Variations of the tautomeric preferences and π-electron delocalization for the neutral and redox forms of purine when proceeding from the gas phase (DFT) to water (PCM)

Quantum-chemical calculations were performed for all possible nine neutral tautomers of purine and their oxidized and reduced forms in water {PCM//DFT(B3LYP)/6?311+G(d,p)} and compared to those in the gas phase {DFT(B3LYP)/6?311+G(d,p)}. PCM hydration influences geometries, π-electron delocalization, and relative energies of purine tautomers in different ways. Generally, the harmonic oscillator model of electron delocalization (HOMED) indices increase when proceeding from the gas phase to aequeous solution for the neutral and redox forms of purine. Their changes for the neutral and oxidized tautomers are almost parallel to the relative energies showing that aromaticity plays an important role in the tautomeric preferences. Tautomeric stabilities and tautomeric preferences vary when proceeding from the gas phase to water indicating additionally that intra- and intermolecular interactions affect tautomeric equilibria. The tautomeric mixture of neutral purine in the gas phase consists mainly of the N9H tautomer, whereas two tautomers (N9H and N7H) dominate in water. For oxidized purine, N9H is favored in the gas phase, whereas N1H in water. A gain of one electron dramatically changes the relative stabilities of the CH and NH tautomers that C6H and C8H dominate in the tautomeric mixture in the gas phase, whereas N3H in water. These variations show exceptional sensitivity of the tautomeric purine system on environment in the electron-transfer reactions.     

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.     

The tautomeric state of histidines in myoglobin

1H-15N HMQC spectra were collected on 15N-labeled sperm whale myoglobin (Mb) to determine the tautomeric state of its histidines in the neutral form. By analyzing metaquoMb and metcyanoMb data sets collected at various pH values, cross-peaks were assigned to the imidazole rings and their patterns interpreted. Of the nine histidines not interacting with the heme in sperm whale myoglobin, it was found that seven (His-12, His-48, His-81, His-82, His-113, His-116, and His-119) are predominantly in the N epsilon2H form with varying degrees of contribution from the Ndelta1 H form. The eighth, His-24, is in the Ndelta1H state as expected from the solid state structure. 13C correlation spectra were collected to probe the state of the ninth residue (His-36). Tentative interpretation of the data through comparison with horse Mb suggested that this ring is predominantly in the Ndelta1H state. In addition, signals were observed from the histidines associated with the heme (His-64, His-93, and His-97) in the 1H-15N HMQC spectra of the metcyano form. In several cases, the tautomeric state of the imidazole ring could not be derived from inspection of the solid state structure. It was noted that hydrogen bonding of the ring was not unambiguously reflected in the nitrogen chemical shift. With the experimentally determined tautomeric state composition in solution, it will be possible to broaden the scope of other studies focused on the electrostatic contribution of histidines to the thermodynamic properties of myoglobin.     

Deprotonated carboxylic group of amino acids transforms adenine into its rare prototropic tautomers

By UV spectroscopic data for anhydrous DMSO solutions and ab initio HF/6-31G** calculations in vacuum it was shown for the first time that deprotonated amino acid carboxylic group is able to change tautomeric state of a nucleotide base, exactly to convert the N9H ground-state prototropic tautomer of adenine into the N7H and N1H rare ones.     

Schiff base of gossypol with 3,6,9-trioxa-decylamine complexes with monovalent cations studied by mass spectrometry, (1)H-NMR, FTIR, and PM5 semiempirical methods

A new Schiff base of gossypol with 3,6,9-trioxo-decylamine (GSTB) forms stable complexes with monovalent cations. This process of complex formation was studied by electrospray ionization mass spectrometry, (1)H-NMR and FTIR spectroscopy, and the PM5 (parametric method 5) semiempirical method. It is found that GSTB forms 1 : 1 and 1 : 2 complexes with Li(+) and Na(+) and 1 : 1 complexes with K(+), Rb(+), or Cs(+) cations and exists in all these complexes in the enamine-enamine tautomeric form. Moreover, within these complexes only Li(+) cations can fluctuate between the oxygen atoms of trioxo-alkyl chains. All other cations are strongly localized. In the complex of GSTB with two protons localized on the N atoms of the Schiff base, the imine-imine tautomeric form is realized. The complexes of the Schiff base with K(+), Rb(+), or Cs(+) cations are the 1 : 1 type with the oxygen atoms of the trioxo-alkyl chains, as well as the O(1)H or O(1')H group coordinating the cation. The structures of the complexes are calculated by the PM5 semiempirical method and discussed.     

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)     

The effect of tautomeric constant on the specificity of nucleotide incorporation during DNA replication: support for the rare tautomer hypothesis of substitution mutagenesis

The nucleoside analogue dP (6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one) displays ambivalent hydrogen bonding characteristics whereby the imino tautomer of P can base-pair with adenine and its amino tautomer can base-pair with guanine. Fixed imino and amino tautomers of 6-methyl-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one (N-methyl P) have been synthesised and their structures obtained by X-ray crystallography. The tautomeric constant of N-methyl P has been calculated from pK(a) values of the fixed tautomers and the kinetic parameters for the incorporation of its 5'-triphosphate (dPTP) by exonuclease-free Klenow fragment of DNA polymerase I have been determined. A strong correlation between the tautomeric constant and the incorporation specificity of dPTP is found. These results lend support to the proposal that the minor tautomeric forms of the natural bases may play an important role in substitution mutagenesis during DNA replication. Furthermore, they imply that DNA polymerases impose specific steric requirements on the base-pair during nucleotide incorporation.     

An efficient route to novel 4,5-di- and 2,4,5-tri substituted imidazoles from imidazo[1,5-a]-1,3,5-triazine (5,8-diaza-7,9-dideazapurine) derivatives

Two new types of imidazole derivatives: N-(2-R1-5-R2-1H-imidazol-4-yl) thioureas 7a-g and N-(2-R1-5-R2-1H-imidazol-4-yl) formamides 8b,c,g were obtained in high yields by the hydrolytic degradation of 6-R1-8-R2-2-thioxo-2,3-dihydroimidazo[1,5-a]-1,3,5-triazin-4(1H)-ones 5a-g and 6-R1-8-R2-imidazo[1,5-a]-1,3,5-triazin-4(3H)-ones 6b,c,d, respectively. The tautomeric preferences of the new imidazoles were determined.     

Tautomeric state of alpha-sarcin histidines. Ndelta tautomers are a common feature in the active site of extracellular microbial ribonucleases

Extracellular fungal RNases, including ribotoxins such as alpha-sarcin, constitute a family of structurally related proteins represented by RNase T1. The tautomeric preferences of the alpha-sarcin imidazole side chains have been determined by nuclear magnetic resonance and electrostatic calculations. Histidine residues at the active site, H50 and H137, adopt the Ndelta tautomer, which is less common in short peptides, as has been found for RNase T1. Comparison with tautomers predicted from crystal structures of other ribonucleases suggests that two active site histidine residues with the Ndelta tautomer are a conserved feature of microbial ribonucleases and that this is related to their ribonucleolytic function.     

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.     

Colorimetric recognizing of biologically important anions based on anion-induced tautomerism of the sensor

The anion sensing ability of compound 1 based on a novel 2,4-dinitrophenylhydrazone derivative was studied through "naked-eye" experiment, UV-Vis spectral titration, and (1)H NMR titration. Sensor 1 was synthesized through a simple method with high yield. The solution of host 1 turned deep purple from light purple in the presence of the strong basic anions and turned yellow upon addition of the weak basic anions, respectively. The results could be explained on the basis of transfer of the tautomeric equilibrium of sensor 1 induced by anions with different basicity.     

Spectroscopic evidence for participation of the 1',4'-imino tautomer of thiamin diphosphate in catalysis by yeast pyruvate decarboxylase

The 1',4'-iminopyrimidine tautomeric form of the coenzyme thiamin diphosphate (ThDP), implicated in catalysis on the basis of the conformation of enzyme-bound ThDP, has been observed by both ultraviolet absorption and circular dichroism spectroscopy. On yeast pyruvate decarboxylase, the unusual tautomer is observed in an active center variant in which catalysis in the post-decarboxylation regime of the reaction is compromised. In a model system consisting of N1-methyl-4-aminopyrimidinium or N1-methyl-N4-n-butylpyrimidinium salts, on treatment with either NaOH in water, or DBU in DMSO there is an intermediate formed with lambda(max) near 310 nm, and this intermediate reverts back to the starting salt on acidification. Proton NMR chemical shifts are consistent with the intermediate representing the 1-methyl-4-imino tautomer. On the enzyme, the intermediate could be observed by rapid-scan stopped flow with UV detection when reacting holoenzyme of the E477Q active center variant with pyruvate, and by circular dichroism even in the absence of pyruvate. This represents the first direct observation of the imino tautomeric form of ThDP both on the enzyme and in models, although some years ago, this laboratory had already reported some pertinent acid-base properties for its formation [Jordan, F., and Mariam, Y. H. (1978) J. Am. Chem. Soc.100, 2534-2541]. The work also represents the first instance in which a rare tautomer implicated in catalysis is identified and suggests that such tautomeric catalysis may be more common in biology than hitherto recognized.     

Metal-induced point defects in DNA: model and mechanisms

The aim of this work was to study the role of H3O+ and transition-metal (TM) ions in keto-enol and amino-imino tautomeric transitions in DNA base pairs and depurination. In this regard, we discuss the thermodynamic model of ion-DNA interactions and UV display of double-proton transfer (DPT) in GC. The probabilities and energies of rare tautomeric forms of GC pairs in DNA induced by H3O+ and TMwere determined being in the range from0.02 (forMg2+) to 1 ( forCu2+), and from 0 kcal/m (for Cu2+) to 2.3 kcal/m (for Mg2+), respectively. It was shown that 3'ACC5'/5'TGG3' site of DNA double helix, which corresponds to the only triplet 5'UGG3' of RNA that codes the most valuable amino acid tryptophan, is a good target for TM ions to attack. It was also shown that the only way to obtain the tryptophan-coding 5'UGG3' triplet in RNA via transition-type G --> A point mutation caused by TM ions is their interaction with the site of a DNA double helix, which corresponds to 5'CGG3' triplet of RNA that codes arginine.     

Secondary phosphine oxides: tautomerism and chiral recognition monitored by multinuclear NMR spectroscopy of their Rh2[(R)-MTPA]4 adducts

Six secondary phosphine oxides and their tautomeric equilibria as free ligands and in the presence of an equimolar amount of the chiral dirhodium complex Rh* are described and discussed. Discrimination of enantiomers is easily possible by inspecting the (31)P NMR resonances; some (1)H and (13)C NMR resonances are useful as well. H/D exchange of the acidic protons in the phosphine oxides takes place with acetone-d(6), the solvent additive, after some hours but does not obscure the chiral recognition experiment. (103)Rh,(31)P coupling constants are discussed briefly. Decomposition of ligand molecules in 1:1-Rh*-adducts occurs slowly but completely.     

Acid-promoted tautomeric lactonization and oxidation-reduction of pyrroloquinoline quinone (PQQ)

Acid-treatment facilitates PQQ detection by electron ionization mass spectroscopy with a molecular ion at M/e 330 and a base ion formed by triple decarboxylation at M/e 198. Other ions found probably arise through acid-catalyzed tautomeric lactonization of PQQ to PQQ-lactone (PQQL) with subsequent oxidation of PQQL and reduction of PQQ. We propose that a carboxyl group, presumably the 9-carboxyl, attacks a double bond in PQQ, reversibly converting the 4,5-orthoquinone into an 4,5-enediol and forming an isomeric lactone, PQQL, of 330 daltons. The masking of carbonyls may explain the low reactivity of PQQ with carbonyl reagents in acid. Acid-promoted tautomeric lactonization with carbonyl-masking is known to occur with fluoresceins, phenolphthalein and other compounds, but has not been recognized before with PQQ. Acid-treated PQQ demonstrates molecular and other ions derived from reduced PQQ (PQQ(2H] or its lactone at M/e 332 with a base ion at M/e 200. There is compelling evidence for a dehydrogenated lactone, PQQ(-2H)L), at M/e 328 with a base ion at M/e 196. We suggest that PQQ, in tautomeric equilibrium with PQQL, oxidizes PQQL to PQQ(-2H)L (328 daltons), with its concurrent reduction to PQQ(2H) (332 daltons). With acidified D2O, PQQ shows deuterated products with ions at M/e values consistent with lactonization and oxidation-reduction. An analytically useful quinoxaline adduct, formed from PQQ and 2,3-diaminonaphthalene (PQQ-DAN) of 452 daltons, also undergoes acid-tautomerization-lactonization and oxidation-reduction similar to PQQ showing molecular ions at M/e 450, 452 and 454 and decarboxylation-derived strong (base) ions at M/e 318, 320 and 322.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: NMR assignments, pH titration, and hydrogen exchange.

Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the 1H, 13C, and 15N resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 1H epsilon 2 of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 x 10(-5) s-1 at pH*7.04 and 30 degrees C) is retarded by > 10(6)-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at approximately 6.5, as measured from the pH dependence of the 15N- and 1H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral N epsilon 2H tautomeric state under all conditions examined. BCX unfolds at low pH and 30 degrees C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.     

UV spectra of adenine methyl and glycosyl derivatives and their transformation induced by amino acid carboxylic groups

UV absorption spectra of adenine, adenosine and their methyl derivatives were studied in dimethylsuloxide (DMSO). Considerable changes in UV spectra of adenine under methylation at the 1 and 3 positions, and adenosine under methylation at the 1 position attested the essential structural reconstruction of adenine purine ring. Ade and m6Ade were shown to form complexes with deprotonated carboxylic group of amino acids (carboxylate-ion) through two H-bonds involving amino group and N7H imino group, tautomeric transition N9H-->N7H being initiated namely by interaction with carboxylate-ion. Considerable changes in UV spectra of m1Ade, m1A, and m3Ade under interaction with neutral carboxylic group of amino acids were interpreted as a result of proton transfer from amino acid to the base.     

Impact of tautomery of 3-(4H-1,2,4-triazol-3-ylthio)-N-phenylpropanamide on the COX-1 inhibitory mechanism

Earlier, we have reported the synthesis and anti-inflammatory evaluation of different 3-(4H-1,2,4-triazol-3-ylthio)-N-substituted propanamide. In this article, we are reporting the various tautomeric forms of the most active anti-inflammatory compound, 3-(4H-1,2,4-triazol-3-ylthio)-N-phenylpropanamide (6a) and their virtual screening by molecular docking using six principle tautomeric forms. Docking analysis suggested that compound 3-(4H-1,2,4-triazol-3-ylthio)-N-phenylpropanamide (6a) bound with COX-1 selectively and drug receptor complex was stabilized by tautomerism. Noticeably, hydroxy group formed by tautomerism appreciably improve the drug receptor interactions. It was also supervised that the compound 3-(4H-1,2,4-triazol-3-ylthio)-N-phenylpropanamide (6a) docked near the gate of COX-1 active site and might block the conversion of arachidonic acid to prostaglandin (PG) H2 in the active site of COXs. Moreover, we have carried out receptor based electrostatic analysis to clarify the electronic, steric and hydrophobic field requirement of 3-(4H-1,2,4-triazol-3-ylthio)-N-phenylpropanamide (6a) to interact with COX -1 receptor.     

Ni(II) complexes with Schiff bases derived from amino sugars

It was found by 1H and 13C NMR spectroscopy that the Schiff base, 2-deoxy-2-(2-hydroxybenzaldimino)-D-glucopyranose exhibits enol-imine-keto-amine and anomeric equilibria in methanolic, and in dimethyl sulfoxide solutions. The reaction of the Schiff base with nickel acetate gave the bidentate, mononuclear Ni(II) complex that was characterized by spectroscopic methods and by cyclic voltammetry. The coordination of the Schiff base to the metal is through the enol-imine tautomeric form, and the anomeric equilibrium remains in dimethyl sulfoxide solutions. This complex was also obtained by reaction of D-glucosamine with Ni(II) salicylaldehydate. The same reaction was employed for the synthesis of bis-N-[2-deoxy-D-galactopyranosyl-2-(2-hydroxybenzaldiminate)]Ni(II). The small paramagnetic shifts of the 1H NMR resonances of the complexes suggest that paramagnetic species are present in low proportions.     

Quantitative identification of the protonation state of histidines in vitro and in vivo

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments. We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1.