An analysis of the regioselectivity of aromatic hydroxylation and N-oxygenation by cytochrome P450 enzymes.

Quinoline was used to probe the steric and electronic contributions to rates of aromatic oxidation of nitrogen-containing, multiring substrates by cytochrome P450 (P450) enzymes. The regioselectivity of the P450 oxidation of quinoline was determined experimentally by identifying and measuring the ratios of metabolites. The laboratory results were compared with those obtained computationally by modeling the electronic effects for aromatic hydroxylation of the substrate. Calculated values predict 8-hydroxyquinoline to have the lowest relative activation energy, whereas 3-hydroxyquinoline was calculated to have the highest relative activation energy. In contrast, 3-hydroxyquinoline was produced to a much greater extent relative to 8-hydroxyquinoline. The sharp contrast observed between the computationally obtained energies and the ratios of products identified experimentally indicates that steric factors play a role in determining the regioselectivity of P450 enzymes with quinoline. To further probe steric contributions to product formation, isoquinoline was used as a substrate and the results were compared with those obtained with quinoline. Isoquinoline N-oxide was determined to be the major metabolite of isoquinoline with all of the P450 enzymes used. These results provide further evidence for the steric influence on the regioselectivity of P450 enzymes with quinoline.