Evolved P450 mutants as general oxidation catalysts for target synthesis via C-H activation

该项目属于EPSRC的研究主题：医疗保健技术(相关：合成生物学、合成有机化学)；物理科学(相关：催化、合成生物学、合成化学)；制造未来(相关：制造技术)。该项目将集中于使用突变细胞色素P450酶库通过后期氧化合成一系列片段和天然产品。这种方法通过在合成结束时引入氧官能团，允许将重点放在骨架构建过程上，从而减少了对保护基团的需要，并使路线更加高效和环保。我们将探索使用P450来实现不对称氧化，在一系列底物上使用这些酶将有助于建立反应活性图谱，使这些突变体更接近作为一般氧化催化剂的应用。后期官能化已成为一种有效的化学合成方法。它允许合成路线专注于骨骼构建，而不需要保护团体策略。由于含氧官能团在天然产物和药物分子中的普遍存在，羟化反应在后期功能化领域引起了人们的极大兴趣。最近关于后期羟化反应的研究倾向于使用化学催化剂来实现这些转化，虽然可行，但可能昂贵且难以处理。该项目将开发一种不同的催化方法，即利用突变的P450(MP450)酶的库。这些酶可以进化和定制，从而可以开发所需的反应活性，包括与当前化学氧化方法互补的活性。细胞色素P450BM3(CYP102A1)属于P450细胞色素(CYP)超家族血蛋白，作为氧化酶存在于几乎所有生物体中。它们在自然界中的普遍存在使人们对它们的结构和作用机制进行了广泛的研究；因此，人们将注意力转向它们作为化学合成的催化剂。P450催化多种有机底物的氧化，其标志性的转化是C-H键的羟基化。在仅使用分子氧和NADPH的情况下，乙醇产物通过酶活性中心内的C-H活化形成。活性中心的性质使其有可能进入未活化的位置，并为不对称羟化提供了机会。铁锈醇是一种松烷二萜，其研究工作将继续进行，关键的MP450氧化是兴趣的转化。碳骨架将针对一组P450突变体进行筛选，该突变体针对的是一种执行必要的芳香族氧化的酶。将在外消旋混合物和单独的对映体上尝试氧化，以测试动力学拆分。有了MP450进行正确的氧化，其他阿拉伯烷天然产物就可以成为目标，以扩大酶的范围。此外，二萜类的刺五加红素和紫杉醇衍生物‘紫杉二酮’也将成为目标。

This project falls within the EPSRC research themes: Healthcare Technologies (related: synthetic biology, synthetic organic chemistry); Physical Sciences (related: catalysis, synthetic biology, synthetic chemistry); Manufacturing the Future (related: manufacturing technologies).This project will concentrate on the synthesis of a range of fragments and natural products via late stage oxidation using a library of mutant cytochrome P450 enzymes. This approach, by introducing oxy-functionality at the end of the synthesis, allows the focus to be on the skeleton-building process, which alleviates the need for protecting groups and makes the route more efficient and environmentally friendly. The use of P450s to effect asymmetric oxidation will be explored, and the use of these enzymes on a range of subtrates will help to build a profile of reactivity, moving these mutants closer to application as general oxidation catalysts.Late stage functionalisation has emerged as an efficient method of chemical synthesis. It allows the synthetic route to focus on skeleton-building without the need for a protecting group strategy. Hydroxylation is of great interest with the field of late stage functionalisation due to the ubiquity of oxy-functionality in both natural products and drug molecules.Recent work on late stage hydroxylation has tended to use chemical catalysts to achieve these transformations which, while practicable, can be expensive and difficult to dispose of. This project will develop a different catalytic method, one which utilises a library of mutant P450 (mP450) enzymes. These enzymes can be evolved and tailored so the desired reactivity can be developed, including activity complementary to current chemical oxidation methods.Cytochrome P450BM3 (CYP102A1) belongs to the P450 cytochrome (CYP) superfamily of haemproteins, found in almost every organism as oxidase enzymes. Their ubiquity in Nature has led to extensive study into their structure and mechanism of action; as a result, attention has turned to their use as catalysts for chemical synthesis. P450s catalyse the oxidation of a wide range of organic substrates and their signature transformation is the hydroxylation of C-H bonds. Using only molecular oxygen and NADPH, alcohol products are formed via C-H activation within the enzyme active site. The nature of the active site makes it possible to access un-activated positions and gives the opportunity for asymmetric hydroxylation.Work towards ferruginol, an abietane diterpene, will continue, with the key mP450 oxidation being the conversion of interest. The carbon skeleton will be screened against a panel of P450 mutants targeting an enzyme that carries out the necessary aromatic oxidation. The oxidation will be attempted on both the racemic mixture and the separate enantiomers in order to test for kinetic resolution. With a mP450 in hand which carries out the correct oxidation, other abietane natural products could be targeted to expand the scope of the enzyme. In addition, the diterpene eleutherobin and taxol derivative 'taxadienone' will also be targeted.