Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.820799
Title: Cyclic amine functionalisation by engineered cytochrome P450BM3
Author: Li, Yushu
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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Abstract:
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 P450BM3 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 P450BM3 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 P450BM3 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 P450BM3 – 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.
Supervisor: Wong, Luet Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.820799  DOI: Not available
Keywords: organic chemistry ; enzymology ; Bioinorganic chemistry ; Bioengineering
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