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.