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

Abstract

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.

An Enzymatic Route to α‐Tocopherol Synthons: Aromatic Hydroxylation of Pseudocumene and Mesitylene with P450 BM3

“…Assuming the binding modes for 1 a (Figures and ) are in good correlation with the catalytic data, we analyzed product patterns and alterations in chemoselectivity between P450 BM3 WT and variant M3. It is accepted that aromatic hydroxylation of benzenes proceeds through epoxidation of the aromatic ring followed by epoxide ring opening, NIH shift, and rearomatization to the respective phenol (Scheme A) . In addition, methyl‐group shifts were previously observed during the conversion of o ‐xylene with P450 BM3 .…”

A hydroquinone-specific screening system for directed P450 evolution

“…As a first starting variant, we used P450 BM3 AW1 (R47Q, Y51F, I401M) and saturated position 330 to all 20 canonical amino acids. Position 330 was reported to have an influence on the activity of P450 BM3 for aromatic hydroxylations (Dennig et al 2017 ; Munday et al 2017 ). We screened in total 180 variants (theoretical calculated > 95% diversity coverage) (Firth and Patrick 2008 ) with the NpCN assay (Fig.…”

“…Furthermore, quinic acid was produced from glucose in Escherichia coli which was subsequent oxidized to HQ (Ran et al 2001 ). Recently, the heme-dependent monooxygenase P450 BM3 has gained great interest regarding the hydroxylation of diverse benzenes (Dennig et al 2013 ; Munday et al 2017 ) and accepts a huge variety of substrates (Bernhardt 2006 ; Urlacher and Girhard 2012 ; Whitehouse et al 2012 ). P450 BM3 was reported to produce HQs from benzenes and phenols (Dennig et al 2017 ; Sulistyaningdyah et al 2005 ).…”

The Oxidation of Hydrophobic Aromatic Substrates by Using a Variant of the P450 Monooxygenase CYP101B1

“…The two products common to both enzymes were 1‐(3‐methylphenyl)ethanol ( m / z 136.05) and (3‐ethylphenyl)methanol ( m / z 136.05) with the latter being formed in excess in both cases (Scheme ). The additional minor product found in the WT turnover (14 % of total product) was characterised by NMR and MS analysis as 2‐ethyl‐4‐methylphenol ( m / z 136.05, Scheme , Figures S4 and S5 c) …”

“…Isobutylbenzene hydroxylation was shifted to the tertiary β‐carbon; the branching in the alkyl chain must alter the binding orientation of the substrate in the vicinity of the heme iron. The oxidation of o ‐xylene and related substrates occurred without any observation of products arising from a 1,2‐shift and minimal desaturation of alkyl chains (<1 %); reactions that occur with CYP102A1 ,. The ability of CYP101B1 to favour hydroxylation at less reactive positions in certain substrates could make it a useful for future synthetic applications.…”