Chemoenzymatic Synthesis of Tri‐antennary N‐Glycans Terminating in Sialyl‐Lewisx Reveals the Importance of Glycan Complexity for Influenza A Virus Receptor Binding

Abstract Sialyl‐Lewis x (SLe x ) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLe x epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty‐two isomeric asymmetrical tri‐antennary N ‐glycans presenting SLe x ‐Le x epitopes on either the MGAT4 or MGAT5 arm that include putative high‐affinity ligands for E‐selectin. The N ‐glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5′‐diphospho‐ N ‐trifluoracetylglucosamine (UDP‐GlcNHTFA) that can be transferred by branching N ‐acetylglucosaminyltransferases to give, after base treatment, GlcNH 2 ‐containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E‐selectin bound equally well to linear glycans and tri‐antennary N ‐glycans presenting SLe x ‐Le x . On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri‐antennary N ‐glycans. Furthermore, several H5 HAs preferentially bound to N ‐glycan presenting SLe x on the MGAT4 arm. SLe x is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N ‐glycans presenting SLe x .