Cytochrome P450 CYP102A1 (P450 Bm3) variants were used to investigate the products arising from the P450 catalysed oxidation of a range of disubstituted benzenes. The variants used all generated increased levels of metabolites compared to the wild‐type enzyme. With ortho‐halotoluenes up to six different metabolites could be identified whereas the oxidation of 2‐methoxytoluene generated only two aromatic oxidation products. Addition of an ethyl group markedly shifted the selectivity for oxidation to the more reactive benzylic position. Epoxidation of an alkene was also preferred to aromatic oxidation in 2‐methylstyrene. Significant minor products arising from the migration of one substituent to a different position on the benzene ring were formed during certain P450‐catalysed substrate turnovers. For example, 2‐bromo‐6‐methylphenol was formed from the turnover of 2‐bromotoluene and the dearomatisation product 6‐ethyl‐6‐methylcyclohex‐2,4‐dienone was generated from the oxidation of 2‐ethyltoluene. The RLYF/A330P variant altered the product distribution enabling the generation of certain metabolites in higher quantities. Using this variant produced 4‐methyl‐2‐ethylphenol from 3‐ethyltoluene with ≥90 % selectivity and with a biocatalytic activity suitable for scale‐up of the reaction.

The bacterial CYP199A4 enzyme is able to oxidise a narrow range of aromatic acids, which includes 4‐methoxybenzoic acid, efficiently. A serine 244 to aspartate variant was identified with enhanced activity for a wide range of para‐methoxy‐substituted benzenes. Substrates in which the acidic benzoic acid moiety is replaced with a phenol and the amide, aldehyde and bromide analogues were all oxidised with high activity by the S244D mutant (product formation rate >600 nmol nmolCYP−1 min−1) with turnover numbers of up to 20 000. If the carboxylate moiety was modified to a nitro, ketone, boronic acid, hydroxymethyl or nitrile group, these substrates were also oxidised at a significantly higher activity by S244D than the wild‐type enzyme. 3,4‐Dimethoxybenzaldehyde was demethylated selectively and oxidatively to 3‐methoxy‐4‐hydroxybenzaldehyde by the S244D mutant 84‐fold more rapidly than with the wild‐type enzyme. CYP199A4 would have applications in the catalytic regioselective oxidative demethylation of suitably substituted benzene substrates under mild conditions and in the presence of more oxidatively sensitive functional groups, such as an aldehyde.

The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates > 60 nmol.(nmol-CYP) À 1 .min À 1 ) and with total turnover numbers of up to 20,000. Ethyl αhydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to > 98 %). The crystal structure of 4-methoxybenzoic acidbound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.

Analysis and preliminary characterisation of the cytochrome P450 monooxygenases from Frankia sp. EuI1c (Frankia inefficax sp.

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