Department of Pharmacognosy, Semmelweis University, Üllői út. 26, H-1085 Budapest, Hungary.
Directorate of Drug Substance Development, Egis Pharmaceuticals Plc., P.O. Box 100, H-1475 Budapest, Hungary.
Int J Mol Sci. 2023 Jan 22;24(3):2180. doi: 10.3390/ijms24032180.
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 (HO:DO 9:1 / solvent at pH 3.0) to obtain H-N HSQC and H-N 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, H and N 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 N NMR-based method to advance this challenging field of carbohydrate analysis.
人乳寡糖 (HMOs) 是结构复杂的非共轭糖，是乳糖和脂肪之后人乳中第三大固体成分。HMOs 具有有益的健康影响，尤其是对母乳喂养的新生儿，因此它们是研究的前沿领域。尽管 HMO 研究是当今的热门话题，但适用于 HMO 常规研究的现成分析方法仍不完善。NMR 光谱提供了详细的结构信息，可用于指示细微的结构差异，特别是对于异构体碳水化合物。在此，我们提出了一种基于 NMR 的方法，利用 HMO 中 GlcNAc 和/或 Neu5Ac 结构单元的酰胺官能团来鉴定主要的异构体 HMOs。优化了实验条件 (HO:DO 9:1 / 溶剂在 pH 3.0)，以获得上述结构单元在 HMOs 中的 H-N HSQC 和 H-N HSQC-TOCSY NMR 图谱。研究了四对异构体 HMO 对，即 LNT/LNnT、3'SL/6'SL、LNFP II/LNFP III 和 LSTa/LSTb，并提供了完整的 NMR 共振归属。此外，H 和 N NMR 共振被发现可指示各种键合，从而有助于区分异构体三糖、四糖和五糖 HMOs。HMO 产品（从婴儿配方奶粉和膳食补充剂到化妆品）的快速增长无疑需要扩大适用分析方法的范围。因此，我们的工作提供了一种基于 N NMR 的方法，可推动碳水化合物分析这一具有挑战性的领域的发展。
