Enzymatic Synthesis of Disialyllacto‐<i>N</i>‐Tetraose (DSLNT) and Related Human Milk Oligosaccharides Reveals Broad Siglec Recognition of the Atypical Neu5Acα2‐6GlcNAc Motif

Abstract Sialic acids (Sias) are ubiquitously expressed on all types of glycans, typically as terminating residues. They usually link to galactose, N ‐acetylgalactosamine, or other Sia residues, forming ligands of many glycan‐binding proteins. An atypical linkage to the C6 of N ‐acetylglucosamine (GlcNAc) has been identified in human milk oligosaccharides (HMOs, e.g., DSLNT) and tumor‐associated glycoconjugates. Herein, describe the systematic synthesis of these HMOs in an enzymatic modular manner. The synthetic strategy relies on a novel activity of ST6GalNAc6 for efficient construction of the Neu5Acα2‐6GlcNAc linkage, and another 12 specific enzyme modules for sequential HMO assembly. The structures enabled comprehensive exploration of their structure‐function relationships using glycan microarrays, revealing broad yet distinct recognition by Siglecs of the atypical Neu5Acα2‐6GlcNAc motif. The work provides tools and new insight for the functional study and potential applications of Siglecs and HMOs.