Generalizing a Ligation Site at the <i>N</i>-Glycosylation Sequon for Chemical Synthesis of <i>N</i>-Linked Glycopeptides and Glycoproteins

Chemical synthesis can generate homogeneous glycoproteins with well-defined and modifiable glycan structures at designated sites. The precision and flexibility of the chemical synthetic approach provide a solution to the heterogeneity problem of glycopeptides/glycoproteins obtained through biological approaches. In this study, we reported that the conserved <i>N</i>-glycosylation sequon (Asn-Xaa-Ser/Thr) of glycoproteins can serve as a general site for performing Ser/Thr ligation to achieve <i>N</i>-linked glycoprotein synthesis. We developed an N + 2 strategy to prepare the corresponding glycopeptide salicylaldehyde esters for Ser/Thr ligation and demonstrated that Ser/Thr ligation at the sequon was not affected by the steric hindrance brought about by the large-sized glycan structures. The effectiveness of this strategy was showcased by the total synthesis of the glycosylated receptor-binding domain (RBD) of the SARS-CoV-2 spike protein.