STEREOSELECTIVE EPOXIDATION OF ARACHIDONIC-ACID BY CYTOCHROME P-450S 2CAA AND 2C2

In the present study, we determined the stereoselective epoxidation of arachidonic acid by cytochrome P-450 (P-450) 2CAA and P-450 2C2, two arachidonic acid epoxygenases found in rabbit renal cortex, by chiral normal-phase high-performance liquid chromatography (HPLC)-analysis. P urified P-450 2CAA reconstituted with P-450 oxidoreductase, lipid and cytochrome b(5) or microsomes isolated from COS-1 cells expressing P-4 50 2C2 were incubated in the presence of [1-C-14]arachidonic acid. The epoxide metabolites 14,15- and 11,72-epoxyeicosatrienoic acids (EETs) were purified by reverse-phase HPLC and derivatized to methyl (14,15- EET) and pentafluorobenzyl (11,12-EET) esters. Enantiomers of 14,15-EE T-methyl ester and 11,12-EET-pentafluorobenzyl ester were resolved on Chiralcel OB and OD columns, respectively, with a mobile phase of 0.15 % 2-propanol in n-hexane. P-450 2CAA and P-450 2C2 produce 11,12- and 14,15-EETs in distinct ratios but are equally stereoselective at the 1 1,12- position. P-450 2CAA produced 11(S),12(R)-EET with 63% stereosel ectivity, and P-450 2C2 produced the same enantiomer with 61% stereose lectivity. Both enzymes are also stereoselective at the 14,15- positio n, preferentially producing the 14(R),15(S)-EET. P-450 2CAA produces t his enantiomer with 72% selectivity, and P-450 2C2 produces it with 62 % selectivity. The results of this study indicate that P-450 2CAA and P-450 2C2 are not only regioselective but also exhibit a high degree o f stereoselectivity.