Oxidative Release of Natural Glycans (ORNG): Preparation of Truncated N‐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.