Abstract
<jats:p>O-GalNAc glycans on glycoproteins are biologically important and structurally heterogeneous, varying in glycan composition and glycosylation sites. Clustered heterogeneous O-GalNAc glycans are key regulatory factors for immune regulation, cancer progression, and mucosal protection. However, functional studies of heterogeneous O-GalNAc glycopeptides remain limited due to the lack of effective synthetic methods for accessing such structurally diverse glycopeptides. To address this, we report a hybrid chemoenzymatic (HCE) strategy that enables the synthesis of heterogeneous O-GalNAc glycopeptides, providing a general strategy for the synthesis of these challenging molecules. In our HCE workflow, quantitative conversion rates were achieved for the enzymatic reactions even with the presence of bulky protecting groups, Fmoc and tBu. The resulting glyco-amino acids were readily converted in three steps or fewer into ready-to-use building blocks for solid phase peptide synthesis. Using this approach, we successfully synthesized six heterogeneous O-GalNAc glycopeptides derived from CCR1 N-termini, demonstrating that the HCE strategy overcomes major hurdles in accessing diverse O-GalNAc glycopeptides. Importantly, the synthesis of a glycopeptide with adjacent consecutive glycosylation sites confirms the feasibility of our method for constructing dense, complex glycosylation patterns. This work establishes a broadly applicable platform for accessing heterogeneous O-GalNAc glycopeptides and paves the way for functional studies.</jats:p>