Received for publication June 30, 2004.
Revised January 4, 2005.
Accepted for publication January 5, 2005.

Structural and Enzymatic Parameters that Determine Alkyl Dehydrogenation/Hydroxylation of Capsaicinoids by P450 Enzymes

Abstract

Previous studies on the metabolism of capsaicinoids, natural products isolated from chili peppers, demonstrated the production of unique macrocyclic, alkyl dehydrogenated, -, and -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 analogues and cytochrome P450 enzymes. CYP2C9 preferentially catalyzed alkyl dehydrogenation while CYP2E1 and 3A4 catalyzed - and -1 hydroxylation, respectively. Analysis of incubations containing various P450s and structural variants of capsaicin by LC/MS/MS demonstrated similarities in the rate of capsaicinoid metabolism, but marked differences in the formation of specific metabolites. Production of macrocyclic and -1-hydroxylated metabolites from the various capsaicinoids was dependent on the structure of the alkyl terminus and P450 enzyme. A tertiary carbon at the -1 position, coupled to an adjacent unsaturated bond at the -2,3 position, enhanced the formation of the macrocyclic and dehydrogenated metabolites, and were requisite structural features for -1-hydroxylated product formation. Conversely, substrates lacking these structural features were efficiently oxidized to the -hydroxylated metabolite. These data were consistent with our working hypothesis that the metabolism of the alkyl portion of capsaicinoids was governed, in part, by the stability, and propensity to form an intermediate radical, and an "end-on" interaction with the iron-oxo active site of most P450 enzymes. These results provided valuable insights into the mechanisms by which P450s metabolize capsaicinoids and highlight critical chemical features that may also govern the metabolism of structurally related compounds including fatty acids, monoterpenes, and isoprenoids.

Key words: analytical pharmacology/toxicology, cell injury, CYP2C, CYP2E, CYP3A, cytochrome P450, human CYP enzymes, mass spectrometry, respiratory toxicants

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