Chemical reactivity as a tool to study carcinogenicity: reaction between chloroethylene oxide and guanine

Chloroethylene oxide, an ultimate carcinogen of vinyl chloride, reacts with DNA giving rise to 7-(2-oxyethyl)guanine adduct in a nearly quantitative yield. This reaction represents an initial step of carcinogenesis associated with vinyl chloride. From experimental data for this reaction we calculated the second-order rate constant of 0.049 s(-1) M(-1), which corresponds to the activation free energy of 19.5 kcal/mol. We also performed a series of medium high ab initio and density functional theory simulations. Effects of hydration were considered in the framework of the Langevine dipoles solvation model and the solvent reaction field method of Tomasi and co-workers. In silico calculated activation free energies are in a good agreement with the experimental value. This fact presents strong evidence in favor of the validity of the proposed reaction mechanism and points to the applicability of quantum-chemical methods to studies of other reactions associated with carcinogenesis. Insignificant stereoselectivity of the studied reaction was also predicted.     

Determination of Estrogens by High Performance Liquid Chromatography with Pre-Label Derivatization with 2-(4-Carboxy)-5,6-Dimethylbenzimidazole (II): Shortening of Analysis Time and Application to Monitoring Estrogen in Serum From In-Vitro Fertilization Embryo Transfer (IVF-ET) Patients

Abstract

We report a sensitive high performance liquid chromatography (HPLC) method for determination of free and conjugated estrogens (estrone, estradiol and estriol) by a fluorescent pre-labeling regent, 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole, with modification of previous work. The modified method was also tried, in preliminary work, for diagnosis of the in-vitro fertilization embryo transfer (IVF-ET) process. The reagent volumes were changed to one-tenth, derivatization conditions were changed to mild conditions at 40 C, and a solid-phase extraction process by SEP-PAK could be omitted after restudy of reaction conditions. As a result analysis time could be shorted within 40 min. The proposed HPLC method was applied to monitoring of free and conjugated estrogens in the patients who attend in-vitro fertilization embryo transfer (IVF-ET). The subsequent increase of free and conjugated estrone, estradiol and estriol was observed with the progress of follicle growth following ovulation stage in the IVF-ET process. We tried to plot estrogens for assist of clinical diagnosis of IVF-ET. The free estrone: 200-600 pg/ml, estradiol: 200-600 pg/ml and estriol: 100-300 pg/ml, conjugate estrone: 1000-5000 pg/ml, estradiol: 3000-8000 pg/ml and estriol: 2000-7000 pg/ml) in the patients without hormone disease were observed before human chorionic gonadotropin stimulation (hCG) on IVF-ET process. It was expected that free estrogen values, especially E1 and E3 could be use as validation products for diagnosis of hormone disease in IVF-ET process.     

Mechanistic study of the deamination reaction of guanine: a computational study

The mechanism for the deamination of guanine with H(2)O, OH(-), H(2)O/OH(-) and for GuaH(+) with H(2)O has been investigated using ab initio calculations. Optimized geometries of the reactants, transition states, intermediates, and products were determined at RHF/6-31G(d), MP2/6-31G(d), B3LYP/6-31G(d), and B3LYP/6-31+G(d) levels of theory. Energies were also determined at G3MP2, G3MP2B3, G4MP2, and CBS-QB3 levels of theory. Intrinsic reaction coordinate (IRC) calculations were performed to characterize the transition states on the potential energy surface. Thermodynamic properties (ΔE, ΔH, and ΔG), activation energies, enthalpies, and Gibbs free energies of activation were also calculated for each reaction investigated. All pathways yield an initial tetrahedral intermediate and an intermediate in the last step that dissociates to products via a 1,3-proton shift. At the G3MP2 level of theory, deamination with OH(-) was found to have an activation energy barrier of 155 kJ mol(-1) compared to 187 kJ mol(-1) for the reaction with H(2)O and 243 kJ mol(-1) for GuaH(+) with H(2)O. The lowest overall activation energy, 144 kJ mol(-1) at the G3MP2 level, was obtained for the deamination of guanine with H(2)O/OH(-). Due to a lack of experimental results for guanine deamination, a comparison is made with those of cytosine, whose deamination reaction parallels that of guanine.     

The influence of formamide (model of protein unit) on the intramolecular proton transfer in the DNA simple base guanine: A density functional theory study

For the purpose of investigating the influence of protein unit on the intramolecular proton transfer (IPT) reactions in the simple base guanine, a simple model (formamide) of peptides is designed to biological system investigations, and five complexes of formamide–guanine (FG1, FG2, FG3, FG4, and FG5) are determined at the B3LYP/6‐311++G(d,p) level of theory. For comparison, HF and MP2 methods are also used in this paper. The proton transfer (PT) reaction processes of guanine and FGs have been investigated employing the B3LYP/6‐311++G(d,p) level of theory. The selected thermodynamic and kinetic parameters, such as the activation energies (E_a), changes of enthalpy (ΔH) and changes of free energies (ΔG), as well as the equilibrium constants (K_p) for those reaction processes, have also been obtained by calculational means. The calculated results indicate that the assisted and protected effects of formamide on IPT in guanine are site‐dependent. CH1 is the lowest activation energy needed PT process no matter where the formamide molecule is located in. The activation energy of CH1 with formamide in S2 is the lowest one (153.3 KJ/mol), whereas the one of CH5 with formamide in S5 is the highest (318.3 KJ/mol). © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008     

Catalytic involvement of CO2 in the mutagenesis caused by reactions of ONOO(-) with guanine

The catalytic role of CO2 in reactions of ONOO- with guanine, leading to the formation of the mutagenic species 8-oxoguanine (8-oxoG) and 8-nitroguanine anion (8-nitroG-), was investigated by considering the reactions of nitrosoperoxycarbonate anion (ONOOCO2-), an adduct of ONOO- and CO2, with guanine at the B3LYP/6-31G** and B3LYP/AUG-cc-pVDZ levels of density functional theory in gas phase. In order to study bulk solvent effect, single-point energy calculations in aqueous media were carried out for all the species occurring in the reactions at the B3LYP/AUG-cc-pVDZ level of theory, by use of the polarizable continuum model (PCM). Vibrational frequency analysis was performed, and zero-point-energy (ZPE)-corrected total energies and Gibbs free energy changes at 298.15 K were obtained. The genuineness of the calculated transition states was confirmed by visually examining the vibrational modes and also by intrinsic reaction coordinate (IRC) calculations. The reaction between ONOOCO2- and guanine occurring through four different mechanisms leads to the formation of 8-oxoG or its anion, while the reaction between the same two species occurring through a different scheme leads to the formation of 8-nitroG-. It has been shown that the presence of a water molecule along with ONOOCO2- would not affect the reaction mechanisms significantly. Structures of the reactant complexes, product complexes and barrier energies involved in the reactions reveal that CO2 acts as a catalyst for the reaction between ONOO- and guanine. The cause of the catalytic action of CO2 is mainly due to intermediacy of the CO3 radical anion and NO2 radical into which ONOOCO2- is fragmented while reacting with guanine. The relative stabilities of the different product complexes suggest that the mutation caused by ONOO- in the presence of CO2 would mainly involve 8-oxoG.     

Binding of anticancer drug Ru(η 6 -C6H5(CH2)2OH)Cl2(DAPTA) to DNA purine bases and amino acid residues: a theoretical study

The hydrolyzed Ru(η ⁶ -C₆H₅(CH₂)₂OH)Cl₂(DAPTA) (DAPTA = 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) binding to guanine(G), adenine (A), cytosine(C), cysteine (Cys), and histidine (His) residues were explored using the B3LYP hybrid functional and IEF-PCM solvation models. The computed activation barriers for the reactions of diaqua complex were lower than those of chloroaqua complex except for binding to cytosine. For the chloroaqua complex, the activation free energy was lowest when binding to cytosine (10.5 kcal/mol). Whereas, the substitution reaction of diaqua complex binding to cysteine showed the lowest activation free energy with 10.1 kcal/mol, closely followed by histidine (15.8 kcal/mol), adenine (20.1 kcal/mol), cytosine (20.7 kcal/mol), and guanine (24.4 kcal/mol) by turns. It could be deduced that the completely hydrolyzed Ru(η ⁶ -C₆H₅(CH₂)₂OH)Cl₂(DAPTA) compounds might preferentially bind to amino acids residues in vivo. In addition, to simulate the protein and DNA environment in vivo, a detailed investigation of the activation free energies for the substitution reactions in dependence of the dielectric constant ε (4, 24, and 78.39) was systematically performed as well. The calculated results demonstrated that the environmental effect had a little impact on these substitution reactions.     

A determination of antioxidant efficiencies using ESR and computational methods

Using Transition-State Theory, experimental rate constants, determined over a range of temperatures, for reactions of Vitamin E type antioxidants are analysed in terms of their enthalpies and entropies of activation. It is further shown that computational methods may be employed to calculate enthalpies and entropies, and hence Gibbs free energies, for the overall reactions. Within the linear free energy relationship (LFER) assumption, that the Gibbs free energy of activation is proportional to the overall Gibbs free energy change for the reaction, it is possible to rationalise, and even to predict, the relative contributions of enthalpy and entropy for reactions of interest, involving potential antioxidants. A method is devised, involving a competitive reaction between *CH3 radicals and both the spin-trap PBN and the antioxidant, which enables the relatively rapid determination of a relative ordering of activities for a series of potential antioxidant compounds, and also of their rate constants for scavenging *CH3 radicals (relative to the rate constant for addition of *CH3 to PBN).     

Estradiol affinity chromatography: application to purification of murine alpha-fetoprotein

A one-step batch procedure is described for purification of murine alpha-fetoprotein (AFP) by estradiol affinity chromatography. Various ratios of carbodiimide (C), diaminononame (D) and estradiol hemisuccinate (E) were tested to determine optimal conditions for AFP purification. Although yields of AFP ranged from 15 to 44% depending on the reagent ratio employed, AFP isolates free of other protein contaminants were achieved at C:D:E ratios of 10:10:1 with a 29% yield. Both estrone and estradiol proved efficient as elution agents to free AFP bound to the estradiol-Sepharose beads, but higher yields were produced with estrone. After isolation the estrogen-eluted AFP preparations were analyzed by (1) estradiol-binding assays, (2) third-party radiocoprecipitation, (3) inhibition of radioimmunoassay for estrone and estradiol and (4) exchange of unlabeled for radiolabeled estradiol. These results indicated that the steroid remained attached to the eluted AFP molecule.     

Activation barriers for DNA alkylation by carcinogenic methane diazonium ions

Methylation reactions of the DNA bases with the methane diazonium ion, which is the reactive intermediate formed from several carcinogenic methylating agents, were examined. The SN2 transition states of the methylation reactions at N7, N3, and O6 of guanine; N7, N3, and N1 of adenine; N3 and O2 of cytosine; and O2 and O4 of thymine were calculated using the B3LYP density functional method. Solvation effects were examined using the conductor-like polarizable continuum method and the combined discrete/SCRF method. The transition states for reactions at guanine N3, adenine N7, and adenine N1 are influenced by steric interactions between the methane diazonium ion and exocyclic amino groups. Both in the gas phase and in aqueous solution, the methylation reactions at N atoms have transition states that are looser, and generally occur earlier along the reaction pathways than reactions at O atoms. The forming bonds in the transition states in water are 0.03 to 0.13 A shorter than those observed in the gas phase, and the activation energies are 13 to 35 kcal/mol higher. The combined discrete/SCRF solvation energy calculations using base-water complexes with three water molecules yield base solvation energies that are larger than those obtained from the CPCM continuum method, especially for cytosine. Reactivities calculated using barriers obtained with the discrete/SCRF method are consistent with the experimentally observed high reactivity at N7 of guanine.     

Do Photolyases Need To Provide Considerable Activation Energy for the Splitting of Cyclobutane Pyrimidine Dimer Radical Anions?

cis-syn Cyclobutane pyrimidine dimers, major UV-induced DNA lesions, are efficiently repaired by DNA photolyases. The key step of the repair reaction is a light-driven electron transfer from the FADH(-) cofactor to the dimer; the resulting radical anion splits spontaneously. Whether the splitting reaction requires considerable activation energy is still under dispute. Recent reports show that the splitting reaction of a dimer radical anion has a significant activation barrier (0.45 eV), and so photolyases have to provide considerable energy. However, these results contradict observations that cis-syn dimer radical anions split into monomers at -196 degrees C, and that the full process of DNA photoreactivation was fast (1.5-2 ns). To investigate the activation energies of dimer radical anions, three model compounds 1-3 were prepared. These include a covalently linked cyclobutane thymine dimer and a tryptophan residue (1) or a flavin unit (3), and the covalently linked uracil dimer and tryptophan (2). Their properties of photosensitised splitting of the dimer units by tryptophan or flavin unit were investigated over a large temperature range, -196 to 70 degrees C. The activation energies were obtained from the temperature dependency of splitting reactions for 1 and 2, 1.9 kJ mol(-1) and 0.9 kJ mol(-1) for the thymine and uracil dimer radical anions, respectively. These values are much lower than that obtained for E. coli photolyase (0.45 eV), and are surmountable at -196 degrees C. The activation energies provide support for previous observations that repair efficiencies for uracil dimers are higher than thymine dimers, both in enzymatic and model systems. The mechanisms of highly efficient enzymatic DNA repair are discussed.     

Guanine alkylation by ethylene oxide: calculation of chemical reactivity

In this paper we report on calculations of the activation free energy for a chemical reaction between ethylene oxide and guanine. Ethylene oxide is biologically relevant per se and is also a model compound for numerous ultimate carcinogens. Calculations were performed on the medium-high ab initio, DFT, and semiempirical MO levels. Effects of solvation were considered using the Langevine dipole method and solvent reaction field method of Tomasi and co-workers. The calculated activation free energies are in reasonable agreement with the experimental value.     

The transfer of tin and germanium atoms from N-heterocyclic stannylenes and germylenes to diazadienes

New N-heterocyclic stannylenes and germylenes were synthesized by transamination of E[N(SiMe3)2] (E = Ge, Sn) with alpha-amino-aldimines or ethylidene-1,2-diamines and were characterized by spectroscopic methods and in the case of the germylene 10 g by X-ray diffraction. The reactions of several germylenes and stannylenes with diazadienes were studied by using dynamic NMR and computational methods. Experimental and theoretical studies confirmed that metathesis with exchange of the Group 14 atom is feasible for both stannylenes and germylenes, with exchange rates being generally higher for stannylenes. The metathesis of the diazadiene 3 b and the stannylene 1 b follows second-order kinetics and exhibits a sizeable negative entropy of activation. The transfer reaction is inhibited by bulky substituents in both reactants and surprisingly coincides with a suppression of the fragmentation of the stannylene into tin and diazadiene. A connection between oxidative addition and ring fragmentation was also observed in the reaction of 1 f with sulfur. Density functional theory (DFT) calculations suggest that all metathesis reactions proceed via transient spirocyclic [1+4] cycloaddition products, the formation of which is generally endothermic and endergonic. The spirostannanes display a distorted Psi-tbp geometry at the tin atom and their cycloreversion requires low or nearly negligible activation energies; spirogermanes exhibit distorted tetrahedral central atoms and sizeable energy barriers with respect to the same reaction. Complementary studies of cycloadditions of diazadienes to triplet germylenes or stannylenes indicate that these reactions are exothermic. The lowest triplet state in the carbene homologues results from promotion of an electron from an n(N) orbital with pi character rather than the n(C)-sigma orbital as in carbenes, and singlet-triplet excitation energies decrease from carbon to tin. Spirostannanes exhibit a triplet ground-state multiplicity that implies that the energy hypersurfaces for the reactions of singlet and triplet stannylenes with diazadienes intersect; for germylenes, the singlet hypersurface is always lower in energy. A reaction mechanism explaining the different thermal stabilities of N-heterocyclic germylenes and stannylenes, and the coincidence between ring metathesis and thermal decomposition of the latter, is proposed based on the different separation of the singlet and triplet energy hypersurfaces.     

Electron-transfer oxidation properties of DNA bases and DNA oligomers

Kinetics for the thermal and photoinduced electron-transfer oxidation of a series of DNA bases with various oxidants having the known one-electron reduction potentials (E(red)) in an aqueous solution at 298 K were examined, and the resulting electron-transfer rate constants (k(et)) were evaluated in light of the free energy relationship of electron transfer to determine the one-electron oxidation potentials (E(ox)) of DNA bases and the intrinsic barrier of the electron transfer. Although the E(ox) value of GMP at pH 7 is the lowest (1.07 V vs SCE) among the four DNA bases, the highest E(ox) value (CMP) is only 0.19 V higher than that of GMP. The selective oxidation of GMP in the thermal electron-transfer oxidation of GMP results from a significant decrease in the pH dependent oxidation potential due to the deprotonation of GMP*+. The one-electron reduced species of the photosensitizer produced by photoinduced electron transfer are observed as the transient absorption spectra when the free energy change of electron transfer is negative. The rate constants of electron-transfer oxidation of the guanine moieties in DNA oligomers with Fe(bpy)3(3+) and Ru(bpy)3(3+) were also determined using DNA oligomers containing different guanine (G) sequences from 1 to 10 G. The rate constants of electron-transfer oxidation of the guanine moieties in single- and double-stranded DNA oligomers with Fe(bpy)3(2+) and Ru(bpy)3(3+) are dependent on the number of sequential guanine molecules as well as on pH.     

H2O3 as a reactive oxygen species: formation of 8-oxoguanine from its reaction with guanine

Reaction of guanine with H2O3 in the absence and presence of a water molecule leading to the formation of 8-oxoguanine (8-oxoG) was investigated. Initial calculations were performed using imidazole (Im) as a model for the five-membered ring of guanine. The reactant, intermediate, and product complexes as well as transition states were obtained in gas phase at the B3LYP/6-31+G* and B3LYP/AUG-cc-pVDZ levels of theory. In all the cases, except for the reactions involving imidazole, single-point energy calculations were performed in gas phase at the MP2/AUG-cc-pVDZ level of theory. Solvation calculations in aqueous media were carried out using the polarizable continuum model (PCM) of the self-consistent reaction field (SCRF) theory. Vibrational frequency analysis and intrinsic reaction coordinate (IRC) calculations were performed to ensure that the transition states connected the reactant and product complexes properly. Zero-point energy (ZPE)-corrected total energies and Gibbs free energies at 298.15 K in gas phase and aqueous media were obtained. When a reaction of H2O3 in place of H2O2 with guanine is considered, the major barrier energy which is encountered at the first step is almost halved showing that H2O3 would be much more reactive than H2O2. Considering the reaction schemes investigated here and the observed fact that H2O3 is dissociated easily under ambient conditions, it appears that H2O3 would serve as an effective reactive oxygen species.     

黄嘌呤异构体质子转移异构化反应机理的理论研究

采用量子化学密度泛函B3LYP/6-31G(d,p)和M06-2X/6-311++G(d,p)方法对黄嘌呤两种酮式异构体X(1,3,7)与X(1,3,9)间质子转移引起的互变异构反应机理进行了计算研究,获得了异构化反应过程的反应焓﹑活化吉布斯自由能和质子转移反应的速率常数等参数。水相计算采用极化连续(PCM)模型。结果表明,由于可能的氢迁移顺序差异,分子内由X(1,3,7)向X(1,3,9)异构化可能共有16条反应通道,涉及11个中间体和20个过渡态,其主反应通道速控步骤的活化吉布斯自由能为183.10k J/mol,速率常数为5.17×10-20s-1,其余各通道速控步骤活化吉布斯自由能均较高,而且整体水溶剂效应不利于质子转移的发生。     

Quadrupole type mass spectrometric study of the abstraction reaction between hydrogen atoms and ethane

The reactions of photochemically generated deuterium atoms of selected initial translational energy with ethane have been investigated. At each initial energy the relative probability of the atoms undergoing reaction or energy loss on collision with ethane was investigated, and the phenomenological threshold energy was measured as 30+/-5kJmol(-1) for the abstraction from the secondary C-H bonds. The ratio of relative yields per bond, secondary:primary was approximately 3 at the higher energies studied. The correlation of threshold energies with bond dissociation energies, heats of reaction and activation energies is discussed for abstraction reactions with several hydrocarbons.     

New Insights into the Reactivity of Cisplatin with Free and Restrained Nucleophiles: Microsolvation Effects and Base Selectivity in Cisplatin–DNA Interactions

The reactivity of cisplatin towards different nucleophiles has been studied by using density functional theory (DFT). Water was considered first to analyze the factors that govern the transformation of cisplatin into more electrophilic aquated species by using an activation‐strain model. It was found that the selectivity and reactivity of cisplatin is a delicate trade‐off between strain and interaction energies and that the second chloride is a worse leaving group than the first. When similar studies were carried out with imidazole, guanine (G), and adenine (A), it was found that in general the second nucleophilic substitution reactions have lower activation barriers than the first ones. Finally, simulations of the structural restrictions imposed by the DNA scaffold in intra‐ and interstrand processes showed that the geometries of the reaction products are nonoptimal with respect to the unrestrained A and G nucleophiles, although the energetic cost is not considerable under physiological conditions, which thus permits nucleophilic substitution reactions that lead to highly distorted DNA.     

Cytosine catalysis of nitrosative guanine deamination and interstrand cross-link formation

Effects are discussed of the anisotropic DNA environment on nitrosative guanine deamination based on results of an ab initio study of the aggregate 3 formed by guaninediazonium ion 1 and cytosine 2. Within 3, the protonation of 2 by 1 is fast and exothermic and forms 6, an aggregate between betaine 4 (2-diazonium-9H-purin-6-olate) and cytosinium ion 5. Electronic structure analysis of 4 shows that this betaine is not mesoionic; only the negative charge is delocalized in the pi-system while the positive charge resides in the sigma-system. Potential energy surface exploration shows that both dediazoniation and ring-opening of betaine 4 in aggregate 6 are fast and exothermic and lead irreversibly to E-11, the aggregate between (E)-5-cyanoimino-4-oxomethylene-4,5-dihydroimidazole E-10 and 5. The computed pair binding energies for 3, 6, and E-11 greatly exceed the GC pair binding energy. While 1 can be a highly reactive intermediate in reactions of the "free nucleobase" (or its nucleoside and nucleotide), the cyanoimine 10 emerges as the key intermediate in nitrosative guanine deamination in ds-DNA and ds-oligonucleotides. In essence, the complementary nucleobase cytosine provides base catalysis and switches the sequence of deprotonation and dediazoniation. It is argued that this environment-induced switch causes entirely different reaction paths to products as compared to the respective "free nucleobase" chemistry, and the complete consistency is demonstrated of this mechanistic model with all known experimental results. Products might form directly from 10 by addition and ring closure, or their formation might involve water catalysis via 5-cyanoamino-4-imidazolecarboxylic acid 12 and/or 5-carbodiimidyl-4-imidazolecarboxylic acid 13. The pyrimidine ring-opened intermediates 10, 12, and 13 can account for the formations of xanthosine, the pH dependency and the environment dependency of oxanosine formation, the formation of the classical cross-link dG(N(2)())-to-dG(C2), including the known sequence specificity of its formation, and the formation of the structure-isomeric cross-link dG(N1)-to-dG(C2).     

Transition Structures for the Aromatic Claisen Rearrangements by the Molecular Orbital Method

Ab initio calculations were performed for eight Claisen rearrangements, eqs 1-8. Transition-state (TS) structures of [3,3] sigmatropic rearrangements of reactions 1-4 are similar, but their activation energies (E(a)'s) are different, E(a)(1) < E(a)(2) and E(a)(3) < E(a)(4). From the intermediate of reaction 3, a hydrogen is moved intermolecularly to form the product, o-allyl phenol. The lower reactivities of reactions 2 and 4 relative to reactions 1 and 3 are ascribed to large endothermicities in the sigmatropic rearrangements, respectively. Chair-type transition states are more favorable than boat-type transition states in reactions 1-4. The allyl group is released from the in-plane C-X (X = O or N) sigma bond and is captured by the pi-type lone-pair electrons. The sulfur- and phosphorus-containing rearrangements, reactions 5-8, are computed to have smaller activation energies but are to be less exothermic than those of oxgyen- and nitrogen-containing rearrangements.