Facile Formation of β-thioGlcNAc Linkages to Thiol-Containing Sugars, Peptides, and Proteins using a Mutant GH20 Hexosaminidase

Angew Chem Int Ed Engl. 2019 Feb 4;58(6):1632-1637. doi: 10.1002/anie.201809928. Epub 2019 Jan 9.


Thioglycosides are hydrolase-resistant mimics of O-linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. The development of an efficient, enzyme-mediated synthesis of thioglycosides, including S-GlcNAcylated proteins, is reported, using a thioglycoligase derived from a GH20 hexosaminidase from Streptomyces plicatus in which the catalytic acid/base glutamate has been mutated to an alanine (SpHex E314A). This robust, easily-prepared, engineered enzyme uses 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 analogues of biologically important GlcNAcylated peptides and proteins.

Keywords: glycoprotein synthesis; glycosynthase; hexosaminidase; thioglycoligase; thioglycosides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / chemistry*
  • Acetylglucosamine / metabolism
  • Molecular Structure
  • Mutation
  • Peptides / chemistry*
  • Peptides / metabolism
  • Proteins / chemistry*
  • Proteins / metabolism
  • Streptomyces / enzymology
  • Sugars / chemistry*
  • Sugars / metabolism
  • Sulfhydryl Compounds / chemistry*
  • Sulfhydryl Compounds / metabolism
  • beta-N-Acetylhexosaminidases / chemistry*
  • beta-N-Acetylhexosaminidases / genetics
  • beta-N-Acetylhexosaminidases / metabolism


  • Peptides
  • Proteins
  • Sugars
  • Sulfhydryl Compounds
  • beta-N-Acetylhexosaminidases
  • Acetylglucosamine

Supplementary concepts

  • Streptomyces plicatus