Applications of Enzymatic C-H Oxidation in Alkaloid Synthesis

该项目福尔斯属于EPSRC新合成方法和天然产物合成研究领域。对映体纯醇是合成药物和农业化学工业中使用的底物的关键中间体。为了生产这些有价值的手性结构单元，后期官能化是化学合成的有效方法，因为它允许合成路线集中于手性构建而不需要保护基策略。以前在这一领域的工作完成了天然产物的全合成分两个阶段：环化酶阶段组装碳环核心，然后是氧化酶阶段添加所有必要的氧功能。后期羟基化倾向于使用化学催化剂来实现氧化转化，其通常使用苛刻的条件，产生不期望的副产物并以低收率得到外消旋产物。细胞色素P450是50年前发现的一种血红蛋白，它催化大量不同的生物反应，包括生物代谢物的合成、降解和解毒。它们最常见的催化反应是C-H键的氧化，这使得能够进入常规化学试剂无法轻易到达的未活化位点。此外，P450酶可以通过优化活性位点中的氨基酸残基来进化以实现所需的反应性。事实上，细胞色素P450是用于手性底物生物合成的选择酶，因为它们具有广泛的底物范围、优异的官能团耐受性、多样的产物选择性和高转化率，最大化了初始命中的可能性以用于进一步的选择性优化。氧化酶的使用是一种有效的和环境友好的替代传统的化学方法，由于温和的反应条件和显着的区域和立体选择性。本项目探讨了使用工程P450 BM 3突变体作为一般的立体和区域选择性氧化剂保护胺，并使用通过这种手段在天然产物的合成中实现的底物。P450 BM 3酶在广泛底物上的应用将增加对酶在面对不同底物时的活性和选择性的了解，有助于建立反应性特征，并使这些突变体更接近于作为一般氧化催化剂的应用。对于第一个目标天然产物，山莨菪碱将从托品酮起始原料合成。从这里，重点将转移到研究双环和三环内酰胺，选择，因为氮是固有的保护，防止氧化或P450失活，和羰基提供了一个有用的处理功能的α-位置，如果需要。Aspidospermidine是父级成员的广泛类Aspidosperma生物碱。这种天然产物的合成将遵循两阶段过程，即构建环状核心，然后针对P450 BM 3突变体文库筛选三环内酰胺，并鉴定对引入氧具有选择性的突变体。如果以外消旋方式合成碳环骨架，则有可能利用P450突变体实现对映异构体动力学拆分的能力，以提供所需的镜像形式。同样，在筛选P450 BM 3突变体以鉴定那些提供所需反应性的突变体之前，将创建碳骨架。由于所需的氧化是在烯丙基位置，这将是重要的比较酶促氧化与化学试剂可实现的。

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.