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Solid-Phase Synthesis of Caged Luminescent Peptides via Side Chain Anchoring
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  • Daan Sondag
    Daan Sondag
    Institute for Molecules and Materials, Radboud University, Nijmegen 6525 AJ, The Netherlands
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  • Jurriaan J. A. Heming
    Jurriaan J. A. Heming
    Institute for Molecules and Materials, Radboud University, Nijmegen 6525 AJ, The Netherlands
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  • Dennis W. P. M. Löwik
    Dennis W. P. M. Löwik
    Institute for Molecules and Materials, Radboud University, Nijmegen 6525 AJ, The Netherlands
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    Orcidhttps://orcid.org/0000-0002-9284-837X
  • Elena Krivosheeva
    Elena Krivosheeva
    Enzyre BV, Novio Tech Campus, Transistorweg 5-i, Nijmegen 6534 AT, The Netherlands
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    Orcidhttps://orcid.org/0000-0001-8499-4409
  • Denise Lejeune
    Denise Lejeune
    Institute for Molecules and Materials, Radboud University, Nijmegen 6525 AJ, The Netherlands
    Enzyre BV, Novio Tech Campus, Transistorweg 5-i, Nijmegen 6534 AT, The Netherlands
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  • Mark van Geffen
    Mark van Geffen
    Enzyre BV, Novio Tech Campus, Transistorweg 5-i, Nijmegen 6534 AT, The Netherlands
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    Orcidhttps://orcid.org/0000-0003-1748-0007
  • Cornelis van’t Veer
    Cornelis van’t Veer
    Enzyre BV, Novio Tech Campus, Transistorweg 5-i, Nijmegen 6534 AT, The Netherlands
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  • Waander L. van Heerde
    Waander L. van Heerde
    Enzyre BV, Novio Tech Campus, Transistorweg 5-i, Nijmegen 6534 AT, The Netherlands
    Department of Haematology, Radboud University Medical Centre, Nijmegen 6525 GA, The Netherlands
    Haemophilia Treatment Centre, Nijmegen Eindhoven Maastricht (HTC-NEM), Nijmegen 6525 GA, The Netherlands
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  • Marjolijn C. J. Beens
    Marjolijn C. J. Beens
    Symeres BV, Kerkenbos 1013, Nijmegen 6546 BB, The Netherlands
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    Brian H. M. Kuijpers
    Symeres BV, Kerkenbos 1013, Nijmegen 6546 BB, The Netherlands
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    Thomas J. Boltje
    Institute for Molecules and Materials, Radboud University, Nijmegen 6525 AJ, The Netherlands
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    Orcidhttps://orcid.org/0000-0001-9141-8784
  • Floris P. J. T. Rutjes*
    Floris P. J. T. Rutjes
    Institute for Molecules and Materials, Radboud University, Nijmegen 6525 AJ, The Netherlands
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0003-1538-3852
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Bioconjugate Chemistry

Cite this: Bioconjugate Chem. 2023, 34, 12, 2234–2242
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https://doi.org/10.1021/acs.bioconjchem.3c00381
Published December 6, 2023

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Abstract

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The synthesis of caged luminescent peptide substrates remains challenging, especially when libraries of the substrates are required. Most currently available synthetic methods rely on a solution-phase approach, which is less suited for parallel synthesis purposes. We herein present a solid-phase peptide synthesis (SPPS) method for the synthesis of caged aminoluciferin peptides via side chain anchoring of the P1 residue. After the synthesis of a preliminary test library consisting of 40 compounds, the synthetic method was validated and optimized for up to >100 g of resin. Subsequently, two separate larger peptide libraries were synthesized either having a P1 = lysine or arginine residue containing in total 719 novel peptide substrates. The use of a more stable caged nitrile precursor instead of caged aminoluciferin rendered our parallel synthetic approach completely suitable for SPPS and serine protease profiling was demonstrated using late-stage aminoluciferin generation.

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Introduction

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In recent decades, luminescent imaging has emerged as a powerful tool to monitor enzymatic activity. Luminescent imaging generally occurs via the enzymatic oxidation of a small molecule, without any external light source, and proceeds with a high signal-to-noise ratio as the background signal is generally lower as compared to fluorescence imaging. (1) Numerous applications have been reported for luminescent imaging, including sensing reactive oxygen and nitrogen species, imaging of cancer cells, and quantifying in vivo glucose uptake. (2−4) White and co-workers were among the first to report the use of d-luciferin derivative aminoluciferin (aLuc) in proteolytic assays in which the amino function is coupled to the C-terminus of a peptide. (5,6) The free 6′-amino group of aLuc appeared crucial to preserve its luminescent properties (similar to the 6′-hydroxy in d-luciferin), while modification of the amine, such as coupling to a carboxylic acid to form an amide, usually results in quenched luminescence. (7) These quenched luminescent probes have also been referred to as “caged” luciferins and can be used in luminescent enzymatic assays, as upon enzymatic activity the free aLuc is released which can readily be quantified (Scheme 1A). (7−9) After proteolytic activity, the AMP adduct of luciferin is generated upon the action of ATP and Mg2+. The aLuc is subsequently oxidized to the excited oxyluciferin by the catalytic action of luciferase, and consecutive relaxation back into the ground state causes the release of photons. Both keto- and enol-intermediates are thought to be the actual light-generating intermediates in the enzyme pocket, the reaction mechanism found in fireflies is depicted in Scheme 1B. (1) Numerous luminescent probes have been developed according to this uncaging principle and are widely used in combination with a large variety of enzymes among which hydrolases and proteases. (10−13)

Scheme 1

Scheme 1. (A) General Reaction Scheme for an Aminoluciferin (aLuc)-Based Assay; upon Enzymatic Activity, d-Aminoluciferin Is Released from the Caged Substrate, Which Can Subsequently Be Converted into a Proportional Light Signal in the Presence of Luciferase, ATP, Mg2+, and O2 and (B) Luciferase-Catalyzed Luminescence Reaction Mechanism as Found in Fireflies
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In order to extend the applications of luminescence imaging, new caged luciferins are required. Current methods for the preparation of luminescent caged peptides mostly rely on the use of solution-phase synthesis. Generally, a protected peptide is C-terminally activated with reagents such as isobutyl chloroformate (IBCF) in the presence of a base, e.g., N-methylmorpholine (NMM), prior to the addition of the aLuc precursor 6-aminobenzo[d]thiazole-2-carbonitrile (6-ABTC, Figure 1A). (14−16) After the coupling, the side chain protecting groups of the peptide can be cleaved off, and a final condensation reaction with d-cysteine yields the caged luciferin peptide. The use of solid-phase peptide synthesis (SPPS) to construct the caged luciferins would drastically lower the number of steps in solution-phase, eliminate multiple purification steps, and especially allow for a parallel automated workflow. (17) Preliminary work by Kovacs et al. revealed that loading of the carboxylic acid moiety of aLuc onto a solid support and subsequent elongation of the peptide chain was unsuccessful, due to the inherent instability of the thiazoline moiety of aLuc (Figure 1B). (18) The thiazoline ring is prone to oxidation resulting in the corresponding thiazole derivative, a well-known luciferase inhibitor. (19,20)

Figure 1

Figure 1. (A) Conventional solution-phase method for the synthesis of aminoluciferin peptides via C-terminal activation of a protected peptide and the subsequent side chain deprotection and d-cysteine condensation all in solution. (B) Hybrid SPPS method reported by Kovacs et al., loading of the aminoluciferin on a solid support afforded the dehydrogenated product. (C) This work with the side chain anchoring of the P1 amino acid residue on a solid support and the subsequent regular Fmoc-based SPPS chemistry.

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Herein, we present an SPPS method for the synthesis of caged luminescent peptides starting with the side chain anchoring of either a lysine or an ornithine P1 residue (Figure 1C). The ornithine moiety allows for on-resin guanidinylation into the corresponding arginine residue. The introduction of the aLuc moiety after resin cleavage renders our approach compatible with SPPS, especially since the relatively unreactive 6-ABTC moiety is introduced at the P1 C-terminus prior to the solid-phase reactions. We chose luminescent caged peptide substrates bearing an arginine or lysine residue on the P1 position, as the targeted trypsin-like serine proteases prefer positively charged P1 residues. (21,22) With the SPPS method, we aim to accelerate the development of novel anticoagulation therapies and the design of new activity-based probes for various serine proteases.

Results and Discussion

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In order to develop a robust method for the synthesis of luminescent-labeled peptides via SPPS, the stable 6-ABTC (1) aLuc precursor was used throughout the solid-phase synthesis procedure. We were inspired by the side chain anchoring approaches toward caged 7-amino-4-methylcoumarin (AMC) fluorescent peptide probes by Beythien et al. and Hamzé et al., both using an arginine residue at P1. (23,24) The synthesis of C-terminally modified peptides via P1 side chain anchoring has been addressed as demonstrated in numerous literature accounts. (25−28) We chose to address the inherent unreactive character of the amino group of 6-ABTC (1) by coupling to the P1 residue in solution, prior to the solid-phase steps (Scheme 2). Thus, 1 was synthesized on a multigram scale starting from 2-chlorobenzo[d]thiazole according to a known literature procedure. (29) Initial coupling reactions of either Fmoc-Orn(Boc)–OH (2) or Fmoc-Lys(Boc)–OH (3) with 1 under standard peptide coupling conditions (HOBt, DIC/HATU, DIPEA) or C-terminal activation with IBCF and NMM all appeared to be low yields with significant amounts of byproducts, presumably because the acid needs to be highly activated to allow aniline coupling. However, when switching to preactivation of 1 with phosphorus trichloride in pyridine, prior to the addition of 2 or 3, high yields of amino acids 4 and 5 were obtained (Scheme 2). (30,31) The Boc side chain protecting groups were subsequently removed with formic acid, and the residues were lyophilized to obtain the C-terminal functionalized ornithine and lysine building blocks 6 and 7, respectively, with the free side chain amine available for further conjugation.

Scheme 2

Scheme 2. Synthesis of the Luminescent Precursor Amino Acids 6 and 7a
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a(i) 6-ABTC (1, 1 equiv), PCl3 (0.51 equiv), pyr, 3.5 h, 40 °C, 77–98%; (ii) HCO2H, lyophilization, quant.

The building blocks 6 and 7 then had to be attached to an aldehyde-functionalized solid support via reductive amination. (32) Since the nitrile moiety of 1 can be prone to (acidic) hydrolysis, we chose to use the highly acid labile (3-formylindolyl)acetamido methyl polystyrene resin (R1, 100–200 mesh). (23,24) Reductive amination of either 6 or 7 with the aldehyde-functionalized resin was carried out with NaBH3CN in a 1:1 mixture of tetrahydrofuran and trimethyl orthoformate (TMOF) as a water scavenger for 4 h at room temperature (Scheme 3). This afforded the anchored ornithine and lysine resins R2 and R3, respectively. Next, the secondary bound amines R2 and R3 were further functionalized before proceeding with N-terminal peptide elongation. The lysine derivatives (R3, n = 4) were Boc protected using di-tert-butyl dicarbonate and DIPEA in DMF while agitating overnight to afford R5. In order to obtain the arginine derivatives from the ornithine-bound resin, we aimed to guanidinylate the immobilized secondary amine. In our validation library, we employed two equivalents of N,N′-di-Boc-thiourea (8) in DCM overnight with N,N′-diisopropylcarbodiimide (DIC) as desulfurization reagent, which afforded resin R4. (33) DIC was used instead of more commonly used HgCl2, which however is not compatible with SPPS due to the formation of insoluble HgS. Knowing that MeOCH2C(O)-β-Ala-Gly-Arg-aLuc is an excellent substrate for trypsin-like serine proteases such as thrombin, we chose to design our validation library accordingly by synthesizing tripeptides using P2 residues the naturally occurring amino acids in order to study the P2 dependence. (34,35)

Scheme 3

Scheme 3. Validation Solid-Phase Peptide Synthesis Library of Luminescent Caged Peptides via Side Chain Anchoring of Amino Acids 6 or 7 via Reductive Amination with R1 ((3-Formylindolyl)acetamido Methyl Resin)a
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a(i) 6 or 7 (2 equiv), THF/TMOF (1:1), 4 h, rt. (ii) NaBH3CN (2 equiv), AcOH (3.5 equiv), 2 h, rt. (iii) For n = 3: 8 (2 equiv), DIC (2 equiv), DCM, rt, 16 h. For n = 4: Boc2O (3 equiv), DIPEA (3 equiv), DMF, rt, 16 h. (iv) (a) 3% DBU in DMF, DMF (3×); (b) HOBt (3.6 equiv), DIC (3.3 equiv), Fmoc-AA–OH/Cap–OH (methoxy acetic acid) (3 equiv) (3×). (v) DMF. (v) TFA/DCM (1:1, v/v), 2 h

To this extent, the peptide chains of the R4 and R5 resins were elongated via parallel Fmoc SPPS chemistry. The Fmoc deprotections were executed with 3% DBU in DMF, as these conditions in our experience gave considerably better results than the use of 20% piperidine in DMF. The subsequent coupling reactions with the Fmoc-protected amino acids and the N-terminal cap (3 equiv) were conducted using standard coupling conditions (3.6 equiv of HOBt and 3.3 equiv of DIC in DMF). This afforded the functionalized resins R6 and R7, respectively. The peptides were then cleaved from the resin with trifluoroacetic acid (TFA) in DCM (1:1, v/v, addition of 2.5% TIS and EDT for Trt-containing sequences), lyophilized, and purified with RP-HPLC in order to obtain pure 6-ABTC-conjugated peptides, which were all analyzed by ESI–MS and NMR (typical scale of 100 mg unfunctionalized resin per substrate gave 2–5 mg of purified product, corresponding to 5–12% isolated yield). Thus, a tripeptide library consisting of 40 peptides MeOCH2C(O)-β-Ala-P2-Arg/Lys-aLuc was obtained, with P2 being any of the 20 naturally occurring amino acids (Scheme 3).
However, we often obtained a significant amount of thiourea byproduct R8 when using the N,N′-di-Boc-thiourea (8) method instead of the desired protected resin-bound guanidine R4 (Scheme 4). Initial attempts to shorten the guanidinylation reaction time or add fresh reagents did not overcome the formation of the byproduct R8. Neither the use of different activators such as EDC, copper(I), or copper(II) chloride lowered the formation of thiourea R8. (36,37) Different guanidinylation reagents such as N,N′-di-Boc-S-methylisothiourea, 1-[N,N′-(di-Boc)amidino]pyrazole, Goodman’s reagent or cyanuric chloride (TCT) were also evaluated but gave rise to an even lower level of guanidinylation as analyzed by LCMS analysis directly after resin cleavage (data not shown). (38−40) Eventually, we tested the benzotriazole-based reagent 9, as described by Moroder and Musiol in 2001. (41) This reagent could after optimization easily be synthesized in our hands-on multigram scale (as a mixture of the 5′ and 6′-isomers) without the use of toxic HgCl2 which was originally used by the authors. Reagent 9 revealed complete conversion of R2 into R4 and fewer byproducts (in particular R8) were observed compared to the method with reagent 8 and DIC activation.

Scheme 4

Scheme 4. Conversion of Resin-Bound Secondary Amine R2 into the Di-Boc-Protected Guanidine R4 and the Observed Thiourea Byproduct R8 When Using N,N′-Di-Boc-thiourea (8) and DIC Activationa
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aThe use of 9 solely afforded the desired resin-bound product R4.

With the validation library in hand and having optimized the guanidinylation reaction, we continued to assess our method for the synthesis of two larger peptide libraries as proof of our synthetic principle. We aimed to validate our newly developed SPPS method by parallel synthesizing two separate tripeptide libraries with alteration of the P2 and P3 residues for all-natural available amino acids. Multiple batches of ornithine- and lysine-functionalized building blocks 6 and 7 were synthesized and coupled to the (3-formylindolyl)acetamidomethyl resin (R1) via reductive amination to afford R2 and R3. After the reductive amination, the lysine-anchored resin was again temporarily protected with a Boc group, and the ornithine resin was guanidinylated with the benzotriazole-based reagent 9 to afford the key intermediate arginine- or lysine-anchored resins R4 and R5, respectively. The peptide chains were subsequently elongated similar to those for the validation libraries via regular Fmoc chemistry and with the N-termini capped (see the Supporting Information). The cleaved products were purified with RP-HPLC to obtain two libraries with all available natural amino acids (except cysteine) at P2 and P3 thereby providing ultimate proof of this synthetic concept (see: Scheme 5). A total of 719 substrates were obtained via this route, which were all analyzed by LCMS (lysine library average purity: 83%, arginine library: 63%). The purity of the arginine substrate library was generally slightly lower due to the smaller amount of material available (0.03 mmol per substrate) to synthesize all of the target substrates, which sometimes made the purification more challenging as compared to the lysine substrates (0.05 mmol per substrate). Additionally, for the lysine library, it appeared that the RP-HPLC purification was most successful by using a trifluoroacetic acid (TFA)-acidified eluent instead of formic acid (0.1%). The lysine products that were initially purified with the formic acid method revealed some extent of oligomerization after several days, which was not observed for the TFA salts of the purified products. Presumably, due to the weaker acid (pKa HCO2H = 3.7 as compared to pKa TFA = 0.2), the lower extent of protonation of the amine causes the lysine side chain to react with cyanide groups thereby leading to oligomers or due to the volatility of the counterion.

Scheme 5

Scheme 5. General Structures of the Two Large Tripeptide Libraries That Were Synthesized in This Work Starting from the Key Intermediate Resins R4 and R5a
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aP3 = natural amino acid, P2 = natural amino acid and P1 = either arginine (n = 3) or lysine (n = 4). After regular Fmoc SPPS chemistry and subsequent RP-HPLC purification after cleavage, a total amount of 719 peptides was obtained.

Using this approach, we obtained two large tripeptide libraries. These large numbers of compounds directly demonstrate the scalability and applicability of our novel parallel SPPS synthesis methodology and prove that our unique method is highly suitable for automated workflow.
In our experience, the 6-ABTC-functionalized peptides appeared to be significantly more stable than the corresponding aLuc peptides. Hence, we reasoned that the active aLuc substrates could be generated from the nitrile precursors with the selective d-cysteine condensation reaction in the final stage. Since the reaction of d-cysteine and 6-ABTC has extensively been described in the literature and rapidly yields the aLuc functionalized derivative, we hypothesized this reaction could be performed in situ. (42−45)
To provide a preliminary example, we ran our validation peptide library (40 substrates) against the two trypsin-like serine proteases Factor Xa (FXa) and thrombin (FXIIa) in order to demonstrate the protease profiling potential. First, we incubated the 6-ABTC-conjugated peptides with d-cysteine for 30 min at 37 °C in a buffer in order to generate the required aLuc-caged substrates, followed by the addition of protease, ATP, MgCl2, and luciferase. Hereafter, the luminescence was recorded at 37 °C and the area under the curve (AUC) was calculated (see: Supporting Information). The ratio of substrate hydrolysis toward either thrombin or FXa was determined and is depicted in Scheme 6. The results in Scheme 6 demonstrate that both the synthetic approach and the in situ d-cysteine condensation are robust.

Scheme 6

Scheme 6. Total Luminescence of Substrate Candidates (β-AXR/β-AXK) Using Thrombin and Factor Xaa
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a(i) β-AXR/β-AXK substrate (1 equiv) d-cysteine (1.5 equiv), buffer (25 mM HEPES, 125 mM NaCl, 0.5% BSA, pH = 7.4), 37 °C, 30 min. (ii) Protease (thrombin/FXa, final conc 10 nM), ATP (1.5 equiv), MgCl2 (10 equiv), Quantilum recombinant firefly luciferase (final conc 10 μM). Screening of peptide library with the substrates (667 μM) methoxyacetyl-βAXR-6ABTC (in blue) and methoxyacetyl-βAXK-6ABTC (in orange) after the condensation reaction with d-cysteine. The proteolytic activity of the substrates towards both factor Xa and thrombin were determined and their ratio was plotted (ratio 1:1 y = x) against the total luminescence for either thrombin or FXa (x- and y-axis, respectively).

These preliminary data reveal that incorporation of proline at P2 in both libraries gave a significant increase in selectivity toward thrombin, which is consistent with known substrates encountered in the literature. (46) Ultimately, hits obtained from our libraries may be further optimized for use in diagnostic applications in order to assess thrombin and FXa activity with a luminescent readout. This newly developed synthesis method allows for rapid luminescent profiling of substrate specificity of serine proteases and thereby contributes to the development of novel anticoagulation therapies or to the design of new specific activity-based probes for point-of-care applications. The use of the 6-ABTC-functionalized peptides and the subsequent d-cysteine chemistry appears to be a novel technique in order to generate the active aLuc substrates in the final stage, facilitating conventional peptide synthesis prior to the proteolytic assays. The increased stability of the 6-ABTC peptides allows for convenient storage, as the aLuc moiety is prone to oxidation over time and can now be generated in situ, making our method suitable for SPPS.
The results presented in this work clearly demonstrate the synthetic scope, scalability, and feasibility of our novel parallel SPPS method and its potential for profiling serine proteases with a luminescent readout. Currently, we are screening various proteases in combination with the compound libraries to hopefully find optimal enzyme–substrate pairs for potential diagnostic purposes. Potential hits will be resynthesized, validated, and optimized for the desired activity-based assay.

Conclusions

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In short, we developed a suitable SPPS method for the rapid parallel synthesis of C-terminally 6-ABTC modified peptides via side chain anchoring of either a lysine or ornithine P1 residue. The use of 6-ABTC and the late-stage in situ condensation reaction with d-cysteine were key steps in rendering our approach suitable for solid-phase synthesis. The ornithine P1 residues were converted into the corresponding arginine residues via an on-resin guanidinylation reaction, which was optimized for a larger scale. As proof of our synthetic methodology, we synthesized three peptide libraries containing >750 tripeptide compounds. This method represents a major step forward in the ease of synthesizing luminescent peptide libraries, as most frequently these substrates are currently being made using regular solution-phase chemistry via C-terminal activation. We showed that both the synthetic approach and the in situ d-cysteine condensation are robust in order to profile serine proteases, as demonstrated with our validation library screening. We feel that our method can strongly contribute to the synthesis of luminescent caged peptide substrates via a parallel and automated workflow, as we primarily focused on the synthetic aspects and practicalities in this work. The compounds from these libraries are currently being screened in our laboratories for potential use in diagnostic protease assays to ultimately find optimal enzyme–substrate pairs for diagnostic and/or therapeutic applications.

Experimental Procedures

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Synthesis

(9H-Fluoren-9-yl)methyl tert-Butyl (5-((2-Cyanobenzo[d]thiazol-6-yl)amino)-5-oxopentane-1,4-diyl)(S)-dicarbamate (Fmoc-Orn(Boc)-6ABTC, 4)

Was prepared in multiple batches. Typical procedure: 6-aminobenzo[d]thiazole-2-carbonitrile (1, 300 mg, 1.71 mmol) was dissolved in dry pyridine (6 mL) in a flame-dried flask. PCl3 (78 μL, 890 μmol) was added dropwise and the reaction mixture was stirred for 1.5 h. Fmoc-Orn(Boc)–OH (778 mg, 1.71 mmol) was added to dry pyridine (3 mL) and the reaction mixture was stirred for 3 h at 40 °C. The reaction mixture was allowed to cool down to rt, diluted with EtOAc (150 mL), and washed with 10% aqueous citric acid (150 mL). The aqueous phase was re-extracted with EtOAc (75 mL). The combined organic layers were washed with 10% aqueous citric acid (100 mL), saturated aqueous NH4Cl (2 × 100 mL), and brine (100 mL). The combined organic layers were dried with MgSO4, concentrated in vacuo, and purified with silica gel column chromatography (20 → 80% EtOAc/heptane) to afford Fmoc-Orn(Boc)-6ABTC (4, 1.03 g, 98%) as a yellow solid. TLC (EtOAc/heptane, 1:1 v/v) RF = 0.69. 1H NMR (500 MHz, CDCl3): δ 9.26 (s, 1H), 8.66 (d, J = 2.1 Hz, 1H), 8.09 (d, J = 8.9 Hz, 1H), 7.76 (d, J = 7.6 Hz, 2H), 7.60 (t, J = 7.2 Hz, 2H), 7.57–7.53 (m, 1H), 7.39 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 5.5 Hz, 2H), 5.76–5.71 (m, 1H), 4.87–4.83 (m, 1H), 4.68–4.64 (m, 1H), 4.42 (d, J = 7.1 Hz, 2H), 4.22 (t, J = 7.0 Hz, 1H), 3.63–3.50 (m, 1H), 3.16–3.06 (m, 1H), 2.04 (s, 2H), 1.67 (s, 2H), 1.45 (s, 9H). 13C NMR (126 MHz, CDCl3): δ 171.3, 157.6, 148.7, 143.7, 141.4, 136.9, 127.9, 127.2, 125.3, 125.2, 121.0, 120.1, 120.1, 113.2, 111.5, 47.3, 28.5.

(9H-Fluoren-9-yl)methyl (S)-(5-Amino-1-((2-cyanobenzo[d]thiazol-6-yl)amino)-1-oxopentan-2-yl)carbamate (Fmoc-Orn-6ABTC, 6)

Was prepared in multiple batches. Typical procedure: Fmoc-Orn(Boc)-6ABTC (4, 785, 1.28 mmol) was dissolved in HCO2H (10 mL) and stirred overnight at rt. The reaction mixture was concentrated in vacuo and the product was lyophilized overnight to afford the HCO2H salt of Fmoc-Orn-6ABTC (6, 733 mg, quant) as a yellow solid. 1H NMR (400 MHz, CD3OD): δ 8.64 (d, J = 2.1 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.78 (d, J = 7.6 Hz, 2H), 7.70 (dd, J = 9.0, 2.1 Hz, 1H), 7.66 (t, J = 7.5 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.33–7.24 (m, 2H), 4.42 (qd, J = 10.6, 6.7 Hz, 2H), 4.35–4.31 (m, 1H), 4.21 (t, J = 6.7 Hz, 1H), 2.97 (t, J = 7.3 Hz, 2H), 1.88–1.73 (m, 3H). 13C NMR (101 MHz, CD3OD): δ 177.3, 172.9, 169.9, 158.3, 149.9, 145.1, 142.2, 140.4, 138.0, 136.8, 128.8, 128.1, 125.9, 122.3, 120.9, 114.0, 112.9, 67.9, 56.5, 39.7, 30.1, 24.2, 22.1. HRMS (m/z): [M + H]+ calcd for C28H25N5O3S, 512.1756; found, 512.1752.

(9H-Fluoren-9-yl)methyl tert-Butyl (6-((2-Cyanobenzo[d]thiazol-6-yl)amino)-6-oxohexane-1,5-diyl)(S)-dicarbamate (Fmoc-Lys(Boc)-6ABTC, 5)

Was prepared in multiple batches. Typical procedure: 6-aminobenzo[d]thiazole-2-carbonitrile (1, 501 mg, 2.86 mmol) was dissolved in dry pyridine (10 mL) in a flame-dried flask. PCl3 (130 μL, 1.49 μmol) was added dropwise, and the reaction mixture was stirred for 1.5 h. Fmoc-Lys(Boc)–OH (1340 mg, 2.86 mmol) was added to dry pyridine (5 mL) and the reaction mixture was stirred for 3 h at 40 °C. The reaction mixture was allowed to cool down to rt, diluted with EtOAc (150 mL), and washed with 10% aqueous citric acid (150 mL). The aqueous phase was re-extracted with EtOAc (75 mL). The combined organic layers were washed with 10% aqueous citric acid (100 mL), saturated aqueous NH4Cl (2 × 100 mL), and brine (100 mL). The combined organic layers were dried with MgSO4, concentrated in vacuo, and purified with silica gel column chromatography (0 → 70% EtOAc/heptane) to afford Fmoc-Lys(Boc)-6ABTC (5, 1.38 g, 77%) as a yellow solid. TLC (EtOAc/heptane, 9:1 v/v) RF = 0.83. 1H NMR (500 MHz, CD3OD): δ 8.65–8.60 (m, 1H), 8.08 (dd, J = 9.2, 4.3 Hz, 1H), 7.79–7.74 (m, 2H), 7.69–7.63 (m, 3H), 7.39–7.34 (m, 2H), 7.32–7.25 (m, 2H), 4.40–4.36 (m, 2H), 4.28–4.24 (m, 1H), 4.22–4.18 (m, 1H), 3.07–3.00 (m, 2H), 1.89–1.82 (m, 1H), 1.80–1.71 (m, 1H), 1.55–1.47 (m, 4H), 1.39 (s, 9H). 13C NMR (126 MHz, CDCl3): δ 173.7, 158.6, 149.8, 145.2, 145.1, 142.5, 140.5, 138.0, 136.7, 128.7, 128.1, 126.2, 125.9, 122.2, 120.9, 114.0, 112.8, 79.8, 67.9, 57.2, 48.4, 38.4, 32.9, 30.6, 28.7, 24.2.

(9H-Fluoren-9-yl)methyl (S)-(6-Amino-1-((2-cyanobenzo[d]thiazol-6-yl)amino)-1-oxohexan-2-yl)carbamate (Fmoc-Lys-6ABTC, 7)

Was prepared in multiple batches. Typical procedure: Fmoc-Lys(Boc)-6ABTC (5, 1.38 g, 2.21 mmol) was dissolved in HCO2H (10 mL) and stirred overnight at rt. The reaction mixture was concentrated in vacuo and the product was lyophilized overnight to afford the HCO2H salt of Fmoc-Lys-6ABTC (7, 1260 mg, quant) as a yellow solid. 1H NMR (400 MHz, CD3OD): δ 8.61 (d, J = 2.1 Hz, 1H), 8.07 (d, J = 9.0 Hz, 1H), 8.28 (s, 2H), 7.75 (d, J = 7.5 Hz, 2H), 7.70–7.60 (m, 3H), 7.35 (t, J = 7.5 Hz, 2H), 7.26 (t, J = 7.5 Hz, 2H), 4.40 (dd, J = 6.7, 3.4 Hz, 2H), 4.28 (dd, J = 8.9, 5.3 Hz, 1H), 4.19 (t, J = 6.7 Hz, 1H), 2.93–2.88 (m, 2H), 1.94–1.83 (m, 1H), 1.83–1.62 (m, 3H), 1.58–1.40 (m, 2H). 13C NMR (101 MHz, CD3OD): δ 172.0, 157.2, 148.5, 143.7, 141.2, 139.1, 136.6, 135.4, 127.4, 126.7, 124.8, 124.6, 120.9, 119.6, 112.7, 111.6, 66.5, 55.6, 47.0, 39.1, 31.3, 26.8, 22.5. HRMS (m/z): [M + H]+ calcd C29H27N5O3S, 526.1912; found, 526.1909.

N,N′-Di-tert-butoxycarbonyl-5-chloro-1H-benzotriazole-1-carboxamidine and N,N′-Di-tert-butoxycarbonyl-6-chloro-1H-benzotriazole-1-carboxamidine (9) (41,47)

It was prepared in multiple batches. Typical procedure: N,N′-di-Boc-thiourea (7.7 g, 27.9 mmol) and 5-chlorobenzotriazole (4.3 g, 27.9 mmol) were dissolved in anhydrous MeCN (250 mL) and DIPEA (14.6 mL, 83.6 mmol) was added. The reaction mixture was cooled to 0 °C, EDCl (10.7 g, 55.7 mmol) was added, and the reaction was stirred for 18 h at rt. The mixture was diluted with EtOAc (600 mL) and washed with 10% aqueous citric acid (300 mL) and brine (300 mL). The organic layer was dried with MgSO4, concentrated in vacuo, and purified through silica gel column chromatography (0 → 15% EtOAc/heptane) to afford the product as a mixture of the 5′- and 6′ isomers (9, 3.49 g, 32%) as a white solid. TLC (EtOAc/heptane, 1:4 v/v) RF = 0.32 (6′ isomer), 0.27 (5′ isomer). 1H NMR (500 MHz, CDCl3): δ 8.99 (s, 2H), 8.39 (s, 1H), 8.32 (d, J = 8.8 Hz, 1H), 8.09 (d, J = 1.9 Hz, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.63–7.58 (m, 1H), 7.51–7.45 (m, 1H), 1.52 (d, J = 17.5 Hz, 27H). 13C NMR (126 MHz, CDCl3): δ 131.1, 127.4, 121.1, 119.8, 116.3, 115.2, 28.1. HRMS (m/z): [M + Na]+ calcd for C17H22ClN5O4Na, 418.1258; found, 418.1244.

Resin Loading Procedures

Loading of Fmoc-Orn-6ABCT (2): Typical Scale 10 g of Resin

(3-Formylindolyl)acetamidomethyl resin (10.00 g, 7.50 mmol according to loading) was suspended in dry THF/TMOF (1:1, 150 mL) in a flame-dried flask. Fmoc-Orn-6ABTC (6, 3.46 g, 6.75 mmol) was added, and the suspension was agitated for 4 h at rt on a rotary evaporator. NaBH3CN (848 mg, 13.5 mmol) in THF (5 mL) and AcOH (1.5 mL, 26.2 mmol) were added, and the suspension was agitated for 2 h. The resin was washed with DMF (3×) and DCM (3×) before the addition of 9 (2.82 g, 6.75 mmol) and DIPEA (3.26 mL, 18.7 mmol) in DMF (100 mL) and agitated for 18 h. Hereafter, the resin was washed with DMF (3×), DCM (3×), and Et2O (3×). The resin was dried under high vacuum to afford R4 which was used in the next steps using regular Fmoc SPPS chemistry (see the Supporting Information). Multiple batches were prepared with a final loading between 0.20 and 0.22 mmol/g (determined by Fmoc quantification at 301 nm).

Loading of Fmoc-Lys-6ABCT (3): Typical Scale 10 g of Resin

(3-Formylindolyl)acetamidomethyl resin (10.00 g, 7.50 mmol according to loading) was suspended in dry THF/TMOF (1:1, 150 mL) in a flame-dried flask. Fmoc-Lys-6ABTC (7, 3.55 g, 6.75 mmol) was added, and the suspension was agitated for 4 h at rt on a rotary evaporator. NaBH3CN (848 mg, 13.5 mmol) in THF (5 mL) and AcOH (1.5 mL, 26.2 mmol) were added and the suspension was agitated for 2 h. The resin was washed with DMF (3×) and DCM (3×) before the addition of Boc2O (4.91 g, 22.5 mmol) and DIPEA (3.92 mL, 22.5 mmol) in DMF (100 mL), after which the mixture was agitated for 18 h. Hereafter, the resin was washed with DMF (3×), DCM (3×), and Et2O (3×). The resin was dried under high vacuum to afford R5 which was used in the next steps with regular Fmoc SPPS chemistry (see the Supporting Information). Multiple batches were prepared with a final loading between 0.16–0.25 mmol/g (determined by Fmoc quantification at 301 nm).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.3c00381.

  • Material and methods, experimental procedures, and NMR spectra (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Daan Sondag - Institute for Molecules and Materials, Radboud University, Nijmegen 6525 AJ, The Netherlands
    • Jurriaan J. A. Heming - Institute for Molecules and Materials, Radboud University, Nijmegen 6525 AJ, The Netherlands
    • Dennis W. P. M. Löwik - Institute for Molecules and Materials, Radboud University, Nijmegen 6525 AJ, The Netherlands;  Orcidhttps://orcid.org/0000-0002-9284-837X
    • Elena Krivosheeva - Enzyre BV, Novio Tech Campus, Transistorweg 5-i, Nijmegen 6534 AT, The Netherlands;  Orcidhttps://orcid.org/0000-0001-8499-4409
    • Denise Lejeune - Institute for Molecules and Materials, Radboud University, Nijmegen 6525 AJ, The Netherlands;  Enzyre BV, Novio Tech Campus, Transistorweg 5-i, Nijmegen 6534 AT, The Netherlands
    • Mark van Geffen - Enzyre BV, Novio Tech Campus, Transistorweg 5-i, Nijmegen 6534 AT, The Netherlands;  Orcidhttps://orcid.org/0000-0003-1748-0007
    • Cornelis van’t Veer - Enzyre BV, Novio Tech Campus, Transistorweg 5-i, Nijmegen 6534 AT, The Netherlands
    • Waander L. van Heerde - Enzyre BV, Novio Tech Campus, Transistorweg 5-i, Nijmegen 6534 AT, The Netherlands;  Department of Haematology, Radboud University Medical Centre, Nijmegen 6525 GA, The Netherlands;  Haemophilia Treatment Centre, Nijmegen Eindhoven Maastricht (HTC-NEM), Nijmegen 6525 GA, The Netherlands
    • Marjolijn C. J. Beens - Symeres BV, Kerkenbos 1013, Nijmegen 6546 BB, The Netherlands
    • Brian H. M. Kuijpers - Symeres BV, Kerkenbos 1013, Nijmegen 6546 BB, The Netherlands
    • Thomas J. Boltje - Institute for Molecules and Materials, Radboud University, Nijmegen 6525 AJ, The Netherlands;  Orcidhttps://orcid.org/0000-0001-9141-8784
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by grants LIFT 741.018.406 from the Dutch Research Council (NWO) and EFRO 891 from the European Fund for Regional Development (EFRD), both awarded to F.P.J.T.R., T.J.B., and W.L.v.H.

References

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    Condensation of benzothiazole I (R = H) with acetobromoglucose gave the β-glucoside (I, R = 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)(II). Attempted basic deacetylation caused methanolysis of the nitrile group to give Me 6-(β-D-glucopyranosyloxy)-2-benzothiazolecarboximidate. II was treated with D-cysteine, followed by deacetylation to give firefly luciferin β-glucoside (III, R = β-D-glucopyranosyl). Luciferin β-galactoside was similarly prepd. These luciferin β-glycosides are substrates for the corresponding glycohydrolases. Because of the great sensitivity of bioluminescence detection, these substrates provide potentially ultrasensitive assays for the glycohydrolases.
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    Rodriguez-Rios, M.; Megia-Fernandez, A.; Norman, D. J.; Bradley, M. Peptide probes for proteases-innovations and applications for monitoring proteolytic activity. Chem. Soc. Rev. 2022, 51, 2081– 2120,  DOI: 10.1039/D1CS00798J
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    Peptide probes for proteases - innovations and applications for monitoring proteolytic activity
    Rodriguez-Rios, Maria; Megia-Fernandez, Alicia; Norman, Daniel J.; Bradley, Mark
    Chemical Society Reviews (2022), 51 (6), 2081-2120CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. Proteases are excellent biomarkers for a variety of diseases, offer multiple opportunities for diagnostic applications and are valuable targets for therapy. From a chem.-based perspective this review discusses and critiques the most recent advances in the field of substrate-based probes for the detection and anal. of proteolytic activity both in vitro and in vivo.
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    Yang, X.; Qin, X.; Ji, H.; Du, L.; Li, M. Constructing Firefly Luciferin Bioluminescence Probes for in Vivo Imaging. Org. Biomol. Chem. 2022, 20, 1360– 1372,  DOI: 10.1039/D1OB01940F
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    Constructing firefly luciferin bioluminescence probes for in vivo imaging
    Yang, Xingye; Qin, Xiaojun; Ji, Huimin; Du, Lupei; Li, Minyong
    Organic & Biomolecular Chemistry (2022), 20 (7), 1360-1372CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    A review. Bioluminescence imaging (BLI) is a widely applied visual approach for real-time detecting many physiol. and pathol. processes in a variety of biol. systems. Based on the caging strategy, lots of bioluminescent probes have been well developed. While the targets react with recognizable groups, caged luciferins liberate luciferase substrates, which react with luciferase generating a bioluminescent response. Among the various bioluminescent systems, the most widely utilized bioluminescent system is the firefly luciferin system. The H and carboxylic acid of luciferin are critically caged sites. The introduced self-immolative linker extends the applications of probes. Firefly luciferin system probes have been successfully applied for analyzing physiol. processes, monitoring the environment, diagnosing diseases, screening candidate drugs, and evaluating the therapeutic effect. Here, we systematically review the general design strategies of firefly luciferin bioluminescence probes and their applications. Bioluminescence probes provide a new approach for facilitating investigation in a diverse range of fields. It inspires us to explore more robust light emission luciferin and novel design strategies to develop bioluminescent probes.
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    Ren, H.; Xiao, F.; Zhan, K.; Kim, Y. P.; Xie, H.; Xia, Z.; Rao, J. A biocompatible condensation reaction for the labeling of terminal cysteine residues on proteins. Angew. Chem., Int. Ed. 2009, 48, 9658– 9662,  DOI: 10.1002/anie.200903627
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    A Biocompatible Condensation Reaction for the Labeling of Terminal Cysteine Residues on Proteins
    Ren, Hongjun; Xiao, Fei; Zhan, Ke; Kim, Young-Pil; Xie, Hexin; Xia, Zuyong; Rao, Jianghong
    Angewandte Chemie, International Edition (2009), 48 (51), 9658-9662, S9658/1-S9658/16CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A protein-labeling method based on the use of a single amino acid tag - an N-terminal cysteine residue - and small-mol. probes contg. a cyanobenzothiazole (CBT) unit has been used for the specific fluorescence labeling of proteins in vitro and at the surface of live cells. This simple ligation reaction proceeds with a high degree of specificity under physiol. conditions.
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    Dragulescu-Andrasi, A.; Liang, G.; Rao, J. In vivo bioluminescence imaging of furin activity in breast cancer cells using bioluminogenic substrates. Bioconjugate Chem. 2009, 20, 1660– 1666,  DOI: 10.1021/bc9002508
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    In Vivo Bioluminescence Imaging of Furin Activity in Breast Cancer Cells Using Bioluminogenic Substrates
    Dragulescu-Andrasi, Anca; Liang, Gaolin; Rao, Jianghong
    Bioconjugate Chemistry (2009), 20 (8), 1660-1666CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Furin, a proprotein convertases family endoprotease, processes numerous physiol. substrates and is overexpressed in cancer and inflammatory conditions. Noninvasive imaging of furin activity will offer a valuable tool to probe furin function over the course of tumor growth and migration in the same animals in real time and directly assess the inhibition efficacy of drugs in vivo. Here, we report successful bioluminescence imaging of furin activity in xenografted MBA-MB-468 breast cancer tumors in mice with bioluminogenic probes. The probes are conjugates of furin substrate, a consensus amino acid motif R-X-K/R-R (X, any amino acid), with the firefly luciferase substrate D-aminoluciferin. In the presence of the luciferase reporter, the probes are unable to produce bioluminescent emission without furin activation. Blocking expts. with a furin inhibitor and control expts. with a scrambled probe showed that the bioluminescence emission in the presence of firefly luciferase is furin-dependent and specific. After furin activation, a 30-fold increase in the bioluminescent emission was obsd. in vitro, and on av., a 7-8-fold contrast between the probe and control was seen in the same tumor xenografts in mice. Direct imaging of furin activity may facilitate the study of furin function in tumorigenicity and the discovery of new drugs for furin-targeted cancer therapy.
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    Sondag, D.; Merx, J.; Rossing, E.; Boltje, T. J.; Löwik, D. W. P. M.; Nelissen, F. H. T.; van Geffen, M.; van’t Veer, C.; van Heerde, W. L.; Rutjes, F. P. J. T. Luminescent Assay for the Screening of SARS-CoV-2 MPro Inhibitors. ChemBioChem 2022, 23, e202200190  DOI: 10.1002/cbic.202200190
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    Luminescent Assay for the Screening of SARS-CoV-2 MPro Inhibitors
    Sondag, Daan; Merx, Jona; Rossing, Emiel; Boltje, Thomas J.; Lowik, Dennis W. P. M.; Nelissen, Frank H. T.; van Geffen, Mark; van 't Veer, Cornelis; van Heerde, Waander L.; Rutjes, Floris P. J. T.
    ChemBioChem (2022), 23 (15), e202200190CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Since the outbreak of SARS-CoV-2 in Dec. 2019 millions of infections have been reported globally. The viral chymotrypsin-like main protease (MPro) exhibits a crucial role in viral replication and represents a relevant target for antiviral drug development. In order to screen potential MPro inhibitors we developed a luminescent assay using a peptide based probe contg. a cleavage site specific for MPro. This assay was validated showing IC50 values similar to those reported in the literature for known MPro inhibitors and can be used to screen new inhibitors.
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    Merrifield, R. B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149– 2154,  DOI: 10.1021/ja00897a025
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    Solid phase peptide synthesis. I. The synthesis of a tetrapeptide
    Merrifield, R. B.
    Journal of the American Chemical Society (1963), 85 (14), 2149-54CODEN: JACSAT; ISSN:0002-7863.
    A new approach to the chem. synthesis of polypeptides was investigated. It involved the stepwise addition of protected amino acids to a growing peptide chain which was bound by a covalent bond to a solid resin particle. This provided a procedure whereby reagents anti by-products were removed by filtration, and the recrystn. of intermediates was eliminated. The advantages of the new method were speed and simplicity of operation. The feasibility of the idea was demonstrated by the synthesis of the model tetrapeptide L-leucyl-L-alanylglycyl-L-valine. The peptide was identical with a sample prepd. by the standard p-nitrophenyl ester procedure.
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    Kovács, A. K.; Hegyes, P.; Szebeni, G. J.; Bogár, K.; Puskás, L. G.; Tóth, G. K. Synthesis of N-Peptide-6-Amino-d-Luciferin Conjugates with Optimized Fragment Condensation Strategy. Int. J. Pept. Res. Ther. 2019, 25, 1209– 1215,  DOI: 10.1007/s10989-018-9768-8
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    Synthesis of N-Peptide-6-Amino-D-Luciferin Conjugates with Optimized Fragment Condensation Strategy
    Kovacs, Anita K.; Hegyes, Peter; Szebeni, Gabor J.; Bogar, Krisztian; Puskas, Laszlo G.; Toth, Gabor K.
    International Journal of Peptide Research and Therapeutics (2019), 25 (3), 1209-1215CODEN: IJPRFC; ISSN:1573-3149. (Springer)
    The synthesis of peptide-luciferin conjugates has a pivotal role in the development of bioluminescent detection systems that are based on the detn. of protease enzyme activity. This work describes the optimized synthesis of an N-peptide-6-amino-D-luciferin conjugate (Fmoc-Gly-Pro-6-amino-D-luciferin) with a simple fragment condensation method in adequate yields. Fmoc-Gly-Pro-6-amino-D-luciferin was produced from a previously synthesized Fmoc-Gly-Pro-OH and also previously synthesized 6-amino-2-cyanobenzothiazole with an optimized method, to which conjugate cysteine was added in an also improved way. The resulting conjugate was successfully used in a bioluminescent system, in vitro, demonstrating the applicability of the method.
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    White, E. H.; McCapra, F.; Field, G. F. The structure and synthesis of firefly luciferin. J. Am. Chem. Soc. 1963, 85, 337– 343,  DOI: 10.1021/ja00886a019
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    The structure and synthesis of firefly luciferin
    White, Emil H.; McCapra, Frank; Field, George F.
    Journal of the American Chemical Society (1963), 85 (), 337-43CODEN: JACSAT; ISSN:0002-7863.
    Luciferin from Photinus pyralis, was shown to be D-2-(6-hydroxy-2-benzothiazolyl)-δ2-thiazoline-4-carboxylic acid (I). The structure was confirmed by a total synthesis. L-Luciferin, DL-luciferin, dehydroluciferin, and various related benzothiazoles were also synthesized.
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    Fontes, R.; Dukhovich, A.; Sillero, A.; Sillero, M. A. G. Synthesis of dehydroluciferin by firefly luciferase: effect of dehydroluciferin, coenzyme A and nucleoside triphosphates on the luminescent reaction. Biochem. Biophys. Res. Commun. 1997, 237, 445– 450,  DOI: 10.1006/bbrc.1997.7161
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    Synthesis of dehydroluciferin by firefly luciferase: effect of dehydroluciferin, coenzyme A and nucleoside triphosphates on the luminescent reaction
    Fontes, Rui; Dukhovich, Alexey; Sillero, Antonio; Sillero, Maria A. Gunther
    Biochemical and Biophysical Research Communications (1997), 237 (2), 445-450CODEN: BBRCA9; ISSN:0006-291X. (Academic)
    The formation of dehydroluciferin (L) from luciferin (LH2) in the reaction catalyzed by firefly luciferase (EC 1.13.12.7) (I) was studied. The I·LH2-AMP complex may follow 2 different pathways: toward prodn. of light and toward the synthesis of the I·L-AMP complex. This last step has an inhibitory effect on light emission as mols. of the enzyme are trapped in a light unproductive complex. The effects of CoA and nucleoside 5'-triphosphates (NTPs) on light emission were quant. different. CoA combined with the L moiety of the I·L-AMP complex, yielding L-CoA, promoting liberation of free I, and increasing light yield. The NTPs reacted with the AMP moiety of the same complex, generating adenosine(5')tetraphospho(5')nucleoside (Ap4N) and, probably, the I·L complex, and scarcely increasing light prodn. The results are discussed in relation to previous reports by others on I.
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    Evnin, L. B.; Vásquez, J. R.; Craik, C. S. Substrate specificity of trypsin investigated by using a genetic selection. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 6659– 6663,  DOI: 10.1073/pnas.87.17.6659
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    Substrate specificity of trypsin investigated by using a genetic selection
    Evnin, Luke B.; Vasquez, John R.; Craik, Charles S.
    Proceedings of the National Academy of Sciences of the United States of America (1990), 87 (17), 6659-63CODEN: PNASA6; ISSN:0027-8424.
    The structural determinants of the primary substrate specificity of rat anionic trypsin were examd. by using oligonucleotide-directed mutagenesis coupled to a genetic selection. A library was created that encoded trypsins substituted at amino acid positions 189 and 190 at the base of the substrate binding pocket. A genetic selection, with a dynamic range of 5 orders of proteolytic activity, was used to search 90,000 transformants of the library. Rapid screening for arginyl amidolysis and esterolysis confirmed the activity of the purified isolates. Trypsin and 15 mutant trypsins with partially preserved function were identified and characterized kinetically on arginyl and lysyl peptide substrates. Alternative arrangements of amino acids in the substrate binding pocket sustained efficient catalysis. A neg. charge at amino acid position 189 or 190 was shown to be essential for high-level catalysis. With the favored aspartic acid residue at position 189, several amino acids could replace serine at position 190. Modulation of the specificity for arginine and lysine substrates was shown to depend on the amino acid at position 190. The regulatory effect of the amino acid side chain at position 190 on the substrate specificity is also reflected in substrate binding pockets of naturally occurring trypsin homologs.
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    Sondag, D.; Verhoeven, S.; Löwik, D. W. P. M.; van Geffen, M.; Veer, C. v.; van Heerde, W. L.; Boltje, T. J.; Rutjes, F. P. J. T. Activity Sensing of Coagulation and Fibrinolytic Proteases. Chem.─Eur. J. 2023, 29, e202203473  DOI: 10.1002/chem.202203473
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    Activity Sensing of Coagulation and Fibrinolytic Proteases
    Sondag, Daan; Verhoeven, Stijn; Lowik, Dennis W. P. M.; van Geffen, Mark; Veer, Cornelis van't; van Heerde, Waander L.; Boltje, Thomas J.; Rutjes, Floris P. J. T.
    Chemistry - A European Journal (2023), 29 (18), e202203473CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. The blood coagulation cascade is a complex physiol. process involving the action of multiple coupled enzymes, cofactors, and substrates, ultimately leading to clot formation. Serine proteases have a crucial role, and aberrations in their activity can lead to life-threatening bleeding disorders and thrombosis. This review summarizes the essential proteases involved in blood coagulation and fibrinolysis, the endogenous peptide sequences they recognize and hydrolyze, and synthetic peptide probes based on these sequences to measure their activity. The information in this review can contribute to developing novel anticoagulant therapies and specific substrates for point-of-care diagnosis of coagulation pathologies.
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    Beythien, J.; Barthélémy, S.; Schneeberger, P.; White, P. D. A novel solid-phase linker strategy for the side-chain anchoring of arginine: an expeditious route to arginine 7-amido-4-methylcoumarins. Tetrahedron Lett. 2006, 47, 3009– 3012,  DOI: 10.1016/j.tetlet.2006.03.019
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    A novel solid-phase linker strategy for the side-chain anchoring of arginine: an expeditious route to arginine 7-amido-4-methylcoumarins
    Beythien, Joerg; Barthelemy, Sophie; Schneeberger, Peter; White, Peter D.
    Tetrahedron Letters (2006), 47 (18), 3009-3012CODEN: TELEAY; ISSN:0040-4039. (Elsevier B.V.)
    A novel linker strategy for the efficient side-chain anchoring of arginine is described. The utility of this approach was demonstrated by the facile synthesis of arginine-specific fluorogenic peptide substrates by std. Fmoc solid phase peptide synthesis (Fmoc = 9-fluorenylmethyloxycarbonyl) methods.
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    Hamzé, A.; Martinez, J.; Hernandez, J.-F. Solid-phase synthesis of arginine-containing peptides and fluorogenic substrates using a side-chain anchoring approach. J. Org. Chem. 2004, 69, 8394– 8402,  DOI: 10.1021/jo048792t
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    Solid-Phase Synthesis of Arginine-Containing Peptides and Fluorogenic Substrates Using a Side-Chain Anchoring Approach
    Hamze, Abdallah; Martinez, Jean; Hernandez, Jean-Francois
    Journal of Organic Chemistry (2004), 69 (24), 8394-8402CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    Attachment of an amino acid to a solid support by its side chain is sometimes necessary in order to make the α-carboxylic group available for diverse modifications, such as incorporation of fluorophores. In contrast to most other amino acids, anchoring the guanidinium group of an arginine to a resin requires the use of a supplementary linker. To avoid the usually multistep synthesis of such a linker as well as its difficult attachment to the guanidine group, the authors developed a simple method where the guanidine group is built on a Rink amide resin. The strategy followed the steps of guanidine formation: (i) addn. of an isothiocyanate deriv. of ornithine to the amino group of a solid support, yielding Nω-linked thiocitrulline; (ii) S-methylation of thiourea; (iii) guanidinylation using ammonium acetate. Cleavage of the resin generated the arginine-contg. compd., the amine group of the resin becoming part of the guanidine. Thus, this strategy was successfully applied to the synthesis of a series of fluorogenic substrates for trypsin-like serine proteases (Ac-Val-Gly-Leu-Arg-AMC, Ac-Val-Pro-Arg-AMC, Ac-Ile-Glu-Gly-Arg-AMC, Ac-Ala-Lys-Arg-AMC, etc.; AMC = 7-amino-4-methylcoumarin) , which were obtained in high yields and purities. Similarly, substituted arginine derivs., including Nω-methyl- and Nω-ethyl-arginines, were prepd.
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    Boas, U.; Brask, J.; Jensen, K. J. Backbone amide linker in solid-phase synthesis. Chem. Rev. 2009, 109, 2092– 2118,  DOI: 10.1021/cr068206r
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    Backbone Amide Linker in Solid-Phase Synthesis
    Boas, Ulrik; Brask, Jesper; Jensen, Knud J.
    Chemical Reviews (Washington, DC, United States) (2009), 109 (5), 2092-2118CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The present review provides a comprehensive survey of the by now widely used BAL (backbone amide linker) chem. methodol., which extends far beyond applications in peptide chem.
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    Ten Brink, H. T.; Meijer, J. T.; Geel, R. V.; Damen, M.; Löwik, D. W. P. M.; van Hest, J. C. M. Solid-phase synthesis of C-terminally modified peptides. J. Pept. Sci. 2006, 12, 686– 692,  DOI: 10.1002/psc.780
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    Solid-phase synthesis of C-terminally modified peptides
    Ten Brink, Hefziba T.; Meijer, Joris T.; Geel, Remon V.; Damen, Mark; Loewik, Dennis W. P. M.; Van Hest, Jan C. M.
    Journal of Peptide Science (2006), 12 (11), 686-692CODEN: JPSIEI; ISSN:1075-2617. (John Wiley & Sons Ltd.)
    In this paper, a straightforward and generic protocol is presented to label the C-terminus of a peptide with any desired moiety that is functionalized with a primary amine. Amine-functional mols. included are polymers (useful for hybrid polymers), long alkyl chains (used in peptide amphiphiles and stabilization of peptides), propargyl amine and azido propyl-amine (desirable for 'click' chem.), dansyl amine (fluorescent labeling of peptides) and crown ethers (peptide switches/hybrids). In the first part of the procedure, the primary amine is attached to an aldehyde-functional resin via reductive amination. To the secondary amine that is produced, an amino acid sequence is coupled via a std. solid-phase peptide synthesis protocol. Since one procedure can be applied for any given amine-functional moiety, a robust method for C-terminal peptide labeling is obtained.
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    Jensen, K. J.; Alsina, J.; Songster, M. F.; Vágner, J.; Albericio, F.; Barany, G. Backbone Amide Linker (BAL) strategy for solid-phase synthesis of C-terminal-modified and cyclic peptides1, 2, 3. J. Am. Chem. Soc. 1998, 120, 5441– 5452,  DOI: 10.1021/ja974116f
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    Backbone Amide Linker Strategy for Solid-Phase Synthesis of C-Terminal-Modified and Cyclic Peptides
    Jensen, Knud J.; Alsina, Jordi; Songster, Michael F.; Vagner, Josef; Albericio, Fernando; Barany, George
    Journal of the American Chemical Society (1998), 120 (22), 5441-5452CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Peptide targets for synthesis are often desired with C-terminal end groups other than the more usual acid and amide functionalities. Relatively few routes exist for synthesis of C-terminal-modified peptides-including cyclic peptides-by either soln. or solid-phase methods, and known routes are often limited in terms of ease and generality. The authors describe a novel backbone amide linker (BAL) approach, whereby the growing peptide is anchored through a backbone nitrogen, thus allowing considerable flexibility in management of the termini. Initial efforts on BAL have adapted the chem. of the tris(alkoxy)benzylamide system exploited previously with PAL anchors. Aldehyde precursors to PAL, e.g. 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid, were reductively coupled to the prospective C-terminal amino acid α-amino group, which was blocked as a tert-Bu, allyl, or Me ester, or to the appropriately protected C-terminal-modified amino acid deriv. These reductive aminations were carried out either in soln. or on the solid phase and occurred without racemization. The secondary amine intermediates resulting from soln. amination were converted to the 9-fluorenylmethoxycarbonyl (Fmoc)-protected preformed handle derivs., which were then attached to poly(ethylene glycol)-polystyrene graft or copoly(styrene-1%-divinylbenzene) supports and used to assemble peptides by std. Fmoc solid-phase chem. Alternatively, BAL anchors formed by on-resin reductive amination were applied directly. Conditions were optimized to achieve near-quant. acylation at the difficult step to introduce the penultimate residue, and a side reaction involving diketopiperazine formation under some circumstances was prevented by a modified protocol for Nα-protection of the second residue/introduction of the third residue. Examples are provided for the syntheses in high yields and purities of representative peptide acids, alcs., N,N-dialkylamides, aldehydes, esters, and head-to-tail cyclic peptides. These methodologies avoid postsynthetic soln.-phase transformations and are ripe for further extension.
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    Alsina, J.; Jensen, K. J.; Albericio, F.; Barany, G. Solid-Phase Synthesis with Tris (alkoxy) benzyl Backbone Amide Linkage (BAL)[≠]. Chem.─Eur. J. 1999, 5, 2787– 2795,  DOI: 10.1002/(sici)1521-3765(19991001)5:10<2787::aid-chem2787>3.0.co;2-2
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    28
    Solid-phase synthesis with tris(alkoxy)benzyl backbone amide linkage (BAL)
    Alsina, Jordi; Jensen, Knud J.; Albericio, Fernando; Barany, George
    Chemistry - A European Journal (1999), 5 (10), 2787-2795CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH)
    This review with 29 refs. discusses solid-phase synthesis of peptides, small proteins, oligonucleotides, and small org. mols. A crucial part of the overall synthesis plan is the choice of an appropriate "handle" (linker) for attaching the org. reactant to the solid support. The authors describe a novel and general concept for solid-phase synthesis that involves attachment of a backbone amide nitrogen to an appropriate handle. This backbone amide linker (BAL) approach allows for the prepn. of C-terminal-modified and cyclic peptides, small org. mols., and modified amino sugars, as well as combinatorial synthesis applications.
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  29. 29
    Hauser, J. R.; Beard, H. A.; Bayana, M. E.; Jolley, K. E.; Warriner, S. L.; Bon, R. S. Economical and scalable synthesis of 6-amino-2-cyanobenzothiazole. Beilstein J. Org. Chem. 2016, 12, 2019– 2025,  DOI: 10.3762/bjoc.12.189
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    Economical and scalable synthesis of 6-amino-2-cyanobenzothiazole
    Hauser, Jacob R.; Beard, Hester A.; Bayana, Mary E.; Jolley, Katherine E.; Warriner, Stuart L.; Bon, Robin S.
    Beilstein Journal of Organic Chemistry (2016), 12 (), 2019-2025CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)
    2-Cyanobenzothiazoles (CBTs) were the useful building blocks for luciferin derivs., for bioluminescent imaging, handles and for bioorthogonal ligations. An economical and scalable synthesis of 6-amino-2-cyanobenzothiazole based on a cyanation catalyzed by 1,4-diazabicyclo[2.2.2]octane (DABCO) was presented and its advantages for scale-up over previously reported routes was also discussed.
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  30. 30
    Grimmel, H.; Guenther, A.; Morgan, J. F. Phosphazo compounds and their use in preparing amides. J. Am. Chem. Soc. 1946, 68, 539– 542,  DOI: 10.1021/ja01208a001
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    Phosphazo compounds and their rise in preparing amides
    Grimmel, H. W.; Guenther, A.; Morgan, Jack F.
    Journal of the American Chemical Society (1946), 68 (), 539-42CODEN: JACSAT; ISSN:0002-7863.
    Two moles of PhNH2 in 300 mL. PhMe at room temp. or (for slightly sol. amines) at 50-70°, treated dropwise with 55 g. PCl3 in 50 mL. PhMe and refluxed and agitated for 1-2 h., give 67.5% of phenylphosphazoanilide (PhN:PNHPh), m. 251-3°; the 4,4'-di-Me deriv. m. 197-200° (preheated bath), 42%; the 2,2'-di-MeO deriv. m. 138-40°, 32%. If the RN:PNHR is treated with a carboxylic acid, R'CO2H, in PhMe and the mixt. is refluxed for 2 h., HPO2 and N-substituted amides, R'CONHR (I) are formed. The following yields of I (R' = Ph) (%) were obtained from the amines listed and BzOH: PhNH2 85, o-, m-, and p-MeC6H4NH2 72, 80, and 56, m-ClC6H4NH2 75, o-MeOC6H4NH2 76, m-O2NC6H4NH2 70, m-H2NC6H4OH 7 (?), p-H2NC6H4NHPh 30, m- and p-C6H4(NH2)2 11 and 67, (p-H2NC6H4)2 94, (p-H2NC6H4)2CH220, 1-C10H7NH239, p-H2NC6H4SO3H 90, 2-aminopyridine 52 (C5H5N as solvent), cyclohexylamine 68, BuNH2 45, dodecylamine 60-77; o- and p-O2NC6H4NH2 give only tars. p-H2NC5H4NHAc and 1-aminoanthraquinone do not form reactive phosphazo compds., nor do amino carboxylic acids. That the reaction is not peculiar to BzOH was shown by the successful prepn. of the following anilides of various acids: HCO2H, small, AcOH 74, EtCO2H 65, lauric 74, stearic 65, PhCH:CHCO2H 80, HOCH2CO2H 49, (CO2H)2 45, CH2(CO2H)2 65, (CH2CO2H)2 41, pimelic 53, fumaric 34, maleic 16, tartaric 45, BzOH 85, p-O2NC6H4CO2H 83, p-HOC6H4CO2H 74, o-H2NC6H4CO2H 42, o-, m-, and p-C6H4(CO2H)2 73, 55, and 25, 3,2-HOC10H6CO2H 89-100, nicotinic 38. Glycine and alanine did not react. p-H2NC6H4CO2H gave a solid m. above 300°; the o-isomer gave 42% of o-H2NC6H4CONHPh. Na salts of carboxylic acids do not react and the method is not applicable to N-substituted sulfonamides. When an excess of PhNH2 was used in the initial reaction with PCl3, the ultimate yields of BzNHPh were increased to 100%; by also using an excess of BzOH the yield is 115% (based on PCl3). It is also possible to prep. RN:PNHR' from PhN:PCl, though the method is less efficient and more tedious. PhN:PNHPh is readily recrystd. from CHCl3 but the resultant crystals are not identical with the original as shown by x-ray diffraction patterns. Prolonged exposure to light or heat (100° or above) causes slow decompn. though the compds. are stable for months at room temp. or in the dark. X-ray patterns are given also for PhNH2.H3PO3 and (PhNH2)POH.
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    van Heerde, W. L.; van Geffen, M.; Steeghs, D. Novel chemiluminescent substrates for Factor Xa. U.S. Patent 20,210,371,461 A1, 2021.
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    Estep, K. G.; Neipp, C. E.; Stephens Stramiello, L. M.; Adam, M. D.; Allen, M. P.; Robinson, S.; Roskamp, E. J. Indole resin: A versatile new support for the solid-phase synthesis of organic molecules. J. Org. Chem. 1998, 63, 5300– 5301,  DOI: 10.1021/jo9806052
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    Indole Resin: A Versatile New Support for the Solid-Phase Synthesis of Organic Molecules
    Estep, Kimberly G.; Neipp, Christopher E.; Stramiello, Linda M. Stephens; Adam, Mavis D.; Allen, Martin P.; Robinson, Shaughnessy; Roskamp, Eric J.
    Journal of Organic Chemistry (1998), 63 (16), 5300-5301CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    A resin prepd. from (aminomethyl)polystyrene and 3-formyl-1H-indole-1-acetic acid was an ideal support for prepg. libraries of amides, ureas, sulfonamides, guanidines, carbamates, and aniline-contg. products.
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    Yong, Y. F.; Kowalski, J. A.; Thoen, J. C.; Lipton, M. A. A new reagent for solid and solution phase synthesis of protected guanidines from amines. Tetrahedron Lett. 1999, 40, 53– 56,  DOI: 10.1016/S0040-4039(98)80017-8
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    A new reagent for solid and solution phase synthesis of protected guanidines from amines
    Yong, Yaw Fui; Kowalski, Jennifer A.; Thoen, Jason C.; Lipton, Mark A.
    Tetrahedron Letters (1999), 40 (1), 53-56CODEN: TELEAY; ISSN:0040-4039. (Elsevier Science Ltd.)
    A new reagent - 4-nitro-1H-pyrazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine - has been developed to effect the rapid and efficient synthesis of bis(carbamate)-protected guanidines from primary and secondary amines. The reagent is a more electrophilic, and consequently more reactive, deriv. of the literature reagent 1H-pyrazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine. The increased reactivity of the new reagent affords it increased yields in soln. and the need for fewer equiv. when guanylating resin-bound amines.
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    Pinilla, C.; Appel, J.; Blanc, P.; Houghten, R. Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries. BioTechniques 1992, 13, 901– 905
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    Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries
    Pinilla, Clemencia; Appel, Jon R.; Blanc, Philippe; Houghten, Richard A.
    BioTechniques (1992), 13 (6), 901-2, 904-5CODEN: BTNQDO; ISSN:0736-6205.
    A conceptually unique set of individual synthetic peptide combinatorial libraries (SPCLs), termed a positional scanning SPCL (PS-SPCL) are described that can be used for the rapid (i.e., a single day) identification of peptide sequences that bind with high affinity to antibodies, receptors or other acceptor mols. The PS-SPCL described here is made up of six individual positional peptide libraries, each one consisting of hexamers with a single position defined and five positions as mixts. As an example of the utility of such PS-SPCLs, the antigenic determinants recognized by two different monoclonal antibodies were correctly identified upon a single screening.
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  35. 35
    Geffen, M. v.; Loof, A.; Lap, P.; Boezeman, J.; Laros-van Gorkom, B. A. P.; Brons, P.; Verbruggen, B.; Kraaij, M. v.; van Heerde, W. L. A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis. Hematology 2011, 16, 327– 336,  DOI: 10.1179/102453311x13085644680348
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    Ube, H.; Uraguchi, D.; Terada, M. Efficient synthetic protocol for substituted guanidines via copper (I)-mediated intermolecular amination of isothiourea derivatives. J. Organomet. Chem. 2007, 692, 545– 549,  DOI: 10.1016/j.jorganchem.2006.06.046
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    Efficient synthetic protocol for substituted guanidines via copper(I)-mediated intermolecular amination of isothiourea derivatives
    Ube, Hitoshi; Uraguchi, Daisuke; Terada, Masahiro
    Journal of Organometallic Chemistry (2007), 692 (1-3), 545-549CODEN: JORCAI; ISSN:0022-328X. (Elsevier Ltd.)
    Amination of S-methyl-N,N'-bis-Boc-isothiourea with either primary or sterically hindered secondary amines, promoted by copper(I) chloride and K2CO3, gave N,N'-bis-Boc protected guanidines in good to excellent yields under mild reaction conditions.
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    Kelly, B.; Rozas, I. Copper (II) chloride promoted transformation of amines into guanidines and asymmetrical N, N′-disubstituted guanidines. Tetrahedron Lett. 2013, 54, 3982– 3984,  DOI: 10.1016/j.tetlet.2013.05.070
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    Copper(II) chloride promoted transformation of amines into guanidines and asymmetrical N,N'-disubstituted guanidines
    Kelly, Brendan; Rozas, Isabel
    Tetrahedron Letters (2013), 54 (30), 3982-3984CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)
    We present a concise, less-toxic and broadly applicable method for coupling weakly nucleophilic amines with N,N'-di-(tert-butoxycarbonyl)thiourea, N-(tert-butxoycarbonyl), N'-alkyl/arylsubstituted-thioureas and N,N'-di-(tert-butoxycarbonyl)imidazolidine-2-thione in the presence of copper(II) chloride. Subsequent removal of Boc protecting groups affords guanidines, di-substituted guanidines and 2-aminoimidazolines in modest to excellent overall yields.
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    Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. Urethane protected derivatives of 1-guanylpyrazole for the mild and efficient preparation of guanidines. Tetrahedron Lett. 1993, 34, 3389– 3392,  DOI: 10.1016/S0040-4039(00)79163-5
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    Urethane protected derivatives of 1-guanylpyrazole for the mild and efficient preparation of guanidines
    Bernatowicz, Michael S.; Wu, Youling; Matsueda, Gary R.
    Tetrahedron Letters (1993), 34 (21), 3389-92CODEN: TELEAY; ISSN:0040-4039.
    Bis-urethane protected derivs. I (R = tert-butoxy- or benzyloxycarbonyl) of 1-guanylpyrazole were prepd. and found to readily react with relatively unreactive amines R1NH2 (R1 = Ph, CF3CH2, p-O2NC6H4) at room temp. to produce bis-protected guanidines R1NHC(:NR)NHR in good yields. Simultaneous removal of both protecting groups from these products efficiently produced monosubstituted guanidines R1NHC(:NH)NH2.
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  39. 39
    Feichtinger, K.; Sings, H. L.; Baker, T. J.; Matthews, K.; Goodman, M. Triurethane-protected guanidines and triflyldiurethane-protected guanidines: new reagents for guanidinylation reactions. J. Org. Chem. 1998, 63, 8432– 8439,  DOI: 10.1021/jo9814344
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    Triurethane-protected guanidines and triflyldiurethane-protected guanidines: new reagents for guanidinylation reactions
    Feichtinger, Konrad; Sings, Heather L.; Baker, Tracy J.; Matthews, Kenneth; Goodman, Murray
    Journal of Organic Chemistry (1998), 63 (23), 8432-8439CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    New guanidinylation reagents are reported. These reagents consist of N,N',N''-tri-Boc-guanidine and N,N',N''-tri-Cbz-guanidine, which allow for the facile conversion of alcs. to substituted guanidines. A series of arginine analogs were synthesized via condensation of a primary or secondary alc. with the guanidinylation reagents, under Mitsunobu conditions to produce protected alkylated guanidines. In addn., an extended study of the previously reported reagents N,N'-di-Boc-N''-triflylguanidine (I) and N,N'-di-Cbz-N''-triflylguanidine is presented. The triflyldiurethane-protected guanidine I was utilized to guanidinylate primary and secondary amines under mild conditions with high yield in both soln. and on solid phase.
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    Porcheddu, A.; De Luca, L.; Giacomelli, G. A mild and inexpensive procedure for the synthesis of N, N′-di-Boc-protected guanidines. Synlett 2009, 2009, 3368– 3372,  DOI: 10.1055/s-0029-1218365
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    Musiol, H.-J.; Moroder, L. N, N′-Di-tert-butoxycarbonyl-1 H-benzotriazole-1-carboxamidine Derivatives Are Highly Reactive Guanidinylating Reagents. Org. Lett. 2001, 3, 3859– 3861,  DOI: 10.1021/ol010191q
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    N,N'-Di-tert-butoxycarbonyl-1H- benzotriazole-1-carboxamidine Derivatives Are Highly Reactive Guanidinylating Reagents
    Musiol, Hans-Juergen; Moroder, Luis
    Organic Letters (2001), 3 (24), 3859-3861CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
    Electron-withdrawing substituents such as the 5-chloro or 6-nitro substituent enhance the leaving group character of benzotriazole in N,N'-di-tert-butoxycarbonyl-1H-benzotriazole-1-carboxamidines, giving highly efficient reagents for conversion of primary and secondary amines in soln. and in solid phase to diprotected guanidines.
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    Zheng, Z.; Chen, P.; Li, G.; Zhu, Y.; Shi, Z.; Luo, Y.; Zhao, C.; Fu, Z.; Cui, X.; Ji, C. Mechanistic study of CBT-Cys click reaction and its application for identifying bioactive N-terminal cysteine peptides in amniotic fluid. Chem. Sci. 2017, 8, 214– 222,  DOI: 10.1039/c6sc01461e
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    Mechanistic study of CBT-Cys click reaction and its application for identifying bioactive N-terminal cysteine peptides in amniotic fluid
    Zheng, Zhen; Chen, Peiyao; Li, Gongyu; Zhu, Yunxia; Shi, Zhonghua; Luo, Yufeng; Zhao, Chun; Fu, Ziyi; Cui, Xianwei; Ji, Chenbo; Wang, Fuqiang; Huang, Guangming; Liang, Gaolin
    Chemical Science (2017), 8 (1), 214-222CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    CBT-Cys click condensation reaction has a high second-order reaction rate const. and has found wide applicability in recent years. However, its reaction mechanism has not been exptl. validated and its application for identifying bioactive N-terminal Cys peptides in real clin. samples has not been reported. Herein, firstly, by employing induced nanoelectrospray ionization-mass spectrometry (InESI-MS) and a home-built micro-reactor, we successfully intercepted and structurally characterized the crucial intermediate in this click reaction for the first time. With the intermediate, the proposed mechanism of this reaction was corroborated. Moreover, we also applied this MS setup to monitor the reaction in real time and obtained the second-order reaction rate consts. of this reaction at different pH values. After mechanistic study, we applied this click reaction for identifying bioactive N-terminal cysteine peptides in amniotic fluid (AF). Eight unique N-terminal Cys peptides in AF, three of which are located in the functional domain regions of their corresponding proteins, were identified with a false pos. rate less than 1%. One of the three peptides was found able to inhibit the growth of uterine endometrial cancer HEC-1-B cells but not the endometrial normal cells via a typical apoptotic pathway.
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    Liang, G.; Ren, H.; Rao, J. A biocompatible condensation reaction for controlled assembly of nanostructures in living cells. Nat. Chem. 2010, 2, 54– 60,  DOI: 10.1038/nchem.480
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    A biocompatible condensation reaction for controlled assembly of nanostructures in living cells
    Liang, Gaolin; Ren, Hongjun; Rao, Jianghong
    Nature Chemistry (2010), 2 (1), 54-60CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    Through controlled synthesis and mol. assembly, biol. systems are able to organize mols. into supramol. structures that carry out sophisticated processes. Although chemists have reported a few examples of supramol. assembly in, the controlled covalent synthesis of large mols. and structures in vivo has remained challenging. Here the authors report a condensation reaction between 1,2-aminothiol and 2-cyanobenzothiazole that occurs in vitro and in living cells under the control of either pH, disulfide redn. or enzymic cleavage. In vitro, the size and shape of the condensation products, and the nanostructures subsequently assembled, were different in each case and could thus be controlled by tuning the structure of the monomers. Direct imaging of the products obtained in the cells revealed their locations-near the Golgi bodies under enzymic cleavage control-demonstrating the feasibility of a controlled and localized reaction in living cells. This intracellular condensation process enabled the imaging of the proteolytic activity of furin.
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    Proj, M.; Strašek, N.; Pajk, S.; Knez, D.; Sosič, I. Tunable Heteroaromatic Nitriles for Selective Bioorthogonal Click Reaction with Cysteine. Bioconjugate Chem. 2023, 34, 1271– 1281,  DOI: 10.1021/acs.bioconjchem.3c00163
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    Tunable Heteroaromatic Nitriles for Selective Bioorthogonal Click Reaction with Cysteine
    Proj, Matic; Strasek, Nika; Pajk, Stane; Knez, Damijan; Sosic, Izidor
    Bioconjugate Chemistry (2023), 34 (7), 1271-1281CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    The binucleophilic properties of 1,2-aminothiol and its rare occurrence in nature make it a useful reporter for tracking mols. in living systems. The 1,2-aminothiol moiety is present in cysteine, which is a substrate for a biocompatible click reaction with heteroarom. nitriles. Despite the wide range of applications for this reaction, the scope of nitrile substrates has been explored only to a limited extent. In this study, we expand the chem. space of heteroarom. nitriles for bioconjugation under physiol. relevant conditions. We systematically assembled a library of 116 2-cyanobenzimidazoles, 1-methyl-2-cyanobenzimidazoles, 2-cyanobenzothiazoles, and 2-cyanobenzoxazoles contg. electron-donating and electron-withdrawing substituents at all positions of the benzene ring. The compds. were evaluated for their stability, reactivity, and selectivity toward the N-terminal cysteine of model oligopeptides. In comparison to the benchmark 6-hydroxy-2-cyanobenzothiazole or 6-amino-2-cyanobenzothiazole, we provide highly selective and moderately reactive nitriles as well as highly reactive yet less selective analogs with a variety of enabling attachment chemistries to aid future applications in bioconjugation, chem. biol., and nanomaterial science.
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    Sondag, D.; de Kleijne, F. F. J.; Castermans, S.; Chatzakis, I.; van Geffen, M.; van’t Veer, C.; van Heerde, W. L.; Boltje, T. J.; Rutjes, F. P. J. T. Synthesis and Evaluation of Glycosyl Luciferins. Chem.─Eur. J. 2023, 29, e202302547  DOI: 10.1002/chem.202302547
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    Lottenberg, R.; Hall, J. A.; Blinder, M.; Binder, E. P.; Jackson, C. M. The action of thrombin on peptide p-Nitroanilide substrates: Substrate selectivity and examination of hydrolysis under different reaction condtions. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1983, 742, 539– 557,  DOI: 10.1016/0167-4838(83)90272-8
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    The action of thrombin on peptide p-nitroanilide substrates. Substrate selectivity and examination of hydrolysis under different reaction conditions
    Lottenberg, Richard; Hall, Julie A.; Blinder, Morey; Binder, Ellen P.; Jackson, Craig M.
    Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1983), 742 (3), 539-57CODEN: BBAEDZ; ISSN:0167-4838.
    Kinetic parameters for the action of bovine α-thrombin on 24 com. available peptide p-nitroanilides were detd. The selectivity const., kcat/Km, ranged from 3.3 × 101 to 1.1 × 108 M-1 s-1 for the poorest and the best substrates, resp. The best substrates for thrombin were identified as those with arginine in the P1 position, proline or a proline homolog in the P2 position, and an apolar amino acid in the P3 position. A quant. distinction between lysine and arginine in the P1 position and other amino acids in the P2-P4 positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. The effects of NaCl, CaCl2, and polyethylene glycol concns., and pH and temp. on the action of thrombin on selected substrates were assessed. A source of large systematic error in thrombin concn. ests. was identified as resulting from adsorption losses. These losses were eliminated by inclusion of polyethylene glycol in diln. and reaction buffers.
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    Tian, G.; Fedoseev, P.; Van der Eycken, E. V. Hypervalent Iodine (III)-Mediated Cascade Cyclization of Propargylguanidines and Total Syntheses of Kealiinine B and C. Chem.─Eur. J. 2017, 23, 5224– 5227,  DOI: 10.1002/chem.201700934
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    47
    Hypervalent Iodine(III)-Mediated Cascade Cyclization of Propargylguanidines and Total Syntheses of Kealiinine B and C
    Tian, Guilong; Fedoseev, Pavel; Van der Eycken, Erik V.
    Chemistry - A European Journal (2017), 23 (22), 5224-5227CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    An oxidative cascade cyclization of propargylguanidines promoted by phenyliodonium diacetate (PIDA) was developed. The protocol provides an efficient route for the synthesis of the alkaloids kealiinines B (I) and C (II) as well as homologues. The difference in the electronic nature of the acetylene substituent resulted in two ways of the cyclization. A plausible mechanism is proposed based on the exptl. results.
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Cite this: Bioconjugate Chem. 2023, 34, 12, 2234–2242
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https://doi.org/10.1021/acs.bioconjchem.3c00381
Published December 6, 2023

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  • Abstract

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    Scheme 1

    Scheme 1. (A) General Reaction Scheme for an Aminoluciferin (aLuc)-Based Assay; upon Enzymatic Activity, d-Aminoluciferin Is Released from the Caged Substrate, Which Can Subsequently Be Converted into a Proportional Light Signal in the Presence of Luciferase, ATP, Mg2+, and O2 and (B) Luciferase-Catalyzed Luminescence Reaction Mechanism as Found in Fireflies
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    Figure 1

    Figure 1. (A) Conventional solution-phase method for the synthesis of aminoluciferin peptides via C-terminal activation of a protected peptide and the subsequent side chain deprotection and d-cysteine condensation all in solution. (B) Hybrid SPPS method reported by Kovacs et al., loading of the aminoluciferin on a solid support afforded the dehydrogenated product. (C) This work with the side chain anchoring of the P1 amino acid residue on a solid support and the subsequent regular Fmoc-based SPPS chemistry.

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    Scheme 2

    Scheme 2. Synthesis of the Luminescent Precursor Amino Acids 6 and 7a
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    a(i) 6-ABTC (1, 1 equiv), PCl3 (0.51 equiv), pyr, 3.5 h, 40 °C, 77–98%; (ii) HCO2H, lyophilization, quant.

    Scheme 3

    Scheme 3. Validation Solid-Phase Peptide Synthesis Library of Luminescent Caged Peptides via Side Chain Anchoring of Amino Acids 6 or 7 via Reductive Amination with R1 ((3-Formylindolyl)acetamido Methyl Resin)a
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    a(i) 6 or 7 (2 equiv), THF/TMOF (1:1), 4 h, rt. (ii) NaBH3CN (2 equiv), AcOH (3.5 equiv), 2 h, rt. (iii) For n = 3: 8 (2 equiv), DIC (2 equiv), DCM, rt, 16 h. For n = 4: Boc2O (3 equiv), DIPEA (3 equiv), DMF, rt, 16 h. (iv) (a) 3% DBU in DMF, DMF (3×); (b) HOBt (3.6 equiv), DIC (3.3 equiv), Fmoc-AA–OH/Cap–OH (methoxy acetic acid) (3 equiv) (3×). (v) DMF. (v) TFA/DCM (1:1, v/v), 2 h

    Scheme 4

    Scheme 4. Conversion of Resin-Bound Secondary Amine R2 into the Di-Boc-Protected Guanidine R4 and the Observed Thiourea Byproduct R8 When Using N,N′-Di-Boc-thiourea (8) and DIC Activationa
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    aThe use of 9 solely afforded the desired resin-bound product R4.

    Scheme 5

    Scheme 5. General Structures of the Two Large Tripeptide Libraries That Were Synthesized in This Work Starting from the Key Intermediate Resins R4 and R5a
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    aP3 = natural amino acid, P2 = natural amino acid and P1 = either arginine (n = 3) or lysine (n = 4). After regular Fmoc SPPS chemistry and subsequent RP-HPLC purification after cleavage, a total amount of 719 peptides was obtained.

    Scheme 6

    Scheme 6. Total Luminescence of Substrate Candidates (β-AXR/β-AXK) Using Thrombin and Factor Xaa
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    a(i) β-AXR/β-AXK substrate (1 equiv) d-cysteine (1.5 equiv), buffer (25 mM HEPES, 125 mM NaCl, 0.5% BSA, pH = 7.4), 37 °C, 30 min. (ii) Protease (thrombin/FXa, final conc 10 nM), ATP (1.5 equiv), MgCl2 (10 equiv), Quantilum recombinant firefly luciferase (final conc 10 μM). Screening of peptide library with the substrates (667 μM) methoxyacetyl-βAXR-6ABTC (in blue) and methoxyacetyl-βAXK-6ABTC (in orange) after the condensation reaction with d-cysteine. The proteolytic activity of the substrates towards both factor Xa and thrombin were determined and their ratio was plotted (ratio 1:1 y = x) against the total luminescence for either thrombin or FXa (x- and y-axis, respectively).

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      Rodriguez-Rios, M.; Megia-Fernandez, A.; Norman, D. J.; Bradley, M. Peptide probes for proteases-innovations and applications for monitoring proteolytic activity. Chem. Soc. Rev. 2022, 51, 2081– 2120,  DOI: 10.1039/D1CS00798J
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      Peptide probes for proteases - innovations and applications for monitoring proteolytic activity
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      Constructing firefly luciferin bioluminescence probes for in vivo imaging
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      A review. Bioluminescence imaging (BLI) is a widely applied visual approach for real-time detecting many physiol. and pathol. processes in a variety of biol. systems. Based on the caging strategy, lots of bioluminescent probes have been well developed. While the targets react with recognizable groups, caged luciferins liberate luciferase substrates, which react with luciferase generating a bioluminescent response. Among the various bioluminescent systems, the most widely utilized bioluminescent system is the firefly luciferin system. The H and carboxylic acid of luciferin are critically caged sites. The introduced self-immolative linker extends the applications of probes. Firefly luciferin system probes have been successfully applied for analyzing physiol. processes, monitoring the environment, diagnosing diseases, screening candidate drugs, and evaluating the therapeutic effect. Here, we systematically review the general design strategies of firefly luciferin bioluminescence probes and their applications. Bioluminescence probes provide a new approach for facilitating investigation in a diverse range of fields. It inspires us to explore more robust light emission luciferin and novel design strategies to develop bioluminescent probes.
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      A protein-labeling method based on the use of a single amino acid tag - an N-terminal cysteine residue - and small-mol. probes contg. a cyanobenzothiazole (CBT) unit has been used for the specific fluorescence labeling of proteins in vitro and at the surface of live cells. This simple ligation reaction proceeds with a high degree of specificity under physiol. conditions.
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      15
      In Vivo Bioluminescence Imaging of Furin Activity in Breast Cancer Cells Using Bioluminogenic Substrates
      Dragulescu-Andrasi, Anca; Liang, Gaolin; Rao, Jianghong
      Bioconjugate Chemistry (2009), 20 (8), 1660-1666CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Furin, a proprotein convertases family endoprotease, processes numerous physiol. substrates and is overexpressed in cancer and inflammatory conditions. Noninvasive imaging of furin activity will offer a valuable tool to probe furin function over the course of tumor growth and migration in the same animals in real time and directly assess the inhibition efficacy of drugs in vivo. Here, we report successful bioluminescence imaging of furin activity in xenografted MBA-MB-468 breast cancer tumors in mice with bioluminogenic probes. The probes are conjugates of furin substrate, a consensus amino acid motif R-X-K/R-R (X, any amino acid), with the firefly luciferase substrate D-aminoluciferin. In the presence of the luciferase reporter, the probes are unable to produce bioluminescent emission without furin activation. Blocking expts. with a furin inhibitor and control expts. with a scrambled probe showed that the bioluminescence emission in the presence of firefly luciferase is furin-dependent and specific. After furin activation, a 30-fold increase in the bioluminescent emission was obsd. in vitro, and on av., a 7-8-fold contrast between the probe and control was seen in the same tumor xenografts in mice. Direct imaging of furin activity may facilitate the study of furin function in tumorigenicity and the discovery of new drugs for furin-targeted cancer therapy.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXpt1Gqt7o%253D&md5=55b233f364cf02d0535645b971a535be
    16. 16
      Sondag, D.; Merx, J.; Rossing, E.; Boltje, T. J.; Löwik, D. W. P. M.; Nelissen, F. H. T.; van Geffen, M.; van’t Veer, C.; van Heerde, W. L.; Rutjes, F. P. J. T. Luminescent Assay for the Screening of SARS-CoV-2 MPro Inhibitors. ChemBioChem 2022, 23, e202200190  DOI: 10.1002/cbic.202200190
      16
      Luminescent Assay for the Screening of SARS-CoV-2 MPro Inhibitors
      Sondag, Daan; Merx, Jona; Rossing, Emiel; Boltje, Thomas J.; Lowik, Dennis W. P. M.; Nelissen, Frank H. T.; van Geffen, Mark; van 't Veer, Cornelis; van Heerde, Waander L.; Rutjes, Floris P. J. T.
      ChemBioChem (2022), 23 (15), e202200190CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Since the outbreak of SARS-CoV-2 in Dec. 2019 millions of infections have been reported globally. The viral chymotrypsin-like main protease (MPro) exhibits a crucial role in viral replication and represents a relevant target for antiviral drug development. In order to screen potential MPro inhibitors we developed a luminescent assay using a peptide based probe contg. a cleavage site specific for MPro. This assay was validated showing IC50 values similar to those reported in the literature for known MPro inhibitors and can be used to screen new inhibitors.
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    17. 17
      Merrifield, R. B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149– 2154,  DOI: 10.1021/ja00897a025
      17
      Solid phase peptide synthesis. I. The synthesis of a tetrapeptide
      Merrifield, R. B.
      Journal of the American Chemical Society (1963), 85 (14), 2149-54CODEN: JACSAT; ISSN:0002-7863.
      A new approach to the chem. synthesis of polypeptides was investigated. It involved the stepwise addition of protected amino acids to a growing peptide chain which was bound by a covalent bond to a solid resin particle. This provided a procedure whereby reagents anti by-products were removed by filtration, and the recrystn. of intermediates was eliminated. The advantages of the new method were speed and simplicity of operation. The feasibility of the idea was demonstrated by the synthesis of the model tetrapeptide L-leucyl-L-alanylglycyl-L-valine. The peptide was identical with a sample prepd. by the standard p-nitrophenyl ester procedure.
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    18. 18
      Kovács, A. K.; Hegyes, P.; Szebeni, G. J.; Bogár, K.; Puskás, L. G.; Tóth, G. K. Synthesis of N-Peptide-6-Amino-d-Luciferin Conjugates with Optimized Fragment Condensation Strategy. Int. J. Pept. Res. Ther. 2019, 25, 1209– 1215,  DOI: 10.1007/s10989-018-9768-8
      18
      Synthesis of N-Peptide-6-Amino-D-Luciferin Conjugates with Optimized Fragment Condensation Strategy
      Kovacs, Anita K.; Hegyes, Peter; Szebeni, Gabor J.; Bogar, Krisztian; Puskas, Laszlo G.; Toth, Gabor K.
      International Journal of Peptide Research and Therapeutics (2019), 25 (3), 1209-1215CODEN: IJPRFC; ISSN:1573-3149. (Springer)
      The synthesis of peptide-luciferin conjugates has a pivotal role in the development of bioluminescent detection systems that are based on the detn. of protease enzyme activity. This work describes the optimized synthesis of an N-peptide-6-amino-D-luciferin conjugate (Fmoc-Gly-Pro-6-amino-D-luciferin) with a simple fragment condensation method in adequate yields. Fmoc-Gly-Pro-6-amino-D-luciferin was produced from a previously synthesized Fmoc-Gly-Pro-OH and also previously synthesized 6-amino-2-cyanobenzothiazole with an optimized method, to which conjugate cysteine was added in an also improved way. The resulting conjugate was successfully used in a bioluminescent system, in vitro, demonstrating the applicability of the method.
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    19. 19
      White, E. H.; McCapra, F.; Field, G. F. The structure and synthesis of firefly luciferin. J. Am. Chem. Soc. 1963, 85, 337– 343,  DOI: 10.1021/ja00886a019
      19
      The structure and synthesis of firefly luciferin
      White, Emil H.; McCapra, Frank; Field, George F.
      Journal of the American Chemical Society (1963), 85 (), 337-43CODEN: JACSAT; ISSN:0002-7863.
      Luciferin from Photinus pyralis, was shown to be D-2-(6-hydroxy-2-benzothiazolyl)-δ2-thiazoline-4-carboxylic acid (I). The structure was confirmed by a total synthesis. L-Luciferin, DL-luciferin, dehydroluciferin, and various related benzothiazoles were also synthesized.
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    20. 20
      Fontes, R.; Dukhovich, A.; Sillero, A.; Sillero, M. A. G. Synthesis of dehydroluciferin by firefly luciferase: effect of dehydroluciferin, coenzyme A and nucleoside triphosphates on the luminescent reaction. Biochem. Biophys. Res. Commun. 1997, 237, 445– 450,  DOI: 10.1006/bbrc.1997.7161
      20
      Synthesis of dehydroluciferin by firefly luciferase: effect of dehydroluciferin, coenzyme A and nucleoside triphosphates on the luminescent reaction
      Fontes, Rui; Dukhovich, Alexey; Sillero, Antonio; Sillero, Maria A. Gunther
      Biochemical and Biophysical Research Communications (1997), 237 (2), 445-450CODEN: BBRCA9; ISSN:0006-291X. (Academic)
      The formation of dehydroluciferin (L) from luciferin (LH2) in the reaction catalyzed by firefly luciferase (EC 1.13.12.7) (I) was studied. The I·LH2-AMP complex may follow 2 different pathways: toward prodn. of light and toward the synthesis of the I·L-AMP complex. This last step has an inhibitory effect on light emission as mols. of the enzyme are trapped in a light unproductive complex. The effects of CoA and nucleoside 5'-triphosphates (NTPs) on light emission were quant. different. CoA combined with the L moiety of the I·L-AMP complex, yielding L-CoA, promoting liberation of free I, and increasing light yield. The NTPs reacted with the AMP moiety of the same complex, generating adenosine(5')tetraphospho(5')nucleoside (Ap4N) and, probably, the I·L complex, and scarcely increasing light prodn. The results are discussed in relation to previous reports by others on I.
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    21. 21
      Evnin, L. B.; Vásquez, J. R.; Craik, C. S. Substrate specificity of trypsin investigated by using a genetic selection. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 6659– 6663,  DOI: 10.1073/pnas.87.17.6659
      21
      Substrate specificity of trypsin investigated by using a genetic selection
      Evnin, Luke B.; Vasquez, John R.; Craik, Charles S.
      Proceedings of the National Academy of Sciences of the United States of America (1990), 87 (17), 6659-63CODEN: PNASA6; ISSN:0027-8424.
      The structural determinants of the primary substrate specificity of rat anionic trypsin were examd. by using oligonucleotide-directed mutagenesis coupled to a genetic selection. A library was created that encoded trypsins substituted at amino acid positions 189 and 190 at the base of the substrate binding pocket. A genetic selection, with a dynamic range of 5 orders of proteolytic activity, was used to search 90,000 transformants of the library. Rapid screening for arginyl amidolysis and esterolysis confirmed the activity of the purified isolates. Trypsin and 15 mutant trypsins with partially preserved function were identified and characterized kinetically on arginyl and lysyl peptide substrates. Alternative arrangements of amino acids in the substrate binding pocket sustained efficient catalysis. A neg. charge at amino acid position 189 or 190 was shown to be essential for high-level catalysis. With the favored aspartic acid residue at position 189, several amino acids could replace serine at position 190. Modulation of the specificity for arginine and lysine substrates was shown to depend on the amino acid at position 190. The regulatory effect of the amino acid side chain at position 190 on the substrate specificity is also reflected in substrate binding pockets of naturally occurring trypsin homologs.
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    22. 22
      Sondag, D.; Verhoeven, S.; Löwik, D. W. P. M.; van Geffen, M.; Veer, C. v.; van Heerde, W. L.; Boltje, T. J.; Rutjes, F. P. J. T. Activity Sensing of Coagulation and Fibrinolytic Proteases. Chem.─Eur. J. 2023, 29, e202203473  DOI: 10.1002/chem.202203473
      22
      Activity Sensing of Coagulation and Fibrinolytic Proteases
      Sondag, Daan; Verhoeven, Stijn; Lowik, Dennis W. P. M.; van Geffen, Mark; Veer, Cornelis van't; van Heerde, Waander L.; Boltje, Thomas J.; Rutjes, Floris P. J. T.
      Chemistry - A European Journal (2023), 29 (18), e202203473CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. The blood coagulation cascade is a complex physiol. process involving the action of multiple coupled enzymes, cofactors, and substrates, ultimately leading to clot formation. Serine proteases have a crucial role, and aberrations in their activity can lead to life-threatening bleeding disorders and thrombosis. This review summarizes the essential proteases involved in blood coagulation and fibrinolysis, the endogenous peptide sequences they recognize and hydrolyze, and synthetic peptide probes based on these sequences to measure their activity. The information in this review can contribute to developing novel anticoagulant therapies and specific substrates for point-of-care diagnosis of coagulation pathologies.
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    23. 23
      Beythien, J.; Barthélémy, S.; Schneeberger, P.; White, P. D. A novel solid-phase linker strategy for the side-chain anchoring of arginine: an expeditious route to arginine 7-amido-4-methylcoumarins. Tetrahedron Lett. 2006, 47, 3009– 3012,  DOI: 10.1016/j.tetlet.2006.03.019
      23
      A novel solid-phase linker strategy for the side-chain anchoring of arginine: an expeditious route to arginine 7-amido-4-methylcoumarins
      Beythien, Joerg; Barthelemy, Sophie; Schneeberger, Peter; White, Peter D.
      Tetrahedron Letters (2006), 47 (18), 3009-3012CODEN: TELEAY; ISSN:0040-4039. (Elsevier B.V.)
      A novel linker strategy for the efficient side-chain anchoring of arginine is described. The utility of this approach was demonstrated by the facile synthesis of arginine-specific fluorogenic peptide substrates by std. Fmoc solid phase peptide synthesis (Fmoc = 9-fluorenylmethyloxycarbonyl) methods.
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    24. 24
      Hamzé, A.; Martinez, J.; Hernandez, J.-F. Solid-phase synthesis of arginine-containing peptides and fluorogenic substrates using a side-chain anchoring approach. J. Org. Chem. 2004, 69, 8394– 8402,  DOI: 10.1021/jo048792t
      24
      Solid-Phase Synthesis of Arginine-Containing Peptides and Fluorogenic Substrates Using a Side-Chain Anchoring Approach
      Hamze, Abdallah; Martinez, Jean; Hernandez, Jean-Francois
      Journal of Organic Chemistry (2004), 69 (24), 8394-8402CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      Attachment of an amino acid to a solid support by its side chain is sometimes necessary in order to make the α-carboxylic group available for diverse modifications, such as incorporation of fluorophores. In contrast to most other amino acids, anchoring the guanidinium group of an arginine to a resin requires the use of a supplementary linker. To avoid the usually multistep synthesis of such a linker as well as its difficult attachment to the guanidine group, the authors developed a simple method where the guanidine group is built on a Rink amide resin. The strategy followed the steps of guanidine formation: (i) addn. of an isothiocyanate deriv. of ornithine to the amino group of a solid support, yielding Nω-linked thiocitrulline; (ii) S-methylation of thiourea; (iii) guanidinylation using ammonium acetate. Cleavage of the resin generated the arginine-contg. compd., the amine group of the resin becoming part of the guanidine. Thus, this strategy was successfully applied to the synthesis of a series of fluorogenic substrates for trypsin-like serine proteases (Ac-Val-Gly-Leu-Arg-AMC, Ac-Val-Pro-Arg-AMC, Ac-Ile-Glu-Gly-Arg-AMC, Ac-Ala-Lys-Arg-AMC, etc.; AMC = 7-amino-4-methylcoumarin) , which were obtained in high yields and purities. Similarly, substituted arginine derivs., including Nω-methyl- and Nω-ethyl-arginines, were prepd.
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    25. 25
      Boas, U.; Brask, J.; Jensen, K. J. Backbone amide linker in solid-phase synthesis. Chem. Rev. 2009, 109, 2092– 2118,  DOI: 10.1021/cr068206r
      25
      Backbone Amide Linker in Solid-Phase Synthesis
      Boas, Ulrik; Brask, Jesper; Jensen, Knud J.
      Chemical Reviews (Washington, DC, United States) (2009), 109 (5), 2092-2118CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The present review provides a comprehensive survey of the by now widely used BAL (backbone amide linker) chem. methodol., which extends far beyond applications in peptide chem.
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    26. 26
      Ten Brink, H. T.; Meijer, J. T.; Geel, R. V.; Damen, M.; Löwik, D. W. P. M.; van Hest, J. C. M. Solid-phase synthesis of C-terminally modified peptides. J. Pept. Sci. 2006, 12, 686– 692,  DOI: 10.1002/psc.780
      26
      Solid-phase synthesis of C-terminally modified peptides
      Ten Brink, Hefziba T.; Meijer, Joris T.; Geel, Remon V.; Damen, Mark; Loewik, Dennis W. P. M.; Van Hest, Jan C. M.
      Journal of Peptide Science (2006), 12 (11), 686-692CODEN: JPSIEI; ISSN:1075-2617. (John Wiley & Sons Ltd.)
      In this paper, a straightforward and generic protocol is presented to label the C-terminus of a peptide with any desired moiety that is functionalized with a primary amine. Amine-functional mols. included are polymers (useful for hybrid polymers), long alkyl chains (used in peptide amphiphiles and stabilization of peptides), propargyl amine and azido propyl-amine (desirable for 'click' chem.), dansyl amine (fluorescent labeling of peptides) and crown ethers (peptide switches/hybrids). In the first part of the procedure, the primary amine is attached to an aldehyde-functional resin via reductive amination. To the secondary amine that is produced, an amino acid sequence is coupled via a std. solid-phase peptide synthesis protocol. Since one procedure can be applied for any given amine-functional moiety, a robust method for C-terminal peptide labeling is obtained.
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    27. 27
      Jensen, K. J.; Alsina, J.; Songster, M. F.; Vágner, J.; Albericio, F.; Barany, G. Backbone Amide Linker (BAL) strategy for solid-phase synthesis of C-terminal-modified and cyclic peptides1, 2, 3. J. Am. Chem. Soc. 1998, 120, 5441– 5452,  DOI: 10.1021/ja974116f
      27
      Backbone Amide Linker Strategy for Solid-Phase Synthesis of C-Terminal-Modified and Cyclic Peptides
      Jensen, Knud J.; Alsina, Jordi; Songster, Michael F.; Vagner, Josef; Albericio, Fernando; Barany, George
      Journal of the American Chemical Society (1998), 120 (22), 5441-5452CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Peptide targets for synthesis are often desired with C-terminal end groups other than the more usual acid and amide functionalities. Relatively few routes exist for synthesis of C-terminal-modified peptides-including cyclic peptides-by either soln. or solid-phase methods, and known routes are often limited in terms of ease and generality. The authors describe a novel backbone amide linker (BAL) approach, whereby the growing peptide is anchored through a backbone nitrogen, thus allowing considerable flexibility in management of the termini. Initial efforts on BAL have adapted the chem. of the tris(alkoxy)benzylamide system exploited previously with PAL anchors. Aldehyde precursors to PAL, e.g. 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid, were reductively coupled to the prospective C-terminal amino acid α-amino group, which was blocked as a tert-Bu, allyl, or Me ester, or to the appropriately protected C-terminal-modified amino acid deriv. These reductive aminations were carried out either in soln. or on the solid phase and occurred without racemization. The secondary amine intermediates resulting from soln. amination were converted to the 9-fluorenylmethoxycarbonyl (Fmoc)-protected preformed handle derivs., which were then attached to poly(ethylene glycol)-polystyrene graft or copoly(styrene-1%-divinylbenzene) supports and used to assemble peptides by std. Fmoc solid-phase chem. Alternatively, BAL anchors formed by on-resin reductive amination were applied directly. Conditions were optimized to achieve near-quant. acylation at the difficult step to introduce the penultimate residue, and a side reaction involving diketopiperazine formation under some circumstances was prevented by a modified protocol for Nα-protection of the second residue/introduction of the third residue. Examples are provided for the syntheses in high yields and purities of representative peptide acids, alcs., N,N-dialkylamides, aldehydes, esters, and head-to-tail cyclic peptides. These methodologies avoid postsynthetic soln.-phase transformations and are ripe for further extension.
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    28. 28
      Alsina, J.; Jensen, K. J.; Albericio, F.; Barany, G. Solid-Phase Synthesis with Tris (alkoxy) benzyl Backbone Amide Linkage (BAL)[≠]. Chem.─Eur. J. 1999, 5, 2787– 2795,  DOI: 10.1002/(sici)1521-3765(19991001)5:10<2787::aid-chem2787>3.0.co;2-2
      28
      Solid-phase synthesis with tris(alkoxy)benzyl backbone amide linkage (BAL)
      Alsina, Jordi; Jensen, Knud J.; Albericio, Fernando; Barany, George
      Chemistry - A European Journal (1999), 5 (10), 2787-2795CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH)
      This review with 29 refs. discusses solid-phase synthesis of peptides, small proteins, oligonucleotides, and small org. mols. A crucial part of the overall synthesis plan is the choice of an appropriate "handle" (linker) for attaching the org. reactant to the solid support. The authors describe a novel and general concept for solid-phase synthesis that involves attachment of a backbone amide nitrogen to an appropriate handle. This backbone amide linker (BAL) approach allows for the prepn. of C-terminal-modified and cyclic peptides, small org. mols., and modified amino sugars, as well as combinatorial synthesis applications.
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    29. 29
      Hauser, J. R.; Beard, H. A.; Bayana, M. E.; Jolley, K. E.; Warriner, S. L.; Bon, R. S. Economical and scalable synthesis of 6-amino-2-cyanobenzothiazole. Beilstein J. Org. Chem. 2016, 12, 2019– 2025,  DOI: 10.3762/bjoc.12.189
      29
      Economical and scalable synthesis of 6-amino-2-cyanobenzothiazole
      Hauser, Jacob R.; Beard, Hester A.; Bayana, Mary E.; Jolley, Katherine E.; Warriner, Stuart L.; Bon, Robin S.
      Beilstein Journal of Organic Chemistry (2016), 12 (), 2019-2025CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)
      2-Cyanobenzothiazoles (CBTs) were the useful building blocks for luciferin derivs., for bioluminescent imaging, handles and for bioorthogonal ligations. An economical and scalable synthesis of 6-amino-2-cyanobenzothiazole based on a cyanation catalyzed by 1,4-diazabicyclo[2.2.2]octane (DABCO) was presented and its advantages for scale-up over previously reported routes was also discussed.
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    30. 30
      Grimmel, H.; Guenther, A.; Morgan, J. F. Phosphazo compounds and their use in preparing amides. J. Am. Chem. Soc. 1946, 68, 539– 542,  DOI: 10.1021/ja01208a001
      30
      Phosphazo compounds and their rise in preparing amides
      Grimmel, H. W.; Guenther, A.; Morgan, Jack F.
      Journal of the American Chemical Society (1946), 68 (), 539-42CODEN: JACSAT; ISSN:0002-7863.
      Two moles of PhNH2 in 300 mL. PhMe at room temp. or (for slightly sol. amines) at 50-70°, treated dropwise with 55 g. PCl3 in 50 mL. PhMe and refluxed and agitated for 1-2 h., give 67.5% of phenylphosphazoanilide (PhN:PNHPh), m. 251-3°; the 4,4'-di-Me deriv. m. 197-200° (preheated bath), 42%; the 2,2'-di-MeO deriv. m. 138-40°, 32%. If the RN:PNHR is treated with a carboxylic acid, R'CO2H, in PhMe and the mixt. is refluxed for 2 h., HPO2 and N-substituted amides, R'CONHR (I) are formed. The following yields of I (R' = Ph) (%) were obtained from the amines listed and BzOH: PhNH2 85, o-, m-, and p-MeC6H4NH2 72, 80, and 56, m-ClC6H4NH2 75, o-MeOC6H4NH2 76, m-O2NC6H4NH2 70, m-H2NC6H4OH 7 (?), p-H2NC6H4NHPh 30, m- and p-C6H4(NH2)2 11 and 67, (p-H2NC6H4)2 94, (p-H2NC6H4)2CH220, 1-C10H7NH239, p-H2NC6H4SO3H 90, 2-aminopyridine 52 (C5H5N as solvent), cyclohexylamine 68, BuNH2 45, dodecylamine 60-77; o- and p-O2NC6H4NH2 give only tars. p-H2NC5H4NHAc and 1-aminoanthraquinone do not form reactive phosphazo compds., nor do amino carboxylic acids. That the reaction is not peculiar to BzOH was shown by the successful prepn. of the following anilides of various acids: HCO2H, small, AcOH 74, EtCO2H 65, lauric 74, stearic 65, PhCH:CHCO2H 80, HOCH2CO2H 49, (CO2H)2 45, CH2(CO2H)2 65, (CH2CO2H)2 41, pimelic 53, fumaric 34, maleic 16, tartaric 45, BzOH 85, p-O2NC6H4CO2H 83, p-HOC6H4CO2H 74, o-H2NC6H4CO2H 42, o-, m-, and p-C6H4(CO2H)2 73, 55, and 25, 3,2-HOC10H6CO2H 89-100, nicotinic 38. Glycine and alanine did not react. p-H2NC6H4CO2H gave a solid m. above 300°; the o-isomer gave 42% of o-H2NC6H4CONHPh. Na salts of carboxylic acids do not react and the method is not applicable to N-substituted sulfonamides. When an excess of PhNH2 was used in the initial reaction with PCl3, the ultimate yields of BzNHPh were increased to 100%; by also using an excess of BzOH the yield is 115% (based on PCl3). It is also possible to prep. RN:PNHR' from PhN:PCl, though the method is less efficient and more tedious. PhN:PNHPh is readily recrystd. from CHCl3 but the resultant crystals are not identical with the original as shown by x-ray diffraction patterns. Prolonged exposure to light or heat (100° or above) causes slow decompn. though the compds. are stable for months at room temp. or in the dark. X-ray patterns are given also for PhNH2.H3PO3 and (PhNH2)POH.
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    31. 31
      van Heerde, W. L.; van Geffen, M.; Steeghs, D. Novel chemiluminescent substrates for Factor Xa. U.S. Patent 20,210,371,461 A1, 2021.
      There is no corresponding record for this reference.
    32. 32
      Estep, K. G.; Neipp, C. E.; Stephens Stramiello, L. M.; Adam, M. D.; Allen, M. P.; Robinson, S.; Roskamp, E. J. Indole resin: A versatile new support for the solid-phase synthesis of organic molecules. J. Org. Chem. 1998, 63, 5300– 5301,  DOI: 10.1021/jo9806052
      32
      Indole Resin: A Versatile New Support for the Solid-Phase Synthesis of Organic Molecules
      Estep, Kimberly G.; Neipp, Christopher E.; Stramiello, Linda M. Stephens; Adam, Mavis D.; Allen, Martin P.; Robinson, Shaughnessy; Roskamp, Eric J.
      Journal of Organic Chemistry (1998), 63 (16), 5300-5301CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      A resin prepd. from (aminomethyl)polystyrene and 3-formyl-1H-indole-1-acetic acid was an ideal support for prepg. libraries of amides, ureas, sulfonamides, guanidines, carbamates, and aniline-contg. products.
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    33. 33
      Yong, Y. F.; Kowalski, J. A.; Thoen, J. C.; Lipton, M. A. A new reagent for solid and solution phase synthesis of protected guanidines from amines. Tetrahedron Lett. 1999, 40, 53– 56,  DOI: 10.1016/S0040-4039(98)80017-8
      33
      A new reagent for solid and solution phase synthesis of protected guanidines from amines
      Yong, Yaw Fui; Kowalski, Jennifer A.; Thoen, Jason C.; Lipton, Mark A.
      Tetrahedron Letters (1999), 40 (1), 53-56CODEN: TELEAY; ISSN:0040-4039. (Elsevier Science Ltd.)
      A new reagent - 4-nitro-1H-pyrazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine - has been developed to effect the rapid and efficient synthesis of bis(carbamate)-protected guanidines from primary and secondary amines. The reagent is a more electrophilic, and consequently more reactive, deriv. of the literature reagent 1H-pyrazole-1-[N,N'-bis(tert-butoxycarbonyl)]carboxamidine. The increased reactivity of the new reagent affords it increased yields in soln. and the need for fewer equiv. when guanylating resin-bound amines.
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    34. 34
      Pinilla, C.; Appel, J.; Blanc, P.; Houghten, R. Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries. BioTechniques 1992, 13, 901– 905
      34
      Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries
      Pinilla, Clemencia; Appel, Jon R.; Blanc, Philippe; Houghten, Richard A.
      BioTechniques (1992), 13 (6), 901-2, 904-5CODEN: BTNQDO; ISSN:0736-6205.
      A conceptually unique set of individual synthetic peptide combinatorial libraries (SPCLs), termed a positional scanning SPCL (PS-SPCL) are described that can be used for the rapid (i.e., a single day) identification of peptide sequences that bind with high affinity to antibodies, receptors or other acceptor mols. The PS-SPCL described here is made up of six individual positional peptide libraries, each one consisting of hexamers with a single position defined and five positions as mixts. As an example of the utility of such PS-SPCLs, the antigenic determinants recognized by two different monoclonal antibodies were correctly identified upon a single screening.
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    35. 35
      Geffen, M. v.; Loof, A.; Lap, P.; Boezeman, J.; Laros-van Gorkom, B. A. P.; Brons, P.; Verbruggen, B.; Kraaij, M. v.; van Heerde, W. L. A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis. Hematology 2011, 16, 327– 336,  DOI: 10.1179/102453311x13085644680348
      There is no corresponding record for this reference.
    36. 36
      Ube, H.; Uraguchi, D.; Terada, M. Efficient synthetic protocol for substituted guanidines via copper (I)-mediated intermolecular amination of isothiourea derivatives. J. Organomet. Chem. 2007, 692, 545– 549,  DOI: 10.1016/j.jorganchem.2006.06.046
      36
      Efficient synthetic protocol for substituted guanidines via copper(I)-mediated intermolecular amination of isothiourea derivatives
      Ube, Hitoshi; Uraguchi, Daisuke; Terada, Masahiro
      Journal of Organometallic Chemistry (2007), 692 (1-3), 545-549CODEN: JORCAI; ISSN:0022-328X. (Elsevier Ltd.)
      Amination of S-methyl-N,N'-bis-Boc-isothiourea with either primary or sterically hindered secondary amines, promoted by copper(I) chloride and K2CO3, gave N,N'-bis-Boc protected guanidines in good to excellent yields under mild reaction conditions.
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    37. 37
      Kelly, B.; Rozas, I. Copper (II) chloride promoted transformation of amines into guanidines and asymmetrical N, N′-disubstituted guanidines. Tetrahedron Lett. 2013, 54, 3982– 3984,  DOI: 10.1016/j.tetlet.2013.05.070
      37
      Copper(II) chloride promoted transformation of amines into guanidines and asymmetrical N,N'-disubstituted guanidines
      Kelly, Brendan; Rozas, Isabel
      Tetrahedron Letters (2013), 54 (30), 3982-3984CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)
      We present a concise, less-toxic and broadly applicable method for coupling weakly nucleophilic amines with N,N'-di-(tert-butoxycarbonyl)thiourea, N-(tert-butxoycarbonyl), N'-alkyl/arylsubstituted-thioureas and N,N'-di-(tert-butoxycarbonyl)imidazolidine-2-thione in the presence of copper(II) chloride. Subsequent removal of Boc protecting groups affords guanidines, di-substituted guanidines and 2-aminoimidazolines in modest to excellent overall yields.
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    38. 38
      Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. Urethane protected derivatives of 1-guanylpyrazole for the mild and efficient preparation of guanidines. Tetrahedron Lett. 1993, 34, 3389– 3392,  DOI: 10.1016/S0040-4039(00)79163-5
      38
      Urethane protected derivatives of 1-guanylpyrazole for the mild and efficient preparation of guanidines
      Bernatowicz, Michael S.; Wu, Youling; Matsueda, Gary R.
      Tetrahedron Letters (1993), 34 (21), 3389-92CODEN: TELEAY; ISSN:0040-4039.
      Bis-urethane protected derivs. I (R = tert-butoxy- or benzyloxycarbonyl) of 1-guanylpyrazole were prepd. and found to readily react with relatively unreactive amines R1NH2 (R1 = Ph, CF3CH2, p-O2NC6H4) at room temp. to produce bis-protected guanidines R1NHC(:NR)NHR in good yields. Simultaneous removal of both protecting groups from these products efficiently produced monosubstituted guanidines R1NHC(:NH)NH2.
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    39. 39
      Feichtinger, K.; Sings, H. L.; Baker, T. J.; Matthews, K.; Goodman, M. Triurethane-protected guanidines and triflyldiurethane-protected guanidines: new reagents for guanidinylation reactions. J. Org. Chem. 1998, 63, 8432– 8439,  DOI: 10.1021/jo9814344
      39
      Triurethane-protected guanidines and triflyldiurethane-protected guanidines: new reagents for guanidinylation reactions
      Feichtinger, Konrad; Sings, Heather L.; Baker, Tracy J.; Matthews, Kenneth; Goodman, Murray
      Journal of Organic Chemistry (1998), 63 (23), 8432-8439CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      New guanidinylation reagents are reported. These reagents consist of N,N',N''-tri-Boc-guanidine and N,N',N''-tri-Cbz-guanidine, which allow for the facile conversion of alcs. to substituted guanidines. A series of arginine analogs were synthesized via condensation of a primary or secondary alc. with the guanidinylation reagents, under Mitsunobu conditions to produce protected alkylated guanidines. In addn., an extended study of the previously reported reagents N,N'-di-Boc-N''-triflylguanidine (I) and N,N'-di-Cbz-N''-triflylguanidine is presented. The triflyldiurethane-protected guanidine I was utilized to guanidinylate primary and secondary amines under mild conditions with high yield in both soln. and on solid phase.
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    40. 40
      Porcheddu, A.; De Luca, L.; Giacomelli, G. A mild and inexpensive procedure for the synthesis of N, N′-di-Boc-protected guanidines. Synlett 2009, 2009, 3368– 3372,  DOI: 10.1055/s-0029-1218365
      There is no corresponding record for this reference.
    41. 41
      Musiol, H.-J.; Moroder, L. N, N′-Di-tert-butoxycarbonyl-1 H-benzotriazole-1-carboxamidine Derivatives Are Highly Reactive Guanidinylating Reagents. Org. Lett. 2001, 3, 3859– 3861,  DOI: 10.1021/ol010191q
      41
      N,N'-Di-tert-butoxycarbonyl-1H- benzotriazole-1-carboxamidine Derivatives Are Highly Reactive Guanidinylating Reagents
      Musiol, Hans-Juergen; Moroder, Luis
      Organic Letters (2001), 3 (24), 3859-3861CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      Electron-withdrawing substituents such as the 5-chloro or 6-nitro substituent enhance the leaving group character of benzotriazole in N,N'-di-tert-butoxycarbonyl-1H-benzotriazole-1-carboxamidines, giving highly efficient reagents for conversion of primary and secondary amines in soln. and in solid phase to diprotected guanidines.
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    42. 42
      Zheng, Z.; Chen, P.; Li, G.; Zhu, Y.; Shi, Z.; Luo, Y.; Zhao, C.; Fu, Z.; Cui, X.; Ji, C. Mechanistic study of CBT-Cys click reaction and its application for identifying bioactive N-terminal cysteine peptides in amniotic fluid. Chem. Sci. 2017, 8, 214– 222,  DOI: 10.1039/c6sc01461e
      42
      Mechanistic study of CBT-Cys click reaction and its application for identifying bioactive N-terminal cysteine peptides in amniotic fluid
      Zheng, Zhen; Chen, Peiyao; Li, Gongyu; Zhu, Yunxia; Shi, Zhonghua; Luo, Yufeng; Zhao, Chun; Fu, Ziyi; Cui, Xianwei; Ji, Chenbo; Wang, Fuqiang; Huang, Guangming; Liang, Gaolin
      Chemical Science (2017), 8 (1), 214-222CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      CBT-Cys click condensation reaction has a high second-order reaction rate const. and has found wide applicability in recent years. However, its reaction mechanism has not been exptl. validated and its application for identifying bioactive N-terminal Cys peptides in real clin. samples has not been reported. Herein, firstly, by employing induced nanoelectrospray ionization-mass spectrometry (InESI-MS) and a home-built micro-reactor, we successfully intercepted and structurally characterized the crucial intermediate in this click reaction for the first time. With the intermediate, the proposed mechanism of this reaction was corroborated. Moreover, we also applied this MS setup to monitor the reaction in real time and obtained the second-order reaction rate consts. of this reaction at different pH values. After mechanistic study, we applied this click reaction for identifying bioactive N-terminal cysteine peptides in amniotic fluid (AF). Eight unique N-terminal Cys peptides in AF, three of which are located in the functional domain regions of their corresponding proteins, were identified with a false pos. rate less than 1%. One of the three peptides was found able to inhibit the growth of uterine endometrial cancer HEC-1-B cells but not the endometrial normal cells via a typical apoptotic pathway.
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    43. 43
      Liang, G.; Ren, H.; Rao, J. A biocompatible condensation reaction for controlled assembly of nanostructures in living cells. Nat. Chem. 2010, 2, 54– 60,  DOI: 10.1038/nchem.480
      43
      A biocompatible condensation reaction for controlled assembly of nanostructures in living cells
      Liang, Gaolin; Ren, Hongjun; Rao, Jianghong
      Nature Chemistry (2010), 2 (1), 54-60CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      Through controlled synthesis and mol. assembly, biol. systems are able to organize mols. into supramol. structures that carry out sophisticated processes. Although chemists have reported a few examples of supramol. assembly in, the controlled covalent synthesis of large mols. and structures in vivo has remained challenging. Here the authors report a condensation reaction between 1,2-aminothiol and 2-cyanobenzothiazole that occurs in vitro and in living cells under the control of either pH, disulfide redn. or enzymic cleavage. In vitro, the size and shape of the condensation products, and the nanostructures subsequently assembled, were different in each case and could thus be controlled by tuning the structure of the monomers. Direct imaging of the products obtained in the cells revealed their locations-near the Golgi bodies under enzymic cleavage control-demonstrating the feasibility of a controlled and localized reaction in living cells. This intracellular condensation process enabled the imaging of the proteolytic activity of furin.
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    44. 44
      Proj, M.; Strašek, N.; Pajk, S.; Knez, D.; Sosič, I. Tunable Heteroaromatic Nitriles for Selective Bioorthogonal Click Reaction with Cysteine. Bioconjugate Chem. 2023, 34, 1271– 1281,  DOI: 10.1021/acs.bioconjchem.3c00163
      44
      Tunable Heteroaromatic Nitriles for Selective Bioorthogonal Click Reaction with Cysteine
      Proj, Matic; Strasek, Nika; Pajk, Stane; Knez, Damijan; Sosic, Izidor
      Bioconjugate Chemistry (2023), 34 (7), 1271-1281CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      The binucleophilic properties of 1,2-aminothiol and its rare occurrence in nature make it a useful reporter for tracking mols. in living systems. The 1,2-aminothiol moiety is present in cysteine, which is a substrate for a biocompatible click reaction with heteroarom. nitriles. Despite the wide range of applications for this reaction, the scope of nitrile substrates has been explored only to a limited extent. In this study, we expand the chem. space of heteroarom. nitriles for bioconjugation under physiol. relevant conditions. We systematically assembled a library of 116 2-cyanobenzimidazoles, 1-methyl-2-cyanobenzimidazoles, 2-cyanobenzothiazoles, and 2-cyanobenzoxazoles contg. electron-donating and electron-withdrawing substituents at all positions of the benzene ring. The compds. were evaluated for their stability, reactivity, and selectivity toward the N-terminal cysteine of model oligopeptides. In comparison to the benchmark 6-hydroxy-2-cyanobenzothiazole or 6-amino-2-cyanobenzothiazole, we provide highly selective and moderately reactive nitriles as well as highly reactive yet less selective analogs with a variety of enabling attachment chemistries to aid future applications in bioconjugation, chem. biol., and nanomaterial science.
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    45. 45
      Sondag, D.; de Kleijne, F. F. J.; Castermans, S.; Chatzakis, I.; van Geffen, M.; van’t Veer, C.; van Heerde, W. L.; Boltje, T. J.; Rutjes, F. P. J. T. Synthesis and Evaluation of Glycosyl Luciferins. Chem.─Eur. J. 2023, 29, e202302547  DOI: 10.1002/chem.202302547
      There is no corresponding record for this reference.
    46. 46
      Lottenberg, R.; Hall, J. A.; Blinder, M.; Binder, E. P.; Jackson, C. M. The action of thrombin on peptide p-Nitroanilide substrates: Substrate selectivity and examination of hydrolysis under different reaction condtions. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1983, 742, 539– 557,  DOI: 10.1016/0167-4838(83)90272-8
      46
      The action of thrombin on peptide p-nitroanilide substrates. Substrate selectivity and examination of hydrolysis under different reaction conditions
      Lottenberg, Richard; Hall, Julie A.; Blinder, Morey; Binder, Ellen P.; Jackson, Craig M.
      Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1983), 742 (3), 539-57CODEN: BBAEDZ; ISSN:0167-4838.
      Kinetic parameters for the action of bovine α-thrombin on 24 com. available peptide p-nitroanilides were detd. The selectivity const., kcat/Km, ranged from 3.3 × 101 to 1.1 × 108 M-1 s-1 for the poorest and the best substrates, resp. The best substrates for thrombin were identified as those with arginine in the P1 position, proline or a proline homolog in the P2 position, and an apolar amino acid in the P3 position. A quant. distinction between lysine and arginine in the P1 position and other amino acids in the P2-P4 positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. The effects of NaCl, CaCl2, and polyethylene glycol concns., and pH and temp. on the action of thrombin on selected substrates were assessed. A source of large systematic error in thrombin concn. ests. was identified as resulting from adsorption losses. These losses were eliminated by inclusion of polyethylene glycol in diln. and reaction buffers.
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    47. 47
      Tian, G.; Fedoseev, P.; Van der Eycken, E. V. Hypervalent Iodine (III)-Mediated Cascade Cyclization of Propargylguanidines and Total Syntheses of Kealiinine B and C. Chem.─Eur. J. 2017, 23, 5224– 5227,  DOI: 10.1002/chem.201700934
      47
      Hypervalent Iodine(III)-Mediated Cascade Cyclization of Propargylguanidines and Total Syntheses of Kealiinine B and C
      Tian, Guilong; Fedoseev, Pavel; Van der Eycken, Erik V.
      Chemistry - A European Journal (2017), 23 (22), 5224-5227CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      An oxidative cascade cyclization of propargylguanidines promoted by phenyliodonium diacetate (PIDA) was developed. The protocol provides an efficient route for the synthesis of the alkaloids kealiinines B (I) and C (II) as well as homologues. The difference in the electronic nature of the acetylene substituent resulted in two ways of the cyclization. A plausible mechanism is proposed based on the exptl. results.
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