Chemical biology can be defined as the area of science where chemical tools are used to study biological systems. The simplest way this can be achieved is in the identification of compounds which inhibit or modulate a biological pathway and the consequences studied. However, novel tools are required to enable, for example, the development of assays to allow simpler screening of difficult targets such as membrane proteins and protein-protein interactions. A series of kisspeptin analogues were synthesised for the development of a screening platform compatible with G-protein coupled receptors and tagged one bead one compound (OBOC) combinatorial libraries. Fluorescently labelled kisspeptin showed good affinity for GPCR54 and an on-bead version of the peptide, with the required C-terminal amide presented away from the bead was prepared and used for testing possible screening methods. GPCR54 was expressed in a number of formats and a kisspeptin based OBOC library designed and synthesised. Investigation into the C-terminal RF-amide motif of Kisspeptin was also carried out in order to assess the importance of the carbonyl moiety. The corresponding peptide amine was synthesised and the compound biologically assessed. This led to the development of a novel acid labile benzofuranone (ALBA) linker for anchoring amines to a solid support. For the preparation of fluorescent kisspeptin ligands, a novel general synthetic route which gives direct access to single isomer functionalised rhodamine dyes from phthalides has been developed. This circumvents the arduous task of isomer separation usually associated with the synthesis of functionalised rhodamines. The route has been demonstrated with a range of linkage groups and rhodamine types. This rhodamine material was used as a reporter group in various multifunctional reagents synthesised using a trifunctional orthogonally protected backbone (TOBa), which was prepared on a solid support and enables rapid synthesis of trifunctional reagents. This resin takes advantage of protecting group orthogonality and the high yields of peptide bond formation. A series of trifunctional reagents for screening use were prepared using this resin. A proof of concept study was carried out involving the simultaneous labelling and immobilisation of a protein for applications in probing protein-protein interactions. Development of a trifunctional hydroxamic acid containing cross-linker was carried out which takes advantage of its reaction with boronic acids to enable reversible capture on solid support for enrichment of cross-linked peptides. A new benzophenone based heterobifunctional reagent was prepared for protein cross-linking and mass spectrometry analysis. This was shown to give complimentary reactivity to existing cross-linkers, allowing more structural information to be extracted from protein samples.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:700061 |
| Date | January 2015 |
| Creators | Mudd, Gemma Elizabeth |
| Contributors | Auer, Manfred ; Millar, Robert |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/17882 |
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