PhD Scientific Days 2023

KDP Poster session

The Contribution of NMR to the Characterization of Isomeric Human Milk Oligosaccharides

Text of the abstract

Introduction
Human milk oligosaccharides (HMOs) are structurally complex unconjugated glycans that are the third largest solid fraction in human milk, they possess beneficial health effects, especially on breastfed neonates. Although HMOs are in the forefront of research, readily available analytical methods suitable for the routine investigation are still incomplete. NMR spectroscopy provides detailed structural information that can be used to indicate subtle structural differences, particularly for isomeric carbohydrates.
Aims
As most HMOs contain the NMR active nucleus 15N incorporated into N-acetylglucosamine (GlcNAc) and/or N-acetylneuraminic acid (Neu5Ac) moieties, we aimed to introduce the 15N and 1H NMR chemical shifts of GlcNAc or Neu5Ac building blocks into the characterization of HMOs to facilitate the identification of isomeric structures.
Method
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 GlcNAc and Neu5Ac moieties in HMOs. Four isomeric HMO pairs: LNT/ LNnT, 3’-SL/6’-SL, LNFP II/LNFP III and LSTa/ LSTb were investigated.
Results
Complete 1H, 13C, and 15N NMR resonance assignments were achieved for the investigated HMOs. The isomeric HMO pairs showed remarkably different 15N NMR resonances (obtained from the 1H-15N HSQC experiment). The highest chemical shift perturbation was observed between the tetrasaccharides LNT and LNnT bearing a GlcNAc unit, while the 15N NMR chemical shifts of the Neu5Ac moiety in 3’SL and 6'SL trisaccharides were less responsive to the minor structural difference. When considering both 1H and 15N NMR chemical shifts of the GlcNAc and/or Neu5Ac moieties (obtained from the 1H-15N HSQC-TOCSY experiment), the distinction of the structural isomers was unambiguous.
Conclusion
The rapid growth of HMO products undoubtedly requires expanding the range of applicable analytical methods. This NMR-based method offers a straightforward approach for the identification of the most common HMOs and may thereby contribute to the analytical investigation of HMO standards, HMO products, or even human milk.
Funding
Prepared with the professional support of the Doctoral Student Scholarship Program of the Co-operative Doctoral Program of the Ministry of Innovation and Technology, financed by the National Research, Development and Innovation Fund (KDP-1007075).