Systematic Evolution of Decoy Molecules for the Highly Efficient Hydroxylation of Benzene and Small Alkanes Catalyzed by Wild-Type Cytochrome P450BM3

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 N-enanthoyl-l-pipecolyl-l-phenylalanine (C7AM-Pip-Phe), the hydroxylation rate for ethane to ethanol reached 82.7 min^–1 P450BM3^–1.