Studies and application of the enzymes of fluorometabolite biosynthesis in Streptomyces cattleya
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This thesis focuses on studies investigating the structure of intermediates involved in fluorometabolite biosynthesis, and the potential applications of the fluorinase enzyme in positron emission tomography (PET). Chapter 1 introduces the rare natural occurrence of fluorinated compounds. The bacterium Streptomyces cattleya is known to biosynthesise two fluorinated secondary metabolites: the toxin fluoroacetate (FAc) and the antibiotic 4-fluorothreonine (4-FT). The enzymes and intermediates identified on this fluorometabolite biosynthetic pathway in S. cattleya, prior to this research, are discussed in detail. Chapter 2 presents studies towards the unambiguous structural identification of (3R,4S)-5- deoxy-5-fluoro-D-ribulose-1-phosphate (5-FRulP) as the third fluorinated intermediate on the biosynthetic pathway to fluoroacetate and 4-fluorothreonine in S. cattleya. Chapter 3 describes the synthetic routes to key molecules, necessary as reference compounds and substrates, to underpin the subsequent studies in this thesis. In particular, synthetic routes to 5'-deoxy-5'-fluoroadenosine (5'-FDA), 5'-deoxy-5'-fluoroinosine (5'-FDI), 5-deoxy-5-fluoro-D-ribose (5-FDR) and 5-deoxy-5-fluoro-D-xylose (5-FDX) are described. Chapter 4 describes the use of the fluorinase enzyme from S. cattleya as a tool for the synthesis of new [¹⁸F]-labelled sugars with potential application in positron emission tomography (PET). A new route to 5-deoxy-5-[¹⁸F]fluoro-D-ribose ([¹⁸F]FDR) is developed in a two-step enzymatic synthesis. A total of three potential radiotracers ([¹⁸F]FDA, [¹⁸F]FDR and [¹⁸F]FDI) are synthesised using fluorinase-coupled enzyme reactions. In addition, in vitro studies are reported with these [¹⁸F]-labelled sugars to investigate their uptake and potential as PET radiotracers in cancer cells. A preliminary rat imaging study with [¹⁸F]FDA is reported. Chapter 5 details the experimental procedures for the compounds synthesised in this research and the biological procedures for chemo-enzymatic syntheses and protein purification.
Thesis, PhD Doctor of Philosophy