NIH Shift Research Articles

Substrate specificity of soluble methane monooxygenase: Mechanistic implications

Following the example set by studies of the mechanistic aspects of the substrate specificity of various cytochrome P-450 enzymes, we have undertaken a parallel investigation of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme with a broad substrate specificity. Using substrates previously tested with cytochrome P-450 enzymes and using purified enzyme preparations, this work indicates that soluble methane monooxygenase has a similar oxidative reaction mechanism to cytochrome P-450 enzymes. The evidence suggests that soluble methane monooxygenase oxidizes substrates via a nonconcerted reaction mechanism (hydrogen abstraction preceding hydroxylation) with radical or carbocation intermediates. Aromatic hydroxylation proceeds by epoxidation followed by an NIH shift.

Aryl hydroxylation of diclofop by a cytochrome P450 dependent monooxygenase from wheat

In tolerant wheat, diclofop metabolism in vivo was rapid (33% in 6 hr) and was sensitive to cytochrome P450 inhibitors (CO, tetcyclasis, and 1-aminobenzotriazole). Microsomal fractions from etiolated wheat seedling shoots were shown to support the in vitro hydroxylation of diclofop in the presence of molecular oxygen and NADPH. Enzyme activity was strongly inhibited by tetcyclasis, but showed less sensitivity to 1-aminobenzotriazole and CO. The major enzymatic reaction product in vitro was isolated and identified by HPLC and electron impact mass spectrometry as the NIH shift product, 2-[4-(2,5-dichloro-4-hydroxyphenoxy)phenoxy]propanoic acid. The enzyme had a pH optimum of 7.4, a V max of 5.7 nmol/mg protein · h, and an apparent K m of 41.6 μ M.

Microbial models of mammalian metabolism: conversion of warfarin to 4'-hydroxywarfarin using Cunninghamella bainieri.

Warfarin, an anticoagulant and "metabolic probe" for cytochrome P-450 isozyme multiplicity, is metabolized to 4'-hydroxywarfarin, a principle mammalian metabolite, using the fungus Cunninghamella bainieri (UI-3065). The metabolite was isolated from cell suspension cultures and characterized by analytical (TLC, HPLC, GC-MS) and spectral (HRMS, EI-MS, PMR) comparisons with authentic 4'-hydroxywarfarin. The mechanism of aromatic hydroxylation was examined in C. bainieri using 4'-deuterowarfarin. The absence of a primary isotope effect (KH/KD = 1.13), migration and retention of deuterium in the phenolic product [80% migration and retention (M&R)], and inhibition of the hydroxylation by carbon monoxide (93% inhibition in a 50:50 CO:O2 atmosphere) are consistent with a cytochrome P-450-mediated hydroxylation involving the classic NIH shift (arene oxide) pathway.

ChemInform Abstract: Mechanism of Aromatic Hydroxylation in the Fenton and Related Reactions. One‐Electron Oxidation and the NIH Shift.

AbstractHydroxylation of substituted benzenes is studied in relation to the NIH shift.

Mechanism of aromatic hydroxylation in the Fenton and related reactions. One-electron oxidation and the NIH shift

Hydroxylation de benzenes substitues par le reactif de Fenton et oxydations par le peroxodisulfate

Studies on the metabolism of Inabenfide (a new plant growth regulator) in rats. I. Characterization of metabolites in rats.

1. The metabolism of 4'-chloro-2'-(alpha-hydroxybenzyl) isonicotinanilide (Inabenfide, IBF) was studied in the rat. After intraperitoneal administration of IBF to rats, eight metabolites were detected in urine by g.l.c.-mass spectrometry and a stable isotope technique. 2. The major metabolites were hydroxylated IBF, and minor metabolites were dihydrodiol IBF, methylated catechol IBF, IBF ketone, IBF N-oxide and an amine derivative. 3. Of these metabolites, IBF ketone was produced by oxidation of the carbinol between the benzene rings, which is an interesting metabolite since similar oxidation between benzene rings is not well known. 4. Metabolism involving an NIH shift of chlorine and an epoxide-diol pathway for IBF are presented.

Absence of reactive intermediates in the formation of catechol estrogens by rat liver microsomes.

Release of 3H2O from regiospecifically labeled estradiol was measured during 2-hydroxylation of this estrogen by rat liver microsomes. The amount of tritium remaining in the isolated catechol estrogen was also determined. Virtually all the tritium was removed from C-2 during the reaction confirming the absence of an NIH shift. About 20% of the tritium at C-1 was also lost without any such change occurring at C-4 or C-6,7 of the steroid molecule. These findings provide evidence for the formation of an arene oxide or o-semiquinone intermediate during the conversion of estradiol to 2-hydroxyestradiol. No indication of adduct formation at either C-1 or C-4 during this biotransformation was obtained although the 2-hydroxylated product was able to react with a nucleophile such as glutathione. The different regiospecificity of tritium loss in the generation of catechol estrogens and in their subsequent reaction leads to the important conclusion that the reactive intermediates in the two processes must be different. The possible role of catechol estrogens in neoplastic transformation is discussed.

Microbial transformations: Part 4(1) Regioselective para hydroxylation of aromatic rings by the fungus [formula omitted][formula omitted] the metabolism of isopropyl n-phenyl

A specific para hydroxylation of the aromatic ring of isopropyl N-phenyl carbamate 1 by the fungus Beauveria sulfurescens has been achieved. Studies of the deuterium labelled compound show high retention (NIH shift) for this transformation, a fact uhich indicates that the mechanism implied is consistent which the intermediate formation of an arene oxide. Also, formation of a glucoside conjugate of the phenol has been observed.

Methoxybenzo[a]pyrene 4,5-oxides labeled with carbon-13: electronic effects in the NIH shift

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMethoxybenzo[a]pyrene 4,5-oxides labeled with carbon-13: electronic effects in the NIH shiftI. Robert Silverman, Guido H. Daub, and David L. VanderJagtCite this: J. Org. Chem. 1985, 50, 26, 5550–5556Publication Date (Print):December 1, 1985Publication History Published online1 May 2002Published inissue 1 December 1985https://doi.org/10.1021/jo00350a024RIGHTS & PERMISSIONSArticle Views639Altmetric-Citations4LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1005 KB) Get e-Alerts Get e-Alerts

The hydroxylation, dechlorination, and glucuronidation of 4,4′-dichlorobiphenyl (4-DCB) by human hepatic microsomes

Since chlorine placement and the degree of chlorination of the biphenyl nucleus play an important role in the metabolism and ultimate elimination of polychlorinated biphenyls (PCBs), we have studied the metabolism of 4,4′-dichlorobiphenyl (4-DCB) by human hepatic musomes. This low molecular weight PCB congener is substituted at the preferred site of metabolism (para-position). 4-DCB was metabolized by human musomes with a K m of 0.43 μm and a V max of 1.2 pmoles/mg musomal protein/min. Six metabolites were identified: 4,4′-dichloro-3,3′-biphenyldiol, 4′-chloro-3-biphenylol, 4′-chloro-4-biphenylol, 4,4′-dichloro-2-biphenylol, 4,4′-dichloro-3-biphenylol (most abundant), and 3,4′-dichloro-4-biphenylol. [ 4C]-4-DCB equivalents were found to covalently bind to musomal protein. Addition of a 1 mM concentration of reduced glutathione decreased the degree of covalent binding. These data suggest that human musomes metabolize this PCB through an arene oxide and that an “NIH shift” occurs. When UDPGA was added to the incubation, human musomal glucuronosyltransferase catalyzed the formation of the glucuronide of the major metabolite, 4,4′-dichloro-3-biphenylol. These and previous in vitro results show that the biotransformation of PCBs by humans is governed by the same principles established for the in vivo biotransformation of PCBs by the rat, mouse and monkey. That is, PCBs without two adjacent unsubstituted carbon atoms are poorly metabolized and that an unsubstituted para-position facilitates metabolism.

Evidence for an arene oxide-NIH shift pathway in the transformation of naphthalene to 1-naphthol by Bacillus cereus.

Bacillus cereus ATCC 14579 transformed naphthalene predominately to 1-naphthol. Experiments with [14C]naphthalene showed that over a 24 h period, B. cereus oxidized 5.2% of the added naphthalene. 1-Naphthol accounted for approximately 80% of the total metabolites. B. cereus incubated with naphthalene under the presence of 18O2 led to the isolation of 1-naphthol that contained 94% 18O. The metabolism of [1-2H]- and [2-2H]-naphthalene by B. cereus yielded 1-naphthol which retained 95% and 94% deuterium, respectively, as determined by mass spectral analysis. NMR spectroscopic analysis of the deuterated 1-naphthol formed from [1-2H]-naphthalene indicated an NIH shift mechanism in which 19% of the deuterium migrated from the C-1 to the C-2 position. The 18O2 and NIH shift experiments implicate naphthalene-1,2-oxide as an intermediate in the formation of 1-naphthol from naphthalene by B. cereus.

Hydroxylation of para-chlorotoluene by model complexes of cytochrome P-450

Hydroxylation of p-chlorotoluene with heminthiol complexes, Fenton's system and Udenfriend's system was studied and the complexes assessed as models of cytochrome P-450 monooxygenases. Five species of possible hydroxylation products of p-chlorotoluene, namely, p-chlorobenzyl alcohol, 2-chloro-5-methylphenol, p-chlorobenzaldehyde, 4-chloro-2-methylphenol and 5-chloro-2-methylphenol, were studied using high performance liquid chromatography. The oxidation reactions were characterized by the yields of hydroxylation products and the product ratio. The system consisting of hemin and cysteine ethyl ester as well as Udenfriend's system gave relatively high hydroxylation yields and the former only induced a methyl migration during hydroxylation (methyl NIH shift). However, neither Fenton's nor Udenfriend's systems induced a methyl NIH shift. The hemin-thiol complex is thus concluded to be a good chemical model of cytochrome P-450 monooxygenases.

Regioselective dihydroarene oxide formation during ortho-hydroxylation of halogenobenzenes by fungi

ortho-Hydroxylation of chloro- and bromo-benzene occurred in the presence of growing cultures of the fungi Rhizopus arrhizus, Rhizopus stolonifer, and Cunninghamella elegans. The absence of a primary kinetic isotope effect and the presence of the NIH shift are consistent with dihydroarene oxides being initial metabolites. N.m.r. analysis of the deuterium-labelled o-halogenophenol products suggests that enzyme-catalysed epoxidation occurs preferentially at the 2,3-bond.

Isoflavonoid biosynthesis: concerning the aryl migration

Feeding experiments with 13C- or 2H-labelled precursors in CuCl2-treated red clover (Trifolium pratense) seedlings have demonstrated that the isoflavone formononetin (6) and the pterocarpan phytoalexins medicarpin (10) and maackiain (11) are biosynthesized from 2′,4,4′-trihydroxychalcone by a rearrangement process involving an intramolecular migration of the cinnamate-derived aromatic ring. In all three compounds, this is accompanied by retention of the chalcone's β-hydrogen but loss of the α-hydrogen. During the formation of maackiain from formononetin, an NIH shift of 2H as a result of aromatic hydroxylation ortho to the methoxy group was observed. Experiments with 7-hydroxy-4′-methoxy[2-2H2]isoflavanone indicate that this compound may be converted into medicarpin without loss of 2H, thus confirming the existence of a metabolic grid of isoflavones and isoflavanones. The results are best explained in terms of an oxidative process in which a chalcone is converted into an isoflavone as the first-formed isoflavonoid derivative.

HYDROXYLATION OF BENZENES INVOLVING THE NIH SHIFT UTILIZING ELECTRONIC MODEL COMPOUND FOR MONOOXYGENASES

Abstract The reaction of anisole-4-2H or toluene-4-2H with 2-hydroperoxyhexafluoro-2-propanol gave corresponding phenols involving the NIH shift, the extent of which was 57% and 51% respectively. These values were very close to those of cytochrome P450.

ChemInform Abstract: ON THE OXYGENATION OF SEVERAL AROMATIC AND ALIPHATIC COMPOUNDS WITH AQUEOUS FERROUS ION‐MOLECULAR OXYGEN

Hydroxylation was shown to occur readily in the reactions of aromatic compounds such as benzoic acid, nitrobenzene, acetanilide, and phenol with ferrous ion-molecular oxygen in 0.5 M phosphate buffer (pH 6.8) at 40°C for 3 h. The yields of hydroxylated products were higher than those obtained with other reported hydroxylation systems of transition metalmolecular oxygen. The NIH shift was not observed in the title reactions of C (4)-deuterioacetanilide and C (4)-deuteriobenzoic acid. This system was also demonstrated to oxygenate caproic acid to give a mixture of 3-, 4-, and 5-oxocaproic acids in 67% total yield.

Stereochemistry and evidence for an arene oxide-NIH shift pathway in the fungal metabolism of naphthalene

The mechanism of naphthalene oxidation by the filamentous fungus, Cunninghamella elegans is described. C. elegans oxidized naphthalene predominately to trans-1,2-dihydroxy-1,2-dihydronaphthalene. A trans configuration was assigned for the dihydrodiol by nuclear magnetic resonance (NMR) spectroscopy at 500 MHz which showed a large coupling constant (J 1,2) of 11.0 Hz. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydronaphthalene to that formed by mammalian enzyme systems indicated that the fungal dihydrodiol contained 76% (+)-(1 S,2 S)-dihydrodiol as the predominant enantiomer. Other naphthalene metabolites formed by C. elegans were identified as 1-naphthol, 2-naphthol and 4-hydroxy-1-tetralone. Incubation of C. elegans with naphthalene and 18O 2 indicated that the trans-1,2-dihydroxy-1,2-dihydronaphthalene contained one atom of molecular oxygen which indicated a monooxygenase catalyzed reaction while similar incubations with naphthalene and H 2 18O indicated that the oxygen atom in trans-1,2-dihydroxy-1,2-dihydronaphthalene was derived from water. Mass spectral analysis of the acid-catalyzed dehydration products of the dihydrodiol indicated that the naphthalene dihydrodiol forms via the addition of water at the C-2 position of naphthalene-1,2-oxide. Fungal metabolism of [1- 2H] naphthalene yielded 1-naphthol which retained 78% of the deuterium. NMR analysis of the deuterated 1-naphthol indicated an NIH shift mechanism in which deuterium migrated from the C-1 position to the C-2 position. The above results indicate that naphthalene-1,2-oxide is an intermediate in the fungal metabolism of naphthalene and that the fungal enzymes are highly stereo-selective in the formation of trans-1,2-dihydroxy-1,2-dihydronaphthalene.

Mechanistic aspects of biotransformations by the monooxygenase system of Methylosinus trichosporium OB3b

AbstractMechanistic aspects of the mode of action of the soluble monooxygenase system of Methylosinus trichosporium OB3b have been investigated. The hydroxylation of 4‐deuteroanisole in the 4‐position was shown to proceed via an NIH shift with a deuterium retention of 66 %, indicative of an epoxide intermediate. Isotopic studies using ethylbenzene and ethylbertzene‐d10 showed an isotopic effect, kH/kD = 4.5 for benzylic hydroxylation, and an inverse isotopic effect, kH/kD = 0.75 for arene hydroxylation. It is suggested that a stepwise mechanism (hydrogen abstraction and hydroxylation) could be involed.

On the oxygenation of several aromatic and aliphatic compounds with aqueous ferrous ion-molecular oxygen.

Hydroxylation was shown to occur readily in the reactions of aromatic compounds such as benzoic acid, nitrobenzene, acetanilide, and phenol with ferrous ion-molecular oxygen in 0.5 M phosphate buffer (pH 6.8) at 40°C for 3 h. The yields of hydroxylated products were higher than those obtained with other reported hydroxylation systems of transition metalmolecular oxygen. The NIH shift was not observed in the title reactions of C (4)-deuterioacetanilide and C (4)-deuteriobenzoic acid. This system was also demonstrated to oxygenate caproic acid to give a mixture of 3-, 4-, and 5-oxocaproic acids in 67% total yield.

Evidence for a 2,3-epoxide as an intermediate in the microsomal metabolism of 6-nitrobenzo[a]pyrene.

The rat liver microsomal metabolism of 3-deutero-6-nitrobenzo(a)pyrene ([3-2H]6-NO2-BaP) was studied. The metabolites were separated by h.p.l.c. The 500 MHz 1H n.m.r. spectral analysis of the metabolites indicated that the 3-hydroxy-6-NO2-BaP and 6-NO2-BaP-3,9-hydroquinone each retained 33% of the deuterium label at the C-2 position. It is proposed that the migration of deuterium occurs via an NIH shift mechanism. These results indicate that a 2,3-epoxide is a common intermediate.