Combined Chemical and Enzymatic Synthesis of a C-Glycopeptide and Its Inhibitory Activity toward Glycoamidases

A novel chemoenzymatic approach to synthesizing high-mannose-type N -glycopeptide and its C -linked glycopeptide analog is described. The synthesis consists of two steps: a chemical synthesis of GlcNAc-containing peptides and an enzymatic glycosyl transfer of Man 9 GlcNAc to the terminal GlcNAc in the peptides in an aqueous medium containing organic solvents. The essential enzyme used is an endo -β- N -acetyl-glucosaminidase from Arthrobacter protophormiae (Endo-A). This approach should be generally applicable to the synthesis of both natural and designed high-mannose-type glycopeptides. It has been found that, while the natural high-mannose-type N -glycopeptide 2 can be rapidly hydrolyzed by glycoamidases [commonly called N -glycanase or, systematically, peptide- N 4 -( N -acetyl-β- d -glucosaminylasparagine amidase], the synthetic C -glycopeptide 1 with an insertion of a methylene group at the crucial asparagine−GlcNAc linkage is resistant to the enzyme-catalyzed hydrolysis and shows apparent inhibitory activity toward glycoamidases of plant, bacterial, and animal origin, with the K i values ranging from 1 to 160 μM for different enzymes. The C -glycopeptide 1 is the first, broad spectrum inhibitor for glycoamidases, which is expected to be a useful tool in the study of the mechanism and biological functions of the enzymes.