Intermolecular orientations of adenosine-5-monophosphate in aqueous solution as studied by fast fourier ytsndgotm 1H Nmr spectroscopy.
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Adenosine Monophosphate↗
Biomedical subjects
F E Evans
Publications and source records attributed to F E Evans.
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-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 (J1,2) 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% (+)-(1S,2S)-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 18O2 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 H182O indicated that the other 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.
Deuterium↗
Structural and conformational analyses of 8-hydroxy-2'-deoxyguanosine.
Oxidative DNA damage has been shown to involve formation of 8-hydroxy-2'-deoxyguanosine, which may serve as a mispairing lesion during cellular DNA replication. In order to assess the mutagenic potential of this DNA adduct, we examined the possible occurrence of several tautomeric forms and of different base conformations about the deoxyribose. Several spectroscopic and electronic absorption techniques were employed and showed structural changes occurring over a broad pH range. Two pKa's near pH 8 and 12 were observed by pH-solvent partitioning experiments, ultraviolet absorption spectral analyses, and 13C NMR spectroscopic methods. The presence of two pKa's suggested the formation of a dianion, with the second being formed in strong alkali. A comparison of ultraviolet absorption band features of 8-hydroxy-2'-deoxyguanosine with that of different C6,C8-diketo or enol derivatives supported a C8-keto assignment and also provided evidence that this DNA adduct contains a C6-keto group at physiological pH. 13C NMR data showed marked chemical shifts at C6 in solutions of pH 7.4-9.3, indicating the location of the first ionization. Increasing basicity produced further shifts at C5 and C8, indicating the C8 position for the second ionization. Multiple infrared bands were observed in the carbonyl region of the neutral compound, but only a single carbonyl band at 1692 cm-1 remained at pH 9.1, implying a keto group at C8. Determination of the chemical shifts and the nuclear Overhauser enhancements of the N1 and N7 resonances in the proton-decoupled 15N NMR spectrum indicated that both nitrogens were indeed protonated at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)
8-Hydroxy-2'-Deoxyguanosine↗
15N nuclear magnetic resonance studies on the tautomerism of 8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanosine, and other C8-substituted guanine nucleosides.
The favored tautomeric and ionic structures were examined for the oxidative DNA damage adduct 8-hydroxy-2'-deoxyguanosine and its RNA analogue 8-hydroxyguanosine by 15N NMR spectroscopy. In addition, 15N chemical shifts and coupling constants from 13 different guanine nucleosides, including a wide variety of C8 substitutions (OH, SH, Br, OCH2C6H5, OCH3, SCH3, and SO2CH3), have been analyzed with respect to their tautomeric structures. A -98.5-Hz proton-nitrogen coupling constant observed for the N7 resonance of 8-hydroxyguanosine in dimethyl sulfoxide was evidence for 8-keto substitution, which is contrary to the structure implied by the generally used nomenclature. The pH dependence of 15N NMR spectra of 8-hydroxyguanosine in aqueous solution showed downfield shifts of the N1 and N7 resonances that were greater than 50 ppm, which indicated the conversion from a neutral 6,8-diketo to a 6-enolate-8-keto (pKa1 = 8.6) and finally to a 6,8-dienolate structure (pKa2 = 11.7). There was no evidence of an 8-enol substituent in the absence of ionization. It is proposed that the syn conformation of these oxidized bases in duplex DNA and RNA can be further stabilized by abnormal hydrogen bonding or mispairing that involves N7-H. The combined data show that 15N NMR is a sensitive probe to examine tautomerism of the guanine ring system. The analysis indicates that the change from a single to a double bond for the C8 substituent, and the accompanying removal of the normal double bond between N7 and C8 on the imidazole ring system, has no detectable effect on the tautomerism at the N1-O6 site of the pyrimidine ring system for both the 8-keto and 8-thio substitutions. In addition, large differences in electronegativity of the C8 substituents do not alter the N1-O6 tautomerism.
8-Hydroxy-2'-Deoxyguanosine↗
Reaction of a chemotherapeutic agent, 6-mercaptopurine, with a direct-acting, electrophilic carcinogen, benzo[a]pyrene-7,8-diol 9,10-epoxide.
The chemotherapeutic agent 6-mercaptopurine (6-MP) has been shown to react covalently with the ultimate carcinogenic metabolite of benzo[a]pyrene, 7-r,8-t-dihydroxy-9-t,10-t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), in aqueous solution, forming a single adduct. NMR studies of the HPLC-purified product were consistent with its identification as 10(S)-(6'-mercaptopurinyl)-7,8,9-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene. Reaction kinetics were analyzed by using both HPLC separation of the products formed and a spectrophotometric assay for adduct formation. A simple model in which direct reaction between 6-MP and BPDE takes place without formation of a physical complex was found to adequately predict the dependence of product ratios on 6-MP concentration. Variations in the observed rate constant for this reaction with changes in temperature, pH, and buffer concentration were determined and compared to the effects of these variables on the observed rate constant for BPDE hydrolysis. In each case, the processes were affected quite differently, suggesting that different rate-determining steps are involved. The data suggest that the reaction mechanism involves SN2 attack of the anion of 6-MP, formed by ionization of the sulfhydryl group, on carbon 10 of BPDE, resulting in a trans-9,10 reaction product.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
In vitro metabolism and DNA adduct formation from the mutagenic environmental contaminant 2-nitrofluoranthene.
The metabolism and DNA adduct formation by the mutagenic environmental contaminant 2-nitrofluoranthene (2-NFA) were studied. Incubation under aerobic conditions with liver microsomes of rats pretreated with 3-methylcholanthrene yielded trans-7,8-dihydroxy-7,8-dihydro-2-nitrofluoranthene, trans-9,10-dihydroxy-9,10-dihydro-2-nitrofluoranthene, and 7-, 8-, and 9-phenolic metabolites. When the epoxide hydrolase inhibitor 3,3,3-trichloropropylene was present in the incubation, only phenolic metabolites were detected. Under hypoxic conditions, 2-aminofluoranthene was obtained, together with a trace of the ring-oxidized metabolites. The activated metabolite, N-hydroxy-2-aminofluoranthene, was prepared in situ and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA and purification by HPLC, a C8-substituted deoxyguanosine adduct, N-(deoxyguanosin-8-yl)-2-aminofluoranthene, was identified by mass and proton NMR spectral analysis. This adduct was also formed at a level of 10 pmol/mg of DNA when 2-NFA was metabolized by xanthine oxidase, 6 pmol/mg of DNA from incubation with liver microsomes of rats pretreated with 3-methylcholanthrene, and 3-pmol/mg of DNA from metabolism by liver microsomes of rats pretreated with phenobarbital.
Animals↗
Reaction of nontoxic, potentially chemopreventive purinethiols with a direct-acting, electrophilic carcinogen, benzo[a]pyrene-7,8-diol 9,10-epoxide.
Several nontoxic purinethiols have been shown to block the ability of the carcinogen 7-r,8-t-dihydroxy-9-t,10-t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) to bind covalently to DNA in Chinese hamster ovary cells. Two of these compounds also block BPDE-induced tumorigenesis in a two-stage mouse skin carcinogenesis model. The suggested mode of action of the purinethiols is through scavenging the electrophilic carcinogen by way of covalent reaction with the purinethiol. In the present work, we demonstrate that a series of five purinethiols (2,6-dithiopurine, thiopurinol, 6-thioxanthine, 2-mercaptopurine, and 9-methyl-6-mercaptopurine) react covalently in vitro with BPDE. The adducts formed have been characterized by UV-visible spectroscopy, solvent partitioning, and NMR spectroscopy; they result from addition of the thiol moiety at the 10-carbon of BPDE. Studies of the effects of Tris buffer and temperature on product ratios at completion of reaction indicate that the two major reaction pathways, hydrolysis of the epoxide and adduct formation, do not share a common rate-determining step. This suggests that the reaction mechanism for adduct formation is through SN2 attack of the thiol moiety at the 10 position of BPDE. The activation energies for the reaction of 5-purinethiols with various combinations of substituents at the 2 and 6 positions are all very similar, implying closely similar transition states. For compounds with a low pKa (2,6-dithiopurine, 2-mercaptopurine, and 6-thioxanthine) the most important reactant at physiological pH is the thiolate anion. However, for compounds with pKa's above 8, the physiologically important reactions appear to be more complicated.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Synthesis of 3-nitrobenzo[a]pyrene bay-region trans-7,8-diol anti-9,10-epoxide and the corresponding N2-deoxyguanosine adduct.
3-Nitrobenzo[a]pyrene (3-nitro-BaP) is a potent mutagenic environmental contaminant, and its biological activities have been intensively studied. It is significant to prepare its reactive metabolites and the corresponding modified DNA adducts for biological studies. The synthesis of its oxidized proximate metabolite trans-7,8-dihydro-3-nitrobenzo[a]pyrene (3-nitro-BaP-trans-7,8-dihydrodiol, 1), its oxidized ultimate metabolite trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-3- nitrobenzo[a]pyrene (3-nitro-BaP-DE, 2), and the corresponding DNA adduct 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3- nitrobenzo[a]pyrene is described.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Structure of phenolic isomers of 2- and 3-nitrofluoranthene studied by one- and two-dimensional 1H NMR spectroscopy. Comparative analysis of mutagenicity.
The structures of selected phenolic metabolites of 2- and 3-nitrofluoranthene have been analyzed. 1H NMR spectral analysis at 500 MHz using one- and two-dimensional methods proves that the site of hydroxy substitution in two metabolites previously reported as 3-nitrofluoranthen-8-ol and 3-nitrofluoranthen-9-ol had been reversed. A third and previously unidentified metabolite is shown to be 3-nitrofluoranthen-7-ol. Complete analysis of NMR spectral data on 2- and 3-nitrofluoranthene enabled confirmation of the previously reported structures of 2-nitrofluoranthen-8-ol and 2-nitrofluoranthen-9-ol from derived chemical shift substituent effects. Chemical shift data suggest that the nitro group is not strictly coplanar with the aromatic ring system in solution and that metabolism at a distant site can alter the conformation about the C-N bond of the nitro group. Reported mutagenicity data are analyzed relative to imine quinone formation, chemical shift substituent effects, electronegativity effects, and conformation in an attempt to explain the large differences in mutagenicity between the C8 and C9 hydroxy isomers of 2- and 3-nitrofluoranthene.
Chemical Phenomena↗
Nitroreduction of 1- and 3-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene and 1-nitrobenzo[a]pyrene resulting in formation of N2-deoxyguanosinyl adducts through long-range migration.
We recently reported that the reaction of N-hydroxy-3-aminobenzo[a] pyrene with calf thymus DNA produced 6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene as the predominant adduct. The deoxyguanosinyl group of this adduct resides at the C6 position, which is remote from the reaction site, the nitrenium ion. It is significant to determine if formation of this type of DNA adduct is general and whether or not adduct formation is due to an increase in the stabilization of the nitrenium ion by increasing aromaticity. Thus, reduction of 1-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, 3-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, and 1-nitrobenzo[a]pyrene, both chemically and enzymatically, followed by reaction with calf thymus DNA was investigated. DNA was isolated and enzymatically digested, and the resulting modified nucleosides were separated by HPLC. Upon spectral analyses by mass and proton nuclear magnetic resonance spectroscopy, 6-(deoxyguanosin-N2-yl)-1-amino-7,8,9,10-tetrahydrobenzo[a] pyrene, 6-(deoxyguanosin-N2-yl)-3-amino-7,8,9,10-tetrahydrobenzo[a]pyrene, and 6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene were identified, respectively. The same DNA adducts were formed from xanthine oxidase-mediated reductive metabolism of 1-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, 3-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, and 1-nitrobenzo[a]pyrene in the presence of calf thymus DNA. Thee results indicate that formation of N2-deoxyguanosinyl adducts of this type is common and that increasing the aromaticity by increasing the number of aromatic rings is not a decisive factor in directing their formation.
Animals↗
Metabolism of 8-hydroxymethylbenz[a]anthracene by rat liver microsomes. Stereochemistry of dihydrodiol metabolites and the effect of enzyme induction.
Metabolism of the carcinogen 8-hydroxymethylbenz[a]anthracene (8-HOCH2-BA) with liver microsomes from 3-methylcholanthrene-treated rats resulted in 12 identifiable metabolites. Trans-1,2-, 3,4-5,6-, 8,9-, and 10,11-dihydrodiols are among the identified metabolites. The major enantiomers of the trans-dihydrodiols have 1R,2R, 3R,4R, 5R,6R, 8S,9S, and 10R,11R absolute configurations, respectively. Metabolites formed by liver microsomes from untreated as well as 3-methylcholanthrene-, phenobarbital-, and polychlorinated biphenyl-treated immature male Sprague-Dawley rats were quantified by using specifically tritium-labeled [3H-CH2]8-HOCH2-BA as the substrate. The identification of an 8,9-dihydrodiol as a metabolite of 8-HOCH2-BA indicates that a hydroxymethyl substituent does not prevent the enzymatic oxidation at the hydroxymethyl-substituted 8,9-double bond of 8-HOCH2-BA. This is the first example of such a dihydrodiol found from the metabolism of a hydroxymethyl-substituted polycyclic aromatic hydrocarbon by rat liver microsomes.
Animals↗