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  • Primer
  • Published:

Bioorthogonal chemistry

Abstract

Bioorthogonal chemistry represents a class of high-yielding chemical reactions that proceed rapidly and selectively in biological environments without side reactions towards endogenous functional groups. Rooted in the principles of physical organic chemistry, bioorthogonal reactions are intrinsically selective transformations not commonly found in biology. Key reactions include native chemical ligation and the Staudinger ligation, copper-catalysed azide–alkyne cycloaddition, strain-promoted [3 + 2] reactions, tetrazine ligation, metal-catalysed coupling reactions, oxime and hydrazone ligations as well as photoinducible bioorthogonal reactions. Bioorthogonal chemistry has significant overlap with the broader field of ‘click chemistry’ — high-yielding reactions that are wide in scope and simple to perform, as recently exemplified by sulfuryl fluoride exchange chemistry. The underlying mechanisms of these transformations and their optimal conditions are described in this Primer, followed by discussion of how bioorthogonal chemistry has become essential to the fields of biomedical imaging, medicinal chemistry, protein synthesis, polymer science, materials science and surface science. The applications of bioorthogonal chemistry are diverse and include genetic code expansion and metabolic engineering, drug target identification, antibody–drug conjugation and drug delivery. This Primer describes standards for reproducibility and data deposition, outlines how current limitations are driving new research directions and discusses new opportunities for applying bioorthogonal chemistry to emerging problems in biology and biomedicine.

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Fig. 1: Different classes of bioorthogonal reactions.
Fig. 2: The native chemical ligation and oxime and hydrazine ligations.
Fig. 3: The Staudinger ligation types and the copper-catalysed azide–alkyne reaction.
Fig. 4: Cycloaddition-based bioorthogonal chemical reaction types.
Fig. 5: Light-activated click chemistry and metal-catalysed coupling reactions.
Fig. 6: Applications for labelling different molecule types in cells.
Fig. 7: Examples of biorthogonal chemistry applications in vitro and in vivo.

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Elizabeth L. Bell, William Finnigan, … Sabine L. Flitsch

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Acknowledgements

J.A.P. acknowledges support over the years from the Alfred P. Sloan Foundation, the Camille & Henry Dreyfus Foundation and the National Institutes of Health (NIH) (R01 GM126226). J.M.F. acknowledges support from the NIH (R01 GM132460), National Science Foundation (NSF) (DMR 1809612 and 2011824) and Pfizer. Q.L. acknowledges support from the NIH (R35 GM130307) and NSF (CHE-1904558). K.L acknowledges support from the German Science Foundation DFG through programmes SFB1035 and SPP1623.

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Authors

Contributions

Introduction (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Experimentation (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Results (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Applications (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Reproducibility and data deposition (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Limitations and optimizations (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Outlook (J.M.F., S.L.S., D.A.B., R.H., W.L., S.S.N., M.X., C.W.a.E., M.G.F., K.L., Q.L., J.P.P., J.A.P. and M.S.R.); Overview of the Primer (J.M.F.).

Corresponding author

Correspondence to Joseph M. Fox.

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Competing interests

W.L. and C.W.a.E. are employees of Pfizer Inc. M.S.R. is an employee and shareholder of Tagworks Pharmaceuticals. S.L.S., D.A.B., R.H., S.S.N., M.X., M.G.F., K.L., Q.L., J.P.P., J.A.P. and J.M.F. declare no competing interests.

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Nature Reviews Methods Primers thanks J. Clavadetscher, D. Johnson, M. Vrábel, H. Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Glossary

Chemoselective

A chemical reaction that is selective for a certain functional group even in the presence of differing functional groups. Reaction partners in bioorthogonal chemistry are chemoselective for each other, even in biological settings.

Click chemistry

A concept, coined by K. B. Sharpless and colleagues, describing bond-forming reactions that are thermodynamically driven, highly selective and reliable, and proceed in water without toxic by-products. Click reactions are often, but not strictly, bioorthogonal.

Nucleophilic catalysts

Electron-rich additives that increase the reaction rate for certain polar bioorthogonal chemistries. In general, the most effective catalysts target the rate-limiting step for a given transformation. Catalysts must also adhere to the same strict requirements as bioorthogonal reagents (non-toxic, chemoselective and so on) if used in a biological context.

Post-translational modifications

Chemical transformations that occur on reactive side chains (such as lysine, serine and cysteine) of proteins. The identity of the modification can drastically affect the function and activity of the target protein. The modifications can be installed enzymatically or can occur spontaneously in solution.

Solid-phase peptide synthesis

Amino acids are iteratively coupled from the carboxy terminus to the amino terminus on a solid support. Protecting group strategies ensure that only one amide bond is formed at a time, without oligomerization or cross-reactivity with reactive side chains. After cleavage from the solid support, peptides are typically purified through high-performance liquid chromatography.

Schiff base

A subclass of imine compounds characterized by a carbon–nitrogen double bond, with a general formula of R1R2C=NR3, where R3 is not a hydrogen atom. They often arise from the condensation reaction between an amine and a carbonyl, and are classified as secondary ketimines or aldimines.

Reactive oxygen species

Highly reactive forms of oxygen involved in diverse cellular signalling processes, and tightly regulated in cells. For bioorthogonal chemistry, reactive oxygen species arise from the oxidation of copper(I) to copper(II) in water, which generates damaging superoxide or hydroxyl radicals. An accumulation of reactive oxygen species damages nucleic acids, proteins and lipids, and is cytotoxic.

Dienophile

An alkene or alkyne that reacts with a conjugated diene in [4 + 2] cycloadditions. Diels–Alder cycloadditions are enabled by electron-poor dienophiles and electron-rich dienes. Conversely, inverse electron-demand Diels–Alder reactions occur between electron-rich dienophiles and electron-poor dienes.

Photoclick chemistry

Click chemistry in which reactions that are initiated using light as an external stimuli. Photoclick reactions can use light sources ranging from short-wavelength to near-IR light and allow for spatial and temporal control of reactions.

HOMO

(Highest occupied molecular orbital). A molecule’s highest energy molecular orbital containing an electron pair.

LUMO

(Lowest unoccupied molecular orbital). A molecule’s lowest energy molecular orbital not containing an electron. The energies of HOMO and LUMO are related to the reactivity of the molecule and the energy difference between the HOMO and LUMO is termed the HOMO–LUMO gap.

Two-photon upconversion

A molecule is excited from the ground state (S0) to the second excited singlet state (S2) by simultaneous absorption of two photons, via a virtual state. A photon with frequency greater than those of the absorbed photons is emitted upon relaxation from the excited state, that is, two-photon upconversion.

Homogeneous catalysis

The catalyst and reaction mixture are in the same phase.

Heterogeneous catalysis

The catalyst and reaction mixture are in a different phase.

Lewis acid

A chemical species that can accept a pair of non-bonding electrons.

Differential scanning calorimetry

(DSC). A technique that measures heat flow rates to determine phase transitions and quantitative heats of decomposition of a compound of interest. DSC requires only milligram quantities of a sample and provides a rapid measurement of the thermal properties of a compound.

Yoshida correlation

The impact sensitivity and explosive propagation properties of compounds can be derived from differential scanning calorimetry data.

Zymogens

Inactive forms of an enzyme. The enzyme takes its active form following a natural biochemical process such as cleavage, hydrolysis or post-translational modification.

Targeted protein degradation

A technique used for targeting specific proteins for degradation within a cell. Commonly, hetero-bifunctional small-molecule compounds are used for targeting the protein of interest to an E3 ubiquitin ligase protein. This facilitates the polyubiquination and subsequent degradation of the targeted protein.

Self-immolative linker

A class of linker that, when exposed to a certain trigger, is designed to break the payload connecting bonds via an intramolecular process.

Prodrug

A pharmacologically inactive precursor compound that is converted into an active drug through in vivo chemical modification achieved via metabolic/enzymatic processes. Prodrugs are employed to improve pharmacokinetic properties (absorption, distribution, metabolism and elimination) and pharmacodynamics properties (selectivity, reduction of adverse effects) of the active drug molecule.

Sequence-specific polymers

Macromolecules that are monodisperse with defined monomer sequences or block sequences. This requires controlled, sequential addition of subunits using highly efficient bond-forming chemical reactions. Naturally occurring examples of sequence-specific macromolecules include DNA, RNA and protein.

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Scinto, S.L., Bilodeau, D.A., Hincapie, R. et al. Bioorthogonal chemistry. Nat Rev Methods Primers 1, 30 (2021). https://doi.org/10.1038/s43586-021-00028-z

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