Preparative separation of α2,3- and α2,6-sialylated N-glycans via linkage-specific charge modulation

Sialylated glycans are essential biomolecules in glycoscience, with linkage variations-particularly α2,3- and α2,6-linked sialic acids-playing critical roles in glycan-protein interactions and disease processes. While analytical techniques can distinguish these linkages through differential chemical reactivity, such as lactone or ester formation that induces characteristic mass shifts, preparative separation is challenging due to similar chromatographic behavior. We report a charge-based strategy for efficient preparative separation of α2,3- and α2,6-sialylated glycans using weak anion exchange (WAX) chromatography. This approach exploits lactone and ethyl ester formation and their differential hydrolysis rates to modulate net charge, enabling milligram-scale isolation of natural glycans from diverse sources. The purified glycans were immobilized on microarrays to investigate glycan-binding specificities of plant lectins and Siglecs, providing a robust platform for functional glycomics.