Abstract/Summary

Glycosylation refers to the post-translational modification of biological molecules to introduce ornate saccharide structures. Glycans have been linked to varying functions within biological systems ranging from cell stabilisation and adhesion, to host-pathogen interactions and cell messaging. Glycosylation can, therefore, have a significant effect upon the activity or viability of a glycosylated biomolecule. The recent growth in the development of protein based biotherapeutics has also resulted in a rapid increase in the development of tools to understand glycan structure and function. Analysis of glycan structures provides significant analytical challenges, attributed to their high hydrophilicity and lack of optically active regions. To overcome these challenges, derivatisation workflows are commonly employed to modify glycan structure in order to incorporate moieties which facilitate analysis. The work described in this thesis contributes to this area by focusing on the development and application of a series of novel multifunctional labelling strategies that enable modification of the reducing end of glycans using reductive amination. These strategies used derivatives of the commercially available labels 2- aminobenzamide and procainamide. Three multifunctional labels containing additional functionality in the form of a terminal alkyne were prepared. These labels were then compared with commercially available derivatives demonstrating they exhibited comparable Hydrophilic interaction liquid chromatography separation, over similar reaction times, to the commercial derivatives. These workflows were then applied for the multifunctional derivatisation of carbohydrates released from biological sources, and showed comparable separation could be achieved, and sialylation remained intact. This demonstrated that multifunctional labels can be successfully applied to glycan profiling workflows while maintaining further downstream capability. The downstream capability was then investigated though Cu(I) catalysed triazole formation between the multifunctionally labelled carbohydrate and azide conjugation partners. This was achieved though conjugation of labelled heptasaccharide to a library of azides ranging in non-polar surface area aimed at increasing hydrophobicity to impact ionisation efficiency in ESI-Mass spectrometry applications. This demonstrated that the analytical sensitivity for detection of low abundance carbohydrates was possible following reversed phase separation and detection by tandem mass spectrometry. This work therefore paves the way for the structural elucidation of undefined low abundance glycan species. Moreover the use of picomlar scale Cu(I) catalysed triazole formation on biologically derived carbohydrate provides a route towards the generation of neo-glycoconjugates enabling the further illumination of carbohydrate interactions on biological systems.