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.

Fig. 1 |. Different classes of bioorthogonal reactions.

The broad range of bioorthogonal reactions with their associated reactants, key reagents, products and key feature(s) are highlighted here.

Fig. 2 |. The native chemical ligation and oxime and hydrazine ligations.

a | Native chemical ligation is enabled by a catalysed reaction between a thioester and an amino-terminal cysteine residue to afford a native amide bond through a thioester intermediate that undergoes an S,N-acyl shift. b | Using aniline-based catalysts, the oxime and hydrazone ligations occur between a carbonyl group with a hydroxylamine or a hydrazine, respectively. k₂, second-order rate constant.

Fig. 3 |. The Staudinger ligation types and the copper-catalysed azide–alkyne reaction.

a | Both the original and traceless Staudinger ligations enable native amide bond formation between an azide and a carbonyl group using triaryl phosphines to afford the product and a phosphine oxide as a by-product. b | The model ‘click reaction’ between an azide and an alkyne enabled by use of a copper-based catalysis route to afford a triazole product. Ligand examples are provided. k₂, second-order rate constant.

Fig. 4 |. Cycloaddition-based bioorthogonal chemical reaction types.

a | Strained alkynes enable copper-free catalysis of cycloaddition to dipoles such as azides. b | The fastest biorthogonal reaction involves the inverse-electron demand [4 + 2] addition between a tetrazine and a dienophile. BARAC, biarylazacyclooctynone; BCN, bicyclo[2.1.0]nonyne; DIBO, dibenzocyclooctyne; DIFO, difluorinated cyclooctyne; k₂, second-order rate constant; OCT, cyclooctyne; TMTH, 3,3,6,6-tetramethylthiaheptyne.

Fig. 5 |. Light-activated click chemistry and metal-catalysed coupling reactions.

a | Two examples of photoclick chemistry: a photoinduced tetrazole-alkene 1,3-dipolar cycloaddition reaction to generate a pyrazoline adduct (top) and a photo-triggered alkyne-azide cycloaddition reaction (bottom). The cycloalkyne is masked in the dibenzocyclopropenone form. b | Two examples of palladium-catalysed reactions: Suzuki–Miyaura cross-coupling and copper-free Sonogashira cross-coupling (top left and right, respectively), and ruthenium-mediated olefin cross-metathesis (bottom) involving the use of allyl chalcogen-based privileged substrates. BCN, bicyclo[2.1.0]nonyne.

Fig. 6 |. Applications for labelling different molecule types in cells.

a | Model for metabolic engineering for cell labelling and imaging. b | Fluorescence microscopy of CHO cells incubated in the presence (left) or absence (right) of peracetylated N-azidoacetylmannosamine (Ac₄ManNAz) and labelled with a fluorophore by the Staudinger ligation. c | Model for genetic code expansion as a strategy for cell labelling and imaging. d | Fluorescence and direct stochastic optical reconstruction microscopy (dSTORM) super-resolution images of COS-7 cells where microtubule-associated protein was encoded with an unnatural trans-cyclooctene (TCO) amino acid and tetrazine ligation was used to attach a microscopy dye. e | Structured illumination microscopy (SIM) images of Escherichia coli, where N-azidoacetyl-muramic acid (NAM) was metabolically incorporated into the bacterial peptidoglycan and fluorophore-labelled by copper(I)-catalysed azide–alkyne cycloaddition (CuAAC). f | Electron microscopy images of HeLa cells, where azido-choline was metabolically incorporated, and cyclooctyne/azide click chemistry was used to conjugate electron microscopy imaging agents. The arrows indicate sites of endoplasmic reticulum–mitochondria contacts. aaRS, aminoacyl-tRNA synthetase; WF, widefield image. Images in panel b adapted with permission from REF., ACS. Images in panel d reprinted from REF., CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). Images in panel e reprinted from REF., CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). Images in panel f reprinted from REF., Springer Nature Limited.

Fig. 7 |. Examples of biorthogonal chemistry applications in vitro and in vivo.

a | Enrichment strategies for proteome labelling enabled by the copper-catalysed azide–alkyne reaction. b | Examples of bioorthogonal cage molecules. c | The tetrazine ligation as a strategy for pre-targeted radiochemical imaging of cancer. d | Uncaging small-molecule cargo has been applied at tumour sites in animal models. LC-MS/MS, liquid chromatography with tandem mass spectrometry; SPECT, single-photon emission computed tomography; WB, western blot. Middle image in panel c originally published in REF., JNM. Rossin, R., Läppchen, T., Van Den Bosch, S. M., Laforest, R. & Robillard, M. S. Diels–Alder reaction for tumor pretargeting: in vivo chemistry can boost tumor radiation dose compared with directly labeled antibody. J. Nucl. Med. 54, 1989–1995 (2013). © SNMMI. Bottom image in panel c adapted with permission from REF., ACS. Panel d adapted from REF., CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/).