Published papers

Singlet oxygen production efficiency of several laser-excited fullerene derivatives is compared by measuring the 1268 nm emissions; the efficiency was independent of the kind of addends, but it decreased with an increase in the number of addends, and addends at adjacent positions caused a greater decrease than remote addends.

1. Effects of a fullerene C60, derivative, monomalonic acid C60 (MMA C60), on endothelium-containing or denuded aorta of rabbit, trachea and ileum of guinea pig, and stomach (fundus), vas deferens and uterus of rat were studied pharmacologically. 2. MMA C60 (10-5 M) significantly reduced the maximum response of the relaxation induced by acetylcholine in endothelium containing thoracic aorta of rabbit, and the acetylcholine induced relaxation was recovered in the presence of superoxide dismutase (SOD, 250 units/ml). 3. Nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused the relaxation of aorta without endothelium in a concentration dependent manner, and the concentration-response curve was shifted to the right in the presence of MMA C60. This inhibitory effect of the derivative was also masked in the presence of superoxide dismutase (SOD). 4. Sodium nitroprusside-induced relaxation was not affected by either MMA C60 Or SOD. In the other tissues, this C60 derivative had no effect on the responses induced by any agonist. 5. These observations indicate that MMA C60 inhibits the endothelium dependent relaxation induced by acetylcholine but does not affect the agonist-induced contractile response of smooth muscle.

Dimalonic acid C60 (10(-5) M), a new fullerene derivative, produced an augmentation of phenylephrine-induced tone and reduced both the acetylcholine-induced maximum relaxation and the amplitude of substance P (10(-8) M)-induced relaxation in endothelium-containing thoracic aorta of rabbit; the acetylcholine- and substance P-induced relaxation was restored in the presence of superoxide dismutase (250 U/ml). Dimalonic acid C60 (10(-5) M) did not influence the phenylephrine-induced contractile response in the absence of endothelium, but the acetylcholine-induced relaxation was eliminated by removal of the endothelium. Superoxide anion generation, using hypoxanthine (1 mM)/xanthine oxidase (16 mU/ml), reduced the acetylcholine-induced relaxation and produced an augmentation of phenylephrine-induced tone in endothelium-containing strips; these effects were negated by the addition of superoxide dismutase (250 U/ml). A nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused relaxation of aorta without endothelium in a concentration-dependent manner, and the concentration-response curve was shifted to the right in the presence of dimalonic acid C60. This inhibitory effect of dimalonic acid C60 was also masked in the presence of superoxide dismutase. Sodium nitroprusside-induced relaxation was not affected by either dimalonic acid C60 or superoxide dismutase. These observations suggest that dimalonic acid C60 inhibits endothelium (nitric oxide)-dependent agonist-induced relaxation through the production of superoxide.

When various p-substituted phenols (substituent = NO2, CN, CH2OH, COCH3, COPh, COOH, F, Cl, and Br) were incubated with rat liver microsomes, the substituent was eliminated to produce hydroquinone, and the reaction was inhibited by CO and a cytochrome P450-specific inhibitor. In the case of p- cresol (substituent = CH3), p-toluquinol was formed instead of hydroquinone. Experiments using 18O2 proved that the elimination is accompanied with ipso-substitution by the oxygen atom of the active species in cytochrome P450. These results are similar to those in a cytochrome P450 chemical model system (Ohe, T., et al., Tetrahedron Lett. 42, 7681-7684, 1995), implying that the model is a good mimic of cytochrome P450. Substrates that lack a hydroxy group, namely p-substituted toluenes, did not undergo the reaction, thus indicating that a hydroxy group at the p-position to the eliminated substituent is necessary for this pathway. This is the same as the result obtained with the cytochrome P450 model. Finally, to elucidate how the substituent is eliminated, we attempted to detect the product derived from the eliminated group with several substrates. Results indicated that the mechanism of the substituent elimination can be divided into two types: the substituent is eliminated as an anion or as a cation.

A water-soluble fullerene derivative (C60-dimalonic acid, C62(COOH)4) quenches superoxide radical (O2.-), in the xanthine/xanthine oxidase system, and oxidizes reduced cyt. c in the presence of hydrogen peroxide.

Reaction of [60]fullerene in cytochrome P450 (P450) chemical model systems gives several oxidation products; sequential epoxidation occurs, and the second and third oxygen atoms are each introduced at a double bond adjacent to an existing epoxide.

The meso-telrakis(2,6-difluorophenyl)porpriinatoiron(III) chloride-mCPBA system, a chemical model of cytochrome P450, catalyzed substituent elimination from various para-substituted phenols. An experiment using [18O)mCPBA proved that these reactions are accompanied with ipso- substitution by the oxygen atom of the active species of the P450 model. © 1995 Pergamon Elsevier Science Ltd.

Horseradish peroxidase (HRP) catalyzed hydroxylation of benzene with an oxidant when benzene was used as the reaction solvent. HRP immobilized on poly(γ-methyl-L-glutamate) also catalyzed the reaction with higher activity than that of free HRP. The results of the reaction using [18O]hydrogen peroxide show that free and immobilized HRP incorporated the oxygen atom of the reactive species into the product, indicating that both forms of HRP can catalyze hydroxylation of benzene in an analogous manner to that of cytochrome P450. © 1995.

Recently we showed that cytochrome c (cyt.c) has some cytochrome P450 (P450)-like substrate oxidation activity, which was enhanced by immobilization on poly-γ-methyl-L-glutamate. We now report that immobilized cyt.c catalysed hydroxylation of benzene when benzene containing a small amount of water was used as the reaction solvent, whereas free cyt.c did not catalyse the reaction. The EPR spectrum of immobilized cyt.c showed that the immobilization had caused a change in the protein conformation, and this might have led cyt.c to acquire the new activity. The results of the reaction using [ 18O]m-chloroperbenzoic acid (mCPBA) or [18O] molecular dioxygen showed that 59% of the oxygen atom of the product, phenol, was derived from the oxidant, and 33% from molecular dioxygen. When [18O]mCPBA was used as an oxidant, the value of deuterium isotope effect (kH/k D)was 1.08 when 18O was incorporated into the product, and 1.48 when 18O was not incorporated. These results indicate that immobilized cyt.c catalysed hydroxylation of benzene via two major pathways, of which one resembles that of 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-NaBH4/O2 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 18O2 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. © 1994 Academic Press. All rights reserved.

Oxygenation of olefin, N-dealkylation of tertiary amine, oxidation of sulfide, and oxidative cleavage of ether bond were conducted with tetraphenylporphyrinatoiron(III) (Fe3+TPPCl), NaBH4, Me4N·OH, and molecular dioxygen in benzene-methanol solution. Fe3+TPPCl, NaBH4, and molecular dioxygen were essential for these reactions and the yields were decreased when Me4N·OH was absent. Olefins were converted to alcohols, which were not produced from the corresponding epoxides under the same conditions. In styrene oxygenation, an electron-donating substituent on the substrate decreased the reactivity, whereas in N,N-dimethylaniline demethylation, it enhanced the reactivity. Despite the use of the same reagents, the key intermediates of these two reactions are different. Fe2+TPP-σ-alkyl complexes produced from Fe3+TPPCl, olefin, and NaBH4 were identified as intermediates under anaerobic conditions. Fe2+TPP-σ-alkyl complex reacted with molecular dioxygen to give oxygenated products. Examination of the relative reactivities of p-substituted N,N-dimethylanilines in the NaBH4 reaction system revealed first, that the demethylation proceeded via one-electron abstraction, and second, that the reactive species of the demethylation reactions seems to be an iron-oxenoid.

Cytochrome c (cyt.c) was shown to catalyze cytochrome P450 (P450)-like oxidative reactions, such as N-, and 0-demethylation, S-oxidation, and epoxidation of olefins. A more detailed examination showed that (i) N-methylcarbazole and thioanisole oxidation with H218O2 catalyzed by cyt.c resulted in introduction of 18O into the product, and (ii) during the epoxidation of cis-stilbene catalyzed by cyt.c, the stereochemistry of the substrate was retained and 18O was introduced when H218O2 was used as an oxidant. These results show that cyt.c catalyzed N-demethylation, S-oxidation, and epoxidation in the same manner as P450. To utilize these P450-like reactivities effectively, cyt.c was immobilized on poly-γ-methyl-L-glutamate. Up to 99% of the cyt.c used was immobilized. This immobilized cyt.c catalyzed N-demethylation, S-oxidation, and epoxidation in the same manner as both P450 and free cyt.c, and the activities of these reactions were increased by the immobilization. In N-demethylation of N,N-dimethylaniline with cumene hydroperoxide (CHP) catalyzed by cyt.c, the Vmax for CHP was increased by 4.4-fold by the immobilization of the enzyme, while the Km remained unchanged. Since P450 is involved in the metabolism of many xenobiotics, the above results suggest that immobilized cyt.c may be useful in drug metabolism research. © 1993 Academic Press. All rights reserved.

Polypeptide-bound porphyrinatoiron(III) effectively catalyzed the N,N coupling reaction of N-methylaniline in the presence of NaBH4/O2/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. © 1993 Academic Press. All rights reserved.

Microperoxidase-11 (MP-11), prepared by pepsin digestion of cytochrome c, contains protoheme IX and one proximal histidine ligand. MP-11 oxygenated N-methylcarbazole, methyl phenyl sulfide, and phenacyl phenyl sulfide with 80%, 100%, and 96% 18O atom incorporation from H2 18O2, respectively. These results indicate that the oxygen atom on heme iron of MP-11 immediately rebinds to one-electron-oxidized intermediates of the substrates and that MP-11 catalyzes N-demethylation through a cytochrome P-450-like mechanism rather than a peroxidase-like mechanism. © 1992.

14C-Labeled 1,2,3,4-tetrahydroisoquinoline (TIQ) and 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) were synthesized, and their metabolism and tissue distribution were studied. Both compounds showed similar metabolic patterns. In 24 hr after po administration (50 mg/kg) to rats, 76% of TIQ and 72% of 1MeTIQ were excreted unchanged, and 2.7 and 8.7% were excreted as the 4-hydroxyl derivatives, 4-hydroxy-TIQ and 4-hydroxy-1MeTIQ, respectively. Small amounts of N-methylated metabolites, 2-methyl-TIQ (0.4%) and 2-methyl-1MeTIQ (0.7%) were detected. Isoquinoline (2.5%) also was found as a metabolite of TIQ and 1-methyl-3,4-dihydroisoquinoline (1.0%) was found as a metabolite of 1MeTIQ. The concentration of labeled compounds in the brain was about 4.5-fold higher than the blood concentration at 4 hr after dosing, and over 90% was unchanged TIQ or 1MeTIQ. These data indicate that TIQ and 1MeTIQ easily passed through the blood-brain barrier and were concentrated in the brain. Thus, it appears that TIQ and 1MeTIQ as endogenous or exogenous amines may accumulate in the brain and may be related to the onset of Parkinson's disease.

Heme-undecapeptide, microperoxidase-11, prepared from cytochrome c by pepsin digestion, retains the proximal 5th His-18 ligand. Microperoxidase is thought to be a unique peroxidase which does not have a substrate binding site. Compared with heroin-Cl, mieroperoxidase-11 was an effective catalyst for sulfide oxidation, amine N-demethylation, and olefin oxidation in the presence of H2O2. © 1990.

The reaction of CO2 with superoxide cooxidized sulfides and olefins to the corresponding sulfoxides and epoxides, respectively, in dimethylformamide. The peroxy intermediates, CO4??? and C2O6???, generated in situ from superoxide and CO2, were proposed as the ultimate oxidants in the oxidations. © 1989.

Bovine liver catalase suffered a biphasic inactivation when exposed to NH2Cl. A rapid and irreversible phase of activity loss was followed by a much slower and reversible inactivation. Removal of tightly bound NADPH from the enzyme decreased the extent of the rapid phase; whereas addition of NADPH augmented it. The catalase from Aspergillus niger, which does not contain bound NADPH, was not nearly as sensitive toward NH2Cl as was the liver enzyme and was not sensitized by added NADPH. NADPH is oxidized by NH2Cl, as evidenced by loss of the 340-nm absorption band, but the product of this oxidation was not NADP+. The rapid inactivation of liver catalase by NH2Cl was accompanied by some bleaching of the bands in the visible, while the slow inactivation was coincident with the appearance of a new band at 570 nm. A tentative explanation for these observations is proposed. © 1988.