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Published in final edited form as: Chem. 2023 Jun 5;9(8):2095–2109. doi: 10.1016/j.chempr.2023.05.016

Bioorthogonal chemistry: Bridging chemistry, biology, and medicine

Kaitlin M Hartung 1, Ellen M Sletten 1,*
PMCID: PMC11245302  NIHMSID: NIHMS1957052  PMID: 39006002

SUMMARY

As chemical biologists sought methods to modify and study biomolecules in their native environments, the need for bioorthogonal chemical reactions emerged. These fast and selective reactions between otherwise inert, abiotic functional groups have enabled exploration of some of the most intriguing and challenging questions in chemical biology. Further, the ability to perform organic reactions in cells and organisms has led to important applications in clinical spaces, and one reaction is now an integral part of a phase 2 trial for treating solid tumors. Given that bioorthogonal chemistry was a recipient of the 2022 Nobel Prize, we expect this field to be even more energized. Here, we highlight some of the most recent studies in this sphere and how these set the stage for where bioorthogonal chemistry is headed.

INTRODUCTION

The foundation of chemical biology can be traced back centuries to the use of chemicals to perturb biological processes.1 Today, it is an active field applying chemistry to study biological systems and using biology to enable new chemistries.2 A distinction between chemical biology and biochemistry is the desire to characterize biomolecules in their native environments rather than in vitro, a challenging task given the complexity of living cells and organisms.

A breakthrough toward studying biomolecules in living systems came with the advent of bioorthogonal chemistry: chemical reactions between abiotic functional groups that react selectively with each other but are inert to biological functionalities. Bioorthogonal reactions must exhibit exquisite selectivity, be high yielding, require simple design and execution, feature non-toxic components, and proceed in physiological media and conditions.3 The first three requirements overlap those of click reactions, introduced to the synthetic chemistry community around the same time by Barry Sharpless4; however, the remaining conditions impose additional challenges. Despite this, bioorthogonal chemistry has quickly become a veritable wealth of tools used for probing biological questions once out of reach and has even expanded to technologies related to human health (Figure 1A). Its impact, intertwined with click chemistry, was recognized with the awarding of the 2022 Nobel Prize.

Figure 1. The various roles of bioorthogonal chemistry in chemical biology.

Figure 1.

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(A) Selective reactions that take place in living systems without interacting with native functionalities have enabled detailed studies and discoveries in chemical biology and led to important biomedical advances.

(B) The four standard bioorthogonal and click reactions: the Staudinger ligation, copper-catalyzed azide-alkyne click cycloaddition (CuAAC, *which is generally not considered bioorthogonal because of the toxicity of copper), strain-promoted azide-alkyne cycloaddition (SPAAC), and the inverse electron-demand Diels-Alder (IEDDA) reaction between tetrazines and strained alkenes (tetrazine ligation). General second-order rate constants are listed for bioorthogonal reactions.

An initial inspiration for the pursuit of bioorthogonal reactions was the need for new chemistries in the study of glycans, which were not amenable to molecular biology tools available at the time. Developing such reactions was the quest of Carolyn Bertozzi and her lab in the 1990s. At the turn of the century, Saxon and Bertozzi published the Staudinger ligation, the first of what would become a suite of bioorthogonal reactions (Figure 1B).5 A modification of the classic Staudinger reduction of azides by phosphine reagents, this reaction includes a trap on the phosphine probe that allows for the generation of an amide-linked ligation product. This report also served as the introduction of the azide as an ideal bioorthogonal functional group for modifying biomolecules given that it is not found naturally and is small and non-perturbing. This small but mighty three-atom arrangement has become a staple in chemical biology.

The azide is also a key component of the quintessential click reaction—a formal [3 + 2] cycloaddition between azides and terminal alkynes catalyzed by Cu(I) (copper-catalyzed click chemistry, or CuAAC; Figure 1B), which was reported independently by the Meldal and Sharpless groups in 2002.6,7 Although not bioorthogonal because of the toxicity of Cu(I), CuAAC is a prominent method for labeling azides in fixed cells, lysates, and proteins. More recent advances in ligands have decreased the amount of Cu(I) necessary, allowing CuAAC to be used on live cells and organisms, though the need for three things to come together still limits in vivo applications.8

During the early 2000s, taking inspiration from physical organic chemists Huisgen, Wittig, and Krebs, Bertozzi was also focused on the 1,3-diploar cycloaddition between azides and alkynes.9 Instead of activating alkynes for reactivity with azides by copper catalysis, Bertozzi and co-workers looked to a fundamental chemistry concept: ring strain. The strain-promoted azide-alkyne cycloaddition (SPAAC, or copper-free click chemistry), between a strained cyclooctyne and azide, was thus born (Figure 1B).10 A variety of cyclooctynes for SPAAC have been discovered, enabling the tagging of biomolecules in live cells, zebrafish, and mice.3,8 A few years later, a faster reaction between strained alkenes and tetrazines, new bioorthogonal functional moieties, burst onto the scene (Figure 1B).11,12 This inverse electron-demand Diels-Alder (IEDDA, also known as the tetrazine ligation) reaction quickly became a staple in the bioorthogonal toolbox. With inherent strain energy now taking the place of a discrete catalyst, click and bioorthogonal chemistries converged in a partnership that would propel the field of chemical biology into a new era of development, discovery, and applications.

We begin this perspective by broadly discussing the evolution of bioorthogonal chemistry and highlighting recent new directions likely to be applied in chemical biology. We then venture into the realm of biological inquiry and discovery made possible by bioorthogonal strategies, and finally, we highlight the potential bioorthogonal chemistry holds with regard to medical advances.

DEVELOPMENT AND IMPROVEMENT OF FOUNDATIONAL CHEMISTRIES

Although the four popular reactions introduced above remain standards in chemical biology applications, new and improved reactions are continuously in development, particularly for metal-free reactions. Improvements on old reactions often focus on increasing reaction rates without compromising the size and/or stability of reagents, a tradeoff commonly encountered in the development of bioorthogonal reactions. Other developments incorporate features for increased control over when or where a reaction occurs in living systems, including light-activated reagents and click-and-release technologies.

Building on classic themes: Phosphine reactions and cycloadditions

Widely regarded as the first bioorthogonal reaction, the Staudinger ligation has experienced many modifications. One of the first advancements was a traceless variant where the phosphine reagent is not attached to the amide-bond-containing final product (Figure 2A).13,14 This is a desirable reaction for modifying molecules in living systems because the result is a naturally occurring linkage. Focusing on the final product linkage has not taken center stage in the development of bioorthogonal reactions but represents a subfield of bioorthogonal chemistry that is ripe for development. Reactions that yield natural linkages or known pharmacophores would have potential for in situ synthesis of (bio)therapeutics. Other modifications have featured phosphite or phosphonite reagents, introduced by Hackenberger and co-workers, in place of phosphines15,16 and the formation of a stable iminophosphorane intermediate that is resistant to hydrolysis.17

Figure 2. Expanding classic bioorthogonal chemical reactions.

Figure 2.

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A sampling of the bioorthogonal reactions in use today, including variations of the classic Staudinger reactions, such as a traceless variant (A) and the use of cyclopropenones (B), tetrazine ligations enabled by photocleavage of a protecting group or dihydrotetrazine oxidation promoted by a photocatalyst (C), and cyclooctyne retro-Cope eliminations with hydroxylamines (D). The hydroamination product in (E) can be selectively cleaved with diboron reagents.

Inspired by the ideal properties of the Staudinger ligation—namely, its high selectivity and the small size of azides—the Prescher group developed alternative cyclopropenone chemical reporters that react with phosphines via conjugate additions (Figure 2B).18 Variations of this reaction have enabled multiple applications, including reaction-promoted fluorophore synthesis and orthogonal bioorthogonal reactions.19 Although most of these applications are possible with other bioorthogonal reactions, the facile chemical adaptation of this new reaction showcases the opportunities when selectivity and size are key motivators during reaction development.

Bioorthogonal cycloadditions have also seen extensive optimization and derivation. Newer renditions improve the areas of reactivity, stability, size, hydrophilicity, and orthogonality. Many iterations of alternative cyclooctyne and tetrazine reactive partners have been explored and are included in recent reviews.8,20 Other variations of cycloaddition reactions, including those featuring alternative 1,3-dipoles, have been added to the bioorthogonal toolbox: (imino)sydnones in the strain-promoted (imino) sydnone-alkyne cycloaddition (SPSAC or SPICC),21,22 nitrones in the strain-promoted alkyne-nitrone cycloaddition (SPANC),23,24 nitrile oxides,25,26 nitrile imines,27 and diazo compounds.28 For IEDDA reactions, tetrazine substitutes, such as the more biologically stable triazines,29 have been explored, and cyclopentadienes have been adopted for a classic Diels-Alder bioorthogonal reaction.30 Notably, some of these reactions are orthogonal to each other, enabling multiple bioorthogonal chemistries to be performed at once. We expect the development of orthogonal and bioorthogonal chemistries to be increasingly important as the complexity of systems that are studied with these chemistries increases.

Computation has played an increasingly important role in the discovery and enhancement of bioorthogonal cycloaddition reactions. For example, Houk, Murphy, and co-workers used computational methods to design cyclopentadienes to react with existing biocompatible dienophiles.30 Experimental work confirmed the computed reactivities by showing reaction rates on par with those of SPAAC. More recently, Stuyver and Coley used machine learning to screen thousands of cycloaddition partners for new bioorthogonal reactions.31 Reactions were evaluated for “bioorthogonal potential,” or the likelihood of reacting with theoretical partners over biological functional groups. As computation workflows are optimized and expanded to include other types of potential bioorthogonal reagents, we are likely to see the design of entirely new classes of reactions rather than the optimization of existing reaction partners.

Light-activated bioorthogonal reactions

The growing use of light to activate bioorthogonal reactions adds a degree of spatiotemporal control to chemical biology studies.32 Early examples included “photoclick chemistry,” pioneered by Lin and colleagues, where the irradiation of a tetrazole led to the in situ formation of a nitrile-imine 1,3-dipole that rapidly reacted with alkenes.33 In a similar time frame, Boons, Popik, and co-workers reported a cyclopropenone-masked dibenzocyclooctyne, which upon irradiation with light released CO to reveal the alkyne for reaction with azide.34 More recently, the focus has switched to controlling the location and timing of tetrazine ligations with light. Devaraj and co-workers recently tackled the issue of tetrazine instability in biological settings by adding a photocage to a dihydrotetrazine (Figure 2C; R = photocage).35 After UV-light-activated deprotection, the unreactive dihydrotetrazine was oxidized to a reactive tetrazine, which underwent rapid IEDDA with a trans-cyclooctene probe. The control that this strategy provides, in addition to the stability of the photocaged tetrazine, is likely to find use in future studies, especially as cages responding to longer wavelengths of light—which are more biocompatible—are incorporated. On the theme of using more red-shifted light to control bioorthogonal chemistry, Jia, Li, Fox, and co-workers employed silicon-rhodamine dyes as photocatalysts that promoted the oxidation of dihydrotetrazines to tetrazines (Figure 2C; R = H).36 The photocatalyst is activated by far-red (660 nm) light. Besides providing control, this approach also benefits from low catalyst loadings and the observation that oxidation occurs much faster than singlet oxygen sensitization, which would otherwise damage cells. An elaborated version of this system with a more cell-permeable dihydrotetrazine led to the most reactive tetrazine used to date in living cells (IEDDA rate constant over 106 M−1s−1).37 This combination of control, speed, and biocompatibility promises future applications and further demonstrates the general utility that light brings to the bioorthogonal table.

Fluorogenic bioorthogonal reactions

Given that many applications of bioorthogonal chemistry revolve around imaging, it is beneficial to have methods of reducing background signal. One approach is to use fluorogenic bioorthogonal reactions, designed to produce a probe with signal only after the reaction has occurred. This was first demonstrated with the Staudinger ligation through fluorophore activation upon the formation of phosphine oxide.38 A few iterations of bioorthogonal reactions, including coumarins activated by SPAAC39,40 or SPSAC,41 have been co-opted to be fluorogenic. Prescher and co-workers have also designed a cyclopropenone where, after phosphine attack, the ketene-ylide intermediate undergoes an intramolecular reaction to generate a fluorescent coumarin dye.19 The tetrazine ligation has enjoyed the most success with fluorogenic probes given that tetrazine naturally quenches the fluorescence of red fluorophores.42 Recent work has demonstrated impressive turn-on capabilities for fluorophores emitting at far-red and near-infrared wavelengths.43,44 This red shifting has long been desired such that turn-on probes can be used for in vivo applications, and future fluorogenic reactions will continue the trek across the electromagnetic spectrum by moving to the near- and shortwave-infrared regions.

Click-and-release reactions

Although a key focus of developing bioorthogonal reactions is selectively forming bonds to label biomolecules, there is parallel interest in selectively cleaving bonds in biological contexts through the development of click-and-release, or bioorthogonal cleavage, reactions.45 Some click-and-release reactions arose through a redesign of existing reactions, such as the inclusion of a collapsible carbamate moiety in a trans-cyclooctene scaffold. Upon reaction with a tetrazine, an intermediate spontaneously collapses, releasing a molecule of CO2 and an attached payload (Figure 3A).46 This strategy has expanded over the years and is employed in the first click chemistry performed in human patients (vide infra). Another recent example of click-and-release chemistry utilized the SPICC reaction to release two fluorescent products at once.47 Kim and co-workers expanded their previously disclosed reaction between cyclooctynes and hydroxylamines to include an on-demand cleavage of the enamine N-oxide products (Figure 2D).48 On-demand, as opposed to spontaneous, cleavage adds a new layer of control to reversible chemical modifications. Kim’s work represents an adaptation of classic dynamic covalent chemistry to bioorthogonal chemistries, an area where there is significant potential for further expansion. Future advances will focus on improved yield and rate of cleavage and are likely to also feature the development of other click and on-demand release pairings.

Figure 3. Moving beyond covalent-bond formation.

Figure 3.

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Bringing chemical partners together in living systems is not limited to irreversible covalent bonds.

(A) Several click-and-release reactions where the formed intermediate is designed to spontaneously collapse and release two products have been developed.

(B) Non-covalent approaches using host-guest chemistry principles have seen increased use in labeling living cells.

Non-covalent chemistry

Non-covalent chemistry is growing in popularity as an alternative approach to covalency for forming unnatural associations in living systems (Figure 3B). This often involves the application of host-guest chemistry to cell labeling, which relies on the combined strength of multiple non-covalent interactions.49 Besides having the potential for reversibility, non-covalent chemistry benefits from not being reliant on second-order rate constants or highly activated reagents, two of the major challenges present in the development of bioorthogonal reactions. Instead, the focus is on achieving high binding affinities (Ka). Work from the Papot and Jiang groups recently showed that the host compounds cyclodextrin and amide naphthotubes, respectively, could be used to label living systems through the recognition of appropriate guests.50,51 Our group used known host cucurbit[7]uril to label modified cell-surface sialic acids and demonstrated significant labeling with nanomolar concentrations of host.52 Future work will involve utilizing small guests that can be metabolically incorporated and the design of new hosts specifically for bioorthogonal labeling.

BIOLOGY IN CONTEXT

Since its inception, bioorthogonal chemistry has been applied to answering many biological questions that were previously daunting or fully unanswerable with existing tools. Here, we highlight certain foundational applications and recent studies that showcase the power and future promise of such studies.

Characterizing glycans

As mentioned, the study of glycans is not amendable to traditional molecular biology techniques. To address this, Sletten and Bertozzi introduced a two-step approach, referred to as the chemical reporter strategy (Figure 4A),3 that incorporates an unnatural carbohydrate with a bioorthogonal functional group into glycans by using natural biosynthetic pathways. In a second step, a probe is attached. The need to attach the probe to the unnatural functional group gave rise to the field of bioorthogonal chemistry. Today, the chemical reporter strategy has been applied to many different metabolites and biomolecules; however, glycans continue to be a popular target for advances in bioorthogonal chemistry.

Figure 4. Applying bioorthogonal chemistry to the study and manipulation of living systems.

Figure 4.

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(A) The bioorthogonal chemical reporter strategy.

(B) Scheme of unnatural sugar modification and display. Specific examples of photolabeling and glycoRNA discovery are shown.

(C) Bioorthogonal control over enzyme activation.

(D) CRISPR-guided, SPAAC-enabled control over the formation of chromatin loops.

A recent application using glycan-associated chemical reporters was the mapping of the local interactions of cell-surface sialic acids (Figure 4B).53 In this work, an iridium photocatalyst was appended via a SPAAC reaction rather than attachment of a traditional probe (e.g., fluorophore or biotin). With the photocatalyst installed, the “microenvironment” of sialic-acid-capped glycoproteins in cells was characterized through iridium-catalyzed proximity labeling with diazirines. The proximity labeling revealed a relationship between sialylation and the regulation of the expression of solute carrier proteins in cancerous cervical cells. This approach is amenable to further profiling of the consequences of sialic acid overexpression in various cancers and could be extended to characterizing other cell-surface interactions. More broadly, this report represents the expanding functionality of groups that are attached via the bioorthogonal reaction, a trend that is likely to increase in the coming years.

Bioorthogonal chemistry and the chemical reporter strategy have also led to striking new discoveries regarding glycosylation in terms of localization, dynamics, and biomolecule modification. A marquee example is the recent finding that RNA can be glycosylated (Figure 4B).54 RNA samples from cells grown in the presence of azide-containing sialic acid precursors were found to have high amounts of azides reacting with dibenzocyclooctyne probes. This study revealed a new dimension of RNA structure and function, and further investigations into glycoRNAs are sure to probe the mechanisms of transport and localization as well as other undiscovered functions. As the analytical toolbox for detecting glycosylated biomolecules increases, we expect the metabolic incorporation of chemical reporters followed by bioorthogonal chemistry to unveil many other unique roles of carbohydrates.

Labeling lipids

Lipids are another class of biomolecules whose in-depth exploration has been enhanced by bioorthogonal chemistry. As for glycans, there are no direct genetic techniques for systematically manipulating lipids. Initial applications of bioorthogonal chemistry to lipids included the introduction of complete lipids containing a chemical reporter group for subsequent labeling with fluorophores via click or SPAAC chemistries.55 Shortly after came the pursuit of metabolic approaches; for example, alkyne-functionalized choline was employed as a precursor to phosphatidylcholine lipids,56 the most abundant mammalian lipids, and alkynols were introduced to label phosphatidic acid.57 Both of these studies employed click chemistry to image lipid synthesis. In the case of phosphatidic acid labeling, the scope of accepted chemical reporter groups has expanded to enable labeling with SPAAC58 and tetrazine ligations.59 In the latter work, a fluorogenic tetrazine allowed for real-time imaging and revealed new subcellular information regarding the synthesis of phosphatidic acid. The phosphatidic acid work comparing the utility of click chemistry with that of tetrazine ligation showcased the importance of rapid, fluorogenic bioorthogonal reactions. However, it was a significant challenge to obtain a system that accepted the trans-cyclooctene chemical reporter group required for the fast tetrazine ligation, demonstrating the ongoing need for fast bioorthogonal reactions with small chemical reporter groups.

Imaging biomolecules has been a primary application of bioorthogonal chemistry and the chemical reporter strategy. As more detailed subcellular information is desired, the limits of conventional optical microscopy are reached. One approach to overcoming these limitations is to use super-resolution microscopy. Another is to enlarge the cell through expansion microscopy. The Baskin group has utilized click chemistry and the incorporation of chemical reporters onto lipids to create a variation of expansion microscopy deemed lipid expansion microscopy (LExM).60 In LExM, metabolically labeled lipids are detected by a dual-purpose probe via click reaction. The probe functions as both a fluorescent reporter and a crosslinker with a hydrogel matrix that is expanded. The membranes of cells and organelles can be visualized in striking detail on a standard fluorescence microscope. This version of expansion microscopy completely removes the membrane-permeabilization step that limited previous lipid microscopy endeavors. The unique approach to expansion microscopy based on chemical reporter groups is set for extension to other metabolically labeled biomolecules.

Bioorthogonal chemistry and proteins

Protein chemical biology has benefited greatly from bioorthogonal chemistry both for tracking proteins in natural settings and for improving chemical modifications in proteomics workflows. The introduction of chemical-reporter-modified amino acids to living systems offers a powerful way to investigate protein synthesis. Bioorthogonal non-canonical amino acid tagging (BONCAT) emerged as such a tool.61 Here, cells utilize modified amino acids for protein synthesis, and after cell lysis, new proteins that used the unnatural building blocks can be detected through bioorthogonal reactions. Commonly, an azide- or alkyne-tagged amino acid is used and detected by CuAAC or SPAAC. Although this is no longer a new strategy, BONCAT’s usefulness continues in characterizing protein dynamics under changing conditions, such as infection by microorganisms.62 The technique can also be combined with other labeling strategies for the identification of protein exchange between host and microbe. Elsewhere, bioorthogonal reactions have offered solutions to challenges with older techniques, such as activity-based protein profiling (ABPP), a tool for identifying active enzymes in samples by using specially designed probes featuring an enzyme inhibitor conjugated to a detection tag.63 Disadvantaged in its original form by large tags that disrupted inhibitor function, ABPP was altered to introduce minimally disruptive chemical reporters that could be tagged in cell lysates or live cells and has since seen routine use in diverse proteomics studies.64

Additionally, although proteins have the advantage of being able to be modified with genetic techniques, bioorthogonal chemistry will continue to be an important force for studying these biopolymers. For example, the convergence of genetic techniques and bioorthogonal cleavage reactions has allowed for the selective un-caging of active-site residues in enzymes to allow for controlled enzyme turn-on (Figure 4C).65,66 As orthogonal, bioorthogonal cleavage reactions emerge, the control of multiple enzymes in a single cell could be on the horizon. Further, reversible bioorthogonal cleavage reactions could allow for temporal control.

Interfacing with CRISPR

Bioorthogonal chemistry is well equipped to augment other powerful biological techniques. The pairing of bioorthogonal chemistry and CRISPR led to a greater understanding of regulatory mechanisms at both the genomic and enzymatic levels. Qu and co-workers developed a system that utilizes CRISPR-Cas9/sgRNA technology to bring bioorthogonal reaction partners to specific genomic loci, and their reaction caused a chromatin loop to form (Figure 4D).67 Chromatin folding is a key epigenetic regulatory event, and the ability to control it with the formation of multiple loops at once, shown here for the first time, will improve studies on proper gene regulation. Baskin and co-workers also strengthened a CRISPR genome-wide screen by incorporating a bioorthogonal reaction for the phenotypic selection step.68 The group’s previously mentioned SPAAC-based approach to studying phosphatidic acid was enabled by the promiscuity of phospholipase (PLD), the enzyme that transforms phosphatidylcholine into the acid product. Employing the strategy in a CRISPR interference screen led to the identification of glycogen synthase 3 (GSK3) as a previously unknown regulator of PLD signaling. This application of bioorthogonally introduced tags in CRISPR screens holds potential for identifying regulatory genes in other pathways. Other work combining bioorthogonal chemistry and CRISPR could make use of small reporters on guide RNA for selectively delivering probes to specific genes. Overall, the combination of bioorthogonal chemistry and CRISPR will increase opportunities for discovery and control in biological settings.

BIOMEDICAL APPLICATIONS

Excitingly, we have recently seen the impact of bioorthogonal chemistry extend beyond fundamental science research. The same tools used for investigating biological systems are finding their way into the clinic for diagnosing and even treating diseases. Not only are the products of bioorthogonal chemistry part of clinical trials,69 but an ongoing trial also involves the tetrazine ligation occurring in humans! The current trials most likely represent just the beginning of new medical applications.

Diagnostic profiling

In immunotherapy research, the ability to visualize several different biomarkers quickly in the same sample from a sick patient allows for detailed profiling of disease states, and similarly, biomarker profiling at different time points can paint a picture of disease progress. Toward this end, bioorthogonal chemistry has enabled documentation of changes in the tumor microenvironment over the course of cancer treatment. The Weissleder group developed a platform for single-cell cyclic imaging of samples taken from patients.70 Antibodies for specific biomarkers were connected to different fluorophores via a trans-cyclooctene, allowed to bind, and imaged. A tetrazine reagent was then added to trigger a click-and-release reaction, resulting in a free fluorophore that was washed away. New antibody-fluorophore conjugates for other biomarkers were then introduced and imaged, and the cycle was repeated. These “bioorthogonal recycling reagents” were applied to patient samples and demonstrated rapid destaining, allowing for the quick elaboration of the phenotypic profile of tumors with minimal residual background signal from each cycle. This diagnostic application shows the relevancy of not only classical bioorthogonal reactions but also more contemporary advances, such as click-and-release reactions for clinical purposes. This work featured antibody targeting for biomarkers, but it will be interesting to see similar technology applied via alternative targeting approaches in addition to applications to other cell populations and diseases.

Therapeutic potential

Metabolic incorporation of chemical reporter groups provides an opportunity to selectively tag specific cell types, which can be further elaborated via bioorthogonal chemistry. An example of this is the incorporation of azide-containing d-amino acids, designed to react with dibenzocyclooctyne-displaying probiotics, into the gut microbiota (Figure 5A).71 The SPAAC reaction between gut colonizers and delivered bacteria enhanced retention and demonstrated relief from induced colitis in mice. In the future, one could imagine similar strategies for targeting and treating drug-resistant infections, tuberculosis, or diseases with increased metabolism, such as cancer.

Figure 5. Biomedical avenues of bioorthogonal chemical reactions.

Figure 5.

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(A) Bioorthogonal chemistry increases cell-cell interactions in cell-based therapies.

(B) Bioorthogonal reactions allow for the in situ formation of active drug molecules from delivered fragments.

(C) The model of targeted drug delivery and activation via tetrazine ligation currently in clinical trials.

Bioorthogonal chemistry has also enabled improved theranostic approaches, namely, pretargeting tumor cells with a reporter-labeled antibody that can then be modified with radiolabels through a reaction. Robillard and co-workers demonstrated the promise of this two-step approach by utilizing the IEDDA reaction, which removed the issues of immunogenicity and lengthy engineering requirements of previous pretargeting systems.72 Over the past decade, this approach has been greatly expanded upon, and we refer readers to recent reports and reviews.73,74

Cell therapy represents another avenue for introducing chemical reporter groups that can later be elaborated via bioorthogonal chemistry. This has indeed been applied to one of the most prominent types, chimeric antigen receptor T cell (CAR-T) therapy. For CAR-T therapy, primary patient-derived T cells are genetically modified to target cancer cells, often via viral transduction. A combination of metabolic incorporation of azides into T cells and subsequent SPAAC with a dibenzo-cyclooctyne-polyethyleneimine resulted in enhanced lentiviral transduction and doubled the yield of CAR-T cells.75 In other instances, the properties of the genetically engineered T cells have been improved through surface modification by bioorthogonal chemistry. Yuan and co-workers used SPAAC to append an engineered hyaluronidase (HAse) and an ɑ-PDL1 antibody to azide-decorated CAR-T cells.76 The extracellular-matrix-degrading activity of HAse improved the efficiency of CAR-T penetration into solid tumors, and the antibody was released in the low-pH environment of said tumors to reverse the suppression of the PD1-PDL1 pathway. Ultimately, the power of bioorthogonal chemistry is its ability to be performed in vivo, and thus the above examples do not represent the full potential of the convergence of bioorthogonal chemistry and cell therapy. Pan et al. have demonstrated the in vivo reaction of azide-modified CAR-T cells with cyclooctyne-modified xenograft tumors to increase the efficacy of blood cancer treatment.77 Although not directly therapeutically relevant because the tumor cells were also labeled ex vivo, this work demonstrates that bioorthogonal chemistry can be used as a targeting strategy for cell therapy if the appropriate chemical reporter can be introduced to the cancerous site potentially through the increased metabolism of cancerous cells or implantation of a chemical-reporter-modified hydrogel at the tumor site. Additional future applications could involve in vivo controlled activation or deactivation of CAR-T cells via bioorthogonal reactions.

A challenge in the delivery of some therapeutics is the tradeoff between the size of the drug and its permeability or solubility. The ability to form covalent bonds in situ by using bioorthogonal chemistry allows for the introduction of smaller components of a larger system that can form the active compound after delivery (Figure 5B). Such an idea was tested with proteolysis-targeting chimeras (PROTACs). PROTACs are heterobifunctional molecules featuring a targeting ligand for a protein of interest linked to an E3 ubiquitin ligase recruiter molecule. In a cell, the PROTAC brings a protein of interest and a ubiquitin ligase into close proximity such that the target is ubiquitinated and designated for degradation. Although PROTACs are a distinctly promising approach for controlling dysregulated proteins implicated in many diseases, they are large molecules with non-druglike properties, including low cell permeability and solubility. In a creative approach to solving this issue, Heightman and co-workers labeled the individual targeting ligand and recruiter with bioorthogonal handles (trans-cyclooctene and tetrazine, respectively) such that the whole PROTAC could be formed after the more soluble and permeable components were delivered separately to a cell.78 This in situ drug formation is applicable currently to therapeutics with flexibility in the linkage. Future in situ therapeutic synthesis would benefit from traceless bioorthogonal reactions or those that are designed to result in pharmacophores. Another aspect that would enhance this approach is to develop bioorthogonal reactions that are catalyzed by the intracellular environment (e.g., high glutathione levels) such that only intracellular and not extracellular drug synthesis would occur. The concept of local-environment-promoted click reactions has been demonstrated by the Qu group, who exploited elevated levels of Cu(I) in an Alzheimer’s disease model to catalyze a click reaction for in situ drug formation.79 Overall, as chemical advances to bioorthogonal chemistries are made (vida supra), the scope of in situ therapeutic synthesis will increase.

First use in humans

Finally, as a remarkable sign of the field’s explosion into relevancy on many scales, bioorthogonal chemistry is now being performed in humans (Figure 5C). The tetrazine-trans-cyclooctene reaction is the subject of an ongoing US clinical trial. A two-component cancer treatment system features a tetrazine-displaying biopolymer that is injected once at the tumor site and subsequent dosing of a trans-cyclooctene-caged prodrug form of doxorubicin, a potent chemotherapeutic.80 Functional doxorubicin is freed from the fast click-and-release reaction, which will occur only where the biopolymer is injected, garnering a high degree of control over where the drug is released. This system demonstrates reduced off-target toxicity and is expected to be a generalized cancer treatment independent of differences between cancer types or individual patients. The general approach of targeting specific locales in a patient with one bioorthogonal reaction partner to bring drugs to the disease site has the potential to be used for other disorders as well. One can also imagine using implanted functional biopolymers to target cell therapies to disease sites. Overall, this first instance of bioorthogonal chemistry performed in humans sets an important precedent for future clinical applications, which we will undoubtedly see soon.

Conclusion

There was a time when the thought of performing non-native organic reactions in living systems without disruption would have seemed outlandish and all but impossible. But chemists are no strangers to creativity, and it is fitting that the pursuit of nature’s attributes of selectivity and elegance should lead to chemistries that work in increasingly complex environments. The future of bioorthogonal chemistry is bursting with opportunity, and no doubt the field of chemical biology will continue to evolve and influence applications such as disease diagnosis and treatment.

THE BIGGER PICTURE.

Challenges and opportunities:

  • Bioorthogonal reactions with enhanced reaction rates have been achieved, albeit often at the cost of reagent size or stability. New or improved reactions will benefit from smaller, more stable reagents that still react quickly.

  • To date, these reactions have been extensively utilized for answering biological questions and making new, exciting, and sometimes unexpected discoveries, especially when applied to biomolecules that cannot be studied with other tools, such as genetic approaches. This unraveling of nature’s mysteries is likely to continue as new reactions and approaches are utilized further.

  • As bioorthogonal chemistry developed, it became clear that there was potential for therapeutic applications. Now, the first bioorthogonal reaction in humans is in clinical trials; it is anticipated that more reactions will be elevated to this level soon.

ACKNOWLEDGMENTS

We would like to thank Prairie Hammer and Anthony Spearman for providing valuable feedback during manuscript preparation. Bioorthogonal chemistry work within the group is supported by the NIH under awards DP2GM13268 and T32GM136614.

INCLUSION AND DIVERSITY

We support inclusive, diverse, and equitable conduct of research.

Footnotes

DECLARATION OF INTERESTS

The authors declare no competing interests.

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