Hydroxylamine and methoxyamine mutagenesis: displacement of the tautomeric equilibrium of the promutagen N6-methoxyadenosine by complementary base pairing

The imino-amino tautomeric equilibrium of the promutagenic adenosine analogue N6-methoxy-2',3',5'-tri-O-methyladenosine [OMe6A(Me)3], in solvents of various polarities, has been studied with the aid of 1H and 13C NMR spectroscopy. The high energy barrier (free enthalpy delta G = 80 +/- 5 kJ X mol-1) between the two tautomeric species renders possible direct observation of the independent sets of all 1H and 13C signals from each of them. The equilibrium ranges from 10% imino in CCl4 to 90% in aqueous medium. Thermodynamic parameters, including energy barriers and lifetimes, were calculated from the temperature dependence of the equilibrium. Essentially similar results prevail for the promutagenic N6-hydroxy analogue. The conformations of the sugar moieties, and of the base about the glycosidic bond, for both tautomers are similar to those for adenosine. The conformation of the exocyclic N6-OCH3 group, which determines the ability of each species to form planar associates (hydrogen-bonded base pairs), has also been evaluated. Formation of autoassociates of OMe6A(Me)3 and of heteroassociates with the potentially complementary 2',3',5'-tri-O-methyluridine and -cytidine, in chloroform solution, was also investigated. The amino form base pairs with uridine and the imino form with cytidine. Formation of a complementary base pair by a given tautomeric species was accompanied by an increase of up to 10% in the population of this species and a concomitant decrease in population of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of hydroxylamine mutagenesis: tautomeric shifts and proton exchange between the promutagen N6-methoxyadenosine and cytidine

Whereas the amino, but not imino, tautomer of the promutagen N6-methoxyadenosine (OMe6A) forms planar associates (base pairs) with the potentially complementary uridine [Stolarski, R., Kierdaszuk, B., Hagberg, C.-E., & Shugar, D. (1984) Biochemistry 23, 2906-2913], it has now been found, with the aid of 1H NMR spectroscopic techniques, that only the imino tautomer of OMe6A base pairs with the potentially complementary cytidine. The association constant for such heteroassociates is more than an order of magnitude higher than that for autoassociates of OMe6A. The formation of heteroassociates is accompanied by a marked shift in tautomeric equilibrium of OMe6A, with an increase in the population of the amino form from 18% to as high as 44% and a corresponding decrease in the population of the imino species. Furthermore, the presence of cytidine in a solution of OMe6A appreciably enhances the rate of tautomeric exchange between the two tautomeric forms. Formation of planar heteroassociates between cytidine and the imino form of OMe6A is also accompanied by proton exchange between the cytidine NH2 and the N6-H of the amino form of OMe6A. The rate constants for this exchange and for tautomeric exchange, determined by the saturation transfer technique, have been measured at various concentrations and temperatures. A model is advanced for proton exchange that takes into account the interdependence of tautomeric exchange and proton exchange, as well as the role of auto- and heteroassociates. The relevance of these results to the molecular basis of hydroxylamine and methoxyamine mutagenesis and to the phenomenon of proton exchange in other systems is briefly discussed.     

Tautomerism and conformation of the promutagenic analogue N6-methoxy-2'',3'',5''-tri-O-methyladenosine

N6-Methoxy-2',3',5'-tri-O-methyladenosine crystallizes in space group P2(1)2(1)2(1) with cell dimensions a = 4.693, b = 11.412, c = 31.741 A. Least-squares refinement of diffractometer data converged at R = 0.038. The location of a hydrogen atom at N1 and the observed bond lengths and bond angles indicate unequivocally the imino tautomer of the adenine moiety. The N6-methoxy group is oriented syn to N1 and the glycosidic torsion angle XCN is -3.6 degrees, i.e. in the anti range. The furanose ring has a C2'-exo/C3'- endo pucker (P = 0.9 degrees) and is unusually flattened (tau m = 30.0 degrees). The conformations of the O-methyl groups of the ribose ring are compared with those of monomethylated nucleosides, including the biologically important 2'-O-methyl nucleosides. Evidence is presented for the existence of C-H ... N intermolecular hydrogen bonds between adenine moieties. Bearing in mind that N6-methoxyadenosine is a promutagenic analogue, the results are compared with those for the corresponding promutagenic N4-methoxycytidine. They are also discussed in relation to the tautomerism, the conformation of the N6-methoxy group, and the associated base-pairing abilities in the absence and presence of polymerases.     

Aminequinone-hydroxylquinoneimine tautomeric equilibrium revisited: molecular modeling study of the tautomeric equilibrium and substituent effects in 4-(4-R-phenylamino)naphthalene-1,2-diones

Semi-empirical (AM1 and PM3) and DFT (B3LYP/6-31G(d)) calculations were employed to study the tautomeric equilibrium between the aminequinone A and hydroxylquinoneimine B forms of 4-(4-R-phenylamino)naphthalene-1,2-diones. Substituent effects on the tautomeric equilibrium as well as on geometric and electronic parameters were also determined. In the gas phase the hydroxylquinoneimine B form is the most stable, whereas in water the aminequinone A form becomes more stable. The substituents do not modify the relative energies of the two tautomers. These results are in accordance with experimental data reported in the literature.     

Tautomerism, acid-base properties and conformation of methylated analogues of the promutagenic N4-hydroxycytosine

UV and NMR spectroscopy were employed to study the tautomerism, acid-base properties and conformation of the exocyclic N(4)-OH group in 1-methyl-N(4)-hydroxycytosine (1-mOH(4)C), and its methyl derivatives, viz. the fixed imino forms (1,3-m(2)OH(4)C and 1,3,5-m(3)OH(4)C), the fixed amino form (1,N(4)-m(2)OH(4)C), and analogues sterically constrained to the form syn (1,5-m(2)OH(4)C) or anti (1,3-m(2)OH(4)C) with respect to the ring N(3). Relative to 1,N(4)-m(2)OH(4)C, UV spectroscopy showed that the other analogues were predominantly imino and that all analogues formed a structurally common cation in acid medium, with results pointing to approximately 90% population of the imino species for 1-mOH(4)C and 1,5-m(2)OH(4)C, further supported by NMR spectroscopy. Both exhibited two sequential dissociations in alkaline medium, the first due to N(4)-OH, followed by the N(3)-H. (1)H and (13)C NMR spectroscopy showed 1-mOH(4)C in the conformation syn. With 1,3,5-m(3)OH(4)C, an ;overcrowded' planar molecule with steric constraints to both the syn and anti conformations, a syn-anti equilibrium is observed, with a preference of approximately 75% for the anti rotamer, independently of the polarity of the medium. Exchange between the rotamers is slow on the NMR time-scale, with a minimal barrier to exchange exceeding 100 kJ/mol. In low-polar media, the analogues associate as dimers via O(4)-Hcdots, three dots, centeredO(2) or O(4)-Hcdots, three dots, centeredN(4) hydrogen bonds, with association constants at ambient temperature of 4.6 (1,3-m(2)OH(4)C), 12.8 (anti 1,3,5-m(3)OH(4)C), 36 (1,5-m(2)OH(4)C), 109 (syn 1,3,5-m(3)OH(4)C) M(-1). Implications of the overall findings to the promutagenic activities of OH(4)C and OMe(4)C are examined.     

Synthesis,equilibrium studies and structural characterisation of the Zn(II) complexes with trimethylene-N6,N6'-bisadenine

The new compound trimethylene-N(6),N(6')-bisadenine (L), in which two adenine molecules are linked together by a trimethylene bridge that connects the N(6) atoms, has been prepared. Reaction of L with HgCl(2) and ZnCl(2) in concentrated HCl solution leads to crystalline solids. The X-ray characterisation of the Hg(II) complex (H(2)L)[HgCl(4)].3H(2)O reveals that it is an outer-sphere complex in which the ligand is protonated at N(1) and N(1'). In contrast, the structure of the complex [H(2)L(ZnCl(3))(2)].2H(2)O shows the ligand co-ordinated to two different Zn(II) ions through the N(7) of both adenine fragments, the protons being located on the N(1) atoms. The latter compound constitutes the first crystallographic evidence of an inner sphere complex with bis-adenines and, for this reason, an equilibrium study was carried out on the Zn(II)-L-H(+) system. Potentiometric studies indicate that L is protonated in aqueous solution to form HL(+) and H(2)L(2+) with logK(H) values of 4.42 and 3.35 (25 degrees C, 0.10 M KNO(3)). The data from potentiometric titrations in the presence of Zn(2+) can be analysed considering the formation of the species LZn(2+), HLZn(3+), LZn(2)(4+) and HLZn(2)(5+), whose stability constants exceed the value expected for a monodentate interaction of the metal ion with adenine and suggest the possibility of a polydentate behaviour of L in the pH range 2.5-5.0. In contrast, spectrophotometric titrations carried out under conditions similar to those used in the synthetic work (1 M HCl) can be fitted with a model involving exclusively the H(2)LZn(4+) and H(2)LZn(2)(6+) species with logK(M) values reasonable for the interaction of Zn(II) with the N(7) of the protonated adenine fragments. Despite the H(2)LZn(2)(6+) species has a low stability, the spectrophotometric results are in agreement with its formation under the conditions in which the solid complex was prepared.     

Acid-base, tautomeric and isomeric equilibrium of pyridoxal oximes, pyridoxal-5'-phosphate and some of their analogs

Success has been achieved in detailed understanding of tautomeric and isomeric equilibria and search for the new tautomeric and isomeric forms of oximes of pyridoxal, pyridoxal-5'-phosphate and some of their analogs, their presence is explained. This is due to a careful deconvolution of absorption spectra of different ionic forms of oximes into bands corresponding to separate electronic transitions. The spectroscopical data and the results of quantum-chemical calculations are compared for all the forms of compounds under investigation. As it has been found to be valid for other vitamin B6 derivatives as well, quantum-chemical calculations can be used for analytical purposes.     

Specific interaction of isocytosine and amino acid carboxylic group shifts the base tautomeric equilibrium

By 1H NMR, UV and IR spectroscopies in anhydrous DMSO and quantum-chemical calculations by MNDO/H in vacuum specific interactions of isocytosine with neutral and deprotonated carboxylic groups of amino acids were investigated. In vacuum interaction with carboxylate ion provokes in isoCyt transition from the ground-state enolic form to the high energy N3H-keto tautomer. In DMSO keto tautomer N3H of isoCyt is stabilized but interactions with carboxylate ion essentially shifts equilibrium to enolic form. Neutral carboxylic group forms the most stable complex with the ground-state enolic tautomer in vacuum but in DMSO it proves to shift the keto(N3H)-enolic equilibrium to the right.     

A synthesis of N6,N6,N6-trimethyl-L-lysine dioxalate in gram amounts

A procedure is described for the synthesis of crystalline N6,N6,N6-trimethyl-L-lysine dioxalate in gram amounts starting from the commercially available N2-tert-butoxycarbonyl-N6-benzyloxycarbonyl-L-lysine, which is reacted with methyl iodide in methanol in the presence of potassium hydrogen carbonate after deprotection of the side-chain amino group by catalytic hydrogenation. The work-up involves only filtrations and evaporations.     

The Synthesis of RNA Containing the Modified Nucleotides N 2-Methylguanosine and N 6, N 6-Dimethyladenosine

Abstract

The phosphoramidites of the naturally occurring modified nucleotides N ²-methylguanosine and N ⁶,N ⁶-dimethyladenosine were synthesized and incorporated into short oligoribonucleotides. Described are the syntheses of the phosphoramidites and the procedures used to deprotect oligoribonucleotides in which the O ⁶ of m²G is protected with a 2-(p-nitrophenyl)ethyl group.     

Enzymatic glycosylation using 6-O-acylated sugar donors and acceptors: beta-N-acetylhexosaminidase-catalysed synthesis of 6-O,N,N'-triacetylchitobiose and 6'-O,N,N'-triacetylchitobiose

p-Nitrophenyl 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranoside (5a) was used as the glycosyl donor in a beta-N-acetylhexosaminidase-catalysed (from Penicillium brasilianum) glycosylation of GlcNAc yielding 6'-O,N,N'-triacetylchitobiose (6), while 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranose (3a) served as a selectively protected acceptor in a transglycosylation reaction catalysed by the same enzyme to yield 6-O,N,N'-triacetylchitobiose (4).     

Structural studies of O6-methyldeoxyguanosine and related compounds: a promutagenic DNA lesion by methylating carcinogens.

O6-Methylation of guanine residues in DNA can induce mutations by formation of base mispairing due to the deprotonation of N(1). The electronic, geometric and conformational properties of three N(9)-Substituted O6-methylguanine derivatives, O6-methyldeoxyguanosine (O6mdGuo), O6-methylguanosine (O6mGuo) and O6, 9-dimethylguanine (O6mdGua), were investigated by X-ray and/or NMR studies. O6mdGuo crystallizes in the monoclinic space group P2(1) with cell parameters a = 5.267(1), b = 19.109(2), c = 12.330(2) A, beta = 92.45(1) degrees, V = 1239.8(3) A3, z = 4 (two nucleosides per asymmetric unit), and O6mGua in the monoclinic space group P2(1)/n with cell parameters a = 10.729(2), b = 7.640(1) c = 10.216(1) A, beta = 92.17(2) degrees, V = 836.7(2) A3, z = 4. The geometry and conformation of O6-methylguanine moieties observed in both crystals and are very similar. Furthermore, the molecular dimensions of the O6methylguanine residue resemble more closely those of adenine than those of guanine. The methoxy group is coplanar with the purine ring, the methyl group being cis to N(1). The conformation of O6-methylguanine nucleosides is variable. The glycosidic conformation of O6mdGuo is anti for molecule (a) and high-anti for molecule (b) in the crystal, while that of O6mGuo is syn [Parthasarathy, R & Fridey, S. M. (1986) Carcinogenesis 7, 221-227]. The sugar ring pucker of O6mdGuo is C(2')-endo for molecule (a) and C(1')-exo for molecule (b). The C(4')-C(5') exocyclic bond conformation in O6mdGuo is gauche- for molecule (a) but trans for molecule (b), in contrast with gauche+ for O6mGuo. The hydrogen bonds exhibited by O6-methylguanine derivatives differ from those in guanine derivatives; the amino N(2) and ring N(3) and N(7) atoms of O6-methylguanine residues are involved in hydrogen bonding. 1H-NMR data for O6mdGuo and O6mdGuo reveal the predominance of a C(2')-endo type sugar puckering. In O6mdGuo, however, a contribution of a C(1')-exo sugar puckering is significant. The NOE data also indicate that O6mdGuo molecules exist with nearly equal population for anti (including high anti) and syn glycosidic conformations. These observations and their biological implications are discussed.     

Stacking Interactions of nucleobases: NMR-Investigations I. Selfassociation of N6,N9-Dimethyladenine and N6-Dimethyl-N9-Ethyladenine

The selfassociation of N⁶,N⁹-dimethyladenine and N⁶-dimethyl-N⁹-ethyladenine has been studied by means of NMR technique. The thermodynamic quantities have been calculated using an isodesmic NMR model with three NMR parameters (the monomer shift _M and two complex shifts ₂ and ₃).The dependence of the thermodynamic quantities on the NMR parameters is discussed. Special attention is given to the determination of _M and its temperature dependence.Calculations with ₃=2· ₂ and _M taken independently of temperature result in an average entropy _S =–17.9±1.8 e.u. for N⁶,N⁹-dimethyladenine and _S =–16.7±1.7 e.u. for N⁶-dimethyl-N⁹-ethyladenine and in an average enthalpy _H=–7.2±0.6 kcal· mol^–1 for both substances investigated.Part of the Ph.D. Theses of W. Schimmack and H. Sapper.Dedicated to Professor Dr. A. Schraub on the occasion of his 65th birthday.     

Concerning the width of spark channels with different polarities in submicrosecond sliding discharges in noble gases

Previously, the parameters of submicrosecond (with a duration of <200 ns) multichannel high-current discharges sliding along a ceramic surface in Ne, Ar, and Xe were studied only for the negative polarity of the applied voltage. The experimental data indicate that the channels expand in the transverse direction mainly due to electron drift from the channel surface layer under the action of the electric field perpendicular to the channel axis and subsequent gas ionization by these electrons. To investigate mechanisms for the channel development in a sliding discharge—in particular, to determine the contribution of electron drift—it is necessary to carry out experiments similar to those performed earlier for the opposite polarity of the applied voltage. Here, the results of measurements of the widths of the spark channels of negativeand positive-polarity sliding discharges excited in Ne, Ar, and Xe at pressures of 30 and 100 kPa are presented and discussed. It is shown that, depending on the pressure and sort of gas, the averaged optical width of positive-polarity channels is smaller by a factor of 1.27–1.60 than that of negative-polarity channels. The experimental data are analyzed using the theory of propagation of ionization waves with different polarities in gases. Analysis has shown that electron diffusion contributes insignificantly to channel expansion and that, for both polarities, the channel expansion rate exceeds the electron drift velocity in the transverse electric field near the channel. In the framework of the so-called approximation of nonlocalized initial conditions, the measured ratio between of the widths of negativeand positive-polarity channels and their relation to the electron mobility are explained by the channel expansion governed by both electron drift and primary free electrons produced by a short-term source in a narrow region ahead of the front of the expansion wave. Numerical simulations show that the width of this region is comparable with that of the wave front and is more than one order of magnitude smaller than the observed channel radius. Gas photoionization by the channel radiation can serve as a source of primary electrons.     

Stability and bioactivity of gibberellic acid in different solvents

Summary HPLC chromatograms indicated that transformation of gibberellic acid (GA₃) to the corresponding dicarboxylic acid via iso-GA₃ occurred in weak alkaline solution. The bioactivity of this dicarboxylic acid was about 20% that of GA₃ and above 0.3 M NaOH this compound appeared to decompose. Aqueous solutions of NaOH cannot, therefore, be used as solvent in bioassays of GA₃ activity.     

DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine.

While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C). Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy. Eight of the bacterial DNAs contained m4C. Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile. The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs. Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns. Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes. Instead, some of them have deamination-resistant m4C residues.     

Mutagenic nucleoside analog N4-aminocytidine: metabolism, incorporation into DNA, and mutagenesis in Escherichia coli.

N4-Aminocytidine, a nucleoside analog, is strongly mutagenic to various organisms including Escherichia coli. Using E. coli WP2 (trp), we measured the incorporation of [5-3H]N4-aminocytidine into DNA and at the same time measured the frequency of reversion of the wild type, thereby attempting to correlate the incorporation with mutation induction. First, we observed that N4-aminocytidine uptake by the E. coli cells was as efficient as cytidine uptake. High-pressure liquid chromatographic analysis of nucleoside mixtures obtained by enzymatic digestion of isolated cellular DNA showed that the DNA contained [3H]N4-aminodeoxycytidine, corresponding to 0.01 to 0.07% of the total nucleoside; the content was dependent on the dose of N4-aminocytidine. There was a linear relationship between the N4-aminocytosine content in DNA and the mutation frequency observed. These results constitute strong evidence for the view that the N4-aminocytidine-induced mutation in E. coli is caused by the incorporation of this agent into DNA as N4-aminodeoxycytidine. We also found that the major portion of radioactivity in DNA of cells that had been treated with [5-3H]N4-aminocytidine was in the deoxycytidine fraction. We propose a metabolic pathway for N4-aminocytidine in cells of E. coli. This pathway involves the formation of both N4-aminodeoxycytidine 5'-triphosphate and deoxycytidine 5'-triphosphate; the deoxycytidine 5'-triphosphate formation is initiated by conversion of N4-aminocytidine into uridine. In support of this proposed scheme, a cytidine deaminase preparation obtained from E. coli catalyzed the decomposition of N4-aminocytidine into uridine and hydrazine.