Facile formation of β‐thioGlcNAc linkages to thiol‐containing sugars, peptides, and proteins using a mutant GH20 hexosaminidase

Thioglycosides are hydrolase‐resistant mimics of O‐linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. Traditional chemical syntheses of thioglycoside analogues of O‐GlcNAc‐modified peptides and proteins require lengthy, multi‐step approaches. We report the development of efficient, enzyme‐mediated syntheses of thioglycosides, including S‐GlcNAcylated proteins, using a thioglycoligase derived from a GH20 hexosaminidase from Streptomyces plicatus in which the catalytic acid/base glutamate has been replaced with an alanine (SpHex E314A). This robust, easily‐prepared, engineered enzyme uses readily available GlcNAc and GalNAc donors and couples them to a remarkably diverse set of thiol acceptors. Thioglycoligation using 3‐, 4‐, and 6‐thiosugar acceptors from a variety of sugar families produces S‐linked disaccharides in nearly quantitative yields. The set of possible thiol acceptors also includes cysteine‐containing peptides and proteins, rendering this mutant enzyme a promising catalyst for the production of thio analogs of biologically important GlcNAcylated peptides and proteins.

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