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.