Development of Biocatalysts for Fluorination of Organic Compounds

描述(申请人提供)：1970年，5%的许可药品中至少含有一个氟原子。2005年，这一数字大幅上升到20%，现在据估计，目前正在研发的药物中有25%至少含有一个氟原子。氟对药物发现和功能的重要性怎么强调都不为过。然而，化学氟化的工业方法代表了目前最危险和最有毒的一些化学方法，通常使用NaNO2、HF和F2直接氟化。相比之下，自然界在温和的水环境中进行氟化反应时，通常具有酶反应的高选择性。应用“氟化酶”生物催化剂作为化学合成的补充方法，为制备具有重要生物医学意义的重要含氟化合物提供了更安全、更环保的路线的可能性。我们建议应用一种新的高通量筛选方法来从微生物来源中鉴定和改进新的氟化酶生物催化剂。作为最初的目标，我们将重点关注最近在卡特莱链霉菌中发现的一种氟化酶，该酶已被证明能够形成C-F键，但目前其活性太低，无法用于工业。我们将通过定向进化和从基因组文库中鉴定更多新的氟化酶来证明这种酶的改进，作为分子生物学方法的原理证明，以提供可开发用于工业氟化的有用的生物催化剂。氟化酶还将被用作将F-18同位素结合到适当生物分子中的合成工具，特别是考虑到正电子发射断层扫描(PET)作为一种诊断技术的快速增长，以及作为评估所有药物在临床试验中的生物分布的要求。 新近发现的微生物氟化酶能将无机氟离子转化为有机氟。氟的生物催化掺入为清洁和安全的有机氟生物合成以及将放射性标记的氟[F-18同位素]掺入药物开辟了巨大的生物技术机会。对氟化酶的适用性的广泛兴趣源于对含有氟原子的精细化学品、医药和农用化学实体的巨大商业兴趣，以及F-18同位素在正电子发射断层扫描(PET)医学成像应用中的日益广泛的应用。旨在提高这种氟化酶的效率和用途的研究是独一无二的，随后将有许多应用。

DESCRIPTION (provided by applicant): In 1970 5% of licensed pharmaceuticals contained at least one fluorine atom. In 2005 this figure had risen dramatically to 20% and it is now estimated that 25% of current pipeline drugs now contain at least one fluorine atom. The importance of fluorine to pharmaceutical discovery and functionality cannot be overstated. However, industrial methods of chemical fluorination represent some of the most hazardous and toxic chemical methods currently practiced, generally utilizing NaNO2, HF and direct fluorination with F2. Nature, in contrast carries out fluorination with the typically high selectivity of enzymatic reactions and in a mild aqueous environment. The application of "fluorinase" biocatalysts as a complementary approach to chemical synthesis offers the potential of safer, more environmentally acceptable routes to prepare important fluorinated compounds of biomedical importance. We propose to apply a novel high-throughput screening approach to identify and improve new fluorinase biocatalysts from microbial sources. As the initial target we will focus upon a fluorinase, recently identified in Streptomyces cattleya which has been shown to carry out C-F bond formation but which currently possesses activity too low to be industrially useful. We will demonstrate the improvement of this enzyme by directed evolution and the identification of further novel fluorinases from genomic libraries as the proof-of-principle of molecular biological approaches to provide useful biocatalysts which can be developed for industrial fluorination. The fluorinase will be applied also as a synthetic tool for the incorporation of the F-18 isotope into appropriate bio- molecules, particularly in light of the rapid growth in positron emission tomography (PET) as a diagnostic technique and as a requirement for the assessment of the biodistribution of all drugs progressing through clinical trials. The recently discovered microbial fluorinase enzyme converts inorganic fluoride ion to organic fluorine. Biocatalytic incorporation of fluorine opens up a tremendous biotechnological opportunity for clean and safe organofluorine biosynthesis and also the incorporation of radio-labeled fluorine [F-18 isotope] into drugs. The widespread interest in the fluorinase's applicability arises from the huge commercial interest in the fine chemicals, medicinal and agrochemical entities that contain fluorine atoms and also in the growing application of the F-18 isotope in positron emission tomography (PET) applications for medical imaging. The research directed towards improving the efficiency and utility of this fluorination enzyme is unique and numerous applications would follow.