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Development of New Synthetic Approaches to Biologically-Active Peptides

Verhoork, S (2019) Development of New Synthetic Approaches to Biologically-Active Peptides. Doctoral thesis, Liverpool John Moores University.

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Abstract

Synthetic peptides are widely used in drug research due to their high selectivity and efficaciousness, as well as in chemical biology as tools for modulating protein-protein interactions. While there are a large number of naturally occurring peptides, which can be used for the development of new pharmaceuticals, there is also an increasing interest in rationally designed synthesised and optimised peptides. In the field of chemical biology peptides are used to understand complex biological processes, where gaining knowledge and understanding can lead to improved human health and the development of novel drug targeting strategies. Since the synthesis of the first peptide back in 1882, there have been major changes to the peptide synthesis protocol, leading to robust Fmoc-solid phase peptide synthesis that is used in most laboratories today. However, the peptide purification protocols have been lacking behind, meaning peptide purification is still a major bottleneck in fast, efficient, environmentally friendly peptide synthesis. In this thesis, a new peptide synthesis and purification method has been developed and optimised making use of an acrylamide-scavenging approach. Different strategies to introduce an N-terminal acrylamide cap have been explored, including the use of acryloyl chloride and acrylic anhydride, where the latter was suited for automated SPPS. It was found that replacement of piperidine by DABCO for Fmoc-deprotections was equally efficient and suitable for automated SPPS. The scavenging of acrylamide-tagged deletions was performed making use of a thiol-resin, of which the rate was determined by LCMS in peptide model systems. Upon optimisation of acrylamide-tagging and Fmoc-deprotection conditions, the new purification method was compatible with both manual and automated SPPS, requiring only minor adjustments from standard synthesis protocols. This allowed for the synthesis and purification of the diabetes peptide exenatide (39 amino acids), without making use of preparative-HPLC. An attempt was made to extend this methodology to the synthesis of cyclic thioether peptides; however, this did not provide conclusive results. An alternative approach to cyclising peptides was explored, making use of disulfide crosslinking with hexafluorobenzene in biologically active p53 peptides. Residues of the biologically active pDI peptide that had been shown to be tolerant to substitution were replaced with cysteine (analogues), including D-cysteine, homocysteine and penicillamine. It was found that replacement of L-cysteine by any of the analogues resulted in altered binding affinity and selectivity. Finally, the use of fluorine as a reporter for 19F NMR analysis of biological molecules was investigated. Fluorinated prolines were used to explore protein-protein interactions and dynamics, specifically to study the peptidyl-prolyl isomerase cyclophilin D (CypD). First, short substrates were synthesised, including a section of Bcl2 (8 amino acids) and F0F1 ATPase subunit B (9 amino acids), which formed a proof of principle. Significant line broadening was observed in the 19F NMR spectrum with increased protein concentration, indicating substrate binding. These results were more apparent for the Subunit B substrate, indicating it is a better binder to CypD than the Bcl2 peptide. The synthesis of another CypD substrate, a part of the poly proline motif in p53, was optimised for the incorporation of 4S-fluoroprolines. A very significant finding was that each proline residue had a distinct chemical shift. The native cis-trans ratios of each peptide was determined, after which some changes in the cis-trans ratios were observed in the presence of CypD. For some of the fluorinated proline residues line broadening was observed upon incubation with CypD, indicating the occurrence of protein-protein interactions.

Item Type: Thesis (Doctoral)
Uncontrolled Keywords: Chemistry; Peptides; Chemical Biology; Synthetic Chemistry; Peptide Purification
Subjects: R Medicine > RM Therapeutics. Pharmacology
Divisions: Pharmacy & Biomolecular Sciences
Date Deposited: 20 Sep 2019 11:39
Last Modified: 20 Sep 2019 11:39
DOI or Identification number: 10.24377/LJMU.t.00011319
Supervisors: Coxon, C, Sharples, A and Stewart, C
URI: http://researchonline.ljmu.ac.uk/id/eprint/11319

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