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Title: Small molecule tools for structural and functional studies of 2OG oxygenases
Author: Lesniak, Robert
ISNI:       0000 0004 6497 6088
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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The work described in this thesis focused on members of the Fe(II), 2-oxoglutarate dependent oxygenase family that are involved in the biosynthesis of L-carnitine in mammals and in hypoxia sensing. Work on metallo β-lactamases was also carried out. Nϵ-Trimethyllysine hydroxylase (TMLH) and γ-butyrobetaine hydroxylase (BBOX) catalyse the first and final steps in carnitine biosynthesis, respectively. Chapter 1 provides an overview of the current literature regarding the 2OG oxygenases involved in the biosynthesis of L-carnitine from a therapeutic perspective. A discussion concerning the clinically used inhibitor of carnitine biosynthesis, Mildronate (Meldonium, THP, Met-88), is also included. In Chapter 2 the stereochemical elucidation of the product of TMLH catalysis as (2S,3S)- 3-hydroxy-Nϵ-trimethyllysine is described. This was achieved via the successful implementation of a silver(I) catalysed asymmetric aldol reaction to obtain (2S,3R)-3-hydroxy-Nϵ-trimethyllysine which enabled elucidation of the TMLH catalysed product using 1D and 2D NMR spectroscopic techniques. Chapter 3 explores the use of a 19F labelled cysteine mutant of BBOX from Pseudomonas sp. AK1 to monitor conformational changes upon ligand binding using 19F NMR spectroscopy. The results validate the use of 19F chemical labelling to monitor ligand binding to 2OG-dependent oxygenases and identifies a potential new class of small molecule inhibitor of BBOX. Synthetic work towards novel BBOX inhibitors is described in Appendix I. Chapter 4 describes the design and synthesis of novel, broad-spectrum class B metallo β- lactamase inhibitors. The predicted binding modes of the potent mercaptopropionic acid derivative RL368 and its diastereomer RL369 were validated by X-ray crystallography. RL368, in particular, showed superior inhibition to the known inhibitors L- and D-captopril, and, pleasingly displays cell-based potential as an antibiotic following antimicrobial susceptibility assays against Gram negative pathogens of clinical origin; E. coli, E. aerogenes and K. pneumoniae. Chapter 5 concerns the design, synthesis and implementation of a multifunctional PHD2- selective covalent probe compound RL409, which is aimed at irreversibly binding PHD2. By functionalising a reversibly binding PHD2 inhibitor with electrophilic and photoreactive moieties, it was possible to attach a biotin tag to PHD2 using a copper-free 'click' reaction with the aim to capture PHD2 from cell lysates. Overall the results provide new insights into metallo-enzyme catalysis and describe new ways to inhibit them.
Supervisor: Schofield, Christopher J. Sponsor: Wellcome Trust ; Biological Sciences Research Council (BBSRC) ; British Heart Foundation (BHF)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available