Investigation of tautomeric composition of cytosine by multiphoton excitation spectra. II. Two-photon absorption spectra

For the first time the quantum-mechanical calculations of intensity distribution in the two-photon absorption spectra of the six tautomeric forms of cytosine were performed. It has been confirmed that in the aqueous solution of cytosine (pH 3.0) there exist several tauromeric forms: canonical amino-oxo tautomeric form, cation, as well as cys-imino-oxo and cys-amino-hydroxy tautomers.     

Consequence of one-electron oxidation and one-electron reduction for aniline

Quantum-chemical calculations were performed for all possible isomers of neutral aniline and its redox forms, and intramolecular proton-transfer (prototropy) accompanied by π-electron delocalization was analyzed. One-electron oxidation (PhNH₂ – e → [PhNH₂]^+•) has no important effect on tautomeric preferences. The enamine tautomer is preferred for oxidized aniline similarly as for the neutral molecule. Dramatical changes take place when proceeding from neutral to reduced aniline. One-electron reduction (PhNH₂ + e → [PhNH₂]^-•) favors the imine tautomer. Independently on the state of oxidation, π- and n-electrons are more delocalized for the enamine than imine tautomers. The change of the tautomeric preferences for reduced aniline may partially explain the origin of the CH tautomers for reduced nucleobases (cytosine, adenine, and guanine).     

On tautomerism of the cytosine molecule

Tautomerism of the cytosine molecule is discussed in connection with recent experimental matrix-isolation infrared spectroscopic measurements and recent ab initio calculations of relative stabilities of tautomers and of IR spectra for different tautomeric forms of the compound. Experimental IR spectra in the N-H and O-H stretching regions and in the C = O stretching region are presented for cytosine and for its several derivatives considered as model compounds. This experimental evidence, as well as the quantum-mechanical calculations (including both electron correlation and zero-point vibrational contributions), clearly indicate that two tautomers of cytosine, i.e. the amino-hydroxy and amino-oxo forms with the hydrogen atom at the N(1) position, exist in equilibrium when the cytosine molecule is isolated in an inert environment. The effect of the environment on the relative stabilities of several tautomers is also discussed briefly.     

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.     

Consequences of one-electron oxidation and one-electron reduction for 4-aminopyrimidine—DFT studies

The consequences of one-electron oxidation and one-electron reduction were studied for 4-aminopyrimidine (4APM), which displays prototropic tautomerism. Since experimental techniques are incapable of detecting less than 0.1% of minor tautomers, quantum-chemical calculations [DFT(B3LYP)/6-311+G(d,p)] were carried out for all possible tautomers of neutral 4AMP and its redox forms, 4APM (+ ?) and 4APM (- ?). Four tautomers were considered: one amine and three imine tautomers (two NH and one CH form). Geometric isomerism of the exo?=?NH group was also taken into account. One-electron oxidation (4APM - e → 4APM (+??)) has no significant effect on the tautomeric preferences; it influences solely the composition of the tautomeric mixture. The amine tautomer is favored for both 4APM (+??) and 4APM. An interesting change in the tautomeric preference occurs for 4APM (- ?). One-electron reduction (4APM?+?e → 4APM (- ?)) favors the C5 atom for the labile proton. The preference of the imine CH tautomer in the tautomeric mixture of 4APM (- ?) may partially explain the origin of CH tautomers in nucleobases.     

Charges of phosphate groups. A role in stabilization of 2'-deoxyribonucleotides. A DFT investigation

We have analyzed the relative stabilities and Gibbs tautomeric free energy for tautomeric transitions of neutral 2'-deoxyribonucleotides and its mono- and di-protonated forms. Geometry optimizations of these nucleic acid constituents have been performed at the DFT/B3LYP level using the standard 6-31G(d) basis set. The prediction of relative stabilities, Gibbs tautomeric free energy has been made at the B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level of theory. For each nucleoside four major conformers, i.e., north/anti, north/syn, south/anti, and south/syn have been taken into consideration. We have found the substantial effect of the uncompensated charge on the relative stability of 2'-deoxyribonucleotides. In particular, when the charge of 2'-deoxyribonucleotide anions is completely compensated by protons, the syn conformations have been found to be the global minima due to stabilization provided by intramolecular hydrogen bonds. However, the negative charge that appears due to the successive removal of the protons from the phosphate group destabilizes these syn conformations and stabilizes preferably the south/anti conformations (except of 2'-deoxyguanosine phosphate). Only 2'-deoxyribonucleotides, possessing south/anti and north/anti orientations, containing guanine and cytosine can contribute significantly to the rate of spontaneous point mutations due to the formation of biologically relevant amounts of 'rare' tautomers. However, we found strong influence of uncompensated negative charge for 2'-deoxyribonucleotides which possess syn conformations. Finally we have found that the proton transfer could result in the spontaneous change of 2'-deoxyribonucleotides conformations. We conclude that this phenomenon could be considered as a new way for the stabilization of 'rare' isomers for such DNA bases as cytosine and thymine.     

Hydrogen sulfide formation as well as ethanol production in different media by cysND- and/or cysIJ-inactivated mutant strains of Zymomonas mobilis ZM4

Many bacteria reduce inorganic sulfate to sulfide to satisfy their need for sulfur, one of the most important elements for biological life. But little is known about the metabolic pathways involving hydrogen sulfide (H₂S) in mesophilic bacteria. By genomic sequence analysis, a complete set of genes for the assimilatory sulfate reduction pathway has been identified in the ethanologen Zymomonas mobilis. In this study, the first ATP sulfurylase- and final sulfite reductase-encoding genes cysND and cysIJ, respectively, in the putative pathway from sulfate to sulfite in Z. mobilis ZM4 was singly or doubly inactivated by homologous recombination and a site-specific FLP-FRT recombination. The resultant mutants, ?cysND, ?cysIJ and ?cysND-cat?cysIJ, were unable to produce detectable H₂S in glucose or sucrose-containing rich medium and sweet sorghum juice, in which the wild-type ZM4 produced detectable H₂S. While adding sulfite (SO₃ ^2?) into media impaired the growth of the mutants and ZM4 to varying degrees, the sulfite restored the H₂S formation in the ?cysND in the above media, but not in the ?cysIJ and ?cysND-cat?cysIJ mutants. Although it seemed that the inactivation of cysND and cysIJ did not exert a significant negative effect on the cell growth at least in glucose or sucrose medium, the ethanol production of all mutants was inferior to that of ZM4 in sucrose medium and sweet sorghum juice. In addition, adding l-cysteine to glucose-containing rich media restored H₂S formation of all mutants, indicating the existence of another pathway for producing H₂S in Z. mobilis. All these results would help to further elucidate the metabolic pathways involving H₂S in Z. mobilis and exploit the biotechnological applications of this industrially important bacterium.     

Quantum chemical study of the structure and properties of citrinin

Detailed structures and electronic properties of three tautomeric forms of the toxin citrinin were investigated using several quantum calculation methods. Energetic preference of the predominant p- and o-quinone methide tautomeric forms is dependent on the method of calculation. A previously unstudied carboxylic acid enol tautomer was calculated to be surprisingly stable in vacuo, being within 2.5 kcal mol^? 1 at the B3LYP/6-311++G(2d,2p) level of theory. Despite differences in bond nature and connectivity of tautomers, the natural bond orbital analysis revealed that tautomeric forms share similar natural charges and natural electron configurations. Calculated bond lengths corresponded with experimentally observed values and assignments for the calculated infrared vibrational frequencies are reported.     

Tautomeric energetics of xanthine oxidase substrates: xanthine, 2-oxo-6-methylpurine, and lumazine

The stability of the tautomers of each of the three important substrates of xanthine oxidase, xanthine, 2-oxo-6-methylpurine, and lumazine, was examined by quantum mechanical calculations. The geometries of these tautomers were optimized at the AM1, Hartree-Fock (HF/6-31G), and hybrid Hartree-Fock/density functional theory (B3LYP/6-31G(d)) levels of theory. The single point energies of some of the more stable tautomers for each of the substrates were calculated at the B3LYP/6-311 +G(2d,p) level of theory. The Conductor Polarized Continuum Model (CPCM) was used to evaluate the solvent effects on the relative stabilities of these tautomers. The calculations clearly identify the lowest energy tautomeric form for xanthine and lumazine. On the other hand, there appear to be three tautomers for 2-oxo-6-methylpurine, with only minor energetic differences in vacuo. In water, however, only one of them predominates. The lowest energy tautomers presumably represent the predominant tautomeric forms at the molybdenum center of xanthine oxidase during catalysis. Implications of these computational results are discussed in the context of enzyme catalysis.     

Variable Temperature Infrared Spectroscopy of Cytosine-Guanine Base Pairs: Tautomerism Versus Polarization

Abstract

This report describes an infrared (IR) spectroscopic study of a model cytosine - guanine base pair. This base pair is part of a self-consistent experimental system based on lipophilic ribose derivatives of cytidine (C), guanosine (G) and O⁶-methylguanosine (O⁶MeG) that are soluble in non-aqueous, low dielectric solvents at appreciable concentrations. Previous experiments on this system have revealed different rotation dynamics for the amino bonds within the CG base pair, an observation that could be explained by the presence of rare tautomers (P.O. Lowdin, Reviews of Modern Physics 35, 724 (1963)), or by mutual polarization of the base pairs (L.D. Williams, N.G. Williams and B.R. Shaw, J. Am. Chem. Soc. 112, 829 (1990)). The IR spectra in the OH and NH stretching region indicate formation of hydrogen-bonded CG base pairs and self associates in 1,2-dichlorobenzene over a temperature range from 10 to 290K. Changes in the lineshapes and intensities of the IR bands with temperature correlate with phase transitions of the solvent but no evidence is seen for an OH stretching band that would indicate the formation of hydroxyl tautomers within base pairs. Similarly, the relative intensities of the C=O stretching bands of CG in cyclohexane solution remain constant over this same temperature range, confirming that within the base pair, the tautomeric states of the bases remain essentially unperturbed in the 2-amino/6-keto form of G and the 2-keto/4-amino form of C. The spectra of O⁶-MeG aid in the band assignments, since this molecule is frozen in an equivalent of the 2-amino/6-hydroxyl tautomer, but without the OH group and its associated stretching band. We conclude that the probability of tautomerism does not appear to be sufficient to explain the different rotation dynamics for the two amino bonds of the CG base pair. Rather it is argued that mutual polarization within the base pair, which would increase the bond order of the amino bond of C within the base pair, can explain the results without the formation of unconventional tautomers.     

Density-functional study on the equilibria in the ThDP activation

The equilibria among the various ionization and tautomeric states involved in the activation of ThDP is addressed using high level density functional theory calculations, X3LYP/6-311++G(d,p)//X3LYP(PB)/6-31++G(d,p). This study provides the first theoretically derived thermodynamic data for the internal equilibria in the activation of ThDP. The role of the medium polarity on the geometry and thermodynamics of the diverse equilibria of ThDP is addressed. The media chosen are cyclohexane and water, as paradigms of apolar and polar media. The results suggest that all ionization and tautomeric states are accessible during the catalytic cycle, even in the absence of substrate, being APH⁺ the form required to interconvert the AP and IP tautomers; and the generation of the ylide proceeds via the formation of the IP form. Additionally, the calculated ΔG° values allow to calculate all the equilibrium constants, including the pK_C2 for the thiazolium C2 atom whose ionization is believed to initiate the catalytic cycle.     

Tauto Merism and Ionization of Xanthosine

Abstract

Tautomerism and ionization of xanthosine have been studied by infrared spectroscopy. N-methyl and O-methyl model compounds which are isoelectronic with possible keto and enol tautomers have been synthesized. Comparisons of their spectra with neutral and with ionized xanthosine demonstrate that unionized xanthosine has the diketo form and that on acid dissociation (pK 5.7), the first proton is lost from N3 (rather than N1) to give the 6-keto-2-enolate anion. Specific labelling at the 2- and 6-positions with ¹⁸O confirms these conclusions and supports assignment of the highest frequency bands in the double bond region to carbonyl stretching vibrations. These conclusions are relevant to two different proposed structures for the ordered form of polyxanthylic acid (pH 7), one based upon keto and one upon enol tautomeric forms.     

Muscimol and some aza analogues: molecular orbital calculations relating to their potency as GABA mimetics

Ab initio molecular orbital calculations have been carried out on muscimol (1) and on two aza analogues, 5-aminomethylpyrazol-3-one (2) and 5-aminomethyl-1,2,4-triazol-3-one (3). Fully ionized species were studied in each case including the tautomeric forms of (2) and (3). The calculations reveal that the more stable tautomeric forms of (2) and (3) resemble muscimol in having a low energy barrier to rotation of the aminomethyl group (2–4 kcal mol^?1) but differ substantially from muscimol in the electronic character of the heterocyclic ring. The change in π-electron distribution in the more stable tautomers of (2) and (3) compared with muscimol is summarized by the dipole moment decrease (1) 7·75 au, (2) 6·62 au, (3) 5·81 au. In addition, the frontier molecular orbitals show analogous differences. On the other hand, the less stable tautomers of (2) and (3) are similar to muscimol in π-electron distribution and frontier orbitals, but have a high energy barrier to rotation of the aminomethyl group (11–14 kcal mol^?1). The calculated properties of the molecules are related to the ability of the three compounds to displace [³H]-GABA from the postsynaptic receptors of human cerebellum and it is suggested that the dominant influence on biochemical activity is the π-electron distribution in the heterocyclic ring.     

The effects of tautomerization and protonation on the adenine–cytosine mismatches: a density functional theory study

In the present work, we demonstrate the results of a theoretical study concerned with the question how tautomerization and protonation of adenine affect the various properties of adenine–cytosine mismatches. The calculations, in gas phase and in water, are performed at B3LYP/6-311++G(d,p) level. In gas phase, it is observed that any tautomeric form of investigated mismatches is more stabilized when adenine is protonated. As for the neutral mismatches, the mismatches containing amino form of cytosine and imino form of protonated adenine are more stable. The role of aromaticity on the stability of tautomeric forms of mismatches is investigated by NICS(1)_ZZ index. The stability of mispairs decreases by going from gas phase to water. It can be explained using dipole moment parameter. The influence of hydrogen bonds on the stability of mismatches is examined by atoms in molecules and natural bond orbital analyses. In addition to geometrical parameters and binding energies, the study of the topological properties of electron charge density aids in better understanding of these mispairs.     

Unexpected Shift to a 4-Imino Tautomer Upon N4-Acylation of 5-Methylcytosin-1-yl Nucleoside Analogues

Abstract

In contrast with the behaviour of 5-unsubstituted cytosine nucleoside analogues, 5-methylcytosine derivatives show upon N⁴-benzoylation, commonly used as base protection in oligonucleotide synthesis, a tautomeric change of the base moiety from a 4-amino- into a 4-imino isomer. In the latter form, which is easily diagnosticized by ¹³C NMR and confirmed by X-ray data, the compounds seem to be hydrolytically less stable.     

A Theoretical Study of the cis-syn Pyrimidine Dimers in the Gas Phase and Water Cluster and a Tautomer-Bypass Mechanism for the Origin of UV-induced Mutations

Abstract

A quantum mechanical study of all cis-syn cyclobutane pyrimidine photodimers including the normal and rare tautomeric forms of bases has been performed using the ab initio method at HF/6–31G(d,p), MP2(fc)//HF/6–31G(d,p) and MP2(fc)/6–31G(d,p) levels. A puckering angle of the cyclobutyl ring and twist angle of pyrimidine rings with respect to each other is well described by these calculations. It is predicted that in the gas phase all photodimers containing the rare imino form of cytosine are more stable than those containing its normal form. The Monte Carlo simulations show that the dimer containing the imino form of cytosine is more stabilized by water cluster than that containing its amino forms. The possible biological significance stems from the fact that the cytosine in the dimer directs the incorporation of adenine in the complementary strand during replicative bypass. Data obtained point to the cytosine tautomerism as a possible mechanism for the origin of UV-induced mutation.     

Observation of the keto tautomer of D-fructose in D(2)O using (1)H NMR spectroscopy

D-Fructose was analysed by NMR spectroscopy and previously unidentified (1)H NMR resonances were assigned to the keto and α-pyranose tautomers. The full assignment of shifts for the various fructose tautomers enabled the use of (1)H NMR spectroscopy in studies of the mutarotation (5-25°C) and tautomeric composition at equilibrium (5-50°C). The mutarotation of β-pyranose to furanose tautomers in D(2)O at a concentration of 0.18 M was found to have an activation energy of 62.6 kJmol(-1). At tautomeric equilibrium (20°C in D(2)O) the distribution of the β-pyranose, β-furanose, α-furanose, α-pyranose and the keto tautomers was found to be 68.23%, 22.35%, 6.24%, 2.67% and 0.50%, respectively. This tautomeric composition was not significantly affected by varying concentrations between 0.089 and 0.36 M or acidification to pH 3. Upon equilibrating at 6 temperatures between 5 and 50°C there was a linear relationship between the change in concentration and temperature for all forms.     

DFT studies on one-electron oxidation and one-electron reduction for 2- and 4-aminopyridines

Quantum-chemical calculations {DFT(B3LYP)/6-311+G(d,p)} were performed for all possible tautomers (aromatic and nonaromatic) of neutral 2- and 4-aminopyridines and their oxidized and reduced forms. One-electron oxidation has no important effect on the tautomeric preference for 2-aminopyridine. The amine tautomer is favored. However, oxidation increases the stability of the imine NH tautomer, and its contribution in the tautomeric mixture cannot be neglected. In the case of 4-aminopyridine, one-electron oxidation increases the stability of both the amine and imine NH tautomers. Consequently, they possess very close energies. As major tautomers, they dictate the composition of the tautomeric mixture. The CH tautomers may be considered as very rare forms for both neutral and oxidized aminopyridines. A reverse situation takes place for the reduced forms of aminopyridines. One-electron reduction favors the C3 atom for the labile proton for both aminopyridines. This may partially explain the origin of the CH tautomers for the anionic states of nucleobases containing the exo NH(2) group.     

More than two pyrrole tautomers of mesoporphyrin stabilized by a protein. High resolution optical spectroscopic study.

Mesoporphyrin IX substituted horseradish peroxidase was studied by fluorescence line narrowing and hole burning techniques at cryogenic temperatures. The spectral data reveal that four pyrrole tautomeric configurations of the chromophore are populated within the protein under the influence of irradiation and/or thermal treatment, and the existence of a fifth and a sixth tautomeric configuration is also likely. The relative population of the tautomers changes upon deuterium substitution through modification of the phototransition rate, and also depends on pH, which changes the protonation of neighboring amino acids in the heme pocket. The energy separation of the origins of the tautomers is approximately 100 cm-1. The distribution of barrier heights separating the different tautomeric forms in the ground state and their distribution was determined by temperature cycling hole burning. The distributions can be approximated by Gaussians. The experiment directly yields the distributions on a relative temperature scale, and a model is presented to transform the barrier heights into energy values. It is suggested that the energies for the tautomers are split partially due to the protein crystal field and that the trapping of the tautomeric forms is the consequence of interactions with neighboring amino acids within the heme pocket.     

Use ofN,N′-polymethylenebis(iodoacetamide) derivatives as probes for the detection of conformational differences in tubulin isotypes

Mammalian brain tubulin is anαΒ heterodimer; bothα andΒ exist in 6–7 isotypic forms which differ in their amino acid sequences. By the use of isotype-specific monoclonal antibodies, we have previously shown that we can purify theαΒ _II,αΒ _III, andaΒ _IV tubulin dimers from bovine brain. We have also observed that these isotypes differ in their distributionin vivo and their polymerization and drug-binding propertiesin vitro. We have now explored the question of whether the isotypically purified dimers differ in their overall conformation using as probes compounds of theN,N′-polymethylenebis (iodoacetamide) series which are known to form discrete intrachain cross-links inΒ-tubulin. These compounds have the structure ICH₂CONH(CH₂) _n NHCOCH₂I. One of these cross-links, designatedΒ ^s, is between cys¹² and either cys²⁰¹ or cys²¹¹. The other, designatedΒ ^*, is between cys²³⁹ and cys³⁵⁴. TheΒ ^* cross-link forms inαΒ _II andαΒ _IV but not inαΒ _III; this is not surprising in view of the fact thatαΒ _III has serine at position 239 instead of cysteine. However,αΒ _III is also unable to form theΒ ^s cross-link, although it appears to have all three cysteines which may be involved in the cross-link. This suggests that at least one of the sulfhydryls involved in the cross-link may be inaccessible inαΒ _III. Although bothαΒ _II andαΒ _IV can form theΒ ^s cross-link, the dependence on cross-linker chain length is different.αΒ _II formsΒ ^s with derivatives in whichn=2, 4, 5, 6, and 7 but not with those in whichn=3 or 10. In contrast,αΒ _IV formsΒ ^s with derivatives in whichn=2, 3, 4, 5, 6, 7, and 10. These results imply that theΒ ^s sulfhydryls are slightly more accessible inαΒ _IV and are therefore less dependent on the conformation of the cross-linker to react with it. It appears, therefore, that theαΒ _II,αΒ _III, andαΒ _IV dimers each have unique conformations. This may help to explain the different assembly and drug-binding properties of these dimers.