Extending fluorinase [C-18F]-bond biocatalysis for Positron Emission Tomography (PET)

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Positron emission tomography (PET) is the most sensitive functional imaging method clinically and it application is growing rapidly through the Western world and developing countries, particularly as a diagnostic imaging tool for cancers and degenerative neurological disorders. Many major hospitals and clinical research centres in the Europe, the US and Asia are now commissioning cyclotrons and developing PET research facilities locally. Fluorine-18 is an important isotope for PET. It has a relatively long half-life (109 mins) and is readily generated in a cyclotron in the form of [18F]-fluoride ion, in very high specificic activity (GBq's) from oxygen-18 water. As a consequence new methods to develop C-[18F]F bond formation for PET labelling are in demand, In general the link between fluorine chemistry and pharmaceutical/medical applications is strong. Approximately 20% of all pharmaceutical, since the 1950s, contain a fluorine atom and the bio-distribution of all new pharmaceutical products are required to be explored by PET, as part of clinical trials. Also new PET tracers are in demand as tools for early diagnosis as indicators of disease states. New fluorine chemistry is required to meet the demands of a growing and dynamic PET research community both in the UK and internationally. This proposal aims to develop a novel methodology for incorporating fluoride-18 into relevant orgaganic molecules.In this proposal we aim to exploit a novel enzyme which can form C-F bonds from fluoride ion. The fluorinase enzyme was discovered in 2002 (Nature, 2002, 416, 279) in St Andrews and it has been over-expressed and its structure (X-ray) and mechanism elucidated. The enzyme catalyses the reaction of fluoride ion and S-adenosyl-L-methionine (SAM) to generate 5'-FDA and L-methionine. It has proven to be a chemoselective biotransformation method for generating C-18F bonds from inorganic [18F]-fluoride. The fluorinase is the only example of an enzyme used in PET synthesis and in this regard it offers an entirely new method for incorporating fluorine. In practical terms it has emerged to be particularly appropriate, because PET uses picomolar [18F]-fluoride ion, but the over-expressed fluorinase enzyme is present at mg/ml (microM), and therefore the kinetics favour C-18F synthesis due to a large molar excess of enzyme. This is a research collaboration between the Universities of St Andrews and Aberdeen where the methods will be developed in St Andrews and the radiolabeling protocols carried out at the Aberdeen PET Centre, situated in the Royal Infirmary Hospital. The major focus of the research will concentrate on developing fluorinase based chemistry to prepare novel fluorine-18 small molecules that can be used to attach to proteins through site specific cysteine residues. It is a current objective to find novel methods to radiolabel proteins such as enzymes and antibodies, to monitor their in vivo distribution by imaging. A key strategy will explore substrate analogues for the fluorinase, which carry acetylene residues, such that a 'click' chemistry approach can be adopted.A secondary objective of the research is to exploit an efficient chemo-enzymatic synthesis of [18F]-fluoroacetyl-CoA, developed between St Andrews and Aberdeen, to prepare [18F]-fluorocitrate, [18F]-fluoromalate and [18F]-N-aceylglutamate, as novelties for in vivo imaging. In overview the research will present a range of new methodologies for PET chemistry for the incorporation of the fluorine-18 isotope into molecules of biological relevance. The research in generic and aims to expemplify new methods, for much wider applications by the growing international research community of PET radiochemists.