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

Abstract

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 ) 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 O .

A tailor‐made, self‐sufficient and recyclable monooxygenase catalyst based on coimmobilized cytochrome P450 BM3 and glucose dehydrogenase

“…Nonetheless, a (TTN; mol product per mole enzyme ) of 2,220 was calculated for immobilized Z_P450 BM3 from these experiments. This is a useful value for conversion of a nonnatural substrate (Dennig et al, , Dennig, Busto, Kroutil, & Faber, , Dennig et al, ; Whitehouse et al, ), in particular considering that no further optimization of catalyst or reaction engineering was done. Assuming a coupling efficiency of 50%, we calculate that NADP + (0.8 mM) was regenerated at least 10 times in the reaction.…”

Biocatalytic Oxidation Reactions: A Chemist's Perspective

“…Recent advances in (semi-)rational enzyme engineering, however, demonstrate that this issue can be solved. [39] With peroxygenases, [40] further conversion of the phenol products into phenoxy radicals (and further radical reactions) represents an undesired side reaction, which can be circumvented either by co-administration of antioxidants or, more elegantly,byp rotein engineering. [41] Dioxygenases (DOs,E .C.1.13.11) are very powerful catalysts for the selective cis dihydroxylation of aromatic compounds.S everal hundreds of DO products have been reported.…”

Mimochrome, a metalloporphyrin‐based catalytic Swiss knife†

“…All these reactions occur through carbene or nitrene transfer from an electrophilic heme-carbenoid or -nitrenoid intermediate to a suitable nucleophilic substrate, following a mechanism that resembles the oxygenation of the same substrates mediated by C-I [149]. Directed evolution has also been applied to alter regio-and stereo-selectivity of substrate oxygenation [150][151][152] and fluorination [153], affording artificial biocatalysts for the enzymatic total synthesis of complex molecules [154,155].…”