Applications of Enzymatic C-H Oxidation in Alkaloid Synthesis
Lead Research Organisation:
University of Oxford
Abstract
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.
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.
Planned Impact
This programme is focused on a new cohort-driven approach to the training of next-generation doctoral scientists in the practice of novel and efficient chemical synthesis coupled with an in-depth appreciation of its application to biology and medicine.
This collaborative academic-industrial SBM CDT will have long-term benefit to the chemical industry, including the pharmaceutical, agrochemical and fine chemical sectors. These industries will benefit through: (i) the potential to employ individuals trained with broad and relevant scientific and transferable skills; (ii) new approaches to the investigation of complex biological and medical problems through novel chemistry; and (iii) better and more efficient synthetic methods.
We will link the work of DSTL, and our pharmaceutical and agrochemical partners (GSK, UCB, Vertex, Evotec, Eisai, AstraZeneca, Syngenta, Novartis, Takeda, Sumitomo and Pfizer) through a common theme of synthesis training. The design and synthesis of new compounds is essential for disease treatment and prevention, and for maintaining food security. Synthesis contributes significantly to UK tax revenue and results in sustained employment across a number of sectors. Employers in the finance, law, health, academic, analytical, government, and teaching professions, for example, also recognise the value of the translational skill-sets possessed by synthesis postgraduates, which this programme will provide.
The SBM CDT training programme will adopt an IP-free model to enable completely free exchange of information, know-how and specific expertise between students and supervisors on different projects and across different industrial companies. This will lead to better knowledge creation through unfettered access to information from all academics, partners and students involved in the project. By focussing on basic science, we will engender genuine collaboration leading to enabling technology that will be of use across a wide range of industries.
We will train the next generation of multidisciplinary synthetic chemists with an appreciation of the impact of synthesis in biology and medicine. Their unconstrained view of synthesis will aid in new scientific discoveries leading to new products, which (with appropriate inward investment), can lead to the formation of new companies and new UK employment.
We will, in part through an alliance with the Defence, Science and Technology Laboratory, engage with policy-makers to influence future policy issues involving chemistry such as food security and the rise of antibiotic resistance (both of which are relevant to our programme and are important for society as a whole).
Outreach and public engagement will be a key aspect of our programme; and all students in the proposed SBM CDT will take part in at least one outreach activity. Typical activities include: open days in the Chemistry Department through the 'Outreach Alchemists', engaging with the Oxfordshire Science Festival and participation in the various other activities already in place through the public engagement programme of the Department of Chemistry.
The research output of the students will be disseminated via high impact international publications and lectures; these will be of value to other academics in relevant fields and will be of value in the development of further research funding applications. Outreach activities and research output will also be advertised on a website dedicated to the proposed SBM programme.
This collaborative academic-industrial SBM CDT will have long-term benefit to the chemical industry, including the pharmaceutical, agrochemical and fine chemical sectors. These industries will benefit through: (i) the potential to employ individuals trained with broad and relevant scientific and transferable skills; (ii) new approaches to the investigation of complex biological and medical problems through novel chemistry; and (iii) better and more efficient synthetic methods.
We will link the work of DSTL, and our pharmaceutical and agrochemical partners (GSK, UCB, Vertex, Evotec, Eisai, AstraZeneca, Syngenta, Novartis, Takeda, Sumitomo and Pfizer) through a common theme of synthesis training. The design and synthesis of new compounds is essential for disease treatment and prevention, and for maintaining food security. Synthesis contributes significantly to UK tax revenue and results in sustained employment across a number of sectors. Employers in the finance, law, health, academic, analytical, government, and teaching professions, for example, also recognise the value of the translational skill-sets possessed by synthesis postgraduates, which this programme will provide.
The SBM CDT training programme will adopt an IP-free model to enable completely free exchange of information, know-how and specific expertise between students and supervisors on different projects and across different industrial companies. This will lead to better knowledge creation through unfettered access to information from all academics, partners and students involved in the project. By focussing on basic science, we will engender genuine collaboration leading to enabling technology that will be of use across a wide range of industries.
We will train the next generation of multidisciplinary synthetic chemists with an appreciation of the impact of synthesis in biology and medicine. Their unconstrained view of synthesis will aid in new scientific discoveries leading to new products, which (with appropriate inward investment), can lead to the formation of new companies and new UK employment.
We will, in part through an alliance with the Defence, Science and Technology Laboratory, engage with policy-makers to influence future policy issues involving chemistry such as food security and the rise of antibiotic resistance (both of which are relevant to our programme and are important for society as a whole).
Outreach and public engagement will be a key aspect of our programme; and all students in the proposed SBM CDT will take part in at least one outreach activity. Typical activities include: open days in the Chemistry Department through the 'Outreach Alchemists', engaging with the Oxfordshire Science Festival and participation in the various other activities already in place through the public engagement programme of the Department of Chemistry.
The research output of the students will be disseminated via high impact international publications and lectures; these will be of value to other academics in relevant fields and will be of value in the development of further research funding applications. Outreach activities and research output will also be advertised on a website dedicated to the proposed SBM programme.
Organisations
People |
ORCID iD |
| Jeremy Robertson (Primary Supervisor) | |
| Mary Wilson (Student) |