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

正电子发射断层扫描（PET）是临床上最敏感的功能成像方法，其应用在西方世界和发展中国家迅速增长，特别是作为癌症和退行性神经系统疾病的诊断成像工具。欧洲、美国和亚洲的许多主要医院和临床研究中心目前正在启用回旋加速器，并在当地开发PET研究设施。氟-18是PET的重要同位素。它具有相对较长的半衰期（109分钟），并且在回旋加速器中容易以[18 F]-氟离子的形式从氧-18水以非常高的比活度（GBq's）产生。因此，需要开发用于PET标记的C-[18F]F键形成的新方法。一般来说，氟化学与制药/医疗应用之间的联系很强。自20世纪50年代以来，大约20%的药物含有氟原子，所有新药物产品的生物分布都需要通过PET进行探索，作为临床试验的一部分。此外，需要新的PET示踪剂作为早期诊断的工具，作为疾病状态的指标。新的氟化学需要满足不断增长的和动态的PET研究社区在英国和国际上的需求。本研究的目的是开发一种新的方法，将氟化物-18整合到相关的有机分子中，在本研究中，我们的目标是开发一种新的酶，它可以从氟化物离子形成C-F键。氟酶于2002年在圣安德鲁斯发现（Nature，2002，416，279），并且它已被过度表达，其结构（X射线）和机制被阐明。该酶催化氟离子和S-腺苷-L-甲硫氨酸（SAM）反应生成5 '-FDA和L-甲硫氨酸。它已被证明是一种化学选择性生物转化方法，用于从无机[18F]-氟化物产生C-18F键。氟化酶是PET合成中使用的酶的唯一例子，并且在这方面，它提供了一种引入氟的全新方法。在实践中，它已经变得特别合适，因为PET使用皮摩尔[18F]-氟离子，但过表达的氟化酶以mg/ml（μ M）存在，因此由于酶的摩尔过量，动力学有利于C-18F合成。这是圣安德鲁斯大学和阿伯丁大学之间的研究合作，方法将在圣安德鲁斯开发，放射性标记协议在位于皇家医院的阿伯丁PET中心进行。该研究的主要重点将集中在开发基于氟化酶的化学方法，以制备新型氟-18小分子，这些小分子可用于通过位点特异性半胱氨酸残基连接到蛋白质上。目前的目标是找到放射性标记蛋白质如酶和抗体的新方法，以通过成像监测它们的体内分布。一个关键的策略是探索携带乙炔残基的氟化酶的底物类似物，使得可以采用“点击”化学方法。该研究的第二个目标是利用由St Andrews和Aberdeen开发的[18 F]-氟乙酰辅酶A的有效化学-酶促合成，以制备[18 F]-氟柠檬酸盐、[18 F]-氟苹果酸盐和[18 F]-N-乙酰谷氨酸盐，作为体内成像的新方法。总的来说，这项研究将提出一系列新的PET化学方法，用于将氟-18同位素掺入生物相关分子中。这项研究的目的是通过PET放射化学家日益增长的国际研究社区，为更广泛的应用示范新方法。

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.