Fast Cysteine Bioconjugation Chemistry

Abstract

Cysteine bioconjugation serves as a powerful tool in biological research and has been widely used for chemical modification of proteins, constructing antibody-drug conjugates, and enabling cell imaging studies. Cysteine conjugation reactions with fast kinetics and exquisite selectivity have been under heavy pursuit as they would allow clean protein modification with just stoichiometric amounts of reagents, which minimizes side reactions, simplifies purification and broadens functional group tolerance. In this concept, we summarize the recent advances in fast cysteine bioconjugation, and discuss the mechanism and chemical principles that underlie the high efficiencies of the newly developed cysteine reactive reagents.

oxSTEF Reagents Are Tunable and Versatile Electrophiles for Selective Disulfide-Rebridging of Native Proteins

“…Notably, in the absence of reductants, no modifications were observed following 24 h of incubation (Figure S19), which, in accordance with the previous experiments, indicates that the oxSTEF reagents have no or negligible reactivity towards other residues, in particular lysines. Collectively, this data demonstrates that oxSTEF reagents can perform disulfide rebridging but, interestingly, might also be used for site-selective N -Cys-modification of peptides. ,, …”

“…Collectively, this data demonstrates that oxSTEF reagents can perform disulfide rebridging but, interestingly, might also be used for site-selective N-Cysmodification of peptides. 11,12,44 oxSTEF Reagents Selectively Rebridge Disulfides in Native Proteins. Next, we tested the reagents for disulfide rebridging in native proteins using human growth hormone (hGH) as a model system.…”

oxSTEF reagents are tunable and versatile electrophiles for selective disulfide-rebridging of native proteins

“…Collectively, this data demonstrates that oxSTEF reagents can perform disulfide rebridging, but, interestingly, might also be used for site-selective N-Cys-modification of peptides. 11,43,44 oxSTEF reagents selectively rebridge disulfides in native proteins Next, we tested the reagents for disulfide rebridging in native proteins using human growth hormone (hGH) as a model system. This protein has two naturally occurring disulfide bonds.…”

Tunable Heteroaromatic Nitriles for Selective Bioorthogonal Click Reaction with Cysteine

“…Several electrophilic groups were developed to selectively target proteins with N-terminal Cys, − e.g., thioesters, O -salicylaldehyde esters, activated aldehydes, activated nitriles, 2-((alkylthio)­(aryl)­methylene)­malononitriles, N -hydroxysuccinimide-activated acrylamides, 2-benzylacrylaldehydes, 2-formylphenylboronic acids, − and monosubstituted cyclopropenones . Here, we focused on activated heteroaromatic nitriles, which were found to have a wide range of applicability in recent years. ,− Inspired by the final step in the biosynthesis of d -luciferin, Rao and co-workers utilized a click reaction between 2-cyanobenzothiazole and N-terminal Cys for protein labeling . In the first step, addition of the thiol to the nitrile leads to reversible formation of the thioimidate intermediate (Figure A), which is followed by an intramolecular condensation reaction with amine to form the 2-aminothiazolidine intermediate.…”