Applications of Enzymatic C-H Oxidation in Alkaloid Synthesis

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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.