A Protecting-Group-Free S‑Glycosylation Strategy for Complex S‑Glycopeptide Synthesis.

Protecting-group (PG)-free S-glycosylation offers an attractive strategy for glycopeptide synthesis, yet practical methods for complex peptides remain limited. Here, we report a non-photochemical S-glycosylation strategy that directly couples unprotected glycosyl donors with unprotected cysteine-containing peptides, enabling efficient access to β-S-glycopeptides. The method shows broad compatibility with glycosyl donors and peptide substrates and enables direct glycosylation of a structurally complex 37-mer precursor bearing one free cysteine, four protected cysteines, and oxidation-sensitive functionalities, thereby facilitating the efficient synthesis of sublancin and diverse glycosylated analogues. In contrast to photochemical radical-based S-glycosylation, which produces α-S-glycopeptides and proved unsuitable for the full-length substrate, the present strategy allows efficient late-stage glycosylation of structurally complex peptides. Combined with a fragment-based native chemical ligation approach for α-S-glycoside synthesis, this platform provides stereochemically defined sublancin analogues and reveals that the configuration of the S-glycosidic linkage strongly influences antibacterial activity. These results establish a practical PG-free platform for complex S-glycopeptide synthesis and glycocin structure-activity studies.