A radical clock investigation of microsomal cytochrome P-450 hydroxylation of hydrocarbons. Rate of oxygen rebound

A number of alkyl-substituted cyclopropanes for which the rates of ring opening of the corresponding cyclopropylcarbinyl radicals have been determined (see preceding paper in this issue) have been used as substrates for hydroxylation by phenobarbital-induced, rat liver microsomal cytochrome P-450 at 37 \ub0C. Three of these compounds gave both ring-closed and ring-opened alcohols, thus allowing the rate constant, kOH, for "oxygen rebound" onto the corresponding carbon-centered radicals to be determined. In particular, both trans- (1bH) and cis- (1cH) 1,2-dimethylcyclopropane gave 4-penten-2-ol (2bOH) and 2-methyl-3-buten-1-ol (3bOH) together with the corresponding trans- (1bOH) or cis- (1cOH) 2-methylcyclopropanemethanols. Of much greater importance, for both 1bH and 1cH the ratios of the yields of the secondary-to-primary ring-opened alcohols, i.e., [2bOH]/[3bOH], were the same, within experimental error as the ratio of the rates of ring opening of the corresponding trans- (1b\u2022) and cis- (1c\u2022) methylcyclopropylmethyl radicals in solution at 37 \ub0C. This indicates that when 1b\u2022 and 1c\u2022 are formed from their parent hydrocarbons by H-atom abstraction in the hydrophobic pocket of cytochrome P-450 they are not detectably constrained in their subsequent reactions by their unusual environment. From the ratio of the yields of the unrearranged alcohol to each of the rearranged alcohols we calculate kOH values of 1.5 and 1.6 7 1010 s-1 for 1bH as substrate and values of 1.9 and 1.8 7 1010 s-1 for 1cH as substrate. Consistent with these values we have obtained kOH = 2.2 7 1010 s-1 for bicyclo[2.1.0]pentane as substrate. Substrates such as methylcyclopropane and 1,1-dimethylcyclopropane, for which the corresponding cyclopropylmethyl radicals undergo relatively slow ring opening, yielded only the ring-closed alcohols on oxidation with cytochrome P-450. 1,1,2,2-Tetramethylcyclopropane gave only a trace of a ring-opened alcohol, corresponding to kOH = 2.5 7 1011 s-1 for this substrate. Hexamethylcyclopropane gave no detectable ring-opened alcohol from which observation a limit for kOH > 5 7 1011 s-1 can be calculated. Possible explanations for the unexpected behavior of these last two, relatively bulky, substrates are discussed.Peer reviewed: YesNRC publication: Ye

Kinetics of nitroxide radical trapping. 2. Structural effects

Laser flash photolysis and kinetic competition product demonstrated that in isooctane at ambient temperature the rate constant for coupling of carbon-centered with presistent nitroxides, kT, upon the of steric hindrance to coupling and upon the extent of resonance stabilization of the carbon radical. Sterically induced reductions in the magnitude of kT are observed for changes in both the structure of the nitroxide the structure of the carbon radical. Thus, for any particular carbon kT is largest for the Bredt's rule protected nitroxides, 9-azabicyclo[3.3.1]nonane-N-oxyl (ABNO) and nortropane-N-oxyl, while for the "usual" di-tert-alkyl nitroxides kT decreases along the series, 1,1,3,3-tetra-methylisoindoline-2-oxyl 65 2,2,5,5-tetramethylpiperidin-1-oxyl (Tempo) > di-tert-butyl nitroxide, i.e., kT decreased on going from a five-membered ring to a six-membered ring to a noncyclic structure. Cyclopropyl triphenylmethyl are trapped at the fastest and slowest rates, respectively, the corresponding kT value 3.0 7 109 and 1.2 7 108 M-1 s-1 for ABNO and 2.1 7 109 <1 7 106 M-1 s-1 for Tempo. Steric effects in the carbon radicals are for Tempo than for ABNO. For example, the ratio of kT's for the trapping of nonyl tert-butyl is 1.7 for Tempo but 1.3 for ABNO, while for the trapping of benzyl cumyl the ratio of kT's is 4.1 for Tempo 0.9 for ABNO. The effect of resonance stabilization can be illustrated by the kT values for three sterically unhindered primary radicals, n-nonyl, benzyl, 2-naphthyhmethyl, viz., 1.2 7 109, 4.8 7 108, and 5.7 7 107 M-1 s-1, respectively, for Tempo and 2.2 7 109, 1.2 7 109, and 8.1 7 108 M-1 s-1, respectively, for ABNO.Peer reviewed: YesNRC publication: Ye

The unexpected role of vitamin E (\u3b1-tocopherol) in the peroxidation of human low-density lipoprotein

Peer reviewed: YesNRC publication: Ye

Extraordinary kinetic behavior of the \u3b1-tocopheroxyl (vitamin E) radical

Rate constants which have been reported for the bimolecular self-reaction of \u3b1-tocopheroxyl radicals vary by about 5 orders of magnitude. We have found that the observed bimolecular rate constant can vary by about a factor of 7 during a single, but typical experiment, e.g., in chlorobenzene at 37 \ub0C from ca. 7 x 103 M-1 s-1 initially to ca. 1 x 103 M-1 s-1 finally. The overall reaction involves a disproportionation with the transfer of a hydrogen atom from the 5-methyl group of one radical to the phenoxyl oxygen atom of the other radical forming \u3b1-tocopherol and an o-quinone methide. In the slow regime (which corresponds to the true reaction of two \u3b1-tocopheroxyl radicals) this disproportionation has a deuterium kinetic isotope effect of 3.7. The bizarre kinetic behavior observed with \u3b1-tocopheroxyl radicals has been traced to a very minor impurity which will be present in any normal sample of \u3b1-tocopherol. The impurity in question is a bisphenol in which two \u3b1-tocopherol moieties have become linked through their 5-methyl carbon atoms. This bisphenol is a "natural" impurity in \u3b1-tocopherol since it will be formed upon exposure of \u3b1-tocopherol to air. The coupling of two o-quinone methide molecules yields a spiro-dimer which is then reduced to the bisphenol, probably by unoxidized \u3b1-tocopherol. \ua9 1995 American Chemical Society.Peer reviewed: YesNRC publication: Ye

Kinetics of nitroxide radical trapping. 1. Solvent effects

Solvent effects on the kinetics of the nitroxide radical trapping (NRT) of various carbon-centered radicals have been probed both by using the radical "clock" method and by the laser flash photolysis (LFP) technique. Although the rate constants for NRT, kT, are lower than the diffusion-controlled limit they are, nevertheless, influenced by solvent viscosity. Rate constants arc even more strongly influenced by the ability of the solvent to solvate the nitroxide. Thus, using the 2,2-dimethyl-to 1,1-dimethyl-3-butenyl radical clock rearrangement, 1* \u2192 kC 2*, at 80 \ub0C (kc = 2.4 7 107 s-1) with 1,1,3,3-tetramethyl-isoindole-2-oxyl (TMIO) as the trap, in 32 solvents ranging from to aqueous methanol, it found that log (KT/kT)/M-1) was strongly correlated with the nitroxide's solvation, as gauged by the solvent's effect on the nitrogen hyperfine splitting of a structurally analogous nitroxide ((r) = 0.961 for 26 nonhydroxylic solvents, the hydroxylic solvents forming a separate group). Similar results were obtained at 80 \ub0C with five other radical clocks using smaller solvent sets. Comparison of these radical clock data with the kinetic results obtained by LFP at 18 \ub0 for the NRT of benzyl (22 solvents), n-nonyl (4), and neopentyl (6) by Tempo provides the first unequivocal proof that the kinetics of commonly used alkyl radical clock rearrangements are essentially uninfluenced by solvent properties. Although NRT is primarily an activation-controlled reaction, the magnitude of kT is decreased by an increase in solvent viscosity as is clearly indicated by LFP data for the trapping of benzyl radicals by the sterically unencumbered, Bredt's rule protected nitroxide, 9-azabicyclo[3.3.1]nonane-N-oxyl (ABNO) in saturated hydrocarbons (\u3b7 = 0.3-16 cP). Using a model for a partially diffusion-controlled reaction, we obtained a theoretical diffusion-controlled limiting rate constant, ks 48 3.5 7 109 M-1 s-1, for ABNO/benzyl coupling in a solvent of viscosity \u3b7 = 1 versus an extrapolated zero viscosity or "activation" limit, k 1e = 1.4 7 109 M-1 s-1. The Tempo/benzyl coupling in saturated hydrocarbons is less curtailed by diffusion since the diffusion/activation ratio is higher, viz., ks/k 1e 48 3.0 7 109/0.48 7 1091 (for \u3b7 = 1).Peer reviewed: YesNRC publication: Ye

Calibration of the bicyclo[2.1.0]pent-2-yl radical ring opening and an oxygen rebound rate constant for cytochrome P-450

Peer reviewed: YesNRC publication: Ye

Calibration of a new horologery of fast radical "clocks". Ring-opening rates for ring- and \u3b1-alkyl-substituted cyclopropylcarbinyl radicals and for the bicyclo[2.1.0]pent-2-yl radical

Rate constants have been determined at 37\ub0C for the ring opening of a variety of alkyl-substituted cyclopropylcarbinyl radical "clocks" by nitroxide radical trapping (NRT) using TEMPO. Relative yields of unrearranged and rearranged trialkylhydroxylamines were measured at various TEMPO concentrations, and these data were then combined with absolute rate constants for the reactions of structurally appropriate alkyl radicals with TEMPO as determined by laser flash photolysis. Cyclopropylcarbinyl radicals, including the bicyclo[2.1.0]pent-2-yl radical, were generated by H-atom abstraction from the parent hydrocarbon and, in a few cases, also from the appropriate diacyl peroxide. Twelve substrates yielded sixteen clock reactions because some cyclopropylcarbinyls can undergo two different ring-opening reactions. For six methyl-substituted cyclopropylcarbinyls rate constants ranged from 0.8 7 108 s-1 for 1-methylcyclopropylcarbinyl to 47 7 108 s-1 for pentamethylcyclopropylcarbinyl. Rate constants for the ring opening of cyclopropylcarbinyl, \u3b1-methyl- and \u3b1,\u3b1-dimethylcyclopropylcarbinyl are 1.2, 0.70, and 0.88 7 108 s-1, respectively. Rate constants for H-atom abstraction by tert-butoxyl from various positions in the 12 cyclopropane substrates relative to the rate of H-atom abstraction from cyclopentane have also been determined by using competitive NRT.Peer reviewed: YesNRC publication: Ye

Evidence for reversible ring-opening of the \u3b1-cyclopropylbenzyl radical

Kinetic absorption spectroscopy, EPR, and tributylstannane product data indicate that the \u3b1-cyclopropylbenzyl radical (1a) undergoes reversible ring-opening to the 4-phenylbut-3-enyl radical (2a) and that equilibrium favours the ring-closed form, (1a).Peer reviewed: YesNRC publication: Ye

Calibration of a fast benzylic radical "clock" reaction

Peer reviewed: YesNRC publication: Ye