- Department of Molecular Sciences, Swedish University of Agricultural Sciences
Studies of Hyaluronan and Related Structures by NMR Spectroscopy
Hyaluronan (HA) is a natural polysaccharide in the glycosaminoglycan (GAG) family with repeating units of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA), which are linked by β(1→4) and β(1→3) glycosidic linkages. HA has many important regulatory functions in the human body, but comprehension of its structure-function relationship remains limited. Nuclear magnetic resonance (NMR) spectroscopy is the method of choice for studying complex biomolecules at atomic level and also holds great potential for advancing understanding of HA. This thesis investigated various forms of HA in aqueous solution using NMR spectroscopy, to gain insights into structural and dynamic behaviors.
First, correlations between physicochemical properties of cross-linked HA hydrogels and the dynamic behavior of water in the samples were examined. The results showed that transverse relaxation (T2) of water protons was dominated by the chemical exchange between water protons and exchangeable protons of HA. Based on the temperature profile of T2, it was possible to distinguish samples with different concentrations and, to some extent, hydrogels with different physicochemical properties.
Detailed conformational analysis of the GlcNAc monosaccharide was conducted, focusing on the N-acetyl group. Amide cis conformations were observed for the first time by 1 H NMR. The two torsion angles that define the conformation of the N-acetyl group were evaluated based on the measurement of eight 3 J-couplings. In addition, conformations of the β(1→4) and β(1→3) glycosidic linkages and the N-acetyl group in HA-8mer and HA-20mer were compared. As in GlcNAc, the orientation between H2 and NH was determined to be in anti conformation for both chain lengths. The NMR data supported one predominant conformation of the β(1→3) glycosidic linkage and suggested a conformational equilibrium at the β(1→4) glycosidic linkage. Overall, the glycosidic linkage conformations of the two chain lengths exhibited subtle differences.
To overcome the low natural abundance of the 13C and 15N nuclei, 13C,15Nenriched HA polysaccharide was produced biosynthetically. The level of 13C and 15N isotope enrichment was determined quantitatively by NMR and confirmed by highresolution mass spectrometry.
Hyaluronan; NMR spectroscopy; N-acetylglucosamine; hydrogel; oligosaccharide; polysaccharide; isotope labeling
Acta Universitatis Agriculturae Sueciae
2023, number: 2023:57
ISBN: 978-91-8046-166-5, eISBN: 978-91-8046-167-2
Publisher: Swedish University of Agricultural Sciences
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