Chemoenzymatic Assembly of Branch-Selective Sulfated N- and O-Glycans to Decode Glycan-Binding Protein Recognition

N- and O-glycosylations are essential post-translational modifications involved in numerous physiological and pathological processes. Sulfated N- and O-glycans, commonly found on biologically important glycoproteins, serve as critical mediators of molecular recognition. However, their precise structure-function relationships remain poorly understood, primarily due to the limited accessibility of structurally well-defined glycans bearing branch-specific sulfation patterns. Herein, we report a versatile chemoenzymatic strategy that integrates chemical synthesis of editable glycan scaffolds with enzyme-driven diversification, enabling the efficient construction of a diverse library of branch-specifically sulfated N- and O-glycans, along with their nonsulfated counterparts. Central to the strategy is the streamlined chemical synthesis of editable N- and O-glycan precursors bearing two orthogonally protected glucosamine termini, allowing branch-selective sulfation through flexible protecting-group manipulation. Subsequent modular enzymatic extension readily generates a panel of sulfated glycans with sialylation and fucosylation patterns. These diverse structures enable comprehensive profiling of their interactions with immune-associated lectins and viral proteins using microarray technology, uncovering unique binding specificities modulated by branch-specific sulfation patterns and core glycan architectures. This work establishes a powerful synthetic approach to previously inaccessible complex sulfated N- and O-glycans, providing critical tools to advance glycobiology and biomedical applications.