A bioorthogonal chemical reaction. The reaction of compounds A and B bearing bioorthogonal functional groups proceeds in the presence of all the functionality found within living systems, some examples of which are indicated.

Classic bioconjugation reactions for the modification of Cys and Lys residues. Cys residues can be modified through disulfide exchange, alkylation with iodoacetamide reagents, and Michael addition with maleimides (entries 1–3, respectively). Lys residues can be modified through amide, sulfonamide, urea, and thiourea formation with N-hydroxysuccinimide-activated esters, sulfonyl chlorides, isocyanates, and isothiocyanates (entries 4–7, respectively).

Modern bioconjugation reactions for protein modification. A) Selective modification of Lys, Cys, Tyr, Trp, and the N terminus. B) Modern methods to modify Lys, Cys, Tyr, and Trp. Lys is modified through a reductive amination using an Ir hydride as the reductant (entry 1; bipy = bipyridyl, Cp* = C₅Me₅). Cys is modified through a two-step labeling procedure which involves formation of dehydroalanine and subsequent Michael addition of a thiol (entry 2), or the photochemically promoted thiol-ene reaction (entry 3; AIBN = 2,2′-azobisisobutyronitrile). Tyr is modified by a nickel(II)-mediated radical coupling with magnesium monoperoxyphthalate (MMPP) as a stoichiometric oxidant (entry 4), a three-component Mannich reaction with aldehyde and aniline reagents (entry 5), or a palladium-catalyzed π-allylation (entry 6). Trp modification is performed using a rhodium carbenoid (entry 7). C) Methods for modification of the N terminus. Modification of the N terminus is achieved through transamination with aldehydes, oxidation with periodate, a Pictet–Spengler reaction between an N-terminal tryptophan and an aldehyde, formation of a bicyclic lactam with acyl-aldehyde reagents, or native chemical ligation with thioester reagents (entries 1–5, respectively).

Native chemical ligation and intein-based technologies. A) Native chemical ligation of two peptides: Peptide 1 contains a C-terminal thioester that undergoes thioesterification with the N-terminal cysteine of peptide 2. An S-N acyl transfer results in a native peptide bond. B) Expressed protein ligation: A protein is recombinantly expressed and fused to a mutated intein and a chitin-binding domain to facilitate purification. The intein is mutated so that it forms a thioester but does not undergo S-N acyl exchange, thereby allowing for the recombinant protein to be selectively cleaved from the immobilized chitin by a species containing an N-terminal cysteine. C) Protein-trans splicing: A protein is recombinantly expressed fused to a portion of a split intein (Int_C). The complementary portion of the intein (Int_N) is connected to an unnatural chemical species. When the two inteins associate noncovalently, splicing occurs to give a modified protein.

A),B) Fluorogenic biarsenical reagents for site-specific labeling of recombinant proteins with tetracysteine motifs. A) Fluorescein arsenical hairpin binder (FlAsH). B) Resorufin arsenical hairpin binder (ReAsH). C) Fluorogenic bisboronic acid rhodamine reagent (RhoBo) for labeling proteins containing tetraserine motifs.

Site-specific protein modification through the detection of artificial peptide tags by metal-mediated chelation with chemical reagents. Polyhistidine peptides chelate Ni^II and are detected with Ni-NTA reagents (entry 1) or they chelate Zn^II and are imaged with the fluorogenic dye HisZiFit (entry 2). Tetraaspartate peptides are detected with multinuclear zinc complexes (entry 3). Peptides that have been engineered to bind Tb^III can be visualized following chelation through the luminescent properties of Tb^III (entry 4).

Protein modification through the enzymatic elaboration of peptide tags. Biotin ligase (BirA) catalyzes the attachment of biotin (not shown) or ketobiotin to proteins containing the appropriate peptide substrate (entry 1). Transglutaminase (TGase) catalyzes the attachment of primary amine-containing probes to proteins tagged with polyglutamine sequences (entry 2). Lipoic acid ligase (LplA) or a mutated LplA catalyzes the attachment of an alkyl (entry 3) or aryl azido-lipoic acid derivative (entry 4) to proteins containing the appropriate peptide substrate. The formylglycine-generating enzyme (FGE) catalyzes the transformation of a Cys to a formylglycine in proteins that contain the motif CXPXR (entry 5). Sortase A catalyzes the attachment of a polyglycine-containing probe to proteins that contain an LPXTG motif near the C terminus (entry 6). Phosphopantetheinyl transferases (AcpS or Sfp) catalyze the attachment of a coenzyme A (CoA) probe to proteins containing the appropriate protein/peptide substrate (entry 7). A protein is recombinantly expressed as a fusion with the human repair protein O⁶-alkylguanine-DNA alkyltransferase (hAGT). The enzyme hAGT is alkylated through removal of the benzyl group and probe from O⁶-benzylguanosine derivatives (entry 8). A protein is recombinantly expressed as a fusion with an engineered haloalkane dehalogenase (DhaA). The mutated DhaA enzyme is covalently modified by alkyl chloride probes (entry 9).

Bioorthogonal reactions of aldehydes/ketones. Aldehydes and ketones can condense with aminooxy compounds (top) or hydrazide compounds (bottom) to form stable oxime or hydrazone linkages, respectively.

The Staudinger ligation of azides and triarylphosphines. Triarylphosphine 1 attacks the azido biomolecule, thereby releasing nitrogen from a four-coordinate transition state to yield aza-ylide 2, which undergoes intramolecular attack on the ester, extruding methanol, and resulting in bicycle 3. Upon hydrolysis, oxidation of the phosphine and formation of an amide bond occur to give ligation product 4.

Fluorogenic phosphines for the Staudinger ligation. A) Coumarin-based fluorogenic phosphine that becomes fluorescent upon phosphine oxidation. B) FRET-based fluorogenic phosphine that becomes fluorescent upon release of the quencher.

Peptide coupling by the traceless Staudinger ligation. The phosphine appended to peptide 1 through a C-terminal thioester (9) attacks the azide attached to peptide 2 in a manner analogous to the original Staudinger ligation to yield iminophosphorane 10, which rearranges to 11. Hydrolysis of 11 results in coupling product 12 plus a phosphine oxide by-product.

Bioorthogonal [3+2] cycloadditions of azides and alkynes to form triazoles. Terminal alkynes are activated by Cu^I to undergo cycloaddition with azides under physiological conditions (top). Cyclooctynes react with azides through a strain-promoted [3+2] cycloaddition (bottom).

Cyclooctyne reagents for copper-free click reactions.

Bioorthogonal reactions with alkenes. A) Reaction of oxanorbornadienes and azides to yield triazoles. B) Inverse-demand Diels–Alder reaction of dipyridyl tetrazines and trans-cyclooctenes. C) Inverse-demand Diels–Alder reaction of monoaryl tetrazines and norbornenes. D) Photoinduced 1,3-dipolar cycloaddition of tetrazoles and alkenes. E) Ruthenium-catalyzed cross-metathesis of two alkenes in water.

Incorporation of unnatural amino acids into proteins. A) Global incorporation of an unnatural amino acid using auxotrophic cell lines and promiscuous aminoacyl tRNA synthetases (aaRSs). B) Site-specific incorporation of an unnatural amino acid in vitro by using a transcription/translation system, a chemically synthesized tRNA loaded with the unnatural amino acid, and the gene for the protein of interest mutated at the site of modification. C) Site-specific incorporation of an unnatural amino acid in vivo using mutant tRNA and aaRS as well as the gene for the protein of interest mutated at the site of modification.

Selected amino acids that have been site-specifically incorporated into proteins in vivo.

Activity-based protein profiling using a bioorthogonal chemical reporter. A warhead group is functionalized with a chemical reporter and introduced into cells. Following cell lysis, a chemical labeling reagent facilitates identification or visualization of the modified enzymes.

Metabolic oligosaccharide engineering. Unnatural carbohydrate metabolites are taken up by cells and incorporated into cell-surface glycans and/or cytosolic O-GlcNAcylated proteins.

Unnatural carbohydrate metabolites for use in metabolic oligosaccharide engineering. A) N-Acetylmannosamine (ManNAc) metabolites. B) Sialic acid (Sia) metabolites. C) N-Acetylgalactosamine (GalNAc) metabolites. D) N-Acetylglucosamine (GlcNAc) metabolite. E) Fucose (Fuc) metabolites.

Unnatural metabolites containing bioorthogonal functional groups. A) Fatty acids and isoprenol metabolites for metabolic labeling of lipids. B) Nucleic acid metabolites for the incorporation of alkynes and azides into RNA and DNA.