Biomimetic oxidation of 2-methylimidazole derivative with a chemical model system for cytochrome P-450

A chemical model system for cytochrome P-450, consisting of tetraphenylporphyrin manganese chloride (TPPMnCl) and iodosylbenzene, efficiently oxidized 2-methylimidazole to 2-methylimidazolone. This system was next applied to 4-(2-methyl-1-imidazolyl)-2,2-diphenylbutyramide, a muscarinic acetylcholine receptor antagonist under clinical trial, affording the previously unisolated imidazole ring 5-mono-oxidized derivative that is considered to be the precursor of the main metabolites. This system, which is superior to the copper-ascorbate system, should be applicable to in vitro studies of various drugs containing the 2-methylimidazole moiety.     

以杯芳烃为基体的仿细胞色素P-450单加氧酶模型的合成

用对叔丁基杯[4]芳烃与卟啉羧酸的酰氯化物反应,并引入金属,合成了杯芳烃-金属卟啉仿细胞色素P-450单加氧酶模型A,A及前体化合物6的结构经IR,^1H NMR,MS和元素分析确定,并确证A中杯环呈"锥体"构象     

细胞色素P-450模拟体系的活性中间体-氧配位铬(V)四苯基卟啉 配合物的分离、 表征和氧化反应

氮气保护下二氯甲烷中铬(III)四苯基卟啉衍生物在-40℃与亚碘酰苯反应,分离得氧配位铬(V)四苯基卟啉配合物:O=Cr(V)TPP(Cl)PhI,O=Cr(V)TPP(N~30PhI,O=Cr(V)TPP(p-CH~3O-C~6H~4O)(1/2)PhI。已经元素分析、可见、红外、顺磁、核磁和质谱法结构表征。这些配合物能氧化苯乙烯,环己醇,环己烯和环己烷,可作为细胞色素P-450模拟体系的活性中间体。     

Regioselective oxyfunctionalization of unactivated carbons in steroids by a model of cytochrome P-450: osmiumporphyrin complex/tert-butyl hydroperoxide system

tert-Butyl hydroperoxide catalyzed by (5,10,15,20-tetramesitylporphyrinate) osmium(II) carbonyl [Os(TMP)CO] complex was found to be a highly efficient versatile oxidant for C-H carbons in steroid substrates. When reacted with representative steroids with an estrane, pregnane, 5beta-cholane, or 5alpha-cholestane structure, regioselective oxyfunctionalization and/or oxidative degradation occurred to give a variety of novel and uncommon derivatives in one step.     

Kinetic study on enzymatic s-oxygenation promoted by a reconstituted system with purified cytochrome P-450

Thioanisole derivatives ($\text{1a–e}$) were found to be oxygenated by a reconstituted system of purified cytochrome P-450 to give sulfoxides ($\text{2a–e}$). Logarithms of Vmax were found to be correlated linearly with one electron oxidation potentials of the sulfides (Ep) suggesting the oxygenation to proceed via one electron transfer from sulfides to the active species of the enzyme.     

Pyrene.pyrene complexes at the active site of cytochrome P450 3A4: evidence for a multiple substrate binding site

Cytochrome P450 monooxygenases (CYPs) metabolize nearly all drugs and toxins. Recently, it has become clear that CYPs exhibit both homotropic and heterotropic allosteric kinetics for many substrates. However, the mechanism of cooperative kinetics has not been established for any specific human CYP/substrate combination. Suggested mechanisms include binding of multiple substrates within distinct, static, subsites of a single large active site or binding of multiple substrates within a single fluid active site. CYP3A4 hydroxylates pyrene with positive cooperativity. Therefore, experiments were designed to exploit the fluorescence properties of pyrene, which diagnostically distinguish between pyrene.pyrene complexes versus spatially separated pyrene substrates. Pyrene complexes (excimers) yield an emission spectrum clearly distinct from pyrene monomers. In lipid-free aqueous/glycerol solutions of CYP3A4, addition of pyrene affords a concentration-dependent low-spin to high-spin conversion of the CYP3A4 heme prosthetic group, indicating occupancy of the active site by pyrene. Under the same conditions, in the presence of CYP3A4 but not other heme proteins, the excimer/monomer ratio (E/M) of pyrene was decreased in emission spectra, compared to pyrene alone. However, excitation spectra indicate a CYP3A4-dependent increase in the wavelength shift for the excimer excitation spectrum versus the monomer excitation spectrum, as well as changes in the excimer excitation peak shape and vibronic structure. These changes are reversed by the CYP3A4 substrate testosterone. Together, the results demonstrate that pyrene.pyrene ground-state complexes occupy the CYP3A4 active site, and they provide the first spectroscopic evidence for substrate complexes within a single fluid active site. Functional implications include the possibility that turnover rate, regioselectivity, and stereoselectivity of the reaction are determined by the substrate.substrate complex rather than individual substrates.     

Immobilized metal affinity chromatography as a means of fractionating microsomal cytochrome P-450 isozymes.

Fractionation of microsomal cytochrome P-450s is usually done by chromatography on ion-exchange resins and hydroxyapatite. The resolution of the great number of similar P-450 isozymes, however, requires additional methods based on different separation parameters. For this purpose immobilized-metal affinity chromatography (IMAC) was applied to the separation of P-450 isozymes. The method in its application to rat liver microsomes is described in detail. For method optimization and for the reproducibility of analytical fractionations a completely automatic fast protein liquid chromatographic system especially designed for IMAC is presented. Optimization is done with respect to the choice of the immobilized metal ion and the elution conditions. The chromatographic resolution is markedly enhanced by using segmented vs. linear gradients. The efficiency of P-450 resolution is demonstrated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, verifying the different retention behaviours of the isozymes. However, for all the isozymes analysed so far, reactivity with one particular polyclonal antibody is observed with more than two IMAC fractions of a single run. This may be explained in part by the occurrence of isozymic forms distinguishable by the pattern of chymotryptic peptides. Hence IMAC appears to be suitable for the separation of closely related isozyme forms.     

Immobilization of cytochrome P-450 and electrochemical control of its activity

P-450_cam (camphor-induced cytochrome P-450) was immobilized on an indium tin oxide (ITO) electrode by polypyrrole and its activity was controlled electrochemically. The results showed that P-450_cam was immobilized on the ITO electrode without denaturing and the amount of P-450_cam could be easily controlled. When, the electric potential was swept repeatedly between 0.4 and 0 V, the remarkable decrease of oxygen in the reaction mixture solution was observed only in the presence of camphor. In addition, hydroxycamphor was detected only in the same system by means of gas chromatography/mass spectroscopy. These results suggested that immobilized P-450_cam catalyzed the hydroxylation of camphor by the supply of electron from the electrode. The effect of pH and ionic strength on the activity was examined, and it was found that the high activity expressed at the pH of 6.0 – 7.0 and KCl concentration of 0.1 – 0.2 M . The paper is the first report that P-450 enzyme activity could be controlled artificially. © 1998 John Wiley & Sons, Ltd.     

N-substituted porphyrins formation from carbene iron-porphyrin complexes: a possible pathway for cytochrome P450 heme destruction.

N-substituted porphyrins are formed in high yields upon treatment by CF₃CO₂H or FeCl₃ of the iron-porphyrin complexes obtained by one-electron oxidation of iron-porphyrin-vinylidene carbene complexes.