Tunicamycins are potent antimicrobial agents but are also toxic to mammalian cells, which render them clinically impractical to use to treat infectious diseases. Instead, they have been used extensively as biochemical tools to study the N-linked glycosylation of proteins. However, despite such a routine application, their inhibitory mechanisms are still not clear. The central objective of this thesis was to develop novel tunicamycin analogues that are non-toxic to eukaryotic cells that could serve as potential antimicrobial drug candidates. We hypothesised that if we retain the lipid character of tunicamycin structure and modify the GlcNAc moiety then the antimicrobial activity would be retained but the tunicamycins inhibitory action towards GPT would be abolished, thus diminishing tunicamycins cytotoxicity towards mammalian cells. <b>I - Semi-synthesis of the Tunicamycin Core Scaffolds and Lipid Analogues</b> Semi-synthetic strategies were devised for isolating tunicamycin core scaffolds and for the selective addition of lipid chains at the 10'-N and 2"-N positions of tunicamycin, yielding the first library of novel tunicamycin lipid analogues. <b>II - Biological Evaluations of the Tunicamycin Core Scaffolds and Lipid Analogues</b> For the first time, the antibacterial activity of tunicamycins was shown to be dependent on the presence of a lipid chain. The tunicamycin core scaffolds were shown to lack antibacterial activity and cytotoxicity. More importantly, the library of tunicamycin lipid analogues with lipid chain length from seven to twelve carbons showed titrated antibacterial activity profile. Furthermore, the tunicamycin lipid analogues were not only found to have potent antibacterial and anti-M. tuberculosis activities but were non-cytotoxic compared to tunicamycins. The relative therapeutic index calculated for the tunicamycin lipid analogues was up to several thousand folds more than tunicamycins. <b>III - Investigation of the tunB and tunF Knockout in the tun Gene Cluster</b> The tunB and tunF single knockout mutations were made in the tun gene cluster by PCR-targeting and then heterologously expressed in S. coelicolor. The tunB knockout successfully abolished tunicamycin biosynthesis and showed evidence by MS the first existence of exo-glycal intermediates in sugar biology, further supporting the discovery of TunA as a novel NDP-sugar 5,6-dehydrogenase. <b>IV - Investigation of the TunD and TunE Enzymatic Activities in Tunicamycin Biosynthetic Pathway</b> The recapitulation of TunD glycosyltransferase and TunE deacetylase activities in vitro were attempted. Recombinant TunD was refolded from insoluble TunD inclusion bodies, while TunE was isolated in small quantities. However, no TunD and TunE activities were found using proposed intermediates. The co-translation of the tun gene cluster and the formation of multi-protein complex are proposed to be involved in the tunicamycin biosynthesis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:674870 |
| Date | January 2014 |
| Creators | Wang, Hua |
| Contributors | Davis, Benjamin G. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:05c43287-9f84-45f4-8db4-0fb6c2763816 |
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