Oxidative Release of Natural Glycans (ORNG): Preparation of Truncated <i>N</i>‐glycans for Engineering Glycoproteins

N-glycosylation on asparagine is one of the most common post-translational modifications of proteins. The synthesis of homogeneous N-glycopeptides and glycoproteins is crucial for elucidating the concerted functions of glycans and peptides. Multiple endoglycosidase mutants have been developed to efficiently catalyze the coupling of oxazoline derivatives of N-glycans lacking the innermost N-acetylglucosamine (GlcNAc) (mono-GlcNAc N-glycan) with peptide chains bearing a GlcNAcylated asparagine. However, the bottleneck for N-glycopeptide and glycoprotein synthesis remains access to these complex truncated N-glycan substrates in sufficient diversity and quantity. Here, we report a novel chemical method using modified oxidative release of natural glycans (ORNG) to produce a diverse library of mono-GlcNAc N-glycans and their oxazoline derivatives, paving the way for the facile synthesis of N-glycopeptides and glycoproteins. Using these substrates, homogeneous N-glycosylation of monoclonal antibodies can be efficiently achieved.