Genetic Encoding and Enzymatic Deprotection of a Latent Thiol Side Chain to Enable New Protein Bioconjugation Applications

Abstract The thiol group of the cysteine side chain is arguably most versatile chemical handle in proteins. To expand scope established and commercially available bioconjugation reagents, we genetically encoded a second such functional moiety form latent that can be unmasked under mild physiological conditions. Phenylacetamidomethyl (Phacm) protected homocysteine (HcP) was incorporated its on purified proteins using penicillin G acylase (PGA). enzymatic deprotection depends steric accessibility, but occur efficiently within minutes exposed positions flexible sequences. freshly liberated does not require treatment with reducing agents. We demonstrate potential this approach for protein modification conceptually new schemes regioselective dual labeling, presence preserved disulfide bond formation novel intramolecular thioether crosslink.