β1,6-Selective Enzymatic N-Acetylglucosamination Catalyzed by the Family GH84 N-Acetyl-β-D-glucosaminidase from Bacteroides thetaiotaomicron and its Glycosyl Acceptor Specificity.

Abstract

The chemoenzymatic synthesis of oligosaccharides presents a highly attractive methodology with significant potential for diverse applications, particularly through using various glycosidases. In this study, the O-glycan core 6 disaccharide moiety, GlcNAcβ1-6GalNAc, was successfully synthesized via enzymatic glycosylation using an N-acetyl-β-D-glucosaminidase from Bacteroides thetaiotaomicron (BtOGA), a member of glycoside hydrolase family 84 (GH84), alongside an N-acetyl-D-glucosamine oxazoline derivative (GlcNAc-oxa) as the glycosyl donor. Furthermore, an investigation into glycosyl acceptor recognition in BtOGA-catalyzed enzymatic glycosylation indicated that the presence of an aromatic group at the anomeric position and an axial hydroxy group at the 4-position of the saccharide moiety is crucial for effective recognition of BtOGA as a glycosyl acceptor. The protecting-group-free chemoenzymatic synthesis of the core 6 disaccharide moiety was achieved by integrating the direct synthesis of GlcNAc-oxa thorough Shoda activation method using a water-soluble dehydration condensing agent in an aqueous medium, followed by BtOGA-catalyzed enzymatic glycosylation.