Applications of Enzymatic C-H Oxidation in Alkaloid Synthesis

该项目属于EPSRC新合成方法和天然产物合成研究领域。对映体纯醇是合成用于制药和农化工业的底物的关键中间体。为了生产这些有价值的手性构建块，后期官能化是一种有效的化学合成方法，因为它允许合成路线专注于骨架构建，而不需要保护基团策略。以前在这一领域的工作分两个阶段完成天然产物的全合成：组装碳环核心的环化酶阶段，随后是增加所有必要的氧官能团的氧化物阶段。后期羟化倾向于使用化学催化剂来实现氧化转化，这通常使用苛刻的条件，产生不良的副产物，并以低产率产生外消旋结果。本项目旨在利用突变的P450BM3酶文库探索氧化物酶的方法。大约50年前发现的细胞色素P450是一种血液蛋白，可以催化大量不同的生物反应，包括生物合成、降解和生物代谢产物的解毒。它们最常见的催化反应是C-H键的氧化，这允许进入传统化学试剂不容易到达的未活化位置。此外，P450酶可以通过优化活性部位的氨基酸残基来进化以实现所需的反应活性。事实上，细胞色素P450是生物合成手性底物的首选酶，因为它们具有广泛的底物范围、出色的官能团耐受性、多样化的产品选择性和高转化率，最大限度地增加了进一步选择性优化的初始命中可能性。由于反应条件温和和显著的区域和立体选择性，氧化酶的使用是传统化学方法的一种有效和环境友好的替代方法。本项目探索了工程P450BM3突变体作为受保护胺的一般立体和区域选择性氧化剂的使用，以及通过这种方法获得的底物在天然产物合成中的使用。在广泛的底物上使用P450BM3酶将增加对酶在面对不同底物时的活性和选择性的了解，有助于建立反应性图谱，并使这些突变体更接近作为通用氧化催化剂的应用。对于第一个目标天然产物，山梨碱将从托品酮起始物质合成。从这里，重点将转移到研究双环和三环内酰胺，选择是因为氮本身就不会被氧化或P450失活，而且如果需要，羰基为α-位的功能化提供了一个有用的句柄。这种天然产物的合成将遵循两个阶段的过程：构建环核，然后根据P450BM3突变体文库筛选三环内酰胺，并鉴定选择性引入氧的突变体。如果以外消旋的方式合成碳环骨架，就有可能利用P450突变体的能力来实现对映体的动力学拆分，以提供所需的镜像形式。Phlemadine A是一种具有不同结构的石杉生物碱，包括四元环和九元环。同样，在筛选P450BM3突变体之前，将创建碳骨架，以确定那些具有所需反应活性的突变体。由于所需的氧化反应是在烯丙基位置进行的，因此将酶催化氧化反应与化学试剂所能达到的氧化反应进行比较是很重要的。

This project falls within the EPSRC New Synthetic Methods and Natural Product Synthesis research areas.Enantiomerically pure alcohols are key intermediates in the synthesis of substrates used in the pharmaceutical and agrochemical industries. To produce these valuable chiral building blocks, late stage functionalisation is an efficient method of chemical synthesis, since it allows the synthetic route to focus on skeleton-building without the need for a protecting group strategy. Previous work in this area accomplishes the total synthesis of natural product in two phases: a cyclase phase which assembles the carbocyclic core, followed by an oxidase phase which adds all the necessary oxygen functionality. Late stage hydroxylation tends to use chemical catalysts to achieve the oxidative transformation which typically use harsh conditions, produce undesirable side-products and give racemic outcomes in low yields. This project intends to explore enzymatic methods for the oxidase phase, using a library of mutant P450BM3 enzymes.Discovered about 50 years ago, cytochromes P450 are haemoproteins which catalyse a huge number of diverse biological reactions, including biosynthesis, degradation and detoxification of biological metabolites. Their most commonly catalysed reaction is the oxidation of C-H bonds, which allows access to unactivated sites that could not easily be reached by conventional chemical reagents. Furthermore, P450 enzymes can be evolved to achieve the desired reactivity by optimising the amino acid residues in the active site. Indeed, cytochromes P450 are choice enzymes for biosynthesis of chiral substrate because they have a broad substrate range, excellent functional group tolerance, diverse product selectivity and high conversion, maximising the likelihood of initial hits for further selectivity optimisation. The use of oxidative enzymes is an effective and environmentally benign alternative to traditional chemical methods due to the mild reaction conditions and notable regio- and stereoselectivity.This project explores the use of engineered P450BM3 mutants as general stereo- and regioselective oxidants for protected amines, and the use of substrates achieved by this means in the synthesis of natural products. The use of P450BM3 enzymes on a wide range of substrates will increase knowledge of the enzymes' activity and selectivity when faced with diverse substrates, helping to build a reactivity profile and moving these mutants closer to application as general oxidation catalysts.For the first target natural product, anisodamine will be synthesised from the tropinone starting material. From here, the focus will shift to study bi- and tri-cyclic lactams, chosen because the nitrogen is inherently protected against oxidation or P450-inactivation, and the carbonyl provides a useful handle for functionalisation of the alpha-position, if desired.Aspidospermidine is the parent member of the extensive class of Aspidosperma alkaloids. The synthesis of this natural product will follow the two-phase process of building the cyclic core and then screening the tricyclic lactam against the P450BM3 mutant library and identifying mutants selective for introduction of oxygen. Should the carbocyclic skeleton be synthesised in a racemic manner, there is potential for advantage to be taken of the P450 mutants' ability to effect kinetic resolution of the enantiomers to deliver the desired mirror image form.Phlegmadine A is a Lycopodium alkaloid with an unusual structure, including both four- and a nine- membered rings. Again, the carbon skeleton would be created before screening P450BM3 mutants to identify those that give the desired reactivity. Since the required oxidations are at allylic positions, it will be important to compare the enzymatic oxidation profiles with those achievable with chemical reagents.