Understanding P450 hydroxylation profiles
<p>The enzymatic approach to C-H functionalisation is a promising field within contemporary organic chemistry that complements parallel advances with chemical reagents. The cytochrome P450<sub>BM3</sub>, refined through aeons of evolution, offers a sustainable and elegant methodol...
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| Format: | Thesis |
| Language: | English |
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2020
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| _version_ | 1826298922320003072 |
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| author | Orjonikidze, D |
| author2 | Robertson, J |
| author_facet | Robertson, J Orjonikidze, D |
| author_sort | Orjonikidze, D |
| collection | OXFORD |
| description | <p>The enzymatic approach to C-H functionalisation is a promising field within contemporary organic chemistry that complements parallel advances with chemical reagents. The cytochrome P450<sub>BM3</sub>, refined through aeons of evolution, offers a sustainable and elegant methodology for achieving synthetically valuable hydroxylations at ubiquitous C-H sites. This opens the potential for new synthetic strategies, relying on early-, mid- or late-stage C-H functionalisation. Gaining a working understanding of the regioselectivity profiles arising from such hydroxylation reactions is key to the wider adoption of this as a reliable strategy for molecular assembly.</p>
<p>This thesis describes a practical computational workflow that aims to rationalise selected experimental results obtained in the Wong and Robertson groups for the enzymatic hydroxylation of a bicyclic lactone and a spirocyclic amine derivative. The workflow was developed through three stages of increasing sophistication, starting with an investigation of the validity of an existing MD/docking protocol then building towards a stable and reproducible computational platform. The final iteration involved the preparation in silico of specific enzyme mutants based on the X-ray crystal structure of the Wild Type P450<sub>BM3</sub>, then performing molecular dynamics (MD) relaxation of these mutants to relieve the structural strain introduced by the process of mutagenesis and to account for enzyme solvation. The MD process was repeated for each mutant to produce four independent replicas that were clustered by a specified RMSD criterion for rigid ensemble docking with separate energy-minimised substrate conformers. The resulting docking poses were classified by the Fe=O•••H angle and O•••H distance, highlighting positions prone to hydrogen abstraction.</p>
<p>This approach predicted site of metabolism differences between two highly selective mutants, each of which hydroxylates one of two adjacent C-H positions of an N-protected spirocyclic amine. A new tactic for knowledge-guided mutagenesis, that eliminates the undesired binding pockets, suggested by this computational model, has enabled Wong’s group to achieve > 90% regioselectivity, > 90% stereoselectivity and > 90% conversion rates for the hydroxylation of selected N-protected cyclic amines. Potential avenues of future research are proposed and evaluated in the context of ongoing work within the groups on the further refinement and practical application of this computational methodology.</p> |
| first_indexed | 2024-03-07T04:54:07Z |
| format | Thesis |
| id | oxford-uuid:d600b708-6d1c-4b35-a396-bcdddc47392e |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:54:07Z |
| publishDate | 2020 |
| record_format | dspace |
| spelling | oxford-uuid:d600b708-6d1c-4b35-a396-bcdddc47392e2022-03-27T08:30:11ZUnderstanding P450 hydroxylation profilesThesishttp://purl.org/coar/resource_type/c_bdccuuid:d600b708-6d1c-4b35-a396-bcdddc47392eP450BM3 Som predictionEnglishHyrax Deposit2020Orjonikidze, DRobertson, J <p>The enzymatic approach to C-H functionalisation is a promising field within contemporary organic chemistry that complements parallel advances with chemical reagents. The cytochrome P450<sub>BM3</sub>, refined through aeons of evolution, offers a sustainable and elegant methodology for achieving synthetically valuable hydroxylations at ubiquitous C-H sites. This opens the potential for new synthetic strategies, relying on early-, mid- or late-stage C-H functionalisation. Gaining a working understanding of the regioselectivity profiles arising from such hydroxylation reactions is key to the wider adoption of this as a reliable strategy for molecular assembly.</p> <p>This thesis describes a practical computational workflow that aims to rationalise selected experimental results obtained in the Wong and Robertson groups for the enzymatic hydroxylation of a bicyclic lactone and a spirocyclic amine derivative. The workflow was developed through three stages of increasing sophistication, starting with an investigation of the validity of an existing MD/docking protocol then building towards a stable and reproducible computational platform. The final iteration involved the preparation in silico of specific enzyme mutants based on the X-ray crystal structure of the Wild Type P450<sub>BM3</sub>, then performing molecular dynamics (MD) relaxation of these mutants to relieve the structural strain introduced by the process of mutagenesis and to account for enzyme solvation. The MD process was repeated for each mutant to produce four independent replicas that were clustered by a specified RMSD criterion for rigid ensemble docking with separate energy-minimised substrate conformers. The resulting docking poses were classified by the Fe=O•••H angle and O•••H distance, highlighting positions prone to hydrogen abstraction.</p> <p>This approach predicted site of metabolism differences between two highly selective mutants, each of which hydroxylates one of two adjacent C-H positions of an N-protected spirocyclic amine. A new tactic for knowledge-guided mutagenesis, that eliminates the undesired binding pockets, suggested by this computational model, has enabled Wong’s group to achieve > 90% regioselectivity, > 90% stereoselectivity and > 90% conversion rates for the hydroxylation of selected N-protected cyclic amines. Potential avenues of future research are proposed and evaluated in the context of ongoing work within the groups on the further refinement and practical application of this computational methodology.</p> |
| spellingShingle | P450BM3 Som prediction Orjonikidze, D Understanding P450 hydroxylation profiles |
| title | Understanding P450 hydroxylation profiles |
| title_full | Understanding P450 hydroxylation profiles |
| title_fullStr | Understanding P450 hydroxylation profiles |
| title_full_unstemmed | Understanding P450 hydroxylation profiles |
| title_short | Understanding P450 hydroxylation profiles |
| title_sort | understanding p450 hydroxylation profiles |
| topic | P450BM3 Som prediction |
| work_keys_str_mv | AT orjonikidzed understandingp450hydroxylationprofiles |