Structural elucidation of polysaccharides and investigations of enzymatic syntesis of oligosaccharides using NMR spectroscopy

Abstract

Carbohydrates is a source of complex biomolecules with numerous roles in biology, and elucidation of the structures is imperative for understanding their functions. NMR spectroscopy is an important tool for structure analysis of organic molecules, and for carbohydrates it is often the best option for determining the structures. This thesis is made up of several projects focusing on NMR spectroscopy for carbohydrate structural elucidation as well as transglycosylation studies, and the following sections are small abstracts for each project. All of the projects were part of collaborations with researchers from adjacent fields, and most of the results would not have been obtainable without the use of high-field NMR spectroscopy. The capsular polysaccharide of Streptococcus pneumoniae is responsible for much of its virulence, and as such understanding the structures of the polysaccharides from different serotypes can further the understanding of the pathogen. The capsular polysaccharide structure of a novel serotype in serogroup 7 was elucidated, and the novel serotype turned out to be a mixture of two other serotypes capsular polysaccharides and named 7D in the Danish nomenclature. As one of these two polysaccharides was from serotype 7C, the structure of which was previously undetermined, this was also elucidated. Inonotus obliquus, more commonly known as the chaga mushroom, has been used in Eastern European folk medicine to treat a variety of symptoms, and contains several compounds of medicinal interest such as polyphenols, triterpenoids and polysaccharides, the last of which was subjected to several types of structural analysis, including NMR spectroscopy. The fungal polysaccharides were extracted and purified, and they turned out to be heterogeneous, but different structural trends were determined. β-Lactoglobulin is a major protein present in dairy products which is known to bind different types of molecules, but little is known of its ability to bind carbohydrates. For this purpose several polysaccharides were fragmented into oligosaccharide mixtures to be used for binding studies, and in order to understand the implications of those results the composition and structural analysis of the mixtures is required. Human milk oligosaccharides are important for the immune system of infants, but unfortunately it is barely present in the milk of any domesticated mammal, so a large scale synthesis of human milk oligosaccharides can help improve infant formula. However, these are fairly complex, and when synthesising them via transglycosylation several products can formed, making the structural determination rather troublesome, even with NMR spectroscopy. The transglycosylation products of three different β-acetylglucosaminidases using lactose as acceptor were identified. Dissolution dynamic nuclear polarisation can be used to increase the sensitivity of single scan solution 13C-NMR with up to four orders of magnitude, and using a doubly isotopically labelled βgalactopyranoside the lacZ β-galactosidase was investigated. Using this method, previously undescribed short-lived transglycosylation products of the enzyme were observed, and using kinetic model the hydrolysis and transglycosylation rates were determined.