Highly Selective Hydroxylation of Benzene to Phenol by Wild‐type Cytochrome P450BM3 Assisted by Decoy Molecules

Abstract

Phenol is a key intermediate in industry for the synthesis of drugs, dyes, and functional polymers. Because phenol is currently produced by the cumene process, which involves high energy consumption and significant formation of side products such as acetone and methylstyrene, direct hydroxylation of benzene has attracted much attention as an alternative method of production. The direct hydroxylation of benzene to phenol using a variety of catalysts, electrochemical oxidation systems, and photochemical systems has thus been extensively investigated. In contrast to many oxidation reactions at high temperature, monooxygenases in nature, such as cytochrome P450, efficiently catalyze the oxidation of inert alkanes and aromatic compounds under mild conditions. Thus, various engineered enzymes have been constructed by site-directed mutagenesis, random mutagenesis, and chemical modification. Biocatalysts for exclusive hydroxylation of the benzene ring using natural enzymes, if they could be developed, would be ideal systems for the production of phenol. Herein we report an efficient and selective hydroxylation of benzene to phenol catalyzed by wild-type cytochrome P450BM3 (P450BM3) with the assistance of decoy molecules. We and Zilly et al. have recently developed a simple and unique system for the hydroxylation of gaseous alkanes, such as propane and butane, catalyzed by wild-type P450BM3. Wild-type P450BM3 exclusively catalyzes the hydroxylation of long-alkyl-chain fatty acids and never hydroxylates small alkanes, because the active site of P450BM3 is optimized for the hydroxylation of fatty acids (Figure 1a, upper) and the first step of the catalytic cycle of P450BM3 starts only when a fatty acid binds to the substrate binding site of P450BM3, which is accompanied by the removal of the water molecule coordinated to the heme iron (Figure 1b, left). However, in the presence of perfluorinated carboxylic acids (PFCs) as inert dummy substrates (decoy molecules), gaseous alkanes can be hydroxylated by wild-type P450BM3. The decoy molecules initiate the activation of molecular oxygen in the same manner as long-alkyl-chain fatty acids and induce the generation of compound I (Figure 1b, right). Because the C F bonds of PFCs are not oxidizable, compound I exclusively hydroxylates gaseous alkanes. Moreover, shortalkyl-chain PFCs partially occupy the substrate binding site of P450BM3 to afford a space for small alkanes, which leads to efficient hydroxylation of the small alkanes (Figure 1a, lower). This attractive advantage encouraged us to perform benzene hydroxylation for the selective production of phenol. The hydroxylation of benzene by P450BM3 was examined by employing a series of PFCs (PFC8–PFC12; Table 1). The catalytic turnover rates of phenol formation were very much