| Summary: | <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>
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