A novel enzymatic method for synthesis of glycopeptides carrying natural eukaryotic N-glycans

A novel enzymatic approach was developed for facile production of glycopeptides carrying natural eukaryotic N-glycans. In this approach, peptides can be GlcNAcylated at one or two natural N-glycosylation sites via two-step enzymatic reactions catalyzed by an evolved N-glycosyltransferase (ApNGTQ469A) and a glucosamine N-acetyltransferase (GlmA), respectively. The resulting GlcNAc-peptides were further modified by an endo-beta-N-acetylglucosaminidase M mutant (EndoM(N175Q)) to generate glycopeptides. In three steps of enzymatic catalysis, glycopeptides carrying complex-type N-glycans can be efficiently synthesized.