The selective hydroxylation of benzene to phenol, without the formation of side products resulting from overoxidation, is catalyzed by cytochrome P450BM3 with the assistance of amino acid derivatives as decoy molecules. The catalytic turnover rate and the total turnover number reached 259 min P450BM3 and 40 200 P450BM3 when N-heptyl-l-proline modified with l-phenylalanine (C7-l-Pro-l-Phe) was used as the decoy molecule. This work shows that amino acid derivatives with a totally different structure from fatty acids can be used as decoy molecules for aromatic hydroxylation by wild-type P450BM3. This method for non-native substrate hydroxylation by wild-type P450BM3 has the potential to expand the utility of P450BM3 for biotransformations.

Shorter-alkyl-chain fatty acids such as tridecanoic acid or lauric acid were produced from myristic acid by CYP152 peroxygenases.

Highly effective dipeptidic decoy molecules, which stimulate the direct hydroxylation of benzene by wild-type cytochrome P450BM3, were successfully developed through a rationally designed screening method. Extensive synthesis and step-wise screening of over 600 dipeptide derivatives were performed for the efficient evolution of decoy molecules. In the presence of N-(3-cyclopentyl)propanoyl-L-pipecolyl-L-phenylalanine (3CPPA-Pip-Phe), one of the most effective decoy molecules discovered herein, the catalytic turnover frequency and total turnover number for benzene hydroxylation reached 405 min −1 P450BM3 −1 and 54,500 P450BM3 −1 , respectively. Furthermore, the decoy molecules developed in this work drastically accelerated the hydroxylation of other non-native substrates, such as anisole and toluene, as well as nonaromatic compounds, such as cyclohexane, propane, and ethane. Using Nenanthoyl-L-pipecolyl-L-phenylalanine (C7AM-Pip-Phe), the hydroxylation rate for ethane to ethanol reached 82.7 min −1 P450BM3 −1 .

The benzylic hydroxylation of non-native substrates was catalysed by cytochrome P450BM3, wherein “decoy molecules” controlled the stereoselectivity of the reactions.