS of PGM inhibitors and the determination of enzyme kinetics (Figure 29). (253) Sharrett et al. showed that instead of HPTS 93, aminopyrene trisulfonic acid (APTS) 94 could also be used, which had constant fluorescence intensity from pH 4 to 10 and could be readily decorated with handles for polymerization. They applied it for the continuous detection of glucose (in the mM range) by immobilization of the APTS monomers in hydroxyethyl methacrylate hydrogels. (254)

Figure 29

Figure 29. Sensing mechanism for the selective detection of fructose or glucose-6-phosphate by 91 or 92 and viologen HPTS 93. Reproduced with permission from ref (253). Copyright 2009 Elsevier BV.

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Differentiation between diol-containing compounds remains a major challenge in the development of boronic acid-based probes. Choi et al. sought a rapid method to screen for activity in P450 mutants and, among other approaches, conducted preliminary experiments with probe 82. They found the assay incompatible with solid agar plates and not fast or sensitive enough to detect cis-diols in the nM range in solution. High noise from other diol-containing compounds caused many false-positive identifications of mutants. (23)
One approach to achieve strong binding affinity and high selectivity is to use probes with multiple recognition elements, for instance, by including several boronic acid moieties or other functional groups. (229,255,256) Using this principle, fluorescent probes for the detection of diol-containing molecules, such as glucosamine, (257) digoxin, (258) rifampicin, (258) erythromycin, (258) and d-glucose, (259) have been developed.
As previously mentioned, chiral diols are part of many intermediates, pharmaceuticals, and catalysts. Fast and straightforward (probe-based) methods to determine the enantiomeric or diastereomeric excess (ee, de) and the absolute configuration are desirable. Shcherbakova et al. developed a technique that enabled the determination of ee/de, absolute configuration, and yield of 1,2- or 1,3-diols in high throughput. The assay is based on the self-assembly of 1,2- or 1,3-diols with enantiopure tryptophan methyl ester (or tryptophanol, 95) and 2-formylphenylboronic acid (2-FPBA, 96), resulting in diastereomeric boronates that exhibit different fluorogenic properties depending on the absolute configuration. This approach was applied in determining the optical purity of chiral diols such as atorvastatin. (260,261)

8.2. Oxidative Cleavage

An indirect approach to detect diols is converting them to carbonyl compounds by oxidative cleavage with metaperiodate 97 and subsequently using a carbonyl-specific assay (Figure 30) (cf. section 9). The reaction of 1,2-diols with periodic acid or periodates leads to the formation of a cyclic intermediate, which subsequently collapses to give ketones and/or aldehydes. (217) Doderer et al. used imine-based probe 123 to detect the formed aldehydes colorimetrically. With this approach, they determined the activity of epoxide hydrolase from Streptomyces antibioticus Tü4 toward styrene oxide and 1-butene oxide. (262) Other assays are based on cleavage using stoichiometric amounts of periodate and subsequent titration of the remaining unreacted oxidant with chromogenic or fluorogenic reagents. (263,264) Wahler and Reymond demonstrated that adrenaline 98 could be used this way: it reacted rapidly with the excess of sodium periodate, forming the red dye adrenochrome 99 (Scheme 9a). (263)

Figure 30

Figure 30. Selective detection of diols based on the cleavage reaction by metaperiodate 97 and subsequent application of aldehyde sensing methodology (for aldehyde-selective probes, see section 9).

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Scheme 9

Scheme 9. (a) Oxidation of Adrenaline 98 by Metaperiodate 97, Forming Red-Colored Adrenochrome 99, Which Allows for Determination of Excess 97 (Back Titration of Diol); (263) (b) Structure of Carboxyfluorescein, Which Can Also Be Used as a 97 Indicator; The Mechanism for Its Oxidation Is Not Known
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Kirschner and Bornscheuer used the above approach in the screening of Baeyer–Villiger monooxygenase mutants for enhanced activity and enantioselectivity. The reactions formed acetic acid esters, which were hydrolyzed by an esterase, yielding a 1,2-diol that was detected. (265) Apart from 1,2 diols, this so-called adrenaline test can also be applied with 1,2-amino alcohols, 1,2-diamines, or α-hydroxy ketones. It has been used to assay lipases, esterases, phytases, and epoxide hydrolases. (263,266,267)
Titration of residual periodate was also efficiently performed with carboxyfluorescein 100 (Scheme 9b), which exhibited a decrease in fluorescence upon its reaction with residual periodate. (268) Sialic acids in glycoproteins can also be detected and quantified using periodate, as shown by Matsuno and Suzuki. They converted the released formaldehyde to a fluorescent dihydropyridine derivative with acetoacetanilide 135 (cf. section 9.4) and ammonia. N-acetylneuraminic acid, N-acetylneuraminyllactose, sorbitol, and galactitol all gave enhanced fluorescence, but d-glucose and methyl α-d-glucoside did not; this effect was explained by their slow oxidation at 0 °C. (269)
Recently, Preston-Herrera et al. developed a HTS assay for Rieske dioxygenase activity. These enzymes catalyze the stereo- and regioselective dihydroxylation of aromatic compounds. The assay was based on oxidizing enzymatically formed cis-diols to the dialdehydes with sodium metaperiodate and the subsequent reaction with fluoresceinamine 127 (cf. section 9.2), which led to a fluorescence enhancement at 520 nm (Figure 31). It was applied in HTS of toluene dioxygenase variants to broaden their substrate scope. (270)

Figure 31

Figure 31. (a) Coupled reactions employed by the fluorescence-based assay system for the detection of cis-diol metabolites using fluoresceinamine 127. (b) Concentration-dependent fluorescence response of the assay to the presence of cis-diol metabolites. All studies were performed in triplicate; all measurements demonstrated standard deviation <5%; fluorescence responses normalized to negative control ([I – I0]/I0). Reproduced with permission from ref (270). Copyright 2021 The Royal Society of Chemistry.

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9. Chromo- and Fluorogenic Probes for Aldehydes

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Aldehydes play an essential role in many areas of modern civilization, including their widespread use as food and flavor ingredients (e.g., nutty, fatty, fruity, or grassy notes), as synthetic precursors for fine chemicals and pharmaceuticals, as well as their use as valuable building blocks for the synthesis of macromolecules in the rubber and plastics industry. (671,672) Although aldehydes are highly reactive and often toxic, they are ubiquitous as metabolic intermediates of natural compounds, drugs, and xenobiotics. Volatile aldehydes, such as formaldehyde, acetaldehyde, and acrolein, are prevalent in the exhaust of internal combustion engines, in tobacco smoke, and as industrial pollutants. (634,673)

Figure 32

Figure 32. (top) Summary of general properties of aldehydes, reactivity trends toward aldehyde-selective probes, as well as expected cross-reactivity during screenings. (bottom) Compatible pH range and buffers in reported aldehyde-selective assays. Bold numbers indicate the most commonly used pH values.

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Most microorganisms use aldo-keto reductases (AKRs) or alcohol dehydrogenases (ADHs) to eliminate aldehydes, limit their accumulation, and reduce their toxicity. (674) This homeostatic machinery complicates the biocatalytic synthesis of aldehydes in whole cells. Nevertheless, the drawbacks of typical approaches of organic synthesis to make aldehydes (toxicity of metals; additional steps required for reduction to alcohols or protection; unforgiving, narrow reaction parameters) have motivated the development of enzymatic transformations in recent years. By repurposing natural enzymatic pathways, a broad set of biocatalysts has been identified and applied in the biotransformation of suitable substrates to aldehydes (Scheme 10). These pathways include reduction of carboxylic acids by carboxylic acid reductases (CAR), (271) decarboxylation of α-ketoacids by decarboxylases (KDC), (272) reduction of activated esters by acyl-ACP reductases or acyl-CoA reductases, (273) or the well-studied oxidation of alcohols by alcohol dehydrogenases (ADH) (274) and alcohol oxidases (AO). (275) These biocatalysts now rival classic methods from organic chemistry in their performance, convenience, and safety.

Scheme 10

Scheme 10. Selection of Enzymatic Transformations for the Production of Aldehyde-Containing Products
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While screening for activity in allylic oxidation, the group of Kroutil found an alkene oxidase that is capable of the biocatalytic equivalent of ozonolysis, converting substituted styrene derivatives 101 to the corresponding benzaldehydes 102. (276) Employing a different biocatalytic route toward aromatic aldehydes, Schwendenwein et al. optimized the promiscuous CAR from Nocardia iowensis to improve its activity for sterically demanding 2-substituted benzoic acid derivatives 103. (277) Screening 6000 mutants using a selective ABAO aldehyde assay (126) identified a single-point mutation (Q283P) which led to a 3–7-fold increase in activity toward o-substituted substrates.
Soh et al. used a directed evolution strategy to optimize the decarboxylation of 2-ketoisovalerate 105 catalyzed by a KDC at 60 °C. Facilitating the reaction at this temperature would enable the production of isobutyraldehyde 106 during the decomposition of cellulose in lignocellulolytic thermophiles. Random mutagenesis combined with further modeling was used to identify a mutant with a 4-fold increased half-life at the desired temperature. (278)
Improvements in catalytic utility of alcohol oxidases were achieved by tackling the problem of poor O2 solubility in aqueous media, which limits mass transfer and results in low reaction rates. By running reactions in continuous flow, the group of Hollmann demonstrated the oxidation of trans-hex-2-enol 107 to trans-hex-2-enal 108 with turnover numbers >3 × 105 using an aryl alcohol oxidase from Pleurotus eryngii (PeAAOx). (279)
The high reactivity of aldehydes, higher than many other electrophilic species, makes the detection of these compounds in physiological environments relatively simple. Their reactive nature also means that unwanted reactions occur readily, such as oxidations, reductions, and condensation reactions with cellular material. (675) Often, their volatility requires fast-reacting probes to achieve efficient detection. Additionally, formation of geminal diols at non-neutral pH needs to be taken into account; this is important, especially for the detection of formaldehyde, which exists predominantly in its hydrated form in water. (676) Most applied ratiometric assays rely on the reaction of amines or amino-derivatives with aldehydes, forming imines, hydrazones, or oximes. These primary conjugates are often converted further to stable heterocycles via intermolecular reactions, irreversible rearrangements, or oxidations to prevent their hydrolysis. Among these conjugates, hydrazones or oximes are inherently more stable due to the inductive effect of the adjacent nitrogen or oxygen atoms. (340) Because aldehydes are much more reactive than other carbonyls, only minimal cross-reactivity can be expected with ketones, esters, or amide derivatives. Aromatic or sterically demanding aldehydes react more slowly than aliphatic ones; electron-donating effects can additionally reduce their electrophilicity. The low reactivity can be compensated by using highly nucleophilic (electron-rich) reagents, which sometimes are prone to oxidation (Figure 32).

9.1. Hydrazone Formation

The most widely applied method to detect aldehydes is based on the formation of stable hydrazones (Figure 33). Historically, hydrazines were used to identify (potentially liquid) carbonyl species by derivatization to solid hydrazones, which typically have well-defined and thus extensively catalogued melting points. (280) The drive toward developing fast analytical tools has repurposed these compounds for use in spectroscopic detection systems, (38) which have continuously evolved since their initial application as TLC staining solutions.

Figure 33

Figure 33. Summary of chromo- and fluorogenic turn-on probes for the selective detection of aldehydes based on the formation of hydrazone adducts. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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An example of a reagent used for selective carbonyl detection is dinitrophenylhydrazine (DNPH, 109), also called “Brady’s reagent”. Since its first mention in 1926, (281) it has been widely used to detect the carbonylation of proteins. It requires a strong acid as a catalyst to form hydrazones, which limits the application of such reagents for detection in situ. Unreacted probe has to be separated because of the small Stokes shift. (283) However, deprotonation of the resulting hydrazones with strong bases typically causes an 80 nm bathochromic shift, separating the signal from background created by the unreacted reagent. (38) Solutions of 109 are generally prepared in dilute phosphoric acid (284) or dilute HCl. (285) This method was used to quantify carbonyls in oxidized proteins (284) and to determine the concentration of benzaldehyde and phenylpyruvate in the optimization of the stereoselectivity of a threonine aldolase. (286) The reagent was also used in a HTS of variants of mandelate racemase to detect oxidized α-hydroxy acids. (285,287)
The use of 109 is limited to absorbance in the UV, so its fluorescent benzoxadiazole (BD) analogues have been widely adopted as fluorogenic reporters for aldehydes and ketones; (288) they emerged from the fluorogenic amino-selective NBD-Cl 12. (289,290) Substituting the electron-withdrawing nitro-group with more soluble sulfonamides (ABD-H 111, DBD-H 112) increases reaction rates with carbonyl compounds while maintaining a similar spectral profile. The cleanest reactions with aldehydes were found with NBD-H 110 (commercially available as hydrazine adduct), whereas the sulfonamides reacted more cleanly with ketones.
Generally, the substituted hydrazines presented here are synthesized from commercially available chlorides or fluorides by nucleophilic aromatic substitution with hydrazine. Similar to 109, assays are typically performed in acidic (288,289) solution or with a high excess of the reagent under physiological conditions (pH 7.5 in PBS buffer). (291)
Anilines are known to catalyze the conjugation: (292−294) their low pKa values (Figure 34) make them nucleophilic even at pH 5–7, forming highly reactive imines as intermediates and thus activating the carbonyl group. These species undergo fast conjugations at physiological pH. Aniline derivatives bearing hydrogen-bond donors in o-position were found to additionally boost the reaction by iminium formation. These “catalysts” are typically used in excess to the applied probes to compensate for the slow kinetics caused by the poor solubility and reactivity of assay probes. Furthermore, a high salt concentration was found to be beneficial for the conjugation of such systems. (295)

Figure 34

Figure 34. Plot of the nucleophilicity parameters N of amines versus pKaH in water. (296) Reproduced with permission from refs. (297and298). Copyright 2007 American Chemical Society.

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NBD-H 110 is the most prominent representative of this class of probes. It has been used in HTS to evaluate esterase reactions with vinyl esters as acyl donors in organic solvents. (291) The release of acetaldehyde upon esterification was tracked efficiently using this assay. The sulfonamide analogues (111, 112) have yet to be used in HTS. Nevertheless, they have seen use in the chromatographic separation of unsaturated aliphatic aldehydes with low molecular mass. (162,299)
A long known reagent that is still in regular use is 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald, 113). (300,301) This chromogenic probe reacts with aliphatic and aromatic aldehydes at room temperature to form tetrazinanes, which quickly oxidize to the violet-colored tetrazines 115 (Scheme 11). The coloration requires the oxidation of the tetrazinanes, which can be promoted with H2O2, dilute periodate, or atmospheric oxygen. Vigorous shaking of the sample to enable aeration is often sufficient. (302) The assay is performed under strongly basic conditions (e.g., 0.5–2 M NaOH). (302,286,303)

Scheme 11

Scheme 11. Sensing Mechanism of Purpald 113via the Intermediate Formation of Cyclic Tetrazinanes and Subsequent Oxidation to Fully Aromatized Violet-Colored Tetrazines 115
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Purpald has been extensively used in off-line HTS and protein engineering studies. Lauchli et al. used the reagent for the directed evolution of a terpene synthase: Non-natural isoprenoids released methanol upon cyclization by enzymatic catalysis, (191) which was oxidized to formaldehyde using an alcohol oxidase and subsequently detected using 113 at 550 nm. Another example, from the group of Arnold, used 113 in protein engineering of a P450 monooxygenase. (304) Directed evolution transformed a known epoxidase from the rhodobacterium Labrenzia aggregata (P450LA1) into an efficient anti-Markovnikov alkene oxidase. The aldehydes produced in the reaction were detected using 113.
The group of Fraaije engineered an alcohol oxidase fromPhanerochaete chrysosporium (PcAOX) to enhance the oxidation of glycerol. (305) The cavity-enlarging mutation F101S, which increased kcat to 3 s–1, was identified using an assay based on 113.
Another widely used hydrazine probe for the detection of aldehydes is 3-methyl-2-benzothiazolone hydrazone (MBTH) 114. (306) A mechanistic study revealed that two equivalents of the reagent are required to form the blue, cationic dye: one molecule reacts with the aldehyde (116a) and the other is oxidized (116b). They further react to form the highly conjugated system 117, a dark-blue dye with an absorption maximum at 635 nm (Scheme 12).

Scheme 12

Scheme 12. Sensing Mechanism for MBTH 114
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Concluding from the mechanistic analysis, the chromogenic reaction only happens when there is an excess of 114 that has not reacted with an aldehyde and is available for oxidation. An excess of aldehyde might thus not be detectable. (307) Contrary to 113, the reaction of 114 is performed at acidic pH (0.1 M HCl) and with ferric chloride as the oxidant, forming the blue dye. Modifications to the assay include the addition of sulfamic acid, which prevented the emergence of turbidity during the oxidation process. (308)
In a comparison of three chromogenic reagents for the detection of formaldehyde (Purpald 113, Nash reagent 132 (cf. section 9.4), and MBTH 114), 114 proved to be the only dye that formed stable adducts and prevented further oxidation toward formic acid in the screening of an alcohol oxidase (AO). (303) Even though 114 has not yet found widespread use in protein engineering, it has been applied to determine the concentration of reducing sugars (309,310) and of total aldehyde content in fuel ethanol samples. (311)
Recently, the group of Bane synthesized a dye with a benzocoumarin core and a hydrazine handle. This reagent, BzCH 118, was used for the detection of carbonylation in A549 lung carcinoma cells. (677) The quantification of the carbonyl content in the cells was performed in HEPES buffer for 4.5 h or in growth medium for 30–60 min at room temperature. Upon reaction with an aldehyde, the emission shifted from 430 to 550 nm due to disaggregation of the fluorophores. The conjugated hydrazones additionally exhibited an exceptionally large Stokes shift (195 nm) with no meaningful overlap between its absorption and emission spectra. The emission of the probe was shown to be stable from pH 4 to 8.
Methyl 5-MeO-N-aminoanthranilate 119 is a fluorogenic probe for aldehydes developed by Suchý et al., based on a modified anthranilic acid. It was used to measure the concentration of aldehydes in cells. (312) The reaction with their test substate (malondialdehyde) immediately led to the formation of a blue-green fluorescent species (λem = 480 nm), followed by a rapid decay of the signal (50% within 5 min; Figure 35). Still, initial enhancement of fluorescence with aliphatic aldehydes was between 5- and 20-fold.

Figure 35

Figure 35. (a) 119 as a fluorogenic probe for the detection of MDA. (a) Malondialdehyde (MDA) was added to a 25 μM PBS solution of 119 (pH 7.2) under illumination at 365 nm and photographed at indicated time points. (b) (left) Emission spectra of 1 μM 119 (black) or 119 after 1 min incubation with 5 equiv MDA at 37 °C and excited at 310 nm (blue) or 375 nm (cyan). (right) Formation and rapid decay of short-lived intermediate formed in the reaction between 119 and MDA followed by fluorescence spectroscopy with λex/λem = 375/480 nm. Kinetics of 25 μM 119 only (squares) and 119 + MDA (circles) are shown, with three replicates of 119 + MDA shown (light, medium, and dark cyan). (c) 119 shows broad specificity to aliphatic aldehydes. The enhancement of fluorescence relative to 119 only was recorded with λex/λem 375/480 nm following 1 min of incubation of 1 μM solutions of 119 in PBS (pH 7.2) with 5 equiv of MDA (black bar), simple aliphatic aldehydes (red bars), dialdehydes, and complex aldehydes (blue bars), ketones (purple bars), glutathione (yellow bar), and biogenic metal cations (orange bars). Reproduced with permission from ref (312). Copyright 2019 The Royal Society of Chemistry.

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In PBS buffer, the reagent cyclized to blue fluorescent indazoles, which would not react any further; no such reaction occurred in DMSO. Because the desired reaction is much faster than the side reaction in PBS (e.g., 10-fold faster with hexanal), the probe could still be used to study the peroxidation of lipids in HEK293 cells incubated with diethyl maleate.
The group of Kool developed the fluorogenic hydrazine derivative “DarkZone” 120, preloaded with an aromatic azoaldehyde that quenches fluorescence. (313) Upon reaction with the target aldehyde, the quencher is liberated and the desired turn-on is achieved. A variety of other fluorogenic dyes could be incorporated into the “DarkZone” approach (e.g., BODIPY, DEAC, Cy3, fluorescein, TAMRA). Only reactions with aliphatic aldehydes produced a strong signal because bisaryl hydrazone products (similarly to the structure of the original probe) also caused a quenching of the fluorescence emission. To facilitate conjugation at physiological pH, the authors tested various anilines and identified 5-methoxyanthranilic acid as a nontoxic and efficient catalyst for the transformation. Those tests were performed with a large excess of catalyst and analyte (dye/aldehyde/aniline catalyst in a ratio of 1:4000:10000) in phosphate-buffered systems at pH 7, resulting in an enhancement of up to 30-fold. To demonstrate the applicability of their system, aldehyde production from ethanol in K562 cells was monitored using their acetylated fluorescein analogue, which resulted in a 2.5-fold increase in fluorescence after incubation of the cells for 24 h.
Liu et al. (314) developed hydrazine dyes based on the squaraine (SQ) scaffold, such as 121, which have narrow absorbance and fluorescence spectra in the near-infrared region. (315) The cyclobutene ring is electron-deficient and thus susceptible to nucleophilic attack; this property is extensively used in colorimetric and fluorescent probes for the detection of nucleophiles. (316) The loss of π-conjugation upon the addition of various amines or hydrazine bleaches the blue color. Subsequent addition of aliphatic or aromatic aldehydes reverts the optical properties of the parent SQs, most prominently observed with the SQ–hydrazine system (Figure 36). Using the water-soluble SQOH–hydrazine 121, addition of an excess of formaldehyde was proven to completely scavenge the hydrazine, accompanied by a 23-fold increase in absorption and 86% recovery of the emission intensity of the original squaric acid dye in water.

Figure 36

Figure 36. (top) Scheme of the reaction mechanism of colorimetric and fluorescent detection of aldehydes based on the SQs–N2H4 adduct 121. (bottom) The changes in UV–vis absorption of dye 121 (6.0 mM) upon addition of N2H4 (3.0 mM) and subsequent addition of formaldehyde (10.0 mM). Reproduced with permission from ref (314). Copyright 2019 Elsevier BV.

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9.2. Imine Formation (and Trapping)

This group of reagents uses changes of spectral properties upon formation of Schiff’s bases from amines with aldehydes. Imines are less stable in water than hydrazones. Therefore, most of these probes irreversibly entrap the initially formed imine with intra- or intermolecular nucleophiles (Figure 37). The earliest examples of this class of compounds include pararosaniline 122, its methylated analogues rosaniline (also Schiff’s reagent, 123), magenta II 124, and “new fuchsin” 125.

Figure 37

Figure 37. Summary of chromo- and fluorogenic turn-on probes for the selective detection of aldehydes based on the formation of imines (Schiff’s bases). Subsequent entrapment of these electrophilic imines by intra- or intermolecular nucleophiles makes the reaction irreversible. Abs: wavelengths typically used for UV Absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Methylation has a large effect on the absorption spectra after conjugation to formaldehyde. (317) Robins et al. used NMR spectroscopy to study the colored species and the kinetics of its formation. (318) They found that “careful calibration is required” to obtain accurate results when quantifying aldehydes, with a complicated dependency on the concentrations of the dye, the sulfurous acid (or SO2) used as the nucleophile, and the aldehyde.
Despite these issues, the reagent has been used to determine o-dealkylation activity of 4′,7-dihydroxyisoflavone (daidzein) hydroxylase by detection of the purple formaldehyde adduct using an in vivo assay on LB agar plates (Figure 38). (23)

Figure 38

Figure 38. (a) The color of the aldehyde-sensing probe 122 changes to purple when treated with formaldehyde (detection limit: μM concentrations of formaldehyde in the in vitro system). (b) Color changes on solid agar plate from O-dealkylation activity. Aldehyde appears to diffuse out of the cells; cell color changes are observed in the presence of 122. Reproduced with permission from ref (23). Copyright 2013 Wiley-VCH.

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Amino benzamidoxime (ABAO, 126) reagents are another class of probes using an initial Schiff base formation for the selective detection of carbonyl compounds. According to the postulated mechanism, the intermediately formed imine is trapped by the nitrogen of the oxime. The resulting quinazoline oxides show a substantial shift in absorbance from 360 to 405 nm and strong fluorescence at 490 nm. Kitov et al. (319) were the first to report an in-depth analysis of these detection systems and found a linear free energy relationship (LFER) with the pKa of the respective anilinium species (Figure 39). A p-nitro substituent led to a loss of activity while a p-methoxy group increased the conjugation speed.

Figure 39

Figure 39. Relationship between the measured reaction rates and pKa of the corresponding protonated anilines for seven ABAO analogues 126a–g. (i) Structures of ABAOs 126 and corresponding protonated anilines. (ii) Scatter plot showing relationship between log k and pKa. (iii) Reaction rates measured by NMR in CD3COONa buffer (100 mM) at pD 4.5.(iv) Structures and reaction rates for 126f and 126g. Basicity of aniline nitrogen in 2-aminopyridine / 126f was estimated using ab initio calculations. Reproduced with permission from ref (319). Copyright 2014 American Chemical Society.

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Rudroff et al. used ABAO for a systematic mutation study of a carboxylic acid reductase from Nocardia iowensis (CARNI). (277,320) Using the 3-methoxy substituted MeO-126 reagent as a chromogenic aldehyde probe, 6000 mutants of CARNI mutants created using error-prone PCR (epPCR) were screened for improved activity with a panel of 20 aromatic aldehydes. The best mutant (Q283P) had a 9-fold improvement in KM and worked even with typically unreactive o-substituted benzoic acids. In a similar fashion, the ABAO assay was used in screening a library of 598 clones of Mycobacterium marinum (CARMM) to improve the production of piperonal in Pichia pastoris. (321) The assay is typically performed under acidic conditions (pH 4.5) and was also shown to work at physiological pH, but with lower activity. (322)
Xing et al. demonstrated the use of 5-aminofluorescein 127 as potent fluorogenic probe for the detection of a broad range of aliphatic aldehydes. (323) Using the restored fluorescence emission of the dye upon Schiff base formation, they used 127 to detect microbial oxidation of primary alcohols by the ADH NCIMB 621 from Gluconobacter oxidans at pH 6.0 at mM substrate loadings. The detection was improved by using a biphasic mixture of water and isooctane, which accumulated hydrophobic aldehydes in the organic phase. Building upon these findings, Preston-Herrera et al. applied probe 127 in a HTS assay for activity of a Rieske toluene dioxygenase. (270) They converted the cis-diol metabolites to the corresponding dialdehydes by oxidation with sodium metaperiodate, followed by detection of the dialdehydes with 127 (cf. section 8) (Figure 31). The authors showed that Tris buffer was detrimental for the oxidation due to possible reaction of metaperiodate with the hydroxy groups of the buffer. Reactions performed in HEPES or phosphate buffers worked well, with the highest rates at pH 6.5, which was attributed to acid catalysis of the imine formation. A 10–30-fold enhancement of fluorescence emission was observed using the optimal conditions after 5 h. They then used the assay to evaluate mutants from a site-saturation mutagenesis, identifying the critical active site residue L272 and subsequently applying the assay for the screening of a broad range of toluene derivatives.
Li et al. recently reported the development of the rhodamine derivative dRB-EDA 128, which undergoes ring-opening of its deoxylactam moiety upon reaction with an aldehyde (Scheme 13). (324) Cleavage of the C–N bond restores conjugation and causes an increase in fluorescence (only shown in DMF). Solutions of 128 (1 mg mL–1) exposed to low mM concentrations of formaldehyde showed a strong increase in absorption at 560 nm and fluorescence at 590 nm after 90 min (Figure 40).

Scheme 13

Scheme 13. Sensing Mechanism for dRB-EDA 128: Schiff’s Base Formation with Formaldehyde Triggers the Ring-Opening of the Deoxylactam Moiety
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Figure 40

Figure 40. Chromogenic detection of formaldehyde with 128. (a) UV–absorption spectra of 128 in the presence of formaldehyde (concentration: 0, 3.5, 7, 10.5, 14, 17.5, and 35 mM, from bottom to top). The inset shows the titration curve by absorbance at 560 nm. (b) Visual detection of formaldehyde (0–7 mM as indicated) with 128 in DMF after incubation at room temperature for 90 min. Reproduced with permission from ref (324). Copyright 2011 The Royal Society of Chemistry.

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The detection of simple aliphatic and aromatic aldehydes, such as 4-hydroxybenzaldehyde and hexanaldehyde, was shown. The assay was furthermore applied for the staining of oxidized sialoproteins on the cell surface of L929 cells, which were then analyzed by confocal fluorescence microscopy.

9.3. Imidazole Formation

A frequent design for sensitive assays uses the formation of stable heterocycles that irreversibly trap the analyte while concomitantly enlarging a delocalized electron system. This method has been applied for the detection of aldehydes using a combination of 1,2-diketones and suitable ammonia donors, forming imidazoles (Figure 41). Variations of the long known colored 2,4,5-triphenylimidazole (trivial name: lophine) formed by the condensation of benzil, benzaldehyde, and ammonia (325) have been analyzed as potential chemiluminogens. (326) Two particularly promising derivatives emerged from these studies: 2,2′-furil 129 and 9,10-phenanthrenequinone 130. Both probes were used as derivatization reagents, with sensitivity down to the nM range, for the monitoring of aliphatic aldehydes in human serum. (327,328) Derivatizations typically required incubation at 100 °C for 30 min in concentrated ammonium acetate solutions, making an in situ analysis of aldehydes impossible.

Figure 41

Figure 41. Summary of fluorogenic turn-on probes for the selective detection of aldehydes based on the formation of imidazole structures. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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The aromatic diamine analogue 1,2-diamino-4,5-dimethoxybenzene (DDB, 131) has been reported as an effective reagent for the fluorometric detection of aromatic aldehydes, (329) acrolein, (330) and methylglyoxal. (331) Derivatizations either required strongly basic conditions at room temperature or heating to 80 °C in DMSO. Its application in situ or in protein engineering has not yet been reported.

9.4. 1,4-Dihydropyridine Formation

Aldehydes and ammonia condense with 1,3-diketones to form chromogenic 1,4-dihydropyridines, a reaction also used for the Hantzsch pyridine synthesis (Figure 42). (332) The yellow-colored products (333) often have chromogenic and fluorescent properties, depending on the 1,3-diketone. (334) The simplest diketone, acetylacetone 132, can be used, either in its ketone form (“Nash reagent”) or condensed with ammonia (Fluoral-P), for the detection of aldehydes at room temperature in 2 M ammonium acetate buffer at pH 6. (335,336) Conjugates with formaldehyde showed particularly strong fluorescence, other aliphatic and aromatic aldehydes gave only weak fluorescence in solution (and could only be detected with sensitive detectors).

Figure 42

Figure 42. Summary of fluorogenic turn-on probes for the selective detection of aldehydes based on the formation of 1,4-dihydropyridines. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Sawicki and Carnes hypothesized that the rigidity of the fluorophore might influence signal strength (334) and tested 1,3-cyclohexanedione 133 and dimedone 134, resulting in a 100-fold higher fluorescence than with acetylacetone when detecting substituted aldehyde analytes. The increased steric bulk of these sensitive reagents required that the reaction be incubated at 100 °C for 5 min to reach completion. Furthermore, deprotonation of the products using tetraethylammonium hydroxide resulted in a bathochromic shift of approximately 60 nm (λem = 460 nm, λex = 520 nm).
These assays were used to detect aliphatic and aromatic aldehydes in blood plasma or seawater. (337,338) Anthon and Barrett compared three assays used to detect formaldehyde, all based on the AO-catalyzed oxidation of methanol: the Nash reagent 132, Purpald 113, and MBTH 114. Based on absorbance measurements, the Nash reagent had the lowest sensitivity. (303)
Li et al. used acetoacetanilide (AAA, 135) for the detection of formaldehyde, addressing the problem of low reactivity with sterically demanding diketone reagents. Quantification of μM concentrations was possible at room temperature in 10 min in 4 M ammonium acetate buffer at pH 7.5. The conjugation was selective for formaldehyde; even acetaldehyde did not react with the probe. (339)

9.5. Oxime Formation and Fragmentation

Similar to hydrazine reagents, oxyamines react rapidly with carbonyl compounds, forming stable oximes even at low concentrations. (340,341) These conjugates are stable toward hydrolysis under physiological conditions and have been used for the chemoselective ligation of macromolecular fragments. (342,343) The group of Reymond observed the fragmentation of the oxime bond at pH > 11, similar to the Kemp reaction: (344) Base-induced β-elimination of the aldoximes forms a nitrile and a phenolate (Figure 43). Synthetic modification of known nitrophenol or umbelliferone chromophores with the reactive oxyamine moiety enabled the base-induced release of the dyes (136 and 137), (345) and thus spectroscopic or fluorometric measurement of aldehyde conjugation. The same reaction was catalyzed by BSA at neutral pH, where electron-rich aromatic aldehyde and ketone conjugates remained stable.

Figure 43

Figure 43. Summary of chromo- and fluorogenic turn-on probes for the selective detection of aldehydes based on the formation of unstable oximes. Fragmentation of these intermediates leads to the liberation of colored phenols. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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The unspecific hydrolysis of the oxyamine still proceeded with a significant rate, making the detection of aldehydes problematic. The authors thus opted for a sequential approach: formation of the oxime was stopped by adding acetone in excess and any unreacted probe was thus converted into a stable ketoxime. Only then was the fragmentation of the aldoxime initiated by the addition of base.
This assay was used in screening for the release of formaldehyde by lipase-catalyzed hydrolysis of pivaloyloxymethyl ethers, but a low signal-to-noise ratio caused by the spontaneous hydrolysis of the oxyamine made the system impractical. A similar observation was made by the group of Roelfes during their investigations toward designer enzymes catalyzing hydrazone and oxime bond formation. (346) Contrary to the aforementioned report, base-induced fragmentation of their oxyamine-modified NBD–benzaldehyde adduct 138 caused the formation of the corresponding aldoxime and phenolate. The formation of p-nitrophenolate derivative NBD-O– was used for the quantification of the catalytic efficiency of their mutants (Scheme 14). At 25 °C and pH 7.4 the system was able to discriminate the enzymatically catalyzed process against background hydrolysis of the unreacted probe.

Scheme 14

Scheme 14. Enzyme-Catalyzed Formation of the 138–Benzaldehyde Adduct Could Be Efficiently Traced by Spontaneous Decomposition Forming Colored NBD-O–
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9.6. Aza-Cope Reaction

A recently reported approach for the detection of formaldehyde is based on mechanistic investigations from 1950 by Horowitz and Geissman. (347) Their attempts to achieve double alkylation of allylamines with formaldehyde under acid catalysis remained unsuccessful and only formed cleavage products, which were recently shown to result from a 2-aza-Cope sigmatropic rearrangement (Figure 44). This finding led to the development of a diverse set of probes based on the same mechanism. Although this class of compounds has sparked growing interest over the last years, their reactivity limits applications to the detection of formaldehyde. Recent reviews summarize these and other approaches, including the detection method presented in this section. (357,358)

Figure 44

Figure 44. Summary of fluorogenic turn-on probes for the selective detection of formaldehyde based on the 2-aza-Cope sigmatropic rearrangement of initially formed Schiff’s bases. Fragmentation of these intermediates leads to the liberation of colored phenols. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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The first studies were both based on silicon rhodamine dyes but relied on different fluorogenic mechanisms. One approach built on the finding that spirocyclization renders rhodamine dyes weakly emissive at physiologically relevant pH. (348) Probe FAP-1 139 undergoes ring opening after an aza-Cope rearrangement. Subsequent hydrolysis of the resulting imine yields an aldehyde that can not re-(spiro-)cyclize. (349) This assay could detect formaldehyde at 100 μM concentration by an ∼8-fold increase in fluorescence (1 h, pH 7.4). The other approach uses the same rearrangement to liberate a p-nitrobenzyl group, which is known to quench fluorescence through a donor-excited PET process. (350) The assay using probe FP1 140 could detect formaldehyde at 250 μM concentration by a ∼7-fold increase in fluorescence (3 h, pH 7.4).
Building upon these initial reports, the group of Chang developed their assay toward a general 2-aza-Cope reaction trigger with a self-immolative β-elimination linker. They created the homoallylamine probe 141 that, in response to formaldehyde, unmasked fluorogenic phenols on a fluorophore. (351) By decoration of four different phenol-bearing fluorophores (coumarin, carbofluorescein, resorfurin, and rhodol), they achieved a 2–10-fold increase in fluorescence upon formaldehyde conjugation at 100 μM concentration (2 h, pH 7.4, 25 °C).
Du et al. recently investigated a broad set of N-substituted homoallylamines 142 as probes for formaldehyde. (352)N-p-methoxybenzyl homoallylamine was found, by experiment and computation, to have the highest reactivity in the 2-aza-Cope reaction with formaldehyde. Using similar self-immolative linkers as above, they synthesized five silenced phenol-bearing fluorophores 142, with excitation wavelengths ranging from 445 to 706 nm. Exposure to formaldehyde at 500 μM concentrations under physiological conditions prompted a turn-on factor of 5–125. All probes were successfully applied in the visualization of changes of intracellular formaldehyde in living cells (Figure 45). (352)

Figure 45

Figure 45. Confocal fluorescence images of exogenous formaldehyde (FA) with FFP fluorescent probes 142 in HEK293T cells. (A–D) Cells were loaded with (a) FFP511 (Tokyo Green fluorophore) (20 μM), (b) FFP551 (142) (10 μM), (c) FFP585 (resorufin fluorophore) (5 μM), or FFP706 (hemicyanine fluorophore) (2 μM) for 30 min and treated with FA of varying concentrations (0–1 mM) for 60 min before being imaged by confocal fluorescence microscopy. Scale bars represent 50 μm. Reproduced with permission from ref (352). Copyright 2021 The Royal Society of Chemistry.

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Making these reagents typically requires great synthetic effort, which is impractical for biochemists. Consequently, many probes based on simple synthetic modifications of known fluorophores have been developed in recent years. For instance, He et al. and Xu et al. reported the naphthalene-based probe RFFP/AENO 143, which can be synthesized in a one-pot reaction from commercially available 6-hydroxy-2-naphthaldehyde. (353,354) The use of the reagent as turn-on probe with a 100-fold enhancement of fluorescence within 2.5 h for 1 mM formaldehyde was reported (Figure 46). (354)

Figure 46

Figure 46. Fluorescent responses of 143 (10 μM) in DMF/PBS solution (v/v = 1/4, pH 7.4, 10 mM) upon addition of various small molecular species (5.0 mM). λex/λem = 390/513 nm; slits, 2.5/2.5 nm. Each spectrum was recorded after 3 h at 37 °C. Error bars are ±1SD, n = 3. Inset: (a) The color and (b) fluorescence images of 143 (10 μM) in the presence of various small molecular species (5.0 mM). (354) Reproduced with permission from ref (354). Copyright 2016 Elsevier BV.

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The groups of Lin and Peng reported the synthesis of turn-on probes PDB-FA 144a and BD-CHO 144b, based on NBD as a fluorophore, (355,356) in a three-step procedure from commercial 4-chlorobenzoxadiazole. The probes showed 55-fold (PDB-FA) and 255-fold enhancement (BD-CHO) upon exposure to excess formaldehyde after 2 h at pH 7.4. (357,358)

10. Chromo- and Fluorogenic Probes for Ketones

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Ketones are widespread in nature. This electrophilic functional group is present, for instance, in the carbohydrate family of ketoses, as a polar element in important steroids (e.g., testosterone, progesterone), in fragrance compounds derived from terpenoid units, (359) or as the structurally important methyl ketone motif (360) (e.g., camphor, carvone, pulegone, 2-heptanone). Ketones are also found as biological intermediates and energy storage units in important regulatory systems of cells (Krebs cycle, (361) fatty acid synthesis, (362) ketogenesis (363)).

Figure 47

Figure 47. (top) Summary of general properties of ketones, reactivity trends toward ketone-selective probes as well as expected cross-reactivity during screenings. (bottom) Compatible pH range and buffers in reported ketone-selective assays. Bold pH numbers indicate the most commonly used pH values.

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In industry, ketones are widely used in bulk as solvents (e.g., acetone or methyl ethyl ketone, MEK) or as synthetic intermediates. They have found broad use in biocatalysis as starting materials for the synthesis of chiral alcohol and amines (364) using transaminases (TA), alcohol dehydrogenases (ADH), (365) Baeyer–Villiger monooxygenases (BVMO), (366) or aldolases. (367)
They can be synthesized biochemically from β-ketoacids by decarboxylation using carboxy lyases, (368) using aldolases, (369) from carbinols using TDP-dependent lyases, (370) or using acyl transferases (ATase). (371) These biocatalysts have already been demonstrated as good alternatives in organic synthesis. The group of Hammer recently optimized a ketone synthase for the regioselective anti-Markovnikov oxidation of aromatic alkenes. Starting from a P450 wild-type, 12 rounds of evolution identified 18 mutations that were beneficial for the stereoselective oxidation of styrene derivatives 145 (Scheme 15a). (372)

Scheme 15

Scheme 15. Representative Selection of Enzymatic Transformations for the Production of Carboxylic Acid-Containing Products
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Many applications of ketone synthesis use alcohol dehydrogenases as mild oxidative catalysts. Zhang et al. applied the highly regio- and enantioselective ADH from Bacillus subtilis BDHA for the oxidation of racemic trans-cyclic vicinal diols 147 (Scheme 15b) (373) to (R)-α-hydroxy ketones 148 and (S,S)-cyclic diols 149 in >99%ee at 50% conversion in one pot.
The group of Kroutil identified an ADH from Sphingobium yanoikuyae for the opposite purpose, oxidizing the aliphatic alcohols 150 to the corresponding ketones 151 without stereopreference and without the need for a huge excess of ketones as hydrogen acceptors for cofactor recycling (Scheme 15c). (374) The researchers found that α-Cl, -F, or -MeO substituted ketones were not oxidized, hence limiting the necessity for organic recycling reagents to one molar equivalent only.
González-Granda et al. reported the oxidation of propargylic alcohols 152 using the catalytic system composed of a laccase from Trametes versicolor, the oxy-radical (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), and oxygen as terminal oxidant. (375) Under these mild conditions, the products were obtained in good yields (Scheme 15d).
The group of Kroutil was the first to report an enzymatic Friedel–Crafts acylation of activated arenes 154, a reaction that is typically in the realm of classical organic chemistry, as well as the first biocatalytic equivalent to the Fries rearrangement (the intramolecular analogue of the Friedel–Crafts reaction). (371) They reported conversions of up to 95%, using readily available isopropenyl acetate as acetyl donor and without any CoA-activated reagents (Scheme 15e).
Ketones are much less reactive than most aldehydes due to an increase in steric bulk and the diminished electrophilicity caused by the electron-donating effect of the attached alkyl groups (Figure 47). This relatively low activity makes specific derivatization of ketones challenging; reactivity and selectivity of reagents are inversely correlated. With certain exceptions, approaches are similar to those introduced in the section on aldehydes (cf. section 9).
Ketones react slowly in conjugations under physiological conditions, (293) particularly at low concentration. Most assays require harsh reaction conditions that are incompatible with biological systems. Only a few studies have addressed the challenge of developing selective conjugations for ketones, so far, likely for the following reasons: (i) Aldehydes are generally more important in synthesis due to their broader versatility as chemical building blocks, (ii) It is difficult to match the rates of aldehyde conjugations (under both abiological and physiological conditions). Ketones are mostly used as negative examples to highlight the specificity of carbonyl probes for aldehyde conjugations.

10.1. Hydrazone Formation

The most promising probes to detect ketones use the fast reaction of carbonyl compounds with hydrazines forming stable hydrazones (Figure 48). In principle, the approach is the same as for aldehydes, summarized above (cf. section 9.1), but only a small number of assays showed useful reactivity toward ketones. The long-known reagent DNPH 109 has been used by Yang et al. as an at-line analytical tool and in HTS for the activity of mandelate racemase variants by detection of oxidized α-hydroxy acids. (287) Enantioselective mandelate dehydrogenase D-MDH was used to selectively oxidize the desired enantiomer, which was converted to the hydrazone using 109 and detected at 450 nm after basification with aqueous NaOH (Figure 49).

Figure 48

Figure 48. Summary of chromo- and fluorogenic turn-on probes for the selective detection of ketones based on the formation of hydrazone adducts. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Figure 49

Figure 49. Variation in color of reaction mixtures containing different concentrations of 109 and the validation of the developed HTS method by chiral HPLC analysis. Control, no 109 was added into the reaction mixture; 1–10×, different concentrations of 109 were added into the reaction mixture (1×, 8.4 mg/mL). Reproduced with permission from ref (287). Copyright 2017 Springer-Verlag.

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The BD-probes (NBD-H 110, ABD-H 111. DBD-H 112) form fluorescent derivatives with aliphatic and aromatic ketones at room temperature, but only in ACN. (288) Among these compounds, 110 is the most broadly applied for conjugations with aldehydes and ketones that work even under physiological conditions. (291,376) Crisalli and Kool used anilines to catalyze conjugations at neutral pH. Reactions with aromatic ketones were improved using these catalysts, but they still proceeded slower than reactions with aldehydes. (294,377) Wang et al. showed that increased salt concentrations were beneficial for conjugation rates at physiological pH. (295)
Among the already described fluorogenic hydrazines (cf. section 9.1), methyl 5-MeO-N-aminoanthranilate 119 was tested as a probe for aliphatic ketones and showed a small (unquantified) increase in fluorescence emission upon conjugation. (312)

10.2. Aldol Reaction

When ketones tautomerize to enols, they can participate in aldol reactions (Figure 50). Similar to the hemiaminal formation used for the detection of amines (cf.section 6.7), electron-withdrawing carbonyl groups can be used to trigger a hypsochromic shift of the absorption maximum upon aldol reactions. This method has been explored by the group of Barbas and Tanaka. (378−380) They investigated different designs (compounds 156–158) to detect acetone, which required activation via enamines formation using catalytic amounts of pyrrolidine, arginine, glycine, proline, or the aldolase antibody 38C2. A 1000-fold excess of the substrate was required to afford useful conversions. The triazine aldehyde 156 and the phenanthrene aldehyde 157 showed similar excitation and emission properties (approximately 100–1000-fold turn-on) from pH 5 to 9. Exchange of the linked amide aryl-moieties to the benzimidazole analogue 158 red-shifted the excitation maximum from 260 to 315 nm. In turn, it also lowered the sensitivity of the assay (3–20-fold turn-on) due to the parent aldehydes becoming stronger emitters.

Figure 50

Figure 50. Summary of fluorogenic turn-on probes for the selective detection of acetone based on aldol reactions. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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10.3. Michael Addition

The activation via enamine formation can be also used with Michael acceptor probes in 1,4-additions. The benzimidazole 159 and 6-aminoquinoline 160 have useful turn-on properties upon conjugation to ketones (Figure 51). (379) Exposition of fluorogenic 6-aminoquinoline probe 160 to acetone additionally triggers an immediate cyclization by aldol-condensation with the terminal methyl ketone (Scheme 16). Due to a strong spectral overlap between probe and conjugate, their application is problematic. Both probes are slower than the previously reported aldol sensors (cf. section 10.2) and also require a 1000-fold excess of acetone. Due to the unattractive properties of the screening system, no additional studies were undertaken.

Figure 51

Figure 51. Summary of fluorogenic turn-on probes for the selective detection of acetone based on a Michael addition approach. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Scheme 16

Scheme 16. Michael Addition of Acetone onto 159 Triggers a Subsequent Aldol Reaction Forming Cyclic Hydroxyketones
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10.4. Other Methods

Two additional methods for the detection of ketones are (i) the Janovsky reaction, and (ii) reaction with ABAO 126 (Figure 52).

Figure 52

Figure 52. (top) Chromogenic and fluorogenic detection of ketones bearing an enolizable position via the formation of the purple-colored Janovsky complexes with 3,5-dinitrobenzoic acid 161 under strongly basic conditions. (bottom) Fluorogenic detection of ketones via the formation of fluorescent ABAO-adducts (cf. section 9.2). A methoxy substitution of the ABAO core 126 is necessary to increase the reactivity of the aniline NH2 position.

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The Janovsky reaction requires analytes to form reactive enolates under strongly basic conditions, which then form stable, purple-colored σ-complexes with 3,5-dinitrobenzoic acid 161. This method was used to rapidly evaluate 11 substrates for Baeyer–Villiger monooxygenases by monitoring the decrease of absorbance at 550 nm. (381)
Reaction with ABAO, initially suspected to be unreactive toward ketones, emerged as an effective probe for aliphatic and aromatic ketones. (322) The methoxy analogue MeO-126 reduced long reaction times with its reactivity enhanced by the electron-donating effect of the methoxy group. A set of 24 ketones was tested at pH 5.0 using an equimolar amount of ABAO at 10 mM concentration. Fluorometric evaluation of the reaction after 30 min revealed a ∼2–20-fold turn-on. The assay was applied in enzyme mining and directed evolution of alcohol dehydrogenases (ADHs) for the catalytic oxidation of rhododendrol to raspberry ketone (p-hydroxybenzyl acetone).

11. Chromo- and Fluorogenic Probes for Carboxylic Acids

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Carboxylic acids are found in many compounds in nature, (382) pharmaceuticals, (383) and food constituents. (384) Many structures bearing this functional group are involved in essential physiological processes, such as prostaglandins regulating inflammation and blood pressure (385) or citric acid and succinic acid in the Krebs cycle. (386) Fatty acids are the building blocks of lipids and are involved in energy metabolism, (387) whereas α-amino acids are the building blocks for peptides and proteins. (388)

Figure 53

Figure 53. (top) Summary of general properties of carboxylic acid sensors as well as expected cross-reactivity during screenings. (bottom) Compatible pH range and buffers in reported carboxylic acid-selective assays. Bold pH numbers indicate the most commonly used pH values.

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Various biocatalytic methods for the synthesis of carboxylic acids have been developed, complementing nonenzymatic approaches. (389) These methods include the hydrolysis of nitriles by nitrilases, (390−392) the conversion of aldehydes by hydroxynitrile lyases with subsequent hydrolysis, (393) the hydrolysis of esters by esterases, (394) and the oxidation of alcohols or aldehydes by dehydrogenases and oxidases. (395)
Glieder et al. reported a hydroxynitrile lyase mutant from Prunus amygdalus for the enantioselective synthesis of α-hydroxyacids by the addition of HCN to aldehydes and the subsequent hydrolysis of the intermediately formed cyanohydrins. The enzyme was stable under acidic conditions and enabled the conversion of 2-chlorobenzaldehyde 162 to (R)-2-(2-chlorophenyl)-2-hydroxyacetonitrile 163 at pH 3.4 with 96% yield and 97%ee. Hydrolysis of the intermediate yielded the enantiopure α-hydroxyacid 164 (Scheme 17a). (393)

Scheme 17

Scheme 17. Representative Selection of Enzymatic Transformations for the Production of Carboxylic Acid-Containing Products
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DeSantis et al. identified a nitrilase mutant for production of (R)-4-cyano-3-hydroxybutyric acid 166, an intermediate in the synthesis of the drug Lipitor, by directed evolution. The hydroxybutyric acid 166 could be obtained in 96% yield with 98.5%ee and a volumetric productivity of 619 g L–1d–1 (Scheme 17b). (396)
The reactivity of carboxylic acids is mainly determined by their acidic hydroxy group and carbonyl functionality. Under physiological and basic conditions, carboxylic acids (pKa ∼ 3–5 (397,398)) are deprotonated, negatively charged carboxylates, which render nucleophilic attacks at the carbonyl center impossible (Figure 53). (399) Also, protonated carboxylic acids are much less reactive than derivatives like acyl halides or anhydrides, which generally makes an activation for derivatizations necessary. Therefore, detection methods based on the conversion of an acid to a fluorescent derivative (e.g., hydrazide, hydroxamate) usually involve activation as the first step (e.g., with dicyclohexylcarbodiimide DCC, 167). Many approaches for carboxylic acids involve monitoring of pH shifts by pH indicators (404) and the enzymatic conversion of acids with NADH. (405)
Other methods exploit the properties of carboxylic acids and carboxylates to form intermolecular hydrogen bonds, which enable their detection as adducts with probes that exhibit hydrogen bond donating groups, such as amides, thioureas, or guanidinium moieties. (401,402) Some carboxylate probes are based on the coordination of the anion to a metal ion, e.g., Zn2+ or Cu2+. (403)
A challenge in sensing carboxylic acid is the selective detection and the differentiation of acids. (401,406,407) One method to improve selectivity is introducing multiple recognition sites at the probe. (403,407) Many of the assays require nonaqueous conditions or apolar/aprotic solvents (e.g., chloroform) to prevent competitive interactions between the solvent and the binding sites of the probe. (408−411)

11.1. Activation and Nucleophilic Trapping

Carboxylic acids can be detected by converting them to acid hydrazides with hydrazine derivatives (Figure 54). (412,413) Miwa et al. reported a colorimetric method using 2-nitrophenylhydrazine 168 in ethanolic solution in the presence of 167 as a coupling agent and catalytic amounts of pyridine. The resulting acid hydrazides had a violet color upon basification with KOH, and their absorbance could be measured at about 550 nm. (414)

Figure 54

Figure 54. Summary of chromogenic turn-on probes for the selective detection of carboxylic acids based on the entrapment of activated carboxylic acid species. Abs: wavelengths typically used for UV absorbance measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Another colorimetric method uses derivatization of activated carboxylic acids to hydroxamic acids with hydroxylamine 169, followed by the formation of a colored ferric hydroxamate (Figure 54). (415,416) Based on work by Kasai et al. (417) and Takeuchi et al., (418) He et al. reported a spectrophotometric HTS assay to detect various aliphatic and aromatic carboxylic acids in aqueous solutions at room temperature by converting them to ferric hydroxamates. The carboxylic acids were converted to hydroxamic acids with hydroxylamine perchlorate 169·ClO4 in the presence of 167. Purple ferric hydroxamates were formed upon the addition of an acidic solution of ferric perchlorate. The absorbance of the ferric hydroxamate was measured at 520 nm. He et al. applied this method to assay the nitrile-hydrolyzing enzymes Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401. (419)

Figure 55

Figure 55. Summary of fluorogenic turn-on probes for the selective detection of carboxylic acids based on the coordination to metal centers or hydrogen bond donors. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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11.2. Coordination to Metals and Hydrogen Bond Donors

Formation of complexes with a probe via hydrogen bonding or electrostatic interactions can be used to detect carboxylic acids and carboxylates (Figure 55). (403,420−422) These interactions can induce fluorescence by electronic changes of the probe: Nonbonding electron pairs of amines or adjacent electron-donating groups are known to quench fluorescence by PET processes, which can be restored by coordination of the amines to carboxylic acids. (408,422) Similarly, quenching has been observed by binding of amine probes to Cu2+ ions. By coordination and thus scavenging of the Cu2+ ions by carboxylates, the probe’s fluorescence emission can be effectively restored. (411,423) In some cases, hydrogen bonding or electrostatic interactions of carboxylate and probe lead to a fluorescence response by the displacement of a noncovalently bound fluorescent indicator from the probe (indicator-receptor-complex) by the carboxylate which results in the release of the fluorescent dye. (406,409)

Figure 56

Figure 56. Summary of fluorogenic turn-on probes for the selective detection of carboxylic acids based on nucleophilic addition onto trifluoroacetyl groups. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Cabell et al. developed probe 170 for the detection of citrate in HEPES buffer (pH 7.4), enabling the quantification of citrate in beverages at μM concentration. Probe 170 uses a 1,10-phenanthroline moiety as a fluorophore and guanidinium moieties as recognition sites ensuring effective hydrogen bonding to carboxylates in water. Fluorescence is quenched while phenanthroline binds Cu2+. When tribasic citrate binds to Cu2+ and the two guanidinium groups of the probe, electron transfer from the metal to the fluorophore changes as the metal becomes more electron-rich, which leads to an increase in fluorescence at 365 nm. (423)
Xu et al. developed the Eu3+ complex 171, based on a phenanthroline scaffold with chiral amino alcohol groups as ligands that enabled the selective detection of malate anions in aqueous solutions (0.01 M HEPES, pH 7.4, with 0.3% DMSO). Binding of Eu3+ suppresses the fluorescence at about 380 nm, which gets restored when malate forms a complex with the ligand. Other carboxylates, such as alanine, phenylalanine, mandelate, and tartrate, did not show significant responses. The authors assumed that the selectivity for malate arose from the preorganized structure of 171 and the complementary shapes of probe and anion. The enantiomers of malate gave different responses: 487% enhancement with l-malate and 332% with d-malate. (424)
Burguete et al. reported a detection method for citrate based on the fluorescent anthracene-probe 172 in methanolic aqueous solution (70%, pH 7.5–8.0). Their probe consisted of a nonfluorescent Cu2+ complex of two aminoanthracene moieties, each coupled with an l-phenylalanine, and enabled the detection of citrate in the μM range. The addition of citrate led to a displacement of the anthracene moieties, which resulted in a more than 15-fold increase in fluorescence at 420 nm. Mandelate, tartrate, and malate gave small increases in fluorescence (∼10–15%) compared to citrate; succinate, fumarate, lactate, acetate, and glutarate did not produce any increase. Only l-glutamate, which is similar to citrate in the number of potential coordination sites (two carboxylates and one amino group), showed a similar increase in fluorescence (40%). (425)
Boiocchi et al. developed a probe based on the indicator-displacement approach to detect dicarboxylates in water. The binding of the cryptate complex 175 to the fluorescent indicator carboxy-rhodamine 174via the coordination of the two Cu2+ ions to two carboxylates led to the quenching of the indicator’s (174) fluorescence. A displacement of 174 by selected dicarboxylates restored fluorescence at λem, max = 570 nm. Only 1,4-, 1,5-, and 1,6-dicarboxylates terephthalate, glutarate, and adipate that all have an appropriate spacial distance between their COO– groups (irrespective of their carbon chain length) could coordinate to both Cu2+ ions and displace the indicator. Dicarboxylates with shorter or longer distances (e.g., succinate, pimelate, phthalate, or isophthalate) led to no or only minor fluorescence emission. In the case of the very small oxalate anion, a coordination of two molecules to bind both Cu2+ also resulted in the displacement of the indicator. (409)
Amino acids are known to form stable complexes with Cu2+ ions. (406) Klein and Reymond reported the Cu2+-based probe 176 that was used in HTS of the hydrolysis of N-acetyl-l-methionine by acylase I and of l-leucinamide by aminopeptidase. The complex of the quinacridone-derived ligand and Cu2+, gave no fluorescent signal. Due to the weak chelating effect of this macrocycle, amino acids competed with the ligand and displaced it. The fluorescence of the released quinacridone-derived ligand was measured at 584 nm. The low solubility of 176 in aqueous buffer required the use of organic cosolvent (∼40%), lowering enzymatic activity; alternatively, the probe and solvent could be added after the enzymatic reaction has completed. (426)
In later reports, substitution of the quinacridone-derived ligand with the commercial fluorescein-based ligand calcein 177 was proven to be beneficial due to its increased solubility in aqueous buffers. The probe showed enhanced fluorescence at 530 nm with various amino acids, such as valine, alanine, methionine, leucine, serine, histidine, N-acetylmethionine, and glycine, at μM concentrations. The assay worked at pH 6–10 in various buffers (bis-tris, phosphate, borate, Tris) and mixed solvent systems (0–50% organic), including EtOH, MeOH, ACN, and DMSO. Strongly coordinating buffers (e.g., bis-tris-propane) are incompatible because they bind to metal ions. The assay does not work at low pH as the protonated carboxyl groups of 177 do not coordinate well. Probe 177 enabled HTS for activity of proteases, such as trypsin, chymotrypsin, and subtilisin. (427) Xu et al. validated this approach and used the Cu2+–calcein complex for HTS of more than 2500 leucine dehydrogenase mutants with 2-oxobutyric acid as substrate. They detected 11 mutants with a higher activity than the wild type. (428)

11.3. Nucleophilic Addition onto Trifluoroacetyl Groups

Carboxylates can react with trifluoroacetyl ketones in a nucleophilic addition (Figure 56). As in sections 7.1 and 6.7, addition disrupts electron-withdrawing effect of the carbonyl group on conjugated systems and leads to changes in fluorescence. (429,430) Kim and Ahn developed trifluoroacetophenone derivatives 178 with a terthiophene or a pentathiophene moiety as fluorophores and a trifluoroacetyl group as recognition site for carboxylates. Fluorescence of the terthiophene probe rose 120-fold in the presence of acetate in ACN. Other anions, such as phosphate, fluoride, and cyanide, also enhanced the fluorescence. (430)
The trifluoroacetyl carbonyl moiety can also function as a recognition site for amines (cf. section 6.7), as shown by Ryu et al. in the development of probe 179, which uses two trifluoroacetyl groups for the detection of α-amino carboxylates. Binding of the amine and the carboxylate group to both recognition sites resulted in the formation of a bridged adduct accompanied by an increase in fluorescence. The addition of glycinate resulted in a 110-fold enhancement of fluorescence in ACN. Other α-amino carboxylates caused similar emissions; the α-substituents did not affect the formation of the adduct. Probe 179 discriminated α-amino carboxylates against β- and γ-carboxylates. (429)

12. Chromo- and Fluorogenic Probes for Carboxylic Acid Esters

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Carboxylic acid esters are widely found in nature, (431) pharmaceuticals, (432,433) fine chemicals, (434) agrochemicals, (435) and the food industry. (436) Most are long-chained triglycerides in fats and oils and are of central importance in energy metabolism. (387,437) Short-chained derivatives are typically found in fruit and flower fragrances (e.g., benzyl acetate in jasmine (438) or isoamyl acetate in banana (439)). Due to their flavor-carrying and fragrant nature, they are valuable ingredients in cosmetics, food, and beverages. (440) Moreover, they are important building blocks for polymers, (441) lubricants, (442) plasticizers, (443) and surfactants. (444) Enzymatic approaches for the synthesis of carboxylic acid esters include esterifications and transesterifications catalyzed by esterases and lipases. (445−447)

Figure 57

Figure 57. (top) Summary of general properties of carboxylic acid ester sensors as well as expected cross-reactivity during screenings. Due to the lack of direct reactive probes applicable in aqueous environments, no compatible buffers and pH ranges can be stated.

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Lozano et al. reported a biocatalytic approach for the synthesis of aliphatic esters 182 by esterification of C1–C4 carboxylic acids 180 with fragrant alcohols 181 (isoamyl alcohol, citronellol, geraniol, and nerol) using an immobilized lipase from Candida antarctica (Novozym 435) in the ionic liquid N,N′,N″,N′′′-hexadecyltrimethylammonium bis(trifluoromethylsulfonyl)imide (Scheme 18). The authors optimized the workup procedure and achieved yields >78% for all esters. Geranyl valerate was obtained with the highest product concentration of 0.757 g mL–1. (448)

Scheme 18

Scheme 18. Representative Selection of Enzymatic Transformations for the Production of Carboxylic Acid Ester-Containing Products
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Carboxylic esters are compounds derived from carboxylic acids and alcohols. The electrophilic carbonyl center can react by substitution of the alcohol group with many nucleophiles, such as amines, alkoxylates, and hydroxylamines. Under basic or acidic conditions, esters can also hydrolyze. (400,449) The volatility of many carboxylic acid esters must be considered in quantitative measurements (450) (Figure 57).
The only direct colorimetric approach for the detection of carboxylic acid esters is based on the conversion to hydroxamic acids with hydroxylamine hydrochloride 169 in the presence of a base (Figure 58). (451−453) The subsequent addition of ferric chloride under acidic conditions results in the formation of a ferric hydroxamate complex, which has a typical magenta color. (451,452) We described a similar principle for the detection of carboxylic acids (see above).

Figure 58

Figure 58. Chromogenic detection of carboxylic acid esters via the formation of the hydroxamic acids and subsequent complexation with Fe3+ resulting in purple-colored ferric hydroxamates.

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Esters are inherently reactive toward hydroxylamine (cf. section 11) and do not need activation. Buckles and Thelen tested the scope and limitations of this approach with various aromatic and aliphatic esters. Besides simple carboxylic esters, also polymer esters and glycerides of fatty acids were detected. Carbonates, chloroformates, sulfonates, urethanes, and esters of inorganic acids gave negative results. Coloration was also observed in the presence of other electrophiles, including aldehydes, trihalomethyl compounds, imides, nitriles, and amides. (451) Although only certain amides were shown to give a positive colorimetric response, an extraction step might be useful before analysis to reduce false-positive hits from endogenous proteins.
Based on the same concept, Löbs et al. developed a method for the rapid screening of ethyl acetate production by four different strains of Kluyveromyces marxianus growing on various C5, C6, and C12 carbon sources. The formed esters were extracted with hexane and subsequently converted to ferric hydroxamates at room temperature. The absorbance was measured at 520 nm. All strains exhibited the highest ethyl acetate production employing the C6 sugars glucose and fructose. The assay was also validated for ethyl butyrate, ethyl hexanoate, isoamyl acetate, and ethyl octanoate. (454)

13. Chromo- and Fluorogenic Probes for Epoxides

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Epoxides react with various nucleophiles, oxidizing, and reducing agents due to its high polarity and strain of the three-membered ring. Nucleophilic ring-opening reactions, leading to 1,2-disubstituted products, are of particular synthetic interest. (455,456) Possible products include: amino alcohols, glycol ethers, alcohols, hydroxynitriles, and chlorohydrins. (457)

Figure 59

Figure 59. (top) Summary of general properties of epoxide sensors as well as expected cross-reactivity during screenings. (bottom) Compatible pH range and buffers in reported epoxide-selective assays. Bold pH numbers indicate the most commonly used pH values.

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Epoxides are used as industrial intermediates in the production of pharmaceuticals, (458−461) soaps, (460) lubricants, (459) cosmetics, (459,460) and surfactants. (459) Enantiopure epoxides are particularly valuable chiral functional groups for the production of more complex compounds. (462−464) Simple epoxides, such as ethylene oxide or propylene oxide, are of interest for the production of ethylene glycols, ethanolamines, ethylene glycol ethers, and propylene glycols, polyether polyols, and propylene glycol ethers. (465,466) Enzymatic methods for epoxide synthesis include the oxidation of alkenes by monooxygenases (MO), the conversion of halohydrins by halohydrin epoxidases, and the use of epoxide hydrolases. (214,467)
Halohydrin dehalogenases and epoxidases catalyze the reversible enantioselective dehalogenation of vicinal halohydrins 183 to the corresponding epoxides. Ma et al. employed halohydrin dehalogenase (HHDH, EC 3.8.1.5) in the second step of a two-step biocatalytic process to synthesize hydroxynitriles, which were used as intermediates in an environmentally friendly synthesis of atorvastatin. They exploited the promiscuitiy of halohydrin dehalogenases, accepting cyanide as a nucleophile: after formation of epoxide 184, cyanide readily opened the ring and gave the corresponding β-hydroxynitrile 185 (Scheme 19a). Iterations of DNA shuffling increased the enzyme activity >2500-fold over the wild type, and ethyl (R)-4-cyano-3-hydroxybutanoate 185 was obtained in 92% yield. (468)

Scheme 19

Scheme 19. Representative Selection of Enzymatic Transformations for the Production of Epoxide-Containing Products
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Kong et al. created a library of variants of the epoxide hydrolase from Bacillus megaterium (BmEH) to expand the substrate scope from phenyl glycidyl ether toward bulkier epoxides. By mutation of two active site residues, variants with an improved activity (6–340-fold) for nine β-blocker precursors were discovered. Resolution of the racemic epoxide precursors 186 yielded the (S)-epoxides 187 in 37–45% yield and 96.6–99.5%ee (Scheme 19b). (469)
Lin et al. reported a method for the oxidative dearomatization of indoles yielding asymmetric furoindolines using the flavin-dependent monooxygenase TsrE. The enzyme catalyzed the 2,3-epoxidation of 2-methyl-3-indole-3-acetic acid 188 and the subsequent epoxide opening of 189 in the synthesis of (3aS,8aR)-3a-hydroxy-8a-methyl oxofuroindoline 190, which was obtained in 84% yield and 98%ee (Scheme 19c). (470)
Assays for epoxides are usually based on nucleophilic ring-opening reactions, and detection often happens indirectly. They are mostly based on the conversion of epoxides to aldehydes, thiols, or diols and the application of aldehyde-, thiol-, or diol- detection methods. Detection might be complicated by instability (e.g., to hydrolysis), side reactions, (464,471) and volatility. (456,465,472) Epoxides can undergo reactions with hemoglobin or nucleic acids and are therefore associated with mutagenicity, carcinogenicity, and genotoxicity (473) (Figure 59).

13.1. Nucleophilic Ring-Opening by Pyridine Derivatives

Various epoxide assays are based on ring-opening reactions by nucleophilic pyridine derivates, resulting in colorimetric or fluorometric signals (Figure 60). The commercially available reagent 4-(p-nitrobenzyl)pyridine (NBP, 191) is often applied to detect epoxides. (459,461,473−476) The nucleophilic substitution reaction of 191 with epoxides results in an uncolored intermediate, which can subsequently be converted to the blue-colored dihydropyridine using base. Changes in absorption in the range of 560–620 nm have been used for read-outs. (458,459,473,475) Zocher et al. developed a HTS assay with 191 for microtiter plates to screen for activity of an epoxide hydrolase. (458) Alcalde, Farinas, and Arnold also used an NBP-based HTS assay for directed evolution to improve styrene epoxidation by cytochrome P450 BM-3 139-3. (459) Probe 191 can also be used to detect organophosphorus compounds, ethylenimines, and other alkylating reagents, and side reactions with these compound classes might interfere with the detection of the epoxides. (461)

Figure 60

Figure 60. Summary of chromo- and fluorogenic turn-on probes for the selective detection of epoxides based on ring-opening reactions by nucleophilic pyridine derivatives. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Because 191 is insoluble in water, (473) Nelis and Sinsheimer described a similar fluorometric detection method with high sensitivity and solubility in aqueous systems. Their approach was based on the alkylation of nicotinamide 192 by aliphatic epoxides (Scheme 20). The alkylated amides 194 were converted to dihydropyridine derivatives 195 with acetophenone 193 under basic conditions, followed by a cyclization triggered by formic acid, which resulted in the formation of fluorescent products 196. This method is not specific for epoxides; it detects various electrophilic compounds. Fluorescence of NADP+, itself an alkylated nicotinamide, might interfere, too. (474)

Scheme 20

Scheme 20. Sensing Mechanism for Epoxides Employing the Combination of Nicotinamide 192 and Acetophenone 193
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13.2. Indirect Detection

In many cases, the detection of epoxides is done indirectly by conversion to other functional groups and subsequently detecting those (Figure 61). Sano and Takitani developed a two-stage approach to detect epoxides in ethanolic aqueous solutions. The epoxides were converted to the respective S-alkylated derivatives by ring-opening with sodium sulfide. The resulting thiolates could be efficiently labeled by using a system known for fluorogenic isoindole formation employing o-phthalaldehyde 22 and taurine (cf. section 6.3). (477)

Figure 61

Figure 61. Selective detection of epoxides based on indirect detection methods after initial nucleophilic ring-opening reactions with hydroxide or bisulfide. The resulting intermediates can be detected with thiol sensing assays (cf. section 15), diol sensing assays (cf. section 8), or by using a similar method discussed for the indirect detection of diols by oxidative cleavage with metaperiodate 97 and subsequent detection using aldehyde-sensing assays (cf. section 9).

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Another approach for the indirect detection of epoxides is based on their conversion to diols and the subsequent application of diol-sensitive assays. Doderer et al. described a colorimetric HTA that could be applied for all epoxides with at least one hydrogen substituent. The epoxides were converted to the diols using epoxide hydrolase, which were subsequently cleaved by periodate 97 to the corresponding aldehydes and/or ketones. The formed aldehydes could then be detected by Schiff’s reagent 123. The absorption of the resulting magenta dye was measured at 560 nm. (262) Doderer and Schmid reported another concept for diols based on oxidative cleavage with periodate to the corresponding carbonyls. Titration of the residual periodate 97 with carboxyfluorescein resulted in a detectable decrease in fluorescence. (268) Some of the carbonyl species resulting from diol cleavage already have a strong UV absorbance, allowing their direct detection. Mateo et al. used this property for the determination of styrene oxide by detecting benzaldehyde at 290 nm after hydrolysis and cleavage. They used this method for the fast determination of activity of epoxide hydrolase. (481)
Halohydrin dehalogenases are commonly used as enzymatic catalysts to obtain epoxides from aliphatic or aromatic halohydrins. This and the reverse reaction is accompanied by a change in pH, which enables the indirect detection of epoxides using pH indicators, such as phenol red or bromothymol blue. (478,479) Other assays make use of the direct change in UV absorption or fluorescence upon the conversion of epoxides to their corresponding diols. (472,480) Wixtrom and Hammock reported two such spectrophotometric assays. (472)

14. Chromo- and Fluorogenic Probes for Phenols

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Phenols and their derivatives are an important structural motif ubiquitous in nature in the form of catechols, quinones, lignans, flavones, or in polymeric form in lignin, flavolan, or tannins, among others. These hydroxylated aromatic structures are also crucial in industry (epoxide and phenolic resins, azo dyes, explosives) and as biologically active ingredients (drugs, herbicides). (482)

Figure 62

Figure 62. (top) Summary of general properties of phenol sensors as well as expected cross-reactivity during screenings. (bottom): Compatible pH range and buffers in reported phenol-selective assays. Bold pH numbers indicate the most commonly used pH values.

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Synthesis of hydroxylated aromatic compounds (483) typically requires harsh conditions, which has motivated a shift to enzymatic production of this class of compounds. (484)
With the emergence of novel biocatalysts, namely mono- (MO), di- (DO), or peroxygenases (PO) as well as tyrosinases (TYR), which enable aromatic hydroxylation, the scientific community has strived to discover and develop improved enzymatic catalysts. (484) Lan Tee and Schwaneberg summarized the developments for directed evolutions of oxygenases, its success stories, and challenges in a recent review. (485)
Among many examples for the success of these transformations (Scheme 21), Molina-Espeja et al. applied directed evolution strategies for the optimization of an unspecific peroxygenase from Agrocybe aegerita (AaeUPO1), engineered for the synthesis of 1-naphthol 198. (486,678) The resulting double mutant (G241D-R257K) enabled the regioselective synthesis with a total turnover number of 50 000. The wild-type has also been efficiently applied for the hydroxylation of acetanilide 199 (among other relevant precursors), resulting in the selective production of acetaminophen (paracetamol) 200. (487) The group of Arnold evolved a toluene dioxygenase to improve the turnover of 4-picoline 201, resulting in the discovery of the single-point mutant R459L with 5.6-times higher activity than the wild type. (488)

Scheme 21

Scheme 21. Representative Selection of Enzymatic Transformations for the Production of Phenol-Containing Products
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Phenols are more acidic than alcohols because the anion formed in deprotonation is stabilized by the conjugated electron system of the aryl. The delocalized charge increases the nucleophilicity and the antioxidant properties of phenols (stabilization of radicals compared to benzene). (489) Their qualities as antioxidants are often used as a means to quantifying phenol content in terms of total antioxidant capability (AOC). (490)
Several assays have been developed in the food and live sciences due to the necessity for the assessment of antioxidant properties: the Folin–Ciocalteu (FC) test, (491−493) ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging, (494) ORAC (oxygen radical absorbance capacity), (495) FRAP (ferric-reducing antioxidant power), (496) CuPRAC (cupric ion reducing antioxidant capacity), (496) TEAC (trolox equivalent antioxidant capacity), (497) and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging. (494) None of these established methods are recognized as universal, (496,498,499) and the correct selection for the study of AOC remains difficult.
Reducing power of the matrix can be problematic in detecting phenols, particularly in biological media with many reducing compounds (e.g., ascorbic acid, sulphites, reducing sugars). (500) Hence, reaction-based methods that integrate the phenol into a chromo- or fluorogenic product have proven superior in sensitivity. (501) Due to the direct conjugation of the phenol structures into the final chromophores, large differences in absorbance can be expected for each analyte (Figure 62).

14.1. Electrophilic Aromatic Substitution (SEAr) with Activated Amino Groups

Most methods rely on trapping the nucleophilic phenol or phenolate with a positively charged probe in an electrophilic aromatic substitution (Figure 63). The subsequent oxidation of the dienone typically triggers the large change in color (and/or intensity) and increases the stability of the products (iminoquinones). The most widely applied method uses commercially available 4-aminoantipyrine (4-AAP, 203), initially reported by Emerson in 1943. (502) Its oxidative coupling to phenols produces an intensely amber to red colored complex, (490,503,504) which is most sensitive at pH 9–10. (503,505) Appropriate buffering is necessary to reduce noise, which was typically caused by a distinct drop in pH upon the addition of reagents. (505,506)

Figure 63

Figure 63. Summary of chromogenic turn-on probes for the selective detection of phenols based on the electrophilic aromatic substitution with oxidized amino derivatives. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum. Alk = alkyl, Ar = aryl.

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Typically, K3[Fe(CN)6] or K2S2O8 are used as oxidants, although recently systems consisting of iron(III)-octacarboxyphthalocyanine and t-BuOOH have been demonstrated as fast, water-soluble oxidants. (507) Synthetic optimizations of the parent structures have been proposed with the exchange of alkyl and aryl-side substituents, with little improvement over the 4-AAP assay. (506,508) Only phenols with hydrogen, halides, or alkoxy substituents in para-position are detectable. (506) This assay system has been used for the colorimetric determination of phenolic compounds in pharmaceutical preparations, (503) tetracyclines, (509) and many other examples. (505,510)
Probe 203 was also used in directed evolution of hydroxylases toward aromatic and heterocyclic compounds (501,511) and esterases by the group of Schwaneberg. (512) Reliable detection of phenolic substrates at 50–800 μM was enabled after 30 min incubation. Choi et al. applied the probe in HTS and directed evolution of a tyrosine phenol-lyase. (513)
Probe 204 (“Gibbs reagent”) is a prominent example in this category. It is already preoxidized (aniline is trapped as chloroimide), thus does not require an oxidant. (514) The chloroimide is rapidly hydrolyzed at pH > 8.5, yielding the free imine. Both forms react exclusively at the para-position of the phenol ring. As with 203, only hydrogen, halide, or alkoxy substituents are tolerated. (515,516) Even though decomposition of the quinoneimine was shown to occur in alkaline medium, the formation of the product is favored, and the reaction is not hampered by prolonged reaction times. Full conversion of phenol was reported using a 10-fold excess at pH 10 after approximately 10–20 min at μM concentrations. (517)
204 was used by Ang et al. for the directed evolution of an aniline dioxygenase for bioremediation purposes, targeting the resulting catechol derivatives. (518) Arnold’s group used it in HTS of dioxygenase activity using solid-phase digital imaging (486) and for the evolution of a toluene dioxygenase toward 4-picoline (4-methylpyridine). (488) Here, the initially formed cis-dihydrodiol product was not detected and deemed to be unstable. Its spontaneous rearomatization by dehydration, forming the 3-hydroxy-4-picoline 202, allowed the detection with 204 without using dihydrodiol dehydrogenase or decreasing of pH, which is typically required for this transformation. Bornscheuer et al. also used the reagent to test the selectivity of 28 lipases and esterases in the hydrolysis of butanoates of ortho-, meta-, or para-substituted phenols. (519)
Taking advantage of the well-studied spectroscopic properties of phenol blue, Altahir et al. developed a phenol probe based on p-amino-N,N-dimethylaniline 205. (520) Using a similar protocol as for the 4-AAP assay, the two-electron oxidation of the aniline with K3[Fe(CN)6] under basic conditions enabled the detection of eugenol contained in personal-care products.
MBTH 114, known as a tool to detect aldehydes (cf. section 9.1), has also been efficiently used for the derivatization of phenols. (521) Da Silva Granja et al. evaluated 114 among other methods for the determination of total phenol content (203, FC assay, ABTS assay, DPPH 109 assay) and found it to be equally useful. (500) The reaction with 114 works in dilute H2SO4 (500,521) and at at pH 8–9, (522,523) contrary to the typically neutral to basic conditions required by reagents 203 and 204. Oxidations are typically performed with K3[Fe(CN)6], FeCl3, or K2S2O8. Oxidative coupling mediated by laccase or horseradish peroxidase have also been reported. (524,525) Reactions with phenols are not limited to the para-position, as ortho-positions have also been successfully derivatized. Side reactions can be expected with amines, electron-rich aromatic compounds, and aldehydes (e.g., reducing sugars). Nolan and O’Connor applied the assay for the quantification of a toluene-4-monooxygenase activity producing para-substituted phenols. These were detected after tyrosinase-assisted transformation of the products to 4-substituted catechols. (526)

14.2. Azo Coupling

Phenols can be detected using azo-coupling reactions (Figure 64). Herein, aromatic diazonium salts react as strong electrophiles with activated arenes in electrophilic aromatic substitutions at the ortho- or para-unsubstituted positions. (527) The resulting azo compounds often have vivid colors due to their large conjugated systems and are widely used as dyes and pigments. (528,529)

Figure 64

Figure 64. Summary of chromogenic turn-on probes for the selective detection of phenols based on the formation of azo-dyes. Abs: wavelengths typically used for UV absorbance measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Urbányi and Mollica tested 27 differently substituted aniline derivatives and established a strong dependency of the sensitivity of these probes on the functionality and substitution pattern of their aromatic systems. Electron-poor diazonium salts were shown to form more intensely colored products after fixed reaction times (6 min). Although no underlying kinetic study was undertaken and single points were used as read-outs, the authors concluded that electron-poor systems are the better analytical probes for rapid screenings. (530)
Diazotized sulfanilic acid 206 (Pauly’s or Ehrlich’s diazo reagent) has been used as early as 1883 by Ehrlich for the detection of bilirubin in urine samples (531) and has found many applications in the detection of phenolic derivatives since then. (532−535) Its electron-poor character, however, makes it unstable; fresh preparation before use is necessary.
The emergence of commercially available, stabilized diazonium salts, either by decoration of the aromatic core with electron-donating functionalities or complexation with suitable anions, such as zinc chloride or tetrafluoroborates, enabled broad usage of these reagents (“Fast” salts) for the detection of phenols in food, water, and biological samples. (535−538) The high stability of electron-rich arenes has been systematically studied by Schotten et al. (539) by comparing differently substituted anilines. The formation of reversibly cleavable triazenes with secondary amines was reported to further increase stability.
Currently, many differently substituted diazonium salts are commercially available, with a broad scope of applications in different buffer systems. Johnston and Ashford studied the nine most widely used “Fast” reagents for the quantification of activity of hog liver esterase, using α-naphthyl acetate as substrate. (540) Fast Red GG 207 (p-nitrobenzenediazonium tetrafluoroborate) and Fast Violet B 208 emerged as the best combination of reagents to cover a combined pH range of 3–9.5. Coupling reactions with these probes were reported to be complete in under 5 s at approximately 1 mM concentrations using 5-fold excess naphthol, allowing the determination of initial rates.
It has been reported for 207 (but not for 208) that enzymatic activity might be inhibited by certain diazonium salts or their stabilizers. (541) Purification of commercial material or validation of activity by an orthogonal method might prove useful. Lugg tested combinations of 24 airborne phenols and anilines with 25 Fast salts and thus showed the differences in spectral responses of these assays. (542) The author concluded that probe 210 has universal applicability and a low background. The salt has also been used to quantify phenol in air and urine (543) and o-phenylphenol in aqueous samples. (544)
Structural analogues with red-shifted absorption maxima (Fast Blue RR 209, BB 210, and B 211) have also been used to detect phenol in samples. (535,536) Probe 211 was used by the group of Wood for the directed evolution of a toluene o-monooxygenase. (545) Among other protocols for HTS for aromatic hydroxylations using 203 and 204, Otey and Joern presented a concise summary for the detection of phenols, also using 208 with a large set of differently substituted phenols. (510)

14.3. Mannich Type Reaction

A new approach for a fluorogenic detection of phenols is based on Mannich-type reactions of activated arenes (Betti reaction; Figure 65). (546,547) The group of Tanaka reported the reaction of phenols and peptides bearing tyrosine units with cyclic imines to proceed without the use of catalysts from pH 2–10. (548)

Figure 65

Figure 65. Summary of fluorogenic turn-on probes for the selective detection of phenol based on Mannich-type reactions. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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They designed several fluorogenic reporters 212–215 based on this mechanism. (679) Substitution patterns of the attacking arene are crucial: only ortho-phenol substituted product gave the desired increase in fluorescence. The authors hypothesized that intramolecular interaction between the phenolic hydroxyl group and the amine, or π–π stacking interactions between the phenol and fluorophore aryl moieties, were essential for fluorescence to arise. Using methyl 4-hydroxyphenylacetate and their coumarin probe 212, they demonstrated an approximately 100-fold increase in fluorescence after 70 min at pH 7. The slow reaction required a 1000-fold excess of phenol at μM concentrations. No further investigations and optimization of the reaction conditions were undertaken to test the limits of the system.

15. Chromo- and Fluorogenic Probes for Thiols

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Thiols are essential components of biological systems. (549) Compounds such as glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and coenzyme A are involved in various biological processes including regulation of the oxidation–reduction states of the cells, (550) protein synthesis, (551) signal transduction, (552) detoxification of xenobiotics, (553) fatty acid biosynthesis, (554) and posttranslational modifications. (555) Aberrations of thiol levels in cells are considered to be related to diseases such as Parkinson’s disease, (556) Alzheimer’s disease, (557) osteoporosis, (558) and cancer. (559) Therefore, the development of selective and sensitive detection methods for thiols has become an important research field in clinical diagnosis. (556,559)
Other thiols, e.g., thiophenols, are used in the agrochemical industry for pesticide syntheses, (560,561) in the pharmaceutical industry, (562) and in the food industry as flavoring ingredients (e.g., 2-ethylthiophenol with a roasted, smokey odor). (563)
Thiols are nucleophilic and react with electrophiles. In this regard, their reactivity is highly correlated with their pKa value, as the compounds become stronger nucleophiles when deprotonated. A typical pKa of aromatic thiols is ∼6.5, and ∼8.5 for aliphatic thiols. (564,565) Fluorescence-based assays can be applied for thiol detection and quantification, in addition to chromatography, UV/vis spectroscopy, potentiometry, and mass spectrometry. (566−568) Many of the fluorescence assays use the nucleophilicity of the thiols in reactions such as the Michael addition, cleavage of benzenesulfonamides and benzenesulfonates, cyclization with an aldehydes, cleavage of (O-, S-, Se-)phenyl ethers, cleavage of disulfides, or make use of the high affinity of thiols toward metals. (249,564,569)
Potential challenges in the development/application of fluorescent probes for thiols include cross-reactivity with other nucleophiles (e.g., cyanides, (570,571) amines, and amino acids), (572) requirements for a narrow pH range, (573) and slow reaction rates under physiological conditions (Figure 66). Thiols occur in a large range of concentrations in cells, making selective detection even more difficult; Bennett et al. quantified metabolite concentrations in E. coli and found GSH at 17 mM and Hcy at only 0.37 mM. (29) Additionally, selectivity between thiols can be an issue (e.g., GSH vs Cys/Hcy, or the distinction between Cys/Hcy, GSH, and H2S). (563,565,574−576) Various probes thus require substrates with an amino group in addition to the thiol functionality to produce a color/fluorescence response. In the following section, those probes are labeled with a blue “NH2-required”.

Figure 66

Figure 66. (top) Summary of general properties of thiols, reactivity trends toward thiol-selective probes, and expected cross-reactivity. (bottom) Compatible pH range and buffers in reported thiol-selective assays. Bold pH numbers indicate the most commonly used pH values.

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These probes discriminate GSH and Cys/Hcy based on a reaction sequence: nucleophilic substitution reaction or conjugate addition, and subsequent intramolecular thiol to amine rearrangements or cyclizations. These S–N rearrangements for Cys/Hcy are favored compared to GSH due to lower energy transition states.
Some assays that discriminate Cys/Hcy against GSH are based on the cyclization of Cys and Hcy with aldehyde-containing probes, forming stable five-and six-membered rings. Most development is focused on detecting the thiols already mentioned (Cys, Hcy, GSH, and coenzyme A). Hence many assays were only tested with these substrates.

15.1. Nucleophilic Aromatic Substitution (SNAr)

Thiolates readily react with electron-deficient centers via nucleophilic aromatic substitution (SNAr). Electron-withdrawing groups at the aromatic ring provide the needed electron deficiency for the substitution reaction. Common leaving groups for the thiol-mediated SNAr include phenylsulfonyl moieties, halogenides, O-phenyl ethers, and S-phenyl ethers (Figure 67). (577)

Figure 67

Figure 67. Summary of fluorogenic turn-on probes for the selective detection of thiols based on nucleophilic aromatic substitution reactions (SNAr) with aromatic systems containing electron-withdrawing groups (EWG). Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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In 1968, Ghosh and Whitehouse observed that benzofurazans, e.g., 4-chloro-7-nitro-2,1,3-benzofurazan (NBD-Cl, 12), showed a fluorescent response upon reaction with amino groups. They also noticed that thiol derivatives were slightly fluorescent. (79) Since then, 12 and (aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F, 216), which was developed some years later by Toyo’oka and Imai, (578) have been applied for the detection of thiols and activity screenings of carnitine palmitoyltransferase, citrate synthase, and acetylcholinesterase. In these cases, the commercially available probes showed a fluorescent signal upon reaction with the enzymatically released thiols. (579,580)
Niu et al. showed that 12 could be used for the selective and rapid detection of Cys/Hcy in the presence of GSH under mild conditions in an aqueous acetonitrile (25% ACN). The selectivity for of Cys/Hcy is potentially linked to an intramolecular displacement of the thiolate, leading to the formation of the N-substituted NBD derivative with strong fluorescence. In contrast, the S-substituted derivative with GSH only gave negligible fluorescence. The observation that the S-substituted and not the N-substituted derivative was obtained in the case of GSH was explained by comparing transition states (showing an unfavorable macrocyclic transition state with GSH, and favorable five- or six-membered rings as transition states with Cys and Hcy). An possible explanation for the faster response with Cys than Hcy could be that the formation of five-membered rings is faster than six-membered ones (Scheme 22). (581)

Scheme 22

Scheme 22. Proposed Mechanism for the Reaction of NBD-Cl 12 with Cys, Hcy, and GSH (581)
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The ratiometric BODIPY probe (BODIPY-PPh3,217) was used for the selective detection of GSH over Cys/Hcy by nucleophilic substitution of chlorine with the thiolate. Distinction between GSH and Cys/Hcy was possible because GSH gave a highly fluorescent, sulfur-substituted conjugate, but Cys/Hcy formed weakly fluorescent, amino-substituted BODIPY derivatives upon rearrangement. (582) By adding a triphenylphosphonium moiety, it was possible to target the probe at mitochondria. (583) Hu et al. made use of thiols’ ability to cleave phenyl ethers and developed a nonfluorescent probe with an NBD scaffold (NR-NBD, 218) that enabled rapid discrimination (within a few minutes) between Cys/Hcy and GSH under aqueous conditions by using two excitation wavelengths, accompanied by a color change from blue to green. (584) He et al. enhanced this idea and were the first that expanded the functionality with the probe HMN 219, that simultaneously distinguished between Cys/Hcy, GSH, and hydrogen sulfide (Figure 68). (565)

Figure 68

Figure 68. Design of the probe HMN 219 and the proposed fluorescence signal changes in response to individual or sequential detection of thiols with three well-defined emission bands. Reproduced with permission from ref (565). Copyright 2017 Royal Society of Chemistry.

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S-Phenyl ethers and Se-phenyl ethers can also be used as leaving groups. The bond length of the carbon-heteroatom bond increases from oxyethers over thioethers to selenoethers, with a concomitant decrease in stability. The most stable bond (oxyethers) also makes the reaction rate of O-phenyl ether-based probes generally slow. (575,585,586) However, rates can be increased by adding electron-withdrawing groups at the aromatic ring, for example, in nitrophenyl-O-ethers. (565,586)
In 2015, Kim et al. developed probe 220 for the selective and fast detection of GSH under physiological conditions. A phenylselenide linked to a coumarin probe was used as a leaving group. The electrophilicity of the probe was increased by installing two carbonyl groups adjacent to the reaction site, enabling a fast substitution reaction with the nucleophilic thiol. Subsequently, an intramolecular cyclization forming an iminium group was observed. With 220, detection of GSH in an aqueous DMSO solution was possible within seconds. (587) Later, the authors presented modifications of the probe by substituting the diethylamino group with an N-heterocyclic ring and introduction of an ethyl butanoate group. They envisioned that by restricting the free rotation of the amino group and the additional ethyl butanoate functionality, the low quantum yield and the poor water solubility of 220 would be improved. Although probe 221 (DACP-2) showed a slower response (about 10 min) than 220, it indeed was better soluble in water and had a higher quantum yield (0.48 vs <0.001). It enabled the selective detection of GSH and Cys/Hcy. (588)

15.2. DNBS Cleavage via Nucleophilic Aromatic Substitution (SNAr)

DNBS (2,4-dinitrobenzene sulfonyl) is a commonly used building block in thiol-sensitive probes. Upon reaction with thiolates, the benzenesulfonamide or benzenesulfonate bond is cleaved, SO2 is released, and fluorescence is restored (Figure 69). (589−593) In 2005, Maeda et al. developed the DNBS-based probe BESThio 222, which showed a high increase in fluorescence within minutes upon reaction with thiols under mild aqueous conditions (pH 7.4, 37 °C). At the same time, no fluorescence signal was observed with amines, such as ethylamine or piperidine, or alcohols, such as catechol or glucose, at pH 5.8–7.4. The authors demonstrated that their probe could be used as an alternative to the commonly used colorimetric Ellman’s reagent 232 and applied it in the fast screening of ChE (cholinesterase) inhibitors. Furthermore, they showed that their probe could detect selenols and distinguish between thiols and selenols by adjusting the pH from 7.4 to 5.8 (Figure 70). (594,589)

Figure 69

Figure 69. Summary of fluorogenic turn-on probes for the selective detection of thiols based on 2,4-dinitrobenzene sulfonyl (DNBS) cleavage by nucleophilic aromatic substitution reactions (SNAr) with aromatic systems containing electron-withdrawing groups (EWG). Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Figure 70

Figure 70. Fluorescent probe BESThio 222 and its discrimination between selenols and thiols. Reproduced with permission from ref (594). Copyright 2006 Wiley-VCH.

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Figure 71

Figure 71. Summary of fluorogenic turn-on probes for the selective detection of thiols based on disulfide bond cleavage. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Peng et al. investigated methods to detect the activity of β-lactamase and reported an indirect detection approach based on aggregation-induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT). (595) Fluorophores that exhibit AIE fluoresce strongly in the solid state or as aggregates in solution. The AIE and ESIPT characteristics of salicylaldehyde azine-based fluorophores depend on their hydroxyl groups. In probe DNBS-CSA 223, one hydroxyl groups was blocked by a 2,4-dinitrobenzenesulfonate group. The reaction of 223 with thiols led to their deprotection, which recovered fluorescence by AIE and ESIPT.
The indirect detection method by Peng et al. consisted of three steps. After the enzymatic cleavage of the β-lactam ring of the cefazolin substrate, a spontaneous elimination yielded a thiol, which was detected with DNBS-CSA by releasing CSA (salicylaldehyde azine derivative) and thereby enabling AIE and ESIPT fluorescence. This approach enabled the detection of β-lactamase activity under mild conditions in solution (detection limit 0.5 mU mL–1) and on paper-based test strips. (595)
Yuan et al. described the NIR-fluorescent probe NIR-Thiol 224, with DNBS as a recognition moiety, for the detection and imaging of thiols in living mice. The probe does not fluoresce by itself and showed a fast response to μM concentrations of thiols. The addition of Cys in PBS buffer/ACN (v/v = 7:3; pH 7.4) resulted in a 50-fold increase in fluorescence, reaching the maximum within 60 s. Probe 224 was proven to be stable under assay conditions (24 h) and could be stored as a solid for more than one year. (596)
Challenges in clinical diagnostics motivated the development of fast fluorescent probes for thiols. (579,597−599) Lin et al. developed the NIR DNBS-based probe Hcyc-NO 225 that allowed the almost instantaneous detection (<5 s) of thiols relevant for evaluating cellular oxidative stress, including GSH, Cys, and Hcy. The assay works under mild, aqueous conditions (PBS/DMSO 8:2, pH 7.4) and showed detection limits of 0.08 μM (Cys), 0.20 μM (Hcy), and 0.11 μM (GSH). Although probe 225 readily reacts with thiols from pH 4–8, it was observed that more basic conditions resulted in increased fluorescence. (600)
Lu et al. reported a far-red fluorescent probe based on the iminobenzo[g]coumarin dye and DNBS as recognition site (Cou-DNBS, 226), which reacted quickly even with low amounts (nM) of thiols under basic or neutral conditions. It was used for monitoring intracellular fluctuations in thiol levels, demonstrated by incubating HeLa cells that had been pretreated with H2O2 with the probe and imaging the development of thiol concentration over time. Fluorescence increased from zero (or insignificant noise) to strong signals within 20 min, indicating thiol recovery. (597)
Development of probes that are specific to a particular thiol is challenging. Thiophenols are toxic to aquatic organisms and animals and harmful to human health after prolonged exposure. Therefore, probes that can detect thiophenols and differentiate between those and other thiols are of interest. (590,601−603) Jiang et al. developed the essentially nonfluorescent NBD-Cl 12 modified probe 227, with a benzenesulfonamide reaction site. The selectivity for thiophenols was achieved by relying on the pKa differences between aliphatic thiols (∼8.5) and thiophenols (∼6.5). Thus, thiophenolates could rapidly cleave the benzenesulfonamide bond under neutral conditions, whereas the aliphatic thiols remained in the less reactive neutral form. No fluorescence response was observed for aliphatic thiols, such as t-butyl thioalcohol, GSH, and Cys, or other nucleophiles (e.g., NaCN, BnNH2). The water-soluble probe was synthesized in two steps from 12 with moderate yield and gave a more than 50-fold increase in fluorescence intensity within 10 min of addition of thiophenol or derivatives (two equivalents), such as 4-methyl-, 4-methoxy, or 4-chlorothiophenol. A lower fluorescence enhancement was measured with 4-nitrothiophenol, explained by the strong electron-withdrawing properties of the nitro-group. (590)
The same concept for discrimination between aliphatic and aromatic thiols was utilized by Xiong et al. They developed the fluorogenic and chromogenic probe SQ-DNBS 228, with a rapid response (≤10 min) and high selectivity toward thiophenols. Their squaraine-based probe with DNBS as the reactive site was used to detect and quantify thiophenols in water samples. The far-red/NIR probe showed a high sensitivity (LOD: 9.9 nM), good photostability (>1 h under constant irradiation), and could be applied under neutral or slightly basic aqueous conditions without the need for a cosolvent. Furthermore, the color change from pink to blue upon the addition of thiophenols enabled the convenient detection by the naked eye. (604)
Lanthanide complexes can be useful to eliminate background fluorescence in biological samples emanating from flavins, porphyrins, or nicotinamide adenine dinucleotides. Whereas the biogenic fluorescent signals typically have short decay times (<10 ns), the photoluminescence (PL) of lanthanide complexes (e.g., with Eu3+ or Tb3+) takes 10–1000 μs to decay (1000 to 100 000 longer lasting). By starting the measurement only after the autofluorescence has already decayed, a better signal-to-noise ratio can be obtained. With the DNBS-based Eu3+/Tb3+ complex NSTTA-Ln3+229, Dai et al. quantified thiols at μM concentration in aqueous samples and living cells, using this time-gated acquisition. (592,605,606)
Shang et al. and Fan et al. synthesized two-photon fluorescent DNBS probes. The probe DNEPI 230, by Fan et al., exhibited a fast response (about 2 min) and a high selectivity for Cys over other thiols like Hcy and GSH. (569,607) The probe FR-TP 231, developed by Shang et al., showed high selectivity toward thiophenols, a 155-fold fluorescence enhancement with an emission in the far-red region, and a rapid response (complete within minutes). Probe 231 enabled the detection of thiophenol poisoning in an animal model for thiophenol inhalation, using a two-photon fluorescence microscope.

15.3. Disulfide Cleavage

Thiols can cleave disulfide bonds, which can be used to detect them (Figure 71). (591) Chromogenic DTNB 232 (“Ellman’s reagent”) has been frequently used to quantify thiols and enzymes. (608−610) When thiols react with 232, the disulfide bond is cleaved, and chromogenic 5-thio-2-nitrobenzoate with an absorption maximum at 412 nm is released. Instability at pH > 8 creates issues with high background signal and interferences with absorption from other biomolecules (e.g., hemoglobin) and led to the development of other reagents. (609,611−615)
Based on the work by Adamczyk and Grote, Pullela et al. synthesized fluorescein- and rhodamine-containing probes (DSSA, 233) with a disulfide linker that could be used for detecting thiols in vivo and in vitro in the mM range under aqueous conditions. (616)
Cao et al. synthesized the ratiometric fluorescent probe Ratio-HPSSC 234, based on the tetrakis(4-hydroxyphenyl)porphyrin-coumarin scaffold to detect thiols under neutral conditions in ethanolic (50%) aqueous buffer solutions. A change in emission color from red to blue enabled the visual detection of thiols, with a detection limit for Cys at 0.73 μM. It was shown that the probe was suitable for imaging thiols in living cells. (617)
Wang et al. developed the ratiometric probe 235, with a disulfide bond, which linked a BODIPY moiety as FRET donor and rhodamine as FRET acceptor. It showed a high sensitivity for GSH and enabled detection down to the low mM range. (618)
Chen et al. pursued a different strategy using fluorescent carbon nanoparticles, so-called carbon quantum dots, which exhibit beneficial properties including tunable fluorescence emissions, easy functionalization, and high photochemical stability. They reported a nanoswitch based on fluorescent carbon nanoparticles, which had thiol groups covalently attached at their surface (Figure 72). These quantum dots showed a green fluorescence that vanished when the dots aggregated due to disulfide bond formation. In the presence of thiols, the disulfide bonds were cleaved and fluorescence was restored. This process proved to be reversible as the disulfide linkage and reaggregation could be triggered by H2O2. This concept was applied for evaluating the activity of butyrylcholinesterase (BChE) and for screening butyrylcholinesterase inhibitors using butyrylthiocholine iodide as a substrate. Conversion of the substrate yielded butyrate and thiocholine, which reacted with the quantum dots and thereby restored the signal. The assay could also be used to quantify the activity of AChE by changing the substrate to acetylthiocholine chloride. (611,619) Gong et al. developed another method to determine the activity of BChE and test its inhibitors. Their disulfide containing probe (“FRET chemical probe” 236) enabled measuring BChE activity in serum directly without interference from GSH, explained by the probe’s steric hindrance. (612)

Figure 72

Figure 72. Schematic illustration of redox-controlled fluorescent nanoswitch and detection strategy for BChE activity based on thiol-triggered disulfide cleavage on fluorescent carbon nanoparticles. Reproduced with permission from ref (611). Copyright 2018 American Chemical Society.

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15.4. Diselenide Cleavage

Diselenides (R-Se-Se-R) can be used instead of disulfides as a reactive center to detect thiols. (Figure 73). Diselenide bonds have lower bond energies and are cleaved faster by the nucleophilic attack of thiols than disulfides. (620,621) An notable toxicity of organoselenium compound should be expected due to their oxidizing as well as inhibitory properties for certain thiol-containing enzymes. (622)

Figure 73

Figure 73. Summary of fluorogenic turn-on probes for the selective detection of thiols based on diselenide bond cleavage. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Lou et al. developed the diselenide probe FSeSeF 237, with two fluorescein moieties as signal reporters that were linked by a diselenide bond. Upon cleavage of the diselenide bond, the two fluorescein-containing moieties are released, resulting in strong fluorescence. Probe 237 showed high selectivity for thiols and rapid response (within milliseconds for GSH in PBS buffer pH 7.4). The reaction is reversible in the presence of H2O2, and the probe was thus used for the detection of redox changes mediated by thiols and ROS. (623) Han et al. designed the diselenide-based fluorescent probe Cyo-Dise 238, which showed high selectivity for GSH and could detect it within seconds in HEPES buffer (pH 7.4) by fluorescent response, and the color change from green to red. The ratiometric fluorescent probe 238 was applied for evaluating changes in GSH levels in HepG2 and HL-7702 cells under hypothermic and hyperthermic conditions and in HepG2 and HepG2/DDP xenografts on nude mice. (624)

15.5. Selenium–Nitrogen Bond Cleavage

Thiols are sufficiently nucleophilic to cleave selenium–nitrogen bonds. Based on this reactivity, thiol-detecting probes containing selenium–nitrogen linkages have been developed (Figure 74). In 2007, Tang et al. designed the weakly fluorescent rhodamine-based probe 239 with a Se–N bond, which releases the strongly fluorescent dye rhodamine 6G upon cleavage. They demonstrated that probe 239 could be used for the quantitative detection of GSH (LOD: 1.4 nM) in PBS buffer (pH 7.4). Interestingly, a higher sensitivity was observed for the protein-bound thiols in glutathione reductase or metallothionein than for free thiols, such as Cys, 2-mercaptoethanol, or dithiothreitol. (625)

Figure 74

Figure 74. Summary of fluorogenic turn-on probes for the selective detection of thiols based on selenium–nitrogen bond cleavage. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.in the strip at the bottom representing the visible spectrum.

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The high noise and only moderate enhancement of fluorescence (about 6-fold) prompted Tang et al. to develop a better rhodamine-based probe, Rh-Se-2 240. Structural modification prevented an opening of the spirocycle under screening conditions, thereby reducing noise. Probe 240 reacted fast (within minutes) and selectively with thiols, with a >170-fold enhancement in fluorescence in aqueous solution. In addition to the turn-on response in fluorescence, the color changed from light yellow to yellow-green. (626)
The cyanine-dye-based NIR probes Cy-TfSe 241 and Cy-NiSe 242 developed by Wang et al. showed a fast signal response with thiols in PBS buffer at physiological pH. Fluorescence of these dyes was stable from pH 4.0 to 8.6. (627) Tian et al. designed the colorimetric and fluorescent probe 243, with two Se–N bonds that allowed differentiation of Cys, Hcy, and GSH (Figure 75), whereas GSH and Hcy only cleaved one Se–N bond, Cys cleaved both. Differences in the absorption and fluorescence spectra allowed distinguishing between GSH, Hcy, and Cys simultaneously. Determination of the kinetic constants and equilibrium constants showed that the reactivity of Cys toward the probe is much higher than that of GSH and Hcy, which could explain that only the presence of Cys led to the cleavage of both bonds. (628)

Figure 75

Figure 75. Structure and color changes of 243 in the presence of amino acids (1, Cys; 2, Hcy; 3, GSH; 4, none; 5, other natural amino acids). Reproduced with permission from ref (628) . Copyright 2018 Elsevier BV.

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15.6. Cyclization with Aldehydes

1,2- and 1,3-Aminothiols can form five- or six-membered rings with aldehydes. Based on this mechanism, fluorescent probes with aldehyde moieties for the selective detection of cysteine and homocysteine have been reported (Figure 76). (572,629,630) In 2004, Rusin et al. developed the xanthene-based probe 244, bearing an aldehyde for colorimetric and turn-off fluorometric detection of Cys and Hcy in alkaline aqueous solutions (pH 9.5). (629) The requirement for basic conditions prompted the development of other thiol detecting probes that could be used under physiological conditions. Lee et al. reported the coumarin-based probe 245 with a salicylaldehyde moiety, which showed high selectivity for Cys/Hcy and could be used at neutral pH. (631)

Figure 76

Figure 76. Summary of fluorogenic turn-on probes for the selective detection of thiols based on the cyclization with aldehydes. A subsequent cyclization via an additional NH2 moiety limits the application to analytes containing both functional groups. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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15.7. Conjugate Addition (1,4-Addition)

Thiols readily undergo 1,4-additions to α,β-unsaturated carbonyl compounds due to their soft nucleophilicity. They are softer nucleophiles than amines and alcohols. (632−635) Maleimides are frequently used as reactive centers for thiol-sensitive probes because they react selectively with thiols and quench fluorescence based on n−π* transitions or PET (Figure 77). Upon addition of the thiol, the quenching process is suppressed and fluorescence recovered. (632)

Figure 77

Figure 77. Summary of fluorogenic turn-on probes for the selective detection of thiols-based conjugate additions. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Commercially available N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide (CPM, 246) is often used to detect thiols. (636,637) Aharoni et al. applied 246 in a HTS of serum paraoxonase (PON1) mutants to find variants with improved thiolactonase activity. The gene variants were transformed into E. coli, and the single cells were encapsulated in w/o/w (water-in-oil-in-water) droplets together with the substrate thiobutyrolactone and 246. The droplets were sorted using FACS. With this method, they were able to discover variants with ∼100-fold higher catalytic efficiency than the wild-type PON1. (638)
Marcella and Barb presented a FabD (malonyl-coenzyme A transacylase) and a FabH (ketoacyl synthase III) assay that used 246 to detect the enzymatically released coenzyme A. Interfering thiol-containing holo-ACP in the FabD assay was removed by precipitation with trichloroacetic acid before the probe was added. Fluorescence was measured at 470 nm. They applied the assays for the rapid determination of FabH and FabD activity. (639)
Niu et al. used 246 in an assay for HTS of potent and selective hCBS (human cystathionine β-synthase) inhibitors derived from natural compounds. The assay was based on the conversion of methylcysteine by cystathionine β-synthase (CBS), leading to the release of methanethiol, which was subsequently detected by 246 at physiological pH (Figure 78). With this approach, 6491 compounds were tested, and several compounds that inhibited hCBS activity were discovered, including sikokianin C, which exhibited cytotoxic activity against HT29 cancer cells with an IC50 value (half-maximal inhibitory concentration) of 1.5 uM. The assay also could be used for monitoring hCSE (human cystathionine γ-lyase) activity. (637)

Figure 78

Figure 78. Illustration of the high-throughput assays for cystathionine β-synthase (CBS). (a) Methanethiol (CH3SH) generation catalyzed by CBS using methylcysteine (MCys) as a substrate was determined with the CPM 246. (b) The CBS activity was monitored using 10 mM MCys as the substrate in the presence of 15 μM 246 and 2 μg of hCBS in a 200 μL reaction mixture containing 50 mM HEPES, pH 7.4. The fluorescence of the reaction mixture at 460 nm (λex = 400 nm) was monitored for 600 s. Reproduced with permission from ref (637). Copyright 2017 Royal Society of Chemistry.

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Yi et al. designed the coumarin-based probe CME 247 with a maleimide moiety that showed a rapid response with high selectivity and sensitivity for thiols (<1 min for Cys) in Tris-HCl buffer (pH 7.4). It had a 470-fold increase in quantum yield upon conjugation with GSH. The assay’s sensitivity enabled the determination of GSH at concentrations as low as 0.5 nM. The water-soluble 247 could be efficiently synthesized in three steps and is stable for months in solution and in pure form. Yi et al. successfully used it to develop a high-throughput fluorescence assay for glutathione reductase. (640)
Gao et al. compared the performance of various fluorescent probes in assaying the activity of a histone acetyltransferase and concluded that probes 246 and 247 were well suited in a high-throughput format (fast reaction, fluorescence enhancement of >250-fold with coenzyme A). (555)
Zhou et al. developed the cationic probe 248, consisting of a coumarin core linked to a methylpyridinium moiety by an unsaturated ketone. It enabled the detection of Cys at nM concentration under physiological conditions, based on a new assisted electrostatic attraction strategy, and showed a fast fluorescent and colorimetric response. They assumed that the pyridinium group confers high solubility in water, high reactivity with thiols (due to its electron-withdrawing properties), a fast response (due to electrostatic interactions), and good cell permeability. The nonfluorescent and water-soluble 248 showed a 148-fold increase in fluorescence emission upon the addition of Cys and reached the maximum fluorescence intensity within 1 min. Hcy and GSH reacted much slower with the probe than Cys, explained by the lower steric hindrance, pKa, and electrostatic attraction of Cys. (641)
Langmuir et al. developed several maleimide-containing probes (including 249, 250) that almost instantaneously reacted with thiols, such as ME, GSH, and N-acetyl-l-cysteine, under physiological pH. Upon addition of a thiol to the maleimide, the initially weak fluorescence of the probes was strongly enhanced. (636)
Li et al. reported on the development of the NIR probe NIR-BODIPY-Ac 251 for the selective detection of Cys in the presence of GSH and Hcy under neutral aqueous conditions. It consisted of an acrylate group as a reactive site and an indolium-BODIPY as NIR fluorophore. Upon conjugate addition of Cys/Hcy to the acrylate, an intramolecular cyclization led to the subsequent elimination of a cyclic amide (Scheme 23). This release prompted a strong increase in fluorescence emission. The probe is highly selective for Cys in the presence of Hcy, explained by the formation of a kinetically more favored, seven-membered ring with Cys. The probe was used to detect exogenous and endogenous Cys in in vitro and in vivo. (642)

Scheme 23

Scheme 23. Proposed Response Mechanism for NIR-BODIPY-Ac 251 to Cys or Hcy
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15.8. Sulfur–Metal Interaction

Many assays rely on the high affinity of thiols toward metals and various probes that build upon these interactions with Ag, Cu, Hg, and Au, have been reported (Figure 79). (643−645) Apart from concepts based on displacement, assays also use oxidation and reduction of metals. (646−649) Jung et al. and Wang et al. developed probes 252 and 253 with Cu2+ ensembles for the detection of biogenic thiols (Figure 80). Both probes showed a fast response and a high selectivity toward thiols, in the presence of amino acids, in aqueous solution under neutral or alkaline conditions. It was inferred that in the presence of thiols, a decomplexation of Cu2+ from the iminofluorescein-Cu2+252 probe or the iminocoumarin-Cu2+ probe 253 took place, followed by hydrolysis of the probe to yield the fluorescent fluoresceinaldehyde or coumarinaldehyde. (650,651)

Figure 79

Figure 79. Summary of fluorogenic turn-on probes for the selective detection of thiols-based sulfur–metal interactions. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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Figure 80

Figure 80. Schematic illustration of the thiol detecting chemodosimetric mechanism iminocoumarin-Cu2+ probe 253 in aqueous media. Adapted with permission from ref (651). Copyright 2011 The Royal Society of Chemistry.

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Many assays based on thiol–metal interactions use metal nanoparticles or nanoclusters. Based on silver nanoparticles and fluorescent carbon dots, a colorimetric and fluorescent assay for the activity of AChE with acetylthiocholine as a substrate was developed by Zhao et al. (652) The fluorescence of the carbon dots was quenched by the absorption of the dispersed nanoparticles (inner filter effect). The high affinity of the formed thiocholine for the silver nanoparticles and electrostatic interactions led to an aggregation, reducing inner filter effect and leading to a change of fluorescence (LOD: 0.016 mU mL–1) and color (LOD: 0.021 mU mL–1).
Polyethyleneimine-protected copper nanoclusters, or a water-soluble perylene derivative in combination with Cu2+, were used for the detection of thiols, measuring the activity of AChE, and for screening inhibitors. (653,654) The mechanism for thiols was based on quenching the strong fluorescence of the polyethyleneimine-protected nanoclusters by binding of Cu2+ to the amino groups of polyethyleneimine. In the presence of thiols, however, the high affinity of Cu2+ to thiols led to the formation of thiol–Cu2+ complexes instead, which resulted in an increased fluorescent signal. (653) Similarly, adding Cu2+ quenched the fluorescence of the perylene diimide derivative 254 by binding of Cu2+ to the carboxylic groups. Capture of the Cu2+ ions coordinated to the probe by thiols enhanced the fluorescence. (654) Both probes enabled the determination of AChE activity with a detection limit of 1.38 mU mL–1 and 1.78 mU mL–1, respectively. (653,654)
Jiang et al. developed a sensitive, photoluminescent probe based on cytidine-stabilized Au nanoclusters 255 for rapid measurement of glutathione reductase activity and screening of its inhibitors. Although the photoluminescence of metal nanoclusters is often quenched by thiols, their probe showed enhanced photoluminescence. The authors speculated that the increase might result from the formation of smaller, fluorescent Au species (including Au2) etched by thiols. The probe enabled them to detect GSH at nM concentrations (LOD 2.0 nM). (169)
A displacement assay based on BODIPY-Au nanoparticles 256 was reported by Xu et al. The BODIPY chromophores on the Au nanoparticle (AuNP) surfaces could be displaced by thiols, thereby restoring the fluorescence of the BODIPY molecules (Figure 81). It showed a fast response for Cys and Hcy (∼2 min) in PBS buffer (pH 7.4), high selectivity and sensitivity (LOD: 30 nM for Cys) and was used for imaging thiols in living HeLa cells. (655)

Figure 81

Figure 81. Schematic representation of the thiol sensor 256 based on modulation of the fluorescence quenching of the BODIPY chromophore by AuNPs. Reproduced with permission from ref (655) . Copyright 2016 Elsevier BV.

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15.9. Further Reaction Types

There are other types of thiol-sensing probes, based on reaction types such as 1,2 additions to meso-vinyl BODIPY derivatives, (656) the addition of thiols to squaraine-like compounds, (570) thiol-meditated reduction of quinone, (657) thiol–halogen nucleophilic substitution reactions, (581,658−660) or transesterification of thioesters. (661) Chen et al. developed an NIR fluorescent probe for thiols (CHMC-thiol) that consisted of two reaction sites: the 2,4-dinitrobenzenesulfonate moiety was chosen as a high-sensitivity reaction site, and the less reactive chloro group was chosen as a low-sensitivity site. Low concentrations of cysteine (0–50 μM) led to the cleavage of the benzenesulfonate moiety and resulted in an increase in fluorescence at 680 nm. In the presence of high concentrations of cysteine (50–500 uM), the chlorine was substituted, followed by an intramolecular rearrangement to yield the amino product, and a ratiometric fluorescence response was obtained. This approach enabled the sensing of multiple concentration ranges via different fluorescence signals. (662)

16. Concluding Remarks

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Enzymes have intrigued chemists to unleash their catalytic utility ever since their discovery. They provide an efficient and environmentally benign alternative to conventional organic–synthetic methodology due to their unparalleled selectivity. The surge in recent identifications and the potential for optimizing catalytically active proteins promoted their use in an industrial context. In particular, computationally designed proteins and directed evolution allowed chemists to explore uncharted territory in catalysis.
Directed evolution, however, requires sophisticated screening tools that can cope with the immense numbers of variants created in randomized approaches. Libraries containing millions or billions of variants need to be assessed in a time-effective way while maintaining accuracy in the screening process. The number of potential screening events thus imposes constraints in time and matter on the underlying analytical procedures. Every mechanical manipulation has an impact on throughput, making a direct detection in crude cell lysates the optimal choice. Molecular tools thus need to function in complex environments and reduce errors stemming from reactions with endogenous compounds to a minimum.
Fluorogenic substrates that mimic the natural or desired substrate offer a viable alternative to search for catalytic functionality but decouple the analytical signal from the synthetically relevant transformation. Functional group assays offer a more generalized approach for an optical read-out. By selective interaction with defined structural properties of analytes, these tools generate powerful handles to address the issues outlined above.
Finding and adapting these handles for compatibility to physiological conditions are the biggest challenges in the field of assay development, clearly shown in the distribution of probes presented here for the different substance classes. Some functional groups are more easily detected because of high reactivity (e.g., aldehydes, amines), while others have large numbers of reactive probes due to a strong scientific or applied interest (e.g., thiols). In general, assays that work under “real” screening conditions enable applications in HTS and are thus more likely to be used frequently.
Several limitations of functional group probes exist that limit the number of tools for detecting other (not highly reactive) substance classes in situ: low solubility in water, incompatibility with water, or high/low pH, slow reaction kinetics, especially at low product concentrations, insufficient selectivity, or undesirable optical properties. Nevertheless, there are designs that address all these disadvantages: Addition of PEG groups to 37 or incorporation of charged residues like quaternary ammonium or pyridinium groups to known fluorogenic dyes, such as 29 and 248, have been used to increase their water solubility. Kinetical hurdles of ABAO reagents 126 were overcome by understanding the underlying reaction mechanism. Implementation of a methoxy group into the scaffold rendered the aldehyde selective tools also suitable for more challenging ketone detection. Selectivity issues for efficient discrimination between the GSH, Cys, and H2S were effectively overcome by designing the fluorogenic probe HMN 219, which contained multiple reactive sites. The optical properties of pyrylium probes (cf. section 6.5) were also adjusted by modifications of their fluorophore, essentially making the assays applicable over the whole visible spectrum.
Understanding the mechanisms for the genesis of the optical signal and preserving structural features critical to the desired reactivity aids the development of good probes. Although some probes seem to be applicable for a broad set of analytes (e.g., nucleophiles targeting electrophilic reactive sites), the exact selectivity of most assays is generally not (extensively) characterized. It might thus prove useful for protein engineers to test a range of different assays.
We would like to encourage research groups working on new assays to characterize the selectivity toward various derivatives of the same substance or reactivity class (e.g., nucleophiles, electrophiles, oxidants) and with expected interference from cellular components. Only a small number of probes are commercially available or easily accessible by synthesis, particularly for some functional groups (e.g., alcohols, esters). We hope that this overview of available methods motivates the reader to tackle the remaining challenges, helping protein engineers to advance in their pursuit for new and better catalytic activity.

Author Information

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  • Corresponding Author
  • Authors
    • Sebastian Hecko - Institute of Applied Synthetic Chemistry, OC-163, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria;  Orcidhttps://orcid.org/0000-0002-6210-4425
    • Astrid Schiefer - Institute of Applied Synthetic Chemistry, OC-163, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria;  Orcidhttps://orcid.org/0000-0002-3591-8765
    • Christoffel P. S. Badenhorst - Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis, University of Greifswald, Felix-Hausdorff-Str. 4, 17489 Greifswald, Germany;  Orcidhttps://orcid.org/0000-0002-5874-4577
    • Michael J. Fink - Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St, Cambridge, Massachusetts 02138, United States;  Orcidhttps://orcid.org/0000-0003-0023-8767
    • Marko D. Mihovilovic - Institute of Applied Synthetic Chemistry, OC-163, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria
    • Uwe T. Bornscheuer - Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis, University of Greifswald, Felix-Hausdorff-Str. 4, 17489 Greifswald, Germany;  Orcidhttps://orcid.org/0000-0003-0685-2696
  • Author Contributions

    CRediT: Sebastian Hecko data curation, visualization, writing-original draft, writing-review & editing; Astrid Schiefer data curation, visualization, writing-original draft, writing-review & editing; Christoffel P. S. Badenhorst writing-original draft, writing-review & editing; Marko D. Mihovilovic writing-review & editing; Uwe T. Bornscheuer validation, writing-original draft, writing-review & editing; Michael J. Fink validation, writing-review & editing; Florian Rudroff conceptualization, funding acquisition, project administration, resources, supervision, writing-review & editing.

  • Funding

    APC Funding Statement: Open Access is funded by the Austrian Science Fund (FWF).

  • Notes
    The authors declare no competing financial interest.

Biographies

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Sebastian Hecko received his B.Sc. and Dipl.Ing. in Technical Chemistry from the TU Wien. He then joined the joint research group of Prof. Rudroff and Prof. Mihovilovic at TU Wien, where he obtained his Ph.D. degree in Applied Synthetic Chemistry, developing a high-throughput screening platform for the detection of carbonyl compounds in fluorescence-activated droplet sorting systems. His current research as postdoctoral researcher focuses on the synthesis and application of ultrahigh-throughput assay systems for the directed evolution of carbonyl producing enzymes.

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Astrid Schiefer is currently a Ph.D. student in the research group Bioorganic Synthetic Chemistry at TU Wien. She studied Technical Chemistry at TU Wien and received her master’s degree in 2021. During her master’s thesis, she gained experience in assay development for the application in fluorescence-activated droplet sorting. Her current research focuses on enzyme-assisted methods for C═C bond cleavage.

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Chris P. S. Badenhorst received a B.Sc. in Biochemistry and Microbiology from the University of South Africa. He then joined Prof. Albie van Dijk’s laboratory at the Potchefstroom Campus of the North West University, where he obtained his B.Sc. Honours, M.Sc., and Ph.D. degrees in Biochemistry with a focus on molecular biology and human metabolism. He was subsequently awarded an Alexander von Humboldt Research fellowship to join Uwe Bornscheuer’s group, where he has been working on a variety of enzyme classes and the development of new ultrahigh-throughput screening methods.

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Michael J. Fink works in applied and translational research using chemistry, biotechnology, material science, and informatics. In 2019, he cofounded the startup company Datacule (together with George M. Whitesides) to develop new solutions for digital data storage based on his research at the Department of Chemistry and Chemical Biology, Harvard University (2015–2020). His research interests include information in molecular systems, microbial biotechnology, health-and-disease-related microbiology, forensic analysis, and the functional design of molecules for applications in medicine and public health. He received his undergraduate and graduate education in chemical engineering from TU Wien in Austria (B.Sc. 2008, M.Sc. 2009, Ph.D. 2013).

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Marko D. Mihovilovic graduated in Technical Chemistry at TU Wien in 1993, also receiving his doctorate from the same university in 1996, in the field of Organic Synthetic Chemistry. Postdoc placements as an Erwin Schrödinger scholarship holder then followed at the University of New Brunswick (Canada) and the University of Florida (USA) in the fields of biocatalysis and molecular biology. Returning to TU Wien, he set up his own research group in 1999. He completed his habilitation in 2003 in bio-organic chemistry and was appointed Associate Professor in 2004. Marko Mihovilovic has been Head of the Institute for Applied Synthetic Chemistry at TU Wien since 2013 and was appointed as Full Professor for Bioorganic Synthetic Chemistry in February 2014.

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Uwe T. Bornscheuer studied Chemistry and received his Ph.D. in 1993 at Hannover University followed by a postdoc at Nagoya University (Japan). In 1998, he completed his Habilitation at Stuttgart University about the use of lipases and esterases in organic synthesis. He has been Professor at the Institute of Biochemistry at Greifswald University since 1999. In 2022, he received, beside other awards, the Enzyme Engineering Award. His current research interests are on the discovery and engineering of enzymes from various classes for applications in organic synthesis, for flavors and fragrances, in lipid modification, and the degradation of plastics or complex marine polysaccharides.

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Florian Rudroff obtained his diploma and Ph.D. degree at TU Wien in Organic Chemistry. Afterward, he was awarded an “Erwin Schrödinger fellowship” and went for a postdoctoral stay with the group of Prof. Uwe Sauer at ETH Zürich in the field of Systems Biology. In 2011, he returned to the TU Wien and started his independent scientific career in Systems Biocatalysis. In December 2017, he became Assistant Professor and finished his Habilitation in Bioorganic Chemistry at the Institute of Applied Synthetic Chemistry, TU Wien. In 2020, he was appointed as Associate Professor. His main research interests are enzyme cascade catalysis, photoredox biocatalysis (e.g., CO2 utilization by cyanobacteria), protein engineering, the development of high-throughput platforms (e.g., fluorescence-activated droplet sorting, FADS), and organic synthesis.

Acknowledgments

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This work has been financially supported by the Austrian Science Fund (FWF Project P33687-N) and TU Wien.

Abbreviations

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AA

amino acid

AAOx

aryl alcohol oxidase

AaeAPO

aromatic peroxygenase (Agrocybe aegerita)

AaeUPO

unspecific peroxygenase (Agrocybe aegerita)

ABAO

amino benzamidoxime

ABTS

2,2′-azino-bis(3-ethylbenzothiazoline-6 sulfonic acid)

AChE

acetylcholinesterase

ACN

acetonitrile

ACP

acyl carrier protein

ADH

alcohol dehydrogenase

AHL

amidohydrolase

AIE

aggregation-induced emission

AKR

aldo-keto reductase

Alk

alkyl

AOC

antioxidant capability

AO/AOx

alcohol oxidase

Ar

aryl

ATA

amine transaminase

ATase

acyl transferase

AtHNL

hydroxynitrile lyase (Arabidopsis thaliana)

ATP

adenosine triphosphate

AuNCs

gold nanoclusters

AuNPs

gold nanoparticles

BChE

butyrylcholinesterase

BD

benzoxadiazole

BDHA

alcohol dehydrogenase (Bacillus subtilis)

BINOL

(1,1′-binaphthalene)-2,2′-diol

BmEH

epoxide hydrolase (Bacillus megaterium)

BnNH2

benzylamine

BphA

biphenyl dioxygenase

BSA

bovine serum albumin

(n)BuOH

butanol

BVMO

Baeyer–Villiger monooxygenase

CAR

carboxylic acid reductase

CBS

cystathionine β-synthase

CFE

cell-free extract

ChE

cholinesterase

CHEF

chelation-enhanced fluorescence

CoA/CoASH

coenzyme A

CQD

carbon quantum dot

CS

chitosan

CSA

salicylaldehyde azine derivative

CuPRAC

cupric ion reducing antioxidant capacity

Cys

cysteine

DCC

dicyclohexylcarbodiimide

de

diastereomeric excess

DERA

deoxyribose 5-phosphate aldolase

DMF

N,N-dimethylformamide

DMSO

dimethylsulfoxide

DNA

deoxyribonucleic acid

DNBS

2,4-dinitrophenyl sulfonyl

DNPH

2,4-dinitrophenylhydrazine

DO

dioxygenase

DPPH

1,1-diphenyl-2-picrylhydrazyl

EDG

electron-donating group

ee

enantiomeric excess

EET

electronic energy transfer

EH

epoxide hydrolase

epPCR

error-prone PCR

ESIPT

excited-state intramolecular proton transfer

EtOH

ethanol

EtyGly

ethylene glycol

EWG

electron-withdrawing groups

ε

molar absorption coefficient

FA

formaldehyde

FabD

malonyl-coenzyme A transacylase

FabH

ketoacyl synthase III

FACS

fluorescence-activated cell sorting

FADS

fluorescence-activated droplet sorting

FC test

Folin–Ciocalteau test

FGA

functional group assays

FGI

functional group interconversion

FITC

fluorescein isothiocyanate

FRAP

ferric-reducing antioxidant power

FRET

Förster resonance energy transfer

GC

gas chromatography

GDH

glutamate dehydrogenase

GFP

green fluorescent protein

GSH

glutathione

hCBS

human cystathionine β-synthase

hCES

human cystathionine γ-lyase

Hcy

homocysteine

HHDH

halohydrin dehalogenase

HNL

hxdroxynitrile lyases

HPLC

high-performance liquid chromatography

HTA

high-throughput assay

HPTS

8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt

HTS

high-throughput screening

IC

internal conversion

ICT

intramolecular charge transfer

iPrNH2

isopropylamine

IR

infrared

IRED

Imine reductase

ISC

intersystem crossing

ITC

isothiocyanate

KDC

keto acid decarboxylase

KIVD

2-ketoisovalerate decarboxylase

KM

Michaelis–Menten constant

KpADH

diaromatic ketone reductase

KRED

ketoreductase

KS

ketone synthase

LDH

lactate dehydrogenase

LFER

linear free energy relationship

LG

leaving group

LOD

limit of detection

λabs/abs

absorbance wavelength [nm]

λem/em

emission wavelength [nm]

λex/ex

excitation wavelength [nm]

MAH

mandelamide hydrolase

MAO

monoamine oxidase

MCys

methylcysteine

MDA

malondialdehyde

MDH

mandelate dehydrogenase

ME

mercaptoethanol

MEK

methyl ethyl ketone

MeNH2

methylamine

MeNH3Cl

methylammonium chloride

MeOH

methanol

MO

monooxygenase

MS

mass spectrometry

μMS

microscale mass spectrometry

NAD+/NADH

nicotinamide adenine dinucleotide

NADP+/NADPH

nicotinamide adenine dinucleotide phosphate

NBD

nitrobenzoxadiazole

NCL

native chemical ligation

NHS

N-hydroxysuccinimide

NIR

near-infrared

NOS

reactive nitrogen species

Nuc

nucleophile

ORAC

oxygen radical absorbance capacity

PBS

phosphate-buffered saline

PcAOX

alcohol oxidase (Phanerochaete chrysosporium)

PCR

polymerase chain reaction

PEG

polyethylene glycol

PET

photoinduced electron transfer

PFP

pentafluorophenyl

PGM

phosphoglucomutase

PL

photoluminescence

PLP

pyridoxal phosphate

PMA

phosphomolybdic acid

PO

peroxygenase

(n)PrOH

propanol

PVC

polyvinyl chloride

Φ, QY

quantum yield

RA

reductive aminase

ROS

reactive oxygen species

RSS

reactive sulfur species

SHC

squalene hopene cyclase

SNAr

nucleophilic aromatic substitution

SQ

squaraine

TA

transaminase

t-BuOH

tert-butyl alcohol

TDO

toluene dioxygenase

TDP

thiamine diphosphate

TEAC

trolox equivalent antioxidant capacity

TEMPO

(2,2,6,6-tetramethylpiperidin-1-yl)oxyl

ThDP

thiamine diphosphate

TICT

twisted intramolecular charge transfer

TLC

thin-layer chromatography

p-TsOH

p-toluenesulfonic acid

TsrE

monooxygenase

TTN

total turnover number

TYR

tyrosinase

uHTS

ultra-high-throughput screening

UV

ultraviolet

VIS

visible

w/o/w

water-in-oil-in-water

YerE

ThDP-dependent flavoenzyme (Yersinia pseudotuberculosis)

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    Traditional biochem. methods for enzyme detection are mainly based on antibody-based immunoassays, which lack the ability to monitor the spatiotemporal distribution and, in particular, the in situ activity of enzymes in live cells and in vivo. In this review, we comprehensively summarize recent progress that has been made in the development of small-mol. as well as material-based fluorogenic probes for sensitive detection of the activities of enzymes that are related to a no. of human diseases. The principles utilized to design these probes as well as their applications are reviewed. Specific attention is given to fluorogenic probes that have been developed for anal. of the activities of enzymes including oxidases and reductases, those that act on biomacromols. including DNAs, proteins/peptides/amino acids, carbohydrates and lipids, and those that are responsible for translational modifications. We envision that this review will serve as an ideal ref. for practitioners as well as beginners in relevant research fields.
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    Kaur, K.; Saini, R.; Kumar, A.; Luxami, V.; Kaur, N.; Singh, P.; Kumar, S. Chemodosimeters: An Approach for Detection and Estimation of Biologically and Medically Relevant Metal Ions, Anions and Thiols. Coord. Chem. Rev. 2012, 256 (17), 1992– 2028,  DOI: 10.1016/j.ccr.2012.04.013
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    Chemodosimeters: An approach for detection and estimation of biologically and medically relevant metal ions, anions and thiols
    Kaur, Kuljit; Saini, Rajnish; Kumar, Ashwani; Luxami, Vijay; Kaur, Navneet; Singh, Prabhpreet; Kumar, Subodh
    Coordination Chemistry Reviews (2012), 256 (17-18), 1992-2028CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
    In this review, the applications of chemodosimeters in qual. and quant. estn. of various metal ions viz. Hg2+, Cu2+, Fe3+, Ag+, Pd(0) / Pd2+ / Pd4+, Au3+ / Au+ and anions viz. fluoride and cyanide and thiols were discussed. Their use in bioimaging of analytes in living cells and zebra fish has also been presented. Various chem. transformations viz. adduct formation, substitution, hydrolysis, oxidn. and ring transformation etc. have been used in the design of resp. chemodosimeters for specific analyte. In total review contains 349 refs.
  21. 21
    You, L.; Arnold, F. H. Directed Evolution of Subtilisin E in Bacillus Subtilis to Enhance Total Activity in Aqueous Dimethylformamide. Protein Eng. 1996, 9 (1), 77– 83,  DOI: 10.1093/protein/9.1.77
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    Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide
    You, L.; Arnold, F. H.
    Protein Engineering (1996), 9 (1), 77-83CODEN: PRENE9; ISSN:0269-2139. (Oxford University Press)
    Sequential rounds of error-prone PCR to introduce random mutations and screening of the resultant mutant libraries have been used to enhance the total catalytic activity of subtilisin E significantly in a non-natural environment, aq. DMF. Seven DNA substitutions coding for three new amino acid substitutions were identified in a mutant isolated after two addnl. generations of directed evolution carried out on 10M subtilisin E, previously 'evolved' to increase its specific activity in DMF. A Bacillus subtilis-Escherichia coli shuttle vector was developed in order to increase the size of the mutant library that could be established in B. subtilis, and the stringency of the screening process was increased to reflect total as well as specific activity. This directed evolution approach has been extremely effective for improving enzyme activity in a non-natural environment; the resulting evolved 13M subtilisin exhibits specific catalytic efficiency towards the hydrolysis of a peptide substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, in a 60% DMF soln. that is three times that of the parent 10M and 471 times that of wild type subtilisin E. The total activity of the 13M culture supernatant is enhanced 16-fold over that of the parent 10M.
  22. 22
    Schmidt-Dannert, C.; Arnold, F. H. Directed Evolution of Industrial Enzymes. Trends Biotechnol. 1999, 17 (4), 135– 6,  DOI: 10.1016/S0167-7799(98)01283-9
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    Directed evolution of industrial enzymes
    Schmidt-Dannert, Claudia; Arnold, Frances H.
    Trends in Biotechnology (1999), 17 (4), 135-136CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Science Ltd.)
    There is no expanded citation for this reference.
  23. 23
    Choi, K.-Y.; Jung, E.-O.; Yun, H.; Yang, Y.-H.; Kazlauskas, R. J.; Kim, B.-G. Development of Colorimetric HTS Assay of Cytochrome P450 for Ortho-Specific Hydroxylation, and Engineering of CYP102D1 with Enhanced Catalytic Activity and Regioselectivity. ChemBioChem. 2013, 14 (10), 1231– 8,  DOI: 10.1002/cbic.201300212
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    23
    Development of Colorimetric HTS Assay of Cytochrome P450 for ortho-Specific Hydroxylation, and Engineering of CYP102D1 with Enhanced Catalytic Activity and Regioselectivity
    Choi, Kwon-Young; Jung, Eun-Ok; Yun, Hyungdon; Yang, Yung-Hun; Kazlauskas, Romas J.; Kim, Byung-Gee
    ChemBioChem (2013), 14 (10), 1231-1238CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A current challenge in high-throughput screening (HTS) of hydroxylation reactions by P 450 is a fast and sensitive assay for regioselective hydroxylation against millions of mutants. We have developed a solid-agar plate-based HTS assay for screening ortho-specific hydroxylation of daidzein by sensing formaldehyde generated from the O-dealkylation reaction. This method adopts a colorimetric dye, pararosaniline, which has previously been used as an aldehyde-specific probe within cells. The rationale for this method lies in the fact that the hydroxylation activity at ortho-carbon position to C-OH correlates with a linear relationship to O-dealkylation activity on chem. introduced methoxy group at the corresponding C-OH. As a model system, a 4',7-dihydroxyisoflavone (daidzein) hydroxylase (CYP102D1 F96V/M246I), which catalyzes hydroxylation at ortho positions of the daidzein A/B-ring, was examd. for O-dealkylation activity, by using permethylated daidzein as a surrogate substrate. By using the developed indirect bishydroxylation screening assay, the correlation coeff. between O-dealkylation and bishydroxylation activity for the template enzyme was 0.72. For further application of this assay, satn. mutants at A273/G274/T277 were examd. by mutant screening with a permethylated daidzein analog substrate (A-ring inactivated in order to find enhanced 3'-regioselectivity). The whole-cell biotransformation of daidzein by final screened mutant G1 (A273H/G274E/T277G) showed fourfold increased conversion yield, with 14.3 mgL-1 prodn. titer and greatly increased 3'-regioselectivity (3'/6=11.8). These results show that there is a remarkably high correlation (both in vitro and in vivo), thus suggesting that this assay would be ideal for a primary HTS assay for P 450 reactions.
  24. 24
    Roth, M. Fluorescence Reaction for Amino Acids. Anal. Chem. 1971, 43 (7), 880– 2,  DOI: 10.1021/ac60302a020
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    Fluorescence reaction for amino acids
    Roth, Marc
    Analytical Chemistry (1971), 43 (7), 880-2CODEN: ANCHAM; ISSN:0003-2700.
    Phthalaldehyde reacts with amino acids in alk. medium in the presence of a reducing agent such as 2-mercaptoethanol, by giving rise to strongly fluorescing compds. Optimal wavelengths are λex = 340 nm and λfl = 455 nm. This permits fluorimetric assay of amino acids down to the nmole range. No heating is necessary, and the fluorescence may easily be measured 5 min after mixing of the reagents. The reaction is well suited to the automatic detn. of amino acids after ion exchange fractionation. The sensitivity is much better than with ninhydrin procedures. The imino acids proline and hydroxyproline, however, are not detected by the method.
  25. 25
    Weigele, M.; DeBernardo, S. L.; Tengi, J. P.; Leimgruber, W. Novel Reagent for the Fluorometric Assay of Primary Amines. J. Am. Chem. Soc. 1972, 94 (16), 5927– 8,  DOI: 10.1021/ja00771a084
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    Novel reagent for the fluorometric assay of primary amines
    Weigele, M.; DeBernardo, S. L.; Tengi, J. P.; Leimgruber, W.
    Journal of the American Chemical Society (1972), 94 (16), 5927-8CODEN: JACSAT; ISSN:0002-7863.
    A novel reagent (I) has been synthesized for the fluorometric assay of primary amines (RNH2). Alk. hydrolysis of 3-benzylidene-isochroman-1,4-dione gave 0-(α-hydroxycinnamoyl)benzoic acid (II). Formylation of II with (Me2N)3CH and subsequent acid hydrolysis afforded 4-phenylspiro [furan-2(3H),1'-phthalan]-3,3'-dione (I). The spirolactone I reacts with RNH2 in aq. media at room temp. to give fluorescent pyrrolinones (III). This reaction is of particular value for the assay of amino acids, peptides and other primary amines of biol. importance.
  26. 26
    Fellner, M.; Doughty, L. M.; Jameson, G. N.; Wilbanks, S. M. A Chromogenic Assay of Substrate Depletion by Thiol Dioxygenases. Anal. Biochem. 2014, 459, 56– 60,  DOI: 10.1016/j.ab.2014.05.008
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    A chromogenic assay of substrate depletion by thiol dioxygenases
    Fellner, Matthias; Doughty, Laura M.; Jameson, Guy N. L.; Wilbanks, Sigurd M.
    Analytical Biochemistry (2014), 459 (), 56-60CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the non-heme mono-Fe enzyme, cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed std. kinetic parameters to be detd. for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitated a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addn., the relative specificity of Ellman's reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.
  27. 27
    Santos-Aberturas, J.; Dörr, M.; Waldo, G. S.; Bornscheuer, U. T. In-Depth High-Throughput Screening of Protein Engineering Libraries by Split-GFP Direct Crude Cell Extract Data Normalization. Chem. Biol. 2015, 22 (10), 1406– 14,  DOI: 10.1016/j.chembiol.2015.08.014
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    27
    In-Depth High-Throughput Screening of Protein Engineering Libraries by Split-GFP Direct Crude Cell Extract Data Normalization
    Santos-Aberturas, Javier; Doerr, Mark; Waldo, Geoffrey S.; Bornscheuer, Uwe T.
    Chemistry & Biology (Oxford, United Kingdom) (2015), 22 (10), 1406-1414CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
    Here, we report a widely and generally applicable strategy to obtain reliable information in high-throughput protein screenings of enzyme mutant libraries. The method is based on the usage of the split-GFP technol. for the normalization of the expression level of each individual protein variant combined with activity measurements, thus resolving the important problems assocd. with the different soly. of each mutant and allowing the detection of previously invisible variants. The small size of the employed protein tag (16 amino acids) required for the reconstitution of the GFP fluorescence reduces possible interferences such as enzyme activity variations or soly. disturbances to a min. Specific enzyme activity measurements without purifn., in situ sol. protein expression monitoring, and data normalization are the powerful outputs of this methodol., thus enabling the accurate identification of improved protein variants during high-throughput screening by substantially reducing the occurrence of false negatives and false positives.
  28. 28
    Azad, T.; Tashakor, A.; Hosseinkhani, S. Split-Luciferase Complementary Assay: Applications, Recent Developments, and Future Perspectives. Anal. Bioanal. Chem. 2014, 406 (23), 5541– 60,  DOI: 10.1007/s00216-014-7980-8
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    Split-luciferase complementary assay: applications, recent developments, and future perspectives
    Azad, Taha; Tashakor, Amin; Hosseinkhani, Saman
    Analytical and Bioanalytical Chemistry (2014), 406 (23), 5541-5560CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    A review. Bioluminescent systems are considered as potent reporter systems for bioanal. since they have specific characteristics, such as relatively high quantum yields and photon emission over a wide range of colors from green to red. Biochem. events are mostly accomplished through large protein machines. These mol. complexes are built from a few to many proteins organized through their interactions. These protein-protein interactions are vital to facilitate the biol. activity of cells. The split-luciferase complementation assay makes the study of two or more interacting proteins possible. In this technique, each of the two domains of luciferase is attached to each partner of two interacting proteins. On interaction of those proteins, luciferase fragments are placed close to each other and form a complemented luciferase, which produces a luminescent signal. Split luciferase is an effective tool for assaying biochem. metabolites, where a domain or an intact protein is inserted into an internally fragmented luciferase, resulting in ligand binding, which causes a change in the emitted signals. We review the various applications of this novel luminescent biosensor in studying protein-protein interactions and assaying metabolites involved in anal. biochem., cell communication and cell signaling, mol. biol., and the fate of the whole cell, and show that luciferase-based biosensors are powerful tools that can be applied for diagnostic and therapeutic purposes.
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    Bennett, B. D.; Kimball, E. H.; Gao, M.; Osterhout, R.; Van Dien, S. J.; Rabinowitz, J. D. Absolute Metabolite Concentrations and Implied Enzyme Active Site Occupancy in Escherichia Coli. Nat. Chem. Biol. 2009, 5 (8), 593– 9,  DOI: 10.1038/nchembio.186
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    Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli
    Bennett, Bryson D.; Kimball, Elizabeth H.; Gao, Melissa; Osterhout, Robin; Van Dien, Stephen J.; Rabinowitz, Joshua D.
    Nature Chemical Biology (2009), 5 (8), 593-599CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Abs. metabolite concns. are crit. to a quant. understanding of cellular metab., as concns. impact both the free energies and rates of metabolic reactions. Here we use LC-MS/MS to quantify more than 100 metabolite concns. in aerobic, exponentially growing Escherichia coli with glucose, glycerol, or acetate as the carbon source. The total obsd. intracellular metabolite pool was approx. 300 mM. A small no. of metabolites dominate the metabolome on a molar basis, with glutamate being the most abundant. Metabolite concn. exceeds Km for most substrate-enzyme pairs. An exception is lower glycolysis, where concns. of intermediates are near the Km of their consuming enzymes and all reactions are near equil. This may facilitate efficient flux reversibility given thermodn. and osmotic constraints. The data and analyses presented here highlight the ability to identify organizing metabolic principles from systems-level abs. metabolite concn. data.
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    Zuman, P. Reactions of Orthophthalaldehyde with Nucleophiles. Chem. Rev. 2004, 104 (7), 3217– 38,  DOI: 10.1021/cr0304424
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    Reactions of ortho-Phthalaldehyde with Nucleophiles
    Zuman, Petr
    Chemical Reviews (Washington, DC, United States) (2004), 104 (7), 3217-3238CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    The review discusses the chem. of o-phthalaldehyde (I) in its reactions with various oxygen (water, alcs.), nitrogen (amines, amino acids, hydrazines, amides, etc.), carbon and sulfur (thiols) nucleophiles. The special attention is given to the reactions of I in the presence of two nucleophiles, one being a thiol and another an amino acid or amine. The effects of the nucleophile structure and the reaction medium as well as the kinetics of the product formation and the proposed reaction schemes for these two-nucleophile processes are discussed in detail.
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    Henke, E.; Bornscheuer, U. T. Fluorophoric Assay for the High-Throughput Determination of Amidase Activity. Anal. Chem. 2003, 75 (2), 255– 60,  DOI: 10.1021/ac0258610
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    Fluorophoric Assay for the High-Throughput Determination of Amidase Activity
    Henke, Erik; Bornscheuer, Uwe T.
    Analytical Chemistry (2003), 75 (2), 255-260CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    An assay has been developed for the high-throughput identification of amidase activity. Amines released from the enzyme-catalyzed hydrolysis of corresponding amides were detected by the formation of a fluorescent dye by coupling with 4-nitro-7-chloro-benzo-2-oxa-1,3-diazole (NBD-Cl). Using this format, 22 lipases and esterases were tested for their ability to hydrolyze arom. substituted N-acylamines in a microtiter plate format. Identified active enzymes were further characterized toward a broad range of compds. to det. the influence of substrate structure on activity. For recombinantly produced esterases, it could be shown that the assay works with high reproducibility and sensitivity, even in the presence of amino acids and proteins present in culture media and cell debris.
  32. 32
    Médici, R.; de María, P. D.; Otten, L. G.; Straathof, A. J. J. A High-Throughput Screening Assay for Amino Acid Decarboxylase Activity. Adv. Synth. Catal. 2011, 353 (13), 2369– 76,  DOI: 10.1002/adsc.201100386
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    A high-throughput screening assay for amino acid decarboxylase activity
    Medici, Rosario; Dominguez de Maria, Pablo; Otten, Linda G.; Straathof, Adrie J. J.
    Advanced Synthesis & Catalysis (2011), 353 (13), 2369-2376CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The development of sensitive and easy-to-apply high-throughput screening (HTS) methods is a common need in modern biocatalysis. With these powerful anal. tools in hands, chemists can easily assess enzyme libraries to identify either novel biocatalysts or improved mutants. Within biocatalysis, amino acid decarboxylases are gaining an increased importance, with several diverse applications ranging from the synthesis of bio-commodities to medical applications (e.g., synthesis of enzyme inhibitors at the level of L-DOPA decarboxylase). Here, an efficient and simple fluorometric anal. method for HTS of amino acid decarboxylase activity is reported. The method was valid for the discrimination of a broad range of amino acid/amine pairs such as L-tyrosine/tyramine, L-DOPA/dopamine, 5-hydroxy-L-tryptophan/serotonin, L-histidine/histamine, L-serine/ethanolamine, L-tryptophan/tryptamine, L-glutamic acid/GABA, and L-alanine/ethylamine. The method proved its versatility by using pure substrates, mixts., or enzymic reactions, both coming either from com. enzymes or derived from cell-free (crude) exts. The limit of detection was 13 μM for ethanolamine in the presence of 50 mM L-serine, while z' values were in the range of 0.75-0.93, indicating their suitability for HTS.
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    Choi, J.-Y.; Black, R.; Lee, H.; Di Giovanni, J.; Murphy, R. C.; Ben Mamoun, C.; Voelker, D. R. An Improved and Highly Selective Fluorescence Assay for Measuring Phosphatidylserine Decarboxylase Activity. J. Biol. Chem. 2020, 295, 9211,  DOI: 10.1074/jbc.RA120.013421
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    An improved and highly selective fluorescence assay for measuring phosphatidylserine decarboxylase activity
    Choi, Jae-Yeon; Black, Raymond, III; Lee, Heejung; Di Giovanni, James; Murphy, Robert C.; Mamoun, Choukri Ben; Voelker, Dennis R.
    Journal of Biological Chemistry (2020), 295 (27), 9211-9223CODEN: JBCHA3; ISSN:1083-351X. (American Society for Biochemistry and Molecular Biology)
    Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a crit. step in membrane biogenesis and a potential target for development of antimicrobial and anticancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, the authors described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, the authors report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/β-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochem. methods. Model reactions examg. adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent addns. in 96- or 384- well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.
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    Leslie, A. K.; Li, D.; Koide, K. Amine-Promoted β-Elimination of a β-Aryloxy Aldehyde for Fluorogenic Chemodosimeters. J. Org. Chem. 2011, 76 (16), 6860– 5,  DOI: 10.1021/jo200947e
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    Amine-Promoted β-Elimination of a β-Aryloxy Aldehyde for Fluorogenic Chemodosimeters
    Leslie, Amanda K.; Li, Dan-Dan; Koide, Kazunori
    Journal of Organic Chemistry (2011), 76 (16), 6860-6865CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    The authors previously reported a fluorescent chemodosimeter for ozone (Garner et al., 2009). The β-elimination step after the ozonolysis of the chemodosimeter was too slow to be practical for real-time monitoring of ozone. Primary, secondary, and tertiary amines were examd. at various pHs. It was found that pyrrolidine in pH 9 buffer could accelerate the elimination to generate a fluorescence signal. The elimination step is now sufficiently rapid to monitor ozone exposure in real time. It was also discovered that azetidine was distinctly effective for the same elimination reaction in a pH 6 buffer.
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    Rinde, E.; Troll, W. Colorimetric Assay for Aromatic Amines. Anal. Chem. 1976, 48 (3), 542– 4,  DOI: 10.1021/ac60367a036
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    Colorimetric assay for aromatic amines
    Rinde, Esther; Troll, Walter
    Analytical Chemistry (1976), 48 (3), 542-4CODEN: ANCHAM; ISSN:0003-2700.
    Arom. amines can be detected in the nanomole range with the reagent Fluram, with which they form stable yellow derivs. Fluram in glacial acetic acid reacts only with arom. amines. The reaction is complete in 10 min and can be performed on thin-layer (TLC) chromatograms making possible the specific measurement of arom. amines. The yellow product can be quant. eluted from the TLC plates. Fluram is colorless, and the blanks are zero.
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    Spangenberg, B.; Poole, C. F.; Weins, C. Quantitative Thin-Layer Chromatography: A Practical Survey; Springer Science & Business Media, 2011.
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    Ehmann, A. The Van Urk-Salkowski Reagent ─ a Sensitive and Specific Chromogenic Reagent for Silica Gel Thin-Layer Chromatographic Detection and Identification of Indole Derivatives. J. Chromatogr. A 1977, 132 (2), 267– 76,  DOI: 10.1016/S0021-9673(00)89300-0
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    Jones, L. A.; Holmes, J. C.; Seligman, R. B. Spectrophotometric Studies of Some 2,4-Dinitrophenylhydrazones. Anal. Chem. 1956, 28 (2), 191– 8,  DOI: 10.1021/ac60110a013
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    Spectrophotometric studies of some 2,4-dinitrophenylhydrazones
    Jones, Louis A.; Holmes, Joseph C.; Seligman, Robert B.
    (1956), 28 (), 191-8CODEN: ANCHAM; ISSN:0003-2700.
    Ultraviolet and visible spectra of the 2,4-dinitrophenylhydrazones (I) of aliphatic aldehydes, aliphatic ketones, aromatic aldehydes, aromatic ketones, carbonyls contg. the furan nucleus, and olefinic aldehydes in neutral soln. and in basic soln. are recorded. Infrared spectra of the I are also given. Absorption bands obtained with the KBr-disk technique are much sharper than those obtained with the Nujol null technique. The parent carbonyl compd. can be characterized by the combined use of the ultraviolet and infrared spectra.
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    Zarzycki, P.; Bartoszuk, M.; Radziwon, A. Optimization of Tlc Detection by Phosphomolybdic Acid Staining for Robust Quantification of Cholesterol and Bile Acids. J. Planar Chromatogr. - Mod. TLC 2006, 19 (107), 52– 7,  DOI: 10.1556/JPC.19.2006.1.9
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    Optimization of TLC detection by phosphomolybdic acid staining for robust quantification of cholesterol and bile acids
    Zarzycki, Pawel K.; Bartoszuk, Malgorzata A.; Radziwon, Aneta I.
    Journal of Planar Chromatography--Modern TLC (2006), 19 (107), 52-57CODEN: JPCTE5; ISSN:0933-4173. (Research Institute for Medicinal Plants)
    In this paper we describe a robust and sensitive detection procedure for cholesterol and selected bile acids (cholic acid, lithocholic acid, and sodium taurodeoxycholate) using the common derivatization reagent phosphomolybdic acid (PMA). Visualization conditions were studied and optimized for steroids sepd. on glass TLC and HPTLC plates coated with silica gel (K60WF254S) and octadecylsilane (RP-18W) stationary phases. Spot intensities on the plates were quantified after spraying with PMA in methanol (10% w/v) and heating at temps. from 40 to 120° for times ranging from 2 to 40 min. The best conditions for high signal intensity were detd. by using 3D temp. (X)-time (Y)-anal. signal (Z) maps generated from the raw exptl. data. In contrast with the no. of "universal procedures" described in the literature our study indicated that for robust and sensitive quantification of our components of interest heating should be performed at relatively low temps. (below 100°) and for heating times in excess of 10 min. Particularly robust and sensitive detection of steroids sepd. on glass plates coated with both stationary phases was obsd. for temps. ranging between 50 and 80° and heating for at least 20 min in a simple gravity convection oven.
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    Bergmann, F. Colorimetric Determination of Amides as Hydroxamic Acids. Anal. Chem. 1952, 24 (8), 1367– 9,  DOI: 10.1021/ac60068a033
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    Colorimetric determination of amides as hydroxamic acids
    Bergmann, Felix
    (1952), 24 (), 1367-9CODEN: ANCHAM; ISSN:0003-2700.
    Reaction of amides (I) with alk. HONH2 reagent converts them to hydroxamic acids which can be detd. colorimetrically after development of color with FeCl3 (C.A. 44, 77f). Reaction time and temp. depend on the structure of the I and range from 26 to 100° and 10 to 480 min. The I studied (temp. and time in parentheses) were AcNH2 (60° 120), AcNHMe (60 420), PhNHAc (60° 180), N4-acetylsulfanilamide (60°, 240), acetylglycine (60°, 240), fluoroacetamide (26° 60), HCONH2 (26°, 60), HCONMe2 (26° 240), succinimide (60°, 120), caprolactam (60° 420), asparagine (60°, 180), glutamine (60° 180), glutathione (60°, 120), glycylglycine (60°, 120), nicotinamide (26°, 480), N1-methylnicotinamide methosulfate (26°, 360), nicotinic acid methylamide (60°, 240), nicotinic acid diethylamide (60°, 480), Ca pantothenate (26°, 300), barbitone (100°, 45), pentobarbitone (60°, 300), phenobarbitone (100°, 120), and Na evipan (100°, 30).
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    Gayathri, T. Stains for Developing TLC Plates. Int. J. Adv. Res. Sci. Eng. Technol. 2014, 5 (11), 79– 83
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    Leeuwenkamp, O. R.; van Bennekom, W. P.; van der Mark, E. J.; Bult, A. Nitroprusside, Antihypertensive Drug and Analytical Reagent. Review of (Photo)stability, Pharmacology and Analytical Properties. Pharm. Weekbl. Sci. Ed. 1984, 6 (4), 129– 140,  DOI: 10.1007/BF01954040
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    Nitroprusside, antihypertensive drug and analytical reagent. Review of (photo)stability, pharmacology and analytical properties
    Leeuwenkamp, O. R.; Van Bennekom, W. P.; Van der Mark, E. J.; Bult, A.
    Pharmaceutisch Weekblad, Scientific Edition (1984), 6 (4), 129-40CODEN: PWSEDI; ISSN:0167-6555.
    A review with 103 refs. of phys., chem., anal. and pharmacol. properties of nitroprusside [15078-28-1]. In view of the pharmaceutical applications of nitroprusside, special attention is given to the discussion of the (photo)degrdn., stability of the pharmaceutical formulations, the application as a reagent in pharmaceutical anal. and the redox behavior.
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    Small-mol. fluorescent probes have become powerful tools for using light to advance the study of cell biol., discover new drugs, detect environmental contaminants, and further the detection of cancer. These applications correlate with the expansion of the fluorescent probe research community - small in the late 20th century, now a collection of more than a hundred research groups world-wide. This expansion required the entry of adventurous scientists from many other fields. This tutorial review introduces some important concepts related to fluorescent probe development. It is hoped that it will facilitate further expansion of the field by demystifying it.
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    SIGNIFICANCE: The concentrations of reactive oxygen/nitrogen species (ROS/RNS) are critical to various biochemical processes. Small-molecule fluorescent probes have been widely used to detect and/or quantify ROS/RNS in many redox biology studies and serve as an important complementary to protein-based sensors with unique applications. Recent Advances: New sensing reactions have emerged in probe development, allowing more selective and quantitative detection of ROS/RNS, especially in live cells. Improvements have been made in sensing reactions, fluorophores, and bioavailability of probe molecules. CRITICAL ISSUES: In this review, we will not only summarize redox-related small-molecule fluorescent probes but also lay out the challenges of designing probes to help redox biologists independently evaluate the quality of reported small-molecule fluorescent probes, especially in the chemistry literature. We specifically highlight the advantages of reversibility in sensing reactions and its applications in ratiometric probe design for quantitative measurements in living cells. In addition, we compare the advantages and disadvantages of small-molecule probes and protein-based probes. FUTURE DIRECTIONS: The low physiological relevant concentrations of most ROS/RNS call for new sensing reactions with better selectivity, kinetics, and reversibility; fluorophores with high quantum yield, wide wavelength coverage, and Stokes shifts; and structural design with good aqueous solubility, membrane permeability, low protein interference, and organelle specificity. Antioxid. Redox Signal. 29, 518-540.
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    A review. This account describes a range of strategies for the development of fluorescent probes for detecting reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive (redox-active) sulfur species (RSS). Many ROS/RNS have been implicated in pathol. processes such as Alzheimer's disease (AD), cancer, diabetes mellitus, cardiovascular disease and aging. While many RSS play important roles in maintaining redox homeostasis and serve as antioxidants and act as free radical scavengers. Fluorescence based systems have emerged as one of the best ways to monitor the concns. and locations of these often very short-lived species. Due to the high levels of sensitivity and in particular their ability to be used for temporal and spatial sampling for in vivo imaging applications. As a direct result, there has been a huge surge in the development of fluorescent probes to sensitively and selectively detect ROS, RNS and RSS species within cellular environments. However, cellular environments are extremely complex with more than one species often involved in a given biochem. process. As a result, there has been a rise in the development of dual-responsive fluorescent probes (AND-logic probes) able to monitor the presence of more than one species in a biol. environment. Our aim with this account is to introduce the fluorescent probes we have developed for the in vitro and in vivo measurement of ROS, RNS and RSS. Fluorescence based sensing mechanisms used in the construction of the probes include photoinduced electron transfer (PeT), intramol. charge transfer (ICT), excited-state intramol. proton transfer (ESIPT) and fluorescent-resonance-energy-transfer (FRET). In particular probes for hydrogen peroxide (H2O2), hypochlorous acid (HOCl/ClO-), superoxide (O2•-), peroxynitrite (ONOO-), glutathione (GSH), cysteine (Cys), homocysteine (HCys) and Hydrogen sulfide (H2S) are discussed. In addn., we describe the development of AND logic based systems capable of detecting two species, such as peroxynitrite (ONOO-) and glutathione (GSH). One of the most interesting advances contained in this account is our extension of indicator displacement assays (IDAs) to reaction-based indicator displacement assays (RIAs) In an IDA system, an indicator is allowed to bind reversibly to a receptor. Then, a competitive analyte is introduced into the system, resulting in displacement of the indicator from the host, which in turn modulates the optical signal. With an RIA based systems the indicator is cleaved from a preformed receptor-indicator complex rather than being displaced by the analyte. Nevertheless, without a doubt the most significant result contained in this account is the use of an excited-state intramol. proton transfer (ESIPT) based probe for the simultaneous sensing of fibrous proteins/peptides AND environmental ROS/RNS.
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    A review. Intracellular redox homeostasis provides broad implications in physiol. and pathol. fields. The disruption of redox homeostasis is closely assocd. with some human diseases, such as cancer, neurodegenerative diseases, cardiovascular diseases, diabetes mellitus, and gastrointestinal diseases. Therefore, cells possess an elaborate regulation system to maintain their redox balance and the large or significant redox state changes can be buffered by the redox-active mols. These mols. experience interreaction and interconversion to facilitate the dynamic balance of intracellular redox state, among which three types of representative mols. should be mentioned, including reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS).
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    A review. Fluorogenic probes, small-mol. sensors that unmask brilliant fluorescence upon exposure to specific stimuli, are powerful tools for chem. biol. Those probes that respond to enzymic catalysis illuminate the complex dynamics of biol. processes at a level of spatiotemporal detail and sensitivity unmatched by other techniques. Here, the authors review recent advances in enzyme-activated fluorogenic probes for biol. imaging. The authors organize the authors' survey by enzyme classification, with emphasis on fluorophore masking strategies, modes of enzymic activation, and the breadth of current and future applications. Key challenges such as probe selectivity and spectroscopic requirements are described alongside therapeutic, diagnostic, and theranostic opportunities.
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    α-Chiral primary amines are one among the most valuable and versatile building blocks for the synthesis of numerous amine-containing pharmaceuticals and natural compounds. They also serve as chiral ligands or organo-catalysts for asymmetric catalysis. However, most of the existing chemocatalytic methods toward enantiopure primary amines rely on multistep manipulations on N-substituted substrates, which are not ideally atom-economical and cost-effective. Among the catalytic methods including the asymmetric transformations of the pre-prepared or in situ formed NH imines, biomimetic chemocatalysis inspired by enzymatic transaminations has recently emerged as an appealing and straightforward method to access chiral primary amines. This tutorial review highlights the state-of-the-art catalytic methods for the direct asymmetric synthesis of α-chiral primary amines and demonstrates their utility in the construction of molecular complexities, which may attract extensive attention and inspire applications in synthetic and medicinal chemistry.
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    World journal of microbiology & biotechnology (2017), 33 (11), 199 ISSN:.
    Imine reductases are nicotinamide-dependent enzymes that catalyze the asymmetric reduction of various imines to the corresponding amine products. Owing to the increasing roles of chiral amines and heterocyclic compounds as intermediates for pharmaceuticals, the demand for novel selective synthesis strategies is vitally important. Recent studies have demonstrated the discovery and structural characterization of a number of stereoselective imine reductase enzymes. Here, we highlight recent progress in applying imine reductases for the formation of chiral amines and heterocycles. It particularly focuses on the utilization of imine reductases in reductive aminations of aldehydes and ketones with various amine nucleophiles, one of the most powerful reactions in the synthesis of chiral amines. Second, we report on the synthesis of saturated substituted N-heterocycles by combining them with further biocatalysts, such as carboxylic acid reductases, oxidases or transaminases. Finally, we summarize the latest applications of imine reductases in the promiscuous asymmetric hydrogenation of a highly reactive carbonyl compound and the engineering of the cofactor specificity from NADPH to NADH.
  69. 69
    Gaweska, H.; Fitzpatrick, P. F. Structures and Mechanism of the Monoamine Oxidase Family. Biomol Concepts 2011, 2 (5), 365– 77,  DOI: 10.1515/BMC.2011.030
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    Structures and mechanism of the monoamine oxidase family
    Gaweska, Helena; Fitzpatrick, Paul F.
    Biomolecular Concepts (2011), 2 (5), 365-377CODEN: BCIOB8; ISSN:1868-5021. (Walter de Gruyter GmbH & Co. KG)
    A review. Members of the monoamine oxidase family of flavoproteins catalyze the oxidn. of primary and secondary amines, polyamines, amino acids, and methylated lysine side chains in proteins. The enzymes have similar overall structures, with conserved FAD (FAD)-binding domains and varied substrate-binding sites. Multiple mechanisms have been proposed for the catalytic reactions of these enzymes. The present review compares the structures of different members of the family and the various mechanistic proposals.
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    Parmeggiani, F.; Weise, N. J.; Ahmed, S. T.; Turner, N. J. Synthetic and Therapeutic Applications of Ammonia-Lyases and Aminomutases. Chem. Rev. 2018, 118 (1), 73– 118,  DOI: 10.1021/acs.chemrev.6b00824
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    Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases
    Parmeggiani, Fabio; Weise, Nicholas J.; Ahmed, Syed T.; Turner, Nicholas J.
    Chemical Reviews (Washington, DC, United States) (2018), 118 (1), 73-118CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Ammonia-lyases and aminomutases are mechanistically and structurally diverse enzymes which catalyze the deamination and/or isomerization of amino acids in nature by cleaving or shifting a C-N bond. Of the many protein families in which these enzyme activities are found, only a subset have been employed in the synthesis of optically pure fine chems. or in medical applications. This review covers the natural diversity of these enzymes, highlighting particular enzyme classes that are used within industrial and medical biotechnol. These highlights detail the discovery and mechanistic investigations of these com. relevant enzymes, along with comparisons of their various applications as stand-alone catalysts, components of artificial biosynthetic pathways and biocatalytic or chemoenzymic cascades, and therapeutic tools for the potential treatment of various pathologies.
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    Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.; Jarvis, W. R.; Colbeck, J. C.; Krebber, A.; Fleitz, F. J.; Brands, J. Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture. Science 2010, 329 (5989), 305– 9,  DOI: 10.1126/science.1188934
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    Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture
    Savile, Christopher K.; Janey, Jacob M.; Mundorff, Emily C.; Moore, Jeffrey C.; Tam, Sarena; Jarvis, William R.; Colbeck, Jeffrey C.; Krebber, Anke; Fleitz, Fred J.; Brands, Jos; Devine, Paul N.; Huisman, Gjalt W.; Hughes, Gregory J.
    Science (Washington, DC, United States) (2010), 329 (5989), 305-309CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Pharmaceutical synthesis can benefit greatly from the selectivity gains assocd. with enzymic catalysis. Here, we report an efficient biocatalytic process to replace a recently implemented rhodium-catalyzed asym. enamine hydrogenation for the large-scale manuf. of the antidiabetic compd. sitagliptin. Starting from an enzyme that had the catalytic machinery to perform the desired chem. but lacked any activity toward the prositagliptin ketone, we applied a substrate walking, modeling, and mutation approach to create a transaminase with marginal activity for the synthesis of the chiral amine; this variant was then further engineered via directed evolution for practical application in a manufg. setting. The resultant biocatalysts showed broad applicability toward the synthesis of chiral amines that previously were accessible only via resoln. This work underscores the maturation of biocatalysis to enable efficient, economical, and environmentally benign processes for the manuf. of pharmaceuticals.
  72. 72
    Weiß, M. S.; Pavlidis, I. V.; Spurr, P.; Hanlon, S. P.; Wirz, B.; Iding, H.; Bornscheuer, U. T. Protein-Engineering of an Amine Transaminase for the Stereoselective Synthesis of a Pharmaceutically Relevant Bicyclic Amine. Org. Biomol. Chem. 2016, 14 (43), 10249– 54,  DOI: 10.1039/C6OB02139E
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    72
    Protein-engineering of an amine transaminase for the stereoselective synthesis of a pharmaceutically relevant bicyclic amine
    Weiss, Martin S.; Pavlidis, Ioannis V.; Spurr, Paul; Hanlon, Steven P.; Wirz, Beat; Iding, Hans; Bornscheuer, Uwe T.
    Organic & Biomolecular Chemistry (2016), 14 (43), 10249-10254CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones represents an environmentally benign and economically attractive alternative to transition metal catalyzed asym. synthesis. However, the restrictive substrate scope has limited the conversion typically to non-sterically demanding scaffolds. Recently, we reported on the identification and design of fold class I ATAs that effect a highly selective asym. synthesis of a set of chiral arom. bulky amines from the corresponding ketone precursors in high yield. However, for the specific amine synthetic approach extension targeted here, the selective formation of an exo- vs. endo-isomer, these biocatalysts required addnl. refinement. The chosen substrate (exo-3-amino-8-aza-bicyclo[3.2.1]oct-8-yl-phenyl-methanone), apart from its pharmacol. relevance, is a demanding target for ATAs as the bridged bicyclic ring provides substantial steric challenges. Protein engineering combining rational design and directed evolution enabled the identification of an ATA variant which catalyzes the specific synthesis of the target exo-amine with >99.5% selectivity.
  73. 73
    Ma, E. J.; Siirola, E.; Moore, C.; Kummer, A.; Stoeckli, M.; Faller, M.; Bouquet, C.; Eggimann, F.; Ligibel, M.; Huynh, D. Machine-Directed Evolution of an Imine Reductase for Activity and Stereoselectivity. ACS Catal. 2021, 11 (20), 12433– 45,  DOI: 10.1021/acscatal.1c02786
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    Machine-Directed Evolution of an Imine Reductase for Activity and Stereoselectivity
    Ma, Eric J.; Siirola, Elina; Moore, Charles; Kummer, Arkadij; Stoeckli, Markus; Faller, Michael; Bouquet, Caroline; Eggimann, Fabian; Ligibel, Mathieu; Huynh, Dan; Cutler, Geoffrey; Siegrist, Luca; Lewis, Richard A.; Acker, Anne-Christine; Freund, Ernst; Koch, Elke; Vogel, Markus; Schlingensiepen, Holger; Oakeley, Edward J.; Snajdrova, Radka
    ACS Catalysis (2021), 11 (20), 12433-12445CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    Biocatalysis is an effective tool to access chiral mols. that are otherwise hard to synthesize or purify. Time-efficient processes are needed to develop enzymes that adequately perform the desired chem. We evaluated machine-directed evolution as an enzyme engineering strategy using a moderately stereoselective imine reductase as the model system. We compared machine-directed evolution approaches to deep mutational scanning (DMS) and error-prone PCR. Within one cycle, it was found that machine-directed evolution yielded a library of high-activity mutants with a dramatically shifted activity distribution compared to that of traditional directed evolution. Structure-guided anal. revealed that linear additivity might provide a simple explanation for the effectiveness of machine-directed evolution. The most active and selective enzyme mutant, which was identified through DMS and error-prone PCR, was used for the gram-scale synthesis of the H4 receptor antagonist ZPL389 with full conversion, > 99% ee (R), and a 72% yield.
  74. 74
    Ao, Y.-F.; Hu, H.-J.; Zhao, C.-X.; Chen, P.; Huang, T.; Chen, H.; Wang, Q.-Q.; Wang, D.-X.; Wang, M.-X. Reversal and Amplification of the Enantioselectivity of Biocatalytic Desymmetrization toward meso Heterocyclic Dicarboxamides Enabled by Rational Engineering of Amidase. ACS Catal. 2021, 11 (12), 6900– 7,  DOI: 10.1021/acscatal.1c01220
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    Reversal and amplification of the enantioselectivity of biocatalytic desymmetrization toward meso heterocyclic dicarboxamides enabled by rational engineering of amidase
    Ao, Yu-Fei; Hu, Hui-Juan; Zhao, Cheng-Xin; Chen, Peng; Huang, Tingting; Chen, Hui; Wang, Qi-Qiang; Wang, De-Xian; Wang, Mei-Xiang
    ACS Catalysis (2021), 11 (12), 6900-6907CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    By rational engineering of amidase, the efficient biocatalytic desymmetrization of meso O-heterocyclic dicarboxamides for synthesizing both antipodes of functionalized cyclic motifs was presented. Based on the enzyme-substrate binding model suggested by mol. docking, a rational mutagenesis strategy was established. The reversal and amplification of enantioselectivity of amidase were achieved by generating and testing only 10 variants. This enabled the quick access of both antipodes of products in very good yields and up to 99.5% ee under mild conditions. The engineered biocatalyst exhibits a wide substrate promiscuity and can expand to both N-heterocyclic and carbocyclic dicarboxamides with the retained high efficiency and excellent enantioselectivity. The desymmetrization mechanism for amidase, including the wild-type and the variant, was revealed by mol. dynamics simulations and quantum mech./mol. mech. modeling. It suggests a delicate cooperation between the activation and binding sites by nesting the cyclic skeleton of the substrates.
  75. 75
    Wang, P. F.; Yep, A.; Kenyon, G. L.; McLeish, M. J. Using Directed Evolution to Probe the Substrate Specificity of Mandelamide Hydrolase. Protein Eng. Des. Sel. 2009, 22 (2), 103– 10,  DOI: 10.1093/protein/gzn073
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    Using directed evolution to probe the substrate specificity of mandelamide hydrolase
    Wang, Pan-Fen; Yep, Alejandra; Kenyon, George L.; McLeish, Michael J.
    Protein Engineering, Design & Selection (2009), 22 (2), 103-110CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)
    Mandelamide hydrolase (MAH), a member of the amidase signature family, catalyzes the hydrolysis of mandelamide to mandelate and ammonia. X-ray structures of several members of this family, but not that of MAH, have been reported. These reveal nearly superimposable conformations of the unusual Ser-cisSer-Lys catalytic triad. Conversely, the residues involved in substrate recognition are not conserved, implying that the binding pocket could be modified to change the substrate specificity, perhaps by directed evolution. Here we show that MAH is able to hydrolyze small aliph. substrates such as lactamide, albeit with low efficiency. A selection method to monitor changes in mandelamide/lactamide preference was developed and used to identify several mutations affecting substrate binding. A homol. model places some of these mutations close to the catalytic triad, presumably in the MAH active site. In particular, Gly-202 appears to control the preference for arom. substrates as the G202A variant showed three orders of magnitude decrease in kcat/Km for (R)- and (S)-mandelamide. This redn. in activity increased to six orders of magnitude for the G202V variant.
  76. 76
    Thooft, A. M.; Cassaidy, K.; VanVeller, B. A Small Push-Pull Fluorophore for Turn-On Fluorescence. J. Org. Chem. 2017, 82 (17), 8842– 7,  DOI: 10.1021/acs.joc.7b00939
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    A Small Push-Pull Fluorophore for Turn-on Fluorescence
    Thooft, Andrea Marie; Cassaidy, Kyle; VanVeller, Brett
    Journal of Organic Chemistry (2017), 82 (17), 8842-8847CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    A new class of push-pull dyes is reported based on the structures of benzoxa- and benzothiadiazole heterocycles. This new class of dyes displays red-shifted wavelengths of emission and greater sensitivity to polarity and hydrogen bonding solvents relative to previously known derivs.
  77. 77
    Annenkov, V. V.; Verkhozina, O. N.; Shishlyannikova, T. A.; Danilovtseva, E. N. Application of 4-Chloro-7-Nitrobenzo-2-Oxa-1,3-Diazole in Analysis: Fluorescent Dyes and Unexpected Reaction with Tertiary Amines. Anal. Biochem. 2015, 486, 5– 13,  DOI: 10.1016/j.ab.2015.06.025
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    Application of 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole in analysis: Fluorescent dyes and unexpected reaction with tertiary amines
    Annenkov, Vadim V.; Verkhozina, Olga N.; Shishlyannikova, Tatyana A.; Danilovtseva, Elena N.
    Analytical Biochemistry (2015), 486 (), 5-13CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is widely applied as a fluorescent tagging reagent in biochem., as a derivatization agent in anal. chem., and as a component for design of fluorescent nanoparticles. Four new 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-tagged polyamines contg. two to four amine moieties were synthesized and used as an effective tool for staining of siliceous frustules of the diatom algae and spicules of the siliceous sponges, including fossilized samples. An unexpected reaction between NBD-Cl and tertiary amine groups was found, giving rise to NBD-tagged amines with elimination of an alkyl group. The reaction proceeds through the Meisenheimer complex and quaternary salt, which transform to the product by Hofmann reaction (alkene elimination) or nucleophilic substitution (halogenated compd. formation). In the case of polyamines, NBD-Cl causes chain scissoring, giving a set of NBD-tagged amines. The found NBD-Cl reaction with tertiary amines must be taken into account when using NBD-Cl and similar activated arom. systems for amine derivatization in anal. and biochem. applications. The reaction with polyamines opens the way to libraries of NBD-tagged compds.
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    Sanger, F. The Free Amino Groups of Insulin. Biochem. J. 1945, 39 (5), 507– 15,  DOI: 10.1042/bj0390507
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    Free amino groups of insulin
    Sanger, F.
    Biochemical Journal (1945), 39 (), 507-15CODEN: BIJOAK; ISSN:0264-6021.
    The free amino groups of proteins and peptides readily form derivs. with 2,4-(O2N)2C6H3F (I) at room temp. Since these derivs. are relatively stable to acid hydrolysis, hydrolysis of the substituted protein enables isolation of 2,4-dinitrophenyl amino acid derivs. which are separable by partition chromatography. This, then, forms the basis of a new method which was applied to the identification and estn. of the free amino groups in insulin. For comparison and control, several derivs. of the naturally occurring amino acids had to be synthesized. To a mixt. of 0.2 g. l-phenylalanine and 0.4 g. NaHCO3 in 5 ml. H2O was added a soln. of 0.4 g. I in 10 ml. EtOH. The mixt. was reacted 2 hrs. at room temp., giving 0.27 g. of N-2,4-dinitrophenyl-l-phenylalanine, C15H13O6N3, m. 186°. α-Acetyl-l-lysine (0.48 g.) and 0.75 g. NaHCO3 in 3 ml. H2O reacted for 4 hrs. at reflux temp. with 0.5 g. 2,4-(O2N)2C6H3Cl (II) in 10 ml. EtOH gave 0.7 g. (75%) of α-acetyl-ε-2,4-dinitrophenyl-l-lysine (III), m. 110°. III refluxed for 3 hrs. with 20% HCl gives ε-2,4-dinitrophenyl-l-lysine-HCl, which seps. from dil. HCl as the monohydrate, C12H16O6N4.HCl.H2O, m. 186°. ε-Benzoyl-l-lysine (1.25 g.) treated with 1.0 g. II gives 2 g. of α-2,4-dinitrophenyl-ε-benzoyl-l-lysine (IV). IV (0.5 g.) hydrolyzed in HOAc and HCl gave 60% of α-2,4-dinitrophenyl-l-lysine, C12H16O6N4, m. 260° (decompn.). l-Tyrosine (V) in the presence of excess I gives a compd. which is presumably O,N-bis(2,4 dinitrophenyl)-l-tyrosine (VI). Reaction of V with II gives, in addn. to VI, a compd. which was probably N-2,4-dinitrophenyl-l-tyrosine. N-Acetyl-l-tyrosine (0.55 g.) treated for 4 hrs. with 2.0 g. II gave 0.1 g. of N-acetyl-O-2,4-dinitrophenyl-l-tyrosine (VII), m. 194°. VII hydrolyzed in 20% HCl gave O-2,4-dinitrophenyl-l-tyrosine, as the hydrate, C15H13O7N3.H2O, m. 202°. On treatment of histidine with excess I only the bis(2,4-dinitrophenyl) deriv. is obtained, indicating that I reacts with the imidazole ring. This is confirmed by the slow reaction of I with α-acetylhistidine and rapid reaction with histidine Me ester to give again a bis deriv. I reacts also with SH groups of S-contg. amino acids but a mixt. of products is apparently obtained. The band rates on the chromatogram for various known 2,4-dinitrophenyl amino acids were detd. for various solvent mixts. and compared with those of the derivs. obtained from hydrolyzed dinitrophenylinsulin. No indicator was necessary since all of the derivs. were colored. Isolation of the bands enabled quant. colorimetric estn. of the derivs., using the known compds. as standards. Application of this technique shows that in an insulin submol. of mol. wt. 12,000, six free amino groups are present; 2 of these are located on glycine residues, 2 on phenylalanine residues, and 2 represent the ε-amino groups of lysine. The results suggest that the insulin submol. is made up of 4 open polypeptide chains, 2 of these having terminal glycyl residues and the other 2 terminal phenylalanyl residues, the chains being bound together most probably by -S-S- linkages.
  79. 79
    Ghosh, P. B.; Whitehouse, M. W. 7-Chloro-4-Nitrobenzo-2-Oxa-1,3-Diazole: A New Fluorigenic Reagent for Amino Acids and Other Amines. Biochem. J. 1968, 108 (1), 155– 6,  DOI: 10.1042/bj1080155
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    7-Chloro-4-nitrobenzofurazan: a new fluorigenic reagent for amino acids and other amines
    Ghosh, P. B.; Whitehouse, M. W.
    Biochemical Journal (1968), 108 (1), 155-6CODEN: BIJOAK; ISSN:0264-6021.
    7-Chloro-4-nitrobenzoxadiazole (I), m. 97°, was prepd. by nitrating 4-chlorobenzofurazan, obtained from 2,6-dichloroaniline via the dichloronitrosobenzene. I was non-fluorescent. Strong fluorescence of the I-amine compds. (II) was observed in solvents of low polarity and was excited at 464 mμ. I was more stable and more sol. in aq. solns. than 1-dimethylaminonaphthalene-5-sulfonyl (DNS) chloride. As little as 1 mγ I-glycine/ml. in acetone or EtOAc could be detected. The intensity of fluorescence of I-glycine in moist EtOAc was approx. the same as DNS-glycine in water and > the fluorescence of DNS-glycine in moist acetone. Substituents on the amino group that restrict its conjugation with the benzoxadiazole nucleus caused a drastic loss of fluorescence. Related compds. that were not amine derivs. but had other electron-donor groups in the 7-position also fluoresced, but to a lesser extent than the 7-amino derivs. The fluorescence of some aminobenzoxadiazoles and related compds. is given.
  80. 80
    Watanabe, Y.; Imai, K. High-Performance Liquid Chromatography and Sensitive Detection of Amino Acids Derivatized with 7-Fluoro-4-Nitrobenzo-2-Oxa-1,3-Diazole. Anal. Biochem. 1981, 116 (2), 471– 2,  DOI: 10.1016/0003-2697(81)90390-0
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    High-performance liquid chromatography and sensitive detection of amino acids derivatized with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole
    Watanabe, Yoshihiko; Imai, Kazuhiro
    Analytical Biochemistry (1981), 116 (2), 471-2CODEN: ANBCA2; ISSN:0003-2697.
    7-Fluoro-4-nitrobenzo-2-oxa-1,3-diazole is used as a precolumn fluorescent labeling reagent for high-performance liq. chromatog. of amino acids, including proline and hydroxyproline. The reaction is run at pH 8.0 at 60° for 5 min. The fluorophors (aspartate, glutamate, hydroxyproline, serine, glycine, threonine, alanine, proline) are sepd. on a reversed-phase column (μBondapak C18) with 0.1M phosphate buffer (pH 6.0) contg. 6.75% MeOH and 1.8% THF, and are detected at the level of 10 fmol with excitation at 470 nm and emission at 530 nm.
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    Aly, H.; El-Shafie, A. S.; El-Azazy, M. Utilization of 7-Chloro-4-Nitrobenzo-2-Oxa-1,3-Diazole (NBD-Cl) for Spectrochemical Determination of L-Ornithine: A Multivariate Optimization-Assisted Approach. RSC Adv. 2019, 9 (38), 22106– 15,  DOI: 10.1039/C9RA03311D
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    81
    Utilization of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) for spectrochemical determination of L-ornithine: a multivariate optimization-assisted approach
    Aly, Hend; El-Shafie, Ahmed S.; El-Azazy, Marwa
    RSC Advances (2019), 9 (38), 22106-22115CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    A simple and highly sensitive univariate calibration strategy based on UV-visible (UV-Vis) absorption spectroscopy and assisted by multivariate screening and optimization was utilized for the detn. of L-ornithine (L-ORN) as such and in the alimentary supplements. L-ORN, an OTC marketed amino acid, is widely used for bodybuilding and might be abused by athletes. A nucleophilic substitution reaction using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) was the basis of the current investigation. Plackett-Burman design (PBD) and a response surface optimizer as screening and fine-tuning strategies, resp., were instigated. Four numerical variables, reaction time (RT), temp. (Temp), pH and reagent vol. (RV), and one categorical variable, the dilg. solvent (DS), were considered. Absorbance of the yellow-colored adduct at 469 nm was the response studied. Pareto anal., along with anal. of variance (ANOVA) were used to ascertain the significant variables (screening phase) and their domains (optimization phase). Response transformation and stepwise anal. were employed when necessary. Probability, cube and individual value plots were used to get an insight into the statistical impact of the variables tested. Multiple responses' optimization was performed using Derringer's function. Calibration curves were linear in the range of 5-50 μg mL-1. Job's technique of continuous variation showed that the stoichiometric ratio is 2 : 1 (NBD-Cl : L-ORN). The proposed technique was successfully applied to the dietary supplements of L-ORN, inferring no interference from adjuvants and excipients. Anal. performance of this technique was validated conforming to the ICH stds.
  82. 82
    Walash, M. I.; Metwally, M. E. S.; Eid, M.; El-Shaheny, R. N. Validated Spectrophotometric Methods for Determination of Alendronate Sodium in Tablets through Nucleophilic Aromatic Substitution Reactions. Chem. Cent. J. 2012, 6, 25,  DOI: 10.1186/1752-153X-6-25
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    Validated spectrophotometric methods for determination of Alendronate sodium in tablets through nucleophilic aromatic substitution reactions
    Walash, Mohamed I.; Metwally, Mohamed E.-S.; Eid, Manal; El-Shaheny, Rania N.
    Chemistry Central Journal (2012), 6 (), 25CODEN: CCJHA2; ISSN:1752-153X. (Chemistry Central Ltd.)
    Background: Alendronate (ALD) is a member of the bisphosphonate family which is used for the treatment of osteoporosis, bone metastasis, Paget's disease, hypocalcemia assocd. with malignancy and other conditions that feature bone fragility. ALD is a non-chromophoric compd. so its detn. by conventional spectrophotometric methods is not possible. So two derivatization reactions were proposed for detn. of ALD through the reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and 2,4-dinitrofluorobenzene (DNFB) as chromogenic derivatizing reagents. Results: Three simple and sensitive spectrophotometric methods are described for the detn. of ALD. Method I is based on the reaction of ALD with NBD-Cl. Method II involved heat-catalyzed derivatization of ALD with DNFB, while, Method III is based on micellar-catalyzed reaction of the studied drug with DNFB at room temp. The reactions products were measured at 472, 378 and 374 nm, for Methods I, II and III, resp. Beer's law was obeyed over the concn. ranges of 1.0-20.0, 4.0-40.0 and 1.5-30.0 μg/mL with lower limits of detection of 0.09, 1.06 and 0.06 μg/mL for Methods I, II and III, resp. The proposed methods were applied for quantitation of the studied drug in its pure form with mean percentage recoveries of 100.47 ± 1.12, 100.17 ± 1.21 and 99.23 ± 1.26 for Methods I, II and III, resp. Moreover the proposed methods were successfully applied for detn. of ALD in different tablets. Proposals of the reactions pathways have been postulated. Conclusion: The proposed spectrophotometric methods provided sensitive, specific and inexpensive anal. procedures for detn. of the non-chromophoric drug alendronate either per se or in its tablet dosage forms without interference from common excipients.
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    Benson, S.; Fernandez, A.; Barth, N. D.; de Moliner, F.; Horrocks, M. H.; Herrington, C. S.; Abad, J. L.; Delgado, A.; Kelly, L.; Chang, Z. SCOTfluors: Small, Conjugatable, Orthogonal, and Tunable Fluorophores for in Vivo Imaging of Cell Metabolism. Angew. Chem., Int. Ed. 2019, 58 (21), 6911– 5,  DOI: 10.1002/anie.201900465
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    SCOTfluors: Small, Conjugatable, Orthogonal, and Tunable Fluorophores for In Vivo Imaging of Cell Metabolism
    Benson, Sam; Fernandez, Antonio; Barth, Nicole D.; de Moliner, Fabio; Horrocks, Mathew H.; Herrington, C. Simon; Abad, Jose Luis; Delgado, Antonio; Kelly, Lisa; Chang, Ziyuan; Feng, Yi; Nishiura, Miyako; Hori, Yuichiro; Kikuchi, Kazuya; Vendrell, Marc
    Angewandte Chemie, International Edition (2019), 58 (21), 6911-6915CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The transport and trafficking of metabolites are crit. for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chem. structures that allow direct monitoring of small metabolites under physiol. conditions with high spatial and temporal resoln. Herein, the authors describe SCOTfluors as novel small-sized multicolored fluorophores for real-time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin.
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    Satake, K.; Okuyama, T.; Ohashi, M.; Shinoda, T. The Spectrophotometric Determination of Amine, Amino Acid and Peptide with 2,4,6-Trinitrobenzene 1-Sulfonic Acid. J. Biochem. 1960, 47 (5), 654– 60,  DOI: 10.1093/oxfordjournals.jbchem.a127107
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    The spectrophotometric determination of amines, amino acids, and peptides with 2,4,6-trinitrobenzene-1-sulfonic acid
    Satake, Kazuo; Okuyama, Tsuneo; Ohashi, Mochihiko; Shinoda, Tomotaka
    Journal of Biochemistry (1960), 47 (), 654-60CODEN: JOBIAO; ISSN:0021-924X.
    cf. CA 54, 24470i. 2,4,6-Trinitrobenzene-1-sulfonic acid is shown to react specifically with primary amines and .sbd.NH2 and .dbd.NH groups of amino acids and peptides. The av. extinction coeff. of 7 trinitrophenylated (TNP) amines and 20 TNP amino acids (in the vicinity of absorption peak (340 mμ is chosen for convenience) is detd. as 1.27 X 104 and that of 7 TNP peptides as 1.05 X 104 at N concn.
  85. 85
    Means, G. E.; Congdon, W. I.; Bender, M. L. Reactions of 2,4,6-Trinitrobenzenesulfonate Ion with Amines and Hydroxide Ion. Biochemistry 1972, 11 (19), 3564– 71,  DOI: 10.1021/bi00769a011
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    Reactions of 2,4,6-trinitrobenzenesulfonate ion with amines and hydroxide ion
    Means, Gary E.; Congdon, W. I.; Bender, M. L.
    Biochemistry (1972), 11 (19), 3564-71CODEN: BICHAW; ISSN:0006-2960.
    The reactivity of 2,4,6-trinitrobenzenesulfonate ion (TNBS) with hydroxide ion and a series of primary amines was examd. as a function of pH, nucleophile concn., ionic strength, and temp. Its reaction with hydroxide ion is first order in both hydroxide ion and TNBS with a second-order rate const. of 6.33 ± 0.16 × 10-2 M-1 sec-1 at 25° (μ = 1.0), and appears to follow the usual path for nucleophilic aromatic substitution involving the rate-limiting formation of a tetrasubstituted ring carbon intermediate. The reaction of TNBS with primary amines having pKa values higher than ∼8.7 proceeds by a similar mechanism. Reactivity increases as a function of amine basicity according to the relation log k (M-1 sec-1) = 0.23 pKa - 1.18. Similar amines with lower pKa values follow a complex rate law apparently due to partitioning of the tetra-substituted intermediate between breakdown to products and reversion to reactants. The reaction of TNBS with amines is favored as compared to its reaction with hydroxide ion by low ionic strength, low temp., and pH values at least 0.5 unit below the amine pKa value. Complexation of N-alkylpicramides by sulfite ion involves its addn. to ring carbon number three. The second-order rate const. for complexation of sulfite with N-trinitrophenyl-β-alanine is 5.4 × 103 M-1 sec-1 at pH 8.0 (μ = 0.50) and 25.4°. Reactions of TNBS with proteins are discussed based on these results.
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    Snyder, S. L.; Sobocinski, P. Z. An Improved 2,4,6-Trinitrobenzenesulfonic Acid Method for the Determination of Amines. Anal. Biochem. 1975, 64 (1), 284– 8,  DOI: 10.1016/0003-2697(75)90431-5
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    Improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines
    Snyder, Stephen L.; Sobocinski, Philip Z.
    Analytical Biochemistry (1975), 64 (1), 284-8CODEN: ANBCA2; ISSN:0003-2697.
    A convenient and sensitive procedure was described for the detn. of amines, amino acids, and proteins by use of 2,4,6-trinitrobenzenesulfonic acid (TNBS). In this method, the amino content could be related directly to the absorbance of the trinitrophenylation reaction mixt. after 15-30 min incubation; therefore, it was unnecessary to quench the reaction. The method was ∼2-fold more sensitive than the Lowry protein detn. method and ∼6-fold more sensitive than the ninhydrin method for amino acids.
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    Qi, X.-Y.; Keyhani, N. O.; Lee, Y. C. Spectrophotometric Determination of Hydrazine, Hydrazides, and Their Mixtures with Trinitrobenzenesulfonic Acid. Anal. Biochem. 1988, 175 (1), 139– 44,  DOI: 10.1016/0003-2697(88)90371-5
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    Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid
    Qi, Xiao Yang; Keyhani, Nemat O.; Lee, Yuan Chuan
    Analytical Biochemistry (1988), 175 (1), 139-44CODEN: ANBCA2; ISSN:0003-2697.
    A spectrophotometric method has been developed to measure hydrazine, hydrazides, and their mixts. using a modification of the trinitrobenzenesulfonic acid method (T. Okuyama and K. Satake 1960). After incubation of the sample contg. hydrazine and hydrazide with trinitrobenzenesulfonate at pH 8.5 at room temp. for 40 min, the reaction mixt. was dild. with a Na2CO3-NaHCO3 buffer (0.1M, pH 10.8) rather than with 0.5M HCl. Different chromogens were produced from the reaction of hydrazine (λmax = 570 nm) and hydrazides (λmax = 385 and 500 nm) with trinitrobenzenesulfonic acid. The method allowed simultaneous detn. of hydrazine (5 to 60 nmol) with hydrazide (10 to 120 nmol) in a mixt. with a std. deviation of less than 5%. The presence of amino compds. (except for amino sugars) did not interfere with the measurement of hydrazine or hydrazides. Interference by amino sugars in the detn. of hydrazine or hydrazides was eliminated by pretreatment of the sample with NaBH4 to reduce the amino sugars to 2-amino-2-deoxyhexitols.
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    Morçöl, T.; Subramanian, A.; Velander, W. H. Dot-Blot Analysis of the Degree of Covalent Modification of Proteins and Antibodies at Amino Groups. J. Immunol. Methods 1997, 203 (1), 45– 53,  DOI: 10.1016/S0022-1759(97)00013-6
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    Dot-blot analysis of the degree of covalent modification of proteins and antibodies at amino groups
    Morcol T; Subramanian A; Velander W H
    Journal of immunological methods (1997), 203 (1), 45-53 ISSN:0022-1759.
    The present study describes a rapid and sensitive dot-blot assay approach for determining the degree of covalent modification of amino groups in proteins. N-hydroxy-succinimide ester of acetic acid was used for irreversible, covalent modification of proteins whose reactive primary amino groups were reversibly blocked (or protected) with 2,3-dimethyl-maleic anhydride prior to processing. Immobilon AV affinity membrane was utilized for differential covalent attachment of the proteins to the activated ester on the membrane matrix, primarily through their protected epsilon-amino group of lysins. The efficacy of the method was demonstrated for a murine monoclonal antibody and for two human plasma proteins. The degree of covalent modification of proteins at their amino groups as estimated by the proposed method is compared with that obtained by using the conventional trinitrobenzene sulfonic acid (TNBS) method. Several advantages of the present method over the TNBS method are emphasized. The new method, which requires only nanograms of protein, is shown to be more sensitive than the TNBS method where the limit of detection is in the milligram range. The proposed assay is very specific and facile, and the advantage of small sample size requirement (1 microliter) provides sequential detection of multiple samples facilitating much higher precision in data obtained than that of the TNBS assay.
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    Oliverio, R.; Liberelle, B.; Murschel, F.; Garcia-Ac, A.; Banquy, X.; De Crescenzo, G. Versatile and High-Throughput Strategy for the Quantification of Proteins Bound to Nanoparticles. ACS Appl. Nano Mater. 2020, 3 (10), 10497– 507,  DOI: 10.1021/acsanm.0c02414
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    Versatile and High-Throughput Strategy for the Quantification of Proteins Bound to Nanoparticles
    Oliverio, Romane; Liberelle, Benoit; Murschel, Frederic; Garcia-Ac, Araceli; Banquy, Xavier; De Crescenzo, Gregory
    ACS Applied Nano Materials (2020), 3 (10), 10497-10507CODEN: AANMF6; ISSN:2574-0970. (American Chemical Society)
    Biofunctionalized nanomaterials have been extensively studied as a tool for a wide range of applications in biomedical fields. Despite many existing strategies to conjugate proteins to colloidal particles, detg. the grafting efficiency-i.e., the amt. of protein conjugated to the surface of a nanoparticle (NP)-remains challenging. Formulations for biomedical applications are subjected to strict constraints, and a lack of precise characterization can prevent otherwise promising formulations to be explored further. Here, we propose a simple approach to precisely measure the grafting efficiency of biol. mols. on the surface of three types of widely used NPs: polymeric NPs, inorg. NPs, and metallic NPs. This approach relies on the simultaneous hydrolysis of the grafted protein and the NP degrdn. in acidic conditions, followed by a spectrophotometric quantification of primary amines in soln. This strategy can be applied to any type of protein and does not require any labeling agent. It can be performed in a high-throughput manner as a routine expt. and only requires a conventional oven and a microplate reader.
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    Kim, D.; Cho, S. W.; Jun, Y. W.; Ahn, K. H. Fluorescent Labeling of Lysine Residues in Protein Using 8-Thiomethyl-BODIPY. Bull. Korean Chem. Soc. 2017, 38 (9), 995– 6,  DOI: 10.1002/bkcs.11213
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    90
    Fluorescent Labeling of Lysine Residues in Protein using 8-thiomethyl-BODIPY
    Kim, Dokyoung; Cho, Seo Won; Jun, Yong Woong; Ahn, Kyo Han
    Bulletin of the Korean Chemical Society (2017), 38 (9), 995-996CODEN: BKCSDE; ISSN:0253-2964. (Wiley-VCH Verlag GmbH & Co. KGaA)
    We have disclosed that 8-thiomethyl-BODIPY is promising for labeling of proteins with lysineresidues. The substitution reaction of the thiomethyl moietywith amine groups, here the lysine residue, proceeds fast(within 2 h) under mild conditions (37C) and the resultingproduct shows bright blue fluorescence from the amino-BODIPY moiety (exi/emi = 375/409 nm). The labeled lysozyme retained its activity (96 2.6%), indicative of little structural influence by the compact label.
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    Bell, P. J. L.; Karuso, P. Epicocconone, a Novel Fluorescent Compound from the Fungus Epicoccum Nigrum. J. Am. Chem. Soc. 2003, 125 (31), 9304– 5,  DOI: 10.1021/ja035496+
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    Epicocconone, a novel fluorescent compound from the fungus Epicoccum nigrum
    Bell, Phillip J. L.; Karuso, Peter
    Journal of the American Chemical Society (2003), 125 (31), 9304-9305CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Epicocconone represents a new class of natural fluorescent probes based on a polyketide skeleton isolated from the fungus Epicoccum nigrum. Epicocconone is a small, cell permeable natural product with a high molar absorptivity and a long Stokes' shift that will be useful in biotechnol. applications.
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    Coghlan, D. R.; Mackintosh, J. A.; Karuso, P. Mechanism of Reversible Fluorescent Staining of Protein with Epicocconone. Org. Lett. 2005, 7 (12), 2401– 4,  DOI: 10.1021/ol050665b
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    Mechanism of Reversible Fluorescent Staining of Protein with Epicocconone
    Coghlan, Daniel R.; Mackintosh, James A.; Karuso, Peter
    Organic Letters (2005), 7 (12), 2401-2404CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
    Epicocconone is the active ingredient in Deep Purple Total Protein Stain and responsible for the apparent noncovalent staining of proteins in polyacrylamide gel and electroblots. Reaction of epicocconone with amines has shown that epicocconone reacts reversibly with primary amines to produce a highly fluorescent enamine that is readily hydrolyzed by base or strong acid such as in conditions used in post-electrophoretic anal. such as peptide mass fingerprinting or Edman degrdn.
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    Peixoto, P. A.; Boulangé, A.; Ball, M.; Naudin, B.; Alle, T.; Cosette, P.; Karuso, P.; Franck, X. Design and Synthesis of Epicocconone Analogues with Improved Fluorescence Properties. J. Am. Chem. Soc. 2014, 136 (43), 15248– 56,  DOI: 10.1021/ja506914p
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    Design and Synthesis of Epicocconone Analogues with Improved Fluorescence Properties
    Peixoto, Philippe A.; Boulange, Agathe; Ball, Malcolm; Naudin, Bertrand; Alle, Thibault; Cosette, Pascal; Karuso, Peter; Franck, Xavier
    Journal of the American Chemical Society (2014), 136 (43), 15248-15256CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Epicocconone is a natural latent fluorophore that is widely used in biotechnol. because of its large Stokes shift and lack of fluorescence in its unconjugated state. However, the low photostability and quantum yields of epicocconone have limited its wider use, and in the absence of a total synthesis, this limitation was a long-standing problem. Here the authors report a general strategy for the synthesis of epicocconone analogs that relies on a 2-iodoxybenzoic acid-mediated dearomatization and on the replacement of the triene tail of the natural product by an arom. ring. This design element is general and the synthesis is straightforward, providing ready access to libraries of polyfunctional fluorophores with long Stokes shifts based on the epicocconone core. The structural modifications resulted in analogs with increased photostability and quantum yields compared with the natural product. Staining proteomic gels with these new analogs showed significant lowering of the detection limit and a 30% increase in the no. of low-abundance proteins detected. These epiccoconone analogs will substantially improve the discovery rate of biomarker needles in the proteomic haystack.
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    Chatterjee, S.; Karuso, P.; Boulangé, A.; Franck, X.; Datta, A. Excited State Dynamics of Brightly Fluorescent Second Generation Epicocconone Analogues. J. Phys. Chem. B 2015, 119 (20), 6295– 303,  DOI: 10.1021/acs.jpcb.5b02190
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    Excited State Dynamics of Brightly Fluorescent Second Generation Epicocconone Analogues
    Chatterjee, Soumit; Karuso, Peter; Boulange, Agathe; Franck, Xavier; Datta, Anindya
    Journal of Physical Chemistry B (2015), 119 (20), 6295-6303CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    The natural product epicocconone, owing to its unique fluorescence properties, was developed into a range of products used in biotechnol., esp. proteomics. However, its weak green fluorescence in its native state, while advantageous for proteomics applications, is a disadvantage in other applications that require two-color readouts. Here we report the photophys. characterization of two brightly fluorescent analogs of epicocconone. These analogs, with naphthyl or pyridyl groups replacing the heptatriene chain, resulted in bright fluorescence in both the native state and the long Stokes shifted enamine. Time-resolved fluorescence studies and DFT calcns. were carried out to understand the excited state processes involved in fluorescence. Results showed the p-chloro group on the pyridyl is responsible for the high fluorescence of the native fluorophore. The application of one of these compds. for staining electrophoresis gels is exemplified.
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    Castell, J. V.; Cervera, M.; Marco, R. A Convenient Micromethod for the Assay of Primary Amines and Proteins with Fluorescamine. A Reexamination of the Conditions of Reaction. Anal. Biochem. 1979, 99 (2), 379– 91,  DOI: 10.1016/S0003-2697(79)80022-6
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    95
    A convenient micromethod for the assay of primary amines and proteins with fluorescamine. A reexamination of the conditions of reaction
    Castell, Jose V.; Cervera, Margarita; Marco, Roberto
    Analytical Biochemistry (1979), 99 (2), 379-91CODEN: ANBCA2; ISSN:0003-2697.
    Fluorescamine, a reagent capable of reacting with a wide variety of nucleophiles, only gives fluorescent products when reacted with primary amines. The results of reexamn. of the principal parameters affecting the reaction are presented with emphasis on practical aspects. In particular, the potential competition of other nucleophiles (secondary amines and thiols) and the effect of pH on the reaction and on the fluorescence of the product are presented. Polyalcs., due to their reversible interaction with fluorescamine, affect the velocity without interfering with its extent. The initial hydrolysis product of fluorescamine under mild alk. conditions has been isolated and characterized. Although unreactive with most amines, it is shown that at pH <7 it will react with amines that show abnormal low pH-dependent reactivity with fluorescamine. Histidine is the only amino acid that under these conditions will react giving a fluorescent product. Among other amino group-contg. mols. tested, only polyamines, histamine, and N-amino terminal-free peptides have been found to give this type of reaction. The factors affecting the limit of sensitivity of the reaction also were studied. The very low, but significant, fluorescence of the blanks sets the actual limit of sensitivity of the reaction. A micromethod that reduces the amt. of fluorescamine in the assay under conditions in which the reaction is quant. is described. A similar micromethod can be used with the o-phthalaldehyde reaction. The particular application of fluorescamine in the assay of proteins is compared to other methods. Alk. and acid hydrolysis are shown to decrease the variability of the reaction shown by different proteins, while increasing the sensitivity. The different practical alternatives in the assay of proteins with fluorescamine and o-phthalaldehyde are discussed.
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    Funk, G. M.; Hunt, C. E.; Epps, D. E.; Brown, P. K. Use of a Rapid and Highly Sensitive Fluorescamine-Based Procedure for the Assay of Plasma Lipoproteins. J. Lipid Res. 1986, 27, 792,  DOI: 10.1016/S0022-2275(20)38803-9
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    Use of a rapid and highly sensitive fluorescamine-based procedure for the assay of plasma lipoproteins
    Funk, G. Michael; Hunt, Charles E.; Epps, Dennis E.; Brown, Patricia K.
    Journal of Lipid Research (1986), 27 (7), 792-5CODEN: JLPRAW; ISSN:0022-2275.
    A rapid and sensitive method for detg. protein concs. using fluorescamine was characterized for use in the anal. of intact lipoproteins.. Fluorescence was measured with excitation at 390 nm and emission at 470 nm. There was no interference with the assay due to the presence of lipid-assocd. turbidity or primary amine content. The assay was sensitive to as little as 0.3 μg lipoprotein and yielded similar results when compared to the Lowry method.
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    Udenfriend, S.; Stein, S.; Böhlen, P.; Dairman, W.; Leimgruber, W.; Weigele, M. Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range. Science 1972, 178 (4063), 871,  DOI: 10.1126/science.178.4063.871
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    Fluorescamine. Reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range
    Udenfriend, Sidney; Stein, Stanley; Boehlen, Peter; Dairman, Wallace; Leimgruber, Willy; Weigele, Manfred
    Science (Washington, DC, United States) (1972), 178 (4063), 871-2CODEN: SCIEAS; ISSN:0036-8075.
    Fluorescamine is a new reagent for the detection of primary amines in the picomole range. Its reaction with amines is almost instantaneous at room temp. in aq. media. The products are highly fluorescent, whereas the reagent and its degrdn. products are nonfluorescent. Applications are discussed.
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    Murugayah, S. A.; Warring, S. L.; Gerth, M. L. Optimisation of a High-Throughput Fluorescamine Assay for Detection of N-Acyl-L-Homoserine Lactone Acylase Activity. Anal. Biochem. 2019, 566, 10– 2,  DOI: 10.1016/j.ab.2018.10.029
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    Optimisation of a high-throughput fluorescamine assay for detection of N-acyl-L-homoserine lactone acylase activity
    Murugayah, Shereen A.; Warring, Suzanne L.; Gerth, Monica L.
    Analytical Biochemistry (2019), 566 (), 10-12CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    N-acyl-L-homoserine lactone (AHL) acylases are a well-known group of enzymes that disrupt quorum sensing in Gram-neg. bacteria by degrading AHL signalling mols. This degrdn. of signalling mols. (termed 'quorum quenching') has potential uses in the prevention or redn. of biofilm formation and/or bacterial infections. Therefore, there is a great deal of interest in the identification and characterization of quorum quenching enzymes. Here, we present an optimized fluorescamine-based assay for the detection of AHL acylase activity and demonstrate it can be used in a high-throughput screening format.
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    Ashby, J.; Duan, Y.; Ligans, E.; Tamsi, M.; Zhong, W. High-Throughput Profiling of Nanoparticle-Protein Interactions by Fluorescamine Labeling. Anal. Chem. 2015, 87 (4), 2213– 9,  DOI: 10.1021/ac5036814
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    High-Throughput Profiling of Nanoparticle-Protein Interactions by Fluorescamine Labeling
    Ashby, Jonathan; Duan, Yaokai; Ligans, Erik; Tamsi, Michael; Zhong, Wenwan
    Analytical Chemistry (Washington, DC, United States) (2015), 87 (4), 2213-2219CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A rapid, high throughput fluorescence assay was designed to screen interactions between proteins and nanoparticles. The assay employs fluorescamine, a primary-amine specific fluorogenic dye, to label proteins. Because fluorescamine could specifically target the surface amines on proteins, a conformational change of the protein upon interaction with nanoparticles will result in a change in fluorescence. In the present study, the assay was applied to test the interactions between a selection of proteins and nanoparticles made of polystyrene, silica, or iron oxide. The particles were also different in their hydrodynamic diam., synthesis procedure, or surface modification. Significant labeling differences were detected when the same protein incubated with different particles. Principal component anal. (PCA) on the collected fluorescence profiles revealed clear grouping effects of the particles based on their properties. The results prove that fluorescamine labeling is capable of detecting protein-nanoparticle interactions, and the resulting fluorescence profile is sensitive to differences in nanoparticle's phys. properties. The assay can be carried out in a high-throughput manner, and is rapid with low operation cost. Thus, it is well suited for evaluating interactions between a larger no. of proteins and nanoparticles. Such assessment can help to improve our understanding on the mol. basis that governs the biol. behaviors of nanomaterials. It will also be useful for initial examn. of the bioactivity and reproducibility of nanomaterials employed in biomedical fields.
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    Elbashir, A. A.; Ahmed, A. A.; Ali Ahmed, S. M.; Aboul-Enein, H. Y. 1,2-Naphthoquinone-4-Sulphonic Acid Sodium Salt (NQS) as an Analytical Reagent for the Determination of Pharmaceutical Amine by Spectrophotometry. Appl. Spectrosc. Rev. 2012, 47 (3), 219– 32,  DOI: 10.1080/05704928.2011.639107
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    1,2-Naphthoquinone-4-Sulphonic Acid Sodium Salt (NQS) as an Analytical Reagent for the Determination of Pharmaceutical Amine by Spectrophotometry
    Elbashir, Abdalla Ahmed; Ahmed, Abir Abdalla; Ali Ahmed, Shazalia M.; Aboul-Enein, Hassan Y.
    Applied Spectroscopy Reviews (2012), 47 (3), 219-232CODEN: APSRBB; ISSN:0570-4928. (Taylor & Francis, Inc.)
    A review. Several papers have been presented in recent years regarding the field of application of 1,2-naphthoquinone-4-sulfonic acid sodium salt (NQS) as a chromogenic reagent for the detn. of pharmaceutical amines using spectrophotometry. In this review article, various spectrophotometric methods using NQS as a labeling reagent for detn. of pharmaceutical amines are presented. The application of these methods for the detn. of drugs in pharmaceutical formulations and real samples is discussed.
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    Rosenblatt, D. H.; Hlinka, P.; Epstein, J. Use of 1,2-Naphthoquinone-4-Sulfonate for Estimation of Ethylenimine and Primary Amines. Anal. Chem. 1955, 27 (8), 1290– 3,  DOI: 10.1021/ac60104a024
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    101
    Use of 1,2-naphthoquinone-4-sulfonate for the estimation of ethylenimine and primary amines
    Rosenblatt, David H.; Hlinka, Peter; Epstein, Joseph
    (1955), 27 (), 1290-3CODEN: ANCHAM; ISSN:0003-2700.
    Dil. aq. solns. of ethylenimine and butyl amine react with K 1,2-naphthoquinone-4-sulfonate (I) at a pH of 10.3 to give reddish dyes which can be extd. with CHCl3. The amines can be estd. by measuring the absorbance at 420 and 450 mμ, resp. Ethanolamine also gives a reddish dye, but it can not be extd. with CHCl3. It can, however, be extd. with isoamyl alc. Ethylenimine, butylamine, ethanolamine, diethylamine, N-butylethanolamine, diethanolamine, NH4OH, sulfanilamide, and aniline react with I to give dyes which can be distinguished qualitatively by their colors and solubilities in various org. solvents.
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    Darwish, I. A.; Abdine, H. H.; Amer, S. M.; Al-Rayes, L. I. Simple Spectrophotometric Method for Determination of Paroxetine in Tablets Using 1,2-Naphthoquinone-4-Sulphonate as a Chromogenic Reagent. Int. J. Anal. Chem. 2009, 2009, 237601,  DOI: 10.1155/2009/237601
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    102
    Simple spectrophotometric method for determination of paroxetine in tablets using 1,2-naphthoquinone-4-sulphonate as a chromogenic reagent
    Darwish Ibrahim A; Abdine Heba H; Amer Sawsan M; Al-Rayes Lama I
    International journal of analytical chemistry (2009), 2009 (), 237601 ISSN:.
    Simple and rapid spectrophotometric method has been developed and validated for the determination of paroxetine (PRX) in tablets. The proposed method was based on nucleophilic substitution reaction of PRX with 1,2-naphthoquinone-4-sulphonate (NQS) in an alkaline medium to form an orange-colored product of maximum absorption peak (lambda(max)) at 488 nm. The stoichiometry and kinetics of the reaction were studied, and the reaction mechanism was postulated. Under the optimized reaction conditions, Beer's law correlating the absorbance (A) with PRX concentration (C) was obeyed in the range of 1-8 mug mL(-1). The regression equation for the calibration data was: A = 0.0031 + 0.1609 C, with good correlation coefficients (0.9992). The molar absorptivity (epsilon) was 5.9 x 10(5) L mol(-1) 1 cm(-1). The limits of detection and quantitation were 0.3 and 0.8 mug mL(-1), respectively. The precision of the method was satisfactory; the values of relative standard deviations did not exceed 2%. The proposed method was successfully applied to the determination of PRX in its pharmaceutical tablets with good accuracy and precisions; the label claim percentage was 97.17 +/- 1.06 %. The results obtained by the proposed method were comparable with those obtained by the official method.
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    Longstreet, A. R.; Jo, M.; Chandler, R. R.; Hanson, K.; Zhan, N.; Hrudka, J. J.; Mattoussi, H.; Shatruk, M.; McQuade, D. T. Ylidenemalononitrile Enamines as Fluorescent “Turn-On” Indicators for Primary Amines. J. Am. Chem. Soc. 2014, 136 (44), 15493– 6,  DOI: 10.1021/ja509058u
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    Ylidenemalononitrile Enamines as Fluorescent "Turn-On" Indicators for Primary Amines
    Longstreet, Ashley R.; Jo, Minyoung; Chandler, Rebecca R.; Hanson, Kenneth; Zhan, Naiqian; Hrudka, Jeremy J.; Mattoussi, Hedi; Shatruk, Michael; McQuade, D. Tyler
    Journal of the American Chemical Society (2014), 136 (44), 15493-15496CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Ylidenemalononitrile enamines undergo rapid amine exchange followed by a cyclization with primary amines to yield fluorescent products with emission intensities ≤900 times greater than the starting materials. After identifying the fluorescent species by x-ray crystallog., the rate of amine exchange is substrate dependent and by simple structural variation the fluorescence can be tuned over the entire visible spectrum. The authors further demonstrate their potential application in biomol. labeling.
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    Sathiskumar, U.; Easwaramoorthi, S. Red-Emitting Ratiometric Fluorescence Chemodosimeter for the Discriminative Detection of Aromatic and Aliphatic Amines. ChemistrySelect 2019, 4 (25), 7486– 94,  DOI: 10.1002/slct.201901254
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    Red-Emitting Ratiometric Fluorescence Chemodosimeter for the Discriminative Detection of Aromatic and Aliphatic Amines
    Sathiskumar, Udayadasan; Easwaramoorthi, Shanmugam
    ChemistrySelect (2019), 4 (25), 7486-7494CODEN: CHEMUD; ISSN:2365-6549. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A NIR-emitting fluorescent probe using phenothiazine and 1,3-dimethylbarbituric acid (PMB) connected through methylene bridge was developed in a two-step simple chem. reaction. The fluorescent probes were highly reactive towards various primary amines and the reaction was accompanied by the changes in the optical and fluorescent properties of the PMB characteristic to the amine and thereby can have the potential to be used as a sensor for amines. In addn. to the ratiometric changes in the fluorescence intensity corresponding to the PMB before and after reaction with an amine, the emission spectral max. is specific to the analyte. For, example 1,4-diaminobenzene, 4-methoxyaniline, aniline, hydrazine, ethylamine, ethylenediamine resp. shows the max. at 532, 546, 555, 484, 497, and 488 nm and this unique feature originated due to the involvement of the analyte in the extended π-conjugation with the probe. Sensor strips prepd. using PMB coated filter paper were able to sense the volatile amine gases with shorter response time. Anal. of the strips using the CIELAB 1976 color coordinates provides different signals for hydrazine, aniline, propylamine, and ethylamine gases. The applicability of the sensor was further ascertained by monitoring the decompn. of skin used for leather processing and also proteins engineered with an amino-functionalized unnatural amino acid.
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    Turiák, G.; Volicer, L. Stability of o-Phthalaldehyde─Sulfite Derivatives of Amino Acids and Their Methyl Esters: Electrochemical and Chromatographic Properties. J. Chromatogr. A 1994, 668 (2), 323– 9,  DOI: 10.1016/0021-9673(94)80121-5
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    105
    Stability of o-phthalaldehyde-sulfite derivatives of amino acids and their methyl esters: electrochemical and chromatographic properties
    Turiak, Gyorgy; Volicer, Ladislav
    Journal of Chromatography A (1994), 668 (2), 323-9CODEN: JCRAEY; ISSN:0021-9673.
    RP-HPLC coupled with 16-channel coulometric electrode array detection was used to monitor the decompn. of five amino acid o-phthalaldehyde (OPA)-sulfite derivs. (Ala, Arg, Glu, Ser, Tyr) and their Me ester derivs. as well. At fixed OPA and sulfite concns., inclusion of methanol and EDTA in the derivatization media has increased most effectively the room temp. stability of both derivs. measured at pH 9.2 (amino acids) and pH 8.2 (Me esters). Decreases in product concns. by 6% occurred after >15 h for amino acid derivs. and 8 h for Me ester derivs. The oxidn. potential max. for OPA-sulfite derivs. of amino acids were found at 600 mV while the same Me ester derivs. had 60-120 mV higher max. with the exception of tyrosine. The detector responses are linear in the studied 0.1-10 μM concn. range for both deriv. forms and their detection limit was 100-200 fmol injected on the column. The RP-HPLC retention of amino acid Me ester OPA-sulfite derivs. was very similar to the amino acid OPA-2-mercaptoethanol ones while the more polar amino acid OPA-sulfite derivs. were eluted earlier (k'<1) under the same chromatog. conditions.
  106. 106
    De Montigny, P.; Stobaugh, J. F.; Givens, R. S.; Carlson, R. G.; Srinivasachar, K.; Sternson, L. A.; Higuchi, T. Naphthalene-2,3-Dicarboxyaldehyde/Cyanide Ion: A Rationally Designed Fluorogenic Reagent for Primary Amines. Anal. Chem. 1987, 59 (8), 1096– 101,  DOI: 10.1021/ac00135a007
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    106
    Naphthalene-2,3-dicarboxyaldehyde/cyanide ion: a rationally designed fluorogenic reagent for primary amines
    De Montigny, Pierre; Stobaugh, John F.; Givens, Richard S.; Carlson, Robert G.; Srinivasachar, Kasturi; Sternson, Larry A.; Higuchi, Takeru
    Analytical Chemistry (1987), 59 (8), 1096-101CODEN: ANCHAM; ISSN:0003-2700.
    On the basis of the isoindole formation mechanism in the o-phthalaldehyde/2-mercaptoethanol (OPA/2-ME) derivatization of primary amines and the structure-stability relations for isoindoles, an improved fluorogenic reagent, naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide ion (CN-), was developed. Reaction of NDA/CN- with primary amine in aq. media results in the formation of N-substituted 1-cyanobenz[f]isoindole (CBI) derivs. which have significantly improved stability compared to the corresponding OPA/2-ME derivs. (for glycine greater than 50-fold improvement was realized) and have high quantum efficiencies for fluorescence (Φf = 0.54 in 60% aq. MeCN for the CBI-n-propylamine deriv.) in solvent systems commonly used in liq. chromatog. Parameters in the NDA/CN- derivatization of alanine are defined (i.e., pH and the reagent component concns.) and used in the development of a labeling procedure for amino acid mixts. Gradient elution fractionation of 18 CBI-amino acid derivs. was accomplished in 60 min and permitted detection limits of <200 fmol injected (excitation 246 nm) or <3 pmol injected (excitation 420 nm). The utility of the reagent in assaying amino acid mixts. resulting from the enzymic hydrolysis of the peptides Met-enkephalin and glucagon is demonstrated.
  107. 107
    Mroz, E. A.; Roman, R. J.; Lechene, C. Fluorescence Assay for Picomole Quantities of Ammonia. Kidney Int. 1982, 21 (3), 524– 7,  DOI: 10.1038/ki.1982.56
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    Fluorescence assay for picomole quantities of ammonia
    Mroz, Edmund A.; Roman, Richard J.; Lechene, Claude
    Kidney International (1982), 21 (), 524-7CODEN: KDYIA5; ISSN:0085-2538.
    Nanomole, picomole, and subpicomole amts. of NH3 were detd. fluorometrically in rat tubular fluid and urine by reacting NH3 with o-phthalaldehyde and thioglycolic acid at pH 7.4 by modifying the method of S. Taylor et al. (1974). To det. nanomole quantities of NH3 (1-3-mL final vol.), glass test tubes were used, whereas for the detn. of picomole (2-10-μL vol.) and subpicomole (1-nL vol.) amts. of NH3, siliconized slides were used and the samples were covered with degassed oil to prevent evapn. The method gave better specificity than the method of S. Taylor et al.
  108. 108
    Carlson, R. G.; Srinivasachar, K.; Givens, R. S.; Matuszewski, B. K. New Derivatizing Agents for Amino Acids and Peptides. 1. Facile Synthesis of N-Substituted 1-Cyanobenz[f]Isoindoles and Their Spectroscopic Properties. J. Org. Chem. 1986, 51 (21), 3978– 83,  DOI: 10.1021/jo00371a013
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    108
    New derivatizing agents for amino acids and peptides. 1. Facile synthesis of N-substituted 1-cyanobenz[f]isoindoles and their spectroscopic properties
    Carlson, R. G.; Srinivasachar, K.; Givens, R. S.; Matuszewski, B. K.
    Journal of Organic Chemistry (1986), 51 (21), 3978-83CODEN: JOCEAH; ISSN:0022-3263.
    2,3-Naphthalenedicarboxaldehyde (I, R = CHO) (II) was prepd. as a reagent for the derivatization of primary amines, amino acids, and small peptides as highly fluorescent 2-substituted 1-cyanobenz[f]isoindoles III (R1 = group from amino compd. R1NH2). Thus, o-xylene IV was treated with maleic anhydride in the presence of NaI to give I (R = CO2H), which was reduced with LiAlH4 to give I (R = CH2OH), which was partially oxidized with oxalyl chloride in DMSO/CH2Cl2 to give II. The reaction of II with amino compds. R1NH2 (R1 = Pr, Bu, CMe3, CH2CO2H, DL-CHMeCO2H, CH2CO-Gly-OH, D-CHMeCO-D-Ala-OH) in the presence of NaCN gave the corresponding III. The fluorescent properties of III were detd.
  109. 109
    Bantan-Polak, T.; Kassai, M.; Grant, K. B. A Comparison of Fluorescamine and Naphthalene-2,3-Dicarboxaldehyde Fluorogenic Reagents for Microplate-Based Detection of Amino Acids. Anal. Biochem. 2001, 297 (2), 128– 36,  DOI: 10.1006/abio.2001.5338
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    109
    A comparison of fluorescamine and naphthalene-2,3-dicarboxaldehyde fluorogenic reagents for microplate-based detection of amino acids
    Bantan-Polak, Tjasa; Kassai, Miki; Grant, Kathryn B.
    Analytical Biochemistry (2001), 297 (2), 128-136CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)
    The use of appropriate fluorometric derivatization procedures is of considerable importance for accurate detn. of amino acids in biol. samples and in metal-assisted peptide hydrolysis reactions. It is esp. crit. for the relative fluorescence intensities (RFI) of equal amts. of amino acids to be as similar as possible. While fluorescamine and naphthalene-2,3-dicarboxaldehyde (NDA) have proven to be excellent fluorogenic reagents for amino acid detection, the effects of various factors such as org. solvent, buffer, and pH have never been rigorously evaluated with respect to normalizing the relative fluorescence intensities of individual amino acids. To this end, here we describe optimized fluorescamine and NDA derivatization reactions that enhance the accuracy of microplate-based detection of amino acids. For both fluorescamine and NDA, we have shown that the RFI values of 16 of 19 amino acids are greater than 70%. Although detn. of tryptophan is problematic, this difficulty is overcome by the addn. of β-cyclodextrin to the NDA reaction. In principle, the optimized fluorescamine and NDA microplate procedures reported here can be utilized as complementary techniques for the detection of 19 of 20 naturally occurring amino acids. (c) 2001 Academic Press.
  110. 110
    Hapuarachchi, S.; Aspinwall, C. A. Design, Characterization, and Utilization of a Fast Fluorescence Derivatization Reaction Utilizing o-Phthaldialdehyde Coupled with Fluorescent Thiols. Electrophoresis 2007, 28 (7), 1100– 6,  DOI: 10.1002/elps.200600567
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    110
    Design, characterization, and utilization of a fast fluorescence derivatization reaction utilizing o-phthaldialdehyde coupled with fluorescent thiols
    Hapuarachchi, Suminda; Aspinwall, Craig A.
    Electrophoresis (2007), 28 (7), 1100-1106CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The authors have developed a chem. derivatization scheme for primary amines that couples the fast kinetic properties of o-phthaldialdehyde (OPA) with the photophys. properties of visible, high quantum yield, fluorescent dyes. In this reaction, OPA is used as a crosslinking reagent in the labeling reaction of primary amines in the presence of a fluorescent thiol, 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino)fluorescein (SAMSA fluorescein), thereby incorporating fluorescein (ε = 78 000 M-1, quantum yield of 0.98) into the isoindole product. Detection is based on excitation and emission of the incorporated fluorescein using the 488 nm laser line of an Ar+ laser rather than the UV-excited isoindole, thereby eliminating the UV light sources for detection. Using this method, the authors have quant. labeled biol. important primary amines in less than 10 s. Detection limits for anal. of glutamate, glycine, GABA, and taurine were less than 2 nM. The authors present the characterization of OPA/SAMSA-F reaction and the potential utility of the derivatization reaction for dynamic chem. monitoring of biol. relevant analytes using CE.
  111. 111
    Zhang, L.-Y.; Liu, Y.-M.; Wang, Z.-L.; Cheng, J.-K. Capillary Zone Electrophoresis with Pre-Column NDA Derivatization and Amperometric Detection for the Analysis of Four Aliphatic Diamines. Anal. Chim. Acta 2004, 508 (2), 141– 5,  DOI: 10.1016/j.aca.2003.11.075
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    111
    Capillary zone electrophoresis with pre-column NDA derivatization and amperometric detection for the analysis of four aliphatic diamines
    Zhang, Li-Yao; Liu, Yan-Ming; Wang, Zong-Li; Cheng, Jie-Ke
    Analytica Chimica Acta (2004), 508 (2), 141-145CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
    A method was developed for the anal. of four aliph. diamines by capillary zone electrophoresis using pre-column derivatization with naphthalene-2,3-dicarboxaldehyde (NDA)/CN- and amperometric detection. The pre-column derivatization reaction conditions including the molar ratio of NDA to amines, the cyanide concn., the pH value of derivatization buffer, and the reaction time, were studied. The sepn. of four derivs. of aliph. diamines was optimized by capillary zone electrophoresis (CZE) using end-column amperometric detection with a C fiber microelectrode, at a const. potential of 0.7 V vs. SCE. The optimum conditions for the sepn. were 10 mM Tris-H3PO4 (pH 4.0) for the running buffer soln., 15 kV for the sepn. voltage. The detection limits for diaminopropane, putrescine, cadaverine, diaminohexane were 6.7 × 10-8, 5.1 × 10-8, 1.9 × 10-7 and 3.8 × 10-7 M, resp. (S/N = 3). The proposed method was applied to the detn. of aliph. diamines in a lake water sample by the std. addn. method. The recovery of these amines in H2O was 89.9-107%.
  112. 112
    Fekete, A.; Lahaniatis, M.; Lintelmann, J.; Schmitt-Kopplin, P. Determination of Aliphatic Low-Molecular-Weight and Biogenic Amines by Capillary Zone Electrophoresis. In Capillary Electrophoresis: Methods and Protocols; Humana Press: Totowa, NJ, 2008; pp 65– 91. DOI: 10.1007/978-1-59745-376-9_4
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    Böhmer, A.; Jordan, J.; Tsikas, D. High-Performance Liquid Chromatography Ultraviolet Assay for Human Erythrocytic Catalase Activity by Measuring Glutathione as o-Phthalaldehyde Derivative. Anal. Biochem. 2011, 410 (2), 296– 303,  DOI: 10.1016/j.ab.2010.11.026
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    113
    High-performance liquid chromatography ultraviolet assay for human erythrocytic catalase activity by measuring glutathione as o-phthalaldehyde derivative
    Bohmer Anke; Jordan Jens; Tsikas Dimitrios
    Analytical biochemistry (2011), 410 (2), 296-303 ISSN:.
    The most frequently used catalase (CAT) activity assay is based on the spectrophotometric measurement of hydrogen peroxide (H(2)O(2)) absorbance decrease at 240 nm. Here we report an alternative high-performance liquid chromatography (HPLC) assay for human erythrocytic CAT (heCAT) activity measurement based on glutathione (GSH) analysis as a highly stable, H(2)O(2)-insensitive o-phthalaldehyde (OPA) derivative. The method was developed and validated using an isolated heCAT in phosphate-buffered saline at pH 7.4 and was applied to measure CAT activity in lysed human erythrocytes. heCAT activity was measured at initial concentrations of 5 nM for heCAT, 5mM for H(2)O(2), and 10mM for GSH, and the incubation time was 10 min. Nitrite (NO(2)(-)) was found to be an uncompetitive inhibitor of heCAT activity (IC(50)=9 μM) and of CAT activity in hemolysate (IC(50)∼750 μM). Nitrate (NO(3)(-)) at concentrations up to 100 μM did not inhibit heCAT activity. Azide (N(3)(-)) was found to be a very strong inhibitor of the heCAT (IC(50)=0.2 nM) but a relatively weak CAT inhibitor (IC(50)∼10 μM) in human hemolysates. The novel CAT activity assay works under redox conditions that more closely resemble those prevailing in cells and allows high-throughput analysis despite the required HPLC step.
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    Black, G. W.; Brown, N. L.; Perry, J. J. B.; Randall, P. D.; Turnbull, G.; Zhang, M. A High-Throughput Screening Method for Determining the Substrate Scope of Nitrilases. Chem. Commun. 2015, 51 (13), 2660– 2,  DOI: 10.1039/C4CC06021K
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    A high-throughput screening method for determining the substrate scope of nitrilases
    Black, Gary W.; Brown, Nicola L.; Perry, Justin J. B.; Randall, P. David; Turnbull, Graeme; Zhang, Meng
    Chemical Communications (Cambridge, United Kingdom) (2015), 51 (13), 2660-2662CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    Nitrile compds. are intermediates in the synthesis of pharmaceuticals such as atorvastatin. We have developed a chromogenic reagent to screen for nitrilase activity as an alternative to Nessler's reagent. It produces a semi-quantifiable blue color and hydrolysis of 38 nitrile substrates by 23 nitrilases as cell-free exts. has been shown.
  115. 115
    Mann, S.; Eveleigh, L.; Lequin, O.; Ploux, O. A Microplate Fluorescence Assay for DAPA Aminotransferase by Detection of the Vicinal Diamine 7,8-Diaminopelargonic Acid. Anal. Biochem. 2013, 432 (2), 90– 6,  DOI: 10.1016/j.ab.2012.09.038
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    115
    A microplate fluorescence assay for DAPA aminotransferase by detection of the vicinal diamine 7,8-diaminopelargonic acid
    Mann, Stephane; Eveleigh, Luc; Lequin, Olivier; Ploux, Olivier
    Analytical Biochemistry (2013), 432 (2), 90-96CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    7,8-Diaminopelargonic acid (DAPA) aminotransferase is an enzyme of the biotin biosynthetic pathway that plays an essential role in Mycobacterium tuberculosis virulence. Inhibition of this enzyme is a potential strategy to combat this microorganism, the causative agent of tuberculosis. To identify new inhibitors as potential drugs, a simple enzymic assay for high-throughput screening (HTS) is needed. Several methods for measuring DAPA aminotransferase activity are already available. However, requirements for their implementation for HTS are tedious. We describe here a microplate fluorescence assay for DAPA aminotransferase that is simple, cheap, and sensitive, allowing linear detection of DAPA in the range of 20 nM to 50 μM. The principle of the method is the direct detection in the enzymic reaction mixt. of the vicinal diamine DAPA derivatized with ortho-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). The assay was validated with the known inhibitor desmethyl-KAPA (8-amino-7-oxopelargonic acid) and adapted to microplate for HTS. The structure of the stable fluorescent adduct formed between a vicinal primary diamine and OPA in the presence of 2ME was characterized by mass spectrometry and NMR spectroscopy.
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    Kalkhof, S.; Sinz, A. Chances and Pitfalls of Chemical Cross-Linking with Amine-Reactive N-Hydroxysuccinimide Esters. Anal. Bioanal. Chem. 2008, 392 (1), 305– 12,  DOI: 10.1007/s00216-008-2231-5
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    Chances and pitfalls of chemical cross-linking with amine-reactive N-hydroxysuccinimide esters
    Kalkhof, Stefan; Sinz, Andrea
    Analytical and Bioanalytical Chemistry (2008), 392 (1-2), 305-312CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    In this report the authors summarize their experiences with the reaction products of N-hydroxysuccinimide (NHS) esters, which are widely used for chem. crosslinking of lysine residues in proteins. The authors describe the products, which should be scrutinized during data anal. using customized software when NHS esters are employed for chem. crosslinking. Reaction products of NHS esters were obsd. not only with lysines, but also with serines, tyrosines, and threonines. This report is intended to be a practical guide for those working in the field of chem. crosslinking and mass spectrometry.
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    Gayo, L. M.; Suto, M. J. Use of Pentafluorophenyl Esters for One-Pot Protection/Activation of Amino and Thiol Carboxylic Acids. Tetrahedron Lett. 1996, 37 (28), 4915– 8,  DOI: 10.1016/0040-4039(96)00985-9
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    Use of pentafluorophenyl esters for one-pot protection/activation of amino and thiol carboxylic acids
    Gayo, Leah M.; Suto, Mark J.
    Tetrahedron Letters (1996), 37 (28), 4915-4918CODEN: TELEAY; ISSN:0040-4039. (Elsevier)
    The authors describe a new one-pot procedure for the simultaneous protection and activation of amino and thiol carboxylic acids using pentafluorophenyl trifluoroacetate (TFAPfp) and related pentafluorophenyl reagents (FmocPfp and AcPfp). For example, the treatment of 2-aminobenzoic acid with six equiv. of FmocPfp in DMF with pyridine gave pentafluorophenyl 2-(fluorenylmethoxycarbonylamino)benzoate in 82% yield with high purity. This approach can be used for the construction of combinatorial libraries of pentafluorophenyl esters of N-acylated amino acids or S-acylated mercapto acids.
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    Fang, L.; Demee, M.; Sierra, T.; Kshirsagar, T.; Celebi, A. A.; Yan, B. Kinetics Study of Amine Cleavage Reactions of Various Resin-Bound Thiophenol Esters from Marshall Linker. J. Comb. Chem. 2002, 4 (4), 362– 8,  DOI: 10.1021/cc020010r
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    Kinetics Study of Amine Cleavage Reactions of Various Resin-Bound Thiophenol Esters from Marshall Linker
    Fang, Liling; Demee, Michael; Sierra, Teresa; Kshirsagar, Tushar; Celebi, Azim A.; Yan, Bing
    Journal of Combinatorial Chemistry (2002), 4 (4), 362-368CODEN: JCCHFF; ISSN:1520-4766. (American Chemical Society)
    The kinetics of cleavage reactions of seven resin-bound thiophenol esters with three amines has been studied by single-bead FTIR. The reactivity of these seven thiophenol esters was dependent on their structures and could be summarized as follows: 5-benzimidazolecarboxylic thiophenol ester > alkyl thiophenol ester > arom. thiophenol ester. The reactivity of three amines was summarized as follows: n-butylamine > 3,4-dimethoxyphenethylamine > 1-piperonylpiperazine. The rate of the cleavage reaction increased 2-fold per 10 °C rise in reaction temp. Oxidn. of the thiophenol linker increased the rate of the cleavage reaction by 580-fold.
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    Lavis, L. D. Teaching Old Dyes New Tricks: Biological Probes Built from Fluoresceins and Rhodamines. Annu. Rev. Biochem. 2017, 86, 825– 43,  DOI: 10.1146/annurev-biochem-061516-044839
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    Teaching Old Dyes New Tricks: Biological Probes Built from Fluoresceins and Rhodamines
    Lavis, Luke D.
    Annual Review of Biochemistry (2017), 86 (), 825-843CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
    Small-mol. fluorophores, such as fluorescein and rhodamine derivs., are crit. tools in modern biochem. and biol. research. The field of chem. dyes is old; colored mols. were first discovered in the 1800s, and the fluorescein and rhodamine scaffolds have been known for over a century. Nevertheless, there has been a renaissance in using these dyes to create tools for biochem. and biol. The application of modern chem., biochem., mol. genetics, and optical physics to these old structures enables and drives the development of novel, sophisticated fluorescent dyes. This crit. review focuses on an important example of chem. biol.-the melding of old and new chem. knowledge-leading to useful mols. for advanced biochem. and biol. expts.
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    Pereira, A.; Martins, S.; Caldeira, A. T. Phytochemicals in Human Health, Coumarins as Fluorescent Labels of Biomolecules; IntechOpen, 2019.
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    Boens, N.; Leen, V.; Dehaen, W. Fluorescent Indicators Based on BODIPY. Chem. Soc. Rev. 2012, 41 (3), 1130– 72,  DOI: 10.1039/C1CS15132K
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    Fluorescent indicators based on BODIPY
    Boens, Noel; Leen, Volker; Dehaen, Wim
    Chemical Society Reviews (2012), 41 (3), 1130-1172CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. This crit. review covers the advances made using the 4-bora-3a,4a-diaza-s-indacene (BODIPY) scaffold as a fluorophore in the design, synthesis and application of fluorescent indicators for pH, metal ions, anions, biomols., reactive oxygen species, reactive nitrogen species, redox potential, chem. reactions and various phys. phenomena. The sections of the review describing the criteria for rational design of fluorescent indicators and the math. expressions for analyzing spectrophotometric and fluorometric titrns. are applicable to all fluorescent probes (206 refs.).
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    Han, S.-Y.; Kim, Y.-A. Recent Development of Peptide Coupling Reagents in Organic Synthesis. Tetrahedron 2004, 60 (11), 2447– 67,  DOI: 10.1016/j.tet.2004.01.020
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    Recent development of peptide coupling reagents in organic synthesis
    Han, So-Yeop; Kim, Young-Ah
    Tetrahedron (2004), 60 (11), 2447-2467CODEN: TETRAB; ISSN:0040-4020. (Elsevier Science B.V.)
    A review. Various types of peptide coupling reagents (i.e., phosphonium, uronium, immonium, carbodiimide, imidazolium, organophosphorus, acid halogenating compds., chloroformate, pyridinium, etc.) are discussed for the synthesis of bioactive mols. contg. peptide linkages. Methods used to suppress racemization during the peptide coupling step are presented.
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    Karongo, R.; Ge, M.; Horak, J.; Gross, H.; Kohout, M.; Lindner, W.; Lämmerhofer, M. Rapid Enantioselective Amino Acid Analysis by Ultra-High Performance Liquid Chromatography-Mass Spectrometry Combining 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatization with Core-Shell Quinine Carbamate Anion Exchanger Separation. J. Chromatogr. Open 2021, 1, 100004,  DOI: 10.1016/j.jcoa.2021.100004
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    Cao, L.; Wang, H.; Zhang, H. Analytical Potential of 6-Oxy-(N-Succinimidyl Acetate)-9-(2’-Methoxycarbonyl) Fluorescein for the Determination of Amino Compounds by Capillary Electrophoresis with Laser-Induced Fluorescence Detection. Electrophoresis 2005, 26 (10), 1954– 62,  DOI: 10.1002/elps.200410227
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    124
    Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detection
    Cao, Liwei; Wang, Hong; Zhang, Huashan
    Electrophoresis (2005), 26 (10), 1954-1962CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The anal. potential of a fluorescein analog, 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the 1st time synthesized in the lab., as a labeling reagent for the labeling and detn. of amino compds. by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the anal. possibilities of this approach. The derivatization conditions and sepn. parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30°C for 6 min in boric acid buffer (pH 8.0). The derivs. were baseline-sepd. in 15 min with 25 mM boric acid running buffer (pH 9.0), contg. 24 mM SDS and 12.5% vol./vol. acetonitrile. The concn. detection limit for biogenic amines reaches 8 × 10-11 mol/L (signal-to-noise ratio = 3). The application of CE in the anal. of the SAMF-derivatized amino acids was also exploited. The optimal running buffer for amino acids suggested that weak acidic background electrolyte offered better sepn. than the basic one. The proposed method was applied to the detn. of biogenic amines in 3 different beer samples with satisfying recoveries varying from 92.8% to 104.8%. Finally, comparison of several fluorescein-based probes for amino compds. was discussed. With good labeling reaction, excellent photostability, pH-independent fluorescence (pH 4-9), and the resultant widely suited running buffer pH, SAMF has a great prospect in the detn. of amino compds. in CE.
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    Xia, L.-J.; Guo, X.-F.; Ji, Y.; Chen, L.; Wang, H. A Long-Wavelength Fluorescent Probe for Amino Compounds and Its Application in the Determination of Aliphatic Amines. Anal. Methods 2018, 10 (26), 3188– 96,  DOI: 10.1039/C8AY00706C
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    A long-wavelength fluorescent probe for amino compounds and its application in the determination of aliphatic amines
    Xia, Li-Jun; Guo, Xiao-Feng; Ji, Yan; Chen, Liu; Wang, Hong
    Analytical Methods (2018), 10 (26), 3188-3196CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)
    Herein, a highly reactive long-wavelength fluorescent labeling reagent, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimidyl ester) difluoroboradiaza-s-indacene (DMDSPAB-OSu), was designed and synthesized for amino compds. Using DMDSPAB-OSu as a pre-column derivatization reagent, an HPLC-fluorescence detection method was developed for the detn. of trace amts. of aliph. amines in samples. Prior to anal., amines were derivatized with DMDSPAB-OSu at room temp. in 0.2 mol L-1 pH 8.8 borate buffer for only 5 min, and the baseline sepn. of 11 aliph. amines was achieved within 25 min. Limits of detection between 0.8 to 1.2 nM were obtained at 630 nm with an excitation wavelength of 610 nm. Furthermore, the proposed DMDSPAB-OSu-based HPLC method was applied to the anal. of lake water and human urine with satisfying recoveries ranging from 93.0-109.7%.
  126. 126
    Gao, P. F.; Guo, X. F.; Wang, H.; Zhang, H. S. Determination of Trace Biogenic Amines with 1,3,5,7-Tetramethyl-8-(N-Hydroxysuccinimidyl Butyric Ester)-Difluoroboradiaza-s-Indacene Derivatization Using High-Performance Liquid Chromatography and Fluorescence Detection. J. Sep. Sci. 2011, 34 (12), 1383– 90,  DOI: 10.1002/jssc.201100120
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    Determination of trace biogenic amines with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene derivatization using high-performance liquid chromatography and fluorescence detection
    Gao, Pan-Feng; Guo, Xiao-Feng; Wang, Hong; Zhang, Hua-Shan
    Journal of Separation Science (2011), 34 (12), 1383-1390CODEN: JSSCCJ; ISSN:1615-9306. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A reversed-phase high-performance liq. chromatog. method based on chem. derivatization with fluorescence detection has been developed for analyzing biogenic amines in food and environmental samples. A BODIPY-based fluorescent reagent, 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su), was employed for the derivatization of these biogenic amines at 20°C for 20 min in pH 7.20 borate buffer after careful investigation of the derivatization conditions including reagent concn., buffer soln., reaction temp. and reaction time. Sepn. of biogenic amines with gradient elution was conducted on a C8 column with methanol-tetrahydrofuran-water as mobile phase. The detection limits were obtained in the range from 0.1 to 0.2 nM (signal-to-noise=3). This procedure has been validated using practical samples. The study results demonstrated a potential of employing high-performance liq. chromatog. (HPLC) with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene labeling as a tool for quant. anal. of biogenic amines involved in various matrixes.
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    Mallick, S.; Chandra, F.; Koner, A. L. A Ratiometric Fluorescent Probe for Detection of Biogenic Primary Amines with Nanomolar Sensitivity. Analyst 2016, 141 (3), 827– 31,  DOI: 10.1039/C5AN01911G
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    A ratiometric fluorescent probe for detection of biogenic primary amines with nanomolar sensitivity
    Mallick, Suman; Chandra, Falguni; Koner, Apurba L.
    Analyst (Cambridge, United Kingdom) (2016), 141 (3), 827-831CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    An ultrasensitive ratiometric fluorescent sensor (I) made of an N,N-dimethylaminonaphthalene anhydride moiety for detection of aliph. primary amines is reported. Biogenic amines at nanomolar concn. were detected with the addnl. ability to discriminate between primary, secondary and tertiary amines by using both UV-Visible and fluorescence spectroscopy.
  128. 128
    Vázquez, M. E.; Blanco, J. B.; Imperiali, B. Photophysics and Biological Applications of the Environment-Sensitive Fluorophore 6-N,N-Dimethylamino-2,3-Naphthalimide. J. Am. Chem. Soc. 2005, 127 (4), 1300– 6,  DOI: 10.1021/ja0449168
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    128
    Photophysics and biological applications of the environment-sensitive fluorophore 6-N,N-Dimethylamino-2,3-naphthalimide
    Vazquez, M. Eugenio; Blanco, Juan B.; Imperiali, B.
    Journal of the American Chemical Society (2005), 127 (4), 1300-1306CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    We have synthesized a new environment-sensitive fluorophore, 6-N,N-dimethylamino-2,3-naphthalimide (6DMN). This chromophore exhibits valuable fluorescent properties as a biol. probe with emission in the 500-600 nm range and a marked response to changes in the environment polarity. The 6DMN fluorescence is red-shifted in polar protic environments, with the max. emission intensity shifting more than 100 nm from 491 nm in toluene to 592 nm in water. Addnl., the fluorescence quantum yield decreases more than 100-fold from chloroform (Φ = 0.225) to water (Φ = 0.002). The scope and applications of the 6DMN probe are expanded with the synthesis of an Fmoc-protected amino acid deriv. (5), which contains the fluorophore. This unnatural amino acid has been introduced into several peptides, demonstrating that it can be manipulated under std. solid-phase peptide synthesis conditions. Peptides incorporating the new residue can be implemented for monitoring protein-protein interactions as exemplified in studies with Src homol. 2 (SH2) phosphotyrosine binding domains. The designed peptides exhibit a significant increase in the quantum yield of the long wavelength fluorescence emission band (596 nm) upon binding to selected SH2 domains (e.g., Crk SH2, Abl SH2, and PI3K SH2). The peptides can be used as ratiometric sensors, since the short wavelength band (460 nm) was found almost invariable throughout the titrns.
  129. 129
    Anumula, K. R.; Schulz, R. P.; Back, N. Fluorescent N-Methylanthranilyl (Mantyl) Tag for Peptides: Its Application in Subpicomole Determination of Kinins. Peptides 1992, 13 (4), 663– 9,  DOI: 10.1016/0196-9781(92)90170-8
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    129
    Fluorescent N-methylanthranilyl (Mantyl) tag for peptides: its application in subpicomole determination of kinins
    Anumula, Kalyan R.; Schulz, Raymond P.; Back, Nathan
    Peptides (New York, NY, United States) (1992), 13 (4), 663-9CODEN: PPTDD5; ISSN:0196-9781.
    Highly fluorescent N-methylanthranilyl (Mantyl) peptide derivs. were prepd. by a 1-step reaction with N-methylisatoic anhydride (MIA) for quant. detection in HPLC. Reactions were carried out in an org. medium of acetonitrile-triethylamine, in aq. alk. sodium carbonate and sodium phosphate buffers. 4-Dimethylaminopyridine (DMAP) catalyzed specific mantylation of -NH2 groups of peptides in the org. reaction medium. The DMAP had no effect in the aq. buffered reaction systems. Proline amino-terminal peptides reacted equally well with MIA. Mantyl-bradykinin (Mantyl-BK) had excitation and fluorescence max. at 350 nm and 426 nm in water and water/acetonitrile (ACN)/trifluoroacetic acid (TFA) solvent mixts., resp. Fluorescence intensity increased with an increase in ACN concn. and decreased with an increase in acid content. Mantyl kinins were completely resolved on a C18 reversed-phase HPLC column using an ACN-0.1% TFA gradient and their behavior on the column was similar to having an extra amino acid. Di-Mantyl derivs. obtained with Lys-BK and Met-Lys-BK did not exhibit fluorescence appreciably higher than Mantyl-BK. Fluorescence detection of Mantyl kinins was ∼50-100-fold more sensitive (lower limits of 0.1-0.5 pmol) than UV detection of the phenylisothiocyanate-derivatized kinins under typical HPLC conditions.
  130. 130
    Fessler, A.; Garmon, C.; Heavey, T.; Fowler, A.; Ogle, C. Water-Soluble and UV Traceable Isatoic Anhydride-Based Reagents for Bioconjugation. Org. Biomol. Chem. 2017, 15 (45), 9599– 602,  DOI: 10.1039/C7OB02377D
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    130
    Water-soluble and UV traceable isatoic anhydride-based reagents for bioconjugation
    Fessler, Adam; Garmon, Corey; Heavey, Thomas; Fowler, Anthony; Ogle, Craig
    Organic & Biomolecular Chemistry (2017), 15 (45), 9599-9602CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Isatoic anhydride is elaborated to water sol. bioconjugation reagents that gives functionality and water soly. in one self-cleaning step. This new platform offers high atom economy with carbon dioxide being the only byproduct, and is shown to very quickly and efficiently label proteins in bicarbonate buffered solns.
  131. 131
    Jeon, S.; Kim, T.-I.; Jin, H.; Lee, U.; Bae, J.; Bouffard, J.; Kim, Y. Amine-Reactive Activated Esters of meso-CarboxyBODIPY: Fluorogenic Assays and Labeling of Amines, Amino Acids, and Proteins. J. Am. Chem. Soc. 2020, 142 (20), 9231– 9,  DOI: 10.1021/jacs.9b13982
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    131
    Amine-Reactive Activated Esters of meso-CarboxyBODIPY: Fluorogenic Assays and Labeling of Amines, Amino Acids, and Proteins
    Jeon, Sungjin; Kim, Tae-Il; Jin, Hanyong; Lee, Uisung; Bae, Jeehyeon; Bouffard, Jean; Kim, Youngmi
    Journal of the American Chemical Society (2020), 142 (20), 9231-9239CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Fluorescence-based amine-reactive dyes are highly valuable for the sensing of amines and the labeling of biomols. Although it would be highly desirable, large changes in emission spectra and intensity seldom accompany the conjugation of known amine-reactive dyes to their target mols. On the contrary, amide bond formation between amines and the pentafluorophenyl (2-PFP) and succinimidyl (2-NHS) esters of meso-carboxyBODIPY results in significant changes in emission maxima (Δλ: 70-100 nm) and intensity (up to 3000-fold), enabling the fast (down to 5 min) and selective fluorogenic detection and labeling of amines, amino acids, and proteins. This approach further benefits from the demonstrated versatility and high reliability of activated ester chem., and background hydrolysis is negligible. The large "turn-on" response is a testament of the extreme sensitivity of meso-carboxyBODIPYs to the minimal changes in electronic properties that distinguish esters from amides. Applications to the detection of food spoilage, staining of proteins on electrophoretic gels or in living cells, and the expedited synthesis of organelle-specific fluorescence microscope imaging agents are further demonstrated.
  132. 132
    Lee, U.; Kim, T.-I.; Jeon, S.; Luo, Y.; Cho, S.; Bae, J.; Kim, Y. Native Chemical Ligation-Based Fluorescent Probes for Cysteine and Aminopeptidase N Using meso-Thioester-BODIPY. Eur. J. Chem. 2021, 27 (49), 12545– 51,  DOI: 10.1002/chem.202101990
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    132
    Native Chemical Ligation-Based Fluorescent Probes for Cysteine and Aminopeptidase N Using meso-thioester-BODIPY
    Lee, Uisung; Kim, Tae-Il; Jeon, Sungjin; Luo, Yongyang; Cho, Siyoung; Bae, Jeehyeon; Kim, Youngmi
    Chemistry - A European Journal (2021), 27 (49), 12545-12551CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Meso-Carboxyl-BODIPY responds to small electronic changes resulting from acyl substitution reactions with a marked change in fluorescence. Herein, the minute changes that accompany the thioester to amide conversion encountered in native chem. ligation (NCL) are exploited in the construction of fluorescent "turn-on" probes. Two fluorogenic probes, 1 a and 4, derived from a meso-thioester-BODIPY scaffold, were designed for the selective detection of cysteine (1 a) and aminopeptidase N (4), resp. The arom. (1 a) and aliph. (4) thioesters of meso-carboxyl-BODIPY are nonfluorescent. However, specific analyte-induced conversion to the meso-amide deriv. caused significant spectral changes and a dramatic fluorescence enhancement. Probe 1 a exhibited a large fluorescence "turn-on" response with high selectivity toward cysteine via a tandem NCL reaction. Probe 4 was successfully applied to the monitoring and imaging of endogenous aminopeptidase N in live cancer cells.
  133. 133
    Esnal, I.; Bañuelos, J.; López Arbeloa, I.; Costela, A.; Garcia-Moreno, I.; Garzón, M.; Agarrabeitia, A. R.; José Ortiz, M. Nitro and Amino BODIPYs: Crucial Substituents to Modulate Their Photonic Behavior. RSC Adv. 2013, 3 (5), 1547– 56,  DOI: 10.1039/C2RA22916A
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    133
    Nitro and amino BODIPYS: crucial substituents to modulate their photonic behavior
    Esnal, Ixone; Banuelos, Jorge; Lopez Arbeloa, Inigo; Costela, Angel; Garcia-Moreno, Inmaculada; Garzon, Miguel; Agarrabeitia, Antonia R.; Jose Ortiz, Maria
    RSC Advances (2013), 3 (5), 1547-1556CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    The present work deals with the synthesis and photophys., quantum mech., and lasing characterization of novel BODIPYs bearing amino and nitro groups at different positions in the core. The results emphasize the relevant role on the photophys. and lasing properties, not only of the attached functionality but also of the position in which is grafted, as well as the mol. structure of the indacene core. A wide part of the visible spectrum can be covered by the insertion of an amino group at position 3 (red shift) or 8 (blue shift). Furthermore, the electron withdrawing character of the nitro substituent induces intramol. charge transfer processes, the efficiency of which depends on the position of the nitro group on the BODIPY core. All these exptl. findings can be rationalized with the help of quantum mech. calcns.
  134. 134
    Liu, E. Y.; Jung, S.; Weitz, D. A.; Yi, H.; Choi, C.-H. High-Throughput Double Emulsion-Based Microfluidic Production of Hydrogel Microspheres with Tunable Chemical Functionalities toward Biomolecular Conjugation. Lab Chip 2018, 18 (2), 323– 34,  DOI: 10.1039/C7LC01088E
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    134
    High-throughput double emulsion-based microfluidic production of hydrogel microspheres with tunable chemical functionalities toward biomolecular conjugation
    Liu, Eric Y.; Jung, Sukwon; Weitz, David A.; Yi, Hyunmin; Choi, Chang-Hyung
    Lab on a Chip (2018), 18 (2), 323-334CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
    In this work, we present a simple, efficient, and high-throughput capillary microfluidic approach for controlled fabrication of monodisperse and chem. functional hydrogel microspheres via formation of double emulsion drops with an ultra-thin oil shell as a sacrificial template. Furthermore, we show the utility of chitosan's primary amine as an efficient conjugation handle at physiol. pH due to its low pKa by direct comparison with other primary amines. We also report the utility of these microspheres in biomol. conjugation using model fluorescent proteins, R-phycoerythrin (R-PE) and green fluorescent protein (GFPuv), via tetrazine-trans-cyclooctene (Tz-TCO) ligation for CS-PEG microspheres and carbodiimide chem. for AA-PEG microspheres, resp. The results show rapid coupling of R-PE with the microspheres' functional groups with minimal non-specific adsorption. In-depth protein conjugation kinetics studies with our microspheres highlight the differences in reaction and diffusion of R-PE with CS-PEG and AA-PEG microspheres. Finally, we demonstrate orthogonal one-pot protein conjugation of R-PE and GFPuv with CS-PEG and AA-PEG microspheres via simple size-based encoding. Combined, these results represent a significant advancement in the rapid and reliable fabrication of monodisperse and chem. functional hydrogel microspheres with tunable properties.
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    Katritzky, A. R.; Marson, C. M. Pyrylium Mediated Transformations of Primary Amino Groups into Other Functional Groups. New Synthetic Methods (41). Angew. Chem., Int. Ed. Engl. 1984, 23 (6), 420– 9,  DOI: 10.1002/anie.198404201
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    Wetzl, B. K.; Yarmoluk, S. M.; Craig, D. B.; Wolfbeis, O. S. Chameleon Labels for Staining and Quantifying Proteins. Angew. Chem., Int. Ed. Engl. 2004, 43 (40), 5400– 2,  DOI: 10.1002/anie.200460508
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    Chameleon labels for staining and quantifying proteins
    Wetzl Bianca K; Yarmoluk Sergiy M; Craig Douglas B; Wolfbeis Otto S
    Angewandte Chemie (International ed. in English) (2004), 43 (40), 5400-2 ISSN:1433-7851.
    There is no expanded citation for this reference.
  137. 137
    Azab, H. A.; El-Korashy, S. A.; Anwar, Z. M.; Khairy, G. M.; Steiner, M.-S.; Duerkop, A. High-Throughput Sensing Microtiter Plate for Determination of Biogenic Amines in Seafood Using Fluorescence or Eye-Vision. Analyst 2011, 136 (21), 4492– 9,  DOI: 10.1039/c1an15049a
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    137
    High-throughput sensing microtiter plate for determination of biogenic amines in seafood using fluorescence or eye-vision
    Azab, H. A.; El-Korashy, S. A.; Anwar, Z. M.; Khairy, G. M.; Steiner, Mark-Steven; Duerkop, Axel
    Analyst (Cambridge, United Kingdom) (2011), 136 (21), 4492-4499CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    A new optical sensing microplate was developed for rapid screening for the presence of biogenic amines (BAs) in seafood samples with high sensitivity. The deposition of a sensing spot (contg. a chameleon dye (Py-1) in a polymeric cocktail) on the bottom of the wells of a std. microplate renders the plate a new sensing tool for a rapid and parallel detection of up to 96 (real) samples. This sensing microplate enables (1) a semi-quant. readout of analyte concn. by eye-vision, (2) a rapid fluorescence readout of 96 samples with std. instrumentation in less than two minutes (unlike chromatog. and electrophoretic methods), (3) a statistically robust data evaluation (with 8-12 replicates) and (4) a rapid parallel sample prepn. with std. 8 or 12-channel micropipettes. On reaction with biogenic amines, the dye shows a significant visible color change from blue over green to red color. The appearance of red color favorably coincides with the concn. of BAs that can induce symptoms of poisoning. The linear ranges of fluorescence calibration data for six biogenic amines cover the clin. toxicol. relevant range of BAs that is too low to be detected by the human nose. The LODs range from 0.16 to 0.56 μg mL-1, with correlation coeffs. (r2) between 0.985 and 0.999. Finally, the evolution of spoilage of four fish samples (monitored by detn. of their BA status) and the increase of their total amine content were found to agree well with previous data on time-dependent evolution of BAs in fish.
  138. 138
    Azab, H. A.; El-Korashy, S. A.; Anwar, Z. M.; Khairy, G. M.; Duerkop, A. Reactivity of a Luminescent “Off-On” Pyrylium Dye toward Various Classes of Amines and Its Use in a Fluorescence Sensor Microtiter Plate for Environmental Samples. J. Photochem. Photobiol. A: Chem. 2012, 243, 41– 6,  DOI: 10.1016/j.jphotochem.2012.05.029
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    138
    Reactivity of a luminescent "off-on" pyrylium dye toward various classes of amines and its use in a fluorescence sensor microtiter plate for environmental samples
    Azab, H. A.; El-Korashy, S. A.; Anwar, Z. M.; Khairy, G. M.; Duerkop, Axel
    Journal of Photochemistry and Photobiology, A: Chemistry (2012), 243 (), 41-46CODEN: JPPCEJ; ISSN:1010-6030. (Elsevier B.V.)
    The authors report on a systematic study on the reactivity of the pyrylium dye Py-1 toward various classes of amines (primary aliph., primary arom., secondary, tertiary) and its use in environmental sensing of amines. While most primary aliph. amines react almost quant. with Py-1 within 10 min, sterically hindered primary aliph. amines and primary arom. amines react more slowly. Secondary and tertiary amines just induce a decompn. of the chromophore of Py-1 but do not yield a fluorescent product. This makes Py-1 a suitable receptor and transducer in an optical sensing microplate for rapid screening of primary aliph. amines in water and soil samples. Py-1 is embedded into a polymeric cocktail, which is deposited on the bottom of wells in microtiter plates to yield a high-throughput fluorescence sensing tool. On reaction with primary aliph. amines, a significant fluorescence increase (λexc = 485 nm/λem = 620 nm) of the sensor spots is detected in a std. microplate reader after 10 min incubation at 25°. The linear calibration plots of eight primary aliph. amines are at 0.350-70.0 μg mL-1. The limits of detection (LODs) are 0.119-0.589 μg mL-1 and the limits of quantitation (LOQs) are 0.399-1.965 μg mL-1. Further the sensing plate is suitable for sensing mixts. of primary amines and the total content of amines (TAC) found is a good measure for the sum of the contents of the individual amines present. Finally, the sensing plate was successfully applied to the quant. anal. of primary aliph. amines in 3 samples of water and soil, resp.
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    Bayer, M.; König, S. Pyrylium-Based Dye and Charge Tagging in Proteomics. Electrophoresis 2016, 37 (22), 2953– 8,  DOI: 10.1002/elps.201600318
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    139
    Pyrylium-based dye- and charge-tagging in proteomics
    Bayer, Malte; Koenig, Simone
    Electrophoresis (2016), 37 (22), 2953-2958CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The pyrylium group is a selective reagent for ε-amino groups in proteins. In particular for fluorescence labeling a no. of advantages over traditional NHS-ester chem. were recognized such as the rapid pre-staining procedure. Here, we have investigated the labeling reaction for the fluorogenic pyrylium dye Py-1 using liq. chromatog. coupled to mass spectrometry with the aim of detg. its specificity and possible side products. Peptides contg. no, one, and two lysine residue and a choice of no or one cysteine residue were labeled with Py-1 at yields >30%. Gas phase fragmentation proved both labeling of lysine residues as well of that of the N-terminus also in peptides which contained a lysine residue. Evidence for cysteine labeling was not found, but several other products were detected such as the results of rearrangements with adjacent acidic amino acids. Apart from the use as a fluorogenic label Py-1 recommends itself for N-terminal charge-tagging as alternative to the commonly used quaternary ammonium salts. Predominantly a- and b-type ion series were obsd. for N-terminally labeled peptides. Further applications include chromophore tagging since the labeled product is not only fluorescent but also colored red.
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    Turner, E. H.; Dickerson, J. A.; Ramsay, L. M.; Swearingen, K. E.; Wojcik, R.; Dovichi, N. J. Reaction of Fluorogenic Reagents with Proteins: III. Spectroscopic and Electrophoretic Behavior of Proteins Labeled with Chromeo P503. J. Chromatogr. A 2008, 1194 (2), 253– 6,  DOI: 10.1016/j.chroma.2008.04.046
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    Reaction of fluorogenic reagents with proteins. III. Spectroscopic and electrophoretic behavior of proteins labeled with Chromeo P503
    Turner, Emily H.; Dickerson, Jane A.; Ramsay, Lauren M.; Swearingen, Kristian E.; Wojcik, Roza; Dovichi, Norman J.
    Journal of Chromatography A (2008), 1194 (2), 253-256CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
    The spectroscopic and electrophoretic properties of proteins labeled with Chromeo P503 were investigated. Its photobleaching characteristics were detd. by continually infusing Chromeo P503-labeled α-lactalbumin into a sheath-flow cuvette and monitored fluorescence as a function of laser power. The labeled protein is relatively photo-labile with an optimum excitation power of about 2 mW. The unreacted reagent is weakly fluorescent but present at much higher concn. than the labeled protein. The unreacted reagent undergoes photobleaching at a laser power more than an order of magnitude higher than the labeled protein. One-dimensional capillary electrophoresis anal. of Chromeo P503-labeled α-lactalbumin produced concn. detection limits (3σ) of 12 pM and mass detection limits of 0.7 zmol, but with modest theor. plate counts of 17,000. The reagent was employed for the two-dimensional capillary electrophoresis anal. of a homogenate prepd. from a Barrett's esophagus cell line; the sepn. quality is similar to that produced by 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), a more commonly used reagent.
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    Wolfbeis, O. S. Fluorescent Chameleon Labels for Bioconjugation and Imaging of Proteins, Nucleic Acids, Biogenic Amines and Surface Amino Groups. A Review. Methods Appl. Fluoresc. 2021, 9 (4), 042001,  DOI: 10.1088/2050-6120/ac1a0a
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    Fluorescent chameleon labels for bioconjugation and imaging of proteins, nucleic acids, biogenic amines and surface amino groups. A review
    Wolfbeis, Otto S.
    Methods and Applications in Fluorescence (2021), 9 (4), 042001CODEN: MAFEB2; ISSN:2050-6120. (IOP Publishing Ltd.)
    A review. Chameleon labels (ChLs) possess the unique property of changing (visible) color and fluorescence on binding to amino groups of biomols. MostChLs react with primary aliph. amino groups such as those in lysine or with amino groups artificially introduced into polynucleic acids or saccharides, but someothers also react with secondary amino groups. Under controlled circumstances, the reactions are fairly specific. The review is subdivided into the following sections: (1) An introduction and classification of fluorescent labels; (2) pyrylium labels that undergo shortwave color changes upon labeling, typically from blue to red; (3) polymethine type of labels (that also undergo shortwave color changes, typically from green to blue; (4) various other (less common) chromogenic and fluorogenic systems; (5) hemicyanine labels that undergo longwave color changes, typically from yellow to purple; (6) the application of ChLs to labeling of proteins and oligonucleotides; (7) applications to fluorometric assays and sensing; (8) applications to fluorescence imaging of biomols.; (9) applications in studies on affinity interactions (receptor-ligand binding); (10) applications in surface and interface chem.; and (11) applications in chromatog., electrophoresis and isotachophoresis of biomols.
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    Höfelschweiger, B. K., The Pyrylium Dyes: a New Class of Biolabels. Synthesis, Spectroscopy, and Application as Labels and in General Protein Assay , 2005.
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    Walter, W.; Bode, K. D. Syntheses of Thiocarbamates. Angew. Chem., Int. Ed. Engl. 1967, 6 (4), 281– 93,  DOI: 10.1002/anie.196702811
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    Syntheses of thiocarbamates
    Walter, Wolfgang; Bode, Klaus D.
    Angewandte Chemie, International Edition in English (1967), 6 (4), 281-93CODEN: ACIEAY; ISSN:0570-0833.
    cf. following abstr. A review, with 150 references, of the prepn. of thiocarbamates by the addn. of alcs. and thiols to isothiocyanates, the treatment of amines with compds. of the general formula ClC(S)XR, xanthates, and trithiocarbonates, the treatment of alcs., phenols, and thiols with compds. of the general formula R2NCSCl and [Cl(RS)C:N(R1) R2]+Cl-, and N,N'-thiocarbonyldiimidazole, treatment of thiocyanates and cyanates with H2S, alkylation of metal dithiocarbamates, and addn. to thiocyanic acid.
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    Maddani, M. R.; Prabhu, K. R. A Concise Synthesis of Substituted Thiourea Derivatives in Aqueous Medium. J. Org. Chem. 2010, 75 (7), 2327– 32,  DOI: 10.1021/jo1001593
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    A concise synthesis of substituted thiourea derivatives in aqueous medium
    Maddani, Mahagundappa R.; Prabhu, Kandikere R.
    Journal of Organic Chemistry (2010), 75 (7), 2327-2332CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    An efficient method for the synthesis of sym. and unsym. substituted thiourea derivs. by means of simple condensation between available building blocks such as amines and carbon disulfide in aq. medium is presented. This protocol works smoothly with aliph. primary amines to afford various di- and trisubstituted thiourea derivs. The present method is also useful in synthesizing various substituted 2-mercapto imidazole heterocycles. This method proceeds through a xanthate (amino dithiol deriv.) intermediate, unlike isothiocyanate as in an earlier known method.
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    Reetz, M. T.; Kühling, K. M.; Deege, A.; Hinrichs, H.; Belder, D. Super-High-Throughput Screening of Enantioselective Catalysts by Using Capillary Array Electrophoresis. Angew. Chem., Int. Ed. 2000, 39 (21), 3891– 3,  DOI: 10.1002/1521-3773(20001103)39:21<3891::AID-ANIE3891>3.0.CO;2-1
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    Super-high-throughput screening of enantioselective catalysts by using capillary array electrophoresis
    Reetz, Manfred T.; Kuhling, Klaus M.; Deege, Alfred; Hinrichs, Heike; Belder, Detlev
    Angewandte Chemie, International Edition (2000), 39 (21), 3891-3893CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
    The authors' approach to high-throughput screening of enantioselective catalysts was based on the adaptation of capillary array electrophoresis (MegaBACE system) by using chirally modified electrolytes. As a specific goal the authors chose the ee detn. of chiral amines (I, R = n-pentyl, n-hexyl, c-hexyl, Ph, p-tolyl, and 1-naphthyl) using fluorescein isothiocyanate as the derivatization reagent. The authors' study shows that the capillary array electrophoresis for detn. of enantiomeric purity can be used in a truly high-throughput manner (> 7000 ee detns. per day). This result as well as the optimization of the methods described herein are of great significance for the further development of combinatorial asym. transition metal catalysis and directed evolution of enantioselective enzymes.
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    Dabek-Zlotorzynska, E.; Maruszak, W. Determination of Dimethylamine and Other Low-Molecular-Mass Amines Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection. J. Chromatogr. B Biomed. Appl. 1998, 714 (1), 77– 85,  DOI: 10.1016/S0378-4347(98)00070-X
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    Determination of dimethylamine and other low-molecular-mass amines using capillary electrophoresis with laser-induced fluorescence detection
    Dabek-Zlotorzynska, Ewa; Maruszak, Wioleta
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    The potential of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for the sepn. and detn. of dimethylamine (DMA) and other low-mol.-mass amines involving precolumn derivatization with fluorescein isothiocyanate isomer I (FITC) was studied. Different variables that affect derivatization (pH, FITC concn., reaction time and temp.) and sepn. (buffer concn., addn. of various org. modifiers, applied voltage and length of capillary) were studied. The linearity, reproducibility and reliability of the method were evaluated. The estd. instrumental detection limit for a 2-s pressure injection of the FITC-DMA deriv. was 50 pg/mL (10-9 M), using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, resp. However, for practical reasons, a min. of 5 ng/mL DMA should be subjected to the derivatization. The applicability of the described method to the ext. of atm. aerosol samples was demonstrated.
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    Morales, A. R.; Schafer-Hales, K. J.; Marcus, A. I.; Belfield, K. D. Amine-Reactive Fluorene Probes: Synthesis, Optical Characterization, Bioconjugation, and Two-Photon Fluorescence Imaging. Bioconj. Chem. 2008, 19 (12), 2559– 67,  DOI: 10.1021/bc800415t
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    Amine-Reactive Fluorene Probes: Synthesis, Optical Characterization, Bioconjugation, and Two-Photon Fluorescence Imaging
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    Bioconjugate Chemistry (2008), 19 (12), 2559-2567CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, the authors prepd. two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (I) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (II), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomols., assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivs. were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chem. and photophys. properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chem. stability and photostability. The NCS derivs. had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe I, RGDfK-dye conjugate, and Reelin-dye conjugate, was demonstrated.
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    Planas, O.; Gallavardin, T.; Nonell, S. A Novel Fluoro-Chromogenic Click Reaction for the Labelling of Proteins and Nanoparticles with near-IR Theranostic Agents. Chem. Commun. 2015, 51 (26), 5586– 9,  DOI: 10.1039/C4CC09070E
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    A novel fluoro-chromogenic click reaction for the labelling of proteins and nanoparticles with near-IR theranostic agents
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    Reaction of porphycene isothiocyanates with primary and secondary amines leads to the formation of thiazolo[4,5-c]porphycenes, with a substantial shift in the absorption and fluorescence spectra. The conjugates show fluorescence in the near-IR and are capable of photosensitizing the prodn. of the cytotoxic species singlet oxygen.
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    Wang, R.; Yang, W.-j.; Yue, L.; Pan, W.; Zeng, H.-y. DDQ-Promoted C-S Bond Formation: Synthesis of 2-Aminobenzothiazole Derivatives under Transition-Metal-, Ligand-, and Base-Free Conditions. Synlett 2012, 23 (11), 1643– 8,  DOI: 10.1055/s-0031-1291159
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    Synlett (2012), 23 (11), 1643-1648CODEN: SYNLES; ISSN:0936-5214. (Georg Thieme Verlag)
    A transition-metal-free method for the intramol. S-arylation of o-halobenzothioureas via DDQ-mediated leading to the 2-aminobenzothiazole derivs., e.g., I (R = H, Me, MeO, F; R2 = H, Me, MeO, Cl, Br), is reported. The reactions are performed at room temp. under ligand- and base-free conditions with good to excellent yields.
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    Guo, Y.-J.; Tang, R.-Y.; Zhong, P.; Li, J.-H. Copper-Catalyzed Tandem Reactions of 2-Halobenzenamines with Isothiocyanates under Ligand- and Base-Free Conditions. Tetrahedron Lett. 2010, 51 (4), 649– 52,  DOI: 10.1016/j.tetlet.2009.11.086
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    A ligand-free copper-catalyzed reaction of 2-halobenzenamines with isothiocyanates has been developed for the synthesis of 2-aminobenzothiazoles. In the presence of CuBr and TBAB (tetra-Bu ammonium bromide, additive), a variety of 2-halobenzenamines underwent the reaction with isothiocyanates at 40 °C, affording 2-aminobenzothiazoles, e.g., I (R1 = H, MeO, Cl, NO2; R2 = H, Me, Cl, F), in moderate to excellent yields. It is noteworthy that the reaction is conducted under mild, relatively low catalyst loading, and ligand- and base-free conditions.
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    Sutton, J. M.; Clarke, O. J.; Fernandez, N.; Boyle, R. W. Porphyrin, Chlorin, and Bacteriochlorin Isothiocyanates: Useful Reagents for the Synthesis of Photoactive Bioconjugates. Bioconj. Chem. 2002, 13 (2), 249– 63,  DOI: 10.1021/bc015547x
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    Porphyrin, Chlorin, and Bacteriochlorin Isothiocyanates: Useful Reagents for the Synthesis of Photoactive Bioconjugates
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    Bioconjugate Chemistry (2002), 13 (2), 249-263CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    A novel method for conjugating porphyrins and related mols. to proteins has been developed. The method, which involves synthesizing porphyrins, chlorins, and bacteriochlorins bearing a single amine-reactive isothiocyanate group represents a facile system for protein labeling with these photoactive species. Problems assocd. with the noncovalent binding of porphyrins to proteins are highlighted, and a method for purifying conjugates to yield exclusively covalently bound porphyrin protein species is demonstrated. Biol. activity of porphyrin-bovine serum albumin conjugates formed and purified by these methods is demonstrated using laser scanning confocal microscopy.
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    Feuster, E. K.; Glass, T. E. Detection of Amines and Unprotected Amino Acids in Aqueous Conditions by Formation of Highly Fluorescent Iminium Ions. J. Am. Chem. Soc. 2003, 125 (52), 16174– 5,  DOI: 10.1021/ja036434m
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    Detection of amines and unprotected amino acids in aqueous conditions by formation of highly fluorescent iminium ions
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    Properly substituted coumarin aldehydes can be used to detect amines and amino acids under neutral, high ionic strength conditions by the formation of highly fluorescent iminium ions. The fluorescence of one sensor increases by 26-fold upon the addn. of glycine. This strong florescent response is attributed to hydrogen bonding of the chromophore carbonyl by the acidic iminium proton.
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    Patze, C.; Broedner, K.; Rominger, F.; Trapp, O.; Bunz, U. H. F. Aldehyde Cruciforms: Dosimeters for Primary and Secondary Amines. Eur. J. Chem. 2011, 17 (49), 13720– 5,  DOI: 10.1002/chem.201101871
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    Aldehyde Cruciforms: Dosimeters for Primary and Secondary Amines
    Patze, Christian; Broedner, Kerstin; Rominger, Frank; Trapp, Oliver; Bunz, Uwe H. F.
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    Aldehyde-substituted donor-acceptor cruciforms [1,4-bis(arylethynyl)-2,5-distyrylbenzenes] are useful dosimeters for primary amines, primary diamines, and secondary amines. The 1,n-diamines are particularly reactive towards this dosimeter and can be detected in <100 ppm concn. Using a single aldehyde-functionalized cruciform in seven different solvents allowed to discern fourteen different amines by digital photog. and statistical evaluation of the response patterns extd. as red, green, blue (RGB) values.
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    Kumpf, J.; Bunz, U. H. F. Aldehyde-Appended Distyrylbenzenes: Amine Recognition in Water. Eur. J. Chem. 2012, 18 (29), 8921– 4,  DOI: 10.1002/chem.201200930
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    Aldehyde-Appended Distyrylbenzenes: Amine Recognition in Water
    Kumpf, Jan; Bunz, Uwe H. F.
    Chemistry - A European Journal (2012), 18 (29), 8921-8924, S8921/1-S8921/5CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    We have synthesized two aldehyde-substituted distyrylbenzenes, 3 (I) and 8 (II), and investigated their reaction with amines in water. Both 3 and 8 formed imines or cyclic aminals depending on the structure of the employed amine. Turn on and blue shift of the fluorescence resulted. Both 3 and 8 gave fairly unique color responses towards biogenic amines and amino acids that can undergo aminal or thioacetal formation.
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    Mohr, G. J.; Demuth, C.; Spichiger-Keller, U. E. Application of Chromogenic and Fluorogenic Reactands in the Optical Sensing of Dissolved Aliphatic Amines. Anal. Chem. 1998, 70 (18), 3868– 73,  DOI: 10.1021/ac980279q
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    Application of chromogenic and fluorogenic reactands in the optical sensing of dissolved aliphatic amines
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    Analytical Chemistry (1998), 70 (18), 3868-3873CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A new class of dyes was studied that reversibly interacts with aliph. amines, resulting in changes in absorbance or fluorescence. These dyes, which combine the properties of both a chem. reagent and a ligand, are termed reactands. When embedded in plasticized PVC membranes, the reactand 4-(N,N-dioctylamino)-4'-trifluoroacetylazobenzene (ETHT 4001) shows a significant signal change on exposure to aq. amine solns., with a decrease in absorbance around 490 nm and an increase in absorbance around 420 nm wavelength. This change in absorbance is caused by a conversion of the trifluoroacetyl group of the reactand into a hemiaminal or a zwitterion. Upon interaction with 1-butylamine, the sensor layer exhibits a dynamic range from 1 to 100 mM 1-butylamine, with max. relative signal changes of 90%. The limit of detection with this approach is 0.3 mM. The forward response time (t95) for a decade change in activity is 10 min, and the reverse response time is 5 min. The selectivity of ETHT 4001 toward amines correlates with the lipophilicity of the amines in plasticized PVC. Sterical factors addnl. affect selectivity in that the reactions of secondary, tertiary, and bulky primary amines with the trifluoroacetyl group are hindered. A similar response is obsd. for the fluorescent reactand 4-(N,N-dioctylamino)-4'-trifluoroacetylstilbene (ETHT 4004). When this reactand is exposed to 1-butylamine, a decrease in fluorescence around 580 nm is obsd., which can be ≤90% of the total fluorescence intensity. To prevent interferences from ions and ambient light, the sensor layer is coated with a protective layer of microporous white PTFE.
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    Mohr, G. J. Tailoring the Sensitivity and Spectral Properties of a Chromoreactand for the Detection of Amines and Alcohols. Anal. Chim. Acta 2004, 508 (2), 233– 7,  DOI: 10.1016/j.aca.2003.12.005
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    Tailoring the sensitivity and spectral properties of a chromoreactand for the detection of amines and alcohols
    Mohr, Gerhard J.
    Analytica Chimica Acta (2004), 508 (2), 233-237CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
    The chromoreactand N,N-dioctylamino-4'-trifluoroacetyl-2'-nitroazobenzene (CR-546) is sensitive to amines and alcs. when dissolved in plasticized PVC. The chromoreactand provides an improvement over related reactands in that a nitro group meta to the trifluoroacetyl group enhances the chem. reactivity and, consequently, the sensitivity by a factor of 20 compared to the deriv. without nitro group. Also, the absorbance is shifted by around 40 nm to longer wavelengths, making the sensor membranes compatible with the green LED as a light source for a miniaturized sensor device. The sensor membrane responds to aq. 1-propylamine in the 0.5-50 mM concn. range and exhibits a detection limit of 0.1 mM. Response times are at 5-15 min. A fluorescent ethanol-sensitive membrane was obtained by dissolving the inert fluorophore N,N'-bis(1-hexylheptyl)perylene-3,4:9,10-bis(dicarboximide) together with CR-546 and the catalyst tridodecylmethylammonium chloride in the polymer matrix. The trifluoroacetyl form of CR-546 with its absorbance at ∼560 nm overlaps the emission of the fluorophore. Increasing ethanol concns. cause decreasing absorbance at 560 nm and consequently, increasing luminescence of the fluorophore. The resulting layer was evaluated for its detection of ethanol via changes in luminescence intensity. The calibration is linear at 1-15% (vol./vol.) ethanol with a limit of detection of 0.1% (vol./vol.). Response times are at 20-40 min for both forward and reverse reaction.
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    Mohr, G. J. Chromo- and Fluororeactands: Indicators for Detection of Neutral Analytes by Using Reversible Covalent-Bond Chemistry. Eur. J. Chem. 2004, 10 (5), 1082– 90,  DOI: 10.1002/chem.200305524
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    Chromo- and fluororeactands: indicators for detection of neutral analytes by using reversible covalent-bond chemistry
    Mohr, Gerhard J.
    Chemistry - A European Journal (2004), 10 (5), 1082-1090CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Chromo- and fluororeactands are indicator dyes that allow the optical detection of elec. neutral analytes. Unlike complexing agents such as calixarenes, cyclodextrines or cyclophanes, reactands form a reversible covalent bond with the analyte mol. This chem. reaction causes strong changes in absorbance or fluorescence. In this article reactands for analytes such as amines, alcs., aldehydes, saccharides, carbon dioxide and sulfur dioxide are presented. Methods to enhance the sensitivity of the reactands as well as the operational and shelf lives of the corresponding optical sensors are discussed.
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    Mohr, G. J.; Grummt, U.-W. Comparison of Trifluoroacetyl Monostyryl and Distyryl Dyes: Effects of Chromophore Elongation on the Spectral Properties and Chemical Reactivity. J. Fluoresc. 2006, 16 (2), 185– 90,  DOI: 10.1007/s10895-005-0047-7
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    Comparison of Trifluoroacetyl Monostyryl and Distyryl Dyes: effects of Chromophore Elongation on the Spectral Properties and Chemical Reactivity
    Mohr, Gerhard J.; Grummt, Ulrich-W.
    Journal of Fluorescence (2006), 16 (2), 185-190CODEN: JOFLEN; ISSN:1053-0509. (Springer)
    The trifluoroacetyl distyryl deriv. 1-[4-(2-{4-[2-(4-dibutylaminophenyl)-vinyl]-phenyl}-vinyl)-phenyl]-2,2,2-trifluoroethanone was compared with the related monostyryl deriv. 1-{4-[2-(4-dibutylaminophenyl)-vinyl]-phenyl}-2,2,2-trifluoroethanone with respect to spectral properties and sensitivity to amines. Both trifluoroacetyl derivs. had their absorbance max. at around 445 nm. The fluorescence of the distyryl dye, however, was obsd. at significantly longer wavelengths than that of the monostyryl dye, indicating the effect of structural extension of the chromophore system. Furthermore, the distyryl dye exhibited significantly smaller quantum yields in polar solvents than the monostyryl dye. Both dyes were capable of chem. reacting with amines in that their trifluoroacetyl function was converted into a hemiaminal. Consequently, absorbance and fluorescence of both dyes were shifted to shorter wavelengths. The positions of the fluorescence maxima of the spectra when converting from trifluoroacetyl to hemiaminal form were shifted by an almost identical amt. for both mono- and distyryl deriv. The hemiaminal form of the distyryl deriv., however, exhibited much larger quantum yields in both polar and nonpolar solvents than the hemiaminal form of the monostyryl dye. The structural extension of the chromophore affected the sensitivity to amines by enhancing the chem. reactivity of the distyryl dye over the monostyryl deriv.
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    Mertz, E.; Beil, J. B.; Zimmerman, S. C. Kinetics and Thermodynamics of Amine and Diamine Signaling by a Trifluoroacetyl Azobenzene Reporter Group. Org. Lett. 2003, 5 (17), 3127– 30,  DOI: 10.1021/ol0351605
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    Organic Letters (2003), 5 (17), 3127-3130CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
    (Trifluoroacetyl)azobenzene dyes were previously employed as amine reporter groups (chemosensors) in a dendrimer-based monomol. imprinting system. Kinetic and binding studies with a range of amines and diamines show that the highly selective signaling obsd. for alkane diamines by these imprinted dendrimers arises from a kinetic effect due to intramol. general base-catalyzed carbinolamine formation with the dye itself. The relationship between diamine structure and carbinolamine stability and rate of formation is described.
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    Körsten, S.; Mohr, G. J. Star-Shaped Tripodal Chemosensors for the Detection of Aliphatic Amines. Eur. J. Chem. 2011, 17 (3), 969– 75,  DOI: 10.1002/chem.201000787
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    Xu, Y.; Yu, S.; Wang, Y.; Hu, L.; Zhao, F.; Chen, X.; Li, Y.; Yu, X.; Pu, L. Ratiometric Fluorescence Sensors for 1,2-Diamines Based on Trifluoromethyl Ketones. Eur. J. Org. Chem. 2016, 2016 (35), 5868– 75,  DOI: 10.1002/ejoc.201601157
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    European Journal of Organic Chemistry (2016), 2016 (35), 5868-5875CODEN: EJOCFK; ISSN:1099-0690. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Two BINOL-based trifluoromethyl aryl ketones 3 (I) and 4 (II) show greatly enhanced fluorescence at 375 nm accompanied by a decrease at 500 nm when treated with aliph. 1,2-diamines, esp. ethylenediamine, in org. solvents. Under the same conditions, other monoamines and diamines caused much smaller or no fluorescence response. Thus, both 3 and 4 can be used as highly selective ratiometric fluorescence sensors for ethylenediamine. Compd. 3, the BINOL hydroxyl groups of which are protected, exhibits greater fluorescence enhancement than the corresponding unprotected compd. 4. UV and NMR spectroscopic studies have been conducted to probe the mechanism of the sensor-substrate reaction.
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    Ali, M. F. B.; Kishikawa, N.; Ohyama, K.; Mohamed, H. A.-M.; Abdel-Wadood, H. M.; Mahmoud, A. M.; Imazato, T.; Ueki, Y.; Wada, M.; Kuroda, N. Chromatographic Determination of Low-Molecular Mass Unsaturated Aliphatic Aldehydes with Peroxyoxalate Chemiluminescence Detection after Fluorescence Labeling with 4-(N,N-Dimethylaminosulfonyl)-7-Hydrazino-2,1,3-Benzoxadiazole. J. Chromatogr. B 2014, 953–954, 147– 52,  DOI: 10.1016/j.jchromb.2014.02.009
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    Chromatographic determination of low-molecular mass unsaturated aliphatic aldehydes with peroxyoxalate chemiluminescence detection after fluorescence labeling with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole
    Ali, Marwa Fathy Bakr; Kishikawa, Naoya; Ohyama, Kaname; Mohamed, Horria Abdel-Mageed; Abdel-Wadood, Hanaa Mohamed; Mahmoud, Ashraf Mohamed; Imazato, Takahiro; Ueki, Yukitaka; Wada, Mitsuhiro; Kuroda, Naotaka
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    A highly sensitive, selective and reproducible chromatog. method is described for detn. of low-mol. mass unsatd. aliph. aldehydes in human serum. The method combines fluorescent labeling using 4-(N,N-Dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole with peroxyoxalate chemiluminescence. The derivs. were sepd. on a reversed-phase column C8 isocratically using a mixt. of acetonitrile and 90 mM imidazole-HNO3 buffer (pH 6.4, 1:1, % vol./vol.). The calibration ranges were: 20-420 nM for methylglyoxal, 16-320 nM for acrolein, 15-360 nM for crotonaldehyde and 20-320 nM for trans-2-hexenal. The detection limits were ranged from 4.4 to 6.5 nM (88-130 fmol/injection), the recovery results were within the range of 87.4-103.8% and the intra and inter-day precision results were lower than 5.5%. The proposed validated method has been successfully applied to healthy, diabetic and rheumatic arthritis patients' sera with simple pretreatment method. In conclusion, this new method is suitable for routine anal. of large nos. of clin. samples for assessment of the oxidative stress state in patients.
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    Xu, Y.; Yu, S.; Chen, Q.; Chen, X.; Li, Y.; Yu, X.; Pu, L. Fluorescent Recognition of 1,2-Diamines by a 1,1′-Binaphthyl-Based Trifluoromethyl Ketone. Eur. J. Chem. 2016, 22 (34), 12061– 7,  DOI: 10.1002/chem.201601540
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    Chemistry - A European Journal (2016), 22 (34), 12061-12067CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The fluorescent responses of a 1,1'-binaphthol (BINOL)-based trifluoromethyl aryl ketone (I) toward a variety of amines were studied. The aliph. 1,2-diamines, esp. ethylenediamine, can greatly enhance the fluorescence of this compd., but under the same conditions, other mono- and diamines cause much smaller fluorescent responses. This compd. can be used as a fluorescent sensor for the detection of ethylenediamine at concns. over micromolar levels. UV absorption and NMR spectroscopic methods were used to study the interactions of the sensor with ethylenediamine. The trifluoromethyl ketone of the sensor reacts with ethylenediamine much more favorably than with other amines. The hydroxyl groups of the sensor and those of the hemiaminal adducts formed in the presence of the amines are important for the highly selective fluorescent response.
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    Collados, J. F.; Solà, R.; Harutyunyan, S. R.; Maciá, B. Catalytic Synthesis of Enantiopure Chiral Alcohols Via Addition of Grignard Reagents to Carbonyl Compounds. ACS Catal. 2016, 6 (3), 1952– 70,  DOI: 10.1021/acscatal.5b02832
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    ACS Catalysis (2016), 6 (3), 1952-1970CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    Remarkable progress in the enantioselective addn. of Grignard reagents to carbonyl compds. has been made over the past decade. This enantioselective transformation now allows the use of these challenging reactive nucleophiles for the formation of chiral alcs. using catalytic amts. of chiral ligands. This review summarizes the developments in this area.
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    Asym. alcs. were resolved as 1-α-O-alkyl-2,3-unsatd. hexosides. After sepn. of diastereoisomers, the auxiliary and the enantiomeric alc. were recovered by transglycosidation. Potential applications include resoln. of labile secondary and tertiary alcs., difficult by existing techniques, and enhancement of ees of chiral alcs. produced enzymically or by synthetic catalytic methods.
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    Rong, J.; Pellegrini, T.; Harutyunyan, S. R. Synthesis of Chiral Tertiary Alcohols by CuI-Catalyzed Enantioselective Addition of Organomagnesium Reagents to Ketones. Eur. J. Chem. 2016, 22 (11), 3558– 70,  DOI: 10.1002/chem.201503412
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    Chemistry - A European Journal (2016), 22 (11), 3558-3570CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
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    Chen, B.-S.; Ribeiro de Souza, F. Z. Enzymatic Synthesis of Enantiopure Alcohols: Current State and Perspectives. RSC Adv. 2019, 9 (4), 2102– 15,  DOI: 10.1039/C8RA09004A
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    Chen, Bi-Shuang; Ribeiro de Souza, Fayene Zeferino
    RSC Advances (2019), 9 (4), 2102-2115CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    A review. Enantiomerically pure alcs., as key intermediates, play an essential role in the pharmaceutical, agrochem. and chem. industries. Among the methods used for their prodn., biotechnol. approaches are generally considered a green and effective alternative due to their mild reaction conditions and remarkable enantioselectivity. An increasing no. of enzymic strategies for the synthesis of these compds. has been developed over the years, among which seven primary methodologies can be distinguished as follows: (1) enantioselective water addn. to alkenes, (2) enantioselective aldol addn., (3) enantioselective coupling of ketones with hydrogen cyanide, (4) asym. redn. of carbonyl compds., (5) (dynamic) kinetic resoln. of racemates, (6) enantioselective hydrolysis of epoxides, and (7) stereoselective hydroxylation of unactivated C-H bonds. Some recent reviews have examd. these approaches sep.; however, to date, no review has included all the above mentioned strategies. The aim of this mini-review is to provide an overview of all seven enzymic strategies and draw conclusions on the effect of each approach.
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    Kourist, R.; Dominguez de Maria, P.; Bornscheuer, U. T. Enzymatic Synthesis of Optically Active Tertiary Alcohols: Expanding the Biocatalysis Toolbox. ChemBioChem. 2008, 9 (4), 491– 8,  DOI: 10.1002/cbic.200700688
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    Enzymatic synthesis of optically active tertiary alcohols: expanding the biocatalysis toolbox
    Kourist, Robert; Dominguez de Maria, Pablo; Bornscheuer, Uwe T.
    ChemBioChem (2008), 9 (4), 491-498CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Enantiopure tertiary alcs. are very valuable building blocks for the synthesis of many different natural products and pharmaceuticals. As a consequence, several chem. and enzymic strategies to afford such chiral structures have been described. Promising enzymic approaches with agents such as epoxide hydrolases, dehalogenases and hydroxynitrile lyases have been reported, as well as dihydroxylation by microorganisms. Apart from those valuable options, the hydrolase-based kinetic resoln. of tertiary alcs. has been known for the last three decades, as several wild-type enzymes have been reported to be able to accept these sterically hindered mols. More recently, the existence of an amino acid motif within an enzyme's active site has been identified as highly relevant for the acceptance of such bulky structures. This discovery clearly facilitates the identification of novel biocatalysts for this application. Although several tertiary alcs. were successfully resolved with wild-type biocatalysts, enantioselectivities have often been too low for synthetic purposes. These limitations have recently been overcome by accessing enzymes from the metagenome through directed evolution or by rational protein design. This minireview describes the state of the art in this area, highlighting aspects of basic academic research into the practical application of biocatalysts for the synthesis of optically active tertiary alcs.
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    Jiang, H.; Su, X.; Zhang, Y.; Zhou, J.; Fang, D.; Wang, X. Unexpected Thiols Triggering Photoluminescent Enhancement of Cytidine Stabilized Au Nanoclusters for Sensitive Assays of Glutathione Reductase and Its Inhibitors Screening. Anal. Chem. 2016, 88 (9), 4766– 71,  DOI: 10.1021/acs.analchem.6b00112
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    Unexpected Thiols Triggering Photoluminescent Enhancement of Cytidine Stabilized Au Nanoclusters for Sensitive Assays of Glutathione Reductase and Its Inhibitors Screening
    Jiang, Hui; Su, Xiaoqing; Zhang, Yuanyuan; Zhou, Junyu; Fang, Danjun; Wang, Xuemei
    Analytical Chemistry (Washington, DC, United States) (2016), 88 (9), 4766-4771CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The photoluminescence (PL) of nonthiolate ligand capped Au nanoclusters (NCs) is usually quenched by thiols due to the tight adsorption of thiols to the Au surface and formation of larger non-PL species. However, we here report an unexpected PL enhancement of cytidine stabilized Au (AuCyt) NCs triggered by thiols, such as reduced glutathione (GSH) at sub-μM level, while such phenomena have not been obsd. for Au NCs capped with similar adenosine/cytidine nucleotides. The mass spectroscopic results indicate that this enhancement may be caused by the formation of smaller, but highly fluorescent, Au species etched by thiols. This enables the sensitive detection of GSH from 20 nM to 3 μM, with an ultralow detection limit of 2.0 nM. Moreover, the glutathione reductase (GR) activity can be detd. by the initial rate of GSH prodn., i.e., the max. PL increasing rate, with a linear range of 0.34-17.0 U/L (1 U means redn. of 1.0 μmol of oxidized glutathione per min at pH 7.6 at 25 °C) and a limit of detection of 0.34 U/L. This method allows the accurate assays of GR in clin. serum samples as well as the rapid screening of GR inhibitors, indicating its promising biomedical applications.
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    Andexer, J.; von Langermann, J.; Mell, A.; Bocola, M.; Kragl, U.; Eggert, T.; Pohl, M. An R-Selective Hydroxynitrile Lyase from Arabidopsis Thaliana with an α/β-Hydrolase Fold. Angew. Chem., Int. Ed. 2007, 46 (45), 8679– 81,  DOI: 10.1002/anie.200701455
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    An R-selective hydroxynitrile lyase from arabidopsis thaliana with an α/β-hydrolase fold
    Andexer, Jennifer; von Langermann, Jan; Mell, Annett; Bocola, Marco; Kragl, Udo; Eggert, Thorsten; Pohl, Martina
    Angewandte Chemie, International Edition (2007), 46 (45), 8679-8681CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The noncyanogenic plant Arabidopsis thaliana contains a new hydroxynitrile lyase, which was cloned and characterized. This enzyme is readily available form a recombinant source, has a broad range of substrates, and enantioselectively transforms aliph. and arom. aldehydes as well as ketones into the corresponding R-cyanohydrins.
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    Zheng, Y.-G.; Yin, H.-H.; Yu, D.-F.; Chen, X.; Tang, X.-L.; Zhang, X.-J.; Xue, Y.-P.; Wang, Y.-J.; Liu, Z.-Q. Recent Advances in Biotechnological Applications of Alcohol Dehydrogenases. Appl. Microbiol. Biotechnol. 2017, 101 (3), 987– 1001,  DOI: 10.1007/s00253-016-8083-6
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    Recent advances in biotechnological applications of alcohol dehydrogenases
    Zheng, Yu-Guo; Yin, Huan-Huan; Yu, Dao-Fu; Chen, Xiang; Tang, Xiao-Ling; Zhang, Xiao-Jian; Xue, Ya-Ping; Wang, Ya-Jun; Liu, Zhi-Qiang
    Applied Microbiology and Biotechnology (2017), 101 (3), 987-1001CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    Alc. dehydrogenases (ADHs), which belong to the oxidoreductase superfamily, catalyze the interconversion between alcs. and aldehydes or ketones with high stereoselectivity under mild conditions. ADHs are widely employed as biocatalysts for the dynamic kinetic resoln. of racemic substrates and for the prepn. of enantiomerically pure chems. This review provides an overview of biotechnol. applications for ADHs in the prodn. of chiral pharmaceuticals and fine chems.
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    Huisman, G. W.; Liang, J.; Krebber, A. Practical Chiral Alcohol Manufacture Using Ketoreductases. Curr. Opin. Chem. Biol. 2010, 14 (2), 122– 9,  DOI: 10.1016/j.cbpa.2009.12.003
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    Practical chiral alcohol manufacture using ketoreductases
    Huisman, Gjalt W.; Liang, Jack; Krebber, Anke
    Current Opinion in Chemical Biology (2010), 14 (2), 122-129CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    A review. Over the past two years the application of ketoreductases in the com. synthesis of chiral alcs. has undergone a revolution. Biocatalysts are now often the preferred catalyst for the synthesis of chiral alcs. via ketone redn. and are displacing reagents and chemocatalysts that only recently were considered break-through process solns. themselves. Tailor-made enzymes can now be generated from advanced, non-natural variants using HTP screening and modern mol. biol. techniques. At the same time, global economic and environmental pressures direct industrial process development toward versatile platforms that can be applied to the different stages of product development. We will discuss the technologies that have emerged over the past years that have guided biocatalysis from the bottom of the toolbox, to the power tool of choice.
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    Iding, H.; Siegert, P.; Mesch, K.; Pohl, M. Application of α-Keto Acid Decarboxylases in Biotransformations. Biochim. Biophys. Acta 1998, 1385 (2), 307– 22,  DOI: 10.1016/S0167-4838(98)00076-4
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    Application of α-keto acid decarboxylases in biotransformations
    Iding, H.; Siegert, P.; Mesch, K.; Pohl, M.
    Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1998), 1385 (2), 307-322CODEN: BBAEDZ; ISSN:0167-4838. (Elsevier B.V.)
    A review with 145 refs. The advantages of using enzymes in the synthesis of org. compds. relate to their versatility, high reaction rates, and regio- and stereospecificity and the relatively mild reaction conditions involved. Stereospecificity is esp. important in the synthesis of bioactive mols., as only one of the enantiomeric forms usually manifests bioactivity, whereas the other is often toxic. Although enzymes which catalyze asym. carbon-carbon bond formation are of great importance in bioorg. chem., only a few examples are known for thiamin diphosphate (ThDP)-dependent enzymes, whereas transformations using e.g. aldolases, lipases and lyases are well documented already. The present review surveys recent work on the application of pyruvate decarboxylase and benzoylformate decarboxylase in org. synthesis. These enzymes catalyze the synthesis of chiral α-hydroxy ketones which are versatile building blocks for org. and pharmaceutical chem. Besides the substrate spectra of both enzymes amino acid residues relevant for substrate specificity and enantioselectivity of pyruvate decarboxylase have been investigated by site-directed mutagenesis.
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    Lee, S.-H.; Yeom, S.-J.; Kim, S.-E.; Oh, D.-K. Development of Aldolase-Based Catalysts for the Synthesis of Organic Chemicals. Trends Biotechnol. 2022, 40, 306,  DOI: 10.1016/j.tibtech.2021.08.001
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    Development of aldolase-based catalysts for the synthesis of organic chemicals
    Lee, Seon-Hwa; Yeom, Soo-Jin; Kim, Seong-Eun; Oh, Deok-Kun
    Trends in Biotechnology (2022), 40 (3), 306-319CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
    A review. Aldol chems. are synthesized by condensation reactions between the carbon units of ketones and aldehydes using aldolases. The efficient synthesis of diverse org. chems. requires intrinsic modification of aldolases via engineering and design, as well as extrinsic modification through immobilization or combination with other catalysts. This review describes the development of aldolases, including their engineering and design, and the selection of desired aldolases using high-throughput screening, to enhance their catalytic properties and perform novel reactions. Aldolase-contg. catalysts, which catalyze the aldol reaction combined with other enzymic and/or chem. reactions, can efficiently synthesize diverse complex org. chems. using inexpensive and simple materials as substrates. We also discuss the current challenges and emerging solns. for aldolase-based catalysts.
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    Bracco, P.; Busch, H.; von Langermann, J.; Hanefeld, U. Enantioselective Synthesis of Cyanohydrins Catalysed by Hydroxynitrile Lyases - a Review. Org. Biomol. Chem. 2016, 14 (27), 6375– 89,  DOI: 10.1039/C6OB00934D
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    Enantioselective synthesis of cyanohydrins catalysed by hydroxynitrile lyases - a review
    Bracco, Paula; Busch, Hanna; von Langermann, Jan; Hanefeld, Ulf
    Organic & Biomolecular Chemistry (2016), 14 (27), 6375-6389CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    A review. The first enantioselective synthesis was the selective addn. of cyanide to benzaldehyde catalyzed by a hydroxynitrile lyase (HNL). Since then these enzymes have been developed into a reliable tool in org. synthesis. HNLs to prep. either the (R)- or the (S)-enantiomer of the desired cyanohydrin are available and a wide variety of reaction conditions can be applied. As a result of this, numerous applications of these enzymes in org. synthesis have been described. Here the examples of the last decade are summarized, the enzyme catalyzed step is discussed and the follow-up chem. is shown. This proves HNLs to be part of main stream org. synthesis. Addnl. the newest approaches via immobilization and reaction engineering are introduced.
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    Chen, B.-S.; Otten, L. G.; Hanefeld, U. Stereochemistry of Enzymatic Water Addition to C=C Bonds. Biotechnol. Adv. 2015, 33 (5), 526– 46,  DOI: 10.1016/j.biotechadv.2015.01.007
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    Stereochemistry of enzymatic water addition to C = C bonds
    Chen, Bi-Shuang; Otten, Linda G.; Hanefeld, Ulf
    Biotechnology Advances (2015), 33 (5), 526-546CODEN: BIADDD; ISSN:0734-9750. (Elsevier)
    A review. Water addn. to carbon-carbon double bonds using hydratases is attracting great interest in biochem. Most of the known hydratases are involved in primary metab. and to a lesser extent in secondary metab. New hydratases have recently been added to the toolbox, both from natural sources or artificial metalloenzymes. In order to comprehensively understand how the hydratases are able to catalyze the water addn. to carbon-carbon double bonds, this review will highlight the mechanistic and stereochem. studies of the enzymic water addn. to carbon-carbon double bonds, focusing on the syn/anti-addn. and stereochem. of the reaction.
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    Archelas, A.; Furstoss, R. Synthetic Applications of Epoxide Hydrolases. Curr. Opin. Chem. Biol. 2001, 5 (2), 112– 9,  DOI: 10.1016/S1367-5931(00)00179-4
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    Synthetic applications of epoxide hydrolases
    Archelas, Alain; Furstoss, Roland
    Current Opinion in Chemical Biology (2001), 5 (2), 112-119CODEN: COCBF4; ISSN:1367-5931. (Elsevier Science Ltd.)
    A review with 58 refs. There have been several recent advances in the area of biocatalyzed hydrolytic kinetic resoln. of epoxides using "newly discovered" enzymes (i.e. epoxide hydrolases). These biocatalysts, two of which will become com. available in the near future, appear to be highly promising tools for fine org. synthesis, as they enable the prepn. of various epoxides and/or their corresponding diols in enantiopure form.
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    Freakley, S. J.; Kochius, S.; van Marwijk, J.; Fenner, C.; Lewis, R. J.; Baldenius, K.; Marais, S. S.; Opperman, D. J.; Harrison, S. T. L.; Alcalde, M.; Smit, M. S.; Hutchings, G. J. A Chemo-Enzymatic Oxidation Cascade to Activate C-H Bonds with in Situ Generated H2O2. Nat. Commun. 2019, 10 (1), 4178,  DOI: 10.1038/s41467-019-12120-w
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    A chemo-enzymatic oxidation cascade to activate C-H bonds with in situ generated H2O2
    Freakley Simon J; Lewis Richard J; Hutchings Graham J; Freakley Simon J; Kochius Svenja; van Marwijk Jacqueline; Marais Sarel S; Opperman Diederik J; Smit Martha S; Kochius Svenja; van Marwijk Jacqueline; Fenner Caryn; Marais Sarel S; Opperman Diederik J; Harrison Susan T L; Smit Martha S; Fenner Caryn; Harrison Susan T L; Baldenius Kai; Alcalde Miguel
    Nature communications (2019), 10 (1), 4178 ISSN:.
    Continuous low-level supply or in situ generation of hydrogen peroxide (H2O2) is essential for the stability of unspecific peroxygenases, which are deemed ideal biocatalysts for the selective activation of C-H bonds. To envisage potential large scale applications of combined catalytic systems the reactions need to be simple, efficient and produce minimal by-products. We show that gold-palladium nanoparticles supported on TiO2 or carbon have sufficient activity at ambient temperature and pressure to generate H2O2 from H2 and O2 and supply the oxidant to the engineered unspecific heme-thiolate peroxygenase PaDa-I. This tandem catalyst combination facilitates efficient oxidation of a range of C-H bonds to hydroxylated products in one reaction vessel with only water as a by-product under conditions that could be easily scaled.
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    Wang, Y.; Xiang, Q.; Zhou, Q.; Xu, J.; Pei, D. Mini Review: Advances in 2-Haloacid Dehalogenases. Front. Microbiol. 2021, 12, 758886,  DOI: 10.3389/fmicb.2021.758886
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    Mini Review: Advances in 2-Haloacid Dehalogenases
    Wang Yayue; Xiang Qiao; Zhou Qingfeng; Pei Dongli; Xiang Qiao; Xu Jingliang; Xu Jingliang
    Frontiers in microbiology (2021), 12 (), 758886 ISSN:1664-302X.
    The 2-haloacid dehalogenases (EC 3.8.1.X) are industrially important enzymes that catalyze the cleavage of carbon-halogen bonds in 2-haloalkanoic acids, releasing halogen ions and producing corresponding 2-hydroxyl acids. These enzymes are of particular interest in environmental remediation and environmentally friendly synthesis of optically pure chiral compounds due to their ability to degrade a wide range of halogenated compounds with astonishing efficiency for enantiomer resolution. The 2-haloacid dehalogenases have been extensively studied with regard to their biochemical characterization, protein crystal structures, and catalytic mechanisms. This paper comprehensively reviews the source of isolation, classification, protein structures, reaction mechanisms, biochemical properties, and application of 2-haloacid dehalogenases; current trends and avenues for further development have also been included.
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    Koudelakova, T.; Bidmanova, S.; Dvorak, P.; Pavelka, A.; Chaloupkova, R.; Prokop, Z.; Damborsky, J. Haloalkane Dehalogenases: Biotechnological Applications. Biotechnol. J. 2013, 8 (1), 32– 45,  DOI: 10.1002/biot.201100486
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    Haloalkane dehalogenases: Biotechnological applications
    Koudelakova, Tana; Bidmanova, Sarka; Dvorak, Pavel; Pavelka, Antonin; Chaloupkova, Radka; Prokop, Zbynek; Damborsky, Jiri
    Biotechnology Journal (2013), 8 (1), 32-45CODEN: BJIOAM; ISSN:1860-6768. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Haloalkane dehalogenases (EC 3.8.1.5, HLDs) are α/β-hydrolases which act to cleave carbon-halogen bonds. Due to their unique catalytic mechanism, broad substrate specificity and high robustness, the members of this enzyme family have been employed in several practical applications: (i) biocatalytic prepn. of optically pure building-blocks for org. synthesis; (ii) recycling of byproducts from chem. processes; (iii) bioremediation of toxic environmental pollutants; (iv) decontamination of warfare agents; (v) biosensing of environmental pollutants; and (vi) protein tagging for cell imaging and protein anal. This review discusses the application of HLDs in the context of the biochem. properties of individual enzymes. Further extension of HLD uses within the field of biotechnol. will require currently limiting factors - such as low expression, product inhibition, insufficient enzyme selectivity, low affinity and catalytic efficiency towards selected substrates, and instability in the presence of org. co-solvents - to be overcome. We propose that strategies based on protein engineering and isolation of novel HLDs from extremophilic microorganisms may offer solns.
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    Schallmey, A.; Schallmey, M. Recent Advances on Halohydrin Dehalogenases-from Enzyme Identification to Novel Biocatalytic Applications. Appl. Microbiol. Biotechnol. 2016, 100 (18), 7827– 39,  DOI: 10.1007/s00253-016-7750-y
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    Recent advances on halohydrin dehalogenases- from enzyme identification to novel biocatalytic applications
    Schallmey, Anett; Schallmey, Marcus
    Applied Microbiology and Biotechnology (2016), 100 (18), 7827-7839CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    A review. Halohydrin dehalogenases are industrially relevant enzymes that catalyze the reversible dehalogenation of vicinal haloalcs. with formation of the corresponding epoxides. In the reverse reaction, also other neg. charged nucleophiles such as azide, cyanide, or nitrite are accepted besides halides to open the epoxide ring. Thus, novel C-N, C-C, or C-O bonds can be formed by halohydrin dehalogenases, which makes them attractive biocatalysts for the prodn. of various β-substituted alcs. Despite the fact that only five individual halohydrin dehalogenase enzyme sequences have been known until recently enabling their heterologous prodn., a large no. of different biocatalytic applications have been reported using these enzymes. The recent characterization of specific sequence motifs has facilitated the identification of novel halohydrin dehalogenase sequences available in public databases and has largely increased the no. of recombinantly available enzymes. These will help to extend the biocatalytic repertoire of this enzyme family and to foster novel biotechnol. applications and developments in the future. This review gives a general overview on the halohydrin dehalogenase enzyme family and their biochem. properties and further focuses on recent developments in halohydrin dehalogenase biocatalysis and protein engineering.
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    Belafriekh, A.; Secundo, F.; Serra, S.; Djeghaba, Z. Enantioselective Enzymatic Resolution of Racemic Alcohols by Lipases in Green Organic Solvents. Tetrahedron: Asymmetry 2017, 28 (3), 473– 8,  DOI: 10.1016/j.tetasy.2017.02.004
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    Enantioselective enzymatic resolution of racemic alcohols by lipases in green organic solvents
    Belafriekh, Abderahmane; Secundo, Francesco; Serra, Stefano; Djeghaba, Zeineddine
    Tetrahedron: Asymmetry (2017), 28 (3), 473-478CODEN: TASYE3; ISSN:0957-4166. (Elsevier Ltd.)
    The effects of two eco-friendly solvents, 2-methyltetrahydrofuran (MeTHF) and cyclopentyl Me ether (CPME), on the enzyme activity and enantioselectivity of Novozym 435, Candida rugosa lipase (CRL), Porcine pancreas lipase (PPL), Lipase AK, Lipase PS, and Lipozyme, a series of com. lipases, in the enantioselective transesterfications of racemic menthol, racemic sulcatol and racemic α-cyclogeraniol were studied. Vinyl acetate was chosen as the acyl donor and the reactions were carried out at water activity 0.06. The activity of lipases in CPME was similar to that obsd. in other largely employed org. solvents [toluene and tert-Bu Me ether (MTBE)], and was slightly lower in MeTHF. However, for most of the lipases tested, the enantioselectivity was higher in the eco-friendly solvents. Lipase AK exhibited a high enantioselectivity (E = 232) for the resoln. of racemic menthol but the reaction rate was low. Lipase formulation (the enzyme was frozen and lyophilized in potassium phosphate buffer without and with 5% (w/v) of sucrose, D-mannitol, or methoxy poly(ethylene glycol)) was tested with this lipase in order to improve its activity, which increased up to 4.5 times, compared to the untreated enzyme. CALB was found to be a useful biocatalyst for the resoln. of racemic sulcatol, where high activity and enantioselectivity were obtained (E ≥ 1000). For the resoln. of the racemic primary alc. α-cyclogeraniol, most of the lipases tested were active but not enantioselective, except lipase PS which displayed a moderate enantioselectivity (E = 19). The effect of the presence of a low percentage of two ionic liqs. (ILs) 1-Butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([BMIM][TFSI]) (5% (vol./vol.)) and 1-Butyl-3-methylimidazoliumtetrafluoroborate ([BMIM][BF4]) (1% (vol./vol.)) in the medium was also investigated. Only in the case of CRL the ILs slightly increased the enantioselectivity from E = 91 to E = 103 and E = 120 for [BMIM][TFSI] and [BMIM][BF4], resp. However, in all cases ILs caused a decrease of enzyme activity.
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    Bustos-Jaimes, I.; Hummel, W.; Eggert, T.; Bogo, E.; Puls, M.; Weckbecker, A.; Jaeger, K.-E. A High-Throughput Screening Method for Chiral Alcohols and Its Application to Determine Enantioselectivity of Lipases and Esterases. ChemCatChem. 2009, 1 (4), 445– 8,  DOI: 10.1002/cctc.200900190
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    A High-Throughput Screening Method for Chiral Alcohols and its Application to Determine Enantioselectivity of Lipases and Esterases
    Bustos-Jaimes, Ismael; Hummel, Werner; Eggert, Thorsten; Bogo, Eliane; Puls, Michael; Weckbecker, Andrea; Jaeger, Karl-Erich
    ChemCatChem (2009), 1 (4), 445-448CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Chiral alcs. are valuable intermediates in the synthesis of pharmaceutical, agricultural, and fine chems., which can be produced either by hydrocarbon oxidn., ketone redn., or ester hydrolysis. Nevertheless, these reactions usually produce non-enantiopure compds. For this reason, several methods for the enantioselective synthesis of alcs. have been developed, which range from the synthesis of catalysts by combinatorial chem. to the in vitro directed evolution of enzymes. In any case, high-throughput methods need to be applied to measure the enantiomeric excess [ee] or enantiopurity of the produced alcs. within a large no. of samples. Several methods for high-throughput screening for enantioselectivity of catalysts have been reported, including electrospray ionization coupled to mass spectrometry, HPLC coupled to CD, FTIR spectroscopy, and enzymic methods. Some of these sophisticated methods require, however, isotopically labeled pseudo-enantiomers for the assay and occasionally expensive equipment. Herein, a new colorimetric method is reported for the evaluation of the ee values of alcs. based on enantioselective alc. dehydrogenases (ADHs) coupled to a NADP (NADPH) oxidase (diaphorase) and its successful application in directed evolution for the screening of mutant libraries of lipases for enantioselective ester hydrolysis. The assay is based on the enantioselective oxidn. of alcs. by two different ADHs assayed sep. in parallel assays: the (R)-specific ADH from Lactobacillus kefir (LKADH) and the (S)-specific ADH from Rhodococcus erythropolis (READH), of which enantioselectivities and catalytic properties have been reported. The oxidn. of either (R)-1 or (S)-1 produces NAD(P)H, which is again oxidized to NAD(P) by diaphorase from Clostridium kluyveri with the concomitant redn. of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium (INT) to its corresponding red formazan deriv. The formation of this dye can be easily followed at 492 nm. The reaction is carried out within five minutes, during which the slope of color development over time is linear. The regeneration of the oxidized form of the coenzyme also ensures high reaction rates of ADHs. As in any other coupled assay, the amt. of diaphorase, the coupling enzyme, was kept in excess relative to the ADH enzymes in order to follow first-order kinetics.
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    Chen, B.-S.; Resch, V.; Otten, L. G.; Hanefeld, U. Enantioselective Michael Addition of Water. Eur. J. Chem. 2015, 21 (7), 3020– 30,  DOI: 10.1002/chem.201405579
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    Enantioselective Michael Addition of Water
    Chen, Bi-Shuang; Resch, Verena; Otten, Linda G.; Hanefeld, Ulf
    Chemistry - A European Journal (2015), 21 (7), 3020-3030CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The enantioselective Michael addn. using water as both nucleophile and solvent has to date proved beyond the ability of synthetic chemists. Herein, the direct, enantioselective Michael addn. of water in water to prep. important β-hydroxy carbonyl compds. using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. Deuterium labeling studies demonstrate that a Michael hydratase catalyzes the water addn. exclusively with anti-stereochem.
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    Lehwald, P.; Richter, M.; Röhr, C.; Liu, H.-w.; Müller, M. Enantioselective Intermolecular Aldehyde-Ketone Cross-Coupling through an Enzymatic Carboligation Reaction. Angew. Chem., Int. Ed. 2010, 49 (13), 2389– 92,  DOI: 10.1002/anie.200906181
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    Enantioselective Intermolecular Aldehyde-Ketone Cross-Coupling through an Enzymatic Carboligation Reaction
    Lehwald, Patrizia; Richter, Michael; Roehr, Caroline; Liu, Hung-wen; Mueller, Michael
    Angewandte Chemie, International Edition (2010), 49 (13), 2389-2392, S2389/1-S2389/27CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Asym. aldehyde-ketone cross coupling reaction of pyruvate (as acetaldehyde synthon) with cyclic or acyclic ketones, 1,2-diketones, keto esters, and substituted benzaldehydes (R1COR2 in general) catalyzed by ThDP-dependent flavoenzyme YerE of Yersinia pseudotuberculosis O:VI afforded the tertiary alcs. MeCOC(OH)R1R2 in ee of up to 96%. The 2-keto acid pyruvate was the best donor substrate for the enzyme.
  186. 186
    Hammer, S. C.; Marjanovic, A.; Dominicus, J. M.; Nestl, B. M.; Hauer, B. Squalene Hopene Cyclases Are Protonases for Stereoselective Brønsted Acid Catalysis. Nat. Chem. Biol. 2015, 11 (2), 121– 6,  DOI: 10.1038/nchembio.1719
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    Squalene hopene cyclases are protonases for stereoselective Bronsted acid catalysis
    Hammer, Stephan C.; Marjanovic, Antonija; Dominicus, Joerg M.; Nestl, Bettina M.; Hauer, Bernhard
    Nature Chemical Biology (2015), 11 (2), 121-126CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    For many important reactions catalyzed in chem. labs., the corresponding enzymes are missing, representing a restriction in biocatalysis. Although nature provides highly developed machineries appropriate to catalyze such reactions, their potential is often ignored. This also applies to Bronsted acid catalysis, a powerful method to promote a myriad of chem. transformations. Here, we report on the unique protonation machinery of a squalene hopene cyclase (SHC). Active site engineering of this highly evolvable enzyme yielded a platform for enzymic Bronsted acid catalysis in water. This is illustrated by activation of different functional groups (alkenes, epoxides and carbonyls), enabling the highly stereoselective syntheses of various cyclohexanoids while uncoupling SHC from polycyclization chem. This work highlights the potential of systematic investigation on nature's catalytic machineries to generate unique catalysts.
  187. 187
    Loskot, S. A.; Romney, D. K.; Arnold, F. H.; Stoltz, B. M. Enantioselective Total Synthesis of Nigelladine a Via Late-Stage C-H Oxidation Enabled by an Engineered P450 Enzyme. J. Am. Chem. Soc. 2017, 139 (30), 10196– 9,  DOI: 10.1021/jacs.7b05196
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    Enantioselective Total Synthesis of Nigelladine A via Late-Stage C-H Oxidation Enabled by an Engineered P450 Enzyme
    Loskot, Steven A.; Romney, David K.; Arnold, Frances H.; Stoltz, Brian M.
    Journal of the American Chemical Society (2017), 139 (30), 10196-10199CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    An enantioselective total synthesis of the norditerpenoid alkaloid nigelladine A is described. Strategically, the synthesis relies on a late-stage C-H oxidn. of an advanced intermediate. While traditional chem. methods failed to deliver the desired outcome, an engineered cytochrome P 450 enzyme was employed to effect a chemo- and regioselective allylic C-H oxidn. in the presence of four oxidizable positions. The enzyme variant was readily identified from a focused library of three enzymes, allowing for completion of the synthesis without the need for extensive screening.
  188. 188
    Zanon, J. P.; Peres, M. F. S.; Gattás, E. A. L. Colorimetric Assay of Ethanol Using Alcohol Dehydrogenase from Dry Baker’s Yeast. Enzyme Microb. Technol. 2007, 40 (3), 466– 70,  DOI: 10.1016/j.enzmictec.2006.07.029
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    Colorimetric assay of ethanol using alcohol dehydrogenase from dry baker's yeast
    Zanon, Juliana P.; Peres, Maristela F. S.; Gattas, Edwil A. L.
    Enzyme and Microbial Technology (2007), 40 (3), 466-470CODEN: EMTED2; ISSN:0141-0229. (Elsevier B.V.)
    Alc. dehydrogenases (ADHs) are oxidoreductases present in animal tissues, plants, and microorganisms. These enzymes attract major scientific interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in synthesis, thanks to their broad substrate specificity and stereoselectivity. In the present study, the standardization of the activity of the alc. dehydrogenase from baker's yeast was accomplished, and the pH and temp. stability showed, that the enzyme presented a high stability to pH 6.0-7.0 and the thermal stability were completely maintained up to 50 °C during 1 h. The assays of EtOH (detection range 1-5 mM or 4.6 × 10-2 to 23.0 × 10-2 g/L) in different samples in alc. beverages, presented a max. deviation of only 7.2%. The std. curve and the analytic curve of this method meet the conditions of precision, sensitivity, simplicity, and low cost, required for a useable anal. method.
  189. 189
    Baker, J. L.; Faustoferri, R. C.; Quivey, R. G., Jr. A Modified Chromogenic Assay for Determination of the Ratio of Free Intracellular NAD(+)/NADH in Streptococcus Mutans. Bio Protoc. 2016, 6 (16), e1902,  DOI: 10.21769/BioProtoc.1902
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    Yang, Y.; Liu, J.; Li, Z. Engineering of P450pyr Hydroxylase for the Highly Regio- and Enantioselective Subterminal Hydroxylation of Alkanes. Angew. Chem., Int. Ed. 2014, 53 (12), 3120– 4,  DOI: 10.1002/anie.201311091
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    Engineering of P450pyr Hydroxylase for the Highly Regio- and Enantioselective Subterminal Hydroxylation of Alkanes
    Yang, Yi; Liu, Ji; Li, Zhi
    Angewandte Chemie, International Edition (2014), 53 (12), 3120-3124CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Terminal-selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio- and enantioselectivity. A sensitive colorimetric high-throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative satn. mutagenesis, sextuple-mutant P450pyrSM1 was created for the hydroxylation of n-octane (1) to give (S)-2-octanol (2) with 98 % ee and >99 % subterminal selectivity. The engineered P 450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the C-H activation and functionalization of alkanes and the prepn. of enantiopure alcs. Mol. modeling provided structure-based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple-mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene (3) to afford (S)-1-phenyl-2-propanol (4) with 95 % ee and 98 % subterminal selectivity.
  191. 191
    Lauchli, R.; Rabe, K. S.; Kalbarczyk, K. Z.; Tata, A.; Heel, T.; Kitto, R. Z.; Arnold, F. H. High-Throughput Screening for Terpene-Synthase-Cyclization Activity and Directed Evolution of a Terpene Synthase. Angew. Chem., Int. Ed. 2013, 52 (21), 5571– 4,  DOI: 10.1002/anie.201301362
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    High-Throughput Screening for Terpene-Synthase-Cyclization Activity and Directed Evolution of a Terpene Synthase
    Lauchli, Ryan; Rabe, Kersten S.; Kalbarczyk, Karolina Z.; Tata, Amulya; Heel, Thomas; Kitto, Rebekah Z.; Arnold, Frances H.
    Angewandte Chemie, International Edition (2013), 52 (21), 5571-5574CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A synthetic substrate enables a colorimetric screen for terpene synthase cyclization activity, thereby facilitating the engineering of these enzymes. By using directed evolution, the thermostability of a sesquiterpene synthase was increased without the loss of other properties. The technique also enabled rapid optimization of conditions for expression and stabilization in lysate of another terpene synthase.
  192. 192
    Jung, E.; Park, B. G.; Yoo, H.-W.; Kim, J.; Choi, K.-Y.; Kim, B.-G. Semi-Rational Engineering of CYP153A35 to Enhance Ω-Hydroxylation Activity toward Palmitic Acid. Appl. Microbiol. Biotechnol. 2018, 102 (1), 269– 77,  DOI: 10.1007/s00253-017-8584-y
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    Semi-rational engineering of CYP153A35 to enhance ω-hydroxylation activity toward palmitic acid
    Jung, Eunok; Park, Beom Gi; Yoo, Hee-Wang; Kim, Joonwon; Choi, Kwon-Young; Kim, Byung-Gee
    Applied Microbiology and Biotechnology (2018), 102 (1), 269-277CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed satn. mutagenesis was attempted using a semi-rational approach that combined structure-based computational anal. and subsequent satn. mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P 450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover no. (TTN) and catalytic efficiency (kcat/KM) toward palmitic acid compared to wild-type, resp. However, in whole-cell reaction, D131S mutant showed only 50% improvement in ω-hydroxylated palmitic acid yield compared to the wild type. Docking simulation studies explained that the effect of D131S mutation on the catalytic activity would be mainly caused by the binding pose of fatty acids in the substrate access tunnel of the enzyme. This effect of D131S mutation on the catalytic activity is synergistic with that of the mutations in the active site previously reported.
  193. 193
    Francisco, W. A.; Abu-Soud, H. M.; Baldwin, T. O.; Raushel, F. M. Interaction of Bacterial Luciferase with Aldehyde Substrates and Inhibitors. J. Biol. Chem. 1993, 268 (33), 24734– 41,  DOI: 10.1016/S0021-9258(19)74526-8
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    Interaction of bacterial luciferase with aldehyde substrates and inhibitors
    Francisco, Wilson A.; Abu-Soud, Husam M.; Baldwin, Thomas O.; Raushel, Frank M.
    Journal of Biological Chemistry (1993), 268 (33), 24734-41CODEN: JBCHA3; ISSN:0021-9258.
    Bacterial luciferase catalyzes the reaction of FMNH2, O2, and an aliph. aldehyde to yield the carboxylic acid, FMN, water and blue-green light. The kinetics of the bacterial luciferase reaction were measured by stopped-flow spectrophotometry at pH 7 and 25°C for the series of aldehydes from n-heptanal to n-undecanal. The rate of formation of the 4a-hydroperoxyflavin intermediate was dependent on the aldehyde concn. when mixts. of enzyme, FMNH2, and aldehyde were rapidly mixed with O2. At satg. aldehyde, the rate of formation of this intermediate was 100-fold slower than in the absence of aldehyde, demonstrating that an enzyme·FMNH2·aldehyde complex can be formed. Numerical simulation of the time courses for these expts. supported the formation of this intermediate and its direct reaction with O2. The kinetics of the light emitting reaction were dependent upon the chain length of the aldehyde substrate. Although the initial light intensity and the light emission decay rate were different for each aldehyde, the quantum yield for the reaction was independent of the aldehyde used. Luciferase was inhibited by high levels of the aldehyde substrate when the enzyme was assayed by mixing FMNH2 with an aerobic mixt. of enzyme and aldehyde. The extent of inhibition was dependent on the particular aldehyde used, and the binding affinity of the aldehyde for the free enzyme increased in parallel with the aldehyde chain length. The kinetics of the formation and decay of the various intermediates were also studied in the presence of n-alkylaldehyde analogs. These compds. decreased the rate of formation of the 4a-hydroperoxyflavin intermediate in much the same way as the aldehyde substrate, presumably by the formation of the enzyme·FMNH2·analog ternary complex.
  194. 194
    Minak-Bernero, V.; Bare, R. E.; Haith, C. E.; Grossman, M. J. Detection of Alkanes, Alcohols, and Aldehydes Using Bioluminescence. Biotechnol. Bioeng. 2004, 87 (2), 170– 7,  DOI: 10.1002/bit.20089
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    195
    Detection of alkanes, alcohols, and aldehydes using bioluminescence
    Minak-Bernero, Vera; Bare, Richard E.; Haith, Copper E.; Grossman, Matthew J.
    Biotechnology and Bioengineering (2004), 87 (2), 170-177CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
    We report a novel method for the rapid, sensitive, and quant. detection of alkanes, alcs., and aldehydes that relies on the reaction of bacterial luciferase with an aldehyde, resulting in the emission of light. Primary alcs. with corresponding aldehydes that are within the substrate range of the particular luciferase are detected after conversion to the aldehyde by an alc. dehydrogenase. In addn., alkanes themselves may be detected by conversion to primary alcs. by an alkane hydroxylase, followed by conversion to the aldehyde by alc. dehydrogenase. We developed a rapid bioluminescent method by genetically engineering the genes encoding bacterial luciferase, alc. dehydrogenase, and alkane hydroxylase into a plasmid for simultaneous expression in an E. coli host cell line. Alkanes, alcs., or aldehydes were detected within seconds, with sensitivity in the micromolar range, by measuring the resulting light emission with a microplate reader. We demonstrate the application of this method for the detection of alkanes, alcs., and aldehydes and for the detection of alkane hydroxylase and alc. dehydrogenase activity in vivo. This method is amenable to the high-throughput screening needs required for the identification of novel catalysts.
  195. 195
    Greene, L. E.; Lincoln, R.; Krumova, K.; Cosa, G. Development of a Fluorogenic Reactivity Palette for the Study of Nucleophilic Addition Reactions Based on meso-Formyl BODIPY Dyes. ACS Omega 2017, 2 (12), 8618– 24,  DOI: 10.1021/acsomega.7b01795
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    196
    Development of a Fluorogenic Reactivity Palette for the Study of Nucleophilic Addition Reactions Based on meso-Formyl BODIPY Dyes
    Greene, Lana E.; Lincoln, Richard; Krumova, Katerina; Cosa, Gonzalo
    ACS Omega (2017), 2 (12), 8618-8624CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
    We describe herein a fluorescence-based assay to characterize and report on nucleophilic addn. to carbonyl moieties and highlight the advantages a fluorescence-based assay and multiplex anal. can offer. The assay relies on the fluorogenic properties of meso-formyl boron-dipyrromethene (BODIPY) dyes that become emissive following nucleophilic addn. A reactivity palette is assembled based on the increasing electrophilic character of five meso-formyl BODIPY compds. tested. We show that increasing rates of emission enhancement correlate with the decreasing electrophilic character of BODIPY dyes in the presence of an acid catalyst and a nucleophile. These results are consistent with the rate-limiting step involving activation of the electrophile. Increasing product formation is shown to correlate with the increasing electrophilic character of the BODIPY dyes, as expected based on thermodn. In addn. to providing rates of reaction, anal. of the fluorescence parameters for the reaction mixts., including emission quantum yields and fluorescence lifetimes, enables us to det. the extent of reactant conversion at equil. (in our case the estd. yield of a transient species) and the presence of different products, without the need for isolation. We anticipate that our reactivity palette approach, combined with the in-depth fluorescence anal. discussed herein, will provide guidelines toward developing fluorogenic assays of reactivity offering multiplex information, beyond fluorescence intensity.
  196. 196
    Lincoln, R.; Greene, L. E.; Bain, C.; Flores-Rizo, J. O.; Bohle, D. S.; Cosa, G. When Push Comes to Shove: Unravelling the Mechanism and Scope of Nonemissive meso-Unsaturated BODIPY Dyes. J. Phys. Chem. B 2015, 119 (13), 4758– 65,  DOI: 10.1021/acs.jpcb.5b02080
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    197
    When Push Comes to Shove: Unravelling the Mechanism and Scope of Nonemissive meso-Unsaturated BODIPY Dyes
    Lincoln, Richard; Greene, Lana E.; Bain, Cheryl; Flores-Rizo, Juan O.; Bohle, D. Scott; Cosa, Gonzalo
    Journal of Physical Chemistry B (2015), 119 (13), 4758-4765CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    We report herein spectroscopy and computational results that illustrate an efficient intramol. deactivation pathway for meso-unsatd. boron-dipyrromethene (BODIPY) dyes in their singlet excited state. Our results show that the mechanism hinges on the structural flexibility imparted by the boron atom and on the energetic stabilization conferred by extending the conjugation into the meso substituent, which is otherwise unconjugated in the ground state. Following photoexcitation, rotation along the dihedral angle of the meso-unsatd. group results in its conjugation at the expense of shifting one pyrrole moiety in dipyrrin out of the plane. Internal conversion to an energetically hot, ground-state species efficiently competes with emission. The mechanism applies to meso-vinyl, -formyl, and -iminyl moieties. The presence of Me groups at positions C1 and C7 exacerbates the energetic penalty toward conjugation of the meso groups leading to a small energy gap between relaxed excited state and ground state and undetected emission quantum yields. Importantly, methyls at C1 and C7 prevent nonradiative deactivation in meso-aryl moieties, illustrating that when push comes to shove, the energetic (kinetic) barrier toward reaching conjugation is too large for aryl moieties but low enough for smaller groups to effectively compete with radiative transitions. Wisely chosen meso-unsatd. BODIPY dyes may serve as richly sensitive platforms for the prepn. of novel fluorogenic substrates to monitor chem. reactions or to probe the rigidity of their surrounding environment.
  197. 197
    Sawminathan, S.; Iyer, S. K. A New Imidazole Based Phenanthridine Probe for Ratiometric Fluorescence Monitoring of Methanol in Biodiesel. New J. Chem. 2021, 45 (13), 6033– 41,  DOI: 10.1039/D0NJ06252A
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    198
    A new imidazole based phenanthridine probe for ratiometric fluorescence monitoring of methanol in biodiesel
    Sawminathan, Sathish; Iyer, Sathiyanarayanan Kulathu
    New Journal of Chemistry (2021), 45 (13), 6033-6041CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
    We prepd. and characterized an array of polarity-sensitive fluorescent dyes (7 and 9a, 9b and 9c). Fluorescent dyes are based on imidazole cores, suitably functionalized with a conjugated push-pull system. The photophys. properties of the synthesized materials were elaborately investigated. These new fluorescent probes showed good fluorecence emission, higher .vphi.Fl values and large Stokes shifts in different org. solvents because of their ICT nature. Furthermore, with aldehyde groups as the recognition sites, all the dyes could act as ratiometric fluorescent sensors towards methanol. The selective ratiometric fluorescence response towards methanol is due to the strong intermol. H-bonding between the aldehyde unit and methanol. The detection limit of methanol was found to be at the ppm level. Methanol sensing was confirmed using emission spectroscopy, FTIR studies and theor. calcns. Addnl., these dyes were used for the detection of methanol in biodiesel and in test paper strips.
  198. 198
    Mohr, G. J.; Spichiger-Keller, U. E. Novel Fluorescent Sensor Membranes for Alcohols Based on p-N,N-Dioctylamino-4′-Trifluoroacetylstilbene. Anal. Chim. Acta 1997, 351 (1), 189– 96,  DOI: 10.1016/S0003-2670(97)00365-6
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    Novel fluorescent sensor membranes for alcohols based on p-N,N-Dioctylamino-4'-trifluoroacetylstilbene
    Mohr, Gerhard J.; Spichiger-Keller, Ursula E.
    Analytica Chimica Acta (1997), 351 (1-3), 189-196CODEN: ACACAM; ISSN:0003-2670. (Elsevier)
    A chromogenic ligand was described that reversibly interacts with alcs. in a sensor, resulting in a change in both absorbance and fluorescence. When embedded in membranes composed of plasticized PVC, p-N,N-dioctylamino-4'-trifluoroacetylstilbene (ETH 4004) showed a significant signal change on exposure to aq. EtOH with a decrease in absorbance at 453 nm and an increase in absorbance at 373 nm. At the same time, the fluorescence at 576 nm decreased accordingly when excited at 452 nm. The sensor layer exhibited a dynamic range from 1-50 (vol.%) EtOH, with the highest sensitivity at 5-40 (vol.%). The fluorescence of the sensor membrane was virtually insensitive to changes in pH. However, the magnitude of the relative signal change between plain buffer and buffer-contg. EtOH was pH-dependent. A protective PTFE layer was, therefore, applied to the sensor membrane. This prevented interference from ambient light, pH, and ionic interferents. The response time of PTFE-coated membranes was 3-4 min for forward response, and 5-10 min for reverse response. The selectivity of the dye in the sensor membranes was mainly affected by the lipophilicity of the alcs.
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    Mohr, G. J.; Lehmann, F.; Grummt, U.-W.; Spichiger-Keller, U. E. Fluorescent Ligands for Optical Sensing of Alcohols: Synthesis and Characterisation of p-N,N-Dialkylamino-Trifluoroacetylstilbenes. Anal. Chim. Acta 1997, 344 (3), 215– 25,  DOI: 10.1016/S0003-2670(97)00113-X
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    Fluorescent ligands for optical sensing of alcohols: synthesis and characterization of p-N,N-dialkylamino-trifluoroacetylstilbenes
    Mohr, Gerhard J.; Lehmann, Frank; Grummt, Ulrich-W.; Spichiger-Keller, Ursula E.
    Analytica Chimica Acta (1997), 344 (3), 215-225CODEN: ACACAM; ISSN:0003-2670. (Elsevier)
    Novel fluorescent ligands were synthesized which interact with specific alcs. resulting in a change of absorbance as well as fluorescence. 4-Trifluoroacetyl-4'-(di-N-butylamino)stilbene (ETH 4003) exhibits an absorption at ∼450nm in org. solvents, whereas, upon reaction with primary alcs., the max. shifts to around 360 nm. The fluorescence emission of the pos. solvatochromic dye ranges from 500 to 700 nm and, upon reaction with alcs., is also blue-shifted to around 450 nm. The significant shift of both absorbance and fluorescence is based on the chem. conversion of the trifluoroacetyl group of the dye into the hemiacetal, thus causing a change in the electron distribution of the dye and a spectral shift of ∼100 nm. The reaction is selective for alcs., which are not sterically hindered to approach the trifluoroacetyl group, i.e. for primary alcs. An example for the anal. application of the ligands is given. An optical sensor was prepd. by embedding 4-trifluoroacetyl-4'-(di-N-butylamino)stilbene in plasticized PVC and coating the layer with white PTFE. The microporous PTFE coating provides an optical isolation and prevents interferences of ions. The sensor exhibits a sensitive range from 5-50 vol.% EtOH with max. signal changes to be in the range of 40%. The detection limit is 2 vol.% EtOH.
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    Takano, K.; Sasaki, S.-i.; Citterio, D.; Tamiaki, H.; Suzuki, K. An Oxo-Bacteriochlorin Derivative for Long-Wavelength Fluorescence Ratiometric Alcohol Sensing. Analyst 2010, 135 (9), 2334– 9,  DOI: 10.1039/c0an00173b
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    An oxo-bacteriochlorin derivative for long-wavelength fluorescence ratiometric alcohol sensing
    Takano, Kosuke; Sasaki, Shin-ichi; Citterio, Daniel; Tamiaki, Hitoshi; Suzuki, Koji
    Analyst (Cambridge, United Kingdom) (2010), 135 (9), 2334-2339CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    A fluorescent ratiometric optical chem. sensor (optode) for alc. detection is presented. A lipophilized bacteriochlorin deriv. possessing a trifluoroacetyl group at the 3-position was synthesized for alc. detection. When embedded in a plasticized poly(vinyl chloride) (PVC) membrane, the fluororeceptor shows a significant signal change on exposure to aq. ethanol solns. both in the absorbance and the fluorescence emission. This optode allows the detn. of ethanol concns. in aq. sample solns. by ratiometric fluorometry. The fluorescent indicator responds with increasing fluorescence around 701 nm and decreasing fluorescence around 751 nm towards the presence of ethanol. The optode response is fully reversible in a dynamic measurement range from 0.3% to at least 25% (vol./vol.) ethanol. The limit of detection (LOD) and the limit of quantification (LOQ) of the sensor for ethanol are 0.1% and 0.4% (vol./vol.), resp. The new optode can be employed as an ethanol-sensing device with high sensitivity for beverage and industrial analyses.
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    Zhao, M.; Yue, Y.; Liu, C.; Hui, P.; He, S.; Zhao, L.; Zeng, X. A Colorimetric and Fluorometric Dual-Modal Sensor for Methanol Based on a Functionalized Pentacenequinone Derivative. Chem. Commun. 2018, 54 (60), 8339– 42,  DOI: 10.1039/C8CC04515A
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    A colorimetric and fluorometric dual-modal sensor for methanol based on a functionalized pentacenequinone derivative
    Zhao, Mengting; Yue, Yuxin; Liu, Chang; Hui, Peiyi; He, Song; Zhao, Liancheng; Zeng, Xianshun
    Chemical Communications (Cambridge, United Kingdom) (2018), 54 (60), 8339-8342CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A functionalized pentacenequinone deriv., ZR1 (I), as a highly selective sensor for methanol was prepd. Based on the methanol-induced keto-enol tautomerization of the pentacene-quinone moiety, ZR1 shows specific selectivity toward methanol, which leads to dual-modal detection of methanol with a very low detection limit of 0.038% in ethanol.
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    Roy, S.; Das, S.; Ray, A.; Parui, P. P. Fluorometric Trace Methanol Detection in Ethanol and Isopropanol in a Water Medium for Application in Alcoholic Beverages and Hand Sanitizers. RSC Adv. 2021, 11 (48), 30093– 101,  DOI: 10.1039/D1RA05201B
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    Fluorometric trace methanol detection in ethanol and isopropanol in a water medium for application in alcoholic beverages and hand sanitizers
    Roy, Snigdha; Das, Sanju; Ray, Ambarish; Parui, Partha Pratim
    RSC Advances (2021), 11 (48), 30093-30101CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    Detection of methanol (MeOH) in an ethanol (EtOH)/isopropanol (iPrOH) medium contg. water is crucial to recognize MeOH poisoning in alc. beverages and hand sanitizers. Although chem. sensing methods are very sensitive and easy to perform, the chem. similarities between the alcs. make MeOH detection very challenging particularly in the presence of water. Herein, the fluorometric detection of a trace amt. of MeOH in EtOH/iPrOH in the presence of water using alc. coordinated Al(III)-complexes of an aldehydic phenol ligand contg. a dangling pyrazole unit is described. The presence of MeOH in the EtOH/iPrOH causes a change of the complex geometry from tetrahedral (Td) to octahedral (Oh) due to the replacement of the coordinated EtOH/iPrOH by MeOH mols. The Td-complex exhibited fluorescence but the Oh-species did not, because of the intramol. photo-induced electron transfer (PET). By interacting the Oh species with water, its one MeOH coordination is replaced by a water mol. followed by the proton transfer from the water to pyrazole-N which generates strong fluorescence by inhibiting the PET. In contrast, the water interaction dissocs. the Td-complex to exhibit fluorescence quenching. The water induced reversal of the fluorescence response from the decrease to increase between the absence and presence of MeOH is utilized to detect MeOH in an EtOH/iPrOH medium contg. water with a sensitivity of ∼0.03-0.06% (vol./vol.). The presence of water effected the MeOH detection and allows the estn. of the MeOH contamination in alc. beverages and hand sanitizers contg. large amts. of water.
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    Wu, Z.; Fu, X.; Wang, Y. Click Synthesis of a Triphenylamine-Based Fluorescent Methanol Probe with a Unique D-Π-a Structure. Sens. Actuators B Chem. 2017, 245, 406– 13,  DOI: 10.1016/j.snb.2017.01.164
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    Click synthesis of a triphenylamine-based fluorescent methanol probe with a unique D-π-A structure
    Wu, Zhihua; Fu, Xiaobin; Wang, Yong
    Sensors and Actuators, B: Chemical (2017), 245 (), 406-413CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    Facile probing org. solvents is attracting intense attention due to the growing environmental issues. Herein, for the first time, we report a novel D-π-A structured methanol responsive fluorescence mol. (TTO), synthesized by coupling triphenylamine (electron-donor) and p-toluenesulfonyl (electron-acceptor) via Cu (I) catalytic 1,3-dipolar cycloaddn. (click chem.) forming a triazole bridge. D. functional theory (DFT) calcns. and X-ray single crystal structure reveal that TTO has a highly twisted conformation and unique D-π-A structure which render it exceptional optical properties. TTO exhibits strong fluorescence in methanol (QE > 0.67) but weak fluorescence in other org. solvents (QE < 0.1) including various alcs. and can be used as a highly selective fluorescence probe for methanol identification. Notably, TTO is able to excellently probe methanol from its homolog ethanol in water with a detection limit of below 0.7% (vol. concn.), which demonstrates the promise potential of TTO for practical applications.
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    Kumar, V.; Kumar, A.; Diwan, U.; Singh, M. K.; Upadhyay, K. K. A Radical Approach for Fluorescent Turn ‘On’ Detection, Differentiation and Bioimaging of Methanol. Org. Biomol. Chem. 2015, 13 (33), 8822– 6,  DOI: 10.1039/C5OB01333J
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    A radical approach for fluorescent turn 'on' detection, differentiation and bioimaging of methanol
    Kumar, Virendra; Kumar, Ajit; Diwan, Uzra; Singh, Manish Kumar; Upadhyay, K. K.
    Organic & Biomolecular Chemistry (2015), 13 (33), 8822-8826CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    A simple Schiff base (RC) has been explored as a smart example of fluorescent material for the selective detection, differentiation and bioimaging of methanol. The nucleophilic attack of methanol on the cyclic control unit of RC leads to its opening and formation of a highly fluorescent moiety, RO. The RC displays a good sensitivity for MeOH with a detection limit of 0.042% in water.
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    Kumar, V.; Kundu, S.; Sk, B.; Patra, A. A Naked-Eye Colorimetric Sensor for Methanol and ‘Turn-On’ Fluorescence Detection of Al3+. New J. Chem. 2019, 43 (47), 18582– 9,  DOI: 10.1039/C9NJ04688G
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    207
    A naked-eye colorimetric sensor for methanol and 'turn-on' fluorescence detection of Al3+
    Kumar, Virendra; Kundu, Subhankar; Sk, Bahadur; Patra, Abhijit
    New Journal of Chemistry (2019), 43 (47), 18582-18589CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
    The current study demonstrates a simple and economical synthetic approach for the development of a multifunctional Schiff base compd. (NRSB) through the condensation reaction between 2-hydroxynaphthaldehyde and a five-membered heterocycle 3-aminorhodanine. The rhodanine unit was chosen due to the presence of an active electron-deficient carbonyl group. A facile chem. transformation from the closed to the open form was noticeable for rhodanine derivs. in the presence of a suitable nucleophile or an electron-rich system. Such a unique feature was invoked to develop a versatile mol. platform for the colorimetric sensing of methanol and the fluorometric detection of Al3+ ions. The nucleophilic attack by methanol on NRSB led to the formation of an open-ring compd., NRSB-O, with a distinct change in color from colorless to yellow. NRSB showed noticeable sensitivity towards methanol and the detection limit was found to be 0.43 wt%. The methanol-induced chem. transformation from NRSB to NRSB-O was probed through X-ray diffraction studies. The strong CH-π interaction between the methylene protons of the rhodanine unit and the π-electron cloud of the naphthalene unit (3.03 Å) and the π-π interaction between naphthalene and the rhodanine moiety (3.51 Å and 3.55 Å) lead to a two-dimensional (2D) supramol. structure in the NRSB crystal. NRSB-O possesses strong CH-π interactions between mols. with an intermol. distance of 2.49 Å, leading to 2D-supramol. self-assembly. The crystal structure revealed the scope of the chelation of NRSB-O with metal ions due to the presence of different N, O, and S donor centers. Interestingly, NRSB-O exhibited a turn-on fluorescence response specifically towards Al3+ through the chelation-enhanced fluorescence (CHEF) mechanism over other competitive metal ions. The turn 'on-off' fluorescence switching was demonstrated for multiple cycles through the alternative addn. of Al3+ and EDTA. The low-cost, simple design strategy of NRSB and NRSB-O as delineated in the current study would contribute to further development of task-specific mol. sensors and switches.
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    Patel, R. N. Synthesis of Chiral Pharmaceutical Intermediates by Biocatalysis. Coord. Chem. Rev. 2008, 252 (5), 659– 701,  DOI: 10.1016/j.ccr.2007.10.031
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    Synthesis of chiral pharmaceutical intermediates by biocatalysis
    Patel, Ramesh N.
    Coordination Chemistry Reviews (2008), 252 (5-7), 659-701CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
    A review. Chirality is a key factor in the safety and efficacy of many drug products and thus the prodn. of single enantiomers of drug intermediates has become increasingly important in the pharmaceuticals industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes derived therefrom for the transformation of synthetic chems. with high chemo-, regio- and enantioselectivities. In this article, biocatalytic processes are described for the synthesis of chiral intermediates for pharmaceuticals.
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    Chen, Y.; Chen, C.; Wu, X. Dicarbonyl Reduction by Single Enzyme for the Preparation of Chiral Diols. Chem. Soc. Rev. 2012, 41 (5), 1742– 53,  DOI: 10.1039/c1cs15230k
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    Dicarbonyl reduction by single enzyme for the preparation of chiral diols
    Chen, Yijun; Chen, Chen; Wu, Xuri
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    A review. Chiral diols are a group of key building blocks useful for prepg. a variety of important chiral chems. While the prepn. of optically pure diols is generally not an easy task in synthetic org. chem., three classes of enzymes, namely dicarbonyl reductase, dioxygenase and epoxide hydrolase, display remarkable ability to stereoselectively introduce two hydroxyl groups in a single-step enzymic conversion. In this tutorial review, we pay special attention to dicarbonyl reductases that directly produce chiral diols through the bio-redn. of two carbonyl groups. The dicarbonyl reductases include diketoreductase, α-acetoxy ketone reductase and sepiapterin reductase. We present these exceptional enzymes in the context of source and properties, structure and catalytic mechanism as well as biocatalytic application. In addn. to the broad substrate specificity, the excellent stereoselectivity and high catalytic efficiency of these enzymes have positioned them as valuable biocatalysts. With more sophisticated understanding of the structure-function relationship, the practical utilities of these enzymes assocd. with their interesting chem. will be considerably appreciated over time. Moreover, rational redesign and mol. evolution of these unusual biocatalysts will truly enable their broader applications in the synthesis of chiral diols in the future.
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    Ramasastry, S. S. V.; Zhang, H.; Tanaka, F.; Barbas, C. F. Direct Catalytic Asymmetric Synthesis of anti-1,2-Amino Alcohols and syn-1,2-Diols through Organocatalytic anti-Mannich and syn-Aldol Reactions. J. Am. Chem. Soc. 2007, 129 (2), 288– 9,  DOI: 10.1021/ja0677012
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    Direct Catalytic Asymmetric Synthesis of anti-1,2-Amino Alcohols and syn-1,2-Diols through Organocatalytic anti-Mannich and syn-Aldol Reactions
    Ramasastry, S. S. V.; Zhang, Haile; Tanaka, Fujie; Barbas, Carlos F., III
    Journal of the American Chemical Society (2007), 129 (2), 288-289CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Chiral 1,2-amino alcs. and 1,2-diols are common structural motifs found in a vast array of natural and biol. active mols. Efficient enantioselective syntheses of ketones contg. syn- or anti-1,2-amino alcs. and 1,2-diols have been reported using metal-based strategies. To date, however, approaches involving organocatalysis have been limited to the syntheses of the syn-1,2-amino alcs. and anti-1,2-diols. Herein we disclose simple and efficient routes to highly enantiomerically enriched anti-1,2-amino alcs. and syn-1,2-diols through direct asym. Mannich, Mannich-type, and aldol reactions involving unmodified α-hydroxyketones in reactions catalyzed by primary amine-contg. amino acids. These reactions exploit (Z)-enamines of α-hydroxyketones in their bond-forming transition states. This study compliments and extends our bioorg. approach to asym. synthesis in these two versatile synthon classes. Significantly, these reactions are practical, tolerate wet solvent, and exploit com. available amino acids, such as threonine and tryptophan, and their derivs. as catalysts. These results provide addnl. support for our original hypothesis suggesting that amino acid catalysis played a key role in prebiotic chem. facilitating the asym. synthesis of the mols. of life.
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    Guo, Z.; Shin, I.; Yoon, J. Recognition and Sensing of Various Species Using Boronic Acid Derivatives. Chem. Commun. 2012, 48 (48), 5956– 67,  DOI: 10.1039/c2cc31985c
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    Recognition and sensing of various species using boronic acid derivatives
    Guo, Zhiqian; Shin, Injae; Yoon, Juyoung
    Chemical Communications (Cambridge, United Kingdom) (2012), 48 (48), 5956-5967CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A review. A boronic acid moiety can bind to nucleophilic species, such as fluoride ions and 1,2-diols, and arylboronates are converted to the corresponding phenols by treatment with hydrogen peroxide. Based on these reactivity profiles of boron compds., a variety of boronic acid and boronate-based fluorescent chemosensors have been developed for detecting biol. relevant species. This feature article highlights recent advances that have been made in the development of chemosensors of these types for monitoring carbohydrates, dopamine, fluorides, metal ions and hydrogen peroxide.
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    Hudlicky, T.; Thorpe, A. J. Current Status and Future Perspectives of Cyclohexadiene-cis-Diols in Organic Synthesis: Versatile Intermediates in the Concise Design of Natural Products. Chem. Commun. 1996, 17 (17), 1993– 2000,  DOI: 10.1039/cc9960001993
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    Wingstrand, M. J.; Madsen, C. M.; Clausen, M. H. Rapid Synthesis of Macrocycles from Diol Precursors. Tetrahedron Lett. 2009, 50 (6), 693– 5,  DOI: 10.1016/j.tetlet.2008.11.100
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    Rapid synthesis of macrocycles from diol precursors
    Wingstrand, Magnus J.; Madsen, Charlotte M.; Clausen, Mads H.
    Tetrahedron Letters (2009), 50 (6), 693-695CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)
    A method for the formation of synthetic macrocycles with different ring sizes from diols is presented. Reacting a simple diol precursor with electrophilic reagents leads to a cyclic carbonate, sulfite, or phosphate in a single step in 25-60% yield. Converting the cyclization precursor to a bis-electrophilic iodide or aldehyde enables prepn. of a cyclic sulfide and amine, resp., the latter using a double-reductive amination to induce ring closure.
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    Tang, X.; Wei, J.; Ding, N.; Sun, Y.; Zeng, X.; Hu, L.; Liu, S.; Lei, T.; Lin, L. Chemoselective Hydrogenation of Biomass Derived 5-Hydroxymethylfurfural to Diols: Key Intermediates for Sustainable Chemicals, Materials and Fuels. Renew. Sust. Energy Rev. 2017, 77, 287– 96,  DOI: 10.1016/j.rser.2017.04.013
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    Chemoselective hydrogenation of biomass derived 5-hydroxymethylfurfural to diols: Key intermediates for sustainable chemicals, materials and fuels
    Tang, Xing; Wei, Junnan; Ding, Ning; Sun, Yong; Zeng, Xianhai; Hu, Lei; Liu, Shijie; Lei, Tingzhou; Lin, Lu
    Renewable & Sustainable Energy Reviews (2017), 77 (), 287-296CODEN: RSERFH; ISSN:1364-0321. (Elsevier Ltd.)
    A review. Biomass-derived 5-hydroxymethylfurfural (HMF) is hailed as an all-purposed platform mol. that holds great promise to address a no. of high vol. markets for chems., polymeric materials, and transportation fuels. HMF-derived diols, including 2,5-bishydroxymethylfuran (BHMF), 2,5-bishydroxymethyltetrahydrofuran (BHMTF), and 1,6-hexanediol (1,6-HD), are key intermediates for the catalytic upgrading of HMF in a biorefinery. These diols can be employed as renewable polymeric monomers, and among them BHMF and BHMTF are also attractive precursors for biofuels, such as 2,5-dimethylfuran (DMF), 2,5-bis(alkoxymethyl)furans (BAMFs), and 2,5-dimethyltetrahydrofuran (DMTHF). Hence, gaining more insights into the chemoselective hydrogenation of HMF to these diols is of particular importance. In this review, we comprehensively summarize the advances in the selective hydrogenation of HMF into these diols in terms of the diversity of hydrogen sources, mainly including mol. H2, alcs., formic acid and water, over homogeneous or heterogeneous catalysts. Assessment of the relative merits of different hydrogen sources for the hydrogenation of HMF is performed as well. We also discuss challenges and opportunities in employing these HMF-derived diols for the prodn. of polymeric materials and biofuels.
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    Song, J.; Shao, P.-L.; Wang, J.; Huang, F.; Zhang, X. Asymmetric Hydrogenation of 1,4-Diketones: Facile Synthesis of Enantiopure 1,4-Diarylbutane-1,4-Diols. Chem. Commun. 2021, 58 (2), 262– 5,  DOI: 10.1039/D1CC05359K
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    Asymmetric hydrogenation of 1,4-diketones: facile synthesis of enantiopure 1,4-diarylbutane-1,4-diols
    Song Jingyuan; Song Jingyuan; Shao Pan-Lin; Wang Jiang; Huang Fanping; Zhang Xumu; Shao Pan-Lin; Zhang Xumu
    Chemical communications (Cambridge, England) (2021), 58 (2), 262-265 ISSN:.
    Owing to the biological significance and great synthetic value of 1,4-diarylbutane-1,4-diols and their derivatives, increasingly considerable attention has been paid to developing effective synthetic methods for chiral 1,4-diarylbutane-1,4-diols. We herein report an efficient asymmetric hydrogenation of 1,4-diaryldiketones catalyzed by a chiral iridium complex bearing f-amphox as ligand, furnishing a series of 1,4-diarylbutane-1,4-diols in excellent yields (up to >99%) with exceptional enantioselectivities (up to >99.9% ee) and diastereoselectivities (up to >100 : 1 dr).
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    Faber, K.; Mischitz, M.; Kroutil, W. Microbial Epoxide Hydrolases. Acta Chem. Scand. 1996, 50, 249,  DOI: 10.3891/acta.chem.scand.50-0249
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    Microbial epoxide hydrolases
    Faber, Kurt; Mischitz, martin; Kroutil, Wolfgang
    Acta Chemica Scandinavica (1996), 50 (3), 249-58CODEN: ACHSE7; ISSN:0904-213X. (Munksgaard)
    A review with 66 refs. Chiral epoxides and 1,2-diols, which are central building blocks for the asym. synthesis of bioactive compds., can be obtained by using enzymes, which catalyze the enantioselective hydrolysis of epoxides - epoxide hydrolases. These biocatalysts are more widely distributed in fungi and bacteria than previously expected, and sufficient sources from bacteria, such as Rhodococcus and Myobacterium sp., or fungi, for instance Aspergillus and Beauveria sp. have recently been identified. The reaction proceeds via an SN2-specific opening of the epoxide leading to the formation of the corresponding trans-configurated 1,2-diols. For the resoln. of 2-monosubstituted epoxides and for 2,2-disubstituted substrates fungal cells and several bacteria, resp., have been shown to possess excellent selectivities. In addn., the use of non-natural nucleophiles such as azide or amine provides access to chiral azido- and amino-alcs. The synthetic potential of these enzymes is illustrated with recent examples of kinetic resolns. of epoxides from the literature.
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    Ouellette, R. J.; Rawn, J. D. Alcohols: Reactions and Synthesis. In Organic Chemistry, 2nd ed.; Academic Press, 2018; Chapter 16, pp 463– 505.
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    Gubbels, E.; Heitz, T.; Yamamoto, M.; Chilekar, V.; Zarbakhsh, S.; Gepraegs, M.; Köpnick, H.; Schmidt, M.; Brügging, W.; Rüter, J. Polyesters. Ullmann’s Encyclopedia of Industrial Chemistry Wiley VCH, 2018; 1– 30 DOI: 10.1002/14356007.a21_227.pub2 .
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    Steinreiber, A.; Faber, K. Microbial Epoxide Hydrolases for Preparative Biotransformations. Curr. Opin. Biotechnol. 2001, 12 (6), 552– 8,  DOI: 10.1016/S0958-1669(01)00262-2
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    Microbial epoxide hydrolases for preparative biotransformations
    Steinreiber, Andreas; Faber, Kurt
    Current Opinion in Biotechnology (2001), 12 (6), 552-558CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Science Ltd.)
    A review. Epoxide hydrolases from microbial sources are highly versatile biocatalysts for the asym. hydrolysis of epoxides on a preparative scale. Besides kinetic resoln., which furnishes the corresponding vicinal diol and remaining non-hydrolyzed epoxide in nonracemic form, enantioconvergent processes are possible: these are highly attractive as they lead to the formation of a single enantiomeric diol from a racemic oxirane. The data accumulated over recent years reveal a common picture of the substrate structure selectivity pattern of microbial epoxide hydrolases and indicate that substrates of various structural types can be selectively hydrolyzed with enzymes from certain microbial sources.
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    Gibson, D. T.; Parales, R. E. Aromatic Hydrocarbon Dioxygenases in Environmental Biotechnology. Curr. Opin. Biotechnol. 2000, 11 (3), 236– 43,  DOI: 10.1016/S0958-1669(00)00090-2
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    Aromatic hydrocarbon dioxygenases in environmental biotechnology
    Gibson, David T.; Parales, Rebecca E.
    Current Opinion in Biotechnology (2000), 11 (3), 236-243CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Science Ltd.)
    Review of arom. hydrocarbon dioxygenases and the properties that make them attractive synthons for the prodn. of industrially and medically important chiral chems. and also provide essential information for the development of bioremediation technol., with 65 refs. Arom. hydrocarbon dioxygenases belong to a large family of Rieske non-heme iron oxygenases. The dioxygenases have a broad substrate specificity and catalyze enantiospecific reactions with a wide range of substrates.
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    Boyd, D. R.; Sharma, N. D.; Allen, C. C. R. Aromatic Dioxygenases: Molecular Biocatalysis and Applications. Curr. Opin. Biotechnol. 2001, 12 (6), 564– 73,  DOI: 10.1016/S0958-1669(01)00264-6
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    Boyd, Derek R.; Sharma, Narain D.; Allen, Christopher C. R.
    Current Opinion in Biotechnology (2001), 12 (6), 564-573CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Science Ltd.)
    A review, with refs. Arom. dioxygenases have been found to catalyze single and tandem oxidn. reactions of conjugated polyenes. Rational selection and design of dioxygenases, allied to substrate shape, size and substitution pattern, has been used to control regiochem. and stereochem. during the oxygenation process. The resulting enantiopure bioproducts have been increasingly utilized as precursors for new and alternative routes in chiral synthesis.
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    Zhao, L.; Han, B.; Huang, Z.; Miller, M.; Huang, H.; Malashock, D. S.; Zhu, Z.; Milan, A.; Robertson, D. E.; Weiner, D. P. Epoxide Hydrolase-Catalyzed Enantioselective Synthesis of Chiral 1,2-Diols Via Desymmetrization of meso-Epoxides. J. Am. Chem. Soc. 2004, 126 (36), 11156– 7,  DOI: 10.1021/ja0466210
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    Epoxide Hydrolase-Catalyzed Enantioselective Synthesis of Chiral 1,2-Diols via Desymmetrization of meso-Epoxides
    Zhao, Lishan; Han, Bin; Huang, Zilin; Miller, Mark; Huang, Hongjun; Malashock, Dan S.; Zhu, Zuolin; Milan, Aileen; Robertson, Dan E.; Weiner, David P.; Burk, Mark J.
    Journal of the American Chemical Society (2004), 126 (36), 11156-11157CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The discovery, from nature, of a diverse set of microbial epoxide hydrolases is reported. The utility of a library of epoxide hydrolases in the synthesis of chiral 1,2-diols via desymmetrization of a wide range of meso-epoxides, including cyclic as well as acyclic alkyl- and aryl-substituted substrates, is demonstrated. The chiral (R,R)-diols were furnished with high ee's and yields. The discovery of the first microbial epoxide hydrolases providing access to complementary (S,S)-diols is also described.
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    Makarova, M.; Endoma-Arias, M. A. A.; Dela Paz, H. E.; Simionescu, R.; Hudlicky, T. Chemoenzymatic Total Synthesis of ent-Oxycodone: Second-, Third-, and Fourth-Generation Strategies. J. Am. Chem. Soc. 2019, 141 (27), 10883– 904,  DOI: 10.1021/jacs.9b05033
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    Chemoenzymatic Total Synthesis of ent-Oxycodone: Second-, Third-, and Fourth-Generation Strategies
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    Journal of the American Chemical Society (2019), 141 (27), 10883-10904CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Four distinct approaches to ent-oxycodone were designed and accomplished. All rely on the same starting material, the diene diol derived from phenethyl acetate by the whole-cell fermn. with E. coli JM109 (pDTG601A), a strain that overexpresses toluene dioxygenase. The key step in the first-generation approach involves the construction of the C-9/C-14 bond by a SmI2-mediated cyclization of a keto aldehyde. The second-generation design relies on the use of the Henry reaction to accomplish this task. In both of these syntheses, Parker's cyclization was employed to construct the D-ring. The third-generation synthesis provides an improvement over the second in that the nitrogen atom at C-9 is introduced by azidation of the C-9/C-10 olefin, followed by redn. and lactam formation between the C-9 amine and the Fukuyama-type lactone. Finally, the fourth generation takes advantage of the keto-nitrone reductive coupling to generate the C-9/C-14 linkage. The four generations of the total syntheses of ent-oxycodone were accomplished in 13, 18, 16, and 11 operations (19, 23, 24, and 18 steps), resp. Exptl. and spectral data are provided for all new compds.
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    Ikeda, H.; Sato, E.; Sugai, T.; Ohta, H. Yeast-Mediated Synthesis of Optically Active Diols with C2-Symmetry and (R)-4-Pentanolide. Tetrahedron 1996, 52 (24), 8113– 22,  DOI: 10.1016/0040-4020(96)00373-0
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    Redn. of some diketones and a keto acid with the yeast Pichia farinosa IAM 4682 was examd. The redn. of carbonyl groups proceeded highly selectively in an anti-Prelog fashion to give (R)-alcs. (2R,5R)-2,5-Hexanediol (83% yield, >99% e.e., 95% d.e.), (2R,4R)-2,4-pentanediol (95% yield, >99% e.e., 98% d.e.), and (R)-4-pentanolide (67% yield, >99% e.e.) were very efficiently obtained from the corresponding ketones. Effect of the structure of substrate on the stereochem. course as well as the selectivity were discussed.
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    Gijsen, Harrie J. M.; Wong, Chi-Huey
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    Several 6 substituted 2,4,6-trideoxyhexoses, e.g. I (R = H, OH, OMe, Cl, N3), have been stereoselectively synthesized from three achiral C2-aldehydes via a double aldol condensation catalyzed by 2-deoxyribose-5-phosphate aldolase (DERA, 4.1.2.4). This enzymic polymn. stops after two aldol reactions due to the formation of cyclic hemiacetals. The products can be converted to derivs. of the lactone moiety of mevinic acids and to building blocks useful in the synthesis of skipped-polyol macrolides.
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    Müller, M. Chemoenzymatic Synthesis of Building Blocks for Statin Side Chains. Angew. Chem., Int. Ed. 2005, 44 (3), 362– 5,  DOI: 10.1002/anie.200460852
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    Angewandte Chemie, International Edition (2005), 44 (3), 362-365CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review, with refs. Biocatalysis: Enzymic transformation have become competitive methods for synthesis of complex org. compds. The wide range of possibilities offered by biocatalysis for the synthesis of target mols. are highlighted based on the example of the statins. This class of compds., which includes rosuvastatin, are good inhibitors of cholesterol synthesis and dominate the market for cholesterol-lowering drugs.
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    Wu, X.; Jiang, J.; Chen, Y. Correlation between Intracellular Cofactor Concentrations and Biocatalytic Efficiency: Coexpression of Diketoreductase and Glucose Dehydrogenase for the Preparation of Chiral Diol for Statin Drugs. ACS Catal. 2011, 1 (12), 1661– 4,  DOI: 10.1021/cs200408y
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    Correlation between Intracellular Cofactor Concentrations and Biocatalytic Efficiency: Coexpression of Diketoreductase and Glucose Dehydrogenase for the Preparation of Chiral Diol for Statin Drugs
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    ACS Catalysis (2011), 1 (12), 1661-1664CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    Dynamic change of intracellular nicotinamide cofactor concns., the limiting factor for the bioredns. catalyzed by oxidoreductases, was monitored in Escherichia coli cells coexpressing diketoreductase and glucose dehydrogenase. On the basis of an unexpected observation, a relationship between catalytic efficiency and cofactor concns. was established to optimize the process for the prepn. of a chiral diol for statin drugs. Consequently, compared to previous reactions by E. coli cells expressing diketoreductase alone, exogenous addn. of cofactors was completely eliminated to yield an increase of substrate concn. by 15-fold. The present strategy could be employed in the biocatalytic processes catalyzed by nicotinamide-dependent oxidoreductases.
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    Huffman, M. A.; Fryszkowska, A.; Alvizo, O.; Borra-Garske, M.; Campos, K. R.; Canada, K. A.; Devine, P. N.; Duan, D.; Forstater, J. H.; Grosser, S. T. Design of an in Vitro Biocatalytic Cascade for the Manufacture of Islatravir. Science 2019, 366 (6470), 1255– 9,  DOI: 10.1126/science.aay8484
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    Science (Washington, DC, United States) (2019), 366 (6470), 1255-1259CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    Enzyme-catalyzed reactions have begun to transform pharmaceutical manufg., offering levels of selectivity and tunability that can dramatically improve chem. synthesis. Combining enzymic reactions into multistep biocatalytic cascades brings addnl. benefits. Cascades avoid the waste generated by purifn. of intermediates. They also allow reactions to be linked together to overcome an unfavorable equil. or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the no. of steps of the previously reported routes.
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    Bian, Z.; Liu, A.; Li, Y.; Fang, G.; Yao, Q.; Zhang, G.; Wu, Z. Boronic Acid Sensors with Double Recognition Sites: A Review. Analyst 2020, 145 (3), 719– 44,  DOI: 10.1039/C9AN00741E
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    Boronic acid sensors with double recognition sites: a review
    Bian, Zhancun; Liu, Aiqin; Li, Ying; Fang, Guiqian; Yao, Qingqiang; Zhang, Guimin; Wu, Zhongyu
    Analyst (Cambridge, United Kingdom) (2020), 145 (3), 719-744CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    A review. Boronic acids reversibly and covalently bind to Lewis bases and polyols, which facilitated the development of a large no. of chem. sensors to recognize carbohydrates, catecholamines, ions, hydrogen peroxide, and so on. However, as the binding mechanism of boronic acids and analytes is not very clear, it is still a challenge to discover sensors with high affinity and selectivity. In this review, boronic acid sensors with two recognition sites, including diboronic acid sensors, and monoboronic acid sensors having another group or binding moiety, are summarized. Owing to double recognition sites working synergistically, the binding affinity and selectivity of sensors can be improved significantly. This review may help researchers to sort out the binding rules and develop ideal boronic acid-based sensors.
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    Cao, H.; Heagy, M. D. Fluorescent Chemosensors for Carbohydrates: A Decade’s Worth of Bright Spies for Saccharides in Review. J. Fluoresc. 2004, 14 (5), 569– 84,  DOI: 10.1023/B:JOFL.0000039344.34642.4c
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    Fluorescent Chemosensors for Carbohydrates: A Decade's Worth of Bright Spies for Saccharides in Review
    Cao, Haishi; Heagy, Michael D.
    Journal of Fluorescence (2004), 14 (5), 569-584CODEN: JOFLEN; ISSN:1053-0509. (Kluwer Academic/Plenum Publishers)
    This review provides a chronol. survey of over fifty fluorescent chemosensors for carbohydrates from the period between 1992 to the present. The survey contains only those sensors that are synthetic or chemosensory, utilize boronic acids and display a fluorescence response in the form of intensity changes or shifts in wavelength. With each compd. listed, a description of the saccharide probe is given with regard to concn., excitation and emission wavelengths, pH and solvent mixt. proportions. In addn., the selectivity of each chemosensor is provided as well as the trends in binding consts. Where possible, a description of the fluorescence signaling mechanism is given as well as commentary on the probe's unique features within this class of sensors.
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    Fang, G.; Wang, H.; Bian, Z.; Sun, J.; Liu, A.; Fang, H.; Liu, B.; Yao, Q.; Wu, Z. Recent Development of Boronic Acid-Based Fluorescent Sensors. RSC Adv. 2018, 8 (51), 29400– 27,  DOI: 10.1039/C8RA04503H
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    Recent development of boronic acid-based fluorescent sensors
    Fang, Guiqian; Wang, Hao; Bian, Zhancun; Sun, Jie; Liu, Aiqin; Fang, Hao; Liu, Bo; Yao, Qingqiang; Wu, Zhongyu
    RSC Advances (2018), 8 (51), 29400-29427CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    As Lewis acids, boronic acids can bind with 1,2- or 1,3-diols in aq. soln. reversibly and covalently to form five or six cyclic esters, thus resulting in significant fluorescence changes. Based on this phenomenon, boronic acid compds. have been well developed as sensors to recognize carbohydrates or other substances. Several reviews in this area have been reported before, however, novel boronic acid-based fluorescent sensors have emerged in large nos. in recent years. This paper reviews new boron-based sensors from the last five years that can detect carbohydrates such as glucose, ribose and sialyl Lewis A/X, and other substances including catecholamines, reactive oxygen species, and ionic compds. And emerging electrochem. related fluorescent sensors and functionalized boronic acid as new materials including nanoparticles, smart polymer gels, and quantum dots were also involved. By summarizing and discussing these newly developed sensors, we expect new inspiration in the design of boronic acid-based fluorescent sensors.
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    Wang, W.; Gao, X.; Wang, B. Boronic Acid-Based Sensors. Curr. Org. Chem. 2002, 6 (14), 1285– 317,  DOI: 10.2174/1385272023373446
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    Boronic acid-based sensors
    Wang, Wei; Gao, Xingming; Wang, Binghe
    Current Organic Chemistry (2002), 6 (14), 1285-1317CODEN: CORCFE; ISSN:1385-2728. (Bentham Science Publishers)
    A review. There was a great deal of interest in recent years in using boronic acid as the recognition motif for the development of sensors. Because boronic acids can form tight and reversible complexes with diol compds. such as carbohydrates, the majority of the efforts, led by the Shinkai group, were on the development of sensors for carbohydrates. Boronic acids are also known to selectively recognize fluoride among halides and other anions. Therefore, there also were efforts in using boronic acid compds. for the development fluoride sensors. This paper reviews the progress in this field during the last five years.
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    Wu, X.; Li, Z.; Chen, X.-X.; Fossey, J. S.; James, T. D.; Jiang, Y.-B. Selective Sensing of Saccharides Using Simple Boronic Acids and Their Aggregates. Chem. Soc. Rev. 2013, 42 (20), 8032– 48,  DOI: 10.1039/c3cs60148j
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    Selective sensing of saccharides using simple boronic acids and their aggregates
    Wu, Xin; Li, Zhao; Chen, Xuan-Xuan; Fossey, John S.; James, Tony D.; Jiang, Yun-Bao
    Chemical Society Reviews (2013), 42 (20), 8032-8048CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. The reversible boronic acid-diol interaction empowers boronic acid receptors' saccharide binding capacities, rendering them a class of lectin mimetic, termed "boronlectins". Boronic acids follow lectin functions not just in being able to bind saccharides, but in multivalent saccharide binding that enhances both affinity and selectivity. For almost a decade, efforts were made to achieve and improve selectivity for given saccharide targets, most notably glucose, by properly positioned boronic acids, offering multivalent interactions. Incorporation of several boronic acid groups into a covalent framework or non-covalent assembly of boronic acid are two general methods used to create such smart sensors, of which the latter resembles lectin oligomerization that affords multivalent saccharide-binding architectures. In this review, supramol. selective sensing of saccharides by simple boronic acids in their aggregate forms is discussed, after a brief survey of the general aspects of boronic acid-based saccharide sensing.
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    Arnaud, J.; Audfray, A.; Imberty, A. Binding Sugars: From Natural Lectins to Synthetic Receptors and Engineered Neolectins. Chem. Soc. Rev. 2013, 42 (11), 4798– 813,  DOI: 10.1039/c2cs35435g
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    Binding sugars: from natural lectins to synthetic receptors and engineered neolectins
    Arnaud, Julie; Audfray, Aymeric; Imberty, Anne
    Chemical Society Reviews (2013), 42 (11), 4798-4813CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. The large diversity and complexity of glycan structures together with their crucial role in many biol. or pathol. processes require the development of new high-throughput techniques for analyses. Lectins are classically used for characterizing, imaging or targeting glycoconjugates and, when printed on microarrays, they are very useful tools for profiling glycomes. Development of recombinant lectins gives access to reliable and reproducible material, while engineering of new binding sites on existing scaffolds allows tuning of specificity. From the accumulated knowledge on protein-carbohydrate interactions, it is now possible to use nucleotide and peptide (bio)synthesis for producing new carbohydrate-binding mols. Such a biomimetic approach can also be addressed by boron chem. and supra-mol. chem. for the design of fully artificial glycosensors.
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    Wu, X.; Chen, X.-X.; Jiang, Y.-B. Recent Advances in Boronic Acid-Based Optical Chemosensors. Analyst 2017, 142 (9), 1403– 14,  DOI: 10.1039/C7AN00439G
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    Recent advances in boronic acid-based optical chemosensors
    Wu, Xin; Chen, Xuan-Xuan; Jiang, Yun-Bao
    Analyst (Cambridge, United Kingdom) (2017), 142 (9), 1403-1414CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    A review. Reversible covalent binding of boronic acids with polyols and Lewis bases has facilitated the development of robust chemosensors for many biol. important species under physiol. or environmentally relevant conditions. This minireview covers selected examples of advances reported in this area from 2014 to 2016. While the discovery of new boron-contg. binding motifs and identification of new anal. targets have expanded the utility of boronic acid-based mol. recognition, unconventional sensing strategies such as exploitation of nanoscale self-assembly, multicomponent dynamic covalent assembly, and coupling boronate ester formation with a further chem. reaction have led to significantly improved sensor performance, enabling real-world applications in various areas such as cell biol. and asym. catalysis.
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    Kondo, K.; Shiomi, Y.; Saisho, M.; Harada, T.; Shinkai, S. Specific Complexation of Disaccharides with Diphenyl-3,3′-Diboronic Acid That Can Be Detected by Circular Dichroism. Tetrahedron 1992, 48 (38), 8239– 52,  DOI: 10.1016/S0040-4020(01)80492-0
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    Specific complexation of disaccharides with diphenyl-3,3'-diboronic acid that can be detected by circular dichroism
    Kondo, Kaoru; Shiomi, Yutaka; Saisho, Miwako; Harada, Takaaki; Shinkai, Seiji
    Tetrahedron (1992), 48 (38), 8239-52CODEN: TETRAB; ISSN:0040-4020.
    Diphenyldiboronic acid I was prepd. as a new receptor that can recognize sugar mols. The distance between the two boronic acids in I is designed so that it can selectively form cyclic 1:1 complexes with disaccharides. It was shown that I forms 1:1 complexes with several disaccharides and gives the characteristic exciton coupling in CD spectroscopy owing to immobilization of the two Ph rings. Thus, the abs. configuration was successfully predicted from the sign of the exciton coupling.
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    Yoon, J.; Czarnik, A. W. Fluorescent Chemosensors of Carbohydrates. A Means of Chemically Communicating the Binding of Polyols in Water Based on Chelation-Enhanced Quenching. J. Am. Chem. Soc. 1992, 114 (14), 5874– 5,  DOI: 10.1021/ja00040a067
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    Fluorescent chemosensors of carbohydrates. A means of chemically communicating the binding of polyols in water based on chelation-enhanced quenching
    Yoon, Juyoung; Czarnik, Anthony W.
    Journal of the American Chemical Society (1992), 114 (14), 5874-5CODEN: JACSAT; ISSN:0002-7863.
    2-Anthrylboronic acid (I; pKa 8.8) displays decreased fluorescence in the boronate form compared with its boronic acid form, which is attributed to photoinduced electron transfer. When I complexes to polyols such as fructose, the pKa of the complex decreases; thus, the fluorimetrically detd. pKa of the I:fructose borate ester is 5.9. These properties combined permit polyol complexation in water to be obsd. spectrofluorometrically, which has not been reported previously. Fructose binds with a fluorimetrically detd. Kd of 3.7 mM.
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    Elfeky, S. Novel Bronic Acid-Based Fluorescent Sensor for Sugars and Nucleosides. Curr. Org. Synth. 2011, 8 (6), 872,  DOI: 10.2174/1570179411108060872
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    Novel bronic acid-based fluorescent sensor for sugars and nucleosides
    Elfeky, Souad
    Current Organic Synthesis (2011), 8 (6), 872-880CODEN: COSUC2; ISSN:1570-1794. (Bentham Science Publishers Ltd.)
    Sugar and nucleoside identifications are important for the detection and treatment of dangerous diseases. In this study, a fluorescent boronic acid (3-(5-(dimethylamino)naphthalene-1-sulfonamido)phenyl)boronic acid (DNSBA) was bound to a diol quencher via a boronic ester linkage, and fluorescence recovery upon exposure to saccharides and nucleosides was demonstrated. Further, we successfully detected sugars and nucleosides in buffer systems. DNSBA expresses selectivity toward adenosine, fructose, sorbitol, and tartaric acid. Our investigations into the effects of pH on sensor sensitivity revealed that changes between the neutral and anionic forms of the boronic acid group, induced at high pH and/or in the presence of sugars or nucleosides, induce visible/optimal changes in DNSBA. DNSBA is responsive at pH = 8.21 whereas acidic media do not exhibit sensitivity. DNSBA can thus be useful in clin. applications as a novel sensor for sugars and nucleosides.
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    James, T. D.; Shinkai, S. Artificial Receptors as Chemosensors for Carbohydrates. Host-Guest Chemistry 2002, 218, 159– 200,  DOI: 10.1007/3-540-45010-6_6
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    Springsteen, G.; Wang, B. A Detailed Examination of Boronic Acid-Diol Complexation. Tetrahedron 2002, 58 (26), 5291– 300,  DOI: 10.1016/S0040-4020(02)00489-1
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    A detailed examination of boronic acid-diol complexation
    Springsteen, Greg; Wang, Binghe
    Tetrahedron (2002), 58 (26), 5291-5300CODEN: TETRAB; ISSN:0040-4020. (Elsevier Science Ltd.)
    Boronic acids bind with compds. contg. diol moieties with high affinity through reversible boronate formation. However, the conditions that foster tight binding between the diol and the boronic acid are not well understood. Also, due to the multiple ionic states of both the boronic acid and boronate ester, the equil. consts. reported in the literature have not always been strictly defined, and therefore there is a lack of comparability between the reported values. To address these issues, a method was developed for examg. boronate ester stability using the fluorescent reporter Alizarin Red S. and this system has been used to det. the binding consts. of a series of diols, and as a basis from which to derive a no. of relationships that correlate the various equil. consts. in the literature.
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    Jin, S.; Cheng, Y.; Reid, S.; Li, M.; Wang, B. Carbohydrate Recognition by Boronolectins, Small Molecules, and Lectins. Med. Res. Rev. 2009, 30, 171– 257,  DOI: 10.1002/med.20155
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    Yan, J.; Fang, H.; Wang, B. Boronolectins and Fluorescent Boronolectins: An Examination of the Detailed Chemistry Issues Important for the Design. Med. Res. Rev. 2005, 25 (5), 490– 520,  DOI: 10.1002/med.20038
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    Boronolectins and fluorescent boronolectins: An examination of the detailed chemistry issues important for the design
    Yan, Jun; Fang, Hao; Wang, Binghe
    Medicinal Research Reviews (2005), 25 (5), 490-520CODEN: MRREDD; ISSN:0198-6325. (John Wiley & Sons, Inc.)
    A review. Glycans in the form of glycoproteins or glycolipids play very crit. roles in various biol. and pathol. processes including inflammation, cancer metastasis, immune reactions, embryo development, cell-cell communications and adhesions, blood generation, etc. Proteins (lectins) that can recognize carbohydrates have played very important roles in studying glycobiol. Small mol. mimics of lectins will be very useful in the development of new therapeutics and diagnostics. Along this line, boronic acids have been widely used in the design and synthesis of small org. compds. that mimic the function of lectins. This review examines in detail the factors that are important for the design of boronic acid-based lectin mimics, boronolectins.
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    Gao, X.; Zhang, Y.; Wang, B. New Boronic Acid Fluorescent Reporter Compounds. 2. A Naphthalene-Based On-Off Sensor Functional at Physiological pH. Org. Lett. 2003, 5 (24), 4615– 8,  DOI: 10.1021/ol035783i
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    New boronic acid fluorescent reporter compounds. 2. A naphthalene-based on-off sensor functional at physiological pH
    Gao, Xingming; Zhang, Yanling; Wang, Binghe
    Organic Letters (2003), 5 (24), 4615-4618CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
    A new boronic acid fluorescent on-off reporter compd. (1) was synthesized. This fluorescent sensor shows a 41-fold emission intensity increase upon addn. of 50 mM fructose in 0.1 M aq. phosphate buffer at pH 7.4.
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    Gao, X.; Zhang, Y.; Wang, B. A Highly Fluorescent Water-Soluble Boronic Acid Reporter for Saccharide Sensing That Shows Ratiometric UV Changes and Significant Fluorescence Changes. Tetrahedron 2005, 61 (38), 9111– 7,  DOI: 10.1016/j.tet.2005.07.035
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    A highly fluorescent water-soluble boronic acid reporter for saccharide sensing that shows ratiometric UV changes and significant fluorescence changes
    Gao, Xingming; Zhang, Yanling; Wang, Binghe
    Tetrahedron (2005), 61 (38), 9111-9117CODEN: TETRAB; ISSN:0040-4020. (Elsevier B.V.)
    One water-sol. naphthalene-based fluorescent boronic acid, 6-(dimethylamino)-naphthalene-2-boronic acid (6-DMANBA), has been synthesized. 6-DMANBA shows significant ratiometric UV absorbance changes upon addn. of a sugar. For example, addn. of 50 mM fructose shifted the UV absorption wavelengths of 6-DMANBA from 306 and 251 to 280 and 244 nm, resp. In addn., 6-DMANBA is highly fluorescent with a quantum yield of 89% in the absence of a sugar and shows significant fluorescence intensity changes with the addn. of a saccharide in aq. phosphate buffer at physiol. pH. For example, with the addn. of 50 mM fructose, 6-DMANBA shows an 80% fluorescent intensity decrease at 432 nm. All these spectroscopic properties make compd. 1 unique and useful.
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    Gao, X.; Zhang, Y.; Wang, B. Naphthalene-Based Water-Soluble Fluorescent Boronic Acid Isomers Suitable for Ratiometric and Off-On Sensing of Saccharides at Physiological pH. New J. Chem. 2005, 29 (4), 579– 86,  DOI: 10.1039/b413376e
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    Naphthalene-based water-soluble fluorescent boronic acid isomers suitable for ratiometric and off-on sensing of saccharides at physiological pH
    Gao, Xingming; Zhang, Yanling; Wang, Binghe
    New Journal of Chemistry (2005), 29 (4), 579-586CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
    Two water-sol. naphthalene-based fluorescent boronic acid isomers, 5-(dimethylamino)naphthalene-1-boronic acid (5-DMANBA) and 4-(dimethylamino)naphthalene-1-boronic acid (4-DMANBA), have been synthesized; their fluorescent properties upon binding with carbohydrates were detd. in aq. phosphate buffer at pH 7.4. The difference in substitution pattern between 5-DMANBA and 4-DMANBA leads to significant differences in their fluorescence properties. For example, addn. of fructose (50 mM) to a soln. of 5-DMANBA induced a 61% fluorescence intensity decrease at 513 nm and a 36-fold increase at 433 nm. This revealed that 5-DMANBA is a potential sensor for ratiometric sensing of sugars. The pH titrn. curves of 5-DMANBA in the absence and the presence of fructose (50 mM) showed a 93- and 200-fold fluorescence intensity increase at 433 nm, resp., when pH was increased from 3 to 10. 4-DMANBA, however, does not show ratiometric fluorescent intensity changes, but shows significant fluorescence intensity increase upon addn. of a sugar (41-fold intensity increase with 50 mM fructose). The emission intensity of 4-DMANBA increased by over 170-fold at 445 nm upon changing the pH from 2 to 11.
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    Zhang, Y.; Gao, X.; Hardcastle, K.; Wang, B. Water-Soluble Fluorescent Boronic Acid Compounds for Saccharide Sensing: Substituent Effects on Their Fluorescence Properties. Eur. J. Chem. 2006, 12 (5), 1377– 84,  DOI: 10.1002/chem.200500982
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    Water-soluble fluorescent boronic acid compounds for saccharide sensing: substituent effects on their fluorescence properties
    Zhang, Yanling; Gao, Xingming; Hardcastle, Kenneth; Wang, Binghe
    Chemistry - A European Journal (2006), 12 (5), 1377-1384CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Four new naphthalene-based boronic acid I (R = R1 = H; R = Me, R1 = H, COMe, CH2CO2Bu-t) were synthesized. The effect of various carbohydrates on their fluorescence properties has been studied in aq. phosphate buffer at pH 7.4. Different substitutions on the aniline group of the naphthalene ring resulted in significant differences in fluorescence properties for these four compds. Compd. I (R = Me, R1 = CH2CO2Bu-t) shows ratiometric fluorescence changes upon addn. of a sugar. Compds. I (R = R1 = H; R = Me, R1 = H) do not show ratiometric fluorescence changes but show very large fluorescence intensity changes (about 70-fold fluorescence intensity increase). In addn. to the quantifiable fluorescence property changes upon sugar addn., the fluorescence color changes of I (R = R1 = H; R = Me, R1 = H, CH2CO2Bu-t) are also visible to the naked eye. However, amidation of the aniline nitrogen atom significantly diminishes the fluorescence intensity of compd. I (R = Me, R1 = COMe). The crystal structure of one boronic acid provided some insight into the structural features that are important for the fluorescence properties of these compds. These compds. I show very large fluorescence property changes upon monosaccharide binding. Addn. of fructose (50 mM) to the soln. of I (R = Me, R1 = CH2CO2Bu-t) induced a 4.2-fold fluorescence intensity increase at 440 nm and a 32 % fluorescence intensity decrease at 490 nm. The introduction of an ester functional group on the side chain does not abolish the ability of the fluorophore to show a ratiometric response to binding with a sugar.
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    Yang, W.; Yan, J.; Springsteen, G.; Deeter, S.; Wang, B. A Novel Type of Fluorescent Boronic Acid That Shows Large Fluorescence Intensity Changes Upon Binding with a Carbohydrate in Aqueous Solution at Physiological pH. Bioorg. Med. Chem. Lett. 2003, 13 (6), 1019– 22,  DOI: 10.1016/S0960-894X(03)00086-6
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    A novel type of fluorescent boronic acid that shows large fluorescence intensity changes upon binding with a carbohydrate in aqueous solution at physiological pH
    Yang, Wenqian; Yan, Jun; Springsteen, Greg; Deeter, Susan; Wang, Binghe
    Bioorganic & Medicinal Chemistry Letters (2003), 13 (6), 1019-1022CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science B.V.)
    In this paper we report 8-quinoline boronic acid as a novel type of fluorescent probe for carbohydrates. This boronic acid responds to the binding of a carbohydrate with over 40-fold increases in fluorescence intensity and shows optimal fluorescence change at physiol. pH in aq. soln.
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    Yang, W.; Lin, L.; Wang, B. A New Type of Boronic Acid Fluorescent Reporter Compound for Sugar Recognition. Tetrahedron Lett. 2005, 46 (46), 7981– 4,  DOI: 10.1016/j.tetlet.2005.09.074
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    A new type of boronic acid fluorescent reporter compound for sugar recognition
    Yang, Wenqian; Lin, Li; Wang, Binghe
    Tetrahedron Letters (2005), 46 (46), 7981-7984CODEN: TELEAY; ISSN:0040-4039. (Elsevier B.V.)
    Fluorescent boronic acids that change fluorescent properties upon carbohydrate binding are very useful for the prepn. of fluorescent sensors for sugars. Herein the authors report 5-quinolineboronic acid (5-QBA) that shows significant fluorescent property changes through a unique pKa-switching mechanism upon binding a diol in aq. soln.
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    Cheng, Y.; Ni, N.; Yang, W.; Wang, B. A New Class of Fluorescent Boronic Acids That Have Extraordinarily High Affinities for Diols in Aqueous Solution at Physiological pH. Eur. J. Chem. 2010, 16 (45), 13528– 38,  DOI: 10.1002/chem.201000637
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    A New Class of Fluorescent Boronic Acids That Have Extraordinarily High Affinities for Diols in Aqueous Solution at Physiological pH
    Cheng, Yunfeng; Ni, Nanting; Yang, Wenqian; Wang, Binghe
    Chemistry - A European Journal (2010), 16 (45), 13528-13538, S13528/1-S13528/40CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The boronic acid group is an important recognition moiety for sensor design. Herein, the authors report a series of isoquinolinylboronic acids that have extraordinarily high affinities for diol-contg. compds. at physiol. pH. In addn., 5- and 8-isoquinolinylboronic acids also showed fairly high binding affinities towards D-glucose (Ka=42 and 46 M-1, resp.). For the first time, weak but encouraging binding of cis-cyclohexanediol was found for these boronic acids. Such binding was coupled with significant fluorescence changes. Furthermore, 4- and 6-isoquinolinylboronic acids also showed the ability to complex Me α-D-glucopyranose (Ka=3 and 2 M-1, resp.).
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    Samaniego Lopez, C.; Lago Huvelle, M. A.; Uhrig, M. L.; Coluccio Leskow, F.; Spagnuolo, C. C. Recognition of Saccharides in the NIR Region with a Novel Fluorogenic Boronolectin: In Vitro and Live Cell Labeling. Chem. Commun. 2015, 51 (23), 4895– 8,  DOI: 10.1039/C4CC10425K
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    Recognition of saccharides in the NIR region with a novel fluorogenic boronolectin: in vitro and live cell labeling
    Samaniego Lopez, Cecilia; Lago Huvelle, Maria Amparo; Uhrig, Maria Laura; Coluccio Leskow, Federico; Spagnuolo, Carla C.
    Chemical Communications (Cambridge, United Kingdom) (2015), 51 (23), 4895-4898CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    This work describes a novel mono-boronic acid deriv. of a tricarbocyanine. The probe is a genuine near-IR fluorescence emitter with improved properties such as a large Stokes shift, excellent water soly. and sensitive fluorogenicity upon binding to carbohydrates under physiol. conditions.
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    Wang, L.; Yuan, L.; Zeng, X.; Peng, J.; Ni, Y.; Er, J. C.; Xu, W.; Agrawalla, B. K.; Su, D.; Kim, B. A Multisite-Binding Switchable Fluorescent Probe for Monitoring Mitochondrial ATP Level Fluctuation in Live Cells. Angew. Chem., Int. Ed. 2016, 55 (5), 1773– 6,  DOI: 10.1002/anie.201510003
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    Multisite-Binding Switchable Fluorescent Probe for Monitoring Mitochondrial ATP Level Fluctuation in Live Cells
    Wang, Lu; Yuan, Lin; Zeng, Xian; Peng, Juanjuan; Ni, Yong; Er, Jun Cheng; Xu, Wang; Agrawalla, Bikram Keshari; Su, Dongdong; Kim, Beomsue; Chang, Young-Tae
    Angewandte Chemie, International Edition (2016), 55 (5), 1773-1776CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    ATP, commonly produced in mitochondria, is required by almost all the living organisms; thus fluorescent probes for monitoring mitochondrial ATP level fluctuation are essential and highly desired. Herein, the authors report a multisite-binding switchable fluorescent probe, ATP-Red 1 (I), which selectively and rapidly responds to intracellular concns. of ATP. Live-cell imaging indicated that ATP-Red 1 mainly localized to mitochondria with good biocompatibility and membrane penetration. In particular, with the help of ATP-Red 1, the authors successfully obsd. not only the decreased mitochondrial ATP levels in the presence of KCN and starvation state, but also the increased mitochondrial ATP levels in the early stage of cell apoptosis. ATP-Red 1 is a useful tool for studying ATP-relevant biol. processes.
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    Huang, Y.-J.; Ouyang, W.-J.; Wu, X.; Li, Z.; Fossey, J. S.; James, T. D.; Jiang, Y.-B. Glucose Sensing Via Aggregation and the Use of “Knock-Out” Binding to Improve Selectivity. J. Am. Chem. Soc. 2013, 135 (5), 1700– 3,  DOI: 10.1021/ja311442x
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    Glucose Sensing via Aggregation and the Use of "Knock-Out" Binding To Improve Selectivity
    Huang, Yan-Jun; Ouyang, Wen-Juan; Wu, Xin; Li, Zhao; Fossey, John S.; James, Tony D.; Jiang, Yun-Bao
    Journal of the American Chemical Society (2013), 135 (5), 1700-1703CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Aggregates of an amphiphilic monoboronic acid bearing a hydrophobic pyrene fluorophore were employed for highly modulating, sensitive, and selective ratiometric fluorescent sensing of glucose in aq. soln. The selectivity for glucose was improved by "knock-out" binding of fructose by phenylboronic acid.
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    Vilozny, B.; Schiller, A.; Wessling, R. A.; Singaram, B. Enzyme Assays with Boronic Acid Appended Bipyridinium Salts. Anal. Chim. Acta 2009, 649 (2), 246– 51,  DOI: 10.1016/j.aca.2009.07.032
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    Enzyme assays with boronic acid appended bipyridinium salts
    Vilozny, Boaz; Schiller, Alexander; Wessling, Ritchie A.; Singaram, Bakthan
    Analytica Chimica Acta (2009), 649 (2), 246-251CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    In-vitro fluorescent enzyme assays have been developed for sucrose phosphorylase (SPO) and phosphoglucomutase (PGM). These assays make use of a selective carbohydrate sensing system that detects the unlabeled enzymic products fructose and glucose-6-phosphate. The system comprises 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt as the reporter unit and boronic acid appended viologens as selective receptors with working ranges from 70 μM to 1.0 mM for fructose (SPO) and 190 μM to 2.0 mM for glucose-6-phosphate (PGM). The change in fluorescence can be converted into product concn., allowing initial reaction velocities and Michaelis-Menten kinetics to be calcd. The assays are also carried out in multiwell plate formats, making them suitable for high-throughput screening of enzyme inhibitors. Rapid PGM inhibition screening is demonstrated with EDTA and LiCl. The PGM assay can also be used for enzyme quantification with a detection limit of 50 ng mL-1.
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    Sharrett, Z.; Gamsey, S.; Hirayama, L.; Vilozny, B.; Suri, J. T.; Wessling, R. A.; Singaram, B. Exploring the Use of APTS as a Fluorescent Reporter Dye for Continuous Glucose Sensing. Org. Biomol. Chem. 2009, 7 (7), 1461– 70,  DOI: 10.1039/b821934f
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    Exploring the use of APTS as a fluorescent reporter dye for continuous glucose sensing
    Sharrett, Zachary; Gamsey, Soya; Hirayama, Lacie; Vilozny, Boaz; Suri, Jeff T.; Wessling, Ritchie A.; Singaram, Bakthan
    Organic & Biomolecular Chemistry (2009), 7 (7), 1461-1470CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    The anionic fluorescent dye, aminopyrene trisulfonic acid (APTS), was synthesized and used in a soln.-based two-component glucose-sensing system comprising the dye and a boronic acid-appended viologen. The fluorescence of the dye was quenched in the presence of the viologen and the fluorescence restored upon glucose addn. An important feature of this fluorophore is that it can be covalently bonded to a polymer through the amine group without a significant effect on optical properties. Two APTS derivs., functionalized with polymerizable groups, were synthesized and immobilized in hydroxyethyl methacrylate (HEMA)-based hydrogels. The latter were used to continuously monitor glucose. The fluorescence signal modulation, signal stability, reversibility, reproducibility, and pH sensitivity of the hydrogels were evaluated. The APTS dyes described herein are insensitive to pH changes within the physiol. range, both in soln. and when immobilized in a hydrogel. When APTS is used in conjunction with boronic acid-appended viologens to sense glucose, the system displays some pH sensitivity because of the presence of the boronic acid.
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    James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. A Glucose-Selective Molecular Fluorescence Sensor. Angew. Chem., Int. Ed. 1994, 33 (21), 2207– 9,  DOI: 10.1002/anie.199422071
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    James, T. D.; Sandanayake, K. R. A. S.; Iguchi, R.; Shinkai, S. Novel Saccharide-Photoinduced Electron Transfer Sensors Based on the Interaction of Boronic Acid and Amine. J. Chem. Soc., Chem. Commun. 1995, 117 (35), 8982– 7,  DOI: 10.1021/ja00140a013
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    Tran, T. M.; Alan, Y.; Glass, T. E. A Highly Selective Fluorescent Sensor for Glucosamine. Chem. Commun. 2015, 51 (37), 7915– 8,  DOI: 10.1039/C5CC00415B
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    A highly selective fluorescent sensor for glucosamine
    Tran, Tam Minh; Alan, Yuksel; Glass, Timothy Edward
    Chemical Communications (Cambridge, United Kingdom) (2015), 51 (37), 7915-7918CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A new fluorescent chem. sensor for glucosamine is reported. The sensor is based on a boronic acid-contg. coumarin aldehyde and shows excellent selectivity for glucosamine by forming a boronic ester with the sugar diol as well as an iminium ion with the amine group of glucosamine. The sensor successfully discriminates glucosamine over other similar biomols. in terms of both fluorescence intensity and binding affinity. This method provides a new concept for the design and synthesis of very selective turn-on optical sensors for selective detection of multi-functional biomols.
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    Rout, B.; Unger, L.; Armony, G.; Iron, M. A.; Margulies, D. Medication Detection by a Combinatorial Fluorescent Molecular Sensor. Angew. Chem., Int. Ed. 2012, 51 (50), 12477– 81,  DOI: 10.1002/anie.201206374
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    Medication Detection by a Combinatorial Fluorescent Molecular Sensor
    Rout, Bhimsen; Unger, Linor; Armony, Gad; Iron, Mark A.; Margulies, David
    Angewandte Chemie, International Edition (2012), 51 (50), 12477-12481CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Herein, we describe the design and function of a combinatorial fluorescent sensor that takes mol.-scale diagnostics a step further. The mol. anal. system presented herein combines several recognition elements as well as four emission channels and utilizes distinct photo-phys. processes that enable us to identify a wide range of pharmaceuticals and analyze drug concns. and combinations in urine samples in a high-throughput manner. The development of methods for the verification of drug content at point-of-care has been receiving growing international attention. We therefore selected four drug families commonly assocd. with counterfeiting or medication errors as test cases for our mol. sensor. Macrolides, aminoglycosides, and rifamycins are large families of antibiotics whose counterfeits are highly prevalent in the developing world. Cardiac glycosides, used for treating heart conditions, have been assocd. with substandard medication in developed countries and are often involved in medication errors owing to their narrow therapeutic window and adverse drug interactions. To be able to identify different macrolides, aminoglycosides, cardiac glycosides, and rifamycins with a single fluorescent mol., we designed and synthesized sensor (I).
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    Liu, Y.; Deng, C.; Tang, L.; Qin, A.; Hu, R.; Sun, J. Z.; Tang, B. Z. Specific Detection of D-Glucose by a Tetraphenylethene-Based Fluorescent Sensor. J. Am. Chem. Soc. 2011, 133 (4), 660– 3,  DOI: 10.1021/ja107086y
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    Specific Detection of D-Glucose by a Tetraphenylethene-Based Fluorescent Sensor
    Liu, Yi; Deng, Chunmei; Tang, Li; Qin, Anjun; Hu, Rongrong; Sun, Jing Zhi; Tang, Ben Zhong
    Journal of the American Chemical Society (2011), 133 (4), 660-663CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A conceptually new "light-up" biosensor with a high specificity for D-glucose (Glu) in aq. media has been developed. The emission from a tetraphenylethene (TPE)-cored diboronic acid (I) was greatly boosted when the fluorogen was oligomerized with Glu because of restriction of the intramol. rotations of the aryl rotors of TPE by formation of the oligomer. Little change in the light emission was obsd. when I was mixed with D-fructose, D-galactose, or D-mannose, as these saccharides are unable to oligomerize with the fluorogen.
  260. 260
    Shcherbakova, E. G.; Brega, V.; Lynch, V. M.; James, T. D.; Anzenbacher, P., Jr. High-Throughput Assay for Enantiomeric Excess Determination in 1,2- and 1,3-Diols and Direct Asymmetric Reaction Screening. Eur. J. Chem. 2017, 23 (42), 10222– 9,  DOI: 10.1002/chem.201701923
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    High-Throughput Assay for Enantiomeric Excess Determination in 1,2- and 1,3-Diols and Direct Asymmetric Reaction Screening
    Shcherbakova, Elena G.; Brega, Valentina; Lynch, Vincent M.; James, Tony D.; Anzenbacher, Pavel, Jr.
    Chemistry - A European Journal (2017), 23 (42), 10222-10229CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A simple and efficient method for detn. of the yield, enantiomeric/diasteriomeric excess (ee/de), and abs. configuration of crude chiral diols without the need of work-up and product isolation in a high throughput setting is described. This approach uses a self-assembled iminoboronate ester formed as a product by dynamic covalent self-assembly of a chiral diol with an enantiopure fluorescent amine such as tryptophan Me ester or tryptophanol and 2-formylphenylboronic acid. The resulting diastereomeric boronates display different photophys. properties and allow for fluorescence-based ee detn. of mols. contg. a 1,2- or 1,3-diol moiety. This method was used for the screening of ee in a no. of chiral diols including atorvastatin, a statin used for the treatment of hypercholesterolemia. Noyori asym. hydrogenation of benzil was performed in a highly parallel fashion with errors <1% ee confirming the feasibility of the systematic examn. of crude products from the parallel asym. synthesis in real time and in a high-throughput screening (HTS) fashion.
  261. 261
    Shcherbakova, E. G.; James, T. D.; Anzenbacher, P. High-Throughput Assay for Determining Enantiomeric Excess of Chiral Diols, Amino Alcohols, and Amines and for Direct Asymmetric Reaction Screening. Nat. Protoc. 2020, 15 (7), 2203– 29,  DOI: 10.1038/s41596-020-0329-1
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    263
    High-throughput assay for determining enantiomeric excess of chiral diols, amino alcohols, and amines and for direct asymmetric reaction screening
    Shcherbakova, Elena G.; James, Tony D.; Anzenbacher Jr., Pavel
    Nature Protocols (2020), 15 (7), 2203-2229CODEN: NPARDW; ISSN:1750-2799. (Nature Research)
    A review. Abstr.: Detg. enantiomeric excess (e.e.) in chiral compds. is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for detg. e.e. in mixts. of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcs., and amino-acid esters. The method is based on dynamic self-assembly of com. available chiral amines, 2-formylphenylboronic acid, and chiral diols in acetonitrile to form fluorescent diastereomeric complexes. Each analyte enantiomer engenders a diastereomer with distinct fluorescence wavelength/intensity originating from enantiopure fluorescent ligands. In this assay, enantiomers of amines and amine derivs. assemble with diol-type ligands contg. a binaphthol moiety (BINOL and VANOL), whereas diol enantiomers form complexes with the enantiopure amine-type fluorescent ligand tryptophanol. The differential fluorescence is utilized to det. the amt. of each enantiomer in the mixt. with an error of <1% e.e. This method enables high-throughput real-time evaluation of enantiomeric/diastereomeric excess (e.e./d.e.) and product yield of crude asym. reaction products. The procedure comprises high-throughput liq. dispensing of three components into 384-well plates and recording of fluorescence using an automated plate reader. The approach enables scaling up the screening of combinatorial libraries and, together with parallel synthesis, creates a robust platform for discovering chiral catalysts or auxiliaries for asym. transformations and chiral drug development. The procedure takes ∼4-6 h and requires 10-20 ng of substrate per well. Our fluorescence-based assay offers distinct advantages over existing methods because it is not sensitive to the presence of common additives/impurities or unreacted/incompletely utilized reagents or catalysts.
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    Doderer, K.; Lutz-Wahl, S.; Hauer, B.; Schmid, R. D. Spectrophotometric Assay for Epoxide Hydrolase Activity toward any Epoxide. Anal. Biochem. 2003, 321 (1), 131– 4,  DOI: 10.1016/S0003-2697(03)00399-3
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    Spectrophotometric assay for epoxide hydrolase activity toward any epoxide
    Doderer, Kai; Lutz-Wahl, Sabine; Hauer, Bernhard; Schmid, Rolf D.
    Analytical Biochemistry (2003), 321 (1), 131-134CODEN: ANBCA2; ISSN:0003-2697. (Elsevier Science)
    A universal colorimetric high-throughput assay system for the quantification of the reaction products of epoxide hydrolase activity in bacterial cell exts. is described. The assay is based on the quant. colorimetric detection of ketones and aldehydes with Schiff's reagent contg. fuchsin and sulfurous acid. The test is reliable and sensitive with a detection limit of 5 μmol diol in 100 μL soln. For higher amts. of diol the soln. must be dild. to quantify the diol content. The suggested assay for epoxide hydrolase activity toward any epoxide is sensitive and reproducible and is feasible in crude cell exts.
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    Wahler, D.; Reymond, J.-L. The Adrenaline Test for Enzymes. Angew. Chem., Int. Ed. 2002, 41 (7), 1229– 32,  DOI: 10.1002/1521-3773(20020402)41:7<1229::AID-ANIE1229>3.0.CO;2-5
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    265
    The adrenaline test for enzymes
    Wahler, Denis; Reymond, Jean-Louis
    Angewandte Chemie, International Edition (2002), 41 (7), 1229-1232CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
    A versatile high-throughput enzyme assay is demonstrated which is based on the colorimetric back-titrn. of sodium periodate with L-adrenaline. Enzyme activity is assocd. with the depletion of sodium periodate by the reaction product (P), and indicated by the decrease in the amt. of the red dye adrenochrome produced by the oxidn. of adrenaline by sodium periodate. The assay quantitates vicinal diols, amino alcs., diamines and α-hydroxy ketones. The assay was applied to measure lipases, esterases, phytases and epoxide hydrolases. The versatility of the assay in terms of substrate structures was demonstrated for the epoxide hydrolases from Aspergillus niger and Rhodotorula glutinis.
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    Jie, N.; Yang, D.; Zhang, Q.; Yang, J.; Song, Z. Fluorometric Determination of Periodate with Thiamine and Its Application to the Determination of Ethylene Glycol and Glycerol. Anal. Chim. Acta 1998, 359 (1), 87– 92,  DOI: 10.1016/S0003-2670(97)00657-0
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    266
    Fluorometric determination of periodate with thiamine and its application to the determination of ethylene glycol and glycerol
    Jie, Nianqin; Yang, Duanling; Zhang, Qiang; Yang, Jinghe; Song, Zhongqing
    Analytica Chimica Acta (1998), 359 (1-2), 87-92CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
    The fluorometric detn. of periodate with thiamine (vitamin B1) and its application to the indirect detn. of org. compds. (esp. glycols) were developed. Thiamine is oxidized to thiochrome by potassium periodate, which is accelerated by Mn (II). Thiochrome, a fluorescent compd., has excitation and emission max. at 397 and 454 nm, resp. Under optimum conditions, responses for the periodate detn. were linear between 5.0 × 10-7 - 1.8 × 10-5 mol/L concn. The detection limit, corresponding to a signal-to-noise ratio of 3, was 2.6 × 10-7 mol/L, with a relative std. deviation (n = 5) of 1.0%. The proposed method was applied to the indirect detn. of several org. compds. (ethylene glycol and glycerol, in vegetable oil) via the Malaprade reaction; good results were obtained.
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    Kirschner, A.; Bornscheuer, U. T. Directed Evolution of a Baeyer-Villiger Monooxygenase to Enhance Enantioselectivity. Appl. Microbiol. Biotechnol. 2008, 81 (3), 465– 72,  DOI: 10.1007/s00253-008-1646-4
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    267
    Directed evolution of a Baeyer-Villiger monooxygenase to enhance enantioselectivity
    Kirschner, Anett; Bornscheuer, Uwe T.
    Applied Microbiology and Biotechnology (2008), 81 (3), 465-472CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    The Baeyer-Villiger monooxygenase (BVMO) BmoF1 from Pseudomonas fluorescens DSM 50106 was shown before to enantioselectively oxidize different 4-hydroxy-2-ketones to the corresponding hydroxyalkyl acetates, being the first example of a BVMO-catalyzed kinetic resoln. of aliph. acyclic ketones. However, the wild-type enzyme exhibited only moderate E values (E∼55). Thus, the enantioselectivity was enhanced by means of directed evolution and optimization of reaction conditions since it was found that higher E values (E∼70 for wild-type BmoF1) could already be obtained when performing biotransformations in shake flasks rather than small tubes. In a first step, random mutations were introduced by error-prone polymerase chain reaction, and BmoF1 mutants (>3,500 clones) were screened for improved activity and enantioselectivity using a microtiter-plate-based screening method. Mutations S136L and L252Q were found to increase conversion compared to wild type, while several mutations (H51L, F225Y, S305C, and E308V) were identified enhancing the enantioselectivity to a varying extent (E∼75-90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange site-directed mutagenesis resulting in a double mutant (H51L/S136L) showing both improved conversion and enantioselectivity (E∼86).
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    Wahler, D.; Boujard, O.; Lefèvre, F.; Reymond, J.-L. Adrenaline Profiling of Lipases and Esterases with 1,2-Diol and Carbohydrate Acetates. Tetrahedron 2004, 60 (3), 703– 10,  DOI: 10.1016/j.tet.2003.11.059
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    Adrenaline profiling of lipases and esterases with 1,2-diol and carbohydrate acetates
    Wahler, Denis; Boujard, Olivier; Lefevre, Fabrice; Reymond, Jean-Louis
    Tetrahedron (2004), 60 (3), 703-710CODEN: TETRAB; ISSN:0040-4020. (Elsevier Science B.V.)
    The adrenaline test for enzymes is a general back-titrn. procedure to detect 1,2-diols, 1,2-aminoalcs. and α-hydroxyketones reaction products of enzyme catalysis by colorimetry. The method was used to profile a series of esterases and lipases for their esterolytic activity on a series of carbohydrate and polyol acetates. Substrates were prepd. by peracetylation and used for parallel microtiter-plate anal. of enzyme activities. This method can be used to achieve a rapid and automated characterization of a set of enzymes during HTS screening.
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    Fluxá, V. S.; Wahler, D.; Reymond, J.-L. Enzyme Assay and Activity Fingerprinting of Hydrolases with the Red-Chromogenic Adrenaline Test. Nat. Protoc. 2008, 3 (8), 1270– 7,  DOI: 10.1038/nprot.2008.106
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    Enzyme assay and activity fingerprinting of hydrolases with the red-chromogenic adrenaline test
    Fluxa, Viviana S.; Wahler, Denis; Reymond, Jean-Louis
    Nature Protocols (2008), 3 (8), 1270-1277CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
    The adrenaline test for enzymes is a colorimetric enzyme assay based on the quantification of periodate-sensitive reaction products such as 1,2-diols and 1,2-aminoalcs. by back-titrn. of the oxidant with adrenaline to produce adrenochrome as an easily detectable red product. The test uses com. reagents and is suitable for screening the activity of various hydrolases. It is demonstrated here for testing epoxide hydrolases, lipases and esterases, and for activity fingerprinting of these enzymes across substrate series. The complete assay requires 2-3 h.
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    Doderer, K.; Schmid, R. D. Fluorometric Assay for Determining Epoxide Hydrolase Activity. Biotechnol. Lett. 2004, 26 (10), 835– 9,  DOI: 10.1023/B:BILE.0000025887.36874.33
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    Fluorometric assay for determining epoxide hydrolase activity
    Doderer, Kai; Schmid, Rolf D.
    Biotechnology Letters (2004), 26 (10), 835-839CODEN: BILED3; ISSN:0141-5492. (Kluwer Academic Publishers)
    A rapid screening procedure was developed that enabled the screening of hundreds of enzyme samples or variants for epoxide hydrolase activity toward any substrate. The procedure detected the products of the enzymic reaction via periodate cleavage and the remaining fluorescence of carboxyfluorescein.
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    Matsuno, K.; Suzuki, S. Simple Fluorimetric Method for Quantification of Sialic Acids in Glycoproteins. Anal. Biochem. 2008, 375 (1), 53– 9,  DOI: 10.1016/j.ab.2008.01.002
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    Simple fluorimetric method for quantification of sialic acids in glycoproteins
    Matsuno, Kanae; Suzuki, Shigeo
    Analytical Biochemistry (2008), 375 (1), 53-59CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
    A simple and rapid fluorimetric method was developed for detection and quant. anal. of sialic acids in glycoproteins. Sialic acid residues in glycoproteins were specifically oxidized with periodate at 0° for 45 min. Formaldehyde generated from carbon 9 (C-9) of sialic acid was converted specifically to fluorescent dihydropyridine deriv. with acetoacetanilide and ammonia at room temp. for 10 min. The reaction products indicate intense fluorescence with excitation and emission maxima at 388 and 471 nm, resp. When the reaction was conducted in approx. a 1-mL vol., the linearity of the calibration exhibited between 2 and 180 μg of bovine fetuin, or between 0.3 and 27 nmol of N-acetylneuraminic acid, as a model glycoprotein. The limit of detection, based on three times the std. deviation of the reagent blank, was 0.5 μg of fetuin. The proposed method was applied to detn. of sialic acids in various glycoprotein samples. This proposed method is simple and obviates the heating and extn. steps. It is highly specific to sialic acids in glycoproteins and indicates no fluorescence of neutral glycoproteins.
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    Preston-Herrera, C.; Jackson, A. S.; Bachmann, B. O.; Froese, J. T. Development and Application of a High Throughput Assay System for the Detection of Rieske Dioxygenase Activity. Org. Biomol. Chem. 2021, 19 (4), 775– 84,  DOI: 10.1039/D0OB02412K
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    272
    Development and application of a high throughput assay system for the detection of Rieske dioxygenase activity
    Preston-Herrera, Cristina; Jackson, Aaron S.; Bachmann, Brian O.; Froese, Jordan T.
    Organic & Biomolecular Chemistry (2021), 19 (4), 775-784CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Herein we report the development of a new periodate-based reactive assay system for the fluorescent detection of the cis-diol metabolites produced by Rieske dioxygenases. This sensitive and diastereoselective assay system successfully evaluates the substrate scope of Rieske dioxygenases and dets. the relative activity of a rationally designed Rieske dioxygenase variant library. The high throughput capacity of the assay system enables rapid and efficient substrate scope investigations and screening of large dioxygenase variant libraries.
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    Winkler, M. Carboxylic Acid Reductase Enzymes (CARS). Curr. Opin. Chem. Biol. 2018, 43, 23– 9,  DOI: 10.1016/j.cbpa.2017.10.006
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    Carboxylic acid reductase enzymes (CARs)
    Winkler, Margit
    Current Opinion in Chemical Biology (2018), 43 (), 23-29CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    A review. Carboxylate reductases (CARs) are emerging as valuable catalysts for the selective one-step redn. of carboxylic acids to their corresponding aldehydes. The substrate scope of CARs is exceptionally broad and offers potential for their application in diverse synthetic processes. Two major fields of application are the prepn. of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates. The latest applications of CARs are dominated by in vivo cascades and chemo-enzymic reaction sequences. The challenge to fully exploit product selectivity is discussed. Recent developments in the characterization of CARs are summarized, with a focus on aspects related to the domain architecture and protein sequences of CAR enzymes.
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    Sutiono, S.; Carsten, J.; Sieber, V. Structure-Guided Engineering of α-Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature. ChemSusChem 2018, 11 (18), 3335– 44,  DOI: 10.1002/cssc.201800944
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    274
    Structure-Guided Engineering of α-Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature
    Sutiono, Samuel; Carsten, Joerg; Sieber, Volker
    ChemSusChem (2018), 11 (18), 3335-3344CODEN: CHEMIZ; ISSN:1864-5631. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Branched-chain keto acid decarboxylases (KDCs) are a class of enzymes that catalyze the decarboxylation of α-keto acids. They are key enzymes for prodn. of higher alcs. in vivo and in vitro. However, the two most active KDCs (KivD and KdcA) have only moderate thermostability (<55 °C), which hinders the prodn. of alcs. at high temps. Herein, structure-guided engineering toward improved thermostability of KdcA is outlined. Strategies such as stabilization of the catalytic center, surface engineering, and optimization of dimer interactions were applied. With seven amino acid substitutions, variant 7M.D showed an increase of the temp. at which 50 % of activity remains after one-hour incubation T1h50 by 14.8 °C without compromising its substrate specificity. 7M.D exhibited greater than 400-fold improvement of half-life at 70 °C and greater than 600-fold increase in process stability in the presence of 4 % isobutanol at 50 °C. 7M.D is more promising for the prodn. of higher alcs. in thermophiles (>65 °C) and in cell-free applications.
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    Greenhalgh, J. C.; Fahlberg, S. A.; Pfleger, B. F.; Romero, P. A. Machine Learning-Guided Acyl-ACP Reductase Engineering for Improved in Vivo Fatty Alcohol Production. Nat. Commun. 2021, 12, 5825,  DOI: 10.1038/s41467-021-25831-w
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    Machine learning-guided acyl-ACP reductase engineering for improved in vivo fatty alcohol production
    Greenhalgh, Jonathan C.; Fahlberg, Sarah A.; Pfleger, Brian F.; Romero, Philip A.
    Nature Communications (2021), 12 (1), 5825CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: Alc.-forming fatty acyl reductases (FARs) catalyze the redn. of thioesters to alcs. and are key enzymes for microbial prodn. of fatty alcs. Many metabolic engineering strategies utilize FARs to produce fatty alcs. from intracellular acyl-CoA and acyl-ACP pools; however, enzyme activity, esp. on acyl-ACPs, remains a significant bottleneck to high-flux prodn. Here, we engineer FARs with enhanced activity on acyl-ACP substrates by implementing a machine learning (ML)-driven approach to iteratively search the protein fitness landscape. Over the course of ten design-test-learn rounds, we engineer enzymes that produce over twofold more fatty alcs. than the starting natural sequences. We characterize the top sequence and show that it has an enhanced catalytic rate on palmitoyl-ACP. Finally, we analyze the sequence-function data to identify features, like the net charge near the substrate-binding site, that correlate with in vivo activity. This work demonstrates the power of ML to navigate the fitness landscape of traditionally difficult-to-engineer proteins.
  274. 274
    Sellés Vidal, L.; Kelly, C. L.; Mordaka, P. M.; Heap, J. T. Review of NAD(P)H-Dependent Oxidoreductases: Properties, Engineering and Application. Biochim. Biophys. Acta 2018, 1866 (2), 327– 47,  DOI: 10.1016/j.bbapap.2017.11.005
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    Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application
    Selles Vidal, Lara; Kelly, Ciaran L.; Mordaka, Pawel M.; Heap, John T.
    Biochimica et Biophysica Acta, Proteins and Proteomics (2018), 1866 (2), 327-347CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B.V.)
    A review. NAD(P)H-dependent oxidoreductases catalyze the redn. or oxidn. of a substrate coupled to the oxidn. or redn., resp., of a NAD cofactor NAD(P)H or NAD(P)+. NAD(P)H-dependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilization, the synthesis of chems., biodegrdn. and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.
  275. 275
    Goswami, P.; Chinnadayyala, S. S. R.; Chakraborty, M.; Kumar, A. K.; Kakoti, A. An Overview on Alcohol Oxidases and Their Potential Applications. Appl. Microbiol. Biotechnol. 2013, 97 (10), 4259– 75,  DOI: 10.1007/s00253-013-4842-9
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    277
    An overview on alcohol oxidases and their potential applications
    Goswami, Pranab; Chinnadayyala, Soma Sekhar R.; Chakraborty, Mitun; Kumar, Adepu Kiran; Kakoti, Ankana
    Applied Microbiology and Biotechnology (2013), 97 (10), 4259-4275CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    A review. Alc. oxidases (Alc.: O2 Oxidoreductase; EC 1.1.3.x) are flavoenzymes that catalyze the oxidn. of alcs. to the corresponding carbonyl compds. with a concomitant release of hydrogen peroxide. Based on substrate specificity, alc. oxidases may be categorized broadly into four different groups namely, (a) short chain alc. oxidase (SCAO), (b) long chain alc. oxidase (LCAO), (c) arom. alc. oxidase (AAO), and (d) secondary alc. oxidase (SAO). The sources reported for these enzymes are mostly limited to bacteria, yeast, fungi, plant, insect, and mollusks. However, the quantum of reports for each category of enzymes considerably varies across these sources. The enzymes belonging to SCAO and LCAO are intracellular in nature, whereas AAO and SAO are mostly secreted to the medium. SCAO and LCAO are invariably reported as multimeric proteins with very high holoenzyme mol. masses, but the mol. characteristics of these enzymes are yet to be clearly elucidated. One of the striking features of the alc. oxidases that make them distinct from the widely known alc. dehydrogenase is the avidly bound cofactor to the redox center of these enzymes that obviate the need to supplement cofactor during the catalytic reaction. These flavin-based redox enzymes have gained enormous importance in the development of various industrial processes and products primarily for developing biosensors and prodn. of various industrially useful carbonyl compds. The present review provides an overview on alc. oxidases from different categories focusing research on these oxidases during the last decade along with their potential industrial applications.
  276. 276
    Mang, H.; Gross, J.; Lara, M.; Goessler, C.; Schoemaker, H. E.; Guebitz, G. M.; Kroutil, W. Biocatalytic Single-Step Alkene Cleavage from Aryl Alkenes: An Enzymatic Equivalent to Reductive Ozonization. Angew. Chem., Int. Ed. 2006, 45 (31), 5201– 3,  DOI: 10.1002/anie.200601574
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    Biocatalytic single-step alkene cleavage from aryl alkenes: an enzymatic equivalent to reductive ozonization
    Mang, Harald; Gross, Johannes; Lara, Miguel; Goessler, Christian; Schoemaker, Hans E.; Guebitz, Georg M.; Kroutil, Wolfgang
    Angewandte Chemie, International Edition (2006), 45 (31), 5201-5203CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Innocuous mol. oxygen O2 is the only reagent needed to perform highly chemoselective biocatalytic single-step alkene-cleavage reactions. The products are analogous to those of (reductive) ozonization and related metal-based methods. In contrast neither special equipment nor an addnl. reducing agent is required. The biocatalytic reaction can be performed at ambient temp. Depending on the substrate, aldehydes or ketones are obtained.
  277. 277
    Schwendenwein, D.; Ressmann, A. K.; Doerr, M.; Höhne, M.; Bornscheuer, U. T.; Mihovilovic, M. D.; Rudroff, F.; Winkler, M. Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity Using an Amino Benzamidoxime-Mediated High-Throughput Assay. Adv. Synth. Catal. 2019, 361, 2544,  DOI: 10.1002/adsc.201900155
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    279
    Random mutagenesis-driven improvement of carboxylate reductase activity using an amino benzamidoxime-mediated high-throughput assay
    Schwendenwein, Daniel; Ressmann, Anna K.; Doerr, Mark; Hoehne, Matthias; Bornscheuer, Uwe T.; Mihovilovic, Marko D.; Rudroff, Florian; Winkler, Margit
    Advanced Synthesis & Catalysis (2019), 361 (11), 2544-2549CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Carboxylic acid reductases (CARs) catalyze the direct ATP and NADP (NADPH) dependent redn. of carboxylic acids to their corresponding aldehydes. The identification and improvement of CARs by protein engineering is, however, severely limited by the lack of fast and generic methods to quantify aldehydes. Within this study, we applied a convenient high-throughput assay (HTA) based on amino benzamidoxime (ABAO) that allows the substrate-independent and chemoselective quantification of aldehydes. Random mutagenesis of the well-known CAR from Nocardia iowensis (CARNi) to improve its activity for sterically demanding 2-substituted benzoic acid derivs. was conducted in a Km-dependent fashion, and the HTA applied in the presence of microbial cells. The study identified a hot spot in the active site of CARNi that increased the affinity to 2-methoxybenzoic acid 9-fold upon mutation from glutamine to proline (Q283P). The catalytic performance of CARNiQ283P appeared to be significantly improved also for other substrates such as 2-substituted (2-Cl, 2-Br) as well as 3- and 4-substituted benzoic acids (3-OMe, 4-OMe), and even aliph. octanoic acid.
  278. 278
    Soh, L. M. J.; Mak, W. S.; Lin, P. P.; Mi, L.; Chen, F. Y. H.; Damoiseaux, R.; Siegel, J. B.; Liao, J. C. Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design. ACS Synth. Biol. 2017, 6 (4), 610– 8,  DOI: 10.1021/acssynbio.6b00240
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    280
    Engineering a thermostable keto acid decarboxylase using directed evolution and computationally directed protein design
    Soh, Lemuel M. J.; Mak, Wai Shun; Lin, Paul P.; Mi, Luo; Chen, Frederic Y.-H.; Damoiseaux, Robert; Siegel, Justin B.; Liao, James C.
    ACS Synthetic Biology (2017), 6 (4), 610-618CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcs., like isobutanol. The most active Kdc's are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol prodn. temp. Here, the authors report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60°. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and ∼8000 independent variants were screened. The top single-mutation variants were recombined. To further improve thermostability, 16 designs built using Rosetta Comparative Modeling were screened and the most active was recombined to form the authors' best variant, LLM4. Compared to wild-type Kivd, a 13° increase in melting temp. and a >4-fold increase in half-life at 60° were obsd. LLM4 will be useful for keto acid-derived alc. prodn. in lignocellulosic thermophiles.
  279. 279
    Van Schie, M. M. C. H.; Pedroso De Almeida, T.; Laudadio, G.; Tieves, F.; Fernández-Fueyo, E.; Noël, T.; Arends, I. W. C. E.; Hollmann, F. Biocatalytic Synthesis of the Green Note trans-2-Hexenal in a Continuous-Flow Microreactor. Beilstein J. Org. Chem. 2018, 14, 697– 703,  DOI: 10.3762/bjoc.14.58
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    281
    Biocatalytic synthesis of the Green Note trans-2-hexenal in a continuous-flow microreactor
    van Schie, Morten M. C. H.; Pedroso de Almeida, Tiago; Laudadio, Gabriele; Tieves, Florian; Fernandez-Fueyo, Elena; Noel, Timothy; Arends, Isabel W. C. E.; Hollmann, Frank
    Beilstein Journal of Organic Chemistry (2018), 14 (), 697-703CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)
    The biocatalytic prepn. of trans-hex-2-enal from trans-hex-2-enol using a novel aryl alc. oxidase from Pleurotus eryngii (PeAAOx) is reported. As O2-dependent enzyme PeAAOx-dependent reactions are generally plagued by the poor soly. of O2 in aq. media and mass transfer limitations resulting in poor reaction rates. These limitations were efficiently overcome by conducting the reaction in a flow-reactor setup reaching unpreceded catalytic activities for the enzyme in terms of turnover frequency (up to 38 s-1) and turnover nos. (more than 300000) pointing towards preparative usefulness of the proposed reaction scheme.
  280. 280
    Allen, C. F. The Identification of Carbonyl Compounds by Use of 2,4-Dinitrophenylhydrazine. J. Am. Chem. Soc. 1930, 52 (7), 2955– 9,  DOI: 10.1021/ja01370a058
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    282
    Identification of carbonyl compounds by use of 2,4-dinitrophenylhydrazine
    Allen, Chas. F. H.
    Journal of the American Chemical Society (1930), 52 (), 2955-9CODEN: JACSAT; ISSN:0002-7863.
    An inexpensive method is given for the prepn. of 2,4-(O2N)2C6H3NHNH2. This is a suitable reagent to use in prepg. cryst. derivs. of a large no. of aliphatic compds. contg. a CO group. It may also be used with certain cyclic compds. but the production of solid derivs. is not as general. It is not as useful with α-HO ketones, owing to the difficulty in sepg. the mixts. formed. The following new 2,4-dinitrophenylhydrazones are reported: MeCOPr, yellow-orange, m. 141°; MeCOBμ, red-orange, m. 106°; MeCOAm, yellow-orange, m. 89°; Me hexyl ketone, orange, m. 58°; Me nonyl ketone, orange-yellow, m. 63°; Me undecyl ketone, orange-yellow, m. 69°; iso-PrCOMe, orange-yellow, m. 117°; iso-BuCOMe, orange-red m. 95°; iso-AmCOMe, orange, m. 95°; Me isohexyl ketone, orange-yellow, m. 77°; pinacolin orange-yellow, m. 125°; EtCOEt, orange, m. 156°; PrCOPr, yellow-orange, m. 75°; EtCOPr, yellow-orange, m. 130°; iso-BuCOEt, yellow-orange, m. 75°; allylacetone, orange, m. 104°; methylheptenone, red-orange, m. 81°; pseudoionone, deep red, m. 143° mesityl oxide, carmine, m. 200°; cyclopentanone, orange-yellow, m. 142°; cyclohexanone, orange-yellow, m. 160°; Me cyclohexyl ketone, orange, m. 140°; carvone, carmine, m. 189°; menthone, orange, m. 145°; benzalacetone, orange-red, m. 223°; PhCH:CHCOPh, orange, m. 208°; MeCOPh, orange, m. 237°; α-indanone, red-orange, m. 258°; butyroin, yellow, m. 99°; benzoin, orange-yellow, m. 234°; valeraldehyde, yellow, m. 98°; octaldehyde, yellow, m. 106°; decylaldehyde, yellow, m. 104°; undecylaldehyde, yellow, m. 104°; dodecylaldehyde, yellow, m. 106°; acrolein, red-orange, m. 165°; α-methyl-β-ethylacrolein, carmine, m. 159°; citronellal, yellow, m. 78°; citral I, red-orange, m. 108-10°, citral II, red-orange, m. 96°; α-amylcinnamaldehyde, scarlet, m. 164°; pyruvic acid, lemon, m. 213°; levulinic acid, yellow, m. 92°; Et oxomalonate, lemon, m. 128°; BzCO2Me, orange-yellow, m. 171°. Furoin, furil and benzofuroin gave unsatisfactory results. Anisoin, anisil, piperoin and piperil gave mixts. m. above 200°. Solid derivs. could not be prepd. from Me heptyl, octyl and decyl ketones, Bu2CO, com. ionone, pure α-ionone, fenchone or pulegone.
  281. 281
    Brady, O. L.; Elsmie, G. V. The Use of 2,4-Dinitrophenylhydrazine as a Reagent for Aldehydes and Ketones. Analyst 1926, 51 (599), 77– 8,  DOI: 10.1039/an9265100077
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    The use of 2,4-dinitrophenylhydrazine as a reagent for aldehydes and ketones
    Brady, O. L.; Elsmie, Gladys V.
    (1926), 51 (), 77-8 ISSN:.
    The above reagent is so satisfactory that it is surprising that it has not received more attention. It reacts somewhat tardily and is not very sol. in cold alc. Quite a no. of cryst. ppts. with typical aldehydes were prepared and the chem. compn. was found to agree with the demands of theory. Directions for making the reagent are given.
  282. 282
    Bohlmann, F. Konstitution Und Lichtabsorption, I. Mitteil.: Carbonyl-Derivate. Chem. Ber. 1951, 84 (5–6), 490– 504,  DOI: 10.1002/cber.19510840515
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    Constitution and light absorption. I. Carbonyl derivatives
    Bohlmann, Ferdinand
    Chemische Berichte (1951), 84 (), 490-504CODEN: CHBEAM; ISSN:0009-2940.
    With the exception of the oximes, the compds. used for the characterization of CO compds. are chiefly hydrazine derivs. (Ia). These condensation products have characteristic absorption spectra, the max. of which are shifted toward the longer wave lengths although the conjugation is not lengthened by addnl. double bonds. An attempt is made to find a relationship between the constitution and the light absorption. The absorption spectra of the m- (A) and p-nitrophenylhydrazones (B), 2,4-dinitrophenylhydrazones (C), N-methyl-2,4-dinitrophenylhydrazones (D), semicarbazones (E), thiosemicarbazones (F), N-methylthiosemicarbazones (G), and azobenzenesemicarbazones [1-(p-phenylazophenyl)semicarbazones] (H) of Me2CO, MeCH:CHCHO (I), BzH, hexadienal (II), octatrienal (III), β-ionone (IV), RCH2CH:CMeCHO (V) (R = CMe2.CH2.CH2.CH2.CMe:C- throughout the abstr.), RCH:CHCMe:CHCOMe (VI), RCH:CHCMe:CHCH:CHCOMe (VII), RCH2CH:CMeCH:CHCH:CHCHO (VIII) have been detd. as given in the table. Carbonyl compd., No. of conjugated bonds, Color, A, M.p. °C., λmax. mμ, ε × 10-3, Color, B, M.p. °C., λmax. mμ, ε × 10-3, Color, C, M.p. °C., λmax mμ, ε × 10-3, Color, D, M.p. °C., λmax mμ, ε × 10-3; Free base, .., .., .., .., .., .., .., 230, .., .., .., 352, .., ...., .., 230, ..; , , , , , , , , 386, , , , , , , , 370; Me2CO, .., red, 112-13, 268, 22.8, yellow, 148, 250, 11.9, .., .., 362, .., yellow, 138, 220, ..; , , , , (390), (1.4), , , 392, , , , , , , , 375, 19.1; I, 1, .., .., .., .., orange, 184, 285, 7.7, red, 190, 244, 15.8, yellow, 131, 260, 14.3; , , , , , , , , 400, 30.0, , , 282, 9.0, , , 397, 22.3; , , , , , , , , 273a, , , , 376, 27.3; , , , , , , , , 523a, , , , 273a; , , , , , , , , , , , , 523a; BzH, 1b, orange, 130, 238, 22.1, orange, 190, 230, 14.6, .., .., 378, .., brownish yellow, 206, 290, 10.8; , , , , 334, 33.1, , , 299, 7.2, , , , , , , 401, 20.9; , , , , (393), (1.95), , , 403, 31.2; PhCOMe, .., .., .., .., .., .., .., .., .., .., .., .., .., yellow needles, 157-8, 389, 81.4; II, 2, .., .., .., .., red, 170-1, 235, 16.2, .., .., .., .., yellow-orange, 122-3, 290, 20.8; , , , , , , , , 291, 15.0, , , , , leaflets, , 410, 25.35; , , , , , , , , 330, 10.7; , , , , , , , , 414, 43.8; III, 3, .., .., .., .., .., .., 311, .., .., .., 409, .., orange, 150-2, 316, 28.5; , , , , , , , , 425, , , , , , , , 424, 28.9; IV, 2, .., .., .., .., orange, 161-2, 294, 15.5, red, 126-7, 293, 11.0, yellow, 104-6, 298, 9.5; , , , , , , , , 408, 39.0, , , 387, 27.5, leaflets, , 390, 22.0; V, 1, red, 174-5, 248, 13.8, orange, 169, 283, 12.0, red, 164, 252, 18.5, yellow-orange, 148, 267, 17.8; , , , , 314, 37.1, , , 406, 35.0, , , 290, 11.0, leaflets, , 404, 24.6; , , , , (400), (1.36), , , , , , , 385, 31.5; VI, .., .., .., .., .., .., .., .., .., red, 157, 230, 19.0, ...., .., .., ..; , , , , , , , , , , , , 310, 15.7; , , , , , , , , , , , , 408, 34.0; VII, .., .., .., .., .., .., .., .., .., dark red, 177, 332, 22.0, ...., .., .., ..; , , , , , , , , , , , , 416, 43.0; VIII, .., .., .., .., .., .., .., .., .., red, 233, 265c, 18.5, ...., .., .., ..; , , , , , , , , , , , , 332c, 22.8; , , , , , , , , , , , , 410c, 46.5;a In 10% alc. KOH.;b the C6 ring in this series corresponds to about 1 C:C bond and not 1.5 as in polyene chemistry.;c In CHCl3; all others in MeOH.; Me[2,4-(O2N)2C6H3]NNH2, prepd. from MeNHNH2 and 2,4-(O2N)2C6H3Cl, m. 137°, λmax. 370 mμ, ε 14,200. For the prepn. of Ia, 300 mg. of the appropriate hydrazine is boiled 2-3 min. in 30 cc. EtOH and 0.5 cc. HCl with an equiv. amt. of the CO compd. Replacement of the OH in PhCH:NOH (λmax. 247 mμ) by a NH2 group leads to PhCH:NNH2 (λmax. 273 mμ) with a shift of the max. of 25 mμ toward the red; introduction of an Ac group (PhCH:NNHAc, λmax. 283 mμ), a Bz group (PhCH:NNHBz, λmax. 297 mμ), or a Me group (PhCH:NNHMe) causes a further shift to the red. Introduction of Ph as in PhCH:NNHPh (IX) (λmax. 342 mμ) has a stronger effect, whereas introduction of Br in the para position [PhCH:NNHC5H4Br(p), λmax. 347 mμ] has only little effect upon the shift. The position of the max. of IX is about the same as that of (PhCH:CH)2, indicating that the NH group has about the same effect upon the light absorption as a C:C double bond. In the A and B the max. are further shifted to the red, whereas the C generally absorb more in the shorter wave lengths. The NO2 in the ortho position in C therefore causes a shift of the absorption to the shorter wave lengths. In strongly alk. soln. these differences disappear (I B and I C in 10% alc. KOH have λmax. 523 mμ). With the C several limiting forms are possible because the NO2 groups can react in different ways with the electron pair of the NH group, e.g., X and XI. The effect of the o-NO2 group can be annulled by the introduction of a Me at the α-N, whereby the max. are shifted about 20 mμ toward the longer wave lengths and approach the max. of the B. With the D, however, no change in the position of the max. occurs as compared with those of the B. With the D the formulation XI is impossible, whereas with C the formation of H bridges as in XI is favored, as is shown by Stuart models. The usually observed shift toward the long waves' region is reversed by the H bridges because the π-electron system is decisively influenced by a far-reaching shift of the mesomerism equil. toward XI. With the D a certain steric hindrance by the Me group is noticeable, as shown by a stronger resolution of the chromophoric system. Thus the absorption max. attributed to the partial chromophore, R(CH:CH)nCH:N-, is considerably more pronounced, with a simultaneous decrease of the extinctions. The minor max. are more characteristic than the major max. because they indicate the no. of conjugated double bonds. Unlike the C, the D and the A do not give any color with alc. alkali, indicating that the H at the NH group is necessary for the coloration and that a quinoid formulation must be assumed for the colored alkali salts of C and B, which is impossible with A. With the E, F, and H a shift toward the longer wave lengths occurs. This shift, with the E, is of about the same order as that of an addnl. C:C double bond and has been attributed to an enolization of the urea group: RCH:NNHCONH2 → RCH:NN:HC(OH))NH2. However, this is erroneous because N-Me derivs. of E, where enolization is impossible, show the same behavior. Further methylation of PhCH:NNMeCSNH2 to PhCH:NMeC(SMe):NH has no effect on the absorption max. Introduction of a Ph into the semicarbazone group causes a shift of 10 mμ to the longer wave lengths (H2NCONHPh has λmax. 238 mμ, CO(NHPh)2, λmax. 255 mμ). When the Ph group is sepd. by a CH2 group the influence becomes smaller (Me2C:NNHCONH2 has λmax. 224 mμ, MePhC:NNHCONH2 λmax. 228 mμ). With the dinitrophenylsemicarbazones a resolution of the chromophoric system takes place. A replacement of the (O2N)2C6H3 group by a PhN:N group also causes a resolution of the spectrum. Refluxing 5 g. p-PhN:NC6H4NHCONH2 with 3 cc. N2H4.H2O in 250 cc. EtOH 40 hrs. gives the azobenzenesemicarbazide (XII), brownish yellow crystals, m. above 280° (decompn.), λmax. 238, 356 mμ, ε 14,800, 29,000. Warming 0.3 g. XII with an equiv. amt. of the appropriate CO compd. in 15 cc. EtOH and 0.1 cc. AcOH 15 min. gives the following azobenzenesemicarbazones: of BzH, yellow needles, m. 213°, λmax. 292, 354 mμ, ε 20,000, 32,000; of V, yellow needles, m. 235°, λmax. 273, 354 mμ, ε 30,000, 25,500; of cyclohexanone, yellow crystals, m. 223°, λmax. 242, 355mμ, ε 19,300, 28,900; of IV, orange-yellow leaflets, m. 224°, λmax. 300, 355 mμ, ε 17,300, 32,500. The following derivs. of BzH are also studied (m.p., λmax., and ε × 10-3 in the order given): phenylhydrazone, 152°, 342, 303, 235 mμ, 20.2, 10.3, 12.8; p-bromophenylhydrazone, 127°, 347, 315, 235 mμ, 27.4, 17.3, 12.4; N-methyl(p-nitrophenyl)hydrazone, 137°, 403, 299, 230 mμ, 31.2, 9.2, 16.1; hydrazone, -, 273 mμ, 13.8; acetylhydrazone, 134°, 283 mμ, 26.9; benzoylhydrazone, 206°, 297 mμ, 24.8; semicarbazone, 230°, 282 mμ, 21.0; thiosemicarbazone, 160°, 310 mμ, -; 1-methylthiosemicarbazone, 170°, 312 mμ, 29.4; S-Me isomer, 196°, 300 mμ, 27.6. The following addnl. derivs. of V are prepd.: N-methyl(p-nitrophenyl)hydrazone, yellow crystals, m. 158°, λmax. 406, 284 mμ, ε 31,100, 13,500; 1-methylsemicarbazone, shiny leaflets, m. 168°, λmax. 268 mμ, ε 37,000; 1-methylthiosemicarbazone, m. 183°, λmax. 302 mμ, ε 39,300; semicarbazone, m. 155°, λmax. 265 mμ, ε 33,200; thiosemicarbazone, m. 200°, λmax. 299 mμ, ε 37,800. III semicarbazone m. about 250°, λmax. 303, 319, 334 mμ; VIII semicarbazone m. 186°, λmax. 312, 324, 338 mμ.
  283. 283
    Georgiou, C. D.; Zisimopoulos, D.; Argyropoulou, V.; Kalaitzopoulou, E.; Salachas, G.; Grune, T. Protein and Cell Wall Polysaccharide Carbonyl Determination by a Neutral pH 2,4-Dinitrophenylhydrazine-Based Photometric Assay. Redox Biol. 2018, 17, 128– 42,  DOI: 10.1016/j.redox.2018.04.010
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    285
    Protein and cell wall polysaccharide carbonyl determination by a neutral pH 2,4-dinitrophenylhydrazine-based photometric assay
    Georgiou, Christos D.; Zisimopoulos, Dimitrios; Argyropoulou, Vasiliki; Kalaitzopoulou, Electra; Salachas, George; Grune, Tilman
    Redox Biology (2018), 17 (), 128-142CODEN: RBEIB3; ISSN:2213-2317. (Elsevier B.V.)
    A new 2,4-dinitrophenylhydrazine (DNPH)-based photometric assay is developed for the quantification of carbonyls in protein samples from any biol. source by protein carbonyl-DNPH hydrazone formation at acidic pH in the presence of denaturing urea, and subsequent hydrazone solubilization in the presence of SDS and stabilization from acid hydrolysis at pH 7.0. At this neutral pH, interfering unreacted DNPH is uncharged and its thus increased hydrophobicity permits its 100% effective removal from the solubilizate with Et acetate/hexane wash. The present study elucidates the DNA interference mechanism on the stdDNPH assay, and also develops a standardized protocol for sample protein treatment and fractionation (into cytoplasmic/aq., membrane/lipid-bound, and histone/DNA-bound proteins; see Supplement section V) in order to ensure reproducible carbonyl detn. on defined cell protein fractions, and to eliminate assay interference from protein samples contg. (i) Cys sulfenic acid groups (via their neutralization with dithiothreitol), and (ii) DNA (via its removal by streptomycin sulfate pptn.). Lastly, the ntrDNPH assay dets. carbonyl groups on cell wall polysaccharides, thus paving the way on studies to investigate cell walls acting as antioxidant defense in plants, fungi, bacteria and lichens.
  284. 284
    Mesquita, C. S.; Oliveira, R.; Bento, F.; Geraldo, D.; Rodrigues, J. V.; Marcos, J. C. Simplified 2,4-Dinitrophenylhydrazine Spectrophotometric Assay for Quantification of Carbonyls in Oxidized Proteins. Anal. Biochem. 2014, 458, 69– 71,  DOI: 10.1016/j.ab.2014.04.034
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    286
    Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins
    Mesquita, Cristina S.; Oliveira, Raquel; Bento, Fatima; Geraldo, Dulce; Rodrigues, Joao V.; Marcos, Joao C.
    Analytical Biochemistry (2014), 458 (), 69-71CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    This work proposes a modification of the 2,4-dinitrophenylhydrazine (DNPH) spectrophotometric assay commonly used to evaluate the concn. of carbonyl groups in oxidized proteins. In this approach NaOH is added to the protein soln. after the addn. of DNPH, shifting the max. absorbance wavelength of the derivatized protein from 370 to 450 nm. This reduces the interference of DNPH and allows the direct quantification in the sample soln. without the need for the pptn., washing, and resuspension steps that are carried out in the traditional DNPH method. The two methods were compared under various conditions and are statistically equiv.
  285. 285
    Xue, Y.-P.; Wang, W.; Wang, Y.-J.; Liu, Z.-Q.; Zheng, Y.-G.; Shen, Y.-C. Isolation of Enantioselective α-Hydroxyacid Dehydrogenases Based on a High-Throughput Screening Method. Bioprocess Biosyst. Eng. 2012, 35 (9), 1515– 22,  DOI: 10.1007/s00449-012-0741-1
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    287
    Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method
    Xue, Ya-Ping; Wang, Wei; Wang, Ya-Jun; Liu, Zhi-Qiang; Zheng, Yu-Guo; Shen, Yin-Chu
    Bioprocess and Biosystems Engineering (2012), 35 (9), 1515-1522CODEN: BBEIBV; ISSN:1615-7591. (Springer)
    To isolate enantioselective α-hydroxyacid dehydrogenases (α-HADHs), a high-throughput screening method was established. 2,4-Dinitrophenylhydrazine soln. forms a red-brown complex with ketoacid produced during the α-HADH-mediated oxidn. of α-hydroxyacid. The complex can be easily quantified by spectrophotometric measurement at 458 nm. The enantioselectivity of α-HADH in each strain can be measured with this colorimetric method using (R)- and (S)-α-hydroxyacid concurrently as substrates to evaluate the apparent enantioselectivity (Eapp). The Eapp closely matches the value of true enantioselectivity (E true) detd. by HPLC anal. With this method, a total of 34 stains harboring enantioselective α-HADHs were selected from 526 potential α-HADH-producing microorganisms. Pseudomonas aeruginosa displayed the highest (S)-enantioselective α-HADH activity. This strain appears promising for potential application in industry to produce (R)-α-hydroxyacids. The method described herein represents a useful tool for the high-throughput isolation of enantioselective α-HADHs.
  286. 286
    Chen, Q.; Chen, X.; Feng, J.; Wu, Q.; Zhu, D.; Ma, Y. Improving and Inverting Cβ-Stereoselectivity of Threonine Aldolase Via Substrate-Binding-Guided Mutagenesis and a Stepwise Visual Screening. ACS Catal. 2019, 9 (5), 4462– 9,  DOI: 10.1021/acscatal.9b00859
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    288
    Improving and Inverting Cβ-Stereoselectivity of Threonine Aldolase via Substrate-Binding-Guided Mutagenesis and a Stepwise Visual Screening
    Chen, Qijia; Chen, Xi; Feng, Jinhui; Wu, Qiaqing; Zhu, Dunming; Ma, Yanhe
    ACS Catalysis (2019), 9 (5), 4462-4469CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    Threonine aldolase (TA)-catalyzed aldol condensation is a powerful tool for C-C bond formation under mild conditions, but the low Cβ-stereoselectivity has hampered its wide application. A stepwise visual screening method was developed to measure the activity and stereoselectivity of threonine aldolase-catalyzed aldol condensation by employing a stereoselective phenylserine dehydratase, enabling direct selection of mutants with higher Cβ-stereoselectivity. Mutants of L-PsTA from Pseudomonas sp. with improved or inverted stereoselectivity toward arom. aldehydes were obtained by simultaneously mutating amino acid residues which interact with the amino and hydroxyl groups of the substrate and screening the resulting mutant libraries with this method. The mutation and enzyme-substrate docking studies provided some insights into the regulation of the Cβ-stereoselectivity by the enzyme-substrate interactions. This study offers a tool and useful guidance for further engineering of TAs to address the Cβ-stereoselectivity problem.
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    Yang, C.; Ye, L.; Gu, J.; Yang, X.; Li, A.; Yu, H. Directed Evolution of Mandelate Racemase by a Novel High-Throughput Screening Method. Appl. Microbiol. Biotechnol. 2017, 101 (3), 1063– 72,  DOI: 10.1007/s00253-016-7790-3
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    289
    Directed evolution of mandelate racemase by a novel high-throughput screening method
    Yang, Chengcheng; Ye, Lidan; Gu, Jiali; Yang, Xiaohong; Li, Aipeng; Yu, Hongwei
    Applied Microbiology and Biotechnology (2017), 101 (3), 1063-1072CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    Optically pure Me (R)-o-chloromandelate and (R)-acetyl-o-mandelic acid are key intermediates for the synthesis of (S)-clopidogrel, which could be prepd. with 100% theor. yield by sequential hydrolysis and racemization. At the moment, efficient sequential hydrolysis and racemization are hindered by the low catalytic activity of mandelate racemase (MR) toward (S)-o-chloromandelic acid [(S)-2-CMA]. Here, the authors proposed to improve the catalytic performance of MR toward (S)-2-CMA by directed evolution and developed an enantioselective oxidn. system for high-throughput screening (HTS) of MR libraries. Based on this HTS method, a triple mutant V22I/V29I/Y54F (MRDE1) with 3.5-fold greater relative activity as compared to native MR was obtained. Kinetic anal. indicated that the enhanced catalytic efficiency mainly arose from the elevated kcat. Further insight into the source of improved catalytic activity was gained by mol. simulations, finding that substrate binding and product release were possibly made easier by decreased steric bulk and increased hydrophobicity of substrate-binding sites. In addn., the substrate (S)-2-CMA in the enzyme-substrate complex of MRDE1 seemed to have a lower binding free energy comparing with the complex of wild-type MR. The HTS method developed in this work and the successful directed evolution of MR based on this method provide an example for racemase engineering and may inspire directed evolution of other racemases toward enhanced catalytic performance on non-natural substrates.
  288. 288
    Uzu, S.; Kanda, S.; Imai, K.; Nakashima, K.; Akiyama, S. Fluorogenic Reagents: 4-Aminosulphonyl-7-Hydrazino-2,1,3-Benzoxadiazole, 4-(N,N-Dimethylaminosulphonyl)-7-Hydrazino-2,1,3-Benzoxadiazole and 4-Hydrazino-7-Nitro-2,1,3-Benzoxadiazole Hydrazine for Aldehydes and Ketones. Analyst 1990, 115 (11), 1477– 82,  DOI: 10.1039/an9901501477
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    290
    Fluorogenic reagents: 4-aminosulfonyl-7-hydrazino-2,1,3-benzoxadiazole, 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole and 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine for aldehydes and ketones
    Uzu, Sonoko; Kanda, Susumu; Imai, Kazuhiro; Nakashima, Kenichiro; Akiyama, Shuzo
    Analyst (Cambridge, United Kingdom) (1990), 115 (11), 1477-82CODEN: ANALAO; ISSN:0003-2654.
    Fluorogenic reagents for aldehydes and ketones, viz., 4-aminosulfonyl-7-hydrazino-2,1,3-benzoxadiazole (ABD-H) and 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H) and also purified 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine (NBD-H.NH2NH2) were synthesized. These reagents are not fluorescent; however, their reaction products with aldehydes and ketones fluoresce at wavelengths from 548 to 580 nm with excitation from 450 to 470 nm. Both ABD-H and DBD-H exhibited similar reactivity and were more reactive than NBD-H.NH2NH2. The resp. pseudo-first-order reaction rate consts. for the prodn. of the hydrazone of propionaldehyde with ABD-H, DBD-H and NBD-H.NH2NH2 were 8.9 ×10-2, 7.2 × 10-2 and 4.2 × 10-2 min-1 (the reaction was carried out in 0.0025% trifluoroacetic acid in acetonitrile at room temp., 22°). The detection limits using the manual method (i.e., measurement of fluorescence intensity) for the hydrazones of aldehydes and ketones with ABD-H, DBD-H and NBD-H.NH2NH2 were in the μM range. The substrate blank fluorescence with ABD-H was half of that with DBD-H and NBD-H.NH2NH2. The reaction products were sepd. and analyzed by reversed-phase HPLC with spectrofluorometric detection. The detection limits for propionaldehyde were 1040, 120 and 35.0 fmol with ABD-H, DBD-H and NBD-H.NH2NH2, resp., and those for heptan-4-one were 2690, 560 and 673 fmol, resp. Of the three reagents, DBD-H is recommended for the sensitive detection of ketones and NBD-H.NH2NH2 for the detection of aldehydes. The detection limits for aldehydes and ketones by HPLC were in the sub-pmol to pmol range.
  289. 289
    Uchiyama, S.; Santa, T.; Okiyama, N.; Fukushima, T.; Imai, K. Fluorogenic and Fluorescent Labeling Reagents with a Benzofurazan Skeleton. Biomed. Chromatogr. 2001, 15 (5), 295– 318,  DOI: 10.1002/bmc.75
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    Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton
    Uchiyama, Seiichi; Santa, Tomofumi; Okiyama, Natsuko; Fukushima, Takeshi; Imai, Kazuhiro
    Biomedical Chromatography (2001), 15 (5), 295-318CODEN: BICHE2; ISSN:0269-3879. (John Wiley & Sons Ltd.)
    A review with many refs. Fluorogenic and fluorescent labeling reagents having a benzofurazan (2,1,3-benzoxadiazole) skeleton such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F), ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H), 4-N,N-dimethylaminosulfonyl-7-hydrazino-2,1,3-benzoxadiazole (DBD-H), 4-nitro-7-N-piperazino-2,1,3-benzoxadiazole (NBD-PZ), 4-N,N-dimethylaminosulfonyl-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ), 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) are reviewed in terms of synthetic method, reactivity, fluorescence characteristics, sensitivity and application to analytes.
  290. 290
    Frizon, T. E. A.; Vieira, A. A.; da Silva, F. N.; Saba, S.; Farias, G.; de Souza, B.; Zapp, E.; Lôpo, M. N.; Braga, H. d. C.; Grillo, F. Synthesis of 2,1,3-Benzoxadiazole Derivatives as New Fluorophores─Combined Experimental, Optical, Electro, and Theoretical Study. Front. Chem. 2020, 8, 360,  DOI: 10.3389/fchem.2020.00360
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    292
    Synthesis of 2,1,3-benzoxadiazole derivatives as new fluorophores-combined experimental, optical, electro, and theoretical study
    Frizon, Tiago E. A.; Vieira, Andre A.; Da Silva, Fabricia N.; Saba, Sumbal; Farias, Giliandro; De Souza, Bernardo; Zapp, Eduardo; Lopo, Michell N.; Braga, Hugo De C.; Grillo, Felipe; Curcio, Sergio F.; Cazati, Thiago; Rafique, Jamal
    Frontiers in Chemistry (Lausanne, Switzerland) (2020), 8 (), 00360CODEN: FCLSAA; ISSN:2296-2646. (Frontiers Media S.A.)
    Herein, the synthesis and characterization of fluorophores I (R = 2-ethylhexyl, octyl, decyl, dodecyl) contg. a 2,1,3-benzoxadiazole unit assocd. with a π-conjugated system (D-π-A-π-D) has been reported. These new fluorophores in soln. exhibited an absorption max. at around ~ 419 nm (visible region), as expected for electronic transitions of the π-π* type (ε ~ 2.7*107 L mol-1 cm-1), and strong solvent-dependent fluorescence emission (ΦFL ~ 0.5) located in the bluish-green region. The Stokes' shift of these compds.is ca. 3,779 cm-1, which was attributed to an intramol. charge transfer (ICT) state. In CHCl3 soln., the compds. exhibited longer and shorter lifetimes, which was attributed to the emission of monomeric and aggregated mols., resp. DFT was used to model the electronic structure of I in their excited and ground electronic states. The simulated emission spectra are consistent with the exptl. results, with different solvents leading to a shift in the emission peak and the attribution of a π-π* state with the characteristics of a charge transfer excitation. The thermal properties were analyzed by thermogravimetric anal., and a high max. degrdn. rate occurred at around 300°. Electrochem. studies were also performed in order to det. the band gaps of the mols. The electrochem. band gaps (2.48-2.70 eV) showed strong correlations with the optical band gaps (2.64-2.67 eV).
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    Konarzycka-Bessler, M.; Bornscheuer, U. T. A High-Throughput-Screening Method for Determining the Synthetic Activity of Hydrolases. Angew. Chem., Int. Ed. 2003, 42 (12), 1418– 20,  DOI: 10.1002/anie.200390365
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    A high-throughput-screening method for determining the synthetic activity of hydrolases
    Konarzycka-Bessler, Monika; Bornscheuer, Uwe T.
    Angewandte Chemie, International Edition (2003), 42 (12), 1418-1420CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    An assay format for high-throughput detn. of synthetic activity of hydrolases is described. The method is based on lipase- or esterase-catalyzed transesterification reactions between a vinyl ester and an alc. As a model reaction, the esterification between vinyl laurate and 1-propanol, in the presence of 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H), was studied in microtiterplates. Several lipases and esterases were investigated with this reaction. The enzymes studied were Chirazyme L2 (CAL-B), L3, (CRL), L5M L6 (PSL), L9 (RML), Amano PS (PCL), Amano A (ANL), Amano AK (PFL), Amano AY (CRL), and SDE. The assay is extremely versatile for the detn. of the synthetic activity of lipases or esterases in a high-throughput format and thus enables the rapid identification of active enzymes and/or appropriate reaction conditions.
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    Jencks, W. P. Progress in Physical Organic Chemistry; Index to Reviews, Symposia Volumes and Monographs in Organic Chemistry; Cohen, S. G., Streitwieser, A., Jr., Taft, R. W., Eds.; John Wiley & Sons, 1964; Vol. 2, p 110 DOI: 10.1002/9780470171813
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    Larsen, D.; Pittelkow, M.; Karmakar, S.; Kool, E. T. New Organocatalyst Scaffolds with High Activity in Promoting Hydrazone and Oxime Formation at Neutral pH. Org. Lett. 2015, 17 (2), 274– 7,  DOI: 10.1021/ol503372j
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    New Organocatalyst Scaffolds with High Activity in Promoting Hydrazone and Oxime Formation at Neutral pH
    Larsen, Dennis; Pittelkow, Michael; Karmakar, Saswata; Kool, Eric T.
    Organic Letters (2015), 17 (2), 274-277CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
    The discovery of two new classes of catalysts for hydrazone and oxime formation in water at neutral pH, 2-aminophenols and 2-(aminomethyl)benzimidazoles, is reported. Kinetics studies in aq. solns. at pH 7.4 revealed rate enhancements up to 7-fold greater than with classic aniline catalysis. 2-(Aminomethyl)benzimidazoles are effective catalysts with otherwise challenging aryl ketone substrates.
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    Crisalli, P.; Kool, E. T. Importance of Ortho Proton Donors in Catalysis of Hydrazone Formation. Org. Lett. 2013, 15 (7), 1646– 9,  DOI: 10.1021/ol400427x
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    Importance of ortho Proton Donors in Catalysis of Hydrazone Formation
    Crisalli, Pete; Kool, Eric T.
    Organic Letters (2013), 15 (7), 1646-1649CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
    Anthranilic acids were recently reported as superior catalysts for hydrazone and oxime formation compared to aniline, the classic catalyst for these reactions. Here, alternative proton donors were examd. with varied pKa in an effort to enhance activity at biol. pH. The expts. show that 2-aminobenzenephosphonic acids are superior to anthranilic acids in catalyzing hydrazone formation with common aldehyde substrates.
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    Wang, S.; Nawale, G. N.; Kadekar, S.; Oommen, O. P.; Jena, N. K.; Chakraborty, S.; Hilborn, J.; Varghese, O. P. Saline Accelerates Oxime Reaction with Aldehyde and Keto Substrates at Physiological pH. Sci. Rep. 2018, 8, 2193,  DOI: 10.1038/s41598-018-20735-0
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    Saline Accelerates Oxime Reaction with Aldehyde and Keto Substrates at Physiological pH
    Wang Shujiang; Nawale Ganesh N; Kadekar Sandeep; Hilborn Jons; Varghese Oommen P; Oommen Oommen P; Jena Naresh K; Chakraborty Sudip
    Scientific reports (2018), 8 (1), 2193 ISSN:.
    We have discovered a simple and versatile reaction condition for oxime mediated bioconjugation reaction that could be adapted for both aldehyde and keto substrates. We found that saline accelerated the oxime kinetics in a concentration-dependent manner under physiological conditions. The reaction mechanism is validated by computational studies, and the versatility of the reaction is demonstrated by cell-surface labeling experiments. Saline offers an efficient and non-toxic catalytic option for performing the bioorthogonal-coupling reaction of biomolecules at the physiological pH. This saline mediated bioconjugation reaction represents the most biofriendly, mild and versatile approach for conjugating sensitive biomolecules and does not require any extensive purification step.
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    Mayr, H.; Bug, T.; Gotta, M. F.; Hering, N.; Irrgang, B.; Janker, B.; Kempf, B.; Loos, R.; Ofial, A. R.; Remennikov, G. Reference Scales for the Characterization of Cationic Electrophiles and Neutral Nucleophiles. J. Am. Chem. Soc. 2001, 123 (39), 9500– 12,  DOI: 10.1021/ja010890y
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    Reference scales for the characterization of cationic electrophiles and neutral nucleophiles
    Mayr, Herbert; Bug, Thorsten; Gotta, Matthias F.; Hering, Nicole; Irrgang, Bernhard; Janker, Brigitte; Kempf, Bernhard; Loos, Robert; Ofial, Armin R.; Remennikov, Grigoriy; Schimmel, Holger
    Journal of the American Chemical Society (2001), 123 (39), 9500-9512CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Twenty-three diarylcarbenium ions and 38 π-systems (arenes, alkenes, allyl silanes and stannanes, silyl enol ethers, silyl ketene acetals, and enamines) have been defined as basis sets for establishing general reactivity scales for electrophiles and nucleophiles. The rate consts. of 209 combinations of these benzhydrylium ions and π-nucleophiles, 85 of which are first presented in this article, have been subjected to a correlation anal. to det. the electrophilicity parameters E and the nucleophilicity parameters N and s as defined by the equation log k(20°) = s(N + E) (Mayr, H.; Patz, M. Angew. Chem., Int. Ed. Engl. 1994, 33, 938-957). Though the reactivity scales thus obtained cover more than 16 orders of magnitude, the individual rate consts. are reproduced with a std. deviation of a factor of 1.19 . It is shown that the reactivity parameters thus derived from the reactions of diarylcarbenium ions with π-nucleophiles (Figure 3) are also suitable for characterizing the nucleophilic reactivities of alkynes, metal-π-complexes, and hydride donors and for characterizing the electrophilic reactivities of heterosubstituted and metal-coordinated carbenium ions . Reactivity parameters are, therefore, recommended for the characterization of any new electrophiles and nucleophiles in the reactivity range covered. The linear correlation between the electrophilicity parameters E of benzhydryl cations and the corresponding substituent consts. σ+ provides Hammett σ+ consts. for 10 substituents from -1.19 to -2.11, i.e., in a range with only very few previous entries.
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    Brotzel, F.; Chu, Y. C.; Mayr, H. Nucleophilicities of Primary and Secondary Amines in Water. J. Org. Chem. 2007, 72 (10), 3679– 88,  DOI: 10.1021/jo062586z
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    Nucleophilicities of primary and secondary amines in water
    Brotzel, Frank; Chu, Ying Cheung; Mayr, Herbert
    Journal of Organic Chemistry (2007), 72 (10), 3679-3688CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    The kinetics of the reactions of 26 primary and secondary amines with benzhydrylium ions in water were investigated photometrically. Because the parallel reactions of the benzhydrylium ions with hydroxide and water are much slower, the second-order rate consts. for the reactions of amines with benzhydrylium ions could be detd. reliably. Reactivities of anilines were also studied in acetonitrile soln. Plots of log k2,N for these reactions vs the electrophilicity parameters E of the benzhydrylium ions were linear, which allowed us to derive the nucleophilicity parameters N and s for amines as defined by the equation log k (20°) = s(E + N). Because the slope parameters for the different amines are closely similar; the relative nucleophilicities are almost independent of the electrophiles and can be expressed by the nucleophilicity parameters N. The correlation between nucleophilicity N and pKaH values is poor, and it is found that secondary alkyl amines and anilines are considerably more nucleophilic, while ammonia is much less nucleophilic than expected on the basis of their pKaH values.
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    Brotzel, F., lmu, 2008.
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    Nakashima, K.; Hidaka, Y.; Yoshida, T.; Kuroda, N.; Akiyama, S. High-Performance Liquid Chromatographic Determination of Short-Chain Aliphatic Aldehydes Using 4-(N,N-Dimethylaminosulphonyl)-7-Hydrazino-2,1,3-Benzoxadiazole as a Fluorescence Reagent. J. Chromatogr. B Biomed. Appl. 1994, 661 (2), 205– 10,  DOI: 10.1016/0378-4347(94)00359-9
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    High-performance liquid chromatographic determination of short-chain aliphatic aldehydes using 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole as a fluorescence reagent
    Nakashima, Kenichiro; Hidaka, Youko; Yoshida, Tomomi; Kuroda, Naotaka; Akiyama, Shuzo
    Journal of Chromatography B: Biomedical Sciences and Applications (1994), 661 (2), 205-10CODEN: JCBBEP; ISSN:0378-4347. (Elsevier)
    HPLC detn. of four short-chain aliph. aldehydes using fluorescence detection was carried out with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). DBD-H derivs. with three aliph. aldehydes-formaldehyde, acetaldehyde and propionaldehyde-were synthesized and their fluorescence properties were examd. Relative fluorescence intensities of these compds. in acetonitrile were ∼ten-fold larger than those in aq. acetonitrile. DBD-hydrazones could be sepd. by reversed-phase chromatog. using aq. acetonitrile as eluent and detection at 560 nm with excitation at 445 nm. Submicromolar levels of formaldehyde, acetaldehyde, propionaldehyde and butylaldehyde could be detd. The HPLC procedure using propionaldehyde as internal std. was applied to the measurement of acetaldehyde levels in normal human plasma before and 30 min after ingestion of ethanol.
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    Jacobsen, N. W.; Dickinson, R. G. Spectrometric Assay of Aldehydes as 6-Mercapto-3-Substituted-s-Trizolo (4,3-b)-Tetrazines. Anal. Chem. 1974, 46 (2), 298– 9,  DOI: 10.1021/ac60338a039
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    Spectrometric assay of aldehydes as 6-mercapto-3-substituted-s-trizolo(4,3-b)-tetrazines
    Jacobsen, N. W.; Dickinson, R. G.
    Analytical Chemistry (1974), 46 (2), 298-9CODEN: ANCHAM; ISSN:0003-2700.
    H2CO was detd. photometrically at 549 nm after mixing the sample with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (I) and aerating the mixt. 30 min at 20° to form II (R = H). Beer's law was followed for 0.5-5 ppm H2CO. The relative error was ≤3% for 5-20 ppm H2CO, after suitable diln. Reaction of RCHO (R = Me, Et, Pr, and Ph) with I gave II with absorption max. at 537, 536, 532, and 340 nm, resp. demonstrating the suitability of the method for detns. of other aldehydes.
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    Dickinson, R. G.; Jacobsen, N. W. A New Sensitive and Specific Test for the Detection of Aldehydes: Formation of 6-Mercapto-3-Substituted-S-Triazolo[4,3-b]-s-Tetrazines. J. Chem. Soc. D 1970, (24), 1719– 20,  DOI: 10.1039/c29700001719
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    A new sensitive and specific test for the detection of aldehydes: formation of 6-mercapto-3-substituted-s-triazolo[4,3-b]-s-tetrazines
    Dickinson, Ronald G.; Jacobsen, Noel W.
    Journal of the Chemical Society [Section] D: Chemical Communications (1970), (24), 1719-20CODEN: CCJDAO; ISSN:0577-6171.
    Aldehydes (RCHO) (≥10-4M) can be detected specifically by condensation with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole in 1N NaOH with subsequent aeration to give 3-R-substituted-6-mercapto-s-triazolo[4,3-b]-s-tetrazines (I) which exhibited magenta, purple, or purple-brown colors when R was an aliphatic, aromatic, or an aliphatic or aromatic group with addnl. functional groups, resp. I (R = iso-Pr or Ph) m. 146-7° (decompn.) or 192-3° (decompn.), resp., were prepd. and characterized by uv spectroscopy and NMR.
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    Quesenberry, M. S.; Lee, Y. C. A Rapid Formaldehyde Assay Using Purpald Reagent: Application under Periodation Conditions. Anal. Biochem. 1996, 234 (1), 50– 5,  DOI: 10.1006/abio.1996.0048
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    A rapid formaldehyde assay using Purpald reagent: application under periodation conditions
    Quesenberry, M. S.; Lee, Y. C.
    Analytical Biochemistry (1996), 234 (1), 50-5CODEN: ANBCA2; ISSN:0003-2697. (Academic)
    The reaction of formaldehyde with Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole) in formaldehyde assay works under alk. conditions at room temp., and the sensitivity is superior to other methods. The color development by this reagent, however, requires oxidn. of the adduct with hydrogen peroxide, air oxygen, or dil. periodate. We found that low levels of periodate, commonly used to oxidize specifically terminal vicinal diols to yield formaldehyde, are compatible with color development with the Purpald reagent. We have investigated the conditions required for use of the Purpald reagent, esp. in conjunction with periodate oxidn. reactions. We have used the assay either in test tubes or with microplates, attaining sensitivity of as little as 1 nmol formaldehyde.
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    Anthon, G. E.; Barrett, D. M. Comparison of Three Colorimetric Reagents in the Determination of Methanol with Alcohol Oxidase. Application to the Assay of Pectin Methylesterase. J. Agric. Food Chem. 2004, 52 (12), 3749– 53,  DOI: 10.1021/jf035284w
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    Comparison of Three Colorimetric Reagents in the Determination of Methanol with Alcohol Oxidase. Application to the Assay of Pectin Methylesterase
    Anthon, Gordon E.; Barrett, Diane M.
    Journal of Agricultural and Food Chemistry (2004), 52 (12), 3749-3753CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
    Three colorimetric reagents for the detn. of formaldehyde, the Nash reagent (ammonia plus acetylacetone), Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole), and N-methylbenzothiazolinone-2-hydrazone (MBTH), were compared for the detn. of methanol when used in conjunction with alc. oxidase. The combination of alc. oxidase plus the commonly used Nash reagent was specific for methanol vs. ethanol, but had the lowest sensitivity of the three reagents tested. Substituting Purpald for the Nash reagent increased the sensitivity 3-fold while still maintaining a high (59-fold) selectivity for methanol vs. ethanol. Using MBTH increased the sensitivity still further, but with a loss of the selectivity toward methanol. Since MBTH reacted with aldehydes under neutral conditions, it could be included along with the alc. oxidase to act as an aldehyde trap. This prevented further oxidn. reactions by alc. oxidase and allowed for extended incubations. A procedure for assaying low levels of pectin methylesterase activity that relies on this trapping ability is described. In addn., alc. oxidase plus Purpald is shown to be a simple and sensitive way to measure the methanol released from plant material following the thermal activation of endogenous pectin methylesterase.
  304. 304
    Hammer, S. C.; Kubik, G.; Watkins, E.; Huang, S.; Minges, H.; Arnold, F. H. Anti-Markovnikov Alkene Oxidation by Metal-Oxo-Mediated Enzyme Catalysis. Science 2017, 358 (6360), 215– 8,  DOI: 10.1126/science.aao1482
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    Anti-Markovnikov alkene oxidation by metal-oxo-mediated enzyme catalysis
    Hammer, Stephan C.; Kubik, Grzegorz; Watkins, Ella; Huang, Shan; Minges, Hannah; Arnold, Frances H.
    Science (Washington, DC, United States) (2017), 358 (6360), 215-218CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Catalytic anti-Markovnikov oxidn. of alkene feedstocks could simplify synthetic routes to many important mols. and solve a long-standing challenge in chem. Here we report the engineering of a cytochrome P 450 enzyme by directed evolution to catalyze metal-oxo-mediated anti-Markovnikov oxidn. of styrenes with high efficiency. The enzyme uses dioxygen as the terminal oxidant and achieves selectivity for anti-Markovnikov oxidn. over the kinetically favored alkene epoxidn. by trapping high-energy intermediates and catalyzing an oxo transfer, including an enantioselective 1,2-hydride migration. The anti-Markovnikov oxygenase can be combined with other catalysts in synthetic metabolic pathways to access a variety of challenging anti-Markovnikov functionalization reactions.
  305. 305
    Nguyen, Q.-T.; Romero, E.; Dijkman, W. P.; de Vasconcellos, S. P.; Binda, C.; Mattevi, A.; Fraaije, M. W. Structure-Based Engineering of Phanerochaete Chrysosporium Alcohol Oxidase for Enhanced Oxidative Power toward Glycerol. Biochemistry 2018, 57 (43), 6209– 18,  DOI: 10.1021/acs.biochem.8b00918
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    307
    Structure-Based Engineering of Phanerochaete chrysosporium Alcohol Oxidase for Enhanced Oxidative Power toward Glycerol
    Nguyen, Quoc-Thai; Romero, Elvira; Dijkman, Willem P.; de Vasconcellos, Suzan Pantaroto; Binda, Claudia; Mattevi, Andrea; Fraaije, Marco W.
    Biochemistry (2018), 57 (43), 6209-6218CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Glycerol is a major byproduct of biodiesel prodn. and enzymes that oxidize this compd. have been long sought after. The recently described alc. oxidase from the white-rot basidiomycete Phanerochaete chrysosporium (PcAOX) was reported to feature very mild activity on glycerol. Here we describe the comprehensive structural and biochem. characterization of this enzyme. PcAOX was expressed in Escherichia coli in high yields and displayed high thermostability. Steady-state kinetics revealed that PcAOX is highly active towards methanol, ethanol, and 1-propanol (kcat = 18, 19, and 11 s-1, resp.), but showed very limited activity towards glycerol (kcat = 0.2 s-1 at 2 M substrate). The crystal structure of the homo-octameric PcAOX was detd. at 2.6 Å resoln. The catalytic center is a remarkable solvent-inaccessible cavity located at the re side of the flavin cofactor. Its small size explains the obsd. preference for methanol and ethanol as best substrates. These findings led us to design several cavity-enlarging mutants with significantly improved activity towards glycerol. Among them, the F101S variant had a high kcat value of 3 s-1 retaining a high degree of thermostability. The crystal structure of F101S PcAOX was solved, confirming the site of mutation and the larger substrate-binding pocket. Our data demonstrate that PcAOX is a very promising enzyme for glycerol biotransformation.
  306. 306
    Sawicki, E.; Hauser, T. R.; Stanley, T. W.; Elbert, W. The 3-Methyl-2-Benzothiazolone Hydrazone Test. Sensitive New Methods for the Detection, Rapid Estimation, and Determination of Aliphatic Aldehydes. Anal. Chem. 1961, 33 (1), 93– 6,  DOI: 10.1021/ac60169a028
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    308
    3-Methyl-2-benzothiazolinone hydrazone test-sensitive new methods for the detection, rapid estimation, and determination of aliphatic aldehydes
    Sawicki, Eugene; Hauser, Thomas R.; Stanley, Thomas W.; Elbert, Walter
    (1961), 33 (), 93-6CODEN: ANCHAM; ISSN:0003-2700.
    The reaction of an aliphatic aldehyde with 3-methyl-2-benzothiazolinone hydrazone (I) followed by oxidn. of excess I with FeCl3 in acid soln., results in an intensely brilliant, blue cationic dye. The method is applicable to spot plate, paper, silica gel, and colorimetric procedures. The latter 2 methods may be used quant. and can be applied to the analysis of auto exhaust fumes and polluted air. In the colorimetric method, Beer's law is obeyed from 5-125 γ/100 ml. in the case of HCHO. The identification limit of HCHO by either spot plate or paper techniques is 0.1 γ. Two methods for the synthesis of I are described.
  307. 307
    Zhu, P.; Lin, S.-M. MBTH for aliphatic aldehyde measurement, U.S. Patent US7112448B2, September 26, 2006.
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    Hauser, T. R.; Cummins, R. L. Increasing Sensitivity of 3-Methyl-2-Benzothiazolone Hydrozone Test for Analysis of Aliphatic Aldehydes in Air. Anal. Chem. 1964, 36 (3), 679– 81,  DOI: 10.1021/ac60209a067
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    310
    Increasing sensitivity of 3-methyl-2-benzothiazolinone hydrazone test for analysis of aliphatic aldehydes in air
    Hauser, Thomas R.; Cummins, Rodney L.
    (1964), 36 (3), 679-81CODEN: ANCHAM; ISSN:0003-2700.
    cf. CA 55, 8180a. The sensitivity of the test was increased 6-fold by the incorporation of H2NSO3H in the FeCl3 oxidizing reagent. The modification reduces the vol. of reagent required and eliminates the need to dil. with Me2CO in order to avoid turbidity. The absorbance at 628 mμ follows Beer's law for concns. of HCHO from 0 to 1.70 γ/ml.
  309. 309
    Dean Pakulski, J.; Benner, R. An Improved Method for the Hydrolysis and MBTH Analysis of Dissolved and Particulate Carbohydrates in Seawater. Mar. Chem. 1992, 40 (3), 143– 60,  DOI: 10.1016/0304-4203(92)90020-B
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    Gomez, L. D.; Whitehead, C.; Barakate, A.; Halpin, C.; McQueen-Mason, S. J. Automated Saccharification Assay for Determination of Digestibility in Plant Materials. Biotechnol. Biofuels 2010, 3, 23,  DOI: 10.1186/1754-6834-3-23
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    Automated saccharification assay for determination of digestibility in plant materials
    Gomez Leonardo D; Whitehead Caragh; Barakate Abdellah; Halpin Claire; McQueen-Mason Simon J
    Biotechnology for biofuels (2010), 3 (), 23 ISSN:.
    BACKGROUND: Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies. RESULTS: We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared. CONCLUSIONS: The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies.
  311. 311
    Oliveira, F. S. d.; Leite, B. C. O.; Andrade, M. V. A. S. d.; Korn, M. Determination of Total Aldehydes in Fuel Ethanol by MBTH Method: Sequential Injection Analysis. J. Braz. Chem. Soc. 2005, 16, 87– 92,  DOI: 10.1590/S0103-50532005000100013
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    Suchý, M.; Lazurko, C.; Kirby, A.; Dang, T.; Liu, G.; Shuhendler, A. J. Methyl 5-MeO-N-Aminoanthranilate, a Minimalist Fluorogenic Probe for Sensing Cellular Aldehydic Load. Org. Biomol. Chem. 2019, 17 (7), 1843– 53,  DOI: 10.1039/C8OB02255K
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    314
    Methyl 5-MeO-N-aminoanthranilate, a minimalist fluorogenic probe for sensing cellular aldehydic load
    Suchy, Mojmir; Lazurko, Caitlin; Kirby, Alexia; Dang, Trina; Liu, George; Shuhendler, Adam J.
    Organic & Biomolecular Chemistry (2019), 17 (7), 1843-1853CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Me 5-MeO-N-aminoanthranilate, a fluorogenic probe comprising a single substituted benzene ring has been applied towards the fluorescence detection of endogenous carbonyls through rapid, catalyst-free complexation of these bio-derived markers of cell stress under physiol. conditions. The products formed during the reaction between the probe and aldehydic products of lipid peroxidn., including malondialdehyde and long-chain aliph. aldehydes relevant to the oxidative decompn. of cell membranes, have been evaluated. Live cell imaging of di-Et maleate-induced oxidative stress with or without pretreatment with α-tocopherol was carried out, with the result suggesting that the presented mol. might serve as a minimalist mol. probe capable of cellular "Aldehydic Load" detection by fluorescence microscopy. This work also outlines functional constraints of the fluorogenic probe (i.e. intramol. cyclization), providing a realistic evaluation of Me 5-MeO-N-aminoanthranilate for fluorescence-based aldehyde detection.
  313. 313
    Yuen, L. H.; Saxena, N. S.; Park, H. S.; Weinberg, K.; Kool, E. T. Dark Hydrazone Fluorescence Labeling Agents Enable Imaging of Cellular Aldehydic Load. ACS Chem. Biol. 2016, 11 (8), 2312– 9,  DOI: 10.1021/acschembio.6b00269
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    315
    Dark Hydrazone Fluorescence Labeling Agents Enable Imaging of Cellular Aldehydic Load
    Yuen, Lik Hang; Saxena, Nivedita S.; Park, Hyun Shin; Weinberg, Kenneth; Kool, Eric T.
    ACS Chemical Biology (2016), 11 (8), 2312-2319CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Aldehydes are key intermediates in many cellular processes, from endogenous metabolic pathways like glycolysis to undesired exogenously induced processes such as lipid peroxidn. and DNA interstrand crosslinking. Alkyl aldehydes are well documented to be cytotoxic, affecting the functions of DNA and protein, and their levels are tightly regulated by the oxidative enzyme ALDH2. Mutations in this enzyme are assocd. with cardiac damage, diseases such as Fanconi anemia (FA), and cancer. Many attempts have been made to identify and quantify the overall level of these alkyl aldehydes inside cells, yet there are few practical methods available to detect and monitor these volatile aldehydes in real time. Here, the authors describe a multicolor fluorogenic hydrazone transfer ("DarkZone") system to label alkyl aldehydes, yielding up to 30-fold light-up response in vitro. A cell-permeant DarkZone dye design was applied to detect small-mol. aldehydes in the cellular environment. The new dye design also enabled the monitoring of cellular acetaldehyde prodn. from ethanol over time by flow cytometry, demonstrating the utility of the DarkZone dyes for measuring and imaging the aldehydic load related to human disease.
  314. 314
    Liu, T.; Yang, L.; Zhang, J.; Liu, K.; Ding, L.; Peng, H.; Belfield, K. D.; Fang, Y. Squaraine-Hydrazine Adducts for Fast and Colorimetric Detection of Aldehydes in Aqueous Media. Sens. Actuators B Chem. 2019, 292, 88– 93,  DOI: 10.1016/j.snb.2019.04.138
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    316
    Squaraine-hydrazine adducts for fast and colorimetric detection of aldehydes in aqueous media
    Liu, Taihong; Yang, Lvjie; Zhang, Jing; Liu, Ke; Ding, Liping; Peng, Haonan; Belfield, Kevin D.; Fang, Yu
    Sensors and Actuators, B: Chemical (2019), 292 (), 88-93CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    Capitalizing on the nucleophilic addn. of hydrazine toward the central cyclobutenyl core and affinity of aldehydes to hydrazine group, two water-sol. squaraine dyes (SQOH and SQPY) were synthesized as sensitive colorimetric and fluorescent chemosensors for aldehydes. The color change from blue to colorless and back to blue these sensors underwent was readily obsd., even by the naked eyes. The response was fast (less than 1 s) and the detection limit for formaldehyde as an example was about 60μM. In contrast to the previous nucleophilic attack to squaraines and the other amine refs. studied, this work underwent a whole ON-OFF-ON sensing circle based on the characteristics of hydrazine. Meanwhile, the different photophys. properties of the two squaraines related to their different structures were also demonstrated. Importantly, a possible sensing mechanism is proposed suggesting these types of dyes hold great potential in the area of rapid, sensitive, and convenient detection of aldehydes.
  315. 315
    Liu, T.; Liu, X.; Wang, W.; Luo, Z.; Liu, M.; Zou, S.; Sissa, C.; Painelli, A.; Zhang, Y.; Vengris, M. Systematic Molecular Engineering of a Series of Aniline-Based Squaraine Dyes and Their Structure-Related Properties. J. Phys. Chem. C 2018, 122 (7), 3994– 4008,  DOI: 10.1021/acs.jpcc.7b11997
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    317
    Systematic Molecular Engineering of a Series of Aniline-Based Squaraine Dyes and Their Structure-Related Properties
    Liu, Taihong; Liu, Xinglei; Wang, Weina; Luo, Zhipu; Liu, Muqiong; Zou, Shengli; Sissa, Cristina; Painelli, Anna; Zhang, Yuanwei; Vengris, Mikas; Bondar, Mykhailo V.; Hagan, David J.; Van Stryland, Eric W.; Fang, Yu; Belfield, Kevin D.
    Journal of Physical Chemistry C (2018), 122 (7), 3994-4008CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
    With the objective of developing new near-IR fluorescent probes and understanding the effect mol. structure exerts on phys. properties, a series of aniline-based squaraine dyes with different no. and position of methoxy substituents adjacent to the squaraine core were synthesized and investigated. Using both computational and exptl. methods, we found that the subtle changes of the no. or position of the methoxy substituents influenced the twisting angle of the structure and led to significant variations in optical properties. Moreover, the methoxy substituent also affected aggregation behavior due to steric effects. The X-ray crystal structure of one of the key members of the series, SD-2a, clearly demonstrates the distortion between the four-membered squaraine core and the adjacent aniline ring due to methoxy substitution. Structure-related fast relaxation processes were investigated by femtosecond pump-probe expts. and transient absorption spectra. Quantum chem. calcns. and essential state models were exploited to analyze the primary exptl. results. The comprehensive investigation of structure-related properties of dihydroxylaniline-based squaraine dyes, with systematic substitution of OH by OCH3 functional groups, serves as a guide for the design of novel squaraine dyes for photonics applications.
  316. 316
    Xia, G.; Wang, H. Squaraine Dyes: The Hierarchical Synthesis and Its Application in Optical Detection. J. Photochem. Photobiol. C: Photochem. Rev. 2017, 31, 84– 113,  DOI: 10.1016/j.jphotochemrev.2017.03.001
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    Squaraine dyes: The hierarchical synthesis and its application in optical detection
    Xia, Guomin; Wang, Hongming
    Journal of Photochemistry and Photobiology, C: Photochemistry Reviews (2017), 31 (), 84-113CODEN: JPPCAF; ISSN:1389-5567. (Elsevier B.V.)
    Squaraine dyes, a four-membered ring system with structural rigidity, possess unique photoelec. properties and are marked by their exceptionally sharp and intense absorption assocd. with a strong fluorescent emission in soln. These favorable characteristics have prompted their exploitation in a no. of state of the art applications including photocond., data storage, light-emitting field-effect transistors, solar cells and fluorescent histol. probes. In this review, we first summarize the recently proposed novel methods in the synthesis of these versatile derivs. Subsequently, their extensive applications in the prevalent optical detection of the surrounding medium such as ions, pH, thiol-based compds., biomols. and cell over the past decades are covered and discussed. In addn., different categories for the synthesis and sensing mechanisms for various squaric acid-based chemo-/bio- sensors are illustrated. Finally, the challenges and opportunities in the synthesis and application of these derivs. are also briefly discussed.
  317. 317
    Lichtenstein, S. J.; Nettleton, G. S. Effects of Fuchsin Variants in Aldehyde Fuchsin Staining. J. Histochem. Cytochem. 1980, 28 (7), 683– 8,  DOI: 10.1177/28.7.6156202
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    Effects of fuchsin variants in aldehyde fuchsin staining
    Lichtenstein, Steven J.; Nettleton, G. S.
    Journal of Histochemistry and Cytochemistry (1980), 28 (7), 683-8CODEN: JHCYAS; ISSN:0022-1554.
    Aldehyde fuchsin stains pancreatic B cell granules, hypophyseal basophils, goblet cell mucins, gastric chief cells, hyaline cartilage, and elastica. The role of the fuchsin component of aldehyde fuchsin in its staining reaction indicates: (1) single N-methylation of the fuchsin mol. abolishes staining of unoxidized pancreatic B cells, although it does not prevent reaction of fuchsin with paraldehyde; (2) aldehyde fuchsin is probably a Schiff base condensation product of pararosaniline and acetaldehyde; (3) a Schiff base structure alone cannot account for aldehyde fuchsin staining of unoxidized pancreatic B cells; (4) a fully potent aldehyde fuchsin is possibly a Tris-Schiff base deriv. of pararosaniline.
  318. 318
    Robins, J. H.; Abrams, G. D.; Pincock, J. A. The Structure of Schiff Reagent Aldehyde Adducts and the Mechanism of the Schiff Reaction as Determined by Nuclear Magnetic Resonance Spectroscopy. Can. J. Chem. 1980, 58 (4), 339– 47,  DOI: 10.1139/v80-055
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    The structure of Schiff reagent aldehyde adducts and the mechanism of the Schiff reaction as determined by nuclear magnetic resonance spectroscopy
    Robins, J. H.; Abrams, G. D.; Pincock, J. A.
    Canadian Journal of Chemistry (1980), 58 (4), 339-47CODEN: CJCHAG; ISSN:0008-4042.
    An NMR study of compds. isolated from the Schiff aldehyde reaction between pararosaniline hydrochloride, SO2, and AcH showed that these adducts are α-anilinoalkylsulfonic acids, and not the N-phenylalkylsulfonamides, the structures most often accepted in the literature. In combination with NMR spectra obtained from solns. of the Schiff reagent and the Schiff reaction and with literature spectrophotometric data, this result leads to a reasonable proposal for the mechanism of the color development. The effects of the concns. of the dye, AcH, and esp. SO2 are explained and suggest that the dominant colored species is a 2:1 AcH-dye adduct. The NMR results also reveal a kinetic/thermodn. competition for AcH between the aniline of the dye and the aldehyde carbonyl leading to bisulfite addn. The relation of the Schiff test with AcH and the Feulgen test for aldehydes in biol. samples is also discussed.
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    Kitov, P. I.; Vinals, D. F.; Ng, S.; Tjhung, K. F.; Derda, R. Rapid, Hydrolytically Stable Modification of Aldehyde-Terminated Proteins and Phage Libraries. J. Am. Chem. Soc. 2014, 136 (23), 8149– 52,  DOI: 10.1021/ja5023909
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    Rapid, Hydrolytically Stable Modification of Aldehyde-Terminated Proteins and Phage Libraries
    Kitov, Pavel I.; Vinals, Daniel F.; Ng, Simon; Tjhung, Katrina F.; Derda, Ratmir
    Journal of the American Chemical Society (2014), 136 (23), 8149-8152CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The authors describe the rapid reaction of 2-amino benzamidoxime (ABAO) derivs. with aldehydes in water. The ABAO combines an aniline moiety for iminium-based activation of the aldehyde and a nucleophilic group (Nu:) ortho to the amine for intramol. ring closure. The reaction between ABAO and aldehydes is kinetically similar to oxime formations performed under stoichiometric aniline catalysis. The authors characterized the reaction by both NMR and UV spectroscopy and detd. that the rate-detg. step of the process is formation of a Schiff base, which is followed by rapid intramol. ring closure. The relation between apparent rate const. and pH suggests that a protonated benzamidoxime acts as an internal general acid in Schiff-base formation. The reaction is accelerated by substituents in the arom. ring that increase the basicity of the arom. amine. The rate of up to 40 M-1s-1 between an electron-rich aldehyde and 5-methoxy-ABAO (PMA), which was obsd. at pH 4.5, places this reaction among the fastest known bio-orthogonal reactions. Reaction between M13 phage-displayed library of peptides terminated with an aldehyde moiety and 1 mM biotin-ABAO deriv. reaches completion in 1 h at pH 4.5. Finally, the product of reaction, dihydroquinazoline deriv., shows fluorescence at 490 nm suggesting a possibility of developing fluorogenic aldehyde-reactive probes based on ABAO framework.
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    Ressmann, A. K.; Schwendenwein, D.; Leonhartsberger, S.; Mihovilovic, M. D.; Bornscheuer, U. T.; Winkler, M.; Rudroff, F. Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes. Adv. Synth. Catal. 2019, 361, 2538,  DOI: 10.1002/adsc.201900154
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    Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes
    Ressmann, Anna K.; Schwendenwein, Daniel; Leonhartsberger, Simon; Mihovilovic, Marko D.; Bornscheuer, Uwe T.; Winkler, Margit; Rudroff, Florian
    Advanced Synthesis & Catalysis (2019), 361 (11), 2538-2543CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The selective and direct redn. of carboxylic acids into the corresponding aldehydes by chem. methods is still a challenging task in synthesis. Several reductive and oxidative chem. methods are known to produce aldehydes, but most of them require expensive reagents, special reaction conditions, are 2-step procedures and often lack chemoselectivity. Nature provides an elegant tool, so called carboxylic acid reductases (CARs) for the direct redn. of carboxylic acids to aldehydes. Discovery as well as engineering of novel CAR enzymes necessitates a robust, product selective high-throughput assay (HTA). The authors report a simple and fast HTA that allows the substrate-independent and chemoselective quantification of aldehydes (irresp. of their chem. structure) and is sensitive to the nM range. The HTA was validated by NMR and GC analyses and in microbial cells by reexamn. of the substrate scope of CAR from Nocardia iowensis (CARNi). The results were fully consistent with reported data.
  321. 321
    Horvat, M.; Larch, T.-S.; Rudroff, F.; Winkler, M. Amino Benzamidoxime (ABAO)-Based Assay to Identify Efficient Aldehyde-Producing Pichia Pastoris Clones. Adv. Synth. Catal. 2020, 362 (21), 4673– 9,  DOI: 10.1002/adsc.202000558
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    323
    Amino Benzamidoxime (ABAO)-Based Assay to Identify Efficient Aldehyde-Producing Pichia pastoris Clones
    Horvat, Melissa; Larch, Tanja-Saskia; Rudroff, Florian; Winkler, Margit
    Advanced Synthesis & Catalysis (2020), 362 (21), 4673-4679CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The chemoselective synthesis of aldehydes is a challenging task. Nature provides carboxylic acid reductases (CARs) as elegant tools for the direct redn. of carboxylic acids to their resp. aldehydes. The discovery of new CARs and strains that efficiently produce these enzymes necessitates a robust high-throughput assay with selectivity for aldehydes. We recently reported a simple assay that allows the substrate independent and chemoselective quantification of aldehydes (irresp. of their chem. structure). The assay utilized amino benzamidoxime (ABAO), which forms UV-active and fluorescent dihydroquinazolines. In this study, we adapted the ABAO-assay for the identification and comparison of Pichia pastoris clones with the ability to produce aldehydes from carboxylic acids. Specifically, CAR and PPTase from Mycobacterium marinum (MmCAR and MmPPTase) were co-expressed using different bidirectional promoters (BDPs). A library of 598 clones was screened for piperonal prodn. with the ABAO assay and the results were validated by HPLC quantification. 1 OD unit of the best Pichia pastoris clone 2. A7, regulating MmCAR and MmPPTase expression by two strong constitutive promoters, fully converted 5 mM of piperonylic acid within 2 h.
  322. 322
    Mei, Z.; Zhang, K.; Qu, G.; Li, J.-K.; Liu, B.; Ma, J.-A.; Tu, R.; Sun, Z. High-Throughput Fluorescence Assay for Ketone Detection and Its Applications in Enzyme Mining and Protein Engineering. ACS Omega 2020, 5 (23), 13588– 94,  DOI: 10.1021/acsomega.0c00245
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    High-Throughput Fluorescence Assay for Ketone Detection and Its Applications in Enzyme Mining and Protein Engineering
    Mei, Zelong; Zhang, Kun; Qu, Ge; Li, Jun-Kuan; Liu, Baoyan; Ma, Jun-An; Tu, Ran; Sun, Zhoutong
    ACS Omega (2020), 5 (23), 13588-13594CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
    Ketones are of great importance as building blocks in synthetic org. chem. and biocatalysis. Most ketones cannot easily be quant. assayed due to the lack of visible photometric properties. Effective high-throughput assay (HTA) development is therefore necessary for ketone detn. Inspired by previous works of an aldehyde assay based on 2-amino benzamidoxime derivs., we developed a colorimetric method for rapid a HTA of structurally diverse ketones by using para-methoxy-2-amino benzamidoxime (PMA). This PMA-based method is characterized by high sensitivity manner (μM) with low background, as checked by gas chromatog. (GC). It can be used for quant. monitoring ketones by fluorescence screening in microtiter plates. Furthermore, this HTA method was employed in mining alc. dehydrogenases (ADHs), and in directed evolution aimed at enhancing ADH activity in the catalytic transformation of alcs. to ketones. This work provides a general tool for ketone detection in biocatalyst development.
  323. 323
    Xing, Y.; Wang, S.; Mao, X.; Zhao, X.; Wei, D. An Easy and Efficient Fluorescent Method for Detecting Aldehydes and Its Application in Biotransformation. J. Fluoresc. 2011, 21 (2), 587– 94,  DOI: 10.1007/s10895-010-0746-6
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    325
    An Easy and Efficient Fluorescent Method for Detecting Aldehydes and Its Application in Biotransformation
    Xing, Yanlong; Wang, Shu; Mao, Xiangzhao; Zhao, Xuebo; Wei, Dongzhi
    Journal of Fluorescence (2011), 21 (2), 587-594CODEN: JOFLEN; ISSN:1053-0509. (Springer)
    Water-sol. aldehydes (acetaldehyde, propionaldehyde) and non-water-sol. aldehydes (butyraldehyde and phenylacetaldehyde) were easily detected by an efficient fluorescent method with 5-aminofluorescein as probe. Under optimal detection conditions, 5-aminofluorescein could selectively respond to aldehydes with high sensitivity in comparison with other carbonyl compds. like ketones and acids. Thus, the proposed method was used to monitor microbial oxidn. and succeeded in trapping transiently-produced aldehydes during biotransformation of primary alcs. by Gluconobacter oxydans.
  324. 324
    Li, Z.; Xue, Z.; Wu, Z.; Han, J.; Han, S. Chromo-Fluorogenic Detection of Aldehydes with a Rhodamine Based Sensor Featuring an Intramolecular Deoxylactam. Org. Biomol. Chem. 2011, 9 (22), 7652– 4,  DOI: 10.1039/c1ob06448g
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    Chromo-fluorogenic detection of aldehydes with a rhodamine based sensor featuring an intramolecular deoxylactam
    Li, Zhu; Xue, Zhongwei; Wu, Zhisheng; Han, Jiahuai; Han, Shoufa
    Organic & Biomolecular Chemistry (2011), 9 (22), 7652-7654CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    A chromogenic and fluorogenic detection of aldehydes was achieved via analyte triggered opening of the deoxylactam of N-(rhodamine B)-deoxylactam-ethylenediamine (dRB-EDA). The utility of the sensor was demonstrated by fluorescent labeling of aldehyde-displaying sialoproteins on cell surfaces.
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    Davidson, D.; Weiss, M.; Jelling, M. The Action of Ammonia on Benzil. J. Org. Chem. 1937, 02 (4), 319– 27,  DOI: 10.1021/jo01227a004
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    Action of ammonia on benzil
    Davidson, David; Weiss, Marvin; Jelling, Murray
    Journal of Organic Chemistry (1937), 2 (), 319-27CODEN: JOCEAH; ISSN:0022-3263.
    Laurent (J. prakt. Chem. 35, 461(1845)) showed that NH3 and benzil (I) give 3 compds., termed imabenzil (II), benzilimide (III) and benzilam (IV); Henius (Ann. 228, 339(1885)) found that in EtOH at 130° II disappeared after 1 hr. and on continued heating there resulted IV and lophine (V). It is now shown that III is N-desylbenzamide, PhBzCHNHBz, m. 141° (all m. ps. cor.), which is obtained in 80% yield from the SnCl2 deriv. (VI) of desylamine and BzCl with NaOH. The oxime of III m. 197-203° (a more sol. product, m. 140°, may be an isomeric oxime). From the compn. of II (C35H28N2O3) and its scission by acid into III, I and NH3, it is believed to be 1-benzoyl-5,6-dihydroxy-2,3,5,6-tetraphenyl - 1,2,5,6 - tetrahydropyrazine, CPh:N.C-(OH)Ph.C(OH)Ph.NBz.CHPh. III (0.32 g.) with 1.5 g. NH4OAc in 10 cc. AcOH, refluxed 1 hr., gives 0.28 g. (93%) of V. A mechanism for the action of NH3 on I, which avoids the scission of I as a step and relates the reaction to the formation of α-acylamino acids by the action of NH3 on α-keto acids, is proposed. N-Desyl derivs. of acid amides are converted smoothly into glyoxalines by the action of NH3 in AcOH. III and PhNH2 in AcOH give tetraphenylglyoxaline, m. 221°. VI and Ac2O in C6H6N give a nearly quant. yield of N-desylacetamide, m. 137°; with NH4OAc or PhNH2 in AcOH there results 2-methyl-4,5-diphenylglyoxaline, m. 243°, or the 1,4,5-tri-Ph deriv., m. 197°. Refluxing 1.05 g. I and 5 g. NH4OAc in 25 cc. glacial AcOH for 1 hr. gives 7% of IV and 94% V. I (1.05 g.), 0.13 g. hexamethylene-tetramine, 3 g. NH4OAc and 25 g. AcOH, refluxed 1 hr., give 91% of 4,5-diphenylglyoxaline; paraldehyde gives 96% of the 2-Me deriv., m. 244°, while BzH or hydrobenzamide gives 90-5% V.
  326. 326
    Nakashima, K.; Yamasaki, H.; Kuroda, N.; Akiyama, S. Evaluation of Lophine Derivatives as Chemiluminogens by a Flow-Injection Method. Anal. Chim. Acta 1995, 303 (1), 103– 7,  DOI: 10.1016/0003-2670(94)00360-X
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    Evaluation of lophine derivatives as chemiluminogens by a flow-injection method
    Nakashima, Kenichiro; Yamasaki, Hiromi; Kuroda, Naotaka; Akiyama, Shuzo
    Analytica Chimica Acta (1995), 303 (1), 103-7CODEN: ACACAM; ISSN:0003-2670. (Elsevier)
    A rapid and convenient method for the evaluation of lophine (2,4,5-triphenylimidazole) derivs. as to their chemiluminescence efficiency was developed by using a flow-injection technique. Std. solns. of lophine derivs. were injected into a flow line contg. a carrier soln. (1.5 × 10-4 M cobalt(II) contg. 0.2% hydroxylammonium chloride in 75% methanol), which was successively combined with a flow line contg. 100 mM hydrogen peroxide in H2O and a flow line contg. 1.0M KOH in H2O, and the chemiluminescence generated was monitored. Addn. of hydroxylammonium chloride into a carrier soln. resulted in a significant increase in chemiluminescence. Calibration graphs for eighteen derivs. were prepd. and the relative chemiluminescence intensities were estd. from the slopes of these graphs. Among the derivs., the compds. substituted by OMe at the 2- and 3-positions, and COOH, COCl, CONHNH2 and CONHNHCO(CH2)4Me at the 4-position on the Ph ring at the 2-position of the imidazole skeleton showed large chemiluminescence intensities which are comparable to that of lophine. The detection limit of lophine was 72 fmol per injection at a signal-to-noise ratio of 2.
  327. 327
    El-Maghrabey, M. H.; Kishikawa, N.; Ohyama, K.; Kuroda, N. Analytical Method for Lipoperoxidation Relevant Reactive Aldehydes in Human Sera by High-Performance Liquid Chromatography-Fluorescence Detection. Anal. Biochem. 2014, 464, 36– 42,  DOI: 10.1016/j.ab.2014.07.002
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    329
    Analytical method for lipoperoxidation relevant reactive aldehydes in human sera by high-performance liquid chromatography-fluorescence detection
    El-Maghrabey, Mahmoud H.; Kishikawa, Naoya; Ohyama, Kaname; Kuroda, Naotaka
    Analytical Biochemistry (2014), 464 (), 36-42CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    A validated, simple and sensitive HPLC method was developed for the simultaneous detn. of lipoperoxidn. relevant reactive aldehydes: glyoxal (GO), acrolein (ACR), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in human serum. The studied aldehydes were reacted with 2,2'-furil to form fluorescent difurylimidazole derivs. that were sepd. on a C18 column using gradient elution and fluorescence detection at excitation and emission wavelengths of 250 and 355 nm, resp. The method showed good linearity over the concn. ranges of 0.100-5.00, 0.200-10.0, 0.200-40.0, and 0.400-10.0 nmol/mL for GO, ACR, HNE, and MDA, resp., with detection limits ranging from 0.030 to 0.11 nmol/mL. The percentage RSD of intraday and interday precision did not exceed 5.0 and 6.2%, resp., and the accuracy (%found) ranged from 95.5 to 103%. The proposed method was applied for monitoring the four aldehydes in sera of healthy, diabetic, and rheumatic human subjects with simple pretreatment steps and without interference from endogenous components. By virtue of its high sensitivity and accuracy, our method enabled detection of differences between analytes concns. in sera of human subjects under different clin. conditions.
  328. 328
    Fathy Bakr Ali, M.; Kishikawa, N.; Ohyama, K.; Abdel-Mageed Mohamed, H.; Mohamed Abdel-Wadood, H.; Mohamed Mohamed, A.; Kuroda, N. Chromatographic Determination of Aliphatic Aldehydes in Human Serum after Pre-Column Derivatization Using 2,2′-Furil, a Novel Fluorogenic Reagent. J. Chromatogr. A 2013, 1300, 199– 203,  DOI: 10.1016/j.chroma.2013.03.033
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    330
    Chromatographic determination of aliphatic aldehydes in human serum after pre-column derivatization using 2,2'-furil, a novel fluorogenic reagent
    Fathy Bakr Ali Marwa; Kishikawa Naoya; Ohyama Kaname; Abdel-Mageed Mohamed Horria; Mohamed Abdel-Wadood Hanaa; Mohamed Mohamed Ashraf; Kuroda Naotaka
    Journal of chromatography. A (2013), 1300 (), 199-203 ISSN:.
    A novel, highly sensitive and selective fluorimetric liquid chromatographic method for simultaneous determination of medium chain aliphatic aldehydes was developed. The method was based on the derivatization of aliphatic aldehydes with 1,2-di(2-furyl)-1,2-ethanedione (2,2'-furil), a novel fluorogenic reagent, to form highly fluorescent difurylimidazole derivatives. The fluorescence derivatives were separated in less than 20min on a reversed-phase ODS column using an isocratic elution with a mixture of methanol-water (80:20, v/v%). The detection limits were from 0.19 to 0.50nM (1-10fmol/injection) at a signal-to-noise ratio (S/N) of 3. This method was successfully applied for monitoring of aliphatic aldehydes in healthy human sera by a simple pretreatment procedure without interferences from serum constituents.
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    Nakamura, M.; Toda, M.; Saito, H.; Ohkura, Y. Fluorimetric Determination of Aromatic Aldehydes with 4,5-Dimethoxy-1,2-Diaminobenzene. Anal. Chim. Acta 1982, 134, 39– 45,  DOI: 10.1016/S0003-2670(01)84175-1
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    331
    Fluorimetric determination of aromatic aldehydes with 4,5-dimethoxy-1,2-diaminobenzene
    Nakamura, Masaru; Toda, Mitsuko; Saito, Hiroko; Ohkura, Yosuke
    Analytica Chimica Acta (1982), 134 (), 39-45CODEN: ACACAM; ISSN:0003-2670.
    A sensitive fluorometric method for the detn. of arom. aldehydes is based on their reaction in dil. acid with 4,5-dimethoxy-1,2-diaminobenzene to give a compd. which fluoresces intensely in alk. soln. The fluorescence is stabilized by β-mercaptoethanol. The method is simple, selective (for arom. and arylaliph. aldehydes), and sensitive; almost all the aldehydes can be detd. at concns. of 10-8-10-7M.
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    Imazato, T.; Kanematsu, M.; Kishikawa, N.; Ohyama, K.; Hino, T.; Ueki, Y.; Maehata, E.; Kuroda, N. Determination of Acrolein in Serum by High-Performance Liquid Chromatography with Fluorescence Detection after Pre-Column Fluorogenic Derivatization Using 1,2-Diamino-4,5-Dimethoxybenzene. Biomed. Chromatogr. 2015, 29 (9), 1304– 8,  DOI: 10.1002/bmc.3422
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    332
    Determination of acrolein in serum by high-performance liquid chromatography with fluorescence detection after pre-column fluorogenic derivatization using 1,2-diamino-4,5-dimethoxybenzene
    Imazato, Takahiro; Kanematsu, Mariko; Kishikawa, Naoya; Ohyama, Kaname; Hino, Takako; Ueki, Yukitaka; Maehata, Eisuke; Kuroda, Naotaka
    Biomedical Chromatography (2015), 29 (9), 1304-1308CODEN: BICHE2; ISSN:0269-3879. (John Wiley & Sons Ltd.)
    Acrolein is a major unsatd. aldehyde that is generated during the lipid peroxidn. process. The measurement of acrolein in biol. samples should be useful to est. the degree of lipid peroxidn. and to evaluate the effect of hazardous properties of acrolein on human health. In this study, a highly sensitive and selective high-performance liq. chromatog. with fluorescence detection method was developed for the detn. of acrolein in human serum. The proposed method involves the pre-column fluorogenic derivatization of acrolein with 1,2-diamino-4,5-dimethoxybenzene (DDB) as a reagent. The fluorescent deriv. of acrolein could be detected clearly without any interfering reagent blank peaks because DDB does not have intrinsic fluorescence itself, and the detection limit was 10 nM (signal-to-noise ratio = 3). The proposed method could selectively detect acrolein in human serum with a simple protein pptn. treatment. Copyright © 2015 John Wiley & Sons, Ltd.
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    McLellan, A. C.; Phillips, S. A.; Thornalley, P. J. The Assay of Methylglyoxal in Biological Systems Byderivatization with 1,2-Diamino-4,5-Dimethoxybenzene. Anal. Biochem. 1992, 206 (1), 17– 23,  DOI: 10.1016/S0003-2697(05)80005-3
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    The assay of methylglyoxal in biological systems by derivatization with 1,2-diamino-4,5-dimethoxybenzene
    McLellan, A. C.; Phillips, S. A.; Thornalley, P. J.
    Analytical Biochemistry (1992), 206 (1), 17-23CODEN: ANBCA2; ISSN:0003-2697.
    A procedure for the assay of methylglyoxal in biol. systems is described, together with sample storage, sample processing procedures, and statistical evaluation. Specimen data are presented. Methylglyoxal was assayed by derivatization with 1,2-diamino-4,5-dimethoxybenzene and HPLC of the resulting quinoxaline, 6,7-dimethoxy-2-methylquinoxaline, with spectrophotometric or fluorescence detection. Derivatization, solid-phase extn., and HPLC were performed under acid conditions to prevent the spontaneous formation of methylglyoxal from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate during the assay. The limits of detection in the biol. matrix were 45 pmol (absorbance detection) and 10 pmol (fluorometric detection), the recovery was 58%, and the intra- and interbatch coeffs. of variance were 7.7 and 30.0%, resp. The concn. of methylglyoxal in whole blood from normal healthy human individuals was (mean ± SE, nM) 256 ± 92 (n = 12) and that from diabetic patients was 479 ± 49 (n = 55), showing a significant increase in diabetes mellitus (P < 0.01; Mann-Whitney U test). Sample processing under acidic conditions was essential to avoid interferences. Previous ests. of the concn. of methylglyoxal in biol. samples require re-evaluation.
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    Hantzsch, A. Condensationsprodukte Aus Aldehydammoniak Und Ketonartigen Verbindungen. Ber. Dtsch. Chem. Ges. 1881, 14 (2), 1637– 8,  DOI: 10.1002/cber.18810140214
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    Belman, S. The Fluorimetric Determination of Formaldehyde. Anal. Chim. Acta 1963, 29, 120– 6,  DOI: 10.1016/S0003-2670(00)88591-8
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    The fluorimetric determination of formaldehyde
    Belman, Sidney
    Analytica Chimica Acta (1963), 29 (2), 120-6CODEN: ACACAM; ISSN:0003-2670.
    cf. Nash, CA 48, 503i. To det. HCHO, 0.01-1 γ/ml., mix freshly prepd.; pH 6, 2M NH4OAc-0.02M (freshly distd.) Ac2CH2 with an equal vol. of HCHO soln. and incubate for 10 min. at 58° or for 60 min. at 37°. The lower temp. is preferred for low (<0.05 γ/ml.) HCHO concns. After cooling to 25°, read the fluorescence at 510 mμ. The resp. fluorescence excitation and emission max. are 410 and 510 mμ. The fluorescence is linear at 0.005-0.4 γ/ml. HCHO and deviates slightly from linearity at 0.4-1 γ/ml. The reaction of Ac2CH2, NH4+, and HCHO forms sol. 3,5-diacetyl-1,4-dihydrolutidine (I) with max. at 412 mμ. HCHO > 1 γ/ml. is detd. by measuring the absorbance at 412 mμ. In an application, 2 differently treated samples of deoxyribonucleic acid (DNA) were treated with HCHO, and the isolated DNA was analyzed for bound HCHO as described. Heat-denatured DNA had 85% of its NH2 groups bound to HCHO, while 2-amino-1-naphthol treated DNA had 32% of its NH2 groups bound to HCHO. As little as 1 γ NH3/ml. is detected by the described method, with a M NaOAc, pH 6, 0.8M Ac2CH2-2M HCHO reagent, by mixing with an equal vol. of NH4OH soln., heating at 100° for 10 min., cooling to 25°, and measuring the fluorescence. Cryst. I, m. 190-200°, was phys. characterized. The infrared spectra confirm the structure of I (loc. cit.) in dil. soln., and suggest that a dimer of I primarily exists in concd. soln. and in the solid I.
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    Sawicki, E.; Carnes, R. Spectrophotofluorimetric Determination of Aldehydes with Dimedone and Other Reagents. Mikrochim. Acta 1968, 56 (1), 148– 59,  DOI: 10.1007/BF01216118
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    Compton, B. J.; Purdy, W. C. The Mechanism of the Reaction of the Nash and the Sawicki Aldehyde Reagent. Can. J. Chem. 1980, 58 (21), 2207– 11,  DOI: 10.1139/v80-355
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    The mechanism of the reaction of the Nash and the Sawicki aldehyde reagent
    Compton, Bruce Jon; Purdy, William C.
    Canadian Journal of Chemistry (1980), 58 (21), 2207-11CODEN: CJCHAG; ISSN:0008-4042.
    Synthetic and chromatog. studies are presented on the Nash and Sawicki colorimetric methods for quantitating HCHO [reaction of (MeCO)2CH2 or dimedone with NH4OAc and MeCHO). The actual color-forming agent in the Nash method is an isoable iminodione formed in situ by reaction of the 2 components. Explanation is given for differences between these 2 methods.
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    Compton, B. J.; Purdy, W. C. Fluoral-P, a Member of a Selective Family of Reagents for Aldehydes. Anal. Chim. Acta 1980, 119 (2), 349– 57,  DOI: 10.1016/S0003-2670(01)93636-0
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    Fluoral-P, a member of a selective family of reagents for aldehydes
    Compton, Bruce Jon; Purdy, William C.
    Analytica Chimica Acta (1980), 119 (2), 349-57CODEN: ACACAM; ISSN:0003-2670.
    The compd. 4-amino-3-penten-2-one (Fluoral-P) is introduced as a member of a selective family of reagents for aldehydes. The stability, selectivity, and reactivity of the reagent are described and its utility in automatic detns. of HCHO is demonstrated. The condensation reaction between Fluoral-P and HCHO gives a product detectable by fluorescence. The flow injection system utilizing this reagent can be used to det. HCHO in the lower nm range.
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    Mopper, K.; Stahovec, W. L.; Johnson, L. Trace Analysis of Aldehydes by Reversed-Phase High-Performance Liquid Chromatography and Precolumn Fluorigenic Labeling with 5,5-Dimethyl-1,3-Cyclohexanedione. J. Chromatogr. A 1983, 256, 243– 52,  DOI: 10.1016/S0021-9673(01)88237-6
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    Al-Moniee, M. A.; Koopal, C.; Akmal, N.; van Veen, S.; Zhu, X.; Sanders, P. F.; Sanders, P. F.; Al-Abeedi, F. N.; Amer, A. M. Applicability of Dimedone Assays for the Development of Online Aldehyde Sensor in Seawater Flooding Systems. J. Sens. Sci. Technol. 2016, 6, 101,  DOI: 10.4236/jst.2016.64008
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    Li, Q.; Oshima, M.; Motomizu, S. Flow-Injection Spectrofluorometric Determination of Trace Amounts of Formaldehyde in Water after Derivatization with Acetoacetanilide. Talanta 2007, 72 (5), 1675– 80,  DOI: 10.1016/j.talanta.2007.01.054
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    Flow-injection spectrofluorometric determination of trace amounts of formaldehyde in water after derivatization with acetoacetanilide
    Li, Qiong; Oshima, Mitsuko; Motomizu, Shoji
    Talanta (2007), 72 (5), 1675-1680CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
    A novel fluoro-photometric method, which requires no enrichment procedure, to det. formaldehyde in environmental water was developed. This flow-injection anal. method for the spectrofluorometric detn. of formaldehyde in water is based on the reaction of formaldehyde with acetoacetanilide and NH3. The method exhibited good linearity from 0.50 to 40 × 10-7 M; a limit of detection of 3 × 10-9 M (0.09 ppb) is achievable with a 15/h sample through-put. A main advantage of this method is that the reaction can be conducted at room temp. without a heating system. The effect of various interferences possibly present in actual water samples was examd. Most cations, anions, and org. compds. do not interfere with the formaldehyde detn. in environmental water. The method is very simple, rapid, less expensive, highly sensitive, and can be applied to environmental water (rain, tap, and river water) at low concns. without any enrichment procedure.
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    Kolmel, D. K.; Kool, E. T. Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis. Chem. Rev. 2017, 117 (15), 10358– 76,  DOI: 10.1021/acs.chemrev.7b00090
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    Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis
    Kolmel, Dominik K.; Kool, Eric T.
    Chemical Reviews (Washington, DC, United States) (2017), 117 (15), 10358-10376CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The formation of oximes and hydrazones is employed in numerous scientific fields as a simple and versatile conjugation strategy. This imine-forming reaction is applied in fields as diverse as polymer chem., biomaterials and hydrogels, dynamic combinatorial chem., org. synthesis, and chem. biol. Here we outline chem. developments in this field, with special focus on the past ∼10 years of developments. Recent strategies for installing reactive carbonyl groups and α-nucleophiles into biomols. are described. The basic chem. properties of reactants and products in this reaction are then reviewed, with an eye to understanding the reaction's mechanism and how reactant structure controls rates and equil. in the process. Recent work that has uncovered structural features and new mechanisms for speeding the reaction, sometimes by orders of magnitude, is discussed. We describe recent studies that have identified esp. fast reacting aldehyde/ketone substrates and structural effects that lead to rapid-reacting α-nucleophiles as well. Among the most effective new strategies has been the development of substituents near the reactive aldehyde group that either transfer protons at the transition state or trap the initially formed tetrahedral intermediates. In addn., the recent development of efficient nucleophilic catalysts for the reaction is outlined, improving greatly upon aniline, the classical catalyst for imine formation. A no. of uses of such second- and third-generation catalysts in bioconjugation and in cellular applications are highlighted. While formation of hydrazone and oxime has been traditionally regarded as being limited by slow rates, developments in the past 5 years have resulted in completely overturning this limitation; indeed, the reaction is now one of the fastest and most versatile reactions available for conjugations of biomols. and biomaterials.
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    Kalia, J.; Raines, R. T. Hydrolytic Stability of Hydrazones and Oximes. Angew. Chem., Int. Ed. 2008, 47 (39), 7523– 6,  DOI: 10.1002/anie.200802651
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    Hydrolytic stability of hydrazones and oximes
    Kalia, Jeet; Raines, Ronald T.
    Angewandte Chemie, International Edition (2008), 47 (39), 7523-7526CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Watching C'n'N: Hydrazones and oximes are common conjugates but are labile to hydrolysis. The hydrolytic stabilities of isostructural hydrazones and one oxime were detd. at pD 5.0-9.0. The rate const. for the acid-catalyzed hydrolysis of the oxime was nearly 103-fold lower than those for simple hydrazones, and a trialkylhydrazonium ion (formed after condensation) was even more stable than the oxime.
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    Forget, D.; Boturyn, D.; Defrancq, E.; Lhomme, J.; Dumy, P. Highly Efficient Synthesis of Peptide-Oligonucleotide Conjugates: Chemoselective Oxime and Thiazolidine Formation. Eur. J. Chem. 2001, 7 (18), 3976– 84,  DOI: 10.1002/1521-3765(20010917)7:18<3976::AID-CHEM3976>3.0.CO;2-X
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    Highly efficient synthesis of peptide-oligonucleotide conjugates: chemoselective oxime and thiazolidine formation
    Forget, Damien; Boturyn, Didier; Defrancq, Eric; Lhomme, Jean; Dumy, Pascal
    Chemistry - A European Journal (2001), 7 (18), 3976-3984CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH)
    A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-contg. peptide with an aldehyde-contg. oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatized by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aq. conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with mol. biol. tools, this methodol. opens up new prospects for post-functionalization of high-mol.-wt. DNA structures.
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    Shao, J.; Tam, J. P. Unprotected Peptides as Building Blocks for the Synthesis of Peptide Dendrimers with Oxime, Hydrazone, and Thiazolidine Linkages. J. Am. Chem. Soc. 1995, 117 (14), 3893– 9,  DOI: 10.1021/ja00119a001
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    Unprotected Peptides as Building Blocks for the Synthesis of Peptide Dendrimers with Oxime, Hydrazone, and Thiazolidine Linkages
    Shao, Jun; Tam, James P.
    Journal of the American Chemical Society (1995), 117 (14), 3893-9CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A general approach for forming peptide dendrimers with oxime, hydrazone, and thiazolidine linkages was developed using unprotected peptides as building blocks and selective ligation between an aldehyde and a weak base. To illustrate the generality of this approach, a branched lysine core matrix with an aldehyde was used to ligate four copies of unprotected peptides contg. a weak nucleophilic base such as aminooxy, hydrazide, or cysteine 1,2-aminothiol groups at their N-termini to form synthetic branched proteins. Various parameters affecting the ligations were studied, and optimal conditions gave 12-27-fold rate increases and shortened the reaction time from 24-60 to 2-8 h. Among the three reactions studied, ligation by thiazolidine appeared to be superior to ligation by oxime or hydrazone in reaction rate and product stability. The purified dendrimeric products gave single peaks on reverse phase HPLC and size exclusion HPLC. Their macromol. structures were also characterized by mass spectrometry and amino acid anal. CD spectra of the dendrimers showed that they have an increased ordered helical structure. Ligation reactions using a mutually reactive weak base and aldehyde pair should provide a useful approach for the synthesis of peptide dendrimers and artificial proteins.
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    Kemp, D. S.; Woodward, R. B. The N-Ethylbenzisoxazolium Cation─I: Preparation and Reactions with Nucleophilic Species. Tetrahedron 1965, 21 (11), 3019– 35,  DOI: 10.1016/S0040-4020(01)96921-2
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    N-Ethylbenzisoxazolium cation. I. Preparation and reactions with nucleophilic species
    Kemp, D. S.; Woodward, R. B.
    Tetrahedron (1965), 21 (11), 3019-35CODEN: TETRAB; ISSN:0040-4020.
    cf. CA 55, 14322c. H2NOSO3H (250 g.) and 20 g. Na2SO4 in 800 mL. H2O stirred with 230 g. ο-HOC6H4CHO and the soln. stirred vigorously with 400 mL. H2O and 400 mL. CH2Cl2, the chilled mixt. (ice bath) stirred with addn. of 340 g. NaHCO3 and 300 mL. H2O and the mixt. stirred 1 h. before sepn. of the 2 phases, the aq. layer stirred 2 h. with 80 mL. CH2Cl2 and 30 g. NaHCO3 and the new aq. layer extd. with 150 mL. CH2Cl2, the combined CH2Cl2 exts. dried over MgSO4 and distd. yielded 94.6% benzisoxazole (I), b2 35-8°, n25D 1.5605, λ 6.22 μ, λ 235, 243, 280 mμ (ε 10000, 8100, 2900, alc.), τ 1.2s, 2.3-3.0 m. I (55 g.) and 87 g. purified Et3OBF4 kept in 200 mL. CH2Cl2 to incipient crystn. and cooled in ice 30 min., kept 6 h. at 20° and the cryst. mass washed with CH2Cl2 gave 104 g. salt, m. 109.2-10.2°, recrystd. from MeCNEtOAc to give nonhygroscopic light-sensitive N-ethylbenzisoxazolium fluoroborate (II), m. 109.5-10.2°, λ 6.20, 9-10 μ, λ 258, 297 mμ (ε 13100, 2900), τ 0.2s, 1.8-2.4m, 5.1q. II (0.5 g.) in 20 mL. 0.1N HCl heated 3 h. at 90° and the cooled suspension extd. with 30 mL. CH2Cl2 gave ο-HOC6H4CONHEt, m. 55-6.5°. Kinetic evidence indicated the intermediacy of the benzooxoketenimine (III) in the formation of the amide. Addn. of the N-ethylbenzisoxazolium cation (IV) to aq. solns. of HS-, F-, or CN- or to MeOH solns. of MeO- gave high yields of compds. 2-HOC6H4-CX: NEt (V) (X = F, CN, HS or OMe). Na2S.H2O (40 g.) in 50 mL. H2O and a slight excess of HCl kept 5 min. with 5 g. II in 5 mL. MeCN, and the product extd. with 45 mL. CH2Cl2, passed through a silica gel column and the recrystd. (CCl4-C6H12) product again recrystd. from EtOAc-C6H12 gave 1.7 g. ο-HOC6H4CSNHEt (VI), m. 60.5-1.3°, λ 264 mμ, (ε 9300, H2O, pH 1-6), λ 267, 286 mμ (ε 7800, 5500, pH 11), τ - 1.3s, 1.4s, 2.3-3.3m, 6.4p, 8.8t. VI in CH2Cl2 shaken with aq. AgNO3 and filtered from the pptd. Ag2S yielded 80% ο-HOC6H4CONHEt. KBr (4 g.) in 10 mL. H2O and 10 g. II kept 10 min. and filtered, the filtrate treated with 15 g. KF and extd. with 60 mL. CH2Cl2, the ext. dried over Na2SO4 and evapd., the residual liq. taken up in C5H12 and filtered through a mixt. of NaF and Celite, evapd., and the colorless oil (5.4 g., b1.0 42-4°) recrystd. at -70° from C5H12 gave V (X = F), b1 42-4°, n25D 1.5312, λ 3.0-3.7, 5.93 μ, τ - 1.8s, 2.5-3.4 m, 6.5q, 8.8t, sapond. in 0.1N to ο-HOC6H4CONHEt. NaCN (5 g.) in 20 mL. H2O overlayered with 10 mL. C5H12 and stirred vigorously with addn. of 2 g. II in 5 mL. MeCN, the org. layer and C5H12 washings (20 mL.) dried and evapd. yielded 80% V (X = CN), m. 45-5.7° (C5H2, and sublimation), λ 3.0-3.7, 4.48, 6.1μ, τ -2.6s, 2.0-3.0m, 6.0q, 8.6t, sapond. in 0.1N NaOH to ο-HOC6H4CONHEt, and hydrolyzed in 48% HBr at 20° in 3 days to give ο-HOC6H4COCO2H, m. 39.5°. II in 150 mL. MeOH stirred with addn. of 40 mL. MeOH contg. 0.6 g. Na and the residue on evapn. triturated with 80 mL. C5H12, the ext. evapd., and the residue distd. at 60°/0.1 mm. gave 3.0 g. oil, recrystd. from chilled C5H12 to give V (X = MeO), λ 3.0-3.7, 6.10 μ, τ 0.1s, 2.4-3.4m, 6.5s, q, 8.8t. II (1 g.) in 2 mL. MeCN added with stirring to 2 g. NaN3 in 25 mL. ice-cold H2O and extd. with 20 mL. cold CH2Cl2, the ext. dried over MgSO4 and cooled to -10° gave a lemon-yellow soln., λ 3.0-3.7, 4.70 (N3), 6.1 μ, fading in 30 min. at 20° to give 1-ethyl-2-(2-hydroxyphenyl)tetrazole, m. 140.2-1.2°, λ 283 mμ (ε 3200, H2O, pH 2-7), λ 308 mμ (ε 4500, H2O, pH 13). V (X = F) (0.5 g.) and 10 mL. concd. NH4OH stirred vigorously 4 h. at 20° and the CH2Cl2-washed ppt. recrystd. from MeCN gave 42% V (X = NH) (VI), m. 209° (decompn.), λ 340 mμ (ε 6900, H2O, pH 7-11) λ 288 mμ (ε 2800, H2O, pH 3); picrate, m. 151-5-2.5°, reconverted to VI by base. II (2 g.) in 3 mL. MeCN and 10 g. NaCNO in 25 mL. H2O extd. with 30 mL. CH2Cl2 and the dried (MgSO4) ext. evapd. yielded 80% 4-ethyliminobenz[1,3]oxazine-2,4-dione (VII), m. 193.5-4.0° λ 2.88, 3.01, 5.79, 6.18 μ (CH2Cl2), λ 249, 297 mμ (ε 16600, 4500, H2O, pH 1-10), τ 1.9-3.0m, 6.4q, 8.8t, converted in soln. in MeOH by addn. to aq. NaOH to give a high yield of VI. II (2 g.) in 3 mL. MeCN added to 25 mL. 20% NaCNS and the turbid mixt. extd. with 30 mL. CH2Cl2 gave 80% yield of 2-thio-4-ethyliminobenz[1.3]oxazine-2,4-dione, m. 207.8-8.0°, λ 2.85, 3.02, 6.22 μ, λ 259, 288, 297, 325 mμ (ε 12300, 12300, 1500, 8400, H2O, pH 1-6), λ 250, 290, 297 mμ (ε 10700, 12700, 13300, H2O, pH 11.0), τ 1.8-2.9m, 6.3q, 8.8t, converted by addn. in MeOH to AgNO3 to give VII. NaOAc (2 g.) in 15 mL. H2O adjusted to pH 5.5 with dil. HCl and stirred with gradual addn. of 1 g. powd. II, the mixt. extd. with 15 mL. CH2Cl2, and the ext. washed with 20 mL. 0.5N NaHCO3, evapd., and the residue crystd. from CH2Cl2-C6H12 gave 0.75 g. solid, m. 54-5.2°, recrystd. to give O-acetyl-N-ethylsalicylamide (VIII, R = Me) (IX), m. 55.0-5.6° (EtOAc-C6H12), λ 2.90, 5.66, 5.99 μ, λ 265 mμ (ε 800, H2O, pH 1-6), τ 2.4-3.1, 6.7p, 7.8s, identical with a sample obtained by treatment of ο-AcOC6H4COCl with H2NEt. Similarly was produced VIII (R = Ph), m. 112.1-12.6°, λ 5.75, 5.99 μ, in 88% yield. Analogously treatment of II with MeOCH2CO2H yielded 88% VIII (R = MeOCH2), m. 59.3-60.2° (EtOAc-C6H2), λ 5.60, 5.98 μ (CCl4), λ 265 mμ (ε 950, H2O, pH 1-7). II (3 g.) added as a fine powder with stirring to 5 g. H2NCH2CO2H in 50 mL. H2O, the mixt. treated after 5 min. with 2.5 g. picric acid and 1 g. H2NCH2CO2H in 30 mL. H2O and the dried cryst. ppt. (4.4 g., m. 137-9.0°) recrystd. from MeCN gave VIII (R = CH2NH2) picrate, C17H17N5O10, m. 145.5-7.0° (decompn.), λ 5.63 μ. II (2 g.) and 5 g. H2NCH2CO2H stirred 5 min. in 50 mL. H2O and adjusted to pH 8.5 with N NaOH with simultaneous addn. of 25 mL. Me2CO, kept 2 h. at 20° before concn. to 50% vol. and the suspension adjusted to pH 1 with 3N HCl, extd. with 45 mL. EtOAc and the ext. washed with 10 mL. satd. aq. Na2SO4, the dried ext. evapd., and the amide (0.5 g., m. 143-50°) recrystd. from EtOAc-C6H12 gave salicyloylglycine ethylamide, m. 165.0-5.8°, identical with a sample prepd. by treatment of ο-AcOC6H4COCl with excess H2NCH2CONHEt. The formation of O-acyl-N-ethylsalicylamides from the cation IV and carboxylate anions was rationalized as proceeding from an imino anhydride (X) by an acyl shift. BzNHCH2CO2H (7 g.) in 40 mL. N NaOH adjusted to pH 5 with dil. HCl and stirred rapidly with 7 g. powd. II, extd. with 60 mL. CH2Cl2 and the residue on evapn. extd. with 20 mL. boiling C6H12, the ext. evapd. and the residue recrystd. from abs. alc. gave 70 mg. 2-phenyloxazol-5-one (XI), m. 89.5-90.2°. The C6H12-insol. residue recrystd. from EtOAc-C6H12 and the product (8 g., m. 85.5-7.0°) recrystd. from CH2Cl2-Et2O gave VIII (R = BzNHCH2), m. 87.5-9.0°, λ 5.63, 5.99 μ. XI must be formed from a more highly activated deriv., X (R = BzNHCH2). II (2 g.) in 3 mL. MeCN added to 4 g. (H2N)2CS in 50 mL. H2O contg. 0.1 g. NaOAc and 0.1 g. AcOH and the suspension extd. at 15-min. intervals with 5 portions (15 mL.) of CH2Cl2, the dried ext. evapd. and the residue extd. with 16 mL. boiling C6H12, the ext. filtered hot through Celite and the cryst. solid (1.15 g., m. 78-82°) recrystd. from C6H12 and sublimed gave 2-imino-3-ethyl-4-thiobenz[1,3]oxazine-2,4-dione (XI), m. 83-3.6°, λ 2.93, 5.99 μ, λ 277, 320 mμ (ε 10600, 13200, H2O, pH 3-11), τ1.18-3.2m, 3.4s, 5.4q, 8.7t. XI (0.5 g.) in 10 mL. MeOH boiled and treated with 5 mL. 3N HCl, the cryst. product washed with 50% MeOH and recrystd. from dil. MeOH gave 0.4 g. yellow cubes, m. 106.4-7.3°, recrystd. and sublimed to give 3-ethyl-4-thiobenzo [1,3] oxazine-2,4-dione (XII), m. 107.0-7.4°, λ 5.67 μ, λ 270, 328 mμ (ε 12200, 12200, H2O, pH 1-9), converted by alk. hydrolysis to ο-HOC6H4CSNHEt, m. 60-0.8°. Treatment of XII with acidic AgNO3, gave O,N-carbonyl-N-ethylsalicylamide (XIII), m. 102-3°. XIII (2 g., prepd. by C5H5N-catalyzed reaction of ClCO2Et with ο-HOC6H4CONHEt) and 1.2 g. P2S5 triturated vigorously 10 min. at 175° and the cooled mass extd. with CH2Cl2, the ext. evapd., and the residue recrystd. yielded 65% yellow cubes, recrystd. to give XII. II (3 g.) in 3 mL. MeCN kept 6 min. in 50 mL. H2O contg. 4 g. (H2N)2CS, 0.1 g. NaOAc, and 0.1 g. AcOH, extd. rapidly with 20 mL. CH2Cl2, and the aq. layer adjusted to pH 8 with dil. aq. NaOH, the ppt. washed with H2O and CH2Cl2 and dried gave 0.8 g. 2-imino-4-thiobenz[1,3]oxazine-2,4-dione (XIV), m. 175-205° (decompn.), λ 2.97, 6.03 μ, λ 277, 334 mμ (ε 8500, 13200, H2O, pH 5), μ 277, 321 mμ (ε 6100, 9300, H2O, pH 11), τ 1.3s, 1.6-2.8m, 6.5s, converted by warm acidic AgNO3 to O,N-carbonylsalicylamide, m. 226-8°. Finely powd. II (5 g.) added with stirring to 7 g. recrystd. (H2N)2CS in 100 mL. H2O and the soln. extd. after 5 min. with 30 mL. CH2Cl2, the aq. layer added to 50 mL. satd. picric acid in 30% aq. EtOH and the ppt. washed and dried in vacuo gave 4.8 g. picrate of the cation (XV), decompg. at 140-50°; XV.HCl, λ 5.87 μ, λ 273, 340 mμ (ε 7600, 300, H2O); XV picrate, τ -0.2s, 1.4s, 2.6d, 6.3p, 8.8t. Addn. of XV picrate to aq. acetate buffer gave XI; addn. to bicarbonate buffer generated XI and XIV together with a trace of H2S. IV is able to combine with all nucleophilic elements of the 1st row of the periodic table and at least one member of the 2nd. The ready availability and high yields of the products provide unusual starting materials for the synthesis of new benzo-fused heterocyclic systems.
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    Salahuddin, S.; Renaudet, O.; Reymond, J.-L. Aldehyde Detection by Chromogenic/Fluorogenic Oxime Bond Fragmentation. Org. Biomol. Chem. 2004, 2 (10), 1471– 5,  DOI: 10.1039/b400314d
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    Aldehyde detection by chromogenic/fluorogenic oxime bond fragmentation
    Salahuddin, Syed; Renaudet, Olivier; Reymond, Jean-Louis
    Organic & Biomolecular Chemistry (2004), 2 (10), 1471-1475CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Amination of 4-nitrophenol, umbelliferone and 4-methylumbelliferone gave the corresponding oxyamines. These oxyamines react with aldehydes and ketones to form oximes. In the case of aliph. aldehydes and electron-poor arom. aldehydes, the oximes undergo base-catalyzed fragmentation in aq. buffer in the presence of bovine serum albumin to give the parent phenols, which is the acyclic analog of Kemp's elimination reaction of 5-nitrobenzisoxazole. The process can be used as a spectrophotometric assay for formaldehyde under aq. neutral conditions.
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    Drienovská, I.; Mayer, C.; Dulson, C.; Roelfes, G. A Designer Enzyme for Hydrazone and Oxime Formation Featuring an Unnatural Catalytic Aniline Residue. Nat. Chem. 2018, 10 (9), 946– 52,  DOI: 10.1038/s41557-018-0082-z
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    A designer enzyme for hydrazone and oxime formation featuring an unnatural catalytic aniline residue
    Drienovska, Ivana; Mayer, Clemens; Dulson, Christopher; Roelfes, Gerard
    Nature Chemistry (2018), 10 (9), 946-952CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
    Creating designer enzymes with the ability to catalyze abiol. transformations is a formidable challenge. Efforts toward this goal typically consider only canonical amino acids in the initial design process. However, incorporating unnatural amino acids that feature uniquely reactive side chains could significantly expand the catalytic repertoire of designer enzymes. To explore the potential of such artificial building blocks for enzyme design, here we selected p-aminophenylalanine (pAF) as a potentially novel catalytic residue. We demonstrate that the catalytic activity of the aniline side chain for hydrazone and oxime formation reactions is increased by embedding p-aminophenylalanine into the hydrophobic pore of the multidrug transcriptional regulator LmrR from Lactococcus lactis. Both the recruitment of reactants by the promiscuous binding pocket and a judiciously placed aniline that functions as a catalytic residue contribute to the success of the identified artificial enzyme. We anticipate that our design strategy will prove rewarding to significantly expand the catalytic repertoire of designer enzymes in the future.
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    Horowitz, R. M.; Geissman, T. A. A Cleavage Reaction of α-Allylbenzylamines. J. Am. Chem. Soc. 1950, 72 (4), 1518– 22,  DOI: 10.1021/ja01160a025
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    Cleavage reaction of α-allylbenzylamines
    Horowitz, Robert M.; Geissman, T. A.
    Journal of the American Chemical Society (1950), 72 (), 1518-22CODEN: JACSAT; ISSN:0002-7863.
    PhCH(CH2CH:CH2)CO2H (84.4 g.) and 37.8 g. C5H5N in 250 ml. ether, treated gradually with 57 g. SOCl2 in 50 ml. ether, stirred several hrs., kept overnight, and the residual acid chloride added dropwise to 500 ml. ice-cold concd. NH4OH, give 82% α-allyl-α-phenylacetamide (I), m. 53.5-4°. I (68 g.) in 300 ml. H2O, treated with NaOBr (with ice cooling), gives 90% α-allylbenzylamine (II), b3.5 75°, d420 0.953, nD24 1.5300; the phenylsulfonyl deriv. m. 79.5-80.5°; II and 3-O2NC6H3(CO)2O, heated 5 min. at 170 °, give N-(α-allylbenzyl)-3-nitrophthalimide, m. 98-9°. II yields a picrate, yellow, m. 153°; attempted recrystn. from Me2CO gives the N-isopropylidene deriv. of the picrate, yellow, m. 149-51°. II (10 g.) and 22.8 g. NaHCO3 in 100 ml. H2O and 100 ml. EtOH, treated (20 min.) with 17.1 g. Me2SO4 and boiled 2 hrs., give trimethyl-(α-allyl-benzyl)ammonium Me sulfate (III), m. 137-8°; 0.5 g. III and 20 ml. 1.5 N NaOH on distn. yields PhCH:CHCH:CH2 (identified as the dibromide). II (20 g.), added to 9 g. (HCHO)3, 35 g. 90% HCO2H, and 17 ml. H2O (heated until homogeneous) (temp. rise to 40° in 30 min.), heated 2 hrs. on the water bath, kept overnight, treated with 15.7 g. concd. HCl, and steam-distd., gives 70% BzH; the residue from the steam distn., made alk. and again distd., gives about 10% Me2NCH2CH2CH : CH2 (picrate, m. 110.2-11°). II and HCHO in H2O, heated 3 hrs. on the steam bath, give 56% CH2:CHCH2CH2NH2 (picrate, m. 130.8-7.4°) and 80% BzH. Aq. Me2NH (58 g.), 62 g. 36-8% HCHO, 58 g. BuOH, and excess K2CO3, kept overnight and the crude (butoxymethyl)dimethylamine (b. 138-8.5°) treated with excess CH2:CHCH2MgBr, give CH2:CHCH2CH2NMe2, b. 92°; picrate, m. 110-11.8° and then 114-14.2°; methiodide, hygroscopic, m. 240-1.3°. PhCN (77.8 g.), refluxed several hrs. with 1.6 mols. CH2:CHCH2MgBr, gives 11 g. α,α-diallylbenzylamine (IV), b0.75 87.5-90°, nD25 1.5310; picrate, m. 134-4.5°; phenylsulfonyl deriv., m. 151-2°; thiourea deriv., m. 132.5-3.5° and 142°. IV (3 g.), 3 ml. 36-8% HCHO, and 10 ml. 6 N H2SO4 in 100 ml. H2O, distd. until 35 ml. distillate is formed, give CH2:CHCH3CH2NH2 and allyl Ph ketone, isolated as the 2,4-dinitro-phenylhydrazone, orange-red, m. 201-3°. The probable course of the reactions of II and IV is proposed and its analogy with the rearrangement of CH2:CH(CH2:CHCH2)C(CO2Et)2 is pointed out.
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    Sakabe, M.; Asanuma, D.; Kamiya, M.; Iwatate, R. J.; Hanaoka, K.; Terai, T.; Nagano, T.; Urano, Y. Rational Design of Highly Sensitive Fluorescence Probes for Protease and Glycosidase Based on Precisely Controlled Spirocyclization. J. Am. Chem. Soc. 2013, 135 (1), 409– 14,  DOI: 10.1021/ja309688m
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    Rational Design of Highly Sensitive Fluorescence Probes for Protease and Glycosidase Based on Precisely Controlled Spirocyclization
    Sakabe, Masayo; Asanuma, Daisuke; Kamiya, Mako; Iwatate, Ryu J.; Hanaoka, Kenjiro; Terai, Takuya; Nagano, Tetsuo; Urano, Yasuteru
    Journal of the American Chemical Society (2013), 135 (1), 409-414CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    We have synthesized and evaluated a series of hydroxymethyl rhodamine derivs. and found an intriguing difference of intramol. spirocyclization behavior: the acetylated deriv. of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aq. soln. at physiol. pH, whereas HMRG itself takes an open nonspirocyclic structure. Ac-HMRG is colorless and nonfluorescent, whereas HMRG is strongly fluorescent. On the basis of these findings, we have developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the acetyl group of Ac-HMRG with a substrate moiety of the target enzyme. Specific cleavage of the substrate moiety in the nonfluorescent probe by the target enzyme generates a strong fluorescence signal. To confirm the validity and flexibility of our strategy, we designed and synthesized fluorescence probes for leucine aminopeptidase (Leu-HMRG), fibroblast activation protein (Ac-GlyPro-HMRG), and β-galactosidase (βGal-HMRG). All of these probes were almost nonfluorescent due to the formation of spirocyclic structure, but were converted efficiently to highly fluorescent HMRG by the target enzymes. We confirmed that the probes can be used in living cells. These probes offer great practical advantages, including high sensitivity and rapid response (due to regulation of fluorescence at a single reactive site), as well as resistance to photobleaching, and are expected to be useful for a range of biol. and pathol. investigations.
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    Brewer, T. F.; Chang, C. J. An Aza-Cope Reactivity-Based Fluorescent Probe for Imaging Formaldehyde in Living Cells. J. Am. Chem. Soc. 2015, 137 (34), 10886– 9,  DOI: 10.1021/jacs.5b05340
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    An Aza-Cope Reactivity-Based Fluorescent Probe for Imaging Formaldehyde in Living Cells
    Brewer, Thomas F.; Chang, Christopher J.
    Journal of the American Chemical Society (2015), 137 (34), 10886-10889CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Formaldehyde (FA) is a reactive carbonyl species (RCS) produced in living systems that has been implicated in epigenetics as well as in the pathologies of various cancers, diabetes, and heart, liver, and neurodegenerative diseases. Traditional methods for biol. FA detection rely on sample destruction and/or extensive processing, resulting in a loss of spatiotemporal information. To help address this technol. gap, the authors present the design, synthesis, and biol. evaluation of a fluorescent probe for live-cell FA imaging that relies on a FA-induced aza-Cope rearrangement. Formaldehyde probe-1 (FAP-1, I) is capable of detecting physiol. relevant concns. of FA in aq. buffer and in live cells with high selectivity over potentially competing biol. analytes. Moreover, FAP-1 can visualize endogenous FA produced by lysine-specific demethylase 1 in a breast cancer cell model, presaging the potential utility of this chem. approach to probe RCS biol.
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    Roth, A.; Li, H.; Anorma, C.; Chan, J. A Reaction-Based Fluorescent Probe for Imaging of Formaldehyde in Living Cells. J. Am. Chem. Soc. 2015, 137 (34), 10890– 3,  DOI: 10.1021/jacs.5b05339
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    A Reaction-Based Fluorescent Probe for Imaging of Formaldehyde in Living Cells
    Roth, Aaron; Li, Hao; Anorma, Chelsea; Chan, Jefferson
    Journal of the American Chemical Society (2015), 137 (34), 10890-10893CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Formaldehyde (FA), in the 0.2-0.4 mM range, is produced and maintained endogenously via enzymic pathways. At these levels, FA can promote cell proliferation as well as mediate memory formation. Once elevated, FA stress is known to induce cognitive impairments, memory loss, and neurodegeneration owing to its potent DNA and protein crosslinking mechanisms. Optical imaging is a powerful noninvasive approach used to study FA in living systems; however, biocompatible chem. probes for FA are currently lacking. Herein, the authors report the design, synthesis, and biol. evaluation of Formaldehyde Probe 1 (FP1, I), a new fluorescent indicator based on the 2-aza-Cope sigmatropic rearrangement. The remarkable sensitivity, selectivity, and photostability of FP1 has enabled the authors to visualize FA in live HEK293TN and Neuroscreen-1 cells. The authors envision that FP1 will find widespread applications in the study of FA assocd. with normal and pathol. processes.
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    Bruemmer, K. J.; Walvoord, R. R.; Brewer, T. F.; Burgos-Barragan, G.; Wit, N.; Pontel, L. B.; Patel, K. J.; Chang, C. J. Development of a General Aza-Cope Reaction Trigger Applied to Fluorescence Imaging of Formaldehyde in Living Cells. J. Am. Chem. Soc. 2017, 139 (15), 5338– 50,  DOI: 10.1021/jacs.6b12460
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    Development of a General Aza-Cope Reaction Trigger Applied to Fluorescence Imaging of Formaldehyde in Living Cells
    Bruemmer, Kevin J.; Walvoord, Ryan R.; Brewer, Thomas F.; Burgos-Barragan, Guillermo; Wit, Niek; Pontel, Lucas B.; Patel, Ketan J.; Chang, Christopher J.
    Journal of the American Chemical Society (2017), 139 (15), 5338-5350CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Formaldehyde (FA) is a reactive signaling mol. that is continuously produced through a no. of central biol. pathways spanning epigenetics to one-carbon metab. However, aberrant, elevated levels of FA are implicated in disease states ranging from asthma to neurodegenerative disorders. In this context, fluorescence-based probes for FA imaging are emerging as potentially powerful chem. tools to help disentangle the complexities of FA homeostasis and its physiol. and pathol. contributions. Currently available FA indicators require direct modification of the fluorophore backbone through complex synthetic considerations to enable FA detection, often limiting the generalization of designs to other fluorophore classes. To address this challenge, the authors now present the rational, iterative development of a general reaction-based trigger using 2-aza-Cope reactivity for selective and sensitive detection of FA in living systems. Specifically, the authors developed a homoallylamine functionality that can undergo a subsequent self-immolative β-elimination, creating a FA-responsive trigger that is capable of masking a phenol on a fluorophore or any other potential chem. scaffold for related imaging and/or therapeutic applications. The authors demonstrate the utility of this trigger by creating a series of fluorescent probes for FA with excitation and emission wavelengths that span the UV to visible spectral regions through caging of a variety of dye units. In particular, Formaldehyde Probe 573 (FAP573), based on a resorufin scaffold, is the most red shifted and FA sensitive in this series in terms of signal-to-noise responses and enables identification of alc. dehydrogenase 5 (ADH5) as an enzyme that regulates FA metab. in living cells. The results provide a starting point for the broader use of 2-aza-Cope reactivity for probing and manipulating FA biol.
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    Du, Y.; Zhang, Y.; Huang, M.; Wang, S.; Wang, J.; Liao, K.; Wu, X.; Zhou, Q.; Zhang, X.; Wu, Y.-D. Systematic Investigation of the Aza-Cope Reaction for Fluorescence Imaging of Formaldehyde in Vitro and in Vivo. Chem. Sci. 2021, 12 (41), 13857– 69,  DOI: 10.1039/D1SC04387K
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    Systematic investigation of the aza-Cope reaction for fluorescence imaging of formaldehyde in vitro and in vivo
    Du, Yimeng; Zhang, Yuqing; Huang, Meirong; Wang, Shushu; Wang, Jianzheng; Liao, Kongke; Wu, Xiaojun; Zhou, Qiang; Zhang, Xinhao; Wu, Yun-Dong; Peng, Tao
    Chemical Science (2021), 12 (41), 13857-13869CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Increasing evidence has highlighted the endogenous prodn. of formaldehyde (FA) in a variety of fundamental biol. processes and its involvement in many disease conditions ranging from cancer to neurodegeneration. To examine the physiol. and pathol. relevance and functions of FA, fluorescent probes for FA imaging in live biol. samples are of great significance. Herein we report a systematic investigation of 2-aza-Cope reactions between homoallylamines and FA for identification of a highly efficient 2-aza-Cope reaction moiety and development of fluorescent probes for imaging FA in living systems. By screening a set of N-substituted homoallylamines and comparing them to previously reported homoallylamine structures for reaction with FA, we found that N-p-methoxybenzyl homoallylamine exhibited an optimal 2-aza-Cope reactivity to FA. Theor. calcns. were then performed to demonstrate that the N-substituent on homoallylamine greatly affects the condensation with FA, which is more likely the rate-detg. step. Moreover, the newly identified optimal N-p-methoxybenzyl homoallylamine moiety with a self-immolative β-elimination linker was generally utilized to construct a series of fluorescent probes with varying excitation/emission wavelengths for sensitive and selective detection of FA in aq. solns. and live cells. Among these probes, the near-IR probe FFP706 has been well demonstrated to enable direct fluorescence visualization of steady-state endogenous FA in live mouse brain tissues and elevated FA levels in a mouse model of breast cancer. This study provides the optimal aza-Cope reaction moiety for FA probe development and new chem. tools for fluorescence imaging and biol. investigation of FA in living systems.
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    He, L.; Yang, X.; Liu, Y.; Kong, X.; Lin, W. A Ratiometric Fluorescent Formaldehyde Probe for Bioimaging Applications. Chem. Commun. 2016, 52 (21), 4029– 32,  DOI: 10.1039/C5CC09796G
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    A ratiometric fluorescent formaldehyde probe for bioimaging applications
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    We have described a ratiometric fluorescent formaldehyde probe (RFFP) based on the 6-hydroxy naphthalene chromophore for the first time. The probe is suitable for ratiometric detection of formaldehyde both in the soln. and living biol. samples with two distinct emission bands.
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    Xu, J.; Zhang, Y.; Zeng, L.; Liu, J.; Kinsella, J. M.; Sheng, R. A Simple Naphthalene-Based Fluorescent Probe for High Selective Detection of Formaldehyde in Toffees and HeLa Cells Via Aza-Cope Reaction. Talanta 2016, 160, 645– 52,  DOI: 10.1016/j.talanta.2016.08.010
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    A simple naphthalene-based fluorescent probe for high selective detection of formaldehyde in toffees and HeLa cells via aza-Cope reaction
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    Talanta (2016), 160 (), 645-652CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
    A simple naphthalene-based fluorescent probe (AENO) for formaldehyde (FA) was successfully synthesized, which exhibited a significant fluorescence turn-on response towards FA in aq. soln. The probe could quant. det. the concn. of FA (0-1.0 mM) with excellent selectivity, high sensitivity and low limit of detection (0.57 μM). The sensing mechanism is proposed as 2-aza-Cope rearrangement for AENO after reaction with FA, which was confirmed by 1H NMR, HR-MS, FTIR, UV-visible and fluorescence spectra. The probe has been employed to det. the FA contents in several com. available toffee samples with satisfactory performance. Thus, AENO might be used as a promising tool for quant. detection of FA in food. Furthermore, fluorescence imaging of HeLa cells indicated that the probe was cell membrane permeable and could be used for visualizing/imaging the FA trace/transportation in cancer cells.
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    Yang, X.; He, L.; Xu, K.; Yang, Y.; Lin, W. The Development of an Ict-Based Formaldehyde-Responsive Fluorescence Turn-On Probe with a High Signal-to-Noise Ratio. New J. Chem. 2018, 42 (15), 12361– 4,  DOI: 10.1039/C8NJ02467G
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    New Journal of Chemistry (2018), 42 (15), 12361-12364CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
    An illuminating ICT-based formaldehyde-responsive fluorescent probe (PBD-FA) with long wavelength emission was judiciously designed and synthesized, which is suitable for detecting FA in aq. soln. and living cells, both with significant fluorescence signal-to-noise ratios.
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    Yang, M.; Fan, J.; Du, J.; Long, S.; Wang, J.; Peng, X. Imaging of Formaldehyde in Live Cells and Daphnia Magna Via Aza-Cope Reaction Utilizing Fluorescence Probe with Large Stokes Shifts. Front. Chem. 2018, 6, 488,  DOI: 10.3389/fchem.2018.00488
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    Imaging of formaldehyde in live cells and Daphnia magna via aza-cope reaction utilizing fluorescence probe with large stokes shifts
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    Frontiers in Chemistry (Lausanne, Switzerland) (2018), 6 (), 488CODEN: FCLSAA; ISSN:2296-2646. (Frontiers Media S.A.)
    Formaldehyde (FA), a highly reactive carbonyl species, plays significant role in physiol. and pathol. functions. However, elevated FA will lead to cognitive impairments, memory loss and various neurodegenerative diseases due to its potent DNA and protein crosslinking mechanisms. In this work, a fluorescence probe, BD-CHO, based on benz-2-oxa-1, 3- diazole (BD) skeleton, was designed and synthesized for detection of FA via Aza-Cope reaction with high selectivity and large Stokes shifts (about 118 nm). BD-CHO was successfully applied to monitor the changes FA level in living cells, and kidney tissues of mice. Importantly it was the first time that BD-CHO was used for visualizing exogenous FA changes in Daphnia magna through fluorescence microscopy, demonstrating its potential application for studies of biol. processes assocd. with FA.
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    Bruemmer, K. J.; Brewer, T. F.; Chang, C. J. Fluorescent Probes for Imaging Formaldehyde in Biological Systems. Curr. Opin. Chem. Biol. 2017, 39, 17– 23,  DOI: 10.1016/j.cbpa.2017.04.010
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    Current Opinion in Chemical Biology (2017), 39 (), 17-23CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    Formaldehyde (FA) is a common environmental toxin but is also endogenously produced through a diverse array of essential biol. processes, including mitochondrial one-carbon metab., metabolite oxidn., and nuclear epigenetic modifications. Its high electrophilicity enables reactivity with a wide variety of biol. nucleophiles, which can be beneficial or detrimental to cellular function depending on the context. New methods that enable detection of FA in living systems can help disentangle the signal/stress dichotomy of this simplest reactive carbonyl species (RCS), and fluorescent probes for FA with high selectivity and sensitivity have emerged as promising chem. tools in this regard.
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    Liu, X.; Li, N.; Li, M.; Chen, H.; Zhang, N.; Wang, Y.; Zheng, K. Recent Progress in Fluorescent Probes for Detection of Carbonyl Species: Formaldehyde, Carbon Monoxide and Phosgene. Coord. Chem. Rev. 2020, 404, 213109,  DOI: 10.1016/j.ccr.2019.213109
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    A review. Extensive attention has been paid to develop effective systems for the detection of formaldehyde, carbon monoxide and phosgene due to their extreme toxicity and ready accessibility. Numerous methods have been developed for the design and detection of these substances nowadays, such as electro-fluorescent biosensors, piezoelec. sensors, semiconductor sensors, colorimetric probes, quartz crystal microbalance, Raman spectroscopy, transmission electro-microscopy (TEM), gas chromatog., liq. chromatog. and X-ray diffraction (XRD), but fluorescent probes, which rely on chem. reactions between the probes and the target, provoking a dramatic fluorescence change, often remain the most commonly employed method for detecting such important small mols. This review will cover the most significant developments in fluorescent probes for the detection of the carbonyl species formaldehyde, carbon monoxide and phosgene in recent years (typically the last 10 years), with a special emphasis on their mechanisms and applications.
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    Examples of the biol. occurrence of Me ketones are reviewed. The lack of significant accumulations of these compds. in the biosphere indicates that a recycling of these org. mols. is occurring. Evidence for biodegradation of acetone by mammals and longer Me ketones by microorganisms via terminal Me group oxidn. is discussed. A new mechanism for the subterminal oxidn. of Me ketones by microorganisms is proposed whereby the 1st intermediate produced is an acetate ester which subsequently is cleaved to acetate and a primary alc. 2 C shorter than the original ketone substrate.
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    McNally, M. A.; Hartman, A. L. Ketone Bodies in Epilepsy. J. Neurochem. 2012, 121 (1), 28– 35,  DOI: 10.1111/j.1471-4159.2012.07670.x
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    A review. Seizures that are resistant to std. medications remain a major clin. problem. One underutilized option for patients with medication-resistant seizures is the high-fat, low-carbohydrate ketogenic diet. The diet received its name based on the observation that patients consuming this diet produce ketone bodies (e.g., acetoacetate, β-hydroxybutyrate, and acetone). Although the exact mechanisms of the diet are unknown, ketone bodies have been hypothesized to contribute to the anticonvulsant and antiepileptic effects. In this review, anticonvulsant properties of ketone bodies and the ketogenic diet are discussed (including GABAergic and glutamatergic effects). Because of the importance of ketone body metab. in the early stages of life, the effects of ketone bodies on developing neurons in vitro also are discussed. Understanding how ketone bodies exert their effects will help optimize their use in treating epilepsy and other neurol. disorders.
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    Mutti, F. G.; Knaus, T. Enzymes Applied to the Synthesis of Amines. Biocatalysis for Practitioners 2021, 143– 80,  DOI: 10.1002/9783527824465.ch6
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    Enzymes Applied to the Synthesis of Amines
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    Biocatalysis for Practitioners (2021), (), 143-180CODEN: 70ABFT ISSN:. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A great variety of biocatalytic methods are available for the synthesis of amines, in particular, α-chiral amines. Therefore, Chapter 6 aims to orient nonspecialists to the available biocatalytic methodologies for the synthesis of α-chiral amines with a focus on the enzymes' substrate scope and selectivity as well as the structural diversity of the obtained products. The presented enzymic methods comprise the use of hydrolases, amine oxidases, transaminases, amine dehydrogenases, imine reductases, ammonia lyases, Pictet-Spenglerases, and engineered cytochromes P 450. Classification and reactivity, biocatalytic applications, and selected important examples are discussed for each enzyme category. Methodologies entail the kinetic resoln. or deracemization of racemic amines and the asym. synthesis from prochiral precursors, where applicable. Finally, the last chapter's section illustrates practical procedures. Notably, the presented biocatalytic methods can yield primary, secondary, and tertiary amine products and often with excellent chemo-, regio-, and stereoselectivity under mild reaction conditions.
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    Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Recent Advances in the Biocatalytic Reduction of Ketones and Oxidation of sec-Alcohols. Curr. Opin. Chem. Biol. 2004, 8 (2), 120– 6,  DOI: 10.1016/j.cbpa.2004.02.005
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    Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols
    Kroutil, Wolfgang; Mang, Harald; Edegger, Klaus; Faber, Kurt
    Current Opinion in Chemical Biology (2004), 8 (2), 120-126CODEN: COCBF4; ISSN:1367-5931. (Elsevier Science Ltd.)
    A review. To improve the efficiency and applicability of biocatalytic redox-reactions for asym. ketone-redn. and enantioselective alc.-oxidn. catalyzed by nicotinamide-dependent dehydrogenases/reductases, several achievements for cofactor-recycling have been made during the last two years. First, the use of hydrogenases for NADPH recycling in a two enzyme system. Second, preparative transformations with alc. dehydrogenases coupled with NADH oxidases for NAD+/NADP+ recycling. Third, an exceptional chemo-stable alc. dehydrogenase can efficiently use i-propanol and acetone as cosubstrates for redn. and oxidn., resp., in a single-enzyme system. Novel carbonyl reductases and dehydrogenases derived from plant cells are particularly suited for sterically demanding substrates.
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    Leisch, H.; Morley, K.; Lau, P. C. K. Baeyer- Villiger Monooxygenases: More Than Just Green Chemistry. Chem. Rev. 2011, 111 (7), 4165– 222,  DOI: 10.1021/cr1003437
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    Leisch, Hannes; Morley, Krista; Lau, Peter C. K.
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    This review focuses on the monooxygenase-catalyzed Baeyer-Villiger oxidn. of linear or cyclic ketones as a green chem. tool to address environmental sustainability, a system to study its mol. diversity and catalytic mechanism, industrial scale bioprocess development, and protein engineering to evolve new biotechnol. applications.
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    Dean, S. M.; Greenberg, W. A.; Wong, C. H. Recent Advances in Aldolase-Catalyzed Asymmetric Synthesis. Adv. Synth. Catal. 2007, 349 (8–9), 1308– 20,  DOI: 10.1002/adsc.200700115
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    Recent advances in aldolase-catalyzed asymmetric synthesis
    Dean, Stephen M.; Greenberg, William A.; Wong, Chi-Huey
    Advanced Synthesis & Catalysis (2007), 349 (8+9), 1308-1320CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Here, the authors focus on advances over the last several years in the application of aldolases to org. synthesis. Several new technologies have been implemented to increase the scope and practicality of aldolases as tools for the synthetic chemist. These include directed evolution, discovery of new classes of aldolases in nature and the lab., and substrate and reaction engineering.
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    Walsh, C. T. Biologically Generated Carbon Dioxide: Nature’s Versatile Chemical Strategies for Carboxy Lyases. Nat. Prod. Rep. 2020, 37 (1), 100– 35,  DOI: 10.1039/C9NP00015A
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    Natural Product Reports (2020), 37 (1), 100-135CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
    Covering: up to 2019Metabolic prodn. of CO2 is natural product chem. on a mammoth scale. Just counting humans, among all other respiring organisms, the seven billion people on the planet exhale about 3 billion tons of CO2 per yr. Essentially all of the biogenic CO2 arises by action of discrete families of decarboxylases. The mechanistic routes to CO2 release from carboxylic acid metabolites vary with the electronic demands and structures of specific substrates and illustrate the breadth of chem. employed for C-COO (C-C bond) disconnections. Most commonly decarboxylated are α-keto acid and β-keto acid substrates, the former requiring thiamin-PP as cofactor, the latter typically cofactor-free. The extensive decarboxylation of amino acids, e.g. to neurotransmitter amines, is synonymous with the coenzyme form of vitamin B6, pyridoxal-phosphate, although covalent N-terminal pyruvamide residues serve in some amino acid decarboxylases. All told, five B vitamins (B1, B2, B3, B6, B7), ATP, S-adenosylmethionine, manganese and zinc ions are pressed into service for specific decarboxylase catalyzes. There are addnl. cofactor-independent decarboxylases that operate by distinct chem. routes. Finally, while most decarboxylases use heterolytic ionic mechanisms, a small no. of decarboxylases carry out radical pathways.
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    Liu, M.; Wei, D.; Wen, Z.; Wang, J.-b. Progress in Stereoselective Construction of C-C Bonds Enabled by Aldolases and Hydroxynitrile Lyases. Front. Bioeng. Biotechnol. 2021, 9, 653682,  DOI: 10.3389/fbioe.2021.653682
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    Progress in Stereoselective Construction of C-C Bonds Enabled by Aldolases and Hydroxynitrile Lyases
    Liu Mi; Wei Dan; Wen Zexing; Wang Jian-Bo; Liu Mi; Wei Dan; Wen Zexing; Wang Jian-Bo
    Frontiers in bioengineering and biotechnology (2021), 9 (), 653682 ISSN:2296-4185.
    The creation of C-C bonds is an effective strategy for constructing complex compounds from simple synthetic blocks. Although many methods have been developed for C-C bond construction, the stereoselective creation of new C-C bonds remains a challenge. The selectivities (enantioselectivity, regioselectivity, and chemoselectivity) of biocatalysts are higher than those of chemical catalysts, therefore biocatalysts are excellent candidates for use in stereoselective C-C bond formation. Here, we summarize progress made in the past 10 years in stereoselective C-C bond formation enabled by two classic types of enzyme, aldolases and hydroxynitrile lyases. The information in this review will enable the development of new routes to the stereoselective construction of C-C bonds.
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    Müller, M.; Gocke, D.; Pohl, M. Thiamin Diphosphate in Biological Chemistry: Exploitation of Diverse Thiamin Diphosphate-Dependent Enzymes for Asymmetric Chemoenzymatic Synthesis. FEBS J. 2009, 276 (11), 2894– 904,  DOI: 10.1111/j.1742-4658.2009.07017.x
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    Thiamin diphosphate in biological chemistry: exploitation of diverse thiamin diphosphate-dependent enzymes for asymmetric chemoenzymatic synthesis
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    A review. Thiamin diphosphate (ThDP)-dependent enzymes participate in numerous biosynthetic pathways and catalyze a broad range of reactions, mainly involving the cleavage and formation of C-C bonds. For example, they catalyze the nonoxidative and oxidative decarboxylation of 2-keto acids, produce 2-hydroxy ketones, and transfer activated aldehydes to a variety of acceptors. Moreover, they can also catalyze C-N, C-O, and C-S bond formation. Because of their substrate spectra and different stereospecificities, these enzymes extend the synthetic potential for asym. carboligations appreciably. Different strategies have been developed to identify new members of this promiscuous enzyme class and the reactions they catalyze. This has enabled the authors to introduce solns. for longstanding synthetic problems, such as asym. cross-benzoin condensation. Moreover, through a combination of protein structure anal., enzyme and substrate engineering, and screening methods, the authors have explored addnl. stereochem. routes that have not been described previously for any of these interesting enzymes.
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    Schmidt, N. G.; Pavkov-Keller, T.; Richter, N.; Wiltschi, B.; Gruber, K.; Kroutil, W. Biocatalytic Friedel-Crafts Acylation and Fries Reaction. Angew. Chem., Int. Ed. 2017, 56 (26), 7615– 9,  DOI: 10.1002/anie.201703270
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    Angewandte Chemie, International Edition (2017), 56 (26), 7615-7619CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The Friedel-Crafts acylation is commonly used for the synthesis of aryl ketones, and a biocatalytic version, which may benefit from the chemo- and regioselectivity of enzymes, has not yet been introduced. Described here is a bacterial acyltransferase which can catalyze Friedel-Crafts C-acylation of phenolic substrates in buffer without the need of CoA-activated reagents. Conversions reach up to >99 %, and various C- or O-acyl donors, such as DAPG or isopropenyl acetate, are accepted by this enzyme. Furthermore the enzyme enables a Fries-like rearrangement reaction of resorcinol derivs. These findings open an avenue for the development of alternative and selective C-C bond formation methods.
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    Gergel, S.; Soler, J.; Klein, A.; Schülke, K.; Hauer, B.; Garcia-Borràs, M.; Hammer, S. Directed Evolution of a Ketone Synthase for Efficient and Highly Selective Functionalization of Internal Alkenes by Accessing Reactive Carbocation Intermediates. ChemRxiv 2022. dp94p DOI: 10.26434/chemrxiv-2022-dp94p
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    Zhang, J.; Xu, T.; Li, Z. Enantioselective Biooxidation of Racemic trans-Cyclic Vicinal Diols: One-Pot Synthesis of Both Enantiopure (S,S)-Cyclic Vicinal Diols and (R)-α-Hydroxy Ketones. Adv. Synth. Catal. 2013, 355 (16), 3147– 53,  DOI: 10.1002/adsc.201300301
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    Enantioselective Biooxidation of Racemic trans-Cyclic Vicinal Diols: One-Pot Synthesis of Both Enantiopure (S,S)-Cyclic Vicinal Diols and (R)-α-Hydroxy Ketones
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    Advanced Synthesis & Catalysis (2013), 355 (16), 3147-3153CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Highly regio- and enantioselective alc. dehydrogenases BDHA (2,3-butanediol dehydrogenase from Bacillus subtilis BGSC1A1), CDDHPm (cyclic diol dehydrogenase from Pseudomonas mendocina TA5), and CDDHRh (cyclic diol dehydrogenase from Rhodococcus sp. Moj-3449) were discovered for the oxidn. of racemic trans-cyclic vicinal diols. Recombinant Escherichia coli expressing BDHA was engineered as an efficient whole-cell biocatalyst for the oxidn. of (±)-1,2-cyclopentanediol, 1,2-cyclohexanediol, 1,2-cycloheptane-diol, and 1,2-cyclooctanediol, resp., to give the corresponding (R)-α-hydroxy ketones in >99% ee and (S,S)-cyclic diols in >99% ee at 50% conversion in one pot. Escherichia coli (BDHA-LDH) co-expressing lactate dehydrogenase (LDH) for intracellular regeneration of NAD+ catalyzed the regio- and enantioselective oxidn. of (±)-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene to produce the corresponding (R)-α-hydroxy ketone in >99% ee and (S,S)-cyclic diol in 96% ee at 49% conversion. Preparative biotransformations were also demonstrated. Thus, a novel and useful method for the one-pot synthesis of both vicinal diols and α-hydroxy ketones in high ee was developed via highly regio- and enantioselective oxidns. of the racemic vicinal diols.
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    Lavandera, I.; Kern, A.; Resch, V.; Ferreira-Silva, B.; Glieder, A.; Fabian, W. M. F.; de Wildeman, S.; Kroutil, W. One-Way Biohydrogen Transfer for Oxidation of sec-Alcohols. Org. Lett. 2008, 10 (11), 2155– 8,  DOI: 10.1021/ol800549f
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    Organic Letters (2008), 10 (11), 2155-2158CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
    Quasi-irreversible oxidn. of sec-alcs. was achieved via biocatalytic hydrogen transfer reactions using alc. dehydrogenases employing selected ketones as hydrogen acceptors, which can only be reduced but not oxidized. Thus, only 1 equiv of oxidant was required instead of a large excess. For the oxidn. of both isomers of methylcarbinols a single nonstereoselective short-chain dehydrogenase/reductase from Sphingobium yanoikuyae was identified and overexpressed in E. coli.
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    González-Granda, S.; Méndez-Sánchez, D.; Lavandera, I.; Gotor-Fernández, V. Laccase-Mediated Oxidations of Propargylic Alcohols. Application in the Deracemization of 1-Arylprop-2-Yn-1-Ols in Combination with Alcohol Dehydrogenases. ChemCatChem. 2020, 12 (2), 520– 7,  DOI: 10.1002/cctc.201901543
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    ChemCatChem (2020), 12 (2), 520-527CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The catalytic system composed by the laccase from Trametes versicolor and the oxy-radical TEMPO has been successfully applied in the sustainable oxidn. of fourteen propargylic alcs. The corresponding propargylic ketones were obtained in most cases in quant. conversions (87->99% yield), demonstrating the efficiency of the chemoenzymic methodol. in comparison with traditional chem. oxidants, which usually lead to problems assocd. with the formation of byproducts. Also, the stereoselective redn. of propargylic ketones was studied using alc. dehydrogenases such as the one from Ralstonia species overexpressed in E. coli or the com. available evo-1.1.200, allowing the access to both alc. enantiomers mostly with complete conversions and variable selectivities depending on the arom. pattern substitution (97->99% ee). To demonstrate the compatibility of the laccase-mediated oxidn. and the alc. dehydrogenase-catalyzed bioredn., a deracemization strategy starting from the racemic compds. was developed through a sequential one-pot two-step process, obtaining a selection of (S)- or (R)-1-arylprop-2-yn-1-ols with excellent yields (>98%) and selectivities (>98% ee) depending on the alc. dehydrogenase employed.
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    Key, J. A.; Li, C.; Cairo, C. W. Detection of Cellular Sialic Acid Content Using Nitrobenzoxadiazole Carbonyl-Reactive Chromophores. Bioconj. Chem. 2012, 23 (3), 363– 71,  DOI: 10.1021/bc200276k
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    Key, Jessie A.; Li, Caishun; Cairo, Christopher W.
    Bioconjugate Chemistry (2012), 23 (3), 363-371CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    The selective ligation of hydrazine and amino-oxy compds. with carbonyls has gained popularity as a detection strategy with the recognition of aniline catalysis as a way to accelerate the labeling reaction in water. Aldehydes are a convenient functional group choice since there are few native aldehydes found at the cell surface. Aldehydes can be selectively introduced into sialic acid contg. glycoproteins by treatment with dil. sodium periodate. Thus, the combination of periodate oxidn. with aniline-catalyzed ligation (PAL) has become a viable method for detection of glycoconjugates on live cells. Herein the authors examine two fluorescent nitrobenzoxadiazole dyes for labeling of glycoproteins and cell surface glycoconjugates. The authors introduce a novel 4-aminooxy-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDAO) fluorophore and offer a comparison to com. dyes including the known 4-hydrazino-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDH) and Bodipy FL hydrazide. The authors confirm specificity for sialic acid moieties and that both dyes are suitable for in vitro and in vivo labeling studies using PAL and fluorescence spectroscopy. The dyes examd. here are attractive labeling agents for microscopy, as they can be excited by a 488 nm laser line and can be made in a few synthetic steps. These carbonyl-reactive chromophores provide a one step alternative to avidin-biotin labeling strategies and simplify the detection of sialic acid in cells and glycoproteins.
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    Crisalli, P.; Kool, E. T. Water-Soluble Organocatalysts for Hydrazone and Oxime Formation. J. Org. Chem. 2013, 78 (3), 1184– 9,  DOI: 10.1021/jo302746p
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    Water-Soluble Organocatalysts for Hydrazone and Oxime Formation
    Crisalli, Pete; Kool, Eric T.
    Journal of Organic Chemistry (2013), 78 (3), 1184-1189CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    The formation of oximes and hydrazones is widely used in chem. and biol. as a mol. conjugation strategy for achieving ligation, attachment, and bioconjugation. However, the relatively slow rate of reaction has hindered its utility. Here, we report that simple, com. available anthranilic acids and aminobenzoic acids act as superior catalysts for hydrazone and oxime formation, speeding the reaction considerably over the traditional aniline-catalyzed reaction at neutral pH. This efficient nucleophilic catalysis, involving catalyst-imine intermediates, allows rapid hydrazone/oxime formation even with relatively low concns. of the two reactants. The most efficient catalysts are found to be 5-methoxyanthranilic acid and 3,5-diaminobenzoic acid; we find that they can enhance rates by factors of as much as 1-2 orders of magnitude over the aniline-catalyzed reaction. Evidence based on a range of differently substituted arylamines suggests that the ortho-carboxylate group in the anthranilate catalysts serves to aid in intramol. proton transfer during imine and hydrazone formation.
  378. 378
    Guo, H.-M.; Tanaka, F. A Fluorogenic Aldehyde Bearing a 1,2,3-Triazole Moiety for Monitoring the Progress of Aldol Reactions. J. Org. Chem. 2009, 74 (6), 2417– 24,  DOI: 10.1021/jo900013w
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    380
    A Fluorogenic Aldehyde Bearing a 1,2,3-Triazole Moiety for Monitoring the Progress of Aldol Reactions
    Guo, Hai-Ming; Tanaka, Fujie
    Journal of Organic Chemistry (2009), 74 (6), 2417-2424CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    We have developed a new type of fluorogenic aldehyde bearing a 1,2,3-triazole moiety that is useful for monitoring the progress of aldol reactions through an increase in fluorescence. Whereas 6-methoxy-2-naphthaldehyde was highly fluorescent, the fluorogenic aldehyde, 4-formylbenzene connected to the 6-methoxy-2-naphthyl group through a 1,2,3-triazole moiety, was essentially nonfluorescent in aq. solns. We suggest that the 4-formylphenyl group acts as a quencher to suppress the fluorescence of the 6-methoxy-2-naphthyltriazole moiety. The product of the aldol reaction of this aldehyde does not have a quenching moiety and showed more than 800-fold higher fluorescence than the aldehyde. Assay systems using the fluorogenic aldehyde were validated by screening of aldol catalysts, ranking of the activities of the catalysts, and evaluation of reaction conditions.
  379. 379
    Tanaka, F.; Thayumanavan, R.; Barbas, C. F. Fluorescent Detection of Carbon-Carbon Bond Formation. J. Am. Chem. Soc. 2003, 125 (28), 8523– 8,  DOI: 10.1021/ja034069t
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    Fluorescent detection of carbon-carbon bond formation
    Tanaka, Fujie; Thayumanavan, Rajeswari; Barbas, Carlos F.
    Journal of the American Chemical Society (2003), 125 (28), 8523-8528CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    We have developed a new spectroscopic system for detecting carbon-carbon bond formation by fluorescence to enhance high-throughput catalyst screening and rapid characterization of catalysts on a small scale. Fluorogenic substrates composed of a fluorophore possessing an amino group are readily prepd. as amides of α,β-unsatd. carbonyl compds. and generally exhibit low fluorescence, while Michael or Diels-Alder reactions of these fluorogenic substrates provide products of significantly increased fluorescence. The product's fluorescence is approx. 20- to 100-fold higher than that of the substrate. The assay system was validated by screening potential catalysts of the Michael reaction and in solvent optimization expts. The covalent combination of fluorophores possessing an amino group with α,β-unsatd. carbonyl compds. should provide a diverse range of fluorogenic substrates that may be used to rapidly screen catalysts and to optimize reaction conditions.
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    Tanaka, F.; Mase, N.; Barbas, C. F. Design and Use of Fluorogenic Aldehydes for Monitoring the Progress of Aldehyde Transformations. J. Am. Chem. Soc. 2004, 126 (12), 3692– 3,  DOI: 10.1021/ja049641a
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    Design and Use of Fluorogenic Aldehydes for Monitoring the Progress of Aldehyde Transformations
    Tanaka, Fujie; Mase, Nobuyuki; Barbas, Carlos F., III
    Journal of the American Chemical Society (2004), 126 (12), 3692-3693CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    We describe the first examples of fluorogenic aldehydes useful for monitoring many types of reactions including aldol reactions, allylations, and redns. The fluorogenic aldehydes were constructed by covalent combination of a fluorophore and an aldehyde moiety via a linker. In the resulting single mol., the aldehyde functioned as a quencher of the fluorophore's fluorescence. The reaction product, modified at the aldehyde functionality, no longer served as an effective quencher. The reaction products showed up to ∼80-fold higher fluorescence than the aldehyde reactants. The reaction of the aldolase antibody 38C-2-catalyzed aldol reaction of 4-formyl-N-(9-phenanthrenyl)benzenepropanamide (I) with acetone was monitored using a fluorescence assay method. The aldehydes described here are potentially useful the screening of catalysts in chem. libraries (no data).
  381. 381
    Linares-Pastén, J. A.; Chávez-Lizárraga, G.; Villagomez, R.; Mamo, G.; Hatti-Kaul, R. A Method for Rapid Screening of Ketone Biotransformations: Detection of Whole Cell Baeyer-Villiger Monooxygenase Activity. Enzyme Microb. Technol. 2012, 50 (2), 101– 6,  DOI: 10.1016/j.enzmictec.2011.10.004
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    383
    A method for rapid screening of ketone biotransformations: Detection of whole cell Baeyer-Villiger monooxygenase activity
    Linares-Pasten, Javier A.; Chavez-Lizarraga, Georgina; Villagomez, Rodrigo; Mamo, Gashaw; Hatti-Kaul, Rajni
    Enzyme and Microbial Technology (2012), 50 (2), 101-106CODEN: EMTED2; ISSN:0141-0229. (Elsevier)
    A method for screening of ketone biotransformations was developed and applied to the identification of Baeyer-Villiger monooxygenase (BVMO) activity. The method was based on the formation of a purple colored product on reaction between an enolizable ketone and 3,5-dinitrobenzoic acid (DNB) in alk. soln. Absorbance of the color decreased with the size of the cycloketone ring. The stoichiometric ratio between cycloketone and DNB was 1:1 at max. absorbance. The method was applied for monitoring the consumption of cyclohexanone by bacteria under aerobic conditions, and was found to be potentially useful for both screening assays and quant. measurements of BVMO activity. Compared to other existing methods, this method was faster, less expensive, and amenable for whole cell assays.
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    Georgiana Ileana, B.; Gabriel Lucian, R. Carboxylic Acid - Key Role in Life Sciences; IntechOpen, 2018.
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    Brune, K.; Patrignani, P. New Insights into the Use of Currently Available Non-Steroidal Anti-Inflammatory Drugs. J. Pain Res. 2015, 8, 105– 18,  DOI: 10.2147/JPR.S75160
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    New insights into the use of currently available non-steroidal anti-inflammatory drugs
    Brune Kay; Patrignani Paola
    Journal of pain research (2015), 8 (), 105-18 ISSN:1178-7090.
    Non-steroidal anti-inflammatory drugs (NSAIDs), which act via inhibition of the cyclooxygenase (COX) isozymes, were discovered more than 100 years ago. They remain a key component of the pharmacological management of acute and chronic pain. The COX-1 and COX-2 isozymes have different biological functions; analgesic activity is primarily (although not exclusively) associated with inhibition of COX-2, while different side effects result from the inhibition of COX-1 and COX-2. All available NSAIDs, including acetaminophen and aspirin, are associated with potential side effects, particularly gastrointestinal and cardiovascular effects, related to their relative selectivity for COX-1 and COX-2. Since all NSAIDs exert their therapeutic activity through inhibition of the COX isozymes, strategies are needed to reduce the risks associated with NSAIDs while achieving sufficient pain relief. A better understanding of the inhibitory activity and COX-1/COX-2 selectivity of an NSAID at therapeutic doses, based on pharmacokinetic and pharmacodynamic properties (eg, inhibitory dose, absorption, plasma versus tissue distribution, and elimination), and the impact on drug tolerability and safety can guide the selection of appropriate NSAIDs for pain management. For example, many NSAIDs with moderate to high selectivity for COX-2 versus COX-1 can be administered at doses that maximize efficacy (~80% inhibition of COX-2) while minimizing COX-1 inhibition and associated side effects, such as gastrointestinal toxicity. Acidic NSAIDs with favorable tissue distribution and short plasma half-lives can additionally be dosed to provide near-constant analgesia while minimizing plasma concentrations to permit recovery of COX-mediated prostaglandin production in the vascular wall and other organs. Each patient's clinical background, including gastrointestinal and cardiovascular risk factors, should be taken into account when selecting appropriate NSAIDs. New methods are emerging to assist clinicians in the selection of appropriate NSAIDs and their doses/schedules, such as biomarkers that may predict the response to NSAID treatment in individual patients.
  384. 384
    Murali, N.; Srinivas, K.; Ahring, B. K. Biochemical Production and Separation of Carboxylic Acids for Biorefinery Applications. Fermentation 2017, 3 (2), 22,  DOI: 10.3390/fermentation3020022
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    Biochemical production and separation of carboxylic acids for biorefinery applications
    Murali, Nanditha; Srinivas, Keerthi; Ahring, Birgitte K.
    Fermentation (2017), 3 (2), 22/1-22/25CODEN: FERMC4; ISSN:2311-5637. (MDPI AG)
    Carboxylic acids are traditionally produced from fossil fuels and have significant applications in the chem., pharmaceutical, food, and fuel industries. Significant progress has been made in replacing such fossil fuel sources used for prodn. of carboxylic acids with sustainable and renewable biomass resources. However, the merits and demerits of each carboxylic acid processing platform are dependent on the application of the final product in the industry. There are a no. of studies that indicate that sepn. processes account for over 30% of the total processing costs in such processes. This review focuses on the sustainable processing of biomass resources to produce carboxylic acids. The primary focus of the review will be on a discussion of and comparison between existing biochem. processes for producing lower-chain fatty acids such as acetic-, propionic-, butyric-, and lactic acids. The significance of these acids stems from the recent progress in catalytic upgrading to produce biofuels apart from the current applications of the carboxylic acids in the food, pharmaceutical, and plastics sectors. A significant part of the review will discuss current state-of-art of techniques for sepn. and purifn. of these acids from fermn. broths for further downstream processing to produce high-value products.
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    Simmons, D. L.; Botting, R. M.; Hla, T. Cyclooxygenase Isozymes: The Biology of Prostaglandin Synthesis and Inhibition. Pharmacol. Rev. 2004, 56 (3), 387– 437,  DOI: 10.1124/pr.56.3.3
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    Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition
    Simmons, Daniel L.; Botting, Regina M.; Hla, Timothy
    Pharmacological Reviews (2004), 56 (3), 387-437CODEN: PAREAQ; ISSN:0031-6997. (American Society for Pharmacology and Experimental Therapeutics)
    A review. Nonsteroidal anti-inflammatory drugs (NSAIDs) represent one of the most highly utilized classes of pharmaceutical agents in medicine. All NSAIDs act through inhibiting prostaglandin synthesis, a catalytic activity possessed by two distinct cyclooxygenase (COX) isoenzymes encoded by sep. genes. The discovery of COX-2 launched a new era in NSAID pharmacol., resulting in the synthesis, marketing, and widespread use of COX-2 selective drugs. These pharmaceutical agents have quickly become established as important therapeutic medications with potentially fewer side effects than traditional NSAIDs. Addnl., characterization of the two COX isoenzymes is allowing the discrimination of the roles each play in physiol. processes such as homeostatic maintenance of the gastrointestinal tract, renal function, blood clotting, embryonic implantation, parturition, pain, and fever. Of particular importance has been the investigation of COX-1 and -2 isoenzymic functions in cancer, dysregulation of inflammation, and Alzheimer's disease. More recently, addnl. heterogeneity in COX-related proteins has been described, with the finding of variants of COX-1 and COX-2 enzymes. These variants may function in tissue-specific physiol. and pathophysiol. processes and may represent important new targets for drug therapy.
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    Kumar, P.; Dubey, K. K. Citric Acid Cycle Regulation: Back Bone for Secondary Metabolite Production. In New and Future Developments in Microbial Biotechnology and Bioengineering; Elsevier: Amsterdam, 2019; Chapter 13, pp 165– 81.
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    Nakamura, M. T.; Yudell, B. E.; Loor, J. J. Regulation of Energy Metabolism by Long-Chain Fatty Acids. Prog. Lipid Res. 2014, 53, 124– 44,  DOI: 10.1016/j.plipres.2013.12.001
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    Regulation of energy metabolism by long-chain fatty acids
    Nakamura, Manabu T.; Yudell, Barbara E.; Loor, Juan J.
    Progress in Lipid Research (2014), 53 (), 124-144CODEN: PLIRDW; ISSN:0163-7827. (Elsevier Ltd.)
    A review. In mammals, excess energy is stored primarily as triglycerides, which are mobilized when energy demands arise. This review mainly focuses on the role of long chain fatty acids (LCFAs) in regulating energy metab. as ligands of peroxisome proliferator-activated receptors (PPARs). PPAR-alpha expressed primarily in liver is essential for metabolic adaptation to starvation by inducing genes for beta-oxidn. and ketogenesis and by downregulating energy expenditure through fibroblast growth factor 21. PPAR-delta is highly expressed in skeletal muscle and induces genes for LCFA oxidn. during fasting and endurance exercise. PPAR-delta also regulates glucose metab. and mitochondrial biogenesis by inducing FOXO1 and PGC1-alpha. Genes targeted by PPAR-gamma in adipocytes suggest that PPAR-gamma senses incoming non-esterified LCFAs and induces the pathways to store LCFAs as triglycerides. Adiponectin, another important target of PPAR-gamma may act as a spacer between adipocytes to maintain their metabolic activity and insulin sensitivity. Another topic of this review is effects of skin LCFAs on energy metab. Specific LCFAs are required for the synthesis of skin lipids, which are essential for water barrier and thermal insulation functions of the skin. Disturbance of skin lipid metab. often causes apparent resistance to developing obesity at the expense of normal skin function.
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    Barrett, G. C.; Elmore, D. T. Amino Acids and Peptides; Cambridge University Press: Cambridge, 1998 DOI: 10.1017/CBO9781139163828 .
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    Ogliaruso, M. A.; Wolfe, J. F. Synthesis of Carboxylic Acids. Esters and Their Derivatives; Wiley Online Library, 1979; Vol. 267 DOI: 10.1002/9780470771587.ch7 .
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    Wang, H.; Fan, H.; Sun, H.; Zhao, L.; Wei, D. Process Development for the Production of (R)-(−)-Mandelic Acid by Recombinant Escherichia Coli Cells Harboring Nitrilase from Burkholderia Cenocepacia J2315. Org. Process Res. Dev. 2015, 19 (12), 2012– 6,  DOI: 10.1021/acs.oprd.5b00269
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    392
    Process Development for the Production of (R)-(-)-Mandelic Acid by Recombinant Escherichia coli Cells Harboring Nitrilase from Burkholderia cenocepacia J2315
    Wang, Hualei; Fan, Haiyang; Sun, Huihui; Zhao, Li; Wei, Dongzhi
    Organic Process Research & Development (2015), 19 (12), 2012-2016CODEN: OPRDFK; ISSN:1083-6160. (American Chemical Society)
    (R)-(-)-Mandelic acid is an important chiral building block that is widely used in pharmacy and the prodn. of fine chems. A more advanced method for obtaining (R)-(-)-mandelic acid is direct hydrolysis of the corresponding racemic mandelonitrile. In order to develop a cost-effective process, a highly efficient enantioselective nitrilase BCJ2315 from Burkholderia cenocepacia J2315 was used for the biotransformation of mandelonitrile to (R)-(-)-mandelic acid. The recombinant Escherichia coli M15/BCJ2315 showed high substrate tolerance and could completely hydrolyze up to 250 mM of mandelonitrile. A fed-batch reaction was performed by periodically or continuously dosing the substrate into the reactor to alleviate substrate inhibition in a monophasic buffer system. Finally, the highest substrate loading (2.9 M) was achieved in the continuous fed batch reaction mode, giving (R)-(-)-mandelic acid at the highest concn. (2.3 M, 350 g/L) with 97.4% ee ever reported. The hydrolysis process was easily scaled up to 2 and 10 L, indicating the potential for the industrial prodn. of optically pure (R)-(-)-mandelic acid.
  391. 391
    Martínková, L.; Rucká, L.; Nešvera, J.; Pátek, M. Recent Advances and Challenges in the Heterologous Production of Microbial Nitrilases for Biocatalytic Applications. World J. Microbiol. Biotechnol. 2017, 33, 8,  DOI: 10.1007/s11274-016-2173-6
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    393
    Recent advances and challenges in the heterologous production of microbial nitrilases for biocatalytic applications
    Martinkova Ludmila; Rucka Lenka; Nesvera Jan; Patek Miroslav
    World journal of microbiology & biotechnology (2017), 33 (1), 8 ISSN:.
    The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of nitrilases. By exploiting this resource, the number of experimentally verified nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of nitrilases are classified according to their target products (α-hydroxy acids, α- and β-amino acids, cyano acids, amides). The emerging uses of nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications.
  392. 392
    Xu, Z.; Xiong, N.; Zou, S.-P.; Liu, Y.-X.; liu, Z.-Q.; Xue, Y.-P.; Zheng, Y.-G. Highly Efficient Conversion of 1-Cyanocycloalkaneacetonitrile Using a “Super Nitrilase Mutant”. Bioprocess Biosyst. Eng. 2019, 42 (3), 455– 63,  DOI: 10.1007/s00449-018-2049-2
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    394
    Highly efficient conversion of 1-cyanocycloalkaneacetonitrile using a "super nitrilase mutant"
    Xu, Zhe; Xiong, Neng; Zou, Shu-Ping; Liu, Yu-Xiao; liu, Zhi-Qiang; Xue, Ya-Ping; Zheng, Yu-Guo
    Bioprocess and Biosystems Engineering (2019), 42 (3), 455-463CODEN: BBEIBV; ISSN:1615-7591. (Springer)
    Nitrilase is the member of carbon-nitrogen hydrogen hydrolase superfamily, which has been widely used for the hydrolysis of nitriles into corresponding carboxylic acids. But most nitrilases are plagued by product inhibition in the industrial application. In this study, a "super nitrilase mutant" of nitrilase with high activity, thermostability and improved product tolerance from Acidovorax facilis ZJB09122 was characterized. Then, an efficient process was developed by employing the whole cell of recombinant E. coli for the conversion of high concn. of 1-cyanocyclohexylacetonitrile-to-1-cyanocyclohexaneacetic acid, an important intermediate of gabapentin. Under the optimized conditions, the higher substrate concns. such as 1.3 M, 1.5 M and 1.8 M could be hydrolyzed by 13.58 g DCW/L with outstanding productivity (> 740 g/L/day). This study developed a highly efficient bioprocess for the prepn. of 1-cyanocyclohexaneacetic acid which has the great potential for industrial application.
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    Glieder, A.; Weis, R.; Skranc, W.; Poechlauer, P.; Dreveny, I.; Majer, S.; Wubbolts, M.; Schwab, H.; Gruber, K. Comprehensive Step-by-Step Engineering of an (R)-Hydroxynitrile Lyase for Large-Scale Asymmetric Synthesis. Angew. Chem., Int. Ed. 2003, 42 (39), 4815– 8,  DOI: 10.1002/anie.200352141
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    395
    Comprehensive step-by-step engineering of an (R)-hydroxynitrile lyase for large-scale asymmetric synthesis
    Glieder, Anton; Weis, Roland; Skranc, Wolfgang; Poechlauer, Peter; Dreveny, Ingrid; Majer, Sandra; Wubbolts, Marcel; Schwab, Helmut; Gruber, Karl
    Angewandte Chemie, International Edition (2003), 42 (39), 4815-4818CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The goal of the study was to develop a com. viable (R)-hydroxynitrile lyase to catalyze the enantioselective conversion of 2-chlorobenzaldehyde to (R)-2-chloromandeleonitrile under process conditions with the ultimate goal being prodn. of (R)-2-chloromandelic acid.. The approach taken was to clone the (R)-hydroxynitrile lyase gene from Prunus amygdalus and express it in Pichia pastoris. The recombinant enzyme which was highly glycosylated had twice the specific activity of the native enzyme and demonstrated a high degree of stability at low pH. The enzyme prodn. was increased by replacing the N-terminal leucine with a glutamine and replacing the native plant leader sequence with an α-mating factor leader sequence from Saccharomyces cerevisiae. Site-directed mutagenesis was then employed to minimize steric hindrance in the enzyme active site by replacing alanine 111 or valine 317 with glycine residues. Both mutant enzymes had dramatically lower specific activities for nitrile hydrolysis, but the A111G variant retained a high hydrocyanating activity. Addnl. the turnover frequency of the A111G variant was six times faster than the native enzyme. The results demonstrate the rational application of genetic engineering can significantly improve the properties of enzyme catalysts.
  394. 394
    Jiang, M.; Liu, Y.-J.; Liu, G.-S.; Zhou, W.; Shen, Y.-L.; Gao, B.; Wang, F.-Q.; Wei, D.-Z. Sequential Resolution of (S) and (R)-6-Fluoro-Chroman-2-Carboxylic Acid by Two Esterases in Turn. Green Chem. 2022, 24 (8), 3235– 42,  DOI: 10.1039/D1GC04512A
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    396
    Sequential resolution of (S) and (R)-6-fluoro-chroman-2-carboxylic acid by two esterases in turn
    Jiang, Min; Liu, Yong-Jun; Liu, Guo-Song; Zhou, Wei; Shen, Ya-Ling; Gao, Bei; Wang, Feng-Qing; Wei, Dong-Zhi
    Green Chemistry (2022), 24 (8), 3235-3242CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
    Optically pure 6-fluoro-chroman-2-carboxylic acids (FCCAs), with (S) and (R)-configurations, are pivotal chiral building blocks in the pharmaceutical industry. To date, the prodn. of (S) and (R)-FCCAs mainly depends on chem. resoln., which is a complex, low yield, and highly polluting process. Here, we present a practical enzymic resoln. method of FCCAs based on two esterases, EstS and EstR, isolated from Geobacillus thermocatenulatus. Using the racemic Me 6-fluoro-chroman-2-carboxylate (MFCC) as the substrate in an aq.-toluene biphasic system, (S) and (R)-FCCAs were produced by EstS and EstR catalysis with an enantiomeric excess (ee) value >99% and 95-96%, resp., and the highly enantioselective mechanisms were revealed by mol. simulations. To simplify the resoln. process and enhance the productivity, we further designed an innovative methodol. for the "sequential biphasic batch resoln." of MFCC with immobilized cells. In each batch, only the aq. phase needs to be replaced to sequentially change the immobilized cells of EstS or EstR and recover optically pure FCCAs in turn, while the org. phase was retained and MFCC was continually supplemented after every two batches. In this study, ten batches of sequential resoln. were performed, and 229.3 mM (S)-FCCAs with 96.9% ee, and 224.1 mM (R)-FCCAs with 99.1% ee were obtained in 40 h, affording a 93.5% total mole yield. This is the first reported enzymic resoln. technique of FCCAs, and represents significant advantages over those chem. resoln. methods.
  395. 395
    Dijkman, W. P.; Groothuis, D. E.; Fraaije, M. W. Enzyme-Catalyzed Oxidation of 5-Hydroxymethylfurfural to Furan-2,5-Dicarboxylic Acid. Angew. Chem., Int. Ed. 2014, 53 (25), 6515– 8,  DOI: 10.1002/anie.201402904
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    397
    Enzyme-Catalyzed Oxidation of 5-Hydroxymethylfurfural to Furan-2,5-dicarboxylic Acid
    Dijkman, Willem P.; Groothuis, Daphne E.; Fraaije, Marco W.
    Angewandte Chemie, International Edition (2014), 53 (25), 6515-6518CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Furan-2,5-dicarboxylic acid (FDCA) is a biobased platform chem. for the prodn. of polymers. In the past few years, numerous multistep chem. routes have been reported on the synthesis of FDCA by oxidn. of 5-hydroxymethylfurfural (HMF). Recently we identified an FAD-dependent enzyme which is active towards HMF and related compds. This oxidase has the remarkable capability of oxidizing [5-(hydroxymethyl)furan-2-yl]methanol to FDCA, a reaction involving four consecutive oxidns. The oxidase can produce FDCA from HMF with high yield at ambient temp. and pressure. Examn. of the underlying mechanism shows that the oxidase acts on alc. groups only and depends on the hydration of aldehydes for the oxidn. reaction required to form FDCA.
  396. 396
    DeSantis, G.; Wong, K.; Farwell, B.; Chatman, K.; Zhu, Z.; Tomlinson, G.; Huang, H.; Tan, X.; Bibbs, L.; Chen, P. Creation of a Productive, Highly Enantioselective Nitrilase through Gene Site Saturation Mutagenesis (GSSM). J. Am. Chem. Soc. 2003, 125 (38), 11476– 7,  DOI: 10.1021/ja035742h
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    398
    Creation of a Productive, Highly Enantioselective Nitrilase through Gene Site Saturation Mutagenesis (GSSM)
    DeSantis, Grace; Wong, Kelvin; Farwell, Bob; Chatman, Kelly; Zhu, Zoulin; Tomlinson, Geoff; Huang, Hongjun; Tan, Xuqiu; Bibbs, Lisa; Chen, Pei; Kretz, Keith; Burk, Mark J.
    Journal of the American Chemical Society (2003), 125 (38), 11476-11477CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Gene site satn. mutagenesis (GSSM) technol. is applied for the directed evolution of a nitrilase. The nitrilase effectively catalyzes the desymmetrization of the prochiral substrate 3-hydroxyglutaronitrile to afford (R)-4-cyano-3-hydroxybutyric acid, a precursor to the valuable cholesterol-lowering drug Lipitor. The discovered wild-type enzyme effectively performs the reaction at the industrially relevant 3 M substrate concn. but affords a product enantiomeric excess of only 87.6% ee. Through GSSM, a mutagenesis technique that effects the combinatorial satn. of each amino acid in the protein to each of the other 19 amino acids, combined with a novel high-throughput mass spectroscopy assay, a no. of improved variants were identified, the best of which is the Ala190His mutant that yields product enantiomeric excess of 98.5% at 3 M substrate loading and a volumetric productivity of 619 g L-1 d-1.
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    Franz, R. G. Comparisons of pKa and Log P Values of Some Carboxylic and Phosphonic Acids: Synthesis and Measurement. AAPS PharmSci. 2001, 3 (2), 1,  DOI: 10.1208/ps030210
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    Toth, A. M.; Liptak, M. D.; Phillips, D. L.; Shields, G. C. Accurate Relative Pka Calculations for Carboxylic Acids Using Complete Basis Set and Gaussian-N Models Combined with Continuum Solvation Methods. J. Chem. Phys. 2001, 114 (10), 4595– 606,  DOI: 10.1063/1.1337862
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    Accurate relative pKa calculations for carboxylic acids using complete basis set and Gaussian-n models combined with continuum solvation methods
    Toth, Ann Marie; Liptak, Matthew D.; Phillips, Danielle L.; Shields, George C.
    Journal of Chemical Physics (2001), 114 (10), 4595-4606CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
    The complete basis set methods CBS-4, CBS-QB3, and CBS-APNO, and the Gaussian methods G2 and G3 were used to calc. the gas phase energy differences between six different carboxylic acids and their resp. anions. Two different continuum methods, SM5.42R and CPCM, were used to calc. the free energy differences of solvation for the acids and their anions. Relative pKa values were calcd. for each acid using one of the acids as a ref. point. The CBS-QB3 and CBS-APNO gas phase calcns., combined with the CPCM/HF/6-31+G(d)//HF/6-31G(d) or CPCM/HF/6-31+G(d)//HF/6-31+G(d) continuum solvation calcns. on the lowest energy gas phase conformer, and with the conformationally averaged values, give results accurate to (1)/(2) pKa unit.
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    Vollhardt, K. P. C. Organische Chemie, 3rd ed.; Wiley-VCH: Weinheim, 2000.
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    Bruice, P. Y. Organic Chemistry, 5 ed.; Pearson Prentice Hall: Upper Saddle River, NJ, 2007.
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    Descalzo, A. B.; Rurack, K.; Weisshoff, H.; Martínez-Máñez, R.; Marcos, M. D.; Amorós, P.; Hoffmann, K.; Soto, J. Rational Design of a Chromo- and Fluorogenic Hybrid Chemosensor Material for the Detection of Long-Chain Carboxylates. J. Am. Chem. Soc. 2005, 127 (1), 184– 200,  DOI: 10.1021/ja045683n
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    Rational Design of a Chromo- and Fluorogenic Hybrid Chemosensor Material for the Detection of Long-Chain Carboxylates
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    Journal of the American Chemical Society (2005), 127 (1), 184-200CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A strategy for the rational design of a new optical sensor material for the selective recognition of long-chain carboxylates in water is presented. The approach relies on the combination of structure-property relationships to single out the optimal mol. sensor unit and the tuning of the sensing characteristics of an inorg. support material. A spacer-substituted 7-urea-phenoxazin-3-one was employed as the signaling moiety and a mesoporous trimethylsilylated UVM-7 (MCM-41 type) material served as the solid support. The sensor material shows the advantageous features of both modules that is absorption and emission in the visible spectral range, a fluorescence red-shift and enhancement upon analyte coordination, and the amplification of noncovalent (binding) and hydrogen-bonding (recognition) interactions in the detection event. Besides these basic results that are related to the design and performance of the sensor material, the paper discusses general aspects of amido-substituted phenoxazinone photophysics and addresses some general features of mol. anion recognition chem. in aq. vs nonaq. media, utilizing steady-state and time-resolved optical as well as NMR spectroscopies. Detailed studies on potentially competing biochem. species and a first access to the schematic model of the response of the sensor material as obtained by a combination of fluorescence lifetime distribution anal. and Langmuir-type fitting of the gross binding consts. complement the key issues of the paper.
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    Reddy G, U.; Lo, R.; Roy, S.; Banerjee, T.; Ganguly, B.; Das, A. A New Receptor with a FRET Based Fluorescence Response for Selective Recognition of Fumaric and Maleic Acids in Aqueous Medium. Chem. Commun. 2013, 49 (84), 9818,  DOI: 10.1039/c3cc45051a
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    Fitzmaurice, R. J.; Kyne, G. M.; Douheret, D.; Kilburn, J. D. Synthetic Receptors for Carboxylic Acids and Carboxylates. J. Chem. Soc., Perkin Trans. 1 2002, (7), 841– 64,  DOI: 10.1039/b009041g
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    Synthetic receptors for carboxylic acids and carboxylates
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    Journal of the Chemical Society, Perkin Transactions 1 (2002), (7), 841-864CODEN: JCSPCE; ISSN:1472-7781. (Royal Society of Chemistry)
    A review with refs. up to and including June 2001 on the development of synthetic receptors for substrate (host-guest chem.) with carboxylic acids or carboxylates as functional groups.
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    Janes, L. E.; Löwendahl, A. C.; Kazlauskas, R. J. Quantitative Screening of Hydrolase Libraries Using pH Indicators: Identifying Active and Enantioselective Hydrolases. Eur. J. Chem. 1998, 4 (11), 2324– 31,  DOI: 10.1002/(SICI)1521-3765(19981102)4:11<2324::AID-CHEM2324>3.0.CO;2-I
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    Quantitative screening of hydrolase libraries using pH indicators: identifying active and enantioselective hydrolases
    Janes, Lana E.; Lowendahl, A. Christina; Kazlauskas, Romas J.
    Chemistry - A European Journal (1998), 4 (11), 2324-2331CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH)
    The slowest step in finding a selective hydrolase for synthesis is often the screening step. Researchers must run small test reactions and measure the amts. of stereoisomers formed by HPLC, GC, or NMR. We have developed a colorimetric method to speed up this screening. We quant. detect ester hydrolysis using a pH indicator, 4-nitrophenol. We est. the selectivity by measuring the initial rates of hydrolysis for pure stereoisomers sep. To demonstrate the utility of this method, we screened seventy-two com. enzymes for enantioselective hydrolysis of racemic solketal butyrate, an important chiral building block. First, we eliminated the twenty hydrolases that did not catalyze hydrolysis of either enantiomer. Next, we measured initial rates of hydrolysis of the pure enantiomers of solketal butyrate. For horse-liver esterase, these initial rates differed by a factor of twelve. Subsequent GC expts. confirmed an enantiomeric ratio of fifteen for this hydrolase. Although this enantioselectivity is moderate, it is the highest enantioselectivity reported for a hydrolysis of solketal esters.
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    Baumann, M.; Stürmer, R.; Bornscheuer, U. T. A High-Throughput-Screening Method for the Identification of Active and Enantioselective Hydrolases. Angew. Chem., Int. Ed. 2001, 40 (22), 4201– 4,  DOI: 10.1002/1521-3773(20011119)40:22<4201::AID-ANIE4201>3.0.CO;2-V
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    A high-throughput-screening method for the identification of active and enantioselective hydrolases
    Baumann, Markus; Sturmer, Rainer; Bornscheuer, Uwe T.
    Angewandte Chemie, International Edition (2001), 40 (22), 4201-4204CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
    A new assay system format for screening enantioselective hydrolases, based on a coupled enzymic conversion, is described. To evaluate this assay format, the hydrolysis of (R,S)-α-Ph-Et acetate by a recombinant esterase from Pseudomonas fluorescens was chosen as a model reaction. The applicability of the assay format for the detn. of enantioselectivity values was investigated using 16 lipases and esterases. Using this format, an anal. time of less than 3-4 min per assay was found to be sufficient to det. enantioselectivities. In addn., the assay was found to be rather cheap.
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    Wang, J.; Liu, H.-B.; Tong, Z.; Ha, C.-S. Fluorescent/Luminescent Detection of Natural Amino Acids by Organometallic Systems. Coord. Chem. Rev. 2015, 303, 139– 84,  DOI: 10.1016/j.ccr.2015.05.008
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    Fluorescent/luminescent detection of natural amino acids by organometallic systems
    Wang, Jing; Liu, Hai-Bo; Tong, Zhangfa; Ha, Chang-Sik
    Coordination Chemistry Reviews (2015), 303 (), 139-184CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
    In comparison with other detection technologies, fluorescence/luminescence technol. has become a powerful tool owing to its advantageous features including simplicity, low cost, high sensitivity, quick response time, easy sample prepn., noninvasive and nondestructive nature, etc. Due to the important roles played by 20 natural amino acids in living systems, this review focuses on recent contributions (from the year 2000 until July 2014) regarding the development of fluorescent/luminescent chemosensors and chemodosimeters to detect specific AAs, as well as chiral recognition to discriminate AA enantiomers (i.e., D and L), and pattern recognition to distinguish a range of AAs simultaneously based on fluorescent/luminescent organometallic systems, which include org.-metal complexes and hybrid org.-metal nanoparticles/nanoclusters.
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    Gong, R.; Mu, H.; Sun, Y.; Fang, X.; Xue, P.; Fu, E. The First Fluorescent Sensor for Medium-Chain Fatty Acids in Water: Design, Synthesis and Sensing Properties of an Organic-Inorganic Hybrid Material. J. Mater. Chem. B 2013, 1 (15), 2038– 47,  DOI: 10.1039/c3tb00355h
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    The first fluorescent sensor for medium-chain fatty acids in water: design, synthesis and sensing properties of an organic-inorganic hybrid material
    Gong, Rui; Mu, Honglei; Sun, Yimin; Fang, Xiantao; Xue, Peng; Fu, Enqin
    Journal of Materials Chemistry B: Materials for Biology and Medicine (2013), 1 (15), 2038-2047CODEN: JMCBDV; ISSN:2050-7518. (Royal Society of Chemistry)
    This paper reports the first fluorescent sensor for medium-chain fatty acids in water. The hybrid sensing material (S2) was prepd. by the reaction of a rationally designed "recognition center", N-butyl-4-piperazin-1,8-naphthalimide, with a 3-glycidoxypropyl group which is pre-grafted on the interior of a mesoporous solid (MCM-41). The organo-functionalization of the mesoporous solid (MCM-41) was confirmed by FTIR, 29Si MAS NMR and elemental anal. The results of XRD, N2 phys. adsorption-desorption, SEM and TEM studies proved that the organized structure of the nanoscopic porous solid is preserved after the reactions. The fatty acid-selective signaling behavior of S2 was studied in water at pH = 5.80/7.16/8.00. This material displays much stronger fluorescence enhancement with C8-C12 fatty acids than with the shorter and longer chains in aq. media, both in the absence and in the presence of arom. acids.
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    Kusukawa, T.; Tanaka, S.; Inoue, K. Fluorescent Detection of Amidinium-Carboxylate and Amidinium Formation Using a 1,8-Diphenylnaphthalene-Based Diamidine: Dicarboxylic Acid Recognition with High Fluorescence Efficiency. Tetrahedron 2014, 70 (26), 4049– 56,  DOI: 10.1016/j.tet.2014.03.036
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    Fluorescent detection of amidinium-carboxylate and amidinium formation using a 1,8-diphenylnaphthalene-based diamidine: dicarboxylic acid recognition with high fluorescence efficiency
    Kusukawa, Takahiro; Tanaka, Syugo; Inoue, Kouta
    Tetrahedron (2014), 70 (26), 4049-4056CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)
    A 1,8-diphenylnaphthalene-based diamidine (I) 'turn-on' fluorescent probe for the detection of dicarboxylic acids was designed and synthesized. The fluorescence spectra of the diamidine I with carboxylic acids that showed two different fluorescence bands, which corresponded to the amidinium-carboxylate (λem=410-430 nm) and amidinium (λem=440-470 nm as a broad band, which consisted from two peaks) formation, were confirmed by DOSY NMR and TD-DFT calcns. The complexation of diamidine I with dicarboxylic acids, which have sufficient distances between the two carboxylic groups for binding to the diamidine I (3-adamantanediacetic acid, m-phenylenediacetic acid, and α,ω-dicarboxylic acids (C6-C20)), showed the formation of 1:1 complexes (i.e., amidinium-carboxylate formation). However, for the complexation with monocarboxylic acids and dicarboxylic acids having insufficient distances between the two carboxylic groups (benzoic acid, acetic acid, and α,ω-dicarboxylic acids (C3-C5)), formation of the amidinium (1·2H+) was obsd. Relatively similar binding consts. (10-5) for the complexation of the diamidine 1 with dicarboxylic acids 6, which depend on their chain length (strain), were obsd. due to the flexibility of the 1,8-diphenylnaphthalene unit. Addnl., for the complexation of the diamidine 1 with dicarboxylic acids, higher fluorescence quantum yields (Φfl: up to 80%) were obsd. when compared to the binding of the diamidine 2 (Φfl: up to 35%).
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    Boiocchi, M.; Bonizzoni, M.; Fabbrizzi, L.; Piovani, G.; Taglietti, A. A Dimetallic Cage with a Long Ellipsoidal Cavity for the Fluorescent Detection of Dicarboxylate Anions in Water. Angew. Chem., Int. Ed. 2004, 43 (29), 3847– 52,  DOI: 10.1002/anie.200460036
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    A dimetallic cage with a long ellipsoidal cavity for the fluorescent detection of dicarboxylate anions in water
    Boiocchi, Massimo; Bonizzoni, Marco; Fabbrizzi, Luigi; Piovani, Giulio; Taglietti, Angelo
    Angewandte Chemie, International Edition (2004), 43 (29), 3847-3852CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The dicopper(II) complex of a bistren cage contg. ditolyl spacers (I) binds dicarboxylate anions in aq. solns. The inclusion is highly selective from the distance between the COO- groups. For example, terephthalate is discriminated from isophthalate and phthalate.
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    Metzger, A.; Lynch, V. M.; Anslyn, E. V. A Synthetic Receptor Selective for Citrate. Angew. Chem., Int. Ed. 1997, 36 (8), 862– 5,  DOI: 10.1002/anie.199708621
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    A synthetic receptor selective for citrate
    Metzger, Axel; Lynch, Vincent M.; Anslyn, Eric V.
    Angewandte Chemie, International Edition in English (1997), 36 (8), 862-865CODEN: ACIEAY; ISSN:0570-0833. (VCH)
    A synthetic receptor bearing guanidinium groups embedded in aminodihydroimidazolium groups was prepd. and shown to be a selective receptor for citrate.
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    Yang, X.; Liu, X.; Shen, K.; Zhu, C.; Cheng, Y. A Chiral Perazamacrocyclic Fluorescent Sensor for Cascade Recognition of Cu(II) and the Unmodified α-Amino Acids in Protic Solutions. Org. Lett. 2011, 13 (13), 3510– 3,  DOI: 10.1021/ol2013268
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    A Chiral Perazamacrocyclic Fluorescent Sensor for Cascade Recognition of Cu(II) and the Unmodified α-Amino Acids in Protic Solutions
    Yang, Xia; Liu, Xuechao; Shen, Kang; Zhu, Chengjian; Cheng, Yixiang
    Organic Letters (2011), 13 (13), 3510-3513CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
    A novel chiral perazamacrocyclic fluorescent sensor (I) was designed and synthesized. It can serve as a fluorescent turn-off sensor with high selectivity toward Cu(II) among 14 metal ions. Also, though I exhibits no enantioselectivity, after adding Cu(II), the in situ generated Cu(II)-contg. complex of I (Cu(II)-I) can exhibit remarkable fluorescent enhancement responses and considerable enantioselectivities toward unmodified α-amino acids in protic solns. via a ligand displacement mechanism; i.e. a cascade recognition of Cu(II) and unmodified α-amino acids was achieved.
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    Görög, S.; Laukó, A.; Rényei, M.; Hegedüs, B. New Derivatization Reactions in Pharmaceutical Analysis. J. Pharm. Biomed. Anal. 1983, 1 (4), 497– 506,  DOI: 10.1016/0731-7085(83)80063-6
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    New derivatization reactions in pharmaceutical analysis
    Gorog, S.; Lauko, A.; Renyei, M.; Hegedus, B.
    Journal of Pharmaceutical and Biomedical Analysis (1983), 1 (4), 497-506CODEN: JPBADA; ISSN:0731-7085.
    A review with 34 refs. The use of chem. reactions in conjunction with spectrometric and chromatog. methods is exemplified by the formation of 2-nitrophenylhydrazide derivs. for the spectrometric detn. of carboxylic acids; silylation and trifluoroacetylation of drugs for gas chromatog.; selective redn., of steroid ketones for IR spectrometric identification; the use of epoxidn. in discriminating between satd. and unsatd. steroids in gas chromatog.; increasing the selectivity and sensitivity of the spectrometric and gas-chromatog. detn. of isomeric Δ4- and Δ5-3-ethylenedioxy steroids by treatment with HCl; and the use of the same reagent in the difference spectrometric detn. of 2,5-dimethyl-α-ethylbenzhydrol.
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    Munson, J. W.; Bilous, R. Colorimetric Determination of Aliphatic Acids. J. Pharm. Sci. 1977, 66 (10), 1403– 5,  DOI: 10.1002/jps.2600661013
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    Colorimetric determination of aliphatic acids
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    Journal of Pharmaceutical Sciences (1977), 66 (10), 1403-5CODEN: JPMSAE; ISSN:0022-3549.
    A colorimetric method for the detn. of carboxylic acids based on the dicyclohexylcarbodiimide-coupled reaction of 2-nitrophenylhydrazine and carboxylic acids is described. The product of the reaction was extd. into aq. NaOH to produce a blue color. This method is suitable for the anal. of aliph. acids. Arom. acids did not react under these conditions.
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    Miwa, H.; Yamamoto, M.; Momose, T. Colorimetric Detection and Determination of Carboxylic Acids with 2-Nitrophenylhydrazine Hydrochloride. Chem. Pharm. Bull. (Tokyo) 1980, 28 (2), 599– 605,  DOI: 10.1248/cpb.28.599
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    Hill, U. T. Colorimetric Determination of Fatty Acids and Esters. Ind. Eng. Chem. Anal. Ed. 1946, 18 (5), 317– 9,  DOI: 10.1021/i560153a017
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    Colorimetric determination of fatty acids and esters
    Hill, Uno T.
    Industrial and Engineering Chemistry, Analytical Edition (1946), 18 (), 317-19CODEN: IENAAD; ISSN:0096-4484.
    Fatty acids and esters applied to a metal surface for rust prevention, lubrication, etc., can be rapidly extd., converted to their hydroxamic acids and made to react with ferric ions (cf. Feigl, C.A. 38, 315.4) to form a red or lavender complex (Fe[R.CO(NHO)]3) which follows Beer's law if ferric perchlorate in perchloric acid is used. Percentage transmittancy is detd. at 520 mμ with a spectrophotometer, compared with a blank and the amt. of ester present obtained by reference to a previously prepd. ester vs. transmittancy curve. Acids do not form a hydroxamic acid unless first esterified. Complete directions are given with reagent quantities specific for 0.05 to 1 mg. of extractant, but larger quantities can be used. Comparison with a gravimetric method is good; cottonseed oil is detd. within ±0.01 mg. Transmittancy values can be plotted in terms of the sapon., acid, ester, or acetyl values; curves are given for solns. of ferric hydroxamates from dibutyl sebacate, and cottonseed, palm, sulfurized sperm, and lanolin-base oils.
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    Montgomery, H. A. C.; Dymock, J. F.; Thom, N. S. The Rapid Colorimetric Determination of Organic Acids and Their Salts in Sewage-Sludge Liquor. Analyst 1962, 87 (1041), 949– 55,  DOI: 10.1039/an9628700949
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    The rapid colorimetric determination of organic acids and their salts in sewage-sludge liquor
    Montgomery, H. A. C.; Dymock, Joan F.; Thom, N. S.
    (1962), 87 (), 949-55 ISSN:.
    The colorimetric composite detn. of the caxboxylic acids and their salts in sewage-sludge liquor is described. After removal of suspended matter by filtration with the assistance of filter aid, a 0.5-ml. sample is treated with ethylene glycol in the presence of H2SO4, and the resulting mixt. of esters is detd. colorimetrically at 500 mμ, with 4-cm. cells, by the ferric hydroxamate reaction. For HOAc, the calibration is linear to at least 1%. Since the most intense colors are given by the lower fatty acids, which predominate in sludge liquor, the method gives results similar to existing methods for the detn. of volatile acids. The procedure was applied to the detn. of org. acids in an effluent from a bacon factory.
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    Kasai, Y.; Tanimura, T.; Tamura, Z. J. A. C. Spectrophotometric Determination of Carboxylic Acids by the Formation of Hydroxamic Acids with Dicyclohexylcarbodiimide. Anal. Chem. 1975, 47 (1), 34– 7,  DOI: 10.1021/ac60351a047
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    Spectrophotometric determination of carboxylic acids by the formation of hydroxamic acids with dicyclohexylcarbodiimide
    Kasai, Yasuhiko; Tanimura, Takenori; Tamura, Zenzo
    Analytical Chemistry (1975), 47 (1), 34-7CODEN: ANCHAM; ISSN:0003-2700.
    A new method of spectrophotometric detn. of carboxylic acids was developed. The unique feature of the method is the formation, in a single step, of hydroxamate from carboxylic acid and NH2OH by using dicyclohexylcarbodi-imide (DCC). Hydroxamate was prepd. by adding DCC to the mixt. of carboxylic acid and NH2OH in anhyd. or aq. EtOH at 50 or 60°. Ferric hydroxamate was prepd. with large excess of Fe(ClO4)3 in EtOH soln. of HClO4 and was detd. at 525 nm. The working curves obtained were linear for 0.25-2.5 μmoles benzoic, caprylic, lactic, and p-nitrophenylacetic acid.
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    Takeuchi, T.; Horikawa, R.; Tanimura, T. Spectrophotometry Determination of Carboxylic Acids by Ferric Hydroxamate Formation with Water-Soluble Carbodiimide. Anal. Lett. 1980, 13 (7), 603– 9,  DOI: 10.1080/00032718008077690
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    Spectrophotometric determination of carboxylic acids by ferric hydroxamate formation with water-soluble carbodiimide
    Takeuchi, T.; Horikawa, R.; Tanimura, T.
    Analytical Letters (1980), 13 (A7), 603-9CODEN: ANALBP; ISSN:0003-2719.
    Carboxylic acids in aq. soln. were detd. spectrophotometrically as ferric hydroxamates. A H2O-sol. carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, was used as a coupling agent to form hydroxamic acids. The coupling reaction was carried out at 37° for 30 min in a soln. buffered at pH 5.3 with pyridine and HCl. Calibration curves of HOBz, HOAc, citric acid, and α-ketoglutaric acid were linear for 0.25-8 μmol.
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    He, Y.-C.; Ma, C.-L.; Xu, J.-H.; Zhou, L. A High-Throughput Screening Strategy for Nitrile-Hydrolyzing Enzymes Based on Ferric Hydroxamate Spectrophotometry. Appl. Microbiol. Biotechnol. 2011, 89 (3), 817– 23,  DOI: 10.1007/s00253-010-2977-5
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    A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry
    He, Yu-Cai; Ma, Cui-Luan; Xu, Jian-He; Zhou, Li
    Applied Microbiology and Biotechnology (2011), 89 (3), 817-823CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the prodn. of carboxylic acids and their derivs., and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliph. and arom. carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes.
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    Goswami, S.; Hazra, A.; Das, M. K. Selenodiazole-Fused Diacetamidopyrimidine, a Selective Fluorescence Sensor for Aliphatic Monocarboxylates. Tetrahedron Lett. 2010, 51 (25), 3320– 3,  DOI: 10.1016/j.tetlet.2010.04.085
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    Selenodiazole-fused diacetamidopyrimidine, a selective fluorescence sensor for aliphatic monocarboxylates
    Goswami, Shyamaprosad; Hazra, Anita; Das, Manas Kumar
    Tetrahedron Letters (2010), 51 (25), 3320-3323CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)
    A designed sensor, selenodiazole-fused pyrimidine ring having two acetylamino groups at 2,4-positions was synthesized for selective recognition of aliph. monocarboxylate anions over a wide range of other anions. The recognition study was carried out by UV-visible and fluorescence methods. A significant bathochromic shift of the fluorescence intensity of the receptor in the presence of carboxylate makes the receptor a discriminating sensor for aliph. monocarboxylates.
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    Goswami, S.; Hazra, A.; Chakrabarty, R.; Fun, H.-K. Recognition of Carboxylate Anions and Carboxylic Acids by Selenium-Based New Chromogenic Fluorescent Sensor: A Remarkable Fluorescence Enhancement of Hindered Carboxylates. Org. Lett. 2009, 11 (19), 4350– 3,  DOI: 10.1021/ol901737s
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    Recognition of carboxylate anions and carboxylic acids by selenium-based new chromogenic fluorescent sensor: A remarkable fluorescence enhancement of hindered carboxylates
    Goswami, Shyamaprosad; Hazra, Anita; Chakrabarty, Rinku; Fun, Hoong-Kun
    Organic Letters (2009), 11 (19), 4350-4353CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
    A selenium metal-based new fluorescence sensor 5-pivaloylamino-1,2,5-selenodiazolo[3,4-d]pyrimidin-7-(6H)-one (receptor 1) is reported for the recognition of monocarboxylic acids and carboxylate anions both by UV-visible and fluorescence methods. Receptor 1 recognizes carboxylate anions more than monocarboxylic acids and it is a selective sensor for carboxylates with specially hindered carboxylate anions. The changes of fluorescence intensity are remarkably enhanced with red shift in presence of bulky carboxylate anions. The x-ray crystal structure of receptor 1 with pivalic acid is reported.
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    Galindo, F.; Becerril, J.; Isabel Burguete, M.; Luis, S. V.; Vigara, L. Synthesis and Study of a Cyclophane Displaying Dual Fluorescence Emission: A Novel Ratiometric Sensor for Carboxylic Acids in Organic Medium. Tetrahedron Lett. 2004, 45 (8), 1659– 62,  DOI: 10.1016/j.tetlet.2003.12.116
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    Synthesis and study of a cyclophane displaying dual fluorescence emission: a novel ratiometric sensor for carboxylic acids in organic medium
    Galindo, Francisco; Becerril, Jorge; Isabel Burguete, M.; Luis, Santiago V.; Vigara, Laura
    Tetrahedron Letters (2004), 45 (8), 1659-1662CODEN: TELEAY; ISSN:0040-4039. (Elsevier Science B.V.)
    The synthesis and fluorescent properties of a novel cyclophane contg. two L-valine units and one naphthalene chromophore are described. Synthesis of the macrocycle I was accomplished without using high-diln. methods in moderate to high yields. The fluorescence spectrum of I in neutral dichloromethane shows a band at 390 nm attributable to emission from an exciplex formed between the naphthalene unit and the neighboring amine groups. Addn. of trifluoroacetic acid restores the typical naphthalene emission at 330 nm. Due to the fact that both emissions have similar intensities under the working conditions, the ratio between them can be used to obtain a ratiometric response to carboxylic acids in org. medium.
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    Burguete, M. I.; Galindo, F.; Luis, S. V.; Vigara, L. A Turn-On Fluorescent Indicator for Citrate with Micromolar Sensitivity. Dalton Trans. 2007, (36), 4027– 33,  DOI: 10.1039/b711139h
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    A turn-on fluorescent indicator for citric acid (citrate) has been developed, displaying high emission enhancement (+1500%) and low interference by other carboxylates. The sensor is based on the nonemissive copper(II) complex of a fluorescent amino amide, which, upon addn. of citrate decomplexes to yield the emissive ligand. The detection limit estd. for this new chemosensing system is about 0.5 μM. This novel approach to the anal. of citrate constitutes an alternative ∼102-103 times more sensitive than the std. method based on the enzyme citrate lyase.
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    A fluorometric assay is described which measures enzyme that liberate weak metal ligands, such as amino acids, as their reaction products. The assay uses an orange fluorescent metal-sensor derived from quinacridone which coordinates with a metal ion such as copper and the amino acid product. The quinacridone deriv. can be considered as a pM sensor (pM = -lg[M], where M is free metal ions) by its ability to differentiate free metal ions from weakly chelated metal ions. The fact that product sensing occurs indirectly through the pM value makes the assay applicable to a broad variety of substrates. The kinetics of ligand exchange at copper and nickel are sufficiently fast for the fluorescent pM sensor to response in real time to the reaction progress. Its usefulness is illustrated by the examples presented as the first fluorescence assays for acylase and aminopeptidase.
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    Ryu, D.; Park, E.; Kim, D.-S.; Yan, S.; Lee, J. Y.; Chang, B.-Y.; Ahn, K. H. A Rational Approach to Fluorescence “Turn-On” Sensing of α-Amino-Carboxylates. J. Am. Chem. Soc. 2008, 130 (8), 2394– 5,  DOI: 10.1021/ja078308e
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    Kim, D.-S.; Ahn, K. H. Fluorescence “Turn-On” Sensing of Carboxylate Anions with Oligothiophene-Based o-(Carboxamido)Trifluoroacetophenones. J. Org. Chem. 2008, 73 (17), 6831– 4,  DOI: 10.1021/jo801178y
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    O-(Carboxamido)trifluoroacetophenones contg. ter- or pentathiophene moiety as a fluorophore exhibit fluorescence enhancement upon binding carboxylate anions. Particularly, the terthiophene deriv. shows a large fluorescence enhancement factor (FEF = 120). The enhancement is explained by intramol. H-bonding stabilization of an anion-ionophore adduct, through which a possible quenching process, the n-π* transition from the trifluoroacetophenone moiety, is eliminated.
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    Schrader, J.; Etschmann, M. M. W.; Sell, D.; Hilmer, J. M.; Rabenhorst, J. Applied Biocatalysis for the Synthesis of Natural Flavour Compounds - Current Industrial Processes and Future Prospects. Biotechnol. Lett. 2004, 26 (6), 463– 72,  DOI: 10.1023/B:BILE.0000019576.80594.0e
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    A review, with refs. The industrial application of biocatalysis for the prodn. of natural flavor compds. is illustrated by a discussion of the prodn. of vanillin, γ-decalactone, carboxylic acids, C6 aldehydes and alcs. ( green notes'), esters, and 2-phenylethanol. Modern techniques of mol. biol. and process engineering, such as heterologous expression of genes, site-directed mutagenesis, whole-cell biocatalysis in biphasic systems, and cofactor regeneration for in vitro oxygenation, may result in more biocatalytic processes for the prodn. of flavor compds. in the future.
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    Gotor, V. Biocatalysis Applied to the Preparation of Pharmaceuticals. Org. Process Res. Dev. 2002, 6 (4), 420– 6,  DOI: 10.1021/op020008o
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    Biocatalysis Applied to the Preparation of Pharmaceuticals
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    A review. Biotransformations are now accepted as a methodol. for the prepn. of fine chems. The majority of enzyme-catalyzed reactions involve the use of hydrolases, esp. lipases, in hydrolysis, esterification or aminolysis reactions. Lyases, enzymes much less exploited in org. synthesis, are proving increasingly interesting, esp. the use of (R)-oxynitrilases for the synthesis of optically active cyanohydrins which are intermediates of many compds. of pharmacol. importance. In this account, we report the utility of four kinds of processes which we have carried out recently in our lab. in the field of pharmaceutical chem. Enzymic resoln. of alcs., enzymic acylation of amines, regioselective acylation, and alkoxycarbonylation of natural products, and chemoenzymic synthesis of products of high added value using (R)-oxynitrilase as biocatalyst are described.
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    SÁ, A. G. A.; Meneses, A. C. d.; Araújo, P. H. H. d.; Oliveira, D. d. A Review on Enzymatic Synthesis of Aromatic Esters Used as Flavor Ingredients for Food, Cosmetics and Pharmaceuticals Industries. Trends Food Sci. Technol. 2017, 69, 95– 105,  DOI: 10.1016/j.tifs.2017.09.004
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    A review on enzymatic synthesis of aromatic esters used as flavor ingredients for food, cosmetics and pharmaceuticals industries
    Sa, Amanda Gomes Almeida; Meneses, Alessandra Cristina de; Araujo, Pedro Henrique Hermes de; Oliveira, Debora de
    Trends in Food Science & Technology (2017), 69 (Part_A), 95-105CODEN: TFTEEH; ISSN:0924-2244. (Elsevier Ltd.)
    A review. Many sectors of industry, mainly food, cosmetics and pharmaceutics, have increased their interest in esters due to their flavor property. Flavor esters that possess an arom. ring in their mol. structure are also known as arom. esters. These esters are widely found in nature (fruits and plants) and the synthetic (i.e. via chem.) and natural routes (i.e. via direct extn. from nature or via biotechnol.) are suitable for their biocatalysis. In this context, from the industrial point of view, enzyme-catalyzed reactions are the most economical approach to reach final green products with no toxicity and no harm to human health. The present article gives an overview of the arom. esters synthesis, considering the main effects in the reaction media conditions and enzymes used. This review also describes applied trends in enzymic-catalyzed reactions, pointing alternatives to prodn., like ultrasound-assisted reactions and process optimization of arom. esters. Furthermore, this work presents perspectives concerning the biol. potential of these esters and recent advances in their encapsulation. Lipases play an important role in the arom. esters prodn., with several advantages over synthetic route. Lipase-catalyzed reactions usually follows Ping-Pong Bi-Bi or ternary complex (order Bi-Bi) mechanism. The study of the process parameters and their interaction are very important to understand the system optimization and achieve the max. reaction yield to scale up. Arom. esters can present some biol. activities, in addn. to their fragrances, which increases the interest in the encapsulation of these compds.
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    Panke, S.; Held, M.; Wubbolts, M. Trends and Innovations in Industrial Biocatalysis for the Production of Fine Chemicals. Curr. Opin. Biotechnol. 2004, 15 (4), 272– 9,  DOI: 10.1016/j.copbio.2004.06.011
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    Trends and innovations in industrial biocatalysis for the production of fine chemicals
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    A review. Biocatalysis has become an established technol. for the industrial manuf. of fine chems. In recent years, a multitude of chem. companies have embraced biocatalysis for the manuf. of desired stereoisomers, and new or improved methods for the synthesis of enantiomerically pure α- and β-amino acids, amines, amides, peptides, nitriles, alcs., org. acids and epoxides have emerged. Furthermore, the selectivity and mild operational conditions of biocatalysts are increasingly applied in industry to modify complex target mols. These recent innovations in the manuf. of industrial fine chems. using biocatalysis are discussed from an industrial perspective.
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    Joshi, A.; Singh, S.; Iqbal, Z.; De, S. R. CO Free Esterifications of (Hetero)Arenes Via Transition-Metal-Catalyzed Chelation-Induced C-H Activation: Recent Updates. Tetrahedron 2022, 104, 132601,  DOI: 10.1016/j.tet.2021.132601
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    CO free esterifications of (Hetero)arenes via transition-metal-catalyzed chelation-induced C-H activation: Recent updates
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    Tetrahedron (2022), 104 (), 132601CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)
    A review. Esters are substantial compds. generally found in the area of pharmaceuticals, agrochems., drugs, materials etc., and have been utilized for the formation of many add-value compds. They are also significant structural blocks in different chem. transformations. Thus, highly efficient transition-metal-catalyzed C-H alkoxycarbonylation of (hetero)arenes offers to achieve important skeleton in a single step in contrast to the other conventional strategies using toxic carbon monoxide (CO). This review provides an overview of this emerging field by highlighting all the chelation-induced esters synthesized till date without the involvement of toxic carbon monoxide (CO). The article has been classified according to the sources of esterification agents along with their reaction methodologies, examples, reaction mechanisms, and synthetic applications.
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    Larios, A.; García, H. S.; Oliart, R. M.; Valerio-Alfaro, G. Synthesis of Flavor and Fragrance Esters Using Candida Antarctica Lipase. Appl. Microbiol. Biotechnol. 2004, 65 (4), 373– 6,  DOI: 10.1007/s00253-004-1602-x
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    Synthesis of flavor and fragrance esters using Candida antarctica lipase
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    Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsatd. (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10. Similar results were obsd. in a study of the alc. specificity of the enzyme for esterification of acetic and butyric acids with four alcs.: Bu, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral α-substituted or β-substituted carboxylic acids and Bu alc. was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an org. solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids.
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    Smith, G. A. Fatty Acid, Methyl Ester, and Vegetable Oil Ethoxylates, Biobased Surfactants; AOCS Press, 2019; pp 287– 301.
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    A review. Volatile org. chems. (flavors, aromas) are the sensory principles of many consumer products and govern their acceptance and market success. Flavours from microorganisms compete with the traditional agricultural sources. Screening for overproducers, elucidation of metabolic pathways and precursors and application of conventional bioengineering has resulted in a set of more than 100 com. aroma chems. derived via biotechnol. Various routes may lead to volatile metabolites: De novo synthesis from elementary biochem. units, degrdn. of larger substrates such as lipids, and functionalization of immediate flavor precursor mols. More recently, the field was stimulated by the increasing preference of alienated consumers for products bearing the label "natural", and by the vivid discussion on healthy and "functional" food ingredients. The unmistakable call for sustainable sources and environmentally friendly prodn. is forcing the industry to move towards a greener chem. Progress is expected from the toolbox of genetic engineering which is expected to help in identifying metabolic bottlenecks and in creating novel high-yielding strains. Bioengineering, in a complementary way, provides promising tech. options, such as improved substrate dosage, gas-phase or two-phase reactions and in situ product recovery.
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    Gunatillake, P. A.; Adhikari, R.; Gadegaard, N. Biodegradable Synthetic Polymers for Tissue Engineering. Eur. Cell. Mater. 2003, 5 (1), 1– 16,  DOI: 10.22203/eCM.v005a01
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    A review is given on biodegradable synthetic polymers focusing on their potential in tissue engineering applications. The major classes of polymers are briefly discussed with regard to synthesis, properties and biodegradability, and known degrdn. modes and products are indicated based on studies reported in the literature. A vast majority of biodegradable polymers studied belongs to the polyester family, which includes polyglycolides and polylactides. Some disadvantages of these polymers in tissue engineering applications are their poor biocompatibility, release of acidic degrdn. products, poor processability and loss of mech. properties very early during degrdn. Other degradable polymers such as polyorthoesters, polyanhydrides, polyphosphazenes, and polyurethanes are also discussed and their advantages and disadvantages summarized. With advancements in tissue engineering it has become necessary to develop polymers that meet more demanding requirements. Recent work has focused on developing injectable polymer compns. based on poly(propylene fumarate) and poly(anhydrides) to meet these requirements in orthopedic tissue engineering. Polyurethanes have received recent attention for development of degradable polymers because of their great potential in tailoring polymer structure to achieve mech. properties and biodegradability to suit a variety of applications.
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    Chen, J.; Wang, Y.; Huang, J.; Li, K.; Nie, X. Synthesis of Tung-Oil-Based Triglycidyl Ester Plasticizer and Its Effects on Poly(Vinyl Chloride) Soft Films. ACS Sustain. Chem. Eng. 2018, 6 (1), 642– 51,  DOI: 10.1021/acssuschemeng.7b02989
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    Gumel, A. M.; Annuar, M. S. M.; Heidelberg, T.; Chisti, Y. Lipase Mediated Synthesis of Sugar Fatty Acid Esters. Process Biochem. 2011, 46 (11), 2079– 90,  DOI: 10.1016/j.procbio.2011.07.021
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    Lipase mediated synthesis of sugar fatty acid esters
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    Process Biochemistry (Amsterdam, Netherlands) (2011), 46 (11), 2079-2090CODEN: PBCHE5; ISSN:1359-5113. (Elsevier B.V.)
    This review is concerned with lipase catalyzed synthesis of sugar fatty acid esters in water immiscible org. solvents. Sugar esters are widely used nonionic and nontoxic biosurfactants. Certain sugar esters inhibit microbial growth and have other activities. Lipase mediated synthesis has important advantages over conventional chem. synthesis of sugar esters. Lipase catalyzed synthesis is typically carried out in org. solvents having a low water activity to drive the reaction towards synthesis instead of towards ester hydrolysis. The impact of the various reaction conditions on enzymic synthesis of sugar esters in nonaq. media is discussed. Considered in particular are the solvent effects; the effects of water activity; the influence of the nature and concn. of the reactants (sugars and fatty acids); the influence of temp.; and the effects assocd. with the specific nature of the lipase catalyst used.
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    Badgujar, K. C.; Bhanage, B. M. Application of Lipase Immobilized on the Biocompatible Ternary Blend Polymer Matrix for Synthesis of Citronellyl Acetate in Non-Aqueous Media: Kinetic Modelling Study. Enzyme Microb. Technol. 2014, 57, 16– 25,  DOI: 10.1016/j.enzmictec.2014.01.006
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    Application of lipase immobilized on the biocompatible ternary blend polymer matrix for synthesis of citronellyl acetate in non-aqueous media: Kinetic modelling study
    Badgujar, Kirtikumar C.; Bhanage, Bhalchandra M.
    Enzyme and Microbial Technology (2014), 57 (), 16-25CODEN: EMTED2; ISSN:0141-0229. (Elsevier)
    This work reports the use of new support for immobilization of lipase Burkholderia cepacia (BCL) matrix made up of polylactic acid (PLA), chitosan (CH), and polyvinyl alc. (PVA). Initially lipase from various microbial sources and immobilization support compn. was screened to obtain a robust biocatalyst. Among various biocatalysts prepn., the PLA:PVA:CH:BCL (1:6:1:2) was worked as a robust biocatalyst for the citronellyl acetate synthesis. Various reaction parameters were studied in detail to obtain the suitable reaction conditions for model citronellyl acetate synthesis reaction. Various kinetic parameters such as rmax, K(citronellol), K(citronellol), K(vinyl acetate) were detd. using non-linear regression anal. for the ternary complex as well as bi-bi ping-pong mechanism. The exptl. results and kinetic study showed that citronellyl acetate synthesis catalyzed by immobilized lipase BCL followed the ternary complex mechanism with inhibition by alc. (citronellol). The energy of activation for citronellyl acetate synthesis was found to be lower for immobilized lipase (8.9 kcal/mol) than aggregated lipase (14.8 kcal/mol) enzyme. The developed biocatalyst showed four to fivefold higher catalytic activity and excellent recyclability (up to six cycles) than the aggregated lipase.
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    Stergiou, P.-Y.; Foukis, A.; Filippou, M.; Koukouritaki, M.; Parapouli, M.; Theodorou, L. G.; Hatziloukas, E.; Afendra, A.; Pandey, A.; Papamichael, E. M. Advances in Lipase-Catalyzed Esterification Reactions. Biotechnol. Adv. 2013, 31 (8), 1846– 59,  DOI: 10.1016/j.biotechadv.2013.08.006
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    Advances in lipase-catalyzed esterification reactions
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    Biotechnology Advances (2013), 31 (8), 1846-1859CODEN: BIADDD; ISSN:0734-9750. (Elsevier)
    A review. Lipase-catalyzed esterification reactions are among the most significant chem. and biochem. processes of industrial relevance. Lipases catalyze hydrolysis as well as esterification reactions. Enzyme-catalyzed esterification has acquired increasing attention in many applications, due to the significance of the derived products. More specifically, the lipase-catalyzed esterification reactions attracted research interest during the past decade, due to an increased use of org. esters in biotechnol. and the chem. industry. Lipases, as hydrolyzing agents are active in environments, which contain a min. of two distinct phases, where all reactants are partitioned between these phases, although their distribution is not fixed and changes as the reaction proceeds. The kinetics of the lipase-catalyzed reactions is governed by a no. of factors. This article presents a thorough and descriptive evaluation of the applied trends and perspectives concerning the enzymic esterification, mainly for biofuel prodn.; an emphasis is given on essential factors, which affect the lipase-catalyzed esterification reaction. Moreover, the art of using bacterial and/or fungal strains for whole cell biocatalysis purposes, as well as carrying out catalysis by various forms of purified lipases from bacterial and fungal sources is also reviewed.
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    Gao, W.; Wu, K.; Chen, L.; Fan, H.; Zhao, Z.; Gao, B.; Wang, H.; Wei, D. A Novel Esterase from a Marine Mud Metagenomic Library for Biocatalytic Synthesis of Short-Chain Flavor Esters. Microbial Cell Factories 2016, 15, 41,  DOI: 10.1186/s12934-016-0435-5
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    A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters
    Gao, Wenyuan; Wu, Kai; Chen, Lifeng; Fan, Haiyang; Zhao, Zhiqiang; Gao, Bei; Wang, Hualei; Wei, Dongzhi
    Microbial Cell Factories (2016), 15 (), 41/1-41/12CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)
    Marine mud is an abundant and largely unexplored source of enzymes with unique properties that may be useful for industrial and biotechnol. purposes. However, since most microbes cannot be cultured in the lab., a cultivation-independent metagenomic approach would be advantageous for the identification of novel enzymes. Therefore, with the objective of screening novel lipolytic enzymes, a metagenomic library was constructed using the total genomic DNA extd. from marine mud. Based on functional heterologous expression, 34 clones that showed lipolytic activity were isolated. The 5 clones with the largest halos were identified, and the corresponding genes were successfully overexpressed in Escherichia coli. Mol. anal. revealed that these encoded proteins showed 48-79% similarity with other proteins in the GenBank database. Multiple sequence alignment and phylogenetic tree anal. classified these 5 protein sequences as new members of known families of bacterial lipolytic enzymes. Among them, EST4, which had 316 amino acids with a predicted mol. wt. of 33.8 kDa, was further studied in detail due to its strong hydrolytic activity. Characterization of EST4 indicated that it is an alk. esterase that exhibits its highest hydrolytic activity toward p-nitrophenyl butyrate (specific activity: 1389 U/mg) at 45°C and pH 8.0. The half-life of EST4 was 55 and 46 h at 40 and 45°, resp., indicating a relatively high thermostability. EST4 also showed marked stability in org. solvents, retaining 90% of its initial activity when incubated for 12 h in the presence of hydrophobic alkanes. Furthermore, EST4 was used as an efficient whole-cell biocatalyst for the synthesis of short-chain flavor esters, showing high conversion rate and good tolerance for high substrate concns. (up to 3.0M). These results demonstrated a promising potential for industrial scaling-up to produce short-chain flavor esters at high substrate concns. in non-aq. media. Thus, the authors report unprecedented alc. tolerance and conversion of an esterase biocatalyst identified from a marine mud metagenomic library. The high org. solvent tolerance and thermostability of EST4 suggest that it has great potential as a biocatalyst.
  448. 448
    Lozano, P.; Bernal, J. M.; Navarro, A. A Clean Enzymatic Process for Producing Flavour Esters by Direct Esterification in Switchable Ionic Liquid/Solid Phases. Green Chem. 2012, 14 (11), 3026– 33,  DOI: 10.1039/c2gc36081k
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    450
    A clean enzymatic process for producing flavour esters by direct esterification in switchable ionic liquid/solid phases
    Lozano, Pedro; Bernal, Juana M.; Navarro, Alicia
    Green Chemistry (2012), 14 (11), 3026-3033CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
    A clean biocatalytic approach for producing flavor esters using switchable ionic liq./solid phases as reaction/sepn. media has been developed. The phase behavior of different IL/flavor acetyl ester (geranyl acetate, citronellyl acetate, neryl acetate and isoamyl acetate) mixts. was studied at several concns., resulting for all cases in fully homogeneous liq. media at 50 °C, and solid systems at room temp. By using an iterative centrifugation protocol on the solid IL/flavor ester mixts. at controlled temps., the solid IL phase and the liq. flavor ester phase can be easily sepd. The excellent suitability of an immobilized Candida antarctica lipase B (Novozym 435) catalyst in the esterification reaction between an aliph. carboxylic acid (acetic, propionic, butyric or valeric) and a flavor alc. (isoamyl alc., nerol, citronellol or geraniol) in N,N',N'',N'''-hexadecyltrimethyl-ammonium bis(trifluoromethylsulfonyl)imide ([C16tma][NTf2])IL has been demonstrated, the product yield being improved up to 100% under appropriate reaction conditions (enzyme amt., dehydrating mol. sieves, etc.) at 50 °C. The enzymic synthesis of sixteen different flavor esters was carried out in [C16tma][NTf2] by means of this approach, providing products of up to 0.757 g mL-1 concn. after IL sepn. The residual activity of the enzyme/IL system during seven consecutive operation cycles was shown to be practically unchanged after reuse.
  449. 449
    Otera, J.; Nishikido, J. Esterification: Methods, Reactions, and Applications; John Wiley & Sons, 2009. DOI: 10.1002/9783527627622
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    Beekwilder, J.; Alvarez-Huerta, M.; Neef, E.; Verstappen, F. W. A.; Bouwmeester, H. J.; Aharoni, A. Functional Characterization of Enzymes Forming Volatile Esters from Strawberry and Banana. Plant Physiol. 2004, 135 (4), 1865– 78,  DOI: 10.1104/pp.104.042580
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    Functional characterization of enzymes forming volatile esters from strawberry and banana
    Beekwilder, Jules; Alvarez-Huerta, Mayte; Neef, Evert; Verstappen, Francel W. A.; Bouwmeester, Harro J.; Aharoni, Asaph
    Plant Physiology (2004), 135 (4), 1865-1878CODEN: PLPHAY; ISSN:0032-0889. (American Society of Plant Biologists)
    Volatile esters are flavor components of the majority of fruits. The last step in their biosynthesis is catalyzed by alc. acyltransferases (AATs), which link alcs. to acyl moieties. Full-length cDNAs putatively encoding AATs were isolated from fruit of wild strawberry (Fragaria vesca) and banana (Musa sapientum) and compared to the previously isolated SAAT gene from the cultivated strawberry (Fragaria × ananassa). The potential role of these enzymes in fruit flavor formation was assessed. To this end, recombinant enzymes were produced in Escherichia coli, and their activities were analyzed for a variety of alc. and acyl-CoA substrates. When the results of these activity assays were compared to a phylogenetic anal. of the various members of the acyltransferase family, it was clear that substrate preference could not be predicted on the basis of sequence similarity. In addn., the substrate preference of recombinant enzymes was not necessarily reflected in the representation of esters in the corresponding fruit volatile profiles. This suggests that the specific profile of a given fruit species is to a significant extent detd. by the supply of precursors. To study the in planta activity of an alc. acyltransferase and to assess the potential for metabolic engineering of ester prodn., we generated transgenic petunia (Petunia hybrida) plants over-expressing the SAAT gene. While the expression of SAAT and the activity of the corresponding enzyme were readily detected in transgenic plants, the volatile profile was found to be unaltered. Feeding of isoamyl alc. to explants of transgenic lines resulted in the emission of the corresponding acetyl ester. This confirmed that the availability of alc. substrates is an important parameter to consider when engineering volatile ester formation in plants.
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    Buckles, R. E.; Thelen, C. Qualitative Determination of Carboxylic Esters. Anal. Chem. 1950, 22 (5), 676– 8,  DOI: 10.1021/ac60041a016
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    Detection of carboxylic esters. Scope and limitations of the hydroxamic acid test
    Buckles, Robert E.; Thelen, Charles J.
    (1950), 22 (), 676-8CODEN: ANCHAM; ISSN:0003-2700.
    The hydroxamic acid test (development of color with Fe+++) for carboxylic esters (I) has been studied with I, trihalomethyl compds., acid chlorides and anhydrides, acids, phenols, aldehydes, amides, imides, nitriles, isocyanates, nitro compds., and mixts. contg. I. Qual. procedures for distinguishing between I and the other classes of compds. are given.
  452. 452
    Stern, I.; Shapiro, B. A Rapid and Simple Method for the Determination of Esterified Fatty Acids and for Total Fatty Acids in Blood. J. Clin. Pathol. 1953, 6 (2), 158– 60,  DOI: 10.1136/jcp.6.2.158
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    A rapid and simple method for the determination of esterified fatty acids and for total fatty acids in blood
    STERN I; SHAPIRO B
    Journal of clinical pathology (1953), 6 (2), 158-60 ISSN:0021-9746.
    There is no expanded citation for this reference.
  453. 453
    Davidson, D. Hydroxamic Acids in Qualitative Organic Analysis. J. Chem. Educ. 1940, 17 (2), 81,  DOI: 10.1021/ed017p81
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    Hydroxamic acids in qualitative organic analysis
    Davidson, David
    Journal of Chemical Education (1940), 17 (), 81-4CODEN: JCEDA8; ISSN:0021-9584.
    Hydroxamic acids can be regarded as the n-hydroxy derivs. of the acid amides or as the oximes of the carboxylic acids. In either case, they can also be represented by amine oxide formulas. These tautomeric substances can be prepd. in so many ways and they are so easily detected that the possibility of using them in org. qual. analysis is clear. Thus an ester can be identified as follows: To 1 drop of the sample, add 0.5 ml. of N NH2OH.HCl in MeOH and 0.5 ml. of 2 N KOH in MeOH. KCl will ppt. from the MeOH soln. Heat to boiling, cool, make acid with 0.5 ml. of 2 N aq. HCl and test with a drop of 10% FeCl3 aq. soln. A red color will develop. An alc. can be detected by taking 0.5 ml. AcCl and an equal vol. of dimethylaniline and then 0.5 ml. or 0.5 g. of the sample. The mixt. will sep. into 2 layers. Add ice to decompose excess AcCl and treat 1 drop of the oily ester layer with hydroxylamine, etc., as in the case of esters. Ethers give neg. tests by the above procedures but they can be converted into esters by heating with org. acids in the presence of a little concd. H2SO4. The ester formed can be detected as above. Carboxylic acids can be treated with SOCl2 and when the excess has been removed by evapn. the resulting acyl chloride can be tested as in the case of esters. Phenols can be changed to esters by treatment with AcCl. Nitro compds. can be reduced to an alkylhydroxylamine which gives BzCl a n-alkylhydroxamic acid and this yields a colored complex with FeCl3. Aliphatic amides are converted rapidly to hydroxamic acids by boiling with NH2OH.HCl soln. Cyanates by treatment with NH2OH.HCl can be converted to hydroxamic acids which give a purplish blue complex with FeCl3. 36 references.
  454. 454
    Löbs, A.-K.; Lin, J.-L.; Cook, M.; Wheeldon, I. High Throughput, Colorimetric Screening of Microbial Ester Biosynthesis Reveals High Ethyl Acetate Production from Kluyveromyces Marxianus on C5, C6, and C12 Carbon Sources. Biotechnol. J. 2016, 11 (10), 1274– 81,  DOI: 10.1002/biot.201600060
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    456
    High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources
    Lobs Ann-Kathrin; Lin Jyun-Liang; Cook Megan; Wheeldon Ian
    Biotechnology journal (2016), 11 (10), 1274-1281 ISSN:.
    Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z-factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.
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    Smith, J. G. Synthetically Useful Reactions of Epoxides. Synthesis 1984, 1984, 629– 656,  DOI: 10.1055/s-1984-30921
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    Yudin, A. K. Aziridines and Epoxides in Organic Synthesis; John Wiley & Sons, 2006.
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    Kotik, M.; Archelas, A.; Wohlgemuth, R. Epoxide Hydrolases and Their Application in Organic Synthesis. Curr. Org. Chem. 2012, 16 (4), 451– 82,  DOI: 10.2174/138527212799499840
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    Epoxide hydrolases and their application in organic synthesis
    Kotik, Michael; Archelas, Alain; Wohlgemuth, Roland
    Current Organic Chemistry (2012), 16 (4), 451-482CODEN: CORCFE; ISSN:1385-2728. (Bentham Science Publishers Ltd.)
    A review was given on the enzymic asym. hydrolysis of oxiranes using epoxide hydrolases as catalysts. The no. of synthetic applications of epoxide hydrolases in org. chem. has reached a remarkable level. This has been due to a tremendous amt. of work dedicated to the discovery of novel epoxide hydrolases from various biol. sources, understanding the structure and function of these widespread enzymes and last but not least, the stabilization of these biocatalysts by various means for prodn. purposes.
  458. 458
    Zocher, F.; Enzelberger, M. M.; Bornscheuer, U. T.; Hauer, B. D.; Schmid, R. D. A Colorimetric Assay Suitable for Screening Epoxide Hydrolase Activity. Anal. Chim. Acta 1999, 391 (3), 345– 351,  DOI: 10.1016/S0003-2670(99)00216-0
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    460
    A colorimetric assay suitable for screening epoxide hydrolase activity
    Zocher, Frank; Enzelberger, Markus M.; Bornscheuer, Uwe T.; Hauer, Bernhard; Schmid, Rolf D.
    Analytica Chimica Acta (1999), 391 (3), 345-351CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
    A UV/VIS spectrophotometric microtiter plate and a filter-paper based assay using 4-(p-nitrobenzyl)pyridine (NBP) were developed to det. epoxide hydrolytic activity by measuring the decrease of the epoxide concn. Both systems were applied for screening an expression gene bank of Rhodococcus sp. NCIMB 11216. As a ref., whole cells from Rhodococcus sp. NCIMB 11216 and Beauveria sulfurescens ATCC 7159 exhibiting epoxide hydrolase activity were used. The microtiter plate system was also evaluated for different epoxides and performed in a lab. robotic system for high throughput screening. The microtiter plate assay showed a high sensitivity for the detection of small concns. of epoxides (0.1-1 mg/well) such as styrene oxide, Et phenylglycidate, n-hexane oxide and indene oxide. The filter paper assay was further optimized for styrene oxide. Both assays were suitable to screen within libraries of epoxide hydrolases without interference with other enzymes such as esterases, lipases or proteases. The assay should allow to screen large libraries obtained by directed evolution, strain collections and (expression) gene banks for epoxide hydrolytic activity or to monitor the purifn. process of an epoxide hydrolase.
  459. 459
    Alcalde, M.; Farinas, E. T.; Arnold, F. H. Colorimetric High-Throughput Assay for Alkene Epoxidation Catalyzed by Cytochrome P450 BM-3 Variant 139–3. J. Biomol. Screen. 2004, 9 (2), 141– 6,  DOI: 10.1177/1087057103261913
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    Colorimetric high-throughput assay for alkene epoxidation catalyzed by cytochrome P450 BM-3 variant 139-3
    Alcalde, Miguel; Farinas, Edgardo T.; Arnold, Frances H.
    Journal of Biomolecular Screening (2004), 9 (2), 141-146CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
    Cytochrome P 450 BM-3 variant 139-3 is highly active in the hydroxylation of alkanes and fatty acids (A Glieder, ET Farinas, and FH Arnold, Nature Biotech 2002;20:1135-1139); it also epoxidizes various alkenes, including styrene. Here the authors describe a colorimetric, high-throughput assay suitable for optimizing this latter activity by directed evolution. The product of styrene oxidn. by 139-3, styrene oxide, reacts with the nucleophile γ-(4-nitrobenzyl)pyridine (NBP) to form a purple-colored precursor dye, which can be monitored spectrophotometrically in cell lysates. The sensitivity limit of this assay is 50-100 μM of product, and the detection limit for P 450 BM-3 139-3 is ∼0.2 μM of enzyme. To validate the assay, activities in a small library of random mutants were compared to those detd. using an NADPH depletion assay for initial turnover rates.
  460. 460
    Van Damme, F.; Oomens, A. C. Determination of Residual Free Epoxide in Polyether Polyols by Derivatization with Diethylammonium N,N-Diethyldithiocarbamate and Liquid Chromatography. J. Chromatogr. A 1995, 696 (1), 41– 7,  DOI: 10.1016/0021-9673(94)01270-O
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    Determination of residual free epoxide in polyether polyols by derivatization with diethylammonium N,N-diethyldithiocarbamate and liquid chromatography
    Van Damme, F.; Oomens, A. C.
    Journal of Chromatography A (1995), 696 (1), 41-7CODEN: JCRAEY; ISSN:0021-9673. (Elsevier)
    A normal-phase liq. chromatog. method is described for the detn. of epoxides and particularly residual ethylene oxide and propylene oxide in polyether polyols. The residual epoxides are derivatized with diethylammonium N,N-diethyldithiocarbamate into the corresponding 1-(N,N-diethyldithiocarbamoyl)-2-hydroxyethane and -hydroxypropane. The dithiocarbamoyl esters are extd. into chloroform and sepd. by normal-phase liq. chromatog. Detection is done by UV at 278 nm. Total anal. time is ∼45 min. Linearity (0.1-100 ppm), detection limits (0.5 ppm), recovery from spiked samples (>95%) and interferences were studied.
  461. 461
    Hammock, L. G.; Hammock, B. D.; Casida, J. E. Detection and Analysis of Epoxides with 4-(p-Nitrobenzyl)-Pyridine. Bull. Environ. Contam. Toxicol. 1974, 12 (6), 759– 64,  DOI: 10.1007/BF01685927
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    463
    Detection and analysis of epoxides with 4-(p-nitrobenzyl)pyridine
    Hammock, Lassie G.; Hammock, Bruce D.; Casida, John E.
    Bulletin of Environmental Contamination and Toxicology (1974), 12 (6), 759-64CODEN: BECTA6; ISSN:0007-4861.
    Epoxides were detected on paper and thin-layer chromatograms and colorimetrically detd. at 600 nm by reaction with 4-(p-nitrobenzyl)pyridine (I) to form colored derivs. The developed thin-layer and paper chromatograms were sprayed with 2 wt. % I in Me2CO and by 1 wt. % I in 50% Me2CO contg. 0.5 N K acid phthalate, resp. The sprayed chromatograms were heated at 110° for 5 min and, after cooling, were sprayed with 10 vol. % tetraethylenepentamine in Me2CO to give blue spots on a white background. The sensitivities on thin layer and paper chromatograms were 0.01 - >13 and 0.01 - >6 μmole, resp. The method is insensitive for epoxycycloalkanes and related compds. The colorimetric method is useful in monitoring enzymic reactions with epoxide substrates. Incubation of 4-ethylphenyl 6,7-epoxygeranyl ether (II) with buffer, followed by extn. with pentane and colorimetric detn. of II by using I established that the detection limit is 0.2 μmole II and that Beer's law holds for ≤1.0 μmole II.
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    Pedragosa-Moreau, S.; Morisseau, C.; Baratti, J.; Zylber, J.; Archelas, A.; Furstoss, R. Microbiological Transformations 37. An Enantioconvergent Synthesis of the β-Blocker (R)-NiféNalol Using a Combined Chemoenzymatic Approach. Tetrahedron 1997, 53 (28), 9707– 14,  DOI: 10.1016/S0040-4020(97)00639-X
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    Finney, N. S. Enantioselective Epoxide Hydrolysis: Catalysis Involving Microbes, Mammals and Metals. J. Biol. Chem. 1998, 5 (4), R73– R9,  DOI: 10.1016/S1074-5521(98)90630-5
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    Choi, W. J.; Choi, C. Y. Production of Chiral Epoxides: Epoxide Hydrolase-Catalyzed Enantioselective Hydrolysis. Biotechnol. Bioprocess Eng. 2005, 10 (3), 167,  DOI: 10.1007/BF02932009
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    Production of Chiral epoxides: Epoxide hydrolase-catalyzed enantioselective hydrolysis
    Choi, Won Jae; Choi, Cha Yong
    Biotechnology and Bioprocess Engineering (2005), 10 (3), 167-179CODEN: BBEIAU; ISSN:1226-8372. (Korean Society for Biotechnology and Bioengineering)
    A review. Chiral epoxides are highly valuable intermediates, used for the synthesis of pharmaceutical drugs and agrochems. They have broad scope of market demand because of their applications. A major challenge in modern org. chem. is to generate such compds. in high yields, with high stereo- and regio-selectivities. Epoxide hydrolases (EH) are promising biocatalysts for the prepn. of chiral epoxides and vicinal diols. They exhibit high enantioselectivity for their substrates, and can be effectively used in the resoln. of racemic epoxides through enantioselective hydrolysis. The selective hydrolysis of a racemic epoxide can produce both the corresponding diols and the unreacted epoxides with high enantiomeric excess (ee) value. The potential of microbial EH to produce chiral epoxides and vicinal diol has prompted researchers to explore their use in the synthesis of epoxides and diols with high ee values.
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    Baer, H.; Bergamo, M.; Forlin, A.; Pottenger, L. H.; Lindner, J. Propylene Oxide. In Ullmann’s Encyclopedia of Industrial Chemistry: Wiley-VCH, 2000.
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    Rebsdat, S.; Mayer, D. Ethylene Oxide. In Ullmann’s Encyclopedia of Industrial Chemistry; Wiley-VCH, 2001.
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    Archelas, A.; Furstoss, R. Synthesis of Enantiopure Epoxides through Biocatalytic Approaches. Annu. Rev. Microbiol. 1997, 51 (1), 491– 525,  DOI: 10.1146/annurev.micro.51.1.491
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    Synthesis of enantiopure epoxides through biocatalytic approaches
    Archelas A; Furstoss R
    Annual review of microbiology (1997), 51 (), 491-525 ISSN:0066-4227.
    Enantiopure epoxides, as well as their corresponding vicinal diols, are valuable intermediates in fine organic synthesis, in particular for the preparation of biologically active compounds. The necessity of preparing such target molecules in an optically pure form has triggered much research, leading to the emergence of various new methods based on either conventional chemistry or enzymatically catalyzed reactions. In this review, we focus on the biocatalytic approaches, which include direct epoxidation of olefinic double bonds as well as indirect biocatalytic methods, and which allow for the synthesis of these important chiral building blocks in enantiomerically enriched or even enantiopure form.
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    Ma, S. K.; Gruber, J.; Davis, C.; Newman, L.; Gray, D.; Wang, A.; Grate, J.; Huisman, G. W.; Sheldon, R. A. A Green-by-Design Biocatalytic Process for Atorvastatin Intermediate. Green Chem. 2010, 12 (1), 81– 6,  DOI: 10.1039/B919115C
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    A green-by-design biocatalytic process for atorvastatin intermediate
    Ma, Steven K.; Gruber, John; Davis, Chris; Newman, Lisa; Gray, David; Wang, Alica; Grate, John; Huisman, Gjalt W.; Sheldon, Roger A.
    Green Chemistry (2010), 12 (1), 81-86CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
    The development of a green-by-design, two-step, three-enzyme process for the synthesis of a key intermediate in the manuf. of atorvastatin, the active ingredient of the cholesterol lowering drug Lipitor, is described. The first step involves the biocatalytic redn. of ethyl-4-chloroacetoacetate using a ketoreductase (KRED) in combination with glucose and a NADP-dependent glucose dehydrogenase (GDH) for cofactor regeneration. The (S) ethyl-4-chloro-3-hydroxybutyrate product is obtained in 96% isolated yield and >99.5% e.e. In the second step, a halohydrin dehalogenase (HHDH) is employed to catalyze the replacement of the chloro substituent with cyano by reaction with HCN at neutral pH and ambient temp. The natural enzymes were highly selective but exhibited productivities that were insufficient for large scale application. Consequently, in vitro enzyme evolution using gene shuffling technologies was employed to optimize their performance according to predefined criteria and process parameters. In the case of the HHDH reaction, this afforded a 2500-fold improvement in the volumetric productivity per biocatalyst loading. This enabled the economical and environmentally attractive prodn. of the key hydroxynitrile intermediate. The overall process has an E factor (kg waste per kg product) of 5.8 when process water is not included, and 18 if included.
  469. 469
    Kong, X.-D.; Ma, Q.; Zhou, J.; Zeng, B.-B.; Xu, J.-H. A Smart Library of Epoxide Hydrolase Variants and the Top Hits for Synthesis of (S)-β-Blocker Precursors. Angew. Chem., Int. Ed. Engl. 2014, 53 (26), 6641– 4,  DOI: 10.1002/anie.201402653
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    A smart library of epoxide hydrolase variants and the top hits for synthesis of (S)-β-blocker precursors
    Kong Xu-Dong; Ma Qian; Zhou Jiahai; Zeng Bu-Bing; Xu Jian-He
    Angewandte Chemie (International ed. in English) (2014), 53 (26), 6641-4 ISSN:.
    Microtuning of the enzyme active pocket has led to a smart library of epoxide hydrolase variants with an expanded substrate spectrum covering a series of typical β-blocker precursors. Improved activities of 6- to 430-fold were achieved by redesigning the active site at two predicted hot spots. This study represents a breakthrough in protein engineering of epoxide hydrolases and resulted in enhanced activity toward bulky substrates.
  470. 470
    Lin, Z.; Xue, Y.; Liang, X.-W.; Wang, J.; Lin, S.; Tao, J.; You, S.-L.; Liu, W. Oxidative Indole Dearomatization for Asymmetric Furoindoline Synthesis by a Flavin-Dependent Monooxygenase Involved in the Biosynthesis of Bicyclic Thiopeptide Thiostrepton. Angew. Chem. 2021, 60 (15), 8401– 5,  DOI: 10.1002/anie.202013174
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    472
    Oxidative Indole Dearomatization for Asymmetric Furoindoline Synthesis by a Flavin-Dependent Monooxygenase Involved in the Biosynthesis of Bicyclic Thiopeptide Thiostrepton
    Lin, Zhi; Xue, Yufeng; Liang, Xiao-Wei; Wang, Jian; Lin, Shuangjun; Tao, Jiang; You, Shu-Li; Liu, Wen
    Angewandte Chemie, International Edition (2021), 60 (15), 8401-8405CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The interest in indole dearomatization, which serves as a useful tool in the total synthesis of related alkaloid natural products, has recently been renewed with the intention of developing new methods efficient in both yield and stereoselective control. Here, we report an enzymic approach for the oxidative dearomatization of indoles in the asym. synthesis of a variety of furoindolines with a vicinal quaternary carbon stereogenic center. This approach depends on the activity of a flavin-dependent monooxygenase, TsrE, which is involved in the biosynthesis of bicyclic thiopeptide antibiotic thiostrepton. TsrE catalyzes 2,3-epoxidn. and subsequent epoxide opening in a highly enantioselective manner during the conversion of 2-methyl-indole-3-acetic acid or 2-methyl-tryptophol to furoindoline, with up to >99% conversion and >99% ee under mild reaction conditions. Complementing current chem. methods for oxidative indole dearomatization, the TsrE activity-based approach enriches the toolbox in the asym. synthesis of products possessing a furoindoline skeleton.
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    Agarwal, S. C.; Van Duuren, B. L.; Solomon, J. J.; Kline, S. A. Reaction of Epoxides with 4-Nitrothiophenol. Its Possible Application for Trapping and Characterization of Epoxides. Environ. Sci. Technol. 1980, 14 (10), 1249– 53,  DOI: 10.1021/es60170a012
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    Reaction of epoxides with 4-nitrothiophenol. Its possible application for trapping and characterization of epoxides
    Agarwal, Satish C.; Van Duuren, Benjamin L.; Solomon, Jerome J.; Kline, Stanley A.
    Environmental Science and Technology (1980), 14 (10), 1249-53CODEN: ESTHAG; ISSN:0013-936X.
    Reaction of epoxides with the nucleophile 4-nitrothiophenol [1849-36-1] has been examd. The mono- and diepoxides of aliph. and arom. hydrocarbons undergo facile nucleophilic opening by 4-nitrothiophenol to yield the expected 1,2 adducts cleanly and in good yields. The exptl. conditions provide a suitable method for trapping the atm. epoxides which may be significant in air pollution carcinogenesis. In the chem. ionization mass spectrum, these adducts have an intense protonated mol. ion which will be extremely useful in their detection and characterization by the technique of single ion monitoring.
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    Wixtrom, R. N.; Hammock, B. D. Continuous Spectrophotometric Assays for Cytosolic Epoxide Hydrolase. Anal. Biochem. 1988, 174 (1), 291– 9,  DOI: 10.1016/0003-2697(88)90548-9
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    Continuous spectrophotometric assays for cytosolic epoxide hydrolase
    Wixtrom, Roger N.; Hammock, Bruce D.
    Analytical Biochemistry (1988), 174 (1), 291-9CODEN: ANBCA2; ISSN:0003-2697.
    Two convenient and sensitive continuous spectrophotometric assays for cytosolic epoxide hydrolase (I) are described. The assays are based on the differences in the UV spectra of the epoxide substrates and their diol products. The hydrolysis of 1,2-epoxy-1-(p-nitrophenyl)pentane (ENP5) was accompanied by a decrease in absorbance at 302 nm, whereas the hydration of 1,2-epoxy-1-(2-quinolyl)pentane (EQU5) produced an increase in absorbance at 315.5 nm. The Km and Vmax values for ENP5 and EQU5 with purified mouse liver cytosol I were 1.7 and 25 μM and 11,700 and 8300 nmol/min/mg, resp.; these results compared with previously reported values of 5 μM and 3300 nmol/min/mg for trans-stilbene oxide (TSO) with purified mouse liver I. Thus, both substrates were hydrolyzed significantly faster than TSO, which is currently the most commonly used substrate for measuring cytosolic I activity. No spontaneous hydrolysis of the substrates was detectable under normal assay conditions. The assays were applicable to whole tissue homogenates as well as purified enzyme prepns. p-Nitrostyrene oxide and p-nitrophenyl glycidyl ether were also examd. and found to be very poor substrates for cytosolic I from mouse liver.
  473. 473
    González-Pérez, M.; Gómez-Bombarelli, R.; Arenas-Valgañón, J.; Pérez-Prior, M. T.; García-Santos, M. P.; Calle, E.; Casado, J. Connecting the Chemical and Biological Reactivity of Epoxides. Chem. Res. Toxicol. 2012, 25 (12), 2755– 62,  DOI: 10.1021/tx300389z
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    475
    Connecting the Chemical and Biological Reactivity of Epoxides
    Gonzalez-Perez, Marina; Gomez-Bombarelli, Rafael; Arenas-Valganon, Jorge; Perez-Prior, M. Teresa; Garcia-Santos, M. Pilar; Calle, Emilio; Casado, Julio
    Chemical Research in Toxicology (2012), 25 (12), 2755-2762CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    The chem. reactivity of the mutagenic epoxides (EP) propylene oxide (PO), 1,2-epoxybutane (1,2-EB), and cis- and trans-2,3-epoxybutane (cis- and trans-2,3-EB) with 4-(p-nitrobenzyl)pyridine (NBP), a bionucleophile model for SN2 alkylating agents with high affinity for the guanine-N7 position, was investigated kinetically. It was found that three reactions are involved simultaneously: the alkylation reaction of NBP by EP, which yields the corresponding NBP-EP adducts through an SN2 mechanism, and EP and NBP-EP hydrolysis reactions. PO and 1,2-EB were seen to exhibit a higher alkylating potential than cis- and trans-2,3-EB. From a study of the correlations between the chem. reactivity (kinetic parameters) and the biol. effectiveness of oxiranes, the following conclusions can be drawn: (i) the hydrolysis reactions of epoxides must be taken into account to understand their bioactivity; (ii) the fraction (f) of the alkylating oxirane that forms the adduct and the adduct life (AL) permit the potential of epoxides as bioactive mols. to be rationalized even semiquant.; and (iii) alkylation of DNA by epoxides and the O6-/N7-guanine adduct ratio are directly related to their mutagenicity in vitro.
  474. 474
    Nelis, H. J. C. F.; Sinsheimer, J. E. A Sensitive Fluorimetric Procedure for the Determination of Aliphatic Epoxides under Physiological Conditions. Anal. Biochem. 1981, 115 (1), 151– 7,  DOI: 10.1016/0003-2697(81)90538-8
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    476
    A sensitive fluorimetric procedure for the determination of aliphatic epoxides under physiological conditions
    Nelis, Hans J. C. F.; Sinsheimer, J. E.
    Analytical Biochemistry (1981), 115 (1), 151-7CODEN: ANBCA2; ISSN:0003-2697.
    A highly sensitive fluorometric procedure based on alkylation of nicotinamide [98-92-0] is described for the detn. of aliph. epoxides. Subsequent reaction of the resulting N-alkyl nicotinamides with a ketone in basic medium yields strongly fluorescent products after final acidification. Sensitivity of the assay is in the picomole range with good reproducibility. The alkylation reaction proceeds under physiol. conditions and thus shows potential for the detn. of epoxides in biol. materials. Despite rapid enzymic detoxification, styrene oxide [75-56-9] could be detected directly in 9000 g supernatant liver fractions by the present approach.
  475. 475
    Rink, R.; Fennema, M.; Smids, M.; Dehmel, U.; Janssen, D. B. Primary Structure and Catalytic Mechanism of the Epoxide Hydrolase from Agrobacterium Radiobacter Ad1. J. Biol. Chem. 1997, 272 (23), 14650– 7,  DOI: 10.1074/jbc.272.23.14650
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    477
    Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1
    Rink, Rick; Fennema, Marko; Smids, Minke; Dehmel, Uwe; Janssen, Dick B.
    Journal of Biological Chemistry (1997), 272 (23), 14650-14657CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme. The epoxide hydrolase gene coded for a protein of 294 amino acids with a mol. mass of 34 kDa. An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A. radiobacter CFZ11. The recombinant epoxide hydrolase was expressed up to 40% of the total cellular protein content in Escherichia coli BL21(DE3) and the purified enzyme had a kcat of 21 s-1 with epichlorohydrin. Amino acid sequence similarity of the epoxide hydrolase with eukaryotic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the α/β-hydrolase fold family. This conclusion was supported by secondary structure predictions and anal. of the secondary structure with CD spectroscopy. The catalytic triad residues of epoxide hydrolase are proposed to be Asp107, His275, and Asp246. Replacement of these residues to Ala/Glu, Arg/Gln, and Ala, resp., resulted in a dramatic loss of activity for epichlorohydrin. The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate, as was shown by single turnover expts. with the His275 → Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.
  476. 476
    Elder, D. P.; Snodin, D.; Teasdale, A. Analytical Approaches for the Detection of Epoxides and Hydroperoxides in Active Pharmaceutical Ingredients, Drug Products and Herbals. J. Pharm. Biomed. Anal. 2010, 51 (5), 1015– 23,  DOI: 10.1016/j.jpba.2009.11.023
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    478
    Analytical approaches for the detection of epoxides and hydroperoxides in active pharmaceutical ingredients, drug products, and herbals
    Elder, D. P.; Snodin, D.; Teasdale, A.
    Journal of Pharmaceutical and Biomedical Analysis (2010), 51 (5), 1015-1023CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
    This review summarizes the anal. approaches reported in the literature relating to epoxide and hydroperoxide impurities. It is intended that it should provide guidance for analysts faced by the need to control such impurities, particularly where this is due to concerns relating to their potential genotoxicity. An extensive search of the literature relating to this class of impurities revealed a large no. of refs. relating to anal. of epoxides/hydroperoxides assocd. with herbal remedies. Given the general applicability of the anal. methodol. and due to the widespread use of herbal products the authors decided to include herbal medicines in this review. The review also reflects on the very different approaches taken in terms of the assessment/control of genotoxic impurities for such herbal remedies to that required for pharmaceutical products.
  477. 477
    Sano, A.; Takitani, S. Spectrofluorometric Determination of Common Epoxides with Sodium Sulfide and o-Phthalaldehyde and Taurine Reagents. Anal. Chem. 1985, 57 (8), 1687– 90,  DOI: 10.1021/ac00285a039
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    479
    Spectrofluorometric determination of common epoxides with sodium sulfide and o-phthalaldehyde and taurine reagents
    Sano, Akira; Takitani, Shoji
    Analytical Chemistry (1985), 57 (8), 1687-90CODEN: ANCHAM; ISSN:0003-2700.
    A spectrofluorometric method was developed for the detn. of common epoxides. Epoxides in EtOH soln. gave an intense blue fluorescence (λ(excitation) ∼345 nm and λ(emission) ∼440 nm), after the 1st reaction with aq. Na2S at 55° for 20 min and followed the 2nd reaction with taurine and o-phthalaldehyde reagents at pH 8.3. By the proposed method, 1,2-epoxy-3-phenoxypropane and 1,2-epoxyoctane can be detd. in the ranges 0.05-3 nmol/100 μL and 0.1-8 nmol/100 μL, resp., with relative std. deviations of 1.6-2.9%. Some other alkylating agents also showed fluorescence by this method.
  478. 478
    Tang, L.; Li, Y.; Wang, X. A High-Throughput Colorimetric Assay for Screening Halohydrin Dehalogenase Saturation Mutagenesis Libraries. J. Biotechnol. 2010, 147 (3), 164– 8,  DOI: 10.1016/j.jbiotec.2010.04.002
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    480
    A high-throughput colorimetric assay for screening halohydrin dehalogenase saturation mutagenesis libraries
    Tang, Lixia; Li, Yang; Wang, Xiong
    Journal of Biotechnology (2010), 147 (3-4), 164-168CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
    Here, a high throughput pH indicator-based assay to measure the activity of halohydrin dehalogenases (HheC) is reported. The assay relies upon the absorbance change at 560 nm and the visual color change of phenol red in a weakly buffered system, due to the release of protons from the enzyme-catalyzed ring-closure reactions. The assay can be performed in a microplate format using whole cells, making the assay simple and robust. Thus, it is suitable for library screening. The assay has been further validated using two previously studied HheC variants, D80N and W249F, which exhibit 200-fold lower and 2-fold higher kcat values, resp., toward 1,3-dichloro-2-propanol (1,3-DCP) than the wild-type HheC. In addn., a satn. mutagenesis library of HheC was screened using the developed assay for its ability to efficiently catalyze the conversion of 1,3-DCP. After screening of 500 colonies, one mutant W139C was identified and was further purified and characterized. Kinetic anal. indicates that the resulting mutant shows 2- and 5-fold improvement in kcat value toward 1,3-DCP and (R,S)-p-nitro-2-bromo-1-phenylethanol, resp., although it exhibits higher Km values than the wild-type enzyme. The method described herein represents a useful tool given the need for the high throughput screening of halohydrin dehalogenase mutants.
  479. 479
    Gul, I.; Bogale, T. F.; Deng, J.; Chen, Y.; Fang, R.; Feng, J.; Tang, L. Enzyme-Based Detection of Epoxides Using Colorimetric Assay Integrated with Smartphone Imaging. Biotechnol. Appl. Biochem. 2020, 67 (4), 685– 92,  DOI: 10.1002/bab.1898
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    481
    Enzyme-based detection of epoxides using colorimetric assay integrated with smartphone imaging
    Gul, Ijaz; Bogale, Tadesse Fantaye; Deng, Jiao; Chen, Yong; Fang, Ruiqin; Feng, Juan; Tang, Lixia
    Biotechnology and Applied Biochemistry (2020), 67 (4), 685-692CODEN: BABIEC; ISSN:0885-4513. (Wiley-Blackwell)
    Epoxides are widely used chems., the detn. of which is of paramount importance. Herein, we present an enzyme-based approach for noninstrumental detection of epoxides in std. soln. and environmental samples. Halohydrin dehalogenase (HheC) as a biol. recognition element and epichlorohydrin as a model analyte were evaluated for sensing. The detection is based on the color change of the pH indicator dye bromothymol blue caused by the HheC-catalyzed ring-opening of the epoxide substrate. The color change is then exploited for the detn. of epoxide using a smartphone as an image acquisition and data processing device, eliminating the need for computer-based image anal. software. The color parameters were systematically evaluated to det. the optimum quant. anal. parameter. Under optimal conditions, the proposed enzyme-based detection system showed a linear range of 0.13-2 mM with a detection limit of 0.07 mM and an assay time of 8 Min. In addn., the repeatability expressed as relative std. deviation was found to be below 5% (n = 6). Validation with gas chromatog. analyses showed that the proposed enzyme-based epoxide detection could be an alternative way in the quant. detn. of epoxides, and particularly useful in resource-limited settings.
  480. 480
    Dansette, P. M.; DuBois, G. C.; Jerina, D. M. Continuous Fluorometric Assay of Epoxide Hydrase Activity. Anal. Biochem. 1979, 97 (2), 340– 5,  DOI: 10.1016/0003-2697(79)90083-6
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    482
    Continuous fluorometric assay of epoxide hydrase activity
    Dansette, Patrick M.; DuBois, Garrett C.; Jerina, Donald M.
    Analytical Biochemistry (1979), 97 (2), 340-5CODEN: ANBCA2; ISSN:0003-2697.
    A rapid, continuous, and highly sensitive fluorescence assay is described for the measurement of epoxide hydrase activity. The method is based on the large differences between the fluorescence spectra of certain K-region arene oxides and their corresponding trans-dihydrodiols. Enzymic hydration of K-region arene oxides of phenanthrene, pyrene, benzo[a]pyrene, and 7,12-dimethylbenzo[a]anthracene was studied. The assay was most sensitive with benzo[a]pyrene-4,5-oxide as substrate. With 10 μM benzo[a]pyrene-4,5-oxide, enzymic rates of 30 pmol dihydrodiol/min/mg protein were 3- to 5-fold those of the blank without enzyme. The fluorometric method described was used to study site-directed inhibitors of epoxide hydrase and the stereoselective hydration of racemic arene oxides.
  481. 481
    Mateo, C.; Archelas, A.; Furstoss, R. A Spectrophotometric Assay for Measuring and Detecting an Epoxide Hydrolase Activity. Anal. Biochem. 2003, 314 (1), 135– 41,  DOI: 10.1016/S0003-2697(02)00646-2
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    483
    A spectrophotometric assay for measuring and detecting an epoxide hydrolase activity
    Mateo, Cesar; Archelas, Alain; Furstoss, Roland
    Analytical Biochemistry (2003), 314 (1), 135-141CODEN: ANBCA2; ISSN:0003-2697. (Elsevier Science)
    The development of a novel and simple spectrophotometric assay which allows one to achieve the continuous, rapid, sensitive, and accurate detn. of epoxide hydrolase (I) activity is reported. This assay is based on the elaboration of a coupled enzymic/chem. methodol. which allows quantification of I activity within 3 min, and offers good sensitivity of ∼10 μM min-1. Thus, an arom. epoxide such as styrene oxide is hydrolyzed by I to 1-phenyl-1,2-ethanediol, which in turn is oxidized to benzaldehyde by Na metaperiodate; the benzaldehyde formed can be visualized at very low levels at 290 nm thanks to its very strong UV absorbency (ε = 1356 M-1 cm-1). I activity can therefore be detd. continuously, with great sensitivity and accuracy, simply by measuring, at very low conversion ratio, the increase in UV absorbency. The applicability of this test for the detn. of Aspergillus niger I activity with some other arom. epoxides was shown and some limitations were also explored. This assay should be particularly useful for different applications, e.g., (1) activity localization during purifn. of such enzymes, (2) very rapid detn. of kinetic consts., and (3) high-throughput screening expts.
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    Michałowicz, J.; Duda, W. Phenols - Sources and Toxicity. Poish J. Environ. Stud. 2007, 16 (3), 347– 362
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    Phenols- sources and toxicity
    Michalowicz, J.; Duda, W.
    Polish Journal of Environmental Studies (2007), 16 (3), 347-362CODEN: PJESE2; ISSN:1230-1485. (HARD Publishing Co.)
    A review. Phenols and their derivs. commonly exist in the environment. These compds. are used as the components of dyes, polymers, drugs and other org. substances. The presence of phenols in the ecosystems is also related with prodn. and degrdn. of numerous pesticides and the generation of industrial and municipal sewages. Some phenols are also formed during natural processes. These compds. may be substituted with chlorine atoms, may be nitrated, methylated or alkylated. Both phenols and catechols are harmful ecotoxins. Toxic action of these compds. stems from unspecified toxicity related to hydrophobocity and also to the generation of org. radicals and reactive oxygen species. Phenols and catechols reveal peroxidative capacity, they are hematotoxic and hepatotoxic, provoke mutagenesis and carcinogenesis toward humans and other living organisms.
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    Schmidt, R. J. Industrial Catalytic Processes─Phenol Production. Appl. Catal. A: Gen. 2005, 280 (1), 89– 103,  DOI: 10.1016/j.apcata.2004.08.030
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    Industrial catalytic processes - phenol production
    Schmidt, Robert J.
    Applied Catalysis, A: General (2005), 280 (1), 89-103CODEN: ACAGE4; ISSN:0926-860X. (Elsevier B.V.)
    A review on the development of catalytic processes for phenol prodn.
  484. 484
    Ullrich, R.; Hofrichter, M. Enzymatic Hydroxylation of Aromatic Compounds. Cell. Mol. Life Sci. 2007, 64 (3), 271– 93,  DOI: 10.1007/s00018-007-6362-1
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    Enzymatic hydroxylation of aromatic compounds
    Ullrich, Rene; Hofrichter, Martin
    Cellular and Molecular Life Sciences (2007), 64 (3), 271-293CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
    A review. Selective hydroxylation of arom. compds. is among the most challenging chem. reactions in synthetic chem. and has gained steadily increasing attention during recent years, particularly because of the use of hydroxylated aroms. as precursors for pharmaceuticals. Biocatalytic oxygen transfer by isolated enzymes or whole microbial cells is an elegant and efficient way to achieve selective hydroxylation. This review gives an overview of the different enzymes and mechanisms used to introduce oxygen atoms into arom. mols. using either dioxygen (O2) or hydrogen peroxide (H2O2) as oxygen donors or indirect pathways via free radical intermediates. In this context, the article deals with Rieske-type and α-keto acid-dependent dioxygenases, as well as different non-heme monooxygenases (di-iron, pterin, and flavin enzymes), tyrosinase, laccase, and hydroxyl radical generating systems. The main emphasis is on the heme-contg. enzymes, cytochrome P 450 monooxygenases and peroxidases, including novel extracellular heme-thiolate haloperoxidases (peroxygenases), which are functional hybrids of both types of heme-biocatalysts.
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    Lan Tee, K.; Schwaneberg, U. Directed Evolution of Oxygenases: Screening Systems, Success Stories and Challenges. Comb. Chem. High Throughput Screen. 2007, 10 (3), 197– 217,  DOI: 10.2174/138620707780126723
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    Joern, J. M.; Sakamoto, T.; Arisawa, A.; Arnold, F. H. A Versatile High Throughput Screen for Dioxygenase Activity Using Solid-Phase Digital Imaging. J. Biomol. Screen. 2001, 6 (4), 219– 23,  DOI: 10.1177/108705710100600403
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    A versatile high throughput screen for dioxygenase activity using solid-phase digital imaging
    Joern, John M.; Sakamoto, Takeshi; Arisawa, Akira; Arnold, Frances H.
    Journal of Biomolecular Screening (2001), 6 (4), 219-223CODEN: JBISF3; ISSN:1087-0571. (Mary Ann Liebert, Inc.)
    We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-dihydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aroms. to yield colored products that are quantifiable using a microplate reader or by digital imaging and image anal. The method is reproducible and quant. at product concns. of only 30 μM, with essentially no background from media components. This method is an effective general screen for arom. oxidn. and should be a useful tool for the discovery and directed evolution of oxygenases.
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    Poraj-Kobielska, M.; Kinne, M.; Ullrich, R.; Scheibner, K.; Kayser, G.; Hammel, K. E.; Hofrichter, M. Preparation of Human Drug Metabolites Using Fungal Peroxygenases. Biochem. Pharmacol. 2011, 82 (7), 789– 96,  DOI: 10.1016/j.bcp.2011.06.020
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    Preparation of human drug metabolites using fungal peroxygenases
    Poraj-Kobielska, Marzena; Kinne, Matthias; Ullrich, Rene; Scheibner, Katrin; Kayser, Gernot; Hammel, Kenneth E.; Hofrichter, Martin
    Biochemical Pharmacology (2011), 82 (7), 789-796CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
    The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic org. chemists. Here we report that arom. peroxygenases (APOs; E.C. 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and Coprinellus radians catalyzed the H2O2-dependent selective monooxygenation of diverse drugs, including acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions included the hydroxylation of arom. rings and aliph. side chains, as well as O- and N-dealkylations and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs were obtained in yields greater than 80% and with isomeric purities up to 99%. Oxidns. of tolbutamide, acetanilide and carbamazepine in the presence of H2 18O2 resulted in almost complete incorporation of 18O into the corresponding products, thus establishing that these reactions are peroxygenations. The deethylation of phenacetin-d 1 showed an obsd. intramol. deuterium isotope effect [(k H/k D)obs] of 3.1±0.2, which is consistent with the existence of a cytochrome P 450-like intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful biocatalytic tools to prep. pharmacol. relevant drug metabolites.
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    Sakamoto, T.; Joern, J. M.; Arisawa, A.; Arnold, F. H. Laboratory Evolution of Toluene Dioxygenase to Accept 4-Picoline as a Substrate. Appl. Environ. Microbiol. 2001, 67 (9), 3882,  DOI: 10.1128/AEM.67.9.3882-3887.2001
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    Laboratory evolution of toluene dioxygenase to accept 4-picoline as a substrate
    Sakamoto, Takeshi; Joern, John M.; Arisawa, Akira; Arnold, Frances H.
    Applied and Environmental Microbiology (2001), 67 (9), 3882-3887CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
    Directed evolution is being used by the authors to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here, the authors report on the oxidn. of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by lab. evolution. The biotransformation of 4-picoline proceeds at only ∼4.5% of the rate of the natural reaction on toluene. Random mutagenesis, satn. mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding Thr. Escherichia coli-expressed TDO 3-B38 exhibited 5.6-fold higher activity toward 4-picoline and ∼20% more activity toward toluene than did wild-type TDO. The product of the biotransformation of 4-picoline was 3-hydroxy-4-picoline; no cis-diols of 4-picoline were obsd.
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    Foti, M. C. Antioxidant Properties of Phenols. J. Pharm. Pharmacol. 2010, 59 (12), 1673– 85,  DOI: 10.1211/jpp.59.12.0010
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    del Rosario Garcia-Mateos, M.; Palma-Tenango, M.; Soto-Hernandez, M. Phenolic Compounds-Biological Activity; IntechOpen, 2017.
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    Ainsworth, E. A.; Gillespie, K. M. Estimation of Total Phenolic Content and Other Oxidation Substrates in Plant Tissues Using Folin-Ciocalteu Reagent. Nat. Protoc. 2007, 2 (4), 875– 7,  DOI: 10.1038/nprot.2007.102
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    Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent
    Ainsworth, Elizabeth A.; Gillespie, Kelly M.
    Nature Protocols (2007), 2 (4), 875-877CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
    Non-structural phenolic compds. perform a variety of functions in plants, including acting as antioxidants. The authors describe a microplate-adapted colorimetric total phenolics assay that utilizes Folin-Ciocalteu (F-C) reagent. The F-C assay relies on the transfer of electrons in alk. medium from phenolic compds. to phosphomolybdic/phosphotungstic acid complexes, which are detd. spectroscopically at 765 nm. Although the electron transfer reaction is not specific for phenolic compds., the extn. procedure eliminates approx. 85% of ascorbic acid and other potentially interfering compds. This assay is performed in microcentrifuge tubes and assessed in a 96-well plate reader. At least 64 samples can be processed in 1 d.
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    Singleton, V. L.; Orthofer, R.; Lamuela-Raventós, R. M. Analysis of Total Phenols and Other Oxidation Substrates and Antioxidants by Means of Folin–Ciocalteu Reagent. Methods in Enzymology; Academic Press, 1999; Vol. 299; p 152– 178. DOI: 10.1016/S0076-6879(99)99017-1
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    Platzer, M.; Kiese, S.; Herfellner, T.; Schweiggert-Weisz, U.; Eisner, P. How Does the Phenol Structure Influence the Results of the Folin-Ciocalteu Assay?. Antioxidants 2021, 10 (5), 811,  DOI: 10.3390/antiox10050811
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    496
    How does the phenol structure influence the results of the folin-ciocalteu assay?
    Platzer, Melanie; Kiese, Sandra; Herfellner, Thomas; Schweiggert-Weisz, Ute; Eisner, Peter
    Antioxidants (2021), 10 (5), 811CODEN: ANTIGE; ISSN:2076-3921. (MDPI AG)
    Plants produce a diverse array of secondary metabolites that are generally nonessential but facilitate ecol. interactions. Fruits, vegetables, seeds and nuts can accumulate bioactive secondary metabolites with health-promoting properties, including the potent antioxidant activities of phenolic compds. Several in vitro assays have been developed to measure the polyphenol content and antioxidant activity of plant exts., e.g., the simple and highly popular Folin-Ciocalteu (FC) assay. However, the literature contains a no. of different descriptions of the assay and it is unclear whether the assay measures the polyphenol content or reducing capacity of the sample. To det. the influence of phenolic structures on the outcome of the FC assay, we tested phenols representing different subgroups (phenolic acids, flavonols, flavanols, dihydrochalcones and flavanones). We obsd. different results for each ref. substance and subgroup. Accordingly, we concluded that the FC assay does not measure the polyphenol content of a sample but dets. its reducing capacity instead. Assigning the substances to five structural classes showed that the FC results depend on the no. of fulfilled Bors criteria. If a mol. fulfills none of the Bors criteria, the FC results depend on the no. of OH groups. We did not find a correlation with other single electron transfer assays (e.g., ABTS and DPPH assays). Furthermore, the FC assay was compatible with all five subgroups and should be preferred over the DPPH assay, which is specific for exts. rich in dihydrochalcones or flavanones.
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    Miliauskas, G.; Venskutonis, P. R.; van Beek, T. A. Screening of Radical Scavenging Activity of Some Medicinal and Aromatic Plant Extracts. Food Chem. 2004, 85 (2), 231– 7,  DOI: 10.1016/j.foodchem.2003.05.007
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    Cao, G.; Alessio, H. M.; Cutler, R. G. Oxygen-Radical Absorbance Capacity Assay for Antioxidants. Free Radical Biol. Med. 1993, 14 (3), 303– 11,  DOI: 10.1016/0891-5849(93)90027-R
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    Oxygen-radical absorbance capacity assay for antioxidants
    Cao, Guohua; Alessio, Helaine M.; Cutler, Richard G.
    Free Radical Biology & Medicine (1993), 14 (3), 303-11CODEN: FRBMEH; ISSN:0891-5849.
    A relatively simple but sensitive and reliable method of quantitating the oxygen-radical absorbing capacity (ORAC) of antioxidants in serum using a few μL is described. In this assay system, β-phycoerythrin (β-PE) is used as an indicator protein, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-sol. vitamin E analog) as a control std. Results are expressed as ORAC units, where 1 ORAC unit equals the net protection produced by 1 μM Trolox. The uniqueness of this assay is that total antioxidant capacity of a sample is estd. by taking the oxidn. reaction to completion. At this point all of the nonprotein antioxidants (which include α-tocopherol, vitamin C, β-carotene, uric acid, and bilirubin) and most of the albumin in the sample are oxidized by the peroxyl radical. Results are quantified by measuring the protection produced by antioxidants. This solves many problems assocd. with kinetics or lag-time measurements. A linear correlation of ORAC value with concn. of serum, Trolox, vitamin C, uric acid, and bovine albumin is demonstrated. The coeff. of variation within a run is found to be about 2% and from run to run about 5%. Trolox, α-tocopherol, vitamin C, β-carotene, uric acid, and bilirubin completely protect β-PE from oxidn., while bovine albumin protects β-PE only partially. On a molar basis, the relative peroxyl radical absorbance capacity of Trolox, α-tocopherol acid succinate, uric acid, bilirubin, and vitamin C is 1:1:0.92:0.84:0.52. Bovine albumin per unit wt. has a lower peroxyl absorbing capacity than these antioxidants. However, the serum protein fraction, contg. some lipid-sol. antioxidants, represents the major contributor to the ORAC value found in whole serum. The min. amt. of vitamin C and uric acid which could still be detectable when added to a serum supernatant fraction is 1.5 μg and 0.59 μg, resp., which account for about 1% of the total ORAC value of the serum supernatant fraction.
  496. 496
    Prior, R. L.; Wu, X.; Schaich, K. Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements. J. Agric. Food Chem. 2005, 53 (10), 4290– 302,  DOI: 10.1021/jf0502698
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    499
    Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements
    Prior, Ronald L.; Wu, Xianli; Schaich, Karen
    Journal of Agricultural and Food Chemistry (2005), 53 (10), 4290-4302CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
    A review. Methods available for the measurement of antioxidant capacity are discussed, presenting the general chem. underlying the assays, the types of mols. detected, and the most important advantages and shortcomings of each method. This overview provides a basis and rationale for developing standardized antioxidant capacity methods for the food, nutraceutical, and dietary supplement industries. From evaluation of data presented at the First International Congress on Antioxidant Methods in 2004 and in the literature, as well as consideration of potential end uses of antioxidants, it is proposed that procedures and applications for three assays be considered for standardization: the oxygen radical absorbance capacity (ORAC) assay, the Folin-Ciocalteu method, and possibly the Trolox equiv. antioxidant capacity (TEAC) assay. ORAC represent a hydrogen atom transfer (HAT) reaction mechanism, which is most relevant to human biol. The Folin-Ciocalteu method is an electron transfer (ET) based assay and gives reducing capacity, which has normally been expressed as phenolic contents. The TEAC assay represents a second ET-based method.
  497. 497
    van den Berg, R.; Haenen, G. R. M. M.; van den Berg, H.; Bast, A. Applicability of an Improved Trolox Equivalent Antioxidant Capacity (TEAC) Assay for Evaluation of Antioxidant Capacity Measurements of Mixtures. Food Chem. 1999, 66 (4), 511– 7,  DOI: 10.1016/S0308-8146(99)00089-8
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    500
    Applicability of an improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant capacity measurements of mixtures
    Van den Berg, Robin; Haenen, Guido R. M. M.; Van den Berg, Henk; Bast, Aalt
    Food Chemistry (1999), 66 (4), 511-517CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Science Ltd.)
    The TEAC (Trolox equiv. antioxidant capacity) assay is based on scavenging of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical anions (ABTS-). In this report we describe a modification based on pre-generation of the ABTS radical anions with a thermolabile azo compd., 2,2'-azobis(2-amidinopropane)-2HCl (ABAP). This modification makes the assay less susceptible to artifacts, e.g. influence on the radical generation process. For most antioxidants tested, a biphasic reaction pattern was seen, i.e. a fast and slow scavenging rate. We evaluated application of the assay with both lipophilic and hydrophilic compds. with antioxidant capacity. Several org. solvents, compatible with water, were tested with α-tocopherol, quercetin and β-carotene. It was found that the TEACs differed in various solvents. Under standardized conditions additivity of TEACs obtained from individual antioxidants could be demonstrated. This might enable application of the assay for the identification of "unknown" antioxidants.
  498. 498
    Dudonné, S.; Vitrac, X.; Coutière, P.; Woillez, M.; Mérillon, J.-M. Comparative Study of Antioxidant Properties and Total Phenolic Content of 30 Plant Extracts of Industrial Interest Using DPPH, ABTS, FRAP, SOD, and ORAC Assays. J. Agric. Food Chem. 2009, 57 (5), 1768– 74,  DOI: 10.1021/jf803011r
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    501
    Comparative Study of Antioxidant Properties and Total Phenolic Content of 30 Plant Extracts of Industrial Interest Using DPPH, ABTS, FRAP, SOD, and ORAC Assays
    Dudonne, Stephanie; Vitrac, Xavier; Coutiere, Philippe; Woillez, Marion; Merillon, Jean-Michel
    Journal of Agricultural and Food Chemistry (2009), 57 (5), 1768-1774CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
    Aq. exts. of 30 plants were investigated for their antioxidant properties using DPPH and ABTS radical scavenging capacity assay, oxygen radical absorbance capacity (ORAC) assay, superoxide dismutase (SOD) assay, and ferric reducing antioxidant potential (FRAP) assay. Total phenolic content was also detd. by the Folin-Ciocalteu method. Antioxidant properties and total phenolic content differed significantly among selected plants. It was found that oak (Quercus robur), pine (Pinus maritima), and cinnamon (Cinnamomum zeylanicum) aq. exts. possessed the highest antioxidant capacities in most of the methods used, and thus could be potential rich sources of natural antioxidants. These exts. presented the highest phenolic content (300-400 mg GAE/g). Mate (Ilex paraguariensis) and clove (Eugenia caryophyllus clovis) aq. exts. also showed strong antioxidant properties and a high phenolic content (about 200 mg GAE/g). A significant relationship between antioxidant capacity and total phenolic content was found, indicating that phenolic compds. are the major contributors to the antioxidant properties of these plants.
  499. 499
    Thaipong, K.; Boonprakob, U.; Crosby, K.; Cisneros-Zevallos, L.; Hawkins Byrne, D. Comparison of ABTS, DPPH, FRAP, and ORAC Assays for Estimating Antioxidant Activity from Guava Fruit Extracts. J. Food Compos. Anal. 2006, 19 (6), 669– 75,  DOI: 10.1016/j.jfca.2006.01.003
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    502
    Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts
    Thaipong, Kriengsak; Boonprakob, Unaroj; Crosby, Kevin; Cisneros-Zevallos, Luis; Hawkins Byrne, David
    Journal of Food Composition and Analysis (2006), 19 (6-7), 669-675CODEN: JFCAEE; ISSN:0889-1575. (Elsevier)
    Guava fruit exts. were analyzed for antioxidant activity measured in methanol ext. (AOAM), antioxidant activity measured in dichloromethane ext. (AOAD), ascorbic acid, total phenolics, and total carotenoids contents. The ABTS, DPPH, and FRAP assays were used for detg. both AOAM and AOAD, whereas the ORAC was used for detg. only AOAM. Averaged AOAM [μM Trolox equiv. (TE)/g fresh mass (FM)] were 31.1, 25.2, 26.1, and 21.3 as detd. by the ABTS, DPPH, FRAP, and ORAC assays, resp. Averaged AOAD (μM TE/g FM) were 0.44, 0.27, and 0.16 as detd. by the ABTS, DPPH, and FRAP assays, resp. AOAM detd. by all assays were well correlated with ascorbic acid (0.61≤r≤0.92) and total phenolics (0.81≤r≤0.97) and also among themselves (0.68≤r≤0.97) but had neg. correlation with total carotenoids (-0.67≤r≤-0.81).
  500. 500
    da Silva Granja, B.; Honório de Mendonça Filho, J. R.; de Souza Oliveira, W.; Caldas Santos, J. C. Exploring MBTH as a Spectrophotometric Probe for the Determination of Total Phenolic Compounds in Beverage Samples. Anal. Methods 2018, 10 (19), 2197– 204,  DOI: 10.1039/C8AY00464A
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    Wong, T. S.; Wu, N.; Roccatano, D.; Zacharias, M.; Schwaneberg, U. Sensitive Assay for Laboratory Evolution of Hydroxylases toward Aromatic and Heterocyclic Compounds. J. Biomol. Screen. 2005, 10 (3), 246– 52,  DOI: 10.1177/1087057104273336
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    504
    Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds
    Wong, T. S.; Wu, N.; Roccatano, D.; Zacharias, M.; Schwaneberg, U.
    Journal of Biomolecular Screening (2005), 10 (3), 246-252CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
    Powerful directed evolution methods have been developed for tailoring proteins to our needs in industrial applications. Here, the authors report a medium-throughput assay system designed for screening mutant libraries of oxygenases capable of inserting a hydroxyl group into a C-H bond of arom. or O-heterocyclic compds. and for exploring the substrate profile of oxygenases. The assay system is based on 4-aminoantipyrine (4-AAP), a colorimetric phenol detection reagent. By using 2 detection wavelengths (509 nm and 600 nm), the authors achieved a linear response from 50 to 800 μM phenol and std. deviations below 11% in 96-well plate assays. The monooxygenase P 450 BM-3 and its F87A mutant were used as a model system for medium-throughput assay development, identification of novel substrates (e.g., phenoxytoluene, phenylallyether, and coumarone), and discovery of P 450 BM-3 F87A mutants with 8-fold improvement in 3-phenoxytoluene hydroxylation activity. This activity increase was achieved by screening a satn. mutagenesis library of amino acid position Y51 using the 4-AAP protocol in the 96-well format.
  502. 502
    Emerson, E. The Condensation of Aminoantipyrine. II. A New Color Test for Phenolic Compounds. J. Org. Chem. 1943, 8 (5), 417– 428,  DOI: 10.1021/jo01193a004
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    505
    The condensation of aminoantipyrine. II. A new color test for phenolic compounds
    Emerson, Edgar; Beacham, Harry H.; Beegle, Lindley C.
    Journal of Organic Chemistry (1943), 8 (), 417-28CODEN: JOCEAH; ISSN:0022-3263.
    cf. C. A. 33, 594.1. Aminoantipyrine (I) in the presence of a mild oxidizing agent in slightly alk. soln. gives intensely colored compds. with phenols. A large no. of phenols, naphthols, hydroxypyridines, hydroxyquinolines, barbiturates, uracils, hydantoins and imidazolones are tested and the results given in numerous tables. From the results it is concluded that at least 1 free OH group must be present, that substituents in the p-position to the OH group prevent the reaction with the exception of halogen, CO2H, SO3H, OH and OMe groups which are probably expelled, that a NO2 group in the ο-position prevents the reaction while one in the m-position prevents the test but not completely, and that the coupling of I with the phenol takes place in the p-position rather than in the ο-position. 1-Naphthols give red, 2-naphthols green ppts., sol. in CHCl3. Only the 3-hydroxypyridine gives a pos. test. The color given by vitamin B6 is extremely evanescent and is inhibited by the presence of borate ion (cf. Scudi, C. A. 35, 4792.7). 2- and 4-Hydroxyquinolines do not give a color test because the p-positions to the OH groups are blocked. Barbituric acid, 1,3-diphenylbarbituric acid and 2,4-diamino-6-hydroxypyrimidine give red compds. Barbital, 2-ethylmercapto-4-methyluracil, 4-methyluracil, 1,4-dimethyl- and 1,3,4-trimethyluracil, 2-benzyl-5-imidazolone and 2-benzyl-4-imidazolone fail to give a color test. The test is carried out by dissolving 10-20 mg. of the compd. in 10 cc. H2O contg. 2-3 drops 6 N NH4OH. The soln. is then divided into 2 parts. To 1 part, 2 drops of a 2% I soln., and to both parts, several drops of an 8% K3Fe(CN)6 soln. are added. When the color is developed in full, the soln. is shaken with CHCl3.
  503. 503
    Johnson, C. A.; Savidge, R. A. The Determination of Phenolic Compounds in Pharmaceutical Preparations Using 4-Aminohenazone. J. Pharm. Pharmacol. 2011, 10, 171T– 81T,  DOI: 10.1111/j.2042-7158.1958.tb10396.x
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    Varvounis, G.; Fiamegos, Y.; Pilidis, G. Pyrazol-3-Ones. Part III: Reactivity of the Ring Substituents. In Advances in Heterocyclic Chemistry; Academic Press, 2007; Vol. 95; p 27– 141
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    Dannis, M. Determination of Phenols by the Amino-Antipyrine Method. Sewage Ind. Waste 1951, 23, 1516– 1522
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    508
    Determination of phenols by the aminoantipyridine method
    Dannis, Michael
    Sewage and Industrial Wastes (1951), 23 (), 1516-22CODEN: SIWAAQ; ISSN:0096-364X.
    The method offers a quick detn. for phenol in industrial wastes and is sensitive to 0.1 p.p.m. The detailed procedure is given.
  506. 506
    Fiamegos, Y. C.; Stalikas, C. D.; Pilidis, G. A.; Karayannis, M. I. Synthesis and Analytical Applications of 4-Aminopyrazolone Derivatives as Chromogenic Agents for the Spectrophotometric Determination of Phenols. Anal. Chim. Acta 2000, 403 (1), 315– 23,  DOI: 10.1016/S0003-2670(99)00644-3
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    509
    Synthesis and analytical applications of 4-aminopyrazolone derivatives as chromogenic agents for the spectrophotometric determination of phenols
    Fiamegos, Y. C.; Stalikas, C. D.; Pilidis, G. A.; Karayannis, M. I.
    Analytica Chimica Acta (2000), 403 (1-2), 315-323CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
    The synthesis of simple, extensively conjugated chromophores as substitutes for 4-aminoantipyrine (4-AAP) capable of causing hyperchromic shifts when coupled with phenols is described. The 4-aminopyrazolone derivs. synthesized are subsequently applied for the spectrophotometric assay of phenolic compds. and the advantages of each of the proposed systems are highlighted. No improvement is made on the detection of para-substituted phenols as well as polychlorophenols (e.g. pentachlorophenol). An increase in the sensitivity of coupling products was obtained for certain phenolic compds. as indicated from the comparison of the individual responses. The proposed methods have analogous anal. features and in some cases show considerable improvements compared to the 4-AAP method. Beer's law is obeyed over a wide dynamic range (up to ∼500 μg/l) and the relative std. deviations are <3.4%. The methods are applied satisfactorily for phenol assay in real samples, however, taking into account the intrinsic differences of the methods in the responses to the various phenolic compds.
  507. 507
    Li, D.; Ge, S.; Huang, J.; Gong, J.; Yan, P.; Lu, W.; Tiana, G.; Ding, L. Fast Chromogenic Identification of Phenolic Pollutants Via Homogeneous Oxidation with t-BuOOH in the Presence of Iron (III) Octacarboxyphthalocyanine. Catal. Commun. 2014, 45, 95– 9,  DOI: 10.1016/j.catcom.2013.10.038
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    510
    Fast chromogenic identification of phenolic pollutants via homogeneous oxidation with t-BuOOH in the presence of iron(III) octacarboxyphthalocyanine
    Li, Dapeng; Ge, Suxiang; Huang, Jun; Gong, Jingjing; Yan, Ping; Lu, Weiyu; Tiana, Guizhong; Ding, Liyun
    Catalysis Communications (2014), 45 (), 95-99CODEN: CCAOAC; ISSN:1566-7367. (Elsevier B.V.)
    An efficient method was developed for fast chromogenic identification of some phenolic pollutants (phenol, 2-chlorophenol and 1-naphthol) via tert-Bu hydroperoxide (t-BuOOH) oxidn. in the presence of water-sol. iron(III) octacarboxyphthalocyanine complexes (catalyst) and 4-aminoantipyrine (chromogenic reagent). Among them, the chromogenic reaction of 2-chlorophenol could be completed rapidly, just within 6 min. The catalytic mechanism and detailed dye formation process were proposed on the basis of control expts. under different conditions. Potentially, the catalytic system composing of iron(III) octacarboxyphthalocyanine and t-BuOOH is promising for the application in fast chromogenic assay of phenolic pollutants.
  508. 508
    Fiamegos, Y.; Stalikas, C. D.; Pilidis, G. A. 4-Aminoantipyrine Spectrophotometric Method of Phenol Analysis: Study of the Reaction Products Via Liquid Chromatography with Diode-Array and Mass Spectrometric Detection. Anal. Chim. Acta 2002, 467 (1–2), 105– 14,  DOI: 10.1016/S0003-2670(02)00072-7
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    511
    4-Aminoantipyrine spectrophotometric method of phenol analysis. Study of the reaction products via liquid chromatography with diode-array and mass spectrometric detection
    Fiamegos, Y.; Stalikas, C.; Pilidis, G.
    Analytica Chimica Acta (2002), 467 (1-2), 105-114CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
    The synthesis of new pyrazolone mols. and their application as chromogenic agents in phenol anal. by the 4-aminoantipyrine method was described by the authors' group in previous published papers. A fully detailed mechanism is given therein based on exptl. and theor. data. The study of the reaction products and byproducts via liq. chromatog. with diode array and mass spectrometric detection is presented, sorting out the spectrophotometric detn. of phenols.
  509. 509
    Ayad, M.; El-Sadek, M.; Mostaffa, S. 4-Aminoantipyrine as an Analytical Reagent for the Colorimetric Determination of Tetracycline and Oxytetracycline. Anal. Lett. 1986, 19 (21–22), 2169– 81,  DOI: 10.1080/00032718608080875
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    512
    4-Aminoantipyrine as an analytical reagent for the colorimetric determination of tetracycline and oxytetracycline
    Ayad, M.; El-Sadek, M.; Mostaffa, S.
    Analytical Letters (1986), 19 (21-22), 2169-81CODEN: ANALBP; ISSN:0003-2719.
    A spectrophotometric method is proposed for the detn. of tetracycline (I) [60-54-8] and oxytetracycline (II) [79-57-2] in dosage forms. The suggested method depends on the reaction with 4-aminoantipyrine [83-07-8] in the presence of an alk. oxidizing agent. A red antipyrine dye is produced and its absorbance was measured at 435 and 440 nm for I and II, resp. The reaction ratio was detd. Variables such as pH, temp., reagent concn., and stability of the color produced were evaluated to permit selection of the most advantageous technique. Beer's law was obeyed over the concn. range 0.04-0.12 and 0.04-0.16 mg/mL for I and II, resp.
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    Otey, C. R.; Joern, J. M. High-Throughput Screen for Aromatic Hydroxylation, Directed Enzyme Evolution: Screening and Selection Methods; Humana Press: Totowa, NJ, 2003; p 141– 8.
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    Santos, G. d. A.; Dhoke, G. V.; Davari, M. D.; Ruff, A. J.; Schwaneberg, U. Directed Evolution of P450 BM3 Towards Functionalization of Aromatic O-Heterocycles. Int. J. Mol. Sci. 2019, 20 (13), 3353,  DOI: 10.3390/ijms20133353
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    Lülsdorf, N.; Vojcic, L.; Hellmuth, H.; Weber, T. T.; Mußmann, N.; Martinez, R.; Schwaneberg, U. A First Continuous 4-Aminoantipyrine (4-AAP)-Based Screening System for Directed Esterase Evolution. Appl. Microbiol. Biotechnol. 2015, 99 (12), 5237– 46,  DOI: 10.1007/s00253-015-6612-3
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    515
    A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution
    Luelsdorf, Nina; Vojcic, Ljubica; Hellmuth, Hendrik; Weber, Thomas T.; Mussmann, Nina; Martinez, Ronny; Schwaneberg, Ulrich
    Applied Microbiology and Biotechnology (2015), 99 (12), 5237-5246CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    Esterases hydrolyze ester bonds with an often high stereoselectivity as well as regioselectivity and are therefore industrially employed in the synthesis of pharmaceuticals, in food processing, and in laundry detergents. Continuous screening systems based on p-nitrophenyl- (e.g., p-nitrophenyl acetate) or umbelliferyl-esters are commonly used in directed esterase evolution campaigns. Ongoing challenges in directed esterase evolution are screening formats which offer a broad substrate spectrum, esp. for complex arom. substrates. In this report, a novel continuous high throughput screening system for indirect monitoring of esterolytic activity was developed and validated by detection of phenols employing Ph benzoate as substrate and p-nitrobenzyl esterase (pNBEBL from Bacillus licheniformis) as catalyst. The released phenol directly reacts with 4-aminoantipyrine yielding the red compd. 1,5-dimethyl-4-(4-oxo-cyclohexa-2,5-dienylidenamino)-2-phenyl-1,2-dihydro-pyrazol-3-one. In this continuous B. licheniformis esterase activity detection system (cBLE-4AAP), the product formation is followed through an increase in absorbance at 509 nm. The cBLE-4AAP screening system was optimized in 96-well microtiter plate format in respect to std. deviation (5 %), linear detection range (15 to 250 μM), lower detection limit (15 μM), and pH (7.4 to 10.4). The cBLE-4AAP screening system was validated by screening a random epPCR pNBEBL mutagenesis library (2000 clones) for improved esterase activity at elevated temps. Finally, the variant T3 (Ser378Pro) was identified which nearly retains its specific activity at room temp. (WT 1036 U/mg and T3 929 U/mg) and shows compared to WT a 4.7-fold improved residual activity after thermal treatment (30 min incubation at 69.4 °C; WT 170 U/mg to T3 804 U/mg).
  513. 513
    Choi, S.-L.; Rha, E.-G.; Kim, D.-Y.; Song, J.-J.; Hong, S.-P.; Sung, M.-H.; Lee, S.-G. High Throughput Screening and Directed Evolution of Tyrosine Phenol-Lyase. Microbiol. Biotechnol. Lett. 2006, 34, 58– 62
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    Quintana, M. G.; Didion, C.; Dalton, H. Colorimetric Method for a Rapid Detection of Oxygenated Aromatic Biotransformation Products. Biotechnol. Technol. 1997, 11 (8), 585– 7,  DOI: 10.1023/A:1018499024466
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    Colorimetric method for a rapid detection of oxygenated aromatic biotransformation products
    Quintana, M. G.; Didion, C.; Dalton, H.
    Biotechnology Techniques (1997), 11 (8), 585-587CODEN: BTECE6; ISSN:0951-208X. (Chapman and Hall)
    The quant. prodn. of the oxygenated products from the biotransformation of arom. substrates can be detected using a very simple and rapid colorimetric test. The method is based on Gibbs' reagent (2,6-dichloroquinone-4-chloroimide) and has been developed for routine spectrophotometric or microtiter plate assay allowing the detection of products with a sensitivity of ≥5 μM.
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    Dacre, J. C. Nonspecificity of the Gibbs Reaction. Anal. Chem. 1971, 43 (4), 589– 91,  DOI: 10.1021/ac60299a015
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    Nonspecificity of the Gibbs reaction
    Dacre, Jack C.
    Analytical Chemistry (1971), 43 (4), 589-91CODEN: ANCHAM; ISSN:0003-2700.
    The wavelengths of max. absorption and molar absorptivities are given for the products of the Gibbs reaction at pH 9.24 of 16 p-alkoxyphenols, 9 p-halophenols, p-hydroxybenzaldehydes, p-hydroxybenzyl alcs., and p-hydroxybenzoic acids, to demonstrate the nonspecificity of the Gibbs reagent.
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    Josephy, P. D.; Van Damme, A. Reaction of Gibbs Reagent with Para-Substituted Phenols. Anal. Chem. 1984, 56 (4), 813– 4,  DOI: 10.1021/ac00268a052
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    Reaction of Gibbs reagent with para-substituted phenols
    Josephy, P. David; Van Damme, Anjel
    Analytical Chemistry (1984), 56 (4), 813-14CODEN: ANCHAM; ISSN:0003-2700.
    Certain p-substituted phenols (alkoxy, halogen) react with Gibbs reagent (I) to give 2,6-dichloroindophenol. The para substituent is cleaved during the reaction. The product yield is detd. for various substituted phenols, and a mechanism for the reaction is proposed.
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    Svobodová, D.; Křenek, P.; Fraenkl, M.; Gasparič, J. The Colour Reaction of Phenols with the Gibbs Reagent. Microchim. Acta 1978, 70 (3), 197– 211,  DOI: 10.1007/BF01201610
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    Ang, E. L.; Obbard, J. P.; Zhao, H. Directed Evolution of Aniline Dioxygenase for Enhanced Bioremediation of Aromatic Amines. Appl. Microbiol. Biotechnol. 2009, 81 (6), 1063– 70,  DOI: 10.1007/s00253-008-1710-0
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    521
    Directed evolution of aniline dioxygenase for enhanced bioremediation of aromatic amines
    Ang, Ee Lui; Obbard, Jeffrey P.; Zhao, Huimin
    Applied Microbiology and Biotechnology (2009), 81 (6), 1063-1070CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    The objective of this study was to enhance the activity of aniline dioxygenase (I), a multi-component Rieske non-heme iron dioxygenase isolated from Acinetobacter sp. strain YAA, so as to create an enhanced biocatalyst for the bioremediation of arom. amines. Previously, mutation V205A was found to widen the substrate specificity of I to accept 2-isopropylaniline (2IPA) for which the wild-type enzyme had no activity. Using mutant V205A as the parent and applying one round of satn. mutagenesis followed by a round of random mutagenesis, the activity of the final mutant, 3-R21, was increased by 8.9-, 98.0-, and 2.0-fold for aniline, 2,4-dimethylaniline (24DMA), and 2-isopropylaniline (2IPA), resp., over mutant V205A. In particular, the activity of mutant 3-R21 for 24DMA, which is a carcinogenic arom. amine pollutant, was increased by 3.5-fold over wild-type I, while the activity for aniline was restored to the wild-type level, thus yielding a mutant I with enhanced activity and capable of hydroxylating a wider range of arom. amines than the wild-type enzyme.
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    Bornscheuer, U. T.; Ordoñez, G. R.; Hidalgo, A.; Gollin, A.; Lyon, J.; Hitchman, T. S.; Weiner, D. P. Selectivity of Lipases and Esterases Towards Phenol Esters. J. Mol. Catal. B: Enzym. 2005, 36 (1), 8– 13,  DOI: 10.1016/j.molcatb.2005.07.004
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    Selectivity of lipases and esterases towards phenol esters
    Bornscheuer, Uwe T.; Ordonez, Gonzalo Rodriguez; Hidalgo, Aurelio; Gollin, Anita; Lyon, Jonny; Hitchman, Timothy S.; Weiner, David P.
    Journal of Molecular Catalysis B: Enzymatic (2005), 36 (1-6), 8-13CODEN: JMCEF8; ISSN:1381-1177. (Elsevier B.V.)
    The selectivity of 28 lipases and esterases in the hydrolysis of butanoates of o-, m- or p-substituted phenols was investigated in a microtiter plate format. The phenols released during enzyme-catalyzed hydrolysis were converted in situ with Gibbs' reagent to form a blue indophenol complex, which was quantified spectrophotometrically at 600 nm. Substantial differences in rates were found, which exhibits that the type and position of the substituent at the alkyl group has a strong influence on the selectivity of the enzymes. For various enzymes, the p-nitro deriv. was the best substrate, whereas for other enzymes the m-Cl-deriv. was preferentially hydrolyzed. Anal. of the data using the Hammett equation showed that sometimes the obsd. changes followed a predictable trend, but in several cases the result is very unexpected.
  520. 520
    Altahir, B. M.; Abdulazeez, O.; Dikran, S. B.; Taylor, K. E. Determination of Eugenol in Personal-Care Products by Dispersive Liquid-Liquid Microextraction Followed by Spectrophotometry Using p-Amino-N,N-Dimethylaniline as a Derivatizing Agent. Indones. J. Chem. 2020, 21, 192,  DOI: 10.22146/ijc.56198
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    Gasparic, J.; Svobodova, D.; Pospisilova, M. Investigation of the Colour Reaction of Phenols with the MBTH Reagent. Identification of Organic Compounds. LXXXVI. Microchim. Acta 1977, 67, 241– 50,  DOI: 10.1007/BF01213034
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    Pospíšilová, M.; Polášek, M.; Svobodová, D. Spectrophotometric Study of Reactions of Substituted Phenols with MBTH in Alkaline Medium: The Effect of Phenol Structure on the Formation of Analytically Useful Coloured Products. Microchimica Acta 1998, 129 (3), 201– 8,  DOI: 10.1007/BF01244742
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    Kamata, E. Color Reactions of 3-Methyl-2-Benzothiazolone Hydrazone with Phenol Derivatives. I. The Spectrophotometric Microdetermination of Catechol, Hydroquinone and Resorcinol. Bull. Chem. Soc. Jpn. 1964, 37 (11), 1674– 7,  DOI: 10.1246/bcsj.37.1674
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    Color reactions of 3-methyl-2-benzothiazolone hydrazone with phenol derivatives. I. The spectrophotometric microdetermination of catechol, hydroquinone, and resorcinol
    Kamata, Eijiro
    Bulletin of the Chemical Society of Japan (1964), 37 (11), 1674-7CODEN: BCSJA8; ISSN:0009-2673.
    Two methods are given for the detn. of catechol: (1) Add 4 ml. of 0.5% 3-methyl-2-benzothiazolone hydrazone (I)-MeOH soln. and 2 ml. of NH4OH to 4 ml. of test soln.; allow the mixt. to stand for 15 min., and read the absorbance at 510 mμ. (2) Add 2 ml. of 0.4% aq. I, 1.6 ml. of 10% Mohr's salt, and 0.4 ml. of 1% FeCl3 to 4 ml. of test soln., and 2 ml. Me2CO, after 5 min., and read the absorbance at 550 mμ within 30 min. To detn. hydroquinone (II), add 3 ml. of 0.5% I-MeOH soln. and 1 ml. NH4OH to 3 ml. of test soln., after 1 min. add 3 ml. Me2CO, and read the absorbance at 610 mμ within 3 min. Methods are given also for the detn. of II and resorcinol (III) in a mixt. of the 2 and of III alone.
  524. 524
    Setti, L.; Giuliani, S.; Spinozzi, G.; Pifferi, P. G. Laccase Catalyzed-Oxidative Coupling of 3-Methyl 2-Benzothiazolinone Hydrazone and Methoxyphenols. Enzyme Microb. Technol. 1999, 25 (3), 285– 9,  DOI: 10.1016/S0141-0229(99)00059-9
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    527
    Laccase catalyzed-oxidative coupling of 3-methyl 2-benzothiazolinone hydrazone and methoxyphenols
    Setti, Leonardo; Giuliani, Silvia; Spinozzi, Giovanni; Pifferi, Pier Giorgio
    Enzyme and Microbial Technology (1999), 25 (3-5), 285-289CODEN: EMTED2; ISSN:0141-0229. (Elsevier Science Ireland Ltd.)
    The reaction between o-, m-, and p-methoxyphenols and 3-methyl-2-benzothiazolinone hydrazone was studied in the presence of laccase from Pyricularia oryzae. The findings show that laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) catalyzes the oxidative coupling reaction between MBTH and phenols producing red colored azo-dye compds. On the basis of kinetic parameters and optimum pH values, the mechanisms of the oxidative coupling reactions with the different phenols are discussed. The results suggest that the reaction is initialized by the enzymic 3-methyl-2-benzothiazolinone hydrazone activation, which undergoes electrophilic substitution with m-methoxyphenol in soln., enzymic activated guaiacol, and chelated p-methoxyphenol at the catalytic site of the laccase. The current study also demonstrates the possibility of using the azo-dye formed in the presence of guaiacol for measuring laccase activity following a similar assaying method as that used for measuring peroxidase activity. This type of assay permits the detn. of laccase at micromolar levels by fixed time method.
  525. 525
    Setti, L.; Scali, S.; Angeli, I. D.; Pifferi, P. G. Horseradish Peroxidase-Catalyzed Oxidative Coupling of 3-Methyl 2-Benzothiazolinone Hydrazone and Methoxyphenols. Enzyme Microb. Technol. 1998, 22 (8), 656– 61,  DOI: 10.1016/S0141-0229(97)00259-7
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    528
    Horseradish peroxidase-catalyzed oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone and methoxyphenols
    Setti, Leonardo; Scali, Sara; Angeli, Igor Degli; Pifferi, Pier Giorgio
    Enzyme and Microbial Technology (1998), 22 (8), 656-661CODEN: EMTED2; ISSN:0141-0229. (Elsevier Science Inc.)
    The reaction between o-, m-, and p-methoxyphenols and 3-methyl-2-benzothiazolinone hydrazone (MBTH) is studied in the presence of horseradish peroxidase (HRP) and H2O2 as oxidative agent. The findings indicate that enzyme (H2O2 oxidoreductase; EC 1.11.1.7) catalyzes an oxidative coupling reaction between MBTH and phenols which produces azo dye compds. On the basis of kinetic parameters and optimum pH values, a mechanism in which both MBTH and phenols seem to be activated by the HRP for achieving the oxidative coupling is proposed. Furthermore, in the current study, we have evaluated the possibility that these azo dyes may be useful in the measurement of peroxidase activity. The method is based on the obsd. increase in the absorbance at 502 nm (8,355 cm-1 M-1 of extinction molar coeff.) due to the formation of a red azo dye compd. resulting from the peroxidase-catalyzed oxidative coupling of MBTH and o-methoxyphenol (guaiacol). Using this assay system, HRP can be detd. in picomolar levels by a fixed time method.
  526. 526
    Nolan, L. C.; O’Connor, K. E. A Spectrophotometric Method for the Quantification of an Enzyme Activity Producing 4-Substituted Phenols: Determination of Toluene-4-Monooxygenase Activity. Anal. Biochem. 2005, 344 (2), 224– 31,  DOI: 10.1016/j.ab.2005.05.032
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    529
    A spectrophotometric method for the quantification of an enzyme activity producing 4-substituted phenols: Determination of toluene-4-monooxygenase activity
    Nolan, Louise C.; O'Connor, Kevin E.
    Analytical Biochemistry (2005), 344 (2), 224-231CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
    A spectrophotometric method for the quant. detn. of an enzyme activity resulting in the accumulation of 4-substituted phenols is described in this article. Toluene-4-monooxygenase (T4MO) activity in whole cells of Pseudomonas mendocina KR1 is used to demonstrate this method. This spectrophotometric assay is based on the coupling of T4MO activity with tyrosinase activity. The 4-substituted phenol, produced by the action of T4MO on the arom. ring of a substituted arene, is a substrate for tyrosinase, which converts phenols to o-quinones. The latter react with the nucleophile 3-methyl-2-benzothiazolinone hydrazone (MBTH) to produce intensely colored products that absorb light maximally at different wavelengths, depending on the phenolic substrate used. The incubation of whole cells of P. mendocina KRI with fluorobenzene resulted in the accumulation of 4-fluorophenol. The coupling of T4MO activity with tyrosinase activity in the presence of fluorobenzene resulted in the formation of a colored product absorbing maximally at 480 nm. The molar absorptivity (ε) value for the o-quinone-MBTH adduct formed from 4-fluorophenol was detd. exptl. to be 12,827 M-1 cm-1 with a linear range of quantification between 2.5 and 75 μM. The whole cell assay was run as a continuous indirect assay. The initial rates of T4MO activity toward fluorobenzene, as detd. spectrophotometrically, were 61.8 nmol/min/mg P. mendocina KR1 protein (using mushroom tyrosinase), 64.9 nmol/min/mg P. mendocina KR1 protein (using cell exts. Pseudomonas putida F6), and, as detd. by HPLC anal., 62.6 nmol/min/mg P. mendocina KR1 protein.
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    Regitz, M. Diazo Compounds: Properties and Synthesis; Elsevier, 2012.
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    Chudgar, R. J. Azo Dyes; Kirk–Othmer Encyclopedia of Chemical Technology, 2000.
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    de Keijzer, M. The Delight of Modern Organic Pigment Creations, Issues in Contemporary Oil Paint; Springer International Publishing: Cham, 2014; p 45– 73.
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    Urbányi, T.; Mollica, J. A. Potential Diazo Reagents for Colorimetric Determination. J. Pharm. Sci. 1968, 57 (7), 1257– 60,  DOI: 10.1002/jps.2600570746
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    Potential diazo reagents for colorimetric determination
    Urbanyi, Tibor; Mollica, Joseph A.
    Journal of Pharmaceutical Sciences (1968), 57 (7), 1257-60CODEN: JPMSAE; ISSN:0022-3549.
    A method for detg. estrogens is based on their ability to couple with diazotized amines. The diazonium derivs. of 27 substituted aromatic amines were investigated as reagents for colorimetric detn. The phenolic moiety used in the study was estradiol. Their applicability was detd. on the basis of speed, sensitivity, color stability and reproducibility of the coupling reaction. With these criteria, a diazotization product of 4-amino-6-chloro-m-benzenedisulfonamide appeared to be the most suitable colorimetric reagent for compds. contg. a phenolic OH group.
  531. 531
    Ehrlich, P. Uber Eine Neue Harnprobe. Dtsch. Med. Wochenschr. 1883, 9 (1), 11,  DOI: 10.1055/s-0029-1196984
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    Hassan, S. M.; Walash, M. I.; El-Sayed, S. M.; Abou Ouf, A. M. Colorimetric Determination of Certain Phenol Derivatives in Pharmaceutical Preparations. J. Assoc. Off. Anal. Chem. 1981, 64 (6), 1442– 5,  DOI: 10.1093/jaoac/64.6.1442
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    535
    Colorimetric determination of certain phenol derivatives in pharmaceutical preparations
    Hassan, Sayed M.; Walash, Mohammed I.; El-Sayed, Sanaa M.; Ouf, Abdel Malek Abou
    Journal - Association of Official Analytical Chemists (1981), 64 (6), 1442-5CODEN: JANCA2; ISSN:0004-5756.
    acetaminophen (I) [103-90-2] and oxyphenbutazone (II) [129-20-4] are detd. using colorimetry methods. These methods are based on coupling between the phenolic compd. and the diazonium salts of both sulfanilic acid and p-nitroaniline; the optimum conditions for the reactions were studied. For I, the reaction products with diazosulfanilic acid and diazo-p-nitroaniline show max. absorbance at 480 nm and 425 nm, resp. The mean percentage recoveries for authentic samples were 99.5 and 100.6, resp. For II, the obtained colors showed max. at 385 nm with diazosulfanilic acid and 490 nm with diazo-p-nitroaniline reactions. The mean percentage recoveries for authentic samples were 99.8 and 100.1, resp. The proposed methods were successfully applied to the anal. of com. prepns.; results were statistically compared with those of other methods.
  533. 533
    Swaminathan, M. Chemical Estimation of Vitamin B6 in Foods by Means of the Diazo Reaction and the Phenol Reagent. Nature 1940, 145 (3681), 780,  DOI: 10.1038/145780a0
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    536
    Chemical estimation of vitamin B6 in foods by means of the diazo reaction and the phenol reagent
    Swaminathan, M.
    Nature (London, United Kingdom) (1940), 145 (), 780CODEN: NATUAS; ISSN:0028-0836.
    The method makes use of the diazo reaction (diazotized sulfanilic acid) or the phenol reagent. The azo color formed is estd. colorimetrically. The method is applicable to all foods. As little as 10 γ of vitamin B6 is easily estd.
  534. 534
    Whitlock, L. R.; Siggia, S.; Smola, J. E. Spectrophotometric Analysis of Phenols and of Sulfonates by Formation of an Azo Dye. Anal. Chem. 1972, 44 (3), 532– 6,  DOI: 10.1021/ac60311a021
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    537
    Spectrophotometric analysis of phenols and of sulfonates by formation of an azo dye
    Whitlock, L. Ronald; Siggia, Sidney; Smola, Janice E.
    Analytical Chemistry (1972), 44 (3), 532-6CODEN: ANCHAM; ISSN:0003-2700.
    Phenols and sulfonic acids are detd. by a method based on the coupling reaction between a diazotized amine and the phenol. The concn. of the azo dye formed was measured by visible spectrophotometry. Sulfonic acids and their salts were converted to phenols via alkali fusion at 360° by using KOH, prior to coupling. Diazotized sulfanilic acid and p-phenylazoaniline, a previously unreported reagent, were particularly useful reagents for detn. of phenols. With the latter, the optimum pH and reaction time for the phenols were remarkably similar and the molar absorptivities of the dyes formed were significantly higher. The coupling reaction conditions for a wide variety of phenols and the molar absorptivities and absorption max. for the azo dyes are presented. The limit of detection was about 1 μmole phenol/l. soln. and the min. amt. detectable was 0.3 μg. This method for sulfonic acid detn. will extend the useful range of application of the alkali fusion method to include polysulfonates and higher mol. wt. sulfonates. Alkali fusion of halogenated sulfonates resulted in substitution of both the halogen and sulfonate groups. To help det. which sulfonates can be analyzed by the fusion procedure, the thermal stabilities of a large no. of sulfonates and several phenolates are given.
  535. 535
    Abdel Azeem, S. M.; Al Mohesen, I. A.; Ibrahim, A. M. H. Analysis of Total Phenolic Compounds in Tea and Fruits Using Diazotized Aminobenzenes Colorimetric Spots. Food Chem. 2020, 332, 127392,  DOI: 10.1016/j.foodchem.2020.127392
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    538
    Analysis of total phenolic compounds in tea and fruits using diazotized aminobenzenes colorimetric spots
    Abdel Azeem, Sami M.; Al Mohesen, Ibrahem A.; Ibrahim, Ahmed M. H.
    Food Chemistry (2020), 332 (), 127392CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)
    The present work describes a novel and rapid approach for evaluating total phenolic compds. (TPCs) in tea and fruits using colorimetric spots and the digital image-based (DIB) method. Colorimetric spots were formed by reacting diazotized aminobenzenes namely sulfanilic acid, sulfanilamide, or aniline with TPCs in the ext. to form an azo dye. The limit of detection (LOD) was 6.5, 5.5, or 5.1 mg GAE (gallic acid equiv.) L-1 and the anal. range was 25-500, 20-500, or 18-200 mg GAE L-1, resp. Correlation with the Folin-Ciocalteu assay was significant (Pearson coeff., R = 0.970-0.991) while the antioxidant activity assay was moderate to high (R = 0.737-0.977). The method developed was successfully applied to the anal. of tea and fruits and showed RSD (n = 3) not exceeding 9.6, 8.5, and 9.7%, resp. Ecol., the DIB method developed could det. the variation of TPCs within cultivars and was found to be strongly dependent on the growing environment.
  536. 536
    Lester, G. E.; Lewers, K. S.; Medina, M. B.; Saftner, R. A. Comparative Analysis of Strawberry Total Phenolics Via Fast Blue BB Vs. Folin-Ciocalteu: Assay Interference by Ascorbic Acid. J. Food Compos. Anal. 2012, 27 (1), 102– 7,  DOI: 10.1016/j.jfca.2012.05.003
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    539
    Comparative analysis of strawberry total phenolics via Fast Blue BB vs. Folin-Ciocalteu: Assay interference by ascorbic acid
    Lester, Gene E.; Lewers, Kim S.; Medina, Marjorie B.; Saftner, Robert A.
    Journal of Food Composition and Analysis (2012), 27 (1), 102-107CODEN: JFCAEE; ISSN:0889-1575. (Elsevier Inc.)
    Unblemished fully ripe fruit from five day-neutral strawberry cultivars were harvested on two sep. dates and evaluated for ascorbic acid (AsA), fruit sugars, and phenolic compn. Individual phenolics were detd. by HPLC, and total phenolics by Folin-Ciocalteu (F-C) and by a new' assay: Fast Blue BB (FBBB), which detects phenolics directly. FBBB reported an av. 2.9-fold greater concn. of total phenolics than F-C, had a significant correlation (r = 0.80; P = 0.001) with total phenolics via HPLC and did not interact with AsA or sugars, whereas F-C, an indirect detection assay for total phenolics, appeared to under-report total phenolic concns., had no significant correlation (r = 0.20) with total phenolics via HPLC or with sugars, but had a significant correlation (r = 0.64; P = 0.05) with total AsA. Results from this study indicated that previous studies of strawberry fruit, using the std. indirect F-C assay, have greatly underestimated the total phenolics content and that this assay should be replaced in future studies by the FBBB assay.
  537. 537
    Pompella, A.; Comporti, M. The Use of 3-Hydroxy-2-Naphthoic Acid Hydrazide and Fast Blue B for the Histochemical Detection of Lipid Peroxidation in Animal Tissues─a Microphotometric Study. Histochemistry 1991, 95, 255– 262,  DOI: 10.1007/BF00744997
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    540
    The use of 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B for the histochemical detection of lipid peroxidation in animal tissues - a microphotometric study
    Pompella, A.; Comporti, M.
    Histochemistry (1991), 95 (3), 255-62CODEN: HCMYAL; ISSN:0301-5564.
    The title procedure was modified to prevent exposure of tissue sections to lipid solvents. In fresh rat or mouse liver cryostat sections exposed in vitro to various prooxidant conditions (NADPH-Fe2+, NADPH-ADP/Fe3+, BrCCl3-NADPH), a close correlation was found between the intensity of the 3-hydroxy-2-naphthoic acid/Fast Blue B (NAH-FBB) (blue-violet) stain and the amt. of malondialdehyde (used as biochem. index of lipid peroxidn.) released in the incubation medium. Stain intensities obtained with NAH-FBB reaction were several-fold higher than those obtainable with direct Schiff reaction (a previously used procedure) and better paralleled in the the appearance of lipid peroxidn. in tissue. By means of selective delipidation it was obsd. that the NAH-FBB reaction is more efficient than the Schiff reaction in detecting protein and phospholipid-assocd. lipid peroxidn.-derived carbonyl functions. The ability of the NAH-FBB reaction to reveal lipid peroxidn. occurring in tissues in vivo was verified with animals intoxicated with prooxidant toxins, i.e., the haloalkanes BrCCl3 and CCl4, and the glutathione-depleting agent bromobenzene. In livers from haloalkane-treated rats, NAH-FBB positivity provided with te specific absorption spectrum was obsd. in centrolobular regions. In bromobenzene-poisoned mice, NAH-FBB positivity with specific absorption was found, besides the liver, also in kidney (tubular epithelium) and lung (bronchiolar epithelium). This reaction is suggested for the discrimination of cell types undergoing lipid peroxidn. in vivo.
  538. 538
    Tłuścik, F.; Kazubek, A.; Mejbaum-Katzenellenbogen, W. Alkylresorcinols in Rye (Secale Cereale L.) Grains. Vi. Colorimetric Micromethod for the Determination of Alkylresorcinols with the Use of Diazonium Salt, Fast Blue B. Acta Soc. Bot. Polym. 1981, 50, 645,  DOI: 10.5586/asbp.1981.086
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    Alkylresorcinols in rye (Secale cereale L.) grain. VI. Colorimetric micromethod for the determination of alkylresorcinols with the use of the diazonium salt, Fast Blue B
    Tluscik, Franciszek; Kozubek, Arkadiusz; Mejbaum-Katzenellenbogen, Wanda
    Acta Societatis Botanicorum Poloniae (1981), 50 (4), 645-51CODEN: ASBNA2; ISSN:0001-6977.
    A Fast Blue B soln. was added to a Me2CO ext. of rye or wheat grains and the absorbance was read after 1-2 h at 520 nm. A calibration curve was prepd. by using 5-n-pentadecylresorcinol as a std. Almost all of the measured absorbance was due to 5-n-alkylresorcinols.
  539. 539
    Schotten, C.; Leprevost, S. K.; Yong, L. M.; Hughes, C. E.; Harris, K. D. M.; Browne, D. L. Comparison of the Thermal Stabilities of Diazonium Salts and Their Corresponding Triazenes. Org. Process Res. Dev. 2020, 24 (10), 2336– 41,  DOI: 10.1021/acs.oprd.0c00162
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    542
    Comparison of the Thermal Stabilities of Diazonium Salts and Their Corresponding Triazenes
    Schotten, Christiane; Leprevost, Samy K.; Yong, Low Ming; Hughes, Colan E.; Harris, Kenneth D. M.; Browne, Duncan L.
    Organic Process Research & Development (2020), 24 (10), 2336-2341CODEN: OPRDFK; ISSN:1083-6160. (American Chemical Society)
    A range of diazonium salts and their corresponding triazenes have been prepd. in order to directly compare their relative thermal stabilities (via initial decompn. temp.) from differential scanning calorimetry (DSC) data. A structure-stability relationship has been explored to investigate trends in stability, depending on the arom. substituent and the structure of the secondary amine component of the diazonium salts and triazenes. All of the triazenes investigated show significantly greater stability (many are stable above 200°C) compared with the corresponding diazonium salts, which show varying stabilities.
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    Johnston, K. J.; Ashford, A. E. A Simultaneous-Coupling Azo Dye Method for the Quantitative Assay of Esterase Using α-Naphthyl Acetate as Substrate. Histochem. J. 1980, 12 (2), 221– 34,  DOI: 10.1007/BF01024552
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    A simultaneous-coupling azo dye method for the quantitative assay of esterase using α-naphthyl acetate as substrate
    Johnston, K. J.; Ashford, A. E.
    Histochemical Journal (1980), 12 (2), 221-34CODEN: HISJAE; ISSN:0018-2214.
    A simultaneous-coupling azo dye method is described for the measurement of esterase activity using the histochem. substrate, α-naphthyl acetate. By the choice of 2 diazonium salts with optimal coupling characteristics, the reaction can be carried out at any pH between 3.0 and 9.5. The azo dye is maintained in soln. for spectrophotometric measurements with bovine serum albumin. The simultaneous-coupling method is compared with an assay based on the direct measurement of released α-naphthol by its UV absorbance in a pH study of hog liver esterase. There is good agreement between the data obtained by both methods.
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    Pearse, A. G. E. Histochemistry, Theoretical and Applied; Harcourt Brace/Churchill Livingstone, 1968.
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    Lugg, G. A. Stabilized Diazonium Salts as Analytical Reagents for the Determination of Air-Borne Phenols and Amines. Anal. Chem. 1963, 35 (7), 899– 904,  DOI: 10.1021/ac60200a039
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    Stabilized diazonium salts as analytical reagents for the determination of airborne phenols and amines
    Lugg, G. A.
    (1963), 35 (), 899-904CODEN: ANCHAM; ISSN:0003-2700.
    Com. diazonium salts were investigated as reagents for the spectrophotometric detn. of 24 toxic phenols and amines in air. Optimum reaction conditions were detd. after spot testing with 25 diazo reagents at 4 buffer concns. (pH 4, 0.05M KH phthalate, pH 7, 2M NH4OAc, pH 9, 0.05M Na borate, and pH 12.8, 2M Na2CO3). Charge a bubbler of the midget impinger type with 5 ml. of solvent (H2O or Me cellosolve, depending upon the compd. tested), sample at 1 l./min. for 2 min., add buffer and the relevant diazo compd. (these are listed for each test material), prep. a standard, and read both solns. against a blank. The vapor of a representative selection of compds. was sampled in each case at least 80% of the amt. recovered was collected in the 1st bubbler Interference is mainly from accompanying nitro compds. Sensitivity varies, but the compds. can be detd. in air at concns. of interest in hygiene studies.
  543. 543
    Buchwald, H. The Colorimetric Determination of Phenol in Air and Urine with a Stabilized Diazonium Salt. Ann. Occup. Hyg. 1966, 9, 7– 14,  DOI: 10.1093/annhyg/9.1.7
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    546
    Colorimetric determination of phenol in air and urine
    Buchwald, H.
    Annals of Occupational Hygiene (1966), 9 (1), 7-14CODEN: AOHYA3; ISSN:0003-4878.
    Optimum conditions were evaluated for the detn. of phenol with Brentamine Fast GG, the stabilized diazonium salt of p-nitroaniline. Equally efficient air sampling was obtained by 2 U-tubes contg. 10 g. silica gel (40-60 mesh chromatographic grade) connected in series and 2 fritted glass bubblers contg. 5% wt./vol. NaOH soln., but the U tubes are more convenient. The silica gel is placed in N NaOH, allowed to stand 10 min., and then analyzed. Phenol, o-, and m-cresols are sepd. from a soln. contg. 10 ml. urine, 2 ml. H2SO4, and 7 ml. H2O by steam distillation. The first 100 ml. of distillate contains >98% of the phenols present. To a sample contg. 10-100 γ phenol adjusted to pH 10.1 with Na2CO3-NaHCO3 buffer, add 2.5 ml. 0.5% filtered diazo reagent and dil. to 25 ml. Wait 10 min. and read the absorbance at 490 mμ. The color is stable for 1 hr., but the diazo stock soln. must be freshly prepd. every few hrs. o- and μ-Cresols, if present, are also detd. with the phenol.
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    Harvey, D.; Penketh, G. E. The Determination of Small Amounts of o-Phenylphenol. Analyst 1957, 82 (976), 498– 503,  DOI: 10.1039/an9578200498
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    547
    Determination of small amounts of o-phenylphenol
    Harvey, D.; Penketh, G. E.
    (1957), 82 (), 498-503 ISSN:.
    o-Phenylphenol (I) after isolation can be measured by ultraviolet absorption at 284 and 295 mμ in cyclohexane or 300 and 320 mμ in NaOH. To use the hypochromic effect measure at 285 and 315 mμ in NaOH and then in HCl soln. The differences in optical ds. are independent of background. Two wave lengths are used to detect interferences. In alc. NaOH I fluoresces at 0.5 γ/ml. but interferences are serious so that errors of ±20% are found. For a colorimetric detn. neutralize the soln. to pH 7-9 and dil. so that 1 ml. contains 1-10 γ I. To 10 ml. add 1 ml. 1% gum acacia soln., 1 ml. 50% NaOAc soln., and 1 ml. fresh 0.5% Brentamine Fast Red GG soln. After 1 min. add 2 ml. 20% Na2CO3 soln. and measure at 494 mμ against a blank. Methods for isolating I from disinfectant salts and formulations, citrus fruits, timber, paper, and canvas are described.
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    Canada, K. A.; Iwashita, S.; Shim, H.; Wood, T. K. Directed Evolution of Toluene ortho-Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation. J. Bacteriol. 2002, 184 (2), 344– 9,  DOI: 10.1128/JB.184.2.344-349.2002
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    548
    Directed evolution of toluene ortho-monooxygenase for enhanced 1-naphthol synthesis and chlorinated ethene degradation
    Canada, Keith A.; Iwashita, Sachiyo; Shim, Hojae; Wood, Thomas K.
    Journal of Bacteriology (2002), 184 (2), 344-349CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chem. manufg. intermediate. Directed evolution was used to increase the activity of toluene o-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidn. When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5 vs. 1.39 nmol/min/mg protein), 1,1-dichloroethylene, and trans-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was 6-fold faster than that mediated by the wild-type enzyme at a concn. of 0.1 mM (0.19 vs. 0.029 nmol/min/mg protein), whereas at 5 mM, the mutant enzyme was active (0.07 nmol/min/mg protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with >97% 1-naphthol formed. The beneficial mutation of TOM-Green was the substitution of Val to Ala in position 106 of the α-subunit of the hydroxylase, which appears to act as a smaller "gate" to the di-iron active center. This hypothesis was supported by the ability of E. coli expressing TOM-Green to oxidize the 3-ring polyarom. compds., phenanthrene, fluorene, and anthracene, faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chem.
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    Joshi, N. S.; Whitaker, L. R.; Francis, M. B. A Three-Component Mannich-Type Reaction for Selective Tyrosine Bioconjugation. J. Am. Chem. Soc. 2004, 126 (49), 15942– 3,  DOI: 10.1021/ja0439017
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    549
    A Three-Component Mannich-Type Reaction for Selective Tyrosine Bioconjugation
    Joshi, Neel S.; Whitaker, Leanna R.; Francis, Matthew B.
    Journal of the American Chemical Society (2004), 126 (49), 15942-15943CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A new selective bioconjugation reaction is described for the modification of tyrosine residues on protein substrates. The reaction uses imines formed in situ from aldehydes and electron-rich anilines to modify phenolic side chains through a Mannich-type electrophilic arom. substitution pathway. The reaction takes place under mild pH and temp. conditions and can modify protein substrates at concns. as low as 20 μM. Using an efficient fluorescence-based assay, we demonstrated the reaction using a no. of aldehydes and protein targets. Importantly, proteins lacking surface-accessible tyrosines remained unmodified. It was also demonstrated that enzymic activity is preserved under the mild reaction conditions. This strategy represents one of the first carbon-carbon bond-forming reactions for protein modification and provides an important complement to more commonly used lysine- and cysteine-based methods.
  547. 547
    Cardellicchio, C.; Capozzi, M. A. M.; Naso, F. The Betti Base: The Awakening of a Sleeping Beauty. Tetrahedron: Asymmetry 2010, 21 (5), 507– 17,  DOI: 10.1016/j.tetasy.2010.03.020
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    550
    The Betti base. The awakening of a sleeping beauty
    Cardellicchio, Cosimo; Capozzi, Maria Annunziata M.; Naso, Francesco
    Tetrahedron: Asymmetry (2010), 21 (5), 507-517CODEN: TASYE3; ISSN:0957-4166. (Elsevier Ltd.)
    A review. The multicomponent reaction between 2-naphthol, aryl aldehydes and ammonia or amines yields aminobenzylnaphthols in a process known as the Betti reaction, which was first uncovered at the beginning of the 20th century. The aminobenzylnaphthols could be easily resolved into their enantiomers. After a long silence, the results of our research a decade ago on this useful reaction and on the chiral materials produced has stimulated further work in a no. of other labs. As a result, novel applications of the Betti reaction to produce new chiral aminobenzylnaphthols were reported together with the evaluation of these chiral bases in asym. synthesis. Herein, the authors presented a selection of the relevant studies on this topic.
  548. 548
    Minakawa, M.; Guo, H.-M.; Tanaka, F. Imines That React with Phenols in Water over a Wide pH Range. J. Org. Chem. 2008, 73 (21), 8669– 72,  DOI: 10.1021/jo8017389
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    551
    Imines that React with Phenols in Water over a Wide pH Range
    Minakawa, Maki; Guo, Hai-Ming; Tanaka, Fujie
    Journal of Organic Chemistry (2008), 73 (21), 8669-8672CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    Cyclic imine derivs. that react with phenols, including tyrosine residues of peptides, have been developed. Reactions of the imines I-III with phenols proceeded in water over a wide pH range (pH 2-10) at room temp. to 37 °C and afforded Mannich products without the need of addnl. catalysts.
  549. 549
    Kanaoka, Y. Organic Fluorescence Reagents in the Study of Enzymes and Proteins. Angew. Chem., Int. Ed. 1977, 16 (3), 137– 47,  DOI: 10.1002/anie.197701371
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    552
    Organic fluorescence reagents in the study of enzymes and proteins
    Kanaoka Y
    Angewandte Chemie (International ed. in English) (1977), 16 (3), 137-47 ISSN:1433-7851.
    There is no expanded citation for this reference.
  550. 550
    Davis, W.; Ronai, Z.; Tew, K. D. Cellular Thiols and Reactive Oxygen Species in Drug-Induced Apoptosis. J. Pharmacol. Exp. Ther. 2001, 296, 1– 6
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    553
    Cellular thiols and reactive oxygen species in drug-induced apoptosis
    Davis, Warren, Jr.; Ronai, Ze'ev; Tew, Kenneth D.
    Journal of Pharmacology and Experimental Therapeutics (2001), 296 (1), 1-6CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)
    A review with 42 refs. In higher eukaryotes, reactive oxygen species (ROS) are generated during respiration in mitochondria in the course of redn. of mol. oxygen as well as by distinct enzyme systems. ROS have been implicated in the regulation of diverse cellular functions including defense against pathogens, intracellular signaling, transcriptional activation, proliferation, and apoptosis. The redn.-oxidn. (redox) state of the cell is primarily a consequence of the precise balance between the levels of ROS and endogenous thiol buffers present in the cell, such as glutathione and thioredoxin, which protect cells from oxidative damage. Dramatic elevation of ROS, exceeding compensatory changes in the level of the endogenous thiol buffers, may result in the sustained activation of signaling pathways and expression of genes that induce apoptosis in affected cells. Many cytotoxic drugs function selectively to kill cancer cells by the abrogation of proliferative signals, leading to cell death, and numerous reports have demonstrated that ROS are generated following treatment with these drugs. In this review, we will summarize recent contributions to our understanding of the importance of cytotoxic drug-induced modulation of cellular redox status for signaling and transcription leading to activation of apoptotic effector mechanisms.
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    Ball, R. O.; Courtney-Martin, G.; Pencharz, P. B. The in Vivo Sparing of Methionine by Cysteine in Sulfur Amino Acid Requirements in Animal Models and Adult Humans. J. Nutr. 2006, 136 (6), 1682S– 93S,  DOI: 10.1093/jn/136.6.1682S
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    554
    The in vivo sparing of methionine by cysteine in sulfur amino acid requirements in animal models and adult humans
    Ball, Ronald O.; Courtney-Martin, Glenda; Pencharz, Paul B.
    Journal of Nutrition (2006), 136 (6S), 1682S-1693SCODEN: JONUAI; ISSN:0022-3166. (American Society for Nutrition)
    A review. S amino acid metab. was receiving increased attention because of the link to chronic diseases such as cardiovascular disease, Alzheimer's disease, and diabetes. In addn., the role of Cys and optimal intakes for physiol. substrates such as glutathione are currently of considerable interest in human health. Although the dietary indispensability of Met is not in question, the ability of Cys to substitute for a portion of its requirement was the topic of much debate. Met is often the most limiting amino acid in the diets of the developing world's population because of its low concn. in cereal grains. Therefore, the ability of Cys to substitute for Met requirement is not just biol. interesting; it is also of considerable economic and social importance. The primary goal of this review is to discuss the available evidence on the effect of Cys substitution for Met to meet the total sulfur amino acid requirement in adult humans, including an assessment of the methodol. features of expts. with conflicting results. Assessment of the requirement expts. for amino acids with complex metab. such as Met and Cys must begin with a careful definition of requirements and what substitution means. As a result of these definitions, a set of criteria for the intakes of Met that will allow demonstration of the substitution effect were developed. Some recent publications are assessed using these definitions and criteria, and a possible reason for the conflicting results in the literature is proposed. An approach to estg. tolerable upper intakes is also proposed. Research on in vivo S amino acid metab. in humans is tremendously difficult, and therefore, we do not wish to be overly crit. of the high-quality work of the ambitious and highly intelligent men and women who have conducted various studies. Our goal is to objectively review the data for the reader in a logical and comprehensive manner and propose methods that may avoid difficulties in future studies.
  552. 552
    Mishanina, T. V.; Libiad, M.; Banerjee, R. Biogenesis of Reactive Sulfur Species for Signaling by Hydrogen Sulfide Oxidation Pathways. Nat. Chem. Biol. 2015, 11 (7), 457– 64,  DOI: 10.1038/nchembio.1834
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    Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways
    Mishanina, Tatiana V.; Libiad, Marouane; Banerjee, Ruma
    Nature Chemical Biology (2015), 11 (7), 457-464CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    A review. The chem. species involved in H2S signaling remain elusive despite the profound and pleiotropic physiol. effects elicited by this mol. The dominant candidate mechanism for sulfide signaling is persulfidation of target proteins. However, the relatively poor reactivity of H2S toward oxidized thiols, such as disulfides, the low concn. of disulfides in the reducing milieu of the cell and the low steady-state concn. of H2S raise questions about the plausibility of persulfide formation via reaction between an oxidized thiol and a sulfide anion or a reduced thiol and oxidized hydrogen disulfide. In contrast, sulfide oxidn. pathways, considered to be primarily mechanisms for disposing of excess sulfide, generate a series of reactive sulfur species, including persulfides, polysulfides and thiosulfate, that could modify target proteins. We posit that sulfide oxidn. pathways mediate sulfide signaling and that sulfurtransferases ensure target specificity.
  553. 553
    Coleman, J.; Blake-Kalff, M.; Davies, E. Detoxification of Xenobiotics by Plants: Chemical Modification and Vacuolar Compartmentation. Trends Plant Sci. 1997, 2 (4), 144– 51,  DOI: 10.1016/S1360-1385(97)01019-4
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    Tsay, J. T.; Oh, W.; Larson, T. J.; Jackowski, S.; Rock, C. O. Isolation and Characterization of the Beta-Ketoacyl-Acyl Carrier Protein Synthase III Gene (FabH) from Escherichia Coli K-12. J. Biol. Chem. 1992, 267 (10), 6807– 14,  DOI: 10.1016/S0021-9258(19)50498-7
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    557
    Isolation and characterization of the β-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12
    Tsay, Jiu Tsair; Oh, Won; Larson, Timothy J.; Jackowski, Suzanne; Rock, Charles O.
    Journal of Biological Chemistry (1992), 267 (10), 6807-14CODEN: JBCHA3; ISSN:0021-9258.
    β-Ketoacyl-acyl carrier protein (ACP) synthase III catalyzes the condensation of acetyl-CoA with malonyl-ACP in dissocd. (Type II) fatty acid synthase systems. A synthase III mutant was used to localize the structural gene to the 24.5-min region to localize the structural gene to the 24.5-min region of the E. coli chromosome, and the defective synthase III allele was designated fabH1. The fabH gene was identified on a 1.3-kilobase NruI-HindIII chromosomal DNA fragment (plasmid pWO114) that complemented the enzymic defect in fabH1 strains. The NruI-HinIII fragment was sequenced and contained a single open reading frame predicted to encode a 33,517-dalton protein with an isoelec. point of 4.85. The fabH sequence contained an Ala-Cys-Ala tripeptide characteristic of condensing enzyme active sites. A T7 expression system showed that the NruI-HindIII fragment directed the synthesis of a single 34,800-dalton protein. This protein was purified and the order of the N-terminal 30 residues of the protein corresponded exactly to the amino acid structure predicted from the DNA sequence. The purified protein possessed both acetoacetyl-ACP synthase and acetyl-CoA:ACP transacylase activities, and cells harboring plasmid pWO114 overproduced the two activities, supporting the conclusion that a single protein carries out both reactions. Overprodn. of synthase III resulted in a significant increase in shorter-chain fatty acids in the membrane phospholipids. These catalytic properties are consistent with the proposed role of synthase III in the initiation of fatty acid synthesis.
  555. 555
    Gao, T.; Yang, C.; Zheng, Y. G. Comparative Studies of Thiol-Sensitive Fluorogenic Probes for HAT Assays. Anal. Bioanal. Chem. 2013, 405 (4), 1361– 71,  DOI: 10.1007/s00216-012-6522-5
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    558
    Comparative studies of thiol-sensitive fluorogenic probes for HAT assays
    Gao, Tielong; Yang, Chao; Zheng, Yujun George
    Analytical and Bioanalytical Chemistry (2013), 405 (4), 1361-1371CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    Histone acetyltransferases (HATs) catalyze the acetylation of specific lysine residues in histone and nonhistone proteins. Recent studies showed that acetylation is widely distributed among cellular proteins, suggestive of diverse functions of HATs in cellular pathways. Nevertheless, currently available assays for HAT activity study are still quite limited. Here, we evaluated a series of thiol-sensitive fluorogenic compds. for the detection of the enzymic activities of different HAT proteins. Upon conjugation to the thiol group of HSCoA, these mols. gain enhanced quantum yields and strong fluorescence, permitting facile quantitation of HAT activities. We investigated and compared the assay performances of these fluorogenic compds. for their capability as HAT activity reporters, including kinetics of reaction with HSCoA, influence on HAT activity, and fluorescence amplification factors. Our data suggest that CPM and coumarin maleic acid ester are excellent HAT probes owing to their fast reaction kinetics and dramatic fluorescence enhancement during the HAT reaction. Further, the microtiter plate measurements show that this fluorescent approach is robust and well suited for adaptation to high-throughput screening of small mol. inhibitors of HATs, highlighting the value of this assay strategy in new drug discovery.
  556. 556
    Tang, Y.; Jin, L.; Yin, B. A Dual-Selective Fluorescent Probe for GSH and Cys Detection: Emission and pH Dependent Selectivity. Anal. Chim. Acta 2017, 993, 87– 95,  DOI: 10.1016/j.aca.2017.09.028
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    559
    A dual-selective fluorescent probe for GSH and Cys detection: Emission and pH dependent selectivity
    Tang, Yunqiang; Jin, Longyi; Yin, Bingzhu
    Analytica Chimica Acta (2017), 993 (), 87-95CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    A novel fluorescent probe 1 (I) based on acridine orange was developed for the selective detection and bioimaging of biothiols. The probe exhibits higher selectivity and turn-on fluorescence response to cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) than to other amino acids. Importantly, the probe responds to GSH and Cys/Hcy with distinct fluorescence emissions in PBS buffer at pH of 7.4. The Cys/Hcy-triggered tandem SNAr-rearrangement reaction and GSH-induced SNAr reaction with the probe led to the corresponding amino-acridinium and thio-acridinium dyes, resp., which can discriminate GSH from Cys/Hcy through different emission channels. Cys finishes the tandem reaction with the probe and subsequently forms amino-acridinium and Hcy/GSH induces SNAr reaction with the probe to form thio-acridiniums at weakly acidic conditions (pH 6.0), enabling Cys to be discriminated from Hcy/GSH at different emissions. Finally, probe I can selectively probe GSH over Cys and Hcy or Cys over GSH and Hcy in HeLa cells through multicolor imaging.
  557. 557
    Zaric, B. L.; Obradovic, M.; Bajic, V.; Haidara, M. A.; Jovanovic, M.; Isenovic, E. R. Homocysteine and Hyperhomocysteinaemia. Curr. Med. Chem. 2019, 26 (16), 2948– 61,  DOI: 10.2174/0929867325666180313105949
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    560
    Homocysteine and Hyperhomocysteinaemia
    Zaric, Bozidarka L.; Obradovic, Milan; Bajic, Vladan; Haidara, Mohamed A.; Jovanovic, Milos; Isenovic, Esma R.
    Current Medicinal Chemistry (2019), 26 (16), 2948-2961CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)
    A review. Homocysteine (Hcy) is a thiol group contg. the amino acid, which naturally occurs in all humans. Hcy is degraded in the body through two metabolic pathways, while a minor part is excreted through kidneys. The chem. reactions that are necessary for degrdn. of Hcy require the presence of folic acid, vitamins B6 and B12. Consequently, the level of the total Hcy in the serum is influenced by the presence or absence of these vitamins. An elevated level of the Hcy, hyperhomocysteinemia (HHcy) and homocystinuria is connected with occlusive artery disease, esp. in the brain, the heart, and the kidney, in addn. to venous thrombosis, chronic renal failure, megaloblastic anemia, osteoporosis, depression, Alzheimer's disease, pregnancy problems, and others. Elevated Hcy levels are connected with various pathologies both in adult and child population. Causes of HHcy include genetic mutations and enzyme deficiencies in 5, 10-methylenetetrahydrofolate reductase (MTHFR) methionine synthase (MS), and cystathionine β-synthase (CβS). HHcy can be caused by deficiencies in the folate, vitamin B12 and to a lesser extent, deficiency in B6 vitamin what influences methionine metab. Addnl., HHcy can be caused by the rich diet and renal impairment. This review presents literature data from recent research related to Hcy metab. and the etiol. of the Hcy blood level disorder. In addn., we also described various pathol. mechanisms induced by hereditary disturbances or nutritional influences and their assocn. with HHcy induced pathol. in adults and children and treatment of these metabolic disorders.
  558. 558
    van Meurs, J.; Dhonukshe-Rutten, R. A.; Pluijm, S. M.; Van Der Klift, M.; De Jonge, R.; Lindemans, J.; De Groot, L. C.; Hofman, A.; Witteman, J. C.; Van Leeuwen, J. P.; Breteler, M. M.; Lips, P.; Pols, H. A.; Uitterlinden, A. G. Homocysteine Levels and the Risk of Osteoporotic Fracture. N. Engl. J. Med. 2004, 350, 2033– 41,  DOI: 10.1056/NEJMoa032546
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    561
    Homocysteine levels and the risk of osteoporotic fracture
    van Meurs, Joyce B. J.; Dhonukshe-Rutten, Rosalie A. M.; Pluijm, Saskia M. F.; van der Klift, Marjolein; de Jonge, Robert; Lindemans, Jan; de Groot, Lisette C. P. G. M.; Hofman, Albert; Witteman, Jacqueline C. M.; van Leeuwen, Johannes P. T. M.; Breteler, Monique M. B.; Lips, Paul; Pols, Huibert A. P.; Uitterlinden, Andre G.
    New England Journal of Medicine (2004), 350 (20), 2033-2041CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
    Very high plasma homocysteine levels are characteristic of homocystinuria, a rare autosomal recessive disease accompanied by the early onset of generalized osteoporosis. We therefore hypothesized that mildly elevated homocysteine levels might be related to age-related osteoporotic fractures. We therefore hypothesized that mildly elevated homocysteine levels might be related to age-related osteoporotic fractures. In the Rotterdam Study, there were two independent cohorts: 562 subjects in cohort 1, with a mean follow-up period of 8.1 yr; and 553 subjects in cohort 2, with a mean follow-up period of 5.7 yr. In the Longitudinal Aging Study Amsterdam, there was a single cohort of 1291 subjects, with a mean follow-up period of 2.7 yr. Multivariate Cox proportional-hazards regression models were used for anal. of the risk of fracture, with adjustment for age, sex, body-mass index, and other characteristics that may be assocd. with the risk of fracture or with increased homocysteine levels. During 11,253 person-years of follow-up, osteoporotic fractures occurred in 191 subjects. The overall multivariable-adjusted relative risk of fracture was 1.4 (95 percent confidence interval, 1.2 to 1.6) for each increase of 1 SD in the natural-log-transformed homocysteine level. The risk was similar in all three cohorts studied, and it was also similar in men and women. A homocysteine level in the highest age-specific quartile was assocd. with an increase by a factor of 1.9 in the risk of fracture (95 percent confidence interval, 1.4 to 2.6). The assocns. between homocysteine levels and the risk of fracture appeared to be independent of bone mineral d. and other potential risk factors for fracture. An increased homocysteine level appears to be a strong and independent risk factor for osteoporotic fractures in older men and women.
  559. 559
    Lee, D.; Kim, G.; Yin, J.; Yoon, J. An Aryl-Thioether Substituted Nitrobenzothiadiazole Probe for the Selective Detection of Cysteine and Homocysteine. Chem. Commun. 2015, 51 (30), 6518– 20,  DOI: 10.1039/C5CC01071C
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    562
    An aryl-thioether substituted nitrobenzothiadiazole probe for the selective detection of cysteine and homocysteine
    Lee, Dayoung; Kim, Gyoungmi; Yin, Jun; Yoon, Juyoung
    Chemical Communications (Cambridge, United Kingdom) (2015), 51 (30), 6518-6520CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    An aryl-thioether substituted nitrobenzothiadiazole probe was synthesized and employed to detect cysteine and homocysteine selectively in living cells. Interestingly, both cysteine (Cys) and homocysteine (Hcy) promote an enhancement of the fluorescence intensity of the probe at pH 7.4 while only Cys gives rise to this enhancement under weakly acidic conditions (pH 6.0).
  560. 560
    Giannoulis, K. M.; Giokas, D. L.; Tsogas, G. Z.; Vlessidis, A. G. Ligand-Free Gold Nanoparticles as Colorimetric Probes for the Non-Destructive Determination of Total Dithiocarbamate Pesticides after Solid Phase Extraction. Talanta 2014, 119, 276– 83,  DOI: 10.1016/j.talanta.2013.10.063
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    563
    Ligand-free gold nanoparticles as colorimetric probes for the non-destructive determination of total dithiocarbamate pesticides after solid phase extraction
    Giannoulis, Kyriakos M.; Giokas, Dimosthenis L.; Tsogas, George Z.; Vlessidis, Athanasios G.
    Talanta (2014), 119 (), 276-283CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
    In this work, we describe a simple and sensitive non-destructive method for the detn. of the total concn. of dithiocarbamate fungicides (DTCs) in real samples. The proposed method combines for the first time the benefits of an extn. method for sample clean-up and preconcn. with a sensitive colorimetric assay based on gold nanoparticle probes. In this two-step procedure, the target DTCs are isolated from the matrix and preconcd. by solid phase extn. onto com. available C18 sorbents. Following elution, the ext. contg. the target dithiocarbamates, free from most interferences and matrix components, is delivered into an aq. dispersion of plain citrate-capped gold nanoparticles (AuNPs) which aggregate in response to DTCs coordination on AuNPs surface through multiple gold thiolate bonds. This aggregation is evidenced by changes in the spectral properties of the soln. involving a decrease in the original absorbance of Au nanoparticles at 522 nm and the appearance of a new absorption band above 700 nm. An ensuing chromatic shift of the soln. from wine-red to purple-blue is obsd. which is visual by naked eye at concns. as low as 50 μg L-1. Further improvement in the detection limits can be accomplished by scaling-down the method to micro-vol. conditions alleviating the need to preconc. larger sample vols. Overall, by combining sample clean-up and preconcn. with the strong affinity of DTC thiol group for the gold surface, the total concn. of dithiocarbamate pesticides was successfully detd. in various water samples at the low and ultra-low μg L-1 levels without resorting to destructive techniques, sophisticated instrumentation or post-synthetic modification of gold nanoparticles. Method application in real samples showed good anal. features in terms of recoveries (81.0-94.0%), precision (5.6-8.9%) and reproducibility (∼9%) rendering the method as an attractive alternative to current methodologies for the detn. of DTC fungicide residues in samples of environmental interest.
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    Munday, R. Toxicity of Thiols and Disulphides: Involvement of Free-Radical Species. Free Radical Biol. Med. 1989, 7 (6), 659– 73,  DOI: 10.1016/0891-5849(89)90147-0
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    564
    Toxicity of thiols and disulfides: involvement of free-radical species
    Munday, Rex
    Free Radical Biology & Medicine (1989), 7 (6), 659-73CODEN: FRBMEH; ISSN:0891-5849.
    A review with 188 refs. Sulfur is essential to life, and thiols and disulfides play essential roles in cellular biochem. Such compds. are also widely distributed in the food of man and his domestic animals, and they are extensively used in industry. However, many thiols and disulfides have been shown to be toxic. Aliph., arom., and heterocyclic compds. of this type are hemolytic agents in animals, while aminothiols have been shown to induce many cytotoxic effects in vitro and the epidithiodioxopiperazine mycotoxin, sporidesmin, is a potent hepatotoxic agent. Structure-activity relationships among these compds. and factors which modulate their harmful effects are consistent with a toxic mechanism involving redox cycling between the thiol and the corresponding disulfide. Thiyl radicals and active oxygen species are formed in this process, and it is suggested that these substances are responsible for initiating the tissue damage provoked by thiols and disulfides.
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    Cazzola, M.; Calzetta, L.; Page, C.; Rogliani, P.; Matera, M. G. Thiol-Based Drugs in Pulmonary Medicine: Much More Than Mucolytics. Trends Pharmacol. Sci. 2019, 40 (7), 452– 63,  DOI: 10.1016/j.tips.2019.04.015
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    565
    Thiol-Based Drugs in Pulmonary Medicine: Much More than Mucolytics
    Cazzola, Mario; Calzetta, Luigino; Page, Clive; Rogliani, Paola; Matera, Maria Gabriella
    Trends in Pharmacological Sciences (2019), 40 (7), 452-463CODEN: TPHSDY; ISSN:0165-6147. (Elsevier Ltd.)
    Thiol-based drugs are considered as mucolytics because they decrease the viscosity and mostly decrease the elasticity of bronchial secretions by reducing disulfide bonds in proteins. However, they can also act as antioxidant drugs directly through free sulfhydryl groups that serve as a source of reducing equiv., as well as indirectly through the replenishment of intracellular glutathione (GSH) levels. Modulation of neurokinin A levels may also be related to the effect of thiol drugs on oxidative stress. Moreover, thiol-based drugs interfere with inflammatory pathways and modulate human bronchial tone. They might also be considered as therapeutic agents against some types of infection because they reduce bacterial adhesion to the respiratory epithelial cell surface and inhibit biofilm formation, causing biofilm disruption and thereby improving the efficacy of antibiotic therapy.
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    Wang, H.; Wu, X.; Yang, S.; Tian, H.; Liu, Y.; Sun, B. A Rapid and Visible Colorimetric Fluorescent Probe for Benzenethiol Flavor Detection. Food Chem. 2019, 286, 322– 8,  DOI: 10.1016/j.foodchem.2019.02.033
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    566
    A rapid and visible colorimetric fluorescent probe for benzenethiol flavor detection
    Wang, Hao; Wu, Xiaoming; Yang, Shaoxiang; Tian, Hongyu; Liu, Yongguo; Sun, Baoguo
    Food Chemistry (2019), 286 (), 322-328CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)
    Benzenethiols are a class of flavoring ingredients used in the food, pharmaceutical, cosmetics and chem. industries. A rapid and visible colorimetric fluorescent probe was developed for the detection of benzenethiol flavors. It provides rapid quant. detection of benzenethiols at low levels, down to a limit of 10 nM. Test paper contg. the probe changes color according to benzenethiol concn. (from colorless to pink, visible with the naked eye). The probe was also successfully used to test benzenethiol concns. in real food samples. This study demonstrates that this novel probe can be employed as a benzenethiol testing tool.
  564. 564
    Liu, X.-L.; Duan, X.-Y.; Du, X.-J.; Song, Q.-H. Quinolinium-Based Fluorescent Probes for the Detection of Thiophenols in Environmental Samples and Living Cells. Chem.─Asian J. 2012, 7, 2696– 2702,  DOI: 10.1002/asia.201200594
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    567
    Quinolinium-Based Fluorescent Probes for the Detection of Thiophenols in Environmental Samples and Living Cells
    Liu, Xiu-Ling; Duan, Xue-You; Du, Xiao-Jiao; Song, Qin-Hua
    Chemistry - An Asian Journal (2012), 7 (11), 2696-2702, S2696/1-S2696/12CODEN: CAAJBI; ISSN:1861-4728. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A new type of fluorescent probes for thiophenols, 6HQM-DNP (I) and 7HQM-DNP (II), contg. 6- or 7-hydroxy quinonlinium as fluorophore and 2,4-dinitrophenoxy (DNP) as nucleophilic recognition unit were constructed. As ethers, these nonfluorescent probe mols. can release the corresponding fluorescent quinolinium (6HQM and 7HQM) through arom. nucleophilic substitution (SNAr) by thiolate anions from thiophenols. The sensing reaction is highly sensitive (detection limit of 8 nM for 7HQM-DNP) and highly selective to thiophenols over aliph. thiols and other nucleophiles under neutral conditions (pH 7.3). The probes respond rapidly to thiophenols, with 2nd-order rate consts. k = 45 M-1 s-1 for 7HQM-DNP and 24 M-1 s-1 for 6HQM-DNP. Also, the selective detection of thiophenols in living cells by 7HQM-DNP was demonstrated by confocal fluorescence imaging. These quinolinium salts show excellent chem. and thermal stability. In conclusion, this type of probes may find use in the detection of thiophenols in environmental samples and biosystems.
  565. 565
    He, L.; Yang, X.; Xu, K.; Kong, X.; Lin, W. A Multi-Signal Fluorescent Probe for Simultaneously Distinguishing and Sequentially Sensing Cysteine/Homocysteine, Glutathione, and Hydrogen Sulfide in Living Cells. Chem. Sci. 2017, 8 (9), 6257– 65,  DOI: 10.1039/C7SC00423K
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    568
    A multi-signal fluorescent probe for simultaneously distinguishing and sequentially sensing cysteine/homocysteine, glutathione, and hydrogen sulfide in living cells
    He, Longwei; Yang, Xueling; Xu, Kaixin; Kong, Xiuqi; Lin, Weiying
    Chemical Science (2017), 8 (9), 6257-6265CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Biothiols, which have a close network of generation and metabolic pathways among them, are essential reactive sulfur species (RSS) in the cells and play vital roles in human physiol. However, biothiols possess highly similar chem. structures and properties, resulting in it being an enormous challenge to simultaneously discriminate them from each other. Herein, we develop a unique fluorescent probe (HMN) for not only simultaneously distinguishing Cys/Hcy, GSH, and H2S from each other, but also sequentially sensing Cys/Hcy/GSH and H2S using a multi-channel fluorescence mode for the first time. When responding to the resp. biothiols, the robust probe exhibits multiple sets of fluorescence signals at three distinct emission bands (blue-green-red). The new probe can also sense H2S at different concn. levels with changes of fluorescence at the blue and red emission bands. In addn., the novel probe HMN is able to discriminate and sequentially sense biothiols in biol. environments via three-color fluorescence imaging. We expect that the development of the robust probe HMN will provide a powerful strategy to design fluorescent probes for the discrimination and sequential detection of biothiols, and offer a promising tool for exploring the interrelated roles of biothiols in various physiol. and pathol. conditions.
  566. 566
    Wang, N.; Chen, M.; Gao, J.; Ji, X.; He, J.; Zhang, J.; Zhao, W. A Series of BODIPY-Based Probes for the Detection of Cysteine and Homocysteine in Living Cells. Talanta 2019, 195, 281– 9,  DOI: 10.1016/j.talanta.2018.11.066
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    569
    A series of BODIPY-based probes for the detection of cysteine and homocysteine in living cells
    Wang, Nannan; Chen, Miao; Gao, Jinhua; Ji, Xin; He, Jinling; Zhang, Jian; Zhao, Weili
    Talanta (2019), 195 (), 281-289CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
    Biothiols, such as glutathione (GSH), homocysteine (Hcy) and cysteine (Cys), are important biomarkers and play crucial roles in many physiol. processes. Thus, the detection of biothiols is highly important for early diagnosis of diseases and evaluation of disease progression. Herein, new types of BODIPY-based fluorescent probes (probe 1, probe 2 and probe 3) capable of cysteine (Cys)/homocysteine (Hcy) sensing with high selectivity over other amino acids were developed. In addn., the authors further studied the influence of different electronegativity substituents on these probes to sensing Cys/Hcy. Ultimately, the electron withdrawing group on probe 1 can accelerate the probe response to Cys/Hcy, and probe 1 was successfully applied for selective imaging Cys/Hcy in living cells.
  567. 567
    Yao, Z.; Ge, W.; Guo, M.; Xiao, K.; Qiao, Y.; Cao, Z.; Wu, H.-C. Ultrasensitive Detection of Thiophenol Based on a Water-Soluble Pyrenyl Probe. Talanta 2018, 185, 146– 50,  DOI: 10.1016/j.talanta.2018.03.068
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    Ultrasensitive detection of thiophenol based on a water-soluble pyrenyl probe
    Yao, Zhiyi; Ge, Wenqi; Guo, Mingwei; Xiao, Keren; Qiao, Yadong; Cao, Zhong; Wu, Hai-Chen
    Talanta (2018), 185 (), 146-150CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
    We report a simple pyrene-based fluorescent probe, sodium 8-(2,4-dinitrophenoxy)pyrene-1,3,6-trisulfonate (HPTS-DNP), for the ultrasensitive and visual detection of thiophenol in 100% aq. media. The sensing mechanism of this method is based on nucleophilic arom. substitution on HPTS-DNP caused by thiophenol to afford a highly fluorescent product, 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS). The extremely low fluorescence background of HPTS-DNP and high quantum yield of HPTS ensure the superior sensing performance toward thiophenol, including rapid responses, off-on detection mode and excellent sensitivity. The detection limit is as low as 0.49 nmol L-1 according to the measurements with a spectrofluorimeter. This probe also features good selectivity (response ratio of thiophenol to interferents >30), precision (2.93%) and accuracy (102%). This approach could be applied for monitoring the thiophenol concns. in environmental water samples. HPTS-DNP also showed good cell permeation capacity and low cellular cytotoxicity, indicating further application in bioimaging studies.
  568. 568
    Zhang, S.; Cai, F.; Hou, B.; Chen, H.; Gao, C.; Shen, X.-c.; Liang, H. Constructing a Far-Red to near-Infrared Fluorescent Probe for Highly Specific Detection of Cysteine and Its Bioimaging Applications in Living Cells and Zebrafish. New J. Chem. 2019, 43 (17), 6696– 701,  DOI: 10.1039/C9NJ00260J
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    571
    Constructing a far-red to near-infrared fluorescent probe for highly specific detection of cysteine and its bioimaging applications in living cells and zebrafish
    Zhang, Shuping; Cai, Fangyuan; Hou, Bo; Chen, Hua; Gao, Cunji; Shen, Xing-can; Liang, Hong
    New Journal of Chemistry (2019), 43 (17), 6696-6701CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
    Cys is a common and important sulfur-contg. amino acid in living organisms, whose intracellular level changes are assocd. with a variety of diseases. To specifically detect Cys without interference from other thiol species has considerable significance. However, only selective fluorescent probes are available for cysteine detection; other thiol species can also have certain interference in the sensing process. Herein, we report an irreversible fluorescent probe for the specific detection of cysteine without interference from other thiol species both in living cells and zebrafish. The FR-NIR-Cys probe exhibits a large emission wavelength shift (up to 84 nm) and a significant ratio fluorescence enhancement (up to 41-fold) in the far-red and near-IR region. Furthermore, we demonstrate that FR-NIR-Cys with good cell permeability can be employed in the far-red to near-IR imaging of Cys in living cells and zebrafish.
  569. 569
    Fan, L.; Zhang, W.; Wang, X.; Dong, W.; Tong, Y.; Dong, C.; Shuang, S. A Two-Photon Ratiometric Fluorescent Probe for Highly Selective Sensing of Mitochondrial Cysteine in Live Cells. Analyst 2019, 144 (2), 439– 47,  DOI: 10.1039/C8AN01908H
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    A two-photon ratiometric fluorescent probe for highly selective sensing of mitochondrial cysteine in live cells
    Fan, Li; Zhang, Wenjia; Wang, Xiaodong; Dong, Wenjuan; Tong, Yaoli; Dong, Chuan; Shuang, Shaomin
    Analyst (Cambridge, United Kingdom) (2019), 144 (2), 439-447CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    We report herein a two-photon ratiometric fluorescent probe (DNEPI) for mitochondrial cysteine (Cys) detection on the basis of a merocyanine (compd. 1) as the two-photon fluorophore and a 2,4-dinitrobenzensulfonyl (DNBS) unit as the biothiol reaction site. Upon reaction with Cys in DMSO/PBS (1/1, vol./vol.), DNEPI showed a distinct ratiometric fluorescence emission characteristic (F583 nm/F485 nm) linearly proportional to Cys concns. over the range of 2-10 μM, which was attribute to the enhanced intramol. charge transfer (ICT) effect by cleavage of the sulfonic acid ester bond of DNEPI to release compd. 1. More importantly, the probe could detect Cys with a fast response time (within 2 min) and the detection limit was quant. calcd. as 0.29 μM. Furthermore, DNEPI not only exhibited high selectivity toward Cys over other similar biothiols, including homocysteine (Hcy) and glutathione (GSH), but also displayed significant mitochondrial-targeting ability, which were favorable for mitochondrial Cys-selective imaging. Subsequently, application of DNEPI to Cys imaging in live cells was successfully achieved by two-photon fluorescence microscopy, suggesting that the probe proposed here could be used to monitor mitochondrial Cys concn. changes in live cells with negligible interference from other biol. thiols.
  570. 570
    Ros-Lis, J. V.; García, B.; Jiménez, D.; Martínez-Máñez, R.; Sancenón, F.; Soto, J.; Gonzalvo, F.; Valldecabres, M. C. Squaraines as Fluoro-Chromogenic Probes for Thiol-Containing Compounds and Their Application to the Detection of Biorelevant Thiols. J. Am. Chem. Soc. 2004, 126 (13), 4064– 5,  DOI: 10.1021/ja031987i
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    Squaraines as fluoro-chromogenic probes for thiol-containing compounds and their application to the detection of biorelevant thiols
    Ros-Lis, Jose V.; Garcia, Beatriz; Jimenez, Diego; Martinez-Manez, Ramon; Sancenon, Felix; Soto, Juan; Gonzalvo, Fernando; Valldecabres, M. Carmen
    Journal of the American Chemical Society (2004), 126 (13), 4064-4065CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A highly selective colorimetric chemodosimeter for thiol-contg. compds. in aq. solns. is reported. The design protocol makes use of a highly specific reaction between thiols and the electrophilic four-membered ring of highly colored, fluorescent squaraine backbones. At neutral pH selective decoloration and total emission quenching was found due to the rupture of the highly delocalized squaraine framework upon selective nucleophilic addn. of thiol-contg. derivs. The squaraine derivs. have been successfully applied to the detn. of low-mol. mass aminothiols in human plasma. The method utters the high potential applicability of the chemodosimeter approach in the search for new or improved chromogenic selective or specific probes for target guests.
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    Zhang, X.; Li, C.; Cheng, X.; Wang, X.; Zhang, B. A near-Infrared Croconium Dye-Based Colorimetric Chemodosimeter for Biological Thiols and Cyanide Anion. Sens. Actuators B Chem. 2008, 129 (1), 152– 7,  DOI: 10.1016/j.snb.2007.07.094
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    574
    A near-infrared croconium dye-based colorimetric chemodosimeter for biological thiols and cyanide anion
    Zhang, Xuehua; Li, Chao; Cheng, Xuexin; Wang, Xuesong; Zhang, Baowen
    Sensors and Actuators, B: Chemical (2008), 129 (1), 152-157CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    A near-IR colorimetric chemodosimeter based on a croconium dye, 1,3-bis(4-N,N-diethylamino-2-hydroxyl-phenyl) croconine, was developed to selectively and sensitively probe cysteine, homocysteine, or cysteine-glycine at pH 5.7 and cyanide anion at pH 9.0 in aq. ethanol solns.
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    Tanaka, F.; Mase, N.; Barbaa, C. F., III Determination of Cysteine Concentration by Fluorescence Increase: Reaction of Cysteine with a Fluorogenic Aldehyde. Chem. Commun. 2004, 2004, 1762,  DOI: 10.1039/b405642f
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    Jung, H. S.; Ko, K. C.; Kim, G.-H.; Lee, A.-R.; Na, Y.-C.; Kang, C.; Lee, J. Y.; Kim, J. S. Coumarin-Based Thiol Chemosensor: Synthesis, Turn-On Mechanism, and Its Biological Application. Org. Lett. 2011, 13 (6), 1498– 501,  DOI: 10.1021/ol2001864
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    Coumarin-Based Thiol Chemosensor: Synthesis, Turn-On Mechanism, and Its Biological Application
    Jung, Hyo Sung; Ko, Kyoung Chul; Kim, Gun-Hee; Lee, Ah-Rah; Na, Yun-Cheol; Kang, Chulhun; Lee, Jin Yong; Kim, Jong Seung
    Organic Letters (2011), 13 (6), 1498-1501CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
    A new chemodosimetric probe (I) is reported that selectively detects thiols over other relevant biol. species by the turning on of its fluorescence through a Michael type reaction. The fluorogenic process upon its reaction was revealed to be mediated by intramol. charge transfer, as confirmed by time-dependent d. functional theory calcns. The application of probe I to cells is also examd. by confocal microscopy, and its cysteine preference was obsd. by an ex vivo LC-MS anal. of the cellular metabolite.
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    Zhang, Y.; Shao, X.; Wang, Y.; Pan, F.; Kang, R.; Peng, F.; Huang, Z.; Zhang, W.; Zhao, W. Dual Emission Channels for Sensitive Discrimination of Cys/Hcy and GSH in Plasma and Cells. Chem. Commun. 2015, 51 (20), 4245– 8,  DOI: 10.1039/C4CC08687B
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    Dual emission channels for sensitive discrimination of Cys/Hcy and GSH in plasma and cells
    Zhang, Yuanlin; Shao, Xiangmin; Wang, Yue; Pan, Fuchao; Kang, Ruixue; Peng, Fangfang; Huang, Zhentao; Zhang, Weijuan; Zhao, Weili
    Chemical Communications (Cambridge, United Kingdom) (2015), 51 (20), 4245-4248CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A new selective fluorescent and colorimetric chemosensor for the detection of GSH was developed. The discrimination of GSH from Cys and Hcy is achieved through two emission channel detection. The detection limit of probe 1 for GSH reached 10 nM (3 ppb). The excellent sensitivity and selectivity of probe 1 allow the selective detection of GSH over Cys and Hcy, which can be visualized colorimetrically and/or fluorescently. The sensitive detection of GSH allowed for convenient measurement of the GSH content in human plasma. The presence of GSH in cells was demonstrated through cell imaging.
  575. 575
    Niu, L.-Y.; Guan, Y.-S.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. A Turn-On Fluorescent Sensor for the Discrimination of Cystein from Homocystein and Glutathione. Chem. Commun. 2013, 49 (13), 1294– 6,  DOI: 10.1039/c2cc38429a
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    578
    A turn-on fluorescent sensor for the discrimination of cystein from homocystein and glutathione
    Niu, Li-Ya; Guan, Ying-Shi; Chen, Yu-Zhe; Wu, Li-Zhu; Tung, Chen-Ho; Yang, Qing-Zheng
    Chemical Communications (Cambridge, United Kingdom) (2013), 49 (13), 1294-1296CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    The authors report a turn-on fluorescent sensor based on nitrothiophenolate boron dipyrromethene (BODIPY) derivs. for the discrimination of cysteine (Cys) from homocysteine (Hcy) and glutathione (GSH). The sensor was applied for detection of Cys in living cells.
  576. 576
    Chen, W.; Luo, H.; Liu, X.; Foley, J. W.; Song, X. Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo. Anal. Chem. 2016, 88 (7), 3638– 46,  DOI: 10.1021/acs.analchem.5b04333
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    579
    Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo
    Chen, Wenqiang; Luo, Hongchen; Liu, Xingjiang; Foley, James W.; Song, Xiangzhi
    Analytical Chemistry (Washington, DC, United States) (2016), 88 (7), 3638-3646CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are small biomol. thiols that are present in all cells and extracellular fluids of healthy mammals. It is well-known that each plays a sep., critically important role in human physiol. and that abnormal levels of each are predictive of a variety of different disease states. Although a no. of fluorescence-based methods have been developed that can detect biomols. that contain sulfhydryl moieties, few are able to differentiate between GSH and Cys/Hcy. In this report, we demonstrate a broadly applicable approach for the design of fluorescent probes that can achieve this goal. The strategy we employ is to conjugate a fluorescence-quenching 7-nitro-2,1,3-benzoxadiazole (NBD) moiety to a selected fluorophore (Dye) through a sulfhydryl-labile ether linkage to afford nonfluorescent NBD-O-Dye. In the presence of GSH or Cys/Hcy, the ether bond is cleaved with the concomitant generation of both a nonfluorescent NBD-S-R deriv. and a fluorescent dye having a characteristic intense emission band (B1). In the special case of Cys/Hcy, the NBD-S-Cys/Hcy cleavage product can undergo a further, rapid, intramol. Smiles rearrangement to form a new, highly fluorescent NBD-N-Cys/Hcy compd. (band B2); because of geometrical constraints, the GSH derived NBD-S-GSH deriv. cannot undergo a Smiles rearrangement. Thus, the presence of a single B1 or double B1 + B2 signature can be used to detect and differentiate GSH from Cys/Hcy, resp. We demonstrate the broad applicability of our approach by including in our studies members of the Flavone, Bodipy, and Coumarin dye families. Particularly, single excitation wavelength could be applied for the probe NBD-OF in the detection of GSH over Cys/Hcy in both aq. soln. and living cells.
  577. 577
    Mikaliunaite, L.; Green, D. B. Using a 3-Hydroxyflavone Derivative as a Fluorescent Probe for the Indirect Determination of Aminothiols Separated by Ion-Pair HPLC. Anal. Methods 2021, 13 (26), 2915– 25,  DOI: 10.1039/D1AY00499A
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    580
    Using a 3-hydroxyflavone derivative as a fluorescent probe for the indirect determination of aminothiols separated by ion-pair HPLC
    Mikaliunaite, Lina; Green, David B.
    Analytical Methods (2021), 13 (26), 2915-2925CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)
    Homocysteine, cysteine, cysteinyl-glycine, and glutathione are significant biol. aminothiols (ATs) that are marker-mols. in Down syndrome, Alzheimer's disease, or have been implicated as risk factors in atherosclerosis and other vascular diseases, and therefore rapid detn. of these mols. is desirable. After redn. of the disulfides, a widely used method utilizes derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) as a fluorogenic probe prior to reversed-phase HPLC sepn. followed by fluorescence detection. The traditional HPLC detn. of ATs is time consuming and economically expensive. We have developed an ion-pair HPLC method coupled with indirect fluorescence detection after post-column reaction with a 2,4-dinitrobenzenesulfonate deriv. of a 3-hydroxyflavone. The accuracy, precision, post-column temp. and residence time, and limit-of-detection were evaluated. Sample throughput and reduced sample prepn. time of over an hour for the existing methods to less than 20 min for the new method is also demonstrated. No statistical differences in HCy, Cys, or Cys-Gly detns. in plasma samples were obsd. between our method and the traditional HPLC method.
  578. 578
    Toyooka, T.; Imai, K. New Fluorogenic Reagent Having Halogenobenzofurazan Structure for Thiols: 4-(Aminosulfonyl)-7-Fluoro-2,1,3-Benzoxadiazole. Anal. Chem. 1984, 56 (13), 2461– 4,  DOI: 10.1021/ac00277a044
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    581
    New fluorogenic reagent having halogenobenzofurazan structure for thiols: 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole
    Toyooka, Toshimasa; Imai, Kazuhiro
    Analytical Chemistry (1984), 56 (13), 2461-4CODEN: ANCHAM; ISSN:0003-2700.
    4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) was synthesized as a new fluorogenic reagent for thiols. The reaction rate of ABD-F with homocysteine is >30 times faster than that of ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate. The fluorogenic reaction with thiol was completed quant. in 5 min at 50° and pH 8.0. Alanine, proline, and cystine did not react under the same conditions. The fluorescence intensity of the fluorophor was pH dependent with the highest at pH 2. The ABD-thiols obtained by the prelabeling technique were sepd. and detected by reversed-phase HPLC. The detection limits (S/N = 3) for cysteine, glutathione, N-acetylcysteine, homocysteine, and cysteamine were 0.6, 0.4, 1.9, 0.5, and 0.5 pmol, resp.
  579. 579
    Zhao, L.; Zhao, L.; Zhang, C.; Li, Y. A Naked-Eye Visible and Fluorescence ″Turn-On″ Probe for Acetylcholinesterase Assay and the Discrimination of Biothiols. J. Appl. Spectrosc. 2018, 85 (3), 437– 44,  DOI: 10.1007/s10812-018-0669-6
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    582
    A Naked-Eye Visible and Fluorescence "Turn-On" Probe for Acetylcholinesterase Assay and the Discrimination of Biothiols
    Zhao, Lingzhi; Zhao, Liu; Zhang, Chenxiao; Li, Yanqi
    Journal of Applied Spectroscopy (2018), 85 (3), 437-444CODEN: JASYAP; ISSN:0021-9037. (Springer)
    We demonstrate the utility of 4-chloro-7-nitrobenzofurazan (NBD-Cl), a simple, low-cost probe, for colorimetrically and fluorescently detecting and differentiating similar thiol species, including cysteine, homocysteine, and glutathione, as well as for assaying acetylcholinesterase (AChE) activity. This assay is a potential tool for investigating thiol functions in disease and diagnosis, and for screening AChE inhibitors. We envision that this versatile probe, which is com. available and inexpensive, will be useful for further elucidating the roles of thiols in biol. and also shows applicability for anal. of AChE inhibitors in physiol. samples.
  580. 580
    Hassett, R. P.; Crockett, E. L. Endpoint Fluorometric Assays for Determining Activities of Carnitine Palmitoyltransferase and Citrate Synthase. Anal. Biochem. 2000, 287 (1), 176– 9,  DOI: 10.1006/abio.2000.4799
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    583
    Endpoint Fluorometric Assays for Determining Activities of Carnitine Palmitoyltransferase and Citrate Synthase
    Hassett, R. Patrick; Crockett, Elizabeth L.
    Analytical Biochemistry (2000), 287 (1), 176-179CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)
    The authors describe fluorometric procedures for assaying both carnitine palmitoyltransferase (CPT) and citrate synthase (CS). CoA-SH produced in the enzyme-catalyzed reactions is labeled with the thiol-reactive probe 7-fluorobenz-2-oxa- 1,3-diazole-4-sulfonamide (ABD-F). ABD-F has a much higher water soly. than the fluorophore N-(9-acridinyl)maleimide (NAM), which has been used in a continuous assay for CPT and requires dissoln. in a nonaq. solvent (e.g., acetone or DMSO). ABD-F also has a wider sepn. between excitation and emission peaks compared to NAM (390/530 vs 365/440). Advantages of the ABD-F procedure presented here include sensitivity combined with simplicity, and ease of application with microwell plates when a high throughput is desirable and enzymic activities are sufficiently high that a protein sedimentation step is not required. (c) 2000 Academic Press.
  581. 581
    Niu, L.-Y.; Zheng, H.-R.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. Fluorescent Sensors for Selective Detection of Thiols: Expanding the Intramolecular Displacement Based Mechanism to New Chromophores. Analyst 2014, 139 (6), 1389– 95,  DOI: 10.1039/c3an01849k
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    584
    Fluorescent sensors for selective detection of thiols: expanding the intramolecular displacement based mechanism to new chromophores
    Niu, Li-Ya; Zheng, Hai-Rong; Chen, Yu-Zhe; Wu, Li-Zhu; Tung, Chen-Ho; Yang, Qing-Zheng
    Analyst (Cambridge, United Kingdom) (2014), 139 (6), 1389-1395CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    Biol. thiols, including cysteine (Cys), homocystein (Hcy) and glutathione (GSH), play crucial roles in maintaining the appropriate redox status of biol. systems. An abnormal level of biothiols is assocd. with different diseases, therefore, the discrimination between them is of great importance. Herein, we present two fluorescent sensors for selective detection of biothiols based on our recently reported intramol. displacement mechanism. We expanded this mechanism to com. available chromophores, 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) and heptamethine cyanine dye IR-780. The sensors operate by undergoing displacement of chloride by thiolate. The amino groups of Cys/Hcy further replace the thiolate to form amino-substituted products, which exhibit dramatically different photophys. properties compared to sulfur-substituted products from the reaction with GSH. NBD-Cl is highly selective towards Cys/Hcy and exhibits significant fluorescence enhancement. IR-780 showed a variation in its fluorescence ratio towards Cys over other thiols. Both of the sensors can be used for live-cell imaging of Cys. The wide applicability of the mechanism may provide a powerful tool for developing novel fluorescent sensors for selective detection of biothiols.
  582. 582
    Niu, L.-Y.; Guan, Y.-S.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. BODIPY-Based Ratiometric Fluorescent Sensor for Highly Selective Detection of Glutathione over Cysteine and Homocysteine. J. Am. Chem. Soc. 2012, 134 (46), 18928– 31,  DOI: 10.1021/ja309079f
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    585
    BODIPY-Based Ratiometric Fluorescent Sensor for Highly Selective Detection of Glutathione over Cysteine and Homocysteine
    Niu, Li-Ya; Guan, Ying-Shi; Chen, Yu-Zhe; Wu, Li-Zhu; Tung, Chen-Ho; Yang, Qing-Zheng
    Journal of the American Chemical Society (2012), 134 (46), 18928-18931CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The authors report a ratiometric fluorescent sensor based on monochlorinated BODIPY for highly selective detection of glutathione (GSH) over cysteine (Cys)/homocysteine (Hcy). The chlorine of the monochlorinated BODIPY can be rapidly replaced by thiolates of biothiols through thiol-halogen nucleophilic substitution. The amino groups of Cys/Hcy but not GSH further replace the thiolate to form amino-substituted BODIPY. The significantly different photophys. properties of sulfur- and amino-substituted BODIPY enable the discrimination of GSH over Cys and Hcy. The sensor was applied for detection of GSH in living cells.
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    Liu, X.-L.; Niu, L.-Y.; Chen, Y.-Z.; Zheng, M.-L.; Yang, Y.; Yang, Q.-Z. A Mitochondria-Targeting Fluorescent Probe for the Selective Detection of Glutathione in Living Cells. Org. Biomol. Chem. 2017, 15 (5), 1072– 5,  DOI: 10.1039/C6OB02407F
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    586
    A mitochondria-targeting fluorescent probe for the selective detection of glutathione in living cells
    Liu, Xue-Liang; Niu, Li-Ya; Chen, Yu-Zhe; Zheng, Mei-Ling; Yang, Yunxu; Yang, Qing-Zheng
    Organic & Biomolecular Chemistry (2017), 15 (5), 1072-1075CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    The authors report a fluorescent probe, I, for the selective detection of mitochondrial glutathione (GSH). The probe, contg. triphenylphosphine as a mitochondrial targeting group, exhibited ratiometric and selective detection of GSH over Cys/Hcy. The probe was used for imaging mitochondrial GSH in living HeLa cells.
  584. 584
    Hu, Q.; Yu, C.; Xia, X.; Zeng, F.; Wu, S. A Fluorescent Probe for Simultaneous Discrimination of GSH and Cys/Hcy in Human Serum Samples Via Distinctly-Separated Emissions with Independent Excitations. Biosens. Bioelectron. 2016, 81, 341– 8,  DOI: 10.1016/j.bios.2016.03.011
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    587
    A fluorescent probe for simultaneous discrimination of C and Cys/Hcy in human serum samples via distinctly-separated emissions with independent excitations
    Hu, Qinghua; Yu, Changmin; Xia, Xitao; Zeng, Fang; Wu, Shuizhu
    Biosensors & Bioelectronics (2016), 81 (), 341-348CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
    Biothiols like Cys, Hcy and GSH play important roles in various physiol. and pathol. processes, and it is still challenging to simultaneously and discriminatively detect GSH and Cys/Hcy due to their similar structures and reaction activities. Hence, it would be highly desirable to design a fluorescent probe for simultaneously discriminating GSH and Cys/Hcy in biol. samples with no spectral crosstalk, few interferences and rapid response. Herein, through coupling two fluorophores with biothiol-sensitive linker, we developed a fluorescent probe, which has two biothiol-responsive and distinctly-sepd. emissions via independent visible-light excitations, for simultaneously discriminating GSH and Cys/Hcy with near-IR and green emissions. The probe is operable in human serum samples, thus holding promise for diagnostic-related applications. Moreover, the probe shows quite good properties. First, it exhibits a rapid response (within a few minutes) with highly selective and sensitive detection for GSH and Cys/Hcy. Second, it offers an apparent colorimetric and two fluorescence emission signals without spectral crosstalk. Third, it shows low cytotoxicity. Therefore, it would provide a useful method for further elucidating the roles of biothiols as well as for conducting pathol. anal. for diseases involving biothiols.
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    Zhang, J.; Ji, X.; Ren, H.; Zhou, J.; Chen, Z.; Dong, X.; Zhao, W. meso-Heteroaryl BODIPY Dyes as Dual-Responsive Fluorescent Probes for Discrimination of Cys from Hcy and GSH. Sens. Actuators B Chem. 2018, 260, 861– 9,  DOI: 10.1016/j.snb.2018.01.016
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    Meso-heteroaryl BODIPY dyes as dual-responsive fluorescent probes for discrimination of Cys from Hcy and GSH
    Zhang, Jian; Ji, Xin; Ren, Hang; Zhou, Junliang; Chen, Zhongjian; Dong, Xiaochun; Zhao, Weili
    Sensors and Actuators, B: Chemical (2018), 260 (), 861-869CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    Two new BODIPY-based turn-on fluorescent probes (BDP-S-1 and BDP-O-6) contained tetra-Me at 1-, 3-, 5- and 7- positions, as well as O-, or S-aryl substituent at 8-position for the simultaneous and selective detection of Cys and Hcy/GSH from dual emission channels were developed. The spatial steric hindrance of the Me groups at 1- and 7- positions prevented intramol. displacement of sulfur with amino group of Hcy but not of Cys. Based on different thiols-induced SNAr substitution-rearrangement reaction with Cys and Hcy/GSH, leading to the corresponding amino- and thiol-BODIPY dyes with distinct photo-phys. properties, these probes could simultaneously and selectively detect Cys and Hcy/GSH. With the help of laser scanning confocal microscope, we demonstrated that these probes could simultaneously sense Cys and GSH in Hela cells using multicolor imaging.
  586. 586
    Liu, Y.; Yu, Y.; Zhao, Q.; Tang, C.; Zhang, H.; Qin, Y.; Feng, X.; Zhang, J. Fluorescent Probes Based on Nucleophilic Aromatic Substitution Reactions for Reactive Sulfur and Selenium Species: Recent Progress, Applications, and Design Strategies. Coord. Chem. Rev. 2021, 427, 213601,  DOI: 10.1016/j.ccr.2020.213601
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    Fluorescent probes based on nucleophilic aromatic substitution reactions for reactive sulfur and selenium species: Recent progress, applications, and design strategies
    Liu, Yuning; Yu, Yanan; Zhao, Qingyu; Tang, Chaohua; Zhang, Huiyan; Qin, Yuchang; Feng, Xiaohui; Zhang, Junmin
    Coordination Chemistry Reviews (2021), 427 (), 213601CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
    A review. Reactive sulfur species (RSS) and reactive selenium species (RSeS) are important substances for the maintenance of physiol. balance. Imbalance of RSS and RSeS is closely related to a series of human diseases, so it is considered to be an important biomarker in early diagnosis, treatment, and stage monitoring. Fast and accurate quant. anal. of different RSS and RSeS in complex biol. systems may promote the development of personalized diagnosis and treatment in the future. One way to explore the physiol. function of various types of RSS and RSeS in vivo is to detect them at the mol. level, and one of the most effective methods for this is to use fluorescent probes. Nucleophilic arom. substitution (SNAr) reactions are commonly exploited as a detection mechanism for RSS and RSeS in fluorescent probes. In this review, we cover recent progress in fluorescent probes for RSS and RSeS based on SNAr reactions, and discuss their response mechanisms, properties, and applications. Benzenesulfonate, phenyl-O ether, phenyl-S ether, phenyl-Se ether, 7-nitro-2,1,3-benzoxadiazole (NBD), benzoate, and selenium-nitrogen bonds are all good detection groups. Moreover, based on an integration of different reports, we propose the design and synthesis of RSS- and RSeS-selective probes based on SNAr reactions, current challenges, and future research directions, considering the selection of active sites, the effect of substituents on the benzene ring, and the introduction of other functional groups.
  587. 587
    Kim, Y.; Mulay, S. V.; Choi, M.; Yu, S. B.; Jon, S.; Churchill, D. G. Exceptional Time Response, Stability and Selectivity in Doubly-Activated Phenyl Selenium-Based Glutathione-Selective Platform. Chem. Sci. 2015, 6, 5435,  DOI: 10.1039/C5SC02090E
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    Exceptional time response, stability and selectivity in doubly-activated phenyl selenium-based glutathione-selective platform
    Kim, Youngsam; Mulay, Sandip V.; Choi, Minsuk; Yu, Seungyoon B.; Jon, Sangyong; Churchill, David G.
    Chemical Science (2015), 6 (10), 5435-5439CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    A phenyl-selenium-substituted coumarin probe was synthesized for the purpose of achieving highly selective and extremely rapid detection of glutathione (GSH) over cysteine (Cys)/homocysteine (Hcy) without background fluorescence. The fluorescence intensity of the probe with GSH shows a ∼100-fold fluorescent enhancement compared with the signal generated for other closely related amino acids, including Cys and Hcy. Importantly, the substitution reaction with the sulfhydryl group of GSH at the 4-position of the probe, which is doubly-activated by two carbonyl groups, occurs extremely fast, showing subsecond max. fluorescence intensity attainment; equil. was reached within 100 ms (UV-vis). The probe selectivity for GSH was confirmed in Hep3B cells by confocal microscopy imaging.
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    Mulay, S. V.; Kim, Y.; Choi, M.; Lee, D. Y.; Choi, J.; Lee, Y.; Jon, S.; Churchill, D. G. Enhanced Doubly Activated Dual Emission Fluorescent Probes for Selective Imaging of Glutathione or Cysteine in Living Systems. Anal. Chem. 2018, 90 (4), 2648– 54,  DOI: 10.1021/acs.analchem.7b04375
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    591
    Enhanced Doubly Activated Dual Emission Fluorescent Probes for Selective Imaging of Glutathione or Cysteine in Living Systems
    Mulay, Sandip V.; Kim, Youngsam; Choi, Minsuk; Lee, Dong Yun; Choi, Jonghoon; Lee, Yunho; Jon, Sangyong; Churchill, David G.
    Analytical Chemistry (Washington, DC, United States) (2018), 90 (4), 2648-2654CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The development of novel fluorescent probes for monitoring the concn. of various biomols. in living systems has great potential for eventual early diagnosis and disease intervention. Selective detection of competitive species in biol. systems is a great challenge for the design and development of fluorescent probes. To improve on the design of fluorescent coumarin-based biothiol sensing technologies, we have developed herein an enhanced dual emission doubly activated system (DACP-1 and the closely related DACP-2) for the selective detection of glutathione (GSH) through the use of one optical channel and the detection of cysteine (Cys) by another channel. A phenylselenium group present at the 4-position completely quenches the fluorescence of the probe via photoinduced electron transfer to give a nonfluorescent species. Probes are selective for glutathione (GSH) in the red region and for cysteine/homocysteine (Cys/Hcy) in the green region. When they were treated with GSH, DACP-1 and DACP-2 showed strong fluorescence enhancement in comparison to that for closely related species such as amino acids, including Cys/Hcy. Fluorescence quantum yields (ΦF) increased for the red channel (<0.001 to 0.52 (DACP-1) and 0.48 (DACP-2)) and green channel (Cys) (<0.001 to 0.030 (DACP-1) and 0.026 (DACP-2)), resp. Competing fluorescent enhancements upon addn. of closely related species were negligible. Fast responses, improved water soly., and good cell membrane permeability were all properly established with the use of DACP-1 and DACP-2. Live human lung cancer cells and fibroblasts imaged by confocal microscopy, as well as live mice tumor model imaging, confirmed selective detection.
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    Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. 2,4-Dinitrobenzenesulfonyl Fluoresceins as Fluorescent Alternatives to Ellman’s Reagent in Thiol-Quantification Enzyme Assays. Angew. Chem., Int. Ed. 2005, 44 (19), 2922– 5,  DOI: 10.1002/anie.200500114
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    2,4-Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to ellman's reagent in thiol-quantification enzyme assays
    Maeda, Hatsuo; Matsuno, Hiromi; Ushida, Mai; Katayama, Kohei; Saeki, Kanako; Itoh, Norio
    Angewandte Chemie, International Edition (2005), 44 (19), 2922-2925CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Fluorescent sensor for thiols: Deprotection of nonfluorescent 1 by thiols (R'SH) proceeds rapidly and near-quant. in aq. soln. (pH 7.4) to produce highly fluorescent 2. Assays performed in the presence of 1 provide a rapid and simple method for the detn. of inhibitory consts. for inhibitors such as donepezil toward acetyl- and butyrylcholinesterases.
  590. 590
    Jiang, W.; Fu, Q.; Fan, H.; Ho, J.; Wang, W. A Highly Selective Fluorescent Probe for Thiophenols. Angew. Chem., Int. Ed. 2007, 46 (44), 8445– 8,  DOI: 10.1002/anie.200702271
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    A highly selective fluorescent probe for thiophenols
    Jiang, Wei; Fu, Qingquan; Fan, Hongyou; Ho, Joe; Wang, Wei
    Angewandte Chemie, International Edition (2007), 46 (44), 8445-8448CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A rapid response to thiophenols was obtained with 2,4-dinitro-N-[7-nitrobenzo[c]-[1,2,5]oxadiazol-4-yl]benzenesulfonamide probe, which induces a significant (> 50-fold) fluorescence enhancement as a result of cleavage of the electron-withdrawing moiety, thus generating strongly fluorescent mol., 4-amino-7-nitro-2,1,3-benzoxadiazole. No fluorescence was obtained with aliph. thiols, including cysteine and glutathione, or other nucleophiles.
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    Wang, S.; Huang, Y.; Guan, X. Fluorescent Probes for Live Cell Thiol Detection. Molecules 2021, 26 (12), 3575,  DOI: 10.3390/molecules26123575
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    Fluorescent probes for live cell thiol detection
    Wang, Shenggang; Huang, Yue; Guan, Xiangming
    Molecules (2021), 26 (12), 3575CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    A review. Thiols play vital and irreplaceable roles in the biol. system. Abnormality of thiol levels has been linked with various diseases and biol. disorders. Thiols are known to distribute unevenly and change dynamically in the biol. system. Methods that can det. thiols' concn. and distribution in live cells are in high demand. In the last two decades, fluorescent probes have emerged as a powerful tool for achieving that goal for the simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way. They also enable the detn. of intracellular distribution and dynamitic movement of thiols in the intact native environments. This review focuses on some of the major strategies/mechanisms being used for detecting GSH, Cys/Hcy, and other thiols in live cells via fluorescent probes, and how they are applied at the cellular and subcellular levels. The sensing mechanisms (for GSH and Cys/Hcy) and bio-applications of the probes are illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.
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    Dai, Z.; Tian, L.; Ye, Z.; Song, B.; Zhang, R.; Yuan, J. A Lanthanide Complex-Based Ratiometric Luminescence Probe for Time-Gated Luminescence Detection of Intracellular Thiols. Anal. Chem. 2013, 85 (23), 11658– 64,  DOI: 10.1021/ac403370g
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    595
    A Lanthanide Complex-Based Ratiometric Luminescence Probe for Time-Gated Luminescence Detection of Intracellular Thiols
    Dai, Zhichao; Tian, Lu; Ye, Zhiqiang; Song, Bo; Zhang, Run; Yuan, Jingli
    Analytical Chemistry (Washington, DC, United States) (2013), 85 (23), 11658-11664CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A lanthanide complex-based ratiometric luminescence probe, [4'-(2,4-dinitrobenzenesulfonyloxy)-2,2':6',2''-terpyridine-6,6''-diyl] bis-(methylenenitrilo) tetrakis-(acetate)-Eu3+/Tb3+ (NSTTA-Eu3+/Tb3+), has been designed and synthesized for the specific recognition and time-gated luminescence detection of biothiols in physiol. pH aq. media. The probe itself is almost nonluminescent due to the presence of photoinduced electron transfer (PET) from the terpyridine-Ln3+ moiety to the 2,4-dinitrobenzenesulfonyl (DNBS) moiety. In the presence of biothiols, the reaction of NSTTA-Eu3+/Tb3+ with biothiols results in the cleavage of DNBS to afford the deprotonated (4'-hydroxy-2,2':6',2''-terpyridine-6,6''-diyl) bis-(methylenenitrilo) tetrakis-(acetate)-Eu3+/Tb3+ (HTTA-Eu3+/Tb3+), which terminates the PET process. After the reaction, the intensity of Eu3+ emission at 610 nm is unchanged, while that of Tb3+ emission at 540 nm is remarkably increased, which provides a ∼36-fold enhanced intensity ratio of Tb3+ emission to Eu3+ emission (I540/I610). This unique luminescence response allows NSTTA-Eu3+/Tb3+ to be used as a ratiometric probe for the time-gated luminescence detection of biothiols, using the intensity ratio of I540/I610 as a signal. Thus, based on the probe NSTTA-Eu3+/Tb3+, a ratiometric time-gated luminescence detection method for biothiols was established and successfully used for the quant. detection of the total biothiols in several living cell samples.
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    Rong, X.; Xu, Z.-Y.; Yan, J.-W.; Meng, Z.-Z.; Zhu, B.; Zhang, L. Nile-Red-Based Fluorescence Probe for Selective Detection of Biothiols, Computational Study, and Application in Cell Imaging. Molecules 2020, 25 (20), 4718,  DOI: 10.3390/molecules25204718
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    Nile-red-based fluorescence probe for selective detection of biothiols, computational study, and application in cell imaging
    Rong, Xiang; Xu, Zhong-Yong; Yan, Jin-Wu; Meng, Zhi-Zhong; Zhu, Bin; Zhang, Lei
    Molecules (2020), 25 (20), 4718CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    A new colorimetric and fluorescence probe NRSH based on Nile-red chromophore for the detection of biothiols has been developed, exhibiting high selectivity towards biothiols over other interfering species. NRSH shows a blue shift in absorption peak upon reacting with biothiols, from 587 nm to 567 nm, which induces an obvious color change from blue to pink and exhibits a 35-fold fluorescence enhancement at 645 nm in red emission range. NRSH displays rapid (<1 min) response for H2S, which is faster than other biothiols (>5 min). The detection limits of probe NRSH towards biothiols are very low (22.05 nM for H2S, 34.04 nM for Cys, 107.28 nM for GSH and 113.65 nM for Hcy). Furthermore, NRSH is low cytotoxic and can be successfully applied as a bioimaging tool for real-time monitoring biothiols in HeLa cells. In addn., fluorescence mechanism of probe NRSH is further understood by theor. calcns.
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    Maeda, H.; Katayama, K.; Matsuno, H.; Uno, T. 3′-(2,4-Dinitrobenzenesulfonyl)-2′,7′-Dimethylfluorescein as a Fluorescent Probe for Selenols. Angew. Chem., Int. Ed. 2006, 45 (11), 1810– 3,  DOI: 10.1002/anie.200504299
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    597
    3'-(2,4-Dinitrobenzenesulfonyl)-2',7'-dimethylfluorescein as a fluorescent probe for selenols
    Maeda, Hatsuo; Katayama, Kohei; Matsuno, Hiromi; Uno, Tadayuki
    Angewandte Chemie, International Edition (2006), 45 (11), 1810-1813CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The fluorescein deriv. BESThio [3'-(2,4-dinitrobenzenesulfonyl)-2',7'-dimethylfluorescein] operates as a thiol probe at pH 7.4 and functions as a selenol probe at pH 5.8, and can thus be selectively applied to the rapid quantification of selenocysteine and the detn. of the selenocysteine content in selenoproteins such as glutathione peroxidase and thioredoxin reductase with high sensitivity.
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    Peng, L.; Xiao, L.; Ding, Y.; Xiang, Y.; Tong, A. A Simple Design of Fluorescent Probes for Indirect Detection of β-Lactamase Based on AIE and ESIPT Processes. J. Mater. Chem. B 2018, 6 (23), 3922– 6,  DOI: 10.1039/C8TB00414E
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    598
    A simple design of fluorescent probes for indirect detection of β-lactamase based on AIE and ESIPT processes
    Peng, Lu; Xiao, Lu; Ding, Yiwen; Xiang, Yu; Tong, Aijun
    Journal of Materials Chemistry B: Materials for Biology and Medicine (2018), 6 (23), 3922-3926CODEN: JMCBDV; ISSN:2050-7518. (Royal Society of Chemistry)
    A novel fluorescent probe DNBS-CSA is developed for light-up detection of β-lactamase. The probe design is based on an indirect detection approach with three step reactions. β-Lactamase can react with the lactam of its substrate (cefazolin sodium) to produce a secondary amine, initiating a spontaneous elimination reaction and affording a thiol compd. The thiol could further react with the sulfonate group of DNBS-CSA, releasing the salicylaldehyde azine deriv. (CSA) with both aggregation induced emission (AIE) and excited-state intramol. proton transfer (ESIPT) characteristics. Previously reported β-lactamase probes require covalent linkage of the substrate β-lactam ring part to the probe, which makes probe synthesis difficult due to the complicated structure of the β-lactam ring. In contrast, modification of the β-lactam ring is no longer necessary for DNBS-CSA according to our indirect detection approach. The linear range of fluorescence quantification for β-lactamase is 0-10 mU mL-1 in an aq. soln. Moreover, owing to the AIE properties of CSA, detection of β-lactamase with DNBS-CSA on test papers was also achieved.
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    Yuan, L.; Lin, W.; Zhao, S.; Gao, W.; Chen, B.; He, L.; Zhu, S. A Unique Approach to Development of Near-Infrared Fluorescent Sensors for in Vivo Imaging. J. Am. Chem. Soc. 2012, 134 (32), 13510– 23,  DOI: 10.1021/ja305802v
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    A Unique Approach to Development of Near-Infrared Fluorescent Sensors for in Vivo Imaging
    Yuan, Lin; Lin, Weiying; Zhao, Sheng; Gao, Wensha; Chen, Bin; He, Longwei; Zhu, Sasa
    Journal of the American Chemical Society (2012), 134 (32), 13510-13523CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Near-IR (NIR) fluorescent sensors have emerged as promising mol. tools for imaging biomols. in living systems. However, NIR fluorescent sensors are very challenging to be developed. Herein, the authors describe the discovery of a new class of NIR fluorescent dyes represented by 1a/1c/1e, which are superior to the traditional 7-hydroxycoumarin and fluorescein with both absorption and emission in the NIR region while retaining an optically tunable hydroxyl group. Quantum chem. calcns. with the B3LYP exchange functional employing 6-31G(d) basis sets provide insights into the optical property distinctions between 1a/1c/1e and their alkoxy derivs. The unique optical properties of the new type of fluorescent dyes can be exploited as a useful strategy for development of NIR fluorescent sensors. Employing this strategy, two different types of NIR fluorescent sensors, NIR-H2O2 and NIR-thiol, for H2O2 and thiols, resp., were constructed. These novel sensors respond to H2O2 or thiols with a large turn-on NIR fluorescence signal upon excitation in the NIR region. Furthermore, NIR-H2O2 and NIR-thiol are capable of imaging endogenously produced H2O2 and thiols, resp., not only in living cells but also in living mice, demonstrating the value of the new NIR fluorescent sensor design strategy. The new type of NIR dyes presented herein may open up new opportunities for the development of NIR fluorescent sensors based on the hydroxyl functionalized reactive sites for biol. imaging applications in living animals.
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    Lu, Z.; Sun, X.; Wang, M.; Wang, H.; Fan, C.; Lin, W. Rational Design of a Far-Red Fluorescent Probe for Endogenous Biothiol Imbalance Induced by Hydrogen Peroxide in Living Cells and Mice. Bioorg. Chem. 2020, 103, 104173,  DOI: 10.1016/j.bioorg.2020.104173
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    Rational design of a far-red fluorescent probe for endogenous biothiol imbalance induced by hydrogen peroxide in living cells and mice
    Lu, Zhengliang; Sun, Xin; Wang, Mengzhao; Wang, Hongmei; Fan, Chunhua; Lin, Weiying
    Bioorganic Chemistry (2020), 103 (), 104173CODEN: BOCMBM; ISSN:0045-2068. (Elsevier B.V.)
    Intracellular biothiols are correlated with many diseases such as nerve disorder and Parkinson's disease likely due to a redox imbalance. In this work, we designed an ultrafast fluorescent probe (Cou-DNBS) for biothiols with a large Stokes shift (131 nm). The probe was constructed through linking the 2,4-dinitrobenzenesulfonyl moiety as the specially recognizing biothiols site to an iminocoumarin fluorophore Cou-NH obtained by fusing an addnl. benzene ring. The presence of biothiols could ultrafast perform a significant fluorescence emission at 617 nm upon the excitation of 480 with the low limits of detection (2.5 nM for Cys, 1.7 nM for Hcy and 0.84 nM for GSH). HRMS spectra as well as theor. calcns. further evidenced the rationale of recognition mechanism. Furthermore, the probe can successfully visualize endogenous biothiol recovery in living cells damaged by H2O2.
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    Kand, D.; Kalle, A. M.; Varma, S. J.; Talukdar, P. A Chromenoquinoline-Based Fluorescent Off-On Thiol Probe for Bioimaging. Chem. Commun. 2012, 48 (21), 2722– 4,  DOI: 10.1039/c2cc16593g
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    A chromenoquinoline-based fluorescent off-on thiol probe for bioimaging
    Kand, Dnyaneshwar; Kalle, Arunasree Marasanapalli; Varma, Sreejith Jayasree; Talukdar, Pinaki
    Chemical Communications (Cambridge, United Kingdom) (2012), 48 (21), 2722-2724CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A new chromenoquinoline-based fluorescent off-on thiol probe (I) is reported. In aq. buffer solns. at physiol. pH, the probe exhibited 223-fold enhancement in fluorescence intensity by a Michael addn. of cysteine to the maleimide appended to a chromenoquinoline. Cell permeability and live cell imaging of thiols are also demonstrated.
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    Liu, T.; Huo, F.; Li, J.; Chao, J.; Zhang, Y.; Yin, C. A Fast Response and High Sensitivity Thiol Fluorescent Probe in Living Cells. Sens. Actuators B Chem. 2016, 232, 619– 24,  DOI: 10.1016/j.snb.2016.04.014
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    A fast response and high sensitivity thiol fluorescent probe in living cells
    Liu, Tao; Huo, Fangjun; Li, Jianfang; Chao, Jianbin; Zhang, Yongbin; Yin, Caixia
    Sensors and Actuators, B: Chemical (2016), 232 (), 619-624CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    Up to date, the design of highly selective and fast response probes for Cys, Hcy, and GSH is still a challenge and very few examples have been reported so far. In this study, we synthesized a 1,8-naphthalimide-based probe bearing maleimide group to selectively detect thiol. After introduce thiol, the fluorescence intensity of probe showed significant enhancement, but other amino acids did not generate any change. The kinetic study showed that the reaction was complete within 120 s for Cys, Hcy and GSH, indicating that probe reacts rapidly with thiol under the exptl. conditions. Furthermore, the ability of probe to detect thiol in living cells via an enhancement of the fluorescence was proved.
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    Lin, X.; Hu, Y.; Yang, D.; Chen, B. Cyanine-Coumarin Composite NIR Dye Based Instantaneous-Response Probe for Biothiols Detection and Oxidative Stress Assessment of Mitochondria. Dyes Pigm. 2020, 174, 107956,  DOI: 10.1016/j.dyepig.2019.107956
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    Cyanine-coumarin composite NIR dye based instantaneous -response probe for biothiols detection and oxidative stress assessment of mitochondria
    Lin, Xin; Hu, Yanling; Yang, Dongliang; Chen, Bo
    Dyes and Pigments (2020), 174 (), 107956CODEN: DYPIDX; ISSN:0143-7208. (Elsevier Ltd.)
    Developing near-IR (NIR) dye based functional fluorescent probes to anal. the bioactive substances contributes to the understanding of cellular physiopathol. Although there are many fluorescent probes have been developed, the probes can instantaneous response to biothiols is still lacking. In this study, we have designed and synthesized a novel composite NIR dye (Hcyc), which was composed by cyanine and coumarin. Hcyc was further applied to construct a biothiols responsive probe (Hcyc-NO) to assess the oxidative stress status of mitochondria in living cells. The optical properties showed that Hcyc has long absorption and emission wavelength in different solvent system (>720 nm). Interestingly, Hcyc-NO could respond to GSH, Cys, Hcy rapidly (<5 s), the absorption peak of red shifted from 556 nm to 723 nm and fluorescence intensity instantaneously enhanced at 751 nm after adding biothiols. In addn., Hcyc-NO possesses others excellent sensing properties such as high selectivity and sensitivity. Confocal imaging revealed that Hcyc-NO (1μM) could target mitochondria and selective image biothiols (GSH, Cys and Hcy) in vitro. Furthermore, the probe could rapidly reflect oxidative stress induced by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) in vitro and in vivo. These results indicate that Hcyc is an excellent NIR dye, and the probe Hcyc-NO may be an effective tool for instantaneous detection of biothiols and real-time assessing the oxidative stress of cells.
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    Sun, Q.; Yang, S.-H.; Wu, L.; Yang, W.-C.; Yang, G.-F. A Highly Sensitive and Selective Fluorescent Probe for Thiophenol Designed Via a Twist-Blockage Strategy. Anal. Chem. 2016, 88 (4), 2266– 72,  DOI: 10.1021/acs.analchem.5b04029
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    A Highly Sensitive and Selective Fluorescent Probe for Thiophenol Designed via a Twist-Blockage Strategy
    Sun, Qi; Yang, Shu-Hou; Wu, Lei; Yang, Wen-Chao; Yang, Guang-Fu
    Analytical Chemistry (Washington, DC, United States) (2016), 88 (4), 2266-2272CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A benzoquinolizine coumarin-based fluorescent probe was developed for detecting thiophenols, demonstrating the superior fluorescence properties caused by the decay of the twisting effect of N,N-diethylamino group of coumarin. It discriminated thiophenols from various analytes including aliph. thiols with good selectivity and displayed ∼700-fold fluorescence intensity enhancement and a remarkable limit of detection (4.5 nM). The new probe also can be applied to quant. det. the concns. of thiophenol in water samples and living cells.
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    Jiang, W.; Cao, Y.; Liu, Y.; Wang, W. Rational Design of a Highly Selective and Sensitive Fluorescent PET Probe for Discrimination of Thiophenols and Aliphatic Thiols. Chem. Commun. 2010, 46 (11), 1944– 6,  DOI: 10.1039/B926070F
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    Rational design of a highly selective and sensitive fluorescent PET probe for discrimination of thiophenols and aliphatic thiols
    Jiang, Wei; Cao, Yanting; Liu, Yuan; Wang, Wei
    Chemical Communications (Cambridge, United Kingdom) (2010), 46 (11), 1944-1946CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A novel highly sensitive and selective 'off-on' fluorescent probe (I, R1 = R2 = NO2; R1 = Me, R2 = H) for thiophenols was developed by a PET mechanism through a rational design.
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    Li, Y.; Su, W.; Zhou, Z.; Huang, Z.; Wu, C.; Yin, P.; Li, H.; Zhang, Y. A Dual-Response near-Infrared Fluorescent Probe for Rapid Detecting Thiophenol and Its Application in Water Samples and Bio-Imaging. Talanta 2019, 199, 355– 60,  DOI: 10.1016/j.talanta.2019.02.022
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    A dual-response near-IR fluorescent probe for rapid detecting thiophenol and its application in water samples and bio-imaging
    Li, Yaqian; Su, Wei; Zhou, Zile; Huang, Zhen; Wu, Cuiyan; Yin, Peng; Li, Haitao; Zhang, Youyu
    Talanta (2019), 199 (), 355-360CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
    Thiophenol is widely known as a highly toxic substance that can cause serious harm to the environment and health. Rapid and non-destructive detection of thiophenol is of great significance for environmental management. In this work, we designed and synthesized a near-IR (NIR) fluorescent probe, (E)-4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)-2-formylphenyl-2,4-dinitrobenzenesulfonate (DCM-CHO-D)(I), that can respond to thiophenol rapidly (less than 3 min) based on intramol. charge transfer (ICT) mechanism. DCM-CHO-D has high selectivity and sensitivity to the detection of thiophenol. And there is a linear relationship between the fluorescence intensity of DCM-CHO-D and thiophenol concn. in the range of 0-10 μM, the limit of detection (LOD) is as low as 0.22 μM. What's more, DCM-CHO-D can not only be used as an ideal colorimetric tool for detecting thiophenol in water samples, but also image thiophenol in living cells, indicating its potential utility for thiophenol sensing in environmental and biol. samples.
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    Xiong, L.; Ma, J.; Huang, Y.; Wang, Z.; Lu, Z. Highly Sensitive Squaraine-Based Water-Soluble Far-Red/near-Infrared Chromofluorogenic Thiophenol Probe. ACS Sens. 2017, 2 (4), 599– 605,  DOI: 10.1021/acssensors.7b00151
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    Highly Sensitive Squaraine-Based Water-Soluble Far-Red/Near-Infrared Chromofluorogenic Thiophenol Probe
    Xiong, Li; Ma, Jun; Huang, Yan; Wang, Zihe; Lu, Zhiyun
    ACS Sensors (2017), 2 (4), 599-605CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    A squaraine-based far-red/near-IR fluorescent probe (SQ-DNBS) was exploited for thiophenol detection. SQ-DNBS is a colorimetric and "off-on" fluorometric dual-channel "naked-eye" chemosensor showing high selectivity, high sensitivity (detection limit: 9.9 nM), and rapid response to thiophenol in aq. soln. SQ-DNBS also can be used in practical applications for the detection of thiophenol in water samples. Photophys. and spectral characterization results revealed that the probing mechanism of SQ-DNBS toward thiophenol lies in the thiolate-mediated cleavage reaction. Our discovery demonstrates the potential of the arylmethylene-squaraine skeleton as a promising fluorophore unit to construct high-performance far-red/near-IR chemosensors.
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    Song, B.; Wang, G.; Tan, M.; Yuan, J. A Europium(III) Complex as an Efficient Singlet Oxygen Luminescence Probe. J. Am. Chem. Soc. 2006, 128 (41), 13442– 50,  DOI: 10.1021/ja062990f
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    A Europium(III) Complex as an Efficient Singlet Oxygen Luminescence Probe
    Song, Bo; Wang, Guilan; Tan, Mingqian; Yuan, Jingli
    Journal of the American Chemical Society (2006), 128 (41), 13442-13450CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A new europium(III) complex, [4'-(10-methyl-9-anthryl)-2,2':6',2''-terpyridine-6,6''-diyl]bis(methylenenitrilo) tetrakis(acetate)-Eu3+, was designed and synthesized as a highly sensitive and selective time-gated luminescence probe for singlet oxygen (1O2). The new probe is highly water sol. with a large stability const. of ∼1021 and a wide pH available range (pH 3-10), and can specifically react with 1O2 to form its endoperoxide (EP-MTTA-Eu3+) with a high reaction rate const. at 1010 M-1 s-1, accompanied by the remarkable increases of luminescence quantum yield from 0.90% to 13.8% and lifetime from 0.80 to 1.29 ms, resp. The wide applicability of the probe was demonstrated by detection of 1O2 generated from a MoO42-/H2O2 system, a photosensitization system of 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), and a horseradish peroxidase catalyzed aerobic oxidn. system of indole-3-acetic acid (IAA). In addn., it was found that the new probe could be easily transferred into living HeLa cells by incubation with TMPyP. A time-gated luminescence imaging technique that can fully eliminate the short-lived background fluorescence from TMPyP and cell components has been successfully developed for monitoring the time-dependent generation of 1O2 in living cells.
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    Bjartell, A.; Laine, S.; Pettersson, K.; Nilsson, E.; Lövgren, T.; Lilja, H. Time-Resolved Fluorescence in Immunocytochemical Detection of Prostate-Specific Antigen in Prostatic Tissue Sections. Histochem. J. 1999, 31 (1), 45– 52,  DOI: 10.1023/A:1003504115690
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    Time-resolved fluorescence in immunocytochemical detection of prostate-specific antigen in prostatic tissue sections
    Bjartell, Anders; Laine, Sari; Pettersson, Kim; Nilsson, Elise; Lovgren, Timo; Lilja, Hans
    Histochemical Journal (1999), 31 (1), 45-52CODEN: HISJAE; ISSN:0018-2214. (Kluwer Academic Publishers)
    Chelates with fluorescent lanthanides such as europium and terbium are widely used in immunofluorometric assays, e.g. for the measurement of different mol. forms of prostate-specific antigen (PSA) in serum for detection and monitoring of prostate cancer. These chelates have also been introduced as non-radioactive labels in immunocytochem. and in situ hybridization. In the present study, sections of non-malignant prostate were investigated using monoclonal IgGs against PSA. Detection of specific immunostaining employing time-resolved fluorescence with europium-labeled streptavidin was compared with conventional detection by streptavidin conjugated to horse-radish peroxidase. The high PSA concn. in the tissue produced high intensity, specific time-resolved fluorescence signals in the epithelial cells of the prostate gland without disturbance from non-specific tissue autofluorescense. This allowed short exposure times to be used which resulted in insignificant photobleaching. Two of the three europium-chelates evaluated yielded high signal intensities. Counterstaining was found to be optimal with Gill No. 1-Haematoxylin soln. and Merckoglas was the best mounting medium for the europium chelates tested. In conclusion, time-resolved fluorescence imaging is an attractive alternative to conventional detection of streptavidin conjugated to horse-radish peroxidase, as it provides linear, high intensity, specific signals subsequent to the decay of non-specific tissue autofluorescence.
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    Shang, H.; Chen, H.; Tang, Y.; Ma, Y.; Lin, W. Development of a Two-Photon Fluorescent Turn-On Probe with Far-Red Emission for Thiophenols and Its Bioimaging Application in Living Tissues. Biosens. Bioelectron. 2017, 95, 81– 6,  DOI: 10.1016/j.bios.2017.04.017
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    Development of a two-photon fluorescent turn-on probe with far-red emission for thiophenols and its bioimaging application in living tissues
    Shang, Huiming; Chen, Hua; Tang, Yonghe; Ma, Yanyan; Lin, Weiying
    Biosensors & Bioelectronics (2017), 95 (), 81-86CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
    Thiophenol is a highly toxic compd. which is essential in the field of org. synthesis and drug design. However, the accumulation of thiophenols in the environment may cause serious health problems for human bodies ultimately. Therefore, it is crit. to develop efficient methods for visualization of thiophenol species in biol. samples. In this work, an innovative two-photon fluorescent turn-on probe FR-TP with far-red emission for thiophenols based on FR-NH2 fluorophore and 2,4-dinitrophenylsulfonyl recognition site was reported. The new probe can be used for thiophenol detection with large far-red fluorescence enhancement (about 155-fold), rapid response (completed within 100 s), excellent sensitivity (DL 0.363μM), high selectivity, and lower cellular auto-fluorescence interference. Importantly, the probe FR-TP can be successfully employed to visualize thiophenols not only in the living HeLa cells but also in living liver tissues. In addn., through two-photon tissue imaging, the probe was used to monitor and investigate biol. thiophenol poisoning in the animal model of thiophenol inhalation for the first time.
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    Fellner, M.; Doughty, L. M.; Jameson, G. N. L.; Wilbanks, S. M. A Chromogenic Assay of Substrate Depletion by Thiol Dioxygenases. Anal. Biochem. 2014, 459, 56– 60,  DOI: 10.1016/j.ab.2014.05.008
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    A chromogenic assay of substrate depletion by thiol dioxygenases
    Fellner, Matthias; Doughty, Laura M.; Jameson, Guy N. L.; Wilbanks, Sigurd M.
    Analytical Biochemistry (2014), 459 (), 56-60CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the non-heme mono-Fe enzyme, cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed std. kinetic parameters to be detd. for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitated a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addn., the relative specificity of Ellman's reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.
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    Zhu, J.; Dhimitruka, I.; Pei, D. 5-(2-Aminoethyl)Dithio-2-Nitrobenzoate as a More Base-Stable Alternative to Ellman’s Reagent. Org. Lett. 2004, 6 (21), 3809– 12,  DOI: 10.1021/ol048404+
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    5-(2-Aminoethyl)dithio-2-nitrobenzoate as a More Base-Stable Alternative to Ellman's Reagent
    Zhu, Jinge; Dhimitruka, Ilirian; Pei, Dehua
    Organic Letters (2004), 6 (21), 3809-3812CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
    5-(2-Aminoethyl)dithio-2-nitrobenzoate (ADNB) reacts with free thiols with kinetics similar to those of Ellman's reagent but has dramatically improved stability under alk. conditions, making it an excellent alternative to Ellman's reagent for the quantitation of thiol contents and enzymic assays under basic pH conditions.
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    Li, Y.; Lopez, P.; Durand, P.; Ouazzani, J.; Badet, B.; Badet-Denisot, M.-A. An Enzyme-Coupled Assay for Amidotransferase Activity of Glucosamine-6-Phosphate Synthase. Anal. Biochem. 2007, 370 (2), 142– 6,  DOI: 10.1016/j.ab.2007.07.031
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    An enzyme-coupled assay for amidotransferase activity of glucosamine-6-phosphate synthase
    Li, Yanyan; Lopez, Philippe; Durand, Philippe; Ouazzani, Jamal; Badet, Bernard; Badet-Denisot, Marie-Ange
    Analytical Biochemistry (2007), 370 (2), 142-146CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
    An assay for glucosamine-6-phosphate synthase using a yeast glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) as coupling enzyme was developed. GNA1 transfers the acetyl moiety from acetyl-CoA (CoA) to glucosamine-6-phosphate, releasing CoA. The assay measures the prodn. of glucosamine-6-phosphate by either following the consumption of acetyl-CoA spectrophotometrically at 230 nm or quantifying the free thiol with 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent) in a discontinuous manner. This method is simple to perform and can be adapted to a 96-well microtiter plate format, which will facilitate high-throughput inhibitor screening and mechanistic studies using purified GlmS.
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    Chen, G.; Feng, H.; Jiang, X.; Xu, J.; Pan, S.; Qian, Z. Redox-Controlled Fluorescent Nanoswitch Based on Reversible Disulfide and Its Application in Butyrylcholinesterase Activity Assay. Anal. Chem. 2018, 90 (3), 1643– 51,  DOI: 10.1021/acs.analchem.7b02976
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    Redox-Controlled Fluorescent Nanoswitch Based on Reversible Disulfide and Its Application in Butyrylcholinesterase Activity Assay
    Chen, Guilin; Feng, Hui; Jiang, Xiaogan; Xu, Jing; Pan, Saifei; Qian, Zhaosheng
    Analytical Chemistry (Washington, DC, United States) (2018), 90 (3), 1643-1651CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Butyrylcholinesterase (BChE) mainly contributing to plasma cholinesterase activity is an important indicator for routinely diagnosing liver function and organophosphorus poisoning in clin. diagnosis, but its current assays are scarce and frequently suffer from some significant interference and instability. Herein, we report a redox-controlled fluorescence nanoswitch based on reversible disulfide bonds, and further develop a fluorometric assay of BChE via thiol-triggered disaggregation-induced emission. Thiol-functionalized carbon quantum dots (thiol-CQDs) with intense fluorescence is found to be responsive to hydrogen peroxide, and their redox reaction transforms thiol-CQDs to nonfluorescent thiol-CQDs assembly. The thiols inverse this process by a thiol-exchange reaction to turn on the fluorescence. The fluorescence can be reversibly switched by the formation and breaking of disulfide bonds caused by external redox stimuli. The specific thiol-triggered disaggregation-induced emission enables us to assay BChE activity in a fluorescence turn-on and real-time way using butyrylthiocholine iodide as the substrate. As-established BChE assay achieves sufficient sensitivity for practical detn. in plasma, and is capable of avoiding the interference from micormolar glutathione and discriminatively quantifying BChE from its sister enzyme acetylcholinesterase. The first design of reversible redox-controlled nanoswitch based on disulfide expands the application of disulfide chem. in sensing and clin. diagnostics, and this novel BChE assay enriches the detection methods for cholinesterase activity.
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    Gong, M.-M.; Dai, C.-Y.; Severance, S.; Hwang, C.-C.; Fang, B.-K.; Lin, H.-B.; Huang, C.-H.; Ong, C.-W.; Wang, J.-J.; Lee, P.-L. A Bioorthogonally Synthesized and Disulfide-Containing Fluorescence Turn-On Chemical Probe for Measurements of Butyrylcholinesterase Activity and Inhibition in the Presence of Physiological Glutathione. Catalysts 2020, 10 (10), 1169,  DOI: 10.3390/catal10101169
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    A bioorthogonally synthesized and disulfide-containing fluorescence turn-on chemical probe for measurements of butyrylcholinesterase activity and inhibition in the presence of physiological glutathione
    Gong, Ming-Mao; Dai, Chia-Yen; Severance, Scott; Hwang, Chi-Ching; Fang, Bo-Kai; Lin, Heng-Bo; Huang, Chien-Hui; Ong, Chi-Wi; Wang, Jeh-Jeng; Lee, Pei-Lun; Wang, Tzu-Pin
    Catalysts (2020), 10 (10), 1169CODEN: CATACJ; ISSN:2073-4344. (MDPI AG)
    Butyrylcholinesterase (BChE) is a biomarker in human blood. Aberrant BChE activity has been assocd. with human diseases. Here we developed a fluorescence resonance energy transfer (FRET) chem. probe to specifically quantify BChE activity in serum, while simultaneously discriminating against glutathione (GSH). The FRET chem. probe 11 was synthesized from a key trifunctional bicyclononyne exo-6 and derivs. of 5-(2-aminoethylamino)-1-naphthalenesulfonic acid (EDANS) and 4-[4-(dimethylamino)phenylazo]benzoic acid (DABCYL). EDANS fluorescence visualization and kinetic anal. of 11 in the presence of diverse compds. confirmed the outstanding reactivity and specificity of 11 with thiols. The thiol-dependent fluorescence turn-on property of 11 was attributed to a general base-catalyzed SN2 nucleophilic substitution mechanism and independent of metal ions. Moreover, all thiols, except GSH, reacted swiftly with 11. Kinetic studies of 11 in the presence of covalently modified GSH derivs. corroborated that the steric hindrance of 11 imposing on GSH was the likely cause of the distinguished reactivity. Since GSH commonly interferes in assays measuring BChE activity in blood samples, the 11-based fluorescent assay was employed to directly quantify BChE activity without GSH interference, and delivered a linear range of 4.3-182.2 U L-1 for BChE activity with detection limit of 4.3 U L-1, and accurately quantified serum BChE activity in the presence of 10 μM GSH. Finally, the 11-based assay was exploited to det. Ki of 5 nM for tacrine inhibition on BChE catalysis. We are harnessing the modulated characteristics of 6 to synthesize advanced chem. probes able to more sensitively screen for BChE inhibitors and quantify BChE activity in serum.
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    Peng, H.; Chen, W.; Cheng, Y.; Hakuna, L.; Strongin, R.; Wang, B. Thiol Reactive Probes and Chemosensors. Sensors 2012, 12 (11), 15907– 46,  DOI: 10.3390/s121115907
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    Thiol reactive probes and chemosensors
    Peng, Hanjing; Chen, Weixuan; Cheng, Yunfeng; Hakuna, Lovemore; Strongin, Robert; Wang, Binghe
    Sensors (2012), 12 (), 15907-15946CODEN: SENSC9; ISSN:1424-8220. (MDPI AG)
    A review. Thiols are important mols. in the environment and in biol. processes. Cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H2S) play crit. roles in a variety of physiol. and pathol. processes. The selective detection of thiols using reaction-based probes and sensors is very important in basic research and in disease diagnosis. This review focuses on the design of fluorescent and colorimetric probes and sensors for thiol detection. Thiol detection methods include probes and labeling agents based on nucleophilic addn. and substitution, Michael addn., disulfide bond or Se-N bond cleavage, metal-sulfur interactions and more. Probes for H2S are based on nucleophilic cyclization, redn. and metal sulfide formation. Thiol probe and chemosensor design strategies and mechanism of action are discussed in this review.
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    Worek, F.; Mast, U.; Kiderlen, D.; Diepold, C.; Eyer, P. Improved Determination of Acetylcholinesterase Activity in Human Whole Blood. Clin. Chim. Acta 1999, 288 (1), 73– 90,  DOI: 10.1016/S0009-8981(99)00144-8
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    Improved determination of acetylcholinesterase activity in human whole blood
    Worek, F.; Mast, U.; Kiderlen, D.; Diepold, C.; Eyer, P.
    Clinica Chimica Acta (1999), 288 (1-2), 73-90CODEN: CCATAR; ISSN:0009-8981. (Elsevier Science Ireland Ltd.)
    The detn. of erythrocyte acetylcholinesterase (AChE) activity is an appropriate tool for the diagnosis of organophosphate exposure and intoxication. The original colorimetric Ellman procedure is disturbed by a high Hb absorption at 412 nm. In the authors' modified method, the wavelength was changed to 436 nm. This reduced the indicator absorption to 80% and the Hb absorption to 25%. The signal-to-noise ratio was further enhanced by redn. of pH and substrate concn., thus making it possible to measure 3% residual activity. AChE activity was detd. in whole blood samples in the presence of the selective butyrylcholinesterase inhibitor, ethopropazine. Diln. of blood samples (1:100) stopped secondary reactions in the presence of inhibitor (organophosphate) and reactivator (oxime). Normalization of the AChE activity to the Hb content, detd. as cyanmetHb, prevented diln. errors. This modified approach provides a simple way for sensitive and precise detn. of AChE activity in whole blood in the presence of organophosphates even with low-tech equipment.
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    Eyer, P.; Worek, F.; Kiderlen, D.; Sinko, G.; Stuglin, A.; Simeon-Rudolf, V.; Reiner, E. Molar Absorption Coefficients for the Reduced Ellman Reagent: Reassessment. Anal. Biochem. 2003, 312 (2), 224– 7,  DOI: 10.1016/S0003-2697(02)00506-7
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    Molar absorption coefficients for the reduced Ellman reagent: reassessment
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    Analytical Biochemistry (2003), 312 (2), 224-227CODEN: ANBCA2; ISSN:0003-2697. (Elsevier Science)
    The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coeff., 13.6×103 M-1 cm-1, as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for detn. of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temp. increases, while absorbance maxima decrease. Our recommended molar absorption coeffs. at 412 nm are 14.15×103 M-1 cm-1 at 25° and 13.8×103 M-1 cm-1 at 37° (0.1 M phosphate buffer, pH 7.4). Molar absorption coeffs. for other temps. and wavelengths are included in the paper.
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    Pullela, P. K.; Chiku, T.; Carvan, M. J.; Sem, D. S. Fluorescence-Based Detection of Thiols in Vitro and in Vivo Using Dithiol Probes. Anal. Biochem. 2006, 352 (2), 265– 73,  DOI: 10.1016/j.ab.2006.01.047
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    Fluorescence-based detection of thiols in vitro and in vivo using dithiol probes
    Pullela, Phani Kumar; Chiku, Taurai; Carvan, Michael J.; Sem, Daniel S.
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    Thiols play a central role in maintaining biol. homeostasis. Their levels can change dramatically in response to oxidative stress assocd. with toxic insults, bacterial infection, and disease. Therefore, a reagent that can monitor thiol levels both in vitro and in vivo would be useful for assays and as a biomarker. Such a reagent should (i) be selective for thiols, (ii) be able to penetrate cell walls, and (iii) have a low redn. potential so as not to create oxidative stress in a cell. The authors have developed such a fluorescent reagent (DSSA) based on a dithiol linker: (i) the use of a dithiol linker makes it selective for thiols; (ii) the use of fluorophores that populate neutral states at physiol. pH improves cell wall penetration; and (iii) because of the reagent's low redn. potential (-0.60 V), it will not stress cells oxidatively. For example, 5 μM of reagent is responsive to changes in glutathione levels in the physiol. relevant range of 1 to 10 mM, yet this would oxidize less than 1% of cellular glutathione. In Escherichia coli, decreased thiol levels were detected in cells deficient in glutathione synthesis. In zebrafish embryos, the DSSA reagent permitted detection of unusually high thiol levels in the zebrafish chorion.
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    Cao, X.; Lin, W.; Yu, Q. A Ratiometric Fluorescent Probe for Thiols Based on a Tetrakis(4-Hydroxyphenyl)Porphyrin-Coumarin Scaffold. J. Org. Chem. 2011, 76 (18), 7423– 30,  DOI: 10.1021/jo201199k
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    A Ratiometric Fluorescent Probe for Thiols Based on a Tetrakis(4-hydroxyphenyl)porphyrin-Coumarin Scaffold
    Cao, Xiaowei; Lin, Weiying; Yu, Quanxing
    Journal of Organic Chemistry (2011), 76 (18), 7423-7430CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    The authors have designed and synthesized the compd. Ratio-HPSSC, based on a tetrakis(4-hydroxyphenyl)porphyrin-coumarin scaffold, as a new ratiometric fluorescent probe for thiols. The ratiometric probe Ratio-HPSSC is highly selective and sensitive to thiols. Importantly, the novel ratiometric probe exhibited a remarkable change in emission color from red to blue. This key feature allows Ratio-HPSSC to be employed for thiol detection by simple visual inspection. Furthermore, Ratio-HPSSC is suitable for ratiometric fluorescence imaging of thiols in living cells. The authors believe that the new ratiometric probe will find interesting applications in chem., biol., and medicine.
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    Wang, L.; Wang, J.; Xia, S.; Wang, X.; Yu, Y.; Zhou, H.; Liu, H. A FRET-Based near-Infrared Ratiometric Fluorescent Probe for Detection of Mitochondria Biothiol. Talanta 2020, 219, 121296,  DOI: 10.1016/j.talanta.2020.121296
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    A FRET-based near-infrared ratiometric fluorescent probe for detection of mitochondria biothiol
    Wang, Lu; Wang, Jianbo; Xia, Shuai; Wang, Xinxin; Yu, Yating; Zhou, Hongwei; Liu, Haiying
    Talanta (2020), 219 (), 121296CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
    A new fluorescent probe A with BODIPY as FRET donor and near-IR rhodamine as FRET acceptor is constructed through disulfide bonding and use for ratiometric fluorescence detection of biothiol. Due to the efficient fluorescence resonance energy transfer (FRET) from BODIPY donor to near-IR rhodamine acceptor, Probe A only displays near-IR rhodamine fluorescence (λem = 656 nm) under BODIPY excitation at 480 nm. The presence of biothiol leads to BODIPY fluorescence increases (λem = 511 nm) and near-IR rhodamine fluorescence decreases since the disulfide bond of the probe is broken by biothiols, effectively sepg. the donor from the acceptor, thus inhibiting the FRET process. Probe A exhibits remarkable high selectivity and excellent linear relationship from 10 μM to 100 μM of GSH, with low detection limit as 0.26 μM. Cellular imaging expts. shows that the probe is predominantly present in mitochondria and has been successfully applied to detect biothiol concns. changes in mitochondria of living cells.
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    Lim, S. Y.; Shen, W.; Gao, Z. Carbon Quantum Dots and Their Applications. Chem. Soc. Rev. 2015, 44 (1), 362– 81,  DOI: 10.1039/C4CS00269E
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    Carbon quantum dots and their applications
    Lim, Shi Ying; Shen, Wei; Gao, Zhiqiang
    Chemical Society Reviews (2015), 44 (1), 362-381CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. Fluorescent carbon nanoparticles or carbon quantum dots (CQDs) are a new class of carbon nanomaterials that have emerged recently and have garnered much interest as potential competitors to conventional semiconductor quantum dots. In addn. to their comparable optical properties, CQDs have the desired advantages of low toxicity, environmental friendliness low cost and simple synthetic routes. Moreover, surface passivation and functionalization of CQDs allow for the control of their physicochem. properties. Since their discovery, CQDs have found many applications in the fields of chem. sensing, biosensing, bioimaging, nanomedicine, photocatalysis and electrocatalysis. This article reviews the progress in the research and development of CQDs with an emphasis on their synthesis, functionalization and tech. applications along with some discussion on challenges and perspectives in this exciting and promising field.
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    Steinmann, D.; Nauser, T.; Koppenol, W. H. Selenium and Sulfur in Exchange Reactions: A Comparative Study. J. Org. Chem. 2010, 75 (19), 6696– 9,  DOI: 10.1021/jo1011569
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    Selenium and Sulfur in Exchange Reactions: A Comparative Study
    Steinmann, Daniel; Nauser, Thomas; Koppenol, Willem H.
    Journal of Organic Chemistry (2010), 75 (19), 6696-6699CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    Cysteamine reduces selenocystamine to form hemiselenocystamine and then cystamine. The rate consts. are k1 = 1.3 × 105 M-1 s-1; k-1 = 2.6 × 107 M-1 s-1; k2 = 11 M-1 s-1; and k-2 = 1.4 × 103 M-1 s-1, resp. Rate consts. for reactions of cysteine/selenocysteine are similar. Reaction rates of selenium as a nucleophile and as an electrophile are 2-3 and 4 orders of magnitude higher, resp., than those of sulfur. Sulfides and selenides are comparable as leaving groups.
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    Mafireyi, T. J.; Laws, M.; Bassett, J. W.; Cassidy, P. B.; Escobedo, J. O.; Strongin, R. M. A Diselenide Turn-On Fluorescent Probe for the Detection of Thioredoxin Reductase. Angew. Chem., Int. Ed. 2020, 59 (35), 15147– 51,  DOI: 10.1002/anie.202004094
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    A Diselenide Turn-On Fluorescent Probe for the Detection of Thioredoxin Reductase
    Mafireyi, Tendai J.; Laws, Madeleine; Bassett, John W.; Cassidy, Pamela B.; Escobedo, Jorge O.; Strongin, Robert M.
    Angewandte Chemie, International Edition (2020), 59 (35), 15147-15151CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    We report the first diselenide-based probe for the selective detection of thioredoxin reductase (TrxR), an enzyme commonly overexpressed in melanomas. The probe design involves conjugation of a seminaphthorhodafluor dye with a diselenide moiety. TrxR reduces the diselenide bond, triggering a fluorescence turn-on response of the probe. Kinetic studies reveal favorable binding of the probe with TrxR with a Michaelis-Menten const. (Km) of 15.89 μM. Computational docking simulations predict a greater binding affinity to the TrxR active site in comparison to its disulfide analog. In vitro imaging studies further confirmed the diselenide probe exhibited improved signaling of TrxR activity compared to the disulfide analog.
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    Nogueira, C. W.; Barbosa, N. V.; Rocha, J. B. Toxicology and Pharmacology of Synthetic Organoselenium Compounds: An Update. Arch. Toxicol. 2021, 95 (4), 1179– 226,  DOI: 10.1007/s00204-021-03003-5
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    Toxicology and pharmacology of synthetic organoselenium compounds: an update
    Nogueira, Cristina W.; Barbosa, Nilda V.; Rocha, Joao B. T.
    Archives of Toxicology (2021), 95 (4), 1179-1226CODEN: ARTODN; ISSN:0340-5761. (Springer)
    Here, we addressed the pharmacol. and toxicol. of synthetic organoselenium compds. and some naturally occurring organoselenium amino acids. The use of selenium as a tool in org. synthesis and as a pharmacol. agent goes back to the middle of the nineteenth and the beginning of the twentieth centuries. The rediscovery of ebselen and its investigation in clin. trials have motivated the search for new organoselenium mols. with pharmacol. properties. Although ebselen and diselenides have some overlapping pharmacol. properties, their mol. targets are not identical. However, contrary to our early expectations that they could imitate selenoproteins, organoselenium compds. seem to have non-specific modulatory activation of antioxidant pathways and specific inhibitory effects in some thiol-contg. proteins. The thiol-oxidizing properties of organoselenium compds. are considered the mol. basis of their However, the acute use of organoselenium compds. as inhibitors of specific thiol-contg. enzymes can be of therapeutic significance. In summary, the outcomes of the clin. trials of ebselen as a mimetic of lithium or as an inhibitor of SARS-CoV-2 proteases will be important to the field of organoselenium synthesis. The development of computational techniques that could predict rational modifications in the structure of organoselenium compds. to increase their specificity is required to construct a library of thiol-modifying agents with selectivity toward specific target proteins.
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    Lou, Z.; Li, P.; Sun, X.; Yang, S.; Wang, B.; Han, K. A Fluorescent Probe for Rapid Detection of Thiols and Imaging of Thiols Reducing Repair and H2O2 Oxidative Stress Cycles in Living Cells. Chem. Commun. 2013, 49 (4), 391– 3,  DOI: 10.1039/C2CC36839K
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    626
    A fluorescent probe for rapid detection of thiols and imaging of thiols reducing repair and H2O2 oxidative stress cycles in living cells
    Lou, Zhangrong; Li, Peng; Sun, Xiaofei; Yang, Songqiu; Wang, Bingshuai; Han, Keli
    Chemical Communications (Cambridge, United Kingdom) (2013), 49 (4), 391-393CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A diselenide contg. fluorescent probe (I) based on a fluorescein scaffold for thiols was developed. The fluorescent probe exhibited rapid response, high selectivity and reversibility. Confocal fluorescence microscopy was used to visualize the redox changes mediated by thiols and reactive oxygen species in living HeLa cells.
  624. 624
    Han, X.; Song, X.; Yu, F.; Chen, L. A Ratiometric Fluorescent Probe for Imaging and Quantifying Anti-Apoptotic Effects of GSH under Temperature Stress. Chem. Sci. 2017, 8 (10), 6991– 7002,  DOI: 10.1039/C7SC02888A
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    627
    A ratiometric fluorescent probe for imaging and quantifying anti-apoptotic effects of GSH under temperature stress
    Han, Xiaoyue; Song, Xinyu; Yu, Fabiao; Chen, Lingxin
    Chemical Science (2017), 8 (10), 6991-7002CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Hypothermia and hyperthermia are cell stressed states resulting from environmental temp. changes, which can abnormally decrease intracellular glutathione (GSH) concns. and induce apoptosis. As the most abundant intracellular non-protein biothiol, GSH can protect cells from apoptosis. Considering the important roles of GSH in the anti-apoptotic process in cells and in vivo, we strive to develop a powerful chem. tool for the direct detection of GSH concn. changes under temp. stress. Herein, we report a ratiometric fluorescent probe (CyO-Dise) based on a selenium-sulfur exchange reaction for the qual. and quant. detection of GSH concn. fluctuations in cells and in vivo. The probe has been successfully used to assess the changes of GSH levels in HepG2 and HL-7702 cells using the stimulations of hypothermia and hyperthermia. In terms of the anti-apoptotic effect of GSH under hypothermic and hyperthermic conditions, human normal liver HL-7702 cells have stronger abilities to fight against temp. stress than human liver carcinoma HepG2 cells. Hypothermia and hyperthermia can also improve the drug resistance of cis-dichlorodiamineplatinum(II) (DDP)-resistant HepG2/DDP cells. The CyO-Dise probe has been employed to image GSH concn. changes in HepG2 and HepG2/DDP xenografts on nude mice. With the adjuvant therapy effects of hypothermia and hyperthermia, the chemotherapy drug DDP exhibits good ability for the treatment of HepG2 and HepG2/DDP xenografts.
  625. 625
    Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G. A Rhodamine-Based Fluorescent Probe Containing a Se-N Bond for Detecting Thiols and Its Application in Living Cells. J. Am. Chem. Soc. 2007, 129 (38), 11666– 7,  DOI: 10.1021/ja072572q
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    628
    A Rhodamine-Based Fluorescent Probe Containing a Se-N Bond for Detecting Thiols and Its Application in Living Cells
    Tang, Bo; Xing, Yanlong; Li, Ping; Zhang, Ning; Yu, Fabiao; Yang, Guiwen
    Journal of the American Chemical Society (2007), 129 (38), 11666-11667CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Here the authors report a new rhodamine-based fluorescent probe (I) contg. a selenium-nitrogen bond for detecting thiols based on the nucleophilic substitution of sulfhydryl. The probe was successfully applied to the imaging of thiols in both HL-7702 cells and HepG2 cells with high sensitivity and selectivity.
  626. 626
    Tang, B.; Yin, L.; Wang, X.; Chen, Z.; Tong, L.; Xu, K. A Fast-Response, Highly Sensitive and Specific Organoselenium Fluorescent Probe for Thiols and Its Application in Bioimaging. Chem. Commun. 2009, (35), 5293– 5,  DOI: 10.1039/b909542j
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    629
    A fast-response, highly sensitive and specific organoselenium fluorescent probe for thiols and its application in bioimaging
    Tang, Bo; Yin, Lingling; Wang, Xu; Chen, Zhenzhen; Tong, Lili; Xu, Kehua
    Chemical Communications (Cambridge, United Kingdom) (2009), (35), 5293-5295CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A novel organoselenium fluorescent probe (Rh-Se-2, I), which features a high signal-to-noise ratio (up to 170-fold), fast response (5 min) and excellent immunity to interferences, was designed, synthesized and applied to bioimaging.
  627. 627
    Wang, R.; Chen, L.; Liu, P.; Zhang, Q.; Wang, Y. Sensitive near-Infrared Fluorescent Probes for Thiols Based on Se-N Bond Cleavage: Imaging in Living Cells and Tissues. Eur. J. Chem. 2012, 18, 11343,  DOI: 10.1002/chem.201200671
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    630
    Sensitive Near-Infrared Fluorescent Probes for Thiols Based on S-N Bond Cleavage: Imaging in Living Cells and Tissues
    Wang, Rui; Chen, Lingxin; Liu, Ping; Zhang, Qin; Wang, Yunqing
    Chemistry - A European Journal (2012), 18 (36), 11343-11349, S11343/1-S11343/5CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Cy-NiSe and Cy-TfSe were designed and synthesized as sensitive near-IR (NIR) fluorescent probes for detecting thiols on the basis of Se-N bond cleavage both in cells and in tissues. Since a donor-excited photoinduced electron transfer (d-PET) process occurs between the modulator and the fluorophore, Cy-NiSe and Cy-TfSe have weak fluorescence. On titrn. with glutathione, the free dye exhibits significant fluorescence enhancement. The two probes are sensitive and selective for thiols over other relevant biol. species. They can function rapidly at pH 7.4, and their emission lies in the NIR region. Confocal imaging confirms that Cy-NiSe and Cy-TfSe can be used for detecting thiols in living cells and tissues.
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    Tian, Y.; Zhu, B.; Yang, W.; Jing, J.; Zhang, X. A Fluorescent Probe for Differentiating Cys, Hcy and GSH Via a Stepwise Interaction. Sens. Actuators B Chem. 2018, 262, 345– 9,  DOI: 10.1016/j.snb.2018.01.181
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    631
    A fluorescent probe for differentiating Cys, Hcy and GSH via a stepwise interaction
    Tian, Yong; Zhu, Baocun; Yang, Wen; Jing, Jing; Zhang, Xiaoling
    Sensors and Actuators, B: Chemical (2018), 262 (), 345-349CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    It is still challenging to simultaneously distinguish general biothiols (cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) etc.) in complex redox environments, due to the lack of reliable and convenient method. Although a no. of fluorescence and/or UV-vis absorption based measurements have been employed for single or multiple biothiols detection, few of them is capable of recognizing each biothiol with individual signal at the same time. We present a one-step obtained selenadiazole deriv. (1) to distinguish Cys, Hcy and GSH, which not only can produce three new kind of fluorescent compds., but also simultaneously induce different "turn-on" fluorescence emission and red-shifted absorption signals for discriminating each biothiol. With a series of systematically analyses elucidate that probe 1 possesses high sensitivity and good selectivity for the three biothiols. Furthermore, the detection mechanism was also verified via a stepwise selenium-nitrogen (Se-N) bond cleavage process of probe 1 interacting with each biothiol. In the application of tracking in vitro and in vivo biothiols, probe 1 can be acted as a dual-mode fluorescent and colorimetric platform.
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    Rusin, O.; St Luce, N. N.; Agbaria, R. A.; Escobedo, J. O.; Jiang, S.; Warner, I. M.; Dawan, F. B.; Lian, K.; Strongin, R. M. Visual Detection of Cysteine and Homocysteine. J. Am. Chem. Soc. 2004, 126 (2), 438,  DOI: 10.1021/ja036297t
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    Visual detection of cysteine and homocysteine
    Rusin, Oleksandr; St. Luce, Nadia N.; Agbaria, Rezik A.; Escobedo, Jorge O.; Jiang, Shan; Warner, Isiah M.; Dawan, Fareed B.; Lian, Kun; Strongin, Robert M.
    Journal of the American Chemical Society (2004), 126 (2), 438-439CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The detn. of cysteine and homocysteine levels is of great current interest for the monitoring of disease states. A new colorimetric method for the simultaneous detection of L-cysteine and L-homocysteine has been developed. A fluorescein deriv. reacts with the above amino acids, producing their resp. thiazolidines resulting in color changes. Interference from other amino acids and proteins is minimal.
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    Lim, S.; Escobedo, J. O.; Lowry, M.; Xu, X.; Strongin, R. Selective Fluorescence Detection of Cysteine and N-Terminal Cysteine Peptide Residues. Chem. Commun. 2010, 46 (31), 5707– 9,  DOI: 10.1039/c0cc01398f
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    Selective fluorescence detection of cysteine and N-terminal cysteine peptide residues
    Lim, Soojin; Escobedo, Jorge O.; Lowry, Mark; Xu, Xiangyang; Strongin, Robert
    Chemical Communications (Cambridge, United Kingdom) (2010), 46 (31), 5707-5709CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A new fluorogenic fluorescein deriv. contg. an α,β-unsatd. aldehyde moiety produces a selective fluorescent signal enhancement in the presence of cysteine or peptides contg. N-terminal cysteine residues. The mechanism is based on synergistic covalent and supramol. interactions.
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    Lee, K.-S.; Kim, T.-K.; Lee, J. H.; Kim, H.-J.; Hong, J.-I. Fluorescence Turn-On Probe for Homocysteine and Cysteine in Water. Chem. Commun. 2008, (46), 6173– 5,  DOI: 10.1039/b814581d
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    Fluorescence turn-on probe for homocysteine and cysteine in water
    Lee, Kyung-Sik; Kim, Tae-Ki; Lee, Jong Ho; Kim, Hae-Jo; Hong, Jong-In
    Chemical Communications (Cambridge, United Kingdom) (2008), (46), 6173-6175CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A simple fluorescent probe (I) based on an ortho-hydroxy aldehyde-functionalized coumarin showed selective responses to homocysteine and cysteine by fluorescence turn-on.
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    Eun Jun, M.; Roy, B.; Han Ahn, K. “Turn-On” Fluorescent Sensing with “Reactive” Probes. Chem. Commun. 2011, 47 (27), 7583– 601,  DOI: 10.1039/c1cc00014d
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    Hoyle, C. E.; Lowe, A. B.; Bowman, C. N. Thiol-Click Chemistry: A Multifaceted Toolbox for Small Molecule and Polymer Synthesis. Chem. Soc. Rev. 2010, 39 (4), 1355– 87,  DOI: 10.1039/b901979k
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    Thiol-click chemistry: a multifaceted toolbox for small molecule and polymer synthesis
    Hoyle, Charles E.; Lowe, Andrew B.; Bowman, Christopher N.
    Chemical Society Reviews (2010), 39 (4), 1355-1387CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. The merits of thiol-click chem. and its potential for making new forays into chem. synthesis and materials applications are described. Since thiols react to high yields under benign conditions with a vast range of chem. species, their utility extends to a large no. of applications in the chem., biol., phys., materials and engineering fields. This crit. review provides insight into emerging venues for application as well as new mechanistic understanding of this exceptional chem. in its many forms (81 refs.).
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    LoPachin, R. M.; Gavin, T. Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective. Chem. Res. Toxicol. 2014, 27 (7), 1081– 91,  DOI: 10.1021/tx5001046
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    Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective
    LoPachin, Richard M.; Gavin, Terrence
    Chemical Research in Toxicology (2014), 27 (7), 1081-1091CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    A review. Aldehydes are electrophilic compds. to which humans are pervasively exposed. Despite a significant health risk due to exposure, the mechanisms of aldehyde toxicity are poorly understood. This ambiguity is likely due to the structural diversity of aldehyde derivs. and corresponding differences in chem. reactions and biol. targets. To gain mechanistic insight, the authors have used parameters based on the hard and soft, acids and bases (HSAB) theory to profile the different aldehyde subclasses with respect to electronic character (softness, hardness), electrophilic reactivity (electrophilic index), and biol. nucleophilic targets. The authors' analyses indicate that short chain aldehydes and longer chain satd. alkanals are hard electrophiles that cause toxicity by forming adducts with hard biol. nucleophiles, e.g., primary nitrogen groups on lysine residues. In contrast, α,β-unsatd. carbonyl derivs., alkenals, and the α-oxoaldehydes are soft electrophiles that preferentially react with soft nucleophilic thiolate groups on cysteine residues. The aldehydes can therefore be grouped into subclasses according to common electronic characteristics (softness/hardness) and mol. mechanisms of toxicity. As the authors will discuss, the toxic potencies of these subgroups are generally related to corresponding electrophilicities. For some aldehydes, however, predictions of toxicity based on electrophilicity are less accurate due to inherent physicochem. variables that limit target accessibility, e.g., steric hindrance and soly. The unsatd. aldehydes are also members of the conjugated type-2 alkene chem. class that includes α,β-unsatd. amide, ketone, and ester derivs. Type-2 alkenes are electrophiles of varying softness and electrophilicity that share a common mechanism of toxicity. Therefore, exposure to an environmental mixt. of unsatd. carbonyl derivs. could cause "type-2 alkene toxicity" through additive interactions. Finally, the authors propose that environmentally derived aldehydes can accelerate diseases by interacting with endogenous aldehydes generated during oxidative stress. This review provides a basis for understanding aldehyde mechanisms and environmental toxicity through the context of electronic structure, electrophilicity, and nucleophile target selectivity.
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    Chan, J. W.; Hoyle, C. E.; Lowe, A. B.; Bowman, M. Nucleophile-Initiated Thiol-Michael Reactions: Effect of Organocatalyst, Thiol, and Ene. Macromolecules 2010, 43 (15), 6381– 8,  DOI: 10.1021/ma101069c
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    Nucleophile-Initiated Thiol-Michael Reactions: Effect of Organocatalyst, Thiol, and Ene
    Chan, Justin W.; Hoyle, Charles E.; Lowe, Andrew B.; Bowman, Mark
    Macromolecules (Washington, DC, United States) (2010), 43 (15), 6381-6388CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    A detailed evaluation of the kinetics of the thiol-Michael reaction between hexanethiol and hexyl acrylate is described. It is shown that primary amines are more effective catalysts than either secondary or tertiary amines with, for example, quant. conversion being achieved within 500 s in the case of hexylamine with an apparent rate const. of 53.4 mol L-1 s-1 at a catalyst loading of 0.057 mol %. Certain tertiary phosphines, and esp. tri-n-propylphosphine and dimethylphenylphosphine, are shown to be even more effective species even at concns. 2 orders of magnitude lower than employed for hexylamine and performed in soln. with quant. conversions reached within ca. 100 s for both species and apparent rate consts. of 1810 and 431 mol L-1 s-1, resp. The nature of the thiol is also demonstrated to be an important consideration with mercaptoglycolate and mercaptopropionate esters being significantly more reactive than hexanethiol with reactivity mirroring the pKa of the thiols. Likewise, it is shown that the structure of the activated ene is also crucial with the degree of activation and ene-substitution pattern being important features in detg. reactivity. In terms of reaction with hexanethiol in the presence of hexylamine as catalyst, it is shown that propylmaleimide > di-Et fumarate > di-Et maleate > dimethylacrylamide > acrylonitrile > Et crotonate > Et cinnamate > Et methacrylate.
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    Langmuir, M. E.; Yang, J.-R.; Moussa, A. M.; Laura, R.; LeCompte, K. A. New Naphthopyranone Based Fluorescent Thiol Probes. Tetrahedron Lett. 1995, 36 (23), 3989– 92,  DOI: 10.1016/0040-4039(95)00695-9
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    New naphthopyranone based fluorescent thiol probes
    Langmuir, Margaret E.; Yang, Jun-Rui; Moussa, Adel M.; Laura, Richard; LeCompte, Karen A.
    Tetrahedron Letters (1995), 36 (23), 3989-92CODEN: TELEAY; ISSN:0040-4039. (Elsevier)
    Five newly synthesized naphthopyranone-based thiol reactive probes are reported and their photophys. properties are compared with those of their parent naphthopyranones and their glutathione adducts.
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    Niu, W.; Wu, P.; Chen, F.; Wang, J.; Shang, X.; Xu, C. Discovery of Selective Cystathionine β-Synthase Inhibitors by High-Throughput Screening with a Fluorescent Thiol Probe. MedChemComm 2017, 8 (1), 198– 201,  DOI: 10.1039/C6MD00493H
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    640
    Discovery of selective cystathionine β-synthase inhibitors by high-throughput screening with a fluorescent thiol probe
    Niu, Weining; Wu, Ping; Chen, Fei; Wang, Jun; Shang, Xiaoya; Xu, Chunlan
    MedChemComm (2017), 8 (1), 198-201CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)
    A high-throughput assay was developed to identify inhibitors of cystathionine β-synthase (CBS), which is one of three key enzymes involved in H2S biosynthesis. Screening of 6491 natural compds. identified several selective CBS inhibitors, which suppressed the proliferation of HT29 cancer cells, with IC50 values of less than 10 μM.
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    Aharoni, A.; Amitai, G.; Bernath, K.; Magdassi, S.; Tawfik, D. S. High-Throughput Screening of Enzyme Libraries: Thiolactonases Evolved by Fluorescence-Activated Sorting of Single Cells in Emulsion Compartments. Chem. Biol. 2005, 12 (12), 1281– 9,  DOI: 10.1016/j.chembiol.2005.09.012
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    High-Throughput Screening of Enzyme Libraries: Thiolactonases Evolved by Fluorescence-Activated Sorting of Single Cells in Emulsion Compartments
    Aharoni, Amir; Amitai, Gil; Bernath, Kalia; Magdassi, Shlomo; Tawfik, Dan S.
    Chemistry & Biology (Cambridge, MA, United States) (2005), 12 (12), 1281-1289CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
    Single bacterial cells, each expressing a different library variant, were compartmentalized in aq. droplets of water-in-oil (w/o) emulsions, thus maintaining a linkage between a plasmid-borne gene, the encoded enzyme variant, and the fluorescent product this enzyme may generate. Conversion into a double, water-in-oil-in-water (w/o/w) emulsion enabled the sorting of these compartments by FACS, as well as the isolation of living bacteria cells and their enzyme-coding genes. We demonstrate the directed evolution of new enzyme variants by screening >107 serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying >104 enzyme mols., in a vol. of <10 fL (fl), enabled detection and selection despite the fast, spontaneous hydrolysis of the substrate, the very low initial thiolactonase activity of PON1, and the use of difusable fluorescent products.
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    Barb, A. W.; Marcella, A. M. A Rapid Fluorometric Assay for the S-Malonyltransacylase FabD and Other Sulfhydryl Utilizing Enzymes. J. Biol. Methods 2016, 3 (4), e53  DOI: 10.14440/jbm.2016.144
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    Yi, L.; Li, H.; Sun, L.; Liu, L.; Zhang, C.; Xi, Z. A Highly Sensitive Fluorescence Probe for Fast Thiol-Quantification Assay of Glutathione Reductase. Angew. Chem., Int. Ed. 2009, 48 (22), 4034– 7,  DOI: 10.1002/anie.200805693
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    A Highly Sensitive Fluorescence Probe for Fast Thiol-Quantification Assay of Glutathione Reductase
    Yi, Long; Li, Heyang; Sun, Lu; Liu, Liangliang; Zhang, Caihong; Xi, Zhen
    Angewandte Chemie, International Edition (2009), 48 (22), 4034-4037, S4034/1-S4034/17CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Fast detection of cellular thiols in aq. medium was achieved using a newly developed fluorescence probe (see picture). Based on this probe, a high-throughput fluorescence assay for glutathione reductase was developed.
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    Zhou, X.; Jin, X.; Sun, G.; Li, D.; Wu, X. A Cysteine Probe with High Selectivity and Sensitivity Promoted by Response-Assisted Electrostatic Attraction. Chem. Commun. 2012, 48 (70), 8793– 5,  DOI: 10.1039/c2cc33971d
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    A cysteine probe with high selectivity and sensitivity promoted by response-assisted electrostatic attraction
    Zhou, Xin; Jin, Xuejun; Sun, Guangyan; Li, Donghao; Wu, Xue
    Chemical Communications (Cambridge, United Kingdom) (2012), 48 (70), 8793-8795CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A new turn-on fluorescent probe for fast detection of cysteine in physiol. conditions, based on a new response-assisted electrostatic attraction strategy, is reported. The practical utility of the probe in fluorescent protein labeling and subcellular imaging is also demonstrated.
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    Li, S.-J.; Fu, Y.-J.; Li, C.-Y.; Li, Y.-F.; Yi, L.-H.; Ou-Yang, J. A near-Infrared Fluorescent Probe Based on BODIPY Derivative with High Quantum Yield for Selective Detection of Exogenous and Endogenous Cysteine in Biological Samples. Anal. Chim. Acta 2017, 994, 73– 81,  DOI: 10.1016/j.aca.2017.09.031
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    A near-infrared fluorescent probe based on BODIPY derivative with high quantum yield for selective detection of exogenous and endogenous cysteine in biological samples
    Li, Song-Jiao; Fu, Ya-Jun; Li, Chun-Yan; Li, Yong-Fei; Yi, Lan-Hua; Ou-Yang, Juan
    Analytica Chimica Acta (2017), 994 (), 73-81CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    Cysteine (Cys) is involved in cellular growth and Cys deficiency is related with many diseases. So far, a no. of fluorescent probes have been constructed for the detection of Cys successfully. However, the probes are difficult to discriminate Cys from Hcy and the emission wavelength of the probes is in UV or visible range. Herein, a NIR fluorescent probe named NIR-BODIPY-Ac (I) was synthesized and used to detect Cys. The emission wavelength of the probe is at 708 nm that belongs to near-IR (NIR) region by attaching indolium to BODIPY core, which is suitable for bioimaging in vivo. Moreover, the probe exhibits high fluorescence quantum yield (Φ = 0.51) after the addn. of Cys and high sensitivity toward Cys with 81-fold fluorescence enhancement. The linear range of the probe for Cys covers from 0.2 to 30 μM with a detection limit of 0.05 μM. Furthermore, the probe shows high selectivity towards Cys owing to the fact that there is more fast reaction rate between the probe and Cys than that of Hcy. In particular, the NIR fluorescent probe is applied for the detection of exogenous and endogenous Cys in biol. samples such as cell, tissue and mouse with satisfactory results.
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    Li, R.; Lei, C.; Zhao, X.-E.; Gao, Y.; Gao, H.; Zhu, S.; Wang, H. A Label-Free Fluorimetric Detection of Biothiols Based on the Oxidase-Like Activity of Ag+ Ions. Spectrochim. Acta - A: Mol. Biomol. Spectrosc. 2018, 188, 20– 5,  DOI: 10.1016/j.saa.2017.06.056
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    A label-free fluorimetric detection of biothiols based on the oxidase-like activity of Ag+ ions
    Li, Ru; Lei, Cuihua; Zhao, Xian-En; Gao, Yue; Gao, Han; Zhu, Shuyun; Wang, Hua
    Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy (2018), 188 (), 20-25CODEN: SAMCAS; ISSN:1386-1425. (Elsevier B.V.)
    In this work, a label-free and sensitive fluorimetric method has been developed for the detections of biothiols including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), based on the specific biothiol-induced inhibition of the oxidase-like activity of silver ions (Ag+). It is well established that o-phenylenediamine (OPD) can be oxidized by Ag+ ions to generate fluorescent 2,3-diaminophenazine (OPDox). The introduction of biothiols would inhibit the oxidn. of OPD by Ag+ due to the strong coordination between biothiols and Ag+. The changes of fluorescence intensities obtained in the Ag+-OPD system exhibited good linear correlations in the ranges of 0.50-30.0 μM for Cys, 1.0-45.0 μM for Hcy and 0.50-40.0 μM for GSH. The detection limits (S/N = 3) of Cys, Hcy and GSH were 110 nM, 200 nM and 150 nM, resp. Subsequently, the developed fluorimetric method was successfully applied for the detection of biothiols in human serum.
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    Mandani, S.; Sharma, B.; Dey, D.; Sarma, T. K. Carbon Nanodots as Ligand Exchange Probes in Au@C-Dot Nanobeacons for Fluorescent Turn-On Detection of Biothiols. Nanoscale 2015, 7 (5), 1802– 8,  DOI: 10.1039/C4NR05424E
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    Carbon nanodots as ligand exchange probes in Au@C-dot nanobeacons for fluorescent turn-on detection of biothiols
    Mandani, Sonam; Sharma, Bhagwati; Dey, Deepa; Sarma, Tridib K.
    Nanoscale (2015), 7 (5), 1802-1808CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    Au nanoparticle-carbon dot core-shell (Au@C-dot) nanocomposite was synthesized in aq. medium at room temp. using the carbon dots as reducing agents themselves. The carbon nanodots also function as an effective stabilizer by forming a thin layer surrounding Au nanoparticles (Au NPs) similar to self-assembled monolayers. Ligand exchange with thiol contg. biomols. resulted in the release of carbon dots from the Au NP surface leading to an enhancement of fluorescence. Simultaneously the agglomeration of Au NPs stimulated by the interaction of biothiols led to changes in the surface plasmon properties of Au NPs. A detailed spectroscopic investigation revealed a combination of static and dynamic quenching being involved in the process. Thus, the Au nanoparticle-carbon dot composite could be used as a dual colorimetric and fluorometric sensor for biothiols ranging from amino acids, peptides, proteins, enzymes etc. with a detection limit of 50 nM.
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    Fu, Y.; Li, H.; Hu, W.; Zhu, D. Fluorescence Probes for Thiol-Containing Amino Acids and Peptides in Aqueous Solution. Chem. Commun. 2005, (25), 3189– 91,  DOI: 10.1039/b503772g
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    Fluorescence probes for thiol-containing amino acids and peptides in aqueous solution
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    Chemical Communications (Cambridge, United Kingdom) (2005), (25), 3189-3191CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A new fluorescence probe is described using the "chemosensing ensemble" method. The probe shows high selectivity and sensitivity for thiol-contg. amino acids and peptides, and can detect cysteine and homocysteine from healthy to abnormal levels under physiol.-relevant conditions.
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    Nie, L.; Ma, H.; Sun, M.; Li, X.; Su, M.; Liang, S. Direct Chemiluminescence Determination of Cysteine in Human Serum Using Quinine-Ce(IV) System. Talanta 2003, 59 (5), 959– 64,  DOI: 10.1016/S0039-9140(02)00649-5
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    Direct chemiluminescence determination of cysteine in human serum using quinine-Ce(IV) system
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    Talanta (2003), 59 (5), 959-964CODEN: TLNTA2; ISSN:0039-9140. (Elsevier Science B.V.)
    A simple, sensitive and selective chemiluminescence (CL) method was developed for the detn. of cysteine. This method is based on that the weak CL of cysteine oxidized with Ce (IV) can be greatly enhanced by quinine. The calibration curve was linear over the range 3.5 × 10-9-3.5 × 10-6 M with a detection limit of 2.5 × 10-9 M (S/N = 3). The relative std. deviation is 8.4% by 10 replicate detns. of 2.9 × 10-8 M cysteine. Due to high sensitivity, the proposed method can be used directly to det. the total concn. of cysteine in human serum through simply dilg. the sample for a thousand fold. The obtained result was in agreement with that given by amino acid automated analyzer. The present method does not require any sepn., showing a simpler anal. characteristic. The mechanism of the CL reaction is also discussed.
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    Wang, S.; Ma, H.; Li, J.; Chen, X.; Bao, Z.; Sun, S. Direct Determination of Reduced Glutathione in Biological Fluids by Ce(Iv)-Quinine Chemiluminescence. Talanta 2006, 70 (3), 518– 21,  DOI: 10.1016/j.talanta.2005.12.052
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    Direct determination of reduced glutathione in biological fluids by Ce(IV)-quinine chemiluminescence
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    A simple, sensitive and selective chemiluminescence (CL) method is proposed for the detn. of reduced glutathione (GSH) in biol. fluids. This method is based on the fact that the weak CL of GSH oxidized with cerium(IV) can be greatly enhanced by quinine. The optimum conditions for the CL emission were investigated. The calibration curve for GSH was linear over the concn. range of 4.0 × 10-9-4.0 × 10-5 M with a detection limit of 5 × 10-10 M (S/N = 3). The R.S.D. was 4.0% by 11 replicate detns. of 1.0 × 10-7 M GSH. It was also found that GSH and cysteine, the two often-coexisting thiol compds. in biol. samples, exhibited a different CL sensitivity in the Ce(IV)-quinine system (the sensitivity of GSH was higher than that of cysteine). This leads to the successful use of the proposed method for the direct and selective detn. of GSH in rabbit whole blood and rat brain microdialyzate in the presence of cysteine. Moreover, compared to the classical 5,5'-dithiobis(2-nitrobenzoic acid) method, the present one has the advantages of simplicity and high sensitivity.
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    Zou, T.; Lum, C. T.; Chui, S. S.-Y.; Che, C.-M. Gold(III) Complexes Containing N-Heterocyclic Carbene Ligands: Thiol “Switch-On” Fluorescent Probes and Anti-Cancer Agents. Angew. Chem., Int. Ed. 2013, 52 (10), 2930– 3,  DOI: 10.1002/anie.201209787
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    Gold(III) Complexes Containing N-Heterocyclic Carbene Ligands: Thiol "Switch-on" Fluorescent Probes and Anti-Cancer Agents
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    Angewandte Chemie, International Edition (2013), 52 (10), 2930-2933CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A class of Au(III) complexes bearing N-heterocyclic carbene and 2,6-bis(imidazol-2-yl)pyridine or 2,6-bis(benzimidazol-2-yl)pyridine ligands have been developed. These Au(III)-NHC complexes are sensitive towards thiols, which leads to release of fluorescent ligand, and thus can serve as a switch-on probe for thiols in biol. systems. The Au(III) complexes can also suppress tumor growth in mice bearing HeLa xenografts. These Au(III) complexes could be a promising scaffold for the future development of novel switch-on probes and anti-cancer agents.
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    Han, G.-C.; Peng, Y.; Hao, Y.-Q.; Liu, Y.-N.; Zhou, F. Spectrofluorimetric Determination of Total Free Thiols Based on Formation of Complexes of Ce(III) with Disulfide Bonds. Anal. Chim. Acta 2010, 659 (1), 238– 42,  DOI: 10.1016/j.aca.2009.11.057
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    Spectrofluorimetric determination of total free thiols based on formation of complexes of Ce(III) with disulfide bonds
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    Analytica Chimica Acta (2010), 659 (1-2), 238-242CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    A simple, rapid, and sensitive detn. of total free thiol groups in biol. samples using cerium (IV) as a fluorescence probe is reported. The protocol is based on the oxidn. of thiols by Ce(IV) and the formation the Ce(III) disulfide complex, which gives a fluorescence enhancement of Ce(III) at 352 nm. Using glutathione (GSH) and cysteine as model compds., incubation with Ce(IV) at 25° for 6 min results in fluorescence, whose intensity is proportional to the thiol concn. in the range of 1.00-160 nM. The detection limits for GSH and cysteine are 0.05 and 0.08 nM, resp. Other common metal ions and amino acids have little interference to the thiol detection. Cu(II) was used as a fluorescence quencher to eliminate potential interference from tryptophan. The method has been successfully applied to assays of free thiol contents in pig liver tissue samples, with a RSD lower than 2.5% and recovery between 100.6% and 102.3%.
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    Wang, H.; Zhou, G.; Chen, X. An Iminofluorescein-Cu2+ Ensemble Probe for Selective Detection of Thiols. Sens. Actuators B Chem. 2013, 176, 698– 703,  DOI: 10.1016/j.snb.2012.10.006
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    An iminofluorescein-Cu2+ ensemble probe for selective detection of thiols
    Wang, Huilin; Zhou, Guodong; Chen, Xiaoqiang
    Sensors and Actuators, B: Chemical (2013), 176 (), 698-703CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    An iminofluorescein-Cu2+ ensemble probe was prepd. for the detection of thiols in aq. soln. In HEPES buffer, among various amino acids, only cysteine (Cys) induced fluorescence enhancement of Cu2+ ensemble. The mechanism was proposed that addn. of Cys induced decomplexation of Cu2+ from weak-fluorescent ensemble, followed by hydrolytic cleavage of the resulted Schiff base to give a strongly fluorescent fluoresceinaldehyde. By fluorescence titrn. using 2 μM iminofluorescein-Cu2+ ensemble probe, the detection limit for Cys was evaluated to be 9 μM with a signal-to-noise ratio of 3. Furthermore, the confocal microscopy expts. demonstrated that this sensor was potential for the imaging of thiols in living cells.
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    Jung, H. S.; Han, J. H.; Habata, Y.; Kang, C.; Kim, J. S. An Iminocoumarin-Cu(II) Ensemble-Based Chemodosimeter toward Thiols. Chem. Commun. 2011, 47 (18), 5142– 4,  DOI: 10.1039/c1cc10672d
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    An iminocoumarin-Cu(II) ensemble-based chemodosimeter toward thiols
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    Chemical Communications (Cambridge, United Kingdom) (2011), 47 (18), 5142-5144CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    An 'ensemble'-based fluorogenic chemodosimeter 1-Cu(II) (I) for detection of thiols is reported. Complex 1-Cu(II) sensitively senses thiols followed by hydrolysis to give a marked fluorescence enhancement over other amino acids at pH 7.4 under aq. media. Confocal microscopic imaging of complex 1-Cu(II) is also herewith demonstrated for cellular thiol detection in HepG2 cells.
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    Zhao, D.; Chen, C.; Sun, J.; Yang, X. Carbon Dots-Assisted Colorimetric and Fluorometric Dual-Mode Protocol for Acetylcholinesterase Activity and Inhibitors Screening Based on the Inner Filter Effect of Silver Nanoparticles. Analyst 2016, 141 (11), 3280– 8,  DOI: 10.1039/C6AN00514D
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    Carbon dots-assisted colorimetric and fluorometric dual-mode protocol for acetylcholinesterase activity and inhibitors screening based on the inner filter effect of silver nanoparticles
    Zhao, Dan; Chen, Chuanxia; Sun, Jian; Yang, Xiurong
    Analyst (Cambridge, United Kingdom) (2016), 141 (11), 3280-3288CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    The authors proposed an original and versatile dual-readout (colorimetric and fluorometric) protocol by silver nanoparticles (AgNPs) and fluorescent carbon dots (CDs), which was amenable to rapid, ultrasensitive assay of acetylcholinesterase (AChE) activity and its inhibitors. The sensing mechanism was based on the non-fluorescence state of CDs resulting from the inner filter effect (IFE) of AgNPs and the specific AChE-catalyzed hydrolysis of acetylthiocholine (ATCh) into thiocholine (TCh). Herein, the generated pos.-charged and thiol-bearing TCh at trace concn. levels could trigger the aggregation of AgNPs through the known electrostatic and Ag-SH interactions, thereby turning the sensing solns. gray and recovering the IFE-quenched fluorescence simultaneously. Furthermore, the existence of IFE mechanism was conceivably confirmed by combining the zeta potentials, fluorescence spectra, UV-visible spectra, fluorescence lifetime and TEM measurements. As far as the authors know, the present study has reported the first dual-mode proposal for assessing AChE activity by using a CDs-based IFE sensing strategy, where the detection limit was as low as 0.021 mU mL-1 and 0.016 mU mL-1 by colorimetric and fluorometric measurements, resp. However, the proposed assay was feasible to screen AChE inhibitors such as tacrine and carbaryl. Meanwhile, this rationally designed dual-mode sensing platform featured simplicity, rapidity, flexibility and diversity, which was demonstrated by the quant. detection of spiked carbaryl in apple juice samples with satisfactory results.
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    Yang, J.; Song, N.; Lv, X.; Jia, Q. UV-Light-Induced Synthesis of PEI-CuNCs Based on Cu2+-Quenched Fluorescence Turn-On Assay for Sensitive Detection of Biothiols, Acetylcholinesterase Activity and Inhibitor. Sens. Actuators B Chem. 2018, 259, 226– 32,  DOI: 10.1016/j.snb.2017.12.045
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    UV-light-induced synthesis of PEI-CuNCs based on Cu2+-quenched fluorescence turn-on assay for sensitive detection of biothiols, acetylcholinesterase activity and inhibitor
    Yang, Jinlan; Song, Naizhong; Lv, Xueju; Jia, Qiong
    Sensors and Actuators, B: Chemical (2018), 259 (), 226-232CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    Polyethyleneimine-protected copper nanoclusters (PEI-CuNCs) were synthesized and applied to detect Cu2+, biothiols, and acetylcholinesterase (AChE). A facile UV-light-induced method was used in the synthesize process to obtain PEI-CuNCs with high stability and strong fluorescence intensity. Based on the quenching mechanism, a label-free assay for sensing Cu2+ by PEI-CuNCs was developed with the limit of detection (LOD) of 0.12μM. Biothiols with -SH functional groups could react with Cu2+ to form the RSH-Cu2+ complex, making Cu2+-triggered fluorescence quenching turned on. Two biothiols, glutathione and cysteine, were detd. with LODs of 0.26μM and 0.34μM within linear ranges of 0.5-25μM and 1-25μM, resp. AChE could hydrolyze acetylthiocholine into thiocholine (TCh), which reacted with Cu2+ to form TCh-Cu2+ complex and turned on Cu2+-quenched fluorescence. LOD of AChE was calcd. to be 1.38 mU/mL within a linear range from 3 to 200 mU/mL. A typical inhibitor of AChE, tacrine, was also detected by this convenient fluorescence turn-on assay with the corresponding IC50 value (the inhibitor concn. required for 50% inhibition of the enzyme activity) of 53.4 nM. Moreover, the assay was successfully applied to the detection of biothiols and AChE activity in human serum samples. Results demonstrated the great potential of PEI-CuNCs and paved a new avenue for exploring their versatile biosensing applications.
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    Chen, Y.; Liu, W.; Zhang, B.; Suo, Z.; Xing, F.; Feng, L. Sensitive and Reversible Perylene Derivative-Based Fluorescent Probe for Acetylcholinesterase Activity Monitoring and Its Inhibitor. Anal. Biochem. 2020, 607, 113835,  DOI: 10.1016/j.ab.2020.113835
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    Sensitive and reversible perylene derivative-based fluorescent probe for acetylcholinesterase activity monitoring and its inhibitor
    Chen, Yingying; Liu, Wenxia; Zhang, Binbin; Suo, Zhiguang; Xing, Feifei; Feng, Lingyan
    Analytical Biochemistry (2020), 607 (), 113835CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    A reversible fluorescence probe for acetylcholinesterase activity detection was developed based on water sol. perylene deriv., N,N'-di(2-aspartic acid)-perylene-3,4,9,10-tetracarboxylic diimide (PASP). Based on the photo-induced electron transfer (PET), PASP fluorescence in aq. is quenched after combining with copper ions (Cu2+). Acetylcholinesterase (AChE) is known to catalyze the hydrolysis of acetylcholine (ATCh) to produce thiocholine, whose affinity is strong enough to capture Cu2+ by thiol (-SH) group from the complex PASP-Cu, resulting in the fluorescence signal of PASP recovers up to 90%. This optical switch is highly sensitive depended on the coordination and dissocn. between PASP and Cu2+. The authors proposed its application for AChE activity detection, as well as its inhibitor screening. According to the change of fluorescence intensity, quantifying the detection limit of AChE was 1.78 mU·mL-1. Classical inhibitors, tacrine and organophosphate pesticide diazinon, were further evaluated for drug screening. The IC50 value of tacrine is 0.43μM, and the detection limit of diazinon was 0.22μM. Both of these performances were much better than previous results, revealing the authors' probe is sensitive and reversible for screening applications.
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    Xu, J.; Yu, H.; Hu, Y.; Chen, M.; Shao, S. A Gold Nanoparticle-Based Fluorescence Sensor for High Sensitive and Selective Detection of Thiols in Living Cells. Biosens. Bioelectron. 2016, 75, 1– 7,  DOI: 10.1016/j.bios.2015.08.007
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    A gold nanoparticle-based fluorescence sensor for high sensitive and selective detection of thiols in living cells
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    Biosensors & Bioelectronics (2016), 75 (), 1-7CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
    A novel gold nanoparticle (AuNP)-based sensor for detecting thiols in aq. soln. has been developed. Due to the weak N···Au interactions, meso-(4-pyridinyl)-substituted BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes were coordinated to AuNP surfaces, which effectively quenched the fluorescence of org./inorg. hybrid systems. The fluorescent quenching mechanism was mainly ascribed to the highly efficient fluorescent resonance energy transfer (FRET) and the inner filter effect. In the presence of thiols, meso-(4-pyridinyl)-substituted BODIPY chromophore were displaced and released from the AuNP surfaces and thus restored the fluorescence of BODIPY chromophore. The modulation of the fluorescence quenching efficiency of BODIPY-AuNPs in the presence of thiols can achieve a large turn-on fluorescence enhancement (40-fold) in aq. soln. The new AuNP-based fluorescence sensor displayed desired properties such as high specificity, relatively low detection limit (30 nM for Cys), appreciable water soly. and rapid response time (within 2 min for Cys/Hcy). Moreover, the sensor has been successfully applied for monitoring and imaging of intracellular thiols within living HeLa cells.
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    Mu, H.; Miki, K.; Kubo, T.; Otsuka, K.; Ohe, K. Substituted meso-Vinyl-BODIPY as Thiol-Selective Fluorogenic Probes for Sensing Unfolded Proteins in the Endoplasmic Reticulum. Chem. Commun. 2021, 57 (14), 1818– 21,  DOI: 10.1039/D0CC08160D
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    Substituted meso-vinyl-BODIPY as thiol-selective fluorogenic probes for sensing unfolded proteins in the endoplasmic reticulum
    Mu, Huiying; Miki, Koji; Kubo, Takuya; Otsuka, Koji; Ohe, Kouichi
    Chemical Communications (Cambridge, United Kingdom) (2021), 57 (14), 1818-1821CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    A new type of thiol probes based on the meso-vinyl-BODIPY (VB) scaffold were developed. The monochloro-substituted VB1Cl exhibited the largest fluorescence enhancement (>200-fold) as well as high selectivity upon biol. thiol sensing. VB1Cl was successfully applied for reporting the protein unfolding process under ER stress in living cells.
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    Huang, S.-T.; Ting, K.-N.; Wang, K.-L. Development of a Long-Wavelength Fluorescent Probe Based on Quinone-Methide-Type Reaction to Detect Physiologically Significant Thiols. Anal. Chim. Acta 2008, 620 (1), 120– 6,  DOI: 10.1016/j.aca.2008.05.006
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    Development of a long-wavelength fluorescent probe based on quinone-methide-type reaction to detect physiologically significant thiols
    Huang, Sheng-Tung; Ting, Kuo-Neng; Wang, Kun-Li
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    The authors synthesized a new long-wavelength latent fluorimetric probe BCC (I) to detect physiol. significant thiols. The fluorogenic chem. transformation of BCC triggered by thiols is through a tandem reaction, thiol-induced benzoquinone redn., and quinone-methide-type rearrangement reaction, which are spontaneous and irreversible at physiol. temp. in aq. media. The fluorescence signal revealed by this process is specific and exhibited in the near-red spectrum region with emission maxima at 595 nm, and it could be competitively inhibited by thiols scavenger, N-ethylmaleimide. The fluorescent response of BCC is insensitive to various non-thiol amino acids and biol. reductants. This novel fluorimetric probe demonstrates a good relation in detecting thiols in 1-100 μM range, which presents to the applicability for the construction of fiber-optic biosensors in the future clin. diagnostic.
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    Qi, S.; Liu, W.; Zhang, P.; Wu, J.; Zhang, H.; Ren, H.; Ge, J.; Wang, P. A Colorimetric and Ratiometric Fluorescent Probe for Highly Selective Detection of Glutathione in the Mitochondria of Living Cells. Sens. Actuators B Chem. 2018, 270, 459– 65,  DOI: 10.1016/j.snb.2018.05.017
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    A colorimetric and ratiometric fluorescent probe for highly selective detection of glutathione in the mitochondria of living cells
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    Sensors and Actuators, B: Chemical (2018), 270 (), 459-465CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    A new water-sol. fluorescent probe 1 based on hemicyanine dye is designed for selective detection of glutathione (GSH). Probe 1 shows a tremendous colorimetric and ratiometric fluorescence response toward GSH with high selectivity over cysteine/homocysteine. After nucleophilic arom. substitution reaction with GSH, probe 1 undergoes an intramol. aldimine condensation that leads to forming a ring, which is accompanied by 107 and 41nm red-shifts in the absorption and fluorescence spectra, resp. Subsequent expts. in living cells indicate that probe 1 has a good photostability and response to mitochondrial GSH level in a ratiometric manner.
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    Li, X.; Huo, F.; Yue, Y.; Zhang, Y.; Yin, C. A Coumarin-Based “Off-On” Sensor for Fluorescence Selectivily Discriminating GSH from Cys/Hcy and Its Bioimaging in Living Cells. Sens. Actuators B Chem. 2017, 253, 42– 9,  DOI: 10.1016/j.snb.2017.06.120
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    A coumarin-based "off-on" sensor for fluorescence selectivily discriminating GSH from Cys/Hcy and its bioimaging in living cells
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    Glutathione (GSH) is the most abundant intracellular thiol with the concn. in the millimolar range. It has also been reported that GSH plays a crucial role in human pathologies, so it is meaningful to develop an effective probe for GSH. In this work, we report a coumarin-chlorine malononitrile "off-on" sensor for highly selective fluorescence detection of GSH over Cys/Hcy. The chlorine of the probe can be rapidly replaced by thiolates of biothiols through thiol-halogen nucleophilic substitution. The amino groups of Cys/Hcy but not GSH further replace the thiolate to form amino-substituted compd. Subsequently, an intramol. Michael addn. between the α,β-unsatd. malonitrile and thiol group may occur. However, GSH only forms thiol-substituted compd. The significantly different mechanisms enable the discrimination of GSH over Cys/Hcy through fluorescence response. Finally, fluorescence imaging of A549 cells indicated that probe 1 could be used for monitoring GSH in living cells. It would be a useful tool to understand the mechanisms of many related diseases.
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    Long, L.; Lin, W.; Chen, B.; Gao, W.; Yuan, L. Construction of a FRET-Based Ratiometric Fluorescent Thiol Probe. Chem. Commun. 2011, 47 (3), 893– 5,  DOI: 10.1039/C0CC03806G
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    We have rationally constructed a novel FRET-based ratiometric thiol probe suitable for ratiometric imaging in living cells based on the native chem. ligation reaction.
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    Chen, H.; Tang, Y.; Ren, M.; Lin, W. Single near-Infrared Fluorescent Probe with High- and Low-Sensitivity Sites for Sensing Different Concentration Ranges of Biological Thiols with Distinct Modes of Fluorescence Signals. Chem. Sci. 2016, 7 (3), 1896– 903,  DOI: 10.1039/C5SC03591K
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    Inoue, N. Selective Detection of Carcinogenic Aromatic Diamines in Aqueous Solutions Using 4-(N-Chloroformylmethyl-N-Methylamino)-7-Nitro-2,1,3-Benzoxadiazole (NBD-COCl) by HPLC. Anal. Sci. 2017, 33 (12), 1375– 1380
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    Analytical Sciences (2017), 33 (12), 1375-1380CODEN: ANSCEN; ISSN:0910-6340. (Japan Society for Analytical Chemistry)
    Reaction conditions for the selective derivatization of three types of arom. diamines without clean-up in an aq. soln. using 4-(N-chloroformylmethyl-N-methylamino)-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) have been developed for simple, fast, and selective anal. by HPLC. The arom. amines were derivatized to give di-NBD-CO-arom. amines at 35°C for 5 min in a buffer soln. at pH 5 contg. a low acetonitrile concn. (0 - 400 ng/mL, n = 5, r >0.998). The retention times of these di-NBD-CO-arom. amines were <5 min. Sample solns. contg. arom. amines, phenols, and an aliph. amine were also prepd. Under the optimized reaction conditions (i.e., low acetonitrile content and acidic conditions), no derivatization of the phenols and the aliph. amine was obsd. These were related to the differences in the pKa values of the target substances, the org. solvent concn., and the reaction soln. pH. For the model system, a sulfuric acid-impregnated filter was spiked with the arom. amines, phenols, and aliph. amine prior to extn., derivatization, and measurement by HPLC. Only arom. amines were detected quant., with no other compds. being obsd.
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    3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde, a reagent with broad dynamic range for the assay of proteins and lipoproteins in solution
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    This paper describes the application of 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA), a sensitive fluorogenic reagent for detection of amines, to the assay of proteins in soln. The sensitivity and dynamic range of CBQCA in the detn. of protein concn. is modulated by the no. of accessible amines present in the same limitations as other reagents used in fluorogenic assays based on fluorescent amine adducts such as those obtained with fluorescamine or o-phthaldialdehyde, or of other spectrophotometric methods, where the color development is detd. by the abundance in the protein of certain amino acids. However, in comparison to other fluorescence-based protein detection methods, CBQCA has proven to be an extremely sensitive reagent with a very broad, essentially linear dynamic range, capable of detecting from 10 ng to 150 μg of protein (in a 100- to 200-μL assay vol.). The CBQCA reagent also functions well in the presence of substances, such as lipids, known to interfere in many other protein detn. methods.
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    Wu, J.; Chen, Z.; Dovichi, N. J Reaction Rate, Activation Energy, and Detection Limit for the Reaction of 5-Furoylquinoline-3-Carboxaldehyde with Neurotransmitters in Artificial Cerebrospinal Fluid. J. Chromatogr. B Biomed. Appl. 2000, 741 (1), 85,  DOI: 10.1016/S0378-4347(99)00540-X
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    The fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ) has proven valuable in the anal. of proteins and small neurotransmitters. The authors monitored the reaction rate between this reagent and five neurotransmitters at 40° and 65°C in artificial cerebrospinal fluid. The reactions followed pseudo-first order kinetics. The activation energy for the reaction of FQ was 10.6, 10.7, 22.0, 31.4, and 34.4 kJ mol-1 for alanine, taurine, γ-aminobutyric acid (GABA), glutamine, and glutamic acid, resp. At 65°C, the reaction rate was quite similar for alanine, taurine, glutamine, and glutamic acid (1.8 × 10-3 s-1) but was twice as fast for GABA. A reaction time of nearly 1 h was required to quant. convert these neurotransmitters to their fluorescent products at 65°C. Detection limits for the labeled neurotransmitters were 10-9-10-8 M, which corresponded to 0.3-7 amol injected onto the capillary.
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    Pinto, D.; Arriaga, E. A.; Schoenherr, R. M.; Chou, S. S.-H.; Dovichi, N. J. Kinetics and Apparent Activation Energy of the Reaction of the Fluorogenic Reagent 5-Furoylquinoline-3-Carboxaldehyde with Ovalbumin. J. Chromatogr. B 2003, 793 (1), 107,  DOI: 10.1016/S1570-0232(03)00368-4
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    Incomplete labeling of proteins by a derivatizing reagent usually results in the formation of a large no. of products, which can produce unacceptable band broadening during electrophoretic anal. In this paper, we report on the reaction of the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ) with the lysine residues of ovalbumin. Mass spectrometry was first used to det. the distribution in the no. of labels attached to the protein. At room temp., 3.6±1.9 labels were attached after 30 min. The reaction rate and no. of labels increased at elevated temps. At 65°, 6±2.5 labels were attached after 5 min. The apparent activation energy for this reaction is estd. as 48±17 kJ/mol. Based on the mass spectrometry study, the labeling reaction was assumed to consist of two steps. In the first, the protein unfolds to make lysine residues accessible. In the second, the reagents react with the ε-amine of the lysine residues. To test this hypothesis, submicellar capillary electrophoresis and laser-induced fluorescence were used to characterize the reaction mixt. The apparent activation energy was measured for the labeling reaction; the apparent activation energy was 57±12 kJ/mol for reaction performed in the sepn. buffer. Denaturing agents were added to the reaction mixt. The addn. of 2 M thiourea with 6 M urea to the reaction resulted in a modest decrease in the apparent activation energy to 42±2 kJ/mol. The addn. of 2.5 M or higher concn. of ethanol decreased the apparent activation energy to 32±2 kJ/mol. We conclude that the apparent activation energy for protein labeling is dominated by denaturation of the protein, and that the addn. of suitable denaturing reagents can eliminate this contribution to the reaction chem.
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    Arrell, M. S.; Kalman, F. Estimation of Protein Concentration at High Sensitivity Using SDS-Capillary Gel Electrophoresis-Laser Induced Fluorescence Detection with 3-(2-Furoyl)quinoline-2-Carboxaldehyde Protein Labeling. Electrophoresis 2016, 37 (22), 2913,  DOI: 10.1002/elps.201600246
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    Estimation of protein concentration at high sensitivity using SDS-capillary gel electrophoresis-laser induced fluorescence detection with 3-(2-furoyl)quinoline-2-carboxaldehyde protein labeling
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    3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ) is a sensitive fluorogenic dye, used for derivatization of proteins for SDS-capillary gel electrophoresis with laser induced fluorescence detection (SDS-CGE-LIF) at silver staining sensitivity (ng/mL). FQ labels proteins at primary amines, found at lysines and N-termini, which vary in no. and accessibility for different proteins. This work investigates the accuracy of estn. of protein concn. with SDS-CGE-LIF in real biol. samples, where a different protein must be used as a std. 16 purified proteins varying in mol. wt., structure and sequence were labeled with FQ at const. mass concn. applying a commonly used procedure for SDS-CGE-LIF. The fluorescence of these proteins was measured using a spectrofluorometer and found to vary with a relative std. deviation of 36%. This compares favorably with other less sensitive methods for estn. of protein concn. such as SDS-CGE-UV and SDS-PAGE-Coomassie and is vastly superior to the equivalently sensitive silver stain. Investigation into the no. of labels bound with UHPLC-ESI-QTOF-MS revealed large variations in the labeling efficiency (percentage of labels to the no. of labeling sites given by the sequence) for different proteins (from 3-30%). This explains the observation that fluorescence per mol of protein was not proportional to the no. of lysines in the sequence. This article is protected by copyright. All rights reserved.
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    Wu, D.; Liu, Y.; Zheng, F.; Rong, S.-Q.; Yang, T.; Zhao, Y.-K.; Yang, R.-W.; Zou, P.; Wang, G.-T. Detection of Organic Amines Using a Ratiometric Chromophoric Fluorescent Probe with a Significant Emission Shift. J. Chem. Res. 2020, 44, 475– 481,  DOI: 10.1177/1747519820902944
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    Journal of Chemical Research (2020), 44 (7-8), 475-481CODEN: JCROA4; ISSN:2047-6507. (Sage Publications Ltd.)
    Taking advantages of both the well-known α,β-unsatd. structure and the special nucleophilicity of org. amines toward its acceptor moieties, intramol. charge transfer as a signaling mechanism is used to design and synthesize a new ratiometric chromophoric fluorescent probe (BI-CA-ID) with large emission shifts toward org. amines. This probe is employed for the detection of org. amines with high selectivity and sensitivity and a "naked-eye" color change (from red to colorless). UV-visible and fluorescence spectrometric measurements are used to det. detection limits as low as 0.024 and 0.43 μM. Furthermore, nucleophilic addn. of the amine on the α,β-unsatd. of BI-CA-ID indicated that the sensing mechanism occurs via interruption of the π-conjugation.
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    Zhang, J.; Zhou, J.; Xu, G.; Ni, Y. Stereodivergent Evolution of KpADH for the Asymmetric Reduction of Diaryl Ketones with Para-Substituents. Mol. Catal. 2022, 524, 112315,  DOI: 10.1016/j.mcat.2022.112315
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    KpADH is a promising biocatalyst for the synthesis of chiral bulky-bulky alcs. Substrate specificity toward diaryl ketone substrates with different substituents at Ph group was investigated. The results reveal that the para-position of diaryl ketones plays important role in manipulating the stereoselectivity of KpADH. Two vital residues, E214 and T215, were identified through single-point mutagenesis, and satn. mutagenesis was performed to evaluate their contributions to the stereoselectivity. Furthermore, stereodivergent evolution of KpADH was achieved through combinatorial mutagenesis. Among them, E214I/T215S/S237A (ISA) and F161V/S196G/E214G (VGG) exhibited e.e. values of >99% (S) and 99% (R) toward [4-(trifluoromethyl)phenyl]-2-pyridinylmethanone (5a), and e.e. values of >99% (S) and 98% (R) toward (4-methylphenyl)-2-pyridinylmethanone (6a), resp. Kinetic characterization and interaction anal. further prove that the eliminated side-chain collision and enhanced electrostatic interactions are mainly responsible for the increased catalytic efficiency and stereoselectivity. This study provides beneficial mutants with complementary stereoselectivity toward diaryl ketones with para-substituents and also provides guidance for the engineering of homologous alc. dehydrogenases toward bulky-bulky ketones.
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    Ribeaucourt, D.; Bissaro, B.; Lambert, F.; Lafond, M.; Berrin, J.-G. Biocatalytic Oxidation of Fatty Alcohols into Aldehydes for the Flavors and Fragrances Industry. Biotechnol. Adv. 2022, 56, 107787,  DOI: 10.1016/j.biotechadv.2021.107787
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    Biotechnology Advances (2022), 56 (), 107787CODEN: BIADDD; ISSN:0734-9750. (Elsevier Inc.)
    A review. From Egyptian mummies to the Chanel n°5 perfume, fatty aldehydes have long been used and keep impacting our senses in a wide range of foods, beverages and perfumes. Natural sources of fatty aldehydes are threatened by qual. and quant. variability while traditional chem. routes are insufficient to answer the society shift toward more sustainable and natural products. The prodn. of fatty aldehydes using biotechnologies is therefore the most promising alternative for the flavors and fragrances industry. In this review, after drawing the portrait of the origin and characteristics of fragrant fatty aldehydes, we present the three main classes of enzymes that catalyze the reaction of fatty alcs. oxidn. into aldehydes, namely alc. dehydrogenases, flavin-dependent alc. oxidases and copper radical alc. oxidases. The constraints, challenges and opportunities to implement these oxidative enzymes in the flavors and fragrances industry are then discussed. By setting the scene on the biocatalytic prodn. of fatty aldehydes, and providing a crit. assessment of its potential, we expect this review to contribute to the development of biotechnol.-based solns. in the flavors and fragrances industry.
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    A review. Aldehydes are org. compds. that are widespread in nature. They can be formed endogenously by lipid peroxidn. (LPO), carbohydrate or metab. ascorbate autoxidn., amine oxidases, cytochrome P-450s, or myeloperoxidase-catalyzed metabolic activation. This review compares the reactivity of many aldehydes towards biomols. particularly macromols. Furthermore, it includes not only aldehydes of environmental or occupational concerns but also dietary aldehydes and aldehydes formed endogenously by intermediary metab. Drugs that are aldehydes or form reactive aldehyde metabolites that cause side-effect toxicity are also included. The effects of these aldehydes on biol. function, their contribution to human diseases, and the role of nucleic acid and protein carbonylation/oxidn. in mutagenicity and cytotoxicity mechanisms, resp., as well as carbonyl signal transduction and gene expression, are reviewed. Aldehyde metabolic activation and detoxication by metabolizing enzymes are also reviewed, as well as the toxicol. and anticancer therapeutic effects of metabolizing enzyme inhibitors. The human health risks from clin. and animal research studies are reviewed, including aldehydes as haptens in allergenic hypersensitivity diseases, respiratory allergies, and idiosyncratic drug toxicity; the potential carcinogenic risks of the carbonyl body burden; and the toxic effects of aldehydes in liver disease, embryo toxicity/teratogenicity, diabetes/hypertension, sclerosing peritonitis, cerebral ischemia/neurodegenerative diseases, and other aging-assocd. diseases.
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    Comparative risks of aldehyde constituents in cigarette smoke using transient computational fluid dynamics/physiologically based pharmacokinetic models of the rat and human respiratory tracts
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    Ahmed Laskar, A.; Younus, H. Aldehyde Toxicity and Metabolism: The Role of Aldehyde Dehydrogenases in Detoxification, Drug Resistance and Carcinogenesis. Drug Metab. Rev. 2019, 51 (1), 42– 64,  DOI: 10.1080/03602532.2018.1555587
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    A review. Aldehydes are carbonyl compds. found ubiquitously in the environment, derived from both natural and anthropogenic sources. As the aldehydes are reactive species, therefore, they are generally toxic to the body. To reduce the toxicity and pathogenesis related to aldehydes, the human body contains several aldehyde metabolizing enzyme systems including aldehyde oxidases, cytochrome P 450 enzymes, aldo-ketoreductases, alc. dehydrogenases, short-chain dehydrogenases/reductases and aldehyde dehydrogenases (ALDHs). These enzyme systems maintain a low level of aldehydes in the body by catalytically converting them into less-harmful and easily excreted products. The human ALDH (hALDH) superfamily consists of 20 functional ALDH genes identified so far at distinct chromosomal locations, expressing 20 ALDH proteins, which belong to 11 different ALDH families. They are involved in the NAD(P)+-dependent oxidn. of a wide range of exogenous and endogenous aldehydes to their corresponding carboxylic acids. The hALDHs are present in all sub-cellular locations and have a wide tissue distribution. This review gives an account of aldehydes; their source, toxicity and metab., different aldehyde metabolizing enzymes with special emphasis on ALDHs including their biochem., physiol. and pathophysiol. roles in the body.
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    A review with 77 refs. is presented regarding carbinolamines and geminal diols in aq. environmental org. chem. Any aq. process whereby an aldehyde or ketone undergoes nucleophilic substitution can be expected to have some effects from the stability of carbinolamines and geminal diols imparted by the water. A significant body of literature exists on the role of these compds. in aq. org. chem. Nevertheless, carbinolamines are still commonly regarded as short-lived intermediates and geminal diols as exceptions by sophomore org. chem. textbooks, and thus, students come to view them that way. However, in environmental applications these species are ubiquitous and may dominate the observable chem. Even in the field of drinking water chem., these species have been largely ignored. No effort has been made to exploit carbinolamine reaction kinetics or imine formation in anal. chem., such as adding arylamines as catalysts or adjusting pH. Such modifications could lead to improved anal. methods, with advantages in convenience or lower limits of detection and are worthy of exploration.
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    Mukherjee, K.; Chio, T. I.; Gu, H.; Banerjee, A.; Sorrentino, A. M.; Sackett, D. L.; Bane, S. L. Benzocoumarin Hydrazine: A Large Stokes Shift Fluorogenic Sensor for Detecting Carbonyls in Isolated Biomolecules and in Live Cells. ACS Sens. 2017, 2 (1), 128– 134,  DOI: 10.1021/acssensors.6b00616
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    ACS Sensors (2017), 2 (1), 128-134CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    Detection and quantification of biomol. carbonylation, a crit. manifestation of oxidative stress, allows better understanding of assocd. disease states. Existing approaches for such analyses require further processing of cells and tissues, which leads to loss of both spatial and temporal information about carbonylated biomols. in cells. Live cell detection of these species requires sensors that are non-toxic, sufficiently reactive with the biocarbonyl in the intracellular milieu, and detectable with commonly available instrumentation. Presented here is a new fluorescent sensor for biomol. carbonyl detection: a hydrazine deriv. of a benzocoumarin, 7-hydrazinyl-4-methyl-2H-benzo[h]chromen-2-one (BzCH), which meets these requirements. This probe is esp. well suited for live cell studies. It can be excited by a laser line common to many fluorescence microscopes. The emission max. of BzCH undergoes a substantial red shift upon hydrazone formation (from ∼430 to ∼550 nm), which is the result of fluorophore disaggregation. Addnl., the hydrazone exhibits an exceptionally large Stokes shift (∼195 nm). The latter properties eliminate self-quenching of the probe and the need to remove unreacted fluorophore for reliable carbonyl detection. Thus, biomol. carbonylation can be detected and quantified in cells and in cell exts. in a one-step procedure using this probe.
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    Molina-Espeja, P.; Canellas, M.; Plou, F. J.; Hofrichter, M.; Lucas, F.; Guallar, V.; Alcalde, M. Synthesis of 1-Naphthol by a Natural Peroxygenase Engineered by Directed Evolution. ChemBioChem 2016, 17 (4), 341– 349,  DOI: 10.1002/cbic.201500493
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    ChemBioChem (2016), 17 (4), 341-349CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
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    Fluorogenic imines and their precursor amines that can be used for fluorescent visualization of Mannich-type reactions of phenols in aq. buffers have been developed. The precursor amines are aniline derivs. that are covalently conjugated to fluorophores. These amines and their imine derivs. were nonfluorescent or very weakly fluorescent. On the other hand, addn. products of the imines to phenols sh wed more than 100-fold higher fluorescence than the imines and the precursor amines.

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  19. Manfred T. Reetz, Ge Qu, Zhoutong Sun. Engineered enzymes for the synthesis of pharmaceuticals and other high-value products. Nature Synthesis 2024, 3 (1) , 19-32. https://doi.org/10.1038/s44160-023-00417-0
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  24. Nicoletta Cascelli, Vicente Gotor-Fernández, Iván Lavandera, Giovanni Sannia, Vincenzo Lettera. Spectrophotometric Assay for the Detection of 2,5-Diformylfuran and Its Validation through Laccase-Mediated Oxidation of 5-Hydroxymethylfurfural. International Journal of Molecular Sciences 2023, 24 (23) , 16861. https://doi.org/10.3390/ijms242316861
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Cite this: Chem. Rev. 2023, 123, 6, 2832–2901
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  • Abstract

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    Figure 1

    Figure 1. Change of analytical approach depending on screening scale and mutant library size: Increase in library size urges a shift from chromatographic methods to spectroscopic detection for (ultra) high-throughput screenings: (u)HTS. TLC: thin-layer chromatography; GC: gas chromatography; HPLC: high-performance liquid chromatography; (μ)MS: (microscale) mass spectrometry. Created with BioRender.com.

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    Figure 2

    Figure 2. Overview of several ratiometric absorption or fluorescence turn-on strategies applied in directed enzyme evolution in biological systems: (a) direct detection of activity by functional group transformations or cleavage reaction of the target substrate, (b) indirect detection of conversion via dye cofactor recycling with a concomitant increase in signal, (c) triggered enhanced transcription of fluorescent biosensors, (d) formation of the dye in a downstream reaction upon cleavage of a metabolic reporter, detection via suitable recognition domain or subsequent reaction with a secondary chromo-/fluorogenic substrate, (e) induction of Förster resonance energy transfer (FRET) or suppression of photoinduced electron transfer (PET), (f) functional group transformation is detected via subsequent reaction with functional group selective assay component, (g) tandem reaction cascade of a model-substrate (mimic) unmasks chromo-/fluorophore.

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    Scheme 1

    Scheme 1. Representative Reagents Typically Used in Thin-Layer Chromatography (TLC) Staining Solutions
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    Figure 3

    Figure 3. (top) Summary of general properties of amines, reactivity trends toward amine-selective probes, and expected cross-reactivity. (bottom) Compatible pH range and buffers in reported amine-selective assays. Bold numbers indicate the most commonly used pH values.

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    Scheme 2

    Scheme 2. Representative Selection of Enzymatic Transformations for the Production of Amine-Containing Products
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    Figure 4

    Figure 4. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on nucleophilic aromatic substitution (SNAr) with aromatic molecules containing electron-withdrawing groups (EWG). Abs: wavelength typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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    Figure 5

    Figure 5. Structures and spectral properties of SCOTfluors 14a–e. aValues determined in EtOH. bQY (quantum yield) in dioxane using acridine orange, fluorescein, rhodamine 101, and Cy5 as standards. Reproduced with permission from ref (83). Copyright 2019 Wiley-VCH under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/.

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    Figure 6

    Figure 6. Labeling scheme of lysine residues in protein with 16. (a) Normalized absorbance spectra (dashed-lines) and fluorescence emission spectra (solid-lines) of 16 (1 μM) and lysozyme labeled with 16 (5 μg/mL lysozyme, 2 h incubation at 37 °C), measured in HEPES buffer (10 mM, pH 7.4, 0.1% dimethyl sulfoxide). The emission spectra were obtained with excitation at 485 and 375 nm, respectively. The inset photo was taken under UV light (365 nm). (b) A plot of time-dependent (0–4 h) fluorescence intensity at 409 nm. The excitation wavelength was 375 nm. Inset box: lysozyme activity calculated using an assay kit at 2 h incubation point. Means and standard deviations were calculated from triplicate measurements. Reproduced with permission from ref (90). Copyright 2017 Korean Chemical Society; Wiley-VCH.

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    Figure 7

    Figure 7. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on Michael addition type reactions. Abs: wavelength typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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    Figure 8

    Figure 8. Epicocconone 17 is the active ingredient in Deep Purple Total Protein Stain and is responsible for the apparent noncovalent staining of proteins in polyacrylamide gels and electroblots. The reaction of 17 with amines has shown that 17 reacts reversibly with primary amines to produce a highly fluorescent enamine that is readily hydrolyzed by base or strong acid. Such conditions are used in postelectrophoretic analysis, such as peptide mass fingerprinting or Edman degradation. Reproduced with permission from ref (92). Copyright 2005 American Chemical Society.

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    Figure 9

    Figure 9. Normalized emission spectra of ylidenemalononitrile enamines 20a–c in CH2Cl2 at room temperature. Insets show photographs of 1 mM solutions of 20a–c in CH2Cl2 irradiated at 365 nm. Reproduced with permission from ref (103). Copyright 2014 American Chemical Society.

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    Figure 10

    Figure 10. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on isoindole formation with o-phthalaldehyde derivatives and o-diacetylbenzene. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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    Scheme 3

    Scheme 3. Reaction of 22 with Vicinal Diamine Resulting in the Formation of Pyrazino-diisoindole-dione 27
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    Figure 11

    Figure 11. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on amide formation using activated esters. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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    Figure 12

    Figure 12. (top) Synthesis of fluorogenic meso-active-ester-BODIPYs 30a/30b and their conversion to fluorescent meso-amide-BODIPY products upon reaction with amines. (bottom) Absorption (a) and emission (b) spectra of 30b (10 μM) upon treatment with MeNH2 (20 μM) in CH3CN as a function of time (0–5 min) at 25 °C. λex = 470 nm. (inset) Relative fluorescence intensity at 546 nm as a function of incubation time, in the absence (red) and the presence (green) of MeNH2. (c) Relative fluorescence intensity at 546 nm as a function of [MeNH2] (0–50 μM). (inset) Calibration curve of fluorescence intensity at 546 nm vs [MeNH2] (0–6 μM). (d) Photographs of 30b (20 μM) upon addition of MeNH2 (left to right: 0, 3, 5, 10, 20 μM) under ambient light (left) and 365 nm UV irradiation (right). Incubation time = 5 min. Reproduced with permission from ref (131). Copyright 2020 American Chemical Society.

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    Figure 13

    Figure 13. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on pyridinium salt formation with pyrylium salts. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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    Figure 14

    Figure 14. Image of a Py-1 31 sensing microplate reacted with various concentrations of histamine (the numbers indicate the concentrations of histamine in μg mL–1). Reproduced with permission from ref (137). Copyright 2011 The Royal Society of Chemistry.

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    Figure 15

    Figure 15. Colors under daylight and the colors of fluorescence in solution of pH 7.0 under a UV lamp (365 nm) of label 31 and of some other Py labels when conjugated to serum albumin at 37 °C. Note that label 33 does not react but remains blue (at any concentration). Reproduced with permission from ref (141). Copyright 2011 The Royal Society of Chemistry.

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    Figure 16

    Figure 16. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on the formation of thioureas or 2-aminothiazoles from isothiocyanates. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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    Figure 17

    Figure 17. (top) Fluorescence spectra of unbound 9-ITPPo 35 (blue) and its covalent adduct ATAZPo (red) with BSA (a) and gold nanoclusters (AuNCs) (b). (bottom) Absorbance of a mixture of 35 (15 μM) and n-butylamine (3 mM) in toluene: Structures, time curves of concentration, and spectral profiles of the reactant, the intermediate, and the product (from left to right). Reproduced with permission from ref (148). Copyright 2015 The Royal Society of Chemistry.

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    Figure 18

    Figure 18. Summary of chromo- or fluorogenic turn-on probes for the selective detection of amines based on hemiaminal, aminal, or Schiff’s base formation. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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    Figure 19

    Figure 19. Amine-sensing panels of 37 (10 μmol L−1) in water: (1) 37; (2) butylamine; (3) tert-butylamine; (4) benzylamine; (5) cyclohexylamine; (6) ethylene diamine; (7) 1,3-diaminopropane; (8) cadaverine; (9) morpholine; (10) ephedrine; (11) 4-aminopyridine; (12) ethanolamine. Reproduced with permission form ref (154). Copyright 2012 Wiley-VCH.

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    Scheme 4

    Scheme 4. Sensing Mechanism of 36: Chelation of the Formed Iminium Species by the Coumarin Moiety Triggers a Turn-on Response (152)
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    Scheme 5

    Scheme 5. Ozone Sensing Mechanisms Employing 38. (34)
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    Figure 20

    Figure 20. Summary of general properties of alcohols and expected cross-reactivity (bottom).

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    Scheme 6

    Scheme 6. Representative Selection of Enzymatic Transformations for the Production of Alcohol-Containing Products
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    Scheme 7

    Scheme 7. MTT Assay: Detection of NAD(P)H or “Reducing Equivalents” via the Reduction of MTT 55 to the Purple Formazan 56
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    Figure 21

    Figure 21. Summary of chromo- or fluorogenic turn-on probes for the selective detection of alcohols based on hemiacetal formation. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown on the strip at the bottom representing the visible spectrum.

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    Figure 22

    Figure 22. (a) Fluorescence–time trajectories following the reaction of 4.5 μM of compound 57 (R′ = Cl, R′′ = H) and 1.09 M methanol (MeOH), ethanol (EtOH), butanol (BuOH), ethylene glycol (EtyGly), water (H2O), or ethanedithiol at 21 °C in ACN supplemented with 0.333 mM of p-TsOH. (b) Apparent rate constants for various nucleophiles tested were obtained from fitting the intensity–time trajectories in (a). Reproduced with permission from ref (195). Copyright 2017 American Chemical Society.

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    Figure 23

    Figure 23. Photographs showing the color changes of sensor 58 before and after the addition of methanol vapor. The images were taken under UV irradiation (365 nm). Reproduced with permission from ref (197). Copyright 2021 Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

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    Figure 24

    Figure 24. Summary of chromo- or fluorogenic turn-on probes for the selective detection of alcohols based on the coordination to chelation centers. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 25

    Figure 25. Summary of chromo- or fluorogenic turn-on probes for the selective detection of alcohols based on the ring-opening of 3-aminorhodanine derivatives. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 26

    Figure 26. (a) The visible color change of 65 (5 mM) in different solvents: (i) hexane, (ii) toluene, (iii) cyclohexane, (iv) ethyl acetate, (v) chloroform, (vi) dichloromethane, (vii) tetrahydrofuran, (viii) dioxane, (ix) ACN, (x) MeOH, (xi) EtOH, (xii) iPrOH, and (xiii) nBuOH. (b) The absorption spectra of 65 in different solvents, (c) time-dependent absorption spectra (0–30 min) of 5 mM 65 in MeOH; inset: plot of optical density at 470 nm vs time. (d,e) Absorption spectra of 65 (5 mM) at different ratios of ACN:EtOH (0–10%) and ACN:MeOH (0–10%), respectively. Reproduced with permission from ref (205). Copyright 2019 Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

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    Figure 27

    Figure 27. (top) Summary of general properties of diol sensors and expected cross-reactivity. (bottom) Compatible pH range and buffers in reported diol-selective assays. Bold numbers indicate the most commonly used pH values.

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    Scheme 8

    Scheme 8. Representative Selection of Enzymatic Transformations for the Production of Diol-Containing Products
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    Figure 28

    Figure 28. Summary of fluorogenic turn-on probes for the selective detection of diols based on the formation of fluorescent boronate esters. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 29

    Figure 29. Sensing mechanism for the selective detection of fructose or glucose-6-phosphate by 91 or 92 and viologen HPTS 93. Reproduced with permission from ref (253). Copyright 2009 Elsevier BV.

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    Figure 30

    Figure 30. Selective detection of diols based on the cleavage reaction by metaperiodate 97 and subsequent application of aldehyde sensing methodology (for aldehyde-selective probes, see section 9).

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    Scheme 9

    Scheme 9. (a) Oxidation of Adrenaline 98 by Metaperiodate 97, Forming Red-Colored Adrenochrome 99, Which Allows for Determination of Excess 97 (Back Titration of Diol); (263) (b) Structure of Carboxyfluorescein, Which Can Also Be Used as a 97 Indicator; The Mechanism for Its Oxidation Is Not Known
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    Figure 31

    Figure 31. (a) Coupled reactions employed by the fluorescence-based assay system for the detection of cis-diol metabolites using fluoresceinamine 127. (b) Concentration-dependent fluorescence response of the assay to the presence of cis-diol metabolites. All studies were performed in triplicate; all measurements demonstrated standard deviation <5%; fluorescence responses normalized to negative control ([I – I0]/I0). Reproduced with permission from ref (270). Copyright 2021 The Royal Society of Chemistry.

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    Figure 32

    Figure 32. (top) Summary of general properties of aldehydes, reactivity trends toward aldehyde-selective probes, as well as expected cross-reactivity during screenings. (bottom) Compatible pH range and buffers in reported aldehyde-selective assays. Bold numbers indicate the most commonly used pH values.

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    Scheme 10

    Scheme 10. Selection of Enzymatic Transformations for the Production of Aldehyde-Containing Products
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    Figure 33

    Figure 33. Summary of chromo- and fluorogenic turn-on probes for the selective detection of aldehydes based on the formation of hydrazone adducts. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 34

    Figure 34. Plot of the nucleophilicity parameters N of amines versus pKaH in water. (296) Reproduced with permission from refs. (297and298). Copyright 2007 American Chemical Society.

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    Scheme 11

    Scheme 11. Sensing Mechanism of Purpald 113via the Intermediate Formation of Cyclic Tetrazinanes and Subsequent Oxidation to Fully Aromatized Violet-Colored Tetrazines 115
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    Scheme 12

    Scheme 12. Sensing Mechanism for MBTH 114
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    Figure 35

    Figure 35. (a) 119 as a fluorogenic probe for the detection of MDA. (a) Malondialdehyde (MDA) was added to a 25 μM PBS solution of 119 (pH 7.2) under illumination at 365 nm and photographed at indicated time points. (b) (left) Emission spectra of 1 μM 119 (black) or 119 after 1 min incubation with 5 equiv MDA at 37 °C and excited at 310 nm (blue) or 375 nm (cyan). (right) Formation and rapid decay of short-lived intermediate formed in the reaction between 119 and MDA followed by fluorescence spectroscopy with λex/λem = 375/480 nm. Kinetics of 25 μM 119 only (squares) and 119 + MDA (circles) are shown, with three replicates of 119 + MDA shown (light, medium, and dark cyan). (c) 119 shows broad specificity to aliphatic aldehydes. The enhancement of fluorescence relative to 119 only was recorded with λex/λem 375/480 nm following 1 min of incubation of 1 μM solutions of 119 in PBS (pH 7.2) with 5 equiv of MDA (black bar), simple aliphatic aldehydes (red bars), dialdehydes, and complex aldehydes (blue bars), ketones (purple bars), glutathione (yellow bar), and biogenic metal cations (orange bars). Reproduced with permission from ref (312). Copyright 2019 The Royal Society of Chemistry.

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    Figure 36

    Figure 36. (top) Scheme of the reaction mechanism of colorimetric and fluorescent detection of aldehydes based on the SQs–N2H4 adduct 121. (bottom) The changes in UV–vis absorption of dye 121 (6.0 mM) upon addition of N2H4 (3.0 mM) and subsequent addition of formaldehyde (10.0 mM). Reproduced with permission from ref (314). Copyright 2019 Elsevier BV.

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    Figure 37

    Figure 37. Summary of chromo- and fluorogenic turn-on probes for the selective detection of aldehydes based on the formation of imines (Schiff’s bases). Subsequent entrapment of these electrophilic imines by intra- or intermolecular nucleophiles makes the reaction irreversible. Abs: wavelengths typically used for UV Absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 38

    Figure 38. (a) The color of the aldehyde-sensing probe 122 changes to purple when treated with formaldehyde (detection limit: μM concentrations of formaldehyde in the in vitro system). (b) Color changes on solid agar plate from O-dealkylation activity. Aldehyde appears to diffuse out of the cells; cell color changes are observed in the presence of 122. Reproduced with permission from ref (23). Copyright 2013 Wiley-VCH.

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    Figure 39

    Figure 39. Relationship between the measured reaction rates and pKa of the corresponding protonated anilines for seven ABAO analogues 126a–g. (i) Structures of ABAOs 126 and corresponding protonated anilines. (ii) Scatter plot showing relationship between log k and pKa. (iii) Reaction rates measured by NMR in CD3COONa buffer (100 mM) at pD 4.5.(iv) Structures and reaction rates for 126f and 126g. Basicity of aniline nitrogen in 2-aminopyridine / 126f was estimated using ab initio calculations. Reproduced with permission from ref (319). Copyright 2014 American Chemical Society.

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    Scheme 13

    Scheme 13. Sensing Mechanism for dRB-EDA 128: Schiff’s Base Formation with Formaldehyde Triggers the Ring-Opening of the Deoxylactam Moiety
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    Figure 40

    Figure 40. Chromogenic detection of formaldehyde with 128. (a) UV–absorption spectra of 128 in the presence of formaldehyde (concentration: 0, 3.5, 7, 10.5, 14, 17.5, and 35 mM, from bottom to top). The inset shows the titration curve by absorbance at 560 nm. (b) Visual detection of formaldehyde (0–7 mM as indicated) with 128 in DMF after incubation at room temperature for 90 min. Reproduced with permission from ref (324). Copyright 2011 The Royal Society of Chemistry.

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    Figure 41

    Figure 41. Summary of fluorogenic turn-on probes for the selective detection of aldehydes based on the formation of imidazole structures. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 42

    Figure 42. Summary of fluorogenic turn-on probes for the selective detection of aldehydes based on the formation of 1,4-dihydropyridines. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 43

    Figure 43. Summary of chromo- and fluorogenic turn-on probes for the selective detection of aldehydes based on the formation of unstable oximes. Fragmentation of these intermediates leads to the liberation of colored phenols. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Scheme 14

    Scheme 14. Enzyme-Catalyzed Formation of the 138–Benzaldehyde Adduct Could Be Efficiently Traced by Spontaneous Decomposition Forming Colored NBD-O–
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    Figure 44

    Figure 44. Summary of fluorogenic turn-on probes for the selective detection of formaldehyde based on the 2-aza-Cope sigmatropic rearrangement of initially formed Schiff’s bases. Fragmentation of these intermediates leads to the liberation of colored phenols. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 45

    Figure 45. Confocal fluorescence images of exogenous formaldehyde (FA) with FFP fluorescent probes 142 in HEK293T cells. (A–D) Cells were loaded with (a) FFP511 (Tokyo Green fluorophore) (20 μM), (b) FFP551 (142) (10 μM), (c) FFP585 (resorufin fluorophore) (5 μM), or FFP706 (hemicyanine fluorophore) (2 μM) for 30 min and treated with FA of varying concentrations (0–1 mM) for 60 min before being imaged by confocal fluorescence microscopy. Scale bars represent 50 μm. Reproduced with permission from ref (352). Copyright 2021 The Royal Society of Chemistry.

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    Figure 46

    Figure 46. Fluorescent responses of 143 (10 μM) in DMF/PBS solution (v/v = 1/4, pH 7.4, 10 mM) upon addition of various small molecular species (5.0 mM). λex/λem = 390/513 nm; slits, 2.5/2.5 nm. Each spectrum was recorded after 3 h at 37 °C. Error bars are ±1SD, n = 3. Inset: (a) The color and (b) fluorescence images of 143 (10 μM) in the presence of various small molecular species (5.0 mM). (354) Reproduced with permission from ref (354). Copyright 2016 Elsevier BV.

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    Figure 47

    Figure 47. (top) Summary of general properties of ketones, reactivity trends toward ketone-selective probes as well as expected cross-reactivity during screenings. (bottom) Compatible pH range and buffers in reported ketone-selective assays. Bold pH numbers indicate the most commonly used pH values.

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    Scheme 15

    Scheme 15. Representative Selection of Enzymatic Transformations for the Production of Carboxylic Acid-Containing Products
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    Figure 48

    Figure 48. Summary of chromo- and fluorogenic turn-on probes for the selective detection of ketones based on the formation of hydrazone adducts. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 49

    Figure 49. Variation in color of reaction mixtures containing different concentrations of 109 and the validation of the developed HTS method by chiral HPLC analysis. Control, no 109 was added into the reaction mixture; 1–10×, different concentrations of 109 were added into the reaction mixture (1×, 8.4 mg/mL). Reproduced with permission from ref (287). Copyright 2017 Springer-Verlag.

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    Figure 50

    Figure 50. Summary of fluorogenic turn-on probes for the selective detection of acetone based on aldol reactions. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 51

    Figure 51. Summary of fluorogenic turn-on probes for the selective detection of acetone based on a Michael addition approach. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Scheme 16

    Scheme 16. Michael Addition of Acetone onto 159 Triggers a Subsequent Aldol Reaction Forming Cyclic Hydroxyketones
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    Figure 52

    Figure 52. (top) Chromogenic and fluorogenic detection of ketones bearing an enolizable position via the formation of the purple-colored Janovsky complexes with 3,5-dinitrobenzoic acid 161 under strongly basic conditions. (bottom) Fluorogenic detection of ketones via the formation of fluorescent ABAO-adducts (cf. section 9.2). A methoxy substitution of the ABAO core 126 is necessary to increase the reactivity of the aniline NH2 position.

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    Figure 53

    Figure 53. (top) Summary of general properties of carboxylic acid sensors as well as expected cross-reactivity during screenings. (bottom) Compatible pH range and buffers in reported carboxylic acid-selective assays. Bold pH numbers indicate the most commonly used pH values.

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    Scheme 17

    Scheme 17. Representative Selection of Enzymatic Transformations for the Production of Carboxylic Acid-Containing Products
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    Figure 54

    Figure 54. Summary of chromogenic turn-on probes for the selective detection of carboxylic acids based on the entrapment of activated carboxylic acid species. Abs: wavelengths typically used for UV absorbance measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 55

    Figure 55. Summary of fluorogenic turn-on probes for the selective detection of carboxylic acids based on the coordination to metal centers or hydrogen bond donors. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 56

    Figure 56. Summary of fluorogenic turn-on probes for the selective detection of carboxylic acids based on nucleophilic addition onto trifluoroacetyl groups. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 57

    Figure 57. (top) Summary of general properties of carboxylic acid ester sensors as well as expected cross-reactivity during screenings. Due to the lack of direct reactive probes applicable in aqueous environments, no compatible buffers and pH ranges can be stated.

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    Scheme 18

    Scheme 18. Representative Selection of Enzymatic Transformations for the Production of Carboxylic Acid Ester-Containing Products
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    Figure 58

    Figure 58. Chromogenic detection of carboxylic acid esters via the formation of the hydroxamic acids and subsequent complexation with Fe3+ resulting in purple-colored ferric hydroxamates.

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    Figure 59

    Figure 59. (top) Summary of general properties of epoxide sensors as well as expected cross-reactivity during screenings. (bottom) Compatible pH range and buffers in reported epoxide-selective assays. Bold pH numbers indicate the most commonly used pH values.

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    Scheme 19

    Scheme 19. Representative Selection of Enzymatic Transformations for the Production of Epoxide-Containing Products
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    Figure 60

    Figure 60. Summary of chromo- and fluorogenic turn-on probes for the selective detection of epoxides based on ring-opening reactions by nucleophilic pyridine derivatives. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Scheme 20

    Scheme 20. Sensing Mechanism for Epoxides Employing the Combination of Nicotinamide 192 and Acetophenone 193
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    Figure 61

    Figure 61. Selective detection of epoxides based on indirect detection methods after initial nucleophilic ring-opening reactions with hydroxide or bisulfide. The resulting intermediates can be detected with thiol sensing assays (cf. section 15), diol sensing assays (cf. section 8), or by using a similar method discussed for the indirect detection of diols by oxidative cleavage with metaperiodate 97 and subsequent detection using aldehyde-sensing assays (cf. section 9).

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    Figure 62

    Figure 62. (top) Summary of general properties of phenol sensors as well as expected cross-reactivity during screenings. (bottom): Compatible pH range and buffers in reported phenol-selective assays. Bold pH numbers indicate the most commonly used pH values.

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    Scheme 21

    Scheme 21. Representative Selection of Enzymatic Transformations for the Production of Phenol-Containing Products
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    Figure 63

    Figure 63. Summary of chromogenic turn-on probes for the selective detection of phenols based on the electrophilic aromatic substitution with oxidized amino derivatives. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum. Alk = alkyl, Ar = aryl.

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    Figure 64

    Figure 64. Summary of chromogenic turn-on probes for the selective detection of phenols based on the formation of azo-dyes. Abs: wavelengths typically used for UV absorbance measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 65

    Figure 65. Summary of fluorogenic turn-on probes for the selective detection of phenol based on Mannich-type reactions. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 66

    Figure 66. (top) Summary of general properties of thiols, reactivity trends toward thiol-selective probes, and expected cross-reactivity. (bottom) Compatible pH range and buffers in reported thiol-selective assays. Bold pH numbers indicate the most commonly used pH values.

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    Figure 67

    Figure 67. Summary of fluorogenic turn-on probes for the selective detection of thiols based on nucleophilic aromatic substitution reactions (SNAr) with aromatic systems containing electron-withdrawing groups (EWG). Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Scheme 22

    Scheme 22. Proposed Mechanism for the Reaction of NBD-Cl 12 with Cys, Hcy, and GSH (581)
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    Figure 68

    Figure 68. Design of the probe HMN 219 and the proposed fluorescence signal changes in response to individual or sequential detection of thiols with three well-defined emission bands. Reproduced with permission from ref (565). Copyright 2017 Royal Society of Chemistry.

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    Figure 69

    Figure 69. Summary of fluorogenic turn-on probes for the selective detection of thiols based on 2,4-dinitrobenzene sulfonyl (DNBS) cleavage by nucleophilic aromatic substitution reactions (SNAr) with aromatic systems containing electron-withdrawing groups (EWG). Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 70

    Figure 70. Fluorescent probe BESThio 222 and its discrimination between selenols and thiols. Reproduced with permission from ref (594). Copyright 2006 Wiley-VCH.

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    Figure 71

    Figure 71. Summary of fluorogenic turn-on probes for the selective detection of thiols based on disulfide bond cleavage. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 72

    Figure 72. Schematic illustration of redox-controlled fluorescent nanoswitch and detection strategy for BChE activity based on thiol-triggered disulfide cleavage on fluorescent carbon nanoparticles. Reproduced with permission from ref (611). Copyright 2018 American Chemical Society.

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    Figure 73

    Figure 73. Summary of fluorogenic turn-on probes for the selective detection of thiols based on diselenide bond cleavage. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 74

    Figure 74. Summary of fluorogenic turn-on probes for the selective detection of thiols based on selenium–nitrogen bond cleavage. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.in the strip at the bottom representing the visible spectrum.

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    Figure 75

    Figure 75. Structure and color changes of 243 in the presence of amino acids (1, Cys; 2, Hcy; 3, GSH; 4, none; 5, other natural amino acids). Reproduced with permission from ref (628) . Copyright 2018 Elsevier BV.

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    Figure 76

    Figure 76. Summary of fluorogenic turn-on probes for the selective detection of thiols based on the cyclization with aldehydes. A subsequent cyclization via an additional NH2 moiety limits the application to analytes containing both functional groups. Abs: wavelengths typically used for UV absorbance measurements. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 77

    Figure 77. Summary of fluorogenic turn-on probes for the selective detection of thiols-based conjugate additions. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 78

    Figure 78. Illustration of the high-throughput assays for cystathionine β-synthase (CBS). (a) Methanethiol (CH3SH) generation catalyzed by CBS using methylcysteine (MCys) as a substrate was determined with the CPM 246. (b) The CBS activity was monitored using 10 mM MCys as the substrate in the presence of 15 μM 246 and 2 μg of hCBS in a 200 μL reaction mixture containing 50 mM HEPES, pH 7.4. The fluorescence of the reaction mixture at 460 nm (λex = 400 nm) was monitored for 600 s. Reproduced with permission from ref (637). Copyright 2017 Royal Society of Chemistry.

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    Scheme 23

    Scheme 23. Proposed Response Mechanism for NIR-BODIPY-Ac 251 to Cys or Hcy
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    Figure 79

    Figure 79. Summary of fluorogenic turn-on probes for the selective detection of thiols-based sulfur–metal interactions. Ex or em: wavelengths typically used for excitation and emission in fluorescence measurements. The wavelengths of probes in this section are shown in the strip at the bottom representing the visible spectrum.

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    Figure 80

    Figure 80. Schematic illustration of the thiol detecting chemodosimetric mechanism iminocoumarin-Cu2+ probe 253 in aqueous media. Adapted with permission from ref (651). Copyright 2011 The Royal Society of Chemistry.

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    Figure 81

    Figure 81. Schematic representation of the thiol sensor 256 based on modulation of the fluorescence quenching of the BODIPY chromophore by AuNPs. Reproduced with permission from ref (655) . Copyright 2016 Elsevier BV.

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      Chan, Jefferson; Dodani, Sheel C.; Chang, Christopher J.
      Nature Chemistry (2012), 4 (12), 973-984CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      A review. The dynamic chem. diversity of elements, ions and mols. that form the basis of life offers both a challenge and an opportunity for study. Small-mol. fluorescent probes can make use of selective, bioorthogonal chemistries to report on specific analytes in cells and in more complex biol. specimens. These probes offer powerful reagents to interrogate the physiol. and pathol. of reactive chem. species in their native environments with minimal perturbation to living systems. This Review presents a survey of tools and tactics for using such probes to detect biol. important chem. analytes. The authors highlight design criteria for effective chem. tools for use in biol. applications as well as gaps for future exploration.
    15. 15
      Reetz, M. T.; Carballeira, J. D. Iterative Saturation Mutagenesis (ISM) for Rapid Directed Evolution of Functional Enzymes. Nat. Protoc. 2007, 2 (4), 891– 903,  DOI: 10.1038/nprot.2007.72
      15
      Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes
      Reetz, Manfred T.; Carballeira, Jose Daniel
      Nature Protocols (2007), 2 (4), 891-903CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      Iterative satn. mutagenesis (ISM) is a new and efficient method for the directed evolution of functional enzymes. It reduces the necessary mol. biol. work and the screening effort drastically. It is based on a Cartesian view of the protein structure, performing iterative cycles of satn. mutagenesis at rationally chosen sites in an enzyme, a given site being composed of one, two or three amino acid positions. The basis for choosing these sites depends on the nature of the catalytic property to be improved, e.g., enantioselectivity, substrate acceptance or thermostability. In the case of thermostability, sites showing highest B-factors (available from x-ray data) are chosen. The pronounced increase in thermostability of the lipase from Bacillus subtilis (Lip A) as a result of applying ISM is illustrated here.
    16. 16
      Lapetina, S.; Gil-Henn, H. A Guide to Simple, Direct, and Quantitative in Vitro Binding Assays. J. Biol. Methods 2017, 4 (1), e62, DOI: 10.14440/jbm.2017.161 .
      There is no corresponding record for this reference.
    17. 17
      Major, J. Challenges and Opportunities in High Throughput Screening: Implications for New Technologies. J. Biomol. Screen. 1998, 3 (1), 13– 7,  DOI: 10.1177/108705719800300102
      There is no corresponding record for this reference.
    18. 18
      Neun, S.; Kaminski, T. S.; Hollfelder, F. Single-Cell Activity Screening in Microfluidic Droplets. Methods Enzymol. 2019, 628 (5), 95– 112
      18
      Single-cell activity screening in microfluidic droplets
      Neun, Stefanie; Kaminski, Tomasz S.; Hollfelder, Florian
      Methods in Enzymology (2019), 628 (Enzyme Activity in Single Cells), 95-112CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)
      Water-in-oil emulsion droplets can be used as microcompartments to contain single cells that can be subjected to activity assays in this format. Microfluidic devices produce droplets at > kHz rates and can be coupled to modules to, e.g., add reagents, incubate or measure analyte concn. optically (with sensitivities as low as 2 nM). The range of optical assays includes fluorescence and absorbance detection and examples for the use of these technologies for ultrahigh-throughput sorting in directed evolution and functional metagenomics are described.
    19. 19
      Zhang, J.; Chai, X.; He, X.-P.; Kim, H.-J.; Yoon, J.; Tian, H. Fluorogenic Probes for Disease-Relevant Enzymes. Chem. Soc. Rev. 2019, 48 (2), 683– 722,  DOI: 10.1039/C7CS00907K
      19
      Fluorogenic probes for disease-relevant enzymes
      Zhang, Junji; Chai, Xianzhi; He, Xiao-Peng; Kim, Hae-Jo; Yoon, Juyoung; Tian, He
      Chemical Society Reviews (2019), 48 (2), 683-722CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      Traditional biochem. methods for enzyme detection are mainly based on antibody-based immunoassays, which lack the ability to monitor the spatiotemporal distribution and, in particular, the in situ activity of enzymes in live cells and in vivo. In this review, we comprehensively summarize recent progress that has been made in the development of small-mol. as well as material-based fluorogenic probes for sensitive detection of the activities of enzymes that are related to a no. of human diseases. The principles utilized to design these probes as well as their applications are reviewed. Specific attention is given to fluorogenic probes that have been developed for anal. of the activities of enzymes including oxidases and reductases, those that act on biomacromols. including DNAs, proteins/peptides/amino acids, carbohydrates and lipids, and those that are responsible for translational modifications. We envision that this review will serve as an ideal ref. for practitioners as well as beginners in relevant research fields.
    20. 20
      Kaur, K.; Saini, R.; Kumar, A.; Luxami, V.; Kaur, N.; Singh, P.; Kumar, S. Chemodosimeters: An Approach for Detection and Estimation of Biologically and Medically Relevant Metal Ions, Anions and Thiols. Coord. Chem. Rev. 2012, 256 (17), 1992– 2028,  DOI: 10.1016/j.ccr.2012.04.013
      20
      Chemodosimeters: An approach for detection and estimation of biologically and medically relevant metal ions, anions and thiols
      Kaur, Kuljit; Saini, Rajnish; Kumar, Ashwani; Luxami, Vijay; Kaur, Navneet; Singh, Prabhpreet; Kumar, Subodh
      Coordination Chemistry Reviews (2012), 256 (17-18), 1992-2028CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
      In this review, the applications of chemodosimeters in qual. and quant. estn. of various metal ions viz. Hg2+, Cu2+, Fe3+, Ag+, Pd(0) / Pd2+ / Pd4+, Au3+ / Au+ and anions viz. fluoride and cyanide and thiols were discussed. Their use in bioimaging of analytes in living cells and zebra fish has also been presented. Various chem. transformations viz. adduct formation, substitution, hydrolysis, oxidn. and ring transformation etc. have been used in the design of resp. chemodosimeters for specific analyte. In total review contains 349 refs.
    21. 21
      You, L.; Arnold, F. H. Directed Evolution of Subtilisin E in Bacillus Subtilis to Enhance Total Activity in Aqueous Dimethylformamide. Protein Eng. 1996, 9 (1), 77– 83,  DOI: 10.1093/protein/9.1.77
      21
      Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide
      You, L.; Arnold, F. H.
      Protein Engineering (1996), 9 (1), 77-83CODEN: PRENE9; ISSN:0269-2139. (Oxford University Press)
      Sequential rounds of error-prone PCR to introduce random mutations and screening of the resultant mutant libraries have been used to enhance the total catalytic activity of subtilisin E significantly in a non-natural environment, aq. DMF. Seven DNA substitutions coding for three new amino acid substitutions were identified in a mutant isolated after two addnl. generations of directed evolution carried out on 10M subtilisin E, previously 'evolved' to increase its specific activity in DMF. A Bacillus subtilis-Escherichia coli shuttle vector was developed in order to increase the size of the mutant library that could be established in B. subtilis, and the stringency of the screening process was increased to reflect total as well as specific activity. This directed evolution approach has been extremely effective for improving enzyme activity in a non-natural environment; the resulting evolved 13M subtilisin exhibits specific catalytic efficiency towards the hydrolysis of a peptide substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, in a 60% DMF soln. that is three times that of the parent 10M and 471 times that of wild type subtilisin E. The total activity of the 13M culture supernatant is enhanced 16-fold over that of the parent 10M.
    22. 22
      Schmidt-Dannert, C.; Arnold, F. H. Directed Evolution of Industrial Enzymes. Trends Biotechnol. 1999, 17 (4), 135– 6,  DOI: 10.1016/S0167-7799(98)01283-9
      22
      Directed evolution of industrial enzymes
      Schmidt-Dannert, Claudia; Arnold, Frances H.
      Trends in Biotechnology (1999), 17 (4), 135-136CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Science Ltd.)
      There is no expanded citation for this reference.
    23. 23
      Choi, K.-Y.; Jung, E.-O.; Yun, H.; Yang, Y.-H.; Kazlauskas, R. J.; Kim, B.-G. Development of Colorimetric HTS Assay of Cytochrome P450 for Ortho-Specific Hydroxylation, and Engineering of CYP102D1 with Enhanced Catalytic Activity and Regioselectivity. ChemBioChem. 2013, 14 (10), 1231– 8,  DOI: 10.1002/cbic.201300212
      23
      Development of Colorimetric HTS Assay of Cytochrome P450 for ortho-Specific Hydroxylation, and Engineering of CYP102D1 with Enhanced Catalytic Activity and Regioselectivity
      Choi, Kwon-Young; Jung, Eun-Ok; Yun, Hyungdon; Yang, Yung-Hun; Kazlauskas, Romas J.; Kim, Byung-Gee
      ChemBioChem (2013), 14 (10), 1231-1238CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A current challenge in high-throughput screening (HTS) of hydroxylation reactions by P 450 is a fast and sensitive assay for regioselective hydroxylation against millions of mutants. We have developed a solid-agar plate-based HTS assay for screening ortho-specific hydroxylation of daidzein by sensing formaldehyde generated from the O-dealkylation reaction. This method adopts a colorimetric dye, pararosaniline, which has previously been used as an aldehyde-specific probe within cells. The rationale for this method lies in the fact that the hydroxylation activity at ortho-carbon position to C-OH correlates with a linear relationship to O-dealkylation activity on chem. introduced methoxy group at the corresponding C-OH. As a model system, a 4',7-dihydroxyisoflavone (daidzein) hydroxylase (CYP102D1 F96V/M246I), which catalyzes hydroxylation at ortho positions of the daidzein A/B-ring, was examd. for O-dealkylation activity, by using permethylated daidzein as a surrogate substrate. By using the developed indirect bishydroxylation screening assay, the correlation coeff. between O-dealkylation and bishydroxylation activity for the template enzyme was 0.72. For further application of this assay, satn. mutants at A273/G274/T277 were examd. by mutant screening with a permethylated daidzein analog substrate (A-ring inactivated in order to find enhanced 3'-regioselectivity). The whole-cell biotransformation of daidzein by final screened mutant G1 (A273H/G274E/T277G) showed fourfold increased conversion yield, with 14.3 mgL-1 prodn. titer and greatly increased 3'-regioselectivity (3'/6=11.8). These results show that there is a remarkably high correlation (both in vitro and in vivo), thus suggesting that this assay would be ideal for a primary HTS assay for P 450 reactions.
    24. 24
      Roth, M. Fluorescence Reaction for Amino Acids. Anal. Chem. 1971, 43 (7), 880– 2,  DOI: 10.1021/ac60302a020
      24
      Fluorescence reaction for amino acids
      Roth, Marc
      Analytical Chemistry (1971), 43 (7), 880-2CODEN: ANCHAM; ISSN:0003-2700.
      Phthalaldehyde reacts with amino acids in alk. medium in the presence of a reducing agent such as 2-mercaptoethanol, by giving rise to strongly fluorescing compds. Optimal wavelengths are λex = 340 nm and λfl = 455 nm. This permits fluorimetric assay of amino acids down to the nmole range. No heating is necessary, and the fluorescence may easily be measured 5 min after mixing of the reagents. The reaction is well suited to the automatic detn. of amino acids after ion exchange fractionation. The sensitivity is much better than with ninhydrin procedures. The imino acids proline and hydroxyproline, however, are not detected by the method.
    25. 25
      Weigele, M.; DeBernardo, S. L.; Tengi, J. P.; Leimgruber, W. Novel Reagent for the Fluorometric Assay of Primary Amines. J. Am. Chem. Soc. 1972, 94 (16), 5927– 8,  DOI: 10.1021/ja00771a084
      25
      Novel reagent for the fluorometric assay of primary amines
      Weigele, M.; DeBernardo, S. L.; Tengi, J. P.; Leimgruber, W.
      Journal of the American Chemical Society (1972), 94 (16), 5927-8CODEN: JACSAT; ISSN:0002-7863.
      A novel reagent (I) has been synthesized for the fluorometric assay of primary amines (RNH2). Alk. hydrolysis of 3-benzylidene-isochroman-1,4-dione gave 0-(α-hydroxycinnamoyl)benzoic acid (II). Formylation of II with (Me2N)3CH and subsequent acid hydrolysis afforded 4-phenylspiro [furan-2(3H),1'-phthalan]-3,3'-dione (I). The spirolactone I reacts with RNH2 in aq. media at room temp. to give fluorescent pyrrolinones (III). This reaction is of particular value for the assay of amino acids, peptides and other primary amines of biol. importance.
    26. 26
      Fellner, M.; Doughty, L. M.; Jameson, G. N.; Wilbanks, S. M. A Chromogenic Assay of Substrate Depletion by Thiol Dioxygenases. Anal. Biochem. 2014, 459, 56– 60,  DOI: 10.1016/j.ab.2014.05.008
      26
      A chromogenic assay of substrate depletion by thiol dioxygenases
      Fellner, Matthias; Doughty, Laura M.; Jameson, Guy N. L.; Wilbanks, Sigurd M.
      Analytical Biochemistry (2014), 459 (), 56-60CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the non-heme mono-Fe enzyme, cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed std. kinetic parameters to be detd. for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitated a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addn., the relative specificity of Ellman's reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.
    27. 27
      Santos-Aberturas, J.; Dörr, M.; Waldo, G. S.; Bornscheuer, U. T. In-Depth High-Throughput Screening of Protein Engineering Libraries by Split-GFP Direct Crude Cell Extract Data Normalization. Chem. Biol. 2015, 22 (10), 1406– 14,  DOI: 10.1016/j.chembiol.2015.08.014
      27
      In-Depth High-Throughput Screening of Protein Engineering Libraries by Split-GFP Direct Crude Cell Extract Data Normalization
      Santos-Aberturas, Javier; Doerr, Mark; Waldo, Geoffrey S.; Bornscheuer, Uwe T.
      Chemistry & Biology (Oxford, United Kingdom) (2015), 22 (10), 1406-1414CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
      Here, we report a widely and generally applicable strategy to obtain reliable information in high-throughput protein screenings of enzyme mutant libraries. The method is based on the usage of the split-GFP technol. for the normalization of the expression level of each individual protein variant combined with activity measurements, thus resolving the important problems assocd. with the different soly. of each mutant and allowing the detection of previously invisible variants. The small size of the employed protein tag (16 amino acids) required for the reconstitution of the GFP fluorescence reduces possible interferences such as enzyme activity variations or soly. disturbances to a min. Specific enzyme activity measurements without purifn., in situ sol. protein expression monitoring, and data normalization are the powerful outputs of this methodol., thus enabling the accurate identification of improved protein variants during high-throughput screening by substantially reducing the occurrence of false negatives and false positives.
    28. 28
      Azad, T.; Tashakor, A.; Hosseinkhani, S. Split-Luciferase Complementary Assay: Applications, Recent Developments, and Future Perspectives. Anal. Bioanal. Chem. 2014, 406 (23), 5541– 60,  DOI: 10.1007/s00216-014-7980-8
      28
      Split-luciferase complementary assay: applications, recent developments, and future perspectives
      Azad, Taha; Tashakor, Amin; Hosseinkhani, Saman
      Analytical and Bioanalytical Chemistry (2014), 406 (23), 5541-5560CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      A review. Bioluminescent systems are considered as potent reporter systems for bioanal. since they have specific characteristics, such as relatively high quantum yields and photon emission over a wide range of colors from green to red. Biochem. events are mostly accomplished through large protein machines. These mol. complexes are built from a few to many proteins organized through their interactions. These protein-protein interactions are vital to facilitate the biol. activity of cells. The split-luciferase complementation assay makes the study of two or more interacting proteins possible. In this technique, each of the two domains of luciferase is attached to each partner of two interacting proteins. On interaction of those proteins, luciferase fragments are placed close to each other and form a complemented luciferase, which produces a luminescent signal. Split luciferase is an effective tool for assaying biochem. metabolites, where a domain or an intact protein is inserted into an internally fragmented luciferase, resulting in ligand binding, which causes a change in the emitted signals. We review the various applications of this novel luminescent biosensor in studying protein-protein interactions and assaying metabolites involved in anal. biochem., cell communication and cell signaling, mol. biol., and the fate of the whole cell, and show that luciferase-based biosensors are powerful tools that can be applied for diagnostic and therapeutic purposes.
    29. 29
      Bennett, B. D.; Kimball, E. H.; Gao, M.; Osterhout, R.; Van Dien, S. J.; Rabinowitz, J. D. Absolute Metabolite Concentrations and Implied Enzyme Active Site Occupancy in Escherichia Coli. Nat. Chem. Biol. 2009, 5 (8), 593– 9,  DOI: 10.1038/nchembio.186
      29
      Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli
      Bennett, Bryson D.; Kimball, Elizabeth H.; Gao, Melissa; Osterhout, Robin; Van Dien, Stephen J.; Rabinowitz, Joshua D.
      Nature Chemical Biology (2009), 5 (8), 593-599CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Abs. metabolite concns. are crit. to a quant. understanding of cellular metab., as concns. impact both the free energies and rates of metabolic reactions. Here we use LC-MS/MS to quantify more than 100 metabolite concns. in aerobic, exponentially growing Escherichia coli with glucose, glycerol, or acetate as the carbon source. The total obsd. intracellular metabolite pool was approx. 300 mM. A small no. of metabolites dominate the metabolome on a molar basis, with glutamate being the most abundant. Metabolite concn. exceeds Km for most substrate-enzyme pairs. An exception is lower glycolysis, where concns. of intermediates are near the Km of their consuming enzymes and all reactions are near equil. This may facilitate efficient flux reversibility given thermodn. and osmotic constraints. The data and analyses presented here highlight the ability to identify organizing metabolic principles from systems-level abs. metabolite concn. data.
    30. 30
      Zuman, P. Reactions of Orthophthalaldehyde with Nucleophiles. Chem. Rev. 2004, 104 (7), 3217– 38,  DOI: 10.1021/cr0304424
      30
      Reactions of ortho-Phthalaldehyde with Nucleophiles
      Zuman, Petr
      Chemical Reviews (Washington, DC, United States) (2004), 104 (7), 3217-3238CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      The review discusses the chem. of o-phthalaldehyde (I) in its reactions with various oxygen (water, alcs.), nitrogen (amines, amino acids, hydrazines, amides, etc.), carbon and sulfur (thiols) nucleophiles. The special attention is given to the reactions of I in the presence of two nucleophiles, one being a thiol and another an amino acid or amine. The effects of the nucleophile structure and the reaction medium as well as the kinetics of the product formation and the proposed reaction schemes for these two-nucleophile processes are discussed in detail.
    31. 31
      Henke, E.; Bornscheuer, U. T. Fluorophoric Assay for the High-Throughput Determination of Amidase Activity. Anal. Chem. 2003, 75 (2), 255– 60,  DOI: 10.1021/ac0258610
      31
      Fluorophoric Assay for the High-Throughput Determination of Amidase Activity
      Henke, Erik; Bornscheuer, Uwe T.
      Analytical Chemistry (2003), 75 (2), 255-260CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      An assay has been developed for the high-throughput identification of amidase activity. Amines released from the enzyme-catalyzed hydrolysis of corresponding amides were detected by the formation of a fluorescent dye by coupling with 4-nitro-7-chloro-benzo-2-oxa-1,3-diazole (NBD-Cl). Using this format, 22 lipases and esterases were tested for their ability to hydrolyze arom. substituted N-acylamines in a microtiter plate format. Identified active enzymes were further characterized toward a broad range of compds. to det. the influence of substrate structure on activity. For recombinantly produced esterases, it could be shown that the assay works with high reproducibility and sensitivity, even in the presence of amino acids and proteins present in culture media and cell debris.
    32. 32
      Médici, R.; de María, P. D.; Otten, L. G.; Straathof, A. J. J. A High-Throughput Screening Assay for Amino Acid Decarboxylase Activity. Adv. Synth. Catal. 2011, 353 (13), 2369– 76,  DOI: 10.1002/adsc.201100386
      32
      A high-throughput screening assay for amino acid decarboxylase activity
      Medici, Rosario; Dominguez de Maria, Pablo; Otten, Linda G.; Straathof, Adrie J. J.
      Advanced Synthesis & Catalysis (2011), 353 (13), 2369-2376CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The development of sensitive and easy-to-apply high-throughput screening (HTS) methods is a common need in modern biocatalysis. With these powerful anal. tools in hands, chemists can easily assess enzyme libraries to identify either novel biocatalysts or improved mutants. Within biocatalysis, amino acid decarboxylases are gaining an increased importance, with several diverse applications ranging from the synthesis of bio-commodities to medical applications (e.g., synthesis of enzyme inhibitors at the level of L-DOPA decarboxylase). Here, an efficient and simple fluorometric anal. method for HTS of amino acid decarboxylase activity is reported. The method was valid for the discrimination of a broad range of amino acid/amine pairs such as L-tyrosine/tyramine, L-DOPA/dopamine, 5-hydroxy-L-tryptophan/serotonin, L-histidine/histamine, L-serine/ethanolamine, L-tryptophan/tryptamine, L-glutamic acid/GABA, and L-alanine/ethylamine. The method proved its versatility by using pure substrates, mixts., or enzymic reactions, both coming either from com. enzymes or derived from cell-free (crude) exts. The limit of detection was 13 μM for ethanolamine in the presence of 50 mM L-serine, while z' values were in the range of 0.75-0.93, indicating their suitability for HTS.
    33. 33
      Choi, J.-Y.; Black, R.; Lee, H.; Di Giovanni, J.; Murphy, R. C.; Ben Mamoun, C.; Voelker, D. R. An Improved and Highly Selective Fluorescence Assay for Measuring Phosphatidylserine Decarboxylase Activity. J. Biol. Chem. 2020, 295, 9211,  DOI: 10.1074/jbc.RA120.013421
      33
      An improved and highly selective fluorescence assay for measuring phosphatidylserine decarboxylase activity
      Choi, Jae-Yeon; Black, Raymond, III; Lee, Heejung; Di Giovanni, James; Murphy, Robert C.; Mamoun, Choukri Ben; Voelker, Dennis R.
      Journal of Biological Chemistry (2020), 295 (27), 9211-9223CODEN: JBCHA3; ISSN:1083-351X. (American Society for Biochemistry and Molecular Biology)
      Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a crit. step in membrane biogenesis and a potential target for development of antimicrobial and anticancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, the authors described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, the authors report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/β-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochem. methods. Model reactions examg. adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent addns. in 96- or 384- well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.
    34. 34
      Leslie, A. K.; Li, D.; Koide, K. Amine-Promoted β-Elimination of a β-Aryloxy Aldehyde for Fluorogenic Chemodosimeters. J. Org. Chem. 2011, 76 (16), 6860– 5,  DOI: 10.1021/jo200947e
      34
      Amine-Promoted β-Elimination of a β-Aryloxy Aldehyde for Fluorogenic Chemodosimeters
      Leslie, Amanda K.; Li, Dan-Dan; Koide, Kazunori
      Journal of Organic Chemistry (2011), 76 (16), 6860-6865CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      The authors previously reported a fluorescent chemodosimeter for ozone (Garner et al., 2009). The β-elimination step after the ozonolysis of the chemodosimeter was too slow to be practical for real-time monitoring of ozone. Primary, secondary, and tertiary amines were examd. at various pHs. It was found that pyrrolidine in pH 9 buffer could accelerate the elimination to generate a fluorescence signal. The elimination step is now sufficiently rapid to monitor ozone exposure in real time. It was also discovered that azetidine was distinctly effective for the same elimination reaction in a pH 6 buffer.
    35. 35
      Rinde, E.; Troll, W. Colorimetric Assay for Aromatic Amines. Anal. Chem. 1976, 48 (3), 542– 4,  DOI: 10.1021/ac60367a036
      35
      Colorimetric assay for aromatic amines
      Rinde, Esther; Troll, Walter
      Analytical Chemistry (1976), 48 (3), 542-4CODEN: ANCHAM; ISSN:0003-2700.
      Arom. amines can be detected in the nanomole range with the reagent Fluram, with which they form stable yellow derivs. Fluram in glacial acetic acid reacts only with arom. amines. The reaction is complete in 10 min and can be performed on thin-layer (TLC) chromatograms making possible the specific measurement of arom. amines. The yellow product can be quant. eluted from the TLC plates. Fluram is colorless, and the blanks are zero.
    36. 36
      Spangenberg, B.; Poole, C. F.; Weins, C. Quantitative Thin-Layer Chromatography: A Practical Survey; Springer Science & Business Media, 2011.
      There is no corresponding record for this reference.
    37. 37
      Ehmann, A. The Van Urk-Salkowski Reagent ─ a Sensitive and Specific Chromogenic Reagent for Silica Gel Thin-Layer Chromatographic Detection and Identification of Indole Derivatives. J. Chromatogr. A 1977, 132 (2), 267– 76,  DOI: 10.1016/S0021-9673(00)89300-0
      There is no corresponding record for this reference.
    38. 38
      Jones, L. A.; Holmes, J. C.; Seligman, R. B. Spectrophotometric Studies of Some 2,4-Dinitrophenylhydrazones. Anal. Chem. 1956, 28 (2), 191– 8,  DOI: 10.1021/ac60110a013
      38
      Spectrophotometric studies of some 2,4-dinitrophenylhydrazones
      Jones, Louis A.; Holmes, Joseph C.; Seligman, Robert B.
      (1956), 28 (), 191-8CODEN: ANCHAM; ISSN:0003-2700.
      Ultraviolet and visible spectra of the 2,4-dinitrophenylhydrazones (I) of aliphatic aldehydes, aliphatic ketones, aromatic aldehydes, aromatic ketones, carbonyls contg. the furan nucleus, and olefinic aldehydes in neutral soln. and in basic soln. are recorded. Infrared spectra of the I are also given. Absorption bands obtained with the KBr-disk technique are much sharper than those obtained with the Nujol null technique. The parent carbonyl compd. can be characterized by the combined use of the ultraviolet and infrared spectra.
    39. 39
      Zarzycki, P.; Bartoszuk, M.; Radziwon, A. Optimization of Tlc Detection by Phosphomolybdic Acid Staining for Robust Quantification of Cholesterol and Bile Acids. J. Planar Chromatogr. - Mod. TLC 2006, 19 (107), 52– 7,  DOI: 10.1556/JPC.19.2006.1.9
      39
      Optimization of TLC detection by phosphomolybdic acid staining for robust quantification of cholesterol and bile acids
      Zarzycki, Pawel K.; Bartoszuk, Malgorzata A.; Radziwon, Aneta I.
      Journal of Planar Chromatography--Modern TLC (2006), 19 (107), 52-57CODEN: JPCTE5; ISSN:0933-4173. (Research Institute for Medicinal Plants)
      In this paper we describe a robust and sensitive detection procedure for cholesterol and selected bile acids (cholic acid, lithocholic acid, and sodium taurodeoxycholate) using the common derivatization reagent phosphomolybdic acid (PMA). Visualization conditions were studied and optimized for steroids sepd. on glass TLC and HPTLC plates coated with silica gel (K60WF254S) and octadecylsilane (RP-18W) stationary phases. Spot intensities on the plates were quantified after spraying with PMA in methanol (10% w/v) and heating at temps. from 40 to 120° for times ranging from 2 to 40 min. The best conditions for high signal intensity were detd. by using 3D temp. (X)-time (Y)-anal. signal (Z) maps generated from the raw exptl. data. In contrast with the no. of "universal procedures" described in the literature our study indicated that for robust and sensitive quantification of our components of interest heating should be performed at relatively low temps. (below 100°) and for heating times in excess of 10 min. Particularly robust and sensitive detection of steroids sepd. on glass plates coated with both stationary phases was obsd. for temps. ranging between 50 and 80° and heating for at least 20 min in a simple gravity convection oven.
    40. 40
      Bergmann, F. Colorimetric Determination of Amides as Hydroxamic Acids. Anal. Chem. 1952, 24 (8), 1367– 9,  DOI: 10.1021/ac60068a033
      40
      Colorimetric determination of amides as hydroxamic acids
      Bergmann, Felix
      (1952), 24 (), 1367-9CODEN: ANCHAM; ISSN:0003-2700.
      Reaction of amides (I) with alk. HONH2 reagent converts them to hydroxamic acids which can be detd. colorimetrically after development of color with FeCl3 (C.A. 44, 77f). Reaction time and temp. depend on the structure of the I and range from 26 to 100° and 10 to 480 min. The I studied (temp. and time in parentheses) were AcNH2 (60° 120), AcNHMe (60 420), PhNHAc (60° 180), N4-acetylsulfanilamide (60°, 240), acetylglycine (60°, 240), fluoroacetamide (26° 60), HCONH2 (26°, 60), HCONMe2 (26° 240), succinimide (60°, 120), caprolactam (60° 420), asparagine (60°, 180), glutamine (60° 180), glutathione (60°, 120), glycylglycine (60°, 120), nicotinamide (26°, 480), N1-methylnicotinamide methosulfate (26°, 360), nicotinic acid methylamide (60°, 240), nicotinic acid diethylamide (60°, 480), Ca pantothenate (26°, 300), barbitone (100°, 45), pentobarbitone (60°, 300), phenobarbitone (100°, 120), and Na evipan (100°, 30).
    41. 41
      Gayathri, T. Stains for Developing TLC Plates. Int. J. Adv. Res. Sci. Eng. Technol. 2014, 5 (11), 79– 83
      There is no corresponding record for this reference.
    42. 42
      Leeuwenkamp, O. R.; van Bennekom, W. P.; van der Mark, E. J.; Bult, A. Nitroprusside, Antihypertensive Drug and Analytical Reagent. Review of (Photo)stability, Pharmacology and Analytical Properties. Pharm. Weekbl. Sci. Ed. 1984, 6 (4), 129– 140,  DOI: 10.1007/BF01954040
      42
      Nitroprusside, antihypertensive drug and analytical reagent. Review of (photo)stability, pharmacology and analytical properties
      Leeuwenkamp, O. R.; Van Bennekom, W. P.; Van der Mark, E. J.; Bult, A.
      Pharmaceutisch Weekblad, Scientific Edition (1984), 6 (4), 129-40CODEN: PWSEDI; ISSN:0167-6555.
      A review with 103 refs. of phys., chem., anal. and pharmacol. properties of nitroprusside [15078-28-1]. In view of the pharmaceutical applications of nitroprusside, special attention is given to the discussion of the (photo)degrdn., stability of the pharmaceutical formulations, the application as a reagent in pharmaceutical anal. and the redox behavior.
    43. 43
      Lavis, L. D.; Raines, R. T. Bright Building Blocks for Chemical Biology. ACS Chem. Biol. 2014, 9 (4), 855– 66,  DOI: 10.1021/cb500078u
      43
      Bright Building Blocks for Chemical Biology
      Lavis, Luke D.; Raines, Ronald T.
      ACS Chemical Biology (2014), 9 (4), 855-866CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      A review. Small-mol. fluorophores manifest the ability of chem. to solve problems in biol. As we noted in a previous review, the extant collection of fluorescent probes is built on a modest set of "core" scaffolds that evolved during a century of academic and industrial research. Here, we survey traditional and modern synthetic routes to small-mol. fluorophores and highlight recent biol. insights attained with customized fluorescent probes. Our intent is to inspire the design and creation of new high-precision tools that empower chem. biologists.
    44. 44
      Bowen, E. J. Fluorescence and Fluorescence Quenching. Q. Rev. Chem. Soc. 1947, 1 (1), 1– 15,  DOI: 10.1039/qr9470100001
      There is no corresponding record for this reference.
    45. 45
      Fu, Y.; Finney, N. S. Small-Molecule Fluorescent Probes and Their Design. RSC Adv. 2018, 8 (51), 29051– 61,  DOI: 10.1039/C8RA02297F
      45
      Small-molecule fluorescent probes and their design
      Fu, Yanhua; Finney, Nathaniel S.
      RSC Advances (2018), 8 (51), 29051-29061CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      Small-mol. fluorescent probes have become powerful tools for using light to advance the study of cell biol., discover new drugs, detect environmental contaminants, and further the detection of cancer. These applications correlate with the expansion of the fluorescent probe research community - small in the late 20th century, now a collection of more than a hundred research groups world-wide. This expansion required the entry of adventurous scientists from many other fields. This tutorial review introduces some important concepts related to fluorescent probe development. It is hoped that it will facilitate further expansion of the field by demystifying it.
    46. 46
      Grimm, J. B.; Heckman, L. M.; Lavis, L. D. The Chemistry of Small-Molecule Fluorogenic Probes. Prog. Mol. Biol.Transl. Sci. 2013, 113 (1), 1– 34
      46
      The chemistry of small-molecule fluorogenic probes
      Grimm, Jonathan B.; Heckman, Laurel M.; Lavis, Luke D.
      Progress in Molecular Biology and Translational Science (2013), 113 (Fluorescence-Based Biosensors), 1-34CODEN: PNARC5 ISSN:. (Elsevier Inc.)
      A review. Chem. fluorophores find wide use in biol. to detect and visualize different phenomena. A key advantage of small-mol. dyes is the ability to construct compds. where fluorescence is activated by chem. or biochem. processes. Fluorogenic mols., in which fluorescence is activated by enzymic activity, light, or environmental changes, enable advanced bioassays and sophisticated imaging expts. Here, the authors detail the collection of fluorophores and highlight both general strategies and unique approaches that are employed to control fluorescence using chem.
    47. 47
      Daly, B.; Ling, J.; De Silva, A. P. Current Developments in Fluorescent PET (Photoinduced Electron Transfer) Sensors and Switches. Chem. Soc. Rev. 2015, 44 (13), 4203– 11,  DOI: 10.1039/C4CS00334A
      47
      Current developments in fluorescent PET (photoinduced electron transfer) sensors and switches
      Daly, Brian; Ling, Jue; Prasanna de Silva, A.
      Chemical Society Reviews (2015), 44 (13), 4203-4211CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      Following a brief introduction to the principle of fluorescent PET (photoinduced electron transfer) sensors and switches, the outputs of labs. in various countries from the past year or two are categorized and critically discussed. Emphasis is placed on the mol. design and the exptl. outcomes in terms of target-induced fluorescence enhancements and input/output wavelengths. The handling of single targets takes up a major fraction of the review, but the extension to multiple targets is also illustrated. Conceptually new channels of investigation are opened up by the latter approach, e.g. 'lab-on-a-mol.' systems and mol. keypad locks. The growing trends of theor.-fortified design and intracellular application are pointed out.
    48. 48
      Algar, W. R.; Hildebrandt, N.; Vogel, S. S.; Medintz, I. L. Fret as a Biomolecular Research Tool ─ Understanding Its Potential While Avoiding Pitfalls. Nat. Methods 2019, 16 (9), 815– 29,  DOI: 10.1038/s41592-019-0530-8
      48
      FRET as a biomolecular research tool - understanding its potential while avoiding pitfalls
      Algar, W. Russ; Hildebrandt, Niko; Vogel, Steven S.; Medintz, Igor L.
      Nature Methods (2019), 16 (9), 815-829CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      The applications of Forster resonance energy transfer (FRET) grow with each year. However, different FRET techniques are not applied consistently, nor are results uniformly presented, which makes implementing and reproducing FRET expts. challenging. We discuss important considerations for designing and evaluating ensemble FRET expts. Alongside a primer on FRET basics, we provide guidelines for making exptl. design choices such as the donor-acceptor pair, instrumentation and labeling chemistries; selecting control expts. to unambiguously demonstrate FRET and validate that the expts. provide meaningful data about the biomol. process in question; analyzing raw data and assessing the results; and reporting data and exptl. details in a manner that easily allows for reproducibility. Some considerations are also given for FRET assays and FRET imaging, esp. with fluorescent proteins. Our goal is to motivate and empower all biologists to consider FRET for the powerful research tool it can be.
    49. 49
      Misra, R.; Bhattacharyya, S. P. Intramolecular Charge Transfer: Theory and Applications; Wiley VCH, 2018 DOI: 10.1002/9783527801916
      There is no corresponding record for this reference.
    50. 50
      Sedgwick, A. C.; Wu, L.; Han, H.-H.; Bull, S. D.; He, X.-P.; James, T. D.; Sessler, J. L.; Tang, B. Z.; Tian, H.; Yoon, J. Excited-State Intramolecular Proton-Transfer (ESIPT) Based Fluorescence Sensors and Imaging Agents. Chem. Soc. Rev. 2018, 47 (23), 8842– 80,  DOI: 10.1039/C8CS00185E
      50
      Excited-state intramolecular proton-transfer (ESIPT) based fluorescence sensors and imaging agents
      Sedgwick, Adam C.; Wu, Luling; Han, Hai-Hao; Bull, Steven D.; He, Xiao-Peng; James, Tony D.; Sessler, Jonathan L.; Tang, Ben Zhong; Tian, He; Yoon, Juyoung
      Chemical Society Reviews (2018), 47 (23), 8842-8880CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      In this review we will explore recent advances in the design and application of excited-state intramol. proton-transfer (ESIPT) based fluorescent probes. Fluorescence based sensors and imaging agents (probes) are important in biol., physiol., pharmacol., and environmental science for the selective detection of biol. and/or environmentally important species. The development of ESIPT-based fluorescence probes is particularly attractive due to their unique properties, which include a large Stokes shift, environmental sensitivity and potential for ratiometric sensing.
    51. 51
      Hong, Y.; Lam, J. W.; Tang, B. Z. Aggregation-Induced Emission. Chem. Soc. Rev. 2011, 40 (11), 5361– 88,  DOI: 10.1039/c1cs15113d
      51
      Aggregation-induced emission
      Hong, Yuning; Lam, Jacky W. Y.; Tang, Ben Zhong
      Chemical Society Reviews (2011), 40 (11), 5361-5388CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Luminogenic materials with aggregation-induced emission (AIE) attributes have attracted much interest since the debut of the AIE concept in 2001. In this crit. review, recent progress in the area of AIE research is summarized. Typical examples of AIE systems are discussed, from which their structure-property relationships are derived. Through mechanistic decipherment of the photophys. processes, structural design strategies for generating new AIE luminogens are developed. Technol., esp. optoelectronic and biol., applications of the AIE systems are exemplified to illustrate how the novel AIE effect can be utilized for high-tech innovations (183 refs.).
    52. 52
      Grabowski, Z. R.; Rotkiewicz, K.; Rettig, W. Structural Changes Accompanying Intramolecular Electron Transfer: Focus on Twisted Intramolecular Charge-Transfer States and Structures. Chem. Rev. 2003, 103 (10), 3899– 4032,  DOI: 10.1021/cr940745l
      52
      Structural Changes Accompanying Intramolecular Electron Transfer: Focus on Twisted Intramolecular Charge-Transfer States and Structures
      Grabowski, Zbigniew R.; Rotkiewicz, Krystyna; Rettig, Wolfgang
      Chemical Reviews (Washington, DC, United States) (2003), 103 (10), 3899-4031CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      He aim of this review is to summarize, on the background of other types of ICT states, the exptl. and theor. findings concerning the excited- state structures of the much discussed compds. in which the electron donor (D) and the electron acceptor (A) moieties are linked by a formally single bond: the D-A mols. A major part of this review (sections II-IX) concerns the most discussed compd. 4-(dimethylamino)benzonitrile, 1, and its close derivs. and analogs (usually with only a single arom. ring).
    53. 53
      Tanner, P. A.; Zhou, L.; Duan, C.; Wong, K.-L. Misconceptions in Electronic Energy Transfer: Bridging the Gap between Chemistry and Physics. Chem. Soc. Rev. 2018, 47 (14), 5234– 65,  DOI: 10.1039/C8CS00002F
      53
      Misconceptions in electronic energy transfer: bridging the gap between chemistry and physics
      Tanner, Peter A.; Zhou, Lei; Duan, Changkui; Wong, Ka-Leung
      Chemical Society Reviews (2018), 47 (14), 5234-5265CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      Many treatments of energy transfer (ET) phenomena in current literature employ incorrect arguments and formulas and are not quant. enough. This is unfortunate because we witness important breakthroughs from ET expts. in nanoscience. This review aims to clarify basic principles by focusing upon Forster-Dexter elec. dipole-elec. dipole (ED-ED) ET. The roles of ET in upconversion, downconversion and the antenna effect are described and the cliche´s and simple formulas to be avoided in ET studies are highlighted with alternative treatments provided.
    54. 54
      Jiang, X.; Wang, L.; Carroll, S. L.; Chen, J.; Wang, M. C.; Wang, J. Challenges and Opportunities for Small-Molecule Fluorescent Probes in Redox Biology Applications. Antioxid. Redox Signal. 2018, 29 (6), 518– 40,  DOI: 10.1089/ars.2017.7491
      54
      Challenges and Opportunities for Small-Molecule Fluorescent Probes in Redox Biology Applications
      Jiang Xiqian; Wang Lingfei; Carroll Shaina L; Chen Jianwei; Wang Jin; Wang Meng C; Wang Meng C; Wang Jin; Wang Jin
      Antioxidants & redox signaling (2018), 29 (6), 518-540 ISSN:.
      SIGNIFICANCE: The concentrations of reactive oxygen/nitrogen species (ROS/RNS) are critical to various biochemical processes. Small-molecule fluorescent probes have been widely used to detect and/or quantify ROS/RNS in many redox biology studies and serve as an important complementary to protein-based sensors with unique applications. Recent Advances: New sensing reactions have emerged in probe development, allowing more selective and quantitative detection of ROS/RNS, especially in live cells. Improvements have been made in sensing reactions, fluorophores, and bioavailability of probe molecules. CRITICAL ISSUES: In this review, we will not only summarize redox-related small-molecule fluorescent probes but also lay out the challenges of designing probes to help redox biologists independently evaluate the quality of reported small-molecule fluorescent probes, especially in the chemistry literature. We specifically highlight the advantages of reversibility in sensing reactions and its applications in ratiometric probe design for quantitative measurements in living cells. In addition, we compare the advantages and disadvantages of small-molecule probes and protein-based probes. FUTURE DIRECTIONS: The low physiological relevant concentrations of most ROS/RNS call for new sensing reactions with better selectivity, kinetics, and reversibility; fluorophores with high quantum yield, wide wavelength coverage, and Stokes shifts; and structural design with good aqueous solubility, membrane permeability, low protein interference, and organelle specificity. Antioxid. Redox Signal. 29, 518-540.
    55. 55
      Wu, L.; Sedgwick, A. C.; Sun, X.; Bull, S. D.; He, X. P.; James, T. D. Reaction-Based Fluorescent Probes for the Detection and Imaging of Reactive Oxygen, Nitrogen, and Sulfur Species. Acc. Chem. Res. 2019, 52 (9), 2582– 97,  DOI: 10.1021/acs.accounts.9b00302
      55
      Reaction-Based Fluorescent Probes for the Detection and Imaging of Reactive Oxygen, Nitrogen, and Sulfur Species
      Wu, Luling; Sedgwick, Adam C.; Sun, Xiaolong; Bull, Steven D.; He, Xiao-Peng; James, Tony D.
      Accounts of Chemical Research (2019), 52 (9), 2582-2597CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. This account describes a range of strategies for the development of fluorescent probes for detecting reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive (redox-active) sulfur species (RSS). Many ROS/RNS have been implicated in pathol. processes such as Alzheimer's disease (AD), cancer, diabetes mellitus, cardiovascular disease and aging. While many RSS play important roles in maintaining redox homeostasis and serve as antioxidants and act as free radical scavengers. Fluorescence based systems have emerged as one of the best ways to monitor the concns. and locations of these often very short-lived species. Due to the high levels of sensitivity and in particular their ability to be used for temporal and spatial sampling for in vivo imaging applications. As a direct result, there has been a huge surge in the development of fluorescent probes to sensitively and selectively detect ROS, RNS and RSS species within cellular environments. However, cellular environments are extremely complex with more than one species often involved in a given biochem. process. As a result, there has been a rise in the development of dual-responsive fluorescent probes (AND-logic probes) able to monitor the presence of more than one species in a biol. environment. Our aim with this account is to introduce the fluorescent probes we have developed for the in vitro and in vivo measurement of ROS, RNS and RSS. Fluorescence based sensing mechanisms used in the construction of the probes include photoinduced electron transfer (PeT), intramol. charge transfer (ICT), excited-state intramol. proton transfer (ESIPT) and fluorescent-resonance-energy-transfer (FRET). In particular probes for hydrogen peroxide (H2O2), hypochlorous acid (HOCl/ClO-), superoxide (O2•-), peroxynitrite (ONOO-), glutathione (GSH), cysteine (Cys), homocysteine (HCys) and Hydrogen sulfide (H2S) are discussed. In addn., we describe the development of AND logic based systems capable of detecting two species, such as peroxynitrite (ONOO-) and glutathione (GSH). One of the most interesting advances contained in this account is our extension of indicator displacement assays (IDAs) to reaction-based indicator displacement assays (RIAs) In an IDA system, an indicator is allowed to bind reversibly to a receptor. Then, a competitive analyte is introduced into the system, resulting in displacement of the indicator from the host, which in turn modulates the optical signal. With an RIA based systems the indicator is cleaved from a preformed receptor-indicator complex rather than being displaced by the analyte. Nevertheless, without a doubt the most significant result contained in this account is the use of an excited-state intramol. proton transfer (ESIPT) based probe for the simultaneous sensing of fibrous proteins/peptides AND environmental ROS/RNS.
    56. 56
      Jiao, X.; Li, Y.; Niu, J.; Xie, X.; Wang, X.; Tang, B. Small-Molecule Fluorescent Probes for Imaging and Detection of Reactive Oxygen, Nitrogen, and Sulfur Species in Biological Systems. Anal. Chem. 2018, 90 (1), 533– 55,  DOI: 10.1021/acs.analchem.7b04234
      56
      Small-Molecule Fluorescent Probes for Imaging and Detection of Reactive Oxygen, Nitrogen, and Sulfur Species in Biological Systems
      Jiao, Xiaoyun; Li, Yong; Niu, Jinye; Xie, Xilei; Wang, Xu; Tang, Bo
      Analytical Chemistry (Washington, DC, United States) (2018), 90 (1), 533-555CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A review. Intracellular redox homeostasis provides broad implications in physiol. and pathol. fields. The disruption of redox homeostasis is closely assocd. with some human diseases, such as cancer, neurodegenerative diseases, cardiovascular diseases, diabetes mellitus, and gastrointestinal diseases. Therefore, cells possess an elaborate regulation system to maintain their redox balance and the large or significant redox state changes can be buffered by the redox-active mols. These mols. experience interreaction and interconversion to facilitate the dynamic balance of intracellular redox state, among which three types of representative mols. should be mentioned, including reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS).
    57. 57
      Yang, Y.; Zhao, Q.; Feng, W.; Li, F. Luminescent Chemodosimeters for Bioimaging. Chem. Rev. 2013, 113 (1), 192– 270,  DOI: 10.1021/cr2004103
      57
      Luminescent Chemodosimeters for Bioimaging
      Yang, Yuming; Zhao, Qiang; Feng, Wei; Li, Fuyou
      Chemical Reviews (Washington, DC, United States) (2013), 113 (1), 192-270CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Chemodosimeters are a class of sensing system based on analyte-induced irreversible chem. reactions. They exhibit high selectivity, sensitivity, and rapid response. Significant advances have been achieved in the design of chemodosimeters, and many fluorophores have been successfully used as the basis of luminescent chemodosimeters. In this review, the authors have presented the general design principles of luminescent chemodosimeters for bioimaging and summarized recent advances in the detection of metal cations, anions, thiols, reactive oxygen species, reactive nitrogen species, reactive sulfur species, DCP, and amino acids in vitro and in vivo, developed by the authors and other groups.
    58. 58
      Chyan, W.; Raines, R. T. Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging. ACS Chem. Biol. 2018, 13 (7), 1810– 23,  DOI: 10.1021/acschembio.8b00371
      58
      Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging
      Chyan, Wen; Raines, Ronald T.
      ACS Chemical Biology (2018), 13 (7), 1810-1823CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      A review. Fluorogenic probes, small-mol. sensors that unmask brilliant fluorescence upon exposure to specific stimuli, are powerful tools for chem. biol. Those probes that respond to enzymic catalysis illuminate the complex dynamics of biol. processes at a level of spatiotemporal detail and sensitivity unmatched by other techniques. Here, the authors review recent advances in enzyme-activated fluorogenic probes for biol. imaging. The authors organize the authors' survey by enzyme classification, with emphasis on fluorophore masking strategies, modes of enzymic activation, and the breadth of current and future applications. Key challenges such as probe selectivity and spectroscopic requirements are described alongside therapeutic, diagnostic, and theranostic opportunities.
    59. 59
      Lawrence, S. A. Amines: Synthesis, Properties and Applications; Cambridge University Press, 2004.
      There is no corresponding record for this reference.
    60. 60
      Yin, Q.; Shi, Y.; Wang, J.; Zhang, X. Direct Catalytic Asymmetric Synthesis of α-Chiral Primary Amines. Chem. Soc. Rev. 2020, 49 (17), 6141– 53,  DOI: 10.1039/C9CS00921C
      60
      Direct catalytic asymmetric synthesis of α-chiral primary amines
      Yin Qin; Shi Yongjie; Wang Jingxin; Zhang Xumu
      Chemical Society reviews (2020), 49 (17), 6141-6153 ISSN:.
      α-Chiral primary amines are one among the most valuable and versatile building blocks for the synthesis of numerous amine-containing pharmaceuticals and natural compounds. They also serve as chiral ligands or organo-catalysts for asymmetric catalysis. However, most of the existing chemocatalytic methods toward enantiopure primary amines rely on multistep manipulations on N-substituted substrates, which are not ideally atom-economical and cost-effective. Among the catalytic methods including the asymmetric transformations of the pre-prepared or in situ formed NH imines, biomimetic chemocatalysis inspired by enzymatic transaminations has recently emerged as an appealing and straightforward method to access chiral primary amines. This tutorial review highlights the state-of-the-art catalytic methods for the direct asymmetric synthesis of α-chiral primary amines and demonstrates their utility in the construction of molecular complexities, which may attract extensive attention and inspire applications in synthetic and medicinal chemistry.
    61. 61
      Cabré, A.; Verdaguer, X.; Riera, A. Recent Advances in the Enantioselective Synthesis of Chiral Amines Via Transition Metal-Catalyzed Asymmetric Hydrogenation. Chem. Rev. 2022, 122 (1), 269– 339,  DOI: 10.1021/acs.chemrev.1c00496
      61
      Recent Advances in the Enantioselective Synthesis of Chiral Amines via Transition Metal-Catalyzed Asymmetric Hydrogenation
      Cabre, Albert; Verdaguer, Xavier; Riera, Antoni
      Chemical Reviews (Washington, DC, United States) (2022), 122 (1), 269-339CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The present review covers the use of asym. hydrogenation (AH) in the synthesis of chiral amines bearing a stereogenic center either in α, β or γ position with respect to the nitrogen atom, reported from 2010 to 2020. Therefore, an overview of the recent advances in the AH of imines, enamides, enamines, allyl amines and N-heteroarom. compds. Was provided.
    62. 62
      Wu, Z.; Liu, C.; Zhang, Z.; Zheng, R.; Zheng, Y. Amidase as a Versatile Tool in Amide-Bond Cleavage: From Molecular Features to Biotechnological Applications. Biotechnol. Adv. 2020, 43, 107574,  DOI: 10.1016/j.biotechadv.2020.107574
      62
      Amidase as a versatile tool in amide-bond cleavage: From molecular features to biotechnological applications
      Wu, Zheming; Liu, Changfeng; Zhang, Zhaoyu; Zheng, Renchao; Zheng, Yuguo
      Biotechnology Advances (2020), 43 (), 107574CODEN: BIADDD; ISSN:0734-9750. (Elsevier Inc.)
      A review. Amidases (EC 3. 5. 1. X) are versatile biocatalysts for synthesis of chiral carboxylic acids, α-amino acids and amides due to their hydrolytic and acyl transfer activity towards the C-N linkages. They have been extensively exploited and studied during the past years for their high specific activity and excellent enantioselectivity involved in various biotechnol. applications in pharmaceutical and agrochem. industries. Addnl., they have attracted considerable attentions in biodegrdn. and bioremediation owing to environmental pressures. Motivated by industrial demands, crystallog. investigations and catalytic mechanisms of amidases based on structural biol. have witnessed a dramatic promotion in the last two decades. The protein structures showed that different types of amidases have their typical stuctural elements, such as the conserved AS domains in signature amidases and the typical architecture of metal-assocd. active sites in acetamidase/formamidase family amidases. This review provides an overview of recent research advances in various amidases, with a focus on their structural basis of phylogenetics, substrate specificities and catalytic mechanisms as well as their biotechnol. applications. As more crystal structures of amidases are detd., the structure/function relationships of these enzymes will also be further elucidated, which will facilitate mol. engineering and design of amidases to meet industrial requirements.
    63. 63
      Breuer, M.; Ditrich, K.; Habicher, T.; Hauer, B.; Keßeler, M.; Stürmer, R.; Zelinski, T. Industrial Methods for the Production of Optically Active Intermediates. Angew. Chem., Int. Ed. 2004, 43 (7), 788– 824,  DOI: 10.1002/anie.200300599
      63
      Industrial methods for the production of optically active intermediates
      Breuer, Michael; Ditrich, Klaus; Habicher, Tilo; Hauer, Bernhard; Kesseler, Maria; Stuermer, Rainer; Zelinski, Thomas
      Angewandte Chemie, International Edition (2004), 43 (7), 788-824CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Enantiomerically pure amino acids, amino alcs., amines, alcs., and epoxides play an increasingly important role as intermediates in the pharmaceutical industry and agrochem., where both a high degree of purity and large quantities of the compds. are required. The chem. industry has primarily relied upon established chem. methods for the synthesis of these intermediates, but is now turning more and more to enzymic and biotechnol. fermn. processes. For the industrial implementation of many transformations alternative methods are available. The advantages of the individual methods will be discussed herein and exemplified by syntheses of relevant compds.
    64. 64
      Balkenhohl, F.; Ditrich, K.; Hauer, B.; Ladner, W. Optisch Aktive Amine Durch Lipase-Katalysierte Methoxyacetylierung. J. Prakt. Chem. Chem. Ztg. 1997, 339 (1), 381– 4,  DOI: 10.1002/prac.19973390166
      64
      Optically active amines via lipase-catalyzed methoxyacetylation
      Balkenhohl, Friedhelm; Ditrich, Klaus; Hauer, Bernhard; Ladner, Wolfgang
      Journal fuer Praktische Chemie/Chemiker-Zeitung (1997), 339 (4), 381-384CODEN: JPCCEM; ISSN:0941-1216. (Barth)
      Racemic amines can be efficiently resolved by using MeOCH2CO2Et as acylating agent in a lipase-catalyzed reaction. Treating PhMeCHNH2 with MeOCH2CO2Et in the presence of a lipase from Burkholderia plantarii gave 41% (R)-PhMeCHNHCOCH2OMe and 45% (S)-PhMeCHNH2, both with ee >93%.
    65. 65
      Gomm, A.; O’Reilly, E. Transaminases for Chiral Amine Synthesis. Curr. Opin. Chem. Biol. 2018, 43, 106– 12,  DOI: 10.1016/j.cbpa.2017.12.007
      65
      Transaminases for chiral amine synthesis
      Gomm, Andrew; O'Reilly, Elaine
      Current Opinion in Chemical Biology (2018), 43 (), 106-112CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. Amine transaminases are important biocatalysts for the synthesis of chiral primary amines. Unlike many enzymes that have been employed for the synthesis of optically active amines, amine transaminases are capable of asym. synthesis and do not rely on costly cofactors that must be regenerated in situ. However, their application as general catalysts for the prepn. of amines is hampered by a limited substrate scope, substrate and (co)product inhibition and difficulties assocd. with displacing challenging reaction equil. There has been important progress made to overcome these challenges, including the development of enzymes with broader substrate scope and the design of methodol. to effectively displace the reaction equil. Amine transaminases are also being applied in an increasing range of (chemo)enzymic cascades and immobilized for applications in flow.
    66. 66
      Abrahamson, M. J.; Vázquez-Figueroa, E.; Woodall, N. B.; Moore, J. C.; Bommarius, A. S. Development of an Amine Dehydrogenase for Synthesis of Chiral Amines. Angew. Chem., Int. Ed. 2012, 51 (16), 3969– 72,  DOI: 10.1002/anie.201107813
      66
      Development of an Amine Dehydrogenase for Synthesis of Chiral Amines
      Abrahamson, Michael J.; Vazquez-Figueroa, Eduardo; Woodall, Nicholas B.; Moore, Jeffrey C.; Bommarius, Andreas S.
      Angewandte Chemie, International Edition (2012), 51 (16), 3969-3972, S3969/1-S3969/7CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We have successfully developed an amine dehydrogenase, by starting with an existing amino acid dehydrogenase as a template and subsequent active-site-targeted protein engineering. Eleven rounds of protein engineering completely inverted the enzyme's specificity and created amination activity of 0.69 U mg-1 with a corresponding kcat value of 0.46 s-1. In doing so, the native activity toward L-Leu was reduced to immeasurably low levels. The enantioselectivity of the wild-type enzyme was maintained, and yielded (R)-1,3-DMBA with an ee value of 99.8% at 92.5 % conversion. This amine dehydrogenase exhibited activity toward a no. of different substrates. This is the first example of a cofactor-dependent amine dehydrogenase capable of selectively synthesizing chiral amines from a pro-chiral ketone and free ammonia.
    67. 67
      Li, B.-B.; Zhang, J.; Chen, F.-F.; Chen, Q.; Xu, J.-H.; Zheng, G.-W. Direct Reductive Amination of Ketones with Amines by Reductive Aminases. Green Synth. Catal. 2021, 2 (4), 345– 9,  DOI: 10.1016/j.gresc.2021.08.005
      There is no corresponding record for this reference.
    68. 68
      Lenz, M.; Borlinghaus, N.; Weinmann, L.; Nestl, B. M. Recent Advances in Imine Reductase-Catalyzed Reactions. World J. Microbiol. Biotechnol. 2017, 33 (11), 199,  DOI: 10.1007/s11274-017-2365-8
      68
      Recent advances in imine reductase-catalyzed reactions
      Lenz Maike; Borlinghaus Niels; Weinmann Leonie; Nestl Bettina M
      World journal of microbiology & biotechnology (2017), 33 (11), 199 ISSN:.
      Imine reductases are nicotinamide-dependent enzymes that catalyze the asymmetric reduction of various imines to the corresponding amine products. Owing to the increasing roles of chiral amines and heterocyclic compounds as intermediates for pharmaceuticals, the demand for novel selective synthesis strategies is vitally important. Recent studies have demonstrated the discovery and structural characterization of a number of stereoselective imine reductase enzymes. Here, we highlight recent progress in applying imine reductases for the formation of chiral amines and heterocycles. It particularly focuses on the utilization of imine reductases in reductive aminations of aldehydes and ketones with various amine nucleophiles, one of the most powerful reactions in the synthesis of chiral amines. Second, we report on the synthesis of saturated substituted N-heterocycles by combining them with further biocatalysts, such as carboxylic acid reductases, oxidases or transaminases. Finally, we summarize the latest applications of imine reductases in the promiscuous asymmetric hydrogenation of a highly reactive carbonyl compound and the engineering of the cofactor specificity from NADPH to NADH.
    69. 69
      Gaweska, H.; Fitzpatrick, P. F. Structures and Mechanism of the Monoamine Oxidase Family. Biomol Concepts 2011, 2 (5), 365– 77,  DOI: 10.1515/BMC.2011.030
      69
      Structures and mechanism of the monoamine oxidase family
      Gaweska, Helena; Fitzpatrick, Paul F.
      Biomolecular Concepts (2011), 2 (5), 365-377CODEN: BCIOB8; ISSN:1868-5021. (Walter de Gruyter GmbH & Co. KG)
      A review. Members of the monoamine oxidase family of flavoproteins catalyze the oxidn. of primary and secondary amines, polyamines, amino acids, and methylated lysine side chains in proteins. The enzymes have similar overall structures, with conserved FAD (FAD)-binding domains and varied substrate-binding sites. Multiple mechanisms have been proposed for the catalytic reactions of these enzymes. The present review compares the structures of different members of the family and the various mechanistic proposals.
    70. 70
      Parmeggiani, F.; Weise, N. J.; Ahmed, S. T.; Turner, N. J. Synthetic and Therapeutic Applications of Ammonia-Lyases and Aminomutases. Chem. Rev. 2018, 118 (1), 73– 118,  DOI: 10.1021/acs.chemrev.6b00824
      70
      Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases
      Parmeggiani, Fabio; Weise, Nicholas J.; Ahmed, Syed T.; Turner, Nicholas J.
      Chemical Reviews (Washington, DC, United States) (2018), 118 (1), 73-118CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Ammonia-lyases and aminomutases are mechanistically and structurally diverse enzymes which catalyze the deamination and/or isomerization of amino acids in nature by cleaving or shifting a C-N bond. Of the many protein families in which these enzyme activities are found, only a subset have been employed in the synthesis of optically pure fine chems. or in medical applications. This review covers the natural diversity of these enzymes, highlighting particular enzyme classes that are used within industrial and medical biotechnol. These highlights detail the discovery and mechanistic investigations of these com. relevant enzymes, along with comparisons of their various applications as stand-alone catalysts, components of artificial biosynthetic pathways and biocatalytic or chemoenzymic cascades, and therapeutic tools for the potential treatment of various pathologies.
    71. 71
      Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.; Jarvis, W. R.; Colbeck, J. C.; Krebber, A.; Fleitz, F. J.; Brands, J. Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture. Science 2010, 329 (5989), 305– 9,  DOI: 10.1126/science.1188934
      71
      Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture
      Savile, Christopher K.; Janey, Jacob M.; Mundorff, Emily C.; Moore, Jeffrey C.; Tam, Sarena; Jarvis, William R.; Colbeck, Jeffrey C.; Krebber, Anke; Fleitz, Fred J.; Brands, Jos; Devine, Paul N.; Huisman, Gjalt W.; Hughes, Gregory J.
      Science (Washington, DC, United States) (2010), 329 (5989), 305-309CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Pharmaceutical synthesis can benefit greatly from the selectivity gains assocd. with enzymic catalysis. Here, we report an efficient biocatalytic process to replace a recently implemented rhodium-catalyzed asym. enamine hydrogenation for the large-scale manuf. of the antidiabetic compd. sitagliptin. Starting from an enzyme that had the catalytic machinery to perform the desired chem. but lacked any activity toward the prositagliptin ketone, we applied a substrate walking, modeling, and mutation approach to create a transaminase with marginal activity for the synthesis of the chiral amine; this variant was then further engineered via directed evolution for practical application in a manufg. setting. The resultant biocatalysts showed broad applicability toward the synthesis of chiral amines that previously were accessible only via resoln. This work underscores the maturation of biocatalysis to enable efficient, economical, and environmentally benign processes for the manuf. of pharmaceuticals.
    72. 72
      Weiß, M. S.; Pavlidis, I. V.; Spurr, P.; Hanlon, S. P.; Wirz, B.; Iding, H.; Bornscheuer, U. T. Protein-Engineering of an Amine Transaminase for the Stereoselective Synthesis of a Pharmaceutically Relevant Bicyclic Amine. Org. Biomol. Chem. 2016, 14 (43), 10249– 54,  DOI: 10.1039/C6OB02139E
      72
      Protein-engineering of an amine transaminase for the stereoselective synthesis of a pharmaceutically relevant bicyclic amine
      Weiss, Martin S.; Pavlidis, Ioannis V.; Spurr, Paul; Hanlon, Steven P.; Wirz, Beat; Iding, Hans; Bornscheuer, Uwe T.
      Organic & Biomolecular Chemistry (2016), 14 (43), 10249-10254CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones represents an environmentally benign and economically attractive alternative to transition metal catalyzed asym. synthesis. However, the restrictive substrate scope has limited the conversion typically to non-sterically demanding scaffolds. Recently, we reported on the identification and design of fold class I ATAs that effect a highly selective asym. synthesis of a set of chiral arom. bulky amines from the corresponding ketone precursors in high yield. However, for the specific amine synthetic approach extension targeted here, the selective formation of an exo- vs. endo-isomer, these biocatalysts required addnl. refinement. The chosen substrate (exo-3-amino-8-aza-bicyclo[3.2.1]oct-8-yl-phenyl-methanone), apart from its pharmacol. relevance, is a demanding target for ATAs as the bridged bicyclic ring provides substantial steric challenges. Protein engineering combining rational design and directed evolution enabled the identification of an ATA variant which catalyzes the specific synthesis of the target exo-amine with >99.5% selectivity.
    73. 73
      Ma, E. J.; Siirola, E.; Moore, C.; Kummer, A.; Stoeckli, M.; Faller, M.; Bouquet, C.; Eggimann, F.; Ligibel, M.; Huynh, D. Machine-Directed Evolution of an Imine Reductase for Activity and Stereoselectivity. ACS Catal. 2021, 11 (20), 12433– 45,  DOI: 10.1021/acscatal.1c02786
      73
      Machine-Directed Evolution of an Imine Reductase for Activity and Stereoselectivity
      Ma, Eric J.; Siirola, Elina; Moore, Charles; Kummer, Arkadij; Stoeckli, Markus; Faller, Michael; Bouquet, Caroline; Eggimann, Fabian; Ligibel, Mathieu; Huynh, Dan; Cutler, Geoffrey; Siegrist, Luca; Lewis, Richard A.; Acker, Anne-Christine; Freund, Ernst; Koch, Elke; Vogel, Markus; Schlingensiepen, Holger; Oakeley, Edward J.; Snajdrova, Radka
      ACS Catalysis (2021), 11 (20), 12433-12445CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      Biocatalysis is an effective tool to access chiral mols. that are otherwise hard to synthesize or purify. Time-efficient processes are needed to develop enzymes that adequately perform the desired chem. We evaluated machine-directed evolution as an enzyme engineering strategy using a moderately stereoselective imine reductase as the model system. We compared machine-directed evolution approaches to deep mutational scanning (DMS) and error-prone PCR. Within one cycle, it was found that machine-directed evolution yielded a library of high-activity mutants with a dramatically shifted activity distribution compared to that of traditional directed evolution. Structure-guided anal. revealed that linear additivity might provide a simple explanation for the effectiveness of machine-directed evolution. The most active and selective enzyme mutant, which was identified through DMS and error-prone PCR, was used for the gram-scale synthesis of the H4 receptor antagonist ZPL389 with full conversion, > 99% ee (R), and a 72% yield.
    74. 74
      Ao, Y.-F.; Hu, H.-J.; Zhao, C.-X.; Chen, P.; Huang, T.; Chen, H.; Wang, Q.-Q.; Wang, D.-X.; Wang, M.-X. Reversal and Amplification of the Enantioselectivity of Biocatalytic Desymmetrization toward meso Heterocyclic Dicarboxamides Enabled by Rational Engineering of Amidase. ACS Catal. 2021, 11 (12), 6900– 7,  DOI: 10.1021/acscatal.1c01220
      74
      Reversal and amplification of the enantioselectivity of biocatalytic desymmetrization toward meso heterocyclic dicarboxamides enabled by rational engineering of amidase
      Ao, Yu-Fei; Hu, Hui-Juan; Zhao, Cheng-Xin; Chen, Peng; Huang, Tingting; Chen, Hui; Wang, Qi-Qiang; Wang, De-Xian; Wang, Mei-Xiang
      ACS Catalysis (2021), 11 (12), 6900-6907CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      By rational engineering of amidase, the efficient biocatalytic desymmetrization of meso O-heterocyclic dicarboxamides for synthesizing both antipodes of functionalized cyclic motifs was presented. Based on the enzyme-substrate binding model suggested by mol. docking, a rational mutagenesis strategy was established. The reversal and amplification of enantioselectivity of amidase were achieved by generating and testing only 10 variants. This enabled the quick access of both antipodes of products in very good yields and up to 99.5% ee under mild conditions. The engineered biocatalyst exhibits a wide substrate promiscuity and can expand to both N-heterocyclic and carbocyclic dicarboxamides with the retained high efficiency and excellent enantioselectivity. The desymmetrization mechanism for amidase, including the wild-type and the variant, was revealed by mol. dynamics simulations and quantum mech./mol. mech. modeling. It suggests a delicate cooperation between the activation and binding sites by nesting the cyclic skeleton of the substrates.
    75. 75
      Wang, P. F.; Yep, A.; Kenyon, G. L.; McLeish, M. J. Using Directed Evolution to Probe the Substrate Specificity of Mandelamide Hydrolase. Protein Eng. Des. Sel. 2009, 22 (2), 103– 10,  DOI: 10.1093/protein/gzn073
      75
      Using directed evolution to probe the substrate specificity of mandelamide hydrolase
      Wang, Pan-Fen; Yep, Alejandra; Kenyon, George L.; McLeish, Michael J.
      Protein Engineering, Design & Selection (2009), 22 (2), 103-110CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)
      Mandelamide hydrolase (MAH), a member of the amidase signature family, catalyzes the hydrolysis of mandelamide to mandelate and ammonia. X-ray structures of several members of this family, but not that of MAH, have been reported. These reveal nearly superimposable conformations of the unusual Ser-cisSer-Lys catalytic triad. Conversely, the residues involved in substrate recognition are not conserved, implying that the binding pocket could be modified to change the substrate specificity, perhaps by directed evolution. Here we show that MAH is able to hydrolyze small aliph. substrates such as lactamide, albeit with low efficiency. A selection method to monitor changes in mandelamide/lactamide preference was developed and used to identify several mutations affecting substrate binding. A homol. model places some of these mutations close to the catalytic triad, presumably in the MAH active site. In particular, Gly-202 appears to control the preference for arom. substrates as the G202A variant showed three orders of magnitude decrease in kcat/Km for (R)- and (S)-mandelamide. This redn. in activity increased to six orders of magnitude for the G202V variant.
    76. 76
      Thooft, A. M.; Cassaidy, K.; VanVeller, B. A Small Push-Pull Fluorophore for Turn-On Fluorescence. J. Org. Chem. 2017, 82 (17), 8842– 7,  DOI: 10.1021/acs.joc.7b00939
      76
      A Small Push-Pull Fluorophore for Turn-on Fluorescence
      Thooft, Andrea Marie; Cassaidy, Kyle; VanVeller, Brett
      Journal of Organic Chemistry (2017), 82 (17), 8842-8847CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      A new class of push-pull dyes is reported based on the structures of benzoxa- and benzothiadiazole heterocycles. This new class of dyes displays red-shifted wavelengths of emission and greater sensitivity to polarity and hydrogen bonding solvents relative to previously known derivs.
    77. 77
      Annenkov, V. V.; Verkhozina, O. N.; Shishlyannikova, T. A.; Danilovtseva, E. N. Application of 4-Chloro-7-Nitrobenzo-2-Oxa-1,3-Diazole in Analysis: Fluorescent Dyes and Unexpected Reaction with Tertiary Amines. Anal. Biochem. 2015, 486, 5– 13,  DOI: 10.1016/j.ab.2015.06.025
      77
      Application of 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole in analysis: Fluorescent dyes and unexpected reaction with tertiary amines
      Annenkov, Vadim V.; Verkhozina, Olga N.; Shishlyannikova, Tatyana A.; Danilovtseva, Elena N.
      Analytical Biochemistry (2015), 486 (), 5-13CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is widely applied as a fluorescent tagging reagent in biochem., as a derivatization agent in anal. chem., and as a component for design of fluorescent nanoparticles. Four new 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-tagged polyamines contg. two to four amine moieties were synthesized and used as an effective tool for staining of siliceous frustules of the diatom algae and spicules of the siliceous sponges, including fossilized samples. An unexpected reaction between NBD-Cl and tertiary amine groups was found, giving rise to NBD-tagged amines with elimination of an alkyl group. The reaction proceeds through the Meisenheimer complex and quaternary salt, which transform to the product by Hofmann reaction (alkene elimination) or nucleophilic substitution (halogenated compd. formation). In the case of polyamines, NBD-Cl causes chain scissoring, giving a set of NBD-tagged amines. The found NBD-Cl reaction with tertiary amines must be taken into account when using NBD-Cl and similar activated arom. systems for amine derivatization in anal. and biochem. applications. The reaction with polyamines opens the way to libraries of NBD-tagged compds.
    78. 78
      Sanger, F. The Free Amino Groups of Insulin. Biochem. J. 1945, 39 (5), 507– 15,  DOI: 10.1042/bj0390507
      78
      Free amino groups of insulin
      Sanger, F.
      Biochemical Journal (1945), 39 (), 507-15CODEN: BIJOAK; ISSN:0264-6021.
      The free amino groups of proteins and peptides readily form derivs. with 2,4-(O2N)2C6H3F (I) at room temp. Since these derivs. are relatively stable to acid hydrolysis, hydrolysis of the substituted protein enables isolation of 2,4-dinitrophenyl amino acid derivs. which are separable by partition chromatography. This, then, forms the basis of a new method which was applied to the identification and estn. of the free amino groups in insulin. For comparison and control, several derivs. of the naturally occurring amino acids had to be synthesized. To a mixt. of 0.2 g. l-phenylalanine and 0.4 g. NaHCO3 in 5 ml. H2O was added a soln. of 0.4 g. I in 10 ml. EtOH. The mixt. was reacted 2 hrs. at room temp., giving 0.27 g. of N-2,4-dinitrophenyl-l-phenylalanine, C15H13O6N3, m. 186°. α-Acetyl-l-lysine (0.48 g.) and 0.75 g. NaHCO3 in 3 ml. H2O reacted for 4 hrs. at reflux temp. with 0.5 g. 2,4-(O2N)2C6H3Cl (II) in 10 ml. EtOH gave 0.7 g. (75%) of α-acetyl-ε-2,4-dinitrophenyl-l-lysine (III), m. 110°. III refluxed for 3 hrs. with 20% HCl gives ε-2,4-dinitrophenyl-l-lysine-HCl, which seps. from dil. HCl as the monohydrate, C12H16O6N4.HCl.H2O, m. 186°. ε-Benzoyl-l-lysine (1.25 g.) treated with 1.0 g. II gives 2 g. of α-2,4-dinitrophenyl-ε-benzoyl-l-lysine (IV). IV (0.5 g.) hydrolyzed in HOAc and HCl gave 60% of α-2,4-dinitrophenyl-l-lysine, C12H16O6N4, m. 260° (decompn.). l-Tyrosine (V) in the presence of excess I gives a compd. which is presumably O,N-bis(2,4 dinitrophenyl)-l-tyrosine (VI). Reaction of V with II gives, in addn. to VI, a compd. which was probably N-2,4-dinitrophenyl-l-tyrosine. N-Acetyl-l-tyrosine (0.55 g.) treated for 4 hrs. with 2.0 g. II gave 0.1 g. of N-acetyl-O-2,4-dinitrophenyl-l-tyrosine (VII), m. 194°. VII hydrolyzed in 20% HCl gave O-2,4-dinitrophenyl-l-tyrosine, as the hydrate, C15H13O7N3.H2O, m. 202°. On treatment of histidine with excess I only the bis(2,4-dinitrophenyl) deriv. is obtained, indicating that I reacts with the imidazole ring. This is confirmed by the slow reaction of I with α-acetylhistidine and rapid reaction with histidine Me ester to give again a bis deriv. I reacts also with SH groups of S-contg. amino acids but a mixt. of products is apparently obtained. The band rates on the chromatogram for various known 2,4-dinitrophenyl amino acids were detd. for various solvent mixts. and compared with those of the derivs. obtained from hydrolyzed dinitrophenylinsulin. No indicator was necessary since all of the derivs. were colored. Isolation of the bands enabled quant. colorimetric estn. of the derivs., using the known compds. as standards. Application of this technique shows that in an insulin submol. of mol. wt. 12,000, six free amino groups are present; 2 of these are located on glycine residues, 2 on phenylalanine residues, and 2 represent the ε-amino groups of lysine. The results suggest that the insulin submol. is made up of 4 open polypeptide chains, 2 of these having terminal glycyl residues and the other 2 terminal phenylalanyl residues, the chains being bound together most probably by -S-S- linkages.
    79. 79
      Ghosh, P. B.; Whitehouse, M. W. 7-Chloro-4-Nitrobenzo-2-Oxa-1,3-Diazole: A New Fluorigenic Reagent for Amino Acids and Other Amines. Biochem. J. 1968, 108 (1), 155– 6,  DOI: 10.1042/bj1080155
      79
      7-Chloro-4-nitrobenzofurazan: a new fluorigenic reagent for amino acids and other amines
      Ghosh, P. B.; Whitehouse, M. W.
      Biochemical Journal (1968), 108 (1), 155-6CODEN: BIJOAK; ISSN:0264-6021.
      7-Chloro-4-nitrobenzoxadiazole (I), m. 97°, was prepd. by nitrating 4-chlorobenzofurazan, obtained from 2,6-dichloroaniline via the dichloronitrosobenzene. I was non-fluorescent. Strong fluorescence of the I-amine compds. (II) was observed in solvents of low polarity and was excited at 464 mμ. I was more stable and more sol. in aq. solns. than 1-dimethylaminonaphthalene-5-sulfonyl (DNS) chloride. As little as 1 mγ I-glycine/ml. in acetone or EtOAc could be detected. The intensity of fluorescence of I-glycine in moist EtOAc was approx. the same as DNS-glycine in water and > the fluorescence of DNS-glycine in moist acetone. Substituents on the amino group that restrict its conjugation with the benzoxadiazole nucleus caused a drastic loss of fluorescence. Related compds. that were not amine derivs. but had other electron-donor groups in the 7-position also fluoresced, but to a lesser extent than the 7-amino derivs. The fluorescence of some aminobenzoxadiazoles and related compds. is given.
    80. 80
      Watanabe, Y.; Imai, K. High-Performance Liquid Chromatography and Sensitive Detection of Amino Acids Derivatized with 7-Fluoro-4-Nitrobenzo-2-Oxa-1,3-Diazole. Anal. Biochem. 1981, 116 (2), 471– 2,  DOI: 10.1016/0003-2697(81)90390-0
      80
      High-performance liquid chromatography and sensitive detection of amino acids derivatized with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole
      Watanabe, Yoshihiko; Imai, Kazuhiro
      Analytical Biochemistry (1981), 116 (2), 471-2CODEN: ANBCA2; ISSN:0003-2697.
      7-Fluoro-4-nitrobenzo-2-oxa-1,3-diazole is used as a precolumn fluorescent labeling reagent for high-performance liq. chromatog. of amino acids, including proline and hydroxyproline. The reaction is run at pH 8.0 at 60° for 5 min. The fluorophors (aspartate, glutamate, hydroxyproline, serine, glycine, threonine, alanine, proline) are sepd. on a reversed-phase column (μBondapak C18) with 0.1M phosphate buffer (pH 6.0) contg. 6.75% MeOH and 1.8% THF, and are detected at the level of 10 fmol with excitation at 470 nm and emission at 530 nm.
    81. 81
      Aly, H.; El-Shafie, A. S.; El-Azazy, M. Utilization of 7-Chloro-4-Nitrobenzo-2-Oxa-1,3-Diazole (NBD-Cl) for Spectrochemical Determination of L-Ornithine: A Multivariate Optimization-Assisted Approach. RSC Adv. 2019, 9 (38), 22106– 15,  DOI: 10.1039/C9RA03311D
      81
      Utilization of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) for spectrochemical determination of L-ornithine: a multivariate optimization-assisted approach
      Aly, Hend; El-Shafie, Ahmed S.; El-Azazy, Marwa
      RSC Advances (2019), 9 (38), 22106-22115CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      A simple and highly sensitive univariate calibration strategy based on UV-visible (UV-Vis) absorption spectroscopy and assisted by multivariate screening and optimization was utilized for the detn. of L-ornithine (L-ORN) as such and in the alimentary supplements. L-ORN, an OTC marketed amino acid, is widely used for bodybuilding and might be abused by athletes. A nucleophilic substitution reaction using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) was the basis of the current investigation. Plackett-Burman design (PBD) and a response surface optimizer as screening and fine-tuning strategies, resp., were instigated. Four numerical variables, reaction time (RT), temp. (Temp), pH and reagent vol. (RV), and one categorical variable, the dilg. solvent (DS), were considered. Absorbance of the yellow-colored adduct at 469 nm was the response studied. Pareto anal., along with anal. of variance (ANOVA) were used to ascertain the significant variables (screening phase) and their domains (optimization phase). Response transformation and stepwise anal. were employed when necessary. Probability, cube and individual value plots were used to get an insight into the statistical impact of the variables tested. Multiple responses' optimization was performed using Derringer's function. Calibration curves were linear in the range of 5-50 μg mL-1. Job's technique of continuous variation showed that the stoichiometric ratio is 2 : 1 (NBD-Cl : L-ORN). The proposed technique was successfully applied to the dietary supplements of L-ORN, inferring no interference from adjuvants and excipients. Anal. performance of this technique was validated conforming to the ICH stds.
    82. 82
      Walash, M. I.; Metwally, M. E. S.; Eid, M.; El-Shaheny, R. N. Validated Spectrophotometric Methods for Determination of Alendronate Sodium in Tablets through Nucleophilic Aromatic Substitution Reactions. Chem. Cent. J. 2012, 6, 25,  DOI: 10.1186/1752-153X-6-25
      82
      Validated spectrophotometric methods for determination of Alendronate sodium in tablets through nucleophilic aromatic substitution reactions
      Walash, Mohamed I.; Metwally, Mohamed E.-S.; Eid, Manal; El-Shaheny, Rania N.
      Chemistry Central Journal (2012), 6 (), 25CODEN: CCJHA2; ISSN:1752-153X. (Chemistry Central Ltd.)
      Background: Alendronate (ALD) is a member of the bisphosphonate family which is used for the treatment of osteoporosis, bone metastasis, Paget's disease, hypocalcemia assocd. with malignancy and other conditions that feature bone fragility. ALD is a non-chromophoric compd. so its detn. by conventional spectrophotometric methods is not possible. So two derivatization reactions were proposed for detn. of ALD through the reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and 2,4-dinitrofluorobenzene (DNFB) as chromogenic derivatizing reagents. Results: Three simple and sensitive spectrophotometric methods are described for the detn. of ALD. Method I is based on the reaction of ALD with NBD-Cl. Method II involved heat-catalyzed derivatization of ALD with DNFB, while, Method III is based on micellar-catalyzed reaction of the studied drug with DNFB at room temp. The reactions products were measured at 472, 378 and 374 nm, for Methods I, II and III, resp. Beer's law was obeyed over the concn. ranges of 1.0-20.0, 4.0-40.0 and 1.5-30.0 μg/mL with lower limits of detection of 0.09, 1.06 and 0.06 μg/mL for Methods I, II and III, resp. The proposed methods were applied for quantitation of the studied drug in its pure form with mean percentage recoveries of 100.47 ± 1.12, 100.17 ± 1.21 and 99.23 ± 1.26 for Methods I, II and III, resp. Moreover the proposed methods were successfully applied for detn. of ALD in different tablets. Proposals of the reactions pathways have been postulated. Conclusion: The proposed spectrophotometric methods provided sensitive, specific and inexpensive anal. procedures for detn. of the non-chromophoric drug alendronate either per se or in its tablet dosage forms without interference from common excipients.
    83. 83
      Benson, S.; Fernandez, A.; Barth, N. D.; de Moliner, F.; Horrocks, M. H.; Herrington, C. S.; Abad, J. L.; Delgado, A.; Kelly, L.; Chang, Z. SCOTfluors: Small, Conjugatable, Orthogonal, and Tunable Fluorophores for in Vivo Imaging of Cell Metabolism. Angew. Chem., Int. Ed. 2019, 58 (21), 6911– 5,  DOI: 10.1002/anie.201900465
      83
      SCOTfluors: Small, Conjugatable, Orthogonal, and Tunable Fluorophores for In Vivo Imaging of Cell Metabolism
      Benson, Sam; Fernandez, Antonio; Barth, Nicole D.; de Moliner, Fabio; Horrocks, Mathew H.; Herrington, C. Simon; Abad, Jose Luis; Delgado, Antonio; Kelly, Lisa; Chang, Ziyuan; Feng, Yi; Nishiura, Miyako; Hori, Yuichiro; Kikuchi, Kazuya; Vendrell, Marc
      Angewandte Chemie, International Edition (2019), 58 (21), 6911-6915CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The transport and trafficking of metabolites are crit. for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chem. structures that allow direct monitoring of small metabolites under physiol. conditions with high spatial and temporal resoln. Herein, the authors describe SCOTfluors as novel small-sized multicolored fluorophores for real-time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin.
    84. 84
      Satake, K.; Okuyama, T.; Ohashi, M.; Shinoda, T. The Spectrophotometric Determination of Amine, Amino Acid and Peptide with 2,4,6-Trinitrobenzene 1-Sulfonic Acid. J. Biochem. 1960, 47 (5), 654– 60,  DOI: 10.1093/oxfordjournals.jbchem.a127107
      84
      The spectrophotometric determination of amines, amino acids, and peptides with 2,4,6-trinitrobenzene-1-sulfonic acid
      Satake, Kazuo; Okuyama, Tsuneo; Ohashi, Mochihiko; Shinoda, Tomotaka
      Journal of Biochemistry (1960), 47 (), 654-60CODEN: JOBIAO; ISSN:0021-924X.
      cf. CA 54, 24470i. 2,4,6-Trinitrobenzene-1-sulfonic acid is shown to react specifically with primary amines and .sbd.NH2 and .dbd.NH groups of amino acids and peptides. The av. extinction coeff. of 7 trinitrophenylated (TNP) amines and 20 TNP amino acids (in the vicinity of absorption peak (340 mμ is chosen for convenience) is detd. as 1.27 X 104 and that of 7 TNP peptides as 1.05 X 104 at N concn.
    85. 85
      Means, G. E.; Congdon, W. I.; Bender, M. L. Reactions of 2,4,6-Trinitrobenzenesulfonate Ion with Amines and Hydroxide Ion. Biochemistry 1972, 11 (19), 3564– 71,  DOI: 10.1021/bi00769a011
      85
      Reactions of 2,4,6-trinitrobenzenesulfonate ion with amines and hydroxide ion
      Means, Gary E.; Congdon, W. I.; Bender, M. L.
      Biochemistry (1972), 11 (19), 3564-71CODEN: BICHAW; ISSN:0006-2960.
      The reactivity of 2,4,6-trinitrobenzenesulfonate ion (TNBS) with hydroxide ion and a series of primary amines was examd. as a function of pH, nucleophile concn., ionic strength, and temp. Its reaction with hydroxide ion is first order in both hydroxide ion and TNBS with a second-order rate const. of 6.33 ± 0.16 × 10-2 M-1 sec-1 at 25° (μ = 1.0), and appears to follow the usual path for nucleophilic aromatic substitution involving the rate-limiting formation of a tetrasubstituted ring carbon intermediate. The reaction of TNBS with primary amines having pKa values higher than ∼8.7 proceeds by a similar mechanism. Reactivity increases as a function of amine basicity according to the relation log k (M-1 sec-1) = 0.23 pKa - 1.18. Similar amines with lower pKa values follow a complex rate law apparently due to partitioning of the tetra-substituted intermediate between breakdown to products and reversion to reactants. The reaction of TNBS with amines is favored as compared to its reaction with hydroxide ion by low ionic strength, low temp., and pH values at least 0.5 unit below the amine pKa value. Complexation of N-alkylpicramides by sulfite ion involves its addn. to ring carbon number three. The second-order rate const. for complexation of sulfite with N-trinitrophenyl-β-alanine is 5.4 × 103 M-1 sec-1 at pH 8.0 (μ = 0.50) and 25.4°. Reactions of TNBS with proteins are discussed based on these results.
    86. 86
      Snyder, S. L.; Sobocinski, P. Z. An Improved 2,4,6-Trinitrobenzenesulfonic Acid Method for the Determination of Amines. Anal. Biochem. 1975, 64 (1), 284– 8,  DOI: 10.1016/0003-2697(75)90431-5
      86
      Improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines
      Snyder, Stephen L.; Sobocinski, Philip Z.
      Analytical Biochemistry (1975), 64 (1), 284-8CODEN: ANBCA2; ISSN:0003-2697.
      A convenient and sensitive procedure was described for the detn. of amines, amino acids, and proteins by use of 2,4,6-trinitrobenzenesulfonic acid (TNBS). In this method, the amino content could be related directly to the absorbance of the trinitrophenylation reaction mixt. after 15-30 min incubation; therefore, it was unnecessary to quench the reaction. The method was ∼2-fold more sensitive than the Lowry protein detn. method and ∼6-fold more sensitive than the ninhydrin method for amino acids.
    87. 87
      Qi, X.-Y.; Keyhani, N. O.; Lee, Y. C. Spectrophotometric Determination of Hydrazine, Hydrazides, and Their Mixtures with Trinitrobenzenesulfonic Acid. Anal. Biochem. 1988, 175 (1), 139– 44,  DOI: 10.1016/0003-2697(88)90371-5
      87
      Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid
      Qi, Xiao Yang; Keyhani, Nemat O.; Lee, Yuan Chuan
      Analytical Biochemistry (1988), 175 (1), 139-44CODEN: ANBCA2; ISSN:0003-2697.
      A spectrophotometric method has been developed to measure hydrazine, hydrazides, and their mixts. using a modification of the trinitrobenzenesulfonic acid method (T. Okuyama and K. Satake 1960). After incubation of the sample contg. hydrazine and hydrazide with trinitrobenzenesulfonate at pH 8.5 at room temp. for 40 min, the reaction mixt. was dild. with a Na2CO3-NaHCO3 buffer (0.1M, pH 10.8) rather than with 0.5M HCl. Different chromogens were produced from the reaction of hydrazine (λmax = 570 nm) and hydrazides (λmax = 385 and 500 nm) with trinitrobenzenesulfonic acid. The method allowed simultaneous detn. of hydrazine (5 to 60 nmol) with hydrazide (10 to 120 nmol) in a mixt. with a std. deviation of less than 5%. The presence of amino compds. (except for amino sugars) did not interfere with the measurement of hydrazine or hydrazides. Interference by amino sugars in the detn. of hydrazine or hydrazides was eliminated by pretreatment of the sample with NaBH4 to reduce the amino sugars to 2-amino-2-deoxyhexitols.
    88. 88
      Morçöl, T.; Subramanian, A.; Velander, W. H. Dot-Blot Analysis of the Degree of Covalent Modification of Proteins and Antibodies at Amino Groups. J. Immunol. Methods 1997, 203 (1), 45– 53,  DOI: 10.1016/S0022-1759(97)00013-6
      88
      Dot-blot analysis of the degree of covalent modification of proteins and antibodies at amino groups
      Morcol T; Subramanian A; Velander W H
      Journal of immunological methods (1997), 203 (1), 45-53 ISSN:0022-1759.
      The present study describes a rapid and sensitive dot-blot assay approach for determining the degree of covalent modification of amino groups in proteins. N-hydroxy-succinimide ester of acetic acid was used for irreversible, covalent modification of proteins whose reactive primary amino groups were reversibly blocked (or protected) with 2,3-dimethyl-maleic anhydride prior to processing. Immobilon AV affinity membrane was utilized for differential covalent attachment of the proteins to the activated ester on the membrane matrix, primarily through their protected epsilon-amino group of lysins. The efficacy of the method was demonstrated for a murine monoclonal antibody and for two human plasma proteins. The degree of covalent modification of proteins at their amino groups as estimated by the proposed method is compared with that obtained by using the conventional trinitrobenzene sulfonic acid (TNBS) method. Several advantages of the present method over the TNBS method are emphasized. The new method, which requires only nanograms of protein, is shown to be more sensitive than the TNBS method where the limit of detection is in the milligram range. The proposed assay is very specific and facile, and the advantage of small sample size requirement (1 microliter) provides sequential detection of multiple samples facilitating much higher precision in data obtained than that of the TNBS assay.
    89. 89
      Oliverio, R.; Liberelle, B.; Murschel, F.; Garcia-Ac, A.; Banquy, X.; De Crescenzo, G. Versatile and High-Throughput Strategy for the Quantification of Proteins Bound to Nanoparticles. ACS Appl. Nano Mater. 2020, 3 (10), 10497– 507,  DOI: 10.1021/acsanm.0c02414
      89
      Versatile and High-Throughput Strategy for the Quantification of Proteins Bound to Nanoparticles
      Oliverio, Romane; Liberelle, Benoit; Murschel, Frederic; Garcia-Ac, Araceli; Banquy, Xavier; De Crescenzo, Gregory
      ACS Applied Nano Materials (2020), 3 (10), 10497-10507CODEN: AANMF6; ISSN:2574-0970. (American Chemical Society)
      Biofunctionalized nanomaterials have been extensively studied as a tool for a wide range of applications in biomedical fields. Despite many existing strategies to conjugate proteins to colloidal particles, detg. the grafting efficiency-i.e., the amt. of protein conjugated to the surface of a nanoparticle (NP)-remains challenging. Formulations for biomedical applications are subjected to strict constraints, and a lack of precise characterization can prevent otherwise promising formulations to be explored further. Here, we propose a simple approach to precisely measure the grafting efficiency of biol. mols. on the surface of three types of widely used NPs: polymeric NPs, inorg. NPs, and metallic NPs. This approach relies on the simultaneous hydrolysis of the grafted protein and the NP degrdn. in acidic conditions, followed by a spectrophotometric quantification of primary amines in soln. This strategy can be applied to any type of protein and does not require any labeling agent. It can be performed in a high-throughput manner as a routine expt. and only requires a conventional oven and a microplate reader.
    90. 90
      Kim, D.; Cho, S. W.; Jun, Y. W.; Ahn, K. H. Fluorescent Labeling of Lysine Residues in Protein Using 8-Thiomethyl-BODIPY. Bull. Korean Chem. Soc. 2017, 38 (9), 995– 6,  DOI: 10.1002/bkcs.11213
      90
      Fluorescent Labeling of Lysine Residues in Protein using 8-thiomethyl-BODIPY
      Kim, Dokyoung; Cho, Seo Won; Jun, Yong Woong; Ahn, Kyo Han
      Bulletin of the Korean Chemical Society (2017), 38 (9), 995-996CODEN: BKCSDE; ISSN:0253-2964. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We have disclosed that 8-thiomethyl-BODIPY is promising for labeling of proteins with lysineresidues. The substitution reaction of the thiomethyl moietywith amine groups, here the lysine residue, proceeds fast(within 2 h) under mild conditions (37C) and the resultingproduct shows bright blue fluorescence from the amino-BODIPY moiety (exi/emi = 375/409 nm). The labeled lysozyme retained its activity (96 2.6%), indicative of little structural influence by the compact label.
    91. 91
      Bell, P. J. L.; Karuso, P. Epicocconone, a Novel Fluorescent Compound from the Fungus Epicoccum Nigrum. J. Am. Chem. Soc. 2003, 125 (31), 9304– 5,  DOI: 10.1021/ja035496+
      91
      Epicocconone, a novel fluorescent compound from the fungus Epicoccum nigrum
      Bell, Phillip J. L.; Karuso, Peter
      Journal of the American Chemical Society (2003), 125 (31), 9304-9305CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Epicocconone represents a new class of natural fluorescent probes based on a polyketide skeleton isolated from the fungus Epicoccum nigrum. Epicocconone is a small, cell permeable natural product with a high molar absorptivity and a long Stokes' shift that will be useful in biotechnol. applications.
    92. 92
      Coghlan, D. R.; Mackintosh, J. A.; Karuso, P. Mechanism of Reversible Fluorescent Staining of Protein with Epicocconone. Org. Lett. 2005, 7 (12), 2401– 4,  DOI: 10.1021/ol050665b
      92
      Mechanism of Reversible Fluorescent Staining of Protein with Epicocconone
      Coghlan, Daniel R.; Mackintosh, James A.; Karuso, Peter
      Organic Letters (2005), 7 (12), 2401-2404CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      Epicocconone is the active ingredient in Deep Purple Total Protein Stain and responsible for the apparent noncovalent staining of proteins in polyacrylamide gel and electroblots. Reaction of epicocconone with amines has shown that epicocconone reacts reversibly with primary amines to produce a highly fluorescent enamine that is readily hydrolyzed by base or strong acid such as in conditions used in post-electrophoretic anal. such as peptide mass fingerprinting or Edman degrdn.
    93. 93
      Peixoto, P. A.; Boulangé, A.; Ball, M.; Naudin, B.; Alle, T.; Cosette, P.; Karuso, P.; Franck, X. Design and Synthesis of Epicocconone Analogues with Improved Fluorescence Properties. J. Am. Chem. Soc. 2014, 136 (43), 15248– 56,  DOI: 10.1021/ja506914p
      93
      Design and Synthesis of Epicocconone Analogues with Improved Fluorescence Properties
      Peixoto, Philippe A.; Boulange, Agathe; Ball, Malcolm; Naudin, Bertrand; Alle, Thibault; Cosette, Pascal; Karuso, Peter; Franck, Xavier
      Journal of the American Chemical Society (2014), 136 (43), 15248-15256CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Epicocconone is a natural latent fluorophore that is widely used in biotechnol. because of its large Stokes shift and lack of fluorescence in its unconjugated state. However, the low photostability and quantum yields of epicocconone have limited its wider use, and in the absence of a total synthesis, this limitation was a long-standing problem. Here the authors report a general strategy for the synthesis of epicocconone analogs that relies on a 2-iodoxybenzoic acid-mediated dearomatization and on the replacement of the triene tail of the natural product by an arom. ring. This design element is general and the synthesis is straightforward, providing ready access to libraries of polyfunctional fluorophores with long Stokes shifts based on the epicocconone core. The structural modifications resulted in analogs with increased photostability and quantum yields compared with the natural product. Staining proteomic gels with these new analogs showed significant lowering of the detection limit and a 30% increase in the no. of low-abundance proteins detected. These epiccoconone analogs will substantially improve the discovery rate of biomarker needles in the proteomic haystack.
    94. 94
      Chatterjee, S.; Karuso, P.; Boulangé, A.; Franck, X.; Datta, A. Excited State Dynamics of Brightly Fluorescent Second Generation Epicocconone Analogues. J. Phys. Chem. B 2015, 119 (20), 6295– 303,  DOI: 10.1021/acs.jpcb.5b02190
      94
      Excited State Dynamics of Brightly Fluorescent Second Generation Epicocconone Analogues
      Chatterjee, Soumit; Karuso, Peter; Boulange, Agathe; Franck, Xavier; Datta, Anindya
      Journal of Physical Chemistry B (2015), 119 (20), 6295-6303CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      The natural product epicocconone, owing to its unique fluorescence properties, was developed into a range of products used in biotechnol., esp. proteomics. However, its weak green fluorescence in its native state, while advantageous for proteomics applications, is a disadvantage in other applications that require two-color readouts. Here we report the photophys. characterization of two brightly fluorescent analogs of epicocconone. These analogs, with naphthyl or pyridyl groups replacing the heptatriene chain, resulted in bright fluorescence in both the native state and the long Stokes shifted enamine. Time-resolved fluorescence studies and DFT calcns. were carried out to understand the excited state processes involved in fluorescence. Results showed the p-chloro group on the pyridyl is responsible for the high fluorescence of the native fluorophore. The application of one of these compds. for staining electrophoresis gels is exemplified.
    95. 95
      Castell, J. V.; Cervera, M.; Marco, R. A Convenient Micromethod for the Assay of Primary Amines and Proteins with Fluorescamine. A Reexamination of the Conditions of Reaction. Anal. Biochem. 1979, 99 (2), 379– 91,  DOI: 10.1016/S0003-2697(79)80022-6
      95
      A convenient micromethod for the assay of primary amines and proteins with fluorescamine. A reexamination of the conditions of reaction
      Castell, Jose V.; Cervera, Margarita; Marco, Roberto
      Analytical Biochemistry (1979), 99 (2), 379-91CODEN: ANBCA2; ISSN:0003-2697.
      Fluorescamine, a reagent capable of reacting with a wide variety of nucleophiles, only gives fluorescent products when reacted with primary amines. The results of reexamn. of the principal parameters affecting the reaction are presented with emphasis on practical aspects. In particular, the potential competition of other nucleophiles (secondary amines and thiols) and the effect of pH on the reaction and on the fluorescence of the product are presented. Polyalcs., due to their reversible interaction with fluorescamine, affect the velocity without interfering with its extent. The initial hydrolysis product of fluorescamine under mild alk. conditions has been isolated and characterized. Although unreactive with most amines, it is shown that at pH <7 it will react with amines that show abnormal low pH-dependent reactivity with fluorescamine. Histidine is the only amino acid that under these conditions will react giving a fluorescent product. Among other amino group-contg. mols. tested, only polyamines, histamine, and N-amino terminal-free peptides have been found to give this type of reaction. The factors affecting the limit of sensitivity of the reaction also were studied. The very low, but significant, fluorescence of the blanks sets the actual limit of sensitivity of the reaction. A micromethod that reduces the amt. of fluorescamine in the assay under conditions in which the reaction is quant. is described. A similar micromethod can be used with the o-phthalaldehyde reaction. The particular application of fluorescamine in the assay of proteins is compared to other methods. Alk. and acid hydrolysis are shown to decrease the variability of the reaction shown by different proteins, while increasing the sensitivity. The different practical alternatives in the assay of proteins with fluorescamine and o-phthalaldehyde are discussed.
    96. 96
      Funk, G. M.; Hunt, C. E.; Epps, D. E.; Brown, P. K. Use of a Rapid and Highly Sensitive Fluorescamine-Based Procedure for the Assay of Plasma Lipoproteins. J. Lipid Res. 1986, 27, 792,  DOI: 10.1016/S0022-2275(20)38803-9
      96
      Use of a rapid and highly sensitive fluorescamine-based procedure for the assay of plasma lipoproteins
      Funk, G. Michael; Hunt, Charles E.; Epps, Dennis E.; Brown, Patricia K.
      Journal of Lipid Research (1986), 27 (7), 792-5CODEN: JLPRAW; ISSN:0022-2275.
      A rapid and sensitive method for detg. protein concs. using fluorescamine was characterized for use in the anal. of intact lipoproteins.. Fluorescence was measured with excitation at 390 nm and emission at 470 nm. There was no interference with the assay due to the presence of lipid-assocd. turbidity or primary amine content. The assay was sensitive to as little as 0.3 μg lipoprotein and yielded similar results when compared to the Lowry method.
    97. 97
      Udenfriend, S.; Stein, S.; Böhlen, P.; Dairman, W.; Leimgruber, W.; Weigele, M. Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range. Science 1972, 178 (4063), 871,  DOI: 10.1126/science.178.4063.871
      97
      Fluorescamine. Reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range
      Udenfriend, Sidney; Stein, Stanley; Boehlen, Peter; Dairman, Wallace; Leimgruber, Willy; Weigele, Manfred
      Science (Washington, DC, United States) (1972), 178 (4063), 871-2CODEN: SCIEAS; ISSN:0036-8075.
      Fluorescamine is a new reagent for the detection of primary amines in the picomole range. Its reaction with amines is almost instantaneous at room temp. in aq. media. The products are highly fluorescent, whereas the reagent and its degrdn. products are nonfluorescent. Applications are discussed.
    98. 98
      Murugayah, S. A.; Warring, S. L.; Gerth, M. L. Optimisation of a High-Throughput Fluorescamine Assay for Detection of N-Acyl-L-Homoserine Lactone Acylase Activity. Anal. Biochem. 2019, 566, 10– 2,  DOI: 10.1016/j.ab.2018.10.029
      98
      Optimisation of a high-throughput fluorescamine assay for detection of N-acyl-L-homoserine lactone acylase activity
      Murugayah, Shereen A.; Warring, Suzanne L.; Gerth, Monica L.
      Analytical Biochemistry (2019), 566 (), 10-12CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      N-acyl-L-homoserine lactone (AHL) acylases are a well-known group of enzymes that disrupt quorum sensing in Gram-neg. bacteria by degrading AHL signalling mols. This degrdn. of signalling mols. (termed 'quorum quenching') has potential uses in the prevention or redn. of biofilm formation and/or bacterial infections. Therefore, there is a great deal of interest in the identification and characterization of quorum quenching enzymes. Here, we present an optimized fluorescamine-based assay for the detection of AHL acylase activity and demonstrate it can be used in a high-throughput screening format.
    99. 99
      Ashby, J.; Duan, Y.; Ligans, E.; Tamsi, M.; Zhong, W. High-Throughput Profiling of Nanoparticle-Protein Interactions by Fluorescamine Labeling. Anal. Chem. 2015, 87 (4), 2213– 9,  DOI: 10.1021/ac5036814
      99
      High-Throughput Profiling of Nanoparticle-Protein Interactions by Fluorescamine Labeling
      Ashby, Jonathan; Duan, Yaokai; Ligans, Erik; Tamsi, Michael; Zhong, Wenwan
      Analytical Chemistry (Washington, DC, United States) (2015), 87 (4), 2213-2219CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A rapid, high throughput fluorescence assay was designed to screen interactions between proteins and nanoparticles. The assay employs fluorescamine, a primary-amine specific fluorogenic dye, to label proteins. Because fluorescamine could specifically target the surface amines on proteins, a conformational change of the protein upon interaction with nanoparticles will result in a change in fluorescence. In the present study, the assay was applied to test the interactions between a selection of proteins and nanoparticles made of polystyrene, silica, or iron oxide. The particles were also different in their hydrodynamic diam., synthesis procedure, or surface modification. Significant labeling differences were detected when the same protein incubated with different particles. Principal component anal. (PCA) on the collected fluorescence profiles revealed clear grouping effects of the particles based on their properties. The results prove that fluorescamine labeling is capable of detecting protein-nanoparticle interactions, and the resulting fluorescence profile is sensitive to differences in nanoparticle's phys. properties. The assay can be carried out in a high-throughput manner, and is rapid with low operation cost. Thus, it is well suited for evaluating interactions between a larger no. of proteins and nanoparticles. Such assessment can help to improve our understanding on the mol. basis that governs the biol. behaviors of nanomaterials. It will also be useful for initial examn. of the bioactivity and reproducibility of nanomaterials employed in biomedical fields.
    100. 100
      Elbashir, A. A.; Ahmed, A. A.; Ali Ahmed, S. M.; Aboul-Enein, H. Y. 1,2-Naphthoquinone-4-Sulphonic Acid Sodium Salt (NQS) as an Analytical Reagent for the Determination of Pharmaceutical Amine by Spectrophotometry. Appl. Spectrosc. Rev. 2012, 47 (3), 219– 32,  DOI: 10.1080/05704928.2011.639107
      100
      1,2-Naphthoquinone-4-Sulphonic Acid Sodium Salt (NQS) as an Analytical Reagent for the Determination of Pharmaceutical Amine by Spectrophotometry
      Elbashir, Abdalla Ahmed; Ahmed, Abir Abdalla; Ali Ahmed, Shazalia M.; Aboul-Enein, Hassan Y.
      Applied Spectroscopy Reviews (2012), 47 (3), 219-232CODEN: APSRBB; ISSN:0570-4928. (Taylor & Francis, Inc.)
      A review. Several papers have been presented in recent years regarding the field of application of 1,2-naphthoquinone-4-sulfonic acid sodium salt (NQS) as a chromogenic reagent for the detn. of pharmaceutical amines using spectrophotometry. In this review article, various spectrophotometric methods using NQS as a labeling reagent for detn. of pharmaceutical amines are presented. The application of these methods for the detn. of drugs in pharmaceutical formulations and real samples is discussed.
    101. 101
      Rosenblatt, D. H.; Hlinka, P.; Epstein, J. Use of 1,2-Naphthoquinone-4-Sulfonate for Estimation of Ethylenimine and Primary Amines. Anal. Chem. 1955, 27 (8), 1290– 3,  DOI: 10.1021/ac60104a024
      101
      Use of 1,2-naphthoquinone-4-sulfonate for the estimation of ethylenimine and primary amines
      Rosenblatt, David H.; Hlinka, Peter; Epstein, Joseph
      (1955), 27 (), 1290-3CODEN: ANCHAM; ISSN:0003-2700.
      Dil. aq. solns. of ethylenimine and butyl amine react with K 1,2-naphthoquinone-4-sulfonate (I) at a pH of 10.3 to give reddish dyes which can be extd. with CHCl3. The amines can be estd. by measuring the absorbance at 420 and 450 mμ, resp. Ethanolamine also gives a reddish dye, but it can not be extd. with CHCl3. It can, however, be extd. with isoamyl alc. Ethylenimine, butylamine, ethanolamine, diethylamine, N-butylethanolamine, diethanolamine, NH4OH, sulfanilamide, and aniline react with I to give dyes which can be distinguished qualitatively by their colors and solubilities in various org. solvents.
    102. 102
      Darwish, I. A.; Abdine, H. H.; Amer, S. M.; Al-Rayes, L. I. Simple Spectrophotometric Method for Determination of Paroxetine in Tablets Using 1,2-Naphthoquinone-4-Sulphonate as a Chromogenic Reagent. Int. J. Anal. Chem. 2009, 2009, 237601,  DOI: 10.1155/2009/237601
      102
      Simple spectrophotometric method for determination of paroxetine in tablets using 1,2-naphthoquinone-4-sulphonate as a chromogenic reagent
      Darwish Ibrahim A; Abdine Heba H; Amer Sawsan M; Al-Rayes Lama I
      International journal of analytical chemistry (2009), 2009 (), 237601 ISSN:.
      Simple and rapid spectrophotometric method has been developed and validated for the determination of paroxetine (PRX) in tablets. The proposed method was based on nucleophilic substitution reaction of PRX with 1,2-naphthoquinone-4-sulphonate (NQS) in an alkaline medium to form an orange-colored product of maximum absorption peak (lambda(max)) at 488 nm. The stoichiometry and kinetics of the reaction were studied, and the reaction mechanism was postulated. Under the optimized reaction conditions, Beer's law correlating the absorbance (A) with PRX concentration (C) was obeyed in the range of 1-8 mug mL(-1). The regression equation for the calibration data was: A = 0.0031 + 0.1609 C, with good correlation coefficients (0.9992). The molar absorptivity (epsilon) was 5.9 x 10(5) L mol(-1) 1 cm(-1). The limits of detection and quantitation were 0.3 and 0.8 mug mL(-1), respectively. The precision of the method was satisfactory; the values of relative standard deviations did not exceed 2%. The proposed method was successfully applied to the determination of PRX in its pharmaceutical tablets with good accuracy and precisions; the label claim percentage was 97.17 +/- 1.06 %. The results obtained by the proposed method were comparable with those obtained by the official method.
    103. 103
      Longstreet, A. R.; Jo, M.; Chandler, R. R.; Hanson, K.; Zhan, N.; Hrudka, J. J.; Mattoussi, H.; Shatruk, M.; McQuade, D. T. Ylidenemalononitrile Enamines as Fluorescent “Turn-On” Indicators for Primary Amines. J. Am. Chem. Soc. 2014, 136 (44), 15493– 6,  DOI: 10.1021/ja509058u
      103
      Ylidenemalononitrile Enamines as Fluorescent "Turn-On" Indicators for Primary Amines
      Longstreet, Ashley R.; Jo, Minyoung; Chandler, Rebecca R.; Hanson, Kenneth; Zhan, Naiqian; Hrudka, Jeremy J.; Mattoussi, Hedi; Shatruk, Michael; McQuade, D. Tyler
      Journal of the American Chemical Society (2014), 136 (44), 15493-15496CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Ylidenemalononitrile enamines undergo rapid amine exchange followed by a cyclization with primary amines to yield fluorescent products with emission intensities ≤900 times greater than the starting materials. After identifying the fluorescent species by x-ray crystallog., the rate of amine exchange is substrate dependent and by simple structural variation the fluorescence can be tuned over the entire visible spectrum. The authors further demonstrate their potential application in biomol. labeling.
    104. 104
      Sathiskumar, U.; Easwaramoorthi, S. Red-Emitting Ratiometric Fluorescence Chemodosimeter for the Discriminative Detection of Aromatic and Aliphatic Amines. ChemistrySelect 2019, 4 (25), 7486– 94,  DOI: 10.1002/slct.201901254
      104
      Red-Emitting Ratiometric Fluorescence Chemodosimeter for the Discriminative Detection of Aromatic and Aliphatic Amines
      Sathiskumar, Udayadasan; Easwaramoorthi, Shanmugam
      ChemistrySelect (2019), 4 (25), 7486-7494CODEN: CHEMUD; ISSN:2365-6549. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A NIR-emitting fluorescent probe using phenothiazine and 1,3-dimethylbarbituric acid (PMB) connected through methylene bridge was developed in a two-step simple chem. reaction. The fluorescent probes were highly reactive towards various primary amines and the reaction was accompanied by the changes in the optical and fluorescent properties of the PMB characteristic to the amine and thereby can have the potential to be used as a sensor for amines. In addn. to the ratiometric changes in the fluorescence intensity corresponding to the PMB before and after reaction with an amine, the emission spectral max. is specific to the analyte. For, example 1,4-diaminobenzene, 4-methoxyaniline, aniline, hydrazine, ethylamine, ethylenediamine resp. shows the max. at 532, 546, 555, 484, 497, and 488 nm and this unique feature originated due to the involvement of the analyte in the extended π-conjugation with the probe. Sensor strips prepd. using PMB coated filter paper were able to sense the volatile amine gases with shorter response time. Anal. of the strips using the CIELAB 1976 color coordinates provides different signals for hydrazine, aniline, propylamine, and ethylamine gases. The applicability of the sensor was further ascertained by monitoring the decompn. of skin used for leather processing and also proteins engineered with an amino-functionalized unnatural amino acid.
    105. 105
      Turiák, G.; Volicer, L. Stability of o-Phthalaldehyde─Sulfite Derivatives of Amino Acids and Their Methyl Esters: Electrochemical and Chromatographic Properties. J. Chromatogr. A 1994, 668 (2), 323– 9,  DOI: 10.1016/0021-9673(94)80121-5
      105
      Stability of o-phthalaldehyde-sulfite derivatives of amino acids and their methyl esters: electrochemical and chromatographic properties
      Turiak, Gyorgy; Volicer, Ladislav
      Journal of Chromatography A (1994), 668 (2), 323-9CODEN: JCRAEY; ISSN:0021-9673.
      RP-HPLC coupled with 16-channel coulometric electrode array detection was used to monitor the decompn. of five amino acid o-phthalaldehyde (OPA)-sulfite derivs. (Ala, Arg, Glu, Ser, Tyr) and their Me ester derivs. as well. At fixed OPA and sulfite concns., inclusion of methanol and EDTA in the derivatization media has increased most effectively the room temp. stability of both derivs. measured at pH 9.2 (amino acids) and pH 8.2 (Me esters). Decreases in product concns. by 6% occurred after >15 h for amino acid derivs. and 8 h for Me ester derivs. The oxidn. potential max. for OPA-sulfite derivs. of amino acids were found at 600 mV while the same Me ester derivs. had 60-120 mV higher max. with the exception of tyrosine. The detector responses are linear in the studied 0.1-10 μM concn. range for both deriv. forms and their detection limit was 100-200 fmol injected on the column. The RP-HPLC retention of amino acid Me ester OPA-sulfite derivs. was very similar to the amino acid OPA-2-mercaptoethanol ones while the more polar amino acid OPA-sulfite derivs. were eluted earlier (k'<1) under the same chromatog. conditions.
    106. 106
      De Montigny, P.; Stobaugh, J. F.; Givens, R. S.; Carlson, R. G.; Srinivasachar, K.; Sternson, L. A.; Higuchi, T. Naphthalene-2,3-Dicarboxyaldehyde/Cyanide Ion: A Rationally Designed Fluorogenic Reagent for Primary Amines. Anal. Chem. 1987, 59 (8), 1096– 101,  DOI: 10.1021/ac00135a007
      106
      Naphthalene-2,3-dicarboxyaldehyde/cyanide ion: a rationally designed fluorogenic reagent for primary amines
      De Montigny, Pierre; Stobaugh, John F.; Givens, Richard S.; Carlson, Robert G.; Srinivasachar, Kasturi; Sternson, Larry A.; Higuchi, Takeru
      Analytical Chemistry (1987), 59 (8), 1096-101CODEN: ANCHAM; ISSN:0003-2700.
      On the basis of the isoindole formation mechanism in the o-phthalaldehyde/2-mercaptoethanol (OPA/2-ME) derivatization of primary amines and the structure-stability relations for isoindoles, an improved fluorogenic reagent, naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide ion (CN-), was developed. Reaction of NDA/CN- with primary amine in aq. media results in the formation of N-substituted 1-cyanobenz[f]isoindole (CBI) derivs. which have significantly improved stability compared to the corresponding OPA/2-ME derivs. (for glycine greater than 50-fold improvement was realized) and have high quantum efficiencies for fluorescence (Φf = 0.54 in 60% aq. MeCN for the CBI-n-propylamine deriv.) in solvent systems commonly used in liq. chromatog. Parameters in the NDA/CN- derivatization of alanine are defined (i.e., pH and the reagent component concns.) and used in the development of a labeling procedure for amino acid mixts. Gradient elution fractionation of 18 CBI-amino acid derivs. was accomplished in 60 min and permitted detection limits of <200 fmol injected (excitation 246 nm) or <3 pmol injected (excitation 420 nm). The utility of the reagent in assaying amino acid mixts. resulting from the enzymic hydrolysis of the peptides Met-enkephalin and glucagon is demonstrated.
    107. 107
      Mroz, E. A.; Roman, R. J.; Lechene, C. Fluorescence Assay for Picomole Quantities of Ammonia. Kidney Int. 1982, 21 (3), 524– 7,  DOI: 10.1038/ki.1982.56
      107
      Fluorescence assay for picomole quantities of ammonia
      Mroz, Edmund A.; Roman, Richard J.; Lechene, Claude
      Kidney International (1982), 21 (), 524-7CODEN: KDYIA5; ISSN:0085-2538.
      Nanomole, picomole, and subpicomole amts. of NH3 were detd. fluorometrically in rat tubular fluid and urine by reacting NH3 with o-phthalaldehyde and thioglycolic acid at pH 7.4 by modifying the method of S. Taylor et al. (1974). To det. nanomole quantities of NH3 (1-3-mL final vol.), glass test tubes were used, whereas for the detn. of picomole (2-10-μL vol.) and subpicomole (1-nL vol.) amts. of NH3, siliconized slides were used and the samples were covered with degassed oil to prevent evapn. The method gave better specificity than the method of S. Taylor et al.
    108. 108
      Carlson, R. G.; Srinivasachar, K.; Givens, R. S.; Matuszewski, B. K. New Derivatizing Agents for Amino Acids and Peptides. 1. Facile Synthesis of N-Substituted 1-Cyanobenz[f]Isoindoles and Their Spectroscopic Properties. J. Org. Chem. 1986, 51 (21), 3978– 83,  DOI: 10.1021/jo00371a013
      108
      New derivatizing agents for amino acids and peptides. 1. Facile synthesis of N-substituted 1-cyanobenz[f]isoindoles and their spectroscopic properties
      Carlson, R. G.; Srinivasachar, K.; Givens, R. S.; Matuszewski, B. K.
      Journal of Organic Chemistry (1986), 51 (21), 3978-83CODEN: JOCEAH; ISSN:0022-3263.
      2,3-Naphthalenedicarboxaldehyde (I, R = CHO) (II) was prepd. as a reagent for the derivatization of primary amines, amino acids, and small peptides as highly fluorescent 2-substituted 1-cyanobenz[f]isoindoles III (R1 = group from amino compd. R1NH2). Thus, o-xylene IV was treated with maleic anhydride in the presence of NaI to give I (R = CO2H), which was reduced with LiAlH4 to give I (R = CH2OH), which was partially oxidized with oxalyl chloride in DMSO/CH2Cl2 to give II. The reaction of II with amino compds. R1NH2 (R1 = Pr, Bu, CMe3, CH2CO2H, DL-CHMeCO2H, CH2CO-Gly-OH, D-CHMeCO-D-Ala-OH) in the presence of NaCN gave the corresponding III. The fluorescent properties of III were detd.
    109. 109
      Bantan-Polak, T.; Kassai, M.; Grant, K. B. A Comparison of Fluorescamine and Naphthalene-2,3-Dicarboxaldehyde Fluorogenic Reagents for Microplate-Based Detection of Amino Acids. Anal. Biochem. 2001, 297 (2), 128– 36,  DOI: 10.1006/abio.2001.5338
      109
      A comparison of fluorescamine and naphthalene-2,3-dicarboxaldehyde fluorogenic reagents for microplate-based detection of amino acids
      Bantan-Polak, Tjasa; Kassai, Miki; Grant, Kathryn B.
      Analytical Biochemistry (2001), 297 (2), 128-136CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)
      The use of appropriate fluorometric derivatization procedures is of considerable importance for accurate detn. of amino acids in biol. samples and in metal-assisted peptide hydrolysis reactions. It is esp. crit. for the relative fluorescence intensities (RFI) of equal amts. of amino acids to be as similar as possible. While fluorescamine and naphthalene-2,3-dicarboxaldehyde (NDA) have proven to be excellent fluorogenic reagents for amino acid detection, the effects of various factors such as org. solvent, buffer, and pH have never been rigorously evaluated with respect to normalizing the relative fluorescence intensities of individual amino acids. To this end, here we describe optimized fluorescamine and NDA derivatization reactions that enhance the accuracy of microplate-based detection of amino acids. For both fluorescamine and NDA, we have shown that the RFI values of 16 of 19 amino acids are greater than 70%. Although detn. of tryptophan is problematic, this difficulty is overcome by the addn. of β-cyclodextrin to the NDA reaction. In principle, the optimized fluorescamine and NDA microplate procedures reported here can be utilized as complementary techniques for the detection of 19 of 20 naturally occurring amino acids. (c) 2001 Academic Press.
    110. 110
      Hapuarachchi, S.; Aspinwall, C. A. Design, Characterization, and Utilization of a Fast Fluorescence Derivatization Reaction Utilizing o-Phthaldialdehyde Coupled with Fluorescent Thiols. Electrophoresis 2007, 28 (7), 1100– 6,  DOI: 10.1002/elps.200600567
      110
      Design, characterization, and utilization of a fast fluorescence derivatization reaction utilizing o-phthaldialdehyde coupled with fluorescent thiols
      Hapuarachchi, Suminda; Aspinwall, Craig A.
      Electrophoresis (2007), 28 (7), 1100-1106CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The authors have developed a chem. derivatization scheme for primary amines that couples the fast kinetic properties of o-phthaldialdehyde (OPA) with the photophys. properties of visible, high quantum yield, fluorescent dyes. In this reaction, OPA is used as a crosslinking reagent in the labeling reaction of primary amines in the presence of a fluorescent thiol, 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino)fluorescein (SAMSA fluorescein), thereby incorporating fluorescein (ε = 78 000 M-1, quantum yield of 0.98) into the isoindole product. Detection is based on excitation and emission of the incorporated fluorescein using the 488 nm laser line of an Ar+ laser rather than the UV-excited isoindole, thereby eliminating the UV light sources for detection. Using this method, the authors have quant. labeled biol. important primary amines in less than 10 s. Detection limits for anal. of glutamate, glycine, GABA, and taurine were less than 2 nM. The authors present the characterization of OPA/SAMSA-F reaction and the potential utility of the derivatization reaction for dynamic chem. monitoring of biol. relevant analytes using CE.
    111. 111
      Zhang, L.-Y.; Liu, Y.-M.; Wang, Z.-L.; Cheng, J.-K. Capillary Zone Electrophoresis with Pre-Column NDA Derivatization and Amperometric Detection for the Analysis of Four Aliphatic Diamines. Anal. Chim. Acta 2004, 508 (2), 141– 5,  DOI: 10.1016/j.aca.2003.11.075
      111
      Capillary zone electrophoresis with pre-column NDA derivatization and amperometric detection for the analysis of four aliphatic diamines
      Zhang, Li-Yao; Liu, Yan-Ming; Wang, Zong-Li; Cheng, Jie-Ke
      Analytica Chimica Acta (2004), 508 (2), 141-145CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
      A method was developed for the anal. of four aliph. diamines by capillary zone electrophoresis using pre-column derivatization with naphthalene-2,3-dicarboxaldehyde (NDA)/CN- and amperometric detection. The pre-column derivatization reaction conditions including the molar ratio of NDA to amines, the cyanide concn., the pH value of derivatization buffer, and the reaction time, were studied. The sepn. of four derivs. of aliph. diamines was optimized by capillary zone electrophoresis (CZE) using end-column amperometric detection with a C fiber microelectrode, at a const. potential of 0.7 V vs. SCE. The optimum conditions for the sepn. were 10 mM Tris-H3PO4 (pH 4.0) for the running buffer soln., 15 kV for the sepn. voltage. The detection limits for diaminopropane, putrescine, cadaverine, diaminohexane were 6.7 × 10-8, 5.1 × 10-8, 1.9 × 10-7 and 3.8 × 10-7 M, resp. (S/N = 3). The proposed method was applied to the detn. of aliph. diamines in a lake water sample by the std. addn. method. The recovery of these amines in H2O was 89.9-107%.
    112. 112
      Fekete, A.; Lahaniatis, M.; Lintelmann, J.; Schmitt-Kopplin, P. Determination of Aliphatic Low-Molecular-Weight and Biogenic Amines by Capillary Zone Electrophoresis. In Capillary Electrophoresis: Methods and Protocols; Humana Press: Totowa, NJ, 2008; pp 65– 91. DOI: 10.1007/978-1-59745-376-9_4
      There is no corresponding record for this reference.
    113. 113
      Böhmer, A.; Jordan, J.; Tsikas, D. High-Performance Liquid Chromatography Ultraviolet Assay for Human Erythrocytic Catalase Activity by Measuring Glutathione as o-Phthalaldehyde Derivative. Anal. Biochem. 2011, 410 (2), 296– 303,  DOI: 10.1016/j.ab.2010.11.026
      113
      High-performance liquid chromatography ultraviolet assay for human erythrocytic catalase activity by measuring glutathione as o-phthalaldehyde derivative
      Bohmer Anke; Jordan Jens; Tsikas Dimitrios
      Analytical biochemistry (2011), 410 (2), 296-303 ISSN:.
      The most frequently used catalase (CAT) activity assay is based on the spectrophotometric measurement of hydrogen peroxide (H(2)O(2)) absorbance decrease at 240 nm. Here we report an alternative high-performance liquid chromatography (HPLC) assay for human erythrocytic CAT (heCAT) activity measurement based on glutathione (GSH) analysis as a highly stable, H(2)O(2)-insensitive o-phthalaldehyde (OPA) derivative. The method was developed and validated using an isolated heCAT in phosphate-buffered saline at pH 7.4 and was applied to measure CAT activity in lysed human erythrocytes. heCAT activity was measured at initial concentrations of 5 nM for heCAT, 5mM for H(2)O(2), and 10mM for GSH, and the incubation time was 10 min. Nitrite (NO(2)(-)) was found to be an uncompetitive inhibitor of heCAT activity (IC(50)=9 μM) and of CAT activity in hemolysate (IC(50)∼750 μM). Nitrate (NO(3)(-)) at concentrations up to 100 μM did not inhibit heCAT activity. Azide (N(3)(-)) was found to be a very strong inhibitor of the heCAT (IC(50)=0.2 nM) but a relatively weak CAT inhibitor (IC(50)∼10 μM) in human hemolysates. The novel CAT activity assay works under redox conditions that more closely resemble those prevailing in cells and allows high-throughput analysis despite the required HPLC step.
    114. 114
      Black, G. W.; Brown, N. L.; Perry, J. J. B.; Randall, P. D.; Turnbull, G.; Zhang, M. A High-Throughput Screening Method for Determining the Substrate Scope of Nitrilases. Chem. Commun. 2015, 51 (13), 2660– 2,  DOI: 10.1039/C4CC06021K
      114
      A high-throughput screening method for determining the substrate scope of nitrilases
      Black, Gary W.; Brown, Nicola L.; Perry, Justin J. B.; Randall, P. David; Turnbull, Graeme; Zhang, Meng
      Chemical Communications (Cambridge, United Kingdom) (2015), 51 (13), 2660-2662CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Nitrile compds. are intermediates in the synthesis of pharmaceuticals such as atorvastatin. We have developed a chromogenic reagent to screen for nitrilase activity as an alternative to Nessler's reagent. It produces a semi-quantifiable blue color and hydrolysis of 38 nitrile substrates by 23 nitrilases as cell-free exts. has been shown.
    115. 115
      Mann, S.; Eveleigh, L.; Lequin, O.; Ploux, O. A Microplate Fluorescence Assay for DAPA Aminotransferase by Detection of the Vicinal Diamine 7,8-Diaminopelargonic Acid. Anal. Biochem. 2013, 432 (2), 90– 6,  DOI: 10.1016/j.ab.2012.09.038
      115
      A microplate fluorescence assay for DAPA aminotransferase by detection of the vicinal diamine 7,8-diaminopelargonic acid
      Mann, Stephane; Eveleigh, Luc; Lequin, Olivier; Ploux, Olivier
      Analytical Biochemistry (2013), 432 (2), 90-96CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      7,8-Diaminopelargonic acid (DAPA) aminotransferase is an enzyme of the biotin biosynthetic pathway that plays an essential role in Mycobacterium tuberculosis virulence. Inhibition of this enzyme is a potential strategy to combat this microorganism, the causative agent of tuberculosis. To identify new inhibitors as potential drugs, a simple enzymic assay for high-throughput screening (HTS) is needed. Several methods for measuring DAPA aminotransferase activity are already available. However, requirements for their implementation for HTS are tedious. We describe here a microplate fluorescence assay for DAPA aminotransferase that is simple, cheap, and sensitive, allowing linear detection of DAPA in the range of 20 nM to 50 μM. The principle of the method is the direct detection in the enzymic reaction mixt. of the vicinal diamine DAPA derivatized with ortho-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). The assay was validated with the known inhibitor desmethyl-KAPA (8-amino-7-oxopelargonic acid) and adapted to microplate for HTS. The structure of the stable fluorescent adduct formed between a vicinal primary diamine and OPA in the presence of 2ME was characterized by mass spectrometry and NMR spectroscopy.
    116. 116
      Kalkhof, S.; Sinz, A. Chances and Pitfalls of Chemical Cross-Linking with Amine-Reactive N-Hydroxysuccinimide Esters. Anal. Bioanal. Chem. 2008, 392 (1), 305– 12,  DOI: 10.1007/s00216-008-2231-5
      116
      Chances and pitfalls of chemical cross-linking with amine-reactive N-hydroxysuccinimide esters
      Kalkhof, Stefan; Sinz, Andrea
      Analytical and Bioanalytical Chemistry (2008), 392 (1-2), 305-312CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      In this report the authors summarize their experiences with the reaction products of N-hydroxysuccinimide (NHS) esters, which are widely used for chem. crosslinking of lysine residues in proteins. The authors describe the products, which should be scrutinized during data anal. using customized software when NHS esters are employed for chem. crosslinking. Reaction products of NHS esters were obsd. not only with lysines, but also with serines, tyrosines, and threonines. This report is intended to be a practical guide for those working in the field of chem. crosslinking and mass spectrometry.
    117. 117
      Gayo, L. M.; Suto, M. J. Use of Pentafluorophenyl Esters for One-Pot Protection/Activation of Amino and Thiol Carboxylic Acids. Tetrahedron Lett. 1996, 37 (28), 4915– 8,  DOI: 10.1016/0040-4039(96)00985-9
      117
      Use of pentafluorophenyl esters for one-pot protection/activation of amino and thiol carboxylic acids
      Gayo, Leah M.; Suto, Mark J.
      Tetrahedron Letters (1996), 37 (28), 4915-4918CODEN: TELEAY; ISSN:0040-4039. (Elsevier)
      The authors describe a new one-pot procedure for the simultaneous protection and activation of amino and thiol carboxylic acids using pentafluorophenyl trifluoroacetate (TFAPfp) and related pentafluorophenyl reagents (FmocPfp and AcPfp). For example, the treatment of 2-aminobenzoic acid with six equiv. of FmocPfp in DMF with pyridine gave pentafluorophenyl 2-(fluorenylmethoxycarbonylamino)benzoate in 82% yield with high purity. This approach can be used for the construction of combinatorial libraries of pentafluorophenyl esters of N-acylated amino acids or S-acylated mercapto acids.
    118. 118
      Fang, L.; Demee, M.; Sierra, T.; Kshirsagar, T.; Celebi, A. A.; Yan, B. Kinetics Study of Amine Cleavage Reactions of Various Resin-Bound Thiophenol Esters from Marshall Linker. J. Comb. Chem. 2002, 4 (4), 362– 8,  DOI: 10.1021/cc020010r
      118
      Kinetics Study of Amine Cleavage Reactions of Various Resin-Bound Thiophenol Esters from Marshall Linker
      Fang, Liling; Demee, Michael; Sierra, Teresa; Kshirsagar, Tushar; Celebi, Azim A.; Yan, Bing
      Journal of Combinatorial Chemistry (2002), 4 (4), 362-368CODEN: JCCHFF; ISSN:1520-4766. (American Chemical Society)
      The kinetics of cleavage reactions of seven resin-bound thiophenol esters with three amines has been studied by single-bead FTIR. The reactivity of these seven thiophenol esters was dependent on their structures and could be summarized as follows: 5-benzimidazolecarboxylic thiophenol ester > alkyl thiophenol ester > arom. thiophenol ester. The reactivity of three amines was summarized as follows: n-butylamine > 3,4-dimethoxyphenethylamine > 1-piperonylpiperazine. The rate of the cleavage reaction increased 2-fold per 10 °C rise in reaction temp. Oxidn. of the thiophenol linker increased the rate of the cleavage reaction by 580-fold.
    119. 119
      Lavis, L. D. Teaching Old Dyes New Tricks: Biological Probes Built from Fluoresceins and Rhodamines. Annu. Rev. Biochem. 2017, 86, 825– 43,  DOI: 10.1146/annurev-biochem-061516-044839
      119
      Teaching Old Dyes New Tricks: Biological Probes Built from Fluoresceins and Rhodamines
      Lavis, Luke D.
      Annual Review of Biochemistry (2017), 86 (), 825-843CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
      Small-mol. fluorophores, such as fluorescein and rhodamine derivs., are crit. tools in modern biochem. and biol. research. The field of chem. dyes is old; colored mols. were first discovered in the 1800s, and the fluorescein and rhodamine scaffolds have been known for over a century. Nevertheless, there has been a renaissance in using these dyes to create tools for biochem. and biol. The application of modern chem., biochem., mol. genetics, and optical physics to these old structures enables and drives the development of novel, sophisticated fluorescent dyes. This crit. review focuses on an important example of chem. biol.-the melding of old and new chem. knowledge-leading to useful mols. for advanced biochem. and biol. expts.
    120. 120
      Pereira, A.; Martins, S.; Caldeira, A. T. Phytochemicals in Human Health, Coumarins as Fluorescent Labels of Biomolecules; IntechOpen, 2019.
      There is no corresponding record for this reference.
    121. 121
      Boens, N.; Leen, V.; Dehaen, W. Fluorescent Indicators Based on BODIPY. Chem. Soc. Rev. 2012, 41 (3), 1130– 72,  DOI: 10.1039/C1CS15132K
      121
      Fluorescent indicators based on BODIPY
      Boens, Noel; Leen, Volker; Dehaen, Wim
      Chemical Society Reviews (2012), 41 (3), 1130-1172CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. This crit. review covers the advances made using the 4-bora-3a,4a-diaza-s-indacene (BODIPY) scaffold as a fluorophore in the design, synthesis and application of fluorescent indicators for pH, metal ions, anions, biomols., reactive oxygen species, reactive nitrogen species, redox potential, chem. reactions and various phys. phenomena. The sections of the review describing the criteria for rational design of fluorescent indicators and the math. expressions for analyzing spectrophotometric and fluorometric titrns. are applicable to all fluorescent probes (206 refs.).
    122. 122
      Han, S.-Y.; Kim, Y.-A. Recent Development of Peptide Coupling Reagents in Organic Synthesis. Tetrahedron 2004, 60 (11), 2447– 67,  DOI: 10.1016/j.tet.2004.01.020
      122
      Recent development of peptide coupling reagents in organic synthesis
      Han, So-Yeop; Kim, Young-Ah
      Tetrahedron (2004), 60 (11), 2447-2467CODEN: TETRAB; ISSN:0040-4020. (Elsevier Science B.V.)
      A review. Various types of peptide coupling reagents (i.e., phosphonium, uronium, immonium, carbodiimide, imidazolium, organophosphorus, acid halogenating compds., chloroformate, pyridinium, etc.) are discussed for the synthesis of bioactive mols. contg. peptide linkages. Methods used to suppress racemization during the peptide coupling step are presented.
    123. 123
      Karongo, R.; Ge, M.; Horak, J.; Gross, H.; Kohout, M.; Lindner, W.; Lämmerhofer, M. Rapid Enantioselective Amino Acid Analysis by Ultra-High Performance Liquid Chromatography-Mass Spectrometry Combining 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatization with Core-Shell Quinine Carbamate Anion Exchanger Separation. J. Chromatogr. Open 2021, 1, 100004,  DOI: 10.1016/j.jcoa.2021.100004
      There is no corresponding record for this reference.
    124. 124
      Cao, L.; Wang, H.; Zhang, H. Analytical Potential of 6-Oxy-(N-Succinimidyl Acetate)-9-(2’-Methoxycarbonyl) Fluorescein for the Determination of Amino Compounds by Capillary Electrophoresis with Laser-Induced Fluorescence Detection. Electrophoresis 2005, 26 (10), 1954– 62,  DOI: 10.1002/elps.200410227
      124
      Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detection
      Cao, Liwei; Wang, Hong; Zhang, Huashan
      Electrophoresis (2005), 26 (10), 1954-1962CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The anal. potential of a fluorescein analog, 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the 1st time synthesized in the lab., as a labeling reagent for the labeling and detn. of amino compds. by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the anal. possibilities of this approach. The derivatization conditions and sepn. parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30°C for 6 min in boric acid buffer (pH 8.0). The derivs. were baseline-sepd. in 15 min with 25 mM boric acid running buffer (pH 9.0), contg. 24 mM SDS and 12.5% vol./vol. acetonitrile. The concn. detection limit for biogenic amines reaches 8 × 10-11 mol/L (signal-to-noise ratio = 3). The application of CE in the anal. of the SAMF-derivatized amino acids was also exploited. The optimal running buffer for amino acids suggested that weak acidic background electrolyte offered better sepn. than the basic one. The proposed method was applied to the detn. of biogenic amines in 3 different beer samples with satisfying recoveries varying from 92.8% to 104.8%. Finally, comparison of several fluorescein-based probes for amino compds. was discussed. With good labeling reaction, excellent photostability, pH-independent fluorescence (pH 4-9), and the resultant widely suited running buffer pH, SAMF has a great prospect in the detn. of amino compds. in CE.
    125. 125
      Xia, L.-J.; Guo, X.-F.; Ji, Y.; Chen, L.; Wang, H. A Long-Wavelength Fluorescent Probe for Amino Compounds and Its Application in the Determination of Aliphatic Amines. Anal. Methods 2018, 10 (26), 3188– 96,  DOI: 10.1039/C8AY00706C
      125
      A long-wavelength fluorescent probe for amino compounds and its application in the determination of aliphatic amines
      Xia, Li-Jun; Guo, Xiao-Feng; Ji, Yan; Chen, Liu; Wang, Hong
      Analytical Methods (2018), 10 (26), 3188-3196CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)
      Herein, a highly reactive long-wavelength fluorescent labeling reagent, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimidyl ester) difluoroboradiaza-s-indacene (DMDSPAB-OSu), was designed and synthesized for amino compds. Using DMDSPAB-OSu as a pre-column derivatization reagent, an HPLC-fluorescence detection method was developed for the detn. of trace amts. of aliph. amines in samples. Prior to anal., amines were derivatized with DMDSPAB-OSu at room temp. in 0.2 mol L-1 pH 8.8 borate buffer for only 5 min, and the baseline sepn. of 11 aliph. amines was achieved within 25 min. Limits of detection between 0.8 to 1.2 nM were obtained at 630 nm with an excitation wavelength of 610 nm. Furthermore, the proposed DMDSPAB-OSu-based HPLC method was applied to the anal. of lake water and human urine with satisfying recoveries ranging from 93.0-109.7%.
    126. 126
      Gao, P. F.; Guo, X. F.; Wang, H.; Zhang, H. S. Determination of Trace Biogenic Amines with 1,3,5,7-Tetramethyl-8-(N-Hydroxysuccinimidyl Butyric Ester)-Difluoroboradiaza-s-Indacene Derivatization Using High-Performance Liquid Chromatography and Fluorescence Detection. J. Sep. Sci. 2011, 34 (12), 1383– 90,  DOI: 10.1002/jssc.201100120
      126
      Determination of trace biogenic amines with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene derivatization using high-performance liquid chromatography and fluorescence detection
      Gao, Pan-Feng; Guo, Xiao-Feng; Wang, Hong; Zhang, Hua-Shan
      Journal of Separation Science (2011), 34 (12), 1383-1390CODEN: JSSCCJ; ISSN:1615-9306. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A reversed-phase high-performance liq. chromatog. method based on chem. derivatization with fluorescence detection has been developed for analyzing biogenic amines in food and environmental samples. A BODIPY-based fluorescent reagent, 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su), was employed for the derivatization of these biogenic amines at 20°C for 20 min in pH 7.20 borate buffer after careful investigation of the derivatization conditions including reagent concn., buffer soln., reaction temp. and reaction time. Sepn. of biogenic amines with gradient elution was conducted on a C8 column with methanol-tetrahydrofuran-water as mobile phase. The detection limits were obtained in the range from 0.1 to 0.2 nM (signal-to-noise=3). This procedure has been validated using practical samples. The study results demonstrated a potential of employing high-performance liq. chromatog. (HPLC) with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene labeling as a tool for quant. anal. of biogenic amines involved in various matrixes.
    127. 127
      Mallick, S.; Chandra, F.; Koner, A. L. A Ratiometric Fluorescent Probe for Detection of Biogenic Primary Amines with Nanomolar Sensitivity. Analyst 2016, 141 (3), 827– 31,  DOI: 10.1039/C5AN01911G
      127
      A ratiometric fluorescent probe for detection of biogenic primary amines with nanomolar sensitivity
      Mallick, Suman; Chandra, Falguni; Koner, Apurba L.
      Analyst (Cambridge, United Kingdom) (2016), 141 (3), 827-831CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      An ultrasensitive ratiometric fluorescent sensor (I) made of an N,N-dimethylaminonaphthalene anhydride moiety for detection of aliph. primary amines is reported. Biogenic amines at nanomolar concn. were detected with the addnl. ability to discriminate between primary, secondary and tertiary amines by using both UV-Visible and fluorescence spectroscopy.
    128. 128
      Vázquez, M. E.; Blanco, J. B.; Imperiali, B. Photophysics and Biological Applications of the Environment-Sensitive Fluorophore 6-N,N-Dimethylamino-2,3-Naphthalimide. J. Am. Chem. Soc. 2005, 127 (4), 1300– 6,  DOI: 10.1021/ja0449168
      128
      Photophysics and biological applications of the environment-sensitive fluorophore 6-N,N-Dimethylamino-2,3-naphthalimide
      Vazquez, M. Eugenio; Blanco, Juan B.; Imperiali, B.
      Journal of the American Chemical Society (2005), 127 (4), 1300-1306CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We have synthesized a new environment-sensitive fluorophore, 6-N,N-dimethylamino-2,3-naphthalimide (6DMN). This chromophore exhibits valuable fluorescent properties as a biol. probe with emission in the 500-600 nm range and a marked response to changes in the environment polarity. The 6DMN fluorescence is red-shifted in polar protic environments, with the max. emission intensity shifting more than 100 nm from 491 nm in toluene to 592 nm in water. Addnl., the fluorescence quantum yield decreases more than 100-fold from chloroform (Φ = 0.225) to water (Φ = 0.002). The scope and applications of the 6DMN probe are expanded with the synthesis of an Fmoc-protected amino acid deriv. (5), which contains the fluorophore. This unnatural amino acid has been introduced into several peptides, demonstrating that it can be manipulated under std. solid-phase peptide synthesis conditions. Peptides incorporating the new residue can be implemented for monitoring protein-protein interactions as exemplified in studies with Src homol. 2 (SH2) phosphotyrosine binding domains. The designed peptides exhibit a significant increase in the quantum yield of the long wavelength fluorescence emission band (596 nm) upon binding to selected SH2 domains (e.g., Crk SH2, Abl SH2, and PI3K SH2). The peptides can be used as ratiometric sensors, since the short wavelength band (460 nm) was found almost invariable throughout the titrns.
    129. 129
      Anumula, K. R.; Schulz, R. P.; Back, N. Fluorescent N-Methylanthranilyl (Mantyl) Tag for Peptides: Its Application in Subpicomole Determination of Kinins. Peptides 1992, 13 (4), 663– 9,  DOI: 10.1016/0196-9781(92)90170-8
      129
      Fluorescent N-methylanthranilyl (Mantyl) tag for peptides: its application in subpicomole determination of kinins
      Anumula, Kalyan R.; Schulz, Raymond P.; Back, Nathan
      Peptides (New York, NY, United States) (1992), 13 (4), 663-9CODEN: PPTDD5; ISSN:0196-9781.
      Highly fluorescent N-methylanthranilyl (Mantyl) peptide derivs. were prepd. by a 1-step reaction with N-methylisatoic anhydride (MIA) for quant. detection in HPLC. Reactions were carried out in an org. medium of acetonitrile-triethylamine, in aq. alk. sodium carbonate and sodium phosphate buffers. 4-Dimethylaminopyridine (DMAP) catalyzed specific mantylation of -NH2 groups of peptides in the org. reaction medium. The DMAP had no effect in the aq. buffered reaction systems. Proline amino-terminal peptides reacted equally well with MIA. Mantyl-bradykinin (Mantyl-BK) had excitation and fluorescence max. at 350 nm and 426 nm in water and water/acetonitrile (ACN)/trifluoroacetic acid (TFA) solvent mixts., resp. Fluorescence intensity increased with an increase in ACN concn. and decreased with an increase in acid content. Mantyl kinins were completely resolved on a C18 reversed-phase HPLC column using an ACN-0.1% TFA gradient and their behavior on the column was similar to having an extra amino acid. Di-Mantyl derivs. obtained with Lys-BK and Met-Lys-BK did not exhibit fluorescence appreciably higher than Mantyl-BK. Fluorescence detection of Mantyl kinins was ∼50-100-fold more sensitive (lower limits of 0.1-0.5 pmol) than UV detection of the phenylisothiocyanate-derivatized kinins under typical HPLC conditions.
    130. 130
      Fessler, A.; Garmon, C.; Heavey, T.; Fowler, A.; Ogle, C. Water-Soluble and UV Traceable Isatoic Anhydride-Based Reagents for Bioconjugation. Org. Biomol. Chem. 2017, 15 (45), 9599– 602,  DOI: 10.1039/C7OB02377D
      130
      Water-soluble and UV traceable isatoic anhydride-based reagents for bioconjugation
      Fessler, Adam; Garmon, Corey; Heavey, Thomas; Fowler, Anthony; Ogle, Craig
      Organic & Biomolecular Chemistry (2017), 15 (45), 9599-9602CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Isatoic anhydride is elaborated to water sol. bioconjugation reagents that gives functionality and water soly. in one self-cleaning step. This new platform offers high atom economy with carbon dioxide being the only byproduct, and is shown to very quickly and efficiently label proteins in bicarbonate buffered solns.
    131. 131
      Jeon, S.; Kim, T.-I.; Jin, H.; Lee, U.; Bae, J.; Bouffard, J.; Kim, Y. Amine-Reactive Activated Esters of meso-CarboxyBODIPY: Fluorogenic Assays and Labeling of Amines, Amino Acids, and Proteins. J. Am. Chem. Soc. 2020, 142 (20), 9231– 9,  DOI: 10.1021/jacs.9b13982
      131
      Amine-Reactive Activated Esters of meso-CarboxyBODIPY: Fluorogenic Assays and Labeling of Amines, Amino Acids, and Proteins
      Jeon, Sungjin; Kim, Tae-Il; Jin, Hanyong; Lee, Uisung; Bae, Jeehyeon; Bouffard, Jean; Kim, Youngmi
      Journal of the American Chemical Society (2020), 142 (20), 9231-9239CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Fluorescence-based amine-reactive dyes are highly valuable for the sensing of amines and the labeling of biomols. Although it would be highly desirable, large changes in emission spectra and intensity seldom accompany the conjugation of known amine-reactive dyes to their target mols. On the contrary, amide bond formation between amines and the pentafluorophenyl (2-PFP) and succinimidyl (2-NHS) esters of meso-carboxyBODIPY results in significant changes in emission maxima (Δλ: 70-100 nm) and intensity (up to 3000-fold), enabling the fast (down to 5 min) and selective fluorogenic detection and labeling of amines, amino acids, and proteins. This approach further benefits from the demonstrated versatility and high reliability of activated ester chem., and background hydrolysis is negligible. The large "turn-on" response is a testament of the extreme sensitivity of meso-carboxyBODIPYs to the minimal changes in electronic properties that distinguish esters from amides. Applications to the detection of food spoilage, staining of proteins on electrophoretic gels or in living cells, and the expedited synthesis of organelle-specific fluorescence microscope imaging agents are further demonstrated.
    132. 132
      Lee, U.; Kim, T.-I.; Jeon, S.; Luo, Y.; Cho, S.; Bae, J.; Kim, Y. Native Chemical Ligation-Based Fluorescent Probes for Cysteine and Aminopeptidase N Using meso-Thioester-BODIPY. Eur. J. Chem. 2021, 27 (49), 12545– 51,  DOI: 10.1002/chem.202101990
      132
      Native Chemical Ligation-Based Fluorescent Probes for Cysteine and Aminopeptidase N Using meso-thioester-BODIPY
      Lee, Uisung; Kim, Tae-Il; Jeon, Sungjin; Luo, Yongyang; Cho, Siyoung; Bae, Jeehyeon; Kim, Youngmi
      Chemistry - A European Journal (2021), 27 (49), 12545-12551CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Meso-Carboxyl-BODIPY responds to small electronic changes resulting from acyl substitution reactions with a marked change in fluorescence. Herein, the minute changes that accompany the thioester to amide conversion encountered in native chem. ligation (NCL) are exploited in the construction of fluorescent "turn-on" probes. Two fluorogenic probes, 1 a and 4, derived from a meso-thioester-BODIPY scaffold, were designed for the selective detection of cysteine (1 a) and aminopeptidase N (4), resp. The arom. (1 a) and aliph. (4) thioesters of meso-carboxyl-BODIPY are nonfluorescent. However, specific analyte-induced conversion to the meso-amide deriv. caused significant spectral changes and a dramatic fluorescence enhancement. Probe 1 a exhibited a large fluorescence "turn-on" response with high selectivity toward cysteine via a tandem NCL reaction. Probe 4 was successfully applied to the monitoring and imaging of endogenous aminopeptidase N in live cancer cells.
    133. 133
      Esnal, I.; Bañuelos, J.; López Arbeloa, I.; Costela, A.; Garcia-Moreno, I.; Garzón, M.; Agarrabeitia, A. R.; José Ortiz, M. Nitro and Amino BODIPYs: Crucial Substituents to Modulate Their Photonic Behavior. RSC Adv. 2013, 3 (5), 1547– 56,  DOI: 10.1039/C2RA22916A
      133
      Nitro and amino BODIPYS: crucial substituents to modulate their photonic behavior
      Esnal, Ixone; Banuelos, Jorge; Lopez Arbeloa, Inigo; Costela, Angel; Garcia-Moreno, Inmaculada; Garzon, Miguel; Agarrabeitia, Antonia R.; Jose Ortiz, Maria
      RSC Advances (2013), 3 (5), 1547-1556CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      The present work deals with the synthesis and photophys., quantum mech., and lasing characterization of novel BODIPYs bearing amino and nitro groups at different positions in the core. The results emphasize the relevant role on the photophys. and lasing properties, not only of the attached functionality but also of the position in which is grafted, as well as the mol. structure of the indacene core. A wide part of the visible spectrum can be covered by the insertion of an amino group at position 3 (red shift) or 8 (blue shift). Furthermore, the electron withdrawing character of the nitro substituent induces intramol. charge transfer processes, the efficiency of which depends on the position of the nitro group on the BODIPY core. All these exptl. findings can be rationalized with the help of quantum mech. calcns.
    134. 134
      Liu, E. Y.; Jung, S.; Weitz, D. A.; Yi, H.; Choi, C.-H. High-Throughput Double Emulsion-Based Microfluidic Production of Hydrogel Microspheres with Tunable Chemical Functionalities toward Biomolecular Conjugation. Lab Chip 2018, 18 (2), 323– 34,  DOI: 10.1039/C7LC01088E
      134
      High-throughput double emulsion-based microfluidic production of hydrogel microspheres with tunable chemical functionalities toward biomolecular conjugation
      Liu, Eric Y.; Jung, Sukwon; Weitz, David A.; Yi, Hyunmin; Choi, Chang-Hyung
      Lab on a Chip (2018), 18 (2), 323-334CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)
      In this work, we present a simple, efficient, and high-throughput capillary microfluidic approach for controlled fabrication of monodisperse and chem. functional hydrogel microspheres via formation of double emulsion drops with an ultra-thin oil shell as a sacrificial template. Furthermore, we show the utility of chitosan's primary amine as an efficient conjugation handle at physiol. pH due to its low pKa by direct comparison with other primary amines. We also report the utility of these microspheres in biomol. conjugation using model fluorescent proteins, R-phycoerythrin (R-PE) and green fluorescent protein (GFPuv), via tetrazine-trans-cyclooctene (Tz-TCO) ligation for CS-PEG microspheres and carbodiimide chem. for AA-PEG microspheres, resp. The results show rapid coupling of R-PE with the microspheres' functional groups with minimal non-specific adsorption. In-depth protein conjugation kinetics studies with our microspheres highlight the differences in reaction and diffusion of R-PE with CS-PEG and AA-PEG microspheres. Finally, we demonstrate orthogonal one-pot protein conjugation of R-PE and GFPuv with CS-PEG and AA-PEG microspheres via simple size-based encoding. Combined, these results represent a significant advancement in the rapid and reliable fabrication of monodisperse and chem. functional hydrogel microspheres with tunable properties.
    135. 135
      Katritzky, A. R.; Marson, C. M. Pyrylium Mediated Transformations of Primary Amino Groups into Other Functional Groups. New Synthetic Methods (41). Angew. Chem., Int. Ed. Engl. 1984, 23 (6), 420– 9,  DOI: 10.1002/anie.198404201
      There is no corresponding record for this reference.
    136. 136
      Wetzl, B. K.; Yarmoluk, S. M.; Craig, D. B.; Wolfbeis, O. S. Chameleon Labels for Staining and Quantifying Proteins. Angew. Chem., Int. Ed. Engl. 2004, 43 (40), 5400– 2,  DOI: 10.1002/anie.200460508
      136
      Chameleon labels for staining and quantifying proteins
      Wetzl Bianca K; Yarmoluk Sergiy M; Craig Douglas B; Wolfbeis Otto S
      Angewandte Chemie (International ed. in English) (2004), 43 (40), 5400-2 ISSN:1433-7851.
      There is no expanded citation for this reference.
    137. 137
      Azab, H. A.; El-Korashy, S. A.; Anwar, Z. M.; Khairy, G. M.; Steiner, M.-S.; Duerkop, A. High-Throughput Sensing Microtiter Plate for Determination of Biogenic Amines in Seafood Using Fluorescence or Eye-Vision. Analyst 2011, 136 (21), 4492– 9,  DOI: 10.1039/c1an15049a
      137
      High-throughput sensing microtiter plate for determination of biogenic amines in seafood using fluorescence or eye-vision
      Azab, H. A.; El-Korashy, S. A.; Anwar, Z. M.; Khairy, G. M.; Steiner, Mark-Steven; Duerkop, Axel
      Analyst (Cambridge, United Kingdom) (2011), 136 (21), 4492-4499CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      A new optical sensing microplate was developed for rapid screening for the presence of biogenic amines (BAs) in seafood samples with high sensitivity. The deposition of a sensing spot (contg. a chameleon dye (Py-1) in a polymeric cocktail) on the bottom of the wells of a std. microplate renders the plate a new sensing tool for a rapid and parallel detection of up to 96 (real) samples. This sensing microplate enables (1) a semi-quant. readout of analyte concn. by eye-vision, (2) a rapid fluorescence readout of 96 samples with std. instrumentation in less than two minutes (unlike chromatog. and electrophoretic methods), (3) a statistically robust data evaluation (with 8-12 replicates) and (4) a rapid parallel sample prepn. with std. 8 or 12-channel micropipettes. On reaction with biogenic amines, the dye shows a significant visible color change from blue over green to red color. The appearance of red color favorably coincides with the concn. of BAs that can induce symptoms of poisoning. The linear ranges of fluorescence calibration data for six biogenic amines cover the clin. toxicol. relevant range of BAs that is too low to be detected by the human nose. The LODs range from 0.16 to 0.56 μg mL-1, with correlation coeffs. (r2) between 0.985 and 0.999. Finally, the evolution of spoilage of four fish samples (monitored by detn. of their BA status) and the increase of their total amine content were found to agree well with previous data on time-dependent evolution of BAs in fish.
    138. 138
      Azab, H. A.; El-Korashy, S. A.; Anwar, Z. M.; Khairy, G. M.; Duerkop, A. Reactivity of a Luminescent “Off-On” Pyrylium Dye toward Various Classes of Amines and Its Use in a Fluorescence Sensor Microtiter Plate for Environmental Samples. J. Photochem. Photobiol. A: Chem. 2012, 243, 41– 6,  DOI: 10.1016/j.jphotochem.2012.05.029
      138
      Reactivity of a luminescent "off-on" pyrylium dye toward various classes of amines and its use in a fluorescence sensor microtiter plate for environmental samples
      Azab, H. A.; El-Korashy, S. A.; Anwar, Z. M.; Khairy, G. M.; Duerkop, Axel
      Journal of Photochemistry and Photobiology, A: Chemistry (2012), 243 (), 41-46CODEN: JPPCEJ; ISSN:1010-6030. (Elsevier B.V.)
      The authors report on a systematic study on the reactivity of the pyrylium dye Py-1 toward various classes of amines (primary aliph., primary arom., secondary, tertiary) and its use in environmental sensing of amines. While most primary aliph. amines react almost quant. with Py-1 within 10 min, sterically hindered primary aliph. amines and primary arom. amines react more slowly. Secondary and tertiary amines just induce a decompn. of the chromophore of Py-1 but do not yield a fluorescent product. This makes Py-1 a suitable receptor and transducer in an optical sensing microplate for rapid screening of primary aliph. amines in water and soil samples. Py-1 is embedded into a polymeric cocktail, which is deposited on the bottom of wells in microtiter plates to yield a high-throughput fluorescence sensing tool. On reaction with primary aliph. amines, a significant fluorescence increase (λexc = 485 nm/λem = 620 nm) of the sensor spots is detected in a std. microplate reader after 10 min incubation at 25°. The linear calibration plots of eight primary aliph. amines are at 0.350-70.0 μg mL-1. The limits of detection (LODs) are 0.119-0.589 μg mL-1 and the limits of quantitation (LOQs) are 0.399-1.965 μg mL-1. Further the sensing plate is suitable for sensing mixts. of primary amines and the total content of amines (TAC) found is a good measure for the sum of the contents of the individual amines present. Finally, the sensing plate was successfully applied to the quant. anal. of primary aliph. amines in 3 samples of water and soil, resp.
    139. 139
      Bayer, M.; König, S. Pyrylium-Based Dye and Charge Tagging in Proteomics. Electrophoresis 2016, 37 (22), 2953– 8,  DOI: 10.1002/elps.201600318
      139
      Pyrylium-based dye- and charge-tagging in proteomics
      Bayer, Malte; Koenig, Simone
      Electrophoresis (2016), 37 (22), 2953-2958CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The pyrylium group is a selective reagent for ε-amino groups in proteins. In particular for fluorescence labeling a no. of advantages over traditional NHS-ester chem. were recognized such as the rapid pre-staining procedure. Here, we have investigated the labeling reaction for the fluorogenic pyrylium dye Py-1 using liq. chromatog. coupled to mass spectrometry with the aim of detg. its specificity and possible side products. Peptides contg. no, one, and two lysine residue and a choice of no or one cysteine residue were labeled with Py-1 at yields >30%. Gas phase fragmentation proved both labeling of lysine residues as well of that of the N-terminus also in peptides which contained a lysine residue. Evidence for cysteine labeling was not found, but several other products were detected such as the results of rearrangements with adjacent acidic amino acids. Apart from the use as a fluorogenic label Py-1 recommends itself for N-terminal charge-tagging as alternative to the commonly used quaternary ammonium salts. Predominantly a- and b-type ion series were obsd. for N-terminally labeled peptides. Further applications include chromophore tagging since the labeled product is not only fluorescent but also colored red.
    140. 140
      Turner, E. H.; Dickerson, J. A.; Ramsay, L. M.; Swearingen, K. E.; Wojcik, R.; Dovichi, N. J. Reaction of Fluorogenic Reagents with Proteins: III. Spectroscopic and Electrophoretic Behavior of Proteins Labeled with Chromeo P503. J. Chromatogr. A 2008, 1194 (2), 253– 6,  DOI: 10.1016/j.chroma.2008.04.046
      140
      Reaction of fluorogenic reagents with proteins. III. Spectroscopic and electrophoretic behavior of proteins labeled with Chromeo P503
      Turner, Emily H.; Dickerson, Jane A.; Ramsay, Lauren M.; Swearingen, Kristian E.; Wojcik, Roza; Dovichi, Norman J.
      Journal of Chromatography A (2008), 1194 (2), 253-256CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
      The spectroscopic and electrophoretic properties of proteins labeled with Chromeo P503 were investigated. Its photobleaching characteristics were detd. by continually infusing Chromeo P503-labeled α-lactalbumin into a sheath-flow cuvette and monitored fluorescence as a function of laser power. The labeled protein is relatively photo-labile with an optimum excitation power of about 2 mW. The unreacted reagent is weakly fluorescent but present at much higher concn. than the labeled protein. The unreacted reagent undergoes photobleaching at a laser power more than an order of magnitude higher than the labeled protein. One-dimensional capillary electrophoresis anal. of Chromeo P503-labeled α-lactalbumin produced concn. detection limits (3σ) of 12 pM and mass detection limits of 0.7 zmol, but with modest theor. plate counts of 17,000. The reagent was employed for the two-dimensional capillary electrophoresis anal. of a homogenate prepd. from a Barrett's esophagus cell line; the sepn. quality is similar to that produced by 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), a more commonly used reagent.
    141. 141
      Wolfbeis, O. S. Fluorescent Chameleon Labels for Bioconjugation and Imaging of Proteins, Nucleic Acids, Biogenic Amines and Surface Amino Groups. A Review. Methods Appl. Fluoresc. 2021, 9 (4), 042001,  DOI: 10.1088/2050-6120/ac1a0a
      141
      Fluorescent chameleon labels for bioconjugation and imaging of proteins, nucleic acids, biogenic amines and surface amino groups. A review
      Wolfbeis, Otto S.
      Methods and Applications in Fluorescence (2021), 9 (4), 042001CODEN: MAFEB2; ISSN:2050-6120. (IOP Publishing Ltd.)
      A review. Chameleon labels (ChLs) possess the unique property of changing (visible) color and fluorescence on binding to amino groups of biomols. MostChLs react with primary aliph. amino groups such as those in lysine or with amino groups artificially introduced into polynucleic acids or saccharides, but someothers also react with secondary amino groups. Under controlled circumstances, the reactions are fairly specific. The review is subdivided into the following sections: (1) An introduction and classification of fluorescent labels; (2) pyrylium labels that undergo shortwave color changes upon labeling, typically from blue to red; (3) polymethine type of labels (that also undergo shortwave color changes, typically from green to blue; (4) various other (less common) chromogenic and fluorogenic systems; (5) hemicyanine labels that undergo longwave color changes, typically from yellow to purple; (6) the application of ChLs to labeling of proteins and oligonucleotides; (7) applications to fluorometric assays and sensing; (8) applications to fluorescence imaging of biomols.; (9) applications in studies on affinity interactions (receptor-ligand binding); (10) applications in surface and interface chem.; and (11) applications in chromatog., electrophoresis and isotachophoresis of biomols.
    142. 142
      Höfelschweiger, B. K., The Pyrylium Dyes: a New Class of Biolabels. Synthesis, Spectroscopy, and Application as Labels and in General Protein Assay , 2005.
      There is no corresponding record for this reference.
    143. 143
      Walter, W.; Bode, K. D. Syntheses of Thiocarbamates. Angew. Chem., Int. Ed. Engl. 1967, 6 (4), 281– 93,  DOI: 10.1002/anie.196702811
      143
      Syntheses of thiocarbamates
      Walter, Wolfgang; Bode, Klaus D.
      Angewandte Chemie, International Edition in English (1967), 6 (4), 281-93CODEN: ACIEAY; ISSN:0570-0833.
      cf. following abstr. A review, with 150 references, of the prepn. of thiocarbamates by the addn. of alcs. and thiols to isothiocyanates, the treatment of amines with compds. of the general formula ClC(S)XR, xanthates, and trithiocarbonates, the treatment of alcs., phenols, and thiols with compds. of the general formula R2NCSCl and [Cl(RS)C:N(R1) R2]+Cl-, and N,N'-thiocarbonyldiimidazole, treatment of thiocyanates and cyanates with H2S, alkylation of metal dithiocarbamates, and addn. to thiocyanic acid.
    144. 144
      Maddani, M. R.; Prabhu, K. R. A Concise Synthesis of Substituted Thiourea Derivatives in Aqueous Medium. J. Org. Chem. 2010, 75 (7), 2327– 32,  DOI: 10.1021/jo1001593
      144
      A concise synthesis of substituted thiourea derivatives in aqueous medium
      Maddani, Mahagundappa R.; Prabhu, Kandikere R.
      Journal of Organic Chemistry (2010), 75 (7), 2327-2332CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      An efficient method for the synthesis of sym. and unsym. substituted thiourea derivs. by means of simple condensation between available building blocks such as amines and carbon disulfide in aq. medium is presented. This protocol works smoothly with aliph. primary amines to afford various di- and trisubstituted thiourea derivs. The present method is also useful in synthesizing various substituted 2-mercapto imidazole heterocycles. This method proceeds through a xanthate (amino dithiol deriv.) intermediate, unlike isothiocyanate as in an earlier known method.
    145. 145
      Reetz, M. T.; Kühling, K. M.; Deege, A.; Hinrichs, H.; Belder, D. Super-High-Throughput Screening of Enantioselective Catalysts by Using Capillary Array Electrophoresis. Angew. Chem., Int. Ed. 2000, 39 (21), 3891– 3,  DOI: 10.1002/1521-3773(20001103)39:21<3891::AID-ANIE3891>3.0.CO;2-1
      145
      Super-high-throughput screening of enantioselective catalysts by using capillary array electrophoresis
      Reetz, Manfred T.; Kuhling, Klaus M.; Deege, Alfred; Hinrichs, Heike; Belder, Detlev
      Angewandte Chemie, International Edition (2000), 39 (21), 3891-3893CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
      The authors' approach to high-throughput screening of enantioselective catalysts was based on the adaptation of capillary array electrophoresis (MegaBACE system) by using chirally modified electrolytes. As a specific goal the authors chose the ee detn. of chiral amines (I, R = n-pentyl, n-hexyl, c-hexyl, Ph, p-tolyl, and 1-naphthyl) using fluorescein isothiocyanate as the derivatization reagent. The authors' study shows that the capillary array electrophoresis for detn. of enantiomeric purity can be used in a truly high-throughput manner (> 7000 ee detns. per day). This result as well as the optimization of the methods described herein are of great significance for the further development of combinatorial asym. transition metal catalysis and directed evolution of enantioselective enzymes.
    146. 146
      Dabek-Zlotorzynska, E.; Maruszak, W. Determination of Dimethylamine and Other Low-Molecular-Mass Amines Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection. J. Chromatogr. B Biomed. Appl. 1998, 714 (1), 77– 85,  DOI: 10.1016/S0378-4347(98)00070-X
      146
      Determination of dimethylamine and other low-molecular-mass amines using capillary electrophoresis with laser-induced fluorescence detection
      Dabek-Zlotorzynska, Ewa; Maruszak, Wioleta
      Journal of Chromatography B: Biomedical Sciences and Applications (1998), 714 (1), 77-85CODEN: JCBBEP; ISSN:0378-4347. (Elsevier Science B.V.)
      The potential of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for the sepn. and detn. of dimethylamine (DMA) and other low-mol.-mass amines involving precolumn derivatization with fluorescein isothiocyanate isomer I (FITC) was studied. Different variables that affect derivatization (pH, FITC concn., reaction time and temp.) and sepn. (buffer concn., addn. of various org. modifiers, applied voltage and length of capillary) were studied. The linearity, reproducibility and reliability of the method were evaluated. The estd. instrumental detection limit for a 2-s pressure injection of the FITC-DMA deriv. was 50 pg/mL (10-9 M), using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, resp. However, for practical reasons, a min. of 5 ng/mL DMA should be subjected to the derivatization. The applicability of the described method to the ext. of atm. aerosol samples was demonstrated.
    147. 147
      Morales, A. R.; Schafer-Hales, K. J.; Marcus, A. I.; Belfield, K. D. Amine-Reactive Fluorene Probes: Synthesis, Optical Characterization, Bioconjugation, and Two-Photon Fluorescence Imaging. Bioconj. Chem. 2008, 19 (12), 2559– 67,  DOI: 10.1021/bc800415t
      147
      Amine-Reactive Fluorene Probes: Synthesis, Optical Characterization, Bioconjugation, and Two-Photon Fluorescence Imaging
      Morales, Alma R.; Schafer-Hales, Katherine J.; Marcus, Adam I.; Belfield, Kevin D.
      Bioconjugate Chemistry (2008), 19 (12), 2559-2567CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, the authors prepd. two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (I) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (II), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomols., assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivs. were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chem. and photophys. properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chem. stability and photostability. The NCS derivs. had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe I, RGDfK-dye conjugate, and Reelin-dye conjugate, was demonstrated.
    148. 148
      Planas, O.; Gallavardin, T.; Nonell, S. A Novel Fluoro-Chromogenic Click Reaction for the Labelling of Proteins and Nanoparticles with near-IR Theranostic Agents. Chem. Commun. 2015, 51 (26), 5586– 9,  DOI: 10.1039/C4CC09070E
      148
      A novel fluoro-chromogenic click reaction for the labelling of proteins and nanoparticles with near-IR theranostic agents
      Planas, Oriol; Gallavardin, Thibault; Nonell, Santi
      Chemical Communications (Cambridge, United Kingdom) (2015), 51 (26), 5586-5589CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Reaction of porphycene isothiocyanates with primary and secondary amines leads to the formation of thiazolo[4,5-c]porphycenes, with a substantial shift in the absorption and fluorescence spectra. The conjugates show fluorescence in the near-IR and are capable of photosensitizing the prodn. of the cytotoxic species singlet oxygen.
    149. 149
      Wang, R.; Yang, W.-j.; Yue, L.; Pan, W.; Zeng, H.-y. DDQ-Promoted C-S Bond Formation: Synthesis of 2-Aminobenzothiazole Derivatives under Transition-Metal-, Ligand-, and Base-Free Conditions. Synlett 2012, 23 (11), 1643– 8,  DOI: 10.1055/s-0031-1291159
      149
      DDQ-promoted C-S bond formation: synthesis of 2-aminobenzothiazole derivatives under transition-metal-, ligand-, and base-free conditions
      Wang, Rui; Yang, Wen-juan; Yue, Liang; Pan, Wei; Zeng, Hong-yao
      Synlett (2012), 23 (11), 1643-1648CODEN: SYNLES; ISSN:0936-5214. (Georg Thieme Verlag)
      A transition-metal-free method for the intramol. S-arylation of o-halobenzothioureas via DDQ-mediated leading to the 2-aminobenzothiazole derivs., e.g., I (R = H, Me, MeO, F; R2 = H, Me, MeO, Cl, Br), is reported. The reactions are performed at room temp. under ligand- and base-free conditions with good to excellent yields.
    150. 150
      Guo, Y.-J.; Tang, R.-Y.; Zhong, P.; Li, J.-H. Copper-Catalyzed Tandem Reactions of 2-Halobenzenamines with Isothiocyanates under Ligand- and Base-Free Conditions. Tetrahedron Lett. 2010, 51 (4), 649– 52,  DOI: 10.1016/j.tetlet.2009.11.086
      150
      Copper-catalyzed tandem reactions of 2-halobenzenamines with isothiocyanates under ligand- and base-free conditions
      Guo, Yan-Jin; Tang, Ri-Yuan; Zhong, Ping; Li, Jin-Heng
      Tetrahedron Letters (2010), 51 (4), 649-652CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)
      A ligand-free copper-catalyzed reaction of 2-halobenzenamines with isothiocyanates has been developed for the synthesis of 2-aminobenzothiazoles. In the presence of CuBr and TBAB (tetra-Bu ammonium bromide, additive), a variety of 2-halobenzenamines underwent the reaction with isothiocyanates at 40 °C, affording 2-aminobenzothiazoles, e.g., I (R1 = H, MeO, Cl, NO2; R2 = H, Me, Cl, F), in moderate to excellent yields. It is noteworthy that the reaction is conducted under mild, relatively low catalyst loading, and ligand- and base-free conditions.
    151. 151
      Sutton, J. M.; Clarke, O. J.; Fernandez, N.; Boyle, R. W. Porphyrin, Chlorin, and Bacteriochlorin Isothiocyanates: Useful Reagents for the Synthesis of Photoactive Bioconjugates. Bioconj. Chem. 2002, 13 (2), 249– 63,  DOI: 10.1021/bc015547x
      151
      Porphyrin, Chlorin, and Bacteriochlorin Isothiocyanates: Useful Reagents for the Synthesis of Photoactive Bioconjugates
      Sutton, J. M.; Clarke, O. J.; Fernandez, N.; Boyle, R. W.
      Bioconjugate Chemistry (2002), 13 (2), 249-263CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      A novel method for conjugating porphyrins and related mols. to proteins has been developed. The method, which involves synthesizing porphyrins, chlorins, and bacteriochlorins bearing a single amine-reactive isothiocyanate group represents a facile system for protein labeling with these photoactive species. Problems assocd. with the noncovalent binding of porphyrins to proteins are highlighted, and a method for purifying conjugates to yield exclusively covalently bound porphyrin protein species is demonstrated. Biol. activity of porphyrin-bovine serum albumin conjugates formed and purified by these methods is demonstrated using laser scanning confocal microscopy.
    152. 152
      Feuster, E. K.; Glass, T. E. Detection of Amines and Unprotected Amino Acids in Aqueous Conditions by Formation of Highly Fluorescent Iminium Ions. J. Am. Chem. Soc. 2003, 125 (52), 16174– 5,  DOI: 10.1021/ja036434m
      152
      Detection of amines and unprotected amino acids in aqueous conditions by formation of highly fluorescent iminium ions
      Feuster, Ellen K.; Glass, Timothy E.
      Journal of the American Chemical Society (2003), 125 (52), 16174-16175CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Properly substituted coumarin aldehydes can be used to detect amines and amino acids under neutral, high ionic strength conditions by the formation of highly fluorescent iminium ions. The fluorescence of one sensor increases by 26-fold upon the addn. of glycine. This strong florescent response is attributed to hydrogen bonding of the chromophore carbonyl by the acidic iminium proton.
    153. 153
      Patze, C.; Broedner, K.; Rominger, F.; Trapp, O.; Bunz, U. H. F. Aldehyde Cruciforms: Dosimeters for Primary and Secondary Amines. Eur. J. Chem. 2011, 17 (49), 13720– 5,  DOI: 10.1002/chem.201101871
      153
      Aldehyde Cruciforms: Dosimeters for Primary and Secondary Amines
      Patze, Christian; Broedner, Kerstin; Rominger, Frank; Trapp, Oliver; Bunz, Uwe H. F.
      Chemistry - A European Journal (2011), 17 (49), 13720-13725, S13720/1-S13720/9CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Aldehyde-substituted donor-acceptor cruciforms [1,4-bis(arylethynyl)-2,5-distyrylbenzenes] are useful dosimeters for primary amines, primary diamines, and secondary amines. The 1,n-diamines are particularly reactive towards this dosimeter and can be detected in <100 ppm concn. Using a single aldehyde-functionalized cruciform in seven different solvents allowed to discern fourteen different amines by digital photog. and statistical evaluation of the response patterns extd. as red, green, blue (RGB) values.
    154. 154
      Kumpf, J.; Bunz, U. H. F. Aldehyde-Appended Distyrylbenzenes: Amine Recognition in Water. Eur. J. Chem. 2012, 18 (29), 8921– 4,  DOI: 10.1002/chem.201200930
      154
      Aldehyde-Appended Distyrylbenzenes: Amine Recognition in Water
      Kumpf, Jan; Bunz, Uwe H. F.
      Chemistry - A European Journal (2012), 18 (29), 8921-8924, S8921/1-S8921/5CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We have synthesized two aldehyde-substituted distyrylbenzenes, 3 (I) and 8 (II), and investigated their reaction with amines in water. Both 3 and 8 formed imines or cyclic aminals depending on the structure of the employed amine. Turn on and blue shift of the fluorescence resulted. Both 3 and 8 gave fairly unique color responses towards biogenic amines and amino acids that can undergo aminal or thioacetal formation.
    155. 155
      Mohr, G. J.; Demuth, C.; Spichiger-Keller, U. E. Application of Chromogenic and Fluorogenic Reactands in the Optical Sensing of Dissolved Aliphatic Amines. Anal. Chem. 1998, 70 (18), 3868– 73,  DOI: 10.1021/ac980279q
      155
      Application of chromogenic and fluorogenic reactands in the optical sensing of dissolved aliphatic amines
      Mohr, Gerhard J.; Demuth, Caspar; Spichiger-Keller, Ursula E.
      Analytical Chemistry (1998), 70 (18), 3868-3873CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A new class of dyes was studied that reversibly interacts with aliph. amines, resulting in changes in absorbance or fluorescence. These dyes, which combine the properties of both a chem. reagent and a ligand, are termed reactands. When embedded in plasticized PVC membranes, the reactand 4-(N,N-dioctylamino)-4'-trifluoroacetylazobenzene (ETHT 4001) shows a significant signal change on exposure to aq. amine solns., with a decrease in absorbance around 490 nm and an increase in absorbance around 420 nm wavelength. This change in absorbance is caused by a conversion of the trifluoroacetyl group of the reactand into a hemiaminal or a zwitterion. Upon interaction with 1-butylamine, the sensor layer exhibits a dynamic range from 1 to 100 mM 1-butylamine, with max. relative signal changes of 90%. The limit of detection with this approach is 0.3 mM. The forward response time (t95) for a decade change in activity is 10 min, and the reverse response time is 5 min. The selectivity of ETHT 4001 toward amines correlates with the lipophilicity of the amines in plasticized PVC. Sterical factors addnl. affect selectivity in that the reactions of secondary, tertiary, and bulky primary amines with the trifluoroacetyl group are hindered. A similar response is obsd. for the fluorescent reactand 4-(N,N-dioctylamino)-4'-trifluoroacetylstilbene (ETHT 4004). When this reactand is exposed to 1-butylamine, a decrease in fluorescence around 580 nm is obsd., which can be ≤90% of the total fluorescence intensity. To prevent interferences from ions and ambient light, the sensor layer is coated with a protective layer of microporous white PTFE.
    156. 156
      Mohr, G. J. Tailoring the Sensitivity and Spectral Properties of a Chromoreactand for the Detection of Amines and Alcohols. Anal. Chim. Acta 2004, 508 (2), 233– 7,  DOI: 10.1016/j.aca.2003.12.005
      156
      Tailoring the sensitivity and spectral properties of a chromoreactand for the detection of amines and alcohols
      Mohr, Gerhard J.
      Analytica Chimica Acta (2004), 508 (2), 233-237CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
      The chromoreactand N,N-dioctylamino-4'-trifluoroacetyl-2'-nitroazobenzene (CR-546) is sensitive to amines and alcs. when dissolved in plasticized PVC. The chromoreactand provides an improvement over related reactands in that a nitro group meta to the trifluoroacetyl group enhances the chem. reactivity and, consequently, the sensitivity by a factor of 20 compared to the deriv. without nitro group. Also, the absorbance is shifted by around 40 nm to longer wavelengths, making the sensor membranes compatible with the green LED as a light source for a miniaturized sensor device. The sensor membrane responds to aq. 1-propylamine in the 0.5-50 mM concn. range and exhibits a detection limit of 0.1 mM. Response times are at 5-15 min. A fluorescent ethanol-sensitive membrane was obtained by dissolving the inert fluorophore N,N'-bis(1-hexylheptyl)perylene-3,4:9,10-bis(dicarboximide) together with CR-546 and the catalyst tridodecylmethylammonium chloride in the polymer matrix. The trifluoroacetyl form of CR-546 with its absorbance at ∼560 nm overlaps the emission of the fluorophore. Increasing ethanol concns. cause decreasing absorbance at 560 nm and consequently, increasing luminescence of the fluorophore. The resulting layer was evaluated for its detection of ethanol via changes in luminescence intensity. The calibration is linear at 1-15% (vol./vol.) ethanol with a limit of detection of 0.1% (vol./vol.). Response times are at 20-40 min for both forward and reverse reaction.
    157. 157
      Mohr, G. J. Chromo- and Fluororeactands: Indicators for Detection of Neutral Analytes by Using Reversible Covalent-Bond Chemistry. Eur. J. Chem. 2004, 10 (5), 1082– 90,  DOI: 10.1002/chem.200305524
      157
      Chromo- and fluororeactands: indicators for detection of neutral analytes by using reversible covalent-bond chemistry
      Mohr, Gerhard J.
      Chemistry - A European Journal (2004), 10 (5), 1082-1090CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Chromo- and fluororeactands are indicator dyes that allow the optical detection of elec. neutral analytes. Unlike complexing agents such as calixarenes, cyclodextrines or cyclophanes, reactands form a reversible covalent bond with the analyte mol. This chem. reaction causes strong changes in absorbance or fluorescence. In this article reactands for analytes such as amines, alcs., aldehydes, saccharides, carbon dioxide and sulfur dioxide are presented. Methods to enhance the sensitivity of the reactands as well as the operational and shelf lives of the corresponding optical sensors are discussed.
    158. 158
      Mohr, G. J.; Grummt, U.-W. Comparison of Trifluoroacetyl Monostyryl and Distyryl Dyes: Effects of Chromophore Elongation on the Spectral Properties and Chemical Reactivity. J. Fluoresc. 2006, 16 (2), 185– 90,  DOI: 10.1007/s10895-005-0047-7
      158
      Comparison of Trifluoroacetyl Monostyryl and Distyryl Dyes: effects of Chromophore Elongation on the Spectral Properties and Chemical Reactivity
      Mohr, Gerhard J.; Grummt, Ulrich-W.
      Journal of Fluorescence (2006), 16 (2), 185-190CODEN: JOFLEN; ISSN:1053-0509. (Springer)
      The trifluoroacetyl distyryl deriv. 1-[4-(2-{4-[2-(4-dibutylaminophenyl)-vinyl]-phenyl}-vinyl)-phenyl]-2,2,2-trifluoroethanone was compared with the related monostyryl deriv. 1-{4-[2-(4-dibutylaminophenyl)-vinyl]-phenyl}-2,2,2-trifluoroethanone with respect to spectral properties and sensitivity to amines. Both trifluoroacetyl derivs. had their absorbance max. at around 445 nm. The fluorescence of the distyryl dye, however, was obsd. at significantly longer wavelengths than that of the monostyryl dye, indicating the effect of structural extension of the chromophore system. Furthermore, the distyryl dye exhibited significantly smaller quantum yields in polar solvents than the monostyryl dye. Both dyes were capable of chem. reacting with amines in that their trifluoroacetyl function was converted into a hemiaminal. Consequently, absorbance and fluorescence of both dyes were shifted to shorter wavelengths. The positions of the fluorescence maxima of the spectra when converting from trifluoroacetyl to hemiaminal form were shifted by an almost identical amt. for both mono- and distyryl deriv. The hemiaminal form of the distyryl deriv., however, exhibited much larger quantum yields in both polar and nonpolar solvents than the hemiaminal form of the monostyryl dye. The structural extension of the chromophore affected the sensitivity to amines by enhancing the chem. reactivity of the distyryl dye over the monostyryl deriv.
    159. 159
      Mertz, E.; Beil, J. B.; Zimmerman, S. C. Kinetics and Thermodynamics of Amine and Diamine Signaling by a Trifluoroacetyl Azobenzene Reporter Group. Org. Lett. 2003, 5 (17), 3127– 30,  DOI: 10.1021/ol0351605
      159
      Kinetics and Thermodynamics of Amine and Diamine Signaling by a Trifluoroacetyl Azobenzene Reporter Group
      Mertz, Eric; Beil, James B.; Zimmerman, Steven C.
      Organic Letters (2003), 5 (17), 3127-3130CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      (Trifluoroacetyl)azobenzene dyes were previously employed as amine reporter groups (chemosensors) in a dendrimer-based monomol. imprinting system. Kinetic and binding studies with a range of amines and diamines show that the highly selective signaling obsd. for alkane diamines by these imprinted dendrimers arises from a kinetic effect due to intramol. general base-catalyzed carbinolamine formation with the dye itself. The relationship between diamine structure and carbinolamine stability and rate of formation is described.
    160. 160
      Körsten, S.; Mohr, G. J. Star-Shaped Tripodal Chemosensors for the Detection of Aliphatic Amines. Eur. J. Chem. 2011, 17 (3), 969– 75,  DOI: 10.1002/chem.201000787
      There is no corresponding record for this reference.
    161. 161
      Xu, Y.; Yu, S.; Wang, Y.; Hu, L.; Zhao, F.; Chen, X.; Li, Y.; Yu, X.; Pu, L. Ratiometric Fluorescence Sensors for 1,2-Diamines Based on Trifluoromethyl Ketones. Eur. J. Org. Chem. 2016, 2016 (35), 5868– 75,  DOI: 10.1002/ejoc.201601157
      161
      Ratiometric Fluorescence Sensors for 1,2-Diamines Based on Trifluoromethyl Ketones
      Xu, Yimang; Yu, Shanshan; Wang, Yachen; Hu, Lingling; Zhao, Feng; Chen, Xuemin; Li, Yinan; Yu, Xiaoqi; Pu, Lin
      European Journal of Organic Chemistry (2016), 2016 (35), 5868-5875CODEN: EJOCFK; ISSN:1099-0690. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Two BINOL-based trifluoromethyl aryl ketones 3 (I) and 4 (II) show greatly enhanced fluorescence at 375 nm accompanied by a decrease at 500 nm when treated with aliph. 1,2-diamines, esp. ethylenediamine, in org. solvents. Under the same conditions, other monoamines and diamines caused much smaller or no fluorescence response. Thus, both 3 and 4 can be used as highly selective ratiometric fluorescence sensors for ethylenediamine. Compd. 3, the BINOL hydroxyl groups of which are protected, exhibits greater fluorescence enhancement than the corresponding unprotected compd. 4. UV and NMR spectroscopic studies have been conducted to probe the mechanism of the sensor-substrate reaction.
    162. 162
      Ali, M. F. B.; Kishikawa, N.; Ohyama, K.; Mohamed, H. A.-M.; Abdel-Wadood, H. M.; Mahmoud, A. M.; Imazato, T.; Ueki, Y.; Wada, M.; Kuroda, N. Chromatographic Determination of Low-Molecular Mass Unsaturated Aliphatic Aldehydes with Peroxyoxalate Chemiluminescence Detection after Fluorescence Labeling with 4-(N,N-Dimethylaminosulfonyl)-7-Hydrazino-2,1,3-Benzoxadiazole. J. Chromatogr. B 2014, 953–954, 147– 52,  DOI: 10.1016/j.jchromb.2014.02.009
      162
      Chromatographic determination of low-molecular mass unsaturated aliphatic aldehydes with peroxyoxalate chemiluminescence detection after fluorescence labeling with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole
      Ali, Marwa Fathy Bakr; Kishikawa, Naoya; Ohyama, Kaname; Mohamed, Horria Abdel-Mageed; Abdel-Wadood, Hanaa Mohamed; Mahmoud, Ashraf Mohamed; Imazato, Takahiro; Ueki, Yukitaka; Wada, Mitsuhiro; Kuroda, Naotaka
      Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2014), 953-954 (), 147-152CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)
      A highly sensitive, selective and reproducible chromatog. method is described for detn. of low-mol. mass unsatd. aliph. aldehydes in human serum. The method combines fluorescent labeling using 4-(N,N-Dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole with peroxyoxalate chemiluminescence. The derivs. were sepd. on a reversed-phase column C8 isocratically using a mixt. of acetonitrile and 90 mM imidazole-HNO3 buffer (pH 6.4, 1:1, % vol./vol.). The calibration ranges were: 20-420 nM for methylglyoxal, 16-320 nM for acrolein, 15-360 nM for crotonaldehyde and 20-320 nM for trans-2-hexenal. The detection limits were ranged from 4.4 to 6.5 nM (88-130 fmol/injection), the recovery results were within the range of 87.4-103.8% and the intra and inter-day precision results were lower than 5.5%. The proposed validated method has been successfully applied to healthy, diabetic and rheumatic arthritis patients' sera with simple pretreatment method. In conclusion, this new method is suitable for routine anal. of large nos. of clin. samples for assessment of the oxidative stress state in patients.
    163. 163
      Xu, Y.; Yu, S.; Chen, Q.; Chen, X.; Li, Y.; Yu, X.; Pu, L. Fluorescent Recognition of 1,2-Diamines by a 1,1′-Binaphthyl-Based Trifluoromethyl Ketone. Eur. J. Chem. 2016, 22 (34), 12061– 7,  DOI: 10.1002/chem.201601540
      163
      Fluorescent Recognition of 1,2-Diamines by a 1,1'-Binaphthyl-Based Trifluoromethyl Ketone
      Xu, Yimang; Yu, Shanshan; Chen, Qi; Chen, Xuemin; Li, Yinan; Yu, Xiaoqi; Pu, Lin
      Chemistry - A European Journal (2016), 22 (34), 12061-12067CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The fluorescent responses of a 1,1'-binaphthol (BINOL)-based trifluoromethyl aryl ketone (I) toward a variety of amines were studied. The aliph. 1,2-diamines, esp. ethylenediamine, can greatly enhance the fluorescence of this compd., but under the same conditions, other mono- and diamines cause much smaller fluorescent responses. This compd. can be used as a fluorescent sensor for the detection of ethylenediamine at concns. over micromolar levels. UV absorption and NMR spectroscopic methods were used to study the interactions of the sensor with ethylenediamine. The trifluoromethyl ketone of the sensor reacts with ethylenediamine much more favorably than with other amines. The hydroxyl groups of the sensor and those of the hemiaminal adducts formed in the presence of the amines are important for the highly selective fluorescent response.
    164. 164
      Collados, J. F.; Solà, R.; Harutyunyan, S. R.; Maciá, B. Catalytic Synthesis of Enantiopure Chiral Alcohols Via Addition of Grignard Reagents to Carbonyl Compounds. ACS Catal. 2016, 6 (3), 1952– 70,  DOI: 10.1021/acscatal.5b02832
      164
      Catalytic Synthesis of Enantiopure Chiral Alcohols via Addition of Grignard Reagents to Carbonyl Compounds
      Collados, Juan F.; Sola, Ricard; Harutyunyan, Syuzanna R.; Macia, Beatriz
      ACS Catalysis (2016), 6 (3), 1952-1970CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      Remarkable progress in the enantioselective addn. of Grignard reagents to carbonyl compds. has been made over the past decade. This enantioselective transformation now allows the use of these challenging reactive nucleophiles for the formation of chiral alcs. using catalytic amts. of chiral ligands. This review summarizes the developments in this area.
    165. 165
      Malic, N.; Moorhoff, C.; Sage, V.; Saylik, D.; Teoh, E.; Scott, J. L.; Strauss, C. R. Toward Preparative Resolution of Chiral Alcohols by an Organic Chemical Method. New J. Chem. 2010, 34 (3), 398– 402,  DOI: 10.1039/b9nj00768g
      165
      Toward preparative resolution of chiral alcohols by an organic chemical method
      Malic, Nino; Moorhoff, Cornelis; Sage, Valerie; Saylik, Dilek; Teoh, Euneace; Scott, Janet L.; Strauss, Christopher R.
      New Journal of Chemistry (2010), 34 (3), 398-402CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
      Asym. alcs. were resolved as 1-α-O-alkyl-2,3-unsatd. hexosides. After sepn. of diastereoisomers, the auxiliary and the enantiomeric alc. were recovered by transglycosidation. Potential applications include resoln. of labile secondary and tertiary alcs., difficult by existing techniques, and enhancement of ees of chiral alcs. produced enzymically or by synthetic catalytic methods.
    166. 166
      Rong, J.; Pellegrini, T.; Harutyunyan, S. R. Synthesis of Chiral Tertiary Alcohols by CuI-Catalyzed Enantioselective Addition of Organomagnesium Reagents to Ketones. Eur. J. Chem. 2016, 22 (11), 3558– 70,  DOI: 10.1002/chem.201503412
      166
      Synthesis of Chiral Tertiary Alcohols by CuI-Catalyzed Enantioselective Addition of Organomagnesium Reagents to Ketones
      Rong, Jiawei; Pellegrini, Tilde; Harutyunyan, Syuzanna R.
      Chemistry - A European Journal (2016), 22 (11), 3558-3570CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Catalytic enantioselective addn. of organometallic nucleophiles to ketones is among the most straightforward approaches to the synthesis of chiral tertiary alcs. The first such catalytic methodologies using the highly reactive organomagnesium reagents, which are the preferred organometallic reagents in terms of cost, availability, atom efficiency, and structural diversity, were developed only during the last five years. This Concept article highlights the fundamental breakthrough that made the development of methodologies for highly enantioselective Cu(I)-catalyzed alkylation of ketones using organomagnesium reagents possible.
    167. 167
      Chen, B.-S.; Ribeiro de Souza, F. Z. Enzymatic Synthesis of Enantiopure Alcohols: Current State and Perspectives. RSC Adv. 2019, 9 (4), 2102– 15,  DOI: 10.1039/C8RA09004A
      167
      Enzymatic synthesis of enantiopure alcohols: current state and perspectives
      Chen, Bi-Shuang; Ribeiro de Souza, Fayene Zeferino
      RSC Advances (2019), 9 (4), 2102-2115CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      A review. Enantiomerically pure alcs., as key intermediates, play an essential role in the pharmaceutical, agrochem. and chem. industries. Among the methods used for their prodn., biotechnol. approaches are generally considered a green and effective alternative due to their mild reaction conditions and remarkable enantioselectivity. An increasing no. of enzymic strategies for the synthesis of these compds. has been developed over the years, among which seven primary methodologies can be distinguished as follows: (1) enantioselective water addn. to alkenes, (2) enantioselective aldol addn., (3) enantioselective coupling of ketones with hydrogen cyanide, (4) asym. redn. of carbonyl compds., (5) (dynamic) kinetic resoln. of racemates, (6) enantioselective hydrolysis of epoxides, and (7) stereoselective hydroxylation of unactivated C-H bonds. Some recent reviews have examd. these approaches sep.; however, to date, no review has included all the above mentioned strategies. The aim of this mini-review is to provide an overview of all seven enzymic strategies and draw conclusions on the effect of each approach.
    168. 168
      Kourist, R.; Dominguez de Maria, P.; Bornscheuer, U. T. Enzymatic Synthesis of Optically Active Tertiary Alcohols: Expanding the Biocatalysis Toolbox. ChemBioChem. 2008, 9 (4), 491– 8,  DOI: 10.1002/cbic.200700688
      168
      Enzymatic synthesis of optically active tertiary alcohols: expanding the biocatalysis toolbox
      Kourist, Robert; Dominguez de Maria, Pablo; Bornscheuer, Uwe T.
      ChemBioChem (2008), 9 (4), 491-498CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Enantiopure tertiary alcs. are very valuable building blocks for the synthesis of many different natural products and pharmaceuticals. As a consequence, several chem. and enzymic strategies to afford such chiral structures have been described. Promising enzymic approaches with agents such as epoxide hydrolases, dehalogenases and hydroxynitrile lyases have been reported, as well as dihydroxylation by microorganisms. Apart from those valuable options, the hydrolase-based kinetic resoln. of tertiary alcs. has been known for the last three decades, as several wild-type enzymes have been reported to be able to accept these sterically hindered mols. More recently, the existence of an amino acid motif within an enzyme's active site has been identified as highly relevant for the acceptance of such bulky structures. This discovery clearly facilitates the identification of novel biocatalysts for this application. Although several tertiary alcs. were successfully resolved with wild-type biocatalysts, enantioselectivities have often been too low for synthetic purposes. These limitations have recently been overcome by accessing enzymes from the metagenome through directed evolution or by rational protein design. This minireview describes the state of the art in this area, highlighting aspects of basic academic research into the practical application of biocatalysts for the synthesis of optically active tertiary alcs.
    169. 169
      Jiang, H.; Su, X.; Zhang, Y.; Zhou, J.; Fang, D.; Wang, X. Unexpected Thiols Triggering Photoluminescent Enhancement of Cytidine Stabilized Au Nanoclusters for Sensitive Assays of Glutathione Reductase and Its Inhibitors Screening. Anal. Chem. 2016, 88 (9), 4766– 71,  DOI: 10.1021/acs.analchem.6b00112
      169
      Unexpected Thiols Triggering Photoluminescent Enhancement of Cytidine Stabilized Au Nanoclusters for Sensitive Assays of Glutathione Reductase and Its Inhibitors Screening
      Jiang, Hui; Su, Xiaoqing; Zhang, Yuanyuan; Zhou, Junyu; Fang, Danjun; Wang, Xuemei
      Analytical Chemistry (Washington, DC, United States) (2016), 88 (9), 4766-4771CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      The photoluminescence (PL) of nonthiolate ligand capped Au nanoclusters (NCs) is usually quenched by thiols due to the tight adsorption of thiols to the Au surface and formation of larger non-PL species. However, we here report an unexpected PL enhancement of cytidine stabilized Au (AuCyt) NCs triggered by thiols, such as reduced glutathione (GSH) at sub-μM level, while such phenomena have not been obsd. for Au NCs capped with similar adenosine/cytidine nucleotides. The mass spectroscopic results indicate that this enhancement may be caused by the formation of smaller, but highly fluorescent, Au species etched by thiols. This enables the sensitive detection of GSH from 20 nM to 3 μM, with an ultralow detection limit of 2.0 nM. Moreover, the glutathione reductase (GR) activity can be detd. by the initial rate of GSH prodn., i.e., the max. PL increasing rate, with a linear range of 0.34-17.0 U/L (1 U means redn. of 1.0 μmol of oxidized glutathione per min at pH 7.6 at 25 °C) and a limit of detection of 0.34 U/L. This method allows the accurate assays of GR in clin. serum samples as well as the rapid screening of GR inhibitors, indicating its promising biomedical applications.
    170. 170
      Andexer, J.; von Langermann, J.; Mell, A.; Bocola, M.; Kragl, U.; Eggert, T.; Pohl, M. An R-Selective Hydroxynitrile Lyase from Arabidopsis Thaliana with an α/β-Hydrolase Fold. Angew. Chem., Int. Ed. 2007, 46 (45), 8679– 81,  DOI: 10.1002/anie.200701455
      170
      An R-selective hydroxynitrile lyase from arabidopsis thaliana with an α/β-hydrolase fold
      Andexer, Jennifer; von Langermann, Jan; Mell, Annett; Bocola, Marco; Kragl, Udo; Eggert, Thorsten; Pohl, Martina
      Angewandte Chemie, International Edition (2007), 46 (45), 8679-8681CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The noncyanogenic plant Arabidopsis thaliana contains a new hydroxynitrile lyase, which was cloned and characterized. This enzyme is readily available form a recombinant source, has a broad range of substrates, and enantioselectively transforms aliph. and arom. aldehydes as well as ketones into the corresponding R-cyanohydrins.
    171. 171
      Zheng, Y.-G.; Yin, H.-H.; Yu, D.-F.; Chen, X.; Tang, X.-L.; Zhang, X.-J.; Xue, Y.-P.; Wang, Y.-J.; Liu, Z.-Q. Recent Advances in Biotechnological Applications of Alcohol Dehydrogenases. Appl. Microbiol. Biotechnol. 2017, 101 (3), 987– 1001,  DOI: 10.1007/s00253-016-8083-6
      171
      Recent advances in biotechnological applications of alcohol dehydrogenases
      Zheng, Yu-Guo; Yin, Huan-Huan; Yu, Dao-Fu; Chen, Xiang; Tang, Xiao-Ling; Zhang, Xiao-Jian; Xue, Ya-Ping; Wang, Ya-Jun; Liu, Zhi-Qiang
      Applied Microbiology and Biotechnology (2017), 101 (3), 987-1001CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      Alc. dehydrogenases (ADHs), which belong to the oxidoreductase superfamily, catalyze the interconversion between alcs. and aldehydes or ketones with high stereoselectivity under mild conditions. ADHs are widely employed as biocatalysts for the dynamic kinetic resoln. of racemic substrates and for the prepn. of enantiomerically pure chems. This review provides an overview of biotechnol. applications for ADHs in the prodn. of chiral pharmaceuticals and fine chems.
    172. 172
      Huisman, G. W.; Liang, J.; Krebber, A. Practical Chiral Alcohol Manufacture Using Ketoreductases. Curr. Opin. Chem. Biol. 2010, 14 (2), 122– 9,  DOI: 10.1016/j.cbpa.2009.12.003
      172
      Practical chiral alcohol manufacture using ketoreductases
      Huisman, Gjalt W.; Liang, Jack; Krebber, Anke
      Current Opinion in Chemical Biology (2010), 14 (2), 122-129CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. Over the past two years the application of ketoreductases in the com. synthesis of chiral alcs. has undergone a revolution. Biocatalysts are now often the preferred catalyst for the synthesis of chiral alcs. via ketone redn. and are displacing reagents and chemocatalysts that only recently were considered break-through process solns. themselves. Tailor-made enzymes can now be generated from advanced, non-natural variants using HTP screening and modern mol. biol. techniques. At the same time, global economic and environmental pressures direct industrial process development toward versatile platforms that can be applied to the different stages of product development. We will discuss the technologies that have emerged over the past years that have guided biocatalysis from the bottom of the toolbox, to the power tool of choice.
    173. 173
      Iding, H.; Siegert, P.; Mesch, K.; Pohl, M. Application of α-Keto Acid Decarboxylases in Biotransformations. Biochim. Biophys. Acta 1998, 1385 (2), 307– 22,  DOI: 10.1016/S0167-4838(98)00076-4
      173
      Application of α-keto acid decarboxylases in biotransformations
      Iding, H.; Siegert, P.; Mesch, K.; Pohl, M.
      Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1998), 1385 (2), 307-322CODEN: BBAEDZ; ISSN:0167-4838. (Elsevier B.V.)
      A review with 145 refs. The advantages of using enzymes in the synthesis of org. compds. relate to their versatility, high reaction rates, and regio- and stereospecificity and the relatively mild reaction conditions involved. Stereospecificity is esp. important in the synthesis of bioactive mols., as only one of the enantiomeric forms usually manifests bioactivity, whereas the other is often toxic. Although enzymes which catalyze asym. carbon-carbon bond formation are of great importance in bioorg. chem., only a few examples are known for thiamin diphosphate (ThDP)-dependent enzymes, whereas transformations using e.g. aldolases, lipases and lyases are well documented already. The present review surveys recent work on the application of pyruvate decarboxylase and benzoylformate decarboxylase in org. synthesis. These enzymes catalyze the synthesis of chiral α-hydroxy ketones which are versatile building blocks for org. and pharmaceutical chem. Besides the substrate spectra of both enzymes amino acid residues relevant for substrate specificity and enantioselectivity of pyruvate decarboxylase have been investigated by site-directed mutagenesis.
    174. 174
      Lee, S.-H.; Yeom, S.-J.; Kim, S.-E.; Oh, D.-K. Development of Aldolase-Based Catalysts for the Synthesis of Organic Chemicals. Trends Biotechnol. 2022, 40, 306,  DOI: 10.1016/j.tibtech.2021.08.001
      174
      Development of aldolase-based catalysts for the synthesis of organic chemicals
      Lee, Seon-Hwa; Yeom, Soo-Jin; Kim, Seong-Eun; Oh, Deok-Kun
      Trends in Biotechnology (2022), 40 (3), 306-319CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
      A review. Aldol chems. are synthesized by condensation reactions between the carbon units of ketones and aldehydes using aldolases. The efficient synthesis of diverse org. chems. requires intrinsic modification of aldolases via engineering and design, as well as extrinsic modification through immobilization or combination with other catalysts. This review describes the development of aldolases, including their engineering and design, and the selection of desired aldolases using high-throughput screening, to enhance their catalytic properties and perform novel reactions. Aldolase-contg. catalysts, which catalyze the aldol reaction combined with other enzymic and/or chem. reactions, can efficiently synthesize diverse complex org. chems. using inexpensive and simple materials as substrates. We also discuss the current challenges and emerging solns. for aldolase-based catalysts.
    175. 175
      Bracco, P.; Busch, H.; von Langermann, J.; Hanefeld, U. Enantioselective Synthesis of Cyanohydrins Catalysed by Hydroxynitrile Lyases - a Review. Org. Biomol. Chem. 2016, 14 (27), 6375– 89,  DOI: 10.1039/C6OB00934D
      175
      Enantioselective synthesis of cyanohydrins catalysed by hydroxynitrile lyases - a review
      Bracco, Paula; Busch, Hanna; von Langermann, Jan; Hanefeld, Ulf
      Organic & Biomolecular Chemistry (2016), 14 (27), 6375-6389CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      A review. The first enantioselective synthesis was the selective addn. of cyanide to benzaldehyde catalyzed by a hydroxynitrile lyase (HNL). Since then these enzymes have been developed into a reliable tool in org. synthesis. HNLs to prep. either the (R)- or the (S)-enantiomer of the desired cyanohydrin are available and a wide variety of reaction conditions can be applied. As a result of this, numerous applications of these enzymes in org. synthesis have been described. Here the examples of the last decade are summarized, the enzyme catalyzed step is discussed and the follow-up chem. is shown. This proves HNLs to be part of main stream org. synthesis. Addnl. the newest approaches via immobilization and reaction engineering are introduced.
    176. 176
      Chen, B.-S.; Otten, L. G.; Hanefeld, U. Stereochemistry of Enzymatic Water Addition to C=C Bonds. Biotechnol. Adv. 2015, 33 (5), 526– 46,  DOI: 10.1016/j.biotechadv.2015.01.007
      176
      Stereochemistry of enzymatic water addition to C = C bonds
      Chen, Bi-Shuang; Otten, Linda G.; Hanefeld, Ulf
      Biotechnology Advances (2015), 33 (5), 526-546CODEN: BIADDD; ISSN:0734-9750. (Elsevier)
      A review. Water addn. to carbon-carbon double bonds using hydratases is attracting great interest in biochem. Most of the known hydratases are involved in primary metab. and to a lesser extent in secondary metab. New hydratases have recently been added to the toolbox, both from natural sources or artificial metalloenzymes. In order to comprehensively understand how the hydratases are able to catalyze the water addn. to carbon-carbon double bonds, this review will highlight the mechanistic and stereochem. studies of the enzymic water addn. to carbon-carbon double bonds, focusing on the syn/anti-addn. and stereochem. of the reaction.
    177. 177
      Archelas, A.; Furstoss, R. Synthetic Applications of Epoxide Hydrolases. Curr. Opin. Chem. Biol. 2001, 5 (2), 112– 9,  DOI: 10.1016/S1367-5931(00)00179-4
      177
      Synthetic applications of epoxide hydrolases
      Archelas, Alain; Furstoss, Roland
      Current Opinion in Chemical Biology (2001), 5 (2), 112-119CODEN: COCBF4; ISSN:1367-5931. (Elsevier Science Ltd.)
      A review with 58 refs. There have been several recent advances in the area of biocatalyzed hydrolytic kinetic resoln. of epoxides using "newly discovered" enzymes (i.e. epoxide hydrolases). These biocatalysts, two of which will become com. available in the near future, appear to be highly promising tools for fine org. synthesis, as they enable the prepn. of various epoxides and/or their corresponding diols in enantiopure form.
    178. 178
      Freakley, S. J.; Kochius, S.; van Marwijk, J.; Fenner, C.; Lewis, R. J.; Baldenius, K.; Marais, S. S.; Opperman, D. J.; Harrison, S. T. L.; Alcalde, M.; Smit, M. S.; Hutchings, G. J. A Chemo-Enzymatic Oxidation Cascade to Activate C-H Bonds with in Situ Generated H2O2. Nat. Commun. 2019, 10 (1), 4178,  DOI: 10.1038/s41467-019-12120-w
      178
      A chemo-enzymatic oxidation cascade to activate C-H bonds with in situ generated H2O2
      Freakley Simon J; Lewis Richard J; Hutchings Graham J; Freakley Simon J; Kochius Svenja; van Marwijk Jacqueline; Marais Sarel S; Opperman Diederik J; Smit Martha S; Kochius Svenja; van Marwijk Jacqueline; Fenner Caryn; Marais Sarel S; Opperman Diederik J; Harrison Susan T L; Smit Martha S; Fenner Caryn; Harrison Susan T L; Baldenius Kai; Alcalde Miguel
      Nature communications (2019), 10 (1), 4178 ISSN:.
      Continuous low-level supply or in situ generation of hydrogen peroxide (H2O2) is essential for the stability of unspecific peroxygenases, which are deemed ideal biocatalysts for the selective activation of C-H bonds. To envisage potential large scale applications of combined catalytic systems the reactions need to be simple, efficient and produce minimal by-products. We show that gold-palladium nanoparticles supported on TiO2 or carbon have sufficient activity at ambient temperature and pressure to generate H2O2 from H2 and O2 and supply the oxidant to the engineered unspecific heme-thiolate peroxygenase PaDa-I. This tandem catalyst combination facilitates efficient oxidation of a range of C-H bonds to hydroxylated products in one reaction vessel with only water as a by-product under conditions that could be easily scaled.
    179. 179
      Wang, Y.; Xiang, Q.; Zhou, Q.; Xu, J.; Pei, D. Mini Review: Advances in 2-Haloacid Dehalogenases. Front. Microbiol. 2021, 12, 758886,  DOI: 10.3389/fmicb.2021.758886
      179
      Mini Review: Advances in 2-Haloacid Dehalogenases
      Wang Yayue; Xiang Qiao; Zhou Qingfeng; Pei Dongli; Xiang Qiao; Xu Jingliang; Xu Jingliang
      Frontiers in microbiology (2021), 12 (), 758886 ISSN:1664-302X.
      The 2-haloacid dehalogenases (EC 3.8.1.X) are industrially important enzymes that catalyze the cleavage of carbon-halogen bonds in 2-haloalkanoic acids, releasing halogen ions and producing corresponding 2-hydroxyl acids. These enzymes are of particular interest in environmental remediation and environmentally friendly synthesis of optically pure chiral compounds due to their ability to degrade a wide range of halogenated compounds with astonishing efficiency for enantiomer resolution. The 2-haloacid dehalogenases have been extensively studied with regard to their biochemical characterization, protein crystal structures, and catalytic mechanisms. This paper comprehensively reviews the source of isolation, classification, protein structures, reaction mechanisms, biochemical properties, and application of 2-haloacid dehalogenases; current trends and avenues for further development have also been included.
    180. 180
      Koudelakova, T.; Bidmanova, S.; Dvorak, P.; Pavelka, A.; Chaloupkova, R.; Prokop, Z.; Damborsky, J. Haloalkane Dehalogenases: Biotechnological Applications. Biotechnol. J. 2013, 8 (1), 32– 45,  DOI: 10.1002/biot.201100486
      180
      Haloalkane dehalogenases: Biotechnological applications
      Koudelakova, Tana; Bidmanova, Sarka; Dvorak, Pavel; Pavelka, Antonin; Chaloupkova, Radka; Prokop, Zbynek; Damborsky, Jiri
      Biotechnology Journal (2013), 8 (1), 32-45CODEN: BJIOAM; ISSN:1860-6768. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Haloalkane dehalogenases (EC 3.8.1.5, HLDs) are α/β-hydrolases which act to cleave carbon-halogen bonds. Due to their unique catalytic mechanism, broad substrate specificity and high robustness, the members of this enzyme family have been employed in several practical applications: (i) biocatalytic prepn. of optically pure building-blocks for org. synthesis; (ii) recycling of byproducts from chem. processes; (iii) bioremediation of toxic environmental pollutants; (iv) decontamination of warfare agents; (v) biosensing of environmental pollutants; and (vi) protein tagging for cell imaging and protein anal. This review discusses the application of HLDs in the context of the biochem. properties of individual enzymes. Further extension of HLD uses within the field of biotechnol. will require currently limiting factors - such as low expression, product inhibition, insufficient enzyme selectivity, low affinity and catalytic efficiency towards selected substrates, and instability in the presence of org. co-solvents - to be overcome. We propose that strategies based on protein engineering and isolation of novel HLDs from extremophilic microorganisms may offer solns.
    181. 181
      Schallmey, A.; Schallmey, M. Recent Advances on Halohydrin Dehalogenases-from Enzyme Identification to Novel Biocatalytic Applications. Appl. Microbiol. Biotechnol. 2016, 100 (18), 7827– 39,  DOI: 10.1007/s00253-016-7750-y
      181
      Recent advances on halohydrin dehalogenases- from enzyme identification to novel biocatalytic applications
      Schallmey, Anett; Schallmey, Marcus
      Applied Microbiology and Biotechnology (2016), 100 (18), 7827-7839CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      A review. Halohydrin dehalogenases are industrially relevant enzymes that catalyze the reversible dehalogenation of vicinal haloalcs. with formation of the corresponding epoxides. In the reverse reaction, also other neg. charged nucleophiles such as azide, cyanide, or nitrite are accepted besides halides to open the epoxide ring. Thus, novel C-N, C-C, or C-O bonds can be formed by halohydrin dehalogenases, which makes them attractive biocatalysts for the prodn. of various β-substituted alcs. Despite the fact that only five individual halohydrin dehalogenase enzyme sequences have been known until recently enabling their heterologous prodn., a large no. of different biocatalytic applications have been reported using these enzymes. The recent characterization of specific sequence motifs has facilitated the identification of novel halohydrin dehalogenase sequences available in public databases and has largely increased the no. of recombinantly available enzymes. These will help to extend the biocatalytic repertoire of this enzyme family and to foster novel biotechnol. applications and developments in the future. This review gives a general overview on the halohydrin dehalogenase enzyme family and their biochem. properties and further focuses on recent developments in halohydrin dehalogenase biocatalysis and protein engineering.
    182. 182
      Belafriekh, A.; Secundo, F.; Serra, S.; Djeghaba, Z. Enantioselective Enzymatic Resolution of Racemic Alcohols by Lipases in Green Organic Solvents. Tetrahedron: Asymmetry 2017, 28 (3), 473– 8,  DOI: 10.1016/j.tetasy.2017.02.004
      182
      Enantioselective enzymatic resolution of racemic alcohols by lipases in green organic solvents
      Belafriekh, Abderahmane; Secundo, Francesco; Serra, Stefano; Djeghaba, Zeineddine
      Tetrahedron: Asymmetry (2017), 28 (3), 473-478CODEN: TASYE3; ISSN:0957-4166. (Elsevier Ltd.)
      The effects of two eco-friendly solvents, 2-methyltetrahydrofuran (MeTHF) and cyclopentyl Me ether (CPME), on the enzyme activity and enantioselectivity of Novozym 435, Candida rugosa lipase (CRL), Porcine pancreas lipase (PPL), Lipase AK, Lipase PS, and Lipozyme, a series of com. lipases, in the enantioselective transesterfications of racemic menthol, racemic sulcatol and racemic α-cyclogeraniol were studied. Vinyl acetate was chosen as the acyl donor and the reactions were carried out at water activity 0.06. The activity of lipases in CPME was similar to that obsd. in other largely employed org. solvents [toluene and tert-Bu Me ether (MTBE)], and was slightly lower in MeTHF. However, for most of the lipases tested, the enantioselectivity was higher in the eco-friendly solvents. Lipase AK exhibited a high enantioselectivity (E = 232) for the resoln. of racemic menthol but the reaction rate was low. Lipase formulation (the enzyme was frozen and lyophilized in potassium phosphate buffer without and with 5% (w/v) of sucrose, D-mannitol, or methoxy poly(ethylene glycol)) was tested with this lipase in order to improve its activity, which increased up to 4.5 times, compared to the untreated enzyme. CALB was found to be a useful biocatalyst for the resoln. of racemic sulcatol, where high activity and enantioselectivity were obtained (E ≥ 1000). For the resoln. of the racemic primary alc. α-cyclogeraniol, most of the lipases tested were active but not enantioselective, except lipase PS which displayed a moderate enantioselectivity (E = 19). The effect of the presence of a low percentage of two ionic liqs. (ILs) 1-Butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([BMIM][TFSI]) (5% (vol./vol.)) and 1-Butyl-3-methylimidazoliumtetrafluoroborate ([BMIM][BF4]) (1% (vol./vol.)) in the medium was also investigated. Only in the case of CRL the ILs slightly increased the enantioselectivity from E = 91 to E = 103 and E = 120 for [BMIM][TFSI] and [BMIM][BF4], resp. However, in all cases ILs caused a decrease of enzyme activity.
    183. 183
      Bustos-Jaimes, I.; Hummel, W.; Eggert, T.; Bogo, E.; Puls, M.; Weckbecker, A.; Jaeger, K.-E. A High-Throughput Screening Method for Chiral Alcohols and Its Application to Determine Enantioselectivity of Lipases and Esterases. ChemCatChem. 2009, 1 (4), 445– 8,  DOI: 10.1002/cctc.200900190
      183
      A High-Throughput Screening Method for Chiral Alcohols and its Application to Determine Enantioselectivity of Lipases and Esterases
      Bustos-Jaimes, Ismael; Hummel, Werner; Eggert, Thorsten; Bogo, Eliane; Puls, Michael; Weckbecker, Andrea; Jaeger, Karl-Erich
      ChemCatChem (2009), 1 (4), 445-448CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Chiral alcs. are valuable intermediates in the synthesis of pharmaceutical, agricultural, and fine chems., which can be produced either by hydrocarbon oxidn., ketone redn., or ester hydrolysis. Nevertheless, these reactions usually produce non-enantiopure compds. For this reason, several methods for the enantioselective synthesis of alcs. have been developed, which range from the synthesis of catalysts by combinatorial chem. to the in vitro directed evolution of enzymes. In any case, high-throughput methods need to be applied to measure the enantiomeric excess [ee] or enantiopurity of the produced alcs. within a large no. of samples. Several methods for high-throughput screening for enantioselectivity of catalysts have been reported, including electrospray ionization coupled to mass spectrometry, HPLC coupled to CD, FTIR spectroscopy, and enzymic methods. Some of these sophisticated methods require, however, isotopically labeled pseudo-enantiomers for the assay and occasionally expensive equipment. Herein, a new colorimetric method is reported for the evaluation of the ee values of alcs. based on enantioselective alc. dehydrogenases (ADHs) coupled to a NADP (NADPH) oxidase (diaphorase) and its successful application in directed evolution for the screening of mutant libraries of lipases for enantioselective ester hydrolysis. The assay is based on the enantioselective oxidn. of alcs. by two different ADHs assayed sep. in parallel assays: the (R)-specific ADH from Lactobacillus kefir (LKADH) and the (S)-specific ADH from Rhodococcus erythropolis (READH), of which enantioselectivities and catalytic properties have been reported. The oxidn. of either (R)-1 or (S)-1 produces NAD(P)H, which is again oxidized to NAD(P) by diaphorase from Clostridium kluyveri with the concomitant redn. of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium (INT) to its corresponding red formazan deriv. The formation of this dye can be easily followed at 492 nm. The reaction is carried out within five minutes, during which the slope of color development over time is linear. The regeneration of the oxidized form of the coenzyme also ensures high reaction rates of ADHs. As in any other coupled assay, the amt. of diaphorase, the coupling enzyme, was kept in excess relative to the ADH enzymes in order to follow first-order kinetics.
    184. 184
      Chen, B.-S.; Resch, V.; Otten, L. G.; Hanefeld, U. Enantioselective Michael Addition of Water. Eur. J. Chem. 2015, 21 (7), 3020– 30,  DOI: 10.1002/chem.201405579
      185
      Enantioselective Michael Addition of Water
      Chen, Bi-Shuang; Resch, Verena; Otten, Linda G.; Hanefeld, Ulf
      Chemistry - A European Journal (2015), 21 (7), 3020-3030CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The enantioselective Michael addn. using water as both nucleophile and solvent has to date proved beyond the ability of synthetic chemists. Herein, the direct, enantioselective Michael addn. of water in water to prep. important β-hydroxy carbonyl compds. using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. Deuterium labeling studies demonstrate that a Michael hydratase catalyzes the water addn. exclusively with anti-stereochem.
    185. 185
      Lehwald, P.; Richter, M.; Röhr, C.; Liu, H.-w.; Müller, M. Enantioselective Intermolecular Aldehyde-Ketone Cross-Coupling through an Enzymatic Carboligation Reaction. Angew. Chem., Int. Ed. 2010, 49 (13), 2389– 92,  DOI: 10.1002/anie.200906181
      186
      Enantioselective Intermolecular Aldehyde-Ketone Cross-Coupling through an Enzymatic Carboligation Reaction
      Lehwald, Patrizia; Richter, Michael; Roehr, Caroline; Liu, Hung-wen; Mueller, Michael
      Angewandte Chemie, International Edition (2010), 49 (13), 2389-2392, S2389/1-S2389/27CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Asym. aldehyde-ketone cross coupling reaction of pyruvate (as acetaldehyde synthon) with cyclic or acyclic ketones, 1,2-diketones, keto esters, and substituted benzaldehydes (R1COR2 in general) catalyzed by ThDP-dependent flavoenzyme YerE of Yersinia pseudotuberculosis O:VI afforded the tertiary alcs. MeCOC(OH)R1R2 in ee of up to 96%. The 2-keto acid pyruvate was the best donor substrate for the enzyme.
    186. 186
      Hammer, S. C.; Marjanovic, A.; Dominicus, J. M.; Nestl, B. M.; Hauer, B. Squalene Hopene Cyclases Are Protonases for Stereoselective Brønsted Acid Catalysis. Nat. Chem. Biol. 2015, 11 (2), 121– 6,  DOI: 10.1038/nchembio.1719
      187
      Squalene hopene cyclases are protonases for stereoselective Bronsted acid catalysis
      Hammer, Stephan C.; Marjanovic, Antonija; Dominicus, Joerg M.; Nestl, Bettina M.; Hauer, Bernhard
      Nature Chemical Biology (2015), 11 (2), 121-126CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      For many important reactions catalyzed in chem. labs., the corresponding enzymes are missing, representing a restriction in biocatalysis. Although nature provides highly developed machineries appropriate to catalyze such reactions, their potential is often ignored. This also applies to Bronsted acid catalysis, a powerful method to promote a myriad of chem. transformations. Here, we report on the unique protonation machinery of a squalene hopene cyclase (SHC). Active site engineering of this highly evolvable enzyme yielded a platform for enzymic Bronsted acid catalysis in water. This is illustrated by activation of different functional groups (alkenes, epoxides and carbonyls), enabling the highly stereoselective syntheses of various cyclohexanoids while uncoupling SHC from polycyclization chem. This work highlights the potential of systematic investigation on nature's catalytic machineries to generate unique catalysts.
    187. 187
      Loskot, S. A.; Romney, D. K.; Arnold, F. H.; Stoltz, B. M. Enantioselective Total Synthesis of Nigelladine a Via Late-Stage C-H Oxidation Enabled by an Engineered P450 Enzyme. J. Am. Chem. Soc. 2017, 139 (30), 10196– 9,  DOI: 10.1021/jacs.7b05196
      188
      Enantioselective Total Synthesis of Nigelladine A via Late-Stage C-H Oxidation Enabled by an Engineered P450 Enzyme
      Loskot, Steven A.; Romney, David K.; Arnold, Frances H.; Stoltz, Brian M.
      Journal of the American Chemical Society (2017), 139 (30), 10196-10199CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      An enantioselective total synthesis of the norditerpenoid alkaloid nigelladine A is described. Strategically, the synthesis relies on a late-stage C-H oxidn. of an advanced intermediate. While traditional chem. methods failed to deliver the desired outcome, an engineered cytochrome P 450 enzyme was employed to effect a chemo- and regioselective allylic C-H oxidn. in the presence of four oxidizable positions. The enzyme variant was readily identified from a focused library of three enzymes, allowing for completion of the synthesis without the need for extensive screening.
    188. 188
      Zanon, J. P.; Peres, M. F. S.; Gattás, E. A. L. Colorimetric Assay of Ethanol Using Alcohol Dehydrogenase from Dry Baker’s Yeast. Enzyme Microb. Technol. 2007, 40 (3), 466– 70,  DOI: 10.1016/j.enzmictec.2006.07.029
      189
      Colorimetric assay of ethanol using alcohol dehydrogenase from dry baker's yeast
      Zanon, Juliana P.; Peres, Maristela F. S.; Gattas, Edwil A. L.
      Enzyme and Microbial Technology (2007), 40 (3), 466-470CODEN: EMTED2; ISSN:0141-0229. (Elsevier B.V.)
      Alc. dehydrogenases (ADHs) are oxidoreductases present in animal tissues, plants, and microorganisms. These enzymes attract major scientific interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in synthesis, thanks to their broad substrate specificity and stereoselectivity. In the present study, the standardization of the activity of the alc. dehydrogenase from baker's yeast was accomplished, and the pH and temp. stability showed, that the enzyme presented a high stability to pH 6.0-7.0 and the thermal stability were completely maintained up to 50 °C during 1 h. The assays of EtOH (detection range 1-5 mM or 4.6 × 10-2 to 23.0 × 10-2 g/L) in different samples in alc. beverages, presented a max. deviation of only 7.2%. The std. curve and the analytic curve of this method meet the conditions of precision, sensitivity, simplicity, and low cost, required for a useable anal. method.
    189. 189
      Baker, J. L.; Faustoferri, R. C.; Quivey, R. G., Jr. A Modified Chromogenic Assay for Determination of the Ratio of Free Intracellular NAD(+)/NADH in Streptococcus Mutans. Bio Protoc. 2016, 6 (16), e1902,  DOI: 10.21769/BioProtoc.1902
      There is no corresponding record for this reference.
    190. 190
      Yang, Y.; Liu, J.; Li, Z. Engineering of P450pyr Hydroxylase for the Highly Regio- and Enantioselective Subterminal Hydroxylation of Alkanes. Angew. Chem., Int. Ed. 2014, 53 (12), 3120– 4,  DOI: 10.1002/anie.201311091
      191
      Engineering of P450pyr Hydroxylase for the Highly Regio- and Enantioselective Subterminal Hydroxylation of Alkanes
      Yang, Yi; Liu, Ji; Li, Zhi
      Angewandte Chemie, International Edition (2014), 53 (12), 3120-3124CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Terminal-selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio- and enantioselectivity. A sensitive colorimetric high-throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative satn. mutagenesis, sextuple-mutant P450pyrSM1 was created for the hydroxylation of n-octane (1) to give (S)-2-octanol (2) with 98 % ee and >99 % subterminal selectivity. The engineered P 450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the C-H activation and functionalization of alkanes and the prepn. of enantiopure alcs. Mol. modeling provided structure-based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple-mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene (3) to afford (S)-1-phenyl-2-propanol (4) with 95 % ee and 98 % subterminal selectivity.
    191. 191
      Lauchli, R.; Rabe, K. S.; Kalbarczyk, K. Z.; Tata, A.; Heel, T.; Kitto, R. Z.; Arnold, F. H. High-Throughput Screening for Terpene-Synthase-Cyclization Activity and Directed Evolution of a Terpene Synthase. Angew. Chem., Int. Ed. 2013, 52 (21), 5571– 4,  DOI: 10.1002/anie.201301362
      192
      High-Throughput Screening for Terpene-Synthase-Cyclization Activity and Directed Evolution of a Terpene Synthase
      Lauchli, Ryan; Rabe, Kersten S.; Kalbarczyk, Karolina Z.; Tata, Amulya; Heel, Thomas; Kitto, Rebekah Z.; Arnold, Frances H.
      Angewandte Chemie, International Edition (2013), 52 (21), 5571-5574CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A synthetic substrate enables a colorimetric screen for terpene synthase cyclization activity, thereby facilitating the engineering of these enzymes. By using directed evolution, the thermostability of a sesquiterpene synthase was increased without the loss of other properties. The technique also enabled rapid optimization of conditions for expression and stabilization in lysate of another terpene synthase.
    192. 192
      Jung, E.; Park, B. G.; Yoo, H.-W.; Kim, J.; Choi, K.-Y.; Kim, B.-G. Semi-Rational Engineering of CYP153A35 to Enhance Ω-Hydroxylation Activity toward Palmitic Acid. Appl. Microbiol. Biotechnol. 2018, 102 (1), 269– 77,  DOI: 10.1007/s00253-017-8584-y
      193
      Semi-rational engineering of CYP153A35 to enhance ω-hydroxylation activity toward palmitic acid
      Jung, Eunok; Park, Beom Gi; Yoo, Hee-Wang; Kim, Joonwon; Choi, Kwon-Young; Kim, Byung-Gee
      Applied Microbiology and Biotechnology (2018), 102 (1), 269-277CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed satn. mutagenesis was attempted using a semi-rational approach that combined structure-based computational anal. and subsequent satn. mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P 450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover no. (TTN) and catalytic efficiency (kcat/KM) toward palmitic acid compared to wild-type, resp. However, in whole-cell reaction, D131S mutant showed only 50% improvement in ω-hydroxylated palmitic acid yield compared to the wild type. Docking simulation studies explained that the effect of D131S mutation on the catalytic activity would be mainly caused by the binding pose of fatty acids in the substrate access tunnel of the enzyme. This effect of D131S mutation on the catalytic activity is synergistic with that of the mutations in the active site previously reported.
    193. 193
      Francisco, W. A.; Abu-Soud, H. M.; Baldwin, T. O.; Raushel, F. M. Interaction of Bacterial Luciferase with Aldehyde Substrates and Inhibitors. J. Biol. Chem. 1993, 268 (33), 24734– 41,  DOI: 10.1016/S0021-9258(19)74526-8
      194
      Interaction of bacterial luciferase with aldehyde substrates and inhibitors
      Francisco, Wilson A.; Abu-Soud, Husam M.; Baldwin, Thomas O.; Raushel, Frank M.
      Journal of Biological Chemistry (1993), 268 (33), 24734-41CODEN: JBCHA3; ISSN:0021-9258.
      Bacterial luciferase catalyzes the reaction of FMNH2, O2, and an aliph. aldehyde to yield the carboxylic acid, FMN, water and blue-green light. The kinetics of the bacterial luciferase reaction were measured by stopped-flow spectrophotometry at pH 7 and 25°C for the series of aldehydes from n-heptanal to n-undecanal. The rate of formation of the 4a-hydroperoxyflavin intermediate was dependent on the aldehyde concn. when mixts. of enzyme, FMNH2, and aldehyde were rapidly mixed with O2. At satg. aldehyde, the rate of formation of this intermediate was 100-fold slower than in the absence of aldehyde, demonstrating that an enzyme·FMNH2·aldehyde complex can be formed. Numerical simulation of the time courses for these expts. supported the formation of this intermediate and its direct reaction with O2. The kinetics of the light emitting reaction were dependent upon the chain length of the aldehyde substrate. Although the initial light intensity and the light emission decay rate were different for each aldehyde, the quantum yield for the reaction was independent of the aldehyde used. Luciferase was inhibited by high levels of the aldehyde substrate when the enzyme was assayed by mixing FMNH2 with an aerobic mixt. of enzyme and aldehyde. The extent of inhibition was dependent on the particular aldehyde used, and the binding affinity of the aldehyde for the free enzyme increased in parallel with the aldehyde chain length. The kinetics of the formation and decay of the various intermediates were also studied in the presence of n-alkylaldehyde analogs. These compds. decreased the rate of formation of the 4a-hydroperoxyflavin intermediate in much the same way as the aldehyde substrate, presumably by the formation of the enzyme·FMNH2·analog ternary complex.
    194. 194
      Minak-Bernero, V.; Bare, R. E.; Haith, C. E.; Grossman, M. J. Detection of Alkanes, Alcohols, and Aldehydes Using Bioluminescence. Biotechnol. Bioeng. 2004, 87 (2), 170– 7,  DOI: 10.1002/bit.20089
      195
      Detection of alkanes, alcohols, and aldehydes using bioluminescence
      Minak-Bernero, Vera; Bare, Richard E.; Haith, Copper E.; Grossman, Matthew J.
      Biotechnology and Bioengineering (2004), 87 (2), 170-177CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
      We report a novel method for the rapid, sensitive, and quant. detection of alkanes, alcs., and aldehydes that relies on the reaction of bacterial luciferase with an aldehyde, resulting in the emission of light. Primary alcs. with corresponding aldehydes that are within the substrate range of the particular luciferase are detected after conversion to the aldehyde by an alc. dehydrogenase. In addn., alkanes themselves may be detected by conversion to primary alcs. by an alkane hydroxylase, followed by conversion to the aldehyde by alc. dehydrogenase. We developed a rapid bioluminescent method by genetically engineering the genes encoding bacterial luciferase, alc. dehydrogenase, and alkane hydroxylase into a plasmid for simultaneous expression in an E. coli host cell line. Alkanes, alcs., or aldehydes were detected within seconds, with sensitivity in the micromolar range, by measuring the resulting light emission with a microplate reader. We demonstrate the application of this method for the detection of alkanes, alcs., and aldehydes and for the detection of alkane hydroxylase and alc. dehydrogenase activity in vivo. This method is amenable to the high-throughput screening needs required for the identification of novel catalysts.
    195. 195
      Greene, L. E.; Lincoln, R.; Krumova, K.; Cosa, G. Development of a Fluorogenic Reactivity Palette for the Study of Nucleophilic Addition Reactions Based on meso-Formyl BODIPY Dyes. ACS Omega 2017, 2 (12), 8618– 24,  DOI: 10.1021/acsomega.7b01795
      196
      Development of a Fluorogenic Reactivity Palette for the Study of Nucleophilic Addition Reactions Based on meso-Formyl BODIPY Dyes
      Greene, Lana E.; Lincoln, Richard; Krumova, Katerina; Cosa, Gonzalo
      ACS Omega (2017), 2 (12), 8618-8624CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
      We describe herein a fluorescence-based assay to characterize and report on nucleophilic addn. to carbonyl moieties and highlight the advantages a fluorescence-based assay and multiplex anal. can offer. The assay relies on the fluorogenic properties of meso-formyl boron-dipyrromethene (BODIPY) dyes that become emissive following nucleophilic addn. A reactivity palette is assembled based on the increasing electrophilic character of five meso-formyl BODIPY compds. tested. We show that increasing rates of emission enhancement correlate with the decreasing electrophilic character of BODIPY dyes in the presence of an acid catalyst and a nucleophile. These results are consistent with the rate-limiting step involving activation of the electrophile. Increasing product formation is shown to correlate with the increasing electrophilic character of the BODIPY dyes, as expected based on thermodn. In addn. to providing rates of reaction, anal. of the fluorescence parameters for the reaction mixts., including emission quantum yields and fluorescence lifetimes, enables us to det. the extent of reactant conversion at equil. (in our case the estd. yield of a transient species) and the presence of different products, without the need for isolation. We anticipate that our reactivity palette approach, combined with the in-depth fluorescence anal. discussed herein, will provide guidelines toward developing fluorogenic assays of reactivity offering multiplex information, beyond fluorescence intensity.
    196. 196
      Lincoln, R.; Greene, L. E.; Bain, C.; Flores-Rizo, J. O.; Bohle, D. S.; Cosa, G. When Push Comes to Shove: Unravelling the Mechanism and Scope of Nonemissive meso-Unsaturated BODIPY Dyes. J. Phys. Chem. B 2015, 119 (13), 4758– 65,  DOI: 10.1021/acs.jpcb.5b02080
      197
      When Push Comes to Shove: Unravelling the Mechanism and Scope of Nonemissive meso-Unsaturated BODIPY Dyes
      Lincoln, Richard; Greene, Lana E.; Bain, Cheryl; Flores-Rizo, Juan O.; Bohle, D. Scott; Cosa, Gonzalo
      Journal of Physical Chemistry B (2015), 119 (13), 4758-4765CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      We report herein spectroscopy and computational results that illustrate an efficient intramol. deactivation pathway for meso-unsatd. boron-dipyrromethene (BODIPY) dyes in their singlet excited state. Our results show that the mechanism hinges on the structural flexibility imparted by the boron atom and on the energetic stabilization conferred by extending the conjugation into the meso substituent, which is otherwise unconjugated in the ground state. Following photoexcitation, rotation along the dihedral angle of the meso-unsatd. group results in its conjugation at the expense of shifting one pyrrole moiety in dipyrrin out of the plane. Internal conversion to an energetically hot, ground-state species efficiently competes with emission. The mechanism applies to meso-vinyl, -formyl, and -iminyl moieties. The presence of Me groups at positions C1 and C7 exacerbates the energetic penalty toward conjugation of the meso groups leading to a small energy gap between relaxed excited state and ground state and undetected emission quantum yields. Importantly, methyls at C1 and C7 prevent nonradiative deactivation in meso-aryl moieties, illustrating that when push comes to shove, the energetic (kinetic) barrier toward reaching conjugation is too large for aryl moieties but low enough for smaller groups to effectively compete with radiative transitions. Wisely chosen meso-unsatd. BODIPY dyes may serve as richly sensitive platforms for the prepn. of novel fluorogenic substrates to monitor chem. reactions or to probe the rigidity of their surrounding environment.
    197. 197
      Sawminathan, S.; Iyer, S. K. A New Imidazole Based Phenanthridine Probe for Ratiometric Fluorescence Monitoring of Methanol in Biodiesel. New J. Chem. 2021, 45 (13), 6033– 41,  DOI: 10.1039/D0NJ06252A
      198
      A new imidazole based phenanthridine probe for ratiometric fluorescence monitoring of methanol in biodiesel
      Sawminathan, Sathish; Iyer, Sathiyanarayanan Kulathu
      New Journal of Chemistry (2021), 45 (13), 6033-6041CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
      We prepd. and characterized an array of polarity-sensitive fluorescent dyes (7 and 9a, 9b and 9c). Fluorescent dyes are based on imidazole cores, suitably functionalized with a conjugated push-pull system. The photophys. properties of the synthesized materials were elaborately investigated. These new fluorescent probes showed good fluorecence emission, higher .vphi.Fl values and large Stokes shifts in different org. solvents because of their ICT nature. Furthermore, with aldehyde groups as the recognition sites, all the dyes could act as ratiometric fluorescent sensors towards methanol. The selective ratiometric fluorescence response towards methanol is due to the strong intermol. H-bonding between the aldehyde unit and methanol. The detection limit of methanol was found to be at the ppm level. Methanol sensing was confirmed using emission spectroscopy, FTIR studies and theor. calcns. Addnl., these dyes were used for the detection of methanol in biodiesel and in test paper strips.
    198. 198
      Mohr, G. J.; Spichiger-Keller, U. E. Novel Fluorescent Sensor Membranes for Alcohols Based on p-N,N-Dioctylamino-4′-Trifluoroacetylstilbene. Anal. Chim. Acta 1997, 351 (1), 189– 96,  DOI: 10.1016/S0003-2670(97)00365-6
      199
      Novel fluorescent sensor membranes for alcohols based on p-N,N-Dioctylamino-4'-trifluoroacetylstilbene
      Mohr, Gerhard J.; Spichiger-Keller, Ursula E.
      Analytica Chimica Acta (1997), 351 (1-3), 189-196CODEN: ACACAM; ISSN:0003-2670. (Elsevier)
      A chromogenic ligand was described that reversibly interacts with alcs. in a sensor, resulting in a change in both absorbance and fluorescence. When embedded in membranes composed of plasticized PVC, p-N,N-dioctylamino-4'-trifluoroacetylstilbene (ETH 4004) showed a significant signal change on exposure to aq. EtOH with a decrease in absorbance at 453 nm and an increase in absorbance at 373 nm. At the same time, the fluorescence at 576 nm decreased accordingly when excited at 452 nm. The sensor layer exhibited a dynamic range from 1-50 (vol.%) EtOH, with the highest sensitivity at 5-40 (vol.%). The fluorescence of the sensor membrane was virtually insensitive to changes in pH. However, the magnitude of the relative signal change between plain buffer and buffer-contg. EtOH was pH-dependent. A protective PTFE layer was, therefore, applied to the sensor membrane. This prevented interference from ambient light, pH, and ionic interferents. The response time of PTFE-coated membranes was 3-4 min for forward response, and 5-10 min for reverse response. The selectivity of the dye in the sensor membranes was mainly affected by the lipophilicity of the alcs.
    199. 199
      Mohr, G. J.; Lehmann, F.; Grummt, U.-W.; Spichiger-Keller, U. E. Fluorescent Ligands for Optical Sensing of Alcohols: Synthesis and Characterisation of p-N,N-Dialkylamino-Trifluoroacetylstilbenes. Anal. Chim. Acta 1997, 344 (3), 215– 25,  DOI: 10.1016/S0003-2670(97)00113-X
      200
      Fluorescent ligands for optical sensing of alcohols: synthesis and characterization of p-N,N-dialkylamino-trifluoroacetylstilbenes
      Mohr, Gerhard J.; Lehmann, Frank; Grummt, Ulrich-W.; Spichiger-Keller, Ursula E.
      Analytica Chimica Acta (1997), 344 (3), 215-225CODEN: ACACAM; ISSN:0003-2670. (Elsevier)
      Novel fluorescent ligands were synthesized which interact with specific alcs. resulting in a change of absorbance as well as fluorescence. 4-Trifluoroacetyl-4'-(di-N-butylamino)stilbene (ETH 4003) exhibits an absorption at ∼450nm in org. solvents, whereas, upon reaction with primary alcs., the max. shifts to around 360 nm. The fluorescence emission of the pos. solvatochromic dye ranges from 500 to 700 nm and, upon reaction with alcs., is also blue-shifted to around 450 nm. The significant shift of both absorbance and fluorescence is based on the chem. conversion of the trifluoroacetyl group of the dye into the hemiacetal, thus causing a change in the electron distribution of the dye and a spectral shift of ∼100 nm. The reaction is selective for alcs., which are not sterically hindered to approach the trifluoroacetyl group, i.e. for primary alcs. An example for the anal. application of the ligands is given. An optical sensor was prepd. by embedding 4-trifluoroacetyl-4'-(di-N-butylamino)stilbene in plasticized PVC and coating the layer with white PTFE. The microporous PTFE coating provides an optical isolation and prevents interferences of ions. The sensor exhibits a sensitive range from 5-50 vol.% EtOH with max. signal changes to be in the range of 40%. The detection limit is 2 vol.% EtOH.
    200. 200
      Takano, K.; Sasaki, S.-i.; Citterio, D.; Tamiaki, H.; Suzuki, K. An Oxo-Bacteriochlorin Derivative for Long-Wavelength Fluorescence Ratiometric Alcohol Sensing. Analyst 2010, 135 (9), 2334– 9,  DOI: 10.1039/c0an00173b
      201
      An oxo-bacteriochlorin derivative for long-wavelength fluorescence ratiometric alcohol sensing
      Takano, Kosuke; Sasaki, Shin-ichi; Citterio, Daniel; Tamiaki, Hitoshi; Suzuki, Koji
      Analyst (Cambridge, United Kingdom) (2010), 135 (9), 2334-2339CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      A fluorescent ratiometric optical chem. sensor (optode) for alc. detection is presented. A lipophilized bacteriochlorin deriv. possessing a trifluoroacetyl group at the 3-position was synthesized for alc. detection. When embedded in a plasticized poly(vinyl chloride) (PVC) membrane, the fluororeceptor shows a significant signal change on exposure to aq. ethanol solns. both in the absorbance and the fluorescence emission. This optode allows the detn. of ethanol concns. in aq. sample solns. by ratiometric fluorometry. The fluorescent indicator responds with increasing fluorescence around 701 nm and decreasing fluorescence around 751 nm towards the presence of ethanol. The optode response is fully reversible in a dynamic measurement range from 0.3% to at least 25% (vol./vol.) ethanol. The limit of detection (LOD) and the limit of quantification (LOQ) of the sensor for ethanol are 0.1% and 0.4% (vol./vol.), resp. The new optode can be employed as an ethanol-sensing device with high sensitivity for beverage and industrial analyses.
    201. 201
      Zhao, M.; Yue, Y.; Liu, C.; Hui, P.; He, S.; Zhao, L.; Zeng, X. A Colorimetric and Fluorometric Dual-Modal Sensor for Methanol Based on a Functionalized Pentacenequinone Derivative. Chem. Commun. 2018, 54 (60), 8339– 42,  DOI: 10.1039/C8CC04515A
      202
      A colorimetric and fluorometric dual-modal sensor for methanol based on a functionalized pentacenequinone derivative
      Zhao, Mengting; Yue, Yuxin; Liu, Chang; Hui, Peiyi; He, Song; Zhao, Liancheng; Zeng, Xianshun
      Chemical Communications (Cambridge, United Kingdom) (2018), 54 (60), 8339-8342CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A functionalized pentacenequinone deriv., ZR1 (I), as a highly selective sensor for methanol was prepd. Based on the methanol-induced keto-enol tautomerization of the pentacene-quinone moiety, ZR1 shows specific selectivity toward methanol, which leads to dual-modal detection of methanol with a very low detection limit of 0.038% in ethanol.
    202. 202
      Roy, S.; Das, S.; Ray, A.; Parui, P. P. Fluorometric Trace Methanol Detection in Ethanol and Isopropanol in a Water Medium for Application in Alcoholic Beverages and Hand Sanitizers. RSC Adv. 2021, 11 (48), 30093– 101,  DOI: 10.1039/D1RA05201B
      203
      Fluorometric trace methanol detection in ethanol and isopropanol in a water medium for application in alcoholic beverages and hand sanitizers
      Roy, Snigdha; Das, Sanju; Ray, Ambarish; Parui, Partha Pratim
      RSC Advances (2021), 11 (48), 30093-30101CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      Detection of methanol (MeOH) in an ethanol (EtOH)/isopropanol (iPrOH) medium contg. water is crucial to recognize MeOH poisoning in alc. beverages and hand sanitizers. Although chem. sensing methods are very sensitive and easy to perform, the chem. similarities between the alcs. make MeOH detection very challenging particularly in the presence of water. Herein, the fluorometric detection of a trace amt. of MeOH in EtOH/iPrOH in the presence of water using alc. coordinated Al(III)-complexes of an aldehydic phenol ligand contg. a dangling pyrazole unit is described. The presence of MeOH in the EtOH/iPrOH causes a change of the complex geometry from tetrahedral (Td) to octahedral (Oh) due to the replacement of the coordinated EtOH/iPrOH by MeOH mols. The Td-complex exhibited fluorescence but the Oh-species did not, because of the intramol. photo-induced electron transfer (PET). By interacting the Oh species with water, its one MeOH coordination is replaced by a water mol. followed by the proton transfer from the water to pyrazole-N which generates strong fluorescence by inhibiting the PET. In contrast, the water interaction dissocs. the Td-complex to exhibit fluorescence quenching. The water induced reversal of the fluorescence response from the decrease to increase between the absence and presence of MeOH is utilized to detect MeOH in an EtOH/iPrOH medium contg. water with a sensitivity of ∼0.03-0.06% (vol./vol.). The presence of water effected the MeOH detection and allows the estn. of the MeOH contamination in alc. beverages and hand sanitizers contg. large amts. of water.
    203. 203
      Wu, Z.; Fu, X.; Wang, Y. Click Synthesis of a Triphenylamine-Based Fluorescent Methanol Probe with a Unique D-Π-a Structure. Sens. Actuators B Chem. 2017, 245, 406– 13,  DOI: 10.1016/j.snb.2017.01.164
      204
      Click synthesis of a triphenylamine-based fluorescent methanol probe with a unique D-π-A structure
      Wu, Zhihua; Fu, Xiaobin; Wang, Yong
      Sensors and Actuators, B: Chemical (2017), 245 (), 406-413CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      Facile probing org. solvents is attracting intense attention due to the growing environmental issues. Herein, for the first time, we report a novel D-π-A structured methanol responsive fluorescence mol. (TTO), synthesized by coupling triphenylamine (electron-donor) and p-toluenesulfonyl (electron-acceptor) via Cu (I) catalytic 1,3-dipolar cycloaddn. (click chem.) forming a triazole bridge. D. functional theory (DFT) calcns. and X-ray single crystal structure reveal that TTO has a highly twisted conformation and unique D-π-A structure which render it exceptional optical properties. TTO exhibits strong fluorescence in methanol (QE > 0.67) but weak fluorescence in other org. solvents (QE < 0.1) including various alcs. and can be used as a highly selective fluorescence probe for methanol identification. Notably, TTO is able to excellently probe methanol from its homolog ethanol in water with a detection limit of below 0.7% (vol. concn.), which demonstrates the promise potential of TTO for practical applications.
    204. 204
      Kumar, V.; Kumar, A.; Diwan, U.; Singh, M. K.; Upadhyay, K. K. A Radical Approach for Fluorescent Turn ‘On’ Detection, Differentiation and Bioimaging of Methanol. Org. Biomol. Chem. 2015, 13 (33), 8822– 6,  DOI: 10.1039/C5OB01333J
      205
      A radical approach for fluorescent turn 'on' detection, differentiation and bioimaging of methanol
      Kumar, Virendra; Kumar, Ajit; Diwan, Uzra; Singh, Manish Kumar; Upadhyay, K. K.
      Organic & Biomolecular Chemistry (2015), 13 (33), 8822-8826CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      A simple Schiff base (RC) has been explored as a smart example of fluorescent material for the selective detection, differentiation and bioimaging of methanol. The nucleophilic attack of methanol on the cyclic control unit of RC leads to its opening and formation of a highly fluorescent moiety, RO. The RC displays a good sensitivity for MeOH with a detection limit of 0.042% in water.
    205. 205
      Kumar, V.; Kundu, S.; Sk, B.; Patra, A. A Naked-Eye Colorimetric Sensor for Methanol and ‘Turn-On’ Fluorescence Detection of Al3+. New J. Chem. 2019, 43 (47), 18582– 9,  DOI: 10.1039/C9NJ04688G
      207
      A naked-eye colorimetric sensor for methanol and 'turn-on' fluorescence detection of Al3+
      Kumar, Virendra; Kundu, Subhankar; Sk, Bahadur; Patra, Abhijit
      New Journal of Chemistry (2019), 43 (47), 18582-18589CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
      The current study demonstrates a simple and economical synthetic approach for the development of a multifunctional Schiff base compd. (NRSB) through the condensation reaction between 2-hydroxynaphthaldehyde and a five-membered heterocycle 3-aminorhodanine. The rhodanine unit was chosen due to the presence of an active electron-deficient carbonyl group. A facile chem. transformation from the closed to the open form was noticeable for rhodanine derivs. in the presence of a suitable nucleophile or an electron-rich system. Such a unique feature was invoked to develop a versatile mol. platform for the colorimetric sensing of methanol and the fluorometric detection of Al3+ ions. The nucleophilic attack by methanol on NRSB led to the formation of an open-ring compd., NRSB-O, with a distinct change in color from colorless to yellow. NRSB showed noticeable sensitivity towards methanol and the detection limit was found to be 0.43 wt%. The methanol-induced chem. transformation from NRSB to NRSB-O was probed through X-ray diffraction studies. The strong CH-π interaction between the methylene protons of the rhodanine unit and the π-electron cloud of the naphthalene unit (3.03 Å) and the π-π interaction between naphthalene and the rhodanine moiety (3.51 Å and 3.55 Å) lead to a two-dimensional (2D) supramol. structure in the NRSB crystal. NRSB-O possesses strong CH-π interactions between mols. with an intermol. distance of 2.49 Å, leading to 2D-supramol. self-assembly. The crystal structure revealed the scope of the chelation of NRSB-O with metal ions due to the presence of different N, O, and S donor centers. Interestingly, NRSB-O exhibited a turn-on fluorescence response specifically towards Al3+ through the chelation-enhanced fluorescence (CHEF) mechanism over other competitive metal ions. The turn 'on-off' fluorescence switching was demonstrated for multiple cycles through the alternative addn. of Al3+ and EDTA. The low-cost, simple design strategy of NRSB and NRSB-O as delineated in the current study would contribute to further development of task-specific mol. sensors and switches.
    206. 206
      Patel, R. N. Synthesis of Chiral Pharmaceutical Intermediates by Biocatalysis. Coord. Chem. Rev. 2008, 252 (5), 659– 701,  DOI: 10.1016/j.ccr.2007.10.031
      208
      Synthesis of chiral pharmaceutical intermediates by biocatalysis
      Patel, Ramesh N.
      Coordination Chemistry Reviews (2008), 252 (5-7), 659-701CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
      A review. Chirality is a key factor in the safety and efficacy of many drug products and thus the prodn. of single enantiomers of drug intermediates has become increasingly important in the pharmaceuticals industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes derived therefrom for the transformation of synthetic chems. with high chemo-, regio- and enantioselectivities. In this article, biocatalytic processes are described for the synthesis of chiral intermediates for pharmaceuticals.
    207. 207
      Chen, Y.; Chen, C.; Wu, X. Dicarbonyl Reduction by Single Enzyme for the Preparation of Chiral Diols. Chem. Soc. Rev. 2012, 41 (5), 1742– 53,  DOI: 10.1039/c1cs15230k
      209
      Dicarbonyl reduction by single enzyme for the preparation of chiral diols
      Chen, Yijun; Chen, Chen; Wu, Xuri
      Chemical Society Reviews (2012), 41 (5), 1742-1753CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Chiral diols are a group of key building blocks useful for prepg. a variety of important chiral chems. While the prepn. of optically pure diols is generally not an easy task in synthetic org. chem., three classes of enzymes, namely dicarbonyl reductase, dioxygenase and epoxide hydrolase, display remarkable ability to stereoselectively introduce two hydroxyl groups in a single-step enzymic conversion. In this tutorial review, we pay special attention to dicarbonyl reductases that directly produce chiral diols through the bio-redn. of two carbonyl groups. The dicarbonyl reductases include diketoreductase, α-acetoxy ketone reductase and sepiapterin reductase. We present these exceptional enzymes in the context of source and properties, structure and catalytic mechanism as well as biocatalytic application. In addn. to the broad substrate specificity, the excellent stereoselectivity and high catalytic efficiency of these enzymes have positioned them as valuable biocatalysts. With more sophisticated understanding of the structure-function relationship, the practical utilities of these enzymes assocd. with their interesting chem. will be considerably appreciated over time. Moreover, rational redesign and mol. evolution of these unusual biocatalysts will truly enable their broader applications in the synthesis of chiral diols in the future.
    208. 208
      Ramasastry, S. S. V.; Zhang, H.; Tanaka, F.; Barbas, C. F. Direct Catalytic Asymmetric Synthesis of anti-1,2-Amino Alcohols and syn-1,2-Diols through Organocatalytic anti-Mannich and syn-Aldol Reactions. J. Am. Chem. Soc. 2007, 129 (2), 288– 9,  DOI: 10.1021/ja0677012
      210
      Direct Catalytic Asymmetric Synthesis of anti-1,2-Amino Alcohols and syn-1,2-Diols through Organocatalytic anti-Mannich and syn-Aldol Reactions
      Ramasastry, S. S. V.; Zhang, Haile; Tanaka, Fujie; Barbas, Carlos F., III
      Journal of the American Chemical Society (2007), 129 (2), 288-289CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Chiral 1,2-amino alcs. and 1,2-diols are common structural motifs found in a vast array of natural and biol. active mols. Efficient enantioselective syntheses of ketones contg. syn- or anti-1,2-amino alcs. and 1,2-diols have been reported using metal-based strategies. To date, however, approaches involving organocatalysis have been limited to the syntheses of the syn-1,2-amino alcs. and anti-1,2-diols. Herein we disclose simple and efficient routes to highly enantiomerically enriched anti-1,2-amino alcs. and syn-1,2-diols through direct asym. Mannich, Mannich-type, and aldol reactions involving unmodified α-hydroxyketones in reactions catalyzed by primary amine-contg. amino acids. These reactions exploit (Z)-enamines of α-hydroxyketones in their bond-forming transition states. This study compliments and extends our bioorg. approach to asym. synthesis in these two versatile synthon classes. Significantly, these reactions are practical, tolerate wet solvent, and exploit com. available amino acids, such as threonine and tryptophan, and their derivs. as catalysts. These results provide addnl. support for our original hypothesis suggesting that amino acid catalysis played a key role in prebiotic chem. facilitating the asym. synthesis of the mols. of life.
    209. 209
      Guo, Z.; Shin, I.; Yoon, J. Recognition and Sensing of Various Species Using Boronic Acid Derivatives. Chem. Commun. 2012, 48 (48), 5956– 67,  DOI: 10.1039/c2cc31985c
      211
      Recognition and sensing of various species using boronic acid derivatives
      Guo, Zhiqian; Shin, Injae; Yoon, Juyoung
      Chemical Communications (Cambridge, United Kingdom) (2012), 48 (48), 5956-5967CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A review. A boronic acid moiety can bind to nucleophilic species, such as fluoride ions and 1,2-diols, and arylboronates are converted to the corresponding phenols by treatment with hydrogen peroxide. Based on these reactivity profiles of boron compds., a variety of boronic acid and boronate-based fluorescent chemosensors have been developed for detecting biol. relevant species. This feature article highlights recent advances that have been made in the development of chemosensors of these types for monitoring carbohydrates, dopamine, fluorides, metal ions and hydrogen peroxide.
    210. 210
      Hudlicky, T.; Thorpe, A. J. Current Status and Future Perspectives of Cyclohexadiene-cis-Diols in Organic Synthesis: Versatile Intermediates in the Concise Design of Natural Products. Chem. Commun. 1996, 17 (17), 1993– 2000,  DOI: 10.1039/cc9960001993
      There is no corresponding record for this reference.
    211. 211
      Wingstrand, M. J.; Madsen, C. M.; Clausen, M. H. Rapid Synthesis of Macrocycles from Diol Precursors. Tetrahedron Lett. 2009, 50 (6), 693– 5,  DOI: 10.1016/j.tetlet.2008.11.100
      213
      Rapid synthesis of macrocycles from diol precursors
      Wingstrand, Magnus J.; Madsen, Charlotte M.; Clausen, Mads H.
      Tetrahedron Letters (2009), 50 (6), 693-695CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)
      A method for the formation of synthetic macrocycles with different ring sizes from diols is presented. Reacting a simple diol precursor with electrophilic reagents leads to a cyclic carbonate, sulfite, or phosphate in a single step in 25-60% yield. Converting the cyclization precursor to a bis-electrophilic iodide or aldehyde enables prepn. of a cyclic sulfide and amine, resp., the latter using a double-reductive amination to induce ring closure.
    212. 212
      Tang, X.; Wei, J.; Ding, N.; Sun, Y.; Zeng, X.; Hu, L.; Liu, S.; Lei, T.; Lin, L. Chemoselective Hydrogenation of Biomass Derived 5-Hydroxymethylfurfural to Diols: Key Intermediates for Sustainable Chemicals, Materials and Fuels. Renew. Sust. Energy Rev. 2017, 77, 287– 96,  DOI: 10.1016/j.rser.2017.04.013
      214
      Chemoselective hydrogenation of biomass derived 5-hydroxymethylfurfural to diols: Key intermediates for sustainable chemicals, materials and fuels
      Tang, Xing; Wei, Junnan; Ding, Ning; Sun, Yong; Zeng, Xianhai; Hu, Lei; Liu, Shijie; Lei, Tingzhou; Lin, Lu
      Renewable & Sustainable Energy Reviews (2017), 77 (), 287-296CODEN: RSERFH; ISSN:1364-0321. (Elsevier Ltd.)
      A review. Biomass-derived 5-hydroxymethylfurfural (HMF) is hailed as an all-purposed platform mol. that holds great promise to address a no. of high vol. markets for chems., polymeric materials, and transportation fuels. HMF-derived diols, including 2,5-bishydroxymethylfuran (BHMF), 2,5-bishydroxymethyltetrahydrofuran (BHMTF), and 1,6-hexanediol (1,6-HD), are key intermediates for the catalytic upgrading of HMF in a biorefinery. These diols can be employed as renewable polymeric monomers, and among them BHMF and BHMTF are also attractive precursors for biofuels, such as 2,5-dimethylfuran (DMF), 2,5-bis(alkoxymethyl)furans (BAMFs), and 2,5-dimethyltetrahydrofuran (DMTHF). Hence, gaining more insights into the chemoselective hydrogenation of HMF to these diols is of particular importance. In this review, we comprehensively summarize the advances in the selective hydrogenation of HMF into these diols in terms of the diversity of hydrogen sources, mainly including mol. H2, alcs., formic acid and water, over homogeneous or heterogeneous catalysts. Assessment of the relative merits of different hydrogen sources for the hydrogenation of HMF is performed as well. We also discuss challenges and opportunities in employing these HMF-derived diols for the prodn. of polymeric materials and biofuels.
    213. 213
      Song, J.; Shao, P.-L.; Wang, J.; Huang, F.; Zhang, X. Asymmetric Hydrogenation of 1,4-Diketones: Facile Synthesis of Enantiopure 1,4-Diarylbutane-1,4-Diols. Chem. Commun. 2021, 58 (2), 262– 5,  DOI: 10.1039/D1CC05359K
      215
      Asymmetric hydrogenation of 1,4-diketones: facile synthesis of enantiopure 1,4-diarylbutane-1,4-diols
      Song Jingyuan; Song Jingyuan; Shao Pan-Lin; Wang Jiang; Huang Fanping; Zhang Xumu; Shao Pan-Lin; Zhang Xumu
      Chemical communications (Cambridge, England) (2021), 58 (2), 262-265 ISSN:.
      Owing to the biological significance and great synthetic value of 1,4-diarylbutane-1,4-diols and their derivatives, increasingly considerable attention has been paid to developing effective synthetic methods for chiral 1,4-diarylbutane-1,4-diols. We herein report an efficient asymmetric hydrogenation of 1,4-diaryldiketones catalyzed by a chiral iridium complex bearing f-amphox as ligand, furnishing a series of 1,4-diarylbutane-1,4-diols in excellent yields (up to >99%) with exceptional enantioselectivities (up to >99.9% ee) and diastereoselectivities (up to >100 : 1 dr).
    214. 214
      Faber, K.; Mischitz, M.; Kroutil, W. Microbial Epoxide Hydrolases. Acta Chem. Scand. 1996, 50, 249,  DOI: 10.3891/acta.chem.scand.50-0249
      216
      Microbial epoxide hydrolases
      Faber, Kurt; Mischitz, martin; Kroutil, Wolfgang
      Acta Chemica Scandinavica (1996), 50 (3), 249-58CODEN: ACHSE7; ISSN:0904-213X. (Munksgaard)
      A review with 66 refs. Chiral epoxides and 1,2-diols, which are central building blocks for the asym. synthesis of bioactive compds., can be obtained by using enzymes, which catalyze the enantioselective hydrolysis of epoxides - epoxide hydrolases. These biocatalysts are more widely distributed in fungi and bacteria than previously expected, and sufficient sources from bacteria, such as Rhodococcus and Myobacterium sp., or fungi, for instance Aspergillus and Beauveria sp. have recently been identified. The reaction proceeds via an SN2-specific opening of the epoxide leading to the formation of the corresponding trans-configurated 1,2-diols. For the resoln. of 2-monosubstituted epoxides and for 2,2-disubstituted substrates fungal cells and several bacteria, resp., have been shown to possess excellent selectivities. In addn., the use of non-natural nucleophiles such as azide or amine provides access to chiral azido- and amino-alcs. The synthetic potential of these enzymes is illustrated with recent examples of kinetic resolns. of epoxides from the literature.
    215. 215
      Ouellette, R. J.; Rawn, J. D. Alcohols: Reactions and Synthesis. In Organic Chemistry, 2nd ed.; Academic Press, 2018; Chapter 16, pp 463– 505.
      There is no corresponding record for this reference.
    216. 216
      Rinner, U. Chiral Pool Synthesis. In Chiral Pool Syntheses from cis-Cyclohexadiene Diols, Comprehensive Chirality; Elsevier: Amsterdam, 2012; Chapter 2.9, pp 240– 67.
      There is no corresponding record for this reference.
    217. 217
      Bruice, P. Y. Organic Chemistry; 7. ed.; Pearson Education: Harlow, 2014.
      There is no corresponding record for this reference.
    218. 218
      Gubbels, E.; Heitz, T.; Yamamoto, M.; Chilekar, V.; Zarbakhsh, S.; Gepraegs, M.; Köpnick, H.; Schmidt, M.; Brügging, W.; Rüter, J. Polyesters. Ullmann’s Encyclopedia of Industrial Chemistry Wiley VCH, 2018; 1– 30 DOI: 10.1002/14356007.a21_227.pub2 .
      There is no corresponding record for this reference.
    219. 219
      Steinreiber, A.; Faber, K. Microbial Epoxide Hydrolases for Preparative Biotransformations. Curr. Opin. Biotechnol. 2001, 12 (6), 552– 8,  DOI: 10.1016/S0958-1669(01)00262-2
      221
      Microbial epoxide hydrolases for preparative biotransformations
      Steinreiber, Andreas; Faber, Kurt
      Current Opinion in Biotechnology (2001), 12 (6), 552-558CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Science Ltd.)
      A review. Epoxide hydrolases from microbial sources are highly versatile biocatalysts for the asym. hydrolysis of epoxides on a preparative scale. Besides kinetic resoln., which furnishes the corresponding vicinal diol and remaining non-hydrolyzed epoxide in nonracemic form, enantioconvergent processes are possible: these are highly attractive as they lead to the formation of a single enantiomeric diol from a racemic oxirane. The data accumulated over recent years reveal a common picture of the substrate structure selectivity pattern of microbial epoxide hydrolases and indicate that substrates of various structural types can be selectively hydrolyzed with enzymes from certain microbial sources.
    220. 220
      Gibson, D. T.; Parales, R. E. Aromatic Hydrocarbon Dioxygenases in Environmental Biotechnology. Curr. Opin. Biotechnol. 2000, 11 (3), 236– 43,  DOI: 10.1016/S0958-1669(00)00090-2
      222
      Aromatic hydrocarbon dioxygenases in environmental biotechnology
      Gibson, David T.; Parales, Rebecca E.
      Current Opinion in Biotechnology (2000), 11 (3), 236-243CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Science Ltd.)
      Review of arom. hydrocarbon dioxygenases and the properties that make them attractive synthons for the prodn. of industrially and medically important chiral chems. and also provide essential information for the development of bioremediation technol., with 65 refs. Arom. hydrocarbon dioxygenases belong to a large family of Rieske non-heme iron oxygenases. The dioxygenases have a broad substrate specificity and catalyze enantiospecific reactions with a wide range of substrates.
    221. 221
      Boyd, D. R.; Sharma, N. D.; Allen, C. C. R. Aromatic Dioxygenases: Molecular Biocatalysis and Applications. Curr. Opin. Biotechnol. 2001, 12 (6), 564– 73,  DOI: 10.1016/S0958-1669(01)00264-6
      223
      Aromatic dioxygenases: molecular biocatalysis and applications
      Boyd, Derek R.; Sharma, Narain D.; Allen, Christopher C. R.
      Current Opinion in Biotechnology (2001), 12 (6), 564-573CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Science Ltd.)
      A review, with refs. Arom. dioxygenases have been found to catalyze single and tandem oxidn. reactions of conjugated polyenes. Rational selection and design of dioxygenases, allied to substrate shape, size and substitution pattern, has been used to control regiochem. and stereochem. during the oxygenation process. The resulting enantiopure bioproducts have been increasingly utilized as precursors for new and alternative routes in chiral synthesis.
    222. 222
      Zhao, L.; Han, B.; Huang, Z.; Miller, M.; Huang, H.; Malashock, D. S.; Zhu, Z.; Milan, A.; Robertson, D. E.; Weiner, D. P. Epoxide Hydrolase-Catalyzed Enantioselective Synthesis of Chiral 1,2-Diols Via Desymmetrization of meso-Epoxides. J. Am. Chem. Soc. 2004, 126 (36), 11156– 7,  DOI: 10.1021/ja0466210
      224
      Epoxide Hydrolase-Catalyzed Enantioselective Synthesis of Chiral 1,2-Diols via Desymmetrization of meso-Epoxides
      Zhao, Lishan; Han, Bin; Huang, Zilin; Miller, Mark; Huang, Hongjun; Malashock, Dan S.; Zhu, Zuolin; Milan, Aileen; Robertson, Dan E.; Weiner, David P.; Burk, Mark J.
      Journal of the American Chemical Society (2004), 126 (36), 11156-11157CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The discovery, from nature, of a diverse set of microbial epoxide hydrolases is reported. The utility of a library of epoxide hydrolases in the synthesis of chiral 1,2-diols via desymmetrization of a wide range of meso-epoxides, including cyclic as well as acyclic alkyl- and aryl-substituted substrates, is demonstrated. The chiral (R,R)-diols were furnished with high ee's and yields. The discovery of the first microbial epoxide hydrolases providing access to complementary (S,S)-diols is also described.
    223. 223
      Makarova, M.; Endoma-Arias, M. A. A.; Dela Paz, H. E.; Simionescu, R.; Hudlicky, T. Chemoenzymatic Total Synthesis of ent-Oxycodone: Second-, Third-, and Fourth-Generation Strategies. J. Am. Chem. Soc. 2019, 141 (27), 10883– 904,  DOI: 10.1021/jacs.9b05033
      225
      Chemoenzymatic Total Synthesis of ent-Oxycodone: Second-, Third-, and Fourth-Generation Strategies
      Makarova, Mariia; Endoma-Arias, Mary Ann A.; Dela Paz, Helen E.; Simionescu, Razvan; Hudlicky, Tomas
      Journal of the American Chemical Society (2019), 141 (27), 10883-10904CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Four distinct approaches to ent-oxycodone were designed and accomplished. All rely on the same starting material, the diene diol derived from phenethyl acetate by the whole-cell fermn. with E. coli JM109 (pDTG601A), a strain that overexpresses toluene dioxygenase. The key step in the first-generation approach involves the construction of the C-9/C-14 bond by a SmI2-mediated cyclization of a keto aldehyde. The second-generation design relies on the use of the Henry reaction to accomplish this task. In both of these syntheses, Parker's cyclization was employed to construct the D-ring. The third-generation synthesis provides an improvement over the second in that the nitrogen atom at C-9 is introduced by azidation of the C-9/C-10 olefin, followed by redn. and lactam formation between the C-9 amine and the Fukuyama-type lactone. Finally, the fourth generation takes advantage of the keto-nitrone reductive coupling to generate the C-9/C-14 linkage. The four generations of the total syntheses of ent-oxycodone were accomplished in 13, 18, 16, and 11 operations (19, 23, 24, and 18 steps), resp. Exptl. and spectral data are provided for all new compds.
    224. 224
      Ikeda, H.; Sato, E.; Sugai, T.; Ohta, H. Yeast-Mediated Synthesis of Optically Active Diols with C2-Symmetry and (R)-4-Pentanolide. Tetrahedron 1996, 52 (24), 8113– 22,  DOI: 10.1016/0040-4020(96)00373-0
      226
      Yeast-mediated synthesis of optically active diols with C2-symmetry and (R)-4-pentanolide
      Ikeda, Hajime; Sato, Eriko; Sugai, Takeshi; Ohta, Hiromichi
      Tetrahedron (1996), 52 (24), 8113-8122CODEN: TETRAB; ISSN:0040-4020. (Elsevier)
      Redn. of some diketones and a keto acid with the yeast Pichia farinosa IAM 4682 was examd. The redn. of carbonyl groups proceeded highly selectively in an anti-Prelog fashion to give (R)-alcs. (2R,5R)-2,5-Hexanediol (83% yield, >99% e.e., 95% d.e.), (2R,4R)-2,4-pentanediol (95% yield, >99% e.e., 98% d.e.), and (R)-4-pentanolide (67% yield, >99% e.e.) were very efficiently obtained from the corresponding ketones. Effect of the structure of substrate on the stereochem. course as well as the selectivity were discussed.
    225. 225
      Gijsen, H. J. M.; Wong, C.-H. Unprecedented Asymmetric Aldol Reactions with Three Aldehyde Substrates Catalyzed by 2-Deoxyribose-5-Phosphate Aldolase. J. Am. Chem. Soc. 1994, 116 (18), 8422– 3,  DOI: 10.1021/ja00097a082
      227
      Unprecedented Asymmetric Aldol Reactions with Three Aldehyde Substrates Catalyzed by 2-Deoxyribose-5-phosphate Aldolase
      Gijsen, Harrie J. M.; Wong, Chi-Huey
      Journal of the American Chemical Society (1994), 116 (18), 8422-3CODEN: JACSAT; ISSN:0002-7863.
      Several 6 substituted 2,4,6-trideoxyhexoses, e.g. I (R = H, OH, OMe, Cl, N3), have been stereoselectively synthesized from three achiral C2-aldehydes via a double aldol condensation catalyzed by 2-deoxyribose-5-phosphate aldolase (DERA, 4.1.2.4). This enzymic polymn. stops after two aldol reactions due to the formation of cyclic hemiacetals. The products can be converted to derivs. of the lactone moiety of mevinic acids and to building blocks useful in the synthesis of skipped-polyol macrolides.
    226. 226
      Müller, M. Chemoenzymatic Synthesis of Building Blocks for Statin Side Chains. Angew. Chem., Int. Ed. 2005, 44 (3), 362– 5,  DOI: 10.1002/anie.200460852
      228
      Chemoenzymatic synthesis of building blocks for statin side chains
      Mueller, Michael
      Angewandte Chemie, International Edition (2005), 44 (3), 362-365CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review, with refs. Biocatalysis: Enzymic transformation have become competitive methods for synthesis of complex org. compds. The wide range of possibilities offered by biocatalysis for the synthesis of target mols. are highlighted based on the example of the statins. This class of compds., which includes rosuvastatin, are good inhibitors of cholesterol synthesis and dominate the market for cholesterol-lowering drugs.
    227. 227
      Wu, X.; Jiang, J.; Chen, Y. Correlation between Intracellular Cofactor Concentrations and Biocatalytic Efficiency: Coexpression of Diketoreductase and Glucose Dehydrogenase for the Preparation of Chiral Diol for Statin Drugs. ACS Catal. 2011, 1 (12), 1661– 4,  DOI: 10.1021/cs200408y
      229
      Correlation between Intracellular Cofactor Concentrations and Biocatalytic Efficiency: Coexpression of Diketoreductase and Glucose Dehydrogenase for the Preparation of Chiral Diol for Statin Drugs
      Wu, Xuri; Jiang, Jinpeng; Chen, Yijun
      ACS Catalysis (2011), 1 (12), 1661-1664CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      Dynamic change of intracellular nicotinamide cofactor concns., the limiting factor for the bioredns. catalyzed by oxidoreductases, was monitored in Escherichia coli cells coexpressing diketoreductase and glucose dehydrogenase. On the basis of an unexpected observation, a relationship between catalytic efficiency and cofactor concns. was established to optimize the process for the prepn. of a chiral diol for statin drugs. Consequently, compared to previous reactions by E. coli cells expressing diketoreductase alone, exogenous addn. of cofactors was completely eliminated to yield an increase of substrate concn. by 15-fold. The present strategy could be employed in the biocatalytic processes catalyzed by nicotinamide-dependent oxidoreductases.
    228. 228
      Huffman, M. A.; Fryszkowska, A.; Alvizo, O.; Borra-Garske, M.; Campos, K. R.; Canada, K. A.; Devine, P. N.; Duan, D.; Forstater, J. H.; Grosser, S. T. Design of an in Vitro Biocatalytic Cascade for the Manufacture of Islatravir. Science 2019, 366 (6470), 1255– 9,  DOI: 10.1126/science.aay8484
      230
      Design of an in vitro biocatalytic cascade for the manufacture of islatravir
      Huffman, Mark A.; Fryszkowska, Anna; Alvizo, Oscar; Borra-Garske, Margie; Campos, Kevin R.; Canada, Keith A.; Devine, Paul N.; Duan, Da; Forstater, Jacob H.; Grosser, Shane T.; Halsey, Holst M.; Hughes, Gregory J.; Jo, Junyong; Joyce, Leo A.; Kolev, Joshua N.; Liang, Jack; Maloney, Kevin M.; Mann, Benjamin F.; Marshall, Nicholas M.; McLaughlin, Mark; Moore, Jeffrey C.; Murphy, Grant S.; Nawrat, Christopher C.; Nazor, Jovana; Novick, Scott; Patel, Niki R.; Rodriguez-Granillo, Agustina; Robaire, Sandra A.; Sherer, Edward C.; Truppo, Matthew D.; Whittaker, Aaron M.; Verma, Deeptak; Xiao, Li; Xu, Yingju; Yang, Hao
      Science (Washington, DC, United States) (2019), 366 (6470), 1255-1259CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      Enzyme-catalyzed reactions have begun to transform pharmaceutical manufg., offering levels of selectivity and tunability that can dramatically improve chem. synthesis. Combining enzymic reactions into multistep biocatalytic cascades brings addnl. benefits. Cascades avoid the waste generated by purifn. of intermediates. They also allow reactions to be linked together to overcome an unfavorable equil. or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the no. of steps of the previously reported routes.
    229. 229
      Bian, Z.; Liu, A.; Li, Y.; Fang, G.; Yao, Q.; Zhang, G.; Wu, Z. Boronic Acid Sensors with Double Recognition Sites: A Review. Analyst 2020, 145 (3), 719– 44,  DOI: 10.1039/C9AN00741E
      231
      Boronic acid sensors with double recognition sites: a review
      Bian, Zhancun; Liu, Aiqin; Li, Ying; Fang, Guiqian; Yao, Qingqiang; Zhang, Guimin; Wu, Zhongyu
      Analyst (Cambridge, United Kingdom) (2020), 145 (3), 719-744CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      A review. Boronic acids reversibly and covalently bind to Lewis bases and polyols, which facilitated the development of a large no. of chem. sensors to recognize carbohydrates, catecholamines, ions, hydrogen peroxide, and so on. However, as the binding mechanism of boronic acids and analytes is not very clear, it is still a challenge to discover sensors with high affinity and selectivity. In this review, boronic acid sensors with two recognition sites, including diboronic acid sensors, and monoboronic acid sensors having another group or binding moiety, are summarized. Owing to double recognition sites working synergistically, the binding affinity and selectivity of sensors can be improved significantly. This review may help researchers to sort out the binding rules and develop ideal boronic acid-based sensors.
    230. 230
      Cao, H.; Heagy, M. D. Fluorescent Chemosensors for Carbohydrates: A Decade’s Worth of Bright Spies for Saccharides in Review. J. Fluoresc. 2004, 14 (5), 569– 84,  DOI: 10.1023/B:JOFL.0000039344.34642.4c
      232
      Fluorescent Chemosensors for Carbohydrates: A Decade's Worth of Bright Spies for Saccharides in Review
      Cao, Haishi; Heagy, Michael D.
      Journal of Fluorescence (2004), 14 (5), 569-584CODEN: JOFLEN; ISSN:1053-0509. (Kluwer Academic/Plenum Publishers)
      This review provides a chronol. survey of over fifty fluorescent chemosensors for carbohydrates from the period between 1992 to the present. The survey contains only those sensors that are synthetic or chemosensory, utilize boronic acids and display a fluorescence response in the form of intensity changes or shifts in wavelength. With each compd. listed, a description of the saccharide probe is given with regard to concn., excitation and emission wavelengths, pH and solvent mixt. proportions. In addn., the selectivity of each chemosensor is provided as well as the trends in binding consts. Where possible, a description of the fluorescence signaling mechanism is given as well as commentary on the probe's unique features within this class of sensors.
    231. 231
      Fang, G.; Wang, H.; Bian, Z.; Sun, J.; Liu, A.; Fang, H.; Liu, B.; Yao, Q.; Wu, Z. Recent Development of Boronic Acid-Based Fluorescent Sensors. RSC Adv. 2018, 8 (51), 29400– 27,  DOI: 10.1039/C8RA04503H
      233
      Recent development of boronic acid-based fluorescent sensors
      Fang, Guiqian; Wang, Hao; Bian, Zhancun; Sun, Jie; Liu, Aiqin; Fang, Hao; Liu, Bo; Yao, Qingqiang; Wu, Zhongyu
      RSC Advances (2018), 8 (51), 29400-29427CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      As Lewis acids, boronic acids can bind with 1,2- or 1,3-diols in aq. soln. reversibly and covalently to form five or six cyclic esters, thus resulting in significant fluorescence changes. Based on this phenomenon, boronic acid compds. have been well developed as sensors to recognize carbohydrates or other substances. Several reviews in this area have been reported before, however, novel boronic acid-based fluorescent sensors have emerged in large nos. in recent years. This paper reviews new boron-based sensors from the last five years that can detect carbohydrates such as glucose, ribose and sialyl Lewis A/X, and other substances including catecholamines, reactive oxygen species, and ionic compds. And emerging electrochem. related fluorescent sensors and functionalized boronic acid as new materials including nanoparticles, smart polymer gels, and quantum dots were also involved. By summarizing and discussing these newly developed sensors, we expect new inspiration in the design of boronic acid-based fluorescent sensors.
    232. 232
      Wang, W.; Gao, X.; Wang, B. Boronic Acid-Based Sensors. Curr. Org. Chem. 2002, 6 (14), 1285– 317,  DOI: 10.2174/1385272023373446
      234
      Boronic acid-based sensors
      Wang, Wei; Gao, Xingming; Wang, Binghe
      Current Organic Chemistry (2002), 6 (14), 1285-1317CODEN: CORCFE; ISSN:1385-2728. (Bentham Science Publishers)
      A review. There was a great deal of interest in recent years in using boronic acid as the recognition motif for the development of sensors. Because boronic acids can form tight and reversible complexes with diol compds. such as carbohydrates, the majority of the efforts, led by the Shinkai group, were on the development of sensors for carbohydrates. Boronic acids are also known to selectively recognize fluoride among halides and other anions. Therefore, there also were efforts in using boronic acid compds. for the development fluoride sensors. This paper reviews the progress in this field during the last five years.
    233. 233
      Wu, X.; Li, Z.; Chen, X.-X.; Fossey, J. S.; James, T. D.; Jiang, Y.-B. Selective Sensing of Saccharides Using Simple Boronic Acids and Their Aggregates. Chem. Soc. Rev. 2013, 42 (20), 8032– 48,  DOI: 10.1039/c3cs60148j
      235
      Selective sensing of saccharides using simple boronic acids and their aggregates
      Wu, Xin; Li, Zhao; Chen, Xuan-Xuan; Fossey, John S.; James, Tony D.; Jiang, Yun-Bao
      Chemical Society Reviews (2013), 42 (20), 8032-8048CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. The reversible boronic acid-diol interaction empowers boronic acid receptors' saccharide binding capacities, rendering them a class of lectin mimetic, termed "boronlectins". Boronic acids follow lectin functions not just in being able to bind saccharides, but in multivalent saccharide binding that enhances both affinity and selectivity. For almost a decade, efforts were made to achieve and improve selectivity for given saccharide targets, most notably glucose, by properly positioned boronic acids, offering multivalent interactions. Incorporation of several boronic acid groups into a covalent framework or non-covalent assembly of boronic acid are two general methods used to create such smart sensors, of which the latter resembles lectin oligomerization that affords multivalent saccharide-binding architectures. In this review, supramol. selective sensing of saccharides by simple boronic acids in their aggregate forms is discussed, after a brief survey of the general aspects of boronic acid-based saccharide sensing.
    234. 234
      Arnaud, J.; Audfray, A.; Imberty, A. Binding Sugars: From Natural Lectins to Synthetic Receptors and Engineered Neolectins. Chem. Soc. Rev. 2013, 42 (11), 4798– 813,  DOI: 10.1039/c2cs35435g
      236
      Binding sugars: from natural lectins to synthetic receptors and engineered neolectins
      Arnaud, Julie; Audfray, Aymeric; Imberty, Anne
      Chemical Society Reviews (2013), 42 (11), 4798-4813CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. The large diversity and complexity of glycan structures together with their crucial role in many biol. or pathol. processes require the development of new high-throughput techniques for analyses. Lectins are classically used for characterizing, imaging or targeting glycoconjugates and, when printed on microarrays, they are very useful tools for profiling glycomes. Development of recombinant lectins gives access to reliable and reproducible material, while engineering of new binding sites on existing scaffolds allows tuning of specificity. From the accumulated knowledge on protein-carbohydrate interactions, it is now possible to use nucleotide and peptide (bio)synthesis for producing new carbohydrate-binding mols. Such a biomimetic approach can also be addressed by boron chem. and supra-mol. chem. for the design of fully artificial glycosensors.
    235. 235
      Wu, X.; Chen, X.-X.; Jiang, Y.-B. Recent Advances in Boronic Acid-Based Optical Chemosensors. Analyst 2017, 142 (9), 1403– 14,  DOI: 10.1039/C7AN00439G
      237
      Recent advances in boronic acid-based optical chemosensors
      Wu, Xin; Chen, Xuan-Xuan; Jiang, Yun-Bao
      Analyst (Cambridge, United Kingdom) (2017), 142 (9), 1403-1414CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      A review. Reversible covalent binding of boronic acids with polyols and Lewis bases has facilitated the development of robust chemosensors for many biol. important species under physiol. or environmentally relevant conditions. This minireview covers selected examples of advances reported in this area from 2014 to 2016. While the discovery of new boron-contg. binding motifs and identification of new anal. targets have expanded the utility of boronic acid-based mol. recognition, unconventional sensing strategies such as exploitation of nanoscale self-assembly, multicomponent dynamic covalent assembly, and coupling boronate ester formation with a further chem. reaction have led to significantly improved sensor performance, enabling real-world applications in various areas such as cell biol. and asym. catalysis.
    236. 236
      Kondo, K.; Shiomi, Y.; Saisho, M.; Harada, T.; Shinkai, S. Specific Complexation of Disaccharides with Diphenyl-3,3′-Diboronic Acid That Can Be Detected by Circular Dichroism. Tetrahedron 1992, 48 (38), 8239– 52,  DOI: 10.1016/S0040-4020(01)80492-0
      238
      Specific complexation of disaccharides with diphenyl-3,3'-diboronic acid that can be detected by circular dichroism
      Kondo, Kaoru; Shiomi, Yutaka; Saisho, Miwako; Harada, Takaaki; Shinkai, Seiji
      Tetrahedron (1992), 48 (38), 8239-52CODEN: TETRAB; ISSN:0040-4020.
      Diphenyldiboronic acid I was prepd. as a new receptor that can recognize sugar mols. The distance between the two boronic acids in I is designed so that it can selectively form cyclic 1:1 complexes with disaccharides. It was shown that I forms 1:1 complexes with several disaccharides and gives the characteristic exciton coupling in CD spectroscopy owing to immobilization of the two Ph rings. Thus, the abs. configuration was successfully predicted from the sign of the exciton coupling.
    237. 237
      Yoon, J.; Czarnik, A. W. Fluorescent Chemosensors of Carbohydrates. A Means of Chemically Communicating the Binding of Polyols in Water Based on Chelation-Enhanced Quenching. J. Am. Chem. Soc. 1992, 114 (14), 5874– 5,  DOI: 10.1021/ja00040a067
      239
      Fluorescent chemosensors of carbohydrates. A means of chemically communicating the binding of polyols in water based on chelation-enhanced quenching
      Yoon, Juyoung; Czarnik, Anthony W.
      Journal of the American Chemical Society (1992), 114 (14), 5874-5CODEN: JACSAT; ISSN:0002-7863.
      2-Anthrylboronic acid (I; pKa 8.8) displays decreased fluorescence in the boronate form compared with its boronic acid form, which is attributed to photoinduced electron transfer. When I complexes to polyols such as fructose, the pKa of the complex decreases; thus, the fluorimetrically detd. pKa of the I:fructose borate ester is 5.9. These properties combined permit polyol complexation in water to be obsd. spectrofluorometrically, which has not been reported previously. Fructose binds with a fluorimetrically detd. Kd of 3.7 mM.
    238. 238
      Elfeky, S. Novel Bronic Acid-Based Fluorescent Sensor for Sugars and Nucleosides. Curr. Org. Synth. 2011, 8 (6), 872,  DOI: 10.2174/1570179411108060872
      240
      Novel bronic acid-based fluorescent sensor for sugars and nucleosides
      Elfeky, Souad
      Current Organic Synthesis (2011), 8 (6), 872-880CODEN: COSUC2; ISSN:1570-1794. (Bentham Science Publishers Ltd.)
      Sugar and nucleoside identifications are important for the detection and treatment of dangerous diseases. In this study, a fluorescent boronic acid (3-(5-(dimethylamino)naphthalene-1-sulfonamido)phenyl)boronic acid (DNSBA) was bound to a diol quencher via a boronic ester linkage, and fluorescence recovery upon exposure to saccharides and nucleosides was demonstrated. Further, we successfully detected sugars and nucleosides in buffer systems. DNSBA expresses selectivity toward adenosine, fructose, sorbitol, and tartaric acid. Our investigations into the effects of pH on sensor sensitivity revealed that changes between the neutral and anionic forms of the boronic acid group, induced at high pH and/or in the presence of sugars or nucleosides, induce visible/optimal changes in DNSBA. DNSBA is responsive at pH = 8.21 whereas acidic media do not exhibit sensitivity. DNSBA can thus be useful in clin. applications as a novel sensor for sugars and nucleosides.
    239. 239
      James, T. D.; Shinkai, S. Artificial Receptors as Chemosensors for Carbohydrates. Host-Guest Chemistry 2002, 218, 159– 200,  DOI: 10.1007/3-540-45010-6_6
      There is no corresponding record for this reference.
    240. 240
      Springsteen, G.; Wang, B. A Detailed Examination of Boronic Acid-Diol Complexation. Tetrahedron 2002, 58 (26), 5291– 300,  DOI: 10.1016/S0040-4020(02)00489-1
      242
      A detailed examination of boronic acid-diol complexation
      Springsteen, Greg; Wang, Binghe
      Tetrahedron (2002), 58 (26), 5291-5300CODEN: TETRAB; ISSN:0040-4020. (Elsevier Science Ltd.)
      Boronic acids bind with compds. contg. diol moieties with high affinity through reversible boronate formation. However, the conditions that foster tight binding between the diol and the boronic acid are not well understood. Also, due to the multiple ionic states of both the boronic acid and boronate ester, the equil. consts. reported in the literature have not always been strictly defined, and therefore there is a lack of comparability between the reported values. To address these issues, a method was developed for examg. boronate ester stability using the fluorescent reporter Alizarin Red S. and this system has been used to det. the binding consts. of a series of diols, and as a basis from which to derive a no. of relationships that correlate the various equil. consts. in the literature.
    241. 241
      Jin, S.; Cheng, Y.; Reid, S.; Li, M.; Wang, B. Carbohydrate Recognition by Boronolectins, Small Molecules, and Lectins. Med. Res. Rev. 2009, 30, 171– 257,  DOI: 10.1002/med.20155
      There is no corresponding record for this reference.
    242. 242
      Yan, J.; Fang, H.; Wang, B. Boronolectins and Fluorescent Boronolectins: An Examination of the Detailed Chemistry Issues Important for the Design. Med. Res. Rev. 2005, 25 (5), 490– 520,  DOI: 10.1002/med.20038
      244
      Boronolectins and fluorescent boronolectins: An examination of the detailed chemistry issues important for the design
      Yan, Jun; Fang, Hao; Wang, Binghe
      Medicinal Research Reviews (2005), 25 (5), 490-520CODEN: MRREDD; ISSN:0198-6325. (John Wiley & Sons, Inc.)
      A review. Glycans in the form of glycoproteins or glycolipids play very crit. roles in various biol. and pathol. processes including inflammation, cancer metastasis, immune reactions, embryo development, cell-cell communications and adhesions, blood generation, etc. Proteins (lectins) that can recognize carbohydrates have played very important roles in studying glycobiol. Small mol. mimics of lectins will be very useful in the development of new therapeutics and diagnostics. Along this line, boronic acids have been widely used in the design and synthesis of small org. compds. that mimic the function of lectins. This review examines in detail the factors that are important for the design of boronic acid-based lectin mimics, boronolectins.
    243. 243
      Gao, X.; Zhang, Y.; Wang, B. New Boronic Acid Fluorescent Reporter Compounds. 2. A Naphthalene-Based On-Off Sensor Functional at Physiological pH. Org. Lett. 2003, 5 (24), 4615– 8,  DOI: 10.1021/ol035783i
      245
      New boronic acid fluorescent reporter compounds. 2. A naphthalene-based on-off sensor functional at physiological pH
      Gao, Xingming; Zhang, Yanling; Wang, Binghe
      Organic Letters (2003), 5 (24), 4615-4618CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      A new boronic acid fluorescent on-off reporter compd. (1) was synthesized. This fluorescent sensor shows a 41-fold emission intensity increase upon addn. of 50 mM fructose in 0.1 M aq. phosphate buffer at pH 7.4.
    244. 244
      Gao, X.; Zhang, Y.; Wang, B. A Highly Fluorescent Water-Soluble Boronic Acid Reporter for Saccharide Sensing That Shows Ratiometric UV Changes and Significant Fluorescence Changes. Tetrahedron 2005, 61 (38), 9111– 7,  DOI: 10.1016/j.tet.2005.07.035
      246
      A highly fluorescent water-soluble boronic acid reporter for saccharide sensing that shows ratiometric UV changes and significant fluorescence changes
      Gao, Xingming; Zhang, Yanling; Wang, Binghe
      Tetrahedron (2005), 61 (38), 9111-9117CODEN: TETRAB; ISSN:0040-4020. (Elsevier B.V.)
      One water-sol. naphthalene-based fluorescent boronic acid, 6-(dimethylamino)-naphthalene-2-boronic acid (6-DMANBA), has been synthesized. 6-DMANBA shows significant ratiometric UV absorbance changes upon addn. of a sugar. For example, addn. of 50 mM fructose shifted the UV absorption wavelengths of 6-DMANBA from 306 and 251 to 280 and 244 nm, resp. In addn., 6-DMANBA is highly fluorescent with a quantum yield of 89% in the absence of a sugar and shows significant fluorescence intensity changes with the addn. of a saccharide in aq. phosphate buffer at physiol. pH. For example, with the addn. of 50 mM fructose, 6-DMANBA shows an 80% fluorescent intensity decrease at 432 nm. All these spectroscopic properties make compd. 1 unique and useful.
    245. 245
      Gao, X.; Zhang, Y.; Wang, B. Naphthalene-Based Water-Soluble Fluorescent Boronic Acid Isomers Suitable for Ratiometric and Off-On Sensing of Saccharides at Physiological pH. New J. Chem. 2005, 29 (4), 579– 86,  DOI: 10.1039/b413376e
      247
      Naphthalene-based water-soluble fluorescent boronic acid isomers suitable for ratiometric and off-on sensing of saccharides at physiological pH
      Gao, Xingming; Zhang, Yanling; Wang, Binghe
      New Journal of Chemistry (2005), 29 (4), 579-586CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
      Two water-sol. naphthalene-based fluorescent boronic acid isomers, 5-(dimethylamino)naphthalene-1-boronic acid (5-DMANBA) and 4-(dimethylamino)naphthalene-1-boronic acid (4-DMANBA), have been synthesized; their fluorescent properties upon binding with carbohydrates were detd. in aq. phosphate buffer at pH 7.4. The difference in substitution pattern between 5-DMANBA and 4-DMANBA leads to significant differences in their fluorescence properties. For example, addn. of fructose (50 mM) to a soln. of 5-DMANBA induced a 61% fluorescence intensity decrease at 513 nm and a 36-fold increase at 433 nm. This revealed that 5-DMANBA is a potential sensor for ratiometric sensing of sugars. The pH titrn. curves of 5-DMANBA in the absence and the presence of fructose (50 mM) showed a 93- and 200-fold fluorescence intensity increase at 433 nm, resp., when pH was increased from 3 to 10. 4-DMANBA, however, does not show ratiometric fluorescent intensity changes, but shows significant fluorescence intensity increase upon addn. of a sugar (41-fold intensity increase with 50 mM fructose). The emission intensity of 4-DMANBA increased by over 170-fold at 445 nm upon changing the pH from 2 to 11.
    246. 246
      Zhang, Y.; Gao, X.; Hardcastle, K.; Wang, B. Water-Soluble Fluorescent Boronic Acid Compounds for Saccharide Sensing: Substituent Effects on Their Fluorescence Properties. Eur. J. Chem. 2006, 12 (5), 1377– 84,  DOI: 10.1002/chem.200500982
      248
      Water-soluble fluorescent boronic acid compounds for saccharide sensing: substituent effects on their fluorescence properties
      Zhang, Yanling; Gao, Xingming; Hardcastle, Kenneth; Wang, Binghe
      Chemistry - A European Journal (2006), 12 (5), 1377-1384CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Four new naphthalene-based boronic acid I (R = R1 = H; R = Me, R1 = H, COMe, CH2CO2Bu-t) were synthesized. The effect of various carbohydrates on their fluorescence properties has been studied in aq. phosphate buffer at pH 7.4. Different substitutions on the aniline group of the naphthalene ring resulted in significant differences in fluorescence properties for these four compds. Compd. I (R = Me, R1 = CH2CO2Bu-t) shows ratiometric fluorescence changes upon addn. of a sugar. Compds. I (R = R1 = H; R = Me, R1 = H) do not show ratiometric fluorescence changes but show very large fluorescence intensity changes (about 70-fold fluorescence intensity increase). In addn. to the quantifiable fluorescence property changes upon sugar addn., the fluorescence color changes of I (R = R1 = H; R = Me, R1 = H, CH2CO2Bu-t) are also visible to the naked eye. However, amidation of the aniline nitrogen atom significantly diminishes the fluorescence intensity of compd. I (R = Me, R1 = COMe). The crystal structure of one boronic acid provided some insight into the structural features that are important for the fluorescence properties of these compds. These compds. I show very large fluorescence property changes upon monosaccharide binding. Addn. of fructose (50 mM) to the soln. of I (R = Me, R1 = CH2CO2Bu-t) induced a 4.2-fold fluorescence intensity increase at 440 nm and a 32 % fluorescence intensity decrease at 490 nm. The introduction of an ester functional group on the side chain does not abolish the ability of the fluorophore to show a ratiometric response to binding with a sugar.
    247. 247
      Yang, W.; Yan, J.; Springsteen, G.; Deeter, S.; Wang, B. A Novel Type of Fluorescent Boronic Acid That Shows Large Fluorescence Intensity Changes Upon Binding with a Carbohydrate in Aqueous Solution at Physiological pH. Bioorg. Med. Chem. Lett. 2003, 13 (6), 1019– 22,  DOI: 10.1016/S0960-894X(03)00086-6
      249
      A novel type of fluorescent boronic acid that shows large fluorescence intensity changes upon binding with a carbohydrate in aqueous solution at physiological pH
      Yang, Wenqian; Yan, Jun; Springsteen, Greg; Deeter, Susan; Wang, Binghe
      Bioorganic & Medicinal Chemistry Letters (2003), 13 (6), 1019-1022CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science B.V.)
      In this paper we report 8-quinoline boronic acid as a novel type of fluorescent probe for carbohydrates. This boronic acid responds to the binding of a carbohydrate with over 40-fold increases in fluorescence intensity and shows optimal fluorescence change at physiol. pH in aq. soln.
    248. 248
      Yang, W.; Lin, L.; Wang, B. A New Type of Boronic Acid Fluorescent Reporter Compound for Sugar Recognition. Tetrahedron Lett. 2005, 46 (46), 7981– 4,  DOI: 10.1016/j.tetlet.2005.09.074
      250
      A new type of boronic acid fluorescent reporter compound for sugar recognition
      Yang, Wenqian; Lin, Li; Wang, Binghe
      Tetrahedron Letters (2005), 46 (46), 7981-7984CODEN: TELEAY; ISSN:0040-4039. (Elsevier B.V.)
      Fluorescent boronic acids that change fluorescent properties upon carbohydrate binding are very useful for the prepn. of fluorescent sensors for sugars. Herein the authors report 5-quinolineboronic acid (5-QBA) that shows significant fluorescent property changes through a unique pKa-switching mechanism upon binding a diol in aq. soln.
    249. 249
      Cheng, Y.; Ni, N.; Yang, W.; Wang, B. A New Class of Fluorescent Boronic Acids That Have Extraordinarily High Affinities for Diols in Aqueous Solution at Physiological pH. Eur. J. Chem. 2010, 16 (45), 13528– 38,  DOI: 10.1002/chem.201000637
      251
      A New Class of Fluorescent Boronic Acids That Have Extraordinarily High Affinities for Diols in Aqueous Solution at Physiological pH
      Cheng, Yunfeng; Ni, Nanting; Yang, Wenqian; Wang, Binghe
      Chemistry - A European Journal (2010), 16 (45), 13528-13538, S13528/1-S13528/40CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The boronic acid group is an important recognition moiety for sensor design. Herein, the authors report a series of isoquinolinylboronic acids that have extraordinarily high affinities for diol-contg. compds. at physiol. pH. In addn., 5- and 8-isoquinolinylboronic acids also showed fairly high binding affinities towards D-glucose (Ka=42 and 46 M-1, resp.). For the first time, weak but encouraging binding of cis-cyclohexanediol was found for these boronic acids. Such binding was coupled with significant fluorescence changes. Furthermore, 4- and 6-isoquinolinylboronic acids also showed the ability to complex Me α-D-glucopyranose (Ka=3 and 2 M-1, resp.).
    250. 250
      Samaniego Lopez, C.; Lago Huvelle, M. A.; Uhrig, M. L.; Coluccio Leskow, F.; Spagnuolo, C. C. Recognition of Saccharides in the NIR Region with a Novel Fluorogenic Boronolectin: In Vitro and Live Cell Labeling. Chem. Commun. 2015, 51 (23), 4895– 8,  DOI: 10.1039/C4CC10425K
      252
      Recognition of saccharides in the NIR region with a novel fluorogenic boronolectin: in vitro and live cell labeling
      Samaniego Lopez, Cecilia; Lago Huvelle, Maria Amparo; Uhrig, Maria Laura; Coluccio Leskow, Federico; Spagnuolo, Carla C.
      Chemical Communications (Cambridge, United Kingdom) (2015), 51 (23), 4895-4898CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      This work describes a novel mono-boronic acid deriv. of a tricarbocyanine. The probe is a genuine near-IR fluorescence emitter with improved properties such as a large Stokes shift, excellent water soly. and sensitive fluorogenicity upon binding to carbohydrates under physiol. conditions.
    251. 251
      Wang, L.; Yuan, L.; Zeng, X.; Peng, J.; Ni, Y.; Er, J. C.; Xu, W.; Agrawalla, B. K.; Su, D.; Kim, B. A Multisite-Binding Switchable Fluorescent Probe for Monitoring Mitochondrial ATP Level Fluctuation in Live Cells. Angew. Chem., Int. Ed. 2016, 55 (5), 1773– 6,  DOI: 10.1002/anie.201510003
      253
      Multisite-Binding Switchable Fluorescent Probe for Monitoring Mitochondrial ATP Level Fluctuation in Live Cells
      Wang, Lu; Yuan, Lin; Zeng, Xian; Peng, Juanjuan; Ni, Yong; Er, Jun Cheng; Xu, Wang; Agrawalla, Bikram Keshari; Su, Dongdong; Kim, Beomsue; Chang, Young-Tae
      Angewandte Chemie, International Edition (2016), 55 (5), 1773-1776CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      ATP, commonly produced in mitochondria, is required by almost all the living organisms; thus fluorescent probes for monitoring mitochondrial ATP level fluctuation are essential and highly desired. Herein, the authors report a multisite-binding switchable fluorescent probe, ATP-Red 1 (I), which selectively and rapidly responds to intracellular concns. of ATP. Live-cell imaging indicated that ATP-Red 1 mainly localized to mitochondria with good biocompatibility and membrane penetration. In particular, with the help of ATP-Red 1, the authors successfully obsd. not only the decreased mitochondrial ATP levels in the presence of KCN and starvation state, but also the increased mitochondrial ATP levels in the early stage of cell apoptosis. ATP-Red 1 is a useful tool for studying ATP-relevant biol. processes.
    252. 252
      Huang, Y.-J.; Ouyang, W.-J.; Wu, X.; Li, Z.; Fossey, J. S.; James, T. D.; Jiang, Y.-B. Glucose Sensing Via Aggregation and the Use of “Knock-Out” Binding to Improve Selectivity. J. Am. Chem. Soc. 2013, 135 (5), 1700– 3,  DOI: 10.1021/ja311442x
      254
      Glucose Sensing via Aggregation and the Use of "Knock-Out" Binding To Improve Selectivity
      Huang, Yan-Jun; Ouyang, Wen-Juan; Wu, Xin; Li, Zhao; Fossey, John S.; James, Tony D.; Jiang, Yun-Bao
      Journal of the American Chemical Society (2013), 135 (5), 1700-1703CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Aggregates of an amphiphilic monoboronic acid bearing a hydrophobic pyrene fluorophore were employed for highly modulating, sensitive, and selective ratiometric fluorescent sensing of glucose in aq. soln. The selectivity for glucose was improved by "knock-out" binding of fructose by phenylboronic acid.
    253. 253
      Vilozny, B.; Schiller, A.; Wessling, R. A.; Singaram, B. Enzyme Assays with Boronic Acid Appended Bipyridinium Salts. Anal. Chim. Acta 2009, 649 (2), 246– 51,  DOI: 10.1016/j.aca.2009.07.032
      255
      Enzyme assays with boronic acid appended bipyridinium salts
      Vilozny, Boaz; Schiller, Alexander; Wessling, Ritchie A.; Singaram, Bakthan
      Analytica Chimica Acta (2009), 649 (2), 246-251CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      In-vitro fluorescent enzyme assays have been developed for sucrose phosphorylase (SPO) and phosphoglucomutase (PGM). These assays make use of a selective carbohydrate sensing system that detects the unlabeled enzymic products fructose and glucose-6-phosphate. The system comprises 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt as the reporter unit and boronic acid appended viologens as selective receptors with working ranges from 70 μM to 1.0 mM for fructose (SPO) and 190 μM to 2.0 mM for glucose-6-phosphate (PGM). The change in fluorescence can be converted into product concn., allowing initial reaction velocities and Michaelis-Menten kinetics to be calcd. The assays are also carried out in multiwell plate formats, making them suitable for high-throughput screening of enzyme inhibitors. Rapid PGM inhibition screening is demonstrated with EDTA and LiCl. The PGM assay can also be used for enzyme quantification with a detection limit of 50 ng mL-1.
    254. 254
      Sharrett, Z.; Gamsey, S.; Hirayama, L.; Vilozny, B.; Suri, J. T.; Wessling, R. A.; Singaram, B. Exploring the Use of APTS as a Fluorescent Reporter Dye for Continuous Glucose Sensing. Org. Biomol. Chem. 2009, 7 (7), 1461– 70,  DOI: 10.1039/b821934f
      256
      Exploring the use of APTS as a fluorescent reporter dye for continuous glucose sensing
      Sharrett, Zachary; Gamsey, Soya; Hirayama, Lacie; Vilozny, Boaz; Suri, Jeff T.; Wessling, Ritchie A.; Singaram, Bakthan
      Organic & Biomolecular Chemistry (2009), 7 (7), 1461-1470CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      The anionic fluorescent dye, aminopyrene trisulfonic acid (APTS), was synthesized and used in a soln.-based two-component glucose-sensing system comprising the dye and a boronic acid-appended viologen. The fluorescence of the dye was quenched in the presence of the viologen and the fluorescence restored upon glucose addn. An important feature of this fluorophore is that it can be covalently bonded to a polymer through the amine group without a significant effect on optical properties. Two APTS derivs., functionalized with polymerizable groups, were synthesized and immobilized in hydroxyethyl methacrylate (HEMA)-based hydrogels. The latter were used to continuously monitor glucose. The fluorescence signal modulation, signal stability, reversibility, reproducibility, and pH sensitivity of the hydrogels were evaluated. The APTS dyes described herein are insensitive to pH changes within the physiol. range, both in soln. and when immobilized in a hydrogel. When APTS is used in conjunction with boronic acid-appended viologens to sense glucose, the system displays some pH sensitivity because of the presence of the boronic acid.
    255. 255
      James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. A Glucose-Selective Molecular Fluorescence Sensor. Angew. Chem., Int. Ed. 1994, 33 (21), 2207– 9,  DOI: 10.1002/anie.199422071
      There is no corresponding record for this reference.
    256. 256
      James, T. D.; Sandanayake, K. R. A. S.; Iguchi, R.; Shinkai, S. Novel Saccharide-Photoinduced Electron Transfer Sensors Based on the Interaction of Boronic Acid and Amine. J. Chem. Soc., Chem. Commun. 1995, 117 (35), 8982– 7,  DOI: 10.1021/ja00140a013
      There is no corresponding record for this reference.
    257. 257
      Tran, T. M.; Alan, Y.; Glass, T. E. A Highly Selective Fluorescent Sensor for Glucosamine. Chem. Commun. 2015, 51 (37), 7915– 8,  DOI: 10.1039/C5CC00415B
      259
      A highly selective fluorescent sensor for glucosamine
      Tran, Tam Minh; Alan, Yuksel; Glass, Timothy Edward
      Chemical Communications (Cambridge, United Kingdom) (2015), 51 (37), 7915-7918CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A new fluorescent chem. sensor for glucosamine is reported. The sensor is based on a boronic acid-contg. coumarin aldehyde and shows excellent selectivity for glucosamine by forming a boronic ester with the sugar diol as well as an iminium ion with the amine group of glucosamine. The sensor successfully discriminates glucosamine over other similar biomols. in terms of both fluorescence intensity and binding affinity. This method provides a new concept for the design and synthesis of very selective turn-on optical sensors for selective detection of multi-functional biomols.
    258. 258
      Rout, B.; Unger, L.; Armony, G.; Iron, M. A.; Margulies, D. Medication Detection by a Combinatorial Fluorescent Molecular Sensor. Angew. Chem., Int. Ed. 2012, 51 (50), 12477– 81,  DOI: 10.1002/anie.201206374
      260
      Medication Detection by a Combinatorial Fluorescent Molecular Sensor
      Rout, Bhimsen; Unger, Linor; Armony, Gad; Iron, Mark A.; Margulies, David
      Angewandte Chemie, International Edition (2012), 51 (50), 12477-12481CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Herein, we describe the design and function of a combinatorial fluorescent sensor that takes mol.-scale diagnostics a step further. The mol. anal. system presented herein combines several recognition elements as well as four emission channels and utilizes distinct photo-phys. processes that enable us to identify a wide range of pharmaceuticals and analyze drug concns. and combinations in urine samples in a high-throughput manner. The development of methods for the verification of drug content at point-of-care has been receiving growing international attention. We therefore selected four drug families commonly assocd. with counterfeiting or medication errors as test cases for our mol. sensor. Macrolides, aminoglycosides, and rifamycins are large families of antibiotics whose counterfeits are highly prevalent in the developing world. Cardiac glycosides, used for treating heart conditions, have been assocd. with substandard medication in developed countries and are often involved in medication errors owing to their narrow therapeutic window and adverse drug interactions. To be able to identify different macrolides, aminoglycosides, cardiac glycosides, and rifamycins with a single fluorescent mol., we designed and synthesized sensor (I).
    259. 259
      Liu, Y.; Deng, C.; Tang, L.; Qin, A.; Hu, R.; Sun, J. Z.; Tang, B. Z. Specific Detection of D-Glucose by a Tetraphenylethene-Based Fluorescent Sensor. J. Am. Chem. Soc. 2011, 133 (4), 660– 3,  DOI: 10.1021/ja107086y
      261
      Specific Detection of D-Glucose by a Tetraphenylethene-Based Fluorescent Sensor
      Liu, Yi; Deng, Chunmei; Tang, Li; Qin, Anjun; Hu, Rongrong; Sun, Jing Zhi; Tang, Ben Zhong
      Journal of the American Chemical Society (2011), 133 (4), 660-663CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A conceptually new "light-up" biosensor with a high specificity for D-glucose (Glu) in aq. media has been developed. The emission from a tetraphenylethene (TPE)-cored diboronic acid (I) was greatly boosted when the fluorogen was oligomerized with Glu because of restriction of the intramol. rotations of the aryl rotors of TPE by formation of the oligomer. Little change in the light emission was obsd. when I was mixed with D-fructose, D-galactose, or D-mannose, as these saccharides are unable to oligomerize with the fluorogen.
    260. 260
      Shcherbakova, E. G.; Brega, V.; Lynch, V. M.; James, T. D.; Anzenbacher, P., Jr. High-Throughput Assay for Enantiomeric Excess Determination in 1,2- and 1,3-Diols and Direct Asymmetric Reaction Screening. Eur. J. Chem. 2017, 23 (42), 10222– 9,  DOI: 10.1002/chem.201701923
      262
      High-Throughput Assay for Enantiomeric Excess Determination in 1,2- and 1,3-Diols and Direct Asymmetric Reaction Screening
      Shcherbakova, Elena G.; Brega, Valentina; Lynch, Vincent M.; James, Tony D.; Anzenbacher, Pavel, Jr.
      Chemistry - A European Journal (2017), 23 (42), 10222-10229CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A simple and efficient method for detn. of the yield, enantiomeric/diasteriomeric excess (ee/de), and abs. configuration of crude chiral diols without the need of work-up and product isolation in a high throughput setting is described. This approach uses a self-assembled iminoboronate ester formed as a product by dynamic covalent self-assembly of a chiral diol with an enantiopure fluorescent amine such as tryptophan Me ester or tryptophanol and 2-formylphenylboronic acid. The resulting diastereomeric boronates display different photophys. properties and allow for fluorescence-based ee detn. of mols. contg. a 1,2- or 1,3-diol moiety. This method was used for the screening of ee in a no. of chiral diols including atorvastatin, a statin used for the treatment of hypercholesterolemia. Noyori asym. hydrogenation of benzil was performed in a highly parallel fashion with errors <1% ee confirming the feasibility of the systematic examn. of crude products from the parallel asym. synthesis in real time and in a high-throughput screening (HTS) fashion.
    261. 261
      Shcherbakova, E. G.; James, T. D.; Anzenbacher, P. High-Throughput Assay for Determining Enantiomeric Excess of Chiral Diols, Amino Alcohols, and Amines and for Direct Asymmetric Reaction Screening. Nat. Protoc. 2020, 15 (7), 2203– 29,  DOI: 10.1038/s41596-020-0329-1
      263
      High-throughput assay for determining enantiomeric excess of chiral diols, amino alcohols, and amines and for direct asymmetric reaction screening
      Shcherbakova, Elena G.; James, Tony D.; Anzenbacher Jr., Pavel
      Nature Protocols (2020), 15 (7), 2203-2229CODEN: NPARDW; ISSN:1750-2799. (Nature Research)
      A review. Abstr.: Detg. enantiomeric excess (e.e.) in chiral compds. is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for detg. e.e. in mixts. of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcs., and amino-acid esters. The method is based on dynamic self-assembly of com. available chiral amines, 2-formylphenylboronic acid, and chiral diols in acetonitrile to form fluorescent diastereomeric complexes. Each analyte enantiomer engenders a diastereomer with distinct fluorescence wavelength/intensity originating from enantiopure fluorescent ligands. In this assay, enantiomers of amines and amine derivs. assemble with diol-type ligands contg. a binaphthol moiety (BINOL and VANOL), whereas diol enantiomers form complexes with the enantiopure amine-type fluorescent ligand tryptophanol. The differential fluorescence is utilized to det. the amt. of each enantiomer in the mixt. with an error of <1% e.e. This method enables high-throughput real-time evaluation of enantiomeric/diastereomeric excess (e.e./d.e.) and product yield of crude asym. reaction products. The procedure comprises high-throughput liq. dispensing of three components into 384-well plates and recording of fluorescence using an automated plate reader. The approach enables scaling up the screening of combinatorial libraries and, together with parallel synthesis, creates a robust platform for discovering chiral catalysts or auxiliaries for asym. transformations and chiral drug development. The procedure takes ∼4-6 h and requires 10-20 ng of substrate per well. Our fluorescence-based assay offers distinct advantages over existing methods because it is not sensitive to the presence of common additives/impurities or unreacted/incompletely utilized reagents or catalysts.
    262. 262
      Doderer, K.; Lutz-Wahl, S.; Hauer, B.; Schmid, R. D. Spectrophotometric Assay for Epoxide Hydrolase Activity toward any Epoxide. Anal. Biochem. 2003, 321 (1), 131– 4,  DOI: 10.1016/S0003-2697(03)00399-3
      264
      Spectrophotometric assay for epoxide hydrolase activity toward any epoxide
      Doderer, Kai; Lutz-Wahl, Sabine; Hauer, Bernhard; Schmid, Rolf D.
      Analytical Biochemistry (2003), 321 (1), 131-134CODEN: ANBCA2; ISSN:0003-2697. (Elsevier Science)
      A universal colorimetric high-throughput assay system for the quantification of the reaction products of epoxide hydrolase activity in bacterial cell exts. is described. The assay is based on the quant. colorimetric detection of ketones and aldehydes with Schiff's reagent contg. fuchsin and sulfurous acid. The test is reliable and sensitive with a detection limit of 5 μmol diol in 100 μL soln. For higher amts. of diol the soln. must be dild. to quantify the diol content. The suggested assay for epoxide hydrolase activity toward any epoxide is sensitive and reproducible and is feasible in crude cell exts.
    263. 263
      Wahler, D.; Reymond, J.-L. The Adrenaline Test for Enzymes. Angew. Chem., Int. Ed. 2002, 41 (7), 1229– 32,  DOI: 10.1002/1521-3773(20020402)41:7<1229::AID-ANIE1229>3.0.CO;2-5
      265
      The adrenaline test for enzymes
      Wahler, Denis; Reymond, Jean-Louis
      Angewandte Chemie, International Edition (2002), 41 (7), 1229-1232CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
      A versatile high-throughput enzyme assay is demonstrated which is based on the colorimetric back-titrn. of sodium periodate with L-adrenaline. Enzyme activity is assocd. with the depletion of sodium periodate by the reaction product (P), and indicated by the decrease in the amt. of the red dye adrenochrome produced by the oxidn. of adrenaline by sodium periodate. The assay quantitates vicinal diols, amino alcs., diamines and α-hydroxy ketones. The assay was applied to measure lipases, esterases, phytases and epoxide hydrolases. The versatility of the assay in terms of substrate structures was demonstrated for the epoxide hydrolases from Aspergillus niger and Rhodotorula glutinis.
    264. 264
      Jie, N.; Yang, D.; Zhang, Q.; Yang, J.; Song, Z. Fluorometric Determination of Periodate with Thiamine and Its Application to the Determination of Ethylene Glycol and Glycerol. Anal. Chim. Acta 1998, 359 (1), 87– 92,  DOI: 10.1016/S0003-2670(97)00657-0
      266
      Fluorometric determination of periodate with thiamine and its application to the determination of ethylene glycol and glycerol
      Jie, Nianqin; Yang, Duanling; Zhang, Qiang; Yang, Jinghe; Song, Zhongqing
      Analytica Chimica Acta (1998), 359 (1-2), 87-92CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
      The fluorometric detn. of periodate with thiamine (vitamin B1) and its application to the indirect detn. of org. compds. (esp. glycols) were developed. Thiamine is oxidized to thiochrome by potassium periodate, which is accelerated by Mn (II). Thiochrome, a fluorescent compd., has excitation and emission max. at 397 and 454 nm, resp. Under optimum conditions, responses for the periodate detn. were linear between 5.0 × 10-7 - 1.8 × 10-5 mol/L concn. The detection limit, corresponding to a signal-to-noise ratio of 3, was 2.6 × 10-7 mol/L, with a relative std. deviation (n = 5) of 1.0%. The proposed method was applied to the indirect detn. of several org. compds. (ethylene glycol and glycerol, in vegetable oil) via the Malaprade reaction; good results were obtained.
    265. 265
      Kirschner, A.; Bornscheuer, U. T. Directed Evolution of a Baeyer-Villiger Monooxygenase to Enhance Enantioselectivity. Appl. Microbiol. Biotechnol. 2008, 81 (3), 465– 72,  DOI: 10.1007/s00253-008-1646-4
      267
      Directed evolution of a Baeyer-Villiger monooxygenase to enhance enantioselectivity
      Kirschner, Anett; Bornscheuer, Uwe T.
      Applied Microbiology and Biotechnology (2008), 81 (3), 465-472CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      The Baeyer-Villiger monooxygenase (BVMO) BmoF1 from Pseudomonas fluorescens DSM 50106 was shown before to enantioselectively oxidize different 4-hydroxy-2-ketones to the corresponding hydroxyalkyl acetates, being the first example of a BVMO-catalyzed kinetic resoln. of aliph. acyclic ketones. However, the wild-type enzyme exhibited only moderate E values (E∼55). Thus, the enantioselectivity was enhanced by means of directed evolution and optimization of reaction conditions since it was found that higher E values (E∼70 for wild-type BmoF1) could already be obtained when performing biotransformations in shake flasks rather than small tubes. In a first step, random mutations were introduced by error-prone polymerase chain reaction, and BmoF1 mutants (>3,500 clones) were screened for improved activity and enantioselectivity using a microtiter-plate-based screening method. Mutations S136L and L252Q were found to increase conversion compared to wild type, while several mutations (H51L, F225Y, S305C, and E308V) were identified enhancing the enantioselectivity to a varying extent (E∼75-90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange site-directed mutagenesis resulting in a double mutant (H51L/S136L) showing both improved conversion and enantioselectivity (E∼86).
    266. 266
      Wahler, D.; Boujard, O.; Lefèvre, F.; Reymond, J.-L. Adrenaline Profiling of Lipases and Esterases with 1,2-Diol and Carbohydrate Acetates. Tetrahedron 2004, 60 (3), 703– 10,  DOI: 10.1016/j.tet.2003.11.059
      268
      Adrenaline profiling of lipases and esterases with 1,2-diol and carbohydrate acetates
      Wahler, Denis; Boujard, Olivier; Lefevre, Fabrice; Reymond, Jean-Louis
      Tetrahedron (2004), 60 (3), 703-710CODEN: TETRAB; ISSN:0040-4020. (Elsevier Science B.V.)
      The adrenaline test for enzymes is a general back-titrn. procedure to detect 1,2-diols, 1,2-aminoalcs. and α-hydroxyketones reaction products of enzyme catalysis by colorimetry. The method was used to profile a series of esterases and lipases for their esterolytic activity on a series of carbohydrate and polyol acetates. Substrates were prepd. by peracetylation and used for parallel microtiter-plate anal. of enzyme activities. This method can be used to achieve a rapid and automated characterization of a set of enzymes during HTS screening.
    267. 267
      Fluxá, V. S.; Wahler, D.; Reymond, J.-L. Enzyme Assay and Activity Fingerprinting of Hydrolases with the Red-Chromogenic Adrenaline Test. Nat. Protoc. 2008, 3 (8), 1270– 7,  DOI: 10.1038/nprot.2008.106
      269
      Enzyme assay and activity fingerprinting of hydrolases with the red-chromogenic adrenaline test
      Fluxa, Viviana S.; Wahler, Denis; Reymond, Jean-Louis
      Nature Protocols (2008), 3 (8), 1270-1277CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      The adrenaline test for enzymes is a colorimetric enzyme assay based on the quantification of periodate-sensitive reaction products such as 1,2-diols and 1,2-aminoalcs. by back-titrn. of the oxidant with adrenaline to produce adrenochrome as an easily detectable red product. The test uses com. reagents and is suitable for screening the activity of various hydrolases. It is demonstrated here for testing epoxide hydrolases, lipases and esterases, and for activity fingerprinting of these enzymes across substrate series. The complete assay requires 2-3 h.
    268. 268
      Doderer, K.; Schmid, R. D. Fluorometric Assay for Determining Epoxide Hydrolase Activity. Biotechnol. Lett. 2004, 26 (10), 835– 9,  DOI: 10.1023/B:BILE.0000025887.36874.33
      270
      Fluorometric assay for determining epoxide hydrolase activity
      Doderer, Kai; Schmid, Rolf D.
      Biotechnology Letters (2004), 26 (10), 835-839CODEN: BILED3; ISSN:0141-5492. (Kluwer Academic Publishers)
      A rapid screening procedure was developed that enabled the screening of hundreds of enzyme samples or variants for epoxide hydrolase activity toward any substrate. The procedure detected the products of the enzymic reaction via periodate cleavage and the remaining fluorescence of carboxyfluorescein.
    269. 269
      Matsuno, K.; Suzuki, S. Simple Fluorimetric Method for Quantification of Sialic Acids in Glycoproteins. Anal. Biochem. 2008, 375 (1), 53– 9,  DOI: 10.1016/j.ab.2008.01.002
      271
      Simple fluorimetric method for quantification of sialic acids in glycoproteins
      Matsuno, Kanae; Suzuki, Shigeo
      Analytical Biochemistry (2008), 375 (1), 53-59CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
      A simple and rapid fluorimetric method was developed for detection and quant. anal. of sialic acids in glycoproteins. Sialic acid residues in glycoproteins were specifically oxidized with periodate at 0° for 45 min. Formaldehyde generated from carbon 9 (C-9) of sialic acid was converted specifically to fluorescent dihydropyridine deriv. with acetoacetanilide and ammonia at room temp. for 10 min. The reaction products indicate intense fluorescence with excitation and emission maxima at 388 and 471 nm, resp. When the reaction was conducted in approx. a 1-mL vol., the linearity of the calibration exhibited between 2 and 180 μg of bovine fetuin, or between 0.3 and 27 nmol of N-acetylneuraminic acid, as a model glycoprotein. The limit of detection, based on three times the std. deviation of the reagent blank, was 0.5 μg of fetuin. The proposed method was applied to detn. of sialic acids in various glycoprotein samples. This proposed method is simple and obviates the heating and extn. steps. It is highly specific to sialic acids in glycoproteins and indicates no fluorescence of neutral glycoproteins.
    270. 270
      Preston-Herrera, C.; Jackson, A. S.; Bachmann, B. O.; Froese, J. T. Development and Application of a High Throughput Assay System for the Detection of Rieske Dioxygenase Activity. Org. Biomol. Chem. 2021, 19 (4), 775– 84,  DOI: 10.1039/D0OB02412K
      272
      Development and application of a high throughput assay system for the detection of Rieske dioxygenase activity
      Preston-Herrera, Cristina; Jackson, Aaron S.; Bachmann, Brian O.; Froese, Jordan T.
      Organic & Biomolecular Chemistry (2021), 19 (4), 775-784CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Herein we report the development of a new periodate-based reactive assay system for the fluorescent detection of the cis-diol metabolites produced by Rieske dioxygenases. This sensitive and diastereoselective assay system successfully evaluates the substrate scope of Rieske dioxygenases and dets. the relative activity of a rationally designed Rieske dioxygenase variant library. The high throughput capacity of the assay system enables rapid and efficient substrate scope investigations and screening of large dioxygenase variant libraries.
    271. 271
      Winkler, M. Carboxylic Acid Reductase Enzymes (CARS). Curr. Opin. Chem. Biol. 2018, 43, 23– 9,  DOI: 10.1016/j.cbpa.2017.10.006
      273
      Carboxylic acid reductase enzymes (CARs)
      Winkler, Margit
      Current Opinion in Chemical Biology (2018), 43 (), 23-29CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. Carboxylate reductases (CARs) are emerging as valuable catalysts for the selective one-step redn. of carboxylic acids to their corresponding aldehydes. The substrate scope of CARs is exceptionally broad and offers potential for their application in diverse synthetic processes. Two major fields of application are the prepn. of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates. The latest applications of CARs are dominated by in vivo cascades and chemo-enzymic reaction sequences. The challenge to fully exploit product selectivity is discussed. Recent developments in the characterization of CARs are summarized, with a focus on aspects related to the domain architecture and protein sequences of CAR enzymes.
    272. 272
      Sutiono, S.; Carsten, J.; Sieber, V. Structure-Guided Engineering of α-Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature. ChemSusChem 2018, 11 (18), 3335– 44,  DOI: 10.1002/cssc.201800944
      274
      Structure-Guided Engineering of α-Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature
      Sutiono, Samuel; Carsten, Joerg; Sieber, Volker
      ChemSusChem (2018), 11 (18), 3335-3344CODEN: CHEMIZ; ISSN:1864-5631. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Branched-chain keto acid decarboxylases (KDCs) are a class of enzymes that catalyze the decarboxylation of α-keto acids. They are key enzymes for prodn. of higher alcs. in vivo and in vitro. However, the two most active KDCs (KivD and KdcA) have only moderate thermostability (<55 °C), which hinders the prodn. of alcs. at high temps. Herein, structure-guided engineering toward improved thermostability of KdcA is outlined. Strategies such as stabilization of the catalytic center, surface engineering, and optimization of dimer interactions were applied. With seven amino acid substitutions, variant 7M.D showed an increase of the temp. at which 50 % of activity remains after one-hour incubation T1h50 by 14.8 °C without compromising its substrate specificity. 7M.D exhibited greater than 400-fold improvement of half-life at 70 °C and greater than 600-fold increase in process stability in the presence of 4 % isobutanol at 50 °C. 7M.D is more promising for the prodn. of higher alcs. in thermophiles (>65 °C) and in cell-free applications.
    273. 273
      Greenhalgh, J. C.; Fahlberg, S. A.; Pfleger, B. F.; Romero, P. A. Machine Learning-Guided Acyl-ACP Reductase Engineering for Improved in Vivo Fatty Alcohol Production. Nat. Commun. 2021, 12, 5825,  DOI: 10.1038/s41467-021-25831-w
      275
      Machine learning-guided acyl-ACP reductase engineering for improved in vivo fatty alcohol production
      Greenhalgh, Jonathan C.; Fahlberg, Sarah A.; Pfleger, Brian F.; Romero, Philip A.
      Nature Communications (2021), 12 (1), 5825CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: Alc.-forming fatty acyl reductases (FARs) catalyze the redn. of thioesters to alcs. and are key enzymes for microbial prodn. of fatty alcs. Many metabolic engineering strategies utilize FARs to produce fatty alcs. from intracellular acyl-CoA and acyl-ACP pools; however, enzyme activity, esp. on acyl-ACPs, remains a significant bottleneck to high-flux prodn. Here, we engineer FARs with enhanced activity on acyl-ACP substrates by implementing a machine learning (ML)-driven approach to iteratively search the protein fitness landscape. Over the course of ten design-test-learn rounds, we engineer enzymes that produce over twofold more fatty alcs. than the starting natural sequences. We characterize the top sequence and show that it has an enhanced catalytic rate on palmitoyl-ACP. Finally, we analyze the sequence-function data to identify features, like the net charge near the substrate-binding site, that correlate with in vivo activity. This work demonstrates the power of ML to navigate the fitness landscape of traditionally difficult-to-engineer proteins.
    274. 274
      Sellés Vidal, L.; Kelly, C. L.; Mordaka, P. M.; Heap, J. T. Review of NAD(P)H-Dependent Oxidoreductases: Properties, Engineering and Application. Biochim. Biophys. Acta 2018, 1866 (2), 327– 47,  DOI: 10.1016/j.bbapap.2017.11.005
      276
      Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application
      Selles Vidal, Lara; Kelly, Ciaran L.; Mordaka, Pawel M.; Heap, John T.
      Biochimica et Biophysica Acta, Proteins and Proteomics (2018), 1866 (2), 327-347CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B.V.)
      A review. NAD(P)H-dependent oxidoreductases catalyze the redn. or oxidn. of a substrate coupled to the oxidn. or redn., resp., of a NAD cofactor NAD(P)H or NAD(P)+. NAD(P)H-dependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilization, the synthesis of chems., biodegrdn. and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.
    275. 275
      Goswami, P.; Chinnadayyala, S. S. R.; Chakraborty, M.; Kumar, A. K.; Kakoti, A. An Overview on Alcohol Oxidases and Their Potential Applications. Appl. Microbiol. Biotechnol. 2013, 97 (10), 4259– 75,  DOI: 10.1007/s00253-013-4842-9
      277
      An overview on alcohol oxidases and their potential applications
      Goswami, Pranab; Chinnadayyala, Soma Sekhar R.; Chakraborty, Mitun; Kumar, Adepu Kiran; Kakoti, Ankana
      Applied Microbiology and Biotechnology (2013), 97 (10), 4259-4275CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      A review. Alc. oxidases (Alc.: O2 Oxidoreductase; EC 1.1.3.x) are flavoenzymes that catalyze the oxidn. of alcs. to the corresponding carbonyl compds. with a concomitant release of hydrogen peroxide. Based on substrate specificity, alc. oxidases may be categorized broadly into four different groups namely, (a) short chain alc. oxidase (SCAO), (b) long chain alc. oxidase (LCAO), (c) arom. alc. oxidase (AAO), and (d) secondary alc. oxidase (SAO). The sources reported for these enzymes are mostly limited to bacteria, yeast, fungi, plant, insect, and mollusks. However, the quantum of reports for each category of enzymes considerably varies across these sources. The enzymes belonging to SCAO and LCAO are intracellular in nature, whereas AAO and SAO are mostly secreted to the medium. SCAO and LCAO are invariably reported as multimeric proteins with very high holoenzyme mol. masses, but the mol. characteristics of these enzymes are yet to be clearly elucidated. One of the striking features of the alc. oxidases that make them distinct from the widely known alc. dehydrogenase is the avidly bound cofactor to the redox center of these enzymes that obviate the need to supplement cofactor during the catalytic reaction. These flavin-based redox enzymes have gained enormous importance in the development of various industrial processes and products primarily for developing biosensors and prodn. of various industrially useful carbonyl compds. The present review provides an overview on alc. oxidases from different categories focusing research on these oxidases during the last decade along with their potential industrial applications.
    276. 276
      Mang, H.; Gross, J.; Lara, M.; Goessler, C.; Schoemaker, H. E.; Guebitz, G. M.; Kroutil, W. Biocatalytic Single-Step Alkene Cleavage from Aryl Alkenes: An Enzymatic Equivalent to Reductive Ozonization. Angew. Chem., Int. Ed. 2006, 45 (31), 5201– 3,  DOI: 10.1002/anie.200601574
      278
      Biocatalytic single-step alkene cleavage from aryl alkenes: an enzymatic equivalent to reductive ozonization
      Mang, Harald; Gross, Johannes; Lara, Miguel; Goessler, Christian; Schoemaker, Hans E.; Guebitz, Georg M.; Kroutil, Wolfgang
      Angewandte Chemie, International Edition (2006), 45 (31), 5201-5203CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Innocuous mol. oxygen O2 is the only reagent needed to perform highly chemoselective biocatalytic single-step alkene-cleavage reactions. The products are analogous to those of (reductive) ozonization and related metal-based methods. In contrast neither special equipment nor an addnl. reducing agent is required. The biocatalytic reaction can be performed at ambient temp. Depending on the substrate, aldehydes or ketones are obtained.
    277. 277
      Schwendenwein, D.; Ressmann, A. K.; Doerr, M.; Höhne, M.; Bornscheuer, U. T.; Mihovilovic, M. D.; Rudroff, F.; Winkler, M. Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity Using an Amino Benzamidoxime-Mediated High-Throughput Assay. Adv. Synth. Catal. 2019, 361, 2544,  DOI: 10.1002/adsc.201900155
      279
      Random mutagenesis-driven improvement of carboxylate reductase activity using an amino benzamidoxime-mediated high-throughput assay
      Schwendenwein, Daniel; Ressmann, Anna K.; Doerr, Mark; Hoehne, Matthias; Bornscheuer, Uwe T.; Mihovilovic, Marko D.; Rudroff, Florian; Winkler, Margit
      Advanced Synthesis & Catalysis (2019), 361 (11), 2544-2549CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Carboxylic acid reductases (CARs) catalyze the direct ATP and NADP (NADPH) dependent redn. of carboxylic acids to their corresponding aldehydes. The identification and improvement of CARs by protein engineering is, however, severely limited by the lack of fast and generic methods to quantify aldehydes. Within this study, we applied a convenient high-throughput assay (HTA) based on amino benzamidoxime (ABAO) that allows the substrate-independent and chemoselective quantification of aldehydes. Random mutagenesis of the well-known CAR from Nocardia iowensis (CARNi) to improve its activity for sterically demanding 2-substituted benzoic acid derivs. was conducted in a Km-dependent fashion, and the HTA applied in the presence of microbial cells. The study identified a hot spot in the active site of CARNi that increased the affinity to 2-methoxybenzoic acid 9-fold upon mutation from glutamine to proline (Q283P). The catalytic performance of CARNiQ283P appeared to be significantly improved also for other substrates such as 2-substituted (2-Cl, 2-Br) as well as 3- and 4-substituted benzoic acids (3-OMe, 4-OMe), and even aliph. octanoic acid.
    278. 278
      Soh, L. M. J.; Mak, W. S.; Lin, P. P.; Mi, L.; Chen, F. Y. H.; Damoiseaux, R.; Siegel, J. B.; Liao, J. C. Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design. ACS Synth. Biol. 2017, 6 (4), 610– 8,  DOI: 10.1021/acssynbio.6b00240
      280
      Engineering a thermostable keto acid decarboxylase using directed evolution and computationally directed protein design
      Soh, Lemuel M. J.; Mak, Wai Shun; Lin, Paul P.; Mi, Luo; Chen, Frederic Y.-H.; Damoiseaux, Robert; Siegel, Justin B.; Liao, James C.
      ACS Synthetic Biology (2017), 6 (4), 610-618CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcs., like isobutanol. The most active Kdc's are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol prodn. temp. Here, the authors report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60°. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and ∼8000 independent variants were screened. The top single-mutation variants were recombined. To further improve thermostability, 16 designs built using Rosetta Comparative Modeling were screened and the most active was recombined to form the authors' best variant, LLM4. Compared to wild-type Kivd, a 13° increase in melting temp. and a >4-fold increase in half-life at 60° were obsd. LLM4 will be useful for keto acid-derived alc. prodn. in lignocellulosic thermophiles.
    279. 279
      Van Schie, M. M. C. H.; Pedroso De Almeida, T.; Laudadio, G.; Tieves, F.; Fernández-Fueyo, E.; Noël, T.; Arends, I. W. C. E.; Hollmann, F. Biocatalytic Synthesis of the Green Note trans-2-Hexenal in a Continuous-Flow Microreactor. Beilstein J. Org. Chem. 2018, 14, 697– 703,  DOI: 10.3762/bjoc.14.58
      281
      Biocatalytic synthesis of the Green Note trans-2-hexenal in a continuous-flow microreactor
      van Schie, Morten M. C. H.; Pedroso de Almeida, Tiago; Laudadio, Gabriele; Tieves, Florian; Fernandez-Fueyo, Elena; Noel, Timothy; Arends, Isabel W. C. E.; Hollmann, Frank
      Beilstein Journal of Organic Chemistry (2018), 14 (), 697-703CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)
      The biocatalytic prepn. of trans-hex-2-enal from trans-hex-2-enol using a novel aryl alc. oxidase from Pleurotus eryngii (PeAAOx) is reported. As O2-dependent enzyme PeAAOx-dependent reactions are generally plagued by the poor soly. of O2 in aq. media and mass transfer limitations resulting in poor reaction rates. These limitations were efficiently overcome by conducting the reaction in a flow-reactor setup reaching unpreceded catalytic activities for the enzyme in terms of turnover frequency (up to 38 s-1) and turnover nos. (more than 300000) pointing towards preparative usefulness of the proposed reaction scheme.
    280. 280
      Allen, C. F. The Identification of Carbonyl Compounds by Use of 2,4-Dinitrophenylhydrazine. J. Am. Chem. Soc. 1930, 52 (7), 2955– 9,  DOI: 10.1021/ja01370a058
      282
      Identification of carbonyl compounds by use of 2,4-dinitrophenylhydrazine
      Allen, Chas. F. H.
      Journal of the American Chemical Society (1930), 52 (), 2955-9CODEN: JACSAT; ISSN:0002-7863.
      An inexpensive method is given for the prepn. of 2,4-(O2N)2C6H3NHNH2. This is a suitable reagent to use in prepg. cryst. derivs. of a large no. of aliphatic compds. contg. a CO group. It may also be used with certain cyclic compds. but the production of solid derivs. is not as general. It is not as useful with α-HO ketones, owing to the difficulty in sepg. the mixts. formed. The following new 2,4-dinitrophenylhydrazones are reported: MeCOPr, yellow-orange, m. 141°; MeCOBμ, red-orange, m. 106°; MeCOAm, yellow-orange, m. 89°; Me hexyl ketone, orange, m. 58°; Me nonyl ketone, orange-yellow, m. 63°; Me undecyl ketone, orange-yellow, m. 69°; iso-PrCOMe, orange-yellow, m. 117°; iso-BuCOMe, orange-red m. 95°; iso-AmCOMe, orange, m. 95°; Me isohexyl ketone, orange-yellow, m. 77°; pinacolin orange-yellow, m. 125°; EtCOEt, orange, m. 156°; PrCOPr, yellow-orange, m. 75°; EtCOPr, yellow-orange, m. 130°; iso-BuCOEt, yellow-orange, m. 75°; allylacetone, orange, m. 104°; methylheptenone, red-orange, m. 81°; pseudoionone, deep red, m. 143° mesityl oxide, carmine, m. 200°; cyclopentanone, orange-yellow, m. 142°; cyclohexanone, orange-yellow, m. 160°; Me cyclohexyl ketone, orange, m. 140°; carvone, carmine, m. 189°; menthone, orange, m. 145°; benzalacetone, orange-red, m. 223°; PhCH:CHCOPh, orange, m. 208°; MeCOPh, orange, m. 237°; α-indanone, red-orange, m. 258°; butyroin, yellow, m. 99°; benzoin, orange-yellow, m. 234°; valeraldehyde, yellow, m. 98°; octaldehyde, yellow, m. 106°; decylaldehyde, yellow, m. 104°; undecylaldehyde, yellow, m. 104°; dodecylaldehyde, yellow, m. 106°; acrolein, red-orange, m. 165°; α-methyl-β-ethylacrolein, carmine, m. 159°; citronellal, yellow, m. 78°; citral I, red-orange, m. 108-10°, citral II, red-orange, m. 96°; α-amylcinnamaldehyde, scarlet, m. 164°; pyruvic acid, lemon, m. 213°; levulinic acid, yellow, m. 92°; Et oxomalonate, lemon, m. 128°; BzCO2Me, orange-yellow, m. 171°. Furoin, furil and benzofuroin gave unsatisfactory results. Anisoin, anisil, piperoin and piperil gave mixts. m. above 200°. Solid derivs. could not be prepd. from Me heptyl, octyl and decyl ketones, Bu2CO, com. ionone, pure α-ionone, fenchone or pulegone.
    281. 281
      Brady, O. L.; Elsmie, G. V. The Use of 2,4-Dinitrophenylhydrazine as a Reagent for Aldehydes and Ketones. Analyst 1926, 51 (599), 77– 8,  DOI: 10.1039/an9265100077
      283
      The use of 2,4-dinitrophenylhydrazine as a reagent for aldehydes and ketones
      Brady, O. L.; Elsmie, Gladys V.
      (1926), 51 (), 77-8 ISSN:.
      The above reagent is so satisfactory that it is surprising that it has not received more attention. It reacts somewhat tardily and is not very sol. in cold alc. Quite a no. of cryst. ppts. with typical aldehydes were prepared and the chem. compn. was found to agree with the demands of theory. Directions for making the reagent are given.
    282. 282
      Bohlmann, F. Konstitution Und Lichtabsorption, I. Mitteil.: Carbonyl-Derivate. Chem. Ber. 1951, 84 (5–6), 490– 504,  DOI: 10.1002/cber.19510840515
      284
      Constitution and light absorption. I. Carbonyl derivatives
      Bohlmann, Ferdinand
      Chemische Berichte (1951), 84 (), 490-504CODEN: CHBEAM; ISSN:0009-2940.
      With the exception of the oximes, the compds. used for the characterization of CO compds. are chiefly hydrazine derivs. (Ia). These condensation products have characteristic absorption spectra, the max. of which are shifted toward the longer wave lengths although the conjugation is not lengthened by addnl. double bonds. An attempt is made to find a relationship between the constitution and the light absorption. The absorption spectra of the m- (A) and p-nitrophenylhydrazones (B), 2,4-dinitrophenylhydrazones (C), N-methyl-2,4-dinitrophenylhydrazones (D), semicarbazones (E), thiosemicarbazones (F), N-methylthiosemicarbazones (G), and azobenzenesemicarbazones [1-(p-phenylazophenyl)semicarbazones] (H) of Me2CO, MeCH:CHCHO (I), BzH, hexadienal (II), octatrienal (III), β-ionone (IV), RCH2CH:CMeCHO (V) (R = CMe2.CH2.CH2.CH2.CMe:C- throughout the abstr.), RCH:CHCMe:CHCOMe (VI), RCH:CHCMe:CHCH:CHCOMe (VII), RCH2CH:CMeCH:CHCH:CHCHO (VIII) have been detd. as given in the table. Carbonyl compd., No. of conjugated bonds, Color, A, M.p. °C., λmax. mμ, ε × 10-3, Color, B, M.p. °C., λmax. mμ, ε × 10-3, Color, C, M.p. °C., λmax mμ, ε × 10-3, Color, D, M.p. °C., λmax mμ, ε × 10-3; Free base, .., .., .., .., .., .., .., 230, .., .., .., 352, .., ...., .., 230, ..; , , , , , , , , 386, , , , , , , , 370; Me2CO, .., red, 112-13, 268, 22.8, yellow, 148, 250, 11.9, .., .., 362, .., yellow, 138, 220, ..; , , , , (390), (1.4), , , 392, , , , , , , , 375, 19.1; I, 1, .., .., .., .., orange, 184, 285, 7.7, red, 190, 244, 15.8, yellow, 131, 260, 14.3; , , , , , , , , 400, 30.0, , , 282, 9.0, , , 397, 22.3; , , , , , , , , 273a, , , , 376, 27.3; , , , , , , , , 523a, , , , 273a; , , , , , , , , , , , , 523a; BzH, 1b, orange, 130, 238, 22.1, orange, 190, 230, 14.6, .., .., 378, .., brownish yellow, 206, 290, 10.8; , , , , 334, 33.1, , , 299, 7.2, , , , , , , 401, 20.9; , , , , (393), (1.95), , , 403, 31.2; PhCOMe, .., .., .., .., .., .., .., .., .., .., .., .., .., yellow needles, 157-8, 389, 81.4; II, 2, .., .., .., .., red, 170-1, 235, 16.2, .., .., .., .., yellow-orange, 122-3, 290, 20.8; , , , , , , , , 291, 15.0, , , , , leaflets, , 410, 25.35; , , , , , , , , 330, 10.7; , , , , , , , , 414, 43.8; III, 3, .., .., .., .., .., .., 311, .., .., .., 409, .., orange, 150-2, 316, 28.5; , , , , , , , , 425, , , , , , , , 424, 28.9; IV, 2, .., .., .., .., orange, 161-2, 294, 15.5, red, 126-7, 293, 11.0, yellow, 104-6, 298, 9.5; , , , , , , , , 408, 39.0, , , 387, 27.5, leaflets, , 390, 22.0; V, 1, red, 174-5, 248, 13.8, orange, 169, 283, 12.0, red, 164, 252, 18.5, yellow-orange, 148, 267, 17.8; , , , , 314, 37.1, , , 406, 35.0, , , 290, 11.0, leaflets, , 404, 24.6; , , , , (400), (1.36), , , , , , , 385, 31.5; VI, .., .., .., .., .., .., .., .., .., red, 157, 230, 19.0, ...., .., .., ..; , , , , , , , , , , , , 310, 15.7; , , , , , , , , , , , , 408, 34.0; VII, .., .., .., .., .., .., .., .., .., dark red, 177, 332, 22.0, ...., .., .., ..; , , , , , , , , , , , , 416, 43.0; VIII, .., .., .., .., .., .., .., .., .., red, 233, 265c, 18.5, ...., .., .., ..; , , , , , , , , , , , , 332c, 22.8; , , , , , , , , , , , , 410c, 46.5;a In 10% alc. KOH.;b the C6 ring in this series corresponds to about 1 C:C bond and not 1.5 as in polyene chemistry.;c In CHCl3; all others in MeOH.; Me[2,4-(O2N)2C6H3]NNH2, prepd. from MeNHNH2 and 2,4-(O2N)2C6H3Cl, m. 137°, λmax. 370 mμ, ε 14,200. For the prepn. of Ia, 300 mg. of the appropriate hydrazine is boiled 2-3 min. in 30 cc. EtOH and 0.5 cc. HCl with an equiv. amt. of the CO compd. Replacement of the OH in PhCH:NOH (λmax. 247 mμ) by a NH2 group leads to PhCH:NNH2 (λmax. 273 mμ) with a shift of the max. of 25 mμ toward the red; introduction of an Ac group (PhCH:NNHAc, λmax. 283 mμ), a Bz group (PhCH:NNHBz, λmax. 297 mμ), or a Me group (PhCH:NNHMe) causes a further shift to the red. Introduction of Ph as in PhCH:NNHPh (IX) (λmax. 342 mμ) has a stronger effect, whereas introduction of Br in the para position [PhCH:NNHC5H4Br(p), λmax. 347 mμ] has only little effect upon the shift. The position of the max. of IX is about the same as that of (PhCH:CH)2, indicating that the NH group has about the same effect upon the light absorption as a C:C double bond. In the A and B the max. are further shifted to the red, whereas the C generally absorb more in the shorter wave lengths. The NO2 in the ortho position in C therefore causes a shift of the absorption to the shorter wave lengths. In strongly alk. soln. these differences disappear (I B and I C in 10% alc. KOH have λmax. 523 mμ). With the C several limiting forms are possible because the NO2 groups can react in different ways with the electron pair of the NH group, e.g., X and XI. The effect of the o-NO2 group can be annulled by the introduction of a Me at the α-N, whereby the max. are shifted about 20 mμ toward the longer wave lengths and approach the max. of the B. With the D, however, no change in the position of the max. occurs as compared with those of the B. With the D the formulation XI is impossible, whereas with C the formation of H bridges as in XI is favored, as is shown by Stuart models. The usually observed shift toward the long waves' region is reversed by the H bridges because the π-electron system is decisively influenced by a far-reaching shift of the mesomerism equil. toward XI. With the D a certain steric hindrance by the Me group is noticeable, as shown by a stronger resolution of the chromophoric system. Thus the absorption max. attributed to the partial chromophore, R(CH:CH)nCH:N-, is considerably more pronounced, with a simultaneous decrease of the extinctions. The minor max. are more characteristic than the major max. because they indicate the no. of conjugated double bonds. Unlike the C, the D and the A do not give any color with alc. alkali, indicating that the H at the NH group is necessary for the coloration and that a quinoid formulation must be assumed for the colored alkali salts of C and B, which is impossible with A. With the E, F, and H a shift toward the longer wave lengths occurs. This shift, with the E, is of about the same order as that of an addnl. C:C double bond and has been attributed to an enolization of the urea group: RCH:NNHCONH2 → RCH:NN:HC(OH))NH2. However, this is erroneous because N-Me derivs. of E, where enolization is impossible, show the same behavior. Further methylation of PhCH:NNMeCSNH2 to PhCH:NMeC(SMe):NH has no effect on the absorption max. Introduction of a Ph into the semicarbazone group causes a shift of 10 mμ to the longer wave lengths (H2NCONHPh has λmax. 238 mμ, CO(NHPh)2, λmax. 255 mμ). When the Ph group is sepd. by a CH2 group the influence becomes smaller (Me2C:NNHCONH2 has λmax. 224 mμ, MePhC:NNHCONH2 λmax. 228 mμ). With the dinitrophenylsemicarbazones a resolution of the chromophoric system takes place. A replacement of the (O2N)2C6H3 group by a PhN:N group also causes a resolution of the spectrum. Refluxing 5 g. p-PhN:NC6H4NHCONH2 with 3 cc. N2H4.H2O in 250 cc. EtOH 40 hrs. gives the azobenzenesemicarbazide (XII), brownish yellow crystals, m. above 280° (decompn.), λmax. 238, 356 mμ, ε 14,800, 29,000. Warming 0.3 g. XII with an equiv. amt. of the appropriate CO compd. in 15 cc. EtOH and 0.1 cc. AcOH 15 min. gives the following azobenzenesemicarbazones: of BzH, yellow needles, m. 213°, λmax. 292, 354 mμ, ε 20,000, 32,000; of V, yellow needles, m. 235°, λmax. 273, 354 mμ, ε 30,000, 25,500; of cyclohexanone, yellow crystals, m. 223°, λmax. 242, 355mμ, ε 19,300, 28,900; of IV, orange-yellow leaflets, m. 224°, λmax. 300, 355 mμ, ε 17,300, 32,500. The following derivs. of BzH are also studied (m.p., λmax., and ε × 10-3 in the order given): phenylhydrazone, 152°, 342, 303, 235 mμ, 20.2, 10.3, 12.8; p-bromophenylhydrazone, 127°, 347, 315, 235 mμ, 27.4, 17.3, 12.4; N-methyl(p-nitrophenyl)hydrazone, 137°, 403, 299, 230 mμ, 31.2, 9.2, 16.1; hydrazone, -, 273 mμ, 13.8; acetylhydrazone, 134°, 283 mμ, 26.9; benzoylhydrazone, 206°, 297 mμ, 24.8; semicarbazone, 230°, 282 mμ, 21.0; thiosemicarbazone, 160°, 310 mμ, -; 1-methylthiosemicarbazone, 170°, 312 mμ, 29.4; S-Me isomer, 196°, 300 mμ, 27.6. The following addnl. derivs. of V are prepd.: N-methyl(p-nitrophenyl)hydrazone, yellow crystals, m. 158°, λmax. 406, 284 mμ, ε 31,100, 13,500; 1-methylsemicarbazone, shiny leaflets, m. 168°, λmax. 268 mμ, ε 37,000; 1-methylthiosemicarbazone, m. 183°, λmax. 302 mμ, ε 39,300; semicarbazone, m. 155°, λmax. 265 mμ, ε 33,200; thiosemicarbazone, m. 200°, λmax. 299 mμ, ε 37,800. III semicarbazone m. about 250°, λmax. 303, 319, 334 mμ; VIII semicarbazone m. 186°, λmax. 312, 324, 338 mμ.
    283. 283
      Georgiou, C. D.; Zisimopoulos, D.; Argyropoulou, V.; Kalaitzopoulou, E.; Salachas, G.; Grune, T. Protein and Cell Wall Polysaccharide Carbonyl Determination by a Neutral pH 2,4-Dinitrophenylhydrazine-Based Photometric Assay. Redox Biol. 2018, 17, 128– 42,  DOI: 10.1016/j.redox.2018.04.010
      285
      Protein and cell wall polysaccharide carbonyl determination by a neutral pH 2,4-dinitrophenylhydrazine-based photometric assay
      Georgiou, Christos D.; Zisimopoulos, Dimitrios; Argyropoulou, Vasiliki; Kalaitzopoulou, Electra; Salachas, George; Grune, Tilman
      Redox Biology (2018), 17 (), 128-142CODEN: RBEIB3; ISSN:2213-2317. (Elsevier B.V.)
      A new 2,4-dinitrophenylhydrazine (DNPH)-based photometric assay is developed for the quantification of carbonyls in protein samples from any biol. source by protein carbonyl-DNPH hydrazone formation at acidic pH in the presence of denaturing urea, and subsequent hydrazone solubilization in the presence of SDS and stabilization from acid hydrolysis at pH 7.0. At this neutral pH, interfering unreacted DNPH is uncharged and its thus increased hydrophobicity permits its 100% effective removal from the solubilizate with Et acetate/hexane wash. The present study elucidates the DNA interference mechanism on the stdDNPH assay, and also develops a standardized protocol for sample protein treatment and fractionation (into cytoplasmic/aq., membrane/lipid-bound, and histone/DNA-bound proteins; see Supplement section V) in order to ensure reproducible carbonyl detn. on defined cell protein fractions, and to eliminate assay interference from protein samples contg. (i) Cys sulfenic acid groups (via their neutralization with dithiothreitol), and (ii) DNA (via its removal by streptomycin sulfate pptn.). Lastly, the ntrDNPH assay dets. carbonyl groups on cell wall polysaccharides, thus paving the way on studies to investigate cell walls acting as antioxidant defense in plants, fungi, bacteria and lichens.
    284. 284
      Mesquita, C. S.; Oliveira, R.; Bento, F.; Geraldo, D.; Rodrigues, J. V.; Marcos, J. C. Simplified 2,4-Dinitrophenylhydrazine Spectrophotometric Assay for Quantification of Carbonyls in Oxidized Proteins. Anal. Biochem. 2014, 458, 69– 71,  DOI: 10.1016/j.ab.2014.04.034
      286
      Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins
      Mesquita, Cristina S.; Oliveira, Raquel; Bento, Fatima; Geraldo, Dulce; Rodrigues, Joao V.; Marcos, Joao C.
      Analytical Biochemistry (2014), 458 (), 69-71CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      This work proposes a modification of the 2,4-dinitrophenylhydrazine (DNPH) spectrophotometric assay commonly used to evaluate the concn. of carbonyl groups in oxidized proteins. In this approach NaOH is added to the protein soln. after the addn. of DNPH, shifting the max. absorbance wavelength of the derivatized protein from 370 to 450 nm. This reduces the interference of DNPH and allows the direct quantification in the sample soln. without the need for the pptn., washing, and resuspension steps that are carried out in the traditional DNPH method. The two methods were compared under various conditions and are statistically equiv.
    285. 285
      Xue, Y.-P.; Wang, W.; Wang, Y.-J.; Liu, Z.-Q.; Zheng, Y.-G.; Shen, Y.-C. Isolation of Enantioselective α-Hydroxyacid Dehydrogenases Based on a High-Throughput Screening Method. Bioprocess Biosyst. Eng. 2012, 35 (9), 1515– 22,  DOI: 10.1007/s00449-012-0741-1
      287
      Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method
      Xue, Ya-Ping; Wang, Wei; Wang, Ya-Jun; Liu, Zhi-Qiang; Zheng, Yu-Guo; Shen, Yin-Chu
      Bioprocess and Biosystems Engineering (2012), 35 (9), 1515-1522CODEN: BBEIBV; ISSN:1615-7591. (Springer)
      To isolate enantioselective α-hydroxyacid dehydrogenases (α-HADHs), a high-throughput screening method was established. 2,4-Dinitrophenylhydrazine soln. forms a red-brown complex with ketoacid produced during the α-HADH-mediated oxidn. of α-hydroxyacid. The complex can be easily quantified by spectrophotometric measurement at 458 nm. The enantioselectivity of α-HADH in each strain can be measured with this colorimetric method using (R)- and (S)-α-hydroxyacid concurrently as substrates to evaluate the apparent enantioselectivity (Eapp). The Eapp closely matches the value of true enantioselectivity (E true) detd. by HPLC anal. With this method, a total of 34 stains harboring enantioselective α-HADHs were selected from 526 potential α-HADH-producing microorganisms. Pseudomonas aeruginosa displayed the highest (S)-enantioselective α-HADH activity. This strain appears promising for potential application in industry to produce (R)-α-hydroxyacids. The method described herein represents a useful tool for the high-throughput isolation of enantioselective α-HADHs.
    286. 286
      Chen, Q.; Chen, X.; Feng, J.; Wu, Q.; Zhu, D.; Ma, Y. Improving and Inverting Cβ-Stereoselectivity of Threonine Aldolase Via Substrate-Binding-Guided Mutagenesis and a Stepwise Visual Screening. ACS Catal. 2019, 9 (5), 4462– 9,  DOI: 10.1021/acscatal.9b00859
      288
      Improving and Inverting Cβ-Stereoselectivity of Threonine Aldolase via Substrate-Binding-Guided Mutagenesis and a Stepwise Visual Screening
      Chen, Qijia; Chen, Xi; Feng, Jinhui; Wu, Qiaqing; Zhu, Dunming; Ma, Yanhe
      ACS Catalysis (2019), 9 (5), 4462-4469CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      Threonine aldolase (TA)-catalyzed aldol condensation is a powerful tool for C-C bond formation under mild conditions, but the low Cβ-stereoselectivity has hampered its wide application. A stepwise visual screening method was developed to measure the activity and stereoselectivity of threonine aldolase-catalyzed aldol condensation by employing a stereoselective phenylserine dehydratase, enabling direct selection of mutants with higher Cβ-stereoselectivity. Mutants of L-PsTA from Pseudomonas sp. with improved or inverted stereoselectivity toward arom. aldehydes were obtained by simultaneously mutating amino acid residues which interact with the amino and hydroxyl groups of the substrate and screening the resulting mutant libraries with this method. The mutation and enzyme-substrate docking studies provided some insights into the regulation of the Cβ-stereoselectivity by the enzyme-substrate interactions. This study offers a tool and useful guidance for further engineering of TAs to address the Cβ-stereoselectivity problem.
    287. 287
      Yang, C.; Ye, L.; Gu, J.; Yang, X.; Li, A.; Yu, H. Directed Evolution of Mandelate Racemase by a Novel High-Throughput Screening Method. Appl. Microbiol. Biotechnol. 2017, 101 (3), 1063– 72,  DOI: 10.1007/s00253-016-7790-3
      289
      Directed evolution of mandelate racemase by a novel high-throughput screening method
      Yang, Chengcheng; Ye, Lidan; Gu, Jiali; Yang, Xiaohong; Li, Aipeng; Yu, Hongwei
      Applied Microbiology and Biotechnology (2017), 101 (3), 1063-1072CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      Optically pure Me (R)-o-chloromandelate and (R)-acetyl-o-mandelic acid are key intermediates for the synthesis of (S)-clopidogrel, which could be prepd. with 100% theor. yield by sequential hydrolysis and racemization. At the moment, efficient sequential hydrolysis and racemization are hindered by the low catalytic activity of mandelate racemase (MR) toward (S)-o-chloromandelic acid [(S)-2-CMA]. Here, the authors proposed to improve the catalytic performance of MR toward (S)-2-CMA by directed evolution and developed an enantioselective oxidn. system for high-throughput screening (HTS) of MR libraries. Based on this HTS method, a triple mutant V22I/V29I/Y54F (MRDE1) with 3.5-fold greater relative activity as compared to native MR was obtained. Kinetic anal. indicated that the enhanced catalytic efficiency mainly arose from the elevated kcat. Further insight into the source of improved catalytic activity was gained by mol. simulations, finding that substrate binding and product release were possibly made easier by decreased steric bulk and increased hydrophobicity of substrate-binding sites. In addn., the substrate (S)-2-CMA in the enzyme-substrate complex of MRDE1 seemed to have a lower binding free energy comparing with the complex of wild-type MR. The HTS method developed in this work and the successful directed evolution of MR based on this method provide an example for racemase engineering and may inspire directed evolution of other racemases toward enhanced catalytic performance on non-natural substrates.
    288. 288
      Uzu, S.; Kanda, S.; Imai, K.; Nakashima, K.; Akiyama, S. Fluorogenic Reagents: 4-Aminosulphonyl-7-Hydrazino-2,1,3-Benzoxadiazole, 4-(N,N-Dimethylaminosulphonyl)-7-Hydrazino-2,1,3-Benzoxadiazole and 4-Hydrazino-7-Nitro-2,1,3-Benzoxadiazole Hydrazine for Aldehydes and Ketones. Analyst 1990, 115 (11), 1477– 82,  DOI: 10.1039/an9901501477
      290
      Fluorogenic reagents: 4-aminosulfonyl-7-hydrazino-2,1,3-benzoxadiazole, 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole and 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine for aldehydes and ketones
      Uzu, Sonoko; Kanda, Susumu; Imai, Kazuhiro; Nakashima, Kenichiro; Akiyama, Shuzo
      Analyst (Cambridge, United Kingdom) (1990), 115 (11), 1477-82CODEN: ANALAO; ISSN:0003-2654.
      Fluorogenic reagents for aldehydes and ketones, viz., 4-aminosulfonyl-7-hydrazino-2,1,3-benzoxadiazole (ABD-H) and 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H) and also purified 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine (NBD-H.NH2NH2) were synthesized. These reagents are not fluorescent; however, their reaction products with aldehydes and ketones fluoresce at wavelengths from 548 to 580 nm with excitation from 450 to 470 nm. Both ABD-H and DBD-H exhibited similar reactivity and were more reactive than NBD-H.NH2NH2. The resp. pseudo-first-order reaction rate consts. for the prodn. of the hydrazone of propionaldehyde with ABD-H, DBD-H and NBD-H.NH2NH2 were 8.9 ×10-2, 7.2 × 10-2 and 4.2 × 10-2 min-1 (the reaction was carried out in 0.0025% trifluoroacetic acid in acetonitrile at room temp., 22°). The detection limits using the manual method (i.e., measurement of fluorescence intensity) for the hydrazones of aldehydes and ketones with ABD-H, DBD-H and NBD-H.NH2NH2 were in the μM range. The substrate blank fluorescence with ABD-H was half of that with DBD-H and NBD-H.NH2NH2. The reaction products were sepd. and analyzed by reversed-phase HPLC with spectrofluorometric detection. The detection limits for propionaldehyde were 1040, 120 and 35.0 fmol with ABD-H, DBD-H and NBD-H.NH2NH2, resp., and those for heptan-4-one were 2690, 560 and 673 fmol, resp. Of the three reagents, DBD-H is recommended for the sensitive detection of ketones and NBD-H.NH2NH2 for the detection of aldehydes. The detection limits for aldehydes and ketones by HPLC were in the sub-pmol to pmol range.
    289. 289
      Uchiyama, S.; Santa, T.; Okiyama, N.; Fukushima, T.; Imai, K. Fluorogenic and Fluorescent Labeling Reagents with a Benzofurazan Skeleton. Biomed. Chromatogr. 2001, 15 (5), 295– 318,  DOI: 10.1002/bmc.75
      291
      Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton
      Uchiyama, Seiichi; Santa, Tomofumi; Okiyama, Natsuko; Fukushima, Takeshi; Imai, Kazuhiro
      Biomedical Chromatography (2001), 15 (5), 295-318CODEN: BICHE2; ISSN:0269-3879. (John Wiley & Sons Ltd.)
      A review with many refs. Fluorogenic and fluorescent labeling reagents having a benzofurazan (2,1,3-benzoxadiazole) skeleton such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F), ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H), 4-N,N-dimethylaminosulfonyl-7-hydrazino-2,1,3-benzoxadiazole (DBD-H), 4-nitro-7-N-piperazino-2,1,3-benzoxadiazole (NBD-PZ), 4-N,N-dimethylaminosulfonyl-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ), 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) are reviewed in terms of synthetic method, reactivity, fluorescence characteristics, sensitivity and application to analytes.
    290. 290
      Frizon, T. E. A.; Vieira, A. A.; da Silva, F. N.; Saba, S.; Farias, G.; de Souza, B.; Zapp, E.; Lôpo, M. N.; Braga, H. d. C.; Grillo, F. Synthesis of 2,1,3-Benzoxadiazole Derivatives as New Fluorophores─Combined Experimental, Optical, Electro, and Theoretical Study. Front. Chem. 2020, 8, 360,  DOI: 10.3389/fchem.2020.00360
      292
      Synthesis of 2,1,3-benzoxadiazole derivatives as new fluorophores-combined experimental, optical, electro, and theoretical study
      Frizon, Tiago E. A.; Vieira, Andre A.; Da Silva, Fabricia N.; Saba, Sumbal; Farias, Giliandro; De Souza, Bernardo; Zapp, Eduardo; Lopo, Michell N.; Braga, Hugo De C.; Grillo, Felipe; Curcio, Sergio F.; Cazati, Thiago; Rafique, Jamal
      Frontiers in Chemistry (Lausanne, Switzerland) (2020), 8 (), 00360CODEN: FCLSAA; ISSN:2296-2646. (Frontiers Media S.A.)
      Herein, the synthesis and characterization of fluorophores I (R = 2-ethylhexyl, octyl, decyl, dodecyl) contg. a 2,1,3-benzoxadiazole unit assocd. with a π-conjugated system (D-π-A-π-D) has been reported. These new fluorophores in soln. exhibited an absorption max. at around ~ 419 nm (visible region), as expected for electronic transitions of the π-π* type (ε ~ 2.7*107 L mol-1 cm-1), and strong solvent-dependent fluorescence emission (ΦFL ~ 0.5) located in the bluish-green region. The Stokes' shift of these compds.is ca. 3,779 cm-1, which was attributed to an intramol. charge transfer (ICT) state. In CHCl3 soln., the compds. exhibited longer and shorter lifetimes, which was attributed to the emission of monomeric and aggregated mols., resp. DFT was used to model the electronic structure of I in their excited and ground electronic states. The simulated emission spectra are consistent with the exptl. results, with different solvents leading to a shift in the emission peak and the attribution of a π-π* state with the characteristics of a charge transfer excitation. The thermal properties were analyzed by thermogravimetric anal., and a high max. degrdn. rate occurred at around 300°. Electrochem. studies were also performed in order to det. the band gaps of the mols. The electrochem. band gaps (2.48-2.70 eV) showed strong correlations with the optical band gaps (2.64-2.67 eV).
    291. 291
      Konarzycka-Bessler, M.; Bornscheuer, U. T. A High-Throughput-Screening Method for Determining the Synthetic Activity of Hydrolases. Angew. Chem., Int. Ed. 2003, 42 (12), 1418– 20,  DOI: 10.1002/anie.200390365
      293
      A high-throughput-screening method for determining the synthetic activity of hydrolases
      Konarzycka-Bessler, Monika; Bornscheuer, Uwe T.
      Angewandte Chemie, International Edition (2003), 42 (12), 1418-1420CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      An assay format for high-throughput detn. of synthetic activity of hydrolases is described. The method is based on lipase- or esterase-catalyzed transesterification reactions between a vinyl ester and an alc. As a model reaction, the esterification between vinyl laurate and 1-propanol, in the presence of 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H), was studied in microtiterplates. Several lipases and esterases were investigated with this reaction. The enzymes studied were Chirazyme L2 (CAL-B), L3, (CRL), L5M L6 (PSL), L9 (RML), Amano PS (PCL), Amano A (ANL), Amano AK (PFL), Amano AY (CRL), and SDE. The assay is extremely versatile for the detn. of the synthetic activity of lipases or esterases in a high-throughput format and thus enables the rapid identification of active enzymes and/or appropriate reaction conditions.
    292. 292
      Jencks, W. P. Progress in Physical Organic Chemistry; Index to Reviews, Symposia Volumes and Monographs in Organic Chemistry; Cohen, S. G., Streitwieser, A., Jr., Taft, R. W., Eds.; John Wiley & Sons, 1964; Vol. 2, p 110 DOI: 10.1002/9780470171813
      There is no corresponding record for this reference.
    293. 293
      Larsen, D.; Pittelkow, M.; Karmakar, S.; Kool, E. T. New Organocatalyst Scaffolds with High Activity in Promoting Hydrazone and Oxime Formation at Neutral pH. Org. Lett. 2015, 17 (2), 274– 7,  DOI: 10.1021/ol503372j
      295
      New Organocatalyst Scaffolds with High Activity in Promoting Hydrazone and Oxime Formation at Neutral pH
      Larsen, Dennis; Pittelkow, Michael; Karmakar, Saswata; Kool, Eric T.
      Organic Letters (2015), 17 (2), 274-277CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
      The discovery of two new classes of catalysts for hydrazone and oxime formation in water at neutral pH, 2-aminophenols and 2-(aminomethyl)benzimidazoles, is reported. Kinetics studies in aq. solns. at pH 7.4 revealed rate enhancements up to 7-fold greater than with classic aniline catalysis. 2-(Aminomethyl)benzimidazoles are effective catalysts with otherwise challenging aryl ketone substrates.
    294. 294
      Crisalli, P.; Kool, E. T. Importance of Ortho Proton Donors in Catalysis of Hydrazone Formation. Org. Lett. 2013, 15 (7), 1646– 9,  DOI: 10.1021/ol400427x
      296
      Importance of ortho Proton Donors in Catalysis of Hydrazone Formation
      Crisalli, Pete; Kool, Eric T.
      Organic Letters (2013), 15 (7), 1646-1649CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
      Anthranilic acids were recently reported as superior catalysts for hydrazone and oxime formation compared to aniline, the classic catalyst for these reactions. Here, alternative proton donors were examd. with varied pKa in an effort to enhance activity at biol. pH. The expts. show that 2-aminobenzenephosphonic acids are superior to anthranilic acids in catalyzing hydrazone formation with common aldehyde substrates.
    295. 295
      Wang, S.; Nawale, G. N.; Kadekar, S.; Oommen, O. P.; Jena, N. K.; Chakraborty, S.; Hilborn, J.; Varghese, O. P. Saline Accelerates Oxime Reaction with Aldehyde and Keto Substrates at Physiological pH. Sci. Rep. 2018, 8, 2193,  DOI: 10.1038/s41598-018-20735-0
      297
      Saline Accelerates Oxime Reaction with Aldehyde and Keto Substrates at Physiological pH
      Wang Shujiang; Nawale Ganesh N; Kadekar Sandeep; Hilborn Jons; Varghese Oommen P; Oommen Oommen P; Jena Naresh K; Chakraborty Sudip
      Scientific reports (2018), 8 (1), 2193 ISSN:.
      We have discovered a simple and versatile reaction condition for oxime mediated bioconjugation reaction that could be adapted for both aldehyde and keto substrates. We found that saline accelerated the oxime kinetics in a concentration-dependent manner under physiological conditions. The reaction mechanism is validated by computational studies, and the versatility of the reaction is demonstrated by cell-surface labeling experiments. Saline offers an efficient and non-toxic catalytic option for performing the bioorthogonal-coupling reaction of biomolecules at the physiological pH. This saline mediated bioconjugation reaction represents the most biofriendly, mild and versatile approach for conjugating sensitive biomolecules and does not require any extensive purification step.
    296. 296
      Mayr, H.; Bug, T.; Gotta, M. F.; Hering, N.; Irrgang, B.; Janker, B.; Kempf, B.; Loos, R.; Ofial, A. R.; Remennikov, G. Reference Scales for the Characterization of Cationic Electrophiles and Neutral Nucleophiles. J. Am. Chem. Soc. 2001, 123 (39), 9500– 12,  DOI: 10.1021/ja010890y
      298
      Reference scales for the characterization of cationic electrophiles and neutral nucleophiles
      Mayr, Herbert; Bug, Thorsten; Gotta, Matthias F.; Hering, Nicole; Irrgang, Bernhard; Janker, Brigitte; Kempf, Bernhard; Loos, Robert; Ofial, Armin R.; Remennikov, Grigoriy; Schimmel, Holger
      Journal of the American Chemical Society (2001), 123 (39), 9500-9512CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Twenty-three diarylcarbenium ions and 38 π-systems (arenes, alkenes, allyl silanes and stannanes, silyl enol ethers, silyl ketene acetals, and enamines) have been defined as basis sets for establishing general reactivity scales for electrophiles and nucleophiles. The rate consts. of 209 combinations of these benzhydrylium ions and π-nucleophiles, 85 of which are first presented in this article, have been subjected to a correlation anal. to det. the electrophilicity parameters E and the nucleophilicity parameters N and s as defined by the equation log k(20°) = s(N + E) (Mayr, H.; Patz, M. Angew. Chem., Int. Ed. Engl. 1994, 33, 938-957). Though the reactivity scales thus obtained cover more than 16 orders of magnitude, the individual rate consts. are reproduced with a std. deviation of a factor of 1.19 . It is shown that the reactivity parameters thus derived from the reactions of diarylcarbenium ions with π-nucleophiles (Figure 3) are also suitable for characterizing the nucleophilic reactivities of alkynes, metal-π-complexes, and hydride donors and for characterizing the electrophilic reactivities of heterosubstituted and metal-coordinated carbenium ions . Reactivity parameters are, therefore, recommended for the characterization of any new electrophiles and nucleophiles in the reactivity range covered. The linear correlation between the electrophilicity parameters E of benzhydryl cations and the corresponding substituent consts. σ+ provides Hammett σ+ consts. for 10 substituents from -1.19 to -2.11, i.e., in a range with only very few previous entries.
    297. 297
      Brotzel, F.; Chu, Y. C.; Mayr, H. Nucleophilicities of Primary and Secondary Amines in Water. J. Org. Chem. 2007, 72 (10), 3679– 88,  DOI: 10.1021/jo062586z
      299
      Nucleophilicities of primary and secondary amines in water
      Brotzel, Frank; Chu, Ying Cheung; Mayr, Herbert
      Journal of Organic Chemistry (2007), 72 (10), 3679-3688CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      The kinetics of the reactions of 26 primary and secondary amines with benzhydrylium ions in water were investigated photometrically. Because the parallel reactions of the benzhydrylium ions with hydroxide and water are much slower, the second-order rate consts. for the reactions of amines with benzhydrylium ions could be detd. reliably. Reactivities of anilines were also studied in acetonitrile soln. Plots of log k2,N for these reactions vs the electrophilicity parameters E of the benzhydrylium ions were linear, which allowed us to derive the nucleophilicity parameters N and s for amines as defined by the equation log k (20°) = s(E + N). Because the slope parameters for the different amines are closely similar; the relative nucleophilicities are almost independent of the electrophiles and can be expressed by the nucleophilicity parameters N. The correlation between nucleophilicity N and pKaH values is poor, and it is found that secondary alkyl amines and anilines are considerably more nucleophilic, while ammonia is much less nucleophilic than expected on the basis of their pKaH values.
    298. 298
      Brotzel, F., lmu, 2008.
      There is no corresponding record for this reference.
    299. 299
      Nakashima, K.; Hidaka, Y.; Yoshida, T.; Kuroda, N.; Akiyama, S. High-Performance Liquid Chromatographic Determination of Short-Chain Aliphatic Aldehydes Using 4-(N,N-Dimethylaminosulphonyl)-7-Hydrazino-2,1,3-Benzoxadiazole as a Fluorescence Reagent. J. Chromatogr. B Biomed. Appl. 1994, 661 (2), 205– 10,  DOI: 10.1016/0378-4347(94)00359-9
      301
      High-performance liquid chromatographic determination of short-chain aliphatic aldehydes using 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole as a fluorescence reagent
      Nakashima, Kenichiro; Hidaka, Youko; Yoshida, Tomomi; Kuroda, Naotaka; Akiyama, Shuzo
      Journal of Chromatography B: Biomedical Sciences and Applications (1994), 661 (2), 205-10CODEN: JCBBEP; ISSN:0378-4347. (Elsevier)
      HPLC detn. of four short-chain aliph. aldehydes using fluorescence detection was carried out with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). DBD-H derivs. with three aliph. aldehydes-formaldehyde, acetaldehyde and propionaldehyde-were synthesized and their fluorescence properties were examd. Relative fluorescence intensities of these compds. in acetonitrile were ∼ten-fold larger than those in aq. acetonitrile. DBD-hydrazones could be sepd. by reversed-phase chromatog. using aq. acetonitrile as eluent and detection at 560 nm with excitation at 445 nm. Submicromolar levels of formaldehyde, acetaldehyde, propionaldehyde and butylaldehyde could be detd. The HPLC procedure using propionaldehyde as internal std. was applied to the measurement of acetaldehyde levels in normal human plasma before and 30 min after ingestion of ethanol.
    300. 300
      Jacobsen, N. W.; Dickinson, R. G. Spectrometric Assay of Aldehydes as 6-Mercapto-3-Substituted-s-Trizolo (4,3-b)-Tetrazines. Anal. Chem. 1974, 46 (2), 298– 9,  DOI: 10.1021/ac60338a039
      302
      Spectrometric assay of aldehydes as 6-mercapto-3-substituted-s-trizolo(4,3-b)-tetrazines
      Jacobsen, N. W.; Dickinson, R. G.
      Analytical Chemistry (1974), 46 (2), 298-9CODEN: ANCHAM; ISSN:0003-2700.
      H2CO was detd. photometrically at 549 nm after mixing the sample with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (I) and aerating the mixt. 30 min at 20° to form II (R = H). Beer's law was followed for 0.5-5 ppm H2CO. The relative error was ≤3% for 5-20 ppm H2CO, after suitable diln. Reaction of RCHO (R = Me, Et, Pr, and Ph) with I gave II with absorption max. at 537, 536, 532, and 340 nm, resp. demonstrating the suitability of the method for detns. of other aldehydes.
    301. 301
      Dickinson, R. G.; Jacobsen, N. W. A New Sensitive and Specific Test for the Detection of Aldehydes: Formation of 6-Mercapto-3-Substituted-S-Triazolo[4,3-b]-s-Tetrazines. J. Chem. Soc. D 1970, (24), 1719– 20,  DOI: 10.1039/c29700001719
      303
      A new sensitive and specific test for the detection of aldehydes: formation of 6-mercapto-3-substituted-s-triazolo[4,3-b]-s-tetrazines
      Dickinson, Ronald G.; Jacobsen, Noel W.
      Journal of the Chemical Society [Section] D: Chemical Communications (1970), (24), 1719-20CODEN: CCJDAO; ISSN:0577-6171.
      Aldehydes (RCHO) (≥10-4M) can be detected specifically by condensation with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole in 1N NaOH with subsequent aeration to give 3-R-substituted-6-mercapto-s-triazolo[4,3-b]-s-tetrazines (I) which exhibited magenta, purple, or purple-brown colors when R was an aliphatic, aromatic, or an aliphatic or aromatic group with addnl. functional groups, resp. I (R = iso-Pr or Ph) m. 146-7° (decompn.) or 192-3° (decompn.), resp., were prepd. and characterized by uv spectroscopy and NMR.
    302. 302
      Quesenberry, M. S.; Lee, Y. C. A Rapid Formaldehyde Assay Using Purpald Reagent: Application under Periodation Conditions. Anal. Biochem. 1996, 234 (1), 50– 5,  DOI: 10.1006/abio.1996.0048
      304
      A rapid formaldehyde assay using Purpald reagent: application under periodation conditions
      Quesenberry, M. S.; Lee, Y. C.
      Analytical Biochemistry (1996), 234 (1), 50-5CODEN: ANBCA2; ISSN:0003-2697. (Academic)
      The reaction of formaldehyde with Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole) in formaldehyde assay works under alk. conditions at room temp., and the sensitivity is superior to other methods. The color development by this reagent, however, requires oxidn. of the adduct with hydrogen peroxide, air oxygen, or dil. periodate. We found that low levels of periodate, commonly used to oxidize specifically terminal vicinal diols to yield formaldehyde, are compatible with color development with the Purpald reagent. We have investigated the conditions required for use of the Purpald reagent, esp. in conjunction with periodate oxidn. reactions. We have used the assay either in test tubes or with microplates, attaining sensitivity of as little as 1 nmol formaldehyde.
    303. 303
      Anthon, G. E.; Barrett, D. M. Comparison of Three Colorimetric Reagents in the Determination of Methanol with Alcohol Oxidase. Application to the Assay of Pectin Methylesterase. J. Agric. Food Chem. 2004, 52 (12), 3749– 53,  DOI: 10.1021/jf035284w
      305
      Comparison of Three Colorimetric Reagents in the Determination of Methanol with Alcohol Oxidase. Application to the Assay of Pectin Methylesterase
      Anthon, Gordon E.; Barrett, Diane M.
      Journal of Agricultural and Food Chemistry (2004), 52 (12), 3749-3753CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      Three colorimetric reagents for the detn. of formaldehyde, the Nash reagent (ammonia plus acetylacetone), Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole), and N-methylbenzothiazolinone-2-hydrazone (MBTH), were compared for the detn. of methanol when used in conjunction with alc. oxidase. The combination of alc. oxidase plus the commonly used Nash reagent was specific for methanol vs. ethanol, but had the lowest sensitivity of the three reagents tested. Substituting Purpald for the Nash reagent increased the sensitivity 3-fold while still maintaining a high (59-fold) selectivity for methanol vs. ethanol. Using MBTH increased the sensitivity still further, but with a loss of the selectivity toward methanol. Since MBTH reacted with aldehydes under neutral conditions, it could be included along with the alc. oxidase to act as an aldehyde trap. This prevented further oxidn. reactions by alc. oxidase and allowed for extended incubations. A procedure for assaying low levels of pectin methylesterase activity that relies on this trapping ability is described. In addn., alc. oxidase plus Purpald is shown to be a simple and sensitive way to measure the methanol released from plant material following the thermal activation of endogenous pectin methylesterase.
    304. 304
      Hammer, S. C.; Kubik, G.; Watkins, E.; Huang, S.; Minges, H.; Arnold, F. H. Anti-Markovnikov Alkene Oxidation by Metal-Oxo-Mediated Enzyme Catalysis. Science 2017, 358 (6360), 215– 8,  DOI: 10.1126/science.aao1482
      306
      Anti-Markovnikov alkene oxidation by metal-oxo-mediated enzyme catalysis
      Hammer, Stephan C.; Kubik, Grzegorz; Watkins, Ella; Huang, Shan; Minges, Hannah; Arnold, Frances H.
      Science (Washington, DC, United States) (2017), 358 (6360), 215-218CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Catalytic anti-Markovnikov oxidn. of alkene feedstocks could simplify synthetic routes to many important mols. and solve a long-standing challenge in chem. Here we report the engineering of a cytochrome P 450 enzyme by directed evolution to catalyze metal-oxo-mediated anti-Markovnikov oxidn. of styrenes with high efficiency. The enzyme uses dioxygen as the terminal oxidant and achieves selectivity for anti-Markovnikov oxidn. over the kinetically favored alkene epoxidn. by trapping high-energy intermediates and catalyzing an oxo transfer, including an enantioselective 1,2-hydride migration. The anti-Markovnikov oxygenase can be combined with other catalysts in synthetic metabolic pathways to access a variety of challenging anti-Markovnikov functionalization reactions.
    305. 305
      Nguyen, Q.-T.; Romero, E.; Dijkman, W. P.; de Vasconcellos, S. P.; Binda, C.; Mattevi, A.; Fraaije, M. W. Structure-Based Engineering of Phanerochaete Chrysosporium Alcohol Oxidase for Enhanced Oxidative Power toward Glycerol. Biochemistry 2018, 57 (43), 6209– 18,  DOI: 10.1021/acs.biochem.8b00918
      307
      Structure-Based Engineering of Phanerochaete chrysosporium Alcohol Oxidase for Enhanced Oxidative Power toward Glycerol
      Nguyen, Quoc-Thai; Romero, Elvira; Dijkman, Willem P.; de Vasconcellos, Suzan Pantaroto; Binda, Claudia; Mattevi, Andrea; Fraaije, Marco W.
      Biochemistry (2018), 57 (43), 6209-6218CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Glycerol is a major byproduct of biodiesel prodn. and enzymes that oxidize this compd. have been long sought after. The recently described alc. oxidase from the white-rot basidiomycete Phanerochaete chrysosporium (PcAOX) was reported to feature very mild activity on glycerol. Here we describe the comprehensive structural and biochem. characterization of this enzyme. PcAOX was expressed in Escherichia coli in high yields and displayed high thermostability. Steady-state kinetics revealed that PcAOX is highly active towards methanol, ethanol, and 1-propanol (kcat = 18, 19, and 11 s-1, resp.), but showed very limited activity towards glycerol (kcat = 0.2 s-1 at 2 M substrate). The crystal structure of the homo-octameric PcAOX was detd. at 2.6 Å resoln. The catalytic center is a remarkable solvent-inaccessible cavity located at the re side of the flavin cofactor. Its small size explains the obsd. preference for methanol and ethanol as best substrates. These findings led us to design several cavity-enlarging mutants with significantly improved activity towards glycerol. Among them, the F101S variant had a high kcat value of 3 s-1 retaining a high degree of thermostability. The crystal structure of F101S PcAOX was solved, confirming the site of mutation and the larger substrate-binding pocket. Our data demonstrate that PcAOX is a very promising enzyme for glycerol biotransformation.
    306. 306
      Sawicki, E.; Hauser, T. R.; Stanley, T. W.; Elbert, W. The 3-Methyl-2-Benzothiazolone Hydrazone Test. Sensitive New Methods for the Detection, Rapid Estimation, and Determination of Aliphatic Aldehydes. Anal. Chem. 1961, 33 (1), 93– 6,  DOI: 10.1021/ac60169a028
      308
      3-Methyl-2-benzothiazolinone hydrazone test-sensitive new methods for the detection, rapid estimation, and determination of aliphatic aldehydes
      Sawicki, Eugene; Hauser, Thomas R.; Stanley, Thomas W.; Elbert, Walter
      (1961), 33 (), 93-6CODEN: ANCHAM; ISSN:0003-2700.
      The reaction of an aliphatic aldehyde with 3-methyl-2-benzothiazolinone hydrazone (I) followed by oxidn. of excess I with FeCl3 in acid soln., results in an intensely brilliant, blue cationic dye. The method is applicable to spot plate, paper, silica gel, and colorimetric procedures. The latter 2 methods may be used quant. and can be applied to the analysis of auto exhaust fumes and polluted air. In the colorimetric method, Beer's law is obeyed from 5-125 γ/100 ml. in the case of HCHO. The identification limit of HCHO by either spot plate or paper techniques is 0.1 γ. Two methods for the synthesis of I are described.
    307. 307
      Zhu, P.; Lin, S.-M. MBTH for aliphatic aldehyde measurement, U.S. Patent US7112448B2, September 26, 2006.
      There is no corresponding record for this reference.
    308. 308
      Hauser, T. R.; Cummins, R. L. Increasing Sensitivity of 3-Methyl-2-Benzothiazolone Hydrozone Test for Analysis of Aliphatic Aldehydes in Air. Anal. Chem. 1964, 36 (3), 679– 81,  DOI: 10.1021/ac60209a067
      310
      Increasing sensitivity of 3-methyl-2-benzothiazolinone hydrazone test for analysis of aliphatic aldehydes in air
      Hauser, Thomas R.; Cummins, Rodney L.
      (1964), 36 (3), 679-81CODEN: ANCHAM; ISSN:0003-2700.
      cf. CA 55, 8180a. The sensitivity of the test was increased 6-fold by the incorporation of H2NSO3H in the FeCl3 oxidizing reagent. The modification reduces the vol. of reagent required and eliminates the need to dil. with Me2CO in order to avoid turbidity. The absorbance at 628 mμ follows Beer's law for concns. of HCHO from 0 to 1.70 γ/ml.
    309. 309
      Dean Pakulski, J.; Benner, R. An Improved Method for the Hydrolysis and MBTH Analysis of Dissolved and Particulate Carbohydrates in Seawater. Mar. Chem. 1992, 40 (3), 143– 60,  DOI: 10.1016/0304-4203(92)90020-B
      There is no corresponding record for this reference.
    310. 310
      Gomez, L. D.; Whitehead, C.; Barakate, A.; Halpin, C.; McQueen-Mason, S. J. Automated Saccharification Assay for Determination of Digestibility in Plant Materials. Biotechnol. Biofuels 2010, 3, 23,  DOI: 10.1186/1754-6834-3-23
      312
      Automated saccharification assay for determination of digestibility in plant materials
      Gomez Leonardo D; Whitehead Caragh; Barakate Abdellah; Halpin Claire; McQueen-Mason Simon J
      Biotechnology for biofuels (2010), 3 (), 23 ISSN:.
      BACKGROUND: Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies. RESULTS: We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared. CONCLUSIONS: The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies.
    311. 311
      Oliveira, F. S. d.; Leite, B. C. O.; Andrade, M. V. A. S. d.; Korn, M. Determination of Total Aldehydes in Fuel Ethanol by MBTH Method: Sequential Injection Analysis. J. Braz. Chem. Soc. 2005, 16, 87– 92,  DOI: 10.1590/S0103-50532005000100013
      There is no corresponding record for this reference.
    312. 312
      Suchý, M.; Lazurko, C.; Kirby, A.; Dang, T.; Liu, G.; Shuhendler, A. J. Methyl 5-MeO-N-Aminoanthranilate, a Minimalist Fluorogenic Probe for Sensing Cellular Aldehydic Load. Org. Biomol. Chem. 2019, 17 (7), 1843– 53,  DOI: 10.1039/C8OB02255K
      314
      Methyl 5-MeO-N-aminoanthranilate, a minimalist fluorogenic probe for sensing cellular aldehydic load
      Suchy, Mojmir; Lazurko, Caitlin; Kirby, Alexia; Dang, Trina; Liu, George; Shuhendler, Adam J.
      Organic & Biomolecular Chemistry (2019), 17 (7), 1843-1853CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Me 5-MeO-N-aminoanthranilate, a fluorogenic probe comprising a single substituted benzene ring has been applied towards the fluorescence detection of endogenous carbonyls through rapid, catalyst-free complexation of these bio-derived markers of cell stress under physiol. conditions. The products formed during the reaction between the probe and aldehydic products of lipid peroxidn., including malondialdehyde and long-chain aliph. aldehydes relevant to the oxidative decompn. of cell membranes, have been evaluated. Live cell imaging of di-Et maleate-induced oxidative stress with or without pretreatment with α-tocopherol was carried out, with the result suggesting that the presented mol. might serve as a minimalist mol. probe capable of cellular "Aldehydic Load" detection by fluorescence microscopy. This work also outlines functional constraints of the fluorogenic probe (i.e. intramol. cyclization), providing a realistic evaluation of Me 5-MeO-N-aminoanthranilate for fluorescence-based aldehyde detection.
    313. 313
      Yuen, L. H.; Saxena, N. S.; Park, H. S.; Weinberg, K.; Kool, E. T. Dark Hydrazone Fluorescence Labeling Agents Enable Imaging of Cellular Aldehydic Load. ACS Chem. Biol. 2016, 11 (8), 2312– 9,  DOI: 10.1021/acschembio.6b00269
      315
      Dark Hydrazone Fluorescence Labeling Agents Enable Imaging of Cellular Aldehydic Load
      Yuen, Lik Hang; Saxena, Nivedita S.; Park, Hyun Shin; Weinberg, Kenneth; Kool, Eric T.
      ACS Chemical Biology (2016), 11 (8), 2312-2319CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Aldehydes are key intermediates in many cellular processes, from endogenous metabolic pathways like glycolysis to undesired exogenously induced processes such as lipid peroxidn. and DNA interstrand crosslinking. Alkyl aldehydes are well documented to be cytotoxic, affecting the functions of DNA and protein, and their levels are tightly regulated by the oxidative enzyme ALDH2. Mutations in this enzyme are assocd. with cardiac damage, diseases such as Fanconi anemia (FA), and cancer. Many attempts have been made to identify and quantify the overall level of these alkyl aldehydes inside cells, yet there are few practical methods available to detect and monitor these volatile aldehydes in real time. Here, the authors describe a multicolor fluorogenic hydrazone transfer ("DarkZone") system to label alkyl aldehydes, yielding up to 30-fold light-up response in vitro. A cell-permeant DarkZone dye design was applied to detect small-mol. aldehydes in the cellular environment. The new dye design also enabled the monitoring of cellular acetaldehyde prodn. from ethanol over time by flow cytometry, demonstrating the utility of the DarkZone dyes for measuring and imaging the aldehydic load related to human disease.
    314. 314
      Liu, T.; Yang, L.; Zhang, J.; Liu, K.; Ding, L.; Peng, H.; Belfield, K. D.; Fang, Y. Squaraine-Hydrazine Adducts for Fast and Colorimetric Detection of Aldehydes in Aqueous Media. Sens. Actuators B Chem. 2019, 292, 88– 93,  DOI: 10.1016/j.snb.2019.04.138
      316
      Squaraine-hydrazine adducts for fast and colorimetric detection of aldehydes in aqueous media
      Liu, Taihong; Yang, Lvjie; Zhang, Jing; Liu, Ke; Ding, Liping; Peng, Haonan; Belfield, Kevin D.; Fang, Yu
      Sensors and Actuators, B: Chemical (2019), 292 (), 88-93CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      Capitalizing on the nucleophilic addn. of hydrazine toward the central cyclobutenyl core and affinity of aldehydes to hydrazine group, two water-sol. squaraine dyes (SQOH and SQPY) were synthesized as sensitive colorimetric and fluorescent chemosensors for aldehydes. The color change from blue to colorless and back to blue these sensors underwent was readily obsd., even by the naked eyes. The response was fast (less than 1 s) and the detection limit for formaldehyde as an example was about 60μM. In contrast to the previous nucleophilic attack to squaraines and the other amine refs. studied, this work underwent a whole ON-OFF-ON sensing circle based on the characteristics of hydrazine. Meanwhile, the different photophys. properties of the two squaraines related to their different structures were also demonstrated. Importantly, a possible sensing mechanism is proposed suggesting these types of dyes hold great potential in the area of rapid, sensitive, and convenient detection of aldehydes.
    315. 315
      Liu, T.; Liu, X.; Wang, W.; Luo, Z.; Liu, M.; Zou, S.; Sissa, C.; Painelli, A.; Zhang, Y.; Vengris, M. Systematic Molecular Engineering of a Series of Aniline-Based Squaraine Dyes and Their Structure-Related Properties. J. Phys. Chem. C 2018, 122 (7), 3994– 4008,  DOI: 10.1021/acs.jpcc.7b11997
      317
      Systematic Molecular Engineering of a Series of Aniline-Based Squaraine Dyes and Their Structure-Related Properties
      Liu, Taihong; Liu, Xinglei; Wang, Weina; Luo, Zhipu; Liu, Muqiong; Zou, Shengli; Sissa, Cristina; Painelli, Anna; Zhang, Yuanwei; Vengris, Mikas; Bondar, Mykhailo V.; Hagan, David J.; Van Stryland, Eric W.; Fang, Yu; Belfield, Kevin D.
      Journal of Physical Chemistry C (2018), 122 (7), 3994-4008CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      With the objective of developing new near-IR fluorescent probes and understanding the effect mol. structure exerts on phys. properties, a series of aniline-based squaraine dyes with different no. and position of methoxy substituents adjacent to the squaraine core were synthesized and investigated. Using both computational and exptl. methods, we found that the subtle changes of the no. or position of the methoxy substituents influenced the twisting angle of the structure and led to significant variations in optical properties. Moreover, the methoxy substituent also affected aggregation behavior due to steric effects. The X-ray crystal structure of one of the key members of the series, SD-2a, clearly demonstrates the distortion between the four-membered squaraine core and the adjacent aniline ring due to methoxy substitution. Structure-related fast relaxation processes were investigated by femtosecond pump-probe expts. and transient absorption spectra. Quantum chem. calcns. and essential state models were exploited to analyze the primary exptl. results. The comprehensive investigation of structure-related properties of dihydroxylaniline-based squaraine dyes, with systematic substitution of OH by OCH3 functional groups, serves as a guide for the design of novel squaraine dyes for photonics applications.
    316. 316
      Xia, G.; Wang, H. Squaraine Dyes: The Hierarchical Synthesis and Its Application in Optical Detection. J. Photochem. Photobiol. C: Photochem. Rev. 2017, 31, 84– 113,  DOI: 10.1016/j.jphotochemrev.2017.03.001
      318
      Squaraine dyes: The hierarchical synthesis and its application in optical detection
      Xia, Guomin; Wang, Hongming
      Journal of Photochemistry and Photobiology, C: Photochemistry Reviews (2017), 31 (), 84-113CODEN: JPPCAF; ISSN:1389-5567. (Elsevier B.V.)
      Squaraine dyes, a four-membered ring system with structural rigidity, possess unique photoelec. properties and are marked by their exceptionally sharp and intense absorption assocd. with a strong fluorescent emission in soln. These favorable characteristics have prompted their exploitation in a no. of state of the art applications including photocond., data storage, light-emitting field-effect transistors, solar cells and fluorescent histol. probes. In this review, we first summarize the recently proposed novel methods in the synthesis of these versatile derivs. Subsequently, their extensive applications in the prevalent optical detection of the surrounding medium such as ions, pH, thiol-based compds., biomols. and cell over the past decades are covered and discussed. In addn., different categories for the synthesis and sensing mechanisms for various squaric acid-based chemo-/bio- sensors are illustrated. Finally, the challenges and opportunities in the synthesis and application of these derivs. are also briefly discussed.
    317. 317
      Lichtenstein, S. J.; Nettleton, G. S. Effects of Fuchsin Variants in Aldehyde Fuchsin Staining. J. Histochem. Cytochem. 1980, 28 (7), 683– 8,  DOI: 10.1177/28.7.6156202
      319
      Effects of fuchsin variants in aldehyde fuchsin staining
      Lichtenstein, Steven J.; Nettleton, G. S.
      Journal of Histochemistry and Cytochemistry (1980), 28 (7), 683-8CODEN: JHCYAS; ISSN:0022-1554.
      Aldehyde fuchsin stains pancreatic B cell granules, hypophyseal basophils, goblet cell mucins, gastric chief cells, hyaline cartilage, and elastica. The role of the fuchsin component of aldehyde fuchsin in its staining reaction indicates: (1) single N-methylation of the fuchsin mol. abolishes staining of unoxidized pancreatic B cells, although it does not prevent reaction of fuchsin with paraldehyde; (2) aldehyde fuchsin is probably a Schiff base condensation product of pararosaniline and acetaldehyde; (3) a Schiff base structure alone cannot account for aldehyde fuchsin staining of unoxidized pancreatic B cells; (4) a fully potent aldehyde fuchsin is possibly a Tris-Schiff base deriv. of pararosaniline.
    318. 318
      Robins, J. H.; Abrams, G. D.; Pincock, J. A. The Structure of Schiff Reagent Aldehyde Adducts and the Mechanism of the Schiff Reaction as Determined by Nuclear Magnetic Resonance Spectroscopy. Can. J. Chem. 1980, 58 (4), 339– 47,  DOI: 10.1139/v80-055
      320
      The structure of Schiff reagent aldehyde adducts and the mechanism of the Schiff reaction as determined by nuclear magnetic resonance spectroscopy
      Robins, J. H.; Abrams, G. D.; Pincock, J. A.
      Canadian Journal of Chemistry (1980), 58 (4), 339-47CODEN: CJCHAG; ISSN:0008-4042.
      An NMR study of compds. isolated from the Schiff aldehyde reaction between pararosaniline hydrochloride, SO2, and AcH showed that these adducts are α-anilinoalkylsulfonic acids, and not the N-phenylalkylsulfonamides, the structures most often accepted in the literature. In combination with NMR spectra obtained from solns. of the Schiff reagent and the Schiff reaction and with literature spectrophotometric data, this result leads to a reasonable proposal for the mechanism of the color development. The effects of the concns. of the dye, AcH, and esp. SO2 are explained and suggest that the dominant colored species is a 2:1 AcH-dye adduct. The NMR results also reveal a kinetic/thermodn. competition for AcH between the aniline of the dye and the aldehyde carbonyl leading to bisulfite addn. The relation of the Schiff test with AcH and the Feulgen test for aldehydes in biol. samples is also discussed.
    319. 319
      Kitov, P. I.; Vinals, D. F.; Ng, S.; Tjhung, K. F.; Derda, R. Rapid, Hydrolytically Stable Modification of Aldehyde-Terminated Proteins and Phage Libraries. J. Am. Chem. Soc. 2014, 136 (23), 8149– 52,  DOI: 10.1021/ja5023909
      321
      Rapid, Hydrolytically Stable Modification of Aldehyde-Terminated Proteins and Phage Libraries
      Kitov, Pavel I.; Vinals, Daniel F.; Ng, Simon; Tjhung, Katrina F.; Derda, Ratmir
      Journal of the American Chemical Society (2014), 136 (23), 8149-8152CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The authors describe the rapid reaction of 2-amino benzamidoxime (ABAO) derivs. with aldehydes in water. The ABAO combines an aniline moiety for iminium-based activation of the aldehyde and a nucleophilic group (Nu:) ortho to the amine for intramol. ring closure. The reaction between ABAO and aldehydes is kinetically similar to oxime formations performed under stoichiometric aniline catalysis. The authors characterized the reaction by both NMR and UV spectroscopy and detd. that the rate-detg. step of the process is formation of a Schiff base, which is followed by rapid intramol. ring closure. The relation between apparent rate const. and pH suggests that a protonated benzamidoxime acts as an internal general acid in Schiff-base formation. The reaction is accelerated by substituents in the arom. ring that increase the basicity of the arom. amine. The rate of up to 40 M-1s-1 between an electron-rich aldehyde and 5-methoxy-ABAO (PMA), which was obsd. at pH 4.5, places this reaction among the fastest known bio-orthogonal reactions. Reaction between M13 phage-displayed library of peptides terminated with an aldehyde moiety and 1 mM biotin-ABAO deriv. reaches completion in 1 h at pH 4.5. Finally, the product of reaction, dihydroquinazoline deriv., shows fluorescence at 490 nm suggesting a possibility of developing fluorogenic aldehyde-reactive probes based on ABAO framework.
    320. 320
      Ressmann, A. K.; Schwendenwein, D.; Leonhartsberger, S.; Mihovilovic, M. D.; Bornscheuer, U. T.; Winkler, M.; Rudroff, F. Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes. Adv. Synth. Catal. 2019, 361, 2538,  DOI: 10.1002/adsc.201900154
      322
      Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes
      Ressmann, Anna K.; Schwendenwein, Daniel; Leonhartsberger, Simon; Mihovilovic, Marko D.; Bornscheuer, Uwe T.; Winkler, Margit; Rudroff, Florian
      Advanced Synthesis & Catalysis (2019), 361 (11), 2538-2543CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The selective and direct redn. of carboxylic acids into the corresponding aldehydes by chem. methods is still a challenging task in synthesis. Several reductive and oxidative chem. methods are known to produce aldehydes, but most of them require expensive reagents, special reaction conditions, are 2-step procedures and often lack chemoselectivity. Nature provides an elegant tool, so called carboxylic acid reductases (CARs) for the direct redn. of carboxylic acids to aldehydes. Discovery as well as engineering of novel CAR enzymes necessitates a robust, product selective high-throughput assay (HTA). The authors report a simple and fast HTA that allows the substrate-independent and chemoselective quantification of aldehydes (irresp. of their chem. structure) and is sensitive to the nM range. The HTA was validated by NMR and GC analyses and in microbial cells by reexamn. of the substrate scope of CAR from Nocardia iowensis (CARNi). The results were fully consistent with reported data.
    321. 321
      Horvat, M.; Larch, T.-S.; Rudroff, F.; Winkler, M. Amino Benzamidoxime (ABAO)-Based Assay to Identify Efficient Aldehyde-Producing Pichia Pastoris Clones. Adv. Synth. Catal. 2020, 362 (21), 4673– 9,  DOI: 10.1002/adsc.202000558
      323
      Amino Benzamidoxime (ABAO)-Based Assay to Identify Efficient Aldehyde-Producing Pichia pastoris Clones
      Horvat, Melissa; Larch, Tanja-Saskia; Rudroff, Florian; Winkler, Margit
      Advanced Synthesis & Catalysis (2020), 362 (21), 4673-4679CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The chemoselective synthesis of aldehydes is a challenging task. Nature provides carboxylic acid reductases (CARs) as elegant tools for the direct redn. of carboxylic acids to their resp. aldehydes. The discovery of new CARs and strains that efficiently produce these enzymes necessitates a robust high-throughput assay with selectivity for aldehydes. We recently reported a simple assay that allows the substrate independent and chemoselective quantification of aldehydes (irresp. of their chem. structure). The assay utilized amino benzamidoxime (ABAO), which forms UV-active and fluorescent dihydroquinazolines. In this study, we adapted the ABAO-assay for the identification and comparison of Pichia pastoris clones with the ability to produce aldehydes from carboxylic acids. Specifically, CAR and PPTase from Mycobacterium marinum (MmCAR and MmPPTase) were co-expressed using different bidirectional promoters (BDPs). A library of 598 clones was screened for piperonal prodn. with the ABAO assay and the results were validated by HPLC quantification. 1 OD unit of the best Pichia pastoris clone 2. A7, regulating MmCAR and MmPPTase expression by two strong constitutive promoters, fully converted 5 mM of piperonylic acid within 2 h.
    322. 322
      Mei, Z.; Zhang, K.; Qu, G.; Li, J.-K.; Liu, B.; Ma, J.-A.; Tu, R.; Sun, Z. High-Throughput Fluorescence Assay for Ketone Detection and Its Applications in Enzyme Mining and Protein Engineering. ACS Omega 2020, 5 (23), 13588– 94,  DOI: 10.1021/acsomega.0c00245
      324
      High-Throughput Fluorescence Assay for Ketone Detection and Its Applications in Enzyme Mining and Protein Engineering
      Mei, Zelong; Zhang, Kun; Qu, Ge; Li, Jun-Kuan; Liu, Baoyan; Ma, Jun-An; Tu, Ran; Sun, Zhoutong
      ACS Omega (2020), 5 (23), 13588-13594CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
      Ketones are of great importance as building blocks in synthetic org. chem. and biocatalysis. Most ketones cannot easily be quant. assayed due to the lack of visible photometric properties. Effective high-throughput assay (HTA) development is therefore necessary for ketone detn. Inspired by previous works of an aldehyde assay based on 2-amino benzamidoxime derivs., we developed a colorimetric method for rapid a HTA of structurally diverse ketones by using para-methoxy-2-amino benzamidoxime (PMA). This PMA-based method is characterized by high sensitivity manner (μM) with low background, as checked by gas chromatog. (GC). It can be used for quant. monitoring ketones by fluorescence screening in microtiter plates. Furthermore, this HTA method was employed in mining alc. dehydrogenases (ADHs), and in directed evolution aimed at enhancing ADH activity in the catalytic transformation of alcs. to ketones. This work provides a general tool for ketone detection in biocatalyst development.
    323. 323
      Xing, Y.; Wang, S.; Mao, X.; Zhao, X.; Wei, D. An Easy and Efficient Fluorescent Method for Detecting Aldehydes and Its Application in Biotransformation. J. Fluoresc. 2011, 21 (2), 587– 94,  DOI: 10.1007/s10895-010-0746-6
      325
      An Easy and Efficient Fluorescent Method for Detecting Aldehydes and Its Application in Biotransformation
      Xing, Yanlong; Wang, Shu; Mao, Xiangzhao; Zhao, Xuebo; Wei, Dongzhi
      Journal of Fluorescence (2011), 21 (2), 587-594CODEN: JOFLEN; ISSN:1053-0509. (Springer)
      Water-sol. aldehydes (acetaldehyde, propionaldehyde) and non-water-sol. aldehydes (butyraldehyde and phenylacetaldehyde) were easily detected by an efficient fluorescent method with 5-aminofluorescein as probe. Under optimal detection conditions, 5-aminofluorescein could selectively respond to aldehydes with high sensitivity in comparison with other carbonyl compds. like ketones and acids. Thus, the proposed method was used to monitor microbial oxidn. and succeeded in trapping transiently-produced aldehydes during biotransformation of primary alcs. by Gluconobacter oxydans.
    324. 324
      Li, Z.; Xue, Z.; Wu, Z.; Han, J.; Han, S. Chromo-Fluorogenic Detection of Aldehydes with a Rhodamine Based Sensor Featuring an Intramolecular Deoxylactam. Org. Biomol. Chem. 2011, 9 (22), 7652– 4,  DOI: 10.1039/c1ob06448g
      326
      Chromo-fluorogenic detection of aldehydes with a rhodamine based sensor featuring an intramolecular deoxylactam
      Li, Zhu; Xue, Zhongwei; Wu, Zhisheng; Han, Jiahuai; Han, Shoufa
      Organic & Biomolecular Chemistry (2011), 9 (22), 7652-7654CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      A chromogenic and fluorogenic detection of aldehydes was achieved via analyte triggered opening of the deoxylactam of N-(rhodamine B)-deoxylactam-ethylenediamine (dRB-EDA). The utility of the sensor was demonstrated by fluorescent labeling of aldehyde-displaying sialoproteins on cell surfaces.
    325. 325
      Davidson, D.; Weiss, M.; Jelling, M. The Action of Ammonia on Benzil. J. Org. Chem. 1937, 02 (4), 319– 27,  DOI: 10.1021/jo01227a004
      327
      Action of ammonia on benzil
      Davidson, David; Weiss, Marvin; Jelling, Murray
      Journal of Organic Chemistry (1937), 2 (), 319-27CODEN: JOCEAH; ISSN:0022-3263.
      Laurent (J. prakt. Chem. 35, 461(1845)) showed that NH3 and benzil (I) give 3 compds., termed imabenzil (II), benzilimide (III) and benzilam (IV); Henius (Ann. 228, 339(1885)) found that in EtOH at 130° II disappeared after 1 hr. and on continued heating there resulted IV and lophine (V). It is now shown that III is N-desylbenzamide, PhBzCHNHBz, m. 141° (all m. ps. cor.), which is obtained in 80% yield from the SnCl2 deriv. (VI) of desylamine and BzCl with NaOH. The oxime of III m. 197-203° (a more sol. product, m. 140°, may be an isomeric oxime). From the compn. of II (C35H28N2O3) and its scission by acid into III, I and NH3, it is believed to be 1-benzoyl-5,6-dihydroxy-2,3,5,6-tetraphenyl - 1,2,5,6 - tetrahydropyrazine, CPh:N.C-(OH)Ph.C(OH)Ph.NBz.CHPh. III (0.32 g.) with 1.5 g. NH4OAc in 10 cc. AcOH, refluxed 1 hr., gives 0.28 g. (93%) of V. A mechanism for the action of NH3 on I, which avoids the scission of I as a step and relates the reaction to the formation of α-acylamino acids by the action of NH3 on α-keto acids, is proposed. N-Desyl derivs. of acid amides are converted smoothly into glyoxalines by the action of NH3 in AcOH. III and PhNH2 in AcOH give tetraphenylglyoxaline, m. 221°. VI and Ac2O in C6H6N give a nearly quant. yield of N-desylacetamide, m. 137°; with NH4OAc or PhNH2 in AcOH there results 2-methyl-4,5-diphenylglyoxaline, m. 243°, or the 1,4,5-tri-Ph deriv., m. 197°. Refluxing 1.05 g. I and 5 g. NH4OAc in 25 cc. glacial AcOH for 1 hr. gives 7% of IV and 94% V. I (1.05 g.), 0.13 g. hexamethylene-tetramine, 3 g. NH4OAc and 25 g. AcOH, refluxed 1 hr., give 91% of 4,5-diphenylglyoxaline; paraldehyde gives 96% of the 2-Me deriv., m. 244°, while BzH or hydrobenzamide gives 90-5% V.
    326. 326
      Nakashima, K.; Yamasaki, H.; Kuroda, N.; Akiyama, S. Evaluation of Lophine Derivatives as Chemiluminogens by a Flow-Injection Method. Anal. Chim. Acta 1995, 303 (1), 103– 7,  DOI: 10.1016/0003-2670(94)00360-X
      328
      Evaluation of lophine derivatives as chemiluminogens by a flow-injection method
      Nakashima, Kenichiro; Yamasaki, Hiromi; Kuroda, Naotaka; Akiyama, Shuzo
      Analytica Chimica Acta (1995), 303 (1), 103-7CODEN: ACACAM; ISSN:0003-2670. (Elsevier)
      A rapid and convenient method for the evaluation of lophine (2,4,5-triphenylimidazole) derivs. as to their chemiluminescence efficiency was developed by using a flow-injection technique. Std. solns. of lophine derivs. were injected into a flow line contg. a carrier soln. (1.5 × 10-4 M cobalt(II) contg. 0.2% hydroxylammonium chloride in 75% methanol), which was successively combined with a flow line contg. 100 mM hydrogen peroxide in H2O and a flow line contg. 1.0M KOH in H2O, and the chemiluminescence generated was monitored. Addn. of hydroxylammonium chloride into a carrier soln. resulted in a significant increase in chemiluminescence. Calibration graphs for eighteen derivs. were prepd. and the relative chemiluminescence intensities were estd. from the slopes of these graphs. Among the derivs., the compds. substituted by OMe at the 2- and 3-positions, and COOH, COCl, CONHNH2 and CONHNHCO(CH2)4Me at the 4-position on the Ph ring at the 2-position of the imidazole skeleton showed large chemiluminescence intensities which are comparable to that of lophine. The detection limit of lophine was 72 fmol per injection at a signal-to-noise ratio of 2.
    327. 327
      El-Maghrabey, M. H.; Kishikawa, N.; Ohyama, K.; Kuroda, N. Analytical Method for Lipoperoxidation Relevant Reactive Aldehydes in Human Sera by High-Performance Liquid Chromatography-Fluorescence Detection. Anal. Biochem. 2014, 464, 36– 42,  DOI: 10.1016/j.ab.2014.07.002
      329
      Analytical method for lipoperoxidation relevant reactive aldehydes in human sera by high-performance liquid chromatography-fluorescence detection
      El-Maghrabey, Mahmoud H.; Kishikawa, Naoya; Ohyama, Kaname; Kuroda, Naotaka
      Analytical Biochemistry (2014), 464 (), 36-42CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      A validated, simple and sensitive HPLC method was developed for the simultaneous detn. of lipoperoxidn. relevant reactive aldehydes: glyoxal (GO), acrolein (ACR), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in human serum. The studied aldehydes were reacted with 2,2'-furil to form fluorescent difurylimidazole derivs. that were sepd. on a C18 column using gradient elution and fluorescence detection at excitation and emission wavelengths of 250 and 355 nm, resp. The method showed good linearity over the concn. ranges of 0.100-5.00, 0.200-10.0, 0.200-40.0, and 0.400-10.0 nmol/mL for GO, ACR, HNE, and MDA, resp., with detection limits ranging from 0.030 to 0.11 nmol/mL. The percentage RSD of intraday and interday precision did not exceed 5.0 and 6.2%, resp., and the accuracy (%found) ranged from 95.5 to 103%. The proposed method was applied for monitoring the four aldehydes in sera of healthy, diabetic, and rheumatic human subjects with simple pretreatment steps and without interference from endogenous components. By virtue of its high sensitivity and accuracy, our method enabled detection of differences between analytes concns. in sera of human subjects under different clin. conditions.
    328. 328
      Fathy Bakr Ali, M.; Kishikawa, N.; Ohyama, K.; Abdel-Mageed Mohamed, H.; Mohamed Abdel-Wadood, H.; Mohamed Mohamed, A.; Kuroda, N. Chromatographic Determination of Aliphatic Aldehydes in Human Serum after Pre-Column Derivatization Using 2,2′-Furil, a Novel Fluorogenic Reagent. J. Chromatogr. A 2013, 1300, 199– 203,  DOI: 10.1016/j.chroma.2013.03.033
      330
      Chromatographic determination of aliphatic aldehydes in human serum after pre-column derivatization using 2,2'-furil, a novel fluorogenic reagent
      Fathy Bakr Ali Marwa; Kishikawa Naoya; Ohyama Kaname; Abdel-Mageed Mohamed Horria; Mohamed Abdel-Wadood Hanaa; Mohamed Mohamed Ashraf; Kuroda Naotaka
      Journal of chromatography. A (2013), 1300 (), 199-203 ISSN:.
      A novel, highly sensitive and selective fluorimetric liquid chromatographic method for simultaneous determination of medium chain aliphatic aldehydes was developed. The method was based on the derivatization of aliphatic aldehydes with 1,2-di(2-furyl)-1,2-ethanedione (2,2'-furil), a novel fluorogenic reagent, to form highly fluorescent difurylimidazole derivatives. The fluorescence derivatives were separated in less than 20min on a reversed-phase ODS column using an isocratic elution with a mixture of methanol-water (80:20, v/v%). The detection limits were from 0.19 to 0.50nM (1-10fmol/injection) at a signal-to-noise ratio (S/N) of 3. This method was successfully applied for monitoring of aliphatic aldehydes in healthy human sera by a simple pretreatment procedure without interferences from serum constituents.
    329. 329
      Nakamura, M.; Toda, M.; Saito, H.; Ohkura, Y. Fluorimetric Determination of Aromatic Aldehydes with 4,5-Dimethoxy-1,2-Diaminobenzene. Anal. Chim. Acta 1982, 134, 39– 45,  DOI: 10.1016/S0003-2670(01)84175-1
      331
      Fluorimetric determination of aromatic aldehydes with 4,5-dimethoxy-1,2-diaminobenzene
      Nakamura, Masaru; Toda, Mitsuko; Saito, Hiroko; Ohkura, Yosuke
      Analytica Chimica Acta (1982), 134 (), 39-45CODEN: ACACAM; ISSN:0003-2670.
      A sensitive fluorometric method for the detn. of arom. aldehydes is based on their reaction in dil. acid with 4,5-dimethoxy-1,2-diaminobenzene to give a compd. which fluoresces intensely in alk. soln. The fluorescence is stabilized by β-mercaptoethanol. The method is simple, selective (for arom. and arylaliph. aldehydes), and sensitive; almost all the aldehydes can be detd. at concns. of 10-8-10-7M.
    330. 330
      Imazato, T.; Kanematsu, M.; Kishikawa, N.; Ohyama, K.; Hino, T.; Ueki, Y.; Maehata, E.; Kuroda, N. Determination of Acrolein in Serum by High-Performance Liquid Chromatography with Fluorescence Detection after Pre-Column Fluorogenic Derivatization Using 1,2-Diamino-4,5-Dimethoxybenzene. Biomed. Chromatogr. 2015, 29 (9), 1304– 8,  DOI: 10.1002/bmc.3422
      332
      Determination of acrolein in serum by high-performance liquid chromatography with fluorescence detection after pre-column fluorogenic derivatization using 1,2-diamino-4,5-dimethoxybenzene
      Imazato, Takahiro; Kanematsu, Mariko; Kishikawa, Naoya; Ohyama, Kaname; Hino, Takako; Ueki, Yukitaka; Maehata, Eisuke; Kuroda, Naotaka
      Biomedical Chromatography (2015), 29 (9), 1304-1308CODEN: BICHE2; ISSN:0269-3879. (John Wiley & Sons Ltd.)
      Acrolein is a major unsatd. aldehyde that is generated during the lipid peroxidn. process. The measurement of acrolein in biol. samples should be useful to est. the degree of lipid peroxidn. and to evaluate the effect of hazardous properties of acrolein on human health. In this study, a highly sensitive and selective high-performance liq. chromatog. with fluorescence detection method was developed for the detn. of acrolein in human serum. The proposed method involves the pre-column fluorogenic derivatization of acrolein with 1,2-diamino-4,5-dimethoxybenzene (DDB) as a reagent. The fluorescent deriv. of acrolein could be detected clearly without any interfering reagent blank peaks because DDB does not have intrinsic fluorescence itself, and the detection limit was 10 nM (signal-to-noise ratio = 3). The proposed method could selectively detect acrolein in human serum with a simple protein pptn. treatment. Copyright © 2015 John Wiley & Sons, Ltd.
    331. 331
      McLellan, A. C.; Phillips, S. A.; Thornalley, P. J. The Assay of Methylglyoxal in Biological Systems Byderivatization with 1,2-Diamino-4,5-Dimethoxybenzene. Anal. Biochem. 1992, 206 (1), 17– 23,  DOI: 10.1016/S0003-2697(05)80005-3
      333
      The assay of methylglyoxal in biological systems by derivatization with 1,2-diamino-4,5-dimethoxybenzene
      McLellan, A. C.; Phillips, S. A.; Thornalley, P. J.
      Analytical Biochemistry (1992), 206 (1), 17-23CODEN: ANBCA2; ISSN:0003-2697.
      A procedure for the assay of methylglyoxal in biol. systems is described, together with sample storage, sample processing procedures, and statistical evaluation. Specimen data are presented. Methylglyoxal was assayed by derivatization with 1,2-diamino-4,5-dimethoxybenzene and HPLC of the resulting quinoxaline, 6,7-dimethoxy-2-methylquinoxaline, with spectrophotometric or fluorescence detection. Derivatization, solid-phase extn., and HPLC were performed under acid conditions to prevent the spontaneous formation of methylglyoxal from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate during the assay. The limits of detection in the biol. matrix were 45 pmol (absorbance detection) and 10 pmol (fluorometric detection), the recovery was 58%, and the intra- and interbatch coeffs. of variance were 7.7 and 30.0%, resp. The concn. of methylglyoxal in whole blood from normal healthy human individuals was (mean ± SE, nM) 256 ± 92 (n = 12) and that from diabetic patients was 479 ± 49 (n = 55), showing a significant increase in diabetes mellitus (P < 0.01; Mann-Whitney U test). Sample processing under acidic conditions was essential to avoid interferences. Previous ests. of the concn. of methylglyoxal in biol. samples require re-evaluation.
    332. 332
      Hantzsch, A. Condensationsprodukte Aus Aldehydammoniak Und Ketonartigen Verbindungen. Ber. Dtsch. Chem. Ges. 1881, 14 (2), 1637– 8,  DOI: 10.1002/cber.18810140214
      There is no corresponding record for this reference.
    333. 333
      Belman, S. The Fluorimetric Determination of Formaldehyde. Anal. Chim. Acta 1963, 29, 120– 6,  DOI: 10.1016/S0003-2670(00)88591-8
      335
      The fluorimetric determination of formaldehyde
      Belman, Sidney
      Analytica Chimica Acta (1963), 29 (2), 120-6CODEN: ACACAM; ISSN:0003-2670.
      cf. Nash, CA 48, 503i. To det. HCHO, 0.01-1 γ/ml., mix freshly prepd.; pH 6, 2M NH4OAc-0.02M (freshly distd.) Ac2CH2 with an equal vol. of HCHO soln. and incubate for 10 min. at 58° or for 60 min. at 37°. The lower temp. is preferred for low (<0.05 γ/ml.) HCHO concns. After cooling to 25°, read the fluorescence at 510 mμ. The resp. fluorescence excitation and emission max. are 410 and 510 mμ. The fluorescence is linear at 0.005-0.4 γ/ml. HCHO and deviates slightly from linearity at 0.4-1 γ/ml. The reaction of Ac2CH2, NH4+, and HCHO forms sol. 3,5-diacetyl-1,4-dihydrolutidine (I) with max. at 412 mμ. HCHO > 1 γ/ml. is detd. by measuring the absorbance at 412 mμ. In an application, 2 differently treated samples of deoxyribonucleic acid (DNA) were treated with HCHO, and the isolated DNA was analyzed for bound HCHO as described. Heat-denatured DNA had 85% of its NH2 groups bound to HCHO, while 2-amino-1-naphthol treated DNA had 32% of its NH2 groups bound to HCHO. As little as 1 γ NH3/ml. is detected by the described method, with a M NaOAc, pH 6, 0.8M Ac2CH2-2M HCHO reagent, by mixing with an equal vol. of NH4OH soln., heating at 100° for 10 min., cooling to 25°, and measuring the fluorescence. Cryst. I, m. 190-200°, was phys. characterized. The infrared spectra confirm the structure of I (loc. cit.) in dil. soln., and suggest that a dimer of I primarily exists in concd. soln. and in the solid I.
    334. 334
      Sawicki, E.; Carnes, R. Spectrophotofluorimetric Determination of Aldehydes with Dimedone and Other Reagents. Mikrochim. Acta 1968, 56 (1), 148– 59,  DOI: 10.1007/BF01216118
      There is no corresponding record for this reference.
    335. 335
      Compton, B. J.; Purdy, W. C. The Mechanism of the Reaction of the Nash and the Sawicki Aldehyde Reagent. Can. J. Chem. 1980, 58 (21), 2207– 11,  DOI: 10.1139/v80-355
      337
      The mechanism of the reaction of the Nash and the Sawicki aldehyde reagent
      Compton, Bruce Jon; Purdy, William C.
      Canadian Journal of Chemistry (1980), 58 (21), 2207-11CODEN: CJCHAG; ISSN:0008-4042.
      Synthetic and chromatog. studies are presented on the Nash and Sawicki colorimetric methods for quantitating HCHO [reaction of (MeCO)2CH2 or dimedone with NH4OAc and MeCHO). The actual color-forming agent in the Nash method is an isoable iminodione formed in situ by reaction of the 2 components. Explanation is given for differences between these 2 methods.
    336. 336
      Compton, B. J.; Purdy, W. C. Fluoral-P, a Member of a Selective Family of Reagents for Aldehydes. Anal. Chim. Acta 1980, 119 (2), 349– 57,  DOI: 10.1016/S0003-2670(01)93636-0
      338
      Fluoral-P, a member of a selective family of reagents for aldehydes
      Compton, Bruce Jon; Purdy, William C.
      Analytica Chimica Acta (1980), 119 (2), 349-57CODEN: ACACAM; ISSN:0003-2670.
      The compd. 4-amino-3-penten-2-one (Fluoral-P) is introduced as a member of a selective family of reagents for aldehydes. The stability, selectivity, and reactivity of the reagent are described and its utility in automatic detns. of HCHO is demonstrated. The condensation reaction between Fluoral-P and HCHO gives a product detectable by fluorescence. The flow injection system utilizing this reagent can be used to det. HCHO in the lower nm range.
    337. 337
      Mopper, K.; Stahovec, W. L.; Johnson, L. Trace Analysis of Aldehydes by Reversed-Phase High-Performance Liquid Chromatography and Precolumn Fluorigenic Labeling with 5,5-Dimethyl-1,3-Cyclohexanedione. J. Chromatogr. A 1983, 256, 243– 52,  DOI: 10.1016/S0021-9673(01)88237-6
      There is no corresponding record for this reference.
    338. 338
      Al-Moniee, M. A.; Koopal, C.; Akmal, N.; van Veen, S.; Zhu, X.; Sanders, P. F.; Sanders, P. F.; Al-Abeedi, F. N.; Amer, A. M. Applicability of Dimedone Assays for the Development of Online Aldehyde Sensor in Seawater Flooding Systems. J. Sens. Sci. Technol. 2016, 6, 101,  DOI: 10.4236/jst.2016.64008
      There is no corresponding record for this reference.
    339. 339
      Li, Q.; Oshima, M.; Motomizu, S. Flow-Injection Spectrofluorometric Determination of Trace Amounts of Formaldehyde in Water after Derivatization with Acetoacetanilide. Talanta 2007, 72 (5), 1675– 80,  DOI: 10.1016/j.talanta.2007.01.054
      341
      Flow-injection spectrofluorometric determination of trace amounts of formaldehyde in water after derivatization with acetoacetanilide
      Li, Qiong; Oshima, Mitsuko; Motomizu, Shoji
      Talanta (2007), 72 (5), 1675-1680CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      A novel fluoro-photometric method, which requires no enrichment procedure, to det. formaldehyde in environmental water was developed. This flow-injection anal. method for the spectrofluorometric detn. of formaldehyde in water is based on the reaction of formaldehyde with acetoacetanilide and NH3. The method exhibited good linearity from 0.50 to 40 × 10-7 M; a limit of detection of 3 × 10-9 M (0.09 ppb) is achievable with a 15/h sample through-put. A main advantage of this method is that the reaction can be conducted at room temp. without a heating system. The effect of various interferences possibly present in actual water samples was examd. Most cations, anions, and org. compds. do not interfere with the formaldehyde detn. in environmental water. The method is very simple, rapid, less expensive, highly sensitive, and can be applied to environmental water (rain, tap, and river water) at low concns. without any enrichment procedure.
    340. 340
      Kolmel, D. K.; Kool, E. T. Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis. Chem. Rev. 2017, 117 (15), 10358– 76,  DOI: 10.1021/acs.chemrev.7b00090
      342
      Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis
      Kolmel, Dominik K.; Kool, Eric T.
      Chemical Reviews (Washington, DC, United States) (2017), 117 (15), 10358-10376CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The formation of oximes and hydrazones is employed in numerous scientific fields as a simple and versatile conjugation strategy. This imine-forming reaction is applied in fields as diverse as polymer chem., biomaterials and hydrogels, dynamic combinatorial chem., org. synthesis, and chem. biol. Here we outline chem. developments in this field, with special focus on the past ∼10 years of developments. Recent strategies for installing reactive carbonyl groups and α-nucleophiles into biomols. are described. The basic chem. properties of reactants and products in this reaction are then reviewed, with an eye to understanding the reaction's mechanism and how reactant structure controls rates and equil. in the process. Recent work that has uncovered structural features and new mechanisms for speeding the reaction, sometimes by orders of magnitude, is discussed. We describe recent studies that have identified esp. fast reacting aldehyde/ketone substrates and structural effects that lead to rapid-reacting α-nucleophiles as well. Among the most effective new strategies has been the development of substituents near the reactive aldehyde group that either transfer protons at the transition state or trap the initially formed tetrahedral intermediates. In addn., the recent development of efficient nucleophilic catalysts for the reaction is outlined, improving greatly upon aniline, the classical catalyst for imine formation. A no. of uses of such second- and third-generation catalysts in bioconjugation and in cellular applications are highlighted. While formation of hydrazone and oxime has been traditionally regarded as being limited by slow rates, developments in the past 5 years have resulted in completely overturning this limitation; indeed, the reaction is now one of the fastest and most versatile reactions available for conjugations of biomols. and biomaterials.
    341. 341
      Kalia, J.; Raines, R. T. Hydrolytic Stability of Hydrazones and Oximes. Angew. Chem., Int. Ed. 2008, 47 (39), 7523– 6,  DOI: 10.1002/anie.200802651
      343
      Hydrolytic stability of hydrazones and oximes
      Kalia, Jeet; Raines, Ronald T.
      Angewandte Chemie, International Edition (2008), 47 (39), 7523-7526CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Watching C'n'N: Hydrazones and oximes are common conjugates but are labile to hydrolysis. The hydrolytic stabilities of isostructural hydrazones and one oxime were detd. at pD 5.0-9.0. The rate const. for the acid-catalyzed hydrolysis of the oxime was nearly 103-fold lower than those for simple hydrazones, and a trialkylhydrazonium ion (formed after condensation) was even more stable than the oxime.
    342. 342
      Forget, D.; Boturyn, D.; Defrancq, E.; Lhomme, J.; Dumy, P. Highly Efficient Synthesis of Peptide-Oligonucleotide Conjugates: Chemoselective Oxime and Thiazolidine Formation. Eur. J. Chem. 2001, 7 (18), 3976– 84,  DOI: 10.1002/1521-3765(20010917)7:18<3976::AID-CHEM3976>3.0.CO;2-X
      344
      Highly efficient synthesis of peptide-oligonucleotide conjugates: chemoselective oxime and thiazolidine formation
      Forget, Damien; Boturyn, Didier; Defrancq, Eric; Lhomme, Jean; Dumy, Pascal
      Chemistry - A European Journal (2001), 7 (18), 3976-3984CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH)
      A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-contg. peptide with an aldehyde-contg. oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatized by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aq. conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with mol. biol. tools, this methodol. opens up new prospects for post-functionalization of high-mol.-wt. DNA structures.
    343. 343
      Shao, J.; Tam, J. P. Unprotected Peptides as Building Blocks for the Synthesis of Peptide Dendrimers with Oxime, Hydrazone, and Thiazolidine Linkages. J. Am. Chem. Soc. 1995, 117 (14), 3893– 9,  DOI: 10.1021/ja00119a001
      345
      Unprotected Peptides as Building Blocks for the Synthesis of Peptide Dendrimers with Oxime, Hydrazone, and Thiazolidine Linkages
      Shao, Jun; Tam, James P.
      Journal of the American Chemical Society (1995), 117 (14), 3893-9CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A general approach for forming peptide dendrimers with oxime, hydrazone, and thiazolidine linkages was developed using unprotected peptides as building blocks and selective ligation between an aldehyde and a weak base. To illustrate the generality of this approach, a branched lysine core matrix with an aldehyde was used to ligate four copies of unprotected peptides contg. a weak nucleophilic base such as aminooxy, hydrazide, or cysteine 1,2-aminothiol groups at their N-termini to form synthetic branched proteins. Various parameters affecting the ligations were studied, and optimal conditions gave 12-27-fold rate increases and shortened the reaction time from 24-60 to 2-8 h. Among the three reactions studied, ligation by thiazolidine appeared to be superior to ligation by oxime or hydrazone in reaction rate and product stability. The purified dendrimeric products gave single peaks on reverse phase HPLC and size exclusion HPLC. Their macromol. structures were also characterized by mass spectrometry and amino acid anal. CD spectra of the dendrimers showed that they have an increased ordered helical structure. Ligation reactions using a mutually reactive weak base and aldehyde pair should provide a useful approach for the synthesis of peptide dendrimers and artificial proteins.
    344. 344
      Kemp, D. S.; Woodward, R. B. The N-Ethylbenzisoxazolium Cation─I: Preparation and Reactions with Nucleophilic Species. Tetrahedron 1965, 21 (11), 3019– 35,  DOI: 10.1016/S0040-4020(01)96921-2
      346
      N-Ethylbenzisoxazolium cation. I. Preparation and reactions with nucleophilic species
      Kemp, D. S.; Woodward, R. B.
      Tetrahedron (1965), 21 (11), 3019-35CODEN: TETRAB; ISSN:0040-4020.
      cf. CA 55, 14322c. H2NOSO3H (250 g.) and 20 g. Na2SO4 in 800 mL. H2O stirred with 230 g. ο-HOC6H4CHO and the soln. stirred vigorously with 400 mL. H2O and 400 mL. CH2Cl2, the chilled mixt. (ice bath) stirred with addn. of 340 g. NaHCO3 and 300 mL. H2O and the mixt. stirred 1 h. before sepn. of the 2 phases, the aq. layer stirred 2 h. with 80 mL. CH2Cl2 and 30 g. NaHCO3 and the new aq. layer extd. with 150 mL. CH2Cl2, the combined CH2Cl2 exts. dried over MgSO4 and distd. yielded 94.6% benzisoxazole (I), b2 35-8°, n25D 1.5605, λ 6.22 μ, λ 235, 243, 280 mμ (ε 10000, 8100, 2900, alc.), τ 1.2s, 2.3-3.0 m. I (55 g.) and 87 g. purified Et3OBF4 kept in 200 mL. CH2Cl2 to incipient crystn. and cooled in ice 30 min., kept 6 h. at 20° and the cryst. mass washed with CH2Cl2 gave 104 g. salt, m. 109.2-10.2°, recrystd. from MeCNEtOAc to give nonhygroscopic light-sensitive N-ethylbenzisoxazolium fluoroborate (II), m. 109.5-10.2°, λ 6.20, 9-10 μ, λ 258, 297 mμ (ε 13100, 2900), τ 0.2s, 1.8-2.4m, 5.1q. II (0.5 g.) in 20 mL. 0.1N HCl heated 3 h. at 90° and the cooled suspension extd. with 30 mL. CH2Cl2 gave ο-HOC6H4CONHEt, m. 55-6.5°. Kinetic evidence indicated the intermediacy of the benzooxoketenimine (III) in the formation of the amide. Addn. of the N-ethylbenzisoxazolium cation (IV) to aq. solns. of HS-, F-, or CN- or to MeOH solns. of MeO- gave high yields of compds. 2-HOC6H4-CX: NEt (V) (X = F, CN, HS or OMe). Na2S.H2O (40 g.) in 50 mL. H2O and a slight excess of HCl kept 5 min. with 5 g. II in 5 mL. MeCN, and the product extd. with 45 mL. CH2Cl2, passed through a silica gel column and the recrystd. (CCl4-C6H12) product again recrystd. from EtOAc-C6H12 gave 1.7 g. ο-HOC6H4CSNHEt (VI), m. 60.5-1.3°, λ 264 mμ, (ε 9300, H2O, pH 1-6), λ 267, 286 mμ (ε 7800, 5500, pH 11), τ - 1.3s, 1.4s, 2.3-3.3m, 6.4p, 8.8t. VI in CH2Cl2 shaken with aq. AgNO3 and filtered from the pptd. Ag2S yielded 80% ο-HOC6H4CONHEt. KBr (4 g.) in 10 mL. H2O and 10 g. II kept 10 min. and filtered, the filtrate treated with 15 g. KF and extd. with 60 mL. CH2Cl2, the ext. dried over Na2SO4 and evapd., the residual liq. taken up in C5H12 and filtered through a mixt. of NaF and Celite, evapd., and the colorless oil (5.4 g., b1.0 42-4°) recrystd. at -70° from C5H12 gave V (X = F), b1 42-4°, n25D 1.5312, λ 3.0-3.7, 5.93 μ, τ - 1.8s, 2.5-3.4 m, 6.5q, 8.8t, sapond. in 0.1N to ο-HOC6H4CONHEt. NaCN (5 g.) in 20 mL. H2O overlayered with 10 mL. C5H12 and stirred vigorously with addn. of 2 g. II in 5 mL. MeCN, the org. layer and C5H12 washings (20 mL.) dried and evapd. yielded 80% V (X = CN), m. 45-5.7° (C5H2, and sublimation), λ 3.0-3.7, 4.48, 6.1μ, τ -2.6s, 2.0-3.0m, 6.0q, 8.6t, sapond. in 0.1N NaOH to ο-HOC6H4CONHEt, and hydrolyzed in 48% HBr at 20° in 3 days to give ο-HOC6H4COCO2H, m. 39.5°. II in 150 mL. MeOH stirred with addn. of 40 mL. MeOH contg. 0.6 g. Na and the residue on evapn. triturated with 80 mL. C5H12, the ext. evapd., and the residue distd. at 60°/0.1 mm. gave 3.0 g. oil, recrystd. from chilled C5H12 to give V (X = MeO), λ 3.0-3.7, 6.10 μ, τ 0.1s, 2.4-3.4m, 6.5s, q, 8.8t. II (1 g.) in 2 mL. MeCN added with stirring to 2 g. NaN3 in 25 mL. ice-cold H2O and extd. with 20 mL. cold CH2Cl2, the ext. dried over MgSO4 and cooled to -10° gave a lemon-yellow soln., λ 3.0-3.7, 4.70 (N3), 6.1 μ, fading in 30 min. at 20° to give 1-ethyl-2-(2-hydroxyphenyl)tetrazole, m. 140.2-1.2°, λ 283 mμ (ε 3200, H2O, pH 2-7), λ 308 mμ (ε 4500, H2O, pH 13). V (X = F) (0.5 g.) and 10 mL. concd. NH4OH stirred vigorously 4 h. at 20° and the CH2Cl2-washed ppt. recrystd. from MeCN gave 42% V (X = NH) (VI), m. 209° (decompn.), λ 340 mμ (ε 6900, H2O, pH 7-11) λ 288 mμ (ε 2800, H2O, pH 3); picrate, m. 151-5-2.5°, reconverted to VI by base. II (2 g.) in 3 mL. MeCN and 10 g. NaCNO in 25 mL. H2O extd. with 30 mL. CH2Cl2 and the dried (MgSO4) ext. evapd. yielded 80% 4-ethyliminobenz[1,3]oxazine-2,4-dione (VII), m. 193.5-4.0° λ 2.88, 3.01, 5.79, 6.18 μ (CH2Cl2), λ 249, 297 mμ (ε 16600, 4500, H2O, pH 1-10), τ 1.9-3.0m, 6.4q, 8.8t, converted in soln. in MeOH by addn. to aq. NaOH to give a high yield of VI. II (2 g.) in 3 mL. MeCN added to 25 mL. 20% NaCNS and the turbid mixt. extd. with 30 mL. CH2Cl2 gave 80% yield of 2-thio-4-ethyliminobenz[1.3]oxazine-2,4-dione, m. 207.8-8.0°, λ 2.85, 3.02, 6.22 μ, λ 259, 288, 297, 325 mμ (ε 12300, 12300, 1500, 8400, H2O, pH 1-6), λ 250, 290, 297 mμ (ε 10700, 12700, 13300, H2O, pH 11.0), τ 1.8-2.9m, 6.3q, 8.8t, converted by addn. in MeOH to AgNO3 to give VII. NaOAc (2 g.) in 15 mL. H2O adjusted to pH 5.5 with dil. HCl and stirred with gradual addn. of 1 g. powd. II, the mixt. extd. with 15 mL. CH2Cl2, and the ext. washed with 20 mL. 0.5N NaHCO3, evapd., and the residue crystd. from CH2Cl2-C6H12 gave 0.75 g. solid, m. 54-5.2°, recrystd. to give O-acetyl-N-ethylsalicylamide (VIII, R = Me) (IX), m. 55.0-5.6° (EtOAc-C6H12), λ 2.90, 5.66, 5.99 μ, λ 265 mμ (ε 800, H2O, pH 1-6), τ 2.4-3.1, 6.7p, 7.8s, identical with a sample obtained by treatment of ο-AcOC6H4COCl with H2NEt. Similarly was produced VIII (R = Ph), m. 112.1-12.6°, λ 5.75, 5.99 μ, in 88% yield. Analogously treatment of II with MeOCH2CO2H yielded 88% VIII (R = MeOCH2), m. 59.3-60.2° (EtOAc-C6H2), λ 5.60, 5.98 μ (CCl4), λ 265 mμ (ε 950, H2O, pH 1-7). II (3 g.) added as a fine powder with stirring to 5 g. H2NCH2CO2H in 50 mL. H2O, the mixt. treated after 5 min. with 2.5 g. picric acid and 1 g. H2NCH2CO2H in 30 mL. H2O and the dried cryst. ppt. (4.4 g., m. 137-9.0°) recrystd. from MeCN gave VIII (R = CH2NH2) picrate, C17H17N5O10, m. 145.5-7.0° (decompn.), λ 5.63 μ. II (2 g.) and 5 g. H2NCH2CO2H stirred 5 min. in 50 mL. H2O and adjusted to pH 8.5 with N NaOH with simultaneous addn. of 25 mL. Me2CO, kept 2 h. at 20° before concn. to 50% vol. and the suspension adjusted to pH 1 with 3N HCl, extd. with 45 mL. EtOAc and the ext. washed with 10 mL. satd. aq. Na2SO4, the dried ext. evapd., and the amide (0.5 g., m. 143-50°) recrystd. from EtOAc-C6H12 gave salicyloylglycine ethylamide, m. 165.0-5.8°, identical with a sample prepd. by treatment of ο-AcOC6H4COCl with excess H2NCH2CONHEt. The formation of O-acyl-N-ethylsalicylamides from the cation IV and carboxylate anions was rationalized as proceeding from an imino anhydride (X) by an acyl shift. BzNHCH2CO2H (7 g.) in 40 mL. N NaOH adjusted to pH 5 with dil. HCl and stirred rapidly with 7 g. powd. II, extd. with 60 mL. CH2Cl2 and the residue on evapn. extd. with 20 mL. boiling C6H12, the ext. evapd. and the residue recrystd. from abs. alc. gave 70 mg. 2-phenyloxazol-5-one (XI), m. 89.5-90.2°. The C6H12-insol. residue recrystd. from EtOAc-C6H12 and the product (8 g., m. 85.5-7.0°) recrystd. from CH2Cl2-Et2O gave VIII (R = BzNHCH2), m. 87.5-9.0°, λ 5.63, 5.99 μ. XI must be formed from a more highly activated deriv., X (R = BzNHCH2). II (2 g.) in 3 mL. MeCN added to 4 g. (H2N)2CS in 50 mL. H2O contg. 0.1 g. NaOAc and 0.1 g. AcOH and the suspension extd. at 15-min. intervals with 5 portions (15 mL.) of CH2Cl2, the dried ext. evapd. and the residue extd. with 16 mL. boiling C6H12, the ext. filtered hot through Celite and the cryst. solid (1.15 g., m. 78-82°) recrystd. from C6H12 and sublimed gave 2-imino-3-ethyl-4-thiobenz[1,3]oxazine-2,4-dione (XI), m. 83-3.6°, λ 2.93, 5.99 μ, λ 277, 320 mμ (ε 10600, 13200, H2O, pH 3-11), τ1.18-3.2m, 3.4s, 5.4q, 8.7t. XI (0.5 g.) in 10 mL. MeOH boiled and treated with 5 mL. 3N HCl, the cryst. product washed with 50% MeOH and recrystd. from dil. MeOH gave 0.4 g. yellow cubes, m. 106.4-7.3°, recrystd. and sublimed to give 3-ethyl-4-thiobenzo [1,3] oxazine-2,4-dione (XII), m. 107.0-7.4°, λ 5.67 μ, λ 270, 328 mμ (ε 12200, 12200, H2O, pH 1-9), converted by alk. hydrolysis to ο-HOC6H4CSNHEt, m. 60-0.8°. Treatment of XII with acidic AgNO3, gave O,N-carbonyl-N-ethylsalicylamide (XIII), m. 102-3°. XIII (2 g., prepd. by C5H5N-catalyzed reaction of ClCO2Et with ο-HOC6H4CONHEt) and 1.2 g. P2S5 triturated vigorously 10 min. at 175° and the cooled mass extd. with CH2Cl2, the ext. evapd., and the residue recrystd. yielded 65% yellow cubes, recrystd. to give XII. II (3 g.) in 3 mL. MeCN kept 6 min. in 50 mL. H2O contg. 4 g. (H2N)2CS, 0.1 g. NaOAc, and 0.1 g. AcOH, extd. rapidly with 20 mL. CH2Cl2, and the aq. layer adjusted to pH 8 with dil. aq. NaOH, the ppt. washed with H2O and CH2Cl2 and dried gave 0.8 g. 2-imino-4-thiobenz[1,3]oxazine-2,4-dione (XIV), m. 175-205° (decompn.), λ 2.97, 6.03 μ, λ 277, 334 mμ (ε 8500, 13200, H2O, pH 5), μ 277, 321 mμ (ε 6100, 9300, H2O, pH 11), τ 1.3s, 1.6-2.8m, 6.5s, converted by warm acidic AgNO3 to O,N-carbonylsalicylamide, m. 226-8°. Finely powd. II (5 g.) added with stirring to 7 g. recrystd. (H2N)2CS in 100 mL. H2O and the soln. extd. after 5 min. with 30 mL. CH2Cl2, the aq. layer added to 50 mL. satd. picric acid in 30% aq. EtOH and the ppt. washed and dried in vacuo gave 4.8 g. picrate of the cation (XV), decompg. at 140-50°; XV.HCl, λ 5.87 μ, λ 273, 340 mμ (ε 7600, 300, H2O); XV picrate, τ -0.2s, 1.4s, 2.6d, 6.3p, 8.8t. Addn. of XV picrate to aq. acetate buffer gave XI; addn. to bicarbonate buffer generated XI and XIV together with a trace of H2S. IV is able to combine with all nucleophilic elements of the 1st row of the periodic table and at least one member of the 2nd. The ready availability and high yields of the products provide unusual starting materials for the synthesis of new benzo-fused heterocyclic systems.
    345. 345
      Salahuddin, S.; Renaudet, O.; Reymond, J.-L. Aldehyde Detection by Chromogenic/Fluorogenic Oxime Bond Fragmentation. Org. Biomol. Chem. 2004, 2 (10), 1471– 5,  DOI: 10.1039/b400314d
      347
      Aldehyde detection by chromogenic/fluorogenic oxime bond fragmentation
      Salahuddin, Syed; Renaudet, Olivier; Reymond, Jean-Louis
      Organic & Biomolecular Chemistry (2004), 2 (10), 1471-1475CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Amination of 4-nitrophenol, umbelliferone and 4-methylumbelliferone gave the corresponding oxyamines. These oxyamines react with aldehydes and ketones to form oximes. In the case of aliph. aldehydes and electron-poor arom. aldehydes, the oximes undergo base-catalyzed fragmentation in aq. buffer in the presence of bovine serum albumin to give the parent phenols, which is the acyclic analog of Kemp's elimination reaction of 5-nitrobenzisoxazole. The process can be used as a spectrophotometric assay for formaldehyde under aq. neutral conditions.
    346. 346
      Drienovská, I.; Mayer, C.; Dulson, C.; Roelfes, G. A Designer Enzyme for Hydrazone and Oxime Formation Featuring an Unnatural Catalytic Aniline Residue. Nat. Chem. 2018, 10 (9), 946– 52,  DOI: 10.1038/s41557-018-0082-z
      348
      A designer enzyme for hydrazone and oxime formation featuring an unnatural catalytic aniline residue
      Drienovska, Ivana; Mayer, Clemens; Dulson, Christopher; Roelfes, Gerard
      Nature Chemistry (2018), 10 (9), 946-952CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
      Creating designer enzymes with the ability to catalyze abiol. transformations is a formidable challenge. Efforts toward this goal typically consider only canonical amino acids in the initial design process. However, incorporating unnatural amino acids that feature uniquely reactive side chains could significantly expand the catalytic repertoire of designer enzymes. To explore the potential of such artificial building blocks for enzyme design, here we selected p-aminophenylalanine (pAF) as a potentially novel catalytic residue. We demonstrate that the catalytic activity of the aniline side chain for hydrazone and oxime formation reactions is increased by embedding p-aminophenylalanine into the hydrophobic pore of the multidrug transcriptional regulator LmrR from Lactococcus lactis. Both the recruitment of reactants by the promiscuous binding pocket and a judiciously placed aniline that functions as a catalytic residue contribute to the success of the identified artificial enzyme. We anticipate that our design strategy will prove rewarding to significantly expand the catalytic repertoire of designer enzymes in the future.
    347. 347
      Horowitz, R. M.; Geissman, T. A. A Cleavage Reaction of α-Allylbenzylamines. J. Am. Chem. Soc. 1950, 72 (4), 1518– 22,  DOI: 10.1021/ja01160a025
      349
      Cleavage reaction of α-allylbenzylamines
      Horowitz, Robert M.; Geissman, T. A.
      Journal of the American Chemical Society (1950), 72 (), 1518-22CODEN: JACSAT; ISSN:0002-7863.
      PhCH(CH2CH:CH2)CO2H (84.4 g.) and 37.8 g. C5H5N in 250 ml. ether, treated gradually with 57 g. SOCl2 in 50 ml. ether, stirred several hrs., kept overnight, and the residual acid chloride added dropwise to 500 ml. ice-cold concd. NH4OH, give 82% α-allyl-α-phenylacetamide (I), m. 53.5-4°. I (68 g.) in 300 ml. H2O, treated with NaOBr (with ice cooling), gives 90% α-allylbenzylamine (II), b3.5 75°, d420 0.953, nD24 1.5300; the phenylsulfonyl deriv. m. 79.5-80.5°; II and 3-O2NC6H3(CO)2O, heated 5 min. at 170 °, give N-(α-allylbenzyl)-3-nitrophthalimide, m. 98-9°. II yields a picrate, yellow, m. 153°; attempted recrystn. from Me2CO gives the N-isopropylidene deriv. of the picrate, yellow, m. 149-51°. II (10 g.) and 22.8 g. NaHCO3 in 100 ml. H2O and 100 ml. EtOH, treated (20 min.) with 17.1 g. Me2SO4 and boiled 2 hrs., give trimethyl-(α-allyl-benzyl)ammonium Me sulfate (III), m. 137-8°; 0.5 g. III and 20 ml. 1.5 N NaOH on distn. yields PhCH:CHCH:CH2 (identified as the dibromide). II (20 g.), added to 9 g. (HCHO)3, 35 g. 90% HCO2H, and 17 ml. H2O (heated until homogeneous) (temp. rise to 40° in 30 min.), heated 2 hrs. on the water bath, kept overnight, treated with 15.7 g. concd. HCl, and steam-distd., gives 70% BzH; the residue from the steam distn., made alk. and again distd., gives about 10% Me2NCH2CH2CH : CH2 (picrate, m. 110.2-11°). II and HCHO in H2O, heated 3 hrs. on the steam bath, give 56% CH2:CHCH2CH2NH2 (picrate, m. 130.8-7.4°) and 80% BzH. Aq. Me2NH (58 g.), 62 g. 36-8% HCHO, 58 g. BuOH, and excess K2CO3, kept overnight and the crude (butoxymethyl)dimethylamine (b. 138-8.5°) treated with excess CH2:CHCH2MgBr, give CH2:CHCH2CH2NMe2, b. 92°; picrate, m. 110-11.8° and then 114-14.2°; methiodide, hygroscopic, m. 240-1.3°. PhCN (77.8 g.), refluxed several hrs. with 1.6 mols. CH2:CHCH2MgBr, gives 11 g. α,α-diallylbenzylamine (IV), b0.75 87.5-90°, nD25 1.5310; picrate, m. 134-4.5°; phenylsulfonyl deriv., m. 151-2°; thiourea deriv., m. 132.5-3.5° and 142°. IV (3 g.), 3 ml. 36-8% HCHO, and 10 ml. 6 N H2SO4 in 100 ml. H2O, distd. until 35 ml. distillate is formed, give CH2:CHCH3CH2NH2 and allyl Ph ketone, isolated as the 2,4-dinitro-phenylhydrazone, orange-red, m. 201-3°. The probable course of the reactions of II and IV is proposed and its analogy with the rearrangement of CH2:CH(CH2:CHCH2)C(CO2Et)2 is pointed out.
    348. 348
      Sakabe, M.; Asanuma, D.; Kamiya, M.; Iwatate, R. J.; Hanaoka, K.; Terai, T.; Nagano, T.; Urano, Y. Rational Design of Highly Sensitive Fluorescence Probes for Protease and Glycosidase Based on Precisely Controlled Spirocyclization. J. Am. Chem. Soc. 2013, 135 (1), 409– 14,  DOI: 10.1021/ja309688m
      350
      Rational Design of Highly Sensitive Fluorescence Probes for Protease and Glycosidase Based on Precisely Controlled Spirocyclization
      Sakabe, Masayo; Asanuma, Daisuke; Kamiya, Mako; Iwatate, Ryu J.; Hanaoka, Kenjiro; Terai, Takuya; Nagano, Tetsuo; Urano, Yasuteru
      Journal of the American Chemical Society (2013), 135 (1), 409-414CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We have synthesized and evaluated a series of hydroxymethyl rhodamine derivs. and found an intriguing difference of intramol. spirocyclization behavior: the acetylated deriv. of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aq. soln. at physiol. pH, whereas HMRG itself takes an open nonspirocyclic structure. Ac-HMRG is colorless and nonfluorescent, whereas HMRG is strongly fluorescent. On the basis of these findings, we have developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the acetyl group of Ac-HMRG with a substrate moiety of the target enzyme. Specific cleavage of the substrate moiety in the nonfluorescent probe by the target enzyme generates a strong fluorescence signal. To confirm the validity and flexibility of our strategy, we designed and synthesized fluorescence probes for leucine aminopeptidase (Leu-HMRG), fibroblast activation protein (Ac-GlyPro-HMRG), and β-galactosidase (βGal-HMRG). All of these probes were almost nonfluorescent due to the formation of spirocyclic structure, but were converted efficiently to highly fluorescent HMRG by the target enzymes. We confirmed that the probes can be used in living cells. These probes offer great practical advantages, including high sensitivity and rapid response (due to regulation of fluorescence at a single reactive site), as well as resistance to photobleaching, and are expected to be useful for a range of biol. and pathol. investigations.
    349. 349
      Brewer, T. F.; Chang, C. J. An Aza-Cope Reactivity-Based Fluorescent Probe for Imaging Formaldehyde in Living Cells. J. Am. Chem. Soc. 2015, 137 (34), 10886– 9,  DOI: 10.1021/jacs.5b05340
      351
      An Aza-Cope Reactivity-Based Fluorescent Probe for Imaging Formaldehyde in Living Cells
      Brewer, Thomas F.; Chang, Christopher J.
      Journal of the American Chemical Society (2015), 137 (34), 10886-10889CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Formaldehyde (FA) is a reactive carbonyl species (RCS) produced in living systems that has been implicated in epigenetics as well as in the pathologies of various cancers, diabetes, and heart, liver, and neurodegenerative diseases. Traditional methods for biol. FA detection rely on sample destruction and/or extensive processing, resulting in a loss of spatiotemporal information. To help address this technol. gap, the authors present the design, synthesis, and biol. evaluation of a fluorescent probe for live-cell FA imaging that relies on a FA-induced aza-Cope rearrangement. Formaldehyde probe-1 (FAP-1, I) is capable of detecting physiol. relevant concns. of FA in aq. buffer and in live cells with high selectivity over potentially competing biol. analytes. Moreover, FAP-1 can visualize endogenous FA produced by lysine-specific demethylase 1 in a breast cancer cell model, presaging the potential utility of this chem. approach to probe RCS biol.
    350. 350
      Roth, A.; Li, H.; Anorma, C.; Chan, J. A Reaction-Based Fluorescent Probe for Imaging of Formaldehyde in Living Cells. J. Am. Chem. Soc. 2015, 137 (34), 10890– 3,  DOI: 10.1021/jacs.5b05339
      352
      A Reaction-Based Fluorescent Probe for Imaging of Formaldehyde in Living Cells
      Roth, Aaron; Li, Hao; Anorma, Chelsea; Chan, Jefferson
      Journal of the American Chemical Society (2015), 137 (34), 10890-10893CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Formaldehyde (FA), in the 0.2-0.4 mM range, is produced and maintained endogenously via enzymic pathways. At these levels, FA can promote cell proliferation as well as mediate memory formation. Once elevated, FA stress is known to induce cognitive impairments, memory loss, and neurodegeneration owing to its potent DNA and protein crosslinking mechanisms. Optical imaging is a powerful noninvasive approach used to study FA in living systems; however, biocompatible chem. probes for FA are currently lacking. Herein, the authors report the design, synthesis, and biol. evaluation of Formaldehyde Probe 1 (FP1, I), a new fluorescent indicator based on the 2-aza-Cope sigmatropic rearrangement. The remarkable sensitivity, selectivity, and photostability of FP1 has enabled the authors to visualize FA in live HEK293TN and Neuroscreen-1 cells. The authors envision that FP1 will find widespread applications in the study of FA assocd. with normal and pathol. processes.
    351. 351
      Bruemmer, K. J.; Walvoord, R. R.; Brewer, T. F.; Burgos-Barragan, G.; Wit, N.; Pontel, L. B.; Patel, K. J.; Chang, C. J. Development of a General Aza-Cope Reaction Trigger Applied to Fluorescence Imaging of Formaldehyde in Living Cells. J. Am. Chem. Soc. 2017, 139 (15), 5338– 50,  DOI: 10.1021/jacs.6b12460
      353
      Development of a General Aza-Cope Reaction Trigger Applied to Fluorescence Imaging of Formaldehyde in Living Cells
      Bruemmer, Kevin J.; Walvoord, Ryan R.; Brewer, Thomas F.; Burgos-Barragan, Guillermo; Wit, Niek; Pontel, Lucas B.; Patel, Ketan J.; Chang, Christopher J.
      Journal of the American Chemical Society (2017), 139 (15), 5338-5350CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Formaldehyde (FA) is a reactive signaling mol. that is continuously produced through a no. of central biol. pathways spanning epigenetics to one-carbon metab. However, aberrant, elevated levels of FA are implicated in disease states ranging from asthma to neurodegenerative disorders. In this context, fluorescence-based probes for FA imaging are emerging as potentially powerful chem. tools to help disentangle the complexities of FA homeostasis and its physiol. and pathol. contributions. Currently available FA indicators require direct modification of the fluorophore backbone through complex synthetic considerations to enable FA detection, often limiting the generalization of designs to other fluorophore classes. To address this challenge, the authors now present the rational, iterative development of a general reaction-based trigger using 2-aza-Cope reactivity for selective and sensitive detection of FA in living systems. Specifically, the authors developed a homoallylamine functionality that can undergo a subsequent self-immolative β-elimination, creating a FA-responsive trigger that is capable of masking a phenol on a fluorophore or any other potential chem. scaffold for related imaging and/or therapeutic applications. The authors demonstrate the utility of this trigger by creating a series of fluorescent probes for FA with excitation and emission wavelengths that span the UV to visible spectral regions through caging of a variety of dye units. In particular, Formaldehyde Probe 573 (FAP573), based on a resorufin scaffold, is the most red shifted and FA sensitive in this series in terms of signal-to-noise responses and enables identification of alc. dehydrogenase 5 (ADH5) as an enzyme that regulates FA metab. in living cells. The results provide a starting point for the broader use of 2-aza-Cope reactivity for probing and manipulating FA biol.
    352. 352
      Du, Y.; Zhang, Y.; Huang, M.; Wang, S.; Wang, J.; Liao, K.; Wu, X.; Zhou, Q.; Zhang, X.; Wu, Y.-D. Systematic Investigation of the Aza-Cope Reaction for Fluorescence Imaging of Formaldehyde in Vitro and in Vivo. Chem. Sci. 2021, 12 (41), 13857– 69,  DOI: 10.1039/D1SC04387K
      354
      Systematic investigation of the aza-Cope reaction for fluorescence imaging of formaldehyde in vitro and in vivo
      Du, Yimeng; Zhang, Yuqing; Huang, Meirong; Wang, Shushu; Wang, Jianzheng; Liao, Kongke; Wu, Xiaojun; Zhou, Qiang; Zhang, Xinhao; Wu, Yun-Dong; Peng, Tao
      Chemical Science (2021), 12 (41), 13857-13869CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Increasing evidence has highlighted the endogenous prodn. of formaldehyde (FA) in a variety of fundamental biol. processes and its involvement in many disease conditions ranging from cancer to neurodegeneration. To examine the physiol. and pathol. relevance and functions of FA, fluorescent probes for FA imaging in live biol. samples are of great significance. Herein we report a systematic investigation of 2-aza-Cope reactions between homoallylamines and FA for identification of a highly efficient 2-aza-Cope reaction moiety and development of fluorescent probes for imaging FA in living systems. By screening a set of N-substituted homoallylamines and comparing them to previously reported homoallylamine structures for reaction with FA, we found that N-p-methoxybenzyl homoallylamine exhibited an optimal 2-aza-Cope reactivity to FA. Theor. calcns. were then performed to demonstrate that the N-substituent on homoallylamine greatly affects the condensation with FA, which is more likely the rate-detg. step. Moreover, the newly identified optimal N-p-methoxybenzyl homoallylamine moiety with a self-immolative β-elimination linker was generally utilized to construct a series of fluorescent probes with varying excitation/emission wavelengths for sensitive and selective detection of FA in aq. solns. and live cells. Among these probes, the near-IR probe FFP706 has been well demonstrated to enable direct fluorescence visualization of steady-state endogenous FA in live mouse brain tissues and elevated FA levels in a mouse model of breast cancer. This study provides the optimal aza-Cope reaction moiety for FA probe development and new chem. tools for fluorescence imaging and biol. investigation of FA in living systems.
    353. 353
      He, L.; Yang, X.; Liu, Y.; Kong, X.; Lin, W. A Ratiometric Fluorescent Formaldehyde Probe for Bioimaging Applications. Chem. Commun. 2016, 52 (21), 4029– 32,  DOI: 10.1039/C5CC09796G
      355
      A ratiometric fluorescent formaldehyde probe for bioimaging applications
      He, Longwei; Yang, Xueling; Liu, Yong; Kong, Xiuqi; Lin, Weiying
      Chemical Communications (Cambridge, United Kingdom) (2016), 52 (21), 4029-4032CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      We have described a ratiometric fluorescent formaldehyde probe (RFFP) based on the 6-hydroxy naphthalene chromophore for the first time. The probe is suitable for ratiometric detection of formaldehyde both in the soln. and living biol. samples with two distinct emission bands.
    354. 354
      Xu, J.; Zhang, Y.; Zeng, L.; Liu, J.; Kinsella, J. M.; Sheng, R. A Simple Naphthalene-Based Fluorescent Probe for High Selective Detection of Formaldehyde in Toffees and HeLa Cells Via Aza-Cope Reaction. Talanta 2016, 160, 645– 52,  DOI: 10.1016/j.talanta.2016.08.010
      356
      A simple naphthalene-based fluorescent probe for high selective detection of formaldehyde in toffees and HeLa cells via aza-Cope reaction
      Xu, Junchao; Zhang, Yue; Zeng, Lintao; Liu, Jinbiao; Kinsella, Joseph M.; Sheng, Ruilong
      Talanta (2016), 160 (), 645-652CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      A simple naphthalene-based fluorescent probe (AENO) for formaldehyde (FA) was successfully synthesized, which exhibited a significant fluorescence turn-on response towards FA in aq. soln. The probe could quant. det. the concn. of FA (0-1.0 mM) with excellent selectivity, high sensitivity and low limit of detection (0.57 μM). The sensing mechanism is proposed as 2-aza-Cope rearrangement for AENO after reaction with FA, which was confirmed by 1H NMR, HR-MS, FTIR, UV-visible and fluorescence spectra. The probe has been employed to det. the FA contents in several com. available toffee samples with satisfactory performance. Thus, AENO might be used as a promising tool for quant. detection of FA in food. Furthermore, fluorescence imaging of HeLa cells indicated that the probe was cell membrane permeable and could be used for visualizing/imaging the FA trace/transportation in cancer cells.
    355. 355
      Yang, X.; He, L.; Xu, K.; Yang, Y.; Lin, W. The Development of an Ict-Based Formaldehyde-Responsive Fluorescence Turn-On Probe with a High Signal-to-Noise Ratio. New J. Chem. 2018, 42 (15), 12361– 4,  DOI: 10.1039/C8NJ02467G
      357
      The development of an ICT-based formaldehyde-responsive fluorescence turn-on probe with a high signal-to-noise ratio
      Yang, Xueling; He, Longwei; Xu, Kaixin; Yang, Yunzhen; Lin, Weiying
      New Journal of Chemistry (2018), 42 (15), 12361-12364CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
      An illuminating ICT-based formaldehyde-responsive fluorescent probe (PBD-FA) with long wavelength emission was judiciously designed and synthesized, which is suitable for detecting FA in aq. soln. and living cells, both with significant fluorescence signal-to-noise ratios.
    356. 356
      Yang, M.; Fan, J.; Du, J.; Long, S.; Wang, J.; Peng, X. Imaging of Formaldehyde in Live Cells and Daphnia Magna Via Aza-Cope Reaction Utilizing Fluorescence Probe with Large Stokes Shifts. Front. Chem. 2018, 6, 488,  DOI: 10.3389/fchem.2018.00488
      358
      Imaging of formaldehyde in live cells and Daphnia magna via aza-cope reaction utilizing fluorescence probe with large stokes shifts
      Yang, Mingwang; Fan, Jiangli; Du, Jianjun; Long, Saran; Wang, Jia; Peng, Xiaojun
      Frontiers in Chemistry (Lausanne, Switzerland) (2018), 6 (), 488CODEN: FCLSAA; ISSN:2296-2646. (Frontiers Media S.A.)
      Formaldehyde (FA), a highly reactive carbonyl species, plays significant role in physiol. and pathol. functions. However, elevated FA will lead to cognitive impairments, memory loss and various neurodegenerative diseases due to its potent DNA and protein crosslinking mechanisms. In this work, a fluorescence probe, BD-CHO, based on benz-2-oxa-1, 3- diazole (BD) skeleton, was designed and synthesized for detection of FA via Aza-Cope reaction with high selectivity and large Stokes shifts (about 118 nm). BD-CHO was successfully applied to monitor the changes FA level in living cells, and kidney tissues of mice. Importantly it was the first time that BD-CHO was used for visualizing exogenous FA changes in Daphnia magna through fluorescence microscopy, demonstrating its potential application for studies of biol. processes assocd. with FA.
    357. 357
      Bruemmer, K. J.; Brewer, T. F.; Chang, C. J. Fluorescent Probes for Imaging Formaldehyde in Biological Systems. Curr. Opin. Chem. Biol. 2017, 39, 17– 23,  DOI: 10.1016/j.cbpa.2017.04.010
      359
      Fluorescent probes for imaging formaldehyde in biological systems
      Bruemmer, Kevin J.; Brewer, Thomas F.; Chang, Christopher J.
      Current Opinion in Chemical Biology (2017), 39 (), 17-23CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      Formaldehyde (FA) is a common environmental toxin but is also endogenously produced through a diverse array of essential biol. processes, including mitochondrial one-carbon metab., metabolite oxidn., and nuclear epigenetic modifications. Its high electrophilicity enables reactivity with a wide variety of biol. nucleophiles, which can be beneficial or detrimental to cellular function depending on the context. New methods that enable detection of FA in living systems can help disentangle the signal/stress dichotomy of this simplest reactive carbonyl species (RCS), and fluorescent probes for FA with high selectivity and sensitivity have emerged as promising chem. tools in this regard.
    358. 358
      Liu, X.; Li, N.; Li, M.; Chen, H.; Zhang, N.; Wang, Y.; Zheng, K. Recent Progress in Fluorescent Probes for Detection of Carbonyl Species: Formaldehyde, Carbon Monoxide and Phosgene. Coord. Chem. Rev. 2020, 404, 213109,  DOI: 10.1016/j.ccr.2019.213109
      360
      Recent progress in fluorescent probes for detection of carbonyl species: Formaldehyde, carbon monoxide and phosgene
      Liu, Xiang; Li, Ning; Li, Meng; Chen, Hui; Zhang, Nuonuo; Wang, Yanlan; Zheng, Kaibo
      Coordination Chemistry Reviews (2020), 404 (), 213109CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
      A review. Extensive attention has been paid to develop effective systems for the detection of formaldehyde, carbon monoxide and phosgene due to their extreme toxicity and ready accessibility. Numerous methods have been developed for the design and detection of these substances nowadays, such as electro-fluorescent biosensors, piezoelec. sensors, semiconductor sensors, colorimetric probes, quartz crystal microbalance, Raman spectroscopy, transmission electro-microscopy (TEM), gas chromatog., liq. chromatog. and X-ray diffraction (XRD), but fluorescent probes, which rely on chem. reactions between the probes and the target, provoking a dramatic fluorescence change, often remain the most commonly employed method for detecting such important small mols. This review will cover the most significant developments in fluorescent probes for the detection of the carbonyl species formaldehyde, carbon monoxide and phosgene in recent years (typically the last 10 years), with a special emphasis on their mechanisms and applications.
    359. 359
      Hanson, J. R. Terpenoids and Steroids, 7 ed.; Royal Society of Chemistry, 2007.
      There is no corresponding record for this reference.
    360. 360
      Forney, F. W.; Markovetz, A. J. The Biology of Methyl Ketones. J. Lipid Res. 1971, 12 (4), 383– 95,  DOI: 10.1016/S0022-2275(20)39487-6
      362
      Biology of methyl ketones
      Forney, F. W.; Markovetz, A. J.
      Journal of Lipid Research (1971), 12 (4), 383-95CODEN: JLPRAW; ISSN:0022-2275.
      Examples of the biol. occurrence of Me ketones are reviewed. The lack of significant accumulations of these compds. in the biosphere indicates that a recycling of these org. mols. is occurring. Evidence for biodegradation of acetone by mammals and longer Me ketones by microorganisms via terminal Me group oxidn. is discussed. A new mechanism for the subterminal oxidn. of Me ketones by microorganisms is proposed whereby the 1st intermediate produced is an acetate ester which subsequently is cleaved to acetate and a primary alc. 2 C shorter than the original ketone substrate.
    361. 361
      McNally, M. A.; Hartman, A. L. Ketone Bodies in Epilepsy. J. Neurochem. 2012, 121 (1), 28– 35,  DOI: 10.1111/j.1471-4159.2012.07670.x
      363
      Ketone bodies in epilepsy
      McNally, Melanie A.; Hartman, Adam L.
      Journal of Neurochemistry (2012), 121 (1 & 2), 28-35CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)
      A review. Seizures that are resistant to std. medications remain a major clin. problem. One underutilized option for patients with medication-resistant seizures is the high-fat, low-carbohydrate ketogenic diet. The diet received its name based on the observation that patients consuming this diet produce ketone bodies (e.g., acetoacetate, β-hydroxybutyrate, and acetone). Although the exact mechanisms of the diet are unknown, ketone bodies have been hypothesized to contribute to the anticonvulsant and antiepileptic effects. In this review, anticonvulsant properties of ketone bodies and the ketogenic diet are discussed (including GABAergic and glutamatergic effects). Because of the importance of ketone body metab. in the early stages of life, the effects of ketone bodies on developing neurons in vitro also are discussed. Understanding how ketone bodies exert their effects will help optimize their use in treating epilepsy and other neurol. disorders.
    362. 362
      Wakil, S. J.; Stoops, J. K.; Joshi, V. C. Fatty Acid Synthesis and Its Regulation. Annu. Rev. Biochem. 1983, 52 (1), 537– 79,  DOI: 10.1146/annurev.bi.52.070183.002541
      364
      Fatty acid synthesis and its regulation
      Wakil, Salih J.; Stoops, James K.; Joshi, Vasudev C.
      Annual Review of Biochemistry (1983), 52 (), 537-79CODEN: ARBOAW; ISSN:0066-4154.
      A review with 186 refs. on the title subject, including the functional organization and reaction mechanism of fatty acid synthetase.
    363. 363
      Dhillon, K. K.; Gupta, S. Biochemistry, Ketogenesis; StatPearls: Treasure Island, FL, 2021.
      There is no corresponding record for this reference.
    364. 364
      Mutti, F. G.; Knaus, T. Enzymes Applied to the Synthesis of Amines. Biocatalysis for Practitioners 2021, 143– 80,  DOI: 10.1002/9783527824465.ch6
      366
      Enzymes Applied to the Synthesis of Amines
      Mutti, Francesco G.; Knaus, Tanja
      Biocatalysis for Practitioners (2021), (), 143-180CODEN: 70ABFT ISSN:. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A great variety of biocatalytic methods are available for the synthesis of amines, in particular, α-chiral amines. Therefore, Chapter 6 aims to orient nonspecialists to the available biocatalytic methodologies for the synthesis of α-chiral amines with a focus on the enzymes' substrate scope and selectivity as well as the structural diversity of the obtained products. The presented enzymic methods comprise the use of hydrolases, amine oxidases, transaminases, amine dehydrogenases, imine reductases, ammonia lyases, Pictet-Spenglerases, and engineered cytochromes P 450. Classification and reactivity, biocatalytic applications, and selected important examples are discussed for each enzyme category. Methodologies entail the kinetic resoln. or deracemization of racemic amines and the asym. synthesis from prochiral precursors, where applicable. Finally, the last chapter's section illustrates practical procedures. Notably, the presented biocatalytic methods can yield primary, secondary, and tertiary amine products and often with excellent chemo-, regio-, and stereoselectivity under mild reaction conditions.
    365. 365
      Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Recent Advances in the Biocatalytic Reduction of Ketones and Oxidation of sec-Alcohols. Curr. Opin. Chem. Biol. 2004, 8 (2), 120– 6,  DOI: 10.1016/j.cbpa.2004.02.005
      367
      Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols
      Kroutil, Wolfgang; Mang, Harald; Edegger, Klaus; Faber, Kurt
      Current Opinion in Chemical Biology (2004), 8 (2), 120-126CODEN: COCBF4; ISSN:1367-5931. (Elsevier Science Ltd.)
      A review. To improve the efficiency and applicability of biocatalytic redox-reactions for asym. ketone-redn. and enantioselective alc.-oxidn. catalyzed by nicotinamide-dependent dehydrogenases/reductases, several achievements for cofactor-recycling have been made during the last two years. First, the use of hydrogenases for NADPH recycling in a two enzyme system. Second, preparative transformations with alc. dehydrogenases coupled with NADH oxidases for NAD+/NADP+ recycling. Third, an exceptional chemo-stable alc. dehydrogenase can efficiently use i-propanol and acetone as cosubstrates for redn. and oxidn., resp., in a single-enzyme system. Novel carbonyl reductases and dehydrogenases derived from plant cells are particularly suited for sterically demanding substrates.
    366. 366
      Leisch, H.; Morley, K.; Lau, P. C. K. Baeyer- Villiger Monooxygenases: More Than Just Green Chemistry. Chem. Rev. 2011, 111 (7), 4165– 222,  DOI: 10.1021/cr1003437
      368
      Baeyer-Villiger Monooxygenases: More Than Just Green Chemistry
      Leisch, Hannes; Morley, Krista; Lau, Peter C. K.
      Chemical Reviews (Washington, DC, United States) (2011), 111 (7), 4165-4222CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      This review focuses on the monooxygenase-catalyzed Baeyer-Villiger oxidn. of linear or cyclic ketones as a green chem. tool to address environmental sustainability, a system to study its mol. diversity and catalytic mechanism, industrial scale bioprocess development, and protein engineering to evolve new biotechnol. applications.
    367. 367
      Dean, S. M.; Greenberg, W. A.; Wong, C. H. Recent Advances in Aldolase-Catalyzed Asymmetric Synthesis. Adv. Synth. Catal. 2007, 349 (8–9), 1308– 20,  DOI: 10.1002/adsc.200700115
      369
      Recent advances in aldolase-catalyzed asymmetric synthesis
      Dean, Stephen M.; Greenberg, William A.; Wong, Chi-Huey
      Advanced Synthesis & Catalysis (2007), 349 (8+9), 1308-1320CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Here, the authors focus on advances over the last several years in the application of aldolases to org. synthesis. Several new technologies have been implemented to increase the scope and practicality of aldolases as tools for the synthetic chemist. These include directed evolution, discovery of new classes of aldolases in nature and the lab., and substrate and reaction engineering.
    368. 368
      Walsh, C. T. Biologically Generated Carbon Dioxide: Nature’s Versatile Chemical Strategies for Carboxy Lyases. Nat. Prod. Rep. 2020, 37 (1), 100– 35,  DOI: 10.1039/C9NP00015A
      370
      Biologically generated carbon dioxide: nature's versatile chemical strategies for carboxy lyases
      Walsh, Christopher T.
      Natural Product Reports (2020), 37 (1), 100-135CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
      Covering: up to 2019Metabolic prodn. of CO2 is natural product chem. on a mammoth scale. Just counting humans, among all other respiring organisms, the seven billion people on the planet exhale about 3 billion tons of CO2 per yr. Essentially all of the biogenic CO2 arises by action of discrete families of decarboxylases. The mechanistic routes to CO2 release from carboxylic acid metabolites vary with the electronic demands and structures of specific substrates and illustrate the breadth of chem. employed for C-COO (C-C bond) disconnections. Most commonly decarboxylated are α-keto acid and β-keto acid substrates, the former requiring thiamin-PP as cofactor, the latter typically cofactor-free. The extensive decarboxylation of amino acids, e.g. to neurotransmitter amines, is synonymous with the coenzyme form of vitamin B6, pyridoxal-phosphate, although covalent N-terminal pyruvamide residues serve in some amino acid decarboxylases. All told, five B vitamins (B1, B2, B3, B6, B7), ATP, S-adenosylmethionine, manganese and zinc ions are pressed into service for specific decarboxylase catalyzes. There are addnl. cofactor-independent decarboxylases that operate by distinct chem. routes. Finally, while most decarboxylases use heterolytic ionic mechanisms, a small no. of decarboxylases carry out radical pathways.
    369. 369
      Liu, M.; Wei, D.; Wen, Z.; Wang, J.-b. Progress in Stereoselective Construction of C-C Bonds Enabled by Aldolases and Hydroxynitrile Lyases. Front. Bioeng. Biotechnol. 2021, 9, 653682,  DOI: 10.3389/fbioe.2021.653682
      371
      Progress in Stereoselective Construction of C-C Bonds Enabled by Aldolases and Hydroxynitrile Lyases
      Liu Mi; Wei Dan; Wen Zexing; Wang Jian-Bo; Liu Mi; Wei Dan; Wen Zexing; Wang Jian-Bo
      Frontiers in bioengineering and biotechnology (2021), 9 (), 653682 ISSN:2296-4185.
      The creation of C-C bonds is an effective strategy for constructing complex compounds from simple synthetic blocks. Although many methods have been developed for C-C bond construction, the stereoselective creation of new C-C bonds remains a challenge. The selectivities (enantioselectivity, regioselectivity, and chemoselectivity) of biocatalysts are higher than those of chemical catalysts, therefore biocatalysts are excellent candidates for use in stereoselective C-C bond formation. Here, we summarize progress made in the past 10 years in stereoselective C-C bond formation enabled by two classic types of enzyme, aldolases and hydroxynitrile lyases. The information in this review will enable the development of new routes to the stereoselective construction of C-C bonds.
    370. 370
      Müller, M.; Gocke, D.; Pohl, M. Thiamin Diphosphate in Biological Chemistry: Exploitation of Diverse Thiamin Diphosphate-Dependent Enzymes for Asymmetric Chemoenzymatic Synthesis. FEBS J. 2009, 276 (11), 2894– 904,  DOI: 10.1111/j.1742-4658.2009.07017.x
      372
      Thiamin diphosphate in biological chemistry: exploitation of diverse thiamin diphosphate-dependent enzymes for asymmetric chemoenzymatic synthesis
      Mueller, Michael; Gocke, Doerte; Pohl, Martina
      FEBS Journal (2009), 276 (11), 2894-2904CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      A review. Thiamin diphosphate (ThDP)-dependent enzymes participate in numerous biosynthetic pathways and catalyze a broad range of reactions, mainly involving the cleavage and formation of C-C bonds. For example, they catalyze the nonoxidative and oxidative decarboxylation of 2-keto acids, produce 2-hydroxy ketones, and transfer activated aldehydes to a variety of acceptors. Moreover, they can also catalyze C-N, C-O, and C-S bond formation. Because of their substrate spectra and different stereospecificities, these enzymes extend the synthetic potential for asym. carboligations appreciably. Different strategies have been developed to identify new members of this promiscuous enzyme class and the reactions they catalyze. This has enabled the authors to introduce solns. for longstanding synthetic problems, such as asym. cross-benzoin condensation. Moreover, through a combination of protein structure anal., enzyme and substrate engineering, and screening methods, the authors have explored addnl. stereochem. routes that have not been described previously for any of these interesting enzymes.
    371. 371
      Schmidt, N. G.; Pavkov-Keller, T.; Richter, N.; Wiltschi, B.; Gruber, K.; Kroutil, W. Biocatalytic Friedel-Crafts Acylation and Fries Reaction. Angew. Chem., Int. Ed. 2017, 56 (26), 7615– 9,  DOI: 10.1002/anie.201703270
      373
      Biocatalytic Friedel-Crafts Acylation and Fries Reaction
      Schmidt, Nina G.; Pavkov-Keller, Tea; Richter, Nina; Wiltschi, Birgit; Gruber, Karl; Kroutil, Wolfgang
      Angewandte Chemie, International Edition (2017), 56 (26), 7615-7619CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The Friedel-Crafts acylation is commonly used for the synthesis of aryl ketones, and a biocatalytic version, which may benefit from the chemo- and regioselectivity of enzymes, has not yet been introduced. Described here is a bacterial acyltransferase which can catalyze Friedel-Crafts C-acylation of phenolic substrates in buffer without the need of CoA-activated reagents. Conversions reach up to >99 %, and various C- or O-acyl donors, such as DAPG or isopropenyl acetate, are accepted by this enzyme. Furthermore the enzyme enables a Fries-like rearrangement reaction of resorcinol derivs. These findings open an avenue for the development of alternative and selective C-C bond formation methods.
    372. 372
      Gergel, S.; Soler, J.; Klein, A.; Schülke, K.; Hauer, B.; Garcia-Borràs, M.; Hammer, S. Directed Evolution of a Ketone Synthase for Efficient and Highly Selective Functionalization of Internal Alkenes by Accessing Reactive Carbocation Intermediates. ChemRxiv 2022. dp94p DOI: 10.26434/chemrxiv-2022-dp94p
      There is no corresponding record for this reference.
    373. 373
      Zhang, J.; Xu, T.; Li, Z. Enantioselective Biooxidation of Racemic trans-Cyclic Vicinal Diols: One-Pot Synthesis of Both Enantiopure (S,S)-Cyclic Vicinal Diols and (R)-α-Hydroxy Ketones. Adv. Synth. Catal. 2013, 355 (16), 3147– 53,  DOI: 10.1002/adsc.201300301
      375
      Enantioselective Biooxidation of Racemic trans-Cyclic Vicinal Diols: One-Pot Synthesis of Both Enantiopure (S,S)-Cyclic Vicinal Diols and (R)-α-Hydroxy Ketones
      Zhang, Jiandong; Xu, Tingting; Li, Zhi
      Advanced Synthesis & Catalysis (2013), 355 (16), 3147-3153CODEN: ASCAF7; ISSN:1615-4150. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Highly regio- and enantioselective alc. dehydrogenases BDHA (2,3-butanediol dehydrogenase from Bacillus subtilis BGSC1A1), CDDHPm (cyclic diol dehydrogenase from Pseudomonas mendocina TA5), and CDDHRh (cyclic diol dehydrogenase from Rhodococcus sp. Moj-3449) were discovered for the oxidn. of racemic trans-cyclic vicinal diols. Recombinant Escherichia coli expressing BDHA was engineered as an efficient whole-cell biocatalyst for the oxidn. of (±)-1,2-cyclopentanediol, 1,2-cyclohexanediol, 1,2-cycloheptane-diol, and 1,2-cyclooctanediol, resp., to give the corresponding (R)-α-hydroxy ketones in >99% ee and (S,S)-cyclic diols in >99% ee at 50% conversion in one pot. Escherichia coli (BDHA-LDH) co-expressing lactate dehydrogenase (LDH) for intracellular regeneration of NAD+ catalyzed the regio- and enantioselective oxidn. of (±)-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene to produce the corresponding (R)-α-hydroxy ketone in >99% ee and (S,S)-cyclic diol in 96% ee at 49% conversion. Preparative biotransformations were also demonstrated. Thus, a novel and useful method for the one-pot synthesis of both vicinal diols and α-hydroxy ketones in high ee was developed via highly regio- and enantioselective oxidns. of the racemic vicinal diols.
    374. 374
      Lavandera, I.; Kern, A.; Resch, V.; Ferreira-Silva, B.; Glieder, A.; Fabian, W. M. F.; de Wildeman, S.; Kroutil, W. One-Way Biohydrogen Transfer for Oxidation of sec-Alcohols. Org. Lett. 2008, 10 (11), 2155– 8,  DOI: 10.1021/ol800549f
      376
      One-Way Biohydrogen Transfer for Oxidation of sec-Alcohols
      Lavandera, Ivan; Kern, Alexander; Resch, Verena; Ferreira-Silva, Bianca; Glieder, Anton; Fabian, Walter M. F.; de Wildeman, Stefaan; Kroutil, Wolfgang
      Organic Letters (2008), 10 (11), 2155-2158CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      Quasi-irreversible oxidn. of sec-alcs. was achieved via biocatalytic hydrogen transfer reactions using alc. dehydrogenases employing selected ketones as hydrogen acceptors, which can only be reduced but not oxidized. Thus, only 1 equiv of oxidant was required instead of a large excess. For the oxidn. of both isomers of methylcarbinols a single nonstereoselective short-chain dehydrogenase/reductase from Sphingobium yanoikuyae was identified and overexpressed in E. coli.
    375. 375
      González-Granda, S.; Méndez-Sánchez, D.; Lavandera, I.; Gotor-Fernández, V. Laccase-Mediated Oxidations of Propargylic Alcohols. Application in the Deracemization of 1-Arylprop-2-Yn-1-Ols in Combination with Alcohol Dehydrogenases. ChemCatChem. 2020, 12 (2), 520– 7,  DOI: 10.1002/cctc.201901543
      377
      Laccase-mediated Oxidations of Propargylic Alcohols. Application in the Deracemization of 1-arylprop-2-yn-1-ols in Combination with Alcohol Dehydrogenases
      Gonzalez-Granda, Sergio; Mendez-Sanchez, Daniel; Lavandera, Ivan; Gotor-Fernandez, Vicente
      ChemCatChem (2020), 12 (2), 520-527CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The catalytic system composed by the laccase from Trametes versicolor and the oxy-radical TEMPO has been successfully applied in the sustainable oxidn. of fourteen propargylic alcs. The corresponding propargylic ketones were obtained in most cases in quant. conversions (87->99% yield), demonstrating the efficiency of the chemoenzymic methodol. in comparison with traditional chem. oxidants, which usually lead to problems assocd. with the formation of byproducts. Also, the stereoselective redn. of propargylic ketones was studied using alc. dehydrogenases such as the one from Ralstonia species overexpressed in E. coli or the com. available evo-1.1.200, allowing the access to both alc. enantiomers mostly with complete conversions and variable selectivities depending on the arom. pattern substitution (97->99% ee). To demonstrate the compatibility of the laccase-mediated oxidn. and the alc. dehydrogenase-catalyzed bioredn., a deracemization strategy starting from the racemic compds. was developed through a sequential one-pot two-step process, obtaining a selection of (S)- or (R)-1-arylprop-2-yn-1-ols with excellent yields (>98%) and selectivities (>98% ee) depending on the alc. dehydrogenase employed.
    376. 376
      Key, J. A.; Li, C.; Cairo, C. W. Detection of Cellular Sialic Acid Content Using Nitrobenzoxadiazole Carbonyl-Reactive Chromophores. Bioconj. Chem. 2012, 23 (3), 363– 71,  DOI: 10.1021/bc200276k
      378
      Detection of Cellular Sialic Acid Content Using Nitrobenzoxadiazole Carbonyl-Reactive Chromophores
      Key, Jessie A.; Li, Caishun; Cairo, Christopher W.
      Bioconjugate Chemistry (2012), 23 (3), 363-371CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      The selective ligation of hydrazine and amino-oxy compds. with carbonyls has gained popularity as a detection strategy with the recognition of aniline catalysis as a way to accelerate the labeling reaction in water. Aldehydes are a convenient functional group choice since there are few native aldehydes found at the cell surface. Aldehydes can be selectively introduced into sialic acid contg. glycoproteins by treatment with dil. sodium periodate. Thus, the combination of periodate oxidn. with aniline-catalyzed ligation (PAL) has become a viable method for detection of glycoconjugates on live cells. Herein the authors examine two fluorescent nitrobenzoxadiazole dyes for labeling of glycoproteins and cell surface glycoconjugates. The authors introduce a novel 4-aminooxy-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDAO) fluorophore and offer a comparison to com. dyes including the known 4-hydrazino-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDH) and Bodipy FL hydrazide. The authors confirm specificity for sialic acid moieties and that both dyes are suitable for in vitro and in vivo labeling studies using PAL and fluorescence spectroscopy. The dyes examd. here are attractive labeling agents for microscopy, as they can be excited by a 488 nm laser line and can be made in a few synthetic steps. These carbonyl-reactive chromophores provide a one step alternative to avidin-biotin labeling strategies and simplify the detection of sialic acid in cells and glycoproteins.
    377. 377
      Crisalli, P.; Kool, E. T. Water-Soluble Organocatalysts for Hydrazone and Oxime Formation. J. Org. Chem. 2013, 78 (3), 1184– 9,  DOI: 10.1021/jo302746p
      379
      Water-Soluble Organocatalysts for Hydrazone and Oxime Formation
      Crisalli, Pete; Kool, Eric T.
      Journal of Organic Chemistry (2013), 78 (3), 1184-1189CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      The formation of oximes and hydrazones is widely used in chem. and biol. as a mol. conjugation strategy for achieving ligation, attachment, and bioconjugation. However, the relatively slow rate of reaction has hindered its utility. Here, we report that simple, com. available anthranilic acids and aminobenzoic acids act as superior catalysts for hydrazone and oxime formation, speeding the reaction considerably over the traditional aniline-catalyzed reaction at neutral pH. This efficient nucleophilic catalysis, involving catalyst-imine intermediates, allows rapid hydrazone/oxime formation even with relatively low concns. of the two reactants. The most efficient catalysts are found to be 5-methoxyanthranilic acid and 3,5-diaminobenzoic acid; we find that they can enhance rates by factors of as much as 1-2 orders of magnitude over the aniline-catalyzed reaction. Evidence based on a range of differently substituted arylamines suggests that the ortho-carboxylate group in the anthranilate catalysts serves to aid in intramol. proton transfer during imine and hydrazone formation.
    378. 378
      Guo, H.-M.; Tanaka, F. A Fluorogenic Aldehyde Bearing a 1,2,3-Triazole Moiety for Monitoring the Progress of Aldol Reactions. J. Org. Chem. 2009, 74 (6), 2417– 24,  DOI: 10.1021/jo900013w
      380
      A Fluorogenic Aldehyde Bearing a 1,2,3-Triazole Moiety for Monitoring the Progress of Aldol Reactions
      Guo, Hai-Ming; Tanaka, Fujie
      Journal of Organic Chemistry (2009), 74 (6), 2417-2424CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      We have developed a new type of fluorogenic aldehyde bearing a 1,2,3-triazole moiety that is useful for monitoring the progress of aldol reactions through an increase in fluorescence. Whereas 6-methoxy-2-naphthaldehyde was highly fluorescent, the fluorogenic aldehyde, 4-formylbenzene connected to the 6-methoxy-2-naphthyl group through a 1,2,3-triazole moiety, was essentially nonfluorescent in aq. solns. We suggest that the 4-formylphenyl group acts as a quencher to suppress the fluorescence of the 6-methoxy-2-naphthyltriazole moiety. The product of the aldol reaction of this aldehyde does not have a quenching moiety and showed more than 800-fold higher fluorescence than the aldehyde. Assay systems using the fluorogenic aldehyde were validated by screening of aldol catalysts, ranking of the activities of the catalysts, and evaluation of reaction conditions.
    379. 379
      Tanaka, F.; Thayumanavan, R.; Barbas, C. F. Fluorescent Detection of Carbon-Carbon Bond Formation. J. Am. Chem. Soc. 2003, 125 (28), 8523– 8,  DOI: 10.1021/ja034069t
      381
      Fluorescent detection of carbon-carbon bond formation
      Tanaka, Fujie; Thayumanavan, Rajeswari; Barbas, Carlos F.
      Journal of the American Chemical Society (2003), 125 (28), 8523-8528CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We have developed a new spectroscopic system for detecting carbon-carbon bond formation by fluorescence to enhance high-throughput catalyst screening and rapid characterization of catalysts on a small scale. Fluorogenic substrates composed of a fluorophore possessing an amino group are readily prepd. as amides of α,β-unsatd. carbonyl compds. and generally exhibit low fluorescence, while Michael or Diels-Alder reactions of these fluorogenic substrates provide products of significantly increased fluorescence. The product's fluorescence is approx. 20- to 100-fold higher than that of the substrate. The assay system was validated by screening potential catalysts of the Michael reaction and in solvent optimization expts. The covalent combination of fluorophores possessing an amino group with α,β-unsatd. carbonyl compds. should provide a diverse range of fluorogenic substrates that may be used to rapidly screen catalysts and to optimize reaction conditions.
    380. 380
      Tanaka, F.; Mase, N.; Barbas, C. F. Design and Use of Fluorogenic Aldehydes for Monitoring the Progress of Aldehyde Transformations. J. Am. Chem. Soc. 2004, 126 (12), 3692– 3,  DOI: 10.1021/ja049641a
      382
      Design and Use of Fluorogenic Aldehydes for Monitoring the Progress of Aldehyde Transformations
      Tanaka, Fujie; Mase, Nobuyuki; Barbas, Carlos F., III
      Journal of the American Chemical Society (2004), 126 (12), 3692-3693CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We describe the first examples of fluorogenic aldehydes useful for monitoring many types of reactions including aldol reactions, allylations, and redns. The fluorogenic aldehydes were constructed by covalent combination of a fluorophore and an aldehyde moiety via a linker. In the resulting single mol., the aldehyde functioned as a quencher of the fluorophore's fluorescence. The reaction product, modified at the aldehyde functionality, no longer served as an effective quencher. The reaction products showed up to ∼80-fold higher fluorescence than the aldehyde reactants. The reaction of the aldolase antibody 38C-2-catalyzed aldol reaction of 4-formyl-N-(9-phenanthrenyl)benzenepropanamide (I) with acetone was monitored using a fluorescence assay method. The aldehydes described here are potentially useful the screening of catalysts in chem. libraries (no data).
    381. 381
      Linares-Pastén, J. A.; Chávez-Lizárraga, G.; Villagomez, R.; Mamo, G.; Hatti-Kaul, R. A Method for Rapid Screening of Ketone Biotransformations: Detection of Whole Cell Baeyer-Villiger Monooxygenase Activity. Enzyme Microb. Technol. 2012, 50 (2), 101– 6,  DOI: 10.1016/j.enzmictec.2011.10.004
      383
      A method for rapid screening of ketone biotransformations: Detection of whole cell Baeyer-Villiger monooxygenase activity
      Linares-Pasten, Javier A.; Chavez-Lizarraga, Georgina; Villagomez, Rodrigo; Mamo, Gashaw; Hatti-Kaul, Rajni
      Enzyme and Microbial Technology (2012), 50 (2), 101-106CODEN: EMTED2; ISSN:0141-0229. (Elsevier)
      A method for screening of ketone biotransformations was developed and applied to the identification of Baeyer-Villiger monooxygenase (BVMO) activity. The method was based on the formation of a purple colored product on reaction between an enolizable ketone and 3,5-dinitrobenzoic acid (DNB) in alk. soln. Absorbance of the color decreased with the size of the cycloketone ring. The stoichiometric ratio between cycloketone and DNB was 1:1 at max. absorbance. The method was applied for monitoring the consumption of cyclohexanone by bacteria under aerobic conditions, and was found to be potentially useful for both screening assays and quant. measurements of BVMO activity. Compared to other existing methods, this method was faster, less expensive, and amenable for whole cell assays.
    382. 382
      Georgiana Ileana, B.; Gabriel Lucian, R. Carboxylic Acid - Key Role in Life Sciences; IntechOpen, 2018.
      There is no corresponding record for this reference.
    383. 383
      Brune, K.; Patrignani, P. New Insights into the Use of Currently Available Non-Steroidal Anti-Inflammatory Drugs. J. Pain Res. 2015, 8, 105– 18,  DOI: 10.2147/JPR.S75160
      385
      New insights into the use of currently available non-steroidal anti-inflammatory drugs
      Brune Kay; Patrignani Paola
      Journal of pain research (2015), 8 (), 105-18 ISSN:1178-7090.
      Non-steroidal anti-inflammatory drugs (NSAIDs), which act via inhibition of the cyclooxygenase (COX) isozymes, were discovered more than 100 years ago. They remain a key component of the pharmacological management of acute and chronic pain. The COX-1 and COX-2 isozymes have different biological functions; analgesic activity is primarily (although not exclusively) associated with inhibition of COX-2, while different side effects result from the inhibition of COX-1 and COX-2. All available NSAIDs, including acetaminophen and aspirin, are associated with potential side effects, particularly gastrointestinal and cardiovascular effects, related to their relative selectivity for COX-1 and COX-2. Since all NSAIDs exert their therapeutic activity through inhibition of the COX isozymes, strategies are needed to reduce the risks associated with NSAIDs while achieving sufficient pain relief. A better understanding of the inhibitory activity and COX-1/COX-2 selectivity of an NSAID at therapeutic doses, based on pharmacokinetic and pharmacodynamic properties (eg, inhibitory dose, absorption, plasma versus tissue distribution, and elimination), and the impact on drug tolerability and safety can guide the selection of appropriate NSAIDs for pain management. For example, many NSAIDs with moderate to high selectivity for COX-2 versus COX-1 can be administered at doses that maximize efficacy (~80% inhibition of COX-2) while minimizing COX-1 inhibition and associated side effects, such as gastrointestinal toxicity. Acidic NSAIDs with favorable tissue distribution and short plasma half-lives can additionally be dosed to provide near-constant analgesia while minimizing plasma concentrations to permit recovery of COX-mediated prostaglandin production in the vascular wall and other organs. Each patient's clinical background, including gastrointestinal and cardiovascular risk factors, should be taken into account when selecting appropriate NSAIDs. New methods are emerging to assist clinicians in the selection of appropriate NSAIDs and their doses/schedules, such as biomarkers that may predict the response to NSAID treatment in individual patients.
    384. 384
      Murali, N.; Srinivas, K.; Ahring, B. K. Biochemical Production and Separation of Carboxylic Acids for Biorefinery Applications. Fermentation 2017, 3 (2), 22,  DOI: 10.3390/fermentation3020022
      386
      Biochemical production and separation of carboxylic acids for biorefinery applications
      Murali, Nanditha; Srinivas, Keerthi; Ahring, Birgitte K.
      Fermentation (2017), 3 (2), 22/1-22/25CODEN: FERMC4; ISSN:2311-5637. (MDPI AG)
      Carboxylic acids are traditionally produced from fossil fuels and have significant applications in the chem., pharmaceutical, food, and fuel industries. Significant progress has been made in replacing such fossil fuel sources used for prodn. of carboxylic acids with sustainable and renewable biomass resources. However, the merits and demerits of each carboxylic acid processing platform are dependent on the application of the final product in the industry. There are a no. of studies that indicate that sepn. processes account for over 30% of the total processing costs in such processes. This review focuses on the sustainable processing of biomass resources to produce carboxylic acids. The primary focus of the review will be on a discussion of and comparison between existing biochem. processes for producing lower-chain fatty acids such as acetic-, propionic-, butyric-, and lactic acids. The significance of these acids stems from the recent progress in catalytic upgrading to produce biofuels apart from the current applications of the carboxylic acids in the food, pharmaceutical, and plastics sectors. A significant part of the review will discuss current state-of-art of techniques for sepn. and purifn. of these acids from fermn. broths for further downstream processing to produce high-value products.
    385. 385
      Simmons, D. L.; Botting, R. M.; Hla, T. Cyclooxygenase Isozymes: The Biology of Prostaglandin Synthesis and Inhibition. Pharmacol. Rev. 2004, 56 (3), 387– 437,  DOI: 10.1124/pr.56.3.3
      387
      Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition
      Simmons, Daniel L.; Botting, Regina M.; Hla, Timothy
      Pharmacological Reviews (2004), 56 (3), 387-437CODEN: PAREAQ; ISSN:0031-6997. (American Society for Pharmacology and Experimental Therapeutics)
      A review. Nonsteroidal anti-inflammatory drugs (NSAIDs) represent one of the most highly utilized classes of pharmaceutical agents in medicine. All NSAIDs act through inhibiting prostaglandin synthesis, a catalytic activity possessed by two distinct cyclooxygenase (COX) isoenzymes encoded by sep. genes. The discovery of COX-2 launched a new era in NSAID pharmacol., resulting in the synthesis, marketing, and widespread use of COX-2 selective drugs. These pharmaceutical agents have quickly become established as important therapeutic medications with potentially fewer side effects than traditional NSAIDs. Addnl., characterization of the two COX isoenzymes is allowing the discrimination of the roles each play in physiol. processes such as homeostatic maintenance of the gastrointestinal tract, renal function, blood clotting, embryonic implantation, parturition, pain, and fever. Of particular importance has been the investigation of COX-1 and -2 isoenzymic functions in cancer, dysregulation of inflammation, and Alzheimer's disease. More recently, addnl. heterogeneity in COX-related proteins has been described, with the finding of variants of COX-1 and COX-2 enzymes. These variants may function in tissue-specific physiol. and pathophysiol. processes and may represent important new targets for drug therapy.
    386. 386
      Kumar, P.; Dubey, K. K. Citric Acid Cycle Regulation: Back Bone for Secondary Metabolite Production. In New and Future Developments in Microbial Biotechnology and Bioengineering; Elsevier: Amsterdam, 2019; Chapter 13, pp 165– 81.
      There is no corresponding record for this reference.
    387. 387
      Nakamura, M. T.; Yudell, B. E.; Loor, J. J. Regulation of Energy Metabolism by Long-Chain Fatty Acids. Prog. Lipid Res. 2014, 53, 124– 44,  DOI: 10.1016/j.plipres.2013.12.001
      389
      Regulation of energy metabolism by long-chain fatty acids
      Nakamura, Manabu T.; Yudell, Barbara E.; Loor, Juan J.
      Progress in Lipid Research (2014), 53 (), 124-144CODEN: PLIRDW; ISSN:0163-7827. (Elsevier Ltd.)
      A review. In mammals, excess energy is stored primarily as triglycerides, which are mobilized when energy demands arise. This review mainly focuses on the role of long chain fatty acids (LCFAs) in regulating energy metab. as ligands of peroxisome proliferator-activated receptors (PPARs). PPAR-alpha expressed primarily in liver is essential for metabolic adaptation to starvation by inducing genes for beta-oxidn. and ketogenesis and by downregulating energy expenditure through fibroblast growth factor 21. PPAR-delta is highly expressed in skeletal muscle and induces genes for LCFA oxidn. during fasting and endurance exercise. PPAR-delta also regulates glucose metab. and mitochondrial biogenesis by inducing FOXO1 and PGC1-alpha. Genes targeted by PPAR-gamma in adipocytes suggest that PPAR-gamma senses incoming non-esterified LCFAs and induces the pathways to store LCFAs as triglycerides. Adiponectin, another important target of PPAR-gamma may act as a spacer between adipocytes to maintain their metabolic activity and insulin sensitivity. Another topic of this review is effects of skin LCFAs on energy metab. Specific LCFAs are required for the synthesis of skin lipids, which are essential for water barrier and thermal insulation functions of the skin. Disturbance of skin lipid metab. often causes apparent resistance to developing obesity at the expense of normal skin function.
    388. 388
      Barrett, G. C.; Elmore, D. T. Amino Acids and Peptides; Cambridge University Press: Cambridge, 1998 DOI: 10.1017/CBO9781139163828 .
      There is no corresponding record for this reference.
    389. 389
      Ogliaruso, M. A.; Wolfe, J. F. Synthesis of Carboxylic Acids. Esters and Their Derivatives; Wiley Online Library, 1979; Vol. 267 DOI: 10.1002/9780470771587.ch7 .
      There is no corresponding record for this reference.
    390. 390
      Wang, H.; Fan, H.; Sun, H.; Zhao, L.; Wei, D. Process Development for the Production of (R)-(−)-Mandelic Acid by Recombinant Escherichia Coli Cells Harboring Nitrilase from Burkholderia Cenocepacia J2315. Org. Process Res. Dev. 2015, 19 (12), 2012– 6,  DOI: 10.1021/acs.oprd.5b00269
      392
      Process Development for the Production of (R)-(-)-Mandelic Acid by Recombinant Escherichia coli Cells Harboring Nitrilase from Burkholderia cenocepacia J2315
      Wang, Hualei; Fan, Haiyang; Sun, Huihui; Zhao, Li; Wei, Dongzhi
      Organic Process Research & Development (2015), 19 (12), 2012-2016CODEN: OPRDFK; ISSN:1083-6160. (American Chemical Society)
      (R)-(-)-Mandelic acid is an important chiral building block that is widely used in pharmacy and the prodn. of fine chems. A more advanced method for obtaining (R)-(-)-mandelic acid is direct hydrolysis of the corresponding racemic mandelonitrile. In order to develop a cost-effective process, a highly efficient enantioselective nitrilase BCJ2315 from Burkholderia cenocepacia J2315 was used for the biotransformation of mandelonitrile to (R)-(-)-mandelic acid. The recombinant Escherichia coli M15/BCJ2315 showed high substrate tolerance and could completely hydrolyze up to 250 mM of mandelonitrile. A fed-batch reaction was performed by periodically or continuously dosing the substrate into the reactor to alleviate substrate inhibition in a monophasic buffer system. Finally, the highest substrate loading (2.9 M) was achieved in the continuous fed batch reaction mode, giving (R)-(-)-mandelic acid at the highest concn. (2.3 M, 350 g/L) with 97.4% ee ever reported. The hydrolysis process was easily scaled up to 2 and 10 L, indicating the potential for the industrial prodn. of optically pure (R)-(-)-mandelic acid.
    391. 391
      Martínková, L.; Rucká, L.; Nešvera, J.; Pátek, M. Recent Advances and Challenges in the Heterologous Production of Microbial Nitrilases for Biocatalytic Applications. World J. Microbiol. Biotechnol. 2017, 33, 8,  DOI: 10.1007/s11274-016-2173-6
      393
      Recent advances and challenges in the heterologous production of microbial nitrilases for biocatalytic applications
      Martinkova Ludmila; Rucka Lenka; Nesvera Jan; Patek Miroslav
      World journal of microbiology & biotechnology (2017), 33 (1), 8 ISSN:.
      The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of nitrilases. By exploiting this resource, the number of experimentally verified nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of nitrilases are classified according to their target products (α-hydroxy acids, α- and β-amino acids, cyano acids, amides). The emerging uses of nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications.
    392. 392
      Xu, Z.; Xiong, N.; Zou, S.-P.; Liu, Y.-X.; liu, Z.-Q.; Xue, Y.-P.; Zheng, Y.-G. Highly Efficient Conversion of 1-Cyanocycloalkaneacetonitrile Using a “Super Nitrilase Mutant”. Bioprocess Biosyst. Eng. 2019, 42 (3), 455– 63,  DOI: 10.1007/s00449-018-2049-2
      394
      Highly efficient conversion of 1-cyanocycloalkaneacetonitrile using a "super nitrilase mutant"
      Xu, Zhe; Xiong, Neng; Zou, Shu-Ping; Liu, Yu-Xiao; liu, Zhi-Qiang; Xue, Ya-Ping; Zheng, Yu-Guo
      Bioprocess and Biosystems Engineering (2019), 42 (3), 455-463CODEN: BBEIBV; ISSN:1615-7591. (Springer)
      Nitrilase is the member of carbon-nitrogen hydrogen hydrolase superfamily, which has been widely used for the hydrolysis of nitriles into corresponding carboxylic acids. But most nitrilases are plagued by product inhibition in the industrial application. In this study, a "super nitrilase mutant" of nitrilase with high activity, thermostability and improved product tolerance from Acidovorax facilis ZJB09122 was characterized. Then, an efficient process was developed by employing the whole cell of recombinant E. coli for the conversion of high concn. of 1-cyanocyclohexylacetonitrile-to-1-cyanocyclohexaneacetic acid, an important intermediate of gabapentin. Under the optimized conditions, the higher substrate concns. such as 1.3 M, 1.5 M and 1.8 M could be hydrolyzed by 13.58 g DCW/L with outstanding productivity (> 740 g/L/day). This study developed a highly efficient bioprocess for the prepn. of 1-cyanocyclohexaneacetic acid which has the great potential for industrial application.
    393. 393
      Glieder, A.; Weis, R.; Skranc, W.; Poechlauer, P.; Dreveny, I.; Majer, S.; Wubbolts, M.; Schwab, H.; Gruber, K. Comprehensive Step-by-Step Engineering of an (R)-Hydroxynitrile Lyase for Large-Scale Asymmetric Synthesis. Angew. Chem., Int. Ed. 2003, 42 (39), 4815– 8,  DOI: 10.1002/anie.200352141
      395
      Comprehensive step-by-step engineering of an (R)-hydroxynitrile lyase for large-scale asymmetric synthesis
      Glieder, Anton; Weis, Roland; Skranc, Wolfgang; Poechlauer, Peter; Dreveny, Ingrid; Majer, Sandra; Wubbolts, Marcel; Schwab, Helmut; Gruber, Karl
      Angewandte Chemie, International Edition (2003), 42 (39), 4815-4818CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The goal of the study was to develop a com. viable (R)-hydroxynitrile lyase to catalyze the enantioselective conversion of 2-chlorobenzaldehyde to (R)-2-chloromandeleonitrile under process conditions with the ultimate goal being prodn. of (R)-2-chloromandelic acid.. The approach taken was to clone the (R)-hydroxynitrile lyase gene from Prunus amygdalus and express it in Pichia pastoris. The recombinant enzyme which was highly glycosylated had twice the specific activity of the native enzyme and demonstrated a high degree of stability at low pH. The enzyme prodn. was increased by replacing the N-terminal leucine with a glutamine and replacing the native plant leader sequence with an α-mating factor leader sequence from Saccharomyces cerevisiae. Site-directed mutagenesis was then employed to minimize steric hindrance in the enzyme active site by replacing alanine 111 or valine 317 with glycine residues. Both mutant enzymes had dramatically lower specific activities for nitrile hydrolysis, but the A111G variant retained a high hydrocyanating activity. Addnl. the turnover frequency of the A111G variant was six times faster than the native enzyme. The results demonstrate the rational application of genetic engineering can significantly improve the properties of enzyme catalysts.
    394. 394
      Jiang, M.; Liu, Y.-J.; Liu, G.-S.; Zhou, W.; Shen, Y.-L.; Gao, B.; Wang, F.-Q.; Wei, D.-Z. Sequential Resolution of (S) and (R)-6-Fluoro-Chroman-2-Carboxylic Acid by Two Esterases in Turn. Green Chem. 2022, 24 (8), 3235– 42,  DOI: 10.1039/D1GC04512A
      396
      Sequential resolution of (S) and (R)-6-fluoro-chroman-2-carboxylic acid by two esterases in turn
      Jiang, Min; Liu, Yong-Jun; Liu, Guo-Song; Zhou, Wei; Shen, Ya-Ling; Gao, Bei; Wang, Feng-Qing; Wei, Dong-Zhi
      Green Chemistry (2022), 24 (8), 3235-3242CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
      Optically pure 6-fluoro-chroman-2-carboxylic acids (FCCAs), with (S) and (R)-configurations, are pivotal chiral building blocks in the pharmaceutical industry. To date, the prodn. of (S) and (R)-FCCAs mainly depends on chem. resoln., which is a complex, low yield, and highly polluting process. Here, we present a practical enzymic resoln. method of FCCAs based on two esterases, EstS and EstR, isolated from Geobacillus thermocatenulatus. Using the racemic Me 6-fluoro-chroman-2-carboxylate (MFCC) as the substrate in an aq.-toluene biphasic system, (S) and (R)-FCCAs were produced by EstS and EstR catalysis with an enantiomeric excess (ee) value >99% and 95-96%, resp., and the highly enantioselective mechanisms were revealed by mol. simulations. To simplify the resoln. process and enhance the productivity, we further designed an innovative methodol. for the "sequential biphasic batch resoln." of MFCC with immobilized cells. In each batch, only the aq. phase needs to be replaced to sequentially change the immobilized cells of EstS or EstR and recover optically pure FCCAs in turn, while the org. phase was retained and MFCC was continually supplemented after every two batches. In this study, ten batches of sequential resoln. were performed, and 229.3 mM (S)-FCCAs with 96.9% ee, and 224.1 mM (R)-FCCAs with 99.1% ee were obtained in 40 h, affording a 93.5% total mole yield. This is the first reported enzymic resoln. technique of FCCAs, and represents significant advantages over those chem. resoln. methods.
    395. 395
      Dijkman, W. P.; Groothuis, D. E.; Fraaije, M. W. Enzyme-Catalyzed Oxidation of 5-Hydroxymethylfurfural to Furan-2,5-Dicarboxylic Acid. Angew. Chem., Int. Ed. 2014, 53 (25), 6515– 8,  DOI: 10.1002/anie.201402904
      397
      Enzyme-Catalyzed Oxidation of 5-Hydroxymethylfurfural to Furan-2,5-dicarboxylic Acid
      Dijkman, Willem P.; Groothuis, Daphne E.; Fraaije, Marco W.
      Angewandte Chemie, International Edition (2014), 53 (25), 6515-6518CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Furan-2,5-dicarboxylic acid (FDCA) is a biobased platform chem. for the prodn. of polymers. In the past few years, numerous multistep chem. routes have been reported on the synthesis of FDCA by oxidn. of 5-hydroxymethylfurfural (HMF). Recently we identified an FAD-dependent enzyme which is active towards HMF and related compds. This oxidase has the remarkable capability of oxidizing [5-(hydroxymethyl)furan-2-yl]methanol to FDCA, a reaction involving four consecutive oxidns. The oxidase can produce FDCA from HMF with high yield at ambient temp. and pressure. Examn. of the underlying mechanism shows that the oxidase acts on alc. groups only and depends on the hydration of aldehydes for the oxidn. reaction required to form FDCA.
    396. 396
      DeSantis, G.; Wong, K.; Farwell, B.; Chatman, K.; Zhu, Z.; Tomlinson, G.; Huang, H.; Tan, X.; Bibbs, L.; Chen, P. Creation of a Productive, Highly Enantioselective Nitrilase through Gene Site Saturation Mutagenesis (GSSM). J. Am. Chem. Soc. 2003, 125 (38), 11476– 7,  DOI: 10.1021/ja035742h
      398
      Creation of a Productive, Highly Enantioselective Nitrilase through Gene Site Saturation Mutagenesis (GSSM)
      DeSantis, Grace; Wong, Kelvin; Farwell, Bob; Chatman, Kelly; Zhu, Zoulin; Tomlinson, Geoff; Huang, Hongjun; Tan, Xuqiu; Bibbs, Lisa; Chen, Pei; Kretz, Keith; Burk, Mark J.
      Journal of the American Chemical Society (2003), 125 (38), 11476-11477CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Gene site satn. mutagenesis (GSSM) technol. is applied for the directed evolution of a nitrilase. The nitrilase effectively catalyzes the desymmetrization of the prochiral substrate 3-hydroxyglutaronitrile to afford (R)-4-cyano-3-hydroxybutyric acid, a precursor to the valuable cholesterol-lowering drug Lipitor. The discovered wild-type enzyme effectively performs the reaction at the industrially relevant 3 M substrate concn. but affords a product enantiomeric excess of only 87.6% ee. Through GSSM, a mutagenesis technique that effects the combinatorial satn. of each amino acid in the protein to each of the other 19 amino acids, combined with a novel high-throughput mass spectroscopy assay, a no. of improved variants were identified, the best of which is the Ala190His mutant that yields product enantiomeric excess of 98.5% at 3 M substrate loading and a volumetric productivity of 619 g L-1 d-1.
    397. 397
      Franz, R. G. Comparisons of pKa and Log P Values of Some Carboxylic and Phosphonic Acids: Synthesis and Measurement. AAPS PharmSci. 2001, 3 (2), 1,  DOI: 10.1208/ps030210
      There is no corresponding record for this reference.
    398. 398
      Toth, A. M.; Liptak, M. D.; Phillips, D. L.; Shields, G. C. Accurate Relative Pka Calculations for Carboxylic Acids Using Complete Basis Set and Gaussian-N Models Combined with Continuum Solvation Methods. J. Chem. Phys. 2001, 114 (10), 4595– 606,  DOI: 10.1063/1.1337862
      400
      Accurate relative pKa calculations for carboxylic acids using complete basis set and Gaussian-n models combined with continuum solvation methods
      Toth, Ann Marie; Liptak, Matthew D.; Phillips, Danielle L.; Shields, George C.
      Journal of Chemical Physics (2001), 114 (10), 4595-4606CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      The complete basis set methods CBS-4, CBS-QB3, and CBS-APNO, and the Gaussian methods G2 and G3 were used to calc. the gas phase energy differences between six different carboxylic acids and their resp. anions. Two different continuum methods, SM5.42R and CPCM, were used to calc. the free energy differences of solvation for the acids and their anions. Relative pKa values were calcd. for each acid using one of the acids as a ref. point. The CBS-QB3 and CBS-APNO gas phase calcns., combined with the CPCM/HF/6-31+G(d)//HF/6-31G(d) or CPCM/HF/6-31+G(d)//HF/6-31+G(d) continuum solvation calcns. on the lowest energy gas phase conformer, and with the conformationally averaged values, give results accurate to (1)/(2) pKa unit.
    399. 399
      Vollhardt, K. P. C. Organische Chemie, 3rd ed.; Wiley-VCH: Weinheim, 2000.
      There is no corresponding record for this reference.
    400. 400
      Bruice, P. Y. Organic Chemistry, 5 ed.; Pearson Prentice Hall: Upper Saddle River, NJ, 2007.
      There is no corresponding record for this reference.
    401. 401
      Descalzo, A. B.; Rurack, K.; Weisshoff, H.; Martínez-Máñez, R.; Marcos, M. D.; Amorós, P.; Hoffmann, K.; Soto, J. Rational Design of a Chromo- and Fluorogenic Hybrid Chemosensor Material for the Detection of Long-Chain Carboxylates. J. Am. Chem. Soc. 2005, 127 (1), 184– 200,  DOI: 10.1021/ja045683n
      403
      Rational Design of a Chromo- and Fluorogenic Hybrid Chemosensor Material for the Detection of Long-Chain Carboxylates
      Descalzo, Ana B.; Rurack, Knut; Weisshoff, Hardy; Martinez-Manez, Ramon; Marcos, M. Dolores; Amoros, Pedro; Hoffmann, Katrin; Soto, Juan
      Journal of the American Chemical Society (2005), 127 (1), 184-200CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A strategy for the rational design of a new optical sensor material for the selective recognition of long-chain carboxylates in water is presented. The approach relies on the combination of structure-property relationships to single out the optimal mol. sensor unit and the tuning of the sensing characteristics of an inorg. support material. A spacer-substituted 7-urea-phenoxazin-3-one was employed as the signaling moiety and a mesoporous trimethylsilylated UVM-7 (MCM-41 type) material served as the solid support. The sensor material shows the advantageous features of both modules that is absorption and emission in the visible spectral range, a fluorescence red-shift and enhancement upon analyte coordination, and the amplification of noncovalent (binding) and hydrogen-bonding (recognition) interactions in the detection event. Besides these basic results that are related to the design and performance of the sensor material, the paper discusses general aspects of amido-substituted phenoxazinone photophysics and addresses some general features of mol. anion recognition chem. in aq. vs nonaq. media, utilizing steady-state and time-resolved optical as well as NMR spectroscopies. Detailed studies on potentially competing biochem. species and a first access to the schematic model of the response of the sensor material as obtained by a combination of fluorescence lifetime distribution anal. and Langmuir-type fitting of the gross binding consts. complement the key issues of the paper.
    402. 402
      Reddy G, U.; Lo, R.; Roy, S.; Banerjee, T.; Ganguly, B.; Das, A. A New Receptor with a FRET Based Fluorescence Response for Selective Recognition of Fumaric and Maleic Acids in Aqueous Medium. Chem. Commun. 2013, 49 (84), 9818,  DOI: 10.1039/c3cc45051a
      There is no corresponding record for this reference.
    403. 403
      Fitzmaurice, R. J.; Kyne, G. M.; Douheret, D.; Kilburn, J. D. Synthetic Receptors for Carboxylic Acids and Carboxylates. J. Chem. Soc., Perkin Trans. 1 2002, (7), 841– 64,  DOI: 10.1039/b009041g
      405
      Synthetic receptors for carboxylic acids and carboxylates
      Fitzmaurice, Richard J.; Kyne, Graham M.; Douheret, David; Kilburn, Jeremy D.
      Journal of the Chemical Society, Perkin Transactions 1 (2002), (7), 841-864CODEN: JCSPCE; ISSN:1472-7781. (Royal Society of Chemistry)
      A review with refs. up to and including June 2001 on the development of synthetic receptors for substrate (host-guest chem.) with carboxylic acids or carboxylates as functional groups.
    404. 404
      Janes, L. E.; Löwendahl, A. C.; Kazlauskas, R. J. Quantitative Screening of Hydrolase Libraries Using pH Indicators: Identifying Active and Enantioselective Hydrolases. Eur. J. Chem. 1998, 4 (11), 2324– 31,  DOI: 10.1002/(SICI)1521-3765(19981102)4:11<2324::AID-CHEM2324>3.0.CO;2-I
      406
      Quantitative screening of hydrolase libraries using pH indicators: identifying active and enantioselective hydrolases
      Janes, Lana E.; Lowendahl, A. Christina; Kazlauskas, Romas J.
      Chemistry - A European Journal (1998), 4 (11), 2324-2331CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH)
      The slowest step in finding a selective hydrolase for synthesis is often the screening step. Researchers must run small test reactions and measure the amts. of stereoisomers formed by HPLC, GC, or NMR. We have developed a colorimetric method to speed up this screening. We quant. detect ester hydrolysis using a pH indicator, 4-nitrophenol. We est. the selectivity by measuring the initial rates of hydrolysis for pure stereoisomers sep. To demonstrate the utility of this method, we screened seventy-two com. enzymes for enantioselective hydrolysis of racemic solketal butyrate, an important chiral building block. First, we eliminated the twenty hydrolases that did not catalyze hydrolysis of either enantiomer. Next, we measured initial rates of hydrolysis of the pure enantiomers of solketal butyrate. For horse-liver esterase, these initial rates differed by a factor of twelve. Subsequent GC expts. confirmed an enantiomeric ratio of fifteen for this hydrolase. Although this enantioselectivity is moderate, it is the highest enantioselectivity reported for a hydrolysis of solketal esters.
    405. 405
      Baumann, M.; Stürmer, R.; Bornscheuer, U. T. A High-Throughput-Screening Method for the Identification of Active and Enantioselective Hydrolases. Angew. Chem., Int. Ed. 2001, 40 (22), 4201– 4,  DOI: 10.1002/1521-3773(20011119)40:22<4201::AID-ANIE4201>3.0.CO;2-V
      407
      A high-throughput-screening method for the identification of active and enantioselective hydrolases
      Baumann, Markus; Sturmer, Rainer; Bornscheuer, Uwe T.
      Angewandte Chemie, International Edition (2001), 40 (22), 4201-4204CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
      A new assay system format for screening enantioselective hydrolases, based on a coupled enzymic conversion, is described. To evaluate this assay format, the hydrolysis of (R,S)-α-Ph-Et acetate by a recombinant esterase from Pseudomonas fluorescens was chosen as a model reaction. The applicability of the assay format for the detn. of enantioselectivity values was investigated using 16 lipases and esterases. Using this format, an anal. time of less than 3-4 min per assay was found to be sufficient to det. enantioselectivities. In addn., the assay was found to be rather cheap.
    406. 406
      Wang, J.; Liu, H.-B.; Tong, Z.; Ha, C.-S. Fluorescent/Luminescent Detection of Natural Amino Acids by Organometallic Systems. Coord. Chem. Rev. 2015, 303, 139– 84,  DOI: 10.1016/j.ccr.2015.05.008
      408
      Fluorescent/luminescent detection of natural amino acids by organometallic systems
      Wang, Jing; Liu, Hai-Bo; Tong, Zhangfa; Ha, Chang-Sik
      Coordination Chemistry Reviews (2015), 303 (), 139-184CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
      In comparison with other detection technologies, fluorescence/luminescence technol. has become a powerful tool owing to its advantageous features including simplicity, low cost, high sensitivity, quick response time, easy sample prepn., noninvasive and nondestructive nature, etc. Due to the important roles played by 20 natural amino acids in living systems, this review focuses on recent contributions (from the year 2000 until July 2014) regarding the development of fluorescent/luminescent chemosensors and chemodosimeters to detect specific AAs, as well as chiral recognition to discriminate AA enantiomers (i.e., D and L), and pattern recognition to distinguish a range of AAs simultaneously based on fluorescent/luminescent organometallic systems, which include org.-metal complexes and hybrid org.-metal nanoparticles/nanoclusters.
    407. 407
      Gong, R.; Mu, H.; Sun, Y.; Fang, X.; Xue, P.; Fu, E. The First Fluorescent Sensor for Medium-Chain Fatty Acids in Water: Design, Synthesis and Sensing Properties of an Organic-Inorganic Hybrid Material. J. Mater. Chem. B 2013, 1 (15), 2038– 47,  DOI: 10.1039/c3tb00355h
      409
      The first fluorescent sensor for medium-chain fatty acids in water: design, synthesis and sensing properties of an organic-inorganic hybrid material
      Gong, Rui; Mu, Honglei; Sun, Yimin; Fang, Xiantao; Xue, Peng; Fu, Enqin
      Journal of Materials Chemistry B: Materials for Biology and Medicine (2013), 1 (15), 2038-2047CODEN: JMCBDV; ISSN:2050-7518. (Royal Society of Chemistry)
      This paper reports the first fluorescent sensor for medium-chain fatty acids in water. The hybrid sensing material (S2) was prepd. by the reaction of a rationally designed "recognition center", N-butyl-4-piperazin-1,8-naphthalimide, with a 3-glycidoxypropyl group which is pre-grafted on the interior of a mesoporous solid (MCM-41). The organo-functionalization of the mesoporous solid (MCM-41) was confirmed by FTIR, 29Si MAS NMR and elemental anal. The results of XRD, N2 phys. adsorption-desorption, SEM and TEM studies proved that the organized structure of the nanoscopic porous solid is preserved after the reactions. The fatty acid-selective signaling behavior of S2 was studied in water at pH = 5.80/7.16/8.00. This material displays much stronger fluorescence enhancement with C8-C12 fatty acids than with the shorter and longer chains in aq. media, both in the absence and in the presence of arom. acids.
    408. 408
      Kusukawa, T.; Tanaka, S.; Inoue, K. Fluorescent Detection of Amidinium-Carboxylate and Amidinium Formation Using a 1,8-Diphenylnaphthalene-Based Diamidine: Dicarboxylic Acid Recognition with High Fluorescence Efficiency. Tetrahedron 2014, 70 (26), 4049– 56,  DOI: 10.1016/j.tet.2014.03.036
      410
      Fluorescent detection of amidinium-carboxylate and amidinium formation using a 1,8-diphenylnaphthalene-based diamidine: dicarboxylic acid recognition with high fluorescence efficiency
      Kusukawa, Takahiro; Tanaka, Syugo; Inoue, Kouta
      Tetrahedron (2014), 70 (26), 4049-4056CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)
      A 1,8-diphenylnaphthalene-based diamidine (I) 'turn-on' fluorescent probe for the detection of dicarboxylic acids was designed and synthesized. The fluorescence spectra of the diamidine I with carboxylic acids that showed two different fluorescence bands, which corresponded to the amidinium-carboxylate (λem=410-430 nm) and amidinium (λem=440-470 nm as a broad band, which consisted from two peaks) formation, were confirmed by DOSY NMR and TD-DFT calcns. The complexation of diamidine I with dicarboxylic acids, which have sufficient distances between the two carboxylic groups for binding to the diamidine I (3-adamantanediacetic acid, m-phenylenediacetic acid, and α,ω-dicarboxylic acids (C6-C20)), showed the formation of 1:1 complexes (i.e., amidinium-carboxylate formation). However, for the complexation with monocarboxylic acids and dicarboxylic acids having insufficient distances between the two carboxylic groups (benzoic acid, acetic acid, and α,ω-dicarboxylic acids (C3-C5)), formation of the amidinium (1·2H+) was obsd. Relatively similar binding consts. (10-5) for the complexation of the diamidine 1 with dicarboxylic acids 6, which depend on their chain length (strain), were obsd. due to the flexibility of the 1,8-diphenylnaphthalene unit. Addnl., for the complexation of the diamidine 1 with dicarboxylic acids, higher fluorescence quantum yields (Φfl: up to 80%) were obsd. when compared to the binding of the diamidine 2 (Φfl: up to 35%).
    409. 409
      Boiocchi, M.; Bonizzoni, M.; Fabbrizzi, L.; Piovani, G.; Taglietti, A. A Dimetallic Cage with a Long Ellipsoidal Cavity for the Fluorescent Detection of Dicarboxylate Anions in Water. Angew. Chem., Int. Ed. 2004, 43 (29), 3847– 52,  DOI: 10.1002/anie.200460036
      411
      A dimetallic cage with a long ellipsoidal cavity for the fluorescent detection of dicarboxylate anions in water
      Boiocchi, Massimo; Bonizzoni, Marco; Fabbrizzi, Luigi; Piovani, Giulio; Taglietti, Angelo
      Angewandte Chemie, International Edition (2004), 43 (29), 3847-3852CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The dicopper(II) complex of a bistren cage contg. ditolyl spacers (I) binds dicarboxylate anions in aq. solns. The inclusion is highly selective from the distance between the COO- groups. For example, terephthalate is discriminated from isophthalate and phthalate.
    410. 410
      Metzger, A.; Lynch, V. M.; Anslyn, E. V. A Synthetic Receptor Selective for Citrate. Angew. Chem., Int. Ed. 1997, 36 (8), 862– 5,  DOI: 10.1002/anie.199708621
      412
      A synthetic receptor selective for citrate
      Metzger, Axel; Lynch, Vincent M.; Anslyn, Eric V.
      Angewandte Chemie, International Edition in English (1997), 36 (8), 862-865CODEN: ACIEAY; ISSN:0570-0833. (VCH)
      A synthetic receptor bearing guanidinium groups embedded in aminodihydroimidazolium groups was prepd. and shown to be a selective receptor for citrate.
    411. 411
      Yang, X.; Liu, X.; Shen, K.; Zhu, C.; Cheng, Y. A Chiral Perazamacrocyclic Fluorescent Sensor for Cascade Recognition of Cu(II) and the Unmodified α-Amino Acids in Protic Solutions. Org. Lett. 2011, 13 (13), 3510– 3,  DOI: 10.1021/ol2013268
      413
      A Chiral Perazamacrocyclic Fluorescent Sensor for Cascade Recognition of Cu(II) and the Unmodified α-Amino Acids in Protic Solutions
      Yang, Xia; Liu, Xuechao; Shen, Kang; Zhu, Chengjian; Cheng, Yixiang
      Organic Letters (2011), 13 (13), 3510-3513CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
      A novel chiral perazamacrocyclic fluorescent sensor (I) was designed and synthesized. It can serve as a fluorescent turn-off sensor with high selectivity toward Cu(II) among 14 metal ions. Also, though I exhibits no enantioselectivity, after adding Cu(II), the in situ generated Cu(II)-contg. complex of I (Cu(II)-I) can exhibit remarkable fluorescent enhancement responses and considerable enantioselectivities toward unmodified α-amino acids in protic solns. via a ligand displacement mechanism; i.e. a cascade recognition of Cu(II) and unmodified α-amino acids was achieved.
    412. 412
      Görög, S.; Laukó, A.; Rényei, M.; Hegedüs, B. New Derivatization Reactions in Pharmaceutical Analysis. J. Pharm. Biomed. Anal. 1983, 1 (4), 497– 506,  DOI: 10.1016/0731-7085(83)80063-6
      414
      New derivatization reactions in pharmaceutical analysis
      Gorog, S.; Lauko, A.; Renyei, M.; Hegedus, B.
      Journal of Pharmaceutical and Biomedical Analysis (1983), 1 (4), 497-506CODEN: JPBADA; ISSN:0731-7085.
      A review with 34 refs. The use of chem. reactions in conjunction with spectrometric and chromatog. methods is exemplified by the formation of 2-nitrophenylhydrazide derivs. for the spectrometric detn. of carboxylic acids; silylation and trifluoroacetylation of drugs for gas chromatog.; selective redn., of steroid ketones for IR spectrometric identification; the use of epoxidn. in discriminating between satd. and unsatd. steroids in gas chromatog.; increasing the selectivity and sensitivity of the spectrometric and gas-chromatog. detn. of isomeric Δ4- and Δ5-3-ethylenedioxy steroids by treatment with HCl; and the use of the same reagent in the difference spectrometric detn. of 2,5-dimethyl-α-ethylbenzhydrol.
    413. 413
      Munson, J. W.; Bilous, R. Colorimetric Determination of Aliphatic Acids. J. Pharm. Sci. 1977, 66 (10), 1403– 5,  DOI: 10.1002/jps.2600661013
      415
      Colorimetric determination of aliphatic acids
      Munson, James W.; Bilous, Roman
      Journal of Pharmaceutical Sciences (1977), 66 (10), 1403-5CODEN: JPMSAE; ISSN:0022-3549.
      A colorimetric method for the detn. of carboxylic acids based on the dicyclohexylcarbodiimide-coupled reaction of 2-nitrophenylhydrazine and carboxylic acids is described. The product of the reaction was extd. into aq. NaOH to produce a blue color. This method is suitable for the anal. of aliph. acids. Arom. acids did not react under these conditions.
    414. 414
      Miwa, H.; Yamamoto, M.; Momose, T. Colorimetric Detection and Determination of Carboxylic Acids with 2-Nitrophenylhydrazine Hydrochloride. Chem. Pharm. Bull. (Tokyo) 1980, 28 (2), 599– 605,  DOI: 10.1248/cpb.28.599
      There is no corresponding record for this reference.
    415. 415
      Hill, U. T. Colorimetric Determination of Fatty Acids and Esters. Ind. Eng. Chem. Anal. Ed. 1946, 18 (5), 317– 9,  DOI: 10.1021/i560153a017
      417
      Colorimetric determination of fatty acids and esters
      Hill, Uno T.
      Industrial and Engineering Chemistry, Analytical Edition (1946), 18 (), 317-19CODEN: IENAAD; ISSN:0096-4484.
      Fatty acids and esters applied to a metal surface for rust prevention, lubrication, etc., can be rapidly extd., converted to their hydroxamic acids and made to react with ferric ions (cf. Feigl, C.A. 38, 315.4) to form a red or lavender complex (Fe[R.CO(NHO)]3) which follows Beer's law if ferric perchlorate in perchloric acid is used. Percentage transmittancy is detd. at 520 mμ with a spectrophotometer, compared with a blank and the amt. of ester present obtained by reference to a previously prepd. ester vs. transmittancy curve. Acids do not form a hydroxamic acid unless first esterified. Complete directions are given with reagent quantities specific for 0.05 to 1 mg. of extractant, but larger quantities can be used. Comparison with a gravimetric method is good; cottonseed oil is detd. within ±0.01 mg. Transmittancy values can be plotted in terms of the sapon., acid, ester, or acetyl values; curves are given for solns. of ferric hydroxamates from dibutyl sebacate, and cottonseed, palm, sulfurized sperm, and lanolin-base oils.
    416. 416
      Montgomery, H. A. C.; Dymock, J. F.; Thom, N. S. The Rapid Colorimetric Determination of Organic Acids and Their Salts in Sewage-Sludge Liquor. Analyst 1962, 87 (1041), 949– 55,  DOI: 10.1039/an9628700949
      418
      The rapid colorimetric determination of organic acids and their salts in sewage-sludge liquor
      Montgomery, H. A. C.; Dymock, Joan F.; Thom, N. S.
      (1962), 87 (), 949-55 ISSN:.
      The colorimetric composite detn. of the caxboxylic acids and their salts in sewage-sludge liquor is described. After removal of suspended matter by filtration with the assistance of filter aid, a 0.5-ml. sample is treated with ethylene glycol in the presence of H2SO4, and the resulting mixt. of esters is detd. colorimetrically at 500 mμ, with 4-cm. cells, by the ferric hydroxamate reaction. For HOAc, the calibration is linear to at least 1%. Since the most intense colors are given by the lower fatty acids, which predominate in sludge liquor, the method gives results similar to existing methods for the detn. of volatile acids. The procedure was applied to the detn. of org. acids in an effluent from a bacon factory.
    417. 417
      Kasai, Y.; Tanimura, T.; Tamura, Z. J. A. C. Spectrophotometric Determination of Carboxylic Acids by the Formation of Hydroxamic Acids with Dicyclohexylcarbodiimide. Anal. Chem. 1975, 47 (1), 34– 7,  DOI: 10.1021/ac60351a047
      419
      Spectrophotometric determination of carboxylic acids by the formation of hydroxamic acids with dicyclohexylcarbodiimide
      Kasai, Yasuhiko; Tanimura, Takenori; Tamura, Zenzo
      Analytical Chemistry (1975), 47 (1), 34-7CODEN: ANCHAM; ISSN:0003-2700.
      A new method of spectrophotometric detn. of carboxylic acids was developed. The unique feature of the method is the formation, in a single step, of hydroxamate from carboxylic acid and NH2OH by using dicyclohexylcarbodi-imide (DCC). Hydroxamate was prepd. by adding DCC to the mixt. of carboxylic acid and NH2OH in anhyd. or aq. EtOH at 50 or 60°. Ferric hydroxamate was prepd. with large excess of Fe(ClO4)3 in EtOH soln. of HClO4 and was detd. at 525 nm. The working curves obtained were linear for 0.25-2.5 μmoles benzoic, caprylic, lactic, and p-nitrophenylacetic acid.
    418. 418
      Takeuchi, T.; Horikawa, R.; Tanimura, T. Spectrophotometry Determination of Carboxylic Acids by Ferric Hydroxamate Formation with Water-Soluble Carbodiimide. Anal. Lett. 1980, 13 (7), 603– 9,  DOI: 10.1080/00032718008077690
      420
      Spectrophotometric determination of carboxylic acids by ferric hydroxamate formation with water-soluble carbodiimide
      Takeuchi, T.; Horikawa, R.; Tanimura, T.
      Analytical Letters (1980), 13 (A7), 603-9CODEN: ANALBP; ISSN:0003-2719.
      Carboxylic acids in aq. soln. were detd. spectrophotometrically as ferric hydroxamates. A H2O-sol. carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, was used as a coupling agent to form hydroxamic acids. The coupling reaction was carried out at 37° for 30 min in a soln. buffered at pH 5.3 with pyridine and HCl. Calibration curves of HOBz, HOAc, citric acid, and α-ketoglutaric acid were linear for 0.25-8 μmol.
    419. 419
      He, Y.-C.; Ma, C.-L.; Xu, J.-H.; Zhou, L. A High-Throughput Screening Strategy for Nitrile-Hydrolyzing Enzymes Based on Ferric Hydroxamate Spectrophotometry. Appl. Microbiol. Biotechnol. 2011, 89 (3), 817– 23,  DOI: 10.1007/s00253-010-2977-5
      421
      A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry
      He, Yu-Cai; Ma, Cui-Luan; Xu, Jian-He; Zhou, Li
      Applied Microbiology and Biotechnology (2011), 89 (3), 817-823CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the prodn. of carboxylic acids and their derivs., and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliph. and arom. carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes.
    420. 420
      Goswami, S.; Hazra, A.; Das, M. K. Selenodiazole-Fused Diacetamidopyrimidine, a Selective Fluorescence Sensor for Aliphatic Monocarboxylates. Tetrahedron Lett. 2010, 51 (25), 3320– 3,  DOI: 10.1016/j.tetlet.2010.04.085
      422
      Selenodiazole-fused diacetamidopyrimidine, a selective fluorescence sensor for aliphatic monocarboxylates
      Goswami, Shyamaprosad; Hazra, Anita; Das, Manas Kumar
      Tetrahedron Letters (2010), 51 (25), 3320-3323CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)
      A designed sensor, selenodiazole-fused pyrimidine ring having two acetylamino groups at 2,4-positions was synthesized for selective recognition of aliph. monocarboxylate anions over a wide range of other anions. The recognition study was carried out by UV-visible and fluorescence methods. A significant bathochromic shift of the fluorescence intensity of the receptor in the presence of carboxylate makes the receptor a discriminating sensor for aliph. monocarboxylates.
    421. 421
      Goswami, S.; Hazra, A.; Chakrabarty, R.; Fun, H.-K. Recognition of Carboxylate Anions and Carboxylic Acids by Selenium-Based New Chromogenic Fluorescent Sensor: A Remarkable Fluorescence Enhancement of Hindered Carboxylates. Org. Lett. 2009, 11 (19), 4350– 3,  DOI: 10.1021/ol901737s
      423
      Recognition of carboxylate anions and carboxylic acids by selenium-based new chromogenic fluorescent sensor: A remarkable fluorescence enhancement of hindered carboxylates
      Goswami, Shyamaprosad; Hazra, Anita; Chakrabarty, Rinku; Fun, Hoong-Kun
      Organic Letters (2009), 11 (19), 4350-4353CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      A selenium metal-based new fluorescence sensor 5-pivaloylamino-1,2,5-selenodiazolo[3,4-d]pyrimidin-7-(6H)-one (receptor 1) is reported for the recognition of monocarboxylic acids and carboxylate anions both by UV-visible and fluorescence methods. Receptor 1 recognizes carboxylate anions more than monocarboxylic acids and it is a selective sensor for carboxylates with specially hindered carboxylate anions. The changes of fluorescence intensity are remarkably enhanced with red shift in presence of bulky carboxylate anions. The x-ray crystal structure of receptor 1 with pivalic acid is reported.
    422. 422
      Galindo, F.; Becerril, J.; Isabel Burguete, M.; Luis, S. V.; Vigara, L. Synthesis and Study of a Cyclophane Displaying Dual Fluorescence Emission: A Novel Ratiometric Sensor for Carboxylic Acids in Organic Medium. Tetrahedron Lett. 2004, 45 (8), 1659– 62,  DOI: 10.1016/j.tetlet.2003.12.116
      424
      Synthesis and study of a cyclophane displaying dual fluorescence emission: a novel ratiometric sensor for carboxylic acids in organic medium
      Galindo, Francisco; Becerril, Jorge; Isabel Burguete, M.; Luis, Santiago V.; Vigara, Laura
      Tetrahedron Letters (2004), 45 (8), 1659-1662CODEN: TELEAY; ISSN:0040-4039. (Elsevier Science B.V.)
      The synthesis and fluorescent properties of a novel cyclophane contg. two L-valine units and one naphthalene chromophore are described. Synthesis of the macrocycle I was accomplished without using high-diln. methods in moderate to high yields. The fluorescence spectrum of I in neutral dichloromethane shows a band at 390 nm attributable to emission from an exciplex formed between the naphthalene unit and the neighboring amine groups. Addn. of trifluoroacetic acid restores the typical naphthalene emission at 330 nm. Due to the fact that both emissions have similar intensities under the working conditions, the ratio between them can be used to obtain a ratiometric response to carboxylic acids in org. medium.
    423. 423
      Cabell, L. A.; Best, M. D.; Lavigne, J. J.; Schneider, S. E.; Perreault, D. M.; Monahan, M.-K.; Anslyn, E. V. Metal Triggered Fluorescence Sensing of Citrate Using a Synthetic Receptor. J. Chem. Soc., Perkin trans. II 2001, (3), 315– 23,  DOI: 10.1039/b008694k
      There is no corresponding record for this reference.
    424. 424
      Xu, K.-x.; Xie, X.-m.; Kong, H.-j.; Li, P.; Zhang, J.-l.; Pang, X.-b. Selective Fluorescent Sensors for Malate Anion Using the Complex of Phenanthroline-Based Eu(III) in Aqueous Solution. Sens. Actuators B Chem. 2014, 201, 131– 7,  DOI: 10.1016/j.snb.2014.04.086
      426
      Selective fluorescent sensors for malate anion using the complex of phenanthroline-based Eu(III) in aqueous solution
      Xu, Kuo-xi; Xie, Xin-mei; Kong, Hua-jie; Li, Ping; Zhang, Jing-lai; Pang, Xiao-bin
      Sensors and Actuators, B: Chemical (2014), 201 (), 131-137CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      The compds. L1, L2 and D1 based on phenanthroline Schiff base bearing chiral amino alc. groups had been synthesized and demonstrated. The sensing ability of the compds. in the presence of metal cations (Li+, Na+, K+, Mg2+, Co2+, Mn2+, Zn2+, Cd2+, Hg2+, Al3+, Cr3+, Fe3+, Eu3+) was studied by fluorescent spectroscopy. The exptl. results indicated that compds. L1 and D1 could act as selective fluorescent sensors for Cd2+, Zn2+ with fluorescence enhancement and for the Eu3+ ion with fluorescence quenching in aq. soln. Addnl., the emission properties of the L1-Eu3+ complex could be applied as selective sensor to distinguish malate anion from other carboxylate anions in an aq. soln.
    425. 425
      Burguete, M. I.; Galindo, F.; Luis, S. V.; Vigara, L. A Turn-On Fluorescent Indicator for Citrate with Micromolar Sensitivity. Dalton Trans. 2007, (36), 4027– 33,  DOI: 10.1039/b711139h
      427
      A turn-on fluorescent indicator for citrate with micromolar sensitivity
      Burguete, M. Isabel; Galindo, Francisco; Luis, Santiago V.; Vigara, Laura
      Dalton Transactions (2007), (36), 4027-4033CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
      A turn-on fluorescent indicator for citric acid (citrate) has been developed, displaying high emission enhancement (+1500%) and low interference by other carboxylates. The sensor is based on the nonemissive copper(II) complex of a fluorescent amino amide, which, upon addn. of citrate decomplexes to yield the emissive ligand. The detection limit estd. for this new chemosensing system is about 0.5 μM. This novel approach to the anal. of citrate constitutes an alternative ∼102-103 times more sensitive than the std. method based on the enzyme citrate lyase.
    426. 426
      Klein, G.; Reymond, J.-L. An Enzyme Assay Using pM. Angew. Chem., Int. Ed. 2001, 40 (9), 1771– 3,  DOI: 10.1002/1521-3773(20010504)40:9<1771::AID-ANIE17710>3.0.CO;2-M
      428
      An enzyme assay using pM
      Klein, Gerard; Reymond, Jean-Louis
      Angewandte Chemie, International Edition (2001), 40 (9), 1771-1773CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
      A fluorometric assay is described which measures enzyme that liberate weak metal ligands, such as amino acids, as their reaction products. The assay uses an orange fluorescent metal-sensor derived from quinacridone which coordinates with a metal ion such as copper and the amino acid product. The quinacridone deriv. can be considered as a pM sensor (pM = -lg[M], where M is free metal ions) by its ability to differentiate free metal ions from weakly chelated metal ions. The fact that product sensing occurs indirectly through the pM value makes the assay applicable to a broad variety of substrates. The kinetics of ligand exchange at copper and nickel are sufficiently fast for the fluorescent pM sensor to response in real time to the reaction progress. Its usefulness is illustrated by the examples presented as the first fluorescence assays for acylase and aminopeptidase.
    427. 427
      Dean, K. E. S.; Klein, G.; Renaudet, O.; Reymond, J.-L. A Green Fluorescent Chemosensor for Amino Acids Provides a Versatile High-Throughput Screening (HTS) Assay for Proteases. Bioorg. Med. Chem. Lett. 2003, 13 (10), 1653– 6,  DOI: 10.1016/S0960-894X(03)00280-4
      429
      A green fluorescent chemosensor for amino acids provides a versatile high-throughput screening (HTS) assay for proteases
      Dean, Kathryn E. S.; Klein, Gerard; Renaudet, Olivier; Reymond, Jean-Louis
      Bioorganic & Medicinal Chemistry Letters (2003), 13 (10), 1653-1656CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science B.V.)
      The water sol. fluorescein-based ligand 1 forms a non-fluorescent complex with Cu2+. This complex serves as a fluorescent sensor for amino acids in the 10-3 M concn. range. Since the signal response is very fast, the sensor can be used to detect the hydrolytic activity of various proteases (trypsin, chymotrypsin, subtilisin) on bovine serum albumin as a whole protein substrate, and more generally to follow reactions releasing or removing free amino acids, in real time.
    428. 428
      Xu, J.-M.; Fu, F.-T.; Hu, H.-F.; Zheng, Y.-G. A High-Throughput Screening Method for Amino Acid Dehydrogenase. Anal. Biochem. 2016, 495, 29– 31,  DOI: 10.1016/j.ab.2015.11.012
      430
      A high-throughput screening method for amino acid dehydrogenase
      Xu, Jian-Miao; Fu, Fang-Tian; Hu, Hai-Feng; Zheng, Yu-Guo
      Analytical Biochemistry (2016), 495 (), 29-31CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      A simple and rapid screening method for amino acid dehydrogenase (e.g., leucine dehydrogenase, LDH) has been developed. It relies on a competitive relationship between a non-fluorescent Cu(II)-calcein complex and amino acid (e.g., L-2-aminobutyric acid, L-ABA). When ABA was introduced to a Cu(II)-calcein soln., it bound with the Cu(II) ions and this released calcein from the complex, which was detected as strong fluorescence. The principle of this high-throughput screening method was validated by screening an LDH mutant library. Compared with other methods, this method provided much quicker L-ABA detection and screening for leucine dehydrogenase mutations.
    429. 429
      Ryu, D.; Park, E.; Kim, D.-S.; Yan, S.; Lee, J. Y.; Chang, B.-Y.; Ahn, K. H. A Rational Approach to Fluorescence “Turn-On” Sensing of α-Amino-Carboxylates. J. Am. Chem. Soc. 2008, 130 (8), 2394– 5,  DOI: 10.1021/ja078308e
      431
      A rational approach to fluorescence "turn-on" sensing of α-amino-carboxylates
      Ryu, Dowook; Park, Eunju; Kim, Dae-Sik; Yan, Shihai; Lee, Jin Yong; Chang, Byoung-Yong; Ahn, Kyo Han
      Journal of the American Chemical Society (2008), 130 (8), 2394-2395CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A rational approach to fluorescence turn-on sensing of amino carboxylates is described, which primarily relies on the perturbation of the quenching n-π* transition energy level of a carbonyl ionophore relative to the π-π* transition energy level of an anthracene in the sensor. The anthracene-based bis(trifluoroacetylcarboxanilide) sensor is structurally simple but selectively senses α-amino acids as their carboxylate forms over β- and γ-homologs, by forming a (1:1)-cyclic adduct with a large fluorescence enhancement factor of 110.
    430. 430
      Kim, D.-S.; Ahn, K. H. Fluorescence “Turn-On” Sensing of Carboxylate Anions with Oligothiophene-Based o-(Carboxamido)Trifluoroacetophenones. J. Org. Chem. 2008, 73 (17), 6831– 4,  DOI: 10.1021/jo801178y
      432
      Fluorescence "turn-on" sensing of carboxylate anions with oligothiophene-based o-(carboxamido)trifluoroacetophenones
      Kim, Dae-Sik; Ahn, Kyo Han
      Journal of Organic Chemistry (2008), 73 (17), 6831-6834CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      O-(Carboxamido)trifluoroacetophenones contg. ter- or pentathiophene moiety as a fluorophore exhibit fluorescence enhancement upon binding carboxylate anions. Particularly, the terthiophene deriv. shows a large fluorescence enhancement factor (FEF = 120). The enhancement is explained by intramol. H-bonding stabilization of an anion-ionophore adduct, through which a possible quenching process, the n-π* transition from the trifluoroacetophenone moiety, is eliminated.
    431. 431
      Schrader, J.; Etschmann, M. M. W.; Sell, D.; Hilmer, J. M.; Rabenhorst, J. Applied Biocatalysis for the Synthesis of Natural Flavour Compounds - Current Industrial Processes and Future Prospects. Biotechnol. Lett. 2004, 26 (6), 463– 72,  DOI: 10.1023/B:BILE.0000019576.80594.0e
      433
      Applied biocatalysis for the synthesis of natural flavour compounds - current industrial processes and future prospects
      Schrader, J.; Etschmann, M. M. W.; Sell, D.; Hilmer, J.-M.; Rabenhorst, J.
      Biotechnology Letters (2004), 26 (6), 463-472CODEN: BILED3; ISSN:0141-5492. (Kluwer Academic Publishers)
      A review, with refs. The industrial application of biocatalysis for the prodn. of natural flavor compds. is illustrated by a discussion of the prodn. of vanillin, γ-decalactone, carboxylic acids, C6 aldehydes and alcs. ( green notes'), esters, and 2-phenylethanol. Modern techniques of mol. biol. and process engineering, such as heterologous expression of genes, site-directed mutagenesis, whole-cell biocatalysis in biphasic systems, and cofactor regeneration for in vitro oxygenation, may result in more biocatalytic processes for the prodn. of flavor compds. in the future.
    432. 432
      Gotor, V. Biocatalysis Applied to the Preparation of Pharmaceuticals. Org. Process Res. Dev. 2002, 6 (4), 420– 6,  DOI: 10.1021/op020008o
      434
      Biocatalysis Applied to the Preparation of Pharmaceuticals
      Gotor, Vicente
      Organic Process Research & Development (2002), 6 (4), 420-426CODEN: OPRDFK; ISSN:1083-6160. (American Chemical Society)
      A review. Biotransformations are now accepted as a methodol. for the prepn. of fine chems. The majority of enzyme-catalyzed reactions involve the use of hydrolases, esp. lipases, in hydrolysis, esterification or aminolysis reactions. Lyases, enzymes much less exploited in org. synthesis, are proving increasingly interesting, esp. the use of (R)-oxynitrilases for the synthesis of optically active cyanohydrins which are intermediates of many compds. of pharmacol. importance. In this account, we report the utility of four kinds of processes which we have carried out recently in our lab. in the field of pharmaceutical chem. Enzymic resoln. of alcs., enzymic acylation of amines, regioselective acylation, and alkoxycarbonylation of natural products, and chemoenzymic synthesis of products of high added value using (R)-oxynitrilase as biocatalyst are described.
    433. 433
      SÁ, A. G. A.; Meneses, A. C. d.; Araújo, P. H. H. d.; Oliveira, D. d. A Review on Enzymatic Synthesis of Aromatic Esters Used as Flavor Ingredients for Food, Cosmetics and Pharmaceuticals Industries. Trends Food Sci. Technol. 2017, 69, 95– 105,  DOI: 10.1016/j.tifs.2017.09.004
      435
      A review on enzymatic synthesis of aromatic esters used as flavor ingredients for food, cosmetics and pharmaceuticals industries
      Sa, Amanda Gomes Almeida; Meneses, Alessandra Cristina de; Araujo, Pedro Henrique Hermes de; Oliveira, Debora de
      Trends in Food Science & Technology (2017), 69 (Part_A), 95-105CODEN: TFTEEH; ISSN:0924-2244. (Elsevier Ltd.)
      A review. Many sectors of industry, mainly food, cosmetics and pharmaceutics, have increased their interest in esters due to their flavor property. Flavor esters that possess an arom. ring in their mol. structure are also known as arom. esters. These esters are widely found in nature (fruits and plants) and the synthetic (i.e. via chem.) and natural routes (i.e. via direct extn. from nature or via biotechnol.) are suitable for their biocatalysis. In this context, from the industrial point of view, enzyme-catalyzed reactions are the most economical approach to reach final green products with no toxicity and no harm to human health. The present article gives an overview of the arom. esters synthesis, considering the main effects in the reaction media conditions and enzymes used. This review also describes applied trends in enzymic-catalyzed reactions, pointing alternatives to prodn., like ultrasound-assisted reactions and process optimization of arom. esters. Furthermore, this work presents perspectives concerning the biol. potential of these esters and recent advances in their encapsulation. Lipases play an important role in the arom. esters prodn., with several advantages over synthetic route. Lipase-catalyzed reactions usually follows Ping-Pong Bi-Bi or ternary complex (order Bi-Bi) mechanism. The study of the process parameters and their interaction are very important to understand the system optimization and achieve the max. reaction yield to scale up. Arom. esters can present some biol. activities, in addn. to their fragrances, which increases the interest in the encapsulation of these compds.
    434. 434
      Panke, S.; Held, M.; Wubbolts, M. Trends and Innovations in Industrial Biocatalysis for the Production of Fine Chemicals. Curr. Opin. Biotechnol. 2004, 15 (4), 272– 9,  DOI: 10.1016/j.copbio.2004.06.011
      436
      Trends and innovations in industrial biocatalysis for the production of fine chemicals
      Panke, Sven; Held, Martin; Wubbolts, Marcel
      Current Opinion in Biotechnology (2004), 15 (4), 272-279CODEN: CUOBE3; ISSN:0958-1669. (Elsevier Ltd.)
      A review. Biocatalysis has become an established technol. for the industrial manuf. of fine chems. In recent years, a multitude of chem. companies have embraced biocatalysis for the manuf. of desired stereoisomers, and new or improved methods for the synthesis of enantiomerically pure α- and β-amino acids, amines, amides, peptides, nitriles, alcs., org. acids and epoxides have emerged. Furthermore, the selectivity and mild operational conditions of biocatalysts are increasingly applied in industry to modify complex target mols. These recent innovations in the manuf. of industrial fine chems. using biocatalysis are discussed from an industrial perspective.
    435. 435
      Joshi, A.; Singh, S.; Iqbal, Z.; De, S. R. CO Free Esterifications of (Hetero)Arenes Via Transition-Metal-Catalyzed Chelation-Induced C-H Activation: Recent Updates. Tetrahedron 2022, 104, 132601,  DOI: 10.1016/j.tet.2021.132601
      437
      CO free esterifications of (Hetero)arenes via transition-metal-catalyzed chelation-induced C-H activation: Recent updates
      Joshi, Asha; Singh, Sandeep; Iqbal, Zafar; De, Saroj Ranjan
      Tetrahedron (2022), 104 (), 132601CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)
      A review. Esters are substantial compds. generally found in the area of pharmaceuticals, agrochems., drugs, materials etc., and have been utilized for the formation of many add-value compds. They are also significant structural blocks in different chem. transformations. Thus, highly efficient transition-metal-catalyzed C-H alkoxycarbonylation of (hetero)arenes offers to achieve important skeleton in a single step in contrast to the other conventional strategies using toxic carbon monoxide (CO). This review provides an overview of this emerging field by highlighting all the chelation-induced esters synthesized till date without the involvement of toxic carbon monoxide (CO). The article has been classified according to the sources of esterification agents along with their reaction methodologies, examples, reaction mechanisms, and synthetic applications.
    436. 436
      Larios, A.; García, H. S.; Oliart, R. M.; Valerio-Alfaro, G. Synthesis of Flavor and Fragrance Esters Using Candida Antarctica Lipase. Appl. Microbiol. Biotechnol. 2004, 65 (4), 373– 6,  DOI: 10.1007/s00253-004-1602-x
      438
      Synthesis of flavor and fragrance esters using Candida antarctica lipase
      Larios, Araceli; Garcia, Hugo S.; Oliart, Rosa Maria; Valerio-Alfaro, Gerardo
      Applied Microbiology and Biotechnology (2004), 65 (4), 373-376CODEN: AMBIDG; ISSN:0175-7598. (Springer GmbH)
      Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsatd. (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10. Similar results were obsd. in a study of the alc. specificity of the enzyme for esterification of acetic and butyric acids with four alcs.: Bu, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral α-substituted or β-substituted carboxylic acids and Bu alc. was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an org. solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids.
    437. 437
      Smith, G. A. Fatty Acid, Methyl Ester, and Vegetable Oil Ethoxylates, Biobased Surfactants; AOCS Press, 2019; pp 287– 301.
      There is no corresponding record for this reference.
    438. 438
      Panten, J.; Surburg, H. Flavors and Fragrances, 4. Natural Raw Materials, Ullmann’s Encyclopedia of Industrial Chemistry , 2011; pp 1– 58.
      There is no corresponding record for this reference.
    439. 439
      Johannes, P.; Surburg, H. Flavors and Fragrances, 2. Aliphatic Compounds. In Ullmann’s Encyclopedia of Industrial Chemistry , 2015; pp 1– 55.
      There is no corresponding record for this reference.
    440. 440
      Berger, R. G. Biotechnology of Flavours─the Next Generation. Biotechnol. Lett. 2009, 31 (11), 1651,  DOI: 10.1007/s10529-009-0083-5
      442
      Biotechnology of flavours-the next generation
      Berger, Ralf G.
      Biotechnology Letters (2009), 31 (11), 1651-1659CODEN: BILED3; ISSN:0141-5492. (Springer)
      A review. Volatile org. chems. (flavors, aromas) are the sensory principles of many consumer products and govern their acceptance and market success. Flavours from microorganisms compete with the traditional agricultural sources. Screening for overproducers, elucidation of metabolic pathways and precursors and application of conventional bioengineering has resulted in a set of more than 100 com. aroma chems. derived via biotechnol. Various routes may lead to volatile metabolites: De novo synthesis from elementary biochem. units, degrdn. of larger substrates such as lipids, and functionalization of immediate flavor precursor mols. More recently, the field was stimulated by the increasing preference of alienated consumers for products bearing the label "natural", and by the vivid discussion on healthy and "functional" food ingredients. The unmistakable call for sustainable sources and environmentally friendly prodn. is forcing the industry to move towards a greener chem. Progress is expected from the toolbox of genetic engineering which is expected to help in identifying metabolic bottlenecks and in creating novel high-yielding strains. Bioengineering, in a complementary way, provides promising tech. options, such as improved substrate dosage, gas-phase or two-phase reactions and in situ product recovery.
    441. 441
      Gunatillake, P. A.; Adhikari, R.; Gadegaard, N. Biodegradable Synthetic Polymers for Tissue Engineering. Eur. Cell. Mater. 2003, 5 (1), 1– 16,  DOI: 10.22203/eCM.v005a01
      443
      Biodegradable synthetic polymers for tissue engineering
      Gunatillake, Pathiraja A.; Adhikari, Raju
      European Cells and Materials (2003), 5 (), 1-16CODEN: ECMUBB; ISSN:1473-2262. (University of Wales)
      A review is given on biodegradable synthetic polymers focusing on their potential in tissue engineering applications. The major classes of polymers are briefly discussed with regard to synthesis, properties and biodegradability, and known degrdn. modes and products are indicated based on studies reported in the literature. A vast majority of biodegradable polymers studied belongs to the polyester family, which includes polyglycolides and polylactides. Some disadvantages of these polymers in tissue engineering applications are their poor biocompatibility, release of acidic degrdn. products, poor processability and loss of mech. properties very early during degrdn. Other degradable polymers such as polyorthoesters, polyanhydrides, polyphosphazenes, and polyurethanes are also discussed and their advantages and disadvantages summarized. With advancements in tissue engineering it has become necessary to develop polymers that meet more demanding requirements. Recent work has focused on developing injectable polymer compns. based on poly(propylene fumarate) and poly(anhydrides) to meet these requirements in orthopedic tissue engineering. Polyurethanes have received recent attention for development of degradable polymers because of their great potential in tailoring polymer structure to achieve mech. properties and biodegradability to suit a variety of applications.
    442. 442
      Kodali, D. R. High Performance Ester Lubricants from Natural Oils. Ind. Lubr. Tribol. 2002, 54 (4), 165– 70,  DOI: 10.1108/00368790210431718
      There is no corresponding record for this reference.
    443. 443
      Chen, J.; Wang, Y.; Huang, J.; Li, K.; Nie, X. Synthesis of Tung-Oil-Based Triglycidyl Ester Plasticizer and Its Effects on Poly(Vinyl Chloride) Soft Films. ACS Sustain. Chem. Eng. 2018, 6 (1), 642– 51,  DOI: 10.1021/acssuschemeng.7b02989
      There is no corresponding record for this reference.
    444. 444
      Gumel, A. M.; Annuar, M. S. M.; Heidelberg, T.; Chisti, Y. Lipase Mediated Synthesis of Sugar Fatty Acid Esters. Process Biochem. 2011, 46 (11), 2079– 90,  DOI: 10.1016/j.procbio.2011.07.021
      446
      Lipase mediated synthesis of sugar fatty acid esters
      Gumel, A. M.; Annuar, M. S. M.; Heidelberg, T.; Chisti, Y.
      Process Biochemistry (Amsterdam, Netherlands) (2011), 46 (11), 2079-2090CODEN: PBCHE5; ISSN:1359-5113. (Elsevier B.V.)
      This review is concerned with lipase catalyzed synthesis of sugar fatty acid esters in water immiscible org. solvents. Sugar esters are widely used nonionic and nontoxic biosurfactants. Certain sugar esters inhibit microbial growth and have other activities. Lipase mediated synthesis has important advantages over conventional chem. synthesis of sugar esters. Lipase catalyzed synthesis is typically carried out in org. solvents having a low water activity to drive the reaction towards synthesis instead of towards ester hydrolysis. The impact of the various reaction conditions on enzymic synthesis of sugar esters in nonaq. media is discussed. Considered in particular are the solvent effects; the effects of water activity; the influence of the nature and concn. of the reactants (sugars and fatty acids); the influence of temp.; and the effects assocd. with the specific nature of the lipase catalyst used.
    445. 445
      Badgujar, K. C.; Bhanage, B. M. Application of Lipase Immobilized on the Biocompatible Ternary Blend Polymer Matrix for Synthesis of Citronellyl Acetate in Non-Aqueous Media: Kinetic Modelling Study. Enzyme Microb. Technol. 2014, 57, 16– 25,  DOI: 10.1016/j.enzmictec.2014.01.006
      447
      Application of lipase immobilized on the biocompatible ternary blend polymer matrix for synthesis of citronellyl acetate in non-aqueous media: Kinetic modelling study
      Badgujar, Kirtikumar C.; Bhanage, Bhalchandra M.
      Enzyme and Microbial Technology (2014), 57 (), 16-25CODEN: EMTED2; ISSN:0141-0229. (Elsevier)
      This work reports the use of new support for immobilization of lipase Burkholderia cepacia (BCL) matrix made up of polylactic acid (PLA), chitosan (CH), and polyvinyl alc. (PVA). Initially lipase from various microbial sources and immobilization support compn. was screened to obtain a robust biocatalyst. Among various biocatalysts prepn., the PLA:PVA:CH:BCL (1:6:1:2) was worked as a robust biocatalyst for the citronellyl acetate synthesis. Various reaction parameters were studied in detail to obtain the suitable reaction conditions for model citronellyl acetate synthesis reaction. Various kinetic parameters such as rmax, K(citronellol), K(citronellol), K(vinyl acetate) were detd. using non-linear regression anal. for the ternary complex as well as bi-bi ping-pong mechanism. The exptl. results and kinetic study showed that citronellyl acetate synthesis catalyzed by immobilized lipase BCL followed the ternary complex mechanism with inhibition by alc. (citronellol). The energy of activation for citronellyl acetate synthesis was found to be lower for immobilized lipase (8.9 kcal/mol) than aggregated lipase (14.8 kcal/mol) enzyme. The developed biocatalyst showed four to fivefold higher catalytic activity and excellent recyclability (up to six cycles) than the aggregated lipase.
    446. 446
      Stergiou, P.-Y.; Foukis, A.; Filippou, M.; Koukouritaki, M.; Parapouli, M.; Theodorou, L. G.; Hatziloukas, E.; Afendra, A.; Pandey, A.; Papamichael, E. M. Advances in Lipase-Catalyzed Esterification Reactions. Biotechnol. Adv. 2013, 31 (8), 1846– 59,  DOI: 10.1016/j.biotechadv.2013.08.006
      448
      Advances in lipase-catalyzed esterification reactions
      Stergiou, Panagiota-Yiolanda; Foukis, Athanasios; Filippou, Michalis; Koukouritaki, Maria; Parapouli, Maria; Theodorou, Leonidas G.; Hatziloukas, Efstathios; Afendra, Amalia; Pandey, Ashok; Papamichael, Emmanuel M.
      Biotechnology Advances (2013), 31 (8), 1846-1859CODEN: BIADDD; ISSN:0734-9750. (Elsevier)
      A review. Lipase-catalyzed esterification reactions are among the most significant chem. and biochem. processes of industrial relevance. Lipases catalyze hydrolysis as well as esterification reactions. Enzyme-catalyzed esterification has acquired increasing attention in many applications, due to the significance of the derived products. More specifically, the lipase-catalyzed esterification reactions attracted research interest during the past decade, due to an increased use of org. esters in biotechnol. and the chem. industry. Lipases, as hydrolyzing agents are active in environments, which contain a min. of two distinct phases, where all reactants are partitioned between these phases, although their distribution is not fixed and changes as the reaction proceeds. The kinetics of the lipase-catalyzed reactions is governed by a no. of factors. This article presents a thorough and descriptive evaluation of the applied trends and perspectives concerning the enzymic esterification, mainly for biofuel prodn.; an emphasis is given on essential factors, which affect the lipase-catalyzed esterification reaction. Moreover, the art of using bacterial and/or fungal strains for whole cell biocatalysis purposes, as well as carrying out catalysis by various forms of purified lipases from bacterial and fungal sources is also reviewed.
    447. 447
      Gao, W.; Wu, K.; Chen, L.; Fan, H.; Zhao, Z.; Gao, B.; Wang, H.; Wei, D. A Novel Esterase from a Marine Mud Metagenomic Library for Biocatalytic Synthesis of Short-Chain Flavor Esters. Microbial Cell Factories 2016, 15, 41,  DOI: 10.1186/s12934-016-0435-5
      449
      A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters
      Gao, Wenyuan; Wu, Kai; Chen, Lifeng; Fan, Haiyang; Zhao, Zhiqiang; Gao, Bei; Wang, Hualei; Wei, Dongzhi
      Microbial Cell Factories (2016), 15 (), 41/1-41/12CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)
      Marine mud is an abundant and largely unexplored source of enzymes with unique properties that may be useful for industrial and biotechnol. purposes. However, since most microbes cannot be cultured in the lab., a cultivation-independent metagenomic approach would be advantageous for the identification of novel enzymes. Therefore, with the objective of screening novel lipolytic enzymes, a metagenomic library was constructed using the total genomic DNA extd. from marine mud. Based on functional heterologous expression, 34 clones that showed lipolytic activity were isolated. The 5 clones with the largest halos were identified, and the corresponding genes were successfully overexpressed in Escherichia coli. Mol. anal. revealed that these encoded proteins showed 48-79% similarity with other proteins in the GenBank database. Multiple sequence alignment and phylogenetic tree anal. classified these 5 protein sequences as new members of known families of bacterial lipolytic enzymes. Among them, EST4, which had 316 amino acids with a predicted mol. wt. of 33.8 kDa, was further studied in detail due to its strong hydrolytic activity. Characterization of EST4 indicated that it is an alk. esterase that exhibits its highest hydrolytic activity toward p-nitrophenyl butyrate (specific activity: 1389 U/mg) at 45°C and pH 8.0. The half-life of EST4 was 55 and 46 h at 40 and 45°, resp., indicating a relatively high thermostability. EST4 also showed marked stability in org. solvents, retaining 90% of its initial activity when incubated for 12 h in the presence of hydrophobic alkanes. Furthermore, EST4 was used as an efficient whole-cell biocatalyst for the synthesis of short-chain flavor esters, showing high conversion rate and good tolerance for high substrate concns. (up to 3.0M). These results demonstrated a promising potential for industrial scaling-up to produce short-chain flavor esters at high substrate concns. in non-aq. media. Thus, the authors report unprecedented alc. tolerance and conversion of an esterase biocatalyst identified from a marine mud metagenomic library. The high org. solvent tolerance and thermostability of EST4 suggest that it has great potential as a biocatalyst.
    448. 448
      Lozano, P.; Bernal, J. M.; Navarro, A. A Clean Enzymatic Process for Producing Flavour Esters by Direct Esterification in Switchable Ionic Liquid/Solid Phases. Green Chem. 2012, 14 (11), 3026– 33,  DOI: 10.1039/c2gc36081k
      450
      A clean enzymatic process for producing flavour esters by direct esterification in switchable ionic liquid/solid phases
      Lozano, Pedro; Bernal, Juana M.; Navarro, Alicia
      Green Chemistry (2012), 14 (11), 3026-3033CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
      A clean biocatalytic approach for producing flavor esters using switchable ionic liq./solid phases as reaction/sepn. media has been developed. The phase behavior of different IL/flavor acetyl ester (geranyl acetate, citronellyl acetate, neryl acetate and isoamyl acetate) mixts. was studied at several concns., resulting for all cases in fully homogeneous liq. media at 50 °C, and solid systems at room temp. By using an iterative centrifugation protocol on the solid IL/flavor ester mixts. at controlled temps., the solid IL phase and the liq. flavor ester phase can be easily sepd. The excellent suitability of an immobilized Candida antarctica lipase B (Novozym 435) catalyst in the esterification reaction between an aliph. carboxylic acid (acetic, propionic, butyric or valeric) and a flavor alc. (isoamyl alc., nerol, citronellol or geraniol) in N,N',N'',N'''-hexadecyltrimethyl-ammonium bis(trifluoromethylsulfonyl)imide ([C16tma][NTf2])IL has been demonstrated, the product yield being improved up to 100% under appropriate reaction conditions (enzyme amt., dehydrating mol. sieves, etc.) at 50 °C. The enzymic synthesis of sixteen different flavor esters was carried out in [C16tma][NTf2] by means of this approach, providing products of up to 0.757 g mL-1 concn. after IL sepn. The residual activity of the enzyme/IL system during seven consecutive operation cycles was shown to be practically unchanged after reuse.
    449. 449
      Otera, J.; Nishikido, J. Esterification: Methods, Reactions, and Applications; John Wiley & Sons, 2009. DOI: 10.1002/9783527627622
      There is no corresponding record for this reference.
    450. 450
      Beekwilder, J.; Alvarez-Huerta, M.; Neef, E.; Verstappen, F. W. A.; Bouwmeester, H. J.; Aharoni, A. Functional Characterization of Enzymes Forming Volatile Esters from Strawberry and Banana. Plant Physiol. 2004, 135 (4), 1865– 78,  DOI: 10.1104/pp.104.042580
      452
      Functional characterization of enzymes forming volatile esters from strawberry and banana
      Beekwilder, Jules; Alvarez-Huerta, Mayte; Neef, Evert; Verstappen, Francel W. A.; Bouwmeester, Harro J.; Aharoni, Asaph
      Plant Physiology (2004), 135 (4), 1865-1878CODEN: PLPHAY; ISSN:0032-0889. (American Society of Plant Biologists)
      Volatile esters are flavor components of the majority of fruits. The last step in their biosynthesis is catalyzed by alc. acyltransferases (AATs), which link alcs. to acyl moieties. Full-length cDNAs putatively encoding AATs were isolated from fruit of wild strawberry (Fragaria vesca) and banana (Musa sapientum) and compared to the previously isolated SAAT gene from the cultivated strawberry (Fragaria × ananassa). The potential role of these enzymes in fruit flavor formation was assessed. To this end, recombinant enzymes were produced in Escherichia coli, and their activities were analyzed for a variety of alc. and acyl-CoA substrates. When the results of these activity assays were compared to a phylogenetic anal. of the various members of the acyltransferase family, it was clear that substrate preference could not be predicted on the basis of sequence similarity. In addn., the substrate preference of recombinant enzymes was not necessarily reflected in the representation of esters in the corresponding fruit volatile profiles. This suggests that the specific profile of a given fruit species is to a significant extent detd. by the supply of precursors. To study the in planta activity of an alc. acyltransferase and to assess the potential for metabolic engineering of ester prodn., we generated transgenic petunia (Petunia hybrida) plants over-expressing the SAAT gene. While the expression of SAAT and the activity of the corresponding enzyme were readily detected in transgenic plants, the volatile profile was found to be unaltered. Feeding of isoamyl alc. to explants of transgenic lines resulted in the emission of the corresponding acetyl ester. This confirmed that the availability of alc. substrates is an important parameter to consider when engineering volatile ester formation in plants.
    451. 451
      Buckles, R. E.; Thelen, C. Qualitative Determination of Carboxylic Esters. Anal. Chem. 1950, 22 (5), 676– 8,  DOI: 10.1021/ac60041a016
      453
      Detection of carboxylic esters. Scope and limitations of the hydroxamic acid test
      Buckles, Robert E.; Thelen, Charles J.
      (1950), 22 (), 676-8CODEN: ANCHAM; ISSN:0003-2700.
      The hydroxamic acid test (development of color with Fe+++) for carboxylic esters (I) has been studied with I, trihalomethyl compds., acid chlorides and anhydrides, acids, phenols, aldehydes, amides, imides, nitriles, isocyanates, nitro compds., and mixts. contg. I. Qual. procedures for distinguishing between I and the other classes of compds. are given.
    452. 452
      Stern, I.; Shapiro, B. A Rapid and Simple Method for the Determination of Esterified Fatty Acids and for Total Fatty Acids in Blood. J. Clin. Pathol. 1953, 6 (2), 158– 60,  DOI: 10.1136/jcp.6.2.158
      454
      A rapid and simple method for the determination of esterified fatty acids and for total fatty acids in blood
      STERN I; SHAPIRO B
      Journal of clinical pathology (1953), 6 (2), 158-60 ISSN:0021-9746.
      There is no expanded citation for this reference.
    453. 453
      Davidson, D. Hydroxamic Acids in Qualitative Organic Analysis. J. Chem. Educ. 1940, 17 (2), 81,  DOI: 10.1021/ed017p81
      455
      Hydroxamic acids in qualitative organic analysis
      Davidson, David
      Journal of Chemical Education (1940), 17 (), 81-4CODEN: JCEDA8; ISSN:0021-9584.
      Hydroxamic acids can be regarded as the n-hydroxy derivs. of the acid amides or as the oximes of the carboxylic acids. In either case, they can also be represented by amine oxide formulas. These tautomeric substances can be prepd. in so many ways and they are so easily detected that the possibility of using them in org. qual. analysis is clear. Thus an ester can be identified as follows: To 1 drop of the sample, add 0.5 ml. of N NH2OH.HCl in MeOH and 0.5 ml. of 2 N KOH in MeOH. KCl will ppt. from the MeOH soln. Heat to boiling, cool, make acid with 0.5 ml. of 2 N aq. HCl and test with a drop of 10% FeCl3 aq. soln. A red color will develop. An alc. can be detected by taking 0.5 ml. AcCl and an equal vol. of dimethylaniline and then 0.5 ml. or 0.5 g. of the sample. The mixt. will sep. into 2 layers. Add ice to decompose excess AcCl and treat 1 drop of the oily ester layer with hydroxylamine, etc., as in the case of esters. Ethers give neg. tests by the above procedures but they can be converted into esters by heating with org. acids in the presence of a little concd. H2SO4. The ester formed can be detected as above. Carboxylic acids can be treated with SOCl2 and when the excess has been removed by evapn. the resulting acyl chloride can be tested as in the case of esters. Phenols can be changed to esters by treatment with AcCl. Nitro compds. can be reduced to an alkylhydroxylamine which gives BzCl a n-alkylhydroxamic acid and this yields a colored complex with FeCl3. Aliphatic amides are converted rapidly to hydroxamic acids by boiling with NH2OH.HCl soln. Cyanates by treatment with NH2OH.HCl can be converted to hydroxamic acids which give a purplish blue complex with FeCl3. 36 references.
    454. 454
      Löbs, A.-K.; Lin, J.-L.; Cook, M.; Wheeldon, I. High Throughput, Colorimetric Screening of Microbial Ester Biosynthesis Reveals High Ethyl Acetate Production from Kluyveromyces Marxianus on C5, C6, and C12 Carbon Sources. Biotechnol. J. 2016, 11 (10), 1274– 81,  DOI: 10.1002/biot.201600060
      456
      High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources
      Lobs Ann-Kathrin; Lin Jyun-Liang; Cook Megan; Wheeldon Ian
      Biotechnology journal (2016), 11 (10), 1274-1281 ISSN:.
      Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z-factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.
    455. 455
      Smith, J. G. Synthetically Useful Reactions of Epoxides. Synthesis 1984, 1984, 629– 656,  DOI: 10.1055/s-1984-30921
      There is no corresponding record for this reference.
    456. 456
      Yudin, A. K. Aziridines and Epoxides in Organic Synthesis; John Wiley & Sons, 2006.
      There is no corresponding record for this reference.
    457. 457
      Kotik, M.; Archelas, A.; Wohlgemuth, R. Epoxide Hydrolases and Their Application in Organic Synthesis. Curr. Org. Chem. 2012, 16 (4), 451– 82,  DOI: 10.2174/138527212799499840
      459
      Epoxide hydrolases and their application in organic synthesis
      Kotik, Michael; Archelas, Alain; Wohlgemuth, Roland
      Current Organic Chemistry (2012), 16 (4), 451-482CODEN: CORCFE; ISSN:1385-2728. (Bentham Science Publishers Ltd.)
      A review was given on the enzymic asym. hydrolysis of oxiranes using epoxide hydrolases as catalysts. The no. of synthetic applications of epoxide hydrolases in org. chem. has reached a remarkable level. This has been due to a tremendous amt. of work dedicated to the discovery of novel epoxide hydrolases from various biol. sources, understanding the structure and function of these widespread enzymes and last but not least, the stabilization of these biocatalysts by various means for prodn. purposes.
    458. 458
      Zocher, F.; Enzelberger, M. M.; Bornscheuer, U. T.; Hauer, B. D.; Schmid, R. D. A Colorimetric Assay Suitable for Screening Epoxide Hydrolase Activity. Anal. Chim. Acta 1999, 391 (3), 345– 351,  DOI: 10.1016/S0003-2670(99)00216-0
      460
      A colorimetric assay suitable for screening epoxide hydrolase activity
      Zocher, Frank; Enzelberger, Markus M.; Bornscheuer, Uwe T.; Hauer, Bernhard; Schmid, Rolf D.
      Analytica Chimica Acta (1999), 391 (3), 345-351CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
      A UV/VIS spectrophotometric microtiter plate and a filter-paper based assay using 4-(p-nitrobenzyl)pyridine (NBP) were developed to det. epoxide hydrolytic activity by measuring the decrease of the epoxide concn. Both systems were applied for screening an expression gene bank of Rhodococcus sp. NCIMB 11216. As a ref., whole cells from Rhodococcus sp. NCIMB 11216 and Beauveria sulfurescens ATCC 7159 exhibiting epoxide hydrolase activity were used. The microtiter plate system was also evaluated for different epoxides and performed in a lab. robotic system for high throughput screening. The microtiter plate assay showed a high sensitivity for the detection of small concns. of epoxides (0.1-1 mg/well) such as styrene oxide, Et phenylglycidate, n-hexane oxide and indene oxide. The filter paper assay was further optimized for styrene oxide. Both assays were suitable to screen within libraries of epoxide hydrolases without interference with other enzymes such as esterases, lipases or proteases. The assay should allow to screen large libraries obtained by directed evolution, strain collections and (expression) gene banks for epoxide hydrolytic activity or to monitor the purifn. process of an epoxide hydrolase.
    459. 459
      Alcalde, M.; Farinas, E. T.; Arnold, F. H. Colorimetric High-Throughput Assay for Alkene Epoxidation Catalyzed by Cytochrome P450 BM-3 Variant 139–3. J. Biomol. Screen. 2004, 9 (2), 141– 6,  DOI: 10.1177/1087057103261913
      461
      Colorimetric high-throughput assay for alkene epoxidation catalyzed by cytochrome P450 BM-3 variant 139-3
      Alcalde, Miguel; Farinas, Edgardo T.; Arnold, Frances H.
      Journal of Biomolecular Screening (2004), 9 (2), 141-146CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
      Cytochrome P 450 BM-3 variant 139-3 is highly active in the hydroxylation of alkanes and fatty acids (A Glieder, ET Farinas, and FH Arnold, Nature Biotech 2002;20:1135-1139); it also epoxidizes various alkenes, including styrene. Here the authors describe a colorimetric, high-throughput assay suitable for optimizing this latter activity by directed evolution. The product of styrene oxidn. by 139-3, styrene oxide, reacts with the nucleophile γ-(4-nitrobenzyl)pyridine (NBP) to form a purple-colored precursor dye, which can be monitored spectrophotometrically in cell lysates. The sensitivity limit of this assay is 50-100 μM of product, and the detection limit for P 450 BM-3 139-3 is ∼0.2 μM of enzyme. To validate the assay, activities in a small library of random mutants were compared to those detd. using an NADPH depletion assay for initial turnover rates.
    460. 460
      Van Damme, F.; Oomens, A. C. Determination of Residual Free Epoxide in Polyether Polyols by Derivatization with Diethylammonium N,N-Diethyldithiocarbamate and Liquid Chromatography. J. Chromatogr. A 1995, 696 (1), 41– 7,  DOI: 10.1016/0021-9673(94)01270-O
      462
      Determination of residual free epoxide in polyether polyols by derivatization with diethylammonium N,N-diethyldithiocarbamate and liquid chromatography
      Van Damme, F.; Oomens, A. C.
      Journal of Chromatography A (1995), 696 (1), 41-7CODEN: JCRAEY; ISSN:0021-9673. (Elsevier)
      A normal-phase liq. chromatog. method is described for the detn. of epoxides and particularly residual ethylene oxide and propylene oxide in polyether polyols. The residual epoxides are derivatized with diethylammonium N,N-diethyldithiocarbamate into the corresponding 1-(N,N-diethyldithiocarbamoyl)-2-hydroxyethane and -hydroxypropane. The dithiocarbamoyl esters are extd. into chloroform and sepd. by normal-phase liq. chromatog. Detection is done by UV at 278 nm. Total anal. time is ∼45 min. Linearity (0.1-100 ppm), detection limits (0.5 ppm), recovery from spiked samples (>95%) and interferences were studied.
    461. 461
      Hammock, L. G.; Hammock, B. D.; Casida, J. E. Detection and Analysis of Epoxides with 4-(p-Nitrobenzyl)-Pyridine. Bull. Environ. Contam. Toxicol. 1974, 12 (6), 759– 64,  DOI: 10.1007/BF01685927
      463
      Detection and analysis of epoxides with 4-(p-nitrobenzyl)pyridine
      Hammock, Lassie G.; Hammock, Bruce D.; Casida, John E.
      Bulletin of Environmental Contamination and Toxicology (1974), 12 (6), 759-64CODEN: BECTA6; ISSN:0007-4861.
      Epoxides were detected on paper and thin-layer chromatograms and colorimetrically detd. at 600 nm by reaction with 4-(p-nitrobenzyl)pyridine (I) to form colored derivs. The developed thin-layer and paper chromatograms were sprayed with 2 wt. % I in Me2CO and by 1 wt. % I in 50% Me2CO contg. 0.5 N K acid phthalate, resp. The sprayed chromatograms were heated at 110° for 5 min and, after cooling, were sprayed with 10 vol. % tetraethylenepentamine in Me2CO to give blue spots on a white background. The sensitivities on thin layer and paper chromatograms were 0.01 - >13 and 0.01 - >6 μmole, resp. The method is insensitive for epoxycycloalkanes and related compds. The colorimetric method is useful in monitoring enzymic reactions with epoxide substrates. Incubation of 4-ethylphenyl 6,7-epoxygeranyl ether (II) with buffer, followed by extn. with pentane and colorimetric detn. of II by using I established that the detection limit is 0.2 μmole II and that Beer's law holds for ≤1.0 μmole II.
    462. 462
      Pedragosa-Moreau, S.; Morisseau, C.; Baratti, J.; Zylber, J.; Archelas, A.; Furstoss, R. Microbiological Transformations 37. An Enantioconvergent Synthesis of the β-Blocker (R)-NiféNalol Using a Combined Chemoenzymatic Approach. Tetrahedron 1997, 53 (28), 9707– 14,  DOI: 10.1016/S0040-4020(97)00639-X
      There is no corresponding record for this reference.
    463. 463
      Finney, N. S. Enantioselective Epoxide Hydrolysis: Catalysis Involving Microbes, Mammals and Metals. J. Biol. Chem. 1998, 5 (4), R73– R9,  DOI: 10.1016/S1074-5521(98)90630-5
      There is no corresponding record for this reference.
    464. 464
      Choi, W. J.; Choi, C. Y. Production of Chiral Epoxides: Epoxide Hydrolase-Catalyzed Enantioselective Hydrolysis. Biotechnol. Bioprocess Eng. 2005, 10 (3), 167,  DOI: 10.1007/BF02932009
      466
      Production of Chiral epoxides: Epoxide hydrolase-catalyzed enantioselective hydrolysis
      Choi, Won Jae; Choi, Cha Yong
      Biotechnology and Bioprocess Engineering (2005), 10 (3), 167-179CODEN: BBEIAU; ISSN:1226-8372. (Korean Society for Biotechnology and Bioengineering)
      A review. Chiral epoxides are highly valuable intermediates, used for the synthesis of pharmaceutical drugs and agrochems. They have broad scope of market demand because of their applications. A major challenge in modern org. chem. is to generate such compds. in high yields, with high stereo- and regio-selectivities. Epoxide hydrolases (EH) are promising biocatalysts for the prepn. of chiral epoxides and vicinal diols. They exhibit high enantioselectivity for their substrates, and can be effectively used in the resoln. of racemic epoxides through enantioselective hydrolysis. The selective hydrolysis of a racemic epoxide can produce both the corresponding diols and the unreacted epoxides with high enantiomeric excess (ee) value. The potential of microbial EH to produce chiral epoxides and vicinal diol has prompted researchers to explore their use in the synthesis of epoxides and diols with high ee values.
    465. 465
      Baer, H.; Bergamo, M.; Forlin, A.; Pottenger, L. H.; Lindner, J. Propylene Oxide. In Ullmann’s Encyclopedia of Industrial Chemistry: Wiley-VCH, 2000.
      There is no corresponding record for this reference.
    466. 466
      Rebsdat, S.; Mayer, D. Ethylene Oxide. In Ullmann’s Encyclopedia of Industrial Chemistry; Wiley-VCH, 2001.
      There is no corresponding record for this reference.
    467. 467
      Archelas, A.; Furstoss, R. Synthesis of Enantiopure Epoxides through Biocatalytic Approaches. Annu. Rev. Microbiol. 1997, 51 (1), 491– 525,  DOI: 10.1146/annurev.micro.51.1.491
      469
      Synthesis of enantiopure epoxides through biocatalytic approaches
      Archelas A; Furstoss R
      Annual review of microbiology (1997), 51 (), 491-525 ISSN:0066-4227.
      Enantiopure epoxides, as well as their corresponding vicinal diols, are valuable intermediates in fine organic synthesis, in particular for the preparation of biologically active compounds. The necessity of preparing such target molecules in an optically pure form has triggered much research, leading to the emergence of various new methods based on either conventional chemistry or enzymatically catalyzed reactions. In this review, we focus on the biocatalytic approaches, which include direct epoxidation of olefinic double bonds as well as indirect biocatalytic methods, and which allow for the synthesis of these important chiral building blocks in enantiomerically enriched or even enantiopure form.
    468. 468
      Ma, S. K.; Gruber, J.; Davis, C.; Newman, L.; Gray, D.; Wang, A.; Grate, J.; Huisman, G. W.; Sheldon, R. A. A Green-by-Design Biocatalytic Process for Atorvastatin Intermediate. Green Chem. 2010, 12 (1), 81– 6,  DOI: 10.1039/B919115C
      470
      A green-by-design biocatalytic process for atorvastatin intermediate
      Ma, Steven K.; Gruber, John; Davis, Chris; Newman, Lisa; Gray, David; Wang, Alica; Grate, John; Huisman, Gjalt W.; Sheldon, Roger A.
      Green Chemistry (2010), 12 (1), 81-86CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
      The development of a green-by-design, two-step, three-enzyme process for the synthesis of a key intermediate in the manuf. of atorvastatin, the active ingredient of the cholesterol lowering drug Lipitor, is described. The first step involves the biocatalytic redn. of ethyl-4-chloroacetoacetate using a ketoreductase (KRED) in combination with glucose and a NADP-dependent glucose dehydrogenase (GDH) for cofactor regeneration. The (S) ethyl-4-chloro-3-hydroxybutyrate product is obtained in 96% isolated yield and >99.5% e.e. In the second step, a halohydrin dehalogenase (HHDH) is employed to catalyze the replacement of the chloro substituent with cyano by reaction with HCN at neutral pH and ambient temp. The natural enzymes were highly selective but exhibited productivities that were insufficient for large scale application. Consequently, in vitro enzyme evolution using gene shuffling technologies was employed to optimize their performance according to predefined criteria and process parameters. In the case of the HHDH reaction, this afforded a 2500-fold improvement in the volumetric productivity per biocatalyst loading. This enabled the economical and environmentally attractive prodn. of the key hydroxynitrile intermediate. The overall process has an E factor (kg waste per kg product) of 5.8 when process water is not included, and 18 if included.
    469. 469
      Kong, X.-D.; Ma, Q.; Zhou, J.; Zeng, B.-B.; Xu, J.-H. A Smart Library of Epoxide Hydrolase Variants and the Top Hits for Synthesis of (S)-β-Blocker Precursors. Angew. Chem., Int. Ed. Engl. 2014, 53 (26), 6641– 4,  DOI: 10.1002/anie.201402653
      471
      A smart library of epoxide hydrolase variants and the top hits for synthesis of (S)-β-blocker precursors
      Kong Xu-Dong; Ma Qian; Zhou Jiahai; Zeng Bu-Bing; Xu Jian-He
      Angewandte Chemie (International ed. in English) (2014), 53 (26), 6641-4 ISSN:.
      Microtuning of the enzyme active pocket has led to a smart library of epoxide hydrolase variants with an expanded substrate spectrum covering a series of typical β-blocker precursors. Improved activities of 6- to 430-fold were achieved by redesigning the active site at two predicted hot spots. This study represents a breakthrough in protein engineering of epoxide hydrolases and resulted in enhanced activity toward bulky substrates.
    470. 470
      Lin, Z.; Xue, Y.; Liang, X.-W.; Wang, J.; Lin, S.; Tao, J.; You, S.-L.; Liu, W. Oxidative Indole Dearomatization for Asymmetric Furoindoline Synthesis by a Flavin-Dependent Monooxygenase Involved in the Biosynthesis of Bicyclic Thiopeptide Thiostrepton. Angew. Chem. 2021, 60 (15), 8401– 5,  DOI: 10.1002/anie.202013174
      472
      Oxidative Indole Dearomatization for Asymmetric Furoindoline Synthesis by a Flavin-Dependent Monooxygenase Involved in the Biosynthesis of Bicyclic Thiopeptide Thiostrepton
      Lin, Zhi; Xue, Yufeng; Liang, Xiao-Wei; Wang, Jian; Lin, Shuangjun; Tao, Jiang; You, Shu-Li; Liu, Wen
      Angewandte Chemie, International Edition (2021), 60 (15), 8401-8405CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The interest in indole dearomatization, which serves as a useful tool in the total synthesis of related alkaloid natural products, has recently been renewed with the intention of developing new methods efficient in both yield and stereoselective control. Here, we report an enzymic approach for the oxidative dearomatization of indoles in the asym. synthesis of a variety of furoindolines with a vicinal quaternary carbon stereogenic center. This approach depends on the activity of a flavin-dependent monooxygenase, TsrE, which is involved in the biosynthesis of bicyclic thiopeptide antibiotic thiostrepton. TsrE catalyzes 2,3-epoxidn. and subsequent epoxide opening in a highly enantioselective manner during the conversion of 2-methyl-indole-3-acetic acid or 2-methyl-tryptophol to furoindoline, with up to >99% conversion and >99% ee under mild reaction conditions. Complementing current chem. methods for oxidative indole dearomatization, the TsrE activity-based approach enriches the toolbox in the asym. synthesis of products possessing a furoindoline skeleton.
    471. 471
      Agarwal, S. C.; Van Duuren, B. L.; Solomon, J. J.; Kline, S. A. Reaction of Epoxides with 4-Nitrothiophenol. Its Possible Application for Trapping and Characterization of Epoxides. Environ. Sci. Technol. 1980, 14 (10), 1249– 53,  DOI: 10.1021/es60170a012
      473
      Reaction of epoxides with 4-nitrothiophenol. Its possible application for trapping and characterization of epoxides
      Agarwal, Satish C.; Van Duuren, Benjamin L.; Solomon, Jerome J.; Kline, Stanley A.
      Environmental Science and Technology (1980), 14 (10), 1249-53CODEN: ESTHAG; ISSN:0013-936X.
      Reaction of epoxides with the nucleophile 4-nitrothiophenol [1849-36-1] has been examd. The mono- and diepoxides of aliph. and arom. hydrocarbons undergo facile nucleophilic opening by 4-nitrothiophenol to yield the expected 1,2 adducts cleanly and in good yields. The exptl. conditions provide a suitable method for trapping the atm. epoxides which may be significant in air pollution carcinogenesis. In the chem. ionization mass spectrum, these adducts have an intense protonated mol. ion which will be extremely useful in their detection and characterization by the technique of single ion monitoring.
    472. 472
      Wixtrom, R. N.; Hammock, B. D. Continuous Spectrophotometric Assays for Cytosolic Epoxide Hydrolase. Anal. Biochem. 1988, 174 (1), 291– 9,  DOI: 10.1016/0003-2697(88)90548-9
      474
      Continuous spectrophotometric assays for cytosolic epoxide hydrolase
      Wixtrom, Roger N.; Hammock, Bruce D.
      Analytical Biochemistry (1988), 174 (1), 291-9CODEN: ANBCA2; ISSN:0003-2697.
      Two convenient and sensitive continuous spectrophotometric assays for cytosolic epoxide hydrolase (I) are described. The assays are based on the differences in the UV spectra of the epoxide substrates and their diol products. The hydrolysis of 1,2-epoxy-1-(p-nitrophenyl)pentane (ENP5) was accompanied by a decrease in absorbance at 302 nm, whereas the hydration of 1,2-epoxy-1-(2-quinolyl)pentane (EQU5) produced an increase in absorbance at 315.5 nm. The Km and Vmax values for ENP5 and EQU5 with purified mouse liver cytosol I were 1.7 and 25 μM and 11,700 and 8300 nmol/min/mg, resp.; these results compared with previously reported values of 5 μM and 3300 nmol/min/mg for trans-stilbene oxide (TSO) with purified mouse liver I. Thus, both substrates were hydrolyzed significantly faster than TSO, which is currently the most commonly used substrate for measuring cytosolic I activity. No spontaneous hydrolysis of the substrates was detectable under normal assay conditions. The assays were applicable to whole tissue homogenates as well as purified enzyme prepns. p-Nitrostyrene oxide and p-nitrophenyl glycidyl ether were also examd. and found to be very poor substrates for cytosolic I from mouse liver.
    473. 473
      González-Pérez, M.; Gómez-Bombarelli, R.; Arenas-Valgañón, J.; Pérez-Prior, M. T.; García-Santos, M. P.; Calle, E.; Casado, J. Connecting the Chemical and Biological Reactivity of Epoxides. Chem. Res. Toxicol. 2012, 25 (12), 2755– 62,  DOI: 10.1021/tx300389z
      475
      Connecting the Chemical and Biological Reactivity of Epoxides
      Gonzalez-Perez, Marina; Gomez-Bombarelli, Rafael; Arenas-Valganon, Jorge; Perez-Prior, M. Teresa; Garcia-Santos, M. Pilar; Calle, Emilio; Casado, Julio
      Chemical Research in Toxicology (2012), 25 (12), 2755-2762CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      The chem. reactivity of the mutagenic epoxides (EP) propylene oxide (PO), 1,2-epoxybutane (1,2-EB), and cis- and trans-2,3-epoxybutane (cis- and trans-2,3-EB) with 4-(p-nitrobenzyl)pyridine (NBP), a bionucleophile model for SN2 alkylating agents with high affinity for the guanine-N7 position, was investigated kinetically. It was found that three reactions are involved simultaneously: the alkylation reaction of NBP by EP, which yields the corresponding NBP-EP adducts through an SN2 mechanism, and EP and NBP-EP hydrolysis reactions. PO and 1,2-EB were seen to exhibit a higher alkylating potential than cis- and trans-2,3-EB. From a study of the correlations between the chem. reactivity (kinetic parameters) and the biol. effectiveness of oxiranes, the following conclusions can be drawn: (i) the hydrolysis reactions of epoxides must be taken into account to understand their bioactivity; (ii) the fraction (f) of the alkylating oxirane that forms the adduct and the adduct life (AL) permit the potential of epoxides as bioactive mols. to be rationalized even semiquant.; and (iii) alkylation of DNA by epoxides and the O6-/N7-guanine adduct ratio are directly related to their mutagenicity in vitro.
    474. 474
      Nelis, H. J. C. F.; Sinsheimer, J. E. A Sensitive Fluorimetric Procedure for the Determination of Aliphatic Epoxides under Physiological Conditions. Anal. Biochem. 1981, 115 (1), 151– 7,  DOI: 10.1016/0003-2697(81)90538-8
      476
      A sensitive fluorimetric procedure for the determination of aliphatic epoxides under physiological conditions
      Nelis, Hans J. C. F.; Sinsheimer, J. E.
      Analytical Biochemistry (1981), 115 (1), 151-7CODEN: ANBCA2; ISSN:0003-2697.
      A highly sensitive fluorometric procedure based on alkylation of nicotinamide [98-92-0] is described for the detn. of aliph. epoxides. Subsequent reaction of the resulting N-alkyl nicotinamides with a ketone in basic medium yields strongly fluorescent products after final acidification. Sensitivity of the assay is in the picomole range with good reproducibility. The alkylation reaction proceeds under physiol. conditions and thus shows potential for the detn. of epoxides in biol. materials. Despite rapid enzymic detoxification, styrene oxide [75-56-9] could be detected directly in 9000 g supernatant liver fractions by the present approach.
    475. 475
      Rink, R.; Fennema, M.; Smids, M.; Dehmel, U.; Janssen, D. B. Primary Structure and Catalytic Mechanism of the Epoxide Hydrolase from Agrobacterium Radiobacter Ad1. J. Biol. Chem. 1997, 272 (23), 14650– 7,  DOI: 10.1074/jbc.272.23.14650
      477
      Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1
      Rink, Rick; Fennema, Marko; Smids, Minke; Dehmel, Uwe; Janssen, Dick B.
      Journal of Biological Chemistry (1997), 272 (23), 14650-14657CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme. The epoxide hydrolase gene coded for a protein of 294 amino acids with a mol. mass of 34 kDa. An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A. radiobacter CFZ11. The recombinant epoxide hydrolase was expressed up to 40% of the total cellular protein content in Escherichia coli BL21(DE3) and the purified enzyme had a kcat of 21 s-1 with epichlorohydrin. Amino acid sequence similarity of the epoxide hydrolase with eukaryotic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the α/β-hydrolase fold family. This conclusion was supported by secondary structure predictions and anal. of the secondary structure with CD spectroscopy. The catalytic triad residues of epoxide hydrolase are proposed to be Asp107, His275, and Asp246. Replacement of these residues to Ala/Glu, Arg/Gln, and Ala, resp., resulted in a dramatic loss of activity for epichlorohydrin. The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate, as was shown by single turnover expts. with the His275 → Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.
    476. 476
      Elder, D. P.; Snodin, D.; Teasdale, A. Analytical Approaches for the Detection of Epoxides and Hydroperoxides in Active Pharmaceutical Ingredients, Drug Products and Herbals. J. Pharm. Biomed. Anal. 2010, 51 (5), 1015– 23,  DOI: 10.1016/j.jpba.2009.11.023
      478
      Analytical approaches for the detection of epoxides and hydroperoxides in active pharmaceutical ingredients, drug products, and herbals
      Elder, D. P.; Snodin, D.; Teasdale, A.
      Journal of Pharmaceutical and Biomedical Analysis (2010), 51 (5), 1015-1023CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
      This review summarizes the anal. approaches reported in the literature relating to epoxide and hydroperoxide impurities. It is intended that it should provide guidance for analysts faced by the need to control such impurities, particularly where this is due to concerns relating to their potential genotoxicity. An extensive search of the literature relating to this class of impurities revealed a large no. of refs. relating to anal. of epoxides/hydroperoxides assocd. with herbal remedies. Given the general applicability of the anal. methodol. and due to the widespread use of herbal products the authors decided to include herbal medicines in this review. The review also reflects on the very different approaches taken in terms of the assessment/control of genotoxic impurities for such herbal remedies to that required for pharmaceutical products.
    477. 477
      Sano, A.; Takitani, S. Spectrofluorometric Determination of Common Epoxides with Sodium Sulfide and o-Phthalaldehyde and Taurine Reagents. Anal. Chem. 1985, 57 (8), 1687– 90,  DOI: 10.1021/ac00285a039
      479
      Spectrofluorometric determination of common epoxides with sodium sulfide and o-phthalaldehyde and taurine reagents
      Sano, Akira; Takitani, Shoji
      Analytical Chemistry (1985), 57 (8), 1687-90CODEN: ANCHAM; ISSN:0003-2700.
      A spectrofluorometric method was developed for the detn. of common epoxides. Epoxides in EtOH soln. gave an intense blue fluorescence (λ(excitation) ∼345 nm and λ(emission) ∼440 nm), after the 1st reaction with aq. Na2S at 55° for 20 min and followed the 2nd reaction with taurine and o-phthalaldehyde reagents at pH 8.3. By the proposed method, 1,2-epoxy-3-phenoxypropane and 1,2-epoxyoctane can be detd. in the ranges 0.05-3 nmol/100 μL and 0.1-8 nmol/100 μL, resp., with relative std. deviations of 1.6-2.9%. Some other alkylating agents also showed fluorescence by this method.
    478. 478
      Tang, L.; Li, Y.; Wang, X. A High-Throughput Colorimetric Assay for Screening Halohydrin Dehalogenase Saturation Mutagenesis Libraries. J. Biotechnol. 2010, 147 (3), 164– 8,  DOI: 10.1016/j.jbiotec.2010.04.002
      480
      A high-throughput colorimetric assay for screening halohydrin dehalogenase saturation mutagenesis libraries
      Tang, Lixia; Li, Yang; Wang, Xiong
      Journal of Biotechnology (2010), 147 (3-4), 164-168CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)
      Here, a high throughput pH indicator-based assay to measure the activity of halohydrin dehalogenases (HheC) is reported. The assay relies upon the absorbance change at 560 nm and the visual color change of phenol red in a weakly buffered system, due to the release of protons from the enzyme-catalyzed ring-closure reactions. The assay can be performed in a microplate format using whole cells, making the assay simple and robust. Thus, it is suitable for library screening. The assay has been further validated using two previously studied HheC variants, D80N and W249F, which exhibit 200-fold lower and 2-fold higher kcat values, resp., toward 1,3-dichloro-2-propanol (1,3-DCP) than the wild-type HheC. In addn., a satn. mutagenesis library of HheC was screened using the developed assay for its ability to efficiently catalyze the conversion of 1,3-DCP. After screening of 500 colonies, one mutant W139C was identified and was further purified and characterized. Kinetic anal. indicates that the resulting mutant shows 2- and 5-fold improvement in kcat value toward 1,3-DCP and (R,S)-p-nitro-2-bromo-1-phenylethanol, resp., although it exhibits higher Km values than the wild-type enzyme. The method described herein represents a useful tool given the need for the high throughput screening of halohydrin dehalogenase mutants.
    479. 479
      Gul, I.; Bogale, T. F.; Deng, J.; Chen, Y.; Fang, R.; Feng, J.; Tang, L. Enzyme-Based Detection of Epoxides Using Colorimetric Assay Integrated with Smartphone Imaging. Biotechnol. Appl. Biochem. 2020, 67 (4), 685– 92,  DOI: 10.1002/bab.1898
      481
      Enzyme-based detection of epoxides using colorimetric assay integrated with smartphone imaging
      Gul, Ijaz; Bogale, Tadesse Fantaye; Deng, Jiao; Chen, Yong; Fang, Ruiqin; Feng, Juan; Tang, Lixia
      Biotechnology and Applied Biochemistry (2020), 67 (4), 685-692CODEN: BABIEC; ISSN:0885-4513. (Wiley-Blackwell)
      Epoxides are widely used chems., the detn. of which is of paramount importance. Herein, we present an enzyme-based approach for noninstrumental detection of epoxides in std. soln. and environmental samples. Halohydrin dehalogenase (HheC) as a biol. recognition element and epichlorohydrin as a model analyte were evaluated for sensing. The detection is based on the color change of the pH indicator dye bromothymol blue caused by the HheC-catalyzed ring-opening of the epoxide substrate. The color change is then exploited for the detn. of epoxide using a smartphone as an image acquisition and data processing device, eliminating the need for computer-based image anal. software. The color parameters were systematically evaluated to det. the optimum quant. anal. parameter. Under optimal conditions, the proposed enzyme-based detection system showed a linear range of 0.13-2 mM with a detection limit of 0.07 mM and an assay time of 8 Min. In addn., the repeatability expressed as relative std. deviation was found to be below 5% (n = 6). Validation with gas chromatog. analyses showed that the proposed enzyme-based epoxide detection could be an alternative way in the quant. detn. of epoxides, and particularly useful in resource-limited settings.
    480. 480
      Dansette, P. M.; DuBois, G. C.; Jerina, D. M. Continuous Fluorometric Assay of Epoxide Hydrase Activity. Anal. Biochem. 1979, 97 (2), 340– 5,  DOI: 10.1016/0003-2697(79)90083-6
      482
      Continuous fluorometric assay of epoxide hydrase activity
      Dansette, Patrick M.; DuBois, Garrett C.; Jerina, Donald M.
      Analytical Biochemistry (1979), 97 (2), 340-5CODEN: ANBCA2; ISSN:0003-2697.
      A rapid, continuous, and highly sensitive fluorescence assay is described for the measurement of epoxide hydrase activity. The method is based on the large differences between the fluorescence spectra of certain K-region arene oxides and their corresponding trans-dihydrodiols. Enzymic hydration of K-region arene oxides of phenanthrene, pyrene, benzo[a]pyrene, and 7,12-dimethylbenzo[a]anthracene was studied. The assay was most sensitive with benzo[a]pyrene-4,5-oxide as substrate. With 10 μM benzo[a]pyrene-4,5-oxide, enzymic rates of 30 pmol dihydrodiol/min/mg protein were 3- to 5-fold those of the blank without enzyme. The fluorometric method described was used to study site-directed inhibitors of epoxide hydrase and the stereoselective hydration of racemic arene oxides.
    481. 481
      Mateo, C.; Archelas, A.; Furstoss, R. A Spectrophotometric Assay for Measuring and Detecting an Epoxide Hydrolase Activity. Anal. Biochem. 2003, 314 (1), 135– 41,  DOI: 10.1016/S0003-2697(02)00646-2
      483
      A spectrophotometric assay for measuring and detecting an epoxide hydrolase activity
      Mateo, Cesar; Archelas, Alain; Furstoss, Roland
      Analytical Biochemistry (2003), 314 (1), 135-141CODEN: ANBCA2; ISSN:0003-2697. (Elsevier Science)
      The development of a novel and simple spectrophotometric assay which allows one to achieve the continuous, rapid, sensitive, and accurate detn. of epoxide hydrolase (I) activity is reported. This assay is based on the elaboration of a coupled enzymic/chem. methodol. which allows quantification of I activity within 3 min, and offers good sensitivity of ∼10 μM min-1. Thus, an arom. epoxide such as styrene oxide is hydrolyzed by I to 1-phenyl-1,2-ethanediol, which in turn is oxidized to benzaldehyde by Na metaperiodate; the benzaldehyde formed can be visualized at very low levels at 290 nm thanks to its very strong UV absorbency (ε = 1356 M-1 cm-1). I activity can therefore be detd. continuously, with great sensitivity and accuracy, simply by measuring, at very low conversion ratio, the increase in UV absorbency. The applicability of this test for the detn. of Aspergillus niger I activity with some other arom. epoxides was shown and some limitations were also explored. This assay should be particularly useful for different applications, e.g., (1) activity localization during purifn. of such enzymes, (2) very rapid detn. of kinetic consts., and (3) high-throughput screening expts.
    482. 482
      Michałowicz, J.; Duda, W. Phenols - Sources and Toxicity. Poish J. Environ. Stud. 2007, 16 (3), 347– 362
      484
      Phenols- sources and toxicity
      Michalowicz, J.; Duda, W.
      Polish Journal of Environmental Studies (2007), 16 (3), 347-362CODEN: PJESE2; ISSN:1230-1485. (HARD Publishing Co.)
      A review. Phenols and their derivs. commonly exist in the environment. These compds. are used as the components of dyes, polymers, drugs and other org. substances. The presence of phenols in the ecosystems is also related with prodn. and degrdn. of numerous pesticides and the generation of industrial and municipal sewages. Some phenols are also formed during natural processes. These compds. may be substituted with chlorine atoms, may be nitrated, methylated or alkylated. Both phenols and catechols are harmful ecotoxins. Toxic action of these compds. stems from unspecified toxicity related to hydrophobocity and also to the generation of org. radicals and reactive oxygen species. Phenols and catechols reveal peroxidative capacity, they are hematotoxic and hepatotoxic, provoke mutagenesis and carcinogenesis toward humans and other living organisms.
    483. 483
      Schmidt, R. J. Industrial Catalytic Processes─Phenol Production. Appl. Catal. A: Gen. 2005, 280 (1), 89– 103,  DOI: 10.1016/j.apcata.2004.08.030
      485
      Industrial catalytic processes - phenol production
      Schmidt, Robert J.
      Applied Catalysis, A: General (2005), 280 (1), 89-103CODEN: ACAGE4; ISSN:0926-860X. (Elsevier B.V.)
      A review on the development of catalytic processes for phenol prodn.
    484. 484
      Ullrich, R.; Hofrichter, M. Enzymatic Hydroxylation of Aromatic Compounds. Cell. Mol. Life Sci. 2007, 64 (3), 271– 93,  DOI: 10.1007/s00018-007-6362-1
      486
      Enzymatic hydroxylation of aromatic compounds
      Ullrich, Rene; Hofrichter, Martin
      Cellular and Molecular Life Sciences (2007), 64 (3), 271-293CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
      A review. Selective hydroxylation of arom. compds. is among the most challenging chem. reactions in synthetic chem. and has gained steadily increasing attention during recent years, particularly because of the use of hydroxylated aroms. as precursors for pharmaceuticals. Biocatalytic oxygen transfer by isolated enzymes or whole microbial cells is an elegant and efficient way to achieve selective hydroxylation. This review gives an overview of the different enzymes and mechanisms used to introduce oxygen atoms into arom. mols. using either dioxygen (O2) or hydrogen peroxide (H2O2) as oxygen donors or indirect pathways via free radical intermediates. In this context, the article deals with Rieske-type and α-keto acid-dependent dioxygenases, as well as different non-heme monooxygenases (di-iron, pterin, and flavin enzymes), tyrosinase, laccase, and hydroxyl radical generating systems. The main emphasis is on the heme-contg. enzymes, cytochrome P 450 monooxygenases and peroxidases, including novel extracellular heme-thiolate haloperoxidases (peroxygenases), which are functional hybrids of both types of heme-biocatalysts.
    485. 485
      Lan Tee, K.; Schwaneberg, U. Directed Evolution of Oxygenases: Screening Systems, Success Stories and Challenges. Comb. Chem. High Throughput Screen. 2007, 10 (3), 197– 217,  DOI: 10.2174/138620707780126723
      There is no corresponding record for this reference.
    486. 486
      Joern, J. M.; Sakamoto, T.; Arisawa, A.; Arnold, F. H. A Versatile High Throughput Screen for Dioxygenase Activity Using Solid-Phase Digital Imaging. J. Biomol. Screen. 2001, 6 (4), 219– 23,  DOI: 10.1177/108705710100600403
      488
      A versatile high throughput screen for dioxygenase activity using solid-phase digital imaging
      Joern, John M.; Sakamoto, Takeshi; Arisawa, Akira; Arnold, Frances H.
      Journal of Biomolecular Screening (2001), 6 (4), 219-223CODEN: JBISF3; ISSN:1087-0571. (Mary Ann Liebert, Inc.)
      We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-dihydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aroms. to yield colored products that are quantifiable using a microplate reader or by digital imaging and image anal. The method is reproducible and quant. at product concns. of only 30 μM, with essentially no background from media components. This method is an effective general screen for arom. oxidn. and should be a useful tool for the discovery and directed evolution of oxygenases.
    487. 487
      Poraj-Kobielska, M.; Kinne, M.; Ullrich, R.; Scheibner, K.; Kayser, G.; Hammel, K. E.; Hofrichter, M. Preparation of Human Drug Metabolites Using Fungal Peroxygenases. Biochem. Pharmacol. 2011, 82 (7), 789– 96,  DOI: 10.1016/j.bcp.2011.06.020
      489
      Preparation of human drug metabolites using fungal peroxygenases
      Poraj-Kobielska, Marzena; Kinne, Matthias; Ullrich, Rene; Scheibner, Katrin; Kayser, Gernot; Hammel, Kenneth E.; Hofrichter, Martin
      Biochemical Pharmacology (2011), 82 (7), 789-796CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
      The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic org. chemists. Here we report that arom. peroxygenases (APOs; E.C. 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and Coprinellus radians catalyzed the H2O2-dependent selective monooxygenation of diverse drugs, including acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions included the hydroxylation of arom. rings and aliph. side chains, as well as O- and N-dealkylations and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs were obtained in yields greater than 80% and with isomeric purities up to 99%. Oxidns. of tolbutamide, acetanilide and carbamazepine in the presence of H2 18O2 resulted in almost complete incorporation of 18O into the corresponding products, thus establishing that these reactions are peroxygenations. The deethylation of phenacetin-d 1 showed an obsd. intramol. deuterium isotope effect [(k H/k D)obs] of 3.1±0.2, which is consistent with the existence of a cytochrome P 450-like intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful biocatalytic tools to prep. pharmacol. relevant drug metabolites.
    488. 488
      Sakamoto, T.; Joern, J. M.; Arisawa, A.; Arnold, F. H. Laboratory Evolution of Toluene Dioxygenase to Accept 4-Picoline as a Substrate. Appl. Environ. Microbiol. 2001, 67 (9), 3882,  DOI: 10.1128/AEM.67.9.3882-3887.2001
      490
      Laboratory evolution of toluene dioxygenase to accept 4-picoline as a substrate
      Sakamoto, Takeshi; Joern, John M.; Arisawa, Akira; Arnold, Frances H.
      Applied and Environmental Microbiology (2001), 67 (9), 3882-3887CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
      Directed evolution is being used by the authors to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here, the authors report on the oxidn. of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by lab. evolution. The biotransformation of 4-picoline proceeds at only ∼4.5% of the rate of the natural reaction on toluene. Random mutagenesis, satn. mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding Thr. Escherichia coli-expressed TDO 3-B38 exhibited 5.6-fold higher activity toward 4-picoline and ∼20% more activity toward toluene than did wild-type TDO. The product of the biotransformation of 4-picoline was 3-hydroxy-4-picoline; no cis-diols of 4-picoline were obsd.
    489. 489
      Foti, M. C. Antioxidant Properties of Phenols. J. Pharm. Pharmacol. 2010, 59 (12), 1673– 85,  DOI: 10.1211/jpp.59.12.0010
      There is no corresponding record for this reference.
    490. 490
      del Rosario Garcia-Mateos, M.; Palma-Tenango, M.; Soto-Hernandez, M. Phenolic Compounds-Biological Activity; IntechOpen, 2017.
      There is no corresponding record for this reference.
    491. 491
      Ainsworth, E. A.; Gillespie, K. M. Estimation of Total Phenolic Content and Other Oxidation Substrates in Plant Tissues Using Folin-Ciocalteu Reagent. Nat. Protoc. 2007, 2 (4), 875– 7,  DOI: 10.1038/nprot.2007.102
      494
      Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent
      Ainsworth, Elizabeth A.; Gillespie, Kelly M.
      Nature Protocols (2007), 2 (4), 875-877CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      Non-structural phenolic compds. perform a variety of functions in plants, including acting as antioxidants. The authors describe a microplate-adapted colorimetric total phenolics assay that utilizes Folin-Ciocalteu (F-C) reagent. The F-C assay relies on the transfer of electrons in alk. medium from phenolic compds. to phosphomolybdic/phosphotungstic acid complexes, which are detd. spectroscopically at 765 nm. Although the electron transfer reaction is not specific for phenolic compds., the extn. procedure eliminates approx. 85% of ascorbic acid and other potentially interfering compds. This assay is performed in microcentrifuge tubes and assessed in a 96-well plate reader. At least 64 samples can be processed in 1 d.
    492. 492
      Singleton, V. L.; Orthofer, R.; Lamuela-Raventós, R. M. Analysis of Total Phenols and Other Oxidation Substrates and Antioxidants by Means of Folin–Ciocalteu Reagent. Methods in Enzymology; Academic Press, 1999; Vol. 299; p 152– 178. DOI: 10.1016/S0076-6879(99)99017-1
      There is no corresponding record for this reference.
    493. 493
      Platzer, M.; Kiese, S.; Herfellner, T.; Schweiggert-Weisz, U.; Eisner, P. How Does the Phenol Structure Influence the Results of the Folin-Ciocalteu Assay?. Antioxidants 2021, 10 (5), 811,  DOI: 10.3390/antiox10050811
      496
      How does the phenol structure influence the results of the folin-ciocalteu assay?
      Platzer, Melanie; Kiese, Sandra; Herfellner, Thomas; Schweiggert-Weisz, Ute; Eisner, Peter
      Antioxidants (2021), 10 (5), 811CODEN: ANTIGE; ISSN:2076-3921. (MDPI AG)
      Plants produce a diverse array of secondary metabolites that are generally nonessential but facilitate ecol. interactions. Fruits, vegetables, seeds and nuts can accumulate bioactive secondary metabolites with health-promoting properties, including the potent antioxidant activities of phenolic compds. Several in vitro assays have been developed to measure the polyphenol content and antioxidant activity of plant exts., e.g., the simple and highly popular Folin-Ciocalteu (FC) assay. However, the literature contains a no. of different descriptions of the assay and it is unclear whether the assay measures the polyphenol content or reducing capacity of the sample. To det. the influence of phenolic structures on the outcome of the FC assay, we tested phenols representing different subgroups (phenolic acids, flavonols, flavanols, dihydrochalcones and flavanones). We obsd. different results for each ref. substance and subgroup. Accordingly, we concluded that the FC assay does not measure the polyphenol content of a sample but dets. its reducing capacity instead. Assigning the substances to five structural classes showed that the FC results depend on the no. of fulfilled Bors criteria. If a mol. fulfills none of the Bors criteria, the FC results depend on the no. of OH groups. We did not find a correlation with other single electron transfer assays (e.g., ABTS and DPPH assays). Furthermore, the FC assay was compatible with all five subgroups and should be preferred over the DPPH assay, which is specific for exts. rich in dihydrochalcones or flavanones.
    494. 494
      Miliauskas, G.; Venskutonis, P. R.; van Beek, T. A. Screening of Radical Scavenging Activity of Some Medicinal and Aromatic Plant Extracts. Food Chem. 2004, 85 (2), 231– 7,  DOI: 10.1016/j.foodchem.2003.05.007
      There is no corresponding record for this reference.
    495. 495
      Cao, G.; Alessio, H. M.; Cutler, R. G. Oxygen-Radical Absorbance Capacity Assay for Antioxidants. Free Radical Biol. Med. 1993, 14 (3), 303– 11,  DOI: 10.1016/0891-5849(93)90027-R
      498
      Oxygen-radical absorbance capacity assay for antioxidants
      Cao, Guohua; Alessio, Helaine M.; Cutler, Richard G.
      Free Radical Biology & Medicine (1993), 14 (3), 303-11CODEN: FRBMEH; ISSN:0891-5849.
      A relatively simple but sensitive and reliable method of quantitating the oxygen-radical absorbing capacity (ORAC) of antioxidants in serum using a few μL is described. In this assay system, β-phycoerythrin (β-PE) is used as an indicator protein, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-sol. vitamin E analog) as a control std. Results are expressed as ORAC units, where 1 ORAC unit equals the net protection produced by 1 μM Trolox. The uniqueness of this assay is that total antioxidant capacity of a sample is estd. by taking the oxidn. reaction to completion. At this point all of the nonprotein antioxidants (which include α-tocopherol, vitamin C, β-carotene, uric acid, and bilirubin) and most of the albumin in the sample are oxidized by the peroxyl radical. Results are quantified by measuring the protection produced by antioxidants. This solves many problems assocd. with kinetics or lag-time measurements. A linear correlation of ORAC value with concn. of serum, Trolox, vitamin C, uric acid, and bovine albumin is demonstrated. The coeff. of variation within a run is found to be about 2% and from run to run about 5%. Trolox, α-tocopherol, vitamin C, β-carotene, uric acid, and bilirubin completely protect β-PE from oxidn., while bovine albumin protects β-PE only partially. On a molar basis, the relative peroxyl radical absorbance capacity of Trolox, α-tocopherol acid succinate, uric acid, bilirubin, and vitamin C is 1:1:0.92:0.84:0.52. Bovine albumin per unit wt. has a lower peroxyl absorbing capacity than these antioxidants. However, the serum protein fraction, contg. some lipid-sol. antioxidants, represents the major contributor to the ORAC value found in whole serum. The min. amt. of vitamin C and uric acid which could still be detectable when added to a serum supernatant fraction is 1.5 μg and 0.59 μg, resp., which account for about 1% of the total ORAC value of the serum supernatant fraction.
    496. 496
      Prior, R. L.; Wu, X.; Schaich, K. Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements. J. Agric. Food Chem. 2005, 53 (10), 4290– 302,  DOI: 10.1021/jf0502698
      499
      Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements
      Prior, Ronald L.; Wu, Xianli; Schaich, Karen
      Journal of Agricultural and Food Chemistry (2005), 53 (10), 4290-4302CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      A review. Methods available for the measurement of antioxidant capacity are discussed, presenting the general chem. underlying the assays, the types of mols. detected, and the most important advantages and shortcomings of each method. This overview provides a basis and rationale for developing standardized antioxidant capacity methods for the food, nutraceutical, and dietary supplement industries. From evaluation of data presented at the First International Congress on Antioxidant Methods in 2004 and in the literature, as well as consideration of potential end uses of antioxidants, it is proposed that procedures and applications for three assays be considered for standardization: the oxygen radical absorbance capacity (ORAC) assay, the Folin-Ciocalteu method, and possibly the Trolox equiv. antioxidant capacity (TEAC) assay. ORAC represent a hydrogen atom transfer (HAT) reaction mechanism, which is most relevant to human biol. The Folin-Ciocalteu method is an electron transfer (ET) based assay and gives reducing capacity, which has normally been expressed as phenolic contents. The TEAC assay represents a second ET-based method.
    497. 497
      van den Berg, R.; Haenen, G. R. M. M.; van den Berg, H.; Bast, A. Applicability of an Improved Trolox Equivalent Antioxidant Capacity (TEAC) Assay for Evaluation of Antioxidant Capacity Measurements of Mixtures. Food Chem. 1999, 66 (4), 511– 7,  DOI: 10.1016/S0308-8146(99)00089-8
      500
      Applicability of an improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant capacity measurements of mixtures
      Van den Berg, Robin; Haenen, Guido R. M. M.; Van den Berg, Henk; Bast, Aalt
      Food Chemistry (1999), 66 (4), 511-517CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Science Ltd.)
      The TEAC (Trolox equiv. antioxidant capacity) assay is based on scavenging of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical anions (ABTS-). In this report we describe a modification based on pre-generation of the ABTS radical anions with a thermolabile azo compd., 2,2'-azobis(2-amidinopropane)-2HCl (ABAP). This modification makes the assay less susceptible to artifacts, e.g. influence on the radical generation process. For most antioxidants tested, a biphasic reaction pattern was seen, i.e. a fast and slow scavenging rate. We evaluated application of the assay with both lipophilic and hydrophilic compds. with antioxidant capacity. Several org. solvents, compatible with water, were tested with α-tocopherol, quercetin and β-carotene. It was found that the TEACs differed in various solvents. Under standardized conditions additivity of TEACs obtained from individual antioxidants could be demonstrated. This might enable application of the assay for the identification of "unknown" antioxidants.
    498. 498
      Dudonné, S.; Vitrac, X.; Coutière, P.; Woillez, M.; Mérillon, J.-M. Comparative Study of Antioxidant Properties and Total Phenolic Content of 30 Plant Extracts of Industrial Interest Using DPPH, ABTS, FRAP, SOD, and ORAC Assays. J. Agric. Food Chem. 2009, 57 (5), 1768– 74,  DOI: 10.1021/jf803011r
      501
      Comparative Study of Antioxidant Properties and Total Phenolic Content of 30 Plant Extracts of Industrial Interest Using DPPH, ABTS, FRAP, SOD, and ORAC Assays
      Dudonne, Stephanie; Vitrac, Xavier; Coutiere, Philippe; Woillez, Marion; Merillon, Jean-Michel
      Journal of Agricultural and Food Chemistry (2009), 57 (5), 1768-1774CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      Aq. exts. of 30 plants were investigated for their antioxidant properties using DPPH and ABTS radical scavenging capacity assay, oxygen radical absorbance capacity (ORAC) assay, superoxide dismutase (SOD) assay, and ferric reducing antioxidant potential (FRAP) assay. Total phenolic content was also detd. by the Folin-Ciocalteu method. Antioxidant properties and total phenolic content differed significantly among selected plants. It was found that oak (Quercus robur), pine (Pinus maritima), and cinnamon (Cinnamomum zeylanicum) aq. exts. possessed the highest antioxidant capacities in most of the methods used, and thus could be potential rich sources of natural antioxidants. These exts. presented the highest phenolic content (300-400 mg GAE/g). Mate (Ilex paraguariensis) and clove (Eugenia caryophyllus clovis) aq. exts. also showed strong antioxidant properties and a high phenolic content (about 200 mg GAE/g). A significant relationship between antioxidant capacity and total phenolic content was found, indicating that phenolic compds. are the major contributors to the antioxidant properties of these plants.
    499. 499
      Thaipong, K.; Boonprakob, U.; Crosby, K.; Cisneros-Zevallos, L.; Hawkins Byrne, D. Comparison of ABTS, DPPH, FRAP, and ORAC Assays for Estimating Antioxidant Activity from Guava Fruit Extracts. J. Food Compos. Anal. 2006, 19 (6), 669– 75,  DOI: 10.1016/j.jfca.2006.01.003
      502
      Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts
      Thaipong, Kriengsak; Boonprakob, Unaroj; Crosby, Kevin; Cisneros-Zevallos, Luis; Hawkins Byrne, David
      Journal of Food Composition and Analysis (2006), 19 (6-7), 669-675CODEN: JFCAEE; ISSN:0889-1575. (Elsevier)
      Guava fruit exts. were analyzed for antioxidant activity measured in methanol ext. (AOAM), antioxidant activity measured in dichloromethane ext. (AOAD), ascorbic acid, total phenolics, and total carotenoids contents. The ABTS, DPPH, and FRAP assays were used for detg. both AOAM and AOAD, whereas the ORAC was used for detg. only AOAM. Averaged AOAM [μM Trolox equiv. (TE)/g fresh mass (FM)] were 31.1, 25.2, 26.1, and 21.3 as detd. by the ABTS, DPPH, FRAP, and ORAC assays, resp. Averaged AOAD (μM TE/g FM) were 0.44, 0.27, and 0.16 as detd. by the ABTS, DPPH, and FRAP assays, resp. AOAM detd. by all assays were well correlated with ascorbic acid (0.61≤r≤0.92) and total phenolics (0.81≤r≤0.97) and also among themselves (0.68≤r≤0.97) but had neg. correlation with total carotenoids (-0.67≤r≤-0.81).
    500. 500
      da Silva Granja, B.; Honório de Mendonça Filho, J. R.; de Souza Oliveira, W.; Caldas Santos, J. C. Exploring MBTH as a Spectrophotometric Probe for the Determination of Total Phenolic Compounds in Beverage Samples. Anal. Methods 2018, 10 (19), 2197– 204,  DOI: 10.1039/C8AY00464A
      There is no corresponding record for this reference.
    501. 501
      Wong, T. S.; Wu, N.; Roccatano, D.; Zacharias, M.; Schwaneberg, U. Sensitive Assay for Laboratory Evolution of Hydroxylases toward Aromatic and Heterocyclic Compounds. J. Biomol. Screen. 2005, 10 (3), 246– 52,  DOI: 10.1177/1087057104273336
      504
      Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds
      Wong, T. S.; Wu, N.; Roccatano, D.; Zacharias, M.; Schwaneberg, U.
      Journal of Biomolecular Screening (2005), 10 (3), 246-252CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
      Powerful directed evolution methods have been developed for tailoring proteins to our needs in industrial applications. Here, the authors report a medium-throughput assay system designed for screening mutant libraries of oxygenases capable of inserting a hydroxyl group into a C-H bond of arom. or O-heterocyclic compds. and for exploring the substrate profile of oxygenases. The assay system is based on 4-aminoantipyrine (4-AAP), a colorimetric phenol detection reagent. By using 2 detection wavelengths (509 nm and 600 nm), the authors achieved a linear response from 50 to 800 μM phenol and std. deviations below 11% in 96-well plate assays. The monooxygenase P 450 BM-3 and its F87A mutant were used as a model system for medium-throughput assay development, identification of novel substrates (e.g., phenoxytoluene, phenylallyether, and coumarone), and discovery of P 450 BM-3 F87A mutants with 8-fold improvement in 3-phenoxytoluene hydroxylation activity. This activity increase was achieved by screening a satn. mutagenesis library of amino acid position Y51 using the 4-AAP protocol in the 96-well format.
    502. 502
      Emerson, E. The Condensation of Aminoantipyrine. II. A New Color Test for Phenolic Compounds. J. Org. Chem. 1943, 8 (5), 417– 428,  DOI: 10.1021/jo01193a004
      505
      The condensation of aminoantipyrine. II. A new color test for phenolic compounds
      Emerson, Edgar; Beacham, Harry H.; Beegle, Lindley C.
      Journal of Organic Chemistry (1943), 8 (), 417-28CODEN: JOCEAH; ISSN:0022-3263.
      cf. C. A. 33, 594.1. Aminoantipyrine (I) in the presence of a mild oxidizing agent in slightly alk. soln. gives intensely colored compds. with phenols. A large no. of phenols, naphthols, hydroxypyridines, hydroxyquinolines, barbiturates, uracils, hydantoins and imidazolones are tested and the results given in numerous tables. From the results it is concluded that at least 1 free OH group must be present, that substituents in the p-position to the OH group prevent the reaction with the exception of halogen, CO2H, SO3H, OH and OMe groups which are probably expelled, that a NO2 group in the ο-position prevents the reaction while one in the m-position prevents the test but not completely, and that the coupling of I with the phenol takes place in the p-position rather than in the ο-position. 1-Naphthols give red, 2-naphthols green ppts., sol. in CHCl3. Only the 3-hydroxypyridine gives a pos. test. The color given by vitamin B6 is extremely evanescent and is inhibited by the presence of borate ion (cf. Scudi, C. A. 35, 4792.7). 2- and 4-Hydroxyquinolines do not give a color test because the p-positions to the OH groups are blocked. Barbituric acid, 1,3-diphenylbarbituric acid and 2,4-diamino-6-hydroxypyrimidine give red compds. Barbital, 2-ethylmercapto-4-methyluracil, 4-methyluracil, 1,4-dimethyl- and 1,3,4-trimethyluracil, 2-benzyl-5-imidazolone and 2-benzyl-4-imidazolone fail to give a color test. The test is carried out by dissolving 10-20 mg. of the compd. in 10 cc. H2O contg. 2-3 drops 6 N NH4OH. The soln. is then divided into 2 parts. To 1 part, 2 drops of a 2% I soln., and to both parts, several drops of an 8% K3Fe(CN)6 soln. are added. When the color is developed in full, the soln. is shaken with CHCl3.
    503. 503
      Johnson, C. A.; Savidge, R. A. The Determination of Phenolic Compounds in Pharmaceutical Preparations Using 4-Aminohenazone. J. Pharm. Pharmacol. 2011, 10, 171T– 81T,  DOI: 10.1111/j.2042-7158.1958.tb10396.x
      There is no corresponding record for this reference.
    504. 504
      Varvounis, G.; Fiamegos, Y.; Pilidis, G. Pyrazol-3-Ones. Part III: Reactivity of the Ring Substituents. In Advances in Heterocyclic Chemistry; Academic Press, 2007; Vol. 95; p 27– 141
      There is no corresponding record for this reference.
    505. 505
      Dannis, M. Determination of Phenols by the Amino-Antipyrine Method. Sewage Ind. Waste 1951, 23, 1516– 1522
      508
      Determination of phenols by the aminoantipyridine method
      Dannis, Michael
      Sewage and Industrial Wastes (1951), 23 (), 1516-22CODEN: SIWAAQ; ISSN:0096-364X.
      The method offers a quick detn. for phenol in industrial wastes and is sensitive to 0.1 p.p.m. The detailed procedure is given.
    506. 506
      Fiamegos, Y. C.; Stalikas, C. D.; Pilidis, G. A.; Karayannis, M. I. Synthesis and Analytical Applications of 4-Aminopyrazolone Derivatives as Chromogenic Agents for the Spectrophotometric Determination of Phenols. Anal. Chim. Acta 2000, 403 (1), 315– 23,  DOI: 10.1016/S0003-2670(99)00644-3
      509
      Synthesis and analytical applications of 4-aminopyrazolone derivatives as chromogenic agents for the spectrophotometric determination of phenols
      Fiamegos, Y. C.; Stalikas, C. D.; Pilidis, G. A.; Karayannis, M. I.
      Analytica Chimica Acta (2000), 403 (1-2), 315-323CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
      The synthesis of simple, extensively conjugated chromophores as substitutes for 4-aminoantipyrine (4-AAP) capable of causing hyperchromic shifts when coupled with phenols is described. The 4-aminopyrazolone derivs. synthesized are subsequently applied for the spectrophotometric assay of phenolic compds. and the advantages of each of the proposed systems are highlighted. No improvement is made on the detection of para-substituted phenols as well as polychlorophenols (e.g. pentachlorophenol). An increase in the sensitivity of coupling products was obtained for certain phenolic compds. as indicated from the comparison of the individual responses. The proposed methods have analogous anal. features and in some cases show considerable improvements compared to the 4-AAP method. Beer's law is obeyed over a wide dynamic range (up to ∼500 μg/l) and the relative std. deviations are <3.4%. The methods are applied satisfactorily for phenol assay in real samples, however, taking into account the intrinsic differences of the methods in the responses to the various phenolic compds.
    507. 507
      Li, D.; Ge, S.; Huang, J.; Gong, J.; Yan, P.; Lu, W.; Tiana, G.; Ding, L. Fast Chromogenic Identification of Phenolic Pollutants Via Homogeneous Oxidation with t-BuOOH in the Presence of Iron (III) Octacarboxyphthalocyanine. Catal. Commun. 2014, 45, 95– 9,  DOI: 10.1016/j.catcom.2013.10.038
      510
      Fast chromogenic identification of phenolic pollutants via homogeneous oxidation with t-BuOOH in the presence of iron(III) octacarboxyphthalocyanine
      Li, Dapeng; Ge, Suxiang; Huang, Jun; Gong, Jingjing; Yan, Ping; Lu, Weiyu; Tiana, Guizhong; Ding, Liyun
      Catalysis Communications (2014), 45 (), 95-99CODEN: CCAOAC; ISSN:1566-7367. (Elsevier B.V.)
      An efficient method was developed for fast chromogenic identification of some phenolic pollutants (phenol, 2-chlorophenol and 1-naphthol) via tert-Bu hydroperoxide (t-BuOOH) oxidn. in the presence of water-sol. iron(III) octacarboxyphthalocyanine complexes (catalyst) and 4-aminoantipyrine (chromogenic reagent). Among them, the chromogenic reaction of 2-chlorophenol could be completed rapidly, just within 6 min. The catalytic mechanism and detailed dye formation process were proposed on the basis of control expts. under different conditions. Potentially, the catalytic system composing of iron(III) octacarboxyphthalocyanine and t-BuOOH is promising for the application in fast chromogenic assay of phenolic pollutants.
    508. 508
      Fiamegos, Y.; Stalikas, C. D.; Pilidis, G. A. 4-Aminoantipyrine Spectrophotometric Method of Phenol Analysis: Study of the Reaction Products Via Liquid Chromatography with Diode-Array and Mass Spectrometric Detection. Anal. Chim. Acta 2002, 467 (1–2), 105– 14,  DOI: 10.1016/S0003-2670(02)00072-7
      511
      4-Aminoantipyrine spectrophotometric method of phenol analysis. Study of the reaction products via liquid chromatography with diode-array and mass spectrometric detection
      Fiamegos, Y.; Stalikas, C.; Pilidis, G.
      Analytica Chimica Acta (2002), 467 (1-2), 105-114CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
      The synthesis of new pyrazolone mols. and their application as chromogenic agents in phenol anal. by the 4-aminoantipyrine method was described by the authors' group in previous published papers. A fully detailed mechanism is given therein based on exptl. and theor. data. The study of the reaction products and byproducts via liq. chromatog. with diode array and mass spectrometric detection is presented, sorting out the spectrophotometric detn. of phenols.
    509. 509
      Ayad, M.; El-Sadek, M.; Mostaffa, S. 4-Aminoantipyrine as an Analytical Reagent for the Colorimetric Determination of Tetracycline and Oxytetracycline. Anal. Lett. 1986, 19 (21–22), 2169– 81,  DOI: 10.1080/00032718608080875
      512
      4-Aminoantipyrine as an analytical reagent for the colorimetric determination of tetracycline and oxytetracycline
      Ayad, M.; El-Sadek, M.; Mostaffa, S.
      Analytical Letters (1986), 19 (21-22), 2169-81CODEN: ANALBP; ISSN:0003-2719.
      A spectrophotometric method is proposed for the detn. of tetracycline (I) [60-54-8] and oxytetracycline (II) [79-57-2] in dosage forms. The suggested method depends on the reaction with 4-aminoantipyrine [83-07-8] in the presence of an alk. oxidizing agent. A red antipyrine dye is produced and its absorbance was measured at 435 and 440 nm for I and II, resp. The reaction ratio was detd. Variables such as pH, temp., reagent concn., and stability of the color produced were evaluated to permit selection of the most advantageous technique. Beer's law was obeyed over the concn. range 0.04-0.12 and 0.04-0.16 mg/mL for I and II, resp.
    510. 510
      Otey, C. R.; Joern, J. M. High-Throughput Screen for Aromatic Hydroxylation, Directed Enzyme Evolution: Screening and Selection Methods; Humana Press: Totowa, NJ, 2003; p 141– 8.
      There is no corresponding record for this reference.
    511. 511
      Santos, G. d. A.; Dhoke, G. V.; Davari, M. D.; Ruff, A. J.; Schwaneberg, U. Directed Evolution of P450 BM3 Towards Functionalization of Aromatic O-Heterocycles. Int. J. Mol. Sci. 2019, 20 (13), 3353,  DOI: 10.3390/ijms20133353
      There is no corresponding record for this reference.
    512. 512
      Lülsdorf, N.; Vojcic, L.; Hellmuth, H.; Weber, T. T.; Mußmann, N.; Martinez, R.; Schwaneberg, U. A First Continuous 4-Aminoantipyrine (4-AAP)-Based Screening System for Directed Esterase Evolution. Appl. Microbiol. Biotechnol. 2015, 99 (12), 5237– 46,  DOI: 10.1007/s00253-015-6612-3
      515
      A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution
      Luelsdorf, Nina; Vojcic, Ljubica; Hellmuth, Hendrik; Weber, Thomas T.; Mussmann, Nina; Martinez, Ronny; Schwaneberg, Ulrich
      Applied Microbiology and Biotechnology (2015), 99 (12), 5237-5246CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      Esterases hydrolyze ester bonds with an often high stereoselectivity as well as regioselectivity and are therefore industrially employed in the synthesis of pharmaceuticals, in food processing, and in laundry detergents. Continuous screening systems based on p-nitrophenyl- (e.g., p-nitrophenyl acetate) or umbelliferyl-esters are commonly used in directed esterase evolution campaigns. Ongoing challenges in directed esterase evolution are screening formats which offer a broad substrate spectrum, esp. for complex arom. substrates. In this report, a novel continuous high throughput screening system for indirect monitoring of esterolytic activity was developed and validated by detection of phenols employing Ph benzoate as substrate and p-nitrobenzyl esterase (pNBEBL from Bacillus licheniformis) as catalyst. The released phenol directly reacts with 4-aminoantipyrine yielding the red compd. 1,5-dimethyl-4-(4-oxo-cyclohexa-2,5-dienylidenamino)-2-phenyl-1,2-dihydro-pyrazol-3-one. In this continuous B. licheniformis esterase activity detection system (cBLE-4AAP), the product formation is followed through an increase in absorbance at 509 nm. The cBLE-4AAP screening system was optimized in 96-well microtiter plate format in respect to std. deviation (5 %), linear detection range (15 to 250 μM), lower detection limit (15 μM), and pH (7.4 to 10.4). The cBLE-4AAP screening system was validated by screening a random epPCR pNBEBL mutagenesis library (2000 clones) for improved esterase activity at elevated temps. Finally, the variant T3 (Ser378Pro) was identified which nearly retains its specific activity at room temp. (WT 1036 U/mg and T3 929 U/mg) and shows compared to WT a 4.7-fold improved residual activity after thermal treatment (30 min incubation at 69.4 °C; WT 170 U/mg to T3 804 U/mg).
    513. 513
      Choi, S.-L.; Rha, E.-G.; Kim, D.-Y.; Song, J.-J.; Hong, S.-P.; Sung, M.-H.; Lee, S.-G. High Throughput Screening and Directed Evolution of Tyrosine Phenol-Lyase. Microbiol. Biotechnol. Lett. 2006, 34, 58– 62
      There is no corresponding record for this reference.
    514. 514
      Quintana, M. G.; Didion, C.; Dalton, H. Colorimetric Method for a Rapid Detection of Oxygenated Aromatic Biotransformation Products. Biotechnol. Technol. 1997, 11 (8), 585– 7,  DOI: 10.1023/A:1018499024466
      517
      Colorimetric method for a rapid detection of oxygenated aromatic biotransformation products
      Quintana, M. G.; Didion, C.; Dalton, H.
      Biotechnology Techniques (1997), 11 (8), 585-587CODEN: BTECE6; ISSN:0951-208X. (Chapman and Hall)
      The quant. prodn. of the oxygenated products from the biotransformation of arom. substrates can be detected using a very simple and rapid colorimetric test. The method is based on Gibbs' reagent (2,6-dichloroquinone-4-chloroimide) and has been developed for routine spectrophotometric or microtiter plate assay allowing the detection of products with a sensitivity of ≥5 μM.
    515. 515
      Dacre, J. C. Nonspecificity of the Gibbs Reaction. Anal. Chem. 1971, 43 (4), 589– 91,  DOI: 10.1021/ac60299a015
      518
      Nonspecificity of the Gibbs reaction
      Dacre, Jack C.
      Analytical Chemistry (1971), 43 (4), 589-91CODEN: ANCHAM; ISSN:0003-2700.
      The wavelengths of max. absorption and molar absorptivities are given for the products of the Gibbs reaction at pH 9.24 of 16 p-alkoxyphenols, 9 p-halophenols, p-hydroxybenzaldehydes, p-hydroxybenzyl alcs., and p-hydroxybenzoic acids, to demonstrate the nonspecificity of the Gibbs reagent.
    516. 516
      Josephy, P. D.; Van Damme, A. Reaction of Gibbs Reagent with Para-Substituted Phenols. Anal. Chem. 1984, 56 (4), 813– 4,  DOI: 10.1021/ac00268a052
      519
      Reaction of Gibbs reagent with para-substituted phenols
      Josephy, P. David; Van Damme, Anjel
      Analytical Chemistry (1984), 56 (4), 813-14CODEN: ANCHAM; ISSN:0003-2700.
      Certain p-substituted phenols (alkoxy, halogen) react with Gibbs reagent (I) to give 2,6-dichloroindophenol. The para substituent is cleaved during the reaction. The product yield is detd. for various substituted phenols, and a mechanism for the reaction is proposed.
    517. 517
      Svobodová, D.; Křenek, P.; Fraenkl, M.; Gasparič, J. The Colour Reaction of Phenols with the Gibbs Reagent. Microchim. Acta 1978, 70 (3), 197– 211,  DOI: 10.1007/BF01201610
      There is no corresponding record for this reference.
    518. 518
      Ang, E. L.; Obbard, J. P.; Zhao, H. Directed Evolution of Aniline Dioxygenase for Enhanced Bioremediation of Aromatic Amines. Appl. Microbiol. Biotechnol. 2009, 81 (6), 1063– 70,  DOI: 10.1007/s00253-008-1710-0
      521
      Directed evolution of aniline dioxygenase for enhanced bioremediation of aromatic amines
      Ang, Ee Lui; Obbard, Jeffrey P.; Zhao, Huimin
      Applied Microbiology and Biotechnology (2009), 81 (6), 1063-1070CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      The objective of this study was to enhance the activity of aniline dioxygenase (I), a multi-component Rieske non-heme iron dioxygenase isolated from Acinetobacter sp. strain YAA, so as to create an enhanced biocatalyst for the bioremediation of arom. amines. Previously, mutation V205A was found to widen the substrate specificity of I to accept 2-isopropylaniline (2IPA) for which the wild-type enzyme had no activity. Using mutant V205A as the parent and applying one round of satn. mutagenesis followed by a round of random mutagenesis, the activity of the final mutant, 3-R21, was increased by 8.9-, 98.0-, and 2.0-fold for aniline, 2,4-dimethylaniline (24DMA), and 2-isopropylaniline (2IPA), resp., over mutant V205A. In particular, the activity of mutant 3-R21 for 24DMA, which is a carcinogenic arom. amine pollutant, was increased by 3.5-fold over wild-type I, while the activity for aniline was restored to the wild-type level, thus yielding a mutant I with enhanced activity and capable of hydroxylating a wider range of arom. amines than the wild-type enzyme.
    519. 519
      Bornscheuer, U. T.; Ordoñez, G. R.; Hidalgo, A.; Gollin, A.; Lyon, J.; Hitchman, T. S.; Weiner, D. P. Selectivity of Lipases and Esterases Towards Phenol Esters. J. Mol. Catal. B: Enzym. 2005, 36 (1), 8– 13,  DOI: 10.1016/j.molcatb.2005.07.004
      522
      Selectivity of lipases and esterases towards phenol esters
      Bornscheuer, Uwe T.; Ordonez, Gonzalo Rodriguez; Hidalgo, Aurelio; Gollin, Anita; Lyon, Jonny; Hitchman, Timothy S.; Weiner, David P.
      Journal of Molecular Catalysis B: Enzymatic (2005), 36 (1-6), 8-13CODEN: JMCEF8; ISSN:1381-1177. (Elsevier B.V.)
      The selectivity of 28 lipases and esterases in the hydrolysis of butanoates of o-, m- or p-substituted phenols was investigated in a microtiter plate format. The phenols released during enzyme-catalyzed hydrolysis were converted in situ with Gibbs' reagent to form a blue indophenol complex, which was quantified spectrophotometrically at 600 nm. Substantial differences in rates were found, which exhibits that the type and position of the substituent at the alkyl group has a strong influence on the selectivity of the enzymes. For various enzymes, the p-nitro deriv. was the best substrate, whereas for other enzymes the m-Cl-deriv. was preferentially hydrolyzed. Anal. of the data using the Hammett equation showed that sometimes the obsd. changes followed a predictable trend, but in several cases the result is very unexpected.
    520. 520
      Altahir, B. M.; Abdulazeez, O.; Dikran, S. B.; Taylor, K. E. Determination of Eugenol in Personal-Care Products by Dispersive Liquid-Liquid Microextraction Followed by Spectrophotometry Using p-Amino-N,N-Dimethylaniline as a Derivatizing Agent. Indones. J. Chem. 2020, 21, 192,  DOI: 10.22146/ijc.56198
      There is no corresponding record for this reference.
    521. 521
      Gasparic, J.; Svobodova, D.; Pospisilova, M. Investigation of the Colour Reaction of Phenols with the MBTH Reagent. Identification of Organic Compounds. LXXXVI. Microchim. Acta 1977, 67, 241– 50,  DOI: 10.1007/BF01213034
      There is no corresponding record for this reference.
    522. 522
      Pospíšilová, M.; Polášek, M.; Svobodová, D. Spectrophotometric Study of Reactions of Substituted Phenols with MBTH in Alkaline Medium: The Effect of Phenol Structure on the Formation of Analytically Useful Coloured Products. Microchimica Acta 1998, 129 (3), 201– 8,  DOI: 10.1007/BF01244742
      There is no corresponding record for this reference.
    523. 523
      Kamata, E. Color Reactions of 3-Methyl-2-Benzothiazolone Hydrazone with Phenol Derivatives. I. The Spectrophotometric Microdetermination of Catechol, Hydroquinone and Resorcinol. Bull. Chem. Soc. Jpn. 1964, 37 (11), 1674– 7,  DOI: 10.1246/bcsj.37.1674
      526
      Color reactions of 3-methyl-2-benzothiazolone hydrazone with phenol derivatives. I. The spectrophotometric microdetermination of catechol, hydroquinone, and resorcinol
      Kamata, Eijiro
      Bulletin of the Chemical Society of Japan (1964), 37 (11), 1674-7CODEN: BCSJA8; ISSN:0009-2673.
      Two methods are given for the detn. of catechol: (1) Add 4 ml. of 0.5% 3-methyl-2-benzothiazolone hydrazone (I)-MeOH soln. and 2 ml. of NH4OH to 4 ml. of test soln.; allow the mixt. to stand for 15 min., and read the absorbance at 510 mμ. (2) Add 2 ml. of 0.4% aq. I, 1.6 ml. of 10% Mohr's salt, and 0.4 ml. of 1% FeCl3 to 4 ml. of test soln., and 2 ml. Me2CO, after 5 min., and read the absorbance at 550 mμ within 30 min. To detn. hydroquinone (II), add 3 ml. of 0.5% I-MeOH soln. and 1 ml. NH4OH to 3 ml. of test soln., after 1 min. add 3 ml. Me2CO, and read the absorbance at 610 mμ within 3 min. Methods are given also for the detn. of II and resorcinol (III) in a mixt. of the 2 and of III alone.
    524. 524
      Setti, L.; Giuliani, S.; Spinozzi, G.; Pifferi, P. G. Laccase Catalyzed-Oxidative Coupling of 3-Methyl 2-Benzothiazolinone Hydrazone and Methoxyphenols. Enzyme Microb. Technol. 1999, 25 (3), 285– 9,  DOI: 10.1016/S0141-0229(99)00059-9
      527
      Laccase catalyzed-oxidative coupling of 3-methyl 2-benzothiazolinone hydrazone and methoxyphenols
      Setti, Leonardo; Giuliani, Silvia; Spinozzi, Giovanni; Pifferi, Pier Giorgio
      Enzyme and Microbial Technology (1999), 25 (3-5), 285-289CODEN: EMTED2; ISSN:0141-0229. (Elsevier Science Ireland Ltd.)
      The reaction between o-, m-, and p-methoxyphenols and 3-methyl-2-benzothiazolinone hydrazone was studied in the presence of laccase from Pyricularia oryzae. The findings show that laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) catalyzes the oxidative coupling reaction between MBTH and phenols producing red colored azo-dye compds. On the basis of kinetic parameters and optimum pH values, the mechanisms of the oxidative coupling reactions with the different phenols are discussed. The results suggest that the reaction is initialized by the enzymic 3-methyl-2-benzothiazolinone hydrazone activation, which undergoes electrophilic substitution with m-methoxyphenol in soln., enzymic activated guaiacol, and chelated p-methoxyphenol at the catalytic site of the laccase. The current study also demonstrates the possibility of using the azo-dye formed in the presence of guaiacol for measuring laccase activity following a similar assaying method as that used for measuring peroxidase activity. This type of assay permits the detn. of laccase at micromolar levels by fixed time method.
    525. 525
      Setti, L.; Scali, S.; Angeli, I. D.; Pifferi, P. G. Horseradish Peroxidase-Catalyzed Oxidative Coupling of 3-Methyl 2-Benzothiazolinone Hydrazone and Methoxyphenols. Enzyme Microb. Technol. 1998, 22 (8), 656– 61,  DOI: 10.1016/S0141-0229(97)00259-7
      528
      Horseradish peroxidase-catalyzed oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone and methoxyphenols
      Setti, Leonardo; Scali, Sara; Angeli, Igor Degli; Pifferi, Pier Giorgio
      Enzyme and Microbial Technology (1998), 22 (8), 656-661CODEN: EMTED2; ISSN:0141-0229. (Elsevier Science Inc.)
      The reaction between o-, m-, and p-methoxyphenols and 3-methyl-2-benzothiazolinone hydrazone (MBTH) is studied in the presence of horseradish peroxidase (HRP) and H2O2 as oxidative agent. The findings indicate that enzyme (H2O2 oxidoreductase; EC 1.11.1.7) catalyzes an oxidative coupling reaction between MBTH and phenols which produces azo dye compds. On the basis of kinetic parameters and optimum pH values, a mechanism in which both MBTH and phenols seem to be activated by the HRP for achieving the oxidative coupling is proposed. Furthermore, in the current study, we have evaluated the possibility that these azo dyes may be useful in the measurement of peroxidase activity. The method is based on the obsd. increase in the absorbance at 502 nm (8,355 cm-1 M-1 of extinction molar coeff.) due to the formation of a red azo dye compd. resulting from the peroxidase-catalyzed oxidative coupling of MBTH and o-methoxyphenol (guaiacol). Using this assay system, HRP can be detd. in picomolar levels by a fixed time method.
    526. 526
      Nolan, L. C.; O’Connor, K. E. A Spectrophotometric Method for the Quantification of an Enzyme Activity Producing 4-Substituted Phenols: Determination of Toluene-4-Monooxygenase Activity. Anal. Biochem. 2005, 344 (2), 224– 31,  DOI: 10.1016/j.ab.2005.05.032
      529
      A spectrophotometric method for the quantification of an enzyme activity producing 4-substituted phenols: Determination of toluene-4-monooxygenase activity
      Nolan, Louise C.; O'Connor, Kevin E.
      Analytical Biochemistry (2005), 344 (2), 224-231CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
      A spectrophotometric method for the quant. detn. of an enzyme activity resulting in the accumulation of 4-substituted phenols is described in this article. Toluene-4-monooxygenase (T4MO) activity in whole cells of Pseudomonas mendocina KR1 is used to demonstrate this method. This spectrophotometric assay is based on the coupling of T4MO activity with tyrosinase activity. The 4-substituted phenol, produced by the action of T4MO on the arom. ring of a substituted arene, is a substrate for tyrosinase, which converts phenols to o-quinones. The latter react with the nucleophile 3-methyl-2-benzothiazolinone hydrazone (MBTH) to produce intensely colored products that absorb light maximally at different wavelengths, depending on the phenolic substrate used. The incubation of whole cells of P. mendocina KRI with fluorobenzene resulted in the accumulation of 4-fluorophenol. The coupling of T4MO activity with tyrosinase activity in the presence of fluorobenzene resulted in the formation of a colored product absorbing maximally at 480 nm. The molar absorptivity (ε) value for the o-quinone-MBTH adduct formed from 4-fluorophenol was detd. exptl. to be 12,827 M-1 cm-1 with a linear range of quantification between 2.5 and 75 μM. The whole cell assay was run as a continuous indirect assay. The initial rates of T4MO activity toward fluorobenzene, as detd. spectrophotometrically, were 61.8 nmol/min/mg P. mendocina KR1 protein (using mushroom tyrosinase), 64.9 nmol/min/mg P. mendocina KR1 protein (using cell exts. Pseudomonas putida F6), and, as detd. by HPLC anal., 62.6 nmol/min/mg P. mendocina KR1 protein.
    527. 527
      Regitz, M. Diazo Compounds: Properties and Synthesis; Elsevier, 2012.
      There is no corresponding record for this reference.
    528. 528
      Chudgar, R. J. Azo Dyes; Kirk–Othmer Encyclopedia of Chemical Technology, 2000.
      There is no corresponding record for this reference.
    529. 529
      de Keijzer, M. The Delight of Modern Organic Pigment Creations, Issues in Contemporary Oil Paint; Springer International Publishing: Cham, 2014; p 45– 73.
      There is no corresponding record for this reference.
    530. 530
      Urbányi, T.; Mollica, J. A. Potential Diazo Reagents for Colorimetric Determination. J. Pharm. Sci. 1968, 57 (7), 1257– 60,  DOI: 10.1002/jps.2600570746
      533
      Potential diazo reagents for colorimetric determination
      Urbanyi, Tibor; Mollica, Joseph A.
      Journal of Pharmaceutical Sciences (1968), 57 (7), 1257-60CODEN: JPMSAE; ISSN:0022-3549.
      A method for detg. estrogens is based on their ability to couple with diazotized amines. The diazonium derivs. of 27 substituted aromatic amines were investigated as reagents for colorimetric detn. The phenolic moiety used in the study was estradiol. Their applicability was detd. on the basis of speed, sensitivity, color stability and reproducibility of the coupling reaction. With these criteria, a diazotization product of 4-amino-6-chloro-m-benzenedisulfonamide appeared to be the most suitable colorimetric reagent for compds. contg. a phenolic OH group.
    531. 531
      Ehrlich, P. Uber Eine Neue Harnprobe. Dtsch. Med. Wochenschr. 1883, 9 (1), 11,  DOI: 10.1055/s-0029-1196984
      There is no corresponding record for this reference.
    532. 532
      Hassan, S. M.; Walash, M. I.; El-Sayed, S. M.; Abou Ouf, A. M. Colorimetric Determination of Certain Phenol Derivatives in Pharmaceutical Preparations. J. Assoc. Off. Anal. Chem. 1981, 64 (6), 1442– 5,  DOI: 10.1093/jaoac/64.6.1442
      535
      Colorimetric determination of certain phenol derivatives in pharmaceutical preparations
      Hassan, Sayed M.; Walash, Mohammed I.; El-Sayed, Sanaa M.; Ouf, Abdel Malek Abou
      Journal - Association of Official Analytical Chemists (1981), 64 (6), 1442-5CODEN: JANCA2; ISSN:0004-5756.
      acetaminophen (I) [103-90-2] and oxyphenbutazone (II) [129-20-4] are detd. using colorimetry methods. These methods are based on coupling between the phenolic compd. and the diazonium salts of both sulfanilic acid and p-nitroaniline; the optimum conditions for the reactions were studied. For I, the reaction products with diazosulfanilic acid and diazo-p-nitroaniline show max. absorbance at 480 nm and 425 nm, resp. The mean percentage recoveries for authentic samples were 99.5 and 100.6, resp. For II, the obtained colors showed max. at 385 nm with diazosulfanilic acid and 490 nm with diazo-p-nitroaniline reactions. The mean percentage recoveries for authentic samples were 99.8 and 100.1, resp. The proposed methods were successfully applied to the anal. of com. prepns.; results were statistically compared with those of other methods.
    533. 533
      Swaminathan, M. Chemical Estimation of Vitamin B6 in Foods by Means of the Diazo Reaction and the Phenol Reagent. Nature 1940, 145 (3681), 780,  DOI: 10.1038/145780a0
      536
      Chemical estimation of vitamin B6 in foods by means of the diazo reaction and the phenol reagent
      Swaminathan, M.
      Nature (London, United Kingdom) (1940), 145 (), 780CODEN: NATUAS; ISSN:0028-0836.
      The method makes use of the diazo reaction (diazotized sulfanilic acid) or the phenol reagent. The azo color formed is estd. colorimetrically. The method is applicable to all foods. As little as 10 γ of vitamin B6 is easily estd.
    534. 534
      Whitlock, L. R.; Siggia, S.; Smola, J. E. Spectrophotometric Analysis of Phenols and of Sulfonates by Formation of an Azo Dye. Anal. Chem. 1972, 44 (3), 532– 6,  DOI: 10.1021/ac60311a021
      537
      Spectrophotometric analysis of phenols and of sulfonates by formation of an azo dye
      Whitlock, L. Ronald; Siggia, Sidney; Smola, Janice E.
      Analytical Chemistry (1972), 44 (3), 532-6CODEN: ANCHAM; ISSN:0003-2700.
      Phenols and sulfonic acids are detd. by a method based on the coupling reaction between a diazotized amine and the phenol. The concn. of the azo dye formed was measured by visible spectrophotometry. Sulfonic acids and their salts were converted to phenols via alkali fusion at 360° by using KOH, prior to coupling. Diazotized sulfanilic acid and p-phenylazoaniline, a previously unreported reagent, were particularly useful reagents for detn. of phenols. With the latter, the optimum pH and reaction time for the phenols were remarkably similar and the molar absorptivities of the dyes formed were significantly higher. The coupling reaction conditions for a wide variety of phenols and the molar absorptivities and absorption max. for the azo dyes are presented. The limit of detection was about 1 μmole phenol/l. soln. and the min. amt. detectable was 0.3 μg. This method for sulfonic acid detn. will extend the useful range of application of the alkali fusion method to include polysulfonates and higher mol. wt. sulfonates. Alkali fusion of halogenated sulfonates resulted in substitution of both the halogen and sulfonate groups. To help det. which sulfonates can be analyzed by the fusion procedure, the thermal stabilities of a large no. of sulfonates and several phenolates are given.
    535. 535
      Abdel Azeem, S. M.; Al Mohesen, I. A.; Ibrahim, A. M. H. Analysis of Total Phenolic Compounds in Tea and Fruits Using Diazotized Aminobenzenes Colorimetric Spots. Food Chem. 2020, 332, 127392,  DOI: 10.1016/j.foodchem.2020.127392
      538
      Analysis of total phenolic compounds in tea and fruits using diazotized aminobenzenes colorimetric spots
      Abdel Azeem, Sami M.; Al Mohesen, Ibrahem A.; Ibrahim, Ahmed M. H.
      Food Chemistry (2020), 332 (), 127392CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)
      The present work describes a novel and rapid approach for evaluating total phenolic compds. (TPCs) in tea and fruits using colorimetric spots and the digital image-based (DIB) method. Colorimetric spots were formed by reacting diazotized aminobenzenes namely sulfanilic acid, sulfanilamide, or aniline with TPCs in the ext. to form an azo dye. The limit of detection (LOD) was 6.5, 5.5, or 5.1 mg GAE (gallic acid equiv.) L-1 and the anal. range was 25-500, 20-500, or 18-200 mg GAE L-1, resp. Correlation with the Folin-Ciocalteu assay was significant (Pearson coeff., R = 0.970-0.991) while the antioxidant activity assay was moderate to high (R = 0.737-0.977). The method developed was successfully applied to the anal. of tea and fruits and showed RSD (n = 3) not exceeding 9.6, 8.5, and 9.7%, resp. Ecol., the DIB method developed could det. the variation of TPCs within cultivars and was found to be strongly dependent on the growing environment.
    536. 536
      Lester, G. E.; Lewers, K. S.; Medina, M. B.; Saftner, R. A. Comparative Analysis of Strawberry Total Phenolics Via Fast Blue BB Vs. Folin-Ciocalteu: Assay Interference by Ascorbic Acid. J. Food Compos. Anal. 2012, 27 (1), 102– 7,  DOI: 10.1016/j.jfca.2012.05.003
      539
      Comparative analysis of strawberry total phenolics via Fast Blue BB vs. Folin-Ciocalteu: Assay interference by ascorbic acid
      Lester, Gene E.; Lewers, Kim S.; Medina, Marjorie B.; Saftner, Robert A.
      Journal of Food Composition and Analysis (2012), 27 (1), 102-107CODEN: JFCAEE; ISSN:0889-1575. (Elsevier Inc.)
      Unblemished fully ripe fruit from five day-neutral strawberry cultivars were harvested on two sep. dates and evaluated for ascorbic acid (AsA), fruit sugars, and phenolic compn. Individual phenolics were detd. by HPLC, and total phenolics by Folin-Ciocalteu (F-C) and by a new' assay: Fast Blue BB (FBBB), which detects phenolics directly. FBBB reported an av. 2.9-fold greater concn. of total phenolics than F-C, had a significant correlation (r = 0.80; P = 0.001) with total phenolics via HPLC and did not interact with AsA or sugars, whereas F-C, an indirect detection assay for total phenolics, appeared to under-report total phenolic concns., had no significant correlation (r = 0.20) with total phenolics via HPLC or with sugars, but had a significant correlation (r = 0.64; P = 0.05) with total AsA. Results from this study indicated that previous studies of strawberry fruit, using the std. indirect F-C assay, have greatly underestimated the total phenolics content and that this assay should be replaced in future studies by the FBBB assay.
    537. 537
      Pompella, A.; Comporti, M. The Use of 3-Hydroxy-2-Naphthoic Acid Hydrazide and Fast Blue B for the Histochemical Detection of Lipid Peroxidation in Animal Tissues─a Microphotometric Study. Histochemistry 1991, 95, 255– 262,  DOI: 10.1007/BF00744997
      540
      The use of 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B for the histochemical detection of lipid peroxidation in animal tissues - a microphotometric study
      Pompella, A.; Comporti, M.
      Histochemistry (1991), 95 (3), 255-62CODEN: HCMYAL; ISSN:0301-5564.
      The title procedure was modified to prevent exposure of tissue sections to lipid solvents. In fresh rat or mouse liver cryostat sections exposed in vitro to various prooxidant conditions (NADPH-Fe2+, NADPH-ADP/Fe3+, BrCCl3-NADPH), a close correlation was found between the intensity of the 3-hydroxy-2-naphthoic acid/Fast Blue B (NAH-FBB) (blue-violet) stain and the amt. of malondialdehyde (used as biochem. index of lipid peroxidn.) released in the incubation medium. Stain intensities obtained with NAH-FBB reaction were several-fold higher than those obtainable with direct Schiff reaction (a previously used procedure) and better paralleled in the the appearance of lipid peroxidn. in tissue. By means of selective delipidation it was obsd. that the NAH-FBB reaction is more efficient than the Schiff reaction in detecting protein and phospholipid-assocd. lipid peroxidn.-derived carbonyl functions. The ability of the NAH-FBB reaction to reveal lipid peroxidn. occurring in tissues in vivo was verified with animals intoxicated with prooxidant toxins, i.e., the haloalkanes BrCCl3 and CCl4, and the glutathione-depleting agent bromobenzene. In livers from haloalkane-treated rats, NAH-FBB positivity provided with te specific absorption spectrum was obsd. in centrolobular regions. In bromobenzene-poisoned mice, NAH-FBB positivity with specific absorption was found, besides the liver, also in kidney (tubular epithelium) and lung (bronchiolar epithelium). This reaction is suggested for the discrimination of cell types undergoing lipid peroxidn. in vivo.
    538. 538
      Tłuścik, F.; Kazubek, A.; Mejbaum-Katzenellenbogen, W. Alkylresorcinols in Rye (Secale Cereale L.) Grains. Vi. Colorimetric Micromethod for the Determination of Alkylresorcinols with the Use of Diazonium Salt, Fast Blue B. Acta Soc. Bot. Polym. 1981, 50, 645,  DOI: 10.5586/asbp.1981.086
      541
      Alkylresorcinols in rye (Secale cereale L.) grain. VI. Colorimetric micromethod for the determination of alkylresorcinols with the use of the diazonium salt, Fast Blue B
      Tluscik, Franciszek; Kozubek, Arkadiusz; Mejbaum-Katzenellenbogen, Wanda
      Acta Societatis Botanicorum Poloniae (1981), 50 (4), 645-51CODEN: ASBNA2; ISSN:0001-6977.
      A Fast Blue B soln. was added to a Me2CO ext. of rye or wheat grains and the absorbance was read after 1-2 h at 520 nm. A calibration curve was prepd. by using 5-n-pentadecylresorcinol as a std. Almost all of the measured absorbance was due to 5-n-alkylresorcinols.
    539. 539
      Schotten, C.; Leprevost, S. K.; Yong, L. M.; Hughes, C. E.; Harris, K. D. M.; Browne, D. L. Comparison of the Thermal Stabilities of Diazonium Salts and Their Corresponding Triazenes. Org. Process Res. Dev. 2020, 24 (10), 2336– 41,  DOI: 10.1021/acs.oprd.0c00162
      542
      Comparison of the Thermal Stabilities of Diazonium Salts and Their Corresponding Triazenes
      Schotten, Christiane; Leprevost, Samy K.; Yong, Low Ming; Hughes, Colan E.; Harris, Kenneth D. M.; Browne, Duncan L.
      Organic Process Research & Development (2020), 24 (10), 2336-2341CODEN: OPRDFK; ISSN:1083-6160. (American Chemical Society)
      A range of diazonium salts and their corresponding triazenes have been prepd. in order to directly compare their relative thermal stabilities (via initial decompn. temp.) from differential scanning calorimetry (DSC) data. A structure-stability relationship has been explored to investigate trends in stability, depending on the arom. substituent and the structure of the secondary amine component of the diazonium salts and triazenes. All of the triazenes investigated show significantly greater stability (many are stable above 200°C) compared with the corresponding diazonium salts, which show varying stabilities.
    540. 540
      Johnston, K. J.; Ashford, A. E. A Simultaneous-Coupling Azo Dye Method for the Quantitative Assay of Esterase Using α-Naphthyl Acetate as Substrate. Histochem. J. 1980, 12 (2), 221– 34,  DOI: 10.1007/BF01024552
      543
      A simultaneous-coupling azo dye method for the quantitative assay of esterase using α-naphthyl acetate as substrate
      Johnston, K. J.; Ashford, A. E.
      Histochemical Journal (1980), 12 (2), 221-34CODEN: HISJAE; ISSN:0018-2214.
      A simultaneous-coupling azo dye method is described for the measurement of esterase activity using the histochem. substrate, α-naphthyl acetate. By the choice of 2 diazonium salts with optimal coupling characteristics, the reaction can be carried out at any pH between 3.0 and 9.5. The azo dye is maintained in soln. for spectrophotometric measurements with bovine serum albumin. The simultaneous-coupling method is compared with an assay based on the direct measurement of released α-naphthol by its UV absorbance in a pH study of hog liver esterase. There is good agreement between the data obtained by both methods.
    541. 541
      Pearse, A. G. E. Histochemistry, Theoretical and Applied; Harcourt Brace/Churchill Livingstone, 1968.
      There is no corresponding record for this reference.
    542. 542
      Lugg, G. A. Stabilized Diazonium Salts as Analytical Reagents for the Determination of Air-Borne Phenols and Amines. Anal. Chem. 1963, 35 (7), 899– 904,  DOI: 10.1021/ac60200a039
      545
      Stabilized diazonium salts as analytical reagents for the determination of airborne phenols and amines
      Lugg, G. A.
      (1963), 35 (), 899-904CODEN: ANCHAM; ISSN:0003-2700.
      Com. diazonium salts were investigated as reagents for the spectrophotometric detn. of 24 toxic phenols and amines in air. Optimum reaction conditions were detd. after spot testing with 25 diazo reagents at 4 buffer concns. (pH 4, 0.05M KH phthalate, pH 7, 2M NH4OAc, pH 9, 0.05M Na borate, and pH 12.8, 2M Na2CO3). Charge a bubbler of the midget impinger type with 5 ml. of solvent (H2O or Me cellosolve, depending upon the compd. tested), sample at 1 l./min. for 2 min., add buffer and the relevant diazo compd. (these are listed for each test material), prep. a standard, and read both solns. against a blank. The vapor of a representative selection of compds. was sampled in each case at least 80% of the amt. recovered was collected in the 1st bubbler Interference is mainly from accompanying nitro compds. Sensitivity varies, but the compds. can be detd. in air at concns. of interest in hygiene studies.
    543. 543
      Buchwald, H. The Colorimetric Determination of Phenol in Air and Urine with a Stabilized Diazonium Salt. Ann. Occup. Hyg. 1966, 9, 7– 14,  DOI: 10.1093/annhyg/9.1.7
      546
      Colorimetric determination of phenol in air and urine
      Buchwald, H.
      Annals of Occupational Hygiene (1966), 9 (1), 7-14CODEN: AOHYA3; ISSN:0003-4878.
      Optimum conditions were evaluated for the detn. of phenol with Brentamine Fast GG, the stabilized diazonium salt of p-nitroaniline. Equally efficient air sampling was obtained by 2 U-tubes contg. 10 g. silica gel (40-60 mesh chromatographic grade) connected in series and 2 fritted glass bubblers contg. 5% wt./vol. NaOH soln., but the U tubes are more convenient. The silica gel is placed in N NaOH, allowed to stand 10 min., and then analyzed. Phenol, o-, and m-cresols are sepd. from a soln. contg. 10 ml. urine, 2 ml. H2SO4, and 7 ml. H2O by steam distillation. The first 100 ml. of distillate contains >98% of the phenols present. To a sample contg. 10-100 γ phenol adjusted to pH 10.1 with Na2CO3-NaHCO3 buffer, add 2.5 ml. 0.5% filtered diazo reagent and dil. to 25 ml. Wait 10 min. and read the absorbance at 490 mμ. The color is stable for 1 hr., but the diazo stock soln. must be freshly prepd. every few hrs. o- and μ-Cresols, if present, are also detd. with the phenol.
    544. 544
      Harvey, D.; Penketh, G. E. The Determination of Small Amounts of o-Phenylphenol. Analyst 1957, 82 (976), 498– 503,  DOI: 10.1039/an9578200498
      547
      Determination of small amounts of o-phenylphenol
      Harvey, D.; Penketh, G. E.
      (1957), 82 (), 498-503 ISSN:.
      o-Phenylphenol (I) after isolation can be measured by ultraviolet absorption at 284 and 295 mμ in cyclohexane or 300 and 320 mμ in NaOH. To use the hypochromic effect measure at 285 and 315 mμ in NaOH and then in HCl soln. The differences in optical ds. are independent of background. Two wave lengths are used to detect interferences. In alc. NaOH I fluoresces at 0.5 γ/ml. but interferences are serious so that errors of ±20% are found. For a colorimetric detn. neutralize the soln. to pH 7-9 and dil. so that 1 ml. contains 1-10 γ I. To 10 ml. add 1 ml. 1% gum acacia soln., 1 ml. 50% NaOAc soln., and 1 ml. fresh 0.5% Brentamine Fast Red GG soln. After 1 min. add 2 ml. 20% Na2CO3 soln. and measure at 494 mμ against a blank. Methods for isolating I from disinfectant salts and formulations, citrus fruits, timber, paper, and canvas are described.
    545. 545
      Canada, K. A.; Iwashita, S.; Shim, H.; Wood, T. K. Directed Evolution of Toluene ortho-Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation. J. Bacteriol. 2002, 184 (2), 344– 9,  DOI: 10.1128/JB.184.2.344-349.2002
      548
      Directed evolution of toluene ortho-monooxygenase for enhanced 1-naphthol synthesis and chlorinated ethene degradation
      Canada, Keith A.; Iwashita, Sachiyo; Shim, Hojae; Wood, Thomas K.
      Journal of Bacteriology (2002), 184 (2), 344-349CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chem. manufg. intermediate. Directed evolution was used to increase the activity of toluene o-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidn. When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5 vs. 1.39 nmol/min/mg protein), 1,1-dichloroethylene, and trans-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was 6-fold faster than that mediated by the wild-type enzyme at a concn. of 0.1 mM (0.19 vs. 0.029 nmol/min/mg protein), whereas at 5 mM, the mutant enzyme was active (0.07 nmol/min/mg protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with >97% 1-naphthol formed. The beneficial mutation of TOM-Green was the substitution of Val to Ala in position 106 of the α-subunit of the hydroxylase, which appears to act as a smaller "gate" to the di-iron active center. This hypothesis was supported by the ability of E. coli expressing TOM-Green to oxidize the 3-ring polyarom. compds., phenanthrene, fluorene, and anthracene, faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chem.
    546. 546
      Joshi, N. S.; Whitaker, L. R.; Francis, M. B. A Three-Component Mannich-Type Reaction for Selective Tyrosine Bioconjugation. J. Am. Chem. Soc. 2004, 126 (49), 15942– 3,  DOI: 10.1021/ja0439017
      549
      A Three-Component Mannich-Type Reaction for Selective Tyrosine Bioconjugation
      Joshi, Neel S.; Whitaker, Leanna R.; Francis, Matthew B.
      Journal of the American Chemical Society (2004), 126 (49), 15942-15943CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A new selective bioconjugation reaction is described for the modification of tyrosine residues on protein substrates. The reaction uses imines formed in situ from aldehydes and electron-rich anilines to modify phenolic side chains through a Mannich-type electrophilic arom. substitution pathway. The reaction takes place under mild pH and temp. conditions and can modify protein substrates at concns. as low as 20 μM. Using an efficient fluorescence-based assay, we demonstrated the reaction using a no. of aldehydes and protein targets. Importantly, proteins lacking surface-accessible tyrosines remained unmodified. It was also demonstrated that enzymic activity is preserved under the mild reaction conditions. This strategy represents one of the first carbon-carbon bond-forming reactions for protein modification and provides an important complement to more commonly used lysine- and cysteine-based methods.
    547. 547
      Cardellicchio, C.; Capozzi, M. A. M.; Naso, F. The Betti Base: The Awakening of a Sleeping Beauty. Tetrahedron: Asymmetry 2010, 21 (5), 507– 17,  DOI: 10.1016/j.tetasy.2010.03.020
      550
      The Betti base. The awakening of a sleeping beauty
      Cardellicchio, Cosimo; Capozzi, Maria Annunziata M.; Naso, Francesco
      Tetrahedron: Asymmetry (2010), 21 (5), 507-517CODEN: TASYE3; ISSN:0957-4166. (Elsevier Ltd.)
      A review. The multicomponent reaction between 2-naphthol, aryl aldehydes and ammonia or amines yields aminobenzylnaphthols in a process known as the Betti reaction, which was first uncovered at the beginning of the 20th century. The aminobenzylnaphthols could be easily resolved into their enantiomers. After a long silence, the results of our research a decade ago on this useful reaction and on the chiral materials produced has stimulated further work in a no. of other labs. As a result, novel applications of the Betti reaction to produce new chiral aminobenzylnaphthols were reported together with the evaluation of these chiral bases in asym. synthesis. Herein, the authors presented a selection of the relevant studies on this topic.
    548. 548
      Minakawa, M.; Guo, H.-M.; Tanaka, F. Imines That React with Phenols in Water over a Wide pH Range. J. Org. Chem. 2008, 73 (21), 8669– 72,  DOI: 10.1021/jo8017389
      551
      Imines that React with Phenols in Water over a Wide pH Range
      Minakawa, Maki; Guo, Hai-Ming; Tanaka, Fujie
      Journal of Organic Chemistry (2008), 73 (21), 8669-8672CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      Cyclic imine derivs. that react with phenols, including tyrosine residues of peptides, have been developed. Reactions of the imines I-III with phenols proceeded in water over a wide pH range (pH 2-10) at room temp. to 37 °C and afforded Mannich products without the need of addnl. catalysts.
    549. 549
      Kanaoka, Y. Organic Fluorescence Reagents in the Study of Enzymes and Proteins. Angew. Chem., Int. Ed. 1977, 16 (3), 137– 47,  DOI: 10.1002/anie.197701371
      552
      Organic fluorescence reagents in the study of enzymes and proteins
      Kanaoka Y
      Angewandte Chemie (International ed. in English) (1977), 16 (3), 137-47 ISSN:1433-7851.
      There is no expanded citation for this reference.
    550. 550
      Davis, W.; Ronai, Z.; Tew, K. D. Cellular Thiols and Reactive Oxygen Species in Drug-Induced Apoptosis. J. Pharmacol. Exp. Ther. 2001, 296, 1– 6
      553
      Cellular thiols and reactive oxygen species in drug-induced apoptosis
      Davis, Warren, Jr.; Ronai, Ze'ev; Tew, Kenneth D.
      Journal of Pharmacology and Experimental Therapeutics (2001), 296 (1), 1-6CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)
      A review with 42 refs. In higher eukaryotes, reactive oxygen species (ROS) are generated during respiration in mitochondria in the course of redn. of mol. oxygen as well as by distinct enzyme systems. ROS have been implicated in the regulation of diverse cellular functions including defense against pathogens, intracellular signaling, transcriptional activation, proliferation, and apoptosis. The redn.-oxidn. (redox) state of the cell is primarily a consequence of the precise balance between the levels of ROS and endogenous thiol buffers present in the cell, such as glutathione and thioredoxin, which protect cells from oxidative damage. Dramatic elevation of ROS, exceeding compensatory changes in the level of the endogenous thiol buffers, may result in the sustained activation of signaling pathways and expression of genes that induce apoptosis in affected cells. Many cytotoxic drugs function selectively to kill cancer cells by the abrogation of proliferative signals, leading to cell death, and numerous reports have demonstrated that ROS are generated following treatment with these drugs. In this review, we will summarize recent contributions to our understanding of the importance of cytotoxic drug-induced modulation of cellular redox status for signaling and transcription leading to activation of apoptotic effector mechanisms.
    551. 551
      Ball, R. O.; Courtney-Martin, G.; Pencharz, P. B. The in Vivo Sparing of Methionine by Cysteine in Sulfur Amino Acid Requirements in Animal Models and Adult Humans. J. Nutr. 2006, 136 (6), 1682S– 93S,  DOI: 10.1093/jn/136.6.1682S
      554
      The in vivo sparing of methionine by cysteine in sulfur amino acid requirements in animal models and adult humans
      Ball, Ronald O.; Courtney-Martin, Glenda; Pencharz, Paul B.
      Journal of Nutrition (2006), 136 (6S), 1682S-1693SCODEN: JONUAI; ISSN:0022-3166. (American Society for Nutrition)
      A review. S amino acid metab. was receiving increased attention because of the link to chronic diseases such as cardiovascular disease, Alzheimer's disease, and diabetes. In addn., the role of Cys and optimal intakes for physiol. substrates such as glutathione are currently of considerable interest in human health. Although the dietary indispensability of Met is not in question, the ability of Cys to substitute for a portion of its requirement was the topic of much debate. Met is often the most limiting amino acid in the diets of the developing world's population because of its low concn. in cereal grains. Therefore, the ability of Cys to substitute for Met requirement is not just biol. interesting; it is also of considerable economic and social importance. The primary goal of this review is to discuss the available evidence on the effect of Cys substitution for Met to meet the total sulfur amino acid requirement in adult humans, including an assessment of the methodol. features of expts. with conflicting results. Assessment of the requirement expts. for amino acids with complex metab. such as Met and Cys must begin with a careful definition of requirements and what substitution means. As a result of these definitions, a set of criteria for the intakes of Met that will allow demonstration of the substitution effect were developed. Some recent publications are assessed using these definitions and criteria, and a possible reason for the conflicting results in the literature is proposed. An approach to estg. tolerable upper intakes is also proposed. Research on in vivo S amino acid metab. in humans is tremendously difficult, and therefore, we do not wish to be overly crit. of the high-quality work of the ambitious and highly intelligent men and women who have conducted various studies. Our goal is to objectively review the data for the reader in a logical and comprehensive manner and propose methods that may avoid difficulties in future studies.
    552. 552
      Mishanina, T. V.; Libiad, M.; Banerjee, R. Biogenesis of Reactive Sulfur Species for Signaling by Hydrogen Sulfide Oxidation Pathways. Nat. Chem. Biol. 2015, 11 (7), 457– 64,  DOI: 10.1038/nchembio.1834
      555
      Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways
      Mishanina, Tatiana V.; Libiad, Marouane; Banerjee, Ruma
      Nature Chemical Biology (2015), 11 (7), 457-464CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      A review. The chem. species involved in H2S signaling remain elusive despite the profound and pleiotropic physiol. effects elicited by this mol. The dominant candidate mechanism for sulfide signaling is persulfidation of target proteins. However, the relatively poor reactivity of H2S toward oxidized thiols, such as disulfides, the low concn. of disulfides in the reducing milieu of the cell and the low steady-state concn. of H2S raise questions about the plausibility of persulfide formation via reaction between an oxidized thiol and a sulfide anion or a reduced thiol and oxidized hydrogen disulfide. In contrast, sulfide oxidn. pathways, considered to be primarily mechanisms for disposing of excess sulfide, generate a series of reactive sulfur species, including persulfides, polysulfides and thiosulfate, that could modify target proteins. We posit that sulfide oxidn. pathways mediate sulfide signaling and that sulfurtransferases ensure target specificity.
    553. 553
      Coleman, J.; Blake-Kalff, M.; Davies, E. Detoxification of Xenobiotics by Plants: Chemical Modification and Vacuolar Compartmentation. Trends Plant Sci. 1997, 2 (4), 144– 51,  DOI: 10.1016/S1360-1385(97)01019-4
      There is no corresponding record for this reference.
    554. 554
      Tsay, J. T.; Oh, W.; Larson, T. J.; Jackowski, S.; Rock, C. O. Isolation and Characterization of the Beta-Ketoacyl-Acyl Carrier Protein Synthase III Gene (FabH) from Escherichia Coli K-12. J. Biol. Chem. 1992, 267 (10), 6807– 14,  DOI: 10.1016/S0021-9258(19)50498-7
      557
      Isolation and characterization of the β-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12
      Tsay, Jiu Tsair; Oh, Won; Larson, Timothy J.; Jackowski, Suzanne; Rock, Charles O.
      Journal of Biological Chemistry (1992), 267 (10), 6807-14CODEN: JBCHA3; ISSN:0021-9258.
      β-Ketoacyl-acyl carrier protein (ACP) synthase III catalyzes the condensation of acetyl-CoA with malonyl-ACP in dissocd. (Type II) fatty acid synthase systems. A synthase III mutant was used to localize the structural gene to the 24.5-min region to localize the structural gene to the 24.5-min region of the E. coli chromosome, and the defective synthase III allele was designated fabH1. The fabH gene was identified on a 1.3-kilobase NruI-HindIII chromosomal DNA fragment (plasmid pWO114) that complemented the enzymic defect in fabH1 strains. The NruI-HinIII fragment was sequenced and contained a single open reading frame predicted to encode a 33,517-dalton protein with an isoelec. point of 4.85. The fabH sequence contained an Ala-Cys-Ala tripeptide characteristic of condensing enzyme active sites. A T7 expression system showed that the NruI-HindIII fragment directed the synthesis of a single 34,800-dalton protein. This protein was purified and the order of the N-terminal 30 residues of the protein corresponded exactly to the amino acid structure predicted from the DNA sequence. The purified protein possessed both acetoacetyl-ACP synthase and acetyl-CoA:ACP transacylase activities, and cells harboring plasmid pWO114 overproduced the two activities, supporting the conclusion that a single protein carries out both reactions. Overprodn. of synthase III resulted in a significant increase in shorter-chain fatty acids in the membrane phospholipids. These catalytic properties are consistent with the proposed role of synthase III in the initiation of fatty acid synthesis.
    555. 555
      Gao, T.; Yang, C.; Zheng, Y. G. Comparative Studies of Thiol-Sensitive Fluorogenic Probes for HAT Assays. Anal. Bioanal. Chem. 2013, 405 (4), 1361– 71,  DOI: 10.1007/s00216-012-6522-5
      558
      Comparative studies of thiol-sensitive fluorogenic probes for HAT assays
      Gao, Tielong; Yang, Chao; Zheng, Yujun George
      Analytical and Bioanalytical Chemistry (2013), 405 (4), 1361-1371CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      Histone acetyltransferases (HATs) catalyze the acetylation of specific lysine residues in histone and nonhistone proteins. Recent studies showed that acetylation is widely distributed among cellular proteins, suggestive of diverse functions of HATs in cellular pathways. Nevertheless, currently available assays for HAT activity study are still quite limited. Here, we evaluated a series of thiol-sensitive fluorogenic compds. for the detection of the enzymic activities of different HAT proteins. Upon conjugation to the thiol group of HSCoA, these mols. gain enhanced quantum yields and strong fluorescence, permitting facile quantitation of HAT activities. We investigated and compared the assay performances of these fluorogenic compds. for their capability as HAT activity reporters, including kinetics of reaction with HSCoA, influence on HAT activity, and fluorescence amplification factors. Our data suggest that CPM and coumarin maleic acid ester are excellent HAT probes owing to their fast reaction kinetics and dramatic fluorescence enhancement during the HAT reaction. Further, the microtiter plate measurements show that this fluorescent approach is robust and well suited for adaptation to high-throughput screening of small mol. inhibitors of HATs, highlighting the value of this assay strategy in new drug discovery.
    556. 556
      Tang, Y.; Jin, L.; Yin, B. A Dual-Selective Fluorescent Probe for GSH and Cys Detection: Emission and pH Dependent Selectivity. Anal. Chim. Acta 2017, 993, 87– 95,  DOI: 10.1016/j.aca.2017.09.028
      559
      A dual-selective fluorescent probe for GSH and Cys detection: Emission and pH dependent selectivity
      Tang, Yunqiang; Jin, Longyi; Yin, Bingzhu
      Analytica Chimica Acta (2017), 993 (), 87-95CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      A novel fluorescent probe 1 (I) based on acridine orange was developed for the selective detection and bioimaging of biothiols. The probe exhibits higher selectivity and turn-on fluorescence response to cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) than to other amino acids. Importantly, the probe responds to GSH and Cys/Hcy with distinct fluorescence emissions in PBS buffer at pH of 7.4. The Cys/Hcy-triggered tandem SNAr-rearrangement reaction and GSH-induced SNAr reaction with the probe led to the corresponding amino-acridinium and thio-acridinium dyes, resp., which can discriminate GSH from Cys/Hcy through different emission channels. Cys finishes the tandem reaction with the probe and subsequently forms amino-acridinium and Hcy/GSH induces SNAr reaction with the probe to form thio-acridiniums at weakly acidic conditions (pH 6.0), enabling Cys to be discriminated from Hcy/GSH at different emissions. Finally, probe I can selectively probe GSH over Cys and Hcy or Cys over GSH and Hcy in HeLa cells through multicolor imaging.
    557. 557
      Zaric, B. L.; Obradovic, M.; Bajic, V.; Haidara, M. A.; Jovanovic, M.; Isenovic, E. R. Homocysteine and Hyperhomocysteinaemia. Curr. Med. Chem. 2019, 26 (16), 2948– 61,  DOI: 10.2174/0929867325666180313105949
      560
      Homocysteine and Hyperhomocysteinaemia
      Zaric, Bozidarka L.; Obradovic, Milan; Bajic, Vladan; Haidara, Mohamed A.; Jovanovic, Milos; Isenovic, Esma R.
      Current Medicinal Chemistry (2019), 26 (16), 2948-2961CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)
      A review. Homocysteine (Hcy) is a thiol group contg. the amino acid, which naturally occurs in all humans. Hcy is degraded in the body through two metabolic pathways, while a minor part is excreted through kidneys. The chem. reactions that are necessary for degrdn. of Hcy require the presence of folic acid, vitamins B6 and B12. Consequently, the level of the total Hcy in the serum is influenced by the presence or absence of these vitamins. An elevated level of the Hcy, hyperhomocysteinemia (HHcy) and homocystinuria is connected with occlusive artery disease, esp. in the brain, the heart, and the kidney, in addn. to venous thrombosis, chronic renal failure, megaloblastic anemia, osteoporosis, depression, Alzheimer's disease, pregnancy problems, and others. Elevated Hcy levels are connected with various pathologies both in adult and child population. Causes of HHcy include genetic mutations and enzyme deficiencies in 5, 10-methylenetetrahydrofolate reductase (MTHFR) methionine synthase (MS), and cystathionine β-synthase (CβS). HHcy can be caused by deficiencies in the folate, vitamin B12 and to a lesser extent, deficiency in B6 vitamin what influences methionine metab. Addnl., HHcy can be caused by the rich diet and renal impairment. This review presents literature data from recent research related to Hcy metab. and the etiol. of the Hcy blood level disorder. In addn., we also described various pathol. mechanisms induced by hereditary disturbances or nutritional influences and their assocn. with HHcy induced pathol. in adults and children and treatment of these metabolic disorders.
    558. 558
      van Meurs, J.; Dhonukshe-Rutten, R. A.; Pluijm, S. M.; Van Der Klift, M.; De Jonge, R.; Lindemans, J.; De Groot, L. C.; Hofman, A.; Witteman, J. C.; Van Leeuwen, J. P.; Breteler, M. M.; Lips, P.; Pols, H. A.; Uitterlinden, A. G. Homocysteine Levels and the Risk of Osteoporotic Fracture. N. Engl. J. Med. 2004, 350, 2033– 41,  DOI: 10.1056/NEJMoa032546
      561
      Homocysteine levels and the risk of osteoporotic fracture
      van Meurs, Joyce B. J.; Dhonukshe-Rutten, Rosalie A. M.; Pluijm, Saskia M. F.; van der Klift, Marjolein; de Jonge, Robert; Lindemans, Jan; de Groot, Lisette C. P. G. M.; Hofman, Albert; Witteman, Jacqueline C. M.; van Leeuwen, Johannes P. T. M.; Breteler, Monique M. B.; Lips, Paul; Pols, Huibert A. P.; Uitterlinden, Andre G.
      New England Journal of Medicine (2004), 350 (20), 2033-2041CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
      Very high plasma homocysteine levels are characteristic of homocystinuria, a rare autosomal recessive disease accompanied by the early onset of generalized osteoporosis. We therefore hypothesized that mildly elevated homocysteine levels might be related to age-related osteoporotic fractures. We therefore hypothesized that mildly elevated homocysteine levels might be related to age-related osteoporotic fractures. In the Rotterdam Study, there were two independent cohorts: 562 subjects in cohort 1, with a mean follow-up period of 8.1 yr; and 553 subjects in cohort 2, with a mean follow-up period of 5.7 yr. In the Longitudinal Aging Study Amsterdam, there was a single cohort of 1291 subjects, with a mean follow-up period of 2.7 yr. Multivariate Cox proportional-hazards regression models were used for anal. of the risk of fracture, with adjustment for age, sex, body-mass index, and other characteristics that may be assocd. with the risk of fracture or with increased homocysteine levels. During 11,253 person-years of follow-up, osteoporotic fractures occurred in 191 subjects. The overall multivariable-adjusted relative risk of fracture was 1.4 (95 percent confidence interval, 1.2 to 1.6) for each increase of 1 SD in the natural-log-transformed homocysteine level. The risk was similar in all three cohorts studied, and it was also similar in men and women. A homocysteine level in the highest age-specific quartile was assocd. with an increase by a factor of 1.9 in the risk of fracture (95 percent confidence interval, 1.4 to 2.6). The assocns. between homocysteine levels and the risk of fracture appeared to be independent of bone mineral d. and other potential risk factors for fracture. An increased homocysteine level appears to be a strong and independent risk factor for osteoporotic fractures in older men and women.
    559. 559
      Lee, D.; Kim, G.; Yin, J.; Yoon, J. An Aryl-Thioether Substituted Nitrobenzothiadiazole Probe for the Selective Detection of Cysteine and Homocysteine. Chem. Commun. 2015, 51 (30), 6518– 20,  DOI: 10.1039/C5CC01071C
      562
      An aryl-thioether substituted nitrobenzothiadiazole probe for the selective detection of cysteine and homocysteine
      Lee, Dayoung; Kim, Gyoungmi; Yin, Jun; Yoon, Juyoung
      Chemical Communications (Cambridge, United Kingdom) (2015), 51 (30), 6518-6520CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      An aryl-thioether substituted nitrobenzothiadiazole probe was synthesized and employed to detect cysteine and homocysteine selectively in living cells. Interestingly, both cysteine (Cys) and homocysteine (Hcy) promote an enhancement of the fluorescence intensity of the probe at pH 7.4 while only Cys gives rise to this enhancement under weakly acidic conditions (pH 6.0).
    560. 560
      Giannoulis, K. M.; Giokas, D. L.; Tsogas, G. Z.; Vlessidis, A. G. Ligand-Free Gold Nanoparticles as Colorimetric Probes for the Non-Destructive Determination of Total Dithiocarbamate Pesticides after Solid Phase Extraction. Talanta 2014, 119, 276– 83,  DOI: 10.1016/j.talanta.2013.10.063
      563
      Ligand-free gold nanoparticles as colorimetric probes for the non-destructive determination of total dithiocarbamate pesticides after solid phase extraction
      Giannoulis, Kyriakos M.; Giokas, Dimosthenis L.; Tsogas, George Z.; Vlessidis, Athanasios G.
      Talanta (2014), 119 (), 276-283CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      In this work, we describe a simple and sensitive non-destructive method for the detn. of the total concn. of dithiocarbamate fungicides (DTCs) in real samples. The proposed method combines for the first time the benefits of an extn. method for sample clean-up and preconcn. with a sensitive colorimetric assay based on gold nanoparticle probes. In this two-step procedure, the target DTCs are isolated from the matrix and preconcd. by solid phase extn. onto com. available C18 sorbents. Following elution, the ext. contg. the target dithiocarbamates, free from most interferences and matrix components, is delivered into an aq. dispersion of plain citrate-capped gold nanoparticles (AuNPs) which aggregate in response to DTCs coordination on AuNPs surface through multiple gold thiolate bonds. This aggregation is evidenced by changes in the spectral properties of the soln. involving a decrease in the original absorbance of Au nanoparticles at 522 nm and the appearance of a new absorption band above 700 nm. An ensuing chromatic shift of the soln. from wine-red to purple-blue is obsd. which is visual by naked eye at concns. as low as 50 μg L-1. Further improvement in the detection limits can be accomplished by scaling-down the method to micro-vol. conditions alleviating the need to preconc. larger sample vols. Overall, by combining sample clean-up and preconcn. with the strong affinity of DTC thiol group for the gold surface, the total concn. of dithiocarbamate pesticides was successfully detd. in various water samples at the low and ultra-low μg L-1 levels without resorting to destructive techniques, sophisticated instrumentation or post-synthetic modification of gold nanoparticles. Method application in real samples showed good anal. features in terms of recoveries (81.0-94.0%), precision (5.6-8.9%) and reproducibility (∼9%) rendering the method as an attractive alternative to current methodologies for the detn. of DTC fungicide residues in samples of environmental interest.
    561. 561
      Munday, R. Toxicity of Thiols and Disulphides: Involvement of Free-Radical Species. Free Radical Biol. Med. 1989, 7 (6), 659– 73,  DOI: 10.1016/0891-5849(89)90147-0
      564
      Toxicity of thiols and disulfides: involvement of free-radical species
      Munday, Rex
      Free Radical Biology & Medicine (1989), 7 (6), 659-73CODEN: FRBMEH; ISSN:0891-5849.
      A review with 188 refs. Sulfur is essential to life, and thiols and disulfides play essential roles in cellular biochem. Such compds. are also widely distributed in the food of man and his domestic animals, and they are extensively used in industry. However, many thiols and disulfides have been shown to be toxic. Aliph., arom., and heterocyclic compds. of this type are hemolytic agents in animals, while aminothiols have been shown to induce many cytotoxic effects in vitro and the epidithiodioxopiperazine mycotoxin, sporidesmin, is a potent hepatotoxic agent. Structure-activity relationships among these compds. and factors which modulate their harmful effects are consistent with a toxic mechanism involving redox cycling between the thiol and the corresponding disulfide. Thiyl radicals and active oxygen species are formed in this process, and it is suggested that these substances are responsible for initiating the tissue damage provoked by thiols and disulfides.
    562. 562
      Cazzola, M.; Calzetta, L.; Page, C.; Rogliani, P.; Matera, M. G. Thiol-Based Drugs in Pulmonary Medicine: Much More Than Mucolytics. Trends Pharmacol. Sci. 2019, 40 (7), 452– 63,  DOI: 10.1016/j.tips.2019.04.015
      565
      Thiol-Based Drugs in Pulmonary Medicine: Much More than Mucolytics
      Cazzola, Mario; Calzetta, Luigino; Page, Clive; Rogliani, Paola; Matera, Maria Gabriella
      Trends in Pharmacological Sciences (2019), 40 (7), 452-463CODEN: TPHSDY; ISSN:0165-6147. (Elsevier Ltd.)
      Thiol-based drugs are considered as mucolytics because they decrease the viscosity and mostly decrease the elasticity of bronchial secretions by reducing disulfide bonds in proteins. However, they can also act as antioxidant drugs directly through free sulfhydryl groups that serve as a source of reducing equiv., as well as indirectly through the replenishment of intracellular glutathione (GSH) levels. Modulation of neurokinin A levels may also be related to the effect of thiol drugs on oxidative stress. Moreover, thiol-based drugs interfere with inflammatory pathways and modulate human bronchial tone. They might also be considered as therapeutic agents against some types of infection because they reduce bacterial adhesion to the respiratory epithelial cell surface and inhibit biofilm formation, causing biofilm disruption and thereby improving the efficacy of antibiotic therapy.
    563. 563
      Wang, H.; Wu, X.; Yang, S.; Tian, H.; Liu, Y.; Sun, B. A Rapid and Visible Colorimetric Fluorescent Probe for Benzenethiol Flavor Detection. Food Chem. 2019, 286, 322– 8,  DOI: 10.1016/j.foodchem.2019.02.033
      566
      A rapid and visible colorimetric fluorescent probe for benzenethiol flavor detection
      Wang, Hao; Wu, Xiaoming; Yang, Shaoxiang; Tian, Hongyu; Liu, Yongguo; Sun, Baoguo
      Food Chemistry (2019), 286 (), 322-328CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)
      Benzenethiols are a class of flavoring ingredients used in the food, pharmaceutical, cosmetics and chem. industries. A rapid and visible colorimetric fluorescent probe was developed for the detection of benzenethiol flavors. It provides rapid quant. detection of benzenethiols at low levels, down to a limit of 10 nM. Test paper contg. the probe changes color according to benzenethiol concn. (from colorless to pink, visible with the naked eye). The probe was also successfully used to test benzenethiol concns. in real food samples. This study demonstrates that this novel probe can be employed as a benzenethiol testing tool.
    564. 564
      Liu, X.-L.; Duan, X.-Y.; Du, X.-J.; Song, Q.-H. Quinolinium-Based Fluorescent Probes for the Detection of Thiophenols in Environmental Samples and Living Cells. Chem.─Asian J. 2012, 7, 2696– 2702,  DOI: 10.1002/asia.201200594
      567
      Quinolinium-Based Fluorescent Probes for the Detection of Thiophenols in Environmental Samples and Living Cells
      Liu, Xiu-Ling; Duan, Xue-You; Du, Xiao-Jiao; Song, Qin-Hua
      Chemistry - An Asian Journal (2012), 7 (11), 2696-2702, S2696/1-S2696/12CODEN: CAAJBI; ISSN:1861-4728. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A new type of fluorescent probes for thiophenols, 6HQM-DNP (I) and 7HQM-DNP (II), contg. 6- or 7-hydroxy quinonlinium as fluorophore and 2,4-dinitrophenoxy (DNP) as nucleophilic recognition unit were constructed. As ethers, these nonfluorescent probe mols. can release the corresponding fluorescent quinolinium (6HQM and 7HQM) through arom. nucleophilic substitution (SNAr) by thiolate anions from thiophenols. The sensing reaction is highly sensitive (detection limit of 8 nM for 7HQM-DNP) and highly selective to thiophenols over aliph. thiols and other nucleophiles under neutral conditions (pH 7.3). The probes respond rapidly to thiophenols, with 2nd-order rate consts. k = 45 M-1 s-1 for 7HQM-DNP and 24 M-1 s-1 for 6HQM-DNP. Also, the selective detection of thiophenols in living cells by 7HQM-DNP was demonstrated by confocal fluorescence imaging. These quinolinium salts show excellent chem. and thermal stability. In conclusion, this type of probes may find use in the detection of thiophenols in environmental samples and biosystems.
    565. 565
      He, L.; Yang, X.; Xu, K.; Kong, X.; Lin, W. A Multi-Signal Fluorescent Probe for Simultaneously Distinguishing and Sequentially Sensing Cysteine/Homocysteine, Glutathione, and Hydrogen Sulfide in Living Cells. Chem. Sci. 2017, 8 (9), 6257– 65,  DOI: 10.1039/C7SC00423K
      568
      A multi-signal fluorescent probe for simultaneously distinguishing and sequentially sensing cysteine/homocysteine, glutathione, and hydrogen sulfide in living cells
      He, Longwei; Yang, Xueling; Xu, Kaixin; Kong, Xiuqi; Lin, Weiying
      Chemical Science (2017), 8 (9), 6257-6265CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Biothiols, which have a close network of generation and metabolic pathways among them, are essential reactive sulfur species (RSS) in the cells and play vital roles in human physiol. However, biothiols possess highly similar chem. structures and properties, resulting in it being an enormous challenge to simultaneously discriminate them from each other. Herein, we develop a unique fluorescent probe (HMN) for not only simultaneously distinguishing Cys/Hcy, GSH, and H2S from each other, but also sequentially sensing Cys/Hcy/GSH and H2S using a multi-channel fluorescence mode for the first time. When responding to the resp. biothiols, the robust probe exhibits multiple sets of fluorescence signals at three distinct emission bands (blue-green-red). The new probe can also sense H2S at different concn. levels with changes of fluorescence at the blue and red emission bands. In addn., the novel probe HMN is able to discriminate and sequentially sense biothiols in biol. environments via three-color fluorescence imaging. We expect that the development of the robust probe HMN will provide a powerful strategy to design fluorescent probes for the discrimination and sequential detection of biothiols, and offer a promising tool for exploring the interrelated roles of biothiols in various physiol. and pathol. conditions.
    566. 566
      Wang, N.; Chen, M.; Gao, J.; Ji, X.; He, J.; Zhang, J.; Zhao, W. A Series of BODIPY-Based Probes for the Detection of Cysteine and Homocysteine in Living Cells. Talanta 2019, 195, 281– 9,  DOI: 10.1016/j.talanta.2018.11.066
      569
      A series of BODIPY-based probes for the detection of cysteine and homocysteine in living cells
      Wang, Nannan; Chen, Miao; Gao, Jinhua; Ji, Xin; He, Jinling; Zhang, Jian; Zhao, Weili
      Talanta (2019), 195 (), 281-289CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      Biothiols, such as glutathione (GSH), homocysteine (Hcy) and cysteine (Cys), are important biomarkers and play crucial roles in many physiol. processes. Thus, the detection of biothiols is highly important for early diagnosis of diseases and evaluation of disease progression. Herein, new types of BODIPY-based fluorescent probes (probe 1, probe 2 and probe 3) capable of cysteine (Cys)/homocysteine (Hcy) sensing with high selectivity over other amino acids were developed. In addn., the authors further studied the influence of different electronegativity substituents on these probes to sensing Cys/Hcy. Ultimately, the electron withdrawing group on probe 1 can accelerate the probe response to Cys/Hcy, and probe 1 was successfully applied for selective imaging Cys/Hcy in living cells.
    567. 567
      Yao, Z.; Ge, W.; Guo, M.; Xiao, K.; Qiao, Y.; Cao, Z.; Wu, H.-C. Ultrasensitive Detection of Thiophenol Based on a Water-Soluble Pyrenyl Probe. Talanta 2018, 185, 146– 50,  DOI: 10.1016/j.talanta.2018.03.068
      570
      Ultrasensitive detection of thiophenol based on a water-soluble pyrenyl probe
      Yao, Zhiyi; Ge, Wenqi; Guo, Mingwei; Xiao, Keren; Qiao, Yadong; Cao, Zhong; Wu, Hai-Chen
      Talanta (2018), 185 (), 146-150CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      We report a simple pyrene-based fluorescent probe, sodium 8-(2,4-dinitrophenoxy)pyrene-1,3,6-trisulfonate (HPTS-DNP), for the ultrasensitive and visual detection of thiophenol in 100% aq. media. The sensing mechanism of this method is based on nucleophilic arom. substitution on HPTS-DNP caused by thiophenol to afford a highly fluorescent product, 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS). The extremely low fluorescence background of HPTS-DNP and high quantum yield of HPTS ensure the superior sensing performance toward thiophenol, including rapid responses, off-on detection mode and excellent sensitivity. The detection limit is as low as 0.49 nmol L-1 according to the measurements with a spectrofluorimeter. This probe also features good selectivity (response ratio of thiophenol to interferents >30), precision (2.93%) and accuracy (102%). This approach could be applied for monitoring the thiophenol concns. in environmental water samples. HPTS-DNP also showed good cell permeation capacity and low cellular cytotoxicity, indicating further application in bioimaging studies.
    568. 568
      Zhang, S.; Cai, F.; Hou, B.; Chen, H.; Gao, C.; Shen, X.-c.; Liang, H. Constructing a Far-Red to near-Infrared Fluorescent Probe for Highly Specific Detection of Cysteine and Its Bioimaging Applications in Living Cells and Zebrafish. New J. Chem. 2019, 43 (17), 6696– 701,  DOI: 10.1039/C9NJ00260J
      571
      Constructing a far-red to near-infrared fluorescent probe for highly specific detection of cysteine and its bioimaging applications in living cells and zebrafish
      Zhang, Shuping; Cai, Fangyuan; Hou, Bo; Chen, Hua; Gao, Cunji; Shen, Xing-can; Liang, Hong
      New Journal of Chemistry (2019), 43 (17), 6696-6701CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
      Cys is a common and important sulfur-contg. amino acid in living organisms, whose intracellular level changes are assocd. with a variety of diseases. To specifically detect Cys without interference from other thiol species has considerable significance. However, only selective fluorescent probes are available for cysteine detection; other thiol species can also have certain interference in the sensing process. Herein, we report an irreversible fluorescent probe for the specific detection of cysteine without interference from other thiol species both in living cells and zebrafish. The FR-NIR-Cys probe exhibits a large emission wavelength shift (up to 84 nm) and a significant ratio fluorescence enhancement (up to 41-fold) in the far-red and near-IR region. Furthermore, we demonstrate that FR-NIR-Cys with good cell permeability can be employed in the far-red to near-IR imaging of Cys in living cells and zebrafish.
    569. 569
      Fan, L.; Zhang, W.; Wang, X.; Dong, W.; Tong, Y.; Dong, C.; Shuang, S. A Two-Photon Ratiometric Fluorescent Probe for Highly Selective Sensing of Mitochondrial Cysteine in Live Cells. Analyst 2019, 144 (2), 439– 47,  DOI: 10.1039/C8AN01908H
      572
      A two-photon ratiometric fluorescent probe for highly selective sensing of mitochondrial cysteine in live cells
      Fan, Li; Zhang, Wenjia; Wang, Xiaodong; Dong, Wenjuan; Tong, Yaoli; Dong, Chuan; Shuang, Shaomin
      Analyst (Cambridge, United Kingdom) (2019), 144 (2), 439-447CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      We report herein a two-photon ratiometric fluorescent probe (DNEPI) for mitochondrial cysteine (Cys) detection on the basis of a merocyanine (compd. 1) as the two-photon fluorophore and a 2,4-dinitrobenzensulfonyl (DNBS) unit as the biothiol reaction site. Upon reaction with Cys in DMSO/PBS (1/1, vol./vol.), DNEPI showed a distinct ratiometric fluorescence emission characteristic (F583 nm/F485 nm) linearly proportional to Cys concns. over the range of 2-10 μM, which was attribute to the enhanced intramol. charge transfer (ICT) effect by cleavage of the sulfonic acid ester bond of DNEPI to release compd. 1. More importantly, the probe could detect Cys with a fast response time (within 2 min) and the detection limit was quant. calcd. as 0.29 μM. Furthermore, DNEPI not only exhibited high selectivity toward Cys over other similar biothiols, including homocysteine (Hcy) and glutathione (GSH), but also displayed significant mitochondrial-targeting ability, which were favorable for mitochondrial Cys-selective imaging. Subsequently, application of DNEPI to Cys imaging in live cells was successfully achieved by two-photon fluorescence microscopy, suggesting that the probe proposed here could be used to monitor mitochondrial Cys concn. changes in live cells with negligible interference from other biol. thiols.
    570. 570
      Ros-Lis, J. V.; García, B.; Jiménez, D.; Martínez-Máñez, R.; Sancenón, F.; Soto, J.; Gonzalvo, F.; Valldecabres, M. C. Squaraines as Fluoro-Chromogenic Probes for Thiol-Containing Compounds and Their Application to the Detection of Biorelevant Thiols. J. Am. Chem. Soc. 2004, 126 (13), 4064– 5,  DOI: 10.1021/ja031987i
      573
      Squaraines as fluoro-chromogenic probes for thiol-containing compounds and their application to the detection of biorelevant thiols
      Ros-Lis, Jose V.; Garcia, Beatriz; Jimenez, Diego; Martinez-Manez, Ramon; Sancenon, Felix; Soto, Juan; Gonzalvo, Fernando; Valldecabres, M. Carmen
      Journal of the American Chemical Society (2004), 126 (13), 4064-4065CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A highly selective colorimetric chemodosimeter for thiol-contg. compds. in aq. solns. is reported. The design protocol makes use of a highly specific reaction between thiols and the electrophilic four-membered ring of highly colored, fluorescent squaraine backbones. At neutral pH selective decoloration and total emission quenching was found due to the rupture of the highly delocalized squaraine framework upon selective nucleophilic addn. of thiol-contg. derivs. The squaraine derivs. have been successfully applied to the detn. of low-mol. mass aminothiols in human plasma. The method utters the high potential applicability of the chemodosimeter approach in the search for new or improved chromogenic selective or specific probes for target guests.
    571. 571
      Zhang, X.; Li, C.; Cheng, X.; Wang, X.; Zhang, B. A near-Infrared Croconium Dye-Based Colorimetric Chemodosimeter for Biological Thiols and Cyanide Anion. Sens. Actuators B Chem. 2008, 129 (1), 152– 7,  DOI: 10.1016/j.snb.2007.07.094
      574
      A near-infrared croconium dye-based colorimetric chemodosimeter for biological thiols and cyanide anion
      Zhang, Xuehua; Li, Chao; Cheng, Xuexin; Wang, Xuesong; Zhang, Baowen
      Sensors and Actuators, B: Chemical (2008), 129 (1), 152-157CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      A near-IR colorimetric chemodosimeter based on a croconium dye, 1,3-bis(4-N,N-diethylamino-2-hydroxyl-phenyl) croconine, was developed to selectively and sensitively probe cysteine, homocysteine, or cysteine-glycine at pH 5.7 and cyanide anion at pH 9.0 in aq. ethanol solns.
    572. 572
      Tanaka, F.; Mase, N.; Barbaa, C. F., III Determination of Cysteine Concentration by Fluorescence Increase: Reaction of Cysteine with a Fluorogenic Aldehyde. Chem. Commun. 2004, 2004, 1762,  DOI: 10.1039/b405642f
      There is no corresponding record for this reference.
    573. 573
      Jung, H. S.; Ko, K. C.; Kim, G.-H.; Lee, A.-R.; Na, Y.-C.; Kang, C.; Lee, J. Y.; Kim, J. S. Coumarin-Based Thiol Chemosensor: Synthesis, Turn-On Mechanism, and Its Biological Application. Org. Lett. 2011, 13 (6), 1498– 501,  DOI: 10.1021/ol2001864
      576
      Coumarin-Based Thiol Chemosensor: Synthesis, Turn-On Mechanism, and Its Biological Application
      Jung, Hyo Sung; Ko, Kyoung Chul; Kim, Gun-Hee; Lee, Ah-Rah; Na, Yun-Cheol; Kang, Chulhun; Lee, Jin Yong; Kim, Jong Seung
      Organic Letters (2011), 13 (6), 1498-1501CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
      A new chemodosimetric probe (I) is reported that selectively detects thiols over other relevant biol. species by the turning on of its fluorescence through a Michael type reaction. The fluorogenic process upon its reaction was revealed to be mediated by intramol. charge transfer, as confirmed by time-dependent d. functional theory calcns. The application of probe I to cells is also examd. by confocal microscopy, and its cysteine preference was obsd. by an ex vivo LC-MS anal. of the cellular metabolite.
    574. 574
      Zhang, Y.; Shao, X.; Wang, Y.; Pan, F.; Kang, R.; Peng, F.; Huang, Z.; Zhang, W.; Zhao, W. Dual Emission Channels for Sensitive Discrimination of Cys/Hcy and GSH in Plasma and Cells. Chem. Commun. 2015, 51 (20), 4245– 8,  DOI: 10.1039/C4CC08687B
      577
      Dual emission channels for sensitive discrimination of Cys/Hcy and GSH in plasma and cells
      Zhang, Yuanlin; Shao, Xiangmin; Wang, Yue; Pan, Fuchao; Kang, Ruixue; Peng, Fangfang; Huang, Zhentao; Zhang, Weijuan; Zhao, Weili
      Chemical Communications (Cambridge, United Kingdom) (2015), 51 (20), 4245-4248CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A new selective fluorescent and colorimetric chemosensor for the detection of GSH was developed. The discrimination of GSH from Cys and Hcy is achieved through two emission channel detection. The detection limit of probe 1 for GSH reached 10 nM (3 ppb). The excellent sensitivity and selectivity of probe 1 allow the selective detection of GSH over Cys and Hcy, which can be visualized colorimetrically and/or fluorescently. The sensitive detection of GSH allowed for convenient measurement of the GSH content in human plasma. The presence of GSH in cells was demonstrated through cell imaging.
    575. 575
      Niu, L.-Y.; Guan, Y.-S.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. A Turn-On Fluorescent Sensor for the Discrimination of Cystein from Homocystein and Glutathione. Chem. Commun. 2013, 49 (13), 1294– 6,  DOI: 10.1039/c2cc38429a
      578
      A turn-on fluorescent sensor for the discrimination of cystein from homocystein and glutathione
      Niu, Li-Ya; Guan, Ying-Shi; Chen, Yu-Zhe; Wu, Li-Zhu; Tung, Chen-Ho; Yang, Qing-Zheng
      Chemical Communications (Cambridge, United Kingdom) (2013), 49 (13), 1294-1296CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      The authors report a turn-on fluorescent sensor based on nitrothiophenolate boron dipyrromethene (BODIPY) derivs. for the discrimination of cysteine (Cys) from homocysteine (Hcy) and glutathione (GSH). The sensor was applied for detection of Cys in living cells.
    576. 576
      Chen, W.; Luo, H.; Liu, X.; Foley, J. W.; Song, X. Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo. Anal. Chem. 2016, 88 (7), 3638– 46,  DOI: 10.1021/acs.analchem.5b04333
      579
      Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo
      Chen, Wenqiang; Luo, Hongchen; Liu, Xingjiang; Foley, James W.; Song, Xiangzhi
      Analytical Chemistry (Washington, DC, United States) (2016), 88 (7), 3638-3646CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are small biomol. thiols that are present in all cells and extracellular fluids of healthy mammals. It is well-known that each plays a sep., critically important role in human physiol. and that abnormal levels of each are predictive of a variety of different disease states. Although a no. of fluorescence-based methods have been developed that can detect biomols. that contain sulfhydryl moieties, few are able to differentiate between GSH and Cys/Hcy. In this report, we demonstrate a broadly applicable approach for the design of fluorescent probes that can achieve this goal. The strategy we employ is to conjugate a fluorescence-quenching 7-nitro-2,1,3-benzoxadiazole (NBD) moiety to a selected fluorophore (Dye) through a sulfhydryl-labile ether linkage to afford nonfluorescent NBD-O-Dye. In the presence of GSH or Cys/Hcy, the ether bond is cleaved with the concomitant generation of both a nonfluorescent NBD-S-R deriv. and a fluorescent dye having a characteristic intense emission band (B1). In the special case of Cys/Hcy, the NBD-S-Cys/Hcy cleavage product can undergo a further, rapid, intramol. Smiles rearrangement to form a new, highly fluorescent NBD-N-Cys/Hcy compd. (band B2); because of geometrical constraints, the GSH derived NBD-S-GSH deriv. cannot undergo a Smiles rearrangement. Thus, the presence of a single B1 or double B1 + B2 signature can be used to detect and differentiate GSH from Cys/Hcy, resp. We demonstrate the broad applicability of our approach by including in our studies members of the Flavone, Bodipy, and Coumarin dye families. Particularly, single excitation wavelength could be applied for the probe NBD-OF in the detection of GSH over Cys/Hcy in both aq. soln. and living cells.
    577. 577
      Mikaliunaite, L.; Green, D. B. Using a 3-Hydroxyflavone Derivative as a Fluorescent Probe for the Indirect Determination of Aminothiols Separated by Ion-Pair HPLC. Anal. Methods 2021, 13 (26), 2915– 25,  DOI: 10.1039/D1AY00499A
      580
      Using a 3-hydroxyflavone derivative as a fluorescent probe for the indirect determination of aminothiols separated by ion-pair HPLC
      Mikaliunaite, Lina; Green, David B.
      Analytical Methods (2021), 13 (26), 2915-2925CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)
      Homocysteine, cysteine, cysteinyl-glycine, and glutathione are significant biol. aminothiols (ATs) that are marker-mols. in Down syndrome, Alzheimer's disease, or have been implicated as risk factors in atherosclerosis and other vascular diseases, and therefore rapid detn. of these mols. is desirable. After redn. of the disulfides, a widely used method utilizes derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) as a fluorogenic probe prior to reversed-phase HPLC sepn. followed by fluorescence detection. The traditional HPLC detn. of ATs is time consuming and economically expensive. We have developed an ion-pair HPLC method coupled with indirect fluorescence detection after post-column reaction with a 2,4-dinitrobenzenesulfonate deriv. of a 3-hydroxyflavone. The accuracy, precision, post-column temp. and residence time, and limit-of-detection were evaluated. Sample throughput and reduced sample prepn. time of over an hour for the existing methods to less than 20 min for the new method is also demonstrated. No statistical differences in HCy, Cys, or Cys-Gly detns. in plasma samples were obsd. between our method and the traditional HPLC method.
    578. 578
      Toyooka, T.; Imai, K. New Fluorogenic Reagent Having Halogenobenzofurazan Structure for Thiols: 4-(Aminosulfonyl)-7-Fluoro-2,1,3-Benzoxadiazole. Anal. Chem. 1984, 56 (13), 2461– 4,  DOI: 10.1021/ac00277a044
      581
      New fluorogenic reagent having halogenobenzofurazan structure for thiols: 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole
      Toyooka, Toshimasa; Imai, Kazuhiro
      Analytical Chemistry (1984), 56 (13), 2461-4CODEN: ANCHAM; ISSN:0003-2700.
      4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) was synthesized as a new fluorogenic reagent for thiols. The reaction rate of ABD-F with homocysteine is >30 times faster than that of ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate. The fluorogenic reaction with thiol was completed quant. in 5 min at 50° and pH 8.0. Alanine, proline, and cystine did not react under the same conditions. The fluorescence intensity of the fluorophor was pH dependent with the highest at pH 2. The ABD-thiols obtained by the prelabeling technique were sepd. and detected by reversed-phase HPLC. The detection limits (S/N = 3) for cysteine, glutathione, N-acetylcysteine, homocysteine, and cysteamine were 0.6, 0.4, 1.9, 0.5, and 0.5 pmol, resp.
    579. 579
      Zhao, L.; Zhao, L.; Zhang, C.; Li, Y. A Naked-Eye Visible and Fluorescence ″Turn-On″ Probe for Acetylcholinesterase Assay and the Discrimination of Biothiols. J. Appl. Spectrosc. 2018, 85 (3), 437– 44,  DOI: 10.1007/s10812-018-0669-6
      582
      A Naked-Eye Visible and Fluorescence "Turn-On" Probe for Acetylcholinesterase Assay and the Discrimination of Biothiols
      Zhao, Lingzhi; Zhao, Liu; Zhang, Chenxiao; Li, Yanqi
      Journal of Applied Spectroscopy (2018), 85 (3), 437-444CODEN: JASYAP; ISSN:0021-9037. (Springer)
      We demonstrate the utility of 4-chloro-7-nitrobenzofurazan (NBD-Cl), a simple, low-cost probe, for colorimetrically and fluorescently detecting and differentiating similar thiol species, including cysteine, homocysteine, and glutathione, as well as for assaying acetylcholinesterase (AChE) activity. This assay is a potential tool for investigating thiol functions in disease and diagnosis, and for screening AChE inhibitors. We envision that this versatile probe, which is com. available and inexpensive, will be useful for further elucidating the roles of thiols in biol. and also shows applicability for anal. of AChE inhibitors in physiol. samples.
    580. 580
      Hassett, R. P.; Crockett, E. L. Endpoint Fluorometric Assays for Determining Activities of Carnitine Palmitoyltransferase and Citrate Synthase. Anal. Biochem. 2000, 287 (1), 176– 9,  DOI: 10.1006/abio.2000.4799
      583
      Endpoint Fluorometric Assays for Determining Activities of Carnitine Palmitoyltransferase and Citrate Synthase
      Hassett, R. Patrick; Crockett, Elizabeth L.
      Analytical Biochemistry (2000), 287 (1), 176-179CODEN: ANBCA2; ISSN:0003-2697. (Academic Press)
      The authors describe fluorometric procedures for assaying both carnitine palmitoyltransferase (CPT) and citrate synthase (CS). CoA-SH produced in the enzyme-catalyzed reactions is labeled with the thiol-reactive probe 7-fluorobenz-2-oxa- 1,3-diazole-4-sulfonamide (ABD-F). ABD-F has a much higher water soly. than the fluorophore N-(9-acridinyl)maleimide (NAM), which has been used in a continuous assay for CPT and requires dissoln. in a nonaq. solvent (e.g., acetone or DMSO). ABD-F also has a wider sepn. between excitation and emission peaks compared to NAM (390/530 vs 365/440). Advantages of the ABD-F procedure presented here include sensitivity combined with simplicity, and ease of application with microwell plates when a high throughput is desirable and enzymic activities are sufficiently high that a protein sedimentation step is not required. (c) 2000 Academic Press.
    581. 581
      Niu, L.-Y.; Zheng, H.-R.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. Fluorescent Sensors for Selective Detection of Thiols: Expanding the Intramolecular Displacement Based Mechanism to New Chromophores. Analyst 2014, 139 (6), 1389– 95,  DOI: 10.1039/c3an01849k
      584
      Fluorescent sensors for selective detection of thiols: expanding the intramolecular displacement based mechanism to new chromophores
      Niu, Li-Ya; Zheng, Hai-Rong; Chen, Yu-Zhe; Wu, Li-Zhu; Tung, Chen-Ho; Yang, Qing-Zheng
      Analyst (Cambridge, United Kingdom) (2014), 139 (6), 1389-1395CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      Biol. thiols, including cysteine (Cys), homocystein (Hcy) and glutathione (GSH), play crucial roles in maintaining the appropriate redox status of biol. systems. An abnormal level of biothiols is assocd. with different diseases, therefore, the discrimination between them is of great importance. Herein, we present two fluorescent sensors for selective detection of biothiols based on our recently reported intramol. displacement mechanism. We expanded this mechanism to com. available chromophores, 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) and heptamethine cyanine dye IR-780. The sensors operate by undergoing displacement of chloride by thiolate. The amino groups of Cys/Hcy further replace the thiolate to form amino-substituted products, which exhibit dramatically different photophys. properties compared to sulfur-substituted products from the reaction with GSH. NBD-Cl is highly selective towards Cys/Hcy and exhibits significant fluorescence enhancement. IR-780 showed a variation in its fluorescence ratio towards Cys over other thiols. Both of the sensors can be used for live-cell imaging of Cys. The wide applicability of the mechanism may provide a powerful tool for developing novel fluorescent sensors for selective detection of biothiols.
    582. 582
      Niu, L.-Y.; Guan, Y.-S.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. BODIPY-Based Ratiometric Fluorescent Sensor for Highly Selective Detection of Glutathione over Cysteine and Homocysteine. J. Am. Chem. Soc. 2012, 134 (46), 18928– 31,  DOI: 10.1021/ja309079f
      585
      BODIPY-Based Ratiometric Fluorescent Sensor for Highly Selective Detection of Glutathione over Cysteine and Homocysteine
      Niu, Li-Ya; Guan, Ying-Shi; Chen, Yu-Zhe; Wu, Li-Zhu; Tung, Chen-Ho; Yang, Qing-Zheng
      Journal of the American Chemical Society (2012), 134 (46), 18928-18931CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The authors report a ratiometric fluorescent sensor based on monochlorinated BODIPY for highly selective detection of glutathione (GSH) over cysteine (Cys)/homocysteine (Hcy). The chlorine of the monochlorinated BODIPY can be rapidly replaced by thiolates of biothiols through thiol-halogen nucleophilic substitution. The amino groups of Cys/Hcy but not GSH further replace the thiolate to form amino-substituted BODIPY. The significantly different photophys. properties of sulfur- and amino-substituted BODIPY enable the discrimination of GSH over Cys and Hcy. The sensor was applied for detection of GSH in living cells.
    583. 583
      Liu, X.-L.; Niu, L.-Y.; Chen, Y.-Z.; Zheng, M.-L.; Yang, Y.; Yang, Q.-Z. A Mitochondria-Targeting Fluorescent Probe for the Selective Detection of Glutathione in Living Cells. Org. Biomol. Chem. 2017, 15 (5), 1072– 5,  DOI: 10.1039/C6OB02407F
      586
      A mitochondria-targeting fluorescent probe for the selective detection of glutathione in living cells
      Liu, Xue-Liang; Niu, Li-Ya; Chen, Yu-Zhe; Zheng, Mei-Ling; Yang, Yunxu; Yang, Qing-Zheng
      Organic & Biomolecular Chemistry (2017), 15 (5), 1072-1075CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      The authors report a fluorescent probe, I, for the selective detection of mitochondrial glutathione (GSH). The probe, contg. triphenylphosphine as a mitochondrial targeting group, exhibited ratiometric and selective detection of GSH over Cys/Hcy. The probe was used for imaging mitochondrial GSH in living HeLa cells.
    584. 584
      Hu, Q.; Yu, C.; Xia, X.; Zeng, F.; Wu, S. A Fluorescent Probe for Simultaneous Discrimination of GSH and Cys/Hcy in Human Serum Samples Via Distinctly-Separated Emissions with Independent Excitations. Biosens. Bioelectron. 2016, 81, 341– 8,  DOI: 10.1016/j.bios.2016.03.011
      587
      A fluorescent probe for simultaneous discrimination of C and Cys/Hcy in human serum samples via distinctly-separated emissions with independent excitations
      Hu, Qinghua; Yu, Changmin; Xia, Xitao; Zeng, Fang; Wu, Shuizhu
      Biosensors & Bioelectronics (2016), 81 (), 341-348CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
      Biothiols like Cys, Hcy and GSH play important roles in various physiol. and pathol. processes, and it is still challenging to simultaneously and discriminatively detect GSH and Cys/Hcy due to their similar structures and reaction activities. Hence, it would be highly desirable to design a fluorescent probe for simultaneously discriminating GSH and Cys/Hcy in biol. samples with no spectral crosstalk, few interferences and rapid response. Herein, through coupling two fluorophores with biothiol-sensitive linker, we developed a fluorescent probe, which has two biothiol-responsive and distinctly-sepd. emissions via independent visible-light excitations, for simultaneously discriminating GSH and Cys/Hcy with near-IR and green emissions. The probe is operable in human serum samples, thus holding promise for diagnostic-related applications. Moreover, the probe shows quite good properties. First, it exhibits a rapid response (within a few minutes) with highly selective and sensitive detection for GSH and Cys/Hcy. Second, it offers an apparent colorimetric and two fluorescence emission signals without spectral crosstalk. Third, it shows low cytotoxicity. Therefore, it would provide a useful method for further elucidating the roles of biothiols as well as for conducting pathol. anal. for diseases involving biothiols.
    585. 585
      Zhang, J.; Ji, X.; Ren, H.; Zhou, J.; Chen, Z.; Dong, X.; Zhao, W. meso-Heteroaryl BODIPY Dyes as Dual-Responsive Fluorescent Probes for Discrimination of Cys from Hcy and GSH. Sens. Actuators B Chem. 2018, 260, 861– 9,  DOI: 10.1016/j.snb.2018.01.016
      588
      Meso-heteroaryl BODIPY dyes as dual-responsive fluorescent probes for discrimination of Cys from Hcy and GSH
      Zhang, Jian; Ji, Xin; Ren, Hang; Zhou, Junliang; Chen, Zhongjian; Dong, Xiaochun; Zhao, Weili
      Sensors and Actuators, B: Chemical (2018), 260 (), 861-869CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      Two new BODIPY-based turn-on fluorescent probes (BDP-S-1 and BDP-O-6) contained tetra-Me at 1-, 3-, 5- and 7- positions, as well as O-, or S-aryl substituent at 8-position for the simultaneous and selective detection of Cys and Hcy/GSH from dual emission channels were developed. The spatial steric hindrance of the Me groups at 1- and 7- positions prevented intramol. displacement of sulfur with amino group of Hcy but not of Cys. Based on different thiols-induced SNAr substitution-rearrangement reaction with Cys and Hcy/GSH, leading to the corresponding amino- and thiol-BODIPY dyes with distinct photo-phys. properties, these probes could simultaneously and selectively detect Cys and Hcy/GSH. With the help of laser scanning confocal microscope, we demonstrated that these probes could simultaneously sense Cys and GSH in Hela cells using multicolor imaging.
    586. 586
      Liu, Y.; Yu, Y.; Zhao, Q.; Tang, C.; Zhang, H.; Qin, Y.; Feng, X.; Zhang, J. Fluorescent Probes Based on Nucleophilic Aromatic Substitution Reactions for Reactive Sulfur and Selenium Species: Recent Progress, Applications, and Design Strategies. Coord. Chem. Rev. 2021, 427, 213601,  DOI: 10.1016/j.ccr.2020.213601
      589
      Fluorescent probes based on nucleophilic aromatic substitution reactions for reactive sulfur and selenium species: Recent progress, applications, and design strategies
      Liu, Yuning; Yu, Yanan; Zhao, Qingyu; Tang, Chaohua; Zhang, Huiyan; Qin, Yuchang; Feng, Xiaohui; Zhang, Junmin
      Coordination Chemistry Reviews (2021), 427 (), 213601CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
      A review. Reactive sulfur species (RSS) and reactive selenium species (RSeS) are important substances for the maintenance of physiol. balance. Imbalance of RSS and RSeS is closely related to a series of human diseases, so it is considered to be an important biomarker in early diagnosis, treatment, and stage monitoring. Fast and accurate quant. anal. of different RSS and RSeS in complex biol. systems may promote the development of personalized diagnosis and treatment in the future. One way to explore the physiol. function of various types of RSS and RSeS in vivo is to detect them at the mol. level, and one of the most effective methods for this is to use fluorescent probes. Nucleophilic arom. substitution (SNAr) reactions are commonly exploited as a detection mechanism for RSS and RSeS in fluorescent probes. In this review, we cover recent progress in fluorescent probes for RSS and RSeS based on SNAr reactions, and discuss their response mechanisms, properties, and applications. Benzenesulfonate, phenyl-O ether, phenyl-S ether, phenyl-Se ether, 7-nitro-2,1,3-benzoxadiazole (NBD), benzoate, and selenium-nitrogen bonds are all good detection groups. Moreover, based on an integration of different reports, we propose the design and synthesis of RSS- and RSeS-selective probes based on SNAr reactions, current challenges, and future research directions, considering the selection of active sites, the effect of substituents on the benzene ring, and the introduction of other functional groups.
    587. 587
      Kim, Y.; Mulay, S. V.; Choi, M.; Yu, S. B.; Jon, S.; Churchill, D. G. Exceptional Time Response, Stability and Selectivity in Doubly-Activated Phenyl Selenium-Based Glutathione-Selective Platform. Chem. Sci. 2015, 6, 5435,  DOI: 10.1039/C5SC02090E
      590
      Exceptional time response, stability and selectivity in doubly-activated phenyl selenium-based glutathione-selective platform
      Kim, Youngsam; Mulay, Sandip V.; Choi, Minsuk; Yu, Seungyoon B.; Jon, Sangyong; Churchill, David G.
      Chemical Science (2015), 6 (10), 5435-5439CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      A phenyl-selenium-substituted coumarin probe was synthesized for the purpose of achieving highly selective and extremely rapid detection of glutathione (GSH) over cysteine (Cys)/homocysteine (Hcy) without background fluorescence. The fluorescence intensity of the probe with GSH shows a ∼100-fold fluorescent enhancement compared with the signal generated for other closely related amino acids, including Cys and Hcy. Importantly, the substitution reaction with the sulfhydryl group of GSH at the 4-position of the probe, which is doubly-activated by two carbonyl groups, occurs extremely fast, showing subsecond max. fluorescence intensity attainment; equil. was reached within 100 ms (UV-vis). The probe selectivity for GSH was confirmed in Hep3B cells by confocal microscopy imaging.
    588. 588
      Mulay, S. V.; Kim, Y.; Choi, M.; Lee, D. Y.; Choi, J.; Lee, Y.; Jon, S.; Churchill, D. G. Enhanced Doubly Activated Dual Emission Fluorescent Probes for Selective Imaging of Glutathione or Cysteine in Living Systems. Anal. Chem. 2018, 90 (4), 2648– 54,  DOI: 10.1021/acs.analchem.7b04375
      591
      Enhanced Doubly Activated Dual Emission Fluorescent Probes for Selective Imaging of Glutathione or Cysteine in Living Systems
      Mulay, Sandip V.; Kim, Youngsam; Choi, Minsuk; Lee, Dong Yun; Choi, Jonghoon; Lee, Yunho; Jon, Sangyong; Churchill, David G.
      Analytical Chemistry (Washington, DC, United States) (2018), 90 (4), 2648-2654CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      The development of novel fluorescent probes for monitoring the concn. of various biomols. in living systems has great potential for eventual early diagnosis and disease intervention. Selective detection of competitive species in biol. systems is a great challenge for the design and development of fluorescent probes. To improve on the design of fluorescent coumarin-based biothiol sensing technologies, we have developed herein an enhanced dual emission doubly activated system (DACP-1 and the closely related DACP-2) for the selective detection of glutathione (GSH) through the use of one optical channel and the detection of cysteine (Cys) by another channel. A phenylselenium group present at the 4-position completely quenches the fluorescence of the probe via photoinduced electron transfer to give a nonfluorescent species. Probes are selective for glutathione (GSH) in the red region and for cysteine/homocysteine (Cys/Hcy) in the green region. When they were treated with GSH, DACP-1 and DACP-2 showed strong fluorescence enhancement in comparison to that for closely related species such as amino acids, including Cys/Hcy. Fluorescence quantum yields (ΦF) increased for the red channel (<0.001 to 0.52 (DACP-1) and 0.48 (DACP-2)) and green channel (Cys) (<0.001 to 0.030 (DACP-1) and 0.026 (DACP-2)), resp. Competing fluorescent enhancements upon addn. of closely related species were negligible. Fast responses, improved water soly., and good cell membrane permeability were all properly established with the use of DACP-1 and DACP-2. Live human lung cancer cells and fibroblasts imaged by confocal microscopy, as well as live mice tumor model imaging, confirmed selective detection.
    589. 589
      Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. 2,4-Dinitrobenzenesulfonyl Fluoresceins as Fluorescent Alternatives to Ellman’s Reagent in Thiol-Quantification Enzyme Assays. Angew. Chem., Int. Ed. 2005, 44 (19), 2922– 5,  DOI: 10.1002/anie.200500114
      592
      2,4-Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to ellman's reagent in thiol-quantification enzyme assays
      Maeda, Hatsuo; Matsuno, Hiromi; Ushida, Mai; Katayama, Kohei; Saeki, Kanako; Itoh, Norio
      Angewandte Chemie, International Edition (2005), 44 (19), 2922-2925CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Fluorescent sensor for thiols: Deprotection of nonfluorescent 1 by thiols (R'SH) proceeds rapidly and near-quant. in aq. soln. (pH 7.4) to produce highly fluorescent 2. Assays performed in the presence of 1 provide a rapid and simple method for the detn. of inhibitory consts. for inhibitors such as donepezil toward acetyl- and butyrylcholinesterases.
    590. 590
      Jiang, W.; Fu, Q.; Fan, H.; Ho, J.; Wang, W. A Highly Selective Fluorescent Probe for Thiophenols. Angew. Chem., Int. Ed. 2007, 46 (44), 8445– 8,  DOI: 10.1002/anie.200702271
      593
      A highly selective fluorescent probe for thiophenols
      Jiang, Wei; Fu, Qingquan; Fan, Hongyou; Ho, Joe; Wang, Wei
      Angewandte Chemie, International Edition (2007), 46 (44), 8445-8448CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A rapid response to thiophenols was obtained with 2,4-dinitro-N-[7-nitrobenzo[c]-[1,2,5]oxadiazol-4-yl]benzenesulfonamide probe, which induces a significant (> 50-fold) fluorescence enhancement as a result of cleavage of the electron-withdrawing moiety, thus generating strongly fluorescent mol., 4-amino-7-nitro-2,1,3-benzoxadiazole. No fluorescence was obtained with aliph. thiols, including cysteine and glutathione, or other nucleophiles.
    591. 591
      Wang, S.; Huang, Y.; Guan, X. Fluorescent Probes for Live Cell Thiol Detection. Molecules 2021, 26 (12), 3575,  DOI: 10.3390/molecules26123575
      594
      Fluorescent probes for live cell thiol detection
      Wang, Shenggang; Huang, Yue; Guan, Xiangming
      Molecules (2021), 26 (12), 3575CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      A review. Thiols play vital and irreplaceable roles in the biol. system. Abnormality of thiol levels has been linked with various diseases and biol. disorders. Thiols are known to distribute unevenly and change dynamically in the biol. system. Methods that can det. thiols' concn. and distribution in live cells are in high demand. In the last two decades, fluorescent probes have emerged as a powerful tool for achieving that goal for the simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way. They also enable the detn. of intracellular distribution and dynamitic movement of thiols in the intact native environments. This review focuses on some of the major strategies/mechanisms being used for detecting GSH, Cys/Hcy, and other thiols in live cells via fluorescent probes, and how they are applied at the cellular and subcellular levels. The sensing mechanisms (for GSH and Cys/Hcy) and bio-applications of the probes are illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.
    592. 592
      Dai, Z.; Tian, L.; Ye, Z.; Song, B.; Zhang, R.; Yuan, J. A Lanthanide Complex-Based Ratiometric Luminescence Probe for Time-Gated Luminescence Detection of Intracellular Thiols. Anal. Chem. 2013, 85 (23), 11658– 64,  DOI: 10.1021/ac403370g
      595
      A Lanthanide Complex-Based Ratiometric Luminescence Probe for Time-Gated Luminescence Detection of Intracellular Thiols
      Dai, Zhichao; Tian, Lu; Ye, Zhiqiang; Song, Bo; Zhang, Run; Yuan, Jingli
      Analytical Chemistry (Washington, DC, United States) (2013), 85 (23), 11658-11664CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A lanthanide complex-based ratiometric luminescence probe, [4'-(2,4-dinitrobenzenesulfonyloxy)-2,2':6',2''-terpyridine-6,6''-diyl] bis-(methylenenitrilo) tetrakis-(acetate)-Eu3+/Tb3+ (NSTTA-Eu3+/Tb3+), has been designed and synthesized for the specific recognition and time-gated luminescence detection of biothiols in physiol. pH aq. media. The probe itself is almost nonluminescent due to the presence of photoinduced electron transfer (PET) from the terpyridine-Ln3+ moiety to the 2,4-dinitrobenzenesulfonyl (DNBS) moiety. In the presence of biothiols, the reaction of NSTTA-Eu3+/Tb3+ with biothiols results in the cleavage of DNBS to afford the deprotonated (4'-hydroxy-2,2':6',2''-terpyridine-6,6''-diyl) bis-(methylenenitrilo) tetrakis-(acetate)-Eu3+/Tb3+ (HTTA-Eu3+/Tb3+), which terminates the PET process. After the reaction, the intensity of Eu3+ emission at 610 nm is unchanged, while that of Tb3+ emission at 540 nm is remarkably increased, which provides a ∼36-fold enhanced intensity ratio of Tb3+ emission to Eu3+ emission (I540/I610). This unique luminescence response allows NSTTA-Eu3+/Tb3+ to be used as a ratiometric probe for the time-gated luminescence detection of biothiols, using the intensity ratio of I540/I610 as a signal. Thus, based on the probe NSTTA-Eu3+/Tb3+, a ratiometric time-gated luminescence detection method for biothiols was established and successfully used for the quant. detection of the total biothiols in several living cell samples.
    593. 593
      Rong, X.; Xu, Z.-Y.; Yan, J.-W.; Meng, Z.-Z.; Zhu, B.; Zhang, L. Nile-Red-Based Fluorescence Probe for Selective Detection of Biothiols, Computational Study, and Application in Cell Imaging. Molecules 2020, 25 (20), 4718,  DOI: 10.3390/molecules25204718
      596
      Nile-red-based fluorescence probe for selective detection of biothiols, computational study, and application in cell imaging
      Rong, Xiang; Xu, Zhong-Yong; Yan, Jin-Wu; Meng, Zhi-Zhong; Zhu, Bin; Zhang, Lei
      Molecules (2020), 25 (20), 4718CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      A new colorimetric and fluorescence probe NRSH based on Nile-red chromophore for the detection of biothiols has been developed, exhibiting high selectivity towards biothiols over other interfering species. NRSH shows a blue shift in absorption peak upon reacting with biothiols, from 587 nm to 567 nm, which induces an obvious color change from blue to pink and exhibits a 35-fold fluorescence enhancement at 645 nm in red emission range. NRSH displays rapid (<1 min) response for H2S, which is faster than other biothiols (>5 min). The detection limits of probe NRSH towards biothiols are very low (22.05 nM for H2S, 34.04 nM for Cys, 107.28 nM for GSH and 113.65 nM for Hcy). Furthermore, NRSH is low cytotoxic and can be successfully applied as a bioimaging tool for real-time monitoring biothiols in HeLa cells. In addn., fluorescence mechanism of probe NRSH is further understood by theor. calcns.
    594. 594
      Maeda, H.; Katayama, K.; Matsuno, H.; Uno, T. 3′-(2,4-Dinitrobenzenesulfonyl)-2′,7′-Dimethylfluorescein as a Fluorescent Probe for Selenols. Angew. Chem., Int. Ed. 2006, 45 (11), 1810– 3,  DOI: 10.1002/anie.200504299
      597
      3'-(2,4-Dinitrobenzenesulfonyl)-2',7'-dimethylfluorescein as a fluorescent probe for selenols
      Maeda, Hatsuo; Katayama, Kohei; Matsuno, Hiromi; Uno, Tadayuki
      Angewandte Chemie, International Edition (2006), 45 (11), 1810-1813CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The fluorescein deriv. BESThio [3'-(2,4-dinitrobenzenesulfonyl)-2',7'-dimethylfluorescein] operates as a thiol probe at pH 7.4 and functions as a selenol probe at pH 5.8, and can thus be selectively applied to the rapid quantification of selenocysteine and the detn. of the selenocysteine content in selenoproteins such as glutathione peroxidase and thioredoxin reductase with high sensitivity.
    595. 595
      Peng, L.; Xiao, L.; Ding, Y.; Xiang, Y.; Tong, A. A Simple Design of Fluorescent Probes for Indirect Detection of β-Lactamase Based on AIE and ESIPT Processes. J. Mater. Chem. B 2018, 6 (23), 3922– 6,  DOI: 10.1039/C8TB00414E
      598
      A simple design of fluorescent probes for indirect detection of β-lactamase based on AIE and ESIPT processes
      Peng, Lu; Xiao, Lu; Ding, Yiwen; Xiang, Yu; Tong, Aijun
      Journal of Materials Chemistry B: Materials for Biology and Medicine (2018), 6 (23), 3922-3926CODEN: JMCBDV; ISSN:2050-7518. (Royal Society of Chemistry)
      A novel fluorescent probe DNBS-CSA is developed for light-up detection of β-lactamase. The probe design is based on an indirect detection approach with three step reactions. β-Lactamase can react with the lactam of its substrate (cefazolin sodium) to produce a secondary amine, initiating a spontaneous elimination reaction and affording a thiol compd. The thiol could further react with the sulfonate group of DNBS-CSA, releasing the salicylaldehyde azine deriv. (CSA) with both aggregation induced emission (AIE) and excited-state intramol. proton transfer (ESIPT) characteristics. Previously reported β-lactamase probes require covalent linkage of the substrate β-lactam ring part to the probe, which makes probe synthesis difficult due to the complicated structure of the β-lactam ring. In contrast, modification of the β-lactam ring is no longer necessary for DNBS-CSA according to our indirect detection approach. The linear range of fluorescence quantification for β-lactamase is 0-10 mU mL-1 in an aq. soln. Moreover, owing to the AIE properties of CSA, detection of β-lactamase with DNBS-CSA on test papers was also achieved.
    596. 596
      Yuan, L.; Lin, W.; Zhao, S.; Gao, W.; Chen, B.; He, L.; Zhu, S. A Unique Approach to Development of Near-Infrared Fluorescent Sensors for in Vivo Imaging. J. Am. Chem. Soc. 2012, 134 (32), 13510– 23,  DOI: 10.1021/ja305802v
      599
      A Unique Approach to Development of Near-Infrared Fluorescent Sensors for in Vivo Imaging
      Yuan, Lin; Lin, Weiying; Zhao, Sheng; Gao, Wensha; Chen, Bin; He, Longwei; Zhu, Sasa
      Journal of the American Chemical Society (2012), 134 (32), 13510-13523CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Near-IR (NIR) fluorescent sensors have emerged as promising mol. tools for imaging biomols. in living systems. However, NIR fluorescent sensors are very challenging to be developed. Herein, the authors describe the discovery of a new class of NIR fluorescent dyes represented by 1a/1c/1e, which are superior to the traditional 7-hydroxycoumarin and fluorescein with both absorption and emission in the NIR region while retaining an optically tunable hydroxyl group. Quantum chem. calcns. with the B3LYP exchange functional employing 6-31G(d) basis sets provide insights into the optical property distinctions between 1a/1c/1e and their alkoxy derivs. The unique optical properties of the new type of fluorescent dyes can be exploited as a useful strategy for development of NIR fluorescent sensors. Employing this strategy, two different types of NIR fluorescent sensors, NIR-H2O2 and NIR-thiol, for H2O2 and thiols, resp., were constructed. These novel sensors respond to H2O2 or thiols with a large turn-on NIR fluorescence signal upon excitation in the NIR region. Furthermore, NIR-H2O2 and NIR-thiol are capable of imaging endogenously produced H2O2 and thiols, resp., not only in living cells but also in living mice, demonstrating the value of the new NIR fluorescent sensor design strategy. The new type of NIR dyes presented herein may open up new opportunities for the development of NIR fluorescent sensors based on the hydroxyl functionalized reactive sites for biol. imaging applications in living animals.
    597. 597
      Lu, Z.; Sun, X.; Wang, M.; Wang, H.; Fan, C.; Lin, W. Rational Design of a Far-Red Fluorescent Probe for Endogenous Biothiol Imbalance Induced by Hydrogen Peroxide in Living Cells and Mice. Bioorg. Chem. 2020, 103, 104173,  DOI: 10.1016/j.bioorg.2020.104173
      600
      Rational design of a far-red fluorescent probe for endogenous biothiol imbalance induced by hydrogen peroxide in living cells and mice
      Lu, Zhengliang; Sun, Xin; Wang, Mengzhao; Wang, Hongmei; Fan, Chunhua; Lin, Weiying
      Bioorganic Chemistry (2020), 103 (), 104173CODEN: BOCMBM; ISSN:0045-2068. (Elsevier B.V.)
      Intracellular biothiols are correlated with many diseases such as nerve disorder and Parkinson's disease likely due to a redox imbalance. In this work, we designed an ultrafast fluorescent probe (Cou-DNBS) for biothiols with a large Stokes shift (131 nm). The probe was constructed through linking the 2,4-dinitrobenzenesulfonyl moiety as the specially recognizing biothiols site to an iminocoumarin fluorophore Cou-NH obtained by fusing an addnl. benzene ring. The presence of biothiols could ultrafast perform a significant fluorescence emission at 617 nm upon the excitation of 480 with the low limits of detection (2.5 nM for Cys, 1.7 nM for Hcy and 0.84 nM for GSH). HRMS spectra as well as theor. calcns. further evidenced the rationale of recognition mechanism. Furthermore, the probe can successfully visualize endogenous biothiol recovery in living cells damaged by H2O2.
    598. 598
      Kand, D.; Kalle, A. M.; Varma, S. J.; Talukdar, P. A Chromenoquinoline-Based Fluorescent Off-On Thiol Probe for Bioimaging. Chem. Commun. 2012, 48 (21), 2722– 4,  DOI: 10.1039/c2cc16593g
      601
      A chromenoquinoline-based fluorescent off-on thiol probe for bioimaging
      Kand, Dnyaneshwar; Kalle, Arunasree Marasanapalli; Varma, Sreejith Jayasree; Talukdar, Pinaki
      Chemical Communications (Cambridge, United Kingdom) (2012), 48 (21), 2722-2724CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A new chromenoquinoline-based fluorescent off-on thiol probe (I) is reported. In aq. buffer solns. at physiol. pH, the probe exhibited 223-fold enhancement in fluorescence intensity by a Michael addn. of cysteine to the maleimide appended to a chromenoquinoline. Cell permeability and live cell imaging of thiols are also demonstrated.
    599. 599
      Liu, T.; Huo, F.; Li, J.; Chao, J.; Zhang, Y.; Yin, C. A Fast Response and High Sensitivity Thiol Fluorescent Probe in Living Cells. Sens. Actuators B Chem. 2016, 232, 619– 24,  DOI: 10.1016/j.snb.2016.04.014
      602
      A fast response and high sensitivity thiol fluorescent probe in living cells
      Liu, Tao; Huo, Fangjun; Li, Jianfang; Chao, Jianbin; Zhang, Yongbin; Yin, Caixia
      Sensors and Actuators, B: Chemical (2016), 232 (), 619-624CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      Up to date, the design of highly selective and fast response probes for Cys, Hcy, and GSH is still a challenge and very few examples have been reported so far. In this study, we synthesized a 1,8-naphthalimide-based probe bearing maleimide group to selectively detect thiol. After introduce thiol, the fluorescence intensity of probe showed significant enhancement, but other amino acids did not generate any change. The kinetic study showed that the reaction was complete within 120 s for Cys, Hcy and GSH, indicating that probe reacts rapidly with thiol under the exptl. conditions. Furthermore, the ability of probe to detect thiol in living cells via an enhancement of the fluorescence was proved.
    600. 600
      Lin, X.; Hu, Y.; Yang, D.; Chen, B. Cyanine-Coumarin Composite NIR Dye Based Instantaneous-Response Probe for Biothiols Detection and Oxidative Stress Assessment of Mitochondria. Dyes Pigm. 2020, 174, 107956,  DOI: 10.1016/j.dyepig.2019.107956
      603
      Cyanine-coumarin composite NIR dye based instantaneous -response probe for biothiols detection and oxidative stress assessment of mitochondria
      Lin, Xin; Hu, Yanling; Yang, Dongliang; Chen, Bo
      Dyes and Pigments (2020), 174 (), 107956CODEN: DYPIDX; ISSN:0143-7208. (Elsevier Ltd.)
      Developing near-IR (NIR) dye based functional fluorescent probes to anal. the bioactive substances contributes to the understanding of cellular physiopathol. Although there are many fluorescent probes have been developed, the probes can instantaneous response to biothiols is still lacking. In this study, we have designed and synthesized a novel composite NIR dye (Hcyc), which was composed by cyanine and coumarin. Hcyc was further applied to construct a biothiols responsive probe (Hcyc-NO) to assess the oxidative stress status of mitochondria in living cells. The optical properties showed that Hcyc has long absorption and emission wavelength in different solvent system (>720 nm). Interestingly, Hcyc-NO could respond to GSH, Cys, Hcy rapidly (<5 s), the absorption peak of red shifted from 556 nm to 723 nm and fluorescence intensity instantaneously enhanced at 751 nm after adding biothiols. In addn., Hcyc-NO possesses others excellent sensing properties such as high selectivity and sensitivity. Confocal imaging revealed that Hcyc-NO (1μM) could target mitochondria and selective image biothiols (GSH, Cys and Hcy) in vitro. Furthermore, the probe could rapidly reflect oxidative stress induced by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) in vitro and in vivo. These results indicate that Hcyc is an excellent NIR dye, and the probe Hcyc-NO may be an effective tool for instantaneous detection of biothiols and real-time assessing the oxidative stress of cells.
    601. 601
      Sun, Q.; Yang, S.-H.; Wu, L.; Yang, W.-C.; Yang, G.-F. A Highly Sensitive and Selective Fluorescent Probe for Thiophenol Designed Via a Twist-Blockage Strategy. Anal. Chem. 2016, 88 (4), 2266– 72,  DOI: 10.1021/acs.analchem.5b04029
      604
      A Highly Sensitive and Selective Fluorescent Probe for Thiophenol Designed via a Twist-Blockage Strategy
      Sun, Qi; Yang, Shu-Hou; Wu, Lei; Yang, Wen-Chao; Yang, Guang-Fu
      Analytical Chemistry (Washington, DC, United States) (2016), 88 (4), 2266-2272CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A benzoquinolizine coumarin-based fluorescent probe was developed for detecting thiophenols, demonstrating the superior fluorescence properties caused by the decay of the twisting effect of N,N-diethylamino group of coumarin. It discriminated thiophenols from various analytes including aliph. thiols with good selectivity and displayed ∼700-fold fluorescence intensity enhancement and a remarkable limit of detection (4.5 nM). The new probe also can be applied to quant. det. the concns. of thiophenol in water samples and living cells.
    602. 602
      Jiang, W.; Cao, Y.; Liu, Y.; Wang, W. Rational Design of a Highly Selective and Sensitive Fluorescent PET Probe for Discrimination of Thiophenols and Aliphatic Thiols. Chem. Commun. 2010, 46 (11), 1944– 6,  DOI: 10.1039/B926070F
      605
      Rational design of a highly selective and sensitive fluorescent PET probe for discrimination of thiophenols and aliphatic thiols
      Jiang, Wei; Cao, Yanting; Liu, Yuan; Wang, Wei
      Chemical Communications (Cambridge, United Kingdom) (2010), 46 (11), 1944-1946CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A novel highly sensitive and selective 'off-on' fluorescent probe (I, R1 = R2 = NO2; R1 = Me, R2 = H) for thiophenols was developed by a PET mechanism through a rational design.
    603. 603
      Li, Y.; Su, W.; Zhou, Z.; Huang, Z.; Wu, C.; Yin, P.; Li, H.; Zhang, Y. A Dual-Response near-Infrared Fluorescent Probe for Rapid Detecting Thiophenol and Its Application in Water Samples and Bio-Imaging. Talanta 2019, 199, 355– 60,  DOI: 10.1016/j.talanta.2019.02.022
      606
      A dual-response near-IR fluorescent probe for rapid detecting thiophenol and its application in water samples and bio-imaging
      Li, Yaqian; Su, Wei; Zhou, Zile; Huang, Zhen; Wu, Cuiyan; Yin, Peng; Li, Haitao; Zhang, Youyu
      Talanta (2019), 199 (), 355-360CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      Thiophenol is widely known as a highly toxic substance that can cause serious harm to the environment and health. Rapid and non-destructive detection of thiophenol is of great significance for environmental management. In this work, we designed and synthesized a near-IR (NIR) fluorescent probe, (E)-4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)-2-formylphenyl-2,4-dinitrobenzenesulfonate (DCM-CHO-D)(I), that can respond to thiophenol rapidly (less than 3 min) based on intramol. charge transfer (ICT) mechanism. DCM-CHO-D has high selectivity and sensitivity to the detection of thiophenol. And there is a linear relationship between the fluorescence intensity of DCM-CHO-D and thiophenol concn. in the range of 0-10 μM, the limit of detection (LOD) is as low as 0.22 μM. What's more, DCM-CHO-D can not only be used as an ideal colorimetric tool for detecting thiophenol in water samples, but also image thiophenol in living cells, indicating its potential utility for thiophenol sensing in environmental and biol. samples.
    604. 604
      Xiong, L.; Ma, J.; Huang, Y.; Wang, Z.; Lu, Z. Highly Sensitive Squaraine-Based Water-Soluble Far-Red/near-Infrared Chromofluorogenic Thiophenol Probe. ACS Sens. 2017, 2 (4), 599– 605,  DOI: 10.1021/acssensors.7b00151
      607
      Highly Sensitive Squaraine-Based Water-Soluble Far-Red/Near-Infrared Chromofluorogenic Thiophenol Probe
      Xiong, Li; Ma, Jun; Huang, Yan; Wang, Zihe; Lu, Zhiyun
      ACS Sensors (2017), 2 (4), 599-605CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      A squaraine-based far-red/near-IR fluorescent probe (SQ-DNBS) was exploited for thiophenol detection. SQ-DNBS is a colorimetric and "off-on" fluorometric dual-channel "naked-eye" chemosensor showing high selectivity, high sensitivity (detection limit: 9.9 nM), and rapid response to thiophenol in aq. soln. SQ-DNBS also can be used in practical applications for the detection of thiophenol in water samples. Photophys. and spectral characterization results revealed that the probing mechanism of SQ-DNBS toward thiophenol lies in the thiolate-mediated cleavage reaction. Our discovery demonstrates the potential of the arylmethylene-squaraine skeleton as a promising fluorophore unit to construct high-performance far-red/near-IR chemosensors.
    605. 605
      Song, B.; Wang, G.; Tan, M.; Yuan, J. A Europium(III) Complex as an Efficient Singlet Oxygen Luminescence Probe. J. Am. Chem. Soc. 2006, 128 (41), 13442– 50,  DOI: 10.1021/ja062990f
      608
      A Europium(III) Complex as an Efficient Singlet Oxygen Luminescence Probe
      Song, Bo; Wang, Guilan; Tan, Mingqian; Yuan, Jingli
      Journal of the American Chemical Society (2006), 128 (41), 13442-13450CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A new europium(III) complex, [4'-(10-methyl-9-anthryl)-2,2':6',2''-terpyridine-6,6''-diyl]bis(methylenenitrilo) tetrakis(acetate)-Eu3+, was designed and synthesized as a highly sensitive and selective time-gated luminescence probe for singlet oxygen (1O2). The new probe is highly water sol. with a large stability const. of ∼1021 and a wide pH available range (pH 3-10), and can specifically react with 1O2 to form its endoperoxide (EP-MTTA-Eu3+) with a high reaction rate const. at 1010 M-1 s-1, accompanied by the remarkable increases of luminescence quantum yield from 0.90% to 13.8% and lifetime from 0.80 to 1.29 ms, resp. The wide applicability of the probe was demonstrated by detection of 1O2 generated from a MoO42-/H2O2 system, a photosensitization system of 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), and a horseradish peroxidase catalyzed aerobic oxidn. system of indole-3-acetic acid (IAA). In addn., it was found that the new probe could be easily transferred into living HeLa cells by incubation with TMPyP. A time-gated luminescence imaging technique that can fully eliminate the short-lived background fluorescence from TMPyP and cell components has been successfully developed for monitoring the time-dependent generation of 1O2 in living cells.
    606. 606
      Bjartell, A.; Laine, S.; Pettersson, K.; Nilsson, E.; Lövgren, T.; Lilja, H. Time-Resolved Fluorescence in Immunocytochemical Detection of Prostate-Specific Antigen in Prostatic Tissue Sections. Histochem. J. 1999, 31 (1), 45– 52,  DOI: 10.1023/A:1003504115690
      609
      Time-resolved fluorescence in immunocytochemical detection of prostate-specific antigen in prostatic tissue sections
      Bjartell, Anders; Laine, Sari; Pettersson, Kim; Nilsson, Elise; Lovgren, Timo; Lilja, Hans
      Histochemical Journal (1999), 31 (1), 45-52CODEN: HISJAE; ISSN:0018-2214. (Kluwer Academic Publishers)
      Chelates with fluorescent lanthanides such as europium and terbium are widely used in immunofluorometric assays, e.g. for the measurement of different mol. forms of prostate-specific antigen (PSA) in serum for detection and monitoring of prostate cancer. These chelates have also been introduced as non-radioactive labels in immunocytochem. and in situ hybridization. In the present study, sections of non-malignant prostate were investigated using monoclonal IgGs against PSA. Detection of specific immunostaining employing time-resolved fluorescence with europium-labeled streptavidin was compared with conventional detection by streptavidin conjugated to horse-radish peroxidase. The high PSA concn. in the tissue produced high intensity, specific time-resolved fluorescence signals in the epithelial cells of the prostate gland without disturbance from non-specific tissue autofluorescense. This allowed short exposure times to be used which resulted in insignificant photobleaching. Two of the three europium-chelates evaluated yielded high signal intensities. Counterstaining was found to be optimal with Gill No. 1-Haematoxylin soln. and Merckoglas was the best mounting medium for the europium chelates tested. In conclusion, time-resolved fluorescence imaging is an attractive alternative to conventional detection of streptavidin conjugated to horse-radish peroxidase, as it provides linear, high intensity, specific signals subsequent to the decay of non-specific tissue autofluorescence.
    607. 607
      Shang, H.; Chen, H.; Tang, Y.; Ma, Y.; Lin, W. Development of a Two-Photon Fluorescent Turn-On Probe with Far-Red Emission for Thiophenols and Its Bioimaging Application in Living Tissues. Biosens. Bioelectron. 2017, 95, 81– 6,  DOI: 10.1016/j.bios.2017.04.017
      610
      Development of a two-photon fluorescent turn-on probe with far-red emission for thiophenols and its bioimaging application in living tissues
      Shang, Huiming; Chen, Hua; Tang, Yonghe; Ma, Yanyan; Lin, Weiying
      Biosensors & Bioelectronics (2017), 95 (), 81-86CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
      Thiophenol is a highly toxic compd. which is essential in the field of org. synthesis and drug design. However, the accumulation of thiophenols in the environment may cause serious health problems for human bodies ultimately. Therefore, it is crit. to develop efficient methods for visualization of thiophenol species in biol. samples. In this work, an innovative two-photon fluorescent turn-on probe FR-TP with far-red emission for thiophenols based on FR-NH2 fluorophore and 2,4-dinitrophenylsulfonyl recognition site was reported. The new probe can be used for thiophenol detection with large far-red fluorescence enhancement (about 155-fold), rapid response (completed within 100 s), excellent sensitivity (DL 0.363μM), high selectivity, and lower cellular auto-fluorescence interference. Importantly, the probe FR-TP can be successfully employed to visualize thiophenols not only in the living HeLa cells but also in living liver tissues. In addn., through two-photon tissue imaging, the probe was used to monitor and investigate biol. thiophenol poisoning in the animal model of thiophenol inhalation for the first time.
    608. 608
      Fellner, M.; Doughty, L. M.; Jameson, G. N. L.; Wilbanks, S. M. A Chromogenic Assay of Substrate Depletion by Thiol Dioxygenases. Anal. Biochem. 2014, 459, 56– 60,  DOI: 10.1016/j.ab.2014.05.008
      611
      A chromogenic assay of substrate depletion by thiol dioxygenases
      Fellner, Matthias; Doughty, Laura M.; Jameson, Guy N. L.; Wilbanks, Sigurd M.
      Analytical Biochemistry (2014), 459 (), 56-60CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the non-heme mono-Fe enzyme, cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed std. kinetic parameters to be detd. for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitated a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addn., the relative specificity of Ellman's reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.
    609. 609
      Zhu, J.; Dhimitruka, I.; Pei, D. 5-(2-Aminoethyl)Dithio-2-Nitrobenzoate as a More Base-Stable Alternative to Ellman’s Reagent. Org. Lett. 2004, 6 (21), 3809– 12,  DOI: 10.1021/ol048404+
      612
      5-(2-Aminoethyl)dithio-2-nitrobenzoate as a More Base-Stable Alternative to Ellman's Reagent
      Zhu, Jinge; Dhimitruka, Ilirian; Pei, Dehua
      Organic Letters (2004), 6 (21), 3809-3812CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      5-(2-Aminoethyl)dithio-2-nitrobenzoate (ADNB) reacts with free thiols with kinetics similar to those of Ellman's reagent but has dramatically improved stability under alk. conditions, making it an excellent alternative to Ellman's reagent for the quantitation of thiol contents and enzymic assays under basic pH conditions.
    610. 610
      Li, Y.; Lopez, P.; Durand, P.; Ouazzani, J.; Badet, B.; Badet-Denisot, M.-A. An Enzyme-Coupled Assay for Amidotransferase Activity of Glucosamine-6-Phosphate Synthase. Anal. Biochem. 2007, 370 (2), 142– 6,  DOI: 10.1016/j.ab.2007.07.031
      613
      An enzyme-coupled assay for amidotransferase activity of glucosamine-6-phosphate synthase
      Li, Yanyan; Lopez, Philippe; Durand, Philippe; Ouazzani, Jamal; Badet, Bernard; Badet-Denisot, Marie-Ange
      Analytical Biochemistry (2007), 370 (2), 142-146CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
      An assay for glucosamine-6-phosphate synthase using a yeast glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) as coupling enzyme was developed. GNA1 transfers the acetyl moiety from acetyl-CoA (CoA) to glucosamine-6-phosphate, releasing CoA. The assay measures the prodn. of glucosamine-6-phosphate by either following the consumption of acetyl-CoA spectrophotometrically at 230 nm or quantifying the free thiol with 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent) in a discontinuous manner. This method is simple to perform and can be adapted to a 96-well microtiter plate format, which will facilitate high-throughput inhibitor screening and mechanistic studies using purified GlmS.
    611. 611
      Chen, G.; Feng, H.; Jiang, X.; Xu, J.; Pan, S.; Qian, Z. Redox-Controlled Fluorescent Nanoswitch Based on Reversible Disulfide and Its Application in Butyrylcholinesterase Activity Assay. Anal. Chem. 2018, 90 (3), 1643– 51,  DOI: 10.1021/acs.analchem.7b02976
      614
      Redox-Controlled Fluorescent Nanoswitch Based on Reversible Disulfide and Its Application in Butyrylcholinesterase Activity Assay
      Chen, Guilin; Feng, Hui; Jiang, Xiaogan; Xu, Jing; Pan, Saifei; Qian, Zhaosheng
      Analytical Chemistry (Washington, DC, United States) (2018), 90 (3), 1643-1651CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Butyrylcholinesterase (BChE) mainly contributing to plasma cholinesterase activity is an important indicator for routinely diagnosing liver function and organophosphorus poisoning in clin. diagnosis, but its current assays are scarce and frequently suffer from some significant interference and instability. Herein, we report a redox-controlled fluorescence nanoswitch based on reversible disulfide bonds, and further develop a fluorometric assay of BChE via thiol-triggered disaggregation-induced emission. Thiol-functionalized carbon quantum dots (thiol-CQDs) with intense fluorescence is found to be responsive to hydrogen peroxide, and their redox reaction transforms thiol-CQDs to nonfluorescent thiol-CQDs assembly. The thiols inverse this process by a thiol-exchange reaction to turn on the fluorescence. The fluorescence can be reversibly switched by the formation and breaking of disulfide bonds caused by external redox stimuli. The specific thiol-triggered disaggregation-induced emission enables us to assay BChE activity in a fluorescence turn-on and real-time way using butyrylthiocholine iodide as the substrate. As-established BChE assay achieves sufficient sensitivity for practical detn. in plasma, and is capable of avoiding the interference from micormolar glutathione and discriminatively quantifying BChE from its sister enzyme acetylcholinesterase. The first design of reversible redox-controlled nanoswitch based on disulfide expands the application of disulfide chem. in sensing and clin. diagnostics, and this novel BChE assay enriches the detection methods for cholinesterase activity.
    612. 612
      Gong, M.-M.; Dai, C.-Y.; Severance, S.; Hwang, C.-C.; Fang, B.-K.; Lin, H.-B.; Huang, C.-H.; Ong, C.-W.; Wang, J.-J.; Lee, P.-L. A Bioorthogonally Synthesized and Disulfide-Containing Fluorescence Turn-On Chemical Probe for Measurements of Butyrylcholinesterase Activity and Inhibition in the Presence of Physiological Glutathione. Catalysts 2020, 10 (10), 1169,  DOI: 10.3390/catal10101169
      615
      A bioorthogonally synthesized and disulfide-containing fluorescence turn-on chemical probe for measurements of butyrylcholinesterase activity and inhibition in the presence of physiological glutathione
      Gong, Ming-Mao; Dai, Chia-Yen; Severance, Scott; Hwang, Chi-Ching; Fang, Bo-Kai; Lin, Heng-Bo; Huang, Chien-Hui; Ong, Chi-Wi; Wang, Jeh-Jeng; Lee, Pei-Lun; Wang, Tzu-Pin
      Catalysts (2020), 10 (10), 1169CODEN: CATACJ; ISSN:2073-4344. (MDPI AG)
      Butyrylcholinesterase (BChE) is a biomarker in human blood. Aberrant BChE activity has been assocd. with human diseases. Here we developed a fluorescence resonance energy transfer (FRET) chem. probe to specifically quantify BChE activity in serum, while simultaneously discriminating against glutathione (GSH). The FRET chem. probe 11 was synthesized from a key trifunctional bicyclononyne exo-6 and derivs. of 5-(2-aminoethylamino)-1-naphthalenesulfonic acid (EDANS) and 4-[4-(dimethylamino)phenylazo]benzoic acid (DABCYL). EDANS fluorescence visualization and kinetic anal. of 11 in the presence of diverse compds. confirmed the outstanding reactivity and specificity of 11 with thiols. The thiol-dependent fluorescence turn-on property of 11 was attributed to a general base-catalyzed SN2 nucleophilic substitution mechanism and independent of metal ions. Moreover, all thiols, except GSH, reacted swiftly with 11. Kinetic studies of 11 in the presence of covalently modified GSH derivs. corroborated that the steric hindrance of 11 imposing on GSH was the likely cause of the distinguished reactivity. Since GSH commonly interferes in assays measuring BChE activity in blood samples, the 11-based fluorescent assay was employed to directly quantify BChE activity without GSH interference, and delivered a linear range of 4.3-182.2 U L-1 for BChE activity with detection limit of 4.3 U L-1, and accurately quantified serum BChE activity in the presence of 10 μM GSH. Finally, the 11-based assay was exploited to det. Ki of 5 nM for tacrine inhibition on BChE catalysis. We are harnessing the modulated characteristics of 6 to synthesize advanced chem. probes able to more sensitively screen for BChE inhibitors and quantify BChE activity in serum.
    613. 613
      Peng, H.; Chen, W.; Cheng, Y.; Hakuna, L.; Strongin, R.; Wang, B. Thiol Reactive Probes and Chemosensors. Sensors 2012, 12 (11), 15907– 46,  DOI: 10.3390/s121115907
      616
      Thiol reactive probes and chemosensors
      Peng, Hanjing; Chen, Weixuan; Cheng, Yunfeng; Hakuna, Lovemore; Strongin, Robert; Wang, Binghe
      Sensors (2012), 12 (), 15907-15946CODEN: SENSC9; ISSN:1424-8220. (MDPI AG)
      A review. Thiols are important mols. in the environment and in biol. processes. Cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H2S) play crit. roles in a variety of physiol. and pathol. processes. The selective detection of thiols using reaction-based probes and sensors is very important in basic research and in disease diagnosis. This review focuses on the design of fluorescent and colorimetric probes and sensors for thiol detection. Thiol detection methods include probes and labeling agents based on nucleophilic addn. and substitution, Michael addn., disulfide bond or Se-N bond cleavage, metal-sulfur interactions and more. Probes for H2S are based on nucleophilic cyclization, redn. and metal sulfide formation. Thiol probe and chemosensor design strategies and mechanism of action are discussed in this review.
    614. 614
      Worek, F.; Mast, U.; Kiderlen, D.; Diepold, C.; Eyer, P. Improved Determination of Acetylcholinesterase Activity in Human Whole Blood. Clin. Chim. Acta 1999, 288 (1), 73– 90,  DOI: 10.1016/S0009-8981(99)00144-8
      617
      Improved determination of acetylcholinesterase activity in human whole blood
      Worek, F.; Mast, U.; Kiderlen, D.; Diepold, C.; Eyer, P.
      Clinica Chimica Acta (1999), 288 (1-2), 73-90CODEN: CCATAR; ISSN:0009-8981. (Elsevier Science Ireland Ltd.)
      The detn. of erythrocyte acetylcholinesterase (AChE) activity is an appropriate tool for the diagnosis of organophosphate exposure and intoxication. The original colorimetric Ellman procedure is disturbed by a high Hb absorption at 412 nm. In the authors' modified method, the wavelength was changed to 436 nm. This reduced the indicator absorption to 80% and the Hb absorption to 25%. The signal-to-noise ratio was further enhanced by redn. of pH and substrate concn., thus making it possible to measure 3% residual activity. AChE activity was detd. in whole blood samples in the presence of the selective butyrylcholinesterase inhibitor, ethopropazine. Diln. of blood samples (1:100) stopped secondary reactions in the presence of inhibitor (organophosphate) and reactivator (oxime). Normalization of the AChE activity to the Hb content, detd. as cyanmetHb, prevented diln. errors. This modified approach provides a simple way for sensitive and precise detn. of AChE activity in whole blood in the presence of organophosphates even with low-tech equipment.
    615. 615
      Eyer, P.; Worek, F.; Kiderlen, D.; Sinko, G.; Stuglin, A.; Simeon-Rudolf, V.; Reiner, E. Molar Absorption Coefficients for the Reduced Ellman Reagent: Reassessment. Anal. Biochem. 2003, 312 (2), 224– 7,  DOI: 10.1016/S0003-2697(02)00506-7
      618
      Molar absorption coefficients for the reduced Ellman reagent: reassessment
      Eyer, Peter; Worek, Franz; Kiderlen, Daniela; Sinko, Goran; Stuglin, Anita; Simeon-Rudolf, Vera; Reiner, Elsa
      Analytical Biochemistry (2003), 312 (2), 224-227CODEN: ANBCA2; ISSN:0003-2697. (Elsevier Science)
      The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coeff., 13.6×103 M-1 cm-1, as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for detn. of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temp. increases, while absorbance maxima decrease. Our recommended molar absorption coeffs. at 412 nm are 14.15×103 M-1 cm-1 at 25° and 13.8×103 M-1 cm-1 at 37° (0.1 M phosphate buffer, pH 7.4). Molar absorption coeffs. for other temps. and wavelengths are included in the paper.
    616. 616
      Pullela, P. K.; Chiku, T.; Carvan, M. J.; Sem, D. S. Fluorescence-Based Detection of Thiols in Vitro and in Vivo Using Dithiol Probes. Anal. Biochem. 2006, 352 (2), 265– 73,  DOI: 10.1016/j.ab.2006.01.047
      619
      Fluorescence-based detection of thiols in vitro and in vivo using dithiol probes
      Pullela, Phani Kumar; Chiku, Taurai; Carvan, Michael J.; Sem, Daniel S.
      Analytical Biochemistry (2006), 352 (2), 265-273CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
      Thiols play a central role in maintaining biol. homeostasis. Their levels can change dramatically in response to oxidative stress assocd. with toxic insults, bacterial infection, and disease. Therefore, a reagent that can monitor thiol levels both in vitro and in vivo would be useful for assays and as a biomarker. Such a reagent should (i) be selective for thiols, (ii) be able to penetrate cell walls, and (iii) have a low redn. potential so as not to create oxidative stress in a cell. The authors have developed such a fluorescent reagent (DSSA) based on a dithiol linker: (i) the use of a dithiol linker makes it selective for thiols; (ii) the use of fluorophores that populate neutral states at physiol. pH improves cell wall penetration; and (iii) because of the reagent's low redn. potential (-0.60 V), it will not stress cells oxidatively. For example, 5 μM of reagent is responsive to changes in glutathione levels in the physiol. relevant range of 1 to 10 mM, yet this would oxidize less than 1% of cellular glutathione. In Escherichia coli, decreased thiol levels were detected in cells deficient in glutathione synthesis. In zebrafish embryos, the DSSA reagent permitted detection of unusually high thiol levels in the zebrafish chorion.
    617. 617
      Cao, X.; Lin, W.; Yu, Q. A Ratiometric Fluorescent Probe for Thiols Based on a Tetrakis(4-Hydroxyphenyl)Porphyrin-Coumarin Scaffold. J. Org. Chem. 2011, 76 (18), 7423– 30,  DOI: 10.1021/jo201199k
      620
      A Ratiometric Fluorescent Probe for Thiols Based on a Tetrakis(4-hydroxyphenyl)porphyrin-Coumarin Scaffold
      Cao, Xiaowei; Lin, Weiying; Yu, Quanxing
      Journal of Organic Chemistry (2011), 76 (18), 7423-7430CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      The authors have designed and synthesized the compd. Ratio-HPSSC, based on a tetrakis(4-hydroxyphenyl)porphyrin-coumarin scaffold, as a new ratiometric fluorescent probe for thiols. The ratiometric probe Ratio-HPSSC is highly selective and sensitive to thiols. Importantly, the novel ratiometric probe exhibited a remarkable change in emission color from red to blue. This key feature allows Ratio-HPSSC to be employed for thiol detection by simple visual inspection. Furthermore, Ratio-HPSSC is suitable for ratiometric fluorescence imaging of thiols in living cells. The authors believe that the new ratiometric probe will find interesting applications in chem., biol., and medicine.
    618. 618
      Wang, L.; Wang, J.; Xia, S.; Wang, X.; Yu, Y.; Zhou, H.; Liu, H. A FRET-Based near-Infrared Ratiometric Fluorescent Probe for Detection of Mitochondria Biothiol. Talanta 2020, 219, 121296,  DOI: 10.1016/j.talanta.2020.121296
      621
      A FRET-based near-infrared ratiometric fluorescent probe for detection of mitochondria biothiol
      Wang, Lu; Wang, Jianbo; Xia, Shuai; Wang, Xinxin; Yu, Yating; Zhou, Hongwei; Liu, Haiying
      Talanta (2020), 219 (), 121296CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      A new fluorescent probe A with BODIPY as FRET donor and near-IR rhodamine as FRET acceptor is constructed through disulfide bonding and use for ratiometric fluorescence detection of biothiol. Due to the efficient fluorescence resonance energy transfer (FRET) from BODIPY donor to near-IR rhodamine acceptor, Probe A only displays near-IR rhodamine fluorescence (λem = 656 nm) under BODIPY excitation at 480 nm. The presence of biothiol leads to BODIPY fluorescence increases (λem = 511 nm) and near-IR rhodamine fluorescence decreases since the disulfide bond of the probe is broken by biothiols, effectively sepg. the donor from the acceptor, thus inhibiting the FRET process. Probe A exhibits remarkable high selectivity and excellent linear relationship from 10 μM to 100 μM of GSH, with low detection limit as 0.26 μM. Cellular imaging expts. shows that the probe is predominantly present in mitochondria and has been successfully applied to detect biothiol concns. changes in mitochondria of living cells.
    619. 619
      Lim, S. Y.; Shen, W.; Gao, Z. Carbon Quantum Dots and Their Applications. Chem. Soc. Rev. 2015, 44 (1), 362– 81,  DOI: 10.1039/C4CS00269E
      622
      Carbon quantum dots and their applications
      Lim, Shi Ying; Shen, Wei; Gao, Zhiqiang
      Chemical Society Reviews (2015), 44 (1), 362-381CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Fluorescent carbon nanoparticles or carbon quantum dots (CQDs) are a new class of carbon nanomaterials that have emerged recently and have garnered much interest as potential competitors to conventional semiconductor quantum dots. In addn. to their comparable optical properties, CQDs have the desired advantages of low toxicity, environmental friendliness low cost and simple synthetic routes. Moreover, surface passivation and functionalization of CQDs allow for the control of their physicochem. properties. Since their discovery, CQDs have found many applications in the fields of chem. sensing, biosensing, bioimaging, nanomedicine, photocatalysis and electrocatalysis. This article reviews the progress in the research and development of CQDs with an emphasis on their synthesis, functionalization and tech. applications along with some discussion on challenges and perspectives in this exciting and promising field.
    620. 620
      Steinmann, D.; Nauser, T.; Koppenol, W. H. Selenium and Sulfur in Exchange Reactions: A Comparative Study. J. Org. Chem. 2010, 75 (19), 6696– 9,  DOI: 10.1021/jo1011569
      623
      Selenium and Sulfur in Exchange Reactions: A Comparative Study
      Steinmann, Daniel; Nauser, Thomas; Koppenol, Willem H.
      Journal of Organic Chemistry (2010), 75 (19), 6696-6699CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      Cysteamine reduces selenocystamine to form hemiselenocystamine and then cystamine. The rate consts. are k1 = 1.3 × 105 M-1 s-1; k-1 = 2.6 × 107 M-1 s-1; k2 = 11 M-1 s-1; and k-2 = 1.4 × 103 M-1 s-1, resp. Rate consts. for reactions of cysteine/selenocysteine are similar. Reaction rates of selenium as a nucleophile and as an electrophile are 2-3 and 4 orders of magnitude higher, resp., than those of sulfur. Sulfides and selenides are comparable as leaving groups.
    621. 621
      Mafireyi, T. J.; Laws, M.; Bassett, J. W.; Cassidy, P. B.; Escobedo, J. O.; Strongin, R. M. A Diselenide Turn-On Fluorescent Probe for the Detection of Thioredoxin Reductase. Angew. Chem., Int. Ed. 2020, 59 (35), 15147– 51,  DOI: 10.1002/anie.202004094
      624
      A Diselenide Turn-On Fluorescent Probe for the Detection of Thioredoxin Reductase
      Mafireyi, Tendai J.; Laws, Madeleine; Bassett, John W.; Cassidy, Pamela B.; Escobedo, Jorge O.; Strongin, Robert M.
      Angewandte Chemie, International Edition (2020), 59 (35), 15147-15151CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We report the first diselenide-based probe for the selective detection of thioredoxin reductase (TrxR), an enzyme commonly overexpressed in melanomas. The probe design involves conjugation of a seminaphthorhodafluor dye with a diselenide moiety. TrxR reduces the diselenide bond, triggering a fluorescence turn-on response of the probe. Kinetic studies reveal favorable binding of the probe with TrxR with a Michaelis-Menten const. (Km) of 15.89 μM. Computational docking simulations predict a greater binding affinity to the TrxR active site in comparison to its disulfide analog. In vitro imaging studies further confirmed the diselenide probe exhibited improved signaling of TrxR activity compared to the disulfide analog.
    622. 622
      Nogueira, C. W.; Barbosa, N. V.; Rocha, J. B. Toxicology and Pharmacology of Synthetic Organoselenium Compounds: An Update. Arch. Toxicol. 2021, 95 (4), 1179– 226,  DOI: 10.1007/s00204-021-03003-5
      625
      Toxicology and pharmacology of synthetic organoselenium compounds: an update
      Nogueira, Cristina W.; Barbosa, Nilda V.; Rocha, Joao B. T.
      Archives of Toxicology (2021), 95 (4), 1179-1226CODEN: ARTODN; ISSN:0340-5761. (Springer)
      Here, we addressed the pharmacol. and toxicol. of synthetic organoselenium compds. and some naturally occurring organoselenium amino acids. The use of selenium as a tool in org. synthesis and as a pharmacol. agent goes back to the middle of the nineteenth and the beginning of the twentieth centuries. The rediscovery of ebselen and its investigation in clin. trials have motivated the search for new organoselenium mols. with pharmacol. properties. Although ebselen and diselenides have some overlapping pharmacol. properties, their mol. targets are not identical. However, contrary to our early expectations that they could imitate selenoproteins, organoselenium compds. seem to have non-specific modulatory activation of antioxidant pathways and specific inhibitory effects in some thiol-contg. proteins. The thiol-oxidizing properties of organoselenium compds. are considered the mol. basis of their However, the acute use of organoselenium compds. as inhibitors of specific thiol-contg. enzymes can be of therapeutic significance. In summary, the outcomes of the clin. trials of ebselen as a mimetic of lithium or as an inhibitor of SARS-CoV-2 proteases will be important to the field of organoselenium synthesis. The development of computational techniques that could predict rational modifications in the structure of organoselenium compds. to increase their specificity is required to construct a library of thiol-modifying agents with selectivity toward specific target proteins.
    623. 623
      Lou, Z.; Li, P.; Sun, X.; Yang, S.; Wang, B.; Han, K. A Fluorescent Probe for Rapid Detection of Thiols and Imaging of Thiols Reducing Repair and H2O2 Oxidative Stress Cycles in Living Cells. Chem. Commun. 2013, 49 (4), 391– 3,  DOI: 10.1039/C2CC36839K
      626
      A fluorescent probe for rapid detection of thiols and imaging of thiols reducing repair and H2O2 oxidative stress cycles in living cells
      Lou, Zhangrong; Li, Peng; Sun, Xiaofei; Yang, Songqiu; Wang, Bingshuai; Han, Keli
      Chemical Communications (Cambridge, United Kingdom) (2013), 49 (4), 391-393CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A diselenide contg. fluorescent probe (I) based on a fluorescein scaffold for thiols was developed. The fluorescent probe exhibited rapid response, high selectivity and reversibility. Confocal fluorescence microscopy was used to visualize the redox changes mediated by thiols and reactive oxygen species in living HeLa cells.
    624. 624
      Han, X.; Song, X.; Yu, F.; Chen, L. A Ratiometric Fluorescent Probe for Imaging and Quantifying Anti-Apoptotic Effects of GSH under Temperature Stress. Chem. Sci. 2017, 8 (10), 6991– 7002,  DOI: 10.1039/C7SC02888A
      627
      A ratiometric fluorescent probe for imaging and quantifying anti-apoptotic effects of GSH under temperature stress
      Han, Xiaoyue; Song, Xinyu; Yu, Fabiao; Chen, Lingxin
      Chemical Science (2017), 8 (10), 6991-7002CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Hypothermia and hyperthermia are cell stressed states resulting from environmental temp. changes, which can abnormally decrease intracellular glutathione (GSH) concns. and induce apoptosis. As the most abundant intracellular non-protein biothiol, GSH can protect cells from apoptosis. Considering the important roles of GSH in the anti-apoptotic process in cells and in vivo, we strive to develop a powerful chem. tool for the direct detection of GSH concn. changes under temp. stress. Herein, we report a ratiometric fluorescent probe (CyO-Dise) based on a selenium-sulfur exchange reaction for the qual. and quant. detection of GSH concn. fluctuations in cells and in vivo. The probe has been successfully used to assess the changes of GSH levels in HepG2 and HL-7702 cells using the stimulations of hypothermia and hyperthermia. In terms of the anti-apoptotic effect of GSH under hypothermic and hyperthermic conditions, human normal liver HL-7702 cells have stronger abilities to fight against temp. stress than human liver carcinoma HepG2 cells. Hypothermia and hyperthermia can also improve the drug resistance of cis-dichlorodiamineplatinum(II) (DDP)-resistant HepG2/DDP cells. The CyO-Dise probe has been employed to image GSH concn. changes in HepG2 and HepG2/DDP xenografts on nude mice. With the adjuvant therapy effects of hypothermia and hyperthermia, the chemotherapy drug DDP exhibits good ability for the treatment of HepG2 and HepG2/DDP xenografts.
    625. 625
      Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G. A Rhodamine-Based Fluorescent Probe Containing a Se-N Bond for Detecting Thiols and Its Application in Living Cells. J. Am. Chem. Soc. 2007, 129 (38), 11666– 7,  DOI: 10.1021/ja072572q
      628
      A Rhodamine-Based Fluorescent Probe Containing a Se-N Bond for Detecting Thiols and Its Application in Living Cells
      Tang, Bo; Xing, Yanlong; Li, Ping; Zhang, Ning; Yu, Fabiao; Yang, Guiwen
      Journal of the American Chemical Society (2007), 129 (38), 11666-11667CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Here the authors report a new rhodamine-based fluorescent probe (I) contg. a selenium-nitrogen bond for detecting thiols based on the nucleophilic substitution of sulfhydryl. The probe was successfully applied to the imaging of thiols in both HL-7702 cells and HepG2 cells with high sensitivity and selectivity.
    626. 626
      Tang, B.; Yin, L.; Wang, X.; Chen, Z.; Tong, L.; Xu, K. A Fast-Response, Highly Sensitive and Specific Organoselenium Fluorescent Probe for Thiols and Its Application in Bioimaging. Chem. Commun. 2009, (35), 5293– 5,  DOI: 10.1039/b909542j
      629
      A fast-response, highly sensitive and specific organoselenium fluorescent probe for thiols and its application in bioimaging
      Tang, Bo; Yin, Lingling; Wang, Xu; Chen, Zhenzhen; Tong, Lili; Xu, Kehua
      Chemical Communications (Cambridge, United Kingdom) (2009), (35), 5293-5295CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A novel organoselenium fluorescent probe (Rh-Se-2, I), which features a high signal-to-noise ratio (up to 170-fold), fast response (5 min) and excellent immunity to interferences, was designed, synthesized and applied to bioimaging.
    627. 627
      Wang, R.; Chen, L.; Liu, P.; Zhang, Q.; Wang, Y. Sensitive near-Infrared Fluorescent Probes for Thiols Based on Se-N Bond Cleavage: Imaging in Living Cells and Tissues. Eur. J. Chem. 2012, 18, 11343,  DOI: 10.1002/chem.201200671
      630
      Sensitive Near-Infrared Fluorescent Probes for Thiols Based on S-N Bond Cleavage: Imaging in Living Cells and Tissues
      Wang, Rui; Chen, Lingxin; Liu, Ping; Zhang, Qin; Wang, Yunqing
      Chemistry - A European Journal (2012), 18 (36), 11343-11349, S11343/1-S11343/5CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Cy-NiSe and Cy-TfSe were designed and synthesized as sensitive near-IR (NIR) fluorescent probes for detecting thiols on the basis of Se-N bond cleavage both in cells and in tissues. Since a donor-excited photoinduced electron transfer (d-PET) process occurs between the modulator and the fluorophore, Cy-NiSe and Cy-TfSe have weak fluorescence. On titrn. with glutathione, the free dye exhibits significant fluorescence enhancement. The two probes are sensitive and selective for thiols over other relevant biol. species. They can function rapidly at pH 7.4, and their emission lies in the NIR region. Confocal imaging confirms that Cy-NiSe and Cy-TfSe can be used for detecting thiols in living cells and tissues.
    628. 628
      Tian, Y.; Zhu, B.; Yang, W.; Jing, J.; Zhang, X. A Fluorescent Probe for Differentiating Cys, Hcy and GSH Via a Stepwise Interaction. Sens. Actuators B Chem. 2018, 262, 345– 9,  DOI: 10.1016/j.snb.2018.01.181
      631
      A fluorescent probe for differentiating Cys, Hcy and GSH via a stepwise interaction
      Tian, Yong; Zhu, Baocun; Yang, Wen; Jing, Jing; Zhang, Xiaoling
      Sensors and Actuators, B: Chemical (2018), 262 (), 345-349CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      It is still challenging to simultaneously distinguish general biothiols (cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) etc.) in complex redox environments, due to the lack of reliable and convenient method. Although a no. of fluorescence and/or UV-vis absorption based measurements have been employed for single or multiple biothiols detection, few of them is capable of recognizing each biothiol with individual signal at the same time. We present a one-step obtained selenadiazole deriv. (1) to distinguish Cys, Hcy and GSH, which not only can produce three new kind of fluorescent compds., but also simultaneously induce different "turn-on" fluorescence emission and red-shifted absorption signals for discriminating each biothiol. With a series of systematically analyses elucidate that probe 1 possesses high sensitivity and good selectivity for the three biothiols. Furthermore, the detection mechanism was also verified via a stepwise selenium-nitrogen (Se-N) bond cleavage process of probe 1 interacting with each biothiol. In the application of tracking in vitro and in vivo biothiols, probe 1 can be acted as a dual-mode fluorescent and colorimetric platform.
    629. 629
      Rusin, O.; St Luce, N. N.; Agbaria, R. A.; Escobedo, J. O.; Jiang, S.; Warner, I. M.; Dawan, F. B.; Lian, K.; Strongin, R. M. Visual Detection of Cysteine and Homocysteine. J. Am. Chem. Soc. 2004, 126 (2), 438,  DOI: 10.1021/ja036297t
      632
      Visual detection of cysteine and homocysteine
      Rusin, Oleksandr; St. Luce, Nadia N.; Agbaria, Rezik A.; Escobedo, Jorge O.; Jiang, Shan; Warner, Isiah M.; Dawan, Fareed B.; Lian, Kun; Strongin, Robert M.
      Journal of the American Chemical Society (2004), 126 (2), 438-439CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The detn. of cysteine and homocysteine levels is of great current interest for the monitoring of disease states. A new colorimetric method for the simultaneous detection of L-cysteine and L-homocysteine has been developed. A fluorescein deriv. reacts with the above amino acids, producing their resp. thiazolidines resulting in color changes. Interference from other amino acids and proteins is minimal.
    630. 630
      Lim, S.; Escobedo, J. O.; Lowry, M.; Xu, X.; Strongin, R. Selective Fluorescence Detection of Cysteine and N-Terminal Cysteine Peptide Residues. Chem. Commun. 2010, 46 (31), 5707– 9,  DOI: 10.1039/c0cc01398f
      633
      Selective fluorescence detection of cysteine and N-terminal cysteine peptide residues
      Lim, Soojin; Escobedo, Jorge O.; Lowry, Mark; Xu, Xiangyang; Strongin, Robert
      Chemical Communications (Cambridge, United Kingdom) (2010), 46 (31), 5707-5709CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A new fluorogenic fluorescein deriv. contg. an α,β-unsatd. aldehyde moiety produces a selective fluorescent signal enhancement in the presence of cysteine or peptides contg. N-terminal cysteine residues. The mechanism is based on synergistic covalent and supramol. interactions.
    631. 631
      Lee, K.-S.; Kim, T.-K.; Lee, J. H.; Kim, H.-J.; Hong, J.-I. Fluorescence Turn-On Probe for Homocysteine and Cysteine in Water. Chem. Commun. 2008, (46), 6173– 5,  DOI: 10.1039/b814581d
      634
      Fluorescence turn-on probe for homocysteine and cysteine in water
      Lee, Kyung-Sik; Kim, Tae-Ki; Lee, Jong Ho; Kim, Hae-Jo; Hong, Jong-In
      Chemical Communications (Cambridge, United Kingdom) (2008), (46), 6173-6175CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A simple fluorescent probe (I) based on an ortho-hydroxy aldehyde-functionalized coumarin showed selective responses to homocysteine and cysteine by fluorescence turn-on.
    632. 632
      Eun Jun, M.; Roy, B.; Han Ahn, K. “Turn-On” Fluorescent Sensing with “Reactive” Probes. Chem. Commun. 2011, 47 (27), 7583– 601,  DOI: 10.1039/c1cc00014d
      There is no corresponding record for this reference.
    633. 633
      Hoyle, C. E.; Lowe, A. B.; Bowman, C. N. Thiol-Click Chemistry: A Multifaceted Toolbox for Small Molecule and Polymer Synthesis. Chem. Soc. Rev. 2010, 39 (4), 1355– 87,  DOI: 10.1039/b901979k
      636
      Thiol-click chemistry: a multifaceted toolbox for small molecule and polymer synthesis
      Hoyle, Charles E.; Lowe, Andrew B.; Bowman, Christopher N.
      Chemical Society Reviews (2010), 39 (4), 1355-1387CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. The merits of thiol-click chem. and its potential for making new forays into chem. synthesis and materials applications are described. Since thiols react to high yields under benign conditions with a vast range of chem. species, their utility extends to a large no. of applications in the chem., biol., phys., materials and engineering fields. This crit. review provides insight into emerging venues for application as well as new mechanistic understanding of this exceptional chem. in its many forms (81 refs.).
    634. 634
      LoPachin, R. M.; Gavin, T. Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective. Chem. Res. Toxicol. 2014, 27 (7), 1081– 91,  DOI: 10.1021/tx5001046
      637
      Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective
      LoPachin, Richard M.; Gavin, Terrence
      Chemical Research in Toxicology (2014), 27 (7), 1081-1091CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      A review. Aldehydes are electrophilic compds. to which humans are pervasively exposed. Despite a significant health risk due to exposure, the mechanisms of aldehyde toxicity are poorly understood. This ambiguity is likely due to the structural diversity of aldehyde derivs. and corresponding differences in chem. reactions and biol. targets. To gain mechanistic insight, the authors have used parameters based on the hard and soft, acids and bases (HSAB) theory to profile the different aldehyde subclasses with respect to electronic character (softness, hardness), electrophilic reactivity (electrophilic index), and biol. nucleophilic targets. The authors' analyses indicate that short chain aldehydes and longer chain satd. alkanals are hard electrophiles that cause toxicity by forming adducts with hard biol. nucleophiles, e.g., primary nitrogen groups on lysine residues. In contrast, α,β-unsatd. carbonyl derivs., alkenals, and the α-oxoaldehydes are soft electrophiles that preferentially react with soft nucleophilic thiolate groups on cysteine residues. The aldehydes can therefore be grouped into subclasses according to common electronic characteristics (softness/hardness) and mol. mechanisms of toxicity. As the authors will discuss, the toxic potencies of these subgroups are generally related to corresponding electrophilicities. For some aldehydes, however, predictions of toxicity based on electrophilicity are less accurate due to inherent physicochem. variables that limit target accessibility, e.g., steric hindrance and soly. The unsatd. aldehydes are also members of the conjugated type-2 alkene chem. class that includes α,β-unsatd. amide, ketone, and ester derivs. Type-2 alkenes are electrophiles of varying softness and electrophilicity that share a common mechanism of toxicity. Therefore, exposure to an environmental mixt. of unsatd. carbonyl derivs. could cause "type-2 alkene toxicity" through additive interactions. Finally, the authors propose that environmentally derived aldehydes can accelerate diseases by interacting with endogenous aldehydes generated during oxidative stress. This review provides a basis for understanding aldehyde mechanisms and environmental toxicity through the context of electronic structure, electrophilicity, and nucleophile target selectivity.
    635. 635
      Chan, J. W.; Hoyle, C. E.; Lowe, A. B.; Bowman, M. Nucleophile-Initiated Thiol-Michael Reactions: Effect of Organocatalyst, Thiol, and Ene. Macromolecules 2010, 43 (15), 6381– 8,  DOI: 10.1021/ma101069c
      638
      Nucleophile-Initiated Thiol-Michael Reactions: Effect of Organocatalyst, Thiol, and Ene
      Chan, Justin W.; Hoyle, Charles E.; Lowe, Andrew B.; Bowman, Mark
      Macromolecules (Washington, DC, United States) (2010), 43 (15), 6381-6388CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      A detailed evaluation of the kinetics of the thiol-Michael reaction between hexanethiol and hexyl acrylate is described. It is shown that primary amines are more effective catalysts than either secondary or tertiary amines with, for example, quant. conversion being achieved within 500 s in the case of hexylamine with an apparent rate const. of 53.4 mol L-1 s-1 at a catalyst loading of 0.057 mol %. Certain tertiary phosphines, and esp. tri-n-propylphosphine and dimethylphenylphosphine, are shown to be even more effective species even at concns. 2 orders of magnitude lower than employed for hexylamine and performed in soln. with quant. conversions reached within ca. 100 s for both species and apparent rate consts. of 1810 and 431 mol L-1 s-1, resp. The nature of the thiol is also demonstrated to be an important consideration with mercaptoglycolate and mercaptopropionate esters being significantly more reactive than hexanethiol with reactivity mirroring the pKa of the thiols. Likewise, it is shown that the structure of the activated ene is also crucial with the degree of activation and ene-substitution pattern being important features in detg. reactivity. In terms of reaction with hexanethiol in the presence of hexylamine as catalyst, it is shown that propylmaleimide > di-Et fumarate > di-Et maleate > dimethylacrylamide > acrylonitrile > Et crotonate > Et cinnamate > Et methacrylate.
    636. 636
      Langmuir, M. E.; Yang, J.-R.; Moussa, A. M.; Laura, R.; LeCompte, K. A. New Naphthopyranone Based Fluorescent Thiol Probes. Tetrahedron Lett. 1995, 36 (23), 3989– 92,  DOI: 10.1016/0040-4039(95)00695-9
      639
      New naphthopyranone based fluorescent thiol probes
      Langmuir, Margaret E.; Yang, Jun-Rui; Moussa, Adel M.; Laura, Richard; LeCompte, Karen A.
      Tetrahedron Letters (1995), 36 (23), 3989-92CODEN: TELEAY; ISSN:0040-4039. (Elsevier)
      Five newly synthesized naphthopyranone-based thiol reactive probes are reported and their photophys. properties are compared with those of their parent naphthopyranones and their glutathione adducts.
    637. 637
      Niu, W.; Wu, P.; Chen, F.; Wang, J.; Shang, X.; Xu, C. Discovery of Selective Cystathionine β-Synthase Inhibitors by High-Throughput Screening with a Fluorescent Thiol Probe. MedChemComm 2017, 8 (1), 198– 201,  DOI: 10.1039/C6MD00493H
      640
      Discovery of selective cystathionine β-synthase inhibitors by high-throughput screening with a fluorescent thiol probe
      Niu, Weining; Wu, Ping; Chen, Fei; Wang, Jun; Shang, Xiaoya; Xu, Chunlan
      MedChemComm (2017), 8 (1), 198-201CODEN: MCCEAY; ISSN:2040-2503. (Royal Society of Chemistry)
      A high-throughput assay was developed to identify inhibitors of cystathionine β-synthase (CBS), which is one of three key enzymes involved in H2S biosynthesis. Screening of 6491 natural compds. identified several selective CBS inhibitors, which suppressed the proliferation of HT29 cancer cells, with IC50 values of less than 10 μM.
    638. 638
      Aharoni, A.; Amitai, G.; Bernath, K.; Magdassi, S.; Tawfik, D. S. High-Throughput Screening of Enzyme Libraries: Thiolactonases Evolved by Fluorescence-Activated Sorting of Single Cells in Emulsion Compartments. Chem. Biol. 2005, 12 (12), 1281– 9,  DOI: 10.1016/j.chembiol.2005.09.012
      641
      High-Throughput Screening of Enzyme Libraries: Thiolactonases Evolved by Fluorescence-Activated Sorting of Single Cells in Emulsion Compartments
      Aharoni, Amir; Amitai, Gil; Bernath, Kalia; Magdassi, Shlomo; Tawfik, Dan S.
      Chemistry & Biology (Cambridge, MA, United States) (2005), 12 (12), 1281-1289CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
      Single bacterial cells, each expressing a different library variant, were compartmentalized in aq. droplets of water-in-oil (w/o) emulsions, thus maintaining a linkage between a plasmid-borne gene, the encoded enzyme variant, and the fluorescent product this enzyme may generate. Conversion into a double, water-in-oil-in-water (w/o/w) emulsion enabled the sorting of these compartments by FACS, as well as the isolation of living bacteria cells and their enzyme-coding genes. We demonstrate the directed evolution of new enzyme variants by screening >107 serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying >104 enzyme mols., in a vol. of <10 fL (fl), enabled detection and selection despite the fast, spontaneous hydrolysis of the substrate, the very low initial thiolactonase activity of PON1, and the use of difusable fluorescent products.
    639. 639
      Barb, A. W.; Marcella, A. M. A Rapid Fluorometric Assay for the S-Malonyltransacylase FabD and Other Sulfhydryl Utilizing Enzymes. J. Biol. Methods 2016, 3 (4), e53  DOI: 10.14440/jbm.2016.144
      There is no corresponding record for this reference.
    640. 640
      Yi, L.; Li, H.; Sun, L.; Liu, L.; Zhang, C.; Xi, Z. A Highly Sensitive Fluorescence Probe for Fast Thiol-Quantification Assay of Glutathione Reductase. Angew. Chem., Int. Ed. 2009, 48 (22), 4034– 7,  DOI: 10.1002/anie.200805693
      643
      A Highly Sensitive Fluorescence Probe for Fast Thiol-Quantification Assay of Glutathione Reductase
      Yi, Long; Li, Heyang; Sun, Lu; Liu, Liangliang; Zhang, Caihong; Xi, Zhen
      Angewandte Chemie, International Edition (2009), 48 (22), 4034-4037, S4034/1-S4034/17CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Fast detection of cellular thiols in aq. medium was achieved using a newly developed fluorescence probe (see picture). Based on this probe, a high-throughput fluorescence assay for glutathione reductase was developed.
    641. 641
      Zhou, X.; Jin, X.; Sun, G.; Li, D.; Wu, X. A Cysteine Probe with High Selectivity and Sensitivity Promoted by Response-Assisted Electrostatic Attraction. Chem. Commun. 2012, 48 (70), 8793– 5,  DOI: 10.1039/c2cc33971d
      644
      A cysteine probe with high selectivity and sensitivity promoted by response-assisted electrostatic attraction
      Zhou, Xin; Jin, Xuejun; Sun, Guangyan; Li, Donghao; Wu, Xue
      Chemical Communications (Cambridge, United Kingdom) (2012), 48 (70), 8793-8795CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A new turn-on fluorescent probe for fast detection of cysteine in physiol. conditions, based on a new response-assisted electrostatic attraction strategy, is reported. The practical utility of the probe in fluorescent protein labeling and subcellular imaging is also demonstrated.
    642. 642
      Li, S.-J.; Fu, Y.-J.; Li, C.-Y.; Li, Y.-F.; Yi, L.-H.; Ou-Yang, J. A near-Infrared Fluorescent Probe Based on BODIPY Derivative with High Quantum Yield for Selective Detection of Exogenous and Endogenous Cysteine in Biological Samples. Anal. Chim. Acta 2017, 994, 73– 81,  DOI: 10.1016/j.aca.2017.09.031
      645
      A near-infrared fluorescent probe based on BODIPY derivative with high quantum yield for selective detection of exogenous and endogenous cysteine in biological samples
      Li, Song-Jiao; Fu, Ya-Jun; Li, Chun-Yan; Li, Yong-Fei; Yi, Lan-Hua; Ou-Yang, Juan
      Analytica Chimica Acta (2017), 994 (), 73-81CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      Cysteine (Cys) is involved in cellular growth and Cys deficiency is related with many diseases. So far, a no. of fluorescent probes have been constructed for the detection of Cys successfully. However, the probes are difficult to discriminate Cys from Hcy and the emission wavelength of the probes is in UV or visible range. Herein, a NIR fluorescent probe named NIR-BODIPY-Ac (I) was synthesized and used to detect Cys. The emission wavelength of the probe is at 708 nm that belongs to near-IR (NIR) region by attaching indolium to BODIPY core, which is suitable for bioimaging in vivo. Moreover, the probe exhibits high fluorescence quantum yield (Φ = 0.51) after the addn. of Cys and high sensitivity toward Cys with 81-fold fluorescence enhancement. The linear range of the probe for Cys covers from 0.2 to 30 μM with a detection limit of 0.05 μM. Furthermore, the probe shows high selectivity towards Cys owing to the fact that there is more fast reaction rate between the probe and Cys than that of Hcy. In particular, the NIR fluorescent probe is applied for the detection of exogenous and endogenous Cys in biol. samples such as cell, tissue and mouse with satisfactory results.
    643. 643
      Li, R.; Lei, C.; Zhao, X.-E.; Gao, Y.; Gao, H.; Zhu, S.; Wang, H. A Label-Free Fluorimetric Detection of Biothiols Based on the Oxidase-Like Activity of Ag+ Ions. Spectrochim. Acta - A: Mol. Biomol. Spectrosc. 2018, 188, 20– 5,  DOI: 10.1016/j.saa.2017.06.056
      646
      A label-free fluorimetric detection of biothiols based on the oxidase-like activity of Ag+ ions
      Li, Ru; Lei, Cuihua; Zhao, Xian-En; Gao, Yue; Gao, Han; Zhu, Shuyun; Wang, Hua
      Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy (2018), 188 (), 20-25CODEN: SAMCAS; ISSN:1386-1425. (Elsevier B.V.)
      In this work, a label-free and sensitive fluorimetric method has been developed for the detections of biothiols including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), based on the specific biothiol-induced inhibition of the oxidase-like activity of silver ions (Ag+). It is well established that o-phenylenediamine (OPD) can be oxidized by Ag+ ions to generate fluorescent 2,3-diaminophenazine (OPDox). The introduction of biothiols would inhibit the oxidn. of OPD by Ag+ due to the strong coordination between biothiols and Ag+. The changes of fluorescence intensities obtained in the Ag+-OPD system exhibited good linear correlations in the ranges of 0.50-30.0 μM for Cys, 1.0-45.0 μM for Hcy and 0.50-40.0 μM for GSH. The detection limits (S/N = 3) of Cys, Hcy and GSH were 110 nM, 200 nM and 150 nM, resp. Subsequently, the developed fluorimetric method was successfully applied for the detection of biothiols in human serum.
    644. 644
      Mandani, S.; Sharma, B.; Dey, D.; Sarma, T. K. Carbon Nanodots as Ligand Exchange Probes in Au@C-Dot Nanobeacons for Fluorescent Turn-On Detection of Biothiols. Nanoscale 2015, 7 (5), 1802– 8,  DOI: 10.1039/C4NR05424E
      647
      Carbon nanodots as ligand exchange probes in Au@C-dot nanobeacons for fluorescent turn-on detection of biothiols
      Mandani, Sonam; Sharma, Bhagwati; Dey, Deepa; Sarma, Tridib K.
      Nanoscale (2015), 7 (5), 1802-1808CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      Au nanoparticle-carbon dot core-shell (Au@C-dot) nanocomposite was synthesized in aq. medium at room temp. using the carbon dots as reducing agents themselves. The carbon nanodots also function as an effective stabilizer by forming a thin layer surrounding Au nanoparticles (Au NPs) similar to self-assembled monolayers. Ligand exchange with thiol contg. biomols. resulted in the release of carbon dots from the Au NP surface leading to an enhancement of fluorescence. Simultaneously the agglomeration of Au NPs stimulated by the interaction of biothiols led to changes in the surface plasmon properties of Au NPs. A detailed spectroscopic investigation revealed a combination of static and dynamic quenching being involved in the process. Thus, the Au nanoparticle-carbon dot composite could be used as a dual colorimetric and fluorometric sensor for biothiols ranging from amino acids, peptides, proteins, enzymes etc. with a detection limit of 50 nM.
    645. 645
      Fu, Y.; Li, H.; Hu, W.; Zhu, D. Fluorescence Probes for Thiol-Containing Amino Acids and Peptides in Aqueous Solution. Chem. Commun. 2005, (25), 3189– 91,  DOI: 10.1039/b503772g
      648
      Fluorescence probes for thiol-containing amino acids and peptides in aqueous solution
      Fu, Yanyan; Li, Hongxiang; Hu, Wenping; Zhu, Daoben
      Chemical Communications (Cambridge, United Kingdom) (2005), (25), 3189-3191CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A new fluorescence probe is described using the "chemosensing ensemble" method. The probe shows high selectivity and sensitivity for thiol-contg. amino acids and peptides, and can detect cysteine and homocysteine from healthy to abnormal levels under physiol.-relevant conditions.
    646. 646
      Nie, L.; Ma, H.; Sun, M.; Li, X.; Su, M.; Liang, S. Direct Chemiluminescence Determination of Cysteine in Human Serum Using Quinine-Ce(IV) System. Talanta 2003, 59 (5), 959– 64,  DOI: 10.1016/S0039-9140(02)00649-5
      649
      Direct chemiluminescence determination of cysteine in human serum using quinine-Ce(IV) system
      Nie, Lihua; Ma, Huimin; Sun, Ming; Li, Xiaohua; Su, Meihong; Liang, Shuchuan
      Talanta (2003), 59 (5), 959-964CODEN: TLNTA2; ISSN:0039-9140. (Elsevier Science B.V.)
      A simple, sensitive and selective chemiluminescence (CL) method was developed for the detn. of cysteine. This method is based on that the weak CL of cysteine oxidized with Ce (IV) can be greatly enhanced by quinine. The calibration curve was linear over the range 3.5 × 10-9-3.5 × 10-6 M with a detection limit of 2.5 × 10-9 M (S/N = 3). The relative std. deviation is 8.4% by 10 replicate detns. of 2.9 × 10-8 M cysteine. Due to high sensitivity, the proposed method can be used directly to det. the total concn. of cysteine in human serum through simply dilg. the sample for a thousand fold. The obtained result was in agreement with that given by amino acid automated analyzer. The present method does not require any sepn., showing a simpler anal. characteristic. The mechanism of the CL reaction is also discussed.
    647. 647
      Wang, S.; Ma, H.; Li, J.; Chen, X.; Bao, Z.; Sun, S. Direct Determination of Reduced Glutathione in Biological Fluids by Ce(Iv)-Quinine Chemiluminescence. Talanta 2006, 70 (3), 518– 21,  DOI: 10.1016/j.talanta.2005.12.052
      650
      Direct determination of reduced glutathione in biological fluids by Ce(IV)-quinine chemiluminescence
      Wang, Shujuan; Ma, Huimin; Li, Jun; Chen, Xinqi; Bao, Zhijuan; Sun, Shuna
      Talanta (2006), 70 (3), 518-521CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      A simple, sensitive and selective chemiluminescence (CL) method is proposed for the detn. of reduced glutathione (GSH) in biol. fluids. This method is based on the fact that the weak CL of GSH oxidized with cerium(IV) can be greatly enhanced by quinine. The optimum conditions for the CL emission were investigated. The calibration curve for GSH was linear over the concn. range of 4.0 × 10-9-4.0 × 10-5 M with a detection limit of 5 × 10-10 M (S/N = 3). The R.S.D. was 4.0% by 11 replicate detns. of 1.0 × 10-7 M GSH. It was also found that GSH and cysteine, the two often-coexisting thiol compds. in biol. samples, exhibited a different CL sensitivity in the Ce(IV)-quinine system (the sensitivity of GSH was higher than that of cysteine). This leads to the successful use of the proposed method for the direct and selective detn. of GSH in rabbit whole blood and rat brain microdialyzate in the presence of cysteine. Moreover, compared to the classical 5,5'-dithiobis(2-nitrobenzoic acid) method, the present one has the advantages of simplicity and high sensitivity.
    648. 648
      Zou, T.; Lum, C. T.; Chui, S. S.-Y.; Che, C.-M. Gold(III) Complexes Containing N-Heterocyclic Carbene Ligands: Thiol “Switch-On” Fluorescent Probes and Anti-Cancer Agents. Angew. Chem., Int. Ed. 2013, 52 (10), 2930– 3,  DOI: 10.1002/anie.201209787
      651
      Gold(III) Complexes Containing N-Heterocyclic Carbene Ligands: Thiol "Switch-on" Fluorescent Probes and Anti-Cancer Agents
      Zou, Taotao; Lum, Ching Tung; Chui, Stephen Sin-Yin; Che, Chi-Ming
      Angewandte Chemie, International Edition (2013), 52 (10), 2930-2933CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A class of Au(III) complexes bearing N-heterocyclic carbene and 2,6-bis(imidazol-2-yl)pyridine or 2,6-bis(benzimidazol-2-yl)pyridine ligands have been developed. These Au(III)-NHC complexes are sensitive towards thiols, which leads to release of fluorescent ligand, and thus can serve as a switch-on probe for thiols in biol. systems. The Au(III) complexes can also suppress tumor growth in mice bearing HeLa xenografts. These Au(III) complexes could be a promising scaffold for the future development of novel switch-on probes and anti-cancer agents.
    649. 649
      Han, G.-C.; Peng, Y.; Hao, Y.-Q.; Liu, Y.-N.; Zhou, F. Spectrofluorimetric Determination of Total Free Thiols Based on Formation of Complexes of Ce(III) with Disulfide Bonds. Anal. Chim. Acta 2010, 659 (1), 238– 42,  DOI: 10.1016/j.aca.2009.11.057
      652
      Spectrofluorimetric determination of total free thiols based on formation of complexes of Ce(III) with disulfide bonds
      Han, Guo-Cheng; Peng, Yong; Hao, Yuan-Qiang; Liu, You-Nian; Zhou, Feimeng
      Analytica Chimica Acta (2010), 659 (1-2), 238-242CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      A simple, rapid, and sensitive detn. of total free thiol groups in biol. samples using cerium (IV) as a fluorescence probe is reported. The protocol is based on the oxidn. of thiols by Ce(IV) and the formation the Ce(III) disulfide complex, which gives a fluorescence enhancement of Ce(III) at 352 nm. Using glutathione (GSH) and cysteine as model compds., incubation with Ce(IV) at 25° for 6 min results in fluorescence, whose intensity is proportional to the thiol concn. in the range of 1.00-160 nM. The detection limits for GSH and cysteine are 0.05 and 0.08 nM, resp. Other common metal ions and amino acids have little interference to the thiol detection. Cu(II) was used as a fluorescence quencher to eliminate potential interference from tryptophan. The method has been successfully applied to assays of free thiol contents in pig liver tissue samples, with a RSD lower than 2.5% and recovery between 100.6% and 102.3%.
    650. 650
      Wang, H.; Zhou, G.; Chen, X. An Iminofluorescein-Cu2+ Ensemble Probe for Selective Detection of Thiols. Sens. Actuators B Chem. 2013, 176, 698– 703,  DOI: 10.1016/j.snb.2012.10.006
      653
      An iminofluorescein-Cu2+ ensemble probe for selective detection of thiols
      Wang, Huilin; Zhou, Guodong; Chen, Xiaoqiang
      Sensors and Actuators, B: Chemical (2013), 176 (), 698-703CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      An iminofluorescein-Cu2+ ensemble probe was prepd. for the detection of thiols in aq. soln. In HEPES buffer, among various amino acids, only cysteine (Cys) induced fluorescence enhancement of Cu2+ ensemble. The mechanism was proposed that addn. of Cys induced decomplexation of Cu2+ from weak-fluorescent ensemble, followed by hydrolytic cleavage of the resulted Schiff base to give a strongly fluorescent fluoresceinaldehyde. By fluorescence titrn. using 2 μM iminofluorescein-Cu2+ ensemble probe, the detection limit for Cys was evaluated to be 9 μM with a signal-to-noise ratio of 3. Furthermore, the confocal microscopy expts. demonstrated that this sensor was potential for the imaging of thiols in living cells.
    651. 651
      Jung, H. S.; Han, J. H.; Habata, Y.; Kang, C.; Kim, J. S. An Iminocoumarin-Cu(II) Ensemble-Based Chemodosimeter toward Thiols. Chem. Commun. 2011, 47 (18), 5142– 4,  DOI: 10.1039/c1cc10672d
      654
      An iminocoumarin-Cu(II) ensemble-based chemodosimeter toward thiols
      Jung, Hyo Sung; Han, Ji Hye; Habata, Yoichi; Kang, Chulhun; Kim, Jong Seung
      Chemical Communications (Cambridge, United Kingdom) (2011), 47 (18), 5142-5144CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      An 'ensemble'-based fluorogenic chemodosimeter 1-Cu(II) (I) for detection of thiols is reported. Complex 1-Cu(II) sensitively senses thiols followed by hydrolysis to give a marked fluorescence enhancement over other amino acids at pH 7.4 under aq. media. Confocal microscopic imaging of complex 1-Cu(II) is also herewith demonstrated for cellular thiol detection in HepG2 cells.
    652. 652
      Zhao, D.; Chen, C.; Sun, J.; Yang, X. Carbon Dots-Assisted Colorimetric and Fluorometric Dual-Mode Protocol for Acetylcholinesterase Activity and Inhibitors Screening Based on the Inner Filter Effect of Silver Nanoparticles. Analyst 2016, 141 (11), 3280– 8,  DOI: 10.1039/C6AN00514D
      655
      Carbon dots-assisted colorimetric and fluorometric dual-mode protocol for acetylcholinesterase activity and inhibitors screening based on the inner filter effect of silver nanoparticles
      Zhao, Dan; Chen, Chuanxia; Sun, Jian; Yang, Xiurong
      Analyst (Cambridge, United Kingdom) (2016), 141 (11), 3280-3288CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      The authors proposed an original and versatile dual-readout (colorimetric and fluorometric) protocol by silver nanoparticles (AgNPs) and fluorescent carbon dots (CDs), which was amenable to rapid, ultrasensitive assay of acetylcholinesterase (AChE) activity and its inhibitors. The sensing mechanism was based on the non-fluorescence state of CDs resulting from the inner filter effect (IFE) of AgNPs and the specific AChE-catalyzed hydrolysis of acetylthiocholine (ATCh) into thiocholine (TCh). Herein, the generated pos.-charged and thiol-bearing TCh at trace concn. levels could trigger the aggregation of AgNPs through the known electrostatic and Ag-SH interactions, thereby turning the sensing solns. gray and recovering the IFE-quenched fluorescence simultaneously. Furthermore, the existence of IFE mechanism was conceivably confirmed by combining the zeta potentials, fluorescence spectra, UV-visible spectra, fluorescence lifetime and TEM measurements. As far as the authors know, the present study has reported the first dual-mode proposal for assessing AChE activity by using a CDs-based IFE sensing strategy, where the detection limit was as low as 0.021 mU mL-1 and 0.016 mU mL-1 by colorimetric and fluorometric measurements, resp. However, the proposed assay was feasible to screen AChE inhibitors such as tacrine and carbaryl. Meanwhile, this rationally designed dual-mode sensing platform featured simplicity, rapidity, flexibility and diversity, which was demonstrated by the quant. detection of spiked carbaryl in apple juice samples with satisfactory results.
    653. 653
      Yang, J.; Song, N.; Lv, X.; Jia, Q. UV-Light-Induced Synthesis of PEI-CuNCs Based on Cu2+-Quenched Fluorescence Turn-On Assay for Sensitive Detection of Biothiols, Acetylcholinesterase Activity and Inhibitor. Sens. Actuators B Chem. 2018, 259, 226– 32,  DOI: 10.1016/j.snb.2017.12.045
      656
      UV-light-induced synthesis of PEI-CuNCs based on Cu2+-quenched fluorescence turn-on assay for sensitive detection of biothiols, acetylcholinesterase activity and inhibitor
      Yang, Jinlan; Song, Naizhong; Lv, Xueju; Jia, Qiong
      Sensors and Actuators, B: Chemical (2018), 259 (), 226-232CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      Polyethyleneimine-protected copper nanoclusters (PEI-CuNCs) were synthesized and applied to detect Cu2+, biothiols, and acetylcholinesterase (AChE). A facile UV-light-induced method was used in the synthesize process to obtain PEI-CuNCs with high stability and strong fluorescence intensity. Based on the quenching mechanism, a label-free assay for sensing Cu2+ by PEI-CuNCs was developed with the limit of detection (LOD) of 0.12μM. Biothiols with -SH functional groups could react with Cu2+ to form the RSH-Cu2+ complex, making Cu2+-triggered fluorescence quenching turned on. Two biothiols, glutathione and cysteine, were detd. with LODs of 0.26μM and 0.34μM within linear ranges of 0.5-25μM and 1-25μM, resp. AChE could hydrolyze acetylthiocholine into thiocholine (TCh), which reacted with Cu2+ to form TCh-Cu2+ complex and turned on Cu2+-quenched fluorescence. LOD of AChE was calcd. to be 1.38 mU/mL within a linear range from 3 to 200 mU/mL. A typical inhibitor of AChE, tacrine, was also detected by this convenient fluorescence turn-on assay with the corresponding IC50 value (the inhibitor concn. required for 50% inhibition of the enzyme activity) of 53.4 nM. Moreover, the assay was successfully applied to the detection of biothiols and AChE activity in human serum samples. Results demonstrated the great potential of PEI-CuNCs and paved a new avenue for exploring their versatile biosensing applications.
    654. 654
      Chen, Y.; Liu, W.; Zhang, B.; Suo, Z.; Xing, F.; Feng, L. Sensitive and Reversible Perylene Derivative-Based Fluorescent Probe for Acetylcholinesterase Activity Monitoring and Its Inhibitor. Anal. Biochem. 2020, 607, 113835,  DOI: 10.1016/j.ab.2020.113835
      657
      Sensitive and reversible perylene derivative-based fluorescent probe for acetylcholinesterase activity monitoring and its inhibitor
      Chen, Yingying; Liu, Wenxia; Zhang, Binbin; Suo, Zhiguang; Xing, Feifei; Feng, Lingyan
      Analytical Biochemistry (2020), 607 (), 113835CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      A reversible fluorescence probe for acetylcholinesterase activity detection was developed based on water sol. perylene deriv., N,N'-di(2-aspartic acid)-perylene-3,4,9,10-tetracarboxylic diimide (PASP). Based on the photo-induced electron transfer (PET), PASP fluorescence in aq. is quenched after combining with copper ions (Cu2+). Acetylcholinesterase (AChE) is known to catalyze the hydrolysis of acetylcholine (ATCh) to produce thiocholine, whose affinity is strong enough to capture Cu2+ by thiol (-SH) group from the complex PASP-Cu, resulting in the fluorescence signal of PASP recovers up to 90%. This optical switch is highly sensitive depended on the coordination and dissocn. between PASP and Cu2+. The authors proposed its application for AChE activity detection, as well as its inhibitor screening. According to the change of fluorescence intensity, quantifying the detection limit of AChE was 1.78 mU·mL-1. Classical inhibitors, tacrine and organophosphate pesticide diazinon, were further evaluated for drug screening. The IC50 value of tacrine is 0.43μM, and the detection limit of diazinon was 0.22μM. Both of these performances were much better than previous results, revealing the authors' probe is sensitive and reversible for screening applications.
    655. 655
      Xu, J.; Yu, H.; Hu, Y.; Chen, M.; Shao, S. A Gold Nanoparticle-Based Fluorescence Sensor for High Sensitive and Selective Detection of Thiols in Living Cells. Biosens. Bioelectron. 2016, 75, 1– 7,  DOI: 10.1016/j.bios.2015.08.007
      658
      A gold nanoparticle-based fluorescence sensor for high sensitive and selective detection of thiols in living cells
      Xu, Jian; Yu, Hui; Hu, Yue; Chen, Mingzhong; Shao, Shijun
      Biosensors & Bioelectronics (2016), 75 (), 1-7CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
      A novel gold nanoparticle (AuNP)-based sensor for detecting thiols in aq. soln. has been developed. Due to the weak N···Au interactions, meso-(4-pyridinyl)-substituted BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes were coordinated to AuNP surfaces, which effectively quenched the fluorescence of org./inorg. hybrid systems. The fluorescent quenching mechanism was mainly ascribed to the highly efficient fluorescent resonance energy transfer (FRET) and the inner filter effect. In the presence of thiols, meso-(4-pyridinyl)-substituted BODIPY chromophore were displaced and released from the AuNP surfaces and thus restored the fluorescence of BODIPY chromophore. The modulation of the fluorescence quenching efficiency of BODIPY-AuNPs in the presence of thiols can achieve a large turn-on fluorescence enhancement (40-fold) in aq. soln. The new AuNP-based fluorescence sensor displayed desired properties such as high specificity, relatively low detection limit (30 nM for Cys), appreciable water soly. and rapid response time (within 2 min for Cys/Hcy). Moreover, the sensor has been successfully applied for monitoring and imaging of intracellular thiols within living HeLa cells.
    656. 656
      Mu, H.; Miki, K.; Kubo, T.; Otsuka, K.; Ohe, K. Substituted meso-Vinyl-BODIPY as Thiol-Selective Fluorogenic Probes for Sensing Unfolded Proteins in the Endoplasmic Reticulum. Chem. Commun. 2021, 57 (14), 1818– 21,  DOI: 10.1039/D0CC08160D
      659
      Substituted meso-vinyl-BODIPY as thiol-selective fluorogenic probes for sensing unfolded proteins in the endoplasmic reticulum
      Mu, Huiying; Miki, Koji; Kubo, Takuya; Otsuka, Koji; Ohe, Kouichi
      Chemical Communications (Cambridge, United Kingdom) (2021), 57 (14), 1818-1821CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A new type of thiol probes based on the meso-vinyl-BODIPY (VB) scaffold were developed. The monochloro-substituted VB1Cl exhibited the largest fluorescence enhancement (>200-fold) as well as high selectivity upon biol. thiol sensing. VB1Cl was successfully applied for reporting the protein unfolding process under ER stress in living cells.
    657. 657
      Huang, S.-T.; Ting, K.-N.; Wang, K.-L. Development of a Long-Wavelength Fluorescent Probe Based on Quinone-Methide-Type Reaction to Detect Physiologically Significant Thiols. Anal. Chim. Acta 2008, 620 (1), 120– 6,  DOI: 10.1016/j.aca.2008.05.006
      660
      Development of a long-wavelength fluorescent probe based on quinone-methide-type reaction to detect physiologically significant thiols
      Huang, Sheng-Tung; Ting, Kuo-Neng; Wang, Kun-Li
      Analytica Chimica Acta (2008), 620 (1-2), 120-126CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      The authors synthesized a new long-wavelength latent fluorimetric probe BCC (I) to detect physiol. significant thiols. The fluorogenic chem. transformation of BCC triggered by thiols is through a tandem reaction, thiol-induced benzoquinone redn., and quinone-methide-type rearrangement reaction, which are spontaneous and irreversible at physiol. temp. in aq. media. The fluorescence signal revealed by this process is specific and exhibited in the near-red spectrum region with emission maxima at 595 nm, and it could be competitively inhibited by thiols scavenger, N-ethylmaleimide. The fluorescent response of BCC is insensitive to various non-thiol amino acids and biol. reductants. This novel fluorimetric probe demonstrates a good relation in detecting thiols in 1-100 μM range, which presents to the applicability for the construction of fiber-optic biosensors in the future clin. diagnostic.
    658. 658
      Kosower, N. S.; Kosower, E. M.; Newton, G. L.; Ranney, H. M. Bimane Fluorescent Labels: Labeling of Normal Human Red Cells under Physiological Conditions. Proc. Natl. Acad. Sci. U.S.A 1979, 76 (7), 3382– 6,  DOI: 10.1073/pnas.76.7.3382
      661
      Bimane fluorescent labels: Labeling of normal human red cells under physiological conditions
      Kosower, Nechama S.; Kosower, Edward M.; Newton, Gerald L.; Ranney, Helen M.
      Proceedings of the National Academy of Sciences of the United States of America (1979), 76 (7), 3382-6CODEN: PNASA6; ISSN:0027-8424.
      The bimane fluorescent labels, monobromobimane, dibromobimane, and monobromotrimethylammoniobimane, are derivs. of syn-9,10-dioxabimane:1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione. They efficiently labeled Hb (reactive SH groups), membrane proteins, and glutathione of normal human red cells under physiol. conditions. Monobromobimane and dibromobimane were effective on intact cells whereas cell membranes may be impermeable to the pos. charged monobromotrimethylammoniobimane, the latter being effective only on lysed cells. These bimane labels provide a class of labeling agents that may have wide applicability in biol. materials.
    659. 659
      Qi, S.; Liu, W.; Zhang, P.; Wu, J.; Zhang, H.; Ren, H.; Ge, J.; Wang, P. A Colorimetric and Ratiometric Fluorescent Probe for Highly Selective Detection of Glutathione in the Mitochondria of Living Cells. Sens. Actuators B Chem. 2018, 270, 459– 65,  DOI: 10.1016/j.snb.2018.05.017
      662
      A colorimetric and ratiometric fluorescent probe for highly selective detection of glutathione in the mitochondria of living cells
      Qi, Sujie; Liu, Weimin; Zhang, Panpan; Wu, Jiasheng; Zhang, Hongyan; Ren, Haohui; Ge, Jiechao; Wang, Pengfei
      Sensors and Actuators, B: Chemical (2018), 270 (), 459-465CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      A new water-sol. fluorescent probe 1 based on hemicyanine dye is designed for selective detection of glutathione (GSH). Probe 1 shows a tremendous colorimetric and ratiometric fluorescence response toward GSH with high selectivity over cysteine/homocysteine. After nucleophilic arom. substitution reaction with GSH, probe 1 undergoes an intramol. aldimine condensation that leads to forming a ring, which is accompanied by 107 and 41nm red-shifts in the absorption and fluorescence spectra, resp. Subsequent expts. in living cells indicate that probe 1 has a good photostability and response to mitochondrial GSH level in a ratiometric manner.
    660. 660
      Li, X.; Huo, F.; Yue, Y.; Zhang, Y.; Yin, C. A Coumarin-Based “Off-On” Sensor for Fluorescence Selectivily Discriminating GSH from Cys/Hcy and Its Bioimaging in Living Cells. Sens. Actuators B Chem. 2017, 253, 42– 9,  DOI: 10.1016/j.snb.2017.06.120
      663
      A coumarin-based "off-on" sensor for fluorescence selectivily discriminating GSH from Cys/Hcy and its bioimaging in living cells
      Li, Xiaoqi; Huo, Fangjun; Yue, Yongkang; Zhang, Yongbin; Yin, Caixia
      Sensors and Actuators, B: Chemical (2017), 253 (), 42-49CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      Glutathione (GSH) is the most abundant intracellular thiol with the concn. in the millimolar range. It has also been reported that GSH plays a crucial role in human pathologies, so it is meaningful to develop an effective probe for GSH. In this work, we report a coumarin-chlorine malononitrile "off-on" sensor for highly selective fluorescence detection of GSH over Cys/Hcy. The chlorine of the probe can be rapidly replaced by thiolates of biothiols through thiol-halogen nucleophilic substitution. The amino groups of Cys/Hcy but not GSH further replace the thiolate to form amino-substituted compd. Subsequently, an intramol. Michael addn. between the α,β-unsatd. malonitrile and thiol group may occur. However, GSH only forms thiol-substituted compd. The significantly different mechanisms enable the discrimination of GSH over Cys/Hcy through fluorescence response. Finally, fluorescence imaging of A549 cells indicated that probe 1 could be used for monitoring GSH in living cells. It would be a useful tool to understand the mechanisms of many related diseases.
    661. 661
      Long, L.; Lin, W.; Chen, B.; Gao, W.; Yuan, L. Construction of a FRET-Based Ratiometric Fluorescent Thiol Probe. Chem. Commun. 2011, 47 (3), 893– 5,  DOI: 10.1039/C0CC03806G
      664
      Construction of FRET-based ratiometric fluorescent thiol probe
      Long, Lingliang; Lin, Weiying; Chen, Bingbing; Gao, Wensha; Yuan, Lin
      Chemical Communications (Cambridge, United Kingdom) (2011), 47 (3), 893-895CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      We have rationally constructed a novel FRET-based ratiometric thiol probe suitable for ratiometric imaging in living cells based on the native chem. ligation reaction.
    662. 662
      Chen, H.; Tang, Y.; Ren, M.; Lin, W. Single near-Infrared Fluorescent Probe with High- and Low-Sensitivity Sites for Sensing Different Concentration Ranges of Biological Thiols with Distinct Modes of Fluorescence Signals. Chem. Sci. 2016, 7 (3), 1896– 903,  DOI: 10.1039/C5SC03591K
      665
      Single near-infrared fluorescent probe with high- and low-sensitivity sites for sensing different concentration ranges of biological thiols with distinct modes of fluorescence signals
      Chen, Hua; Tang, Yonghe; Ren, Mingguang; Lin, Weiying
      Chemical Science (2016), 7 (3), 1896-1903CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      We describe a unique approach for the development of an interesting type of the fluorescent probes, which can show different modes of fluorescence signals to distinct concn. ranges of a target of interest. The key points for the design of the new type of the fluorescent probes include the judicious selection of the dye platforms and the corresponding high- and low-sensitivity sites. It is known that the normal concns. of biol. thiols have significant biol. functions. However, up- or down-regulated concns. of thiols may induce several diseases. Therefore, it is highly important to monitor the changes of thiol concns. in living systems. Based on the proposed strategy, we engineer the novel NIR fluorescent probe, CHMC-thiol, which remarkably can display a turn-on signal to the low concn. range of thiols and a ratiometric response to the high concn. range of thiols for the first time. We anticipate that the intriguing strategy formulated herein will be widely useful for the development of concn. range-dependent fluorescent probes.
    663. 663
      Hall, H. K., Jr. Correlation of the Nucleophilic Reactivity of Aliphatic Amines. J. Org. Chem. 1964, 29 (12), 3539– 3544,  DOI: 10.1021/jo01035a024
      667
      Correlation of the nucleophilic reactivity of aliphatic amines
      Hall, H. K., Jr.
      Journal of Organic Chemistry (1964), 29 (12), 3539-44CODEN: JOCEAH; ISSN:0022-3263.
      Data for the reaction of 39 aliphatic amines, piperidines, and piperazines with 15 substrates (e.g. glycidol, CO2, ClCO2Et, 2,4(O2N)2C6H3Cl, EtBr) were correlated by the Swain-Scott equation (CA 48, 548i), but not by the Taft equation. The n-values of the amines were lowered by electron-attracting groups and by alkyl branching (F-strain).
    664. 664
      Inoue, N. Selective Detection of Carcinogenic Aromatic Diamines in Aqueous Solutions Using 4-(N-Chloroformylmethyl-N-Methylamino)-7-Nitro-2,1,3-Benzoxadiazole (NBD-COCl) by HPLC. Anal. Sci. 2017, 33 (12), 1375– 1380
      668
      Selective detection of carcinogenic aromatic diamines in aqueous solutions using 4-(N-chloroformylmethyl-N-methylamino)-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) by HPLC
      Inoue, Naoko
      Analytical Sciences (2017), 33 (12), 1375-1380CODEN: ANSCEN; ISSN:0910-6340. (Japan Society for Analytical Chemistry)
      Reaction conditions for the selective derivatization of three types of arom. diamines without clean-up in an aq. soln. using 4-(N-chloroformylmethyl-N-methylamino)-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) have been developed for simple, fast, and selective anal. by HPLC. The arom. amines were derivatized to give di-NBD-CO-arom. amines at 35°C for 5 min in a buffer soln. at pH 5 contg. a low acetonitrile concn. (0 - 400 ng/mL, n = 5, r >0.998). The retention times of these di-NBD-CO-arom. amines were <5 min. Sample solns. contg. arom. amines, phenols, and an aliph. amine were also prepd. Under the optimized reaction conditions (i.e., low acetonitrile content and acidic conditions), no derivatization of the phenols and the aliph. amine was obsd. These were related to the differences in the pKa values of the target substances, the org. solvent concn., and the reaction soln. pH. For the model system, a sulfuric acid-impregnated filter was spiked with the arom. amines, phenols, and aliph. amine prior to extn., derivatization, and measurement by HPLC. Only arom. amines were detected quant., with no other compds. being obsd.
    665. 665
      You, W. W.; Haugland, R. P.; Ryan, D. K.; Haugland, R. P. 3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde, a Reagent with Broad Dynamic Range for the Assay of Proteins and Lipoproteins in Solution. Anal. Biochem. 1997, 244 (2), 277,  DOI: 10.1006/abio.1996.9920
      669
      3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde, a reagent with broad dynamic range for the assay of proteins and lipoproteins in solution
      You, Wendy W.; Haugland, Rosaria P.; Ryan, Diane K.; Haughland, Richard P.
      Analytical Biochemistry (1997), 244 (2), 277-282CODEN: ANBCA2; ISSN:0003-2697. (Academic)
      This paper describes the application of 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA), a sensitive fluorogenic reagent for detection of amines, to the assay of proteins in soln. The sensitivity and dynamic range of CBQCA in the detn. of protein concn. is modulated by the no. of accessible amines present in the same limitations as other reagents used in fluorogenic assays based on fluorescent amine adducts such as those obtained with fluorescamine or o-phthaldialdehyde, or of other spectrophotometric methods, where the color development is detd. by the abundance in the protein of certain amino acids. However, in comparison to other fluorescence-based protein detection methods, CBQCA has proven to be an extremely sensitive reagent with a very broad, essentially linear dynamic range, capable of detecting from 10 ng to 150 μg of protein (in a 100- to 200-μL assay vol.). The CBQCA reagent also functions well in the presence of substances, such as lipids, known to interfere in many other protein detn. methods.
    666. 666
      Wu, J.; Chen, Z.; Dovichi, N. J Reaction Rate, Activation Energy, and Detection Limit for the Reaction of 5-Furoylquinoline-3-Carboxaldehyde with Neurotransmitters in Artificial Cerebrospinal Fluid. J. Chromatogr. B Biomed. Appl. 2000, 741 (1), 85,  DOI: 10.1016/S0378-4347(99)00540-X
      670
      Reaction rate, activation energy, and detection limit for the reaction of 5-furoylquinoline-3-carboxaldehyde with neurotransmitters in artificial cerebrospinal fluid
      Wu, J.; Chen, Z.; Dovichi, N. J.
      Journal of Chromatography B: Biomedical Sciences and Applications (2000), 741 (1), 85-88CODEN: JCBBEP; ISSN:0378-4347. (Elsevier Science B.V.)
      The fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ) has proven valuable in the anal. of proteins and small neurotransmitters. The authors monitored the reaction rate between this reagent and five neurotransmitters at 40° and 65°C in artificial cerebrospinal fluid. The reactions followed pseudo-first order kinetics. The activation energy for the reaction of FQ was 10.6, 10.7, 22.0, 31.4, and 34.4 kJ mol-1 for alanine, taurine, γ-aminobutyric acid (GABA), glutamine, and glutamic acid, resp. At 65°C, the reaction rate was quite similar for alanine, taurine, glutamine, and glutamic acid (1.8 × 10-3 s-1) but was twice as fast for GABA. A reaction time of nearly 1 h was required to quant. convert these neurotransmitters to their fluorescent products at 65°C. Detection limits for the labeled neurotransmitters were 10-9-10-8 M, which corresponded to 0.3-7 amol injected onto the capillary.
    667. 667
      Pinto, D.; Arriaga, E. A.; Schoenherr, R. M.; Chou, S. S.-H.; Dovichi, N. J. Kinetics and Apparent Activation Energy of the Reaction of the Fluorogenic Reagent 5-Furoylquinoline-3-Carboxaldehyde with Ovalbumin. J. Chromatogr. B 2003, 793 (1), 107,  DOI: 10.1016/S1570-0232(03)00368-4
      671
      Kinetics and apparent activation energy of the reaction of the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde with ovalbumin
      Pinto, Devanand; Arriaga, Edgar A.; Schoenherr, Regine M.; Chou, Shirley Shinn-Huey; Dovichi, Norman J.
      Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2003), 793 (1), 107-114CODEN: JCBAAI; ISSN:1570-0232. (Elsevier Science B.V.)
      Incomplete labeling of proteins by a derivatizing reagent usually results in the formation of a large no. of products, which can produce unacceptable band broadening during electrophoretic anal. In this paper, we report on the reaction of the fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ) with the lysine residues of ovalbumin. Mass spectrometry was first used to det. the distribution in the no. of labels attached to the protein. At room temp., 3.6±1.9 labels were attached after 30 min. The reaction rate and no. of labels increased at elevated temps. At 65°, 6±2.5 labels were attached after 5 min. The apparent activation energy for this reaction is estd. as 48±17 kJ/mol. Based on the mass spectrometry study, the labeling reaction was assumed to consist of two steps. In the first, the protein unfolds to make lysine residues accessible. In the second, the reagents react with the ε-amine of the lysine residues. To test this hypothesis, submicellar capillary electrophoresis and laser-induced fluorescence were used to characterize the reaction mixt. The apparent activation energy was measured for the labeling reaction; the apparent activation energy was 57±12 kJ/mol for reaction performed in the sepn. buffer. Denaturing agents were added to the reaction mixt. The addn. of 2 M thiourea with 6 M urea to the reaction resulted in a modest decrease in the apparent activation energy to 42±2 kJ/mol. The addn. of 2.5 M or higher concn. of ethanol decreased the apparent activation energy to 32±2 kJ/mol. We conclude that the apparent activation energy for protein labeling is dominated by denaturation of the protein, and that the addn. of suitable denaturing reagents can eliminate this contribution to the reaction chem.
    668. 668
      Arrell, M. S.; Kalman, F. Estimation of Protein Concentration at High Sensitivity Using SDS-Capillary Gel Electrophoresis-Laser Induced Fluorescence Detection with 3-(2-Furoyl)quinoline-2-Carboxaldehyde Protein Labeling. Electrophoresis 2016, 37 (22), 2913,  DOI: 10.1002/elps.201600246
      672
      Estimation of protein concentration at high sensitivity using SDS-capillary gel electrophoresis-laser induced fluorescence detection with 3-(2-furoyl)quinoline-2-carboxaldehyde protein labeling
      Arrell, Miriam S.; Kalman, Franka
      Electrophoresis (2016), 37 (22), 2913-2921CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)
      3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ) is a sensitive fluorogenic dye, used for derivatization of proteins for SDS-capillary gel electrophoresis with laser induced fluorescence detection (SDS-CGE-LIF) at silver staining sensitivity (ng/mL). FQ labels proteins at primary amines, found at lysines and N-termini, which vary in no. and accessibility for different proteins. This work investigates the accuracy of estn. of protein concn. with SDS-CGE-LIF in real biol. samples, where a different protein must be used as a std. 16 purified proteins varying in mol. wt., structure and sequence were labeled with FQ at const. mass concn. applying a commonly used procedure for SDS-CGE-LIF. The fluorescence of these proteins was measured using a spectrofluorometer and found to vary with a relative std. deviation of 36%. This compares favorably with other less sensitive methods for estn. of protein concn. such as SDS-CGE-UV and SDS-PAGE-Coomassie and is vastly superior to the equivalently sensitive silver stain. Investigation into the no. of labels bound with UHPLC-ESI-QTOF-MS revealed large variations in the labeling efficiency (percentage of labels to the no. of labeling sites given by the sequence) for different proteins (from 3-30%). This explains the observation that fluorescence per mol of protein was not proportional to the no. of lysines in the sequence. This article is protected by copyright. All rights reserved.
    669. 669
      Wu, D.; Liu, Y.; Zheng, F.; Rong, S.-Q.; Yang, T.; Zhao, Y.-K.; Yang, R.-W.; Zou, P.; Wang, G.-T. Detection of Organic Amines Using a Ratiometric Chromophoric Fluorescent Probe with a Significant Emission Shift. J. Chem. Res. 2020, 44, 475– 481,  DOI: 10.1177/1747519820902944
      673
      Detection of organic amines using a ratiometric chromophoric fluorescent probe with a significant emission shift
      Wu, Dan; Liu, Yi; Zheng, Fei; Rong, Shi-Qi; Yang, Tao; Zhao, Yan-Kun; Yang, Rui-Wu; Zou, Ping; Wang, Guang-Tu
      Journal of Chemical Research (2020), 44 (7-8), 475-481CODEN: JCROA4; ISSN:2047-6507. (Sage Publications Ltd.)
      Taking advantages of both the well-known α,β-unsatd. structure and the special nucleophilicity of org. amines toward its acceptor moieties, intramol. charge transfer as a signaling mechanism is used to design and synthesize a new ratiometric chromophoric fluorescent probe (BI-CA-ID) with large emission shifts toward org. amines. This probe is employed for the detection of org. amines with high selectivity and sensitivity and a "naked-eye" color change (from red to colorless). UV-visible and fluorescence spectrometric measurements are used to det. detection limits as low as 0.024 and 0.43 μM. Furthermore, nucleophilic addn. of the amine on the α,β-unsatd. of BI-CA-ID indicated that the sensing mechanism occurs via interruption of the π-conjugation.
    670. 670
      Zhang, J.; Zhou, J.; Xu, G.; Ni, Y. Stereodivergent Evolution of KpADH for the Asymmetric Reduction of Diaryl Ketones with Para-Substituents. Mol. Catal. 2022, 524, 112315,  DOI: 10.1016/j.mcat.2022.112315
      674
      Stereodivergent evolution of KpADH for the asymmetric reduction of diaryl ketones with para-substituents
      Zhang, Jiacheng; Zhou, Jieyu; Xu, Guochao; Ni, Ye
      Molecular Catalysis (2022), 524 (), 112315CODEN: MCOADH ISSN:. (Elsevier B.V.)
      KpADH is a promising biocatalyst for the synthesis of chiral bulky-bulky alcs. Substrate specificity toward diaryl ketone substrates with different substituents at Ph group was investigated. The results reveal that the para-position of diaryl ketones plays important role in manipulating the stereoselectivity of KpADH. Two vital residues, E214 and T215, were identified through single-point mutagenesis, and satn. mutagenesis was performed to evaluate their contributions to the stereoselectivity. Furthermore, stereodivergent evolution of KpADH was achieved through combinatorial mutagenesis. Among them, E214I/T215S/S237A (ISA) and F161V/S196G/E214G (VGG) exhibited e.e. values of >99% (S) and 99% (R) toward [4-(trifluoromethyl)phenyl]-2-pyridinylmethanone (5a), and e.e. values of >99% (S) and 98% (R) toward (4-methylphenyl)-2-pyridinylmethanone (6a), resp. Kinetic characterization and interaction anal. further prove that the eliminated side-chain collision and enhanced electrostatic interactions are mainly responsible for the increased catalytic efficiency and stereoselectivity. This study provides beneficial mutants with complementary stereoselectivity toward diaryl ketones with para-substituents and also provides guidance for the engineering of homologous alc. dehydrogenases toward bulky-bulky ketones.
    671. 671
      Ribeaucourt, D.; Bissaro, B.; Lambert, F.; Lafond, M.; Berrin, J.-G. Biocatalytic Oxidation of Fatty Alcohols into Aldehydes for the Flavors and Fragrances Industry. Biotechnol. Adv. 2022, 56, 107787,  DOI: 10.1016/j.biotechadv.2021.107787
      675
      Biocatalytic oxidation of fatty alcohols into aldehydes for the flavors and fragrances industry
      Ribeaucourt, David; Bissaro, Bastien; Lambert, Fanny; Lafond, Mickael; Berrin, Jean-Guy
      Biotechnology Advances (2022), 56 (), 107787CODEN: BIADDD; ISSN:0734-9750. (Elsevier Inc.)
      A review. From Egyptian mummies to the Chanel n°5 perfume, fatty aldehydes have long been used and keep impacting our senses in a wide range of foods, beverages and perfumes. Natural sources of fatty aldehydes are threatened by qual. and quant. variability while traditional chem. routes are insufficient to answer the society shift toward more sustainable and natural products. The prodn. of fatty aldehydes using biotechnologies is therefore the most promising alternative for the flavors and fragrances industry. In this review, after drawing the portrait of the origin and characteristics of fragrant fatty aldehydes, we present the three main classes of enzymes that catalyze the reaction of fatty alcs. oxidn. into aldehydes, namely alc. dehydrogenases, flavin-dependent alc. oxidases and copper radical alc. oxidases. The constraints, challenges and opportunities to implement these oxidative enzymes in the flavors and fragrances industry are then discussed. By setting the scene on the biocatalytic prodn. of fatty aldehydes, and providing a crit. assessment of its potential, we expect this review to contribute to the development of biotechnol.-based solns. in the flavors and fragrances industry.
    672. 672
      O'Brien, P. J.; Siraki, A. G.; Shangari, N. Aldehyde Sources, Metabolism, Molecular Toxicity Mechanisms, and Possible Effects on Human Health. Crit. Rev. Toxicol. 2005, 35 (7), 609– 662,  DOI: 10.1080/10408440591002183
      676
      Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health
      O'Brien, Peter; Siraki, Arno; Shangari, Nandita
      Critical Reviews in Toxicology (2005), 35 (7), 609-662CODEN: CRTXB2; ISSN:1040-8444. (Taylor & Francis, Inc.)
      A review. Aldehydes are org. compds. that are widespread in nature. They can be formed endogenously by lipid peroxidn. (LPO), carbohydrate or metab. ascorbate autoxidn., amine oxidases, cytochrome P-450s, or myeloperoxidase-catalyzed metabolic activation. This review compares the reactivity of many aldehydes towards biomols. particularly macromols. Furthermore, it includes not only aldehydes of environmental or occupational concerns but also dietary aldehydes and aldehydes formed endogenously by intermediary metab. Drugs that are aldehydes or form reactive aldehyde metabolites that cause side-effect toxicity are also included. The effects of these aldehydes on biol. function, their contribution to human diseases, and the role of nucleic acid and protein carbonylation/oxidn. in mutagenicity and cytotoxicity mechanisms, resp., as well as carbonyl signal transduction and gene expression, are reviewed. Aldehyde metabolic activation and detoxication by metabolizing enzymes are also reviewed, as well as the toxicol. and anticancer therapeutic effects of metabolizing enzyme inhibitors. The human health risks from clin. and animal research studies are reviewed, including aldehydes as haptens in allergenic hypersensitivity diseases, respiratory allergies, and idiosyncratic drug toxicity; the potential carcinogenic risks of the carbonyl body burden; and the toxic effects of aldehydes in liver disease, embryo toxicity/teratogenicity, diabetes/hypertension, sclerosing peritonitis, cerebral ischemia/neurodegenerative diseases, and other aging-assocd. diseases.
    673. 673
      Corley, R. A.; Kabilan, S.; Kuprat, A. P.; Carson, J. P.; Jacob, R. E.; Minard, K. R.; Teeguarden, J. G.; Timchalk, C.; Pipavath, S.; Glenny, R.; Einstein, D. R. Comparative Risks of Aldehyde Constituents in Cigarette Smoke Using Transient Computational Fluid Dynamics/Physiologically Based Pharmacokinetic Models of the Rat and Human Respiratory Tracts. Toxicol. Sci. 2015, 146 (1), 65– 88,  DOI: 10.1093/toxsci/kfv071
      677
      Comparative risks of aldehyde constituents in cigarette smoke using transient computational fluid dynamics/physiologically based pharmacokinetic models of the rat and human respiratory tracts
      Corley, Richard A.; Kabilan, Senthil; Kuprat, Andrew P.; Carson, James P.; Jacob, Richard E.; Minard, Kevin R.; Teeguarden, Justin G.; Timchalk, Charles; Pipavath, Sudhakar; Glenny, Robb; Einstein, Daniel R.
      Toxicological Sciences (2015), 146 (1), 65-88CODEN: TOSCF2; ISSN:1096-0929. (Oxford University Press)
      Computational fluid dynamics (CFD) modeling is well suited for addressing species-specific anatomy and physiol. in calcg. respiratory tissue exposures to inhaled materials. In this study, we overcame prior CFD model limitations to demonstrate the importance of realistic, transient breathing patterns for predicting site-specific tissue dose. Specifically, extended airway CFD models of the rat and human were coupled with airway region-specific physiol. based pharmacokinetic (PBPK) tissue models to describe the kinetics of 3 reactive constituents of cigarette smoke: acrolein, acetaldehyde and formaldehyde. Simulations of aldehyde no-obsd.-adverse-effect levels for nasal toxicity in the rat were conducted until breath-by-breath tissue concn. profiles reached steady state. Human oral breathing simulations were conducted using representative aldehyde yields from cigarette smoke, measured puff ventilation profiles and nos. of cigarettes smoked per day. As with prior steady-state CFD/PBPK simulations, the anterior respiratory nasal epithelial tissues received the greatest initial uptake rates for each aldehyde in the rat. However, integrated time- and tissue depth-dependent area under the curve (AUC) concns. were typically greater in the anterior dorsal olfactory epithelium using the more realistic transient breathing profiles. For human simulations, oral and laryngeal tissues received the highest local tissue dose with greater penetration to pulmonary tissues than predicted in the rat. Based upon lifetime av. daily dose comparisons of tissue hot-spot AUCs (top 2.5% of surface area-normalized AUCs in each region) and nos. of cigarettes smoked/day, the order of concern for human exposures was acrolein > formaldehyde > acetaldehyde even though acetaldehyde yields were 10-fold greater than formaldehyde and acrolein.
    674. 674
      Ahmed Laskar, A.; Younus, H. Aldehyde Toxicity and Metabolism: The Role of Aldehyde Dehydrogenases in Detoxification, Drug Resistance and Carcinogenesis. Drug Metab. Rev. 2019, 51 (1), 42– 64,  DOI: 10.1080/03602532.2018.1555587
      678
      Aldehyde toxicity and metabolism: the role of aldehyde dehydrogenases in detoxification, drug resistance and carcinogenesis
      Ahmed Laskar, Amaj; Younus, Hina
      Drug Metabolism Reviews (2019), 51 (1), 42-64CODEN: DMTRAR; ISSN:0360-2532. (Taylor & Francis Ltd.)
      A review. Aldehydes are carbonyl compds. found ubiquitously in the environment, derived from both natural and anthropogenic sources. As the aldehydes are reactive species, therefore, they are generally toxic to the body. To reduce the toxicity and pathogenesis related to aldehydes, the human body contains several aldehyde metabolizing enzyme systems including aldehyde oxidases, cytochrome P 450 enzymes, aldo-ketoreductases, alc. dehydrogenases, short-chain dehydrogenases/reductases and aldehyde dehydrogenases (ALDHs). These enzyme systems maintain a low level of aldehydes in the body by catalytically converting them into less-harmful and easily excreted products. The human ALDH (hALDH) superfamily consists of 20 functional ALDH genes identified so far at distinct chromosomal locations, expressing 20 ALDH proteins, which belong to 11 different ALDH families. They are involved in the NAD(P)+-dependent oxidn. of a wide range of exogenous and endogenous aldehydes to their corresponding carboxylic acids. The hALDHs are present in all sub-cellular locations and have a wide tissue distribution. This review gives an account of aldehydes; their source, toxicity and metab., different aldehyde metabolizing enzymes with special emphasis on ALDHs including their biochem., physiol. and pathophysiol. roles in the body.
    675. 675
      Fritz, K. S.; Petersen, D. R. An Overview of the Chemistry and Biology of Reactive Aldehydes. Free Radical Biol. Med. 2013, 59, 85– 91,  DOI: 10.1016/j.freeradbiomed.2012.06.025
      679
      An overview of the chemistry and biology of reactive aldehydes
      Fritz, Kristofer S.; Petersen, Dennis R.
      Free Radical Biology & Medicine (2013), 59 (), 85-91CODEN: FRBMEH; ISSN:0891-5849. (Elsevier B.V.)
      A review. The nonenzymic free radical generation of reactive aldehydes is known to contribute to diseases of sustained oxidative stress including rheumatoid arthritis, atherosclerosis, neurodegeneration, and a no. of liver diseases. At the same time, the accumulation of lipid electrophiles has been demonstrated to play a role in cell signaling events through modification of proteins crit. for cellular homeostasis. Given the broad scope of reactivity profiles and the ability to modify numerous proteomic and genomic processes, new emphasis is being placed on a systems-based anal. of the consequences of electrophilic adduction. This review focuses on the generation and chem. reactivity of lipid-derived aldehydes with a special focus on the homeostatic responses to electrophilic stress.
    676. 676
      Urbansky, E. T. Carbinolamines and Geminal Diols in Aqueous Environmental Organic Chemistry. J. Chem. Ed. 2000, 77 (12), 1644,  DOI: 10.1021/ed077p1644
      680
      Carbinolamines and geminal diols in aqueous environmental organic chemistry
      Urbansky, Edward T.
      Journal of Chemical Education (2000), 77 (12), 1644-1647CODEN: JCEDA8; ISSN:0021-9584. (Division of Chemical Education of the American Chemical Society)
      A review with 77 refs. is presented regarding carbinolamines and geminal diols in aq. environmental org. chem. Any aq. process whereby an aldehyde or ketone undergoes nucleophilic substitution can be expected to have some effects from the stability of carbinolamines and geminal diols imparted by the water. A significant body of literature exists on the role of these compds. in aq. org. chem. Nevertheless, carbinolamines are still commonly regarded as short-lived intermediates and geminal diols as exceptions by sophomore org. chem. textbooks, and thus, students come to view them that way. However, in environmental applications these species are ubiquitous and may dominate the observable chem. Even in the field of drinking water chem., these species have been largely ignored. No effort has been made to exploit carbinolamine reaction kinetics or imine formation in anal. chem., such as adding arylamines as catalysts or adjusting pH. Such modifications could lead to improved anal. methods, with advantages in convenience or lower limits of detection and are worthy of exploration.
    677. 677
      Mukherjee, K.; Chio, T. I.; Gu, H.; Banerjee, A.; Sorrentino, A. M.; Sackett, D. L.; Bane, S. L. Benzocoumarin Hydrazine: A Large Stokes Shift Fluorogenic Sensor for Detecting Carbonyls in Isolated Biomolecules and in Live Cells. ACS Sens. 2017, 2 (1), 128– 134,  DOI: 10.1021/acssensors.6b00616
      681
      Benzocoumarin Hydrazine: A Large Stokes Shift Fluorogenic Sensor for Detecting Carbonyls in Isolated Biomolecules and in Live Cells
      Mukherjee, Kamalika; Chio, Tak Ian; Gu, Han; Banerjee, Abhijit; Sorrentino, Anthony M.; Sackett, Dan L.; Bane, Susan L.
      ACS Sensors (2017), 2 (1), 128-134CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      Detection and quantification of biomol. carbonylation, a crit. manifestation of oxidative stress, allows better understanding of assocd. disease states. Existing approaches for such analyses require further processing of cells and tissues, which leads to loss of both spatial and temporal information about carbonylated biomols. in cells. Live cell detection of these species requires sensors that are non-toxic, sufficiently reactive with the biocarbonyl in the intracellular milieu, and detectable with commonly available instrumentation. Presented here is a new fluorescent sensor for biomol. carbonyl detection: a hydrazine deriv. of a benzocoumarin, 7-hydrazinyl-4-methyl-2H-benzo[h]chromen-2-one (BzCH), which meets these requirements. This probe is esp. well suited for live cell studies. It can be excited by a laser line common to many fluorescence microscopes. The emission max. of BzCH undergoes a substantial red shift upon hydrazone formation (from ∼430 to ∼550 nm), which is the result of fluorophore disaggregation. Addnl., the hydrazone exhibits an exceptionally large Stokes shift (∼195 nm). The latter properties eliminate self-quenching of the probe and the need to remove unreacted fluorophore for reliable carbonyl detection. Thus, biomol. carbonylation can be detected and quantified in cells and in cell exts. in a one-step procedure using this probe.
    678. 678
      Molina-Espeja, P.; Canellas, M.; Plou, F. J.; Hofrichter, M.; Lucas, F.; Guallar, V.; Alcalde, M. Synthesis of 1-Naphthol by a Natural Peroxygenase Engineered by Directed Evolution. ChemBioChem 2016, 17 (4), 341– 349,  DOI: 10.1002/cbic.201500493
      682
      Synthesis of 1-naphththol by a natural peroxygenase engineered by directed evolution
      Molina-Espeja, Patricia; Canellas, Marina; Plou, Francisco J.; Hofrichter, Martin; Lucas, Fatima; Guallar, Victor; Alcalde, Miguel
      ChemBioChem (2016), 17 (4), 341-349CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      There is an increasing interest in enzymes that catalyze the hydroxylation of naphthalene under mild conditions and with minimal requirements. To address this challenge, an extracellular mushroom (Agrocybe aegerita) arom. peroxygenase with mono(per)oxygenase activity was engineered to convert naphthalene selectively into 1-naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene together with quenching of the undesired peroxidative activity on 1-naphthol (one-electron oxidn.). The resulting double mutant (G241D/R257K) obtained from this process was characterized biochem. and computationally. The conformational changes produced by directed evolution improved the substrate's catalytic position. Powered exclusively by catalytic concns. of H2O2, this sol. and stable biocatalyst had a total turnover no. of 50,000, with high regioselectivity (97%) and reduced peroxidative activity.
    679. 679
      Guo, H.-M.; Minakawa, M.; Tanaka, F. Fluorogenic Imines for Fluorescent Detection of Mannich-Type Reactions of Phenols in Water. J. Org. Chem. 2008, 73 (10), 3964– 3966,  DOI: 10.1021/jo8003293
      683
      Fluorogenic Imines for Fluorescent Detection of Mannich-Type Reactions of Phenols in Water
      Guo, Hai-Ming; Minakawa, Maki; Tanaka, Fujie
      Journal of Organic Chemistry (2008), 73 (10), 3964-3966CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      Fluorogenic imines and their precursor amines that can be used for fluorescent visualization of Mannich-type reactions of phenols in aq. buffers have been developed. The precursor amines are aniline derivs. that are covalently conjugated to fluorophores. These amines and their imine derivs. were nonfluorescent or very weakly fluorescent. On the other hand, addn. products of the imines to phenols sh wed more than 100-fold higher fluorescence than the imines and the precursor amines.