London, James 
ORCID: 0000-0002-5823-7057
(2022)
Analysis of Complex Polysaccharides of Biological and Industrial
Relevance via Nuclear Magnetic Resonance Spectroscopy:
Structure, Modification and Protein Interactions.
Doctor of Philosophy thesis, University of Liverpool.
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Abstract
Carbohydrates, as one of the principal classes of biological molecule, are ubiquitous, with natural roles and biopharmaceutical, commercial, and industrial applications resulting from their structures. Variations to these structures have the potential to generate vastly differing biological, physico-chemical, and interaction properties as required. One such structural variation, sulfation, has been observed to affect the ability of multiple carbohydrates to bind proteins whilst also having the potential to alter their commercially relevant physico-chemical properties. The characterisation of carbohydrate structures including any differences present between samples from different sources, and the effect that modifications have is an important asset during the development or analysis of novel samples. The research detailed in this thesis, therefore, was targeted towards the optimisation of NMR-based methods for the analysis of, both starting material and modified, carbohydrate structures. Additional targets included the optimisation of a pyridine sulfur trioxide based sulfation method in recyclable solvents, and the analysis of resulting samples in terms of their interactions with proteins. Improvements were, therefore, made to the nuclear magnetic resonance based protocol for carbohydrate structure characterisation with an increase in resolution approaching, and in some cases at, the natural linewidth. These spectrum resolution improvements generated a greater number of peaks than was previously possible, which were assigned specifically to the individual CH groups present, in 2D 1H,13C-HSQC spectra, for the repeating units of a xyloglucan test-case sample. Application of this protocol to a series of sulfated samples provided a means by which modification could be, indirectly, monitored more specifically for individual hydroxyl groups. The development of an improved sulfation method, suitable for gel forming and water insoluble carbohydrates, resulted in the production of a range of samples that could be used to test the NMR protocol developed. The amended pyridine sulfur trioxide sulfation method developed, with solvation in 1-butyl-3-methylimidazolium chloride instead of the traditional organic solvents, generated a series of variably sulfated samples. In some instances these reactions followed the expected order of hydroxyl group reactivity, i.e. primary -OHs > equatorial secondary -OHs > axial secondary -OHs, however in other reactions modifications did not follow this order. Those reactions that differed from the expected order of hydroxyl group reactivity highlight the potential applicability of this method for the generation of carbohydrates containing novel sulfation patterns. Carbohydrate-protein interactions are involved in many of the biological processes throughout all forms of life. A selection of the sulfated samples produced and analysed were therefore screened to determine their interactions with four proteins, Fibroblast Growth Factors (FGFs) 1 and 2 as well as Cyclophilins (Cyps) B and D, using differential scanning fluorimetry. Of the combinations screened, examples that were shown to form interactions, were analysed via isothermal titration calorimetry to provide thermodynamic data and corroboration of the DSF. The research carried out in this project, therefore, could be used to analyse carbohydrate samples in greater detail than was previously possible, in a manner proven by the analysis of samples sourced from different suppliers, as well as those produced by chemical modification. Equally, the research presented in this thesis has identified a method by which gel forming and water insoluble samples could be modified without requiring a preparation step, that has been shown to produce novel sulfation pattern samples. Screening the samples produced; they have subsequently been shown to bind proteins in interactions that have been previously identified as well as those that haven’t. These could form the basis of any further work that could be carried out.
| Item Type: | Thesis (Doctor of Philosophy) |
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| Divisions: | Faculty of Health and Life Sciences |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 10 Nov 2022 14:39 |
| Last Modified: | 18 Jan 2023 20:40 |
| DOI: | 10.17638/03165135 |
| Supervisors: |
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| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3165135 |
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