Protein S-Glyco-Modification through an Elimination-Addition Mechanism

Per-O-acetylated unnatural monosaccharides containing a bioorthogonal group have been widely used for metabolic glycan labeling (MGL) in live cells for two decades, but it is only recently that we discovered the existence of an artificial S-glycosylation between protein cysteines and per-O-acetylated sugars. 'While efforts are being made to avoid this nonspecific reaction in MGL, the reaction mechanism remains unknown. Here, we present a detailed mechanistic investigation, which unveils the S-glycosylation being an atypical glycosylation termed S-glyco-modification. In alkaline protein microenvironments, per-O-acetylated monosaccharides undergo base-promoted beta-elimination to form thiol-reactive alpha,beta-unsaturated aldehydes, which then react with cysteine residues via Michael addition. This S-glyco-modification produces 3-thiolated sugars in hemiacetal form, rather than typical glycosides. The elimination-addition mechanism guides us to develop 1,6-di-O-propionyl-N-azidoacetylgalactosamine (1,6-Pr(2)GalNAz) as an improved unnatural monosaccharide for MGL.