Traceless 6-O-Lysyl Modification of N-Acetylglucosamine Enables Synthesis and Derivatization of Aggregation-Prone...

Chemical synthesis of N-glycosylated peptides and proteins is essential for understanding glycan-dependent biological functions, but it faces challenges associated with aggregation-prone sequences. Here, we report a lysine-masked GlcNAc (LMG) strategy that leverages a covalently attached lysyl ester to the 6-hydroxyl group of N-acetylglucosamine (GlcNAc) on asparagine or serine residues, resulting in significantly increased solubility of glycopeptides. This LMG unit is fully compatible with Fmoc-based solid-phase peptide synthesis and can be unmasked under mild neutral conditions to reveal the native GlcNAc moiety. The applicability of the LMG strategy is demonstrated in the efficient synthesis and purification of several difficult glycopeptides derived from biologically relevant proteins, such as TMEM106B and MST1R. Importantly, LMG-modified peptides serve as effective substrates in chemoenzymatic N-glycan elaboration and peptide ligation reactions, enabling the chemical synthesis of two homogeneous glycoforms of the PD-1 IgV domain bearing four N-glycosylations. These results establish the LMG strategy as an effective method for accessing structurally complex and aggregation-prone glycopeptides that are valuable for glycoscience investigations.