From mother's milk to structural insights: H-N NMR analysis of Lewis X antigen-bearing oligosaccharides isolated from human milk.

Human milk oligosaccharides (HMOs) are complex unconjugated glycans abundant in breast milk, with vital roles in infant nutrition and health. The structural elucidation of HMOs remains challenging due to their diverse compositions and isomeric complexities. This study presents a novel approach employing H-N NMR correlations to pinpoint and characterize HMOs, LNH, LNnH, and their fucosylated derivatives including those of bearing the Lewis X motif. Isolated from human milk, these hexa-, hepta-, and octasaccharides were extensively analysed by NMR. This enabled their first complete H, C and N resonance assignments. Using H-N HSQC experiments, the distinctive H-N correlations of GlcNAc units within the HMOs provided structural fingerprints, allowing for unequivocal differentiation of isomeric structures and fucosylation patterns. Results demonstrate the potential of H-N NMR spectroscopy in decoding complex HMO structures, offering new perspectives on their detailed structural characterization and contributing to a deeper understanding of their potential benefits.