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The Future of Bioorthogonal Chemistry
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Cite this: ACS Cent. Sci. 2018, 4, 8, 952–959
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https://doi.org/10.1021/acscentsci.8b00251
Published July 23, 2018

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Abstract

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Bioorthogonal reactions have found widespread use in applications ranging from glycan engineering to in vivo imaging. Researchers have devised numerous reactions that can be predictably performed in a biological setting. Depending on the requirements of the intended application, one or more reactions from the available toolkit can be readily deployed. As an increasing number of investigators explore and apply chemical reactions in living systems, it is clear that there are a myriad of ways in which the field may advance. This article presents an outlook on the future of bioorthogonal chemistry. I discuss currently emerging opportunities and speculate on how bioorthogonal reactions might be applied in research and translational settings. I also outline hurdles that must be cleared if progress toward these goals is to be made. Given the incredible past successes of bioorthogonal chemistry and the rapid pace of innovations in the field, the future is undoubtedly very bright.

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Synopsis

Bioorthogonal reactions have found widespread use in chemical biology. This article gives a brief outlook on the future of the field, outlining emerging areas and key challenges to overcome.

Introduction

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Although chemists have been making molecules that interact with life since the dawn of modern chemistry, the actual chemical reactions used to assemble the molecules were kept as far away from life as possible. They were performed in organic solvents where water, and often oxygen, were to be avoided. Impurities were anathema. This all changed with the introduction of bioorthogonal chemistry by Bertozzi and co-workers. (1−3) The concept is elegant. Can we design reactions that are so selective they can be performed reliably even in a complex biological environment? These reactions must proceed efficiently in the presence of the multitude of functional groups found in living systems such nucleophiles, electrophiles, reductants, oxidants, and of course the solvent of life water. Simultaneously, these reactions should have a minimal impact on the biology itself. The transformation bioorthogonal chemistry triggered in the field of chemical biology was monumental. Suddenly, reactions that previous generations performed in refluxing toluene, were now being done in an aqueous mixture of proteins and sugars. Cancer cells and zebrafish replaced round-bottom flasks. (4,5) Bioorthogonal reactions have already made a tremendous scientific impact, helping us understand glycosylation in cells and animals, (6) providing tools for conjugating functional groups to therapeutically relevant proteins such as antibodies, (7) and enabling the assembly of molecular imaging agents in vivo to detect disease. (8)

The transformation bioorthogonal chemistry triggered in the field of chemical biology was monumental. Suddenly, reactions that previous generations performed in refluxing toluene, were now being done in an aqueous mixture of proteins and sugars. Cancer cells and zebrafish replaced round-bottom flasks.

The concept of bioorthogonal chemistry has inspired a generation of chemical biologists to think about how classic organic reactions can be performed in concert with living systems and how such reactions could lead to the development of tools to help understand biology. I think one of the greatest contributions of bioorthogonal chemistry has been its ability to challenge our imagination regarding the kinds of reactions that can be performed in living systems and how this enables us to ask extremely interesting and ambitious questions. Can pharmaceuticals be synthesized inside humans? (9) Can we co-opt bioorthogonal reactions to detect metabolites in situ? (10) How many orthogonal reactions can be performed simultaneously? (11)
Over the last several years, our ability to combine chemistry and biology has accelerated through improved tools and resources. Therefore, I believe there are numerous future prospects for how bioorthogonal chemistry will have an increasing impact on chemical biology and medicine. In this short Outlook, I will describe my opinion of the future of bioorthogonal chemistry and explore what I believe are some outstanding opportunities in the field. I also outline many of the challenges that will need to be overcome for some of these opportunities to be realized.

The Development of New Bioorthogonal Reactions

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Undoubtedly there will be continued development of new bioorthogonal reactions. Bioorthogonal chemistry has encouraged chemists to consider how a vast number of organic transformations might be adapted to work in living systems. In just the last year alone, there has been the introduction of several new bioorthogonal reactions. (12−15) However, while there are a multitude of possible reactions that could be developed into bioorthogonal processes, it is worthwhile pointing out some of the desired properties of new bioorthogonal reactions that would significantly advance the field. For instance, the continued development of very rapid bioorthogonal reactions is desirable. Rapid reactions are useful because they, in principle, allow one to perform bioconjugations on a practical time scale using a lower concentration of reactants. This is important from a cost perspective. It also may not be practical to achieve high concentrations of a reactant, for instance, when working with proteins or attempting to perform reactions in living cells and animals. Previous work has made notable gains in improving the rate of bioorthogonal reactions, perhaps most notably through the development of tetrazine ligations, which have reported second order rate constants often exceeding 1000 M–1 s–1. (16,17) For comparison, Staudinger ligations or strain-promoted cycloadditions typically have reported rates between 1 × 10–3 and 1 M–1 s–1. (4,18,19) However, rate constants greater than 10 000 M–1 s–1 would be extremely useful. While there have been reports of very rapid reactions with rate constants approaching or exceeding these levels, the reactants themselves are often prone to degradation through side reaction, calling the “orthogonality” of these reactions into question. Development of more stable reactants that can still react very rapidly or the development of entirely new reactions will be highly welcome. There have been some recent inroads in these directions. Triazines have been explored as more stable alternatives to tetrazines, though the reaction rates are modest in comparison. (20) Additionally, researchers have shown that modified strained trans-cyclooctenes can avoid cycloisomerization back to the cis isomer, (21,22) a phenomenon that occurs frequently in the presence of thiols and essentially renders the reagents inactive.
Another area where there is room for future improvement is the size and physical properties of the reaction partners. One of the great advantages of the classic azide-cyclooctyne bioorthogonal reaction is the unobtrusive size of the azide functional group. Azides have been shown to be readily incorporated into analogs of biological metabolites, oftentimes with minor effects on overall function. This feature has enabled applications in metabolic imaging of glycans (4,23) and the incorporation and modification of reactive unnatural amino acids. (24,25) On the other hand, the cyclooctyne reactive group is a bulky probe that is hydrophobic and thus might accumulate nonspecifically in membranes and other cellular structures. Future reactions that use two coupling partners that are both of a lower molecular weight comparable to azides would be advantageous, as the reactive functionalities would be expected to have minimum impact on the molecules that they are appended to (Figure 1A). Recent studies exploring smaller bioorthogonal handles such as cyclopropenes (26,27) and diazo groups are promising directions toward this goal. (28)

Figure 1

Figure 1. (A) Examples of bioorthogonal functional groups and their approximate molecular weights. (B) Mutually orthogonal bioorthogonal reactions for metabolic imaging. Tetrazines and cyclooctynes can selectively label cyclopropenes and azides, respectively. In this instance, the application is to visualize metabolic incorporation of two different sugars onto cell surface glycans. Image reproduced from ref (30).

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There has also been growing interest in developing new bioorthogonal reactions that are orthogonal to preexisting bioorthogonal reactions. (11) For instance, several investigators have shown the use of tetrazine/dienophile reactions in parallel with azide/cyclooctyne reactions (Figure 1B). (27,29−31) With careful design of precursors and considering relative rate constants, reactions can be designed to be mutually orthogonal. The benefits of such selectivity are several. For instance, mutually orthogonal reactions enable one to perform metabolic imaging of two different processes by introduction of two differently tagged metabolites. Another exemplary application is the introduction of two reactive unnatural amino acids which can then be tagged using orthogonal chemistries. (32) Such an approach can be used to introduce FRET probes site-specifically on a protein to potentially monitor dynamics. It will be intriguing to see how many different bioorthogonal reactions can be reliably performed simultaneously. It will also be important to understand what the unique applications of such an approach would be and how to implement multiplexed bioorthogonal conjugations for answering biological questions.

Assembling Bioactive Compounds for in Situ Drug Delivery

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The ability to stitch together organic molecules in vivo enables applications that involve the assembly of bioactive small molecules in situ. An exciting future goal might be the in situ assembly of pharmaceuticals at the site of their action. (9) The concept that therapeutic molecules might predictably self-assemble within living systems has been discussed for decades. There have been some notable examples, including the seminal work of Rideout, which demonstrated that aldehyde and hydrazine fragments could assemble into bioactive hydrazones within cells. (33) Since bioorthogonal reactions are robust methods for assembling molecules together, it stands to reason that they might have application toward in situ bioactive small molecule assembly. There is some prior art that suggests this might be feasible. First off, based on the strength and success of fragment based approaches for drug discovery, (34) there has been significant effort in using bioorthogonal chemistry, particularly azide–alkyne cycloadditions, to stitch together drug fragments in situ. Remarkably, it has been found that protein targets can be used to catalyze the formation of their own inhibitors by templating bioorthogonal reactions between reactive fragments that bind in close proximity to one another (Figure 2A). (35) Potent inhibitors for enzymes such as acetylcholinesterase, carbonic anhydrase, and HIV protease have been discovered by screening libraries of compounds bearing reactive handles and analyzing what products are formed through templated reaction. (36−38)

Figure 2

Figure 2. Assembling bioactive molecules in situ using bioorthogonal reactions. (A) A target biomolecule such as an enzyme can bind to two fragments bearing bioorthogonal functional groups. If the fragments are bound such that the functional groups are brought into close proximity to one another, reaction occurs, and a tighter binding compound is formed. This method has been used for in situ discovery of enzyme inhibitors, but it will be interesting to see if such reactions can take place within living cells and in vivo. (B) Researchers have used bioorthogonal reactions to form druglike molecules inside cells. Here, a heterobifunctional compound is formed by tetrazine ligation which enables the degradation of BRD4 through the ubiquitin pathway. Figure reproduced from ref (39).

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In principle, templated synthesis of inhibitors might be extended beyond a tool for drug discovery and be used to trigger drug formation in vivo in response to specific protein targets. (9) There are, of course, several hurdles to realizing this vision. In some notable previous examples of in situ click chemistry, the templated thermal azide–alkyne cycloadditions were sluggish, and reagents were incubated over lengthy time scales. (36) For these reactions to work in vivo, the rate of product formation should be much faster, ideally within hours at physiological temperature. Fortunately, the development of biorthogonal reactions with highly tunable reaction rates (39) should enable control over the rate of fragment assembly such that product would form in short enough time scales to be therapeutically meaningful. Other hurdles include the complexity involved in having to optimize pharmacokinetics and delivery for two reagents versus one. However, the benefits could be enormous since bioactive compounds would in principle only be accumulating at high concentrations in cells that have appreciable concentration of protein targets. This would likely mean lower side effects due to off target engagement and potentially the ability to have a larger therapeutic window, which could be very valuable in the case of delivery of cytotoxic chemotherapeutics. (33) Additionally, the concept of delivering two smaller agents that later assemble to form a larger bioactive compound would represent a novel drug delivery strategy and potentially have advantages in terms of the ability of the smaller precursors to pass through cell membranes and the blood–brain barrier.
Recent work has made progress toward using bioorthogonal reactions to assemble functional therapeutics. There has been recent excitement in proteolysis inducing chimeras (PROTACS), which are heterobifunctional drugs that can trigger the degradation of proteins by linking a target protein with the cellular ubiquitinating machinery. However, the required bifunctional compounds are often of high molecular weight (800–1000 Da) and can have less than desirable pharmacokinetics for penetrating cell membranes or accumulating in the central nervous system. Recently, Astex Pharmaceutical has shown that tetrazine bioorthogonal reactions can be used to stitch together functional PROTACS for degrading oncology targets BRD4 and ERK1/2 within living cells (Figure 2B). (40) They were also able to demonstrate that in situ assembly of two small molecules within a cell showed greater efficacy than simply adding the preassembled molecule. However, a major challenge that remains to be addressed is how to selectively trigger formation within the cell versus outside the cell. In the described approach, bioorthogonal assembly would also rapidly take place in the bloodstream. Templated chemistry or perhaps small molecule targeting to specific cell types might alleviate this problem and lead to future translational applications.

Bioorthogonal Uncaging Reactions

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A recent innovative direction for the coupling of bioorthogonal reactions to biological activity has been the use of bioorthogonal chemistry to uncage substrates, a strategy that has been termed “click to release.” (41) This is an area of accelerating excitement, and applications are continuously expanding. The concept is similar to the well-known uncaging of small molecules in response to light. However, when using light as a stimuli, there is limited depth penetration. Therefore, optical techniques are often restricted to research applications or translational applications that do not require significant depth penetration or systemic activation. In contrast, the use of bioorthogonally reactive small molecules to trigger uncaging offers the potential to activate on the systemic level and in theory could be translatable to patients. Uncaging is typically done by using a reactive handle to mask an amine or alcohol (Figure 3). A prominent example is the use of the highly strained alkene trans-cyclooctene to mask an amine, which is then released upon reaction with a tetrazine. Several investigators have already applied “click to release” strategies to trigger the formation of drugs in vitro and in vivo. (42−45) Furthermore, researchers have shown that, by incorporating a masked lysine residue through site-specific unnatural amino acid incorporation, bioorthogonal reaction can be used to trigger protein activation. (46,47) This strategy provides a mechanism to pharmacologically activate proteins in live animals and could reveal insights on the effects of protein activity on physiology. Additional work has shown that bioorthogonal masks can be used to activate fluorophores for imaging to detect biomolecules such as RNA. (48) Recent studies have even shown the ability to release gases such as carbon monoxide, indicating that bioorthogonal prodrugs of gasotransmitters are on the horizon. (49) I imagine that the use of bioorthogonal strategies to uncage biologically relevant molecules will continue to expand and see continued translation in vivo, potentially to humans. To aid applications, future studies may focus on improving the rate and yield of the release reaction, and recent efforts are promising in this direction. (50)

Figure 3

Figure 3. General examples of bioorthogonal uncaging reactions. Dienophiles act to mask functional groups such as amines and alcohols. Upon bioorthogonal reaction with tetrazine, the functional group is released. This enables the uncaging of drugs, imaging agents, and even enzymes.

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Bioorthogonal Reactions that Lead to Natural Linkages

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Most bioorthogonal reactions result in the formation of unnatural linkages. This makes sense since one would expect that exotic, abiological functional groups would have less chance of interacting with biological systems and thus contribute to the chemoselectivity of the reaction. There has been an interest in using bioorthogonal reactions to create analogs of natural compounds such as sugars, lipids, and nucleic acids. (51−53) However, the presence of unnatural linkages such as triazoles does cause concerns regarding whether the compounds formed are truly analogous in function. Thus, a future avenue of exploration would be the development of bioorthogonal coupling reactions that can be performed in vivo selectively but result in the formation of native linkages such as amides, esters, phosphodiesters, etc. A closely related class of reactions are the native chemical ligation, traceless Staudinger ligation, and associated coupling reactions that lead to amide bond formation. (54−57) The native chemical ligation involves the coupling of N-terminal cysteines to thioesters to form a native cysteine linkage. Unfortunately, these reactions have been for the most part limited to use in in vitro settings with purified components. However, one can imagine that the next generation of reactions will have appropriate selectivity and kinetics to be feasible for use in living cells and perhaps even in vivo. Strategies that template reactants could be one way to improve rates and selectivity, transforming chemoselective ligations into bioorthogonal reactions that can be performed in living systems. (58) Researchers could also take advantage of elegant reactions that use abiotic functional groups that come together to rapidly form peptide linkages. (59) For instance, recently our group has built on selective peptide forming ligations to synthesize bioactive lipids in living cells. (60) With such tools at our disposal, it seems likely that we would be able to use bioorthogonal coupling reactions to form native molecules inside living cells and test the effect of forming these molecules on cellular function. This would have tremendous benefits over genetic approaches, where pleiotropic effects and enzyme promiscuity can obscure observations and make it unclear if the activity observed is actually due to a change in concentration of the molecule of interest. As previously discussed, assembling molecules in situ would represent a novel mechanism of cellular delivery. The ability to shuttle in two molecules of lower molecular weight, which assemble at the site of action, could bypass physiological barriers such as cellular membranes.

a future avenue of exploration would be the development of bioorthogonal coupling reactions that can be performed in vivo selectively but result in the formation of native linkages such as amides, esters, phosphodiesters, etc.

In Situ Formation of Bioorthogonal Handles

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Currently, bioorthogonal functional groups must be synthesized using standard organic chemistry methods and then introduced onto a biological molecule or metabolite, which is then introduced to the living system. This exogenous application of functional groups suffers occasionally from drawbacks including delivery issues, which are potentially problematic for in vivo applications. It would be very interesting if organisms could be engineered to synthesize bioorthogonal handles in situ. Past work from the Schultz lab has demonstrated that organisms can be engineered to synthesize unnatural amino acids, which can subsequently be incorporated into proteins site-specifically. (61) Would it be possible to do this but in a manner that the unnatural amino acid contained a reactive handle such as an azide, tetrazine, or strained dienophile? One ripe area for exploration might be the biosynthesis of strained cyclopropenes, dienophiles that are known to be useful bioorthogonal handles for reaction with tetrazines. Cyclopropenes are found in natural products, for instance, in the fatty acids malvalic acid and sterculic acid. (62) Unfortunately, the full details regarding the biosynthesis of cyclopropene fatty acids are still unclear, though future studies are likely to illuminate the pathway. (63) In the meantime, another approach might be to express enzymes specifically engineered to synthesize bioorthogonal handles. Recent work from the Arnold lab has demonstrated that enzymes can be engineered to catalyze the synthesis of cyclopropenes. (64) Adapting such systems to function in living cells might enable the in situ production of strained dienophiles which can then be incorporated into biological molecules and subsequently labeled.

In Vivo Chemistry

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The ultimate frontier for performing reactions in a biological context would be conducting reactions in living animals and ultimately humans. While performing bioorthogonal reactions on and within living cells has become almost a routine phenomenon, the ability to conduct highly efficient reactions inside multicellular organisms remains nontrivial. Zebrafish are excellent model vertebrate organisms since developing zebrafish are optically transparent, and therefore visualization of bioorthogonal reactions through the use of fluorescent imaging probes is feasible. (5) Furthermore, reagents can be delivered either by injecting compounds directly into zebrafish embryos or bathing developing zebrafish in reactive precursors. However, performing bioorthogonal reactions in mammalian model organisms such as mice presents a slew of daunting challenges. First and foremost are optimizing the pharmacokinetics of the reagents to be reacted. There has to be some matching between the rate of the reaction and the lifetime of the precursors at the desired site of action. (8) For this reason, very rapid reactions are likely to find future application, since in many cases the attainable concentrations achievable are in the micromolar range, and lower still when one considers tracer imaging agents. (39) Pharmacokinetics also dictates whether the reagents even make it to the desired site of action. High doses of reagents have little benefit if a reaction desired should occur at a tumor but the reagents never make it there. In vivo chemistry is complicated by the fact that there are two reagents that will need to be delivered. While this may offer some benefits in terms of triggering the formation of drugs or bioactive compounds (see above), it also presents a number of challenges. For instance, the dosing, pharmacokinetics, stability, and toxicity will need to be optimized for not one but two reagents. However, despite these challenges, recent innovations and encouraging preclinical animal results suggest that these issues are likely surmountable, and one would expect to see applications potentially entering the clinic. (45,65,66)

The ultimate frontier for performing reactions in a biological context would be conducting reactions in living animals and ultimately humans. While performing bioorthogonal reactions on and within living cells has become almost a routine phenomenon, the ability to conduct highly efficient reactions inside multicellular organisms remains nontrivial.

Ultimately, I imagine that bioorthogonal chemistry will go beyond research applications and preclinical testing and will be translated to applications in humans. What are some of the emerging applications that we might see translated initially? Arguably, one of the most developed applications is for pretargeted imaging or therapy using reactive monoclonal antibodies (Figure 4). The rationale for pretargeted approaches, which were pioneered by Goodwin and Meares, is compelling. (67) Monoclonal antibodies are excellent affinity ligands that show exceptional selectivity for homing to tissues expressing target antigens and achieving high signal to background. However, monoclonal antibodies also are cleared very slowly, often taking days for clearance in mice and longer still in humans. This long lifetime of monoclonal antibodies can present problems with trying to use antibody drug conjugates, particularly if the conjugate is a radiopharmaceutical used for imaging or therapy. In the case of imaging, the most commonly used radioisotopes are short-lived radioisotopes such as 18F, which has a half-life of just under 2 h. Unfortunately, labeling of antibodies with 18F is not useful since one needs to wait for days to allow sufficient antibody to clear to achieve appropriate target to background ratios for imaging antibody accumulation. By this time, the vast majority of the radionuclide will have decayed and imaging would not be possible. In a pretargeting approach, one would instead deliver a nonradioactive antibody conjugate bearing a bioorthogonal handle. This antibody will be allowed to accumulate and clear after which point a small molecule chaser with a much quicker clearance rate will be delivered. The chaser would possess the radionuclide and a complementary bioorthogonal handle, enabling reaction to the pretargeted antibody. Because the chaser is a small molecule and clears readily, appropriate target to background ratios in principle can be achieved quickly. Similar benefits can be achieved with therapeutic radiopharmaceuticals to avoid systemic toxicity and potentially maximize dose delivery to the target.

Figure 4

Figure 4. Bioorthogonal pretargeted therapy. Cartoon depicting the direct versus multistep labeling approach. (A) Target cells (blue) are exposed to an affinity ligand directly modified by a chemotherapeutic or imaging agent. The affinity ligand binds, but an excess of long circulating affinity ligand persists, contributing to toxicity or background signal. The latter problem prevents the use of short-lived radioisotopes (e.g., fluorine-18) with commonly used affinity ligands such as antibodies. (B) In the multistep approach, an affinity ligand directly connected to a nontherapeutic bioorthogonally reactive element (trans-cyclooctene) is delivered. Again, background is initially high, but this background is inactive. After time has passed to allow for clearance, a small molecule that reacts with the affinity ligand (tetrazine) and possesses a chemotherapeutic or imaging agent is delivered. The small molecule reacts rapidly with available trans-cyclooctene. The molecule also clears rapidly due to its small size thus lowering background side effects or signal.

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There have already been some outstanding developments toward achieving pretargeting imaging using bioorthogonal reagents. (65,66,68−70) Past work has shown promise using a variety of long-lived and short-lived radioisotopes. Most of these studies utilize fast tetrazine ligations, since very rapid kinetics is likely necessary to achieve efficient in vivo reaction with tracer quantities of imaging agent. However, while early work is encouraging, there are still several potential challenges that will need to be overcome in the future for such techniques to be translated to the clinic. Perhaps most restrictive is the current emphasis on targeting extracellular targets. (8,68) If the target antibody internalizes, then it will be more difficult for the bioorthogonal reaction to take place, as one of the reactants will be trapped within the cell. It would be simpler if the reactions were to take place extracellularly, but this presents the challenge of choosing appropriate targets that do not internalize once bound by antibodies, even after many hours. Additionally, it will be necessary to further improve the stability of the reactive partners while maintaining high rate constants. Recent studies have shown that highly strained alkenes that are often used in such bioorthogonal strategies may be prone to side reaction in physiological media. (21,26) Despite these challenges, current efforts by several clinical and commercial groups suggest that future improvements and clinical translation might lie ahead.

Conclusions

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Bioorthogonal chemistries have enabled researchers to perform controlled chemistry in the presence of biological functional groups and in situ within living cells and organisms. Since its introduction, the toolkit of bioorthogonal reactions has steadily expanded and will definitely grow larger. New reactions will enable new opportunities and applications, particularly as kinetics, stability, and reactive handle size are further optimized. It seems likely that future studies will further emphasize the use of bioorthogonal couplings to trigger the formation of biologically active components, whether by direct in situ synthesis of drugs or through triggered drug release. Finally, continued applications in vivo will take place, and ultimately bioorthogonal reactions will find their way into the clinic. The predictions I have outlined are obviously just a small part of the future of bioorthogonal chemistry. Indeed, I am confident that some of the most exciting applications will come as a surprise and be driven by the need to address pressing biological and medical problems. Given the flexibility and power of the bioorthogonal approach, controlling chemistry within biological systems may soon only be limited by our imaginations.

Author Information

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  • Corresponding Author
    • Notes
      The author declares no competing financial interest.

    Acknowledgments

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    N.K.D. acknowledges the University of California, San Diego, National Institutes of Health, Department of Defense, and the Camille and Henry Dreyfus foundation for supporting research related to bioorthogonal reactions.

    References

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      Science (Washington, DC, United States) (2008), 320 (5876), 664-667CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Glycans are attractive targets for mol. imaging but have been inaccessible because of their incompatibility with genetically encoded reporters. We demonstrated the noninvasive imaging of glycans in live developing zebrafish, using a chem. reporter strategy. Zebrafish embryos were treated with an unnatural sugar to metabolically label their cell-surface glycans with azides. Subsequently, the embryos were reacted with fluorophore conjugates by means of copper-free click chem., enabling the visualization of glycans in vivo at subcellular resoln. during development. At 60 h after fertilization, we obsd. an increase in de novo glycan biosynthesis in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, we performed a spatiotemporal anal. of glycan expression and trafficking and identified patterns that would be undetectable with conventional mol. imaging approaches.
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    6. 6
      Agarwal, P.; Beahm, B. J.; Shieh, P.; Bertozzi, C. R. Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry. Angew. Chem., Int. Ed. 2015, 54, 11504– 11510,  DOI: 10.1002/anie.201504249
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      6
      Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry
      Agarwal, Paresh; Beahm, Brendan J.; Shieh, Peyton; Bertozzi, Carolyn R.
      Angewandte Chemie, International Edition (2015), 54 (39), 11504-11510CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Vertebrate glycans constitute a large, important, and dynamic set of post-translational modifications that are notoriously difficult to manipulate and image. Although the chem. reporter strategy has been used in conjunction with bioorthogonal chem. to image the external glycosylation state of live zebrafish and detect tumor-assocd. glycans in mice, the ability to image glycans systemically within a live organism has remained elusive. Here, the authors report a method that combines the metabolic incorporation of a cyclooctyne-functionalized sialic acid deriv. with a ligation reaction of a fluorogenic tetrazine, allowing for the imaging of sialylated glycoconjugates within live zebrafish embryos.
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    7. 7
      Oller-Salvia, B.; Kym, G.; Chin, J. W. Rapid and Efficient Generation of Stable Antibody-Drug Conjugates via an Encoded Cyclopropene and an Inverse-Electron-Demand Diels-Alder Reaction. Angew. Chem., Int. Ed. 2018, 57, 2831– 2834,  DOI: 10.1002/anie.201712370
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      7
      Rapid and Efficient Generation of Stable Antibody-Drug Conjugates via an Encoded Cyclopropene and an Inverse-Electron-Demand Diels-Alder Reaction
      Oller-Salvia, Benjami; Kym, Gene; Chin, Jason W.
      Angewandte Chemie, International Edition (2018), 57 (11), 2831-2834CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Homogeneous antibody-drug conjugates (ADCs), generated by site-specific toxin linkage, show improved therapeutic indexes with respect to traditional ADCs. However, current methods to produce site-specific conjugates suffer from low protein expression, slow reaction kinetics, and low yields, or are limited to particular conjugation sites. Here we describe high yielding expression systems that efficiently incorporate a cyclopropene deriv. of lysine (CypK) into antibodies through genetic-code expansion. We express trastuzumab bearing CypK and conjugate tetrazine derivs. to the antibody. We show that the dihydropyridazine linkage resulting from the conjugation reaction is stable in serum, and generate an ADC bearing monomethyl auristatin E that selectively kills cells expressing a high level of HER2. Our results demonstrate that CypK is a minimal bioorthogonal handle for the rapid prodn. of stable therapeutic protein conjugates.
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    8. 8
      Devaraj, N. K.; Thurber, G. M.; Keliher, E. J.; Marinelli, B.; Weissleder, R. Reactive polymer enables efficient in vivo bioorthogonal chemistry. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 4762– 4767,  DOI: 10.1073/pnas.1113466109
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      8
      Reactive polymer enables efficient in vivo bioorthogonal chemistry
      Devaraj, Neal K.; Thurber, Greg M.; Keliher, Edmund J.; Marinelli, Brett; Weissleder, Ralph
      Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (13), 4762-4767, S4762/1-S4762/2CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      There has been intense interest in the development of selective bioorthogonal reactions or "click" chem. that can proceed in live animals. Until now however, most reactions still require vast surpluses of reactants because of steep temporal and spatial concn. gradients. Using computational modeling and design of pharmacokinetically optimized reactants, we have developed a predictable method for efficient in vivo click reactions. Specifically, we show that polymer modified tetrazines (PMT) are a key enabler for in vivo bioorthogonal chem. based on the very fast and catalyst-free [4 + 2] tetrazine/trans-cyclooctene cycloaddn. Using fluorescent PMT for cellular resoln. and 18F labeled PMT for whole animal imaging, we show that cancer cell epitopes can be easily reacted in vivo. This generic strategy should help guide the design of future chemistries and find widespread use for different in vivo bioorthogonal applications, particularly in the biomedical sciences.
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    9. 9
      Antonow, D. Fragment-based approaches and the prospect of fragmented prodrugs. Drug Discovery Today 2010, 15, 801– 803,  DOI: 10.1016/j.drudis.2010.08.015
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      9
      Fragment-based approaches and the prospect of fragmented prodrugs
      Antonow Dyeison
      Drug discovery today (2010), 15 (19-20), 801-3 ISSN:.
      There is no expanded citation for this reference.
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    10. 10
      Zengeya, T. T.; Garlick, J. M.; Kulkarni, R. A.; Miley, M.; Roberts, A. M.; Yang, Y.; Crooks, D. R.; Sourbier, C.; Linehan, W. M.; Meier, J. L. Co-opting a Bioorthogonal Reaction for Oncometabolite Detection. J. Am. Chem. Soc. 2016, 138, 15813– 15816,  DOI: 10.1021/jacs.6b09706
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      10
      Co-opting a Bioorthogonal Reaction for Oncometabolite Detection
      Zengeya, Thomas T.; Garlick, Julie M.; Kulkarni, Rhushikesh A.; Miley, Mikayla; Roberts, Allison M.; Yang, Youfeng; Crooks, Daniel R.; Sourbier, Carole; Linehan, W. Marston; Meier, Jordan L.
      Journal of the American Chemical Society (2016), 138 (49), 15813-15816CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Dysregulated metab. is a hallmark of many diseases, including cancer. Methods to fluorescently detect metabolites have the potential to enable new approaches to cancer detection and imaging. However, fluorescent sensing methods for naturally occurring cellular metabolites are relatively unexplored. Here the authors report the development of a chem. approach to detect the oncometabolite fumarate. The authors' strategy exploits a known bioorthogonal reaction, the 1,3-dipolar cycloaddn. of nitrileimines and electron-poor olefins, to detect fumarate via fluorescent pyrazoline cycloadduct formation. The authors demonstrate that hydrazonyl chlorides serve as readily accessible nitrileimine precursors, whose reactivity and spectral properties can be tuned to enable detection of fumarate and other dipolarophile metabolites. Finally, the authors show this reaction can be used to detect enzyme activity changes caused by mutations in fumarate hydratase, which underlie the familial cancer predisposition syndrome hereditary leiomyomatosis and renal cell cancer. The authors' studies define a novel intersection of bioorthogonal chem. and metabolite reactivity that may be harnessed to enable biol. profiling, imaging, and diagnostic applications.
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    11. 11
      Patterson, D. M.; Prescher, J. A. Orthogonal bioorthogonal chemistries. Curr. Opin. Chem. Biol. 2015, 28, 141– 149,  DOI: 10.1016/j.cbpa.2015.07.006
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      11
      Orthogonal bioorthogonal chemistries
      Patterson, David M.; Prescher, Jennifer A.
      Current Opinion in Chemical Biology (2015), 28 (), 141-149CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. Bioorthogonal reactions have long been used to examine individual biomols. in living systems. Studies of multi-component networks demand not only reliable bioorthogonal chemistries, but also combinations of reactions that can be used in tandem. Such orthogonal bioorthogonal transformations have been reported in recent years, and these chemistries are enabling new explorations into biol. This article highlights the development of orthogonal bioorthogonal reactions and their application in multi-target imaging and macromol. assembly. Methods to tune and control orthogonal reactivity are also discussed, along with prospects for identifying new classes of compatible reactions.
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    12. 12
      Yu, J.; Shen, D.; Zhang, H.; Yin, Z. Rapid, Stoichiometric, Site-Specific Modification of Aldehyde-Containing Proteins Using a Tandem Knoevenagel-Intra Michael Addition Reaction. Bioconjugate Chem. 2018, 29, 1016– 1020,  DOI: 10.1021/acs.bioconjchem.8b00086
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      12
      Rapid, Stoichiometric, Site-Specific Modification of Aldehyde-Containing Proteins Using a Tandem Knoevenagel-Intra Michael Addition Reaction
      Yu, Jian; Shen, Da; Zhang, Hanjie; Yin, Zheng
      Bioconjugate Chemistry (2018), 29 (4), 1016-1020CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      A site-specific modification of aldehyde-contg. proteins using a tandem Knoevenagel-intra Michael addn. reaction was developed. The reaction featured fast kinetics (50 M-1s-1) and favorable stoichiometry. Various functionalities could be introduced into the protein with little impact on its function and conformation. The reaction was successfully applied in the labeling of living cells.
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    13. 13
      An, P.; Lewandowski, T. M.; Erbay, T. G.; Liu, P.; Lin, Q. Sterically Shielded, Stabilized Nitrile Imine for Rapid Bioorthogonal Protein Labeling in Live Cells. J. Am. Chem. Soc. 2018, 140, 4860– 4868,  DOI: 10.1021/jacs.8b00126
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      13
      Sterically Shielded, Stabilized Nitrile Imine for Rapid Bioorthogonal Protein Labeling in Live Cells
      An, Peng; Lewandowski, Tracey M.; Erbay, Tugce G.; Liu, Peng; Lin, Qing
      Journal of the American Chemical Society (2018), 140 (14), 4860-4868CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      In pursuit of fast bioorthogonal reactions, reactive moieties have been increasingly employed for selective labeling of biomols. in living systems, posing a challenge in attaining reactivity without sacrificing selectivity. To address this challenge, here the authors report a bioinspired strategy in which mol. shape controls the selectivity of a transient, highly reactive nitrile imine dipole. By tuning the shape of structural pendants attached to the ortho position of the N-aryl ring of diaryltetrazoles, precursors of nitrile imines, the authors discovered a sterically shielded nitrile imine that favors the 1,3-dipolar cycloaddn. over the competing nucleophilic addn. The photogenerated nitrile imine exhibits an extraordinarily long half-life of 102 s in aq. medium, owing to its unique mol. shape that hinders the approach of a nucleophile as shown by DFT calcns. The utility of this sterically shielded nitrile imine in rapid (∼1 min) bioorthogonal labeling of glucagon receptor in live mammalian cells was demonstrated.
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    14. 14
      Matsuo, K.; Nishikawa, Y.; Masuda, M.; Hamachi, I. Live-Cell Protein Sulfonylation Based on Proximity-driven N-Sulfonyl Pyridone Chemistry. Angew. Chem., Int. Ed. 2018, 57, 659– 662,  DOI: 10.1002/anie.201707972
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      14
      Live-Cell Protein Sulfonylation Based on Proximity-driven N-Sulfonyl Pyridone Chemistry
      Matsuo, Kazuya; Nishikawa, Yuki; Masuda, Marie; Hamachi, Itaru
      Angewandte Chemie, International Edition (2018), 57 (3), 659-662CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The development of bioorthogonal approaches for labeling of endogenous proteins under the multimol. crowding conditions of live cells is highly desirable for the anal. and engineering of proteins without using genetic manipulation. N-Sulfonyl pyridone (SP) is reported as a new reactive group for protein sulfonylation. The ligand-directed SP chem. was able to modify not only purified proteins in vitro, but also endogenous ones on the surface of and inside live cells selectively and rapidly, which allowed to convert endogenous proteins to FRET-based biosensors in situ.
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    15. 15
      Fang, Y. Z.; Zhang, H.; Huang, Z.; Scinto, S. L.; Yang, J. C.; Ende, C. W. A.; Dmitrenko, O.; Johnson, D. S.; Fox, J. M. Photochemical syntheses, transformations, and bioorthogonal chemistry of trans-cycloheptene and sila trans-cycloheptene Ag(I) complexes. Chem. Sci. 2018, 9, 1953– 1963,  DOI: 10.1039/C7SC04773H
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      15
      Photochemical syntheses, transformations, and bioorthogonal chemistry of trans-cycloheptene and sila trans-cycloheptene Ag(I) complexes
      Fang, Yinzhi; Zhang, Han; Huang, Zhen; Scinto, Samuel L.; Yang, Jeffrey C.; am Ende, Christopher W.; Dmitrenko, Olga; Johnson, Douglas S.; Fox, Joseph M.
      Chemical Science (2018), 9 (7), 1953-1963CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      A photochem. synthesis of AgNO3 complexes of trans-cycloheptene (TCH) and trans-1-sila-4-cycloheptene (Si-TCH) derivs. is described. A low temp. flow photoreactor was designed to enable the synthesis of carbocyclic TCH derivs. due to their thermal sensitivity in the absence of metal coordination. Unlike the free carbocycles, TCH•AgNO3 complexes can be handled at rt and stored for weeks in the freezer (-18 °C). Si-TCH•AgNO3 complexes are esp. robust, and are bench stable for days at rt, and for months in the freezer. X-ray crystallog. was used to characterize a Si-TCH•AgNO3 complex for the first time. With decomplexation of AgNO3 in situ, metal-free TCO and Si-TCH derivs. can engage in a range of cycloaddn. reactions as well as dihydroxylation reactions. Computation was used to predict that Si-TCH would engage in bioorthogonal reactions that are more rapid than the most reactive trans-cyclooctenes. Metal-free Si-TCH derivs. were shown to display good stability in soln., and to engage in the fastest bioorthogonal reaction reported to date (k2 1.14 x 107 M-1s-1 in 9:1 H2O:MeOH). Utility in bioorthogonal protein labeling in live cells is described, including labeling of GFP with an unnatural tetrazine-contg. amino acid. The reactivity and specificity of the Si-TCH reagents with tetrazines in live mammalian cells was also evaluated using the HaloTag platform. The cell labeling expts. show that Si-TCH derivs. are best suited as probe mols. in the cellular environment.
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    16. 16
      Blackman, M. L.; Royzen, M.; Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity. J. Am. Chem. Soc. 2008, 130, 13518– 13519,  DOI: 10.1021/ja8053805
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      16
      Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity
      Blackman, Melissa L.; Royzen, Maksim; Fox, Joseph M.
      Journal of the American Chemical Society (2008), 130 (41), 13518-13519CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Described is a bioorthogonal reaction that proceeds with unusually fast reaction rates without need for catalysis: the cycloaddn. of s-tetrazine and trans-cyclooctene derivs. The reactions tolerate a broad range of functionality and proceed in high yield in org. solvents, water, cell media, or cell lysate. The rate of the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is very rapid (k2 2000 M-1 s-1). This fast reactivity enables protein modification at low concn.
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    17. 17
      Devaraj, N. K.; Upadhyay, R.; Haun, J. B.; Hilderbrand, S. A.; Weissleder, R. Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition. Angew. Chem., Int. Ed. 2009, 48, 7013– 7016,  DOI: 10.1002/anie.200903233
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      17
      Fast and Sensitive Pretargeted Labeling of Cancer Cells through a Tetrazine/trans-Cyclooctene Cycloaddition
      Devaraj, Neal K.; Upadhyay, Rabi; Haun, Jered B.; Hilderbrand, Scott A.; Weissleder, Ralph
      Angewandte Chemie, International Edition (2009), 48 (38), 7013-7016, S7013/1-S7013/6CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The novel asym. tetrazine I was conjugated to a com. available far-red indocyanine fluorophore, Vivo-Tag 680. This fluorescent tetrazine deriv. was used to image trans-cyclooctene-modified affinity ligands on live cancer cells through a bioorthogonal cycloaddn. with a reaction rate of approx. 6000±200 M-1 s-1 in serum at 37 °C. To maximize the fluorescence signal, up to six trans-cyclooctene moieties were attached to the antibody used to pretarget cells for labeling. Com. available cetuximab was labeled with trans-cyclooctene succinimidyl carbonate and used for pretargeting expts.
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    18. 18
      Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R. A comparative study of bioorthogonal reactions with azides. ACS Chem. Biol. 2006, 1, 644– 648,  DOI: 10.1021/cb6003228
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      18
      A comparative study of bioorthogonal reactions with azides
      Agard, Nicholas J.; Baskin, Jeremy M.; Prescher, Jennifer A.; Lo, Anderson; Bertozzi, Carolyn R.
      ACS Chemical Biology (2006), 1 (10), 644-648CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Detection of metabolites and post-translational modifications can be achieved using the azide as a bioorthogonal chem. reporter. Once introduced into target biomols., either metabolically or through chem. modification, the azide can be tagged with probes using one of three highly selective reactions: the Staudinger ligation, the Cu(I)-catalyzed azide-alkyne cycloaddn., or the strain-promoted [3+2] cycloaddn. Here, the authors compared these chemistries in the context of various biol. applications, including labeling of biomols. in complex lysates and on live cell surfaces. The Cu(I)-catalyzed reaction was most efficient for detecting azides in protein samples but was not compatible with live cells due to the toxicity of the reagents. Both the Staudinger ligation and the strain-promoted [3+2] cycloaddn. using optimized cyclooctynes were effective for tagging azides on live cells. The best reagent for this application was dependent upon the specific structure of the azide. These results provide a guide for biologists in choosing a suitable ligation chem.
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    19. 19
      Dommerholt, J.; van Rooijen, O.; Borrmann, A.; Guerra, C. F.; Bickelhaupt, F. M.; van Delft, F. L. Highly accelerated inverse electron-demand cycloaddition of electron-deficient azides with aliphatic cyclooctynes. Nat. Commun. 2014, 5, 5378,  DOI: 10.1038/ncomms6378
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      19
      Highly accelerated inverse electron-demand cycloaddition of electron-deficient azides with aliphatic cyclooctynes
      Dommerholt, Jan; van Rooijen, Olivia; Borrmann, Annika; Guerra, Celia Fonseca; Bickelhaupt, F. Matthias; van Delft, Floris L.
      Nature Communications (2014), 5 (), 5378CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      Strain-promoted azide-alkyne cycloaddn. (SPAAC) as a conjugation tool has found broad application in material sciences, chem. biol. and even in vivo use. However, despite tremendous effort, SPAAC remains fairly slow (0.2-0.5 M-1 s-1) and efforts to increase reaction rates by tailoring of cyclooctyne structure have suffered from a poor trade-off between cyclooctyne reactivity and stability. We here wish to report tremendous acceleration of strain-promoted cycloaddn. of an aliph. cyclooctyne (bicyclo[6.1.0]non-4-yne, BCN) with electron-deficient aryl azides, with reaction rate consts. reaching 2.0-2.9 M-1 s-1. A remarkable difference in rate consts. of aliph. cyclooctynes vs. benzoannulated cyclooctynes is noted, enabling a next level of orthogonality by a judicious choice of azide-cyclooctyne combinations, which is inter alia applied in one-pot three-component protein labeling. The pivotal role of azide electronegativity is explained by d.-functional theory calcns. and electronic-structure analyses, which indicates an inverse electron-demand mechanism is operative with an aliph. cyclooctyne.
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    20. 20
      Kamber, D. N.; Liang, Y.; Blizzard, R. J.; Liu, F.; Mehl, R. A.; Houk, K. N.; Prescher, J. A. 1,2,4-Triazines Are Versatile Bioorthogonal Reagents. J. Am. Chem. Soc. 2015, 137, 8388– 8391,  DOI: 10.1021/jacs.5b05100
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      20
      1,2,4-Triazines Are Versatile Bioorthogonal Reagents
      Kamber, David N.; Liang, Yong; Blizzard, Robert J.; Liu, Fang; Mehl, Ryan A.; Houk, K. N.; Prescher, Jennifer A.
      Journal of the American Chemical Society (2015), 137 (26), 8388-8391CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A new class of bioorthogonal reagents, 1,2,4-triazines, is described. These scaffolds are stable in biol. media and capable of robust reactivity with trans-cyclooctene (TCO). The enhanced stability of the triazine scaffold enabled its direct use in recombinant protein prodn. The triazine-TCO reaction can also be used in tandem with other bioorthogonal cycloaddn. reactions. These features fill current voids in the bioorthogonal toolkit.
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    21. 21
      Hoffmann, J. E.; Plass, T.; Nikic, I.; Aramburu, I. V.; Koehler, C.; Gillandt, H.; Lemke, E. A.; Schultz, C. Highly Stable trans-Cyclooctene Amino Acids for Live-Cell Labeling. Chem. - Eur. J. 2015, 21, 12266– 12270,  DOI: 10.1002/chem.201501647
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      21
      Highly Stable trans-Cyclooctene Amino Acids for Live-Cell Labeling
      Hoffmann, Jan-Erik; Plass, Tilman; Nikic, Ivana; Valle Aramburu, Iker; Koehler, Christine; Gillandt, Hartmut; Lemke, Edward A.; Schultz, Carsten
      Chemistry - A European Journal (2015), 21 (35), 12266-12270CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Trans-Cyclooctene groups incorporated into proteins via non-canonical amino acids (ncAAs) are emerging as specific handles for bioorthogonal chem. Here, we present a highly improved synthetic access to the axially and the equatorially linked trans-cyclooct-2-ene isomers (1 a,b). We further show that the axially connected isomer has a half-life about 10 times higher than the equatorial isomer and reacts with tetrazines much faster, as detd. by stopped-flow expts. The improved properties resulted in different labeling performance of the insulin receptor on the surface of intact cells.
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    22. 22
      Rossin, R.; van den Bosch, S. M.; Ten Hoeve, W.; Carvelli, M.; Versteegen, R. M.; Lub, J.; Robillard, M. S. Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems. Bioconjugate Chem. 2013, 24, 1210– 1217,  DOI: 10.1021/bc400153y
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      22
      Highly Reactive trans-Cyclooctene Tags with Improved Stability for Diels-Alder Chemistry in Living Systems
      Rossin, Raffaella; van den Bosch, Sandra M.; ten Hoeve, Wolter; Carvelli, Marco; Versteegen, Ron M.; Lub, Johan; Robillard, Marc S.
      Bioconjugate Chemistry (2013), 24 (7), 1210-1217CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      One of the challenges of pretargeted radioimmunotherapy, which centers on the capture of a radiolabeled probe by a preinjected tumor-bound antibody, is the potential immunogenicity of biol. capturing systems. A bioorthogonal chem. approach may circumvent this drawback, but effective in vivo chem. in mice, larger animals, and eventually humans, requires very high reagent reactivity, sufficient stability, and retained selectivity. We report here that the reactivity of the fastest bioorthogonal reaction, the inverse-electron-demand-Diels-Alder cycloaddn. between a tetrazine probe and a trans-cyclooctene-tagged antibody, can be increased 10-fold (k2 = 2.7 × 105 M-1s-1) via the trans-cyclooctene, approaching the speed of biol. interactions, while also increasing its stability. This was enabled by the finding that the trans-cyclooctene tag is probably deactivated through isomerization to the unreactive cis-cyclooctene isomer by interactions with copper-contg. proteins, and that increasing the steric hindrance on the tag can impede this process. Next, we found that the higher reactivity of axial vs. equatorial linked TCO can be augmented by the choice of linker. The new, stabilized, and more reactive tag allowed for improved tumor-to-nontumor ratios in pretargeted tumor-bearing mice.
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    23. 23
      Sletten, E. M.; Bertozzi, C. R. From mechanism to mouse: a tale of two bioorthogonal reactions. Acc. Chem. Res. 2011, 44, 666– 676,  DOI: 10.1021/ar200148z
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      From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions
      Sletten, Ellen M.; Bertozzi, Carolyn R.
      Accounts of Chemical Research (2011), 44 (9), 666-676CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Bioorthogonal reactions are chem. reactions that neither interact with nor interfere with a biol. system. The participating functional groups must be inert to biol. moieties, must selectively reactive with each other under biocompatible conditions, and, for in vivo applications, must be nontoxic to cells and organisms. Addnl., it is helpful if one reactive group is small and therefore minimally perturbing of a biomol. into which it has been introduced either chem. or biosynthetically. Examples from the past decade suggest that a promising strategy for bioorthogonal reaction development begins with an anal. of functional group and reactivity space outside those defined by Nature. Issues such as stability of reactants and products (particularly in water), kinetics, and unwanted side reactivity with biofunctionalities must be addressed, ideally guided by detailed mechanistic studies. Finally, the reaction must be tested in a variety of environments, escalating from aq. media to biomol. solns. to cultured cells and, for the most optimized transformations, to live organisms. Work in our lab. led to the development of two bioorthogonal transformations that exploit the azide as a small, abiotic, and bioinert reaction partner: the Staudinger ligation and strain-promoted azide-alkyne cycloaddn. The Staudinger ligation is based on the classic Staudinger redn. of azides with triarylphosphines first reported in 1919. In the ligation reaction, the intermediate aza-ylide undergoes intramol. reaction with an ester, forming an amide bond faster than aza-ylide hydrolysis would otherwise occur in water. The Staudinger ligation is highly selective and reliably forms its product in environs as demanding as live mice. However, the Staudinger ligation has some liabilities, such as the propensity of phosphine reagents to undergo air oxidn. and the relatively slow kinetics of the reaction. The Staudinger ligation takes advantage of the electrophilicity of the azide; however, the azide can also participate in cycloaddn. reactions. In 1961, Wittig and Krebs noted that the strained, cyclic alkyne cyclooctyne reacts violently when combined neat with Ph azide, forming a triazole product by 1,3-dipolar cycloaddn. This observation stood in stark contrast to the slow kinetics assocd. with 1,3-dipolar cycloaddn. of azides with unstrained, linear alkynes, the conventional Huisgen process. Notably, the reaction of azides with terminal alkynes can be accelerated dramatically by copper catalysis (this highly popular Cu-catalyzed azide-alkyne cycloaddn. (CuAAC) is a quintessential "click" reaction). However, the copper catalysts are too cytotoxic for long-term exposure with live cells or organisms. Thus, for applications of bioorthogonal chem. in living systems, we built upon Wittig and Krebs' observation with the design of cyclooctyne reagents that react rapidly and selectively with biomol.-assocd. azides. This strain-promoted azide-alkyne cycloaddn. is often referred to as "Cu-free click chem.". Mechanistic and theor. studies inspired the design of a series of cyclooctyne compds. bearing fluorine substituents, fused rings, and judiciously situated heteroatoms, with the goals of optimizing azide cycloaddn. kinetics, stability, soly., and pharmacokinetic properties. Cyclooctyne reagents have now been used for labeling azide-modified biomols. on cultured cells and in live Caenorhabditis elegans, zebrafish, and mice. As this special issue testifies, the field of bioorthogonal chem. is firmly established as a challenging frontier of reaction methodol. and an important new instrument for biol. discovery. The above reactions, as well as several newcomers with bioorthogonal attributes, have enabled the high-precision chem. modification of biomols. in vitro, as well as real-time visualization of mols. and processes in cells and live organisms. The consequence is an impressive body of new knowledge and technol., amassed using a relatively small bioorthogonal reaction compendium. Expansion of this toolkit, an effort that is already well underway, is an important objective for chemists and biologists alike.
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      Link, A. J.; Vink, M. K.; Tirrell, D. A. Presentation and detection of azide functionality in bacterial cell surface proteins. J. Am. Chem. Soc. 2004, 126, 10598– 10602,  DOI: 10.1021/ja047629c
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      Presentation and detection of azide functionality in bacterial cell surface proteins
      Link, A. James; Vink, Mandy K. S.; Tirrell, David A.
      Journal of the American Chemical Society (2004), 126 (34), 10598-10602CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      An improved protocol for copper-catalyzed triazole formation on the bacterial cell surface is described. Addn. of highly pure CuBr to cells treated with azidohomoalanine (2) leads to ∼. 10-Fold more extensive cell surface labeling than previously obsd. This highly active catalyst allows detection of the methionine analogs azidoalanine (1), azidonorvaline (3), and azidonorleucine (4) in cell surface proteins. Azidoalanine was previously believed to be silent with regard to the cellular protein synthesis machinery.
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      Tian, F.; Tsao, M. L.; Schultz, P. G. A phage display system with unnatural amino acids. J. Am. Chem. Soc. 2004, 126, 15962– 15963,  DOI: 10.1021/ja045673m
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      A Phage Display System with Unnatural Amino Acids
      Tian, Feng; Tsao, Meng-Lin; Schultz, Peter G.
      Journal of the American Chemical Society (2004), 126 (49), 15962-15963CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We report a general method to display peptide-contg. unnatural amino acids on filamentous M13 phage. Five distinct unnatural amino acids were site-specifically incorporated at the N-terminal of the M13 phage minor coat protein pIII. Phages that contain p-azidophenylalanine can undergo a highly specific azide-alkyne [3+2] cycloaddn. reaction with an alkyne-derivatized fluorophore. The generalization of phage display to include unnatural amino acids should significantly increase the scope of phage display technol.
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      Yang, J.; Seckute, J.; Cole, C. M.; Devaraj, N. K. Live-cell imaging of cyclopropene tags with fluorogenic tetrazine cycloadditions. Angew. Chem., Int. Ed. 2012, 51, 7476– 7479,  DOI: 10.1002/anie.201202122
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      Live-Cell Imaging of Cyclopropene Tags with Fluorogenic Tetrazine Cycloadditions
      Yang, Jun; Seckute, Jolita; Cole, Christian M.; Devaraj, Neal K.
      Angewandte Chemie, International Edition (2012), 51 (30), 7476-7479, S7476/1-S7476/26CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The authors demonstrated the applicability of methylcyclopropene tags as dienophiles for reaction with fluorogenic tetrazines. Through systematic synthetic modifications, the stability, size, and reactivity of the cyclopropene scaffold were optimized. Methylcyclopropene derivs. were developed that react rapidly with tetrazines while retaining their aq. stability and small size. These cyclopropene handles elicited fluorescent responses from quenched tetrazine dyes and were suitable for cellular imaging applications, which was demonstrated by imaging cyclopropene phospholipids distributed in live human breast cancer cells.
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      Patterson, D. M.; Nazarova, L. A.; Xie, B.; Kamber, D. N.; Prescher, J. A. Functionalized cyclopropenes as bioorthogonal chemical reporters. J. Am. Chem. Soc. 2012, 134, 18638– 18643,  DOI: 10.1021/ja3060436
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      Functionalized Cyclopropenes as Bioorthogonal Chemical Reporters
      Patterson, David M.; Nazarova, Lidia A.; Xie, Bryan; Kamber, David N.; Prescher, Jennifer A.
      Journal of the American Chemical Society (2012), 134 (45), 18638-18643CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Chem. reporters are unique functional groups that can be used to label biomols. in living systems. Only a handful of broadly applicable reporters have been identified to date, owing to the rigorous demands placed on these functional groups in biol. settings. We describe here a new chem. reporter-cyclopropene-that can be used to target biomols. in vitro and in live cells. A variety of substituted cyclopropene scaffolds were synthesized and found to be stable in aq. soln. and in the presence of biol. nucleophiles. Furthermore, some of the cyclopropene units were metabolically introduced into cell surface glycans and subsequently detected with covalent probes. The small size and selective reactivity of cyclopropenes will facilitate efforts to tag diverse collections of biomols. in vivo.
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      Andersen, K. A.; Aronoff, M. R.; McGrath, N. A.; Raines, R. T. Diazo groups endure metabolism and enable chemoselectivity in cellulo. J. Am. Chem. Soc. 2015, 137, 2412– 2415,  DOI: 10.1021/ja5095815
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      Diazo Groups Endure Metabolism and Enable Chemoselectivity in Cellulo
      Andersen, Kristen A.; Aronoff, Matthew R.; McGrath, Nicholas A.; Raines, Ronald T.
      Journal of the American Chemical Society (2015), 137 (7), 2412-2415CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The authors introduce a stabilized diazo group as a reporter for chem. biol. ManDiaz, which is a diazo deriv. of N-acetylmannosamine, is found to endure cellular metab. and label the surface of a mammalian cell. There its diazo group can undergo a 1,3-dipolar cycloaddn. with a strained alkyne, providing a signal comparable to that from the azido congener, ManNAz. The chemoselectivity of diazo and alkynyl groups enables dual labeling of cells that is not possible with azido and alkynyl groups. Thus, the diazo group, which is approx. half the size of an azido group, provides unique opportunities for orthogonal labeling of cellular components.
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      Karver, M. R.; Weissleder, R.; Hilderbrand, S. A. Bioorthogonal reaction pairs enable simultaneous, selective, multi-target imaging. Angew. Chem., Int. Ed. 2012, 51, 920– 922,  DOI: 10.1002/anie.201104389
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      Bioorthogonal Reaction Pairs Enable Simultaneous, Selective, Multi-Target Imaging
      Karver, Mark R.; Weissleder, Ralph; Hilderbrand, Scott A.
      Angewandte Chemie, International Edition (2012), 51 (4), 920-922, S920/1-S920/12CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The authors describe the testing and in cell validation of a tetrazine-trans-cyclooctene (TCO) reaction pair that is orthogonal to azide-cyclooctyne cycloaddn. chem. The chosen pairs were able to react concurrently in the same culture to fluorescently label two different cancer cell types. The authors' method uses only small mol. based reagents that also can be readily incorporated into a wide variety of systems and potentially may be used in conjunction with fluorogenic azide and tetrazine substrates to achieve addnl. improvements in specificity and sensitivity. The ability to perform multiple, rapid, simultaneous chem. reactions with a high degree of specificity in chem. complex environments should prove to be a useful tool in chem., biol., and medicine.
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      Cole, C. M.; Yang, J.; Seckute, J.; Devaraj, N. K. Fluorescent live-cell imaging of metabolically incorporated unnatural cyclopropene-mannosamine derivatives. ChemBioChem 2013, 14, 205– 208,  DOI: 10.1002/cbic.201200719
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      Fluorescent Live-Cell Imaging of Metabolically Incorporated Unnatural Cyclopropene-Mannosamine Derivatives
      Cole, Christian M.; Yang, Jun; Seckute, Jolita; Devaraj, Neal K.
      ChemBioChem (2013), 14 (2), 205-208CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      This study reported that unnatural cyclo-propene-mannosamine derivs. can be used to image glycans on live human cancer cell lines. The results showed that recently developed methylcyclopropene bioorthogonal handles can be utilized for metabolic imaging of unnatural mannosamine derivs. on live-cell surfaces. In general, the data exhibit the possible use of cyclopropenes as reactive mini-tags for a myriad of applications in the profiling and imaging of small mols.
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      Spate, A. K.; Busskamp, H.; Niederwieser, A.; Schart, V. F.; Marx, A.; Wittmann, V. Rapid labeling of metabolically engineered cell-surface glycoconjugates with a carbamate-linked cyclopropene reporter. Bioconjugate Chem. 2014, 25, 147– 154,  DOI: 10.1021/bc4004487
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      Rapid labeling of metabolically engineered cell-surface glycoconjugates with a carbamate-linked cyclopropene reporter
      Spate Anne-Katrin; Busskamp Holger; Niederwieser Andrea; Schart Verena F; Marx Andreas; Wittmann Valentin
      Bioconjugate chemistry (2014), 25 (1), 147-54 ISSN:.
      Metabolic oligosaccharide engineering is a valuable tool to monitor cellular carbohydrates. Here, we report the synthesis of a novel N-acyl-mannosamine derivative bearing a methylcyclopropene tag that is attached to the sugar via a carbamate moiety. This derivative undergoes rapid Diels-Alder reaction with inverse electron demand. We demonstrate that the cell's biosynthetic machinery incorporates this non-natural mannosamine derivative into glycoconjugates that can, subsequently, be labeled within less than 10 min with a new sulfo-Cy3-tetrazine conjugate. Using this tetrazine-dye conjugate for the detection of the methylcyclopropene-tagged mannosamine derivative, we could achieve dual labeling of two different metabolically incorporated sugars combining a Diels-Alder reaction with inverse electron demand and a strain-promoted azide-alkyne cycloaddition which are carried out simultaneously in a single step.
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      Sachdeva, A.; Wang, K.; Elliott, T.; Chin, J. W. Concerted, rapid, quantitative, and site-specific dual labeling of proteins. J. Am. Chem. Soc. 2014, 136, 7785– 7788,  DOI: 10.1021/ja4129789
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      Concerted, Rapid, Quantitative, and Site-Specific Dual Labeling of Proteins
      Sachdeva, Amit; Wang, Kaihang; Elliott, Thomas; Chin, Jason W.
      Journal of the American Chemical Society (2014), 136 (22), 7785-7788CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small mols. at physiol. pH, temp., and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-contg. amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quant., site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, resp. Protein double labeling in aq. buffer at physiol. pH, temp., and pressure is quant. in 30 min.
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      Self-assembling cytotoxins
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      Science (Washington, DC, United States) (1986), 233 (4763), 561-3CODEN: SCIEAS; ISSN:0036-8075.
      Decanal [112-31-2] and N-amino-N'-1-octylguanidine (AOG) [66002-80-0], combined at 28 μM each, mediated erythrocyte lysis within 80 min under physiol. conditions. By contrast, no lysis was obsd. after 20 h with decanal (56 μM) or AOG (100 μM) alone. The pronounced synergism obsd. for these chems. and similar reactive pairs of chems. is due to the self-assembly of more cytotoxic hydrazones in situ. Decanal and AOG also exhibit synergistic activity against cultured human cells (HeLa) and bacteria (Escherichia coli J96). This synergism may be useful in the design of cytotoxins that would self-assemble selectively from nontoxic precursors within tumors, while sparing normal tissue.
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      The rise of fragment-based drug discovery
      Murray, Christopher W.; Rees, David C.
      Nature Chemistry (2009), 1 (3), 187-192CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      A review. The search for new drugs is plagued by high attrition rates at all stages in research and development. Chemists have an opportunity to tackle this problem because attrition can be traced back, in part, to the quality of the chem. leads. Fragment-based drug discovery (FBDD) is a new approach, increasingly used in the pharmaceutical industry, for reducing attrition and providing leads for previously intractable biol. targets. FBDD identifies low-mol.-wt. ligands (∼150 Da) that bind to biol. important macromols. The three-dimensional exptl. binding mode of these fragments is detd. using X-ray crystallog. or NMR spectroscopy, and is used to facilitate their optimization into potent mols. with drug-like properties. Compared with high-throughput-screening, the fragment approach requires fewer compds. to be screened, and, despite the lower initial potency of the screening hits, offers more efficient and fruitful optimization campaigns. Here, we review the rise of FBDD, including its application to discovering clin. candidates against targets for which other chem. approaches have struggled.
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      Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Click chemistry for drug development and diverse chemical-biology applications. Chem. Rev. 2013, 113, 4905– 4979,  DOI: 10.1021/cr200409f
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      Click Chemistry for Drug Development and Diverse Chemical-Biology Applications
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      Chemical Reviews (Washington, DC, United States) (2013), 113 (7), 4905-4979CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review with 303 refs. on application of lick chem. reactions in chem., biochem., and pharmacol.
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      Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Click chemistry in situ: acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks. Angew. Chem., Int. Ed. 2002, 41, 1053– 1057,  DOI: 10.1002/1521-3773(20020315)41:6<1053::AID-ANIE1053>3.0.CO;2-4
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      Click chemistry in situ: Acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks
      Lewis, Warren G.; Green, Luke G.; Grynszpan, Flavio; Radic, Zoran; Carlier, Paul R.; Taylor, Palmer; Finn, M. G.; Sharpless, K. Barry
      Angewandte Chemie, International Edition (2002), 41 (6), 1053-1057CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
      Form-fitting chem. in a protein mold is enabled by the use of the 1,3-dipolar cycloaddn. of azides and alkynes. The enzyme acetylcholinesterase preferentially assembles one pair of these reactants, each of which bears a group that binds to adjacent positions on the protein structure, into a 1,2,3-triazole adduct that is the most potent noncovalent inhibitor of the enzyme yet developed.
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      Mocharla, V. P.; Colasson, B.; Lee, L. V.; Roper, S.; Sharpless, K. B.; Wong, C. H.; Kolb, H. C. In situ click chemistry: enzyme-generated inhibitors of carbonic anhydrase II. Angew. Chem., Int. Ed. 2005, 44, 116– 120,  DOI: 10.1002/anie.200461580
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      In situ click chemistry: Enzyme-generated inhibitors of carbonic anhydrase II
      Mocharla, Vani P.; Colasson, Benoit; Lee, Lac V.; Roeper, Stefanie; Sharpless, K. Barry; Wong, Chi-Huey; Kolb, Hartmuth C.
      Angewandte Chemie, International Edition (2005), 44 (1), 116-120CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Target-guided synthesis: Rather than making and screening thousands of compds. for lead discovery, in situ click chem. employs the biol. target itself to assemble its inhibitors by selectively binding and interconnecting reagents within the confines of its binding sites. Subnanomolar inhibitors of carbonic anhydrase II were produced by the enzyme from simple azide and acetylene precursors.
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      Whiting, M.; Muldoon, J.; Lin, Y. C.; Silverman, S. M.; Lindstrom, W.; Olson, A. J.; Kolb, H. C.; Finn, M. G.; Sharpless, K. B.; Elder, J. H.; Fokin, V. V. Inhibitors of HIV-1 protease by using in situ click chemistry. Angew. Chem., Int. Ed. 2006, 45, 1435– 1439,  DOI: 10.1002/anie.200502161
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      Inhibitors of HIV-1 protease by using in situ click chemistry
      Whiting, Matthew; Muldoon, John; Lin, Ying-Chuan; Silverman, Steven M.; Lindstrom, William; Olson, Arthur J.; Kolb, Hartmuth C.; Finn, M. G.; Sharpless, K. Barry; Elder, John H.; Fokin, Valery V.
      Angewandte Chemie, International Edition (2006), 45 (9), 1435-1439CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Twice poor equals potent: HIV-1 Protease assembles its own potent inhibitor through formation of the triazole linkage from azide- and alkyne-contg. fragments that are themselves poor binders.
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      Devaraj, N. K.; Weissleder, R. Biomedical applications of tetrazine cycloadditions. Acc. Chem. Res. 2011, 44, 816– 827,  DOI: 10.1021/ar200037t
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      Biomedical Applications of Tetrazine Cycloadditions
      Devaraj, Neal K.; Weissleder, Ralph
      Accounts of Chemical Research (2011), 44 (9), 816-827CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Disease mechanisms are increasingly being resolved at the mol. level. Biomedical success at this scale creates synthetic opportunities for combining specifically designed orthogonal reactions in applications such as imaging, diagnostics, and therapy. For practical reasons, it would be helpful if bioorthogonal coupling reactions proceeded with extremely rapid kinetics (k > 103 M-1 s-1) and high specificity. Improving kinetics would minimize both the time and amt. of labeling agent required to maintain high coupling yields. In this Account, we discuss our recent efforts to design extremely rapid bioorthogonal coupling reactions between tetrazines and strained alkenes. These selective reactions were first used to covalently couple conjugated tetrazine near-IR-emitting fluorophores to dienophile-modified extracellular proteins on living cancer cells. Confocal fluorescence microscopy demonstrated efficient and selective labeling, and control expts. showed minimal background fluorescence. Multistep techniques were optimized to work with nanomolar concns. of labeling agent over a time scale of minutes: the result was successful real-time imaging of covalent modification. We subsequently discovered fluorogenic probes that increase in fluorescence intensity after the chem. reaction, leading to an improved signal-to-background ratio. Fluorogenic probes were used for intracellular imaging of dienophiles. We further developed strategies to react and image chemotherapeutics, such as trans-cyclooctene taxol analogs, inside living cells. Because the coupling partners are small mols. (<300 Da), they offer unique steric advantages in multistep amplification. We also describe recent success in using tetrazine reactions to label biomarkers on cells with magneto-fluorescent nanoparticles. Two-step protocols that use bioorthogonal chem. can significantly amplify signals over both one-step labeling procedures as well as two-step procedures that use more sterically hindered biotin-avidin interactions. Nanoparticles can be detected with fluorescence or magnetic resonance techniques. These strategies are now being routinely used on clin. samples for biomarker profiling to predict malignancy and patient outcome. Finally, the authors discuss recent results with tetrazine reactions used for in vivo mol. imaging applications. Rapid tetrazine cycloaddns. allow modular labeling of small mols. with the most commonly used positron emission tomog. isotope, 18F. Addnl., recent work has applied this reaction directly in vivo for the pretargeted imaging of solid tumors. Future work with tetrazine cycloaddns. will undoubtedly lead to optimized protocols, improved probes, and addnl. biomedical applications.
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    40. 40
      Lebraud, H.; Wright, D. J.; Johnson, C. N.; Heightman, T. D. Protein Degradation by In-Cell Self-Assembly of Proteolysis Targeting Chimeras. ACS Cent. Sci. 2016, 2, 927– 934,  DOI: 10.1021/acscentsci.6b00280
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      Protein Degradation by In-Cell Self-Assembly of Proteolysis Targeting Chimeras
      Lebraud, Honorine; Wright, David J.; Johnson, Christopher N.; Heightman, Tom D.
      ACS Central Science (2016), 2 (12), 927-934CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      Selective degrdn. of proteins by proteolysis targeting chimeras (PROTACs) offers a promising potential alternative to protein inhibition for therapeutic intervention. Current PROTAC mols. incorporate a ligand for the target protein, a linker, and an E3 ubiquitin ligase recruiting group, which bring together target protein and ubiquitinating machinery. Such hetero-bifunctional mols. require significant linker optimization and possess high mol. wt., which can limit cellular permeation, soly., and other drug-like properties. We show here that the hetero-bifunctional mol. can be formed intracellularly by bio-orthogonal click combination of two smaller precursors. We designed a tetrazine tagged thalidomide deriv. which reacts rapidly with a trans-cyclo-octene tagged ligand of the target protein in cells to form a cereblon E3 ligase recruiting PROTAC mol. The in-cell click-formed proteolysis targeting chimeras (CLIPTACs) were successfully used to degrade two key oncol. targets, BRD4 and ERK1/2. ERK1/2 degrdn. was achieved using a CLIPTAC based on a covalent inhibitor. We expect this approach to be readily extendable to other inhibitor-protein systems because the tagged E3 ligase recruiter is capable of undergoing the click reaction with a suitably tagged ligand of any protein of interest to elicit its degrdn.
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      Versteegen, R. M.; Rossin, R.; ten Hoeve, W.; Janssen, H. M.; Robillard, M. S. Click to release: instantaneous doxorubicin elimination upon tetrazine ligation. Angew. Chem., Int. Ed. 2013, 52, 14112– 14116,  DOI: 10.1002/anie.201305969
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      Click to Release: Instantaneous Doxorubicin Elimination upon Tetrazine Ligation
      Versteegen, Ron M.; Rossin, Raffaella; ten Hoeve, Wolter; Janssen, Henk M.; Robillard, Marc S.
      Angewandte Chemie, International Edition (2013), 52 (52), 14112-14116CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We have developed a new bioorthogonal elimination reaction that enables instantaneous, self-immolative, and traceless release of doxorubicin from trans-cyclooctene following tetrazine ligation. In this proof-of-principle study, the inverse-electron-demand Diels-Alder (inv-DA) pyridazine elimination reached 79 % yield within minutes under ambient conditions at micromolar concns.
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      Xu, M.; Tu, J.; Franzini, R. M. Rapid and efficient tetrazine-induced drug release from highly stable benzonorbornadiene derivatives. Chem. Commun. 2017, 53, 6271– 6274,  DOI: 10.1039/C7CC03477F
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      Rapid and efficient tetrazine-induced drug release from highly stable benzonorbornadiene derivatives
      Xu, Minghao; Tu, Julian; Franzini, Raphael M.
      Chemical Communications (Cambridge, United Kingdom) (2017), 53 (46), 6271-6274CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      A novel class of bioorthogonal release reactions based on benzonorbornadiene derivs. was developed. These carrier mols. are highly stable at physiol. conditions, but react rapidly with 1,2,4,5-tetrazines, and near-quant. release cargo mols. such as drugs and optical reporters.
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      Neumann, K.; Jain, S.; Gambardella, A.; Walker, S. E.; Valero, E.; Lilienkampf, A.; Bradley, M. Tetrazine-Responsive Self-immolative Linkers. ChemBioChem 2017, 18, 91– 95,  DOI: 10.1002/cbic.201600560
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      Tetrazine-Responsive Self-immolative Linkers
      Neumann, Kevin; Jain, Sarthak; Gambardella, Alessia; Walker, Sarah E.; Valero, Elsa; Lilienkampf, Annamaria; Bradley, Mark
      ChemBioChem (2017), 18 (1), 91-95CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Mols. that undergo activation or modulation following the addn. of benign external small-mol. chem. stimuli have numerous applications. Here, we report the highly efficient "decaging" of a variety of moieties by activation of a "self-immolative" linker, by application of water-sol. and stable tetrazine, including the controlled delivery of doxorubicin in a cellular context.
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      Jimenez-Moreno, E.; Guo, Z.; Oliveira, B. L.; Albuquerque, I. S.; Kitowski, A.; Guerreiro, A.; Boutureira, O.; Rodrigues, T.; Jimenez-Oses, G.; Bernardes, G. J. Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells. Angew. Chem., Int. Ed. 2017, 56, 243– 247,  DOI: 10.1002/anie.201609607
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      Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells
      Jimenez-Moreno, Ester; Guo, Zijian; Oliveira, Bruno L.; Albuquerque, Ines S.; Kitowski, Annabel; Guerreiro, Ana; Boutureira, Omar; Rodrigues, Tiago; Jimenez-Oses, Gonzalo; Bernardes, Goncalo J. L.
      Angewandte Chemie, International Edition (2017), 56 (1), 243-247CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The cleavage of a protecting group from a protein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity. Disclosed herein is that vinyl ethers serve as protecting groups for alc.-contg. mols. and as reagents for bioorthogonal bond-cleavage reactions. A vinyl ether moiety was installed in a range of mols., including amino acids, a monosaccharide, a fluorophore, and an analog of the cytotoxic drug duocarmycin. Tetrazine-mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics. Importantly, the nontoxic, vinyl ether duocarmycin double prodrug was successfully decaged in live cells to reinstate cytotoxicity. This bioorthogonal reaction presents broad applicability and may be suitable for in vivo applications.
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      Mejia Oneto, J. M.; Khan, I.; Seebald, L.; Royzen, M. In Vivo Bioorthogonal Chemistry Enables Local Hydrogel and Systemic Pro-Drug To Treat Soft Tissue Sarcoma. ACS Cent. Sci. 2016, 2, 476– 482,  DOI: 10.1021/acscentsci.6b00150
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      In Vivo Bioorthogonal Chemistry Enables Local Hydrogel and Systemic Pro-Drug To Treat Soft Tissue Sarcoma
      Mejia Oneto, Jose M.; Khan, Irfan; Seebald, Leah; Royzen, Maksim
      ACS Central Science (2016), 2 (7), 476-482CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      The ability to activate drugs only at desired locations avoiding systemic immunosuppression and other dose limiting toxicities is highly desirable. Here we present a new approach, named local drug activation, that uses bioorthogonal chem. to conc. and activate systemic small mols. at a location of choice. This method is independent of endogenous cellular or environmental markers and only depends on the presence of a preimplanted biomaterial near a desired site (e.g., tumor). We demonstrate the clear therapeutic benefit with minimal side effects of this approach in mice over systemic therapy using a doxorubicin pro-drug against xenograft tumors of a type of soft tissue sarcoma (HT1080).
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      Zhang, G.; Li, J.; Xie, R.; Fan, X.; Liu, Y.; Zheng, S.; Ge, Y.; Chen, P. R. Bioorthogonal Chemical Activation of Kinases in Living Systems. ACS Cent. Sci. 2016, 2, 325– 331,  DOI: 10.1021/acscentsci.6b00024
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      Bioorthogonal Chemical Activation of Kinases in Living Systems
      Zhang, Gong; Li, Jie; Xie, Ran; Fan, Xinyuan; Liu, Yanjun; Zheng, Siqi; Ge, Yun; Chen, Peng R.
      ACS Central Science (2016), 2 (5), 325-331CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      Selective manipulation of protein kinases under living conditions is highly desirable yet extremely challenging, particularly in a gain-of-function fashion. Here we employ our recently developed bioorthogonal cleavage reaction as a general strategy for intracellular activation of individual kinases. Site-specific incorporation of trans-cyclooctene-caged lysine in place of the conserved catalytic lysine, in conjunction with the cleavage partner dimethyl-tetrazine, allowed efficient lysine decaging with the kinase activity chem. rescued in living systems.
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      Li, J.; Jia, S.; Chen, P. R. Diels-Alder reaction-triggered bioorthogonal protein decaging in living cells. Nat. Chem. Biol. 2014, 10, 1003– 1005,  DOI: 10.1038/nchembio.1656
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      Diels-Alder reaction-triggered bioorthogonal protein decaging in living cells
      Li, Jie; Jia, Shang; Chen, Peng R.
      Nature Chemical Biology (2014), 10 (12), 1003-1005CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Small mols. that specifically activate an intracellular protein of interest are highly desirable. A generally applicable strategy, however, remains elusive. Herein we describe a small mol.-triggered bioorthogonal protein decaging technique that relies on the inverse electron-demand Diels-Alder reaction for eliminating a chem. caged protein side chain within living cells. This method permits the efficient activation of a given protein (for example, an enzyme) in its native cellular context within minutes.
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      Wu, H.; Alexander, S. C.; Jin, S.; Devaraj, N. K. A Bioorthogonal Near-Infrared Fluorogenic Probe for mRNA Detection. J. Am. Chem. Soc. 2016, 138, 11429– 11432,  DOI: 10.1021/jacs.6b01625
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      A Bioorthogonal Near-Infrared Fluorogenic Probe for mRNA Detection
      Wu, Haoxing; Alexander, Seth C.; Jin, Shuaijiang; Devaraj, Neal K.
      Journal of the American Chemical Society (2016), 138 (36), 11429-11432CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      There is significant interest in developing methods that visualize and detect RNA in live cells. Bioorthogonal template driven tetrazine ligations could be a powerful route to visualizing nucleic acids in native cells, yet past work has been limited with respect to the diversity of fluorogens that can be activated via a tetrazine reaction. Herein we report a novel bioorthogonal tetrazine uncaging reaction that harnesses tetrazine reactivity to unmask vinyl ether caged fluorophores spanning the visible spectrum, including a near-IR (NIR) emitting cyanine dye. Vinyl ether caged fluorophores and tetrazine partners are conjugated to high affinity antisense nucleic acid probes, which show highly selective fluorogenic reactivity when annealed to their resp. target RNA sequences. A target sequence in the 3' untranslated region (3' UTR) of an expressed mRNA was successfully detected in live cells employing appropriate nucleic acid probes bearing a tetrazine reactive NIR fluorogen. Given the expansion of tetrazine fluorogenic chem. to NIR dyes, we believe highly selective proximity induced fluorogenic tetrazine reactions could find broad uses in illuminating endogenous biomols. in living cells.
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      Ji, X.; Zhou, C.; Ji, K.; Aghoghovbia, R. E.; Pan, Z.; Chittavong, V.; Ke, B.; Wang, B. Click and Release: A Chemical Strategy toward Developing Gasotransmitter Prodrugs by Using an Intramolecular Diels-Alder Reaction. Angew. Chem., Int. Ed. 2016, 55, 15846– 15851,  DOI: 10.1002/anie.201608732
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      Click and Release: A Chemical Strategy toward Developing Gasotransmitter Prodrugs by Using an Intramolecular Diels-Alder Reaction
      Ji, Xingyue; Zhou, Cheng; Ji, Kaili; Aghoghovbia, Robert E.; Pan, Zhixiang; Chittavong, Vayou; Ke, Bowen; Wang, Binghe
      Angewandte Chemie, International Edition (2016), 55 (51), 15846-15851CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Prodrug strategies have been proven to be a very effective way of addressing delivery problems. Much of the chem. in prodrug development relies on the ability to mask an appropriate functional group, which can be removed under appropriate conditions. However, developing org. prodrugs of gasotransmitters represent unique challenges. This is esp. true with carbon monoxide, which does not have an easy "handle" for bioreversible derivatization. By taking advantage of an intramol. Diels-Alder reaction, we have developed a prodrug strategy for prepns. of org. CO prodrugs (I, where X = O, NR1 and n = 1,2) that are stable during synthesis and storage, and yet readily release CO with tunable release rates under near physiol. conditions. The effectiveness of the CO prodrug system in delivering a sufficient quantity of CO for possible therapeutic applications has been studied using a cell culture anti-inflammatory assay and a colitis animal model. These studies fully demonstrate the proof of concept, and lay a strong foundation for further medicinal chem. work in developing org. CO prodrugs.
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      Carlson, J. C. T.; Mikula, H.; Weissleder, R. Unraveling Tetrazine-Triggered Bioorthogonal Elimination Enables Chemical Tools for Ultrafast Release and Universal Cleavage. J. Am. Chem. Soc. 2018, 140, 3603– 3612,  DOI: 10.1021/jacs.7b11217
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      Unraveling tetrazine-triggered bioorthogonal elimination enables chemical tools for ultrafast release and universal cleavage
      Carlson, Jonathan C. T.; Mikula, Hannes; Weissleder, Ralph
      Journal of the American Chemical Society (2018), 140 (10), 3603-3612CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Recent developments in bond cleavage reactions have expanded the scope of bioorthogonal chem. beyond click ligation and enabled new strategies for probe activation and therapeutic delivery. These applications, however, remain in their infancy, with further innovations needed to achieve the efficiency required for versatile and broadly useful tools in vivo. Among these chemistries, the tetrazine/trans-cyclooctene click-to-release reaction has exemplary kinetics and adaptability but achieves only partial release and is incompletely understood, which has limited its application. Investigating the mechanistic features of this reaction's performance, we discovered profound pH sensitivity, exploited it with acid-functionalized tetrazines that both enhance and markedly accelerate release, and ultimately uncovered an unexpected dead-end isomer as the reason for poor release. Implementing facile methods to prevent formation of this dead end, we have achieved exceptional efficiency, with essentially complete release across the full scope of physiol. pH, potentiating drug-delivery strategies and expanding the dynamic range of bioorthogonal on/off control.
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      El-Sagheer, A. H.; Brown, T. Synthesis and polymerase chain reaction amplification of DNA strands containing an unnatural triazole linkage. J. Am. Chem. Soc. 2009, 131, 3958– 3964,  DOI: 10.1021/ja8065896
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      Synthesis and Polymerase Chain Reaction Amplification of DNA Strands Containing an Unnatural Triazole Linkage
      El-Sagheer, Afaf H.; Brown, Tom
      Journal of the American Chemical Society (2009), 131 (11), 3958-3964CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      DNA strands contg. an unnatural T-triazole-T linkage have been synthesized by click DNA ligation between oligonucleotides with 3'-AZT and 5'-propargylamido dT and amplified efficiently by polymerase chain reaction (PCR) using several different polymerases. DNA sequencing of PCR amplicons and clones in two different sequence contexts revealed the presence of a single thymidine at the ligation site. The remarkable ability of thermostable polymerases to reproducibly copy DNA templates contg. such an unnatural backbone opens up intriguing possibilities in gene synthesis, genetic anal., biol., and nanotechnol.
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      Budin, I.; Devaraj, N. K. Membrane assembly driven by a biomimetic coupling reaction. J. Am. Chem. Soc. 2012, 134, 751– 753,  DOI: 10.1021/ja2076873
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      Membrane Assembly Driven by a Biomimetic Coupling Reaction
      Budin, Itay; Devaraj, Neal K.
      Journal of the American Chemical Society (2012), 134 (2), 751-753CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      One of the major goals of synthetic biol. is the development of non-natural cellular systems. In this work, we describe a catalytic biomimetic coupling reaction capable of driving the de novo self-assembly of phospholipid membranes. Our system features a copper-catalyzed azide-alkyne cycloaddn. that results in the formation of a triazole-contg. phospholipid analog. Concomitant assembly of membranes occurs spontaneously, not requiring preexisting membranes to house catalysts or precursors. The substitution of efficient synthetic reactions for key biochem. processes may offer a general route toward synthetic biol. systems.
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      Cendret, V.; Francois-Heude, M.; Mendez-Ardoy, A.; Moreau, V.; Fernandez, J. M.; Djedaini-Pilard, F. Design and synthesis of a ″click″ high-mannose oligosaccharide mimic emulating Man8 binding affinity towards Con A. Chem. Commun. 2012, 48, 3733– 3735,  DOI: 10.1039/c2cc30773a
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      Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Synthesis of proteins by native chemical ligation. Science 1994, 266, 776– 779,  DOI: 10.1126/science.7973629
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      Synthesis of proteins by native chemical ligation
      Dawson, Philip E.; Muir, Tom W.; Clark-Lewis, Ian; Kent, Stephen B. H.
      Science (Washington, D. C.) (1994), 266 (5186), 776-9CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chem. ligation was demonstrated by the one-step prepn. of a cytokine contg. multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-contg. protein mol. Native chem. ligation is an important step toward the general application of chem. to proteins.
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      de Figueiredo, R. M.; Suppo, J. S.; Campagne, J. M. Nonclassical Routes for Amide Bond Formation. Chem. Rev. 2016, 116, 12029– 12122,  DOI: 10.1021/acs.chemrev.6b00237
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      Nonclassical Routes for Amide Bond Formation
      de Figueiredo Renata Marcia; Suppo Jean-Simon; Campagne Jean-Marc
      Chemical reviews (2016), 116 (19), 12029-12122 ISSN:.
      The present review offers an overview of nonclassical (e.g., with no pre- or in situ activation of a carboxylic acid partner) approaches for the construction of amide bonds. The review aims to comprehensively discuss relevant work, which was mainly done in the field in the last 20 years. Organization of the data follows a subdivision according to substrate classes: catalytic direct formation of amides from carboxylic and amines ( section 2 ); the use of carboxylic acid surrogates ( section 3 ); and the use of amine surrogates ( section 4 ). The ligation strategies (NCL, Staudinger, KAHA, KATs, etc.) that could involve both carboxylic acid and amine surrogates are treated separately in section 5 .
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      Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. High-yielding Staudinger ligation of a phosphinothioester and azide to form a peptide. Org. Lett. 2001, 3, 9– 12,  DOI: 10.1021/ol006739v
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      High-Yielding Staudinger Ligation of a Phosphinothioester and Azide To Form a Peptide
      Nilsson, Bradley L.; Kiessling, Laura L.; Raines, Ronald T.
      Organic Letters (2001), 3 (1), 9-12CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      The Staudinger ligation can be used to couple a peptide with a C-terminal phosphinothioester to another with an N-terminal α-azido group to form a single peptide that contains no residual atoms of the phosphinothiol entity. Here, diphenylphosphinomethanethiol thioesters are shown to give high isolated yields for this transformation. This finding provides precedent for a powerful and versatile new method for the total synthesis of proteins, avoiding the native chem. ligation method's reliance on the presence of a cysteine residue for bond formation.
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      Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. A ″traceless″ Staudinger ligation for the chemoselective synthesis of amide bonds. Org. Lett. 2000, 2, 2141– 2143,  DOI: 10.1021/ol006054v
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      A "Traceless" Staudinger Ligation for the Chemoselective Synthesis of Amide Bonds
      Saxon, Eliana; Armstrong, Joshua I.; Bertozzi, Carolyn R.
      Organic Letters (2000), 2 (14), 2141-2143CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      A novel modification is reported of the authors' previously reported Staudinger ligation that generates an amide bond from an azide and a specifically functionalized phosphine. This method for the selective formation of an amide bond, which does not require the orthogonal protection of distal functional groups, should find general utility in synthetic and biol. chem. 2-AcOC6H4PPh2 and (1-acetyl-2-imidazolyl)diphenylphosphine reacted with an azidonucleoside (5'-azido-N-benzoyl-2',5'-dideoxyadenosine) in wet THF to convert the azido group to an acetylamino group in >95% yield.
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      Erben, A.; Grossmann, T. N.; Seitz, O. DNA-triggered synthesis and bioactivity of proapoptotic peptides. Angew. Chem., Int. Ed. 2011, 50, 2828– 2832,  DOI: 10.1002/anie.201007103
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      DNA-Triggered Synthesis and Bioactivity of Proapoptotic Peptides
      Erben, Anne; Grossmann, Tom N.; Seitz, Oliver
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      Diseases are frequently caused by changes in the genetic infrastructure. In such cases, the disordered state of a diseased cell is encoded in the DNA and reflected in the level and sequence of the expressed RNA mols. The information obtained from nucleic acids may be used to direct mol. therapies only to diseased cells and tissues. In a fascinating approach, disease-specific nucleic acid sequences could be hijacked to trigger the formation or release of drug mols. Herein a reaction system is introduced in which the sequence information of an unstructured DNA template is used to trigger the transfer of an aminoacyl group from a donating thioester-modified peptide-nucleic acid (PNA) conjugate to an acceptor peptidyl-PNA conjugate. It is demonstrated that the template can act as a catalyst which instructs the formation of many product mols. per template mol. The formed peptide-PNA conjugate was designed to interfere with the protein-protein interactions between caspase-9, a protease involved in the initiation of programmed cell death (apoptosis), and the X-linked inhibitor of apoptosis protein XIAP. It is shown that the nucleic acid programmed peptide synthesis allows activation of caspase-9 and a downstream caspase.
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      Rapid ligations with equimolar reactants in water with the potassium acyltrifluoroborate (KAT) amide formation
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      The identification of fast, chemoselective bond-forming reactions is one of the major contemporary challenges in chem. We show that chemoselective amide-forming ligations of potassium acyltrifluoroborates (KATs) and O-carbamoylhydroxylamines proceed in the presence of all unprotected functional groups with a second-order rate const. of 20 M-1 s-1. PEG chains, lipids, biotin, and dyes were introduced onto an unprotected 31-mer peptide (GLP-1 analog) with equimolar ratios of reactants within minutes at 1 mM and within 1 h at 100 μM, even with Mw 20 000 PEG. This conjugation reaction provides a new approach to the synthesis of mols. such as protein-protein and protein-polymer conjugates.
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      We have generated a completely autonomous bacterium with a 21 amino acid genetic code. This bacterium can biosynthesize a nonstandard amino acid from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon. The biosynthetic pathway for the amino acid p-aminophenylalanine (pAF) as well as a unique pAF synthetase and cognate tRNA were added to Escherichia coli. Denaturing gel electrophoresis and mass spectrometric anal. show that pAF is incorporated into myoglobin with fidelity and efficiency rivaling those of the common 20 amino acids. This and other such organisms may provide an opportunity to examine the evolutionary consequences of adding new amino acids to the genetic repertoire, as well as generate proteins with new or enhanced biol. functions.
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      Small carbocycles are structurally rigid and possess high intrinsic energy due to their ring strain. These features lead to broad applications but also create challenges for their construction. We report the engineering of a cytochrome P 450 variant (designated P411) that catalyze the formation of chiral bicyclobutanes, one of the most strained four-membered systems, via successive carbene addn. to unsatd. carbon-carbon bonds. Enzymes that produce cyclopropenes, putative intermediates to the bicyclobutanes, were also identified. These genetically encoded proteins are readily optimized by directed evolution, function in Escherichia coli, and act on structurally diverse substrates with high efficiency and selectivity, providing an effective route to many chiral strained structures. This biotransformation is easily performed at preparative scale, and the resulting strained carbocycles can be derivatized, opening myriad potential applications.
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      Lappchen, T.; Rossin, R.; van Mourik, T. R.; Gruntz, G.; Hoeben, F. J. M.; Versteegen, R. M.; Janssen, H. M.; Lub, J.; Robillard, M. S. DOTA-tetrazine probes with modified linkers for tumor pretargeting. Nucl. Med. Biol. 2017, 55, 19– 26,  DOI: 10.1016/j.nucmedbio.2017.09.001
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      DOTA-tetrazine probes with modified linkers for tumor pretargeting
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      Nuclear medicine and biology (2017), 55 (), 19-26 ISSN:.
      INTRODUCTION: Pretargeted radioimmunoimaging and -therapy approaches building on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have yielded impressive results in recent years and have proven a vital alternative to biological pretargeting systems. After improvement of the TCO-antibody conjugates, we here report on our evaluation of a new series of radiolabeled Tz-probes. METHODS: Four new Tz-probes were synthesized, radiolabeled with lutetium-177, and characterized in vitro in terms of lipophilicity, reactivity, and stability in PBS and mouse serum. The in vivo biodistribution profile and tumor-targeting potential of the probes were evaluated in LS174T tumor-bearing mice pretargeted with TCO-antibody conjugates using non-pretargeted mice as control. RESULTS: Radiolabeling of all probes proceeded in high yields providing the (177)Lu-labeled tetrazines in >95% radiochemical purity without any further purification. In mouse serum, half-lives of the probes varied between 8 and 13 h, with the exception of the most lipophilic probe, [(177)Lu]1b, with a serum half-life of less than 1 h. This probe also showed the fastest blood clearance (t1/2 = 5.4 min), more than 2-fold faster than PEG-linked probes [(177)Lu]3 and [(177)Lu]4, and even 3-fold faster than the other small probes without the PEG-linker, [(177)Lu]1a and [(177)Lu]2. In the pretargeting experiments, tumor uptake of the lead probe [(177)Lu]4 (~6 %ID/g) was most closely approached by [(177)Lu]2, followed by [(177)Lu]3 and [(177)Lu]1a. While all the smaller and more lipophilic probes suffered from increased liver uptake, the PEG-linked probe [(177)Lu]3 with its additional negative charge surprisingly showed the highest kidney uptake among all of the probes. CONCLUSION: The in vitro performance of some of the new tetrazine probes turned out to be comparable to the established lead probe [(177)Lu]Lu-DOTA-PEG11-Tz ([(177)Lu]4). However, tumor pretargeting studies in vivo showed lower tumor uptake and increased uptake in non-target organs.
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      Houghton, J. L.; Membreno, R.; Abdel-Atti, D.; Cunanan, K. M.; Carlin, S.; Scholz, W. W.; Zanzonico, P. B.; Lewis, J. S.; Zeglis, B. M. Establishment of the In Vivo Efficacy of Pretargeted Radioimmunotherapy Utilizing Inverse Electron Demand Diels-Alder Click Chemistry. Mol. Cancer Ther. 2017, 16, 124– 133,  DOI: 10.1158/1535-7163.MCT-16-0503
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      Establishment of the In Vivo Efficacy of Pretargeted Radioimmunotherapy Utilizing Inverse Electron Demand Diels-Alder Click Chemistry
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      Molecular Cancer Therapeutics (2017), 16 (1), 124-133CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
      The pretargeting system based on the inverse electron demand Diels-Alder reaction (IEDDA) between trans-cyclooctene (TCO) and tetrazine (Tz) combines the favorable pharmacokinetic properties of radiolabeled small mols. with the affinity and specificity of antibodies. This strategy has proven to be an efficient method for the molecularly targeted delivery of pharmaceuticals, including isotopes for radiol. imaging. Despite encouraging results from in vivo PET imaging studies, this promising system has yet to be thoroughly evaluated for pretargeted radioimmunotherapy (PRIT). Toward that end, we synthesized two novel 177Lu-labeled tetrazine-bearing radioligands. Next, we compared the usefulness of our ligands for PRIT when paired with TCO-modified 5B1-a human, anti-CA19.9 mAb-in preclin. murine models of pancreatic cancer. The exemplary ligand, 177Lu-DOTA-PEG7-Tz, showed rapid (4.6 ± 0.8% ID/g at 4 h) and persistent (16.8 ± 3.9% ID/g at 120 h) uptake in tumors while concurrently clearing from blood and nontarget tissues. Single-dose therapy studies using 5B1-TCO and varying amts. of 177Lu-DOTA-PEG7-Tz (400, 800, and 1,200μCi) showed that our system elicits a dose-dependent therapeutic response in mice bearing human xenografts. Furthermore, dosimetry calcns. suggest that our approach is amenable to clin. applications with its excellent dosimetric profile in organs of clearance (i.e., liver and kidneys) as well as in dose-limiting tissues, such as red marrow. This study established that a pretargeted methodol. utilizing the IEDDA reaction can rapidly and specifically deliver a radiotherapeutic payload to tumor tissue, thus illustrating its excellent potential for clin. translation.
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      Zeglis, B. M.; Sevak, K. K.; Reiner, T.; Mohindra, P.; Carlin, S. D.; Zanzonico, P.; Weissleder, R.; Lewis, J. S. A pretargeted PET imaging strategy based on bioorthogonal Diels-Alder click chemistry. J. Nucl. Med. 2013, 54, 1389– 1396,  DOI: 10.2967/jnumed.112.115840
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      A pretargeted PET imaging strategy based on bioorthogonal Diels-Alder click chemistry
      Zeglis, Brian M.; Sevak, Kuntal K.; Reiner, Thomas; Mohindra, Priya; Carlin, Sean D.; Zanzonico, Pat; Weissleder, Ralph; Lewis, Jason S.
      Journal of Nuclear Medicine (2013), 54 (8), 1389-1396CODEN: JNMEAQ; ISSN:0161-5505. (Society of Nuclear Medicine and Molecular Imaging)
      The specificity of antibodies have made immunoconjugates promising vectors for the delivery of radioisotopes to cancer cells; however, their long pharmacol. half-lives necessitate the use of radioisotopes with long phys. half-lives, a combination that leads to high radiation doses to patients. Therefore, the development of targeting modalities that harness the advantages of antibodies without their pharmacokinetic limitations is desirable. To this end, we report the development of a methodol. for pretargeted PET imaging based on the bioorthogonal Diels-Alder click reaction between tetrazine and transcyclooctene. Methods: A proof-of-concept system based on the A33 antibody, SW1222 colorectal cancer cells, and 64Cu was used. The huA33 antibody was covalently modified with transcyclooctene, and a NOTA-modified tetrazine was synthesized and radiolabeled with 64Cu. Pretargeted in vivo biodistribution and PET imaging expts. were performed with athymic nude mice bearing A33 antigen-expressing, SW1222 colorectal cancer xenografts. Results: The huA33 antibody was modified with transcyclooctene to produce a conjugate with high immunoreactivity, and the 64Cu-NOTA-labeled tetrazine ligand was synthesized with greater than 99% purity and a specific activity of 9-10 MBq/μg. For in vivo expts., mice bearing SW1222 xenografts were injected with transcyclooctene-modified A33; after allowing 24 h for accumulation of the antibody in the tumor, the mice were injected with 64Cu-NOTA-labeled tetrazine for PET imaging and biodistribution expts. At 12 h after injection, the retention of uptake in the tumor (4.1 ± 0.3 percent injected dose per g), coupled with the fecal excretion of excess radioligand, produced images with high tumor-to-background ratios. PET imaging and biodistribution expts. performed using A33 directly labeled with either 64Cu or 89Zr revealed that although abs. tumor uptake was higher with the directly radiolabeled antibodies, the pretargeted system yielded comparable images and tumor-to-muscle ratios at 12 and 24 h after injection. Further, dosimetry calcns. revealed that the 64Cu pretargeting system resulted in only a fraction of the absorbed background dose of A33 directly labeled with 89Zr (0.0124 mSv/MBq vs. 0.4162 mSv/MBq, resp.). Conclusion: The high quality of the images produced by this pretargeting approach, combined with the ability of the methodol. to dramatically reduce nontarget radiation doses to patients, marks this system as a strong candidate for clin. translation.
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      Rossin, R.; Verkerk, P. R.; van den Bosch, S. M.; Vulders, R. C.; Verel, I.; Lub, J.; Robillard, M. S. In vivo chemistry for pretargeted tumor imaging in live mice. Angew. Chem., Int. Ed. 2010, 49, 3375– 3378,  DOI: 10.1002/anie.200906294
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      In Vivo Chemistry for Pretargeted Tumor Imaging in Live Mice
      Rossin, Raffaella; Renart Verkerk, Pascal; van den Bosch, Sandra M.; Vulders, Roland C. M.; Verel, Iris; Lub, Johan; Robillard, Marc S.
      Angewandte Chemie, International Edition (2010), 49 (19), 3375-3378, S3375/1-S3375/22CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Bioorthogonal chem. reaction between two exogenous moieties in living animal was used for cancer imaging.
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      Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjugate Chem. 2008, 19, 2297– 2299,  DOI: 10.1021/bc8004446
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      Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging
      Devaraj, Neal K.; Weissleder, Ralph; Hilderbrand, Scott A.
      Bioconjugate Chemistry (2008), 19 (12), 2297-2299CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Bioorthogonal tetrazine cycloaddns. have been applied to live cell labeling. Tetrazines react irreversibly with the strained dienophile norbornene forming dihydropyrazine products and dinitrogen. The reaction is high yielding, selective, and fast in aq. media. Her2/neu receptors on live human breast cancer cells were targeted with a monoclonal antibody modified with a norbornene. Tetrazines conjugated to a near-IR fluorochrome selectively and rapidly label the pretargeted antibody in the presence of serum. These findings indicate that this chem. is suitable for in vitro labeling expts., and suggests that it may prove a useful strategy for in vivo pretargeted imaging under numerous modalities.
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    • Abstract

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      Figure 1

      Figure 1. (A) Examples of bioorthogonal functional groups and their approximate molecular weights. (B) Mutually orthogonal bioorthogonal reactions for metabolic imaging. Tetrazines and cyclooctynes can selectively label cyclopropenes and azides, respectively. In this instance, the application is to visualize metabolic incorporation of two different sugars onto cell surface glycans. Image reproduced from ref (30).

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      Figure 2

      Figure 2. Assembling bioactive molecules in situ using bioorthogonal reactions. (A) A target biomolecule such as an enzyme can bind to two fragments bearing bioorthogonal functional groups. If the fragments are bound such that the functional groups are brought into close proximity to one another, reaction occurs, and a tighter binding compound is formed. This method has been used for in situ discovery of enzyme inhibitors, but it will be interesting to see if such reactions can take place within living cells and in vivo. (B) Researchers have used bioorthogonal reactions to form druglike molecules inside cells. Here, a heterobifunctional compound is formed by tetrazine ligation which enables the degradation of BRD4 through the ubiquitin pathway. Figure reproduced from ref (39).

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      Figure 3

      Figure 3. General examples of bioorthogonal uncaging reactions. Dienophiles act to mask functional groups such as amines and alcohols. Upon bioorthogonal reaction with tetrazine, the functional group is released. This enables the uncaging of drugs, imaging agents, and even enzymes.

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      Figure 4

      Figure 4. Bioorthogonal pretargeted therapy. Cartoon depicting the direct versus multistep labeling approach. (A) Target cells (blue) are exposed to an affinity ligand directly modified by a chemotherapeutic or imaging agent. The affinity ligand binds, but an excess of long circulating affinity ligand persists, contributing to toxicity or background signal. The latter problem prevents the use of short-lived radioisotopes (e.g., fluorine-18) with commonly used affinity ligands such as antibodies. (B) In the multistep approach, an affinity ligand directly connected to a nontherapeutic bioorthogonally reactive element (trans-cyclooctene) is delivered. Again, background is initially high, but this background is inactive. After time has passed to allow for clearance, a small molecule that reacts with the affinity ligand (tetrazine) and possesses a chemotherapeutic or imaging agent is delivered. The small molecule reacts rapidly with available trans-cyclooctene. The molecule also clears rapidly due to its small size thus lowering background side effects or signal.

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    • References

      This article references 70 other publications.

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        A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation
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        Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality
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        4
        Copper-free click chemistry for dynamic in vivo imaging
        Baskin, Jeremy M.; Prescher, Jennifer A.; Laughlin, Scott T.; Agard, Nicholas J.; Chang, Pamela V.; Miller, Isaac A.; Lo, Anderson; Codelli, Julian A.; Bertozzi, Carolyn R.
        Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (43), 16793-16797CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
        Dynamic imaging of proteins in live cells is routinely performed by using genetically encoded reporters, an approach that cannot be extended to other classes of biomols. such as glycans and lipids. Here, the authors report a Cu-free variant of click chem. that can label these biomols. rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction. The crit. reagent, a substituted cyclooctyne, possesses ring strain and electron-withdrawing fluorine substituents that together promote the [3+2] dipolar cycloaddn. with azides installed metabolically into biomols. This Cu-free click reaction possesses comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells with no apparent toxicity. With this technique, the authors studied the dynamics of glycan trafficking and identified a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics.
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        5
        In Vivo Imaging of Membrane-Associated Glycans in Developing Zebrafish
        Laughlin, Scott T.; Baskin, Jeremy M.; Amacher, Sharon L.; Bertozzi, Carolyn R.
        Science (Washington, DC, United States) (2008), 320 (5876), 664-667CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
        Glycans are attractive targets for mol. imaging but have been inaccessible because of their incompatibility with genetically encoded reporters. We demonstrated the noninvasive imaging of glycans in live developing zebrafish, using a chem. reporter strategy. Zebrafish embryos were treated with an unnatural sugar to metabolically label their cell-surface glycans with azides. Subsequently, the embryos were reacted with fluorophore conjugates by means of copper-free click chem., enabling the visualization of glycans in vivo at subcellular resoln. during development. At 60 h after fertilization, we obsd. an increase in de novo glycan biosynthesis in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, we performed a spatiotemporal anal. of glycan expression and trafficking and identified patterns that would be undetectable with conventional mol. imaging approaches.
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        Agarwal, P.; Beahm, B. J.; Shieh, P.; Bertozzi, C. R. Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry. Angew. Chem., Int. Ed. 2015, 54, 11504– 11510,  DOI: 10.1002/anie.201504249
        6
        Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry
        Agarwal, Paresh; Beahm, Brendan J.; Shieh, Peyton; Bertozzi, Carolyn R.
        Angewandte Chemie, International Edition (2015), 54 (39), 11504-11510CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        Vertebrate glycans constitute a large, important, and dynamic set of post-translational modifications that are notoriously difficult to manipulate and image. Although the chem. reporter strategy has been used in conjunction with bioorthogonal chem. to image the external glycosylation state of live zebrafish and detect tumor-assocd. glycans in mice, the ability to image glycans systemically within a live organism has remained elusive. Here, the authors report a method that combines the metabolic incorporation of a cyclooctyne-functionalized sialic acid deriv. with a ligation reaction of a fluorogenic tetrazine, allowing for the imaging of sialylated glycoconjugates within live zebrafish embryos.
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        Oller-Salvia, B.; Kym, G.; Chin, J. W. Rapid and Efficient Generation of Stable Antibody-Drug Conjugates via an Encoded Cyclopropene and an Inverse-Electron-Demand Diels-Alder Reaction. Angew. Chem., Int. Ed. 2018, 57, 2831– 2834,  DOI: 10.1002/anie.201712370
        7
        Rapid and Efficient Generation of Stable Antibody-Drug Conjugates via an Encoded Cyclopropene and an Inverse-Electron-Demand Diels-Alder Reaction
        Oller-Salvia, Benjami; Kym, Gene; Chin, Jason W.
        Angewandte Chemie, International Edition (2018), 57 (11), 2831-2834CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        Homogeneous antibody-drug conjugates (ADCs), generated by site-specific toxin linkage, show improved therapeutic indexes with respect to traditional ADCs. However, current methods to produce site-specific conjugates suffer from low protein expression, slow reaction kinetics, and low yields, or are limited to particular conjugation sites. Here we describe high yielding expression systems that efficiently incorporate a cyclopropene deriv. of lysine (CypK) into antibodies through genetic-code expansion. We express trastuzumab bearing CypK and conjugate tetrazine derivs. to the antibody. We show that the dihydropyridazine linkage resulting from the conjugation reaction is stable in serum, and generate an ADC bearing monomethyl auristatin E that selectively kills cells expressing a high level of HER2. Our results demonstrate that CypK is a minimal bioorthogonal handle for the rapid prodn. of stable therapeutic protein conjugates.
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        Reactive polymer enables efficient in vivo bioorthogonal chemistry
        Devaraj, Neal K.; Thurber, Greg M.; Keliher, Edmund J.; Marinelli, Brett; Weissleder, Ralph
        Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (13), 4762-4767, S4762/1-S4762/2CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
        There has been intense interest in the development of selective bioorthogonal reactions or "click" chem. that can proceed in live animals. Until now however, most reactions still require vast surpluses of reactants because of steep temporal and spatial concn. gradients. Using computational modeling and design of pharmacokinetically optimized reactants, we have developed a predictable method for efficient in vivo click reactions. Specifically, we show that polymer modified tetrazines (PMT) are a key enabler for in vivo bioorthogonal chem. based on the very fast and catalyst-free [4 + 2] tetrazine/trans-cyclooctene cycloaddn. Using fluorescent PMT for cellular resoln. and 18F labeled PMT for whole animal imaging, we show that cancer cell epitopes can be easily reacted in vivo. This generic strategy should help guide the design of future chemistries and find widespread use for different in vivo bioorthogonal applications, particularly in the biomedical sciences.
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        Fragment-based approaches and the prospect of fragmented prodrugs
        Antonow Dyeison
        Drug discovery today (2010), 15 (19-20), 801-3 ISSN:.
        There is no expanded citation for this reference.
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      10. 10
        Zengeya, T. T.; Garlick, J. M.; Kulkarni, R. A.; Miley, M.; Roberts, A. M.; Yang, Y.; Crooks, D. R.; Sourbier, C.; Linehan, W. M.; Meier, J. L. Co-opting a Bioorthogonal Reaction for Oncometabolite Detection. J. Am. Chem. Soc. 2016, 138, 15813– 15816,  DOI: 10.1021/jacs.6b09706
        10
        Co-opting a Bioorthogonal Reaction for Oncometabolite Detection
        Zengeya, Thomas T.; Garlick, Julie M.; Kulkarni, Rhushikesh A.; Miley, Mikayla; Roberts, Allison M.; Yang, Youfeng; Crooks, Daniel R.; Sourbier, Carole; Linehan, W. Marston; Meier, Jordan L.
        Journal of the American Chemical Society (2016), 138 (49), 15813-15816CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        Dysregulated metab. is a hallmark of many diseases, including cancer. Methods to fluorescently detect metabolites have the potential to enable new approaches to cancer detection and imaging. However, fluorescent sensing methods for naturally occurring cellular metabolites are relatively unexplored. Here the authors report the development of a chem. approach to detect the oncometabolite fumarate. The authors' strategy exploits a known bioorthogonal reaction, the 1,3-dipolar cycloaddn. of nitrileimines and electron-poor olefins, to detect fumarate via fluorescent pyrazoline cycloadduct formation. The authors demonstrate that hydrazonyl chlorides serve as readily accessible nitrileimine precursors, whose reactivity and spectral properties can be tuned to enable detection of fumarate and other dipolarophile metabolites. Finally, the authors show this reaction can be used to detect enzyme activity changes caused by mutations in fumarate hydratase, which underlie the familial cancer predisposition syndrome hereditary leiomyomatosis and renal cell cancer. The authors' studies define a novel intersection of bioorthogonal chem. and metabolite reactivity that may be harnessed to enable biol. profiling, imaging, and diagnostic applications.
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        Patterson, D. M.; Prescher, J. A. Orthogonal bioorthogonal chemistries. Curr. Opin. Chem. Biol. 2015, 28, 141– 149,  DOI: 10.1016/j.cbpa.2015.07.006
        11
        Orthogonal bioorthogonal chemistries
        Patterson, David M.; Prescher, Jennifer A.
        Current Opinion in Chemical Biology (2015), 28 (), 141-149CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
        A review. Bioorthogonal reactions have long been used to examine individual biomols. in living systems. Studies of multi-component networks demand not only reliable bioorthogonal chemistries, but also combinations of reactions that can be used in tandem. Such orthogonal bioorthogonal transformations have been reported in recent years, and these chemistries are enabling new explorations into biol. This article highlights the development of orthogonal bioorthogonal reactions and their application in multi-target imaging and macromol. assembly. Methods to tune and control orthogonal reactivity are also discussed, along with prospects for identifying new classes of compatible reactions.
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      12. 12
        Yu, J.; Shen, D.; Zhang, H.; Yin, Z. Rapid, Stoichiometric, Site-Specific Modification of Aldehyde-Containing Proteins Using a Tandem Knoevenagel-Intra Michael Addition Reaction. Bioconjugate Chem. 2018, 29, 1016– 1020,  DOI: 10.1021/acs.bioconjchem.8b00086
        12
        Rapid, Stoichiometric, Site-Specific Modification of Aldehyde-Containing Proteins Using a Tandem Knoevenagel-Intra Michael Addition Reaction
        Yu, Jian; Shen, Da; Zhang, Hanjie; Yin, Zheng
        Bioconjugate Chemistry (2018), 29 (4), 1016-1020CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
        A site-specific modification of aldehyde-contg. proteins using a tandem Knoevenagel-intra Michael addn. reaction was developed. The reaction featured fast kinetics (50 M-1s-1) and favorable stoichiometry. Various functionalities could be introduced into the protein with little impact on its function and conformation. The reaction was successfully applied in the labeling of living cells.
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      13. 13
        An, P.; Lewandowski, T. M.; Erbay, T. G.; Liu, P.; Lin, Q. Sterically Shielded, Stabilized Nitrile Imine for Rapid Bioorthogonal Protein Labeling in Live Cells. J. Am. Chem. Soc. 2018, 140, 4860– 4868,  DOI: 10.1021/jacs.8b00126
        13
        Sterically Shielded, Stabilized Nitrile Imine for Rapid Bioorthogonal Protein Labeling in Live Cells
        An, Peng; Lewandowski, Tracey M.; Erbay, Tugce G.; Liu, Peng; Lin, Qing
        Journal of the American Chemical Society (2018), 140 (14), 4860-4868CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        In pursuit of fast bioorthogonal reactions, reactive moieties have been increasingly employed for selective labeling of biomols. in living systems, posing a challenge in attaining reactivity without sacrificing selectivity. To address this challenge, here the authors report a bioinspired strategy in which mol. shape controls the selectivity of a transient, highly reactive nitrile imine dipole. By tuning the shape of structural pendants attached to the ortho position of the N-aryl ring of diaryltetrazoles, precursors of nitrile imines, the authors discovered a sterically shielded nitrile imine that favors the 1,3-dipolar cycloaddn. over the competing nucleophilic addn. The photogenerated nitrile imine exhibits an extraordinarily long half-life of 102 s in aq. medium, owing to its unique mol. shape that hinders the approach of a nucleophile as shown by DFT calcns. The utility of this sterically shielded nitrile imine in rapid (∼1 min) bioorthogonal labeling of glucagon receptor in live mammalian cells was demonstrated.
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      14. 14
        Matsuo, K.; Nishikawa, Y.; Masuda, M.; Hamachi, I. Live-Cell Protein Sulfonylation Based on Proximity-driven N-Sulfonyl Pyridone Chemistry. Angew. Chem., Int. Ed. 2018, 57, 659– 662,  DOI: 10.1002/anie.201707972
        14
        Live-Cell Protein Sulfonylation Based on Proximity-driven N-Sulfonyl Pyridone Chemistry
        Matsuo, Kazuya; Nishikawa, Yuki; Masuda, Marie; Hamachi, Itaru
        Angewandte Chemie, International Edition (2018), 57 (3), 659-662CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        The development of bioorthogonal approaches for labeling of endogenous proteins under the multimol. crowding conditions of live cells is highly desirable for the anal. and engineering of proteins without using genetic manipulation. N-Sulfonyl pyridone (SP) is reported as a new reactive group for protein sulfonylation. The ligand-directed SP chem. was able to modify not only purified proteins in vitro, but also endogenous ones on the surface of and inside live cells selectively and rapidly, which allowed to convert endogenous proteins to FRET-based biosensors in situ.
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      15. 15
        Fang, Y. Z.; Zhang, H.; Huang, Z.; Scinto, S. L.; Yang, J. C.; Ende, C. W. A.; Dmitrenko, O.; Johnson, D. S.; Fox, J. M. Photochemical syntheses, transformations, and bioorthogonal chemistry of trans-cycloheptene and sila trans-cycloheptene Ag(I) complexes. Chem. Sci. 2018, 9, 1953– 1963,  DOI: 10.1039/C7SC04773H
        15
        Photochemical syntheses, transformations, and bioorthogonal chemistry of trans-cycloheptene and sila trans-cycloheptene Ag(I) complexes
        Fang, Yinzhi; Zhang, Han; Huang, Zhen; Scinto, Samuel L.; Yang, Jeffrey C.; am Ende, Christopher W.; Dmitrenko, Olga; Johnson, Douglas S.; Fox, Joseph M.
        Chemical Science (2018), 9 (7), 1953-1963CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
        A photochem. synthesis of AgNO3 complexes of trans-cycloheptene (TCH) and trans-1-sila-4-cycloheptene (Si-TCH) derivs. is described. A low temp. flow photoreactor was designed to enable the synthesis of carbocyclic TCH derivs. due to their thermal sensitivity in the absence of metal coordination. Unlike the free carbocycles, TCH•AgNO3 complexes can be handled at rt and stored for weeks in the freezer (-18 °C). Si-TCH•AgNO3 complexes are esp. robust, and are bench stable for days at rt, and for months in the freezer. X-ray crystallog. was used to characterize a Si-TCH•AgNO3 complex for the first time. With decomplexation of AgNO3 in situ, metal-free TCO and Si-TCH derivs. can engage in a range of cycloaddn. reactions as well as dihydroxylation reactions. Computation was used to predict that Si-TCH would engage in bioorthogonal reactions that are more rapid than the most reactive trans-cyclooctenes. Metal-free Si-TCH derivs. were shown to display good stability in soln., and to engage in the fastest bioorthogonal reaction reported to date (k2 1.14 x 107 M-1s-1 in 9:1 H2O:MeOH). Utility in bioorthogonal protein labeling in live cells is described, including labeling of GFP with an unnatural tetrazine-contg. amino acid. The reactivity and specificity of the Si-TCH reagents with tetrazines in live mammalian cells was also evaluated using the HaloTag platform. The cell labeling expts. show that Si-TCH derivs. are best suited as probe mols. in the cellular environment.
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      16. 16
        Blackman, M. L.; Royzen, M.; Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity. J. Am. Chem. Soc. 2008, 130, 13518– 13519,  DOI: 10.1021/ja8053805
        16
        Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity
        Blackman, Melissa L.; Royzen, Maksim; Fox, Joseph M.
        Journal of the American Chemical Society (2008), 130 (41), 13518-13519CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        Described is a bioorthogonal reaction that proceeds with unusually fast reaction rates without need for catalysis: the cycloaddn. of s-tetrazine and trans-cyclooctene derivs. The reactions tolerate a broad range of functionality and proceed in high yield in org. solvents, water, cell media, or cell lysate. The rate of the ligation between trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is very rapid (k2 2000 M-1 s-1). This fast reactivity enables protein modification at low concn.
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      17. 17
        Devaraj, N. K.; Upadhyay, R.; Haun, J. B.; Hilderbrand, S. A.; Weissleder, R. Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition. Angew. Chem., Int. Ed. 2009, 48, 7013– 7016,  DOI: 10.1002/anie.200903233
        17
        Fast and Sensitive Pretargeted Labeling of Cancer Cells through a Tetrazine/trans-Cyclooctene Cycloaddition
        Devaraj, Neal K.; Upadhyay, Rabi; Haun, Jered B.; Hilderbrand, Scott A.; Weissleder, Ralph
        Angewandte Chemie, International Edition (2009), 48 (38), 7013-7016, S7013/1-S7013/6CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        The novel asym. tetrazine I was conjugated to a com. available far-red indocyanine fluorophore, Vivo-Tag 680. This fluorescent tetrazine deriv. was used to image trans-cyclooctene-modified affinity ligands on live cancer cells through a bioorthogonal cycloaddn. with a reaction rate of approx. 6000±200 M-1 s-1 in serum at 37 °C. To maximize the fluorescence signal, up to six trans-cyclooctene moieties were attached to the antibody used to pretarget cells for labeling. Com. available cetuximab was labeled with trans-cyclooctene succinimidyl carbonate and used for pretargeting expts.
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      18. 18
        Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R. A comparative study of bioorthogonal reactions with azides. ACS Chem. Biol. 2006, 1, 644– 648,  DOI: 10.1021/cb6003228
        18
        A comparative study of bioorthogonal reactions with azides
        Agard, Nicholas J.; Baskin, Jeremy M.; Prescher, Jennifer A.; Lo, Anderson; Bertozzi, Carolyn R.
        ACS Chemical Biology (2006), 1 (10), 644-648CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
        Detection of metabolites and post-translational modifications can be achieved using the azide as a bioorthogonal chem. reporter. Once introduced into target biomols., either metabolically or through chem. modification, the azide can be tagged with probes using one of three highly selective reactions: the Staudinger ligation, the Cu(I)-catalyzed azide-alkyne cycloaddn., or the strain-promoted [3+2] cycloaddn. Here, the authors compared these chemistries in the context of various biol. applications, including labeling of biomols. in complex lysates and on live cell surfaces. The Cu(I)-catalyzed reaction was most efficient for detecting azides in protein samples but was not compatible with live cells due to the toxicity of the reagents. Both the Staudinger ligation and the strain-promoted [3+2] cycloaddn. using optimized cyclooctynes were effective for tagging azides on live cells. The best reagent for this application was dependent upon the specific structure of the azide. These results provide a guide for biologists in choosing a suitable ligation chem.
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      19. 19
        Dommerholt, J.; van Rooijen, O.; Borrmann, A.; Guerra, C. F.; Bickelhaupt, F. M.; van Delft, F. L. Highly accelerated inverse electron-demand cycloaddition of electron-deficient azides with aliphatic cyclooctynes. Nat. Commun. 2014, 5, 5378,  DOI: 10.1038/ncomms6378
        19
        Highly accelerated inverse electron-demand cycloaddition of electron-deficient azides with aliphatic cyclooctynes
        Dommerholt, Jan; van Rooijen, Olivia; Borrmann, Annika; Guerra, Celia Fonseca; Bickelhaupt, F. Matthias; van Delft, Floris L.
        Nature Communications (2014), 5 (), 5378CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
        Strain-promoted azide-alkyne cycloaddn. (SPAAC) as a conjugation tool has found broad application in material sciences, chem. biol. and even in vivo use. However, despite tremendous effort, SPAAC remains fairly slow (0.2-0.5 M-1 s-1) and efforts to increase reaction rates by tailoring of cyclooctyne structure have suffered from a poor trade-off between cyclooctyne reactivity and stability. We here wish to report tremendous acceleration of strain-promoted cycloaddn. of an aliph. cyclooctyne (bicyclo[6.1.0]non-4-yne, BCN) with electron-deficient aryl azides, with reaction rate consts. reaching 2.0-2.9 M-1 s-1. A remarkable difference in rate consts. of aliph. cyclooctynes vs. benzoannulated cyclooctynes is noted, enabling a next level of orthogonality by a judicious choice of azide-cyclooctyne combinations, which is inter alia applied in one-pot three-component protein labeling. The pivotal role of azide electronegativity is explained by d.-functional theory calcns. and electronic-structure analyses, which indicates an inverse electron-demand mechanism is operative with an aliph. cyclooctyne.
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      20. 20
        Kamber, D. N.; Liang, Y.; Blizzard, R. J.; Liu, F.; Mehl, R. A.; Houk, K. N.; Prescher, J. A. 1,2,4-Triazines Are Versatile Bioorthogonal Reagents. J. Am. Chem. Soc. 2015, 137, 8388– 8391,  DOI: 10.1021/jacs.5b05100
        20
        1,2,4-Triazines Are Versatile Bioorthogonal Reagents
        Kamber, David N.; Liang, Yong; Blizzard, Robert J.; Liu, Fang; Mehl, Ryan A.; Houk, K. N.; Prescher, Jennifer A.
        Journal of the American Chemical Society (2015), 137 (26), 8388-8391CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        A new class of bioorthogonal reagents, 1,2,4-triazines, is described. These scaffolds are stable in biol. media and capable of robust reactivity with trans-cyclooctene (TCO). The enhanced stability of the triazine scaffold enabled its direct use in recombinant protein prodn. The triazine-TCO reaction can also be used in tandem with other bioorthogonal cycloaddn. reactions. These features fill current voids in the bioorthogonal toolkit.
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      21. 21
        Hoffmann, J. E.; Plass, T.; Nikic, I.; Aramburu, I. V.; Koehler, C.; Gillandt, H.; Lemke, E. A.; Schultz, C. Highly Stable trans-Cyclooctene Amino Acids for Live-Cell Labeling. Chem. - Eur. J. 2015, 21, 12266– 12270,  DOI: 10.1002/chem.201501647
        21
        Highly Stable trans-Cyclooctene Amino Acids for Live-Cell Labeling
        Hoffmann, Jan-Erik; Plass, Tilman; Nikic, Ivana; Valle Aramburu, Iker; Koehler, Christine; Gillandt, Hartmut; Lemke, Edward A.; Schultz, Carsten
        Chemistry - A European Journal (2015), 21 (35), 12266-12270CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
        Trans-Cyclooctene groups incorporated into proteins via non-canonical amino acids (ncAAs) are emerging as specific handles for bioorthogonal chem. Here, we present a highly improved synthetic access to the axially and the equatorially linked trans-cyclooct-2-ene isomers (1 a,b). We further show that the axially connected isomer has a half-life about 10 times higher than the equatorial isomer and reacts with tetrazines much faster, as detd. by stopped-flow expts. The improved properties resulted in different labeling performance of the insulin receptor on the surface of intact cells.
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      22. 22
        Rossin, R.; van den Bosch, S. M.; Ten Hoeve, W.; Carvelli, M.; Versteegen, R. M.; Lub, J.; Robillard, M. S. Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems. Bioconjugate Chem. 2013, 24, 1210– 1217,  DOI: 10.1021/bc400153y
        22
        Highly Reactive trans-Cyclooctene Tags with Improved Stability for Diels-Alder Chemistry in Living Systems
        Rossin, Raffaella; van den Bosch, Sandra M.; ten Hoeve, Wolter; Carvelli, Marco; Versteegen, Ron M.; Lub, Johan; Robillard, Marc S.
        Bioconjugate Chemistry (2013), 24 (7), 1210-1217CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
        One of the challenges of pretargeted radioimmunotherapy, which centers on the capture of a radiolabeled probe by a preinjected tumor-bound antibody, is the potential immunogenicity of biol. capturing systems. A bioorthogonal chem. approach may circumvent this drawback, but effective in vivo chem. in mice, larger animals, and eventually humans, requires very high reagent reactivity, sufficient stability, and retained selectivity. We report here that the reactivity of the fastest bioorthogonal reaction, the inverse-electron-demand-Diels-Alder cycloaddn. between a tetrazine probe and a trans-cyclooctene-tagged antibody, can be increased 10-fold (k2 = 2.7 × 105 M-1s-1) via the trans-cyclooctene, approaching the speed of biol. interactions, while also increasing its stability. This was enabled by the finding that the trans-cyclooctene tag is probably deactivated through isomerization to the unreactive cis-cyclooctene isomer by interactions with copper-contg. proteins, and that increasing the steric hindrance on the tag can impede this process. Next, we found that the higher reactivity of axial vs. equatorial linked TCO can be augmented by the choice of linker. The new, stabilized, and more reactive tag allowed for improved tumor-to-nontumor ratios in pretargeted tumor-bearing mice.
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      23. 23
        Sletten, E. M.; Bertozzi, C. R. From mechanism to mouse: a tale of two bioorthogonal reactions. Acc. Chem. Res. 2011, 44, 666– 676,  DOI: 10.1021/ar200148z
        23
        From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions
        Sletten, Ellen M.; Bertozzi, Carolyn R.
        Accounts of Chemical Research (2011), 44 (9), 666-676CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
        A review. Bioorthogonal reactions are chem. reactions that neither interact with nor interfere with a biol. system. The participating functional groups must be inert to biol. moieties, must selectively reactive with each other under biocompatible conditions, and, for in vivo applications, must be nontoxic to cells and organisms. Addnl., it is helpful if one reactive group is small and therefore minimally perturbing of a biomol. into which it has been introduced either chem. or biosynthetically. Examples from the past decade suggest that a promising strategy for bioorthogonal reaction development begins with an anal. of functional group and reactivity space outside those defined by Nature. Issues such as stability of reactants and products (particularly in water), kinetics, and unwanted side reactivity with biofunctionalities must be addressed, ideally guided by detailed mechanistic studies. Finally, the reaction must be tested in a variety of environments, escalating from aq. media to biomol. solns. to cultured cells and, for the most optimized transformations, to live organisms. Work in our lab. led to the development of two bioorthogonal transformations that exploit the azide as a small, abiotic, and bioinert reaction partner: the Staudinger ligation and strain-promoted azide-alkyne cycloaddn. The Staudinger ligation is based on the classic Staudinger redn. of azides with triarylphosphines first reported in 1919. In the ligation reaction, the intermediate aza-ylide undergoes intramol. reaction with an ester, forming an amide bond faster than aza-ylide hydrolysis would otherwise occur in water. The Staudinger ligation is highly selective and reliably forms its product in environs as demanding as live mice. However, the Staudinger ligation has some liabilities, such as the propensity of phosphine reagents to undergo air oxidn. and the relatively slow kinetics of the reaction. The Staudinger ligation takes advantage of the electrophilicity of the azide; however, the azide can also participate in cycloaddn. reactions. In 1961, Wittig and Krebs noted that the strained, cyclic alkyne cyclooctyne reacts violently when combined neat with Ph azide, forming a triazole product by 1,3-dipolar cycloaddn. This observation stood in stark contrast to the slow kinetics assocd. with 1,3-dipolar cycloaddn. of azides with unstrained, linear alkynes, the conventional Huisgen process. Notably, the reaction of azides with terminal alkynes can be accelerated dramatically by copper catalysis (this highly popular Cu-catalyzed azide-alkyne cycloaddn. (CuAAC) is a quintessential "click" reaction). However, the copper catalysts are too cytotoxic for long-term exposure with live cells or organisms. Thus, for applications of bioorthogonal chem. in living systems, we built upon Wittig and Krebs' observation with the design of cyclooctyne reagents that react rapidly and selectively with biomol.-assocd. azides. This strain-promoted azide-alkyne cycloaddn. is often referred to as "Cu-free click chem.". Mechanistic and theor. studies inspired the design of a series of cyclooctyne compds. bearing fluorine substituents, fused rings, and judiciously situated heteroatoms, with the goals of optimizing azide cycloaddn. kinetics, stability, soly., and pharmacokinetic properties. Cyclooctyne reagents have now been used for labeling azide-modified biomols. on cultured cells and in live Caenorhabditis elegans, zebrafish, and mice. As this special issue testifies, the field of bioorthogonal chem. is firmly established as a challenging frontier of reaction methodol. and an important new instrument for biol. discovery. The above reactions, as well as several newcomers with bioorthogonal attributes, have enabled the high-precision chem. modification of biomols. in vitro, as well as real-time visualization of mols. and processes in cells and live organisms. The consequence is an impressive body of new knowledge and technol., amassed using a relatively small bioorthogonal reaction compendium. Expansion of this toolkit, an effort that is already well underway, is an important objective for chemists and biologists alike.
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      24. 24
        Link, A. J.; Vink, M. K.; Tirrell, D. A. Presentation and detection of azide functionality in bacterial cell surface proteins. J. Am. Chem. Soc. 2004, 126, 10598– 10602,  DOI: 10.1021/ja047629c
        24
        Presentation and detection of azide functionality in bacterial cell surface proteins
        Link, A. James; Vink, Mandy K. S.; Tirrell, David A.
        Journal of the American Chemical Society (2004), 126 (34), 10598-10602CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        An improved protocol for copper-catalyzed triazole formation on the bacterial cell surface is described. Addn. of highly pure CuBr to cells treated with azidohomoalanine (2) leads to ∼. 10-Fold more extensive cell surface labeling than previously obsd. This highly active catalyst allows detection of the methionine analogs azidoalanine (1), azidonorvaline (3), and azidonorleucine (4) in cell surface proteins. Azidoalanine was previously believed to be silent with regard to the cellular protein synthesis machinery.
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      25. 25
        Tian, F.; Tsao, M. L.; Schultz, P. G. A phage display system with unnatural amino acids. J. Am. Chem. Soc. 2004, 126, 15962– 15963,  DOI: 10.1021/ja045673m
        25
        A Phage Display System with Unnatural Amino Acids
        Tian, Feng; Tsao, Meng-Lin; Schultz, Peter G.
        Journal of the American Chemical Society (2004), 126 (49), 15962-15963CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        We report a general method to display peptide-contg. unnatural amino acids on filamentous M13 phage. Five distinct unnatural amino acids were site-specifically incorporated at the N-terminal of the M13 phage minor coat protein pIII. Phages that contain p-azidophenylalanine can undergo a highly specific azide-alkyne [3+2] cycloaddn. reaction with an alkyne-derivatized fluorophore. The generalization of phage display to include unnatural amino acids should significantly increase the scope of phage display technol.
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      26. 26
        Yang, J.; Seckute, J.; Cole, C. M.; Devaraj, N. K. Live-cell imaging of cyclopropene tags with fluorogenic tetrazine cycloadditions. Angew. Chem., Int. Ed. 2012, 51, 7476– 7479,  DOI: 10.1002/anie.201202122
        26
        Live-Cell Imaging of Cyclopropene Tags with Fluorogenic Tetrazine Cycloadditions
        Yang, Jun; Seckute, Jolita; Cole, Christian M.; Devaraj, Neal K.
        Angewandte Chemie, International Edition (2012), 51 (30), 7476-7479, S7476/1-S7476/26CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        The authors demonstrated the applicability of methylcyclopropene tags as dienophiles for reaction with fluorogenic tetrazines. Through systematic synthetic modifications, the stability, size, and reactivity of the cyclopropene scaffold were optimized. Methylcyclopropene derivs. were developed that react rapidly with tetrazines while retaining their aq. stability and small size. These cyclopropene handles elicited fluorescent responses from quenched tetrazine dyes and were suitable for cellular imaging applications, which was demonstrated by imaging cyclopropene phospholipids distributed in live human breast cancer cells.
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      27. 27
        Patterson, D. M.; Nazarova, L. A.; Xie, B.; Kamber, D. N.; Prescher, J. A. Functionalized cyclopropenes as bioorthogonal chemical reporters. J. Am. Chem. Soc. 2012, 134, 18638– 18643,  DOI: 10.1021/ja3060436
        27
        Functionalized Cyclopropenes as Bioorthogonal Chemical Reporters
        Patterson, David M.; Nazarova, Lidia A.; Xie, Bryan; Kamber, David N.; Prescher, Jennifer A.
        Journal of the American Chemical Society (2012), 134 (45), 18638-18643CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        Chem. reporters are unique functional groups that can be used to label biomols. in living systems. Only a handful of broadly applicable reporters have been identified to date, owing to the rigorous demands placed on these functional groups in biol. settings. We describe here a new chem. reporter-cyclopropene-that can be used to target biomols. in vitro and in live cells. A variety of substituted cyclopropene scaffolds were synthesized and found to be stable in aq. soln. and in the presence of biol. nucleophiles. Furthermore, some of the cyclopropene units were metabolically introduced into cell surface glycans and subsequently detected with covalent probes. The small size and selective reactivity of cyclopropenes will facilitate efforts to tag diverse collections of biomols. in vivo.
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      28. 28
        Andersen, K. A.; Aronoff, M. R.; McGrath, N. A.; Raines, R. T. Diazo groups endure metabolism and enable chemoselectivity in cellulo. J. Am. Chem. Soc. 2015, 137, 2412– 2415,  DOI: 10.1021/ja5095815
        28
        Diazo Groups Endure Metabolism and Enable Chemoselectivity in Cellulo
        Andersen, Kristen A.; Aronoff, Matthew R.; McGrath, Nicholas A.; Raines, Ronald T.
        Journal of the American Chemical Society (2015), 137 (7), 2412-2415CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        The authors introduce a stabilized diazo group as a reporter for chem. biol. ManDiaz, which is a diazo deriv. of N-acetylmannosamine, is found to endure cellular metab. and label the surface of a mammalian cell. There its diazo group can undergo a 1,3-dipolar cycloaddn. with a strained alkyne, providing a signal comparable to that from the azido congener, ManNAz. The chemoselectivity of diazo and alkynyl groups enables dual labeling of cells that is not possible with azido and alkynyl groups. Thus, the diazo group, which is approx. half the size of an azido group, provides unique opportunities for orthogonal labeling of cellular components.
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      29. 29
        Karver, M. R.; Weissleder, R.; Hilderbrand, S. A. Bioorthogonal reaction pairs enable simultaneous, selective, multi-target imaging. Angew. Chem., Int. Ed. 2012, 51, 920– 922,  DOI: 10.1002/anie.201104389
        29
        Bioorthogonal Reaction Pairs Enable Simultaneous, Selective, Multi-Target Imaging
        Karver, Mark R.; Weissleder, Ralph; Hilderbrand, Scott A.
        Angewandte Chemie, International Edition (2012), 51 (4), 920-922, S920/1-S920/12CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        The authors describe the testing and in cell validation of a tetrazine-trans-cyclooctene (TCO) reaction pair that is orthogonal to azide-cyclooctyne cycloaddn. chem. The chosen pairs were able to react concurrently in the same culture to fluorescently label two different cancer cell types. The authors' method uses only small mol. based reagents that also can be readily incorporated into a wide variety of systems and potentially may be used in conjunction with fluorogenic azide and tetrazine substrates to achieve addnl. improvements in specificity and sensitivity. The ability to perform multiple, rapid, simultaneous chem. reactions with a high degree of specificity in chem. complex environments should prove to be a useful tool in chem., biol., and medicine.
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      30. 30
        Cole, C. M.; Yang, J.; Seckute, J.; Devaraj, N. K. Fluorescent live-cell imaging of metabolically incorporated unnatural cyclopropene-mannosamine derivatives. ChemBioChem 2013, 14, 205– 208,  DOI: 10.1002/cbic.201200719
        30
        Fluorescent Live-Cell Imaging of Metabolically Incorporated Unnatural Cyclopropene-Mannosamine Derivatives
        Cole, Christian M.; Yang, Jun; Seckute, Jolita; Devaraj, Neal K.
        ChemBioChem (2013), 14 (2), 205-208CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
        This study reported that unnatural cyclo-propene-mannosamine derivs. can be used to image glycans on live human cancer cell lines. The results showed that recently developed methylcyclopropene bioorthogonal handles can be utilized for metabolic imaging of unnatural mannosamine derivs. on live-cell surfaces. In general, the data exhibit the possible use of cyclopropenes as reactive mini-tags for a myriad of applications in the profiling and imaging of small mols.
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      31. 31
        Spate, A. K.; Busskamp, H.; Niederwieser, A.; Schart, V. F.; Marx, A.; Wittmann, V. Rapid labeling of metabolically engineered cell-surface glycoconjugates with a carbamate-linked cyclopropene reporter. Bioconjugate Chem. 2014, 25, 147– 154,  DOI: 10.1021/bc4004487
        31
        Rapid labeling of metabolically engineered cell-surface glycoconjugates with a carbamate-linked cyclopropene reporter
        Spate Anne-Katrin; Busskamp Holger; Niederwieser Andrea; Schart Verena F; Marx Andreas; Wittmann Valentin
        Bioconjugate chemistry (2014), 25 (1), 147-54 ISSN:.
        Metabolic oligosaccharide engineering is a valuable tool to monitor cellular carbohydrates. Here, we report the synthesis of a novel N-acyl-mannosamine derivative bearing a methylcyclopropene tag that is attached to the sugar via a carbamate moiety. This derivative undergoes rapid Diels-Alder reaction with inverse electron demand. We demonstrate that the cell's biosynthetic machinery incorporates this non-natural mannosamine derivative into glycoconjugates that can, subsequently, be labeled within less than 10 min with a new sulfo-Cy3-tetrazine conjugate. Using this tetrazine-dye conjugate for the detection of the methylcyclopropene-tagged mannosamine derivative, we could achieve dual labeling of two different metabolically incorporated sugars combining a Diels-Alder reaction with inverse electron demand and a strain-promoted azide-alkyne cycloaddition which are carried out simultaneously in a single step.
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      32. 32
        Sachdeva, A.; Wang, K.; Elliott, T.; Chin, J. W. Concerted, rapid, quantitative, and site-specific dual labeling of proteins. J. Am. Chem. Soc. 2014, 136, 7785– 7788,  DOI: 10.1021/ja4129789
        32
        Concerted, Rapid, Quantitative, and Site-Specific Dual Labeling of Proteins
        Sachdeva, Amit; Wang, Kaihang; Elliott, Thomas; Chin, Jason W.
        Journal of the American Chemical Society (2014), 136 (22), 7785-7788CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small mols. at physiol. pH, temp., and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-contg. amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quant., site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, resp. Protein double labeling in aq. buffer at physiol. pH, temp., and pressure is quant. in 30 min.
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      33. 33
        Rideout, D. Self-assembling cytotoxins. Science 1986, 233, 561– 563,  DOI: 10.1126/science.3523757
        33
        Self-assembling cytotoxins
        Rideout, Darryl
        Science (Washington, DC, United States) (1986), 233 (4763), 561-3CODEN: SCIEAS; ISSN:0036-8075.
        Decanal [112-31-2] and N-amino-N'-1-octylguanidine (AOG) [66002-80-0], combined at 28 μM each, mediated erythrocyte lysis within 80 min under physiol. conditions. By contrast, no lysis was obsd. after 20 h with decanal (56 μM) or AOG (100 μM) alone. The pronounced synergism obsd. for these chems. and similar reactive pairs of chems. is due to the self-assembly of more cytotoxic hydrazones in situ. Decanal and AOG also exhibit synergistic activity against cultured human cells (HeLa) and bacteria (Escherichia coli J96). This synergism may be useful in the design of cytotoxins that would self-assemble selectively from nontoxic precursors within tumors, while sparing normal tissue.
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        https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28Xlt12isbc%253D&md5=3abf3eb1b14176e35c5fd91193d5e3bd
      34. 34
        Murray, C. W.; Rees, D. C. The rise of fragment-based drug discovery. Nat. Chem. 2009, 1, 187– 192,  DOI: 10.1038/nchem.217
        34
        The rise of fragment-based drug discovery
        Murray, Christopher W.; Rees, David C.
        Nature Chemistry (2009), 1 (3), 187-192CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
        A review. The search for new drugs is plagued by high attrition rates at all stages in research and development. Chemists have an opportunity to tackle this problem because attrition can be traced back, in part, to the quality of the chem. leads. Fragment-based drug discovery (FBDD) is a new approach, increasingly used in the pharmaceutical industry, for reducing attrition and providing leads for previously intractable biol. targets. FBDD identifies low-mol.-wt. ligands (∼150 Da) that bind to biol. important macromols. The three-dimensional exptl. binding mode of these fragments is detd. using X-ray crystallog. or NMR spectroscopy, and is used to facilitate their optimization into potent mols. with drug-like properties. Compared with high-throughput-screening, the fragment approach requires fewer compds. to be screened, and, despite the lower initial potency of the screening hits, offers more efficient and fruitful optimization campaigns. Here, we review the rise of FBDD, including its application to discovering clin. candidates against targets for which other chem. approaches have struggled.
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      35. 35
        Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Click chemistry for drug development and diverse chemical-biology applications. Chem. Rev. 2013, 113, 4905– 4979,  DOI: 10.1021/cr200409f
        35
        Click Chemistry for Drug Development and Diverse Chemical-Biology Applications
        Thirumurugan, Prakasam; Matosiuk, Dariusz; Jozwiak, Krzysztof
        Chemical Reviews (Washington, DC, United States) (2013), 113 (7), 4905-4979CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
        A review with 303 refs. on application of lick chem. reactions in chem., biochem., and pharmacol.
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      36. 36
        Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Click chemistry in situ: acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks. Angew. Chem., Int. Ed. 2002, 41, 1053– 1057,  DOI: 10.1002/1521-3773(20020315)41:6<1053::AID-ANIE1053>3.0.CO;2-4
        36
        Click chemistry in situ: Acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks
        Lewis, Warren G.; Green, Luke G.; Grynszpan, Flavio; Radic, Zoran; Carlier, Paul R.; Taylor, Palmer; Finn, M. G.; Sharpless, K. Barry
        Angewandte Chemie, International Edition (2002), 41 (6), 1053-1057CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
        Form-fitting chem. in a protein mold is enabled by the use of the 1,3-dipolar cycloaddn. of azides and alkynes. The enzyme acetylcholinesterase preferentially assembles one pair of these reactants, each of which bears a group that binds to adjacent positions on the protein structure, into a 1,2,3-triazole adduct that is the most potent noncovalent inhibitor of the enzyme yet developed.
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      37. 37
        Mocharla, V. P.; Colasson, B.; Lee, L. V.; Roper, S.; Sharpless, K. B.; Wong, C. H.; Kolb, H. C. In situ click chemistry: enzyme-generated inhibitors of carbonic anhydrase II. Angew. Chem., Int. Ed. 2005, 44, 116– 120,  DOI: 10.1002/anie.200461580
        37
        In situ click chemistry: Enzyme-generated inhibitors of carbonic anhydrase II
        Mocharla, Vani P.; Colasson, Benoit; Lee, Lac V.; Roeper, Stefanie; Sharpless, K. Barry; Wong, Chi-Huey; Kolb, Hartmuth C.
        Angewandte Chemie, International Edition (2005), 44 (1), 116-120CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        Target-guided synthesis: Rather than making and screening thousands of compds. for lead discovery, in situ click chem. employs the biol. target itself to assemble its inhibitors by selectively binding and interconnecting reagents within the confines of its binding sites. Subnanomolar inhibitors of carbonic anhydrase II were produced by the enzyme from simple azide and acetylene precursors.
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      38. 38
        Whiting, M.; Muldoon, J.; Lin, Y. C.; Silverman, S. M.; Lindstrom, W.; Olson, A. J.; Kolb, H. C.; Finn, M. G.; Sharpless, K. B.; Elder, J. H.; Fokin, V. V. Inhibitors of HIV-1 protease by using in situ click chemistry. Angew. Chem., Int. Ed. 2006, 45, 1435– 1439,  DOI: 10.1002/anie.200502161
        38
        Inhibitors of HIV-1 protease by using in situ click chemistry
        Whiting, Matthew; Muldoon, John; Lin, Ying-Chuan; Silverman, Steven M.; Lindstrom, William; Olson, Arthur J.; Kolb, Hartmuth C.; Finn, M. G.; Sharpless, K. Barry; Elder, John H.; Fokin, Valery V.
        Angewandte Chemie, International Edition (2006), 45 (9), 1435-1439CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        A review. Twice poor equals potent: HIV-1 Protease assembles its own potent inhibitor through formation of the triazole linkage from azide- and alkyne-contg. fragments that are themselves poor binders.
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      39. 39
        Devaraj, N. K.; Weissleder, R. Biomedical applications of tetrazine cycloadditions. Acc. Chem. Res. 2011, 44, 816– 827,  DOI: 10.1021/ar200037t
        39
        Biomedical Applications of Tetrazine Cycloadditions
        Devaraj, Neal K.; Weissleder, Ralph
        Accounts of Chemical Research (2011), 44 (9), 816-827CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
        A review. Disease mechanisms are increasingly being resolved at the mol. level. Biomedical success at this scale creates synthetic opportunities for combining specifically designed orthogonal reactions in applications such as imaging, diagnostics, and therapy. For practical reasons, it would be helpful if bioorthogonal coupling reactions proceeded with extremely rapid kinetics (k > 103 M-1 s-1) and high specificity. Improving kinetics would minimize both the time and amt. of labeling agent required to maintain high coupling yields. In this Account, we discuss our recent efforts to design extremely rapid bioorthogonal coupling reactions between tetrazines and strained alkenes. These selective reactions were first used to covalently couple conjugated tetrazine near-IR-emitting fluorophores to dienophile-modified extracellular proteins on living cancer cells. Confocal fluorescence microscopy demonstrated efficient and selective labeling, and control expts. showed minimal background fluorescence. Multistep techniques were optimized to work with nanomolar concns. of labeling agent over a time scale of minutes: the result was successful real-time imaging of covalent modification. We subsequently discovered fluorogenic probes that increase in fluorescence intensity after the chem. reaction, leading to an improved signal-to-background ratio. Fluorogenic probes were used for intracellular imaging of dienophiles. We further developed strategies to react and image chemotherapeutics, such as trans-cyclooctene taxol analogs, inside living cells. Because the coupling partners are small mols. (<300 Da), they offer unique steric advantages in multistep amplification. We also describe recent success in using tetrazine reactions to label biomarkers on cells with magneto-fluorescent nanoparticles. Two-step protocols that use bioorthogonal chem. can significantly amplify signals over both one-step labeling procedures as well as two-step procedures that use more sterically hindered biotin-avidin interactions. Nanoparticles can be detected with fluorescence or magnetic resonance techniques. These strategies are now being routinely used on clin. samples for biomarker profiling to predict malignancy and patient outcome. Finally, the authors discuss recent results with tetrazine reactions used for in vivo mol. imaging applications. Rapid tetrazine cycloaddns. allow modular labeling of small mols. with the most commonly used positron emission tomog. isotope, 18F. Addnl., recent work has applied this reaction directly in vivo for the pretargeted imaging of solid tumors. Future work with tetrazine cycloaddns. will undoubtedly lead to optimized protocols, improved probes, and addnl. biomedical applications.
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      40. 40
        Lebraud, H.; Wright, D. J.; Johnson, C. N.; Heightman, T. D. Protein Degradation by In-Cell Self-Assembly of Proteolysis Targeting Chimeras. ACS Cent. Sci. 2016, 2, 927– 934,  DOI: 10.1021/acscentsci.6b00280
        40
        Protein Degradation by In-Cell Self-Assembly of Proteolysis Targeting Chimeras
        Lebraud, Honorine; Wright, David J.; Johnson, Christopher N.; Heightman, Tom D.
        ACS Central Science (2016), 2 (12), 927-934CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
        Selective degrdn. of proteins by proteolysis targeting chimeras (PROTACs) offers a promising potential alternative to protein inhibition for therapeutic intervention. Current PROTAC mols. incorporate a ligand for the target protein, a linker, and an E3 ubiquitin ligase recruiting group, which bring together target protein and ubiquitinating machinery. Such hetero-bifunctional mols. require significant linker optimization and possess high mol. wt., which can limit cellular permeation, soly., and other drug-like properties. We show here that the hetero-bifunctional mol. can be formed intracellularly by bio-orthogonal click combination of two smaller precursors. We designed a tetrazine tagged thalidomide deriv. which reacts rapidly with a trans-cyclo-octene tagged ligand of the target protein in cells to form a cereblon E3 ligase recruiting PROTAC mol. The in-cell click-formed proteolysis targeting chimeras (CLIPTACs) were successfully used to degrade two key oncol. targets, BRD4 and ERK1/2. ERK1/2 degrdn. was achieved using a CLIPTAC based on a covalent inhibitor. We expect this approach to be readily extendable to other inhibitor-protein systems because the tagged E3 ligase recruiter is capable of undergoing the click reaction with a suitably tagged ligand of any protein of interest to elicit its degrdn.
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      41. 41
        Versteegen, R. M.; Rossin, R.; ten Hoeve, W.; Janssen, H. M.; Robillard, M. S. Click to release: instantaneous doxorubicin elimination upon tetrazine ligation. Angew. Chem., Int. Ed. 2013, 52, 14112– 14116,  DOI: 10.1002/anie.201305969
        41
        Click to Release: Instantaneous Doxorubicin Elimination upon Tetrazine Ligation
        Versteegen, Ron M.; Rossin, Raffaella; ten Hoeve, Wolter; Janssen, Henk M.; Robillard, Marc S.
        Angewandte Chemie, International Edition (2013), 52 (52), 14112-14116CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        We have developed a new bioorthogonal elimination reaction that enables instantaneous, self-immolative, and traceless release of doxorubicin from trans-cyclooctene following tetrazine ligation. In this proof-of-principle study, the inverse-electron-demand Diels-Alder (inv-DA) pyridazine elimination reached 79 % yield within minutes under ambient conditions at micromolar concns.
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      42. 42
        Xu, M.; Tu, J.; Franzini, R. M. Rapid and efficient tetrazine-induced drug release from highly stable benzonorbornadiene derivatives. Chem. Commun. 2017, 53, 6271– 6274,  DOI: 10.1039/C7CC03477F
        42
        Rapid and efficient tetrazine-induced drug release from highly stable benzonorbornadiene derivatives
        Xu, Minghao; Tu, Julian; Franzini, Raphael M.
        Chemical Communications (Cambridge, United Kingdom) (2017), 53 (46), 6271-6274CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
        A novel class of bioorthogonal release reactions based on benzonorbornadiene derivs. was developed. These carrier mols. are highly stable at physiol. conditions, but react rapidly with 1,2,4,5-tetrazines, and near-quant. release cargo mols. such as drugs and optical reporters.
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      43. 43
        Neumann, K.; Jain, S.; Gambardella, A.; Walker, S. E.; Valero, E.; Lilienkampf, A.; Bradley, M. Tetrazine-Responsive Self-immolative Linkers. ChemBioChem 2017, 18, 91– 95,  DOI: 10.1002/cbic.201600560
        43
        Tetrazine-Responsive Self-immolative Linkers
        Neumann, Kevin; Jain, Sarthak; Gambardella, Alessia; Walker, Sarah E.; Valero, Elsa; Lilienkampf, Annamaria; Bradley, Mark
        ChemBioChem (2017), 18 (1), 91-95CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
        Mols. that undergo activation or modulation following the addn. of benign external small-mol. chem. stimuli have numerous applications. Here, we report the highly efficient "decaging" of a variety of moieties by activation of a "self-immolative" linker, by application of water-sol. and stable tetrazine, including the controlled delivery of doxorubicin in a cellular context.
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      44. 44
        Jimenez-Moreno, E.; Guo, Z.; Oliveira, B. L.; Albuquerque, I. S.; Kitowski, A.; Guerreiro, A.; Boutureira, O.; Rodrigues, T.; Jimenez-Oses, G.; Bernardes, G. J. Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells. Angew. Chem., Int. Ed. 2017, 56, 243– 247,  DOI: 10.1002/anie.201609607
        44
        Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells
        Jimenez-Moreno, Ester; Guo, Zijian; Oliveira, Bruno L.; Albuquerque, Ines S.; Kitowski, Annabel; Guerreiro, Ana; Boutureira, Omar; Rodrigues, Tiago; Jimenez-Oses, Gonzalo; Bernardes, Goncalo J. L.
        Angewandte Chemie, International Edition (2017), 56 (1), 243-247CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        The cleavage of a protecting group from a protein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity. Disclosed herein is that vinyl ethers serve as protecting groups for alc.-contg. mols. and as reagents for bioorthogonal bond-cleavage reactions. A vinyl ether moiety was installed in a range of mols., including amino acids, a monosaccharide, a fluorophore, and an analog of the cytotoxic drug duocarmycin. Tetrazine-mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics. Importantly, the nontoxic, vinyl ether duocarmycin double prodrug was successfully decaged in live cells to reinstate cytotoxicity. This bioorthogonal reaction presents broad applicability and may be suitable for in vivo applications.
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      45. 45
        Mejia Oneto, J. M.; Khan, I.; Seebald, L.; Royzen, M. In Vivo Bioorthogonal Chemistry Enables Local Hydrogel and Systemic Pro-Drug To Treat Soft Tissue Sarcoma. ACS Cent. Sci. 2016, 2, 476– 482,  DOI: 10.1021/acscentsci.6b00150
        45
        In Vivo Bioorthogonal Chemistry Enables Local Hydrogel and Systemic Pro-Drug To Treat Soft Tissue Sarcoma
        Mejia Oneto, Jose M.; Khan, Irfan; Seebald, Leah; Royzen, Maksim
        ACS Central Science (2016), 2 (7), 476-482CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
        The ability to activate drugs only at desired locations avoiding systemic immunosuppression and other dose limiting toxicities is highly desirable. Here we present a new approach, named local drug activation, that uses bioorthogonal chem. to conc. and activate systemic small mols. at a location of choice. This method is independent of endogenous cellular or environmental markers and only depends on the presence of a preimplanted biomaterial near a desired site (e.g., tumor). We demonstrate the clear therapeutic benefit with minimal side effects of this approach in mice over systemic therapy using a doxorubicin pro-drug against xenograft tumors of a type of soft tissue sarcoma (HT1080).
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      46. 46
        Zhang, G.; Li, J.; Xie, R.; Fan, X.; Liu, Y.; Zheng, S.; Ge, Y.; Chen, P. R. Bioorthogonal Chemical Activation of Kinases in Living Systems. ACS Cent. Sci. 2016, 2, 325– 331,  DOI: 10.1021/acscentsci.6b00024
        46
        Bioorthogonal Chemical Activation of Kinases in Living Systems
        Zhang, Gong; Li, Jie; Xie, Ran; Fan, Xinyuan; Liu, Yanjun; Zheng, Siqi; Ge, Yun; Chen, Peng R.
        ACS Central Science (2016), 2 (5), 325-331CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
        Selective manipulation of protein kinases under living conditions is highly desirable yet extremely challenging, particularly in a gain-of-function fashion. Here we employ our recently developed bioorthogonal cleavage reaction as a general strategy for intracellular activation of individual kinases. Site-specific incorporation of trans-cyclooctene-caged lysine in place of the conserved catalytic lysine, in conjunction with the cleavage partner dimethyl-tetrazine, allowed efficient lysine decaging with the kinase activity chem. rescued in living systems.
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      47. 47
        Li, J.; Jia, S.; Chen, P. R. Diels-Alder reaction-triggered bioorthogonal protein decaging in living cells. Nat. Chem. Biol. 2014, 10, 1003– 1005,  DOI: 10.1038/nchembio.1656
        47
        Diels-Alder reaction-triggered bioorthogonal protein decaging in living cells
        Li, Jie; Jia, Shang; Chen, Peng R.
        Nature Chemical Biology (2014), 10 (12), 1003-1005CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
        Small mols. that specifically activate an intracellular protein of interest are highly desirable. A generally applicable strategy, however, remains elusive. Herein we describe a small mol.-triggered bioorthogonal protein decaging technique that relies on the inverse electron-demand Diels-Alder reaction for eliminating a chem. caged protein side chain within living cells. This method permits the efficient activation of a given protein (for example, an enzyme) in its native cellular context within minutes.
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      48. 48
        Wu, H.; Alexander, S. C.; Jin, S.; Devaraj, N. K. A Bioorthogonal Near-Infrared Fluorogenic Probe for mRNA Detection. J. Am. Chem. Soc. 2016, 138, 11429– 11432,  DOI: 10.1021/jacs.6b01625
        48
        A Bioorthogonal Near-Infrared Fluorogenic Probe for mRNA Detection
        Wu, Haoxing; Alexander, Seth C.; Jin, Shuaijiang; Devaraj, Neal K.
        Journal of the American Chemical Society (2016), 138 (36), 11429-11432CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        There is significant interest in developing methods that visualize and detect RNA in live cells. Bioorthogonal template driven tetrazine ligations could be a powerful route to visualizing nucleic acids in native cells, yet past work has been limited with respect to the diversity of fluorogens that can be activated via a tetrazine reaction. Herein we report a novel bioorthogonal tetrazine uncaging reaction that harnesses tetrazine reactivity to unmask vinyl ether caged fluorophores spanning the visible spectrum, including a near-IR (NIR) emitting cyanine dye. Vinyl ether caged fluorophores and tetrazine partners are conjugated to high affinity antisense nucleic acid probes, which show highly selective fluorogenic reactivity when annealed to their resp. target RNA sequences. A target sequence in the 3' untranslated region (3' UTR) of an expressed mRNA was successfully detected in live cells employing appropriate nucleic acid probes bearing a tetrazine reactive NIR fluorogen. Given the expansion of tetrazine fluorogenic chem. to NIR dyes, we believe highly selective proximity induced fluorogenic tetrazine reactions could find broad uses in illuminating endogenous biomols. in living cells.
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      49. 49
        Ji, X.; Zhou, C.; Ji, K.; Aghoghovbia, R. E.; Pan, Z.; Chittavong, V.; Ke, B.; Wang, B. Click and Release: A Chemical Strategy toward Developing Gasotransmitter Prodrugs by Using an Intramolecular Diels-Alder Reaction. Angew. Chem., Int. Ed. 2016, 55, 15846– 15851,  DOI: 10.1002/anie.201608732
        49
        Click and Release: A Chemical Strategy toward Developing Gasotransmitter Prodrugs by Using an Intramolecular Diels-Alder Reaction
        Ji, Xingyue; Zhou, Cheng; Ji, Kaili; Aghoghovbia, Robert E.; Pan, Zhixiang; Chittavong, Vayou; Ke, Bowen; Wang, Binghe
        Angewandte Chemie, International Edition (2016), 55 (51), 15846-15851CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        Prodrug strategies have been proven to be a very effective way of addressing delivery problems. Much of the chem. in prodrug development relies on the ability to mask an appropriate functional group, which can be removed under appropriate conditions. However, developing org. prodrugs of gasotransmitters represent unique challenges. This is esp. true with carbon monoxide, which does not have an easy "handle" for bioreversible derivatization. By taking advantage of an intramol. Diels-Alder reaction, we have developed a prodrug strategy for prepns. of org. CO prodrugs (I, where X = O, NR1 and n = 1,2) that are stable during synthesis and storage, and yet readily release CO with tunable release rates under near physiol. conditions. The effectiveness of the CO prodrug system in delivering a sufficient quantity of CO for possible therapeutic applications has been studied using a cell culture anti-inflammatory assay and a colitis animal model. These studies fully demonstrate the proof of concept, and lay a strong foundation for further medicinal chem. work in developing org. CO prodrugs.
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      50. 50
        Carlson, J. C. T.; Mikula, H.; Weissleder, R. Unraveling Tetrazine-Triggered Bioorthogonal Elimination Enables Chemical Tools for Ultrafast Release and Universal Cleavage. J. Am. Chem. Soc. 2018, 140, 3603– 3612,  DOI: 10.1021/jacs.7b11217
        50
        Unraveling tetrazine-triggered bioorthogonal elimination enables chemical tools for ultrafast release and universal cleavage
        Carlson, Jonathan C. T.; Mikula, Hannes; Weissleder, Ralph
        Journal of the American Chemical Society (2018), 140 (10), 3603-3612CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        Recent developments in bond cleavage reactions have expanded the scope of bioorthogonal chem. beyond click ligation and enabled new strategies for probe activation and therapeutic delivery. These applications, however, remain in their infancy, with further innovations needed to achieve the efficiency required for versatile and broadly useful tools in vivo. Among these chemistries, the tetrazine/trans-cyclooctene click-to-release reaction has exemplary kinetics and adaptability but achieves only partial release and is incompletely understood, which has limited its application. Investigating the mechanistic features of this reaction's performance, we discovered profound pH sensitivity, exploited it with acid-functionalized tetrazines that both enhance and markedly accelerate release, and ultimately uncovered an unexpected dead-end isomer as the reason for poor release. Implementing facile methods to prevent formation of this dead end, we have achieved exceptional efficiency, with essentially complete release across the full scope of physiol. pH, potentiating drug-delivery strategies and expanding the dynamic range of bioorthogonal on/off control.
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      51. 51
        El-Sagheer, A. H.; Brown, T. Synthesis and polymerase chain reaction amplification of DNA strands containing an unnatural triazole linkage. J. Am. Chem. Soc. 2009, 131, 3958– 3964,  DOI: 10.1021/ja8065896
        51
        Synthesis and Polymerase Chain Reaction Amplification of DNA Strands Containing an Unnatural Triazole Linkage
        El-Sagheer, Afaf H.; Brown, Tom
        Journal of the American Chemical Society (2009), 131 (11), 3958-3964CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        DNA strands contg. an unnatural T-triazole-T linkage have been synthesized by click DNA ligation between oligonucleotides with 3'-AZT and 5'-propargylamido dT and amplified efficiently by polymerase chain reaction (PCR) using several different polymerases. DNA sequencing of PCR amplicons and clones in two different sequence contexts revealed the presence of a single thymidine at the ligation site. The remarkable ability of thermostable polymerases to reproducibly copy DNA templates contg. such an unnatural backbone opens up intriguing possibilities in gene synthesis, genetic anal., biol., and nanotechnol.
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        https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXisVCjtL0%253D&md5=c64505e86e67b410762714ad9af48364
      52. 52
        Budin, I.; Devaraj, N. K. Membrane assembly driven by a biomimetic coupling reaction. J. Am. Chem. Soc. 2012, 134, 751– 753,  DOI: 10.1021/ja2076873
        52
        Membrane Assembly Driven by a Biomimetic Coupling Reaction
        Budin, Itay; Devaraj, Neal K.
        Journal of the American Chemical Society (2012), 134 (2), 751-753CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        One of the major goals of synthetic biol. is the development of non-natural cellular systems. In this work, we describe a catalytic biomimetic coupling reaction capable of driving the de novo self-assembly of phospholipid membranes. Our system features a copper-catalyzed azide-alkyne cycloaddn. that results in the formation of a triazole-contg. phospholipid analog. Concomitant assembly of membranes occurs spontaneously, not requiring preexisting membranes to house catalysts or precursors. The substitution of efficient synthetic reactions for key biochem. processes may offer a general route toward synthetic biol. systems.
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      53. 53
        Cendret, V.; Francois-Heude, M.; Mendez-Ardoy, A.; Moreau, V.; Fernandez, J. M.; Djedaini-Pilard, F. Design and synthesis of a ″click″ high-mannose oligosaccharide mimic emulating Man8 binding affinity towards Con A. Chem. Commun. 2012, 48, 3733– 3735,  DOI: 10.1039/c2cc30773a
        There is no corresponding record for this reference.
      54. 54
        Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Synthesis of proteins by native chemical ligation. Science 1994, 266, 776– 779,  DOI: 10.1126/science.7973629
        54
        Synthesis of proteins by native chemical ligation
        Dawson, Philip E.; Muir, Tom W.; Clark-Lewis, Ian; Kent, Stephen B. H.
        Science (Washington, D. C.) (1994), 266 (5186), 776-9CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
        A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chem. ligation was demonstrated by the one-step prepn. of a cytokine contg. multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-contg. protein mol. Native chem. ligation is an important step toward the general application of chem. to proteins.
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      55. 55
        de Figueiredo, R. M.; Suppo, J. S.; Campagne, J. M. Nonclassical Routes for Amide Bond Formation. Chem. Rev. 2016, 116, 12029– 12122,  DOI: 10.1021/acs.chemrev.6b00237
        55
        Nonclassical Routes for Amide Bond Formation
        de Figueiredo Renata Marcia; Suppo Jean-Simon; Campagne Jean-Marc
        Chemical reviews (2016), 116 (19), 12029-12122 ISSN:.
        The present review offers an overview of nonclassical (e.g., with no pre- or in situ activation of a carboxylic acid partner) approaches for the construction of amide bonds. The review aims to comprehensively discuss relevant work, which was mainly done in the field in the last 20 years. Organization of the data follows a subdivision according to substrate classes: catalytic direct formation of amides from carboxylic and amines ( section 2 ); the use of carboxylic acid surrogates ( section 3 ); and the use of amine surrogates ( section 4 ). The ligation strategies (NCL, Staudinger, KAHA, KATs, etc.) that could involve both carboxylic acid and amine surrogates are treated separately in section 5 .
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      56. 56
        Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. High-yielding Staudinger ligation of a phosphinothioester and azide to form a peptide. Org. Lett. 2001, 3, 9– 12,  DOI: 10.1021/ol006739v
        56
        High-Yielding Staudinger Ligation of a Phosphinothioester and Azide To Form a Peptide
        Nilsson, Bradley L.; Kiessling, Laura L.; Raines, Ronald T.
        Organic Letters (2001), 3 (1), 9-12CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
        The Staudinger ligation can be used to couple a peptide with a C-terminal phosphinothioester to another with an N-terminal α-azido group to form a single peptide that contains no residual atoms of the phosphinothiol entity. Here, diphenylphosphinomethanethiol thioesters are shown to give high isolated yields for this transformation. This finding provides precedent for a powerful and versatile new method for the total synthesis of proteins, avoiding the native chem. ligation method's reliance on the presence of a cysteine residue for bond formation.
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      57. 57
        Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. A ″traceless″ Staudinger ligation for the chemoselective synthesis of amide bonds. Org. Lett. 2000, 2, 2141– 2143,  DOI: 10.1021/ol006054v
        57
        A "Traceless" Staudinger Ligation for the Chemoselective Synthesis of Amide Bonds
        Saxon, Eliana; Armstrong, Joshua I.; Bertozzi, Carolyn R.
        Organic Letters (2000), 2 (14), 2141-2143CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
        A novel modification is reported of the authors' previously reported Staudinger ligation that generates an amide bond from an azide and a specifically functionalized phosphine. This method for the selective formation of an amide bond, which does not require the orthogonal protection of distal functional groups, should find general utility in synthetic and biol. chem. 2-AcOC6H4PPh2 and (1-acetyl-2-imidazolyl)diphenylphosphine reacted with an azidonucleoside (5'-azido-N-benzoyl-2',5'-dideoxyadenosine) in wet THF to convert the azido group to an acetylamino group in >95% yield.
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      58. 58
        Erben, A.; Grossmann, T. N.; Seitz, O. DNA-triggered synthesis and bioactivity of proapoptotic peptides. Angew. Chem., Int. Ed. 2011, 50, 2828– 2832,  DOI: 10.1002/anie.201007103
        58
        DNA-Triggered Synthesis and Bioactivity of Proapoptotic Peptides
        Erben, Anne; Grossmann, Tom N.; Seitz, Oliver
        Angewandte Chemie, International Edition (2011), 50 (12), 2828-2832, S2828/1-S2828/17CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        Diseases are frequently caused by changes in the genetic infrastructure. In such cases, the disordered state of a diseased cell is encoded in the DNA and reflected in the level and sequence of the expressed RNA mols. The information obtained from nucleic acids may be used to direct mol. therapies only to diseased cells and tissues. In a fascinating approach, disease-specific nucleic acid sequences could be hijacked to trigger the formation or release of drug mols. Herein a reaction system is introduced in which the sequence information of an unstructured DNA template is used to trigger the transfer of an aminoacyl group from a donating thioester-modified peptide-nucleic acid (PNA) conjugate to an acceptor peptidyl-PNA conjugate. It is demonstrated that the template can act as a catalyst which instructs the formation of many product mols. per template mol. The formed peptide-PNA conjugate was designed to interfere with the protein-protein interactions between caspase-9, a protease involved in the initiation of programmed cell death (apoptosis), and the X-linked inhibitor of apoptosis protein XIAP. It is shown that the nucleic acid programmed peptide synthesis allows activation of caspase-9 and a downstream caspase.
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      59. 59
        Noda, H.; Eros, G.; Bode, J. W. Rapid ligations with equimolar reactants in water with the potassium acyltrifluoroborate (KAT) amide formation. J. Am. Chem. Soc. 2014, 136, 5611– 5614,  DOI: 10.1021/ja5018442
        59
        Rapid ligations with equimolar reactants in water with the potassium acyltrifluoroborate (KAT) amide formation
        Noda, Hidetoshi; Eros, Gabor; Bode, Jeffrey W.
        Journal of the American Chemical Society (2014), 136 (15), 5611-5614CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        The identification of fast, chemoselective bond-forming reactions is one of the major contemporary challenges in chem. We show that chemoselective amide-forming ligations of potassium acyltrifluoroborates (KATs) and O-carbamoylhydroxylamines proceed in the presence of all unprotected functional groups with a second-order rate const. of 20 M-1 s-1. PEG chains, lipids, biotin, and dyes were introduced onto an unprotected 31-mer peptide (GLP-1 analog) with equimolar ratios of reactants within minutes at 1 mM and within 1 h at 100 μM, even with Mw 20 000 PEG. This conjugation reaction provides a new approach to the synthesis of mols. such as protein-protein and protein-polymer conjugates.
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      60. 60
        Rudd, A. K.; Devaraj, N. K. Traceless Synthesis of Ceramides in Living Cells Reveals Saturation-Dependent Apoptotic Effects. Proc. Natl. Acad. Sci. USA , 2018, In Press. DOI:  DOI: 10.1073/pnas.1804266115 .
        There is no corresponding record for this reference.
      61. 61
        Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.; King, D. S.; Horn, D. M.; Schultz, P. G. Generation of a bacterium with a 21 amino acid genetic code. J. Am. Chem. Soc. 2003, 125, 935– 939,  DOI: 10.1021/ja0284153
        61
        Generation of a Bacterium with a 21 Amino Acid Genetic Code
        Mehl, Ryan A.; Anderson, J. Christopher; Santoro, Stephen W.; Wang, Lei; Martin, Andrew B.; King, David S.; Horn, David M.; Schultz, Peter G.
        Journal of the American Chemical Society (2003), 125 (4), 935-939CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
        We have generated a completely autonomous bacterium with a 21 amino acid genetic code. This bacterium can biosynthesize a nonstandard amino acid from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon. The biosynthetic pathway for the amino acid p-aminophenylalanine (pAF) as well as a unique pAF synthetase and cognate tRNA were added to Escherichia coli. Denaturing gel electrophoresis and mass spectrometric anal. show that pAF is incorporated into myoglobin with fidelity and efficiency rivaling those of the common 20 amino acids. This and other such organisms may provide an opportunity to examine the evolutionary consequences of adding new amino acids to the genetic repertoire, as well as generate proteins with new or enhanced biol. functions.
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      62. 62
        Bohannon, M. B.; Kleiman, R. Cyclopropene fatty acids of selected seed oils from bombacaceae, malvaceae, and sterculiaceae. Lipids 1978, 13, 270– 273,  DOI: 10.1007/BF02533669
        There is no corresponding record for this reference.
      63. 63
        Bao, X.; Katz, S.; Pollard, M.; Ohlrogge, J. Carbocyclic fatty acids in plants: biochemical and molecular genetic characterization of cyclopropane fatty acid synthesis of Sterculiafoetida. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 7172– 7177,  DOI: 10.1073/pnas.092152999
        There is no corresponding record for this reference.
      64. 64
        Chen, K.; Huang, X.; Kan, S. B. J.; Zhang, R. K.; Arnold, F. H. Enzymatic construction of highly strained carbocycles. Science 2018, 360, 71– 75,  DOI: 10.1126/science.aar4239
        64
        Enzymatic construction of highly strained carbocycles
        Chen, Kai; Huang, Xiongyi; Kan, S. B. Jennifer; Zhang, Ruijie K.; Arnold, Frances H.
        Science (Washington, DC, United States) (2018), 360 (6384), 71-75CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
        Small carbocycles are structurally rigid and possess high intrinsic energy due to their ring strain. These features lead to broad applications but also create challenges for their construction. We report the engineering of a cytochrome P 450 variant (designated P411) that catalyze the formation of chiral bicyclobutanes, one of the most strained four-membered systems, via successive carbene addn. to unsatd. carbon-carbon bonds. Enzymes that produce cyclopropenes, putative intermediates to the bicyclobutanes, were also identified. These genetically encoded proteins are readily optimized by directed evolution, function in Escherichia coli, and act on structurally diverse substrates with high efficiency and selectivity, providing an effective route to many chiral strained structures. This biotransformation is easily performed at preparative scale, and the resulting strained carbocycles can be derivatized, opening myriad potential applications.
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      65. 65
        Lappchen, T.; Rossin, R.; van Mourik, T. R.; Gruntz, G.; Hoeben, F. J. M.; Versteegen, R. M.; Janssen, H. M.; Lub, J.; Robillard, M. S. DOTA-tetrazine probes with modified linkers for tumor pretargeting. Nucl. Med. Biol. 2017, 55, 19– 26,  DOI: 10.1016/j.nucmedbio.2017.09.001
        65
        DOTA-tetrazine probes with modified linkers for tumor pretargeting
        Lappchen Tilman; Rossin Raffaella; van Mourik Tiemen R; Gruntz Guillaume; Lub Johan; Hoeben Freek J M; Versteegen Ron M; Janssen Henk M; Robillard Marc S
        Nuclear medicine and biology (2017), 55 (), 19-26 ISSN:.
        INTRODUCTION: Pretargeted radioimmunoimaging and -therapy approaches building on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have yielded impressive results in recent years and have proven a vital alternative to biological pretargeting systems. After improvement of the TCO-antibody conjugates, we here report on our evaluation of a new series of radiolabeled Tz-probes. METHODS: Four new Tz-probes were synthesized, radiolabeled with lutetium-177, and characterized in vitro in terms of lipophilicity, reactivity, and stability in PBS and mouse serum. The in vivo biodistribution profile and tumor-targeting potential of the probes were evaluated in LS174T tumor-bearing mice pretargeted with TCO-antibody conjugates using non-pretargeted mice as control. RESULTS: Radiolabeling of all probes proceeded in high yields providing the (177)Lu-labeled tetrazines in >95% radiochemical purity without any further purification. In mouse serum, half-lives of the probes varied between 8 and 13 h, with the exception of the most lipophilic probe, [(177)Lu]1b, with a serum half-life of less than 1 h. This probe also showed the fastest blood clearance (t1/2 = 5.4 min), more than 2-fold faster than PEG-linked probes [(177)Lu]3 and [(177)Lu]4, and even 3-fold faster than the other small probes without the PEG-linker, [(177)Lu]1a and [(177)Lu]2. In the pretargeting experiments, tumor uptake of the lead probe [(177)Lu]4 (~6 %ID/g) was most closely approached by [(177)Lu]2, followed by [(177)Lu]3 and [(177)Lu]1a. While all the smaller and more lipophilic probes suffered from increased liver uptake, the PEG-linked probe [(177)Lu]3 with its additional negative charge surprisingly showed the highest kidney uptake among all of the probes. CONCLUSION: The in vitro performance of some of the new tetrazine probes turned out to be comparable to the established lead probe [(177)Lu]Lu-DOTA-PEG11-Tz ([(177)Lu]4). However, tumor pretargeting studies in vivo showed lower tumor uptake and increased uptake in non-target organs.
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      66. 66
        Houghton, J. L.; Membreno, R.; Abdel-Atti, D.; Cunanan, K. M.; Carlin, S.; Scholz, W. W.; Zanzonico, P. B.; Lewis, J. S.; Zeglis, B. M. Establishment of the In Vivo Efficacy of Pretargeted Radioimmunotherapy Utilizing Inverse Electron Demand Diels-Alder Click Chemistry. Mol. Cancer Ther. 2017, 16, 124– 133,  DOI: 10.1158/1535-7163.MCT-16-0503
        66
        Establishment of the In Vivo Efficacy of Pretargeted Radioimmunotherapy Utilizing Inverse Electron Demand Diels-Alder Click Chemistry
        Houghton, Jacob L.; Membreno, Rosemery; Abdel-Atti, Dalya; Cunanan, Kristen M.; Carlin, Sean; Scholz, Wolfgang W.; Zanzonico, Pat B.; Lewis, Jason S.; Zeglis, Brian M.
        Molecular Cancer Therapeutics (2017), 16 (1), 124-133CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
        The pretargeting system based on the inverse electron demand Diels-Alder reaction (IEDDA) between trans-cyclooctene (TCO) and tetrazine (Tz) combines the favorable pharmacokinetic properties of radiolabeled small mols. with the affinity and specificity of antibodies. This strategy has proven to be an efficient method for the molecularly targeted delivery of pharmaceuticals, including isotopes for radiol. imaging. Despite encouraging results from in vivo PET imaging studies, this promising system has yet to be thoroughly evaluated for pretargeted radioimmunotherapy (PRIT). Toward that end, we synthesized two novel 177Lu-labeled tetrazine-bearing radioligands. Next, we compared the usefulness of our ligands for PRIT when paired with TCO-modified 5B1-a human, anti-CA19.9 mAb-in preclin. murine models of pancreatic cancer. The exemplary ligand, 177Lu-DOTA-PEG7-Tz, showed rapid (4.6 ± 0.8% ID/g at 4 h) and persistent (16.8 ± 3.9% ID/g at 120 h) uptake in tumors while concurrently clearing from blood and nontarget tissues. Single-dose therapy studies using 5B1-TCO and varying amts. of 177Lu-DOTA-PEG7-Tz (400, 800, and 1,200μCi) showed that our system elicits a dose-dependent therapeutic response in mice bearing human xenografts. Furthermore, dosimetry calcns. suggest that our approach is amenable to clin. applications with its excellent dosimetric profile in organs of clearance (i.e., liver and kidneys) as well as in dose-limiting tissues, such as red marrow. This study established that a pretargeted methodol. utilizing the IEDDA reaction can rapidly and specifically deliver a radiotherapeutic payload to tumor tissue, thus illustrating its excellent potential for clin. translation.
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      67. 67
        Goodwin, D. A.; Meares, C. F.; McCall, M. J.; McTigue, M.; Chaovapong, W. Pre-targeted immunoscintigraphy of murine tumors with indium-111-labeled bifunctional haptens. J. Nucl. Med. 1988, 29 (2), 226– 234
        There is no corresponding record for this reference.
      68. 68
        Zeglis, B. M.; Sevak, K. K.; Reiner, T.; Mohindra, P.; Carlin, S. D.; Zanzonico, P.; Weissleder, R.; Lewis, J. S. A pretargeted PET imaging strategy based on bioorthogonal Diels-Alder click chemistry. J. Nucl. Med. 2013, 54, 1389– 1396,  DOI: 10.2967/jnumed.112.115840
        68
        A pretargeted PET imaging strategy based on bioorthogonal Diels-Alder click chemistry
        Zeglis, Brian M.; Sevak, Kuntal K.; Reiner, Thomas; Mohindra, Priya; Carlin, Sean D.; Zanzonico, Pat; Weissleder, Ralph; Lewis, Jason S.
        Journal of Nuclear Medicine (2013), 54 (8), 1389-1396CODEN: JNMEAQ; ISSN:0161-5505. (Society of Nuclear Medicine and Molecular Imaging)
        The specificity of antibodies have made immunoconjugates promising vectors for the delivery of radioisotopes to cancer cells; however, their long pharmacol. half-lives necessitate the use of radioisotopes with long phys. half-lives, a combination that leads to high radiation doses to patients. Therefore, the development of targeting modalities that harness the advantages of antibodies without their pharmacokinetic limitations is desirable. To this end, we report the development of a methodol. for pretargeted PET imaging based on the bioorthogonal Diels-Alder click reaction between tetrazine and transcyclooctene. Methods: A proof-of-concept system based on the A33 antibody, SW1222 colorectal cancer cells, and 64Cu was used. The huA33 antibody was covalently modified with transcyclooctene, and a NOTA-modified tetrazine was synthesized and radiolabeled with 64Cu. Pretargeted in vivo biodistribution and PET imaging expts. were performed with athymic nude mice bearing A33 antigen-expressing, SW1222 colorectal cancer xenografts. Results: The huA33 antibody was modified with transcyclooctene to produce a conjugate with high immunoreactivity, and the 64Cu-NOTA-labeled tetrazine ligand was synthesized with greater than 99% purity and a specific activity of 9-10 MBq/μg. For in vivo expts., mice bearing SW1222 xenografts were injected with transcyclooctene-modified A33; after allowing 24 h for accumulation of the antibody in the tumor, the mice were injected with 64Cu-NOTA-labeled tetrazine for PET imaging and biodistribution expts. At 12 h after injection, the retention of uptake in the tumor (4.1 ± 0.3 percent injected dose per g), coupled with the fecal excretion of excess radioligand, produced images with high tumor-to-background ratios. PET imaging and biodistribution expts. performed using A33 directly labeled with either 64Cu or 89Zr revealed that although abs. tumor uptake was higher with the directly radiolabeled antibodies, the pretargeted system yielded comparable images and tumor-to-muscle ratios at 12 and 24 h after injection. Further, dosimetry calcns. revealed that the 64Cu pretargeting system resulted in only a fraction of the absorbed background dose of A33 directly labeled with 89Zr (0.0124 mSv/MBq vs. 0.4162 mSv/MBq, resp.). Conclusion: The high quality of the images produced by this pretargeting approach, combined with the ability of the methodol. to dramatically reduce nontarget radiation doses to patients, marks this system as a strong candidate for clin. translation.
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      69. 69
        Rossin, R.; Verkerk, P. R.; van den Bosch, S. M.; Vulders, R. C.; Verel, I.; Lub, J.; Robillard, M. S. In vivo chemistry for pretargeted tumor imaging in live mice. Angew. Chem., Int. Ed. 2010, 49, 3375– 3378,  DOI: 10.1002/anie.200906294
        69
        In Vivo Chemistry for Pretargeted Tumor Imaging in Live Mice
        Rossin, Raffaella; Renart Verkerk, Pascal; van den Bosch, Sandra M.; Vulders, Roland C. M.; Verel, Iris; Lub, Johan; Robillard, Marc S.
        Angewandte Chemie, International Edition (2010), 49 (19), 3375-3378, S3375/1-S3375/22CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
        Bioorthogonal chem. reaction between two exogenous moieties in living animal was used for cancer imaging.
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      70. 70
        Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjugate Chem. 2008, 19, 2297– 2299,  DOI: 10.1021/bc8004446
        70
        Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging
        Devaraj, Neal K.; Weissleder, Ralph; Hilderbrand, Scott A.
        Bioconjugate Chemistry (2008), 19 (12), 2297-2299CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
        Bioorthogonal tetrazine cycloaddns. have been applied to live cell labeling. Tetrazines react irreversibly with the strained dienophile norbornene forming dihydropyrazine products and dinitrogen. The reaction is high yielding, selective, and fast in aq. media. Her2/neu receptors on live human breast cancer cells were targeted with a monoclonal antibody modified with a norbornene. Tetrazines conjugated to a near-IR fluorochrome selectively and rapidly label the pretargeted antibody in the presence of serum. These findings indicate that this chem. is suitable for in vitro labeling expts., and suggests that it may prove a useful strategy for in vivo pretargeted imaging under numerous modalities.
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