Semmelweis Egyetem Elméleti Orvostudományi Központ Hári Pál Előadóterem
2025-07-11
Modern Trends in Pharmaceutical Scientific Research
Dr. Antal István
Dr. Béni Szabolcs
Mazákné Dr. Kraszni Márta
Dr. Timári István
Dr. Tábi Tamás
Dr. Pálla Tamás
Dr. Szalainé Ágoston Bianka
In this thesis, we have conducted a comprehensive structural and isomeric characterization of HMOs using 1H-15N NMR methods. By integrating HSQC and HSQC-TOCSY methodologies, we have demonstrated their effectiveness in distinguishing HMO isomers with high specificity. Our findings contribute to glycoscience by refining analytical techniques that enhance the structural elucidation of complex oligosaccharides.
For the first time, we have applied 2D heteronuclear 15N NMR experiments for the characterization of isomeric HMOs. We systematically characterized the 15N NMR resonances of GlcNAc- and Neu5Ac-containing HMOs, presenting a detailed investigation of nitrogen chemical shifts in these bioactive carbohydrates. By establishing 1H-15N chemical shift correlations, we introduced a novel approach for differentiating structural isomers, particularly in distinguishing trisaccharide, tetrasaccharide, and pentasaccharide HMOs (3’SL, 6’SL, LNFP II, LNFP III, LSTa, LSTb). Our results demonstrate that GlcNAc and Neu5Ac moieties serve as sensitive reporter units, reflecting the chemical environment within different HMOs. Notably, LNT and LNnT exhibited the largest 15N chemical shift perturbations, whereas the Neu5Ac moiety in 3’SL and 6’SL was less responsive to structural variation.
Furthermore, we extended the application of 1H-15N HSQC spectroscopy to larger HMOs isolated from human milk, including hexa-, hepta-, and octasaccharides (LNH, LNnH, MFLNH III, MFLNnH I, MFLNnH II, DFLNnH), revealing critical insights into fucosylation patterns, glycosidic linkage effects, and β-Gal6 branching. Special emphasis was placed on the Lewis X motif, where we demonstrated that GlcNAc units could effectively report on branching and fucosylation patterns, aiding in the structural characterization of Lex-containing HMOs. Our identification of characteristic 1H-15N HSQC spectral regions enables the classification of GlcNAc-containing substructures, facilitating the rapid assignment of HMO compositions in biological samples.
As part of our study, we also explored the potential of ¹H-¹⁵N HSQC techniques for analysing HMO mixtures. While applied to a specific mixture, our findings indicate that this approach could serve as a valuable complementary tool for future investigations into complex oligosaccharide compositions.
Overall, we have established 1H-15N HSQC NMR as a powerful tool for HMO structural characterization, offering enhanced resolution and specificity for isomeric differentiation.
