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

Aromatic hydroxylation of pseudocumene (1 a) and mesitylene (1 b) with P450 BM3 yields key phenolic building blocks for α‐tocopherol synthesis. The P450 BM3 wild‐type (WT) catalyzed selective aromatic hydroxylation of 1 b (94 %), whereas 1 a was hydroxylated to a large extent on benzylic positions (46–64 %). Site‐saturation mutagenesis generated a new P450 BM3 mutant, herein named “variant M3” (R47S, Y51W, A330F, I401M), with significantly increased coupling efficiency (3‐ to 8‐fold) and activity (75‐ to 230‐fold) for the conversion of 1 a and 1 b. Additional π–π interactions introduced by mutation A330F improved not only productivity and coupling efficiency, but also selectivity toward aromatic hydroxylation of 1 a (61 to 75 %). Under continuous nicotinamide adenine dinucleotide phosphate recycling, the novel P450 BM3 variant M3 was able to produce the key tocopherol precursor trimethylhydroquinone (3 a; 35 % selectivity; 0.18 mg mL−1) directly from 1 a. In the case of 1 b, overoxidation leads to dearomatization and the formation of a valuable p‐quinol synthon that can directly serve as an educt for the synthesis of 3 a. Detailed product pattern analysis, substrate docking, and mechanistic considerations support the hypothesis that 1 a binds in an inverted orientation in the active site of P450 BM3 WT, relative to P450 BM3 variant M3, to allow this change in chemoselectivity. This study provides an enzymatic route to key phenolic synthons for α‐tocopherols and the first catalytic and mechanistic insights into direct aromatic hydroxylation and dearomatization of trimethylbenzenes with O2. Supplementing production: Trimethylphenols represent key synthons for α‐tocopherols and α‐tocotrienols (vitamin E), which find broad applications, but natural sources of vitamin E do not cover the annual demand. Aromatic hydroxylation of pseudocumene and mesitylene with P450 BM3 yields key phenolic building blocks for α‐tocopherol synthesis (see scheme). Site‐saturation mutagenesis generated a new P450 BM3 mutant with significantly increased coupling efficiency and activity.