Utilizing the ¹H-¹⁵N NMR Methods for the Characterization of Isomeric Human Milk Oligosaccharides

Human milk oligosaccharides (HMOs) are structurally complex unconjugated glycans that are the third largest solid fraction in human milk after lactose and lipids. HMOs are in the forefront of research since they have been proven to possess beneficial health effects, especially on breast-fed neonates. Although HMO research is a trending topic nowadays, readily available analytical methods suitable for the routine investigation of HMOs are still incomplete. NMR spectroscopy provides detailed structural information that can be used to indicate subtle structural differences, particularly for isomeric carbohydrates. Herein, we propose an NMR-based method to identify the major isomeric HMOs containing GlcNAc and/or Neu5Ac building blocks utilizing their amide functionality. Experimental conditions were optimized (H₂O:D₂O 9:1 v/v solvent at pH 3.0) to obtain ¹H-¹⁵N HSQC and ¹H-¹⁵N HSQC-TOCSY NMR spectra of the aforementioned building blocks in HMOs. Four isomeric HMO pairs, LNT/LNnT, 3'SL/6'SL, LNFP II/LNFP III, and LSTa/LSTb, were investigated, and complete NMR resonance assignments were provided. In addition, ¹H and ¹⁵N NMR resonances were found to be indicative of various linkages, thereby facilitating the distinction of isomeric tri-, tetra-, and pentasaccharide HMOs. The rapid growth of HMO products (from infant formulas and dietary supplements to cosmetics) undoubtedly requires expanding the range of applicable analytical methods. Thus, our work provides a ¹⁵N NMR-based method to advance this challenging field of carbohydrate analysis.