Abstract

Previous studies on the metabolism of capsaicinoids, natural products isolated from chili peppers, demonstrated the production of unique macrocyclic, alkyl dehydrogenated, ω-, and ω-1-hydroxylated products. This study investigated the structural and enzymatic parameters that direct selective alkyl dehydrogenation and hydroxylation of capsaicinoids, using a variety of structurally related capsaicinoid analogs and cytochrome P450 (P450) enzymes. CYP2C9 preferentially catalyzed alkyl dehydrogenation, whereas CYP2E1 and 3A4 catalyzed ω- and ω-1-hydroxylation, respectively. Analysis of incubations containing various P450s and structural variants of capsaicin by liquid chromatography-tandem mass spectrometry demonstrated similarities in the rate of capsaicinoid metabolism, but marked differences in the metabolite profiles. Production of macrocyclic and ω-1-hydroxylated metabolites from the various capsaicinoids was dependent on the structure of the alkyl terminus and P450 enzyme. A tertiary carbon at the ω-1 position, coupled to an adjacent unsaturated bond at the ω-2,3 position, enhanced the formation of the macrocyclic and dehydrogenated metabolites and were requisite structural features for ω-1-hydroxylated product formation. Conversely, substrates lacking these structural features were efficiently oxidized to the ω-hydroxylated metabolite. These data were consistent with our hypothesis that metabolism of the alkyl portion of capsaicinoids was governed, in part, by the stability and propensity to form an intermediate radical and a carbocation, and a direct interaction between the alkyl terminus and the heme of many P450 enzymes. These results provided valuable insights into potential mechanisms by which P450s metabolize capsaicinoids and highlight critical chemical features that may also govern the metabolism of structurally related compounds including fatty acids, monoter-penes, and isoprenoids.