Dissociation energies of deoxyribose nucleotide dimer anions measured using blackbody infrared radiative dissociation

The dissociation kinetics of deprotonated deoxyribose nucleotide dimers were measured using blackbody infrared radiative dissociation. Experiments were performed with noncovalently bound dimers of phosphate, adenosine (dAMP), cytosine (dCMP), guanosine (dGMP), thymidine (dTMP), and the mixed dimers dAMP.dTMP and dGMP.dCMP. The nucleotide dimers fragment through two parallel pathways, resulting in formation of the individual nucleotide or nucleotide + HPO3 ion. Master equation modeling of this kinetic data was used to determine threshold dissociation energies. The dissociation energy of (dGMP.dCMP-H)- is much higher than that for the other nucleotide dimers. This indicates that there is a strong interaction between the nucleobases in this dimer, consistent with the existence of Watson-Crick hydrogen bonding between the base pairs. Molecular mechanics simulations indicate that Watson-Crick hydrogen bonding occurs in the lowest energy structures of (dGMP.dCMP-H)-, but not in (dAMP.dTMP-H)-. The trend in gas phase dissociation energies is similar to the trend in binding energies measured in nonaqueous solutions within experimental error. Finally, the acidity ordering of the nucleotides is determined to be dTMP < dGMP < dCMP < dAMP, where dAMP has the highest acidity (largest delta Gacid).     

超声波破碎-高效液相色谱法定量检测核酸

采用超声波破碎结合高效液相色谱技术,建立了定量检测质粒DNA的方法,测量结果可以溯源至核苷酸标准物质.采用超声波破碎(功率300 W,频率24 kHz)技术将质粒DNA破碎成200～500 bp的小片段DNA,再用蛇毒磷酸二酯酶将其水解为4种核苷酸(dCMP:3.2 min;dTMP:4.7 min; dGMP:5.3...     

四甲基吡啶卟啉与核酸相互作用的稳态和时间分辨光谱

采用稳态吸收和荧光光谱、圆二色谱和皮秒时间分辨荧光光谱手段, 研究了5,10,15,20-四[4-(N-甲基吡啶)]卟啉(TMPyP4)与腺嘌呤(A)、鸟嘌呤(G)、胸腺嘧啶(T)和胞嘧啶(C)等4种碱基, 以及相应的核苷、核苷酸和单链DNA的结合能力和光谱学性质. 研究结果发现, 嘌呤与TMPyP4的结合能力比嘧啶的强. 对于某一碱基系列, 结合能力强弱顺序依次为: 碱基~核苷<核苷酸<单链DNA. 时间分辨荧光谱研究发现, 除鸟嘌呤外, 核酸和TMPyP4复合物的荧光动力学均含有快(1~2 ns)和慢(约10 ns)两个衰减过程, 它们分别是由激基复合体和环境极性对激发态TMPyP4分子的影响所致. 单链DNA能诱导TMPyP4产生诱导圆二色信号, 而单分子(碱基、核苷、核苷酸)则无此功能.     

Separation of N2-ethyl-2'-deoxyguanosine-5'-monophosphate and four native deoxyribonucleoside monophosphates using capillary zone electrophoresis with polyethylene glycol as buffer additive

We investigated the separation of five deoxyribonucleoside monophosphates: 2'-deoxyguanosine-5'-monophosphate (dGMP), 2'-deoxyadenosine-5'-monophosphate (dAMP), 2'-deoxycytosine-5'-monophosphate (dCMP), 2'-deoxythymidine-5'-monophosphate (dTMP) and a dGMP adduct possessing N2-ethyl-guanine, which has been noted in relation to mutagenesis of alcohol, using capillary zone electrophoresis (CZE). The concentration of polyethylene glycol (PEG) as a modifier and the pH of the running solutions can efficiently control the observed separation. Interaction of PEG with analytes was quantitatively evaluated. PEG worked effectively as a hydrophobic selector in these separations. The values of pKa of the acidic-NH-groups in the base moieties of dGMP, dTMP, and the dGMP adduct are close to that of boric acid used as buffer of the running solutions. The control of their charge was facilitated, enabling improved separations. A more sufficient and fast separation was achieved by both optimization of pH of the running solutions and PEG concentration compared with that obtained by pH control alone. On-line concentration using a stacking method followed by the PEG-assisted CZE was briefly studied.     

Stepwise hydration and multibody deprotonation with steep negative temperature dependence in the benzene.+ -water system

We studied the stepwise hydration and solvent-mediated deprotonation of the benzene*+ cation (Bz*+) and found several unusual features. The solvent binding energies DeltaH on-1,n for the reactions Bz*+(H2O)n-1 + H2O --> Bz*+(H2O)n are nearly constant at 9 +/- 1 kcal mol-1 for n = 1 to 8. We observed a remarkable sudden decrease in the entropy of association accompanying the formation of Bz*+(H2O)7 and Bz*+(H2O)8, indicating strong orientational restraint in the hydration shells of these clusters consistent with the formation of cagelike structures. We observed the size-dependent deprotonation of Bz*+ in a cooperative multibody process, where n H2O molecules (n >/= 4) can remove a proton from Bz*+ to form protonated water clusters. We measured, for the first time, the temperature dependence of such a process and found a negative temperature coefficient of a magnitude unprecedented in any chemical reaction, of the form k = AT-67+/- 4, or in an Arrhenius form having an activation energy of -34 +/- 1 kcal mol-1. The temperature effect may be explained by Bz*+ and four H2O molecules needing to be assembled from gas-phase components to form the reactive species. Such large temperature effects may be therefore general in solvent cluster-mediated reactions.     

Stereospecific synthesis and characterization of oligodeoxyribonucleotides containing an N2-(1-carboxyethyl)-2'-deoxyguanosine

Methylglyoxal is a highly reactive alpha-ketoaldehyde that is produced endogenously and present in the environment and foods. It can modify DNA and proteins to form advanced glycation end products (AGEs). Emerging evidence has shown that N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) is a major marker for AGE-linked DNA adducts. Here, we report, for the first time, the preparation of oligodeoxyribonucleotides (ODNs) containing individual diastereomers of N2-CEdG via a postoligomerization synthesis method, which provided authentic substrates for examining the replication and repair of this lesion. In addition, thermodynamic parameters derived from melting temperature data revealed that the two diastereomers of N2-CEdG destabilized significantly the double helix as represented by a 4 kcal/mol increase in Gibbs free energy for duplex formation at 25 degrees C. Primer extension assay results demonstrated that both diastereomers of N2-CEdG could block considerably the replication synthesis mediated by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. Strikingly, the polymerase incorporated incorrect nucleotides, dGMP and dAMP, opposite the lesion more preferentially than the correct nucleotide, dCMP.     

Characterization of carbon monoxide photodissociation from Fe(II)LPO with photoacoustic calorimetry

Lactoperoxidase belongs to a family of mammalian peroxidases that catalyze the oxidation of halides and small organic molecules in the presence of H2O2. We have used photoacoustic calorimetry to characterize thermodynamic parameters associated with ligand dissociation from bovine milk lactoperoxidase. Upon CO photorelease, a prompt (tau < 50 ns) exothermic volume contraction (DeltaH = -20 +/- 7 kcal mol-1 and DeltaH = -2 +/- 1 mL mol-1) was measured at pH 7.0 and 4.0, whereas an endothermic expansion (DeltaH = 30 +/- 13 kcal mol-1 and DeltaV = 9 +/- 2 mL mol(-1)) was observed at pH 10.0 and 7.0 in the presence of 500 mM NaCl. We attribute the observed volume and enthalpy changes to electrostriction arising from changes in the charge distribution associated with a reorganization of the heme binding pocket upon ligand dissociation. It is likely that cleavage of the Fe-CO bond is accompanied by distortion of a salt bridge between Arg557 and the heme propionate group, resulting in the observed electrostriction due to changes in charge distribution.     

Hydrogen atom transfer from iron(II)-tris[2,2'-bi(tetrahydropyrimidine)] to TEMPO: a negative enthalpy of activation predicted by the Marcus equation

The transfer of a hydrogen atom from iron(II)-tris[2,2'-bi(tetrahydropyrimidine)], [FeII(H2bip)3]2+, to the stable nitroxide, TEMPO, was studied by stopped-flow UV-vis spectrophotometry. The products are the deprotonated iron(III) complex [FeIII(H2bip)2(Hbip)]2+ and the hydroxylamine, TEMPO-H. This reaction can also be referred to as proton-coupled electron transfer (PCET). The equilibrium constant for the reaction is close to 1; thus, the reaction can be driven in either direction. The rate constants for the forward and reverse reactions at 298 K are k1 = 260 +/- 30 M-1 s-1 and k-1 = 150 +/- 20 M-1 s-1. Interestingly, the rate constant for the forward reaction decreases as reaction temperature is increased, implying a negative activation enthalpy: DeltaH1 = -2.7 +/- 0.4 kcal mol-1, DeltaS1 = -57 +/- 8 cal mol-1 K-1. Marcus theory predicts this unusual temperature dependence on the basis of independently measured self-exchange rate constants and equilibrium constants: DeltaHcalcd = -3.5 +/- 0.5 kcal mol-1, DeltaScalcd = -42 +/- 10 cal mol-1 K-1. This result illustrates the value of the Marcus approach for these types of reactions. The dominant contributor to the negative activation enthalpy is the favorable enthalpy of reaction, DeltaH1 degrees = -9.4 +/- 0.6 kcal mol-1, rather than the small negative activation enthalpy for the H-atom self-exchange between the iron complexes.     

Effects of electron attachment on C5'-O5' and C1'-N1 bond cleavages of pyrimidine nucleotides: A theoretical study

Sugar-base C(1')-N(1) and phosphate-sugar C(5')-O(5') bond breakings of 2'-deoxycytidine-5'-monophosphates (dCMP) and 2'-deoxythymidine-5'- monophosphates (dTMP) and their radical anions have been explored theoretically at the B3LYP/DZP++ level of theory. Calculations show that the low-energy electrons attachment to the pyrimidine nucleotides results in remarkable structural and chemical bonding changes. Predicted Gibbs free energies of reaction DeltaG for the C(5')-O(5') bond dissociation process of the radical anions are -14.6 and -11.5 kcal mol(-1), respectively, and such dissociation processes may be intrinsically spontaneous in the gas phase. Furthermore, the C(5')-O(5') bond cleavage processes of the anionic dCMP and dTMP were predicted to have activation energies of 6.9 and 8.0 kcal mol(-1) in the gas phase, respectively, much lower than the barriers for the C(1')-N(1) bond breaking process, showing that the C-O bond dissociation in DNA single strand breaks is a dominant process as observed experimentally.     

Large ground-state entropy changes for hydrogen atom transfer reactions of iron complexes

Reported herein are the hydrogen atom transfer (HAT) reactions of two closely related dicationic iron tris(alpha-diimine) complexes. FeII(H2bip) (iron(II) tris[2,2'-bi-1,4,5,6-tetrahydropyrimidine]diperchlorate) and FeII(H2bim) (iron(II) tris[2,2'-bi-2-imidazoline]diperchlorate) both transfer H* to TEMPO (2,2,6,6-tetramethyl-1-piperidinoxyl) to yield the hydroxylamine, TEMPO-H, and the respective deprotonated iron(III) species, FeIII(Hbip) or FeIII(Hbim). The ground-state thermodynamic parameters in MeCN were determined for both systems using both static and kinetic measurements. For FeII(H2bip) + TEMPO, DeltaG degrees = -0.3 +/- 0.2 kcal mol-1, DeltaH degrees = -9.4 +/- 0.6 kcal mol-1, and DeltaS degrees = -30 +/- 2 cal mol-1 K-1. For FeII(H2bim) + TEMPO, DeltaG degrees = 5.0 +/- 0.2 kcal mol-1, DeltaH degrees = -4.1 +/- 0.9 kcal mol-1, and DeltaS degrees = -30 +/- 3 cal mol-1 K-1. The large entropy changes for these reactions, |TDeltaS degrees | = 9 kcal mol-1 at 298 K, are exceptions to the traditional assumption that DeltaS degrees approximately 0 for simple HAT reactions. Various studies indicate that hydrogen bonding, solvent effects, ion pairing, and iron spin equilibria do not make major contributions to the observed DeltaS degrees HAT. Instead, this effect arises primarily from changes in vibrational entropy upon oxidation of the iron center. Measurement of the electron-transfer half-reaction entropy, |DeltaS degrees Fe(H2bim)/ET| = 29 +/- 3 cal mol-1 K-1, is consistent with a vibrational origin. This conclusion is supported by UHF/6-31G* calculations on the simplified reaction [FeII(H2N=CHCH=NH2)2(H2bim)]2+...ONH2 left arrow over right arrow [FeII(H2N=CHCH=NH2)2(Hbim)]2+...HONH2. The discovery that DeltaS degrees HAT can deviate significantly from zero has important implications on the study of HAT and proton-coupled electron-transfer (PCET) reactions. For instance, these results indicate that free energies, rather than enthalpies, should be used to estimate the driving force for HAT when transition-metal centers are involved.     

Kinetic investigations of the process of encapsulation of small hydrocarbons into a cavitand-porphyrin

Exchange of guest molecules into capsule shaped host molecules is the most fundamental process in host-guest chemistry. Several examples of quantitative measurements of guest exchange rates have been reported. However, there have been no reports on the activation energies of these processes. A molecule known as cavitand-porphyrin (H2CP) has been reported to have a flexible host structure capable of facilitating moderate guest exchange rates suitable for kinetic measurements of the guest exchange process with 1H NMR. In this article, various kinetic and thermodynamic parameters related to the process of encapsulation of small hydrocarbons into H2CP in CDCl3 solution were determined by 2D exchange spectroscopy (EXSY): association and dissociation rate constants (k(ass) = 320 M-1 s-1, k(diss) = 1.4 s-1 for methane at 25 degrees C), the corresponding activation energies (E(a,ass) = 27 kJ.mol-1, E(a,diss) = 58 kJ.mol-1), and thermodynamic parameters for each process (DeltaG++(ass) = 59 kJ.mol-1, DeltaG++(diss) = 72 kJ.mol-1, DeltaH++(ass) = 25 kJ.mol-1, DeltaH++(diss) = 55 kJ.mol-1, DeltaS++(ass) = -113 J.K-1.mol-1, and DeltaH++(diss) = 58 J.K-1.mol-1 for methane). The thermodynamic parameters (DeltaG degrees = -13 kJ.mol-1, DeltaH degrees = -31 kJ.mol-1, DeltaS degrees = -60 J.K-1.mol-1 for methane) for this encapsulation equilibrium determined by EXSY were comparable to those for methane determined by 1D 1H NMR titration (DeltaG degrees = -11 kJ.mol-1, DeltaH degrees = -33 kJ.mol-1, DeltaS degrees = -75 J.K-1.mol-1 for methane). In addition, the structure of the methane encapsulation process was revealed by ab initio MO calculations. The activation energies for methane association/dissociation were estimated from MP2 calculations (E(a,ass) = 58.3 kJ.mol-1, E(a,diss) = 89.1 kJ.mol-1, and DeltaH degrees = -30.8 kJ.mol-1). These values are in accord with the experimentally determined values. The observed guest exchange rates and energies are compared with the corresponding values of various reported capsule-shaped hosts.     

Study of neutral Fe(III) complexes of pyridoxal-N-substituted thiosemicarbazone with desolvation-induced spin-state transformation above room temperature

The preparation and characterization of two new neutral ferric complexes with desolvation-induced discontinuous spin-state transformation above room temperature are reported. The compounds, [Fe(Hthpy)(thpy)].CH3OH.3H2O (1) and [Fe(Hmthpy)(mthpy)].2H2O (2), are low-spin (LS) at room temperature and below, whereas their nonsolvated forms are high-spin (HS), exhibiting zero-field splitting. In these complexes, Hthpy, Hmthpy, and thpy, mthpy are the deprotonated forms of pyridoxal thiosemicarbazone and pyridoxal methylthiosemicarbazone, respectively; each is an O,N,S-tridentate ligand. The molecular structures have been determined at 100(1) K using single-crystal X-ray diffraction techniques and resulted in a triclinic system (space group P1) and monoclinic unit cell (space group P21/c) for 1 and 2, respectively. Structures were refined to the final error indices, where RF = 0.0560 for 1 and RF = 0.0522 for 2. The chemical inequivalence of the ligands was clearly established, for the "extra" hydrogen atom on the monodeprotonated ligands (Hthpy, Hmthpy) was found to be bound to the nitrogen of the pyridine ring. The ligands are all of the thiol form; the doubly deprotonated chelates (thpy, mthpy) have C-S bond lengths slightly longer than those of the singly deprotonated forms. There is a three-dimensional network of hydrogen bonds in both compounds. The discontinuous spin-state transformation is accompanied with liberation of solvate molecules. This is evidenced also from DSC analysis. Heat capacity data for the LS and HS phases are tabulated at selected temperatures, the values of the enthalpy and entropy changes connected with the change of spin state were reckoned at DeltaH = 12.5 +/- 0.3 kJ mol-1 and DeltaS = 33.3 +/- 0.8 J mol-1 K-1, respectively, for 1 and DeltaH = 6.5 +/- 0.3 kJ mol-1 and DeltaS = 17.6 +/- 0.8 J mol-1 K-1, respectively, for 2.     

Enthalpy versus entropy-driven binding of bisphosphonates to farnesyl diphosphate synthase

We report the results of an ITC (isothermal titration calorimetry) investigation of the binding of six bisphosphonates to the enzyme farnesyl diphosphate synthase (FPPS; EC 2.5.1.10) from Trypanosoma brucei. The bisphosphonates investigated were zoledronate, risedronate, ibandronate, pamidronate, 2-phenyl-1-hydroxyethane-1,1-bisphosphonate, and 1-(2,2-bisphosphonoethyl)-3-iodo pyridinium. At pH = 7.4, both risedronate and the phenylethane bisphosphonate bind in an enthalpy-driven manner (DeltaH approximately -9 to 10 kcal mol-1), but the other four bisphosphonates bind in an entropy-driven manner (DeltaS varying from 31.2 to 55.1 cal K-1 mol-1). However, at pH = 8.5, zoledronate binding switches from entropy to enthalpy-driven. The DeltaG results are highly correlated with FPPS inhibition results obtained using a radiochemical assay (R2 = 0.85, N = 11, P < 0.001). The DeltaH and DeltaS results are interpreted in terms of a model in which bisphosphonates with charged side chains have positive DeltaH values, due to the enthalpic cost of desolvation (due to strong ion-dipole interactions) and, likewise, a positive DeltaS, due to an increase in water entropy (both ligand and protein associated) on ligand binding to FPPS: the hydrophobic effect. For the neutral side chains (risedronate at pH 7.4, 8.5 and zoledronate at pH 8.5, as well as the phenylethane bisphosphonate), binding is overwhelmingly enthalpy-driven, with the enhanced activity of the basic side chain containing species being attributable to their becoming protonated in the active site. Given the large size of the bisphosphonate market and the potential importance of the development of these compounds for cancer immunotherapy and anti-parasitic chemotherapy, these results are of broad general interest in the context of the development of new, potent, and selective FPPS inhibitors.     

Assay of human erythrocyte pyrimidine and deoxypyrimidine 5'-nucleotidase by isocratic reversed-phase high-performance liquid chromatography

We report a rapid and reproducible assay for activity of human erythrocyte pyrimidine 5'-nucleotidase and deoxypyrimidine 5'-nucleotidase. The nucleotides CMP, UMP, dUMP, dCMP or dTMP are individually incubated 30 min at 37 degrees C with erythrocyte hemolysate and 4 mM magnesium chloride in Tris, pH 7.5. Data are provided for standardization of the reaction with each substrate. Individual nucleoside products are assayed in less than 10 min by reversed-phase high-performance liquid chromatography at 280 nm with 0-14% methanol in 0.01 M potassium dihydrogen phosphate. This is the first report of a high-performance liquid chromatographic assay system which allows quantitation of the activity of pyrimidine 5'-nucleotidase isozymes using five individual pyrimidine and deoxypyrimidine nucleotides as the substrates.     

A molecular NAND gate based on Watson-Crick base pairing

[reaction: see text] A DNA-binding dye, 4',6-diamidino-2-phenylindole (DAPI) signals AT base pairing with a shift in the fluorescence emission spectrum. The signaling follows W-C base-pairing rules, and both dAMP and dTMP are required for the largest spectral shift. Thus, the dye with its two phosphate receptor sites functions as a molecular NAND gate accepting nucleotides as inputs. Moreover, when the observation wavelength is changed from 470 to 411.5 nm, the gate functions in TRANSFER logic.     

Probing the interaction between fluorophores and DNA nucleotides by fluorescence correlation spectroscopy and fluorescence quenching

We have investigated the association interactions between the fluorescent dyes TAMRA, Cy3B and Alexa-546 and the DNA deoxynucleoside monophosphates by means of fluorescence quenching and fluorescence correlation spectroscopy (FCS). The interactions of Cy3B and TAMRA with the nucleotides produce a decrease in the apparent diffusion coefficient of the dyes, which result in a shift toward longer times in the FCS autocorrelation decays. Our results with Cy3B demonstrate the existence of Cy3B-nucleotide interactions that do not affect the fluorescence intensity or lifetime of the dye significantly. The same is true for TAMRA in the presence of dAMP, dCMP and dTMP. In contrast, the diffusion coefficient of Alexa 546 remains practically unchanged even at high concentrations of nucleotide. These results demonstrate that interactions between this dye and the four dNMPs are not significant. The presence of the negatively charged sulfonates and the bulky chlorine atoms in the phenyl group of Alexa 546 possibly prevent strong interactions that are otherwise possible for TAMRA. The characterization of dye-DNA interactions is important in biophysical research because they play an important role in the interpretation of energy transfer experiments, and because they can potentially affect the structure and dynamics of the DNA.     

Ultrafast excited-state deactivation and energy transfer in guanine-cytosine DNA double helices

The DNA double helix poly(dGdC).poly(dGdC) is studied by fluorescence upconversion spectroscopy with femtosecond resolution. It is shown that the excited-state relaxation of the duplex is faster than that of the monomeric components dGMP and dCMP. This contrasts with the behavior of duplexes composed exclusively of adenine-thymine base pairs, for which an overall lengthening of the fluorescence lifetimes with respect to that of an equimolar mixture of dAMP and TMP was reported previously. Despite the difference in the excited-state deactivation rate between the two types of duplexes, the signature of ultrafast energy transfer is present in both of them. It is attested by the decrease of fluorescence anisotropy decay of the duplexes on the subpicosecond time scale, where molecular motions are inhibited, and is corroborated by the fact that their steady-state fluorescence spectra do not change with the excitation wavelength. Energy transfer involves excited states delocalized over at least two bases, whose existence is revealed by the UV absorption spectrum of the duplex, clearly different from that of an equimolar spectrum of dGMP and dCMP.     

A combined nuclear magnetic resonance and computational study of monohydroxyflavones applied to product ion mass spectra

A method is presented for the estimation of 13C-chemical shifts for carbon atoms in protonated and deprotonated molecules; in principle, this method can be applied to ions in general. Experimental 13C-chemical shifts were found to vary linearly with computed atomic charges using the PM3 method. Pseudo-13C-chemical shifts for atoms in protonated and deprotonated molecules can be estimated from computed atomic charges for such atoms using the above linear relationship. The pseudo-13C-chemical shifts obtained were applied to the rationalization of product ion mass spectra of protonated and deprotonated molecules of flavone and 3-, 5-, 6-, 7-, 2'-, 3'-, and 4'-hydroxyflavones, where product ion formation is due to either cross-ring cleavage of the C-ring (retro-Diels-Alder reaction) or to cleavage of a C-ring bond followed by loss of either a small neutral molecule or a radical. The total product ion abundance ratio of C-ring cross cleavage to C-ring bond cleavage, gamma, varied by a factor of 660 for deprotonated monohydroxyflavones, i.e., from 0.014:1 to 9.27:1. The magnitude of gamma, which is dependent on the relative bond orders within the C-ring of the protonated and deprotonated molecules of monohydroxyflavones, can be rationalized on the basis of the magnitudes of the 13C- and 1H-chemical shifts as determined by nuclear magnetic resonance spectroscopy.     

Effects of Gas-Phase Basicity on the Proton Transfer between Organic Bases and Trifluoroacetic Acid in the Gas Phase: Energetics of Charge Solvation and Salt Bridges

The unimolecular dissociation pathways and kinetics of a series of protonated trimer ions consisting of two organic bases and trifluoroacetic acid were investigated using blackbody infrared radiative dissociation. Five bases with gas-phase basicities (GB) ranging from 238.4 to 246.2 kcal/mol were used. Both the dissociation pathways and the threshold dissociation energies depend on the GB of the base. Trimers consisting of the two most basic molecules dissociate to form protonated base monomers with an E(0) ~ 1.4 eV. Trimers consisting of the two least basic molecules dissociate to form protonated base dimers with an E(0) ~ 1.1-1.2 eV. These results indicate that the structures of the trimers change as a function of the GB of the basic molecule. The predominant structure of the protonated trimers consisting of the two most basic molecules is consistent with a salt bridge in which both of the basic molecules are protonated, and the trifluoroacetic acid molecule is deprotonated, whereas the predominant structure of the protonated trimers consisting of the two least basic molecules are consistent with charge-solvated complexes in which the proton is shared. The structure of the trimer consisting of the base of intermediate basicity is less clear; it dissociates to form primarily protonated base dimer, but has an E(0) ~ 1.2 eV. These results are consistent with the structure of this trimer as a salt bridge, but the resulting dissociation A(-). BH(+) product does not appear to be stable as an ion pair in the dissociative transition state.     

Characterisation of estrone-nucleic acid adducts formed by reaction of 3,4-estrone-o-quinone with 2'-deoxynucleosides/deoxynucleotides using capillary liquid chromatography/electrospray ionization mass spectrometry

Xenobiotic and endobiotic molecules can react with DNA leading to formation of so-called DNA adducts. This modified DNA can be repaired enzymatically, but, if not, these modifications are believed to be responsible for the initiation of carcinogenic processes. Hence, we studied the interaction of 2'-deoxynucleosides and 2'-deoxynucleotides with 3,4-estronequinone (3,4-E(1)Q), a metabolite of estrone (E(1)) and a supposed carcinogen. These estrone-nucleic acid adducts were analysed by capillary liquid chromatography (CapLC) coupled to electrospray ionization mass spectrometry (ESI-MS). Knowledge of their behaviour from in vitro studies is a prerequisite for detecting adducts in in vivo studies. Our initial attempts to synthesise nucleos(t)ide adducts of 3,4-E(1)Q in an aprotic solvent (dimethylformamide) yielded no adducts. However, under acidic aqueous conditions, adducts were obtained. With dGuo, a dGuo adduct was found in addition to a Gua adduct. Earlier publications on adduct formation in protic solvents failed to report formation of any adduct with dAdo. A N(3)-Ade adduct was reported upon reaction of 3,4-E(1)Q with Ade base and with DNA. With dAdo, we obtained two nucleoside adducts and six Ade adducts due to loss of 2'-deoxyribose. Thus, contrary to general belief that only 2,3-E(1)Q can form stable adducts, we showed formation of substantial amounts of intact DNA adducts with 3,4-E(1)Q in addition to deglycosylated adducts. Adducts were also obtained with dGMP and dAMP, but no phosphate alkylation was found. Adducts of dCyd, dCMP, dThd, and dTMP were not detected. Using chromatographic-MS data a structural relationship between the 2'-deoxynucleoside, 2'-deoxynucleotide and base adducts was found in the various reaction mixtures. The adducts of dGuo and dGMP reaction mixtures were alkylated at the same N(7)-position of the nucleobase, as indicated by the occurrence of a rapid deglycosylation reaction. In dAdo and dAMP reaction mixtures, 14 adducts were detected; their relationships from the LC and MS data reduced the number of structures to six adenine base alkylated adducts with respect to alkylation between N(1), N(3), N(7) and/or N(6) in the adenine and C(1), C(2) and/or C(6) in 3,4-E(1)Q. We could infer, in addition, whether they had an A ring attachment or a C(6) attachment on the estrone moiety.