Cyclic amine functionalisation by engineered cytochrome P450BM3
<p>Cytochrome P450 enzymes are known for their characteristic activity of C–H bond oxy-functionalisation whereby one oxygen atom of atmospheric dioxygen is inserted in an available carbon-hydrogen bond of their substrates, such as drugs and natural products, to form the alcohol derivative. Cyt...
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| Materyal Türü: | Tez |
| Dil: | English |
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2020
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| _version_ | 1826314493812015104 |
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| author | Li, Y |
| author2 | Wong, L |
| author_facet | Wong, L Li, Y |
| author_sort | Li, Y |
| collection | OXFORD |
| description | <p>Cytochrome P450 enzymes are known for their characteristic activity of C–H bond oxy-functionalisation whereby one oxygen atom of atmospheric dioxygen is inserted in an available carbon-hydrogen bond of their substrates, such as drugs and natural products, to form the alcohol derivative. Cytochrome P450<sub>BM3</sub> from Bacillus megaterium has been extensively engineered to develop biocatalytic pathways to bioactive compounds due to its self-sufficiency, high expression level and promiscuity in substrate recognition. In this thesis, a high proportion of available carbon-hydrogen bonds in cyclic amines and lactams have been shown to be oxidised by engineered P450<sub>BM3</sub> variants with high turnover and reasonable regioselectivity (Figure 1). A combination of substrate engineering via introducing substituents (methoxy and methyl), N-protecting groups (phenyl, benzyl, acyl, Boc, mesyl, tosyl, and isopropylsulfonyl) and auxiliary carbocycles (cyclopentane and cycloheptane), and protein engineering by site-directed and site-saturation mutagenesis at active site residues identified by screening with a 48 P450<sub>BM3</sub> variant library of diverse substrate pocket topology and polarity, have enabled the oxidative diversification of these core motifs in numerous FDA-approved drugs. Unusual activities of P450<sub>BM3</sub> – the Povarov reaction with N-phenyl cyclic amines and N1-C8' coupling (amination) of 6-methoxy-1,2,3,4-tetrahydroquinoline – were also discovered. Biotransformation in vivo and in vitro of these substrates reached 1 g/L/day with 20 mM substrate conversion at turnover numbers of up to 15,000. The research has provided the basis for developing alternative routes to hydroxy and multi-functionalised cyclic amines and lactams, introducing synthetic handles for functional group elaborations of such compounds which are frequently used in fragment-based drug discovery.</p> |
| first_indexed | 2024-03-06T22:44:29Z |
| format | Thesis |
| id | oxford-uuid:5cae742f-b082-4c7c-9cbd-2e4ca3024801 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-09-25T04:33:22Z |
| publishDate | 2020 |
| record_format | dspace |
| spelling | oxford-uuid:5cae742f-b082-4c7c-9cbd-2e4ca30248012024-09-10T08:12:09ZCyclic amine functionalisation by engineered cytochrome P450BM3Thesishttp://purl.org/coar/resource_type/c_db06uuid:5cae742f-b082-4c7c-9cbd-2e4ca3024801Bioinorganic chemistryorganic chemistryenzymologyBioengineeringEnglishHyrax Deposit2020Li, YWong, L<p>Cytochrome P450 enzymes are known for their characteristic activity of C–H bond oxy-functionalisation whereby one oxygen atom of atmospheric dioxygen is inserted in an available carbon-hydrogen bond of their substrates, such as drugs and natural products, to form the alcohol derivative. Cytochrome P450<sub>BM3</sub> from Bacillus megaterium has been extensively engineered to develop biocatalytic pathways to bioactive compounds due to its self-sufficiency, high expression level and promiscuity in substrate recognition. In this thesis, a high proportion of available carbon-hydrogen bonds in cyclic amines and lactams have been shown to be oxidised by engineered P450<sub>BM3</sub> variants with high turnover and reasonable regioselectivity (Figure 1). A combination of substrate engineering via introducing substituents (methoxy and methyl), N-protecting groups (phenyl, benzyl, acyl, Boc, mesyl, tosyl, and isopropylsulfonyl) and auxiliary carbocycles (cyclopentane and cycloheptane), and protein engineering by site-directed and site-saturation mutagenesis at active site residues identified by screening with a 48 P450<sub>BM3</sub> variant library of diverse substrate pocket topology and polarity, have enabled the oxidative diversification of these core motifs in numerous FDA-approved drugs. Unusual activities of P450<sub>BM3</sub> – the Povarov reaction with N-phenyl cyclic amines and N1-C8' coupling (amination) of 6-methoxy-1,2,3,4-tetrahydroquinoline – were also discovered. Biotransformation in vivo and in vitro of these substrates reached 1 g/L/day with 20 mM substrate conversion at turnover numbers of up to 15,000. The research has provided the basis for developing alternative routes to hydroxy and multi-functionalised cyclic amines and lactams, introducing synthetic handles for functional group elaborations of such compounds which are frequently used in fragment-based drug discovery.</p> |
| spellingShingle | Bioinorganic chemistry organic chemistry enzymology Bioengineering Li, Y Cyclic amine functionalisation by engineered cytochrome P450BM3 |
| title | Cyclic amine functionalisation by engineered cytochrome P450BM3 |
| title_full | Cyclic amine functionalisation by engineered cytochrome P450BM3 |
| title_fullStr | Cyclic amine functionalisation by engineered cytochrome P450BM3 |
| title_full_unstemmed | Cyclic amine functionalisation by engineered cytochrome P450BM3 |
| title_short | Cyclic amine functionalisation by engineered cytochrome P450BM3 |
| title_sort | cyclic amine functionalisation by engineered cytochrome p450bm3 |
| topic | Bioinorganic chemistry organic chemistry enzymology Bioengineering |
| work_keys_str_mv | AT liy cyclicaminefunctionalisationbyengineeredcytochromep450bm3 |