Different Geometric Requirements for Cytochrome P450-Catalyzed Aliphatic Versus Aromatic Hydroxylation Results in Chemoselective Oxidation

The cytochrome P450 enzyme, CYP199A4 from Rhodopseudomonas palustris strain HaA2, is able to oxidize para-substituted benzoic acids. This enzyme was used to compare aromatic versus aliphatic C–H bond oxidation, common reactions catalyzed by the P450 superfamily of heme monooxygenases. CYP199A4 was able to bind 4-phenylbenzoic acid and 4-cyclohexylbenzoic acid, and the crystal structures demonstrated that both substrates are bound within the active site in a similar fashion. Despite this, while 4-cyclohexylbenzoic acid was efficiently hydroxylated, no detectable enzyme catalyzed oxidation of the aromatic 4-phenylbenzoic acid was observed. The selectivity of 4-cyclohexylbenzoic acid oxidation favored C–H bond abstraction at one of the β-sites in an enantioselective fashion (66%, 95:5 er), over C–H bond abstraction at the benzylic position (33%). In addition, unlike the oxidation of smaller alkyl-substituted benzoic acids (4-ethyl- and 4-isopropyl-), little or no desaturation of the cyclohexyl ring to give an alkene was detected (∼1%). Molecular dynamics simulations suggested that the cyclohexyl ring of 4-cyclohexylbenzoic acid was able to achieve a suitable orientation to enable efficient C–H bond abstraction and oxidation by the enzyme at the expected positions. In contrast, when the distance and angle of attack were considered, the alignment of the phenyl ring of 4-phenylbenzoic acid rarely attained a productive geometry for aromatic oxidation to occur. Overall, these results illustrate the chemoselectivity that may arise due to the different geometrical requirements for efficient aromatic oxidation versus aliphatic C–H bond hydroxylation by cytochrome P450 enzymes.