Examination of Selectivity in the Oxidation of ortho‐ and meta‐Disubstituted Benzenes by CYP102A1 (P450 Bm3) Variants

• Published in: ChemCatChem 2017-07, Vol.9 (13), p.2512-2522
• Main Authors: Munday, Samuel D., Dezvarei, Shaghayegh, Lau, Ian C.‐K., Bell, Stephen G.
• Format: Article
• Language: English
• Subjects: Alkenes; arenes; Benzene; biocatalysis; Electric power distribution; enzyme catalysis; Epoxidation; Metabolites; Migration; mutagenesis; Oxidation; Selectivity
• ISSN: 1867-3880; 1867-3899
• DOI: 10.1002/cctc.201700116

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. Multiple product choices: Cytochrome P450 CYP102A1 (P450 Bm3) variants are used to catalyze the oxidation of ortho‐and meta‐disubstituted benzenes to a variety of products, with the selective formation of 4‐methyl‐2‐ethylphenol from 3‐ethyltoluene, migration of substituents, and the dearomatization product 6‐ethyl‐6‐methylcyclohex‐2,4‐dienone.