Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.700061
Title: Dyes, linkers, tags and libraries : new tools for systems chemical biology
Author: Mudd, Gemma Elizabeth
ISNI:       0000 0004 5991 5977
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2015
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Abstract:
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.
Supervisor: Auer, Manfred ; Millar, Robert Sponsor: Biotechnology and Biological Sciences Research Council (BBSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.700061  DOI: Not available
Keywords: chemical biology ; rhodamine ; libraries ; tags ; cross-linking ; solid phase synthesis
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