Programmable Chemoenzymatic Assembly of a Bisected and Core‐Fucosylated <i>N</i> ‐Glycan Library Reveals Glycan‐Binding Protein Specificity and Branch Preference

Abstract Bisected and core‐fucosylated N ‐glycans represent a distinct class of complex biomolecules that are implicated in diverse biological and pathological processes. The structural complexity and synthetic challenges of these glycans hinder comprehensive understanding of their biological functions due to limited access to well‐defined samples. Despite advances in the complex N ‐glycan synthesis, the efficient preparation of bisected and core‐fucosylated asymmetric N ‐glycans with various branches and terminal epitopes remains an unmet challenge. In this study, we report a streamlined divergent chemoenzymatic approach for the programmable synthesis of an asymmetric bisected and core‐fucosylated N ‐glycan library, featuring bi‐, tri‐, and tetra‐antennary structures with variable‐length oligo‐LacNAc extensions and various terminal epitopes. This methodology relies on protecting‐group‐controlled branch extension, glycosyltransferase intrinsic branch selectivity and glyco‐epitope blocking effects, enabling the precise installment of each branch with unique epitopes. These structurally diverse N ‐glycans are printed as a microarray to comprehensively investigate structure–function relationships, revealing that glycan‐binding protein specificities are mediated by distinct branching patterns, oligo‐LacNAc chain length, and terminal epitope presentation. This work provides valuable insights into glycan‐protein interactions and highlights the potential of our approach for advancing glycoscience and biomedical applications.