CYTOCHROME P450-CATALYZED HYDROXYLATION OF HYDROCARBONS - KINETIC DEUTERIUM-ISOTOPE EFFECTS FOR THE HYDROXYLATION OF AN ULTRAFAST RADICAL CLOCK

The ultrafast radical clock probe trans-1-methyl-2-phenylcyclopropane (1CH(3)) and its mono-, di-, and trideuteriomethyl analogues were oxid ized by phenobarbital-induced rat liver microsomal enzymes. This cytoc hrome P450-catalyzed hydroxylation of 1CH(3) gave three products: the alcohol trans-(2-phenylcyclopropyl)methanol (2), the rearranged alcoho l 1-phenylbut-3-en-1-ol (3), and the phenol trans-2-(p-hydroxyphenyl)- 1-methylcyclopropane (4). The identification of both the unrearranged and rearranged products of oxidation, 2 and 3, is consistent with the formation of a radical intermediate via a hydrogen atom abstraction fr om the methyl group by the catalytically active iron-oxo center. Hydro xylation of three deuteriomethyl forms of 1CH(3) produced the analogou s deuterated products, although in different amounts of each. Perdeute ration of the methyl group (1CD(3)) disfavored oxidation at the methyl group and caused an increase in the oxidation of the phenyl ring (met abolic switching). By comparing the amounts of alcohols and phenol for med from the individual, noncompetitive oxidation of 1CH(3) and 1CD(3) , the overall (i.e., combined primary and secondary) deuterium kinetic isotope effect (DKIE) was found to be 12.5. Intramolecular DKIEs for 1CHD(2) and 1CH(2)D were 2.9 and 13.2, respectively. From these result s, the primary and secondary DKIEs were calculated to be 7.87 and 1.26 , respectively, values that indicate that there is extensive C-H bond stretching in the transition state for the rate-controlling step in P4 50-catalyzed hydroxylation of 1CH(3). Microsomal incubations performed with optically enriched samples of (R,R)-1CH(3) and (S,S)-1CH(3) and with pseudoracemates composed of equal amounts of an unlabeled enantio mer and a d(3)-labeled enantiomer showed that there was a low selectiv ity for the oxidation of the (R,R) enantiomer and only a small differe nce in the regioselectivity between the two enantiomers for oxidation of the methyl groups vs the aromatic ring.