Cytochrome P-450 Model Systems Research Articles

Spin-forbidden hydrogen atom transfer reactions in a cobalt biimidazoline system

Described here are hydrogen atom transfer (HAT) reactions from high-spin cobalt(II) tris(2,2′-bi-2-imidazoline) (CoIIIIH22bim) to the hydrogen atom acceptors, 2,2,6,6-tetramethyl-1-piperidinyl-oxyl (TEMPO), 2,4,6-tri-tert-butylphenoxyl radical (tBu3ArO˙), and benzoquinone (BQ). The cobalt product is the oxidized and deprotonated, low-spin cobalt(III) complex (CoIIIIIIHbim), and the organic products are TEMPOH, tBu3ArOH, or hydroquinone, respectively. These reactions are formally spin forbidden because the spin state of the reactants is different from that of the products. For instance, quartet CoIIIIH22bim plus doublet RO˙ can have a triplet or quintet ground state, while the CoIIIIIIHbim + ROH product state is a singlet. Kinetics measured in the forward and reverse directions and thermochemical measurements provide a detailed picture of the reactions. The reactions are quite slow: the reaction of 10 mM CoIIIIH22bim with excess TEMPO requires roughly a day at ambient temperatures to reach equilibrium. This is 3400 times slower than the related reaction of the iron analogue FeIIIIH22bim, which is 2 kcal mol−1 more uphill. Mechanistic analyses show that the TEMPO reaction occurs by hydrogen atom transfer (HAT), and this is likely for the tBu3ArO˙ and BQ reactions as well. This is an unusually well defined spin-forbidden HAT system, which serves as a model for more complex multi-spin state HAT processes such as those suggested to occur in cytochrome P450 and metal-oxo model systems. In principle, HAT could occur before, after, or concerted with spin change. Computational studies indicate a reaction mechanism involving pre-equilibrium spin state interconversion of quartet 44CoIIIIH22bim to its doublet excited state 22CoIIIIH22bim, followed by spin-allowed HAT to the organic acceptor. This mechanism is consistent with the available kinetic, thermochemical and spectroscopic measurements. It indicates that the slow rates are due to the large change in geometry between CoIIIIH22bim and CoIIIIIIHbim, rather than any inherent difficulty in changing spin state. The implications of these results for other spin-forbidden or ‘two-state’ HAT processes are discussed.

The solubilization site of 5,10,15,20-tetrakis-(2,6-dichlorophenyl)-porphyrin-Mn(III) in DPPC vesicles: A spectrophotometric and tensiometric study

The solubilization site of 5,10,15,20-tetrakis-(2,6-dichlorophenyl)-porphyrin-Mn(III) (MnTDPPC) in l-α-dipalmitoylphosphatidyl choline (DPPC) vesicles was determined in a systematic UV–vis spectrophotometric study. Measurements of the MnTDPPC absorbance have been conducted at constant temperatures (in the interval 30 °C ≤ T ≤ 48 °C) and varying concentrations of phospholipid. In the entire temperature range, the absorption due to MnTDCCP was found to increase with increasing DPPC concentration, until it reaches a plateau value. The data have been analyzed using a pseudo-phase model yielding the binding constant of MnTDCPP to vesicular aggregates. No significant temperature dependence of the partition coefficient of MnTDCPP between the aqueous phase and the lipophilic domain of DPPC vesicles could be detected in the studied temperature interval (which also includes the main transition temperature of DPPC double layers, T m = 41.3 °C), unambiguously indicating that MnTDCPP is confined in the palisade layer of DPPC bilayers. The nature of the interactions between the different reaction components have been investigated by tensiometric measurements, which provide further support for hydrophobic interaction between MnTDCPP and the DPPC vesicles. This solubilization locus makes this porphyrin – which is used as an important compound in biomimetic models for the cytochrome P450 model systems – accessible for electron donation by suitable, water soluble electron donors.

Biomimetic Oxidation of 2‐Methylimidazole Derivative with a Chemical Model System for Cytochrome P‐450.

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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.

Multiple Active Oxidants in Cytochrome P-450 Model Oxidations

We report evidence for the existence of distinct active oxidizing species in a cytochrome P-450 model system. Competitive oxidations of alkane substrates were carried out using [5,10,15,20-tetrakis...

Electrochemical study of tetraphenylporphinato-iron(III)–thiolate complexes, NH⋯S hydrogen bonding in the cytochrome P-450 model system

Porphinato-iron(III)–thiolate complexes, [FeIII(TPP){S-2-(4-X-C6H4CONH)C6H4}] (TPP=tetraphenylporphyrin, X=H, OMe and NO2), were synthesized as models of cytochrome P-450. The complex, [FeIII(TPP)(S-2-PhCONH–C6H4)], shows a positively shifted Fe(III)/Fe(II) redox potential in dichloromethane at −0.901 V versus Cp2Fe compared with the corresponding redox potential of [FeIII(TPP)(S–Ph)] (−1.120 V vs. Cp2Fe) under the same conditions. Such a positive shift of redox potential is considered to be caused by the formation of an intramolecular NH⋯S hydrogen bond. The existence of this NH⋯S hydrogen bond was confirmed by infrared spectroscopy.

Detection of mutagenicity in Ames test using a metalloporphyrin/oxidant model system for cytochrome P450

A chemical model system for cytochrome P450, a porphyrin and an oxidant, was used in Ames assay as a substitute for S9 mix. In the presence of tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride [Fe(F 5P)Cl] and tert-butyl hydroperoxide ( t-BuOOH), mutagenicity of N-nitrosodibutylamine (NDB) in Salmonella typhimurium TA1535 was detected. The mutagenicity depended on the pre-incubation period, and also on the concentration of an oxidant and of bacteria. In the chemical model system, pH affected the mutagenicity of NDB, which suggested that as observed in an enzymatic activating system, the mutagenicity was due to the labile alkylating species which was derived from NDB activated in the chemical activation system and was sensitive to pH. Under the optimum conditions; a higher concentration of an oxidant, a higher concentration of bacterial culture, and a weakly acidic medium, mutagenicity of N-nitrosodipropylamine in S. typhimurium TA1535 was also detected. Besides N-nitrosodialkylamines, 2-aminofluorene (2-AF) and benzo[ a]pyrene (BaP) were also used as mutagens. Mutagenicity of 2-AF and BaP in S. typhimurium TA1538 were both detected in the same system as used in detecting the mutagenicity of N-nitrosodialkylamines. Ames test using a metalloporphyrin/oxidant model system makes it possible to detect mutagenicity derived from both base pair substitution mutagens and frameshift mutagens without using enzymatic activating system. These results demonstrate that the assay with the chemical model system is useful in detecting unstable unknown active mutagens or investigating the mechanisms of the metabolic pathway of mutagens or carcinogens in a protein-free medium.

Biomimetic metabolism of artelinic acid by chemical cytochrome P-450 model systems.

To study the reaction of artelinic acid with chemical model systems of cytochrome P-450 as a means of obtaining authentic samples of the putative metabolites necessary for identification of the mammalian metabolites of artelinic acid. Artelinic acid was reacted with different organic complexes of iron(II). The reaction products were isolated and characterized by NMR and thermospray mass spectroscopy. Five compounds which are putative metabolites of artelinic acid were isolated from these reactions and unambiguously identified, while the identity of two other compounds await final confirmation. Standards of possible metabolites of artelinic acid can be produced by the reaction of the compound with ferrous complexes that may simulate cytochrome P-450 catalyzed metabolism of xenobiotics. This approach may provide a simple and versatile method for the formation of metabolites of artemisinin compounds which is more advantageous than previous approaches with fungal-based systems.

Application of chemical P-450 model systems to studies on drug metabolism. Part X. Novel hydroxylactonization of γ,δ- and β,γ- unsaturated carboxylic acids with an iron porphyrin–iodosylbenzene system

The oxidative hydroxylactonization of γ,δ- and β,γ-unsaturated carboxylic acids by a chemical cytochrome P-450 model and rat liver microsomal systems has been investigated. In the chemical system using meso-tetrakis(2,6-dichlorphenyl)porphyrin iron chloride [Fe(TDCIPP)Cl] with iodosylbenzene (PhIO), γ,δ-unsaturated carboxylic acids have been converted into δ-hydroxy-γ-lactones in high yield and with high stereoselectivity. As an example of a β,γ-unsaturated carboxylic acid, indomethacin has been converted into the corresponding β-hydroxy γ-lactone. Several experiments directed toward mechanistic elucidation of the lactonization exclude a mechanism occurring via an epoxide intermediate. The products have been used as standards to identify the metabolites in the microsomal oxidation. In the case of indomethacin, the γ-lactone form is detected as a metabolite in the rat liver microsomal system, in a yield of 1.33%; the yield is significantly decreased in the presence of 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A) and under a mixed CO–O2(4:1) atmosphere. Thus, these metabolites are considered to be formed by a cytochrome P-450-dependent reaction.

Enzyme Mimic Displaying Oscillatory Behavior. Oscillating Reduction of Manganese(III) Porphyrin in a Membrane-Bound Cytochrome P-450 Model System

Enzyme Mimic Displaying Oscillatory Behavior. Oscillating Reduction of Manganese(III) Porphyrin in a Membrane-Bound Cytochrome P-450 Model System

Cationic iroporphyrins as catalyst in comparative oxidation of hydrocarbons: homogeneous and supported on inorganic matrices systems

Cationic ironporphyrins in solution and supported on imidazole propyl gel (IPG) and silica gel (SG) as catalyst in cyclohexane hydroxylation using iodosylarene as oxygen donor were studied. FeTM4PyP 5+, 1 as homogeneous catalyst for cyclohexane hydroxylation, in acetonitrile (CH 3CN) and ultrasound stirring, gives cyclohexanol (C-ol) yields of 20%. The FeTM2PyP 5+, 2 as catalyst for cyclohexene oxidation in CH 3CN and ultrasound stirring is a very efficient system, giving 93% of total yield, with an epoxide:alcohol:ketone selectivity of 77:12:11. The supported systems, 1-IPG and 1-SG are particularly efficient in dichloromethane (CH 2Cl 2), giving C-ol yields of 37% and 53%, respectively. These systems consist of polar hemin in isolated sites, which selectively catalyze cyclohexane oxidation in apolar solvent. They represent good cytochrome P-450 model systems. In the same way, the active site of P-450 consists of a polar protohemin in a hydrophobic pocket, promoting selective cyclohexane oxidation. These rigid cationic 1-IPG, 1-SG, 2-IPG, 2-SG, 3-IPG and 3-SG systems can catalyze with very small amounts of ironporphyrins, which correspond to about 30–80 times fewer numbers than the hemin in P-450 catalytic site.

Application of chemical cytochrome P-450 model systems to studies on drug metabolism—VIII. Novel metabolism of carboxylic acids via oxidative decarboxylation

The oxidative decarboxylation of carboxylic acids by the chemical cytochrome P-450 model and rat liver microsomal systems was investigated. In the chemical system using meso-tetrakis(pentafluorophenyl)porphyrin iron chloride [Fe(TPFPP)Cl] with iodosylbenzene (PhIO), α-arylcarboxylic acids and α,α,α-trisubstituted acetic acids are converted to the corresponding one-carbon-reduced alcohol ( I) and carbonyl derivatives ( II) via oxidative decarboxylation. These products were then used as standards to identify the metabolites in vivo and in vitro. Biliary excretion of I a and II a in bile duct-cannulated rats after oral administration of ketoprofen amounted to 0.22 and 0.03% of the dose, respectively. In the case of indomethacin, I b and II b were detected as metabolites in the rat liver microsomal system, in yields of 2.8 and 0.29%, respectively. Further, the yields of I b and II b were decreased in the presence of SKF-525A. Thus, these metabolites were formed by cytochrome P-450-dependent reactions. Metabolites I a , I b, II a and II b had moderate to strong inhibitory activities on arachidonic acid-induced platelet aggregation and cyclooxygenase activity in vitro, comparable to those of the parent compounds.

ChemInform Abstract: Selective Oxygenation of Hydrocarbons and Sulfoxidation of Thioethers by Dioxygen with a Mn‐Porphyrin‐Based Cytochrome P450 Model System Using Zn as Electron Donor.

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Novel Metabolic Pathway of Arylethers by Cytochrome P450: Cleavage of the Oxygen-Aromatic Ring Bond Accompanying ipso-Substitution by the Oxygen-Atom of the Active Species in Cytochrome P450 Models and Cytochrome P450

We have found a novel metabolic pathway of arylethers, involving the cleavage of the oxygen-aromatic ring bond. When p-( p-nitrophenoxy)phenol was utilized as a substrate, cleaved products, p-nitrophenol and p-benzoquinone, were formed in two cytochrome P450 model systems, meso-tetraphenylporphinatoiron(III) chloride-NaBH 4/O 2 system and meso-tetrakis (2,6-difluorophenyl)porphinatoiron(III) chloride- m-chloroperoxybenzoic acid (mCPBA) system. Rat liver microsomes also catalyzed this reaction, which was inhibited by a cytochrome P450-specific inhibitor, and it was confirmed that this cleavage proceeded in vivo. Further, experiments using [ 18O]mCPBA and 18O 2 proved that the cleavage reaction is accompanied with the ipso-substitution by the oxygen atom of the active species in both cytochrome P450 model system and cytochrome P450. When the microsomal reactions of p-( p-nitrophenoxy)phenol analogues which lack a hydroxy group, namely p-nitrophenoxybenzene, p-( p-nitrophenoxy) anisole, and p-( p-nitrophenoxy) toluene, were investigated, the cleavage reaction occurred via p-( p-nitrophenoxy)phenol in the cases of p-nitrophenoxybenzene and p-(p-nitrophenoxy)anisole, indicating that a hydroxy group at the p-position to the ether bond is necessary for this pathway. This metabolic pathway appears to be important, because a diarylether linkage, which is very stable and has generally been thought to resist metabolism, is cleaved and benzoquinone, a highly toxic metabolite, is formed.

Application of Chemical Cytochrome P450 Model Systems to Studies on Drug Metabolism .VI: N, N-Coupling Reaction of N-Methylaniline Catalyzed by Polypeptide-Bound Porphyrinatoiron(III) and Cytochrome P450

Polypeptide-bound porphyrinatoiron(III) effectively catalyzed the N, N coupling reaction of N-methylaniline in the presence of NaBH 4/O 2/tetramethyl-ammonium hydroxide or cumenehydroperoxide, whereas the corresponding non-bound monomer porphyrinatoiron(III) had little catalytic activity. Rat liver microsomes also catalyzed the N, N-coupling which was inhibited by cytochrome P-450 specific inhibitors, SKF-525A and metyrapone. The polypeptide-bound porphinatoiron(III) appears to be an excellent cytochrome P450 model for drug metabolism studies.

Biomimetic oxidation of 21-hydroxy, 21-formyl and 21-carboxylic pregn-4-en-3,20-dione with chemical cytochrome P450 model systems

The electron withdrawing 5,10,15,20-tetra(2′,6′-dichlorophenyl)porphyrin iron (III) chloride [C1 8TPPFe(III)C1] and 5,10,15,20-tetra(2′,3′,4′,5′,6′-pentafluorophenyl)porphyrin iron(III)chloride [F 20TPPFe(III)C1] are more efficient catalysts than sterically hindered 5,10,15,20-tetra (2′,4′,6′-trimethylphenyl)porphyrin iron (III)chloride during the biomimetic oxidation of 21-hydroxypregn-4-en-3,20-dione with CumOOH in the presence of N-methylimidazole.

Rapid partial degradation of DDT by a cytochrome P-450 model system.

Heme compounds, in combination with a reducing agent and oxygen, can express various activities of cytochrome P-450 enzymes. In the present study it was found that a mixture of hemin and excess cysteine was able to degrade the insecticide DDT partially. The major products were three water-soluble, non-toxic conjugates of DDT metabolites with cysteine which had lost two or three of the five chlorine atoms of DDT per molecule and whose structures were elucidated by gas chromatography/mass spectrometry. In 0.05 M NH4HCO3, pH 7.7/ethanol (5:6, by vol.), the degradation reaction catalyzed by the hemin-cysteine model system was at least 8 x 10(4) times faster than the uncatalyzed reaction. In the presence of a designed 24-residues polypeptide or beta-casein, two DDT-binding proteins, an additional fourfold increase in the rate of DDT degradation was observed. Although the concentrations of DDT and cysteine occurring in an organism would be expected to be lower than those in the experiments described, the formation of water-soluble conjugates of DDT metabolites with cysteine (and other amino acids) could also play a role in metabolism and excretion of DDT in vivo.

Regio- and stereochemical studies on the alpha-carbon oxidation of (S)-nicotine by cytochrome P-450 model systems.

Results from previous studies indicate that rabbit liver microsomal cytochrome P-450 catalyzes the C-5' two-electron oxidation of (S)-nicotine stereoselectivity with preferential loss of the pro-(E)-hydrogen atom trans to the pyridine ring. We now have examined the regio- and stereochemical features of the oxidation of (S)-nicotine by peroxides in the presence of various hemoproteins and by electrochemical and photochemical methods. None of these systems gave rise to the stereochemical outcomes observed with the cytochrome P-450 mediated reaction. The results of these studies are interpreted as additional evidence for the formation of a highly ordered complex between (S)-nicotine and cytochrome P-450 that directs the regio- and diasterioselective alpha-carbon oxidation of this substrate.

Radical intermediates in the epoxidation of alkenes by cytochrome P-450 model systems. The design of a hypersensitive radical probe

Radical intermediates in the epoxidation of alkenes by cytochrome P-450 model systems. The design of a hypersensitive radical probe

Cytochrome P-450 model systems: alkene oxidation.

Iron(III) porphyrins efficiently catalyze the epoxidation of alkenes and the hydroxylation of alkanes by iodosylbenzenes and peracids, most likely by the same mechanisms utilized by cytochrome P-450 enzymes. The synthetic catalysts mimic the enzymes in product stereospecificity, 1 the accompanying rearrangements, 2 and the suicide labelling which results in N-alkylhemin formation.3