The Future of Bioorthogonal Chemistry

Abstract

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.

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).

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).

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.

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.