Electrochemical behaviour of mono- and oligonucleotides: Part III. Adsorption parameters of the dilute and compact adsorption layer of adenosine and adenine mononucleotides

Extended systematic studies on the adsorption of protonated adenosine and adenine mononucleotides by the joint application of the small amplitude method out-of-phase a.c. voltammetry and the large amplitude method rapid single sweep voltammetry have established all the data for the quantitative elucidation of the interfacial behaviour of these substances of particular biological significance. Over a wide potential range one has a dilute adsorption layer while within the potential range of maximum adsorption at elevated bulk concentrations formation of a compact film is observed. The adsorption can be described quantitatively for both types of adsorption layers by single and double step Frumkin isotherms, respectively. The resulting adsorption parameters are evaluated and the conclusions on the respective interfacial behaviours, orientations and interactions of these substances are summarized and compared with their corresponding interfacial behaviour when they are integrated as building stones in oligo-and polynucleotides and DNA. Moreover, the adsorption parameters of adenosine typical for a purine-type nucleoside are compared with those of cytidine typical for pyrimidine nucleosides. In a general methodological sense the study is a good example of the particular potentialities of the joint application of the two chosen voltammetric methods for the quantitative elucidation of the involved interfacial behaviour of strongly surface active substances which are able also to undergo electrochemical reduction or oxidation at certain potentials.     

A new reaction of adenine and adenosine

Conclusions A new reaction of adenine and adenosine with hexafluoroacetone, affecting the amidine fragment of their molecules and leading to tricyclic 2, 2,9,9-tetrakis(trifluoromethyl)[1,3, 5]oxadiazine[3,4-i]purines, was conducted.Translated from Izvestiya Akademii Nauk SSSR, No. 11, pp. 2554–2557, November, 1985.     

Methods for the determination of adenosine triphosphate and other adenine nucleotides

The following methods for the determination of adenosine triphosphate reported in the past 25 years are considered: bioluminescence methods with the use of the firefly luciferase enzyme (with sensitivity to 10^?14 M); chromatographic methods (ion-exchange, thin-layer, and high performance liquid chromatography) for the determination of adenine nucleotides in mixtures with other nucleotides, nucleosides, and nitrogen bases; and fluorescence, spectrophotometric, and electrochemical techniques (including those with the use of sensors), which are promising but not commonly used for the determination of adenine nucleotides. The advantages and disadvantages of these methods are demonstrated.     

Cathodic and anodic stripping determination of traces of adenine and adenosine based on accumulation of copper(I) compounds at mercury or amalgam electrodes

In the presence of adenine and adenosine, the copper(II)/copper(Hg) couple splits to the copper(II)/copper(I) and copper(I)/copper(Hg) couples. Sparingly soluble complexes of copper(I) with adenine and adenosine can be accumulated on the electrode surface either by reduction of Cu(II) ions or by oxidation of the copper amalgam electrode. The copper(I)/adenine deposit can be stripped either cathodically or anodically with detection limits of 5×10^?9 and 2×10^?8 mol dm^?3, respectively. The copper(I)/ adenosine complex yields only the cathodic stripping peak with a detection limit of 9×10^?6 mol dm^?3. The stripping peaks obtained for the copper(I)/adenine and copper(I)/ adenosine complexes are better defined and appear over a wider range of pH than the peaks related to the corresponding mercury compounds. Adenosine cannot be determined in the presence of adenine bur adenine can be determined in the presence of moderate amounts of adenosine.     

Negative-ion mass spectrometry of substituted adenine bases and adenosine nucleosides

The negative-ion chemical ionization (ammonia, 5 Pa source pressure) mass spectra of a series of substituted adenine bases, adenosine nucleosides, and the trimethylsilyl derivatives of the nucleosides are described. Selected ions from these spectra were subject to collisionally activated dissociation with mass-analysed ion kinetic energy (CAD/MIKE) analysis of the products and the spectra assessed for information content. In addition to observing strong peaks due to quasimolecular ions and heterocyclic-base ions, it proved possible to differentiate between 2'-, 3'- and 5'-deoxy and between 2'- and 3'-O-methyl isomers. The negative-ion chemical ionization spectra of four methyladenines are essentially identical, but could be clearly distinguished from each other by CAD/MIKE analysis.     

Interaction of porphyrins with adenine and adenosine complexes. Effect of a metal nature

Reactions of complex formation of 5,10,15,20-tetraphenylporphine (H₂TPP) and tetra-tert-butylphthalocyanine (H₂(t-Bu)₄Pc) with adenine and adenosine complexes of d-metals in DMSO and ethanol are studied. It was found that H₂TPP reacts with Cu(II) and Hg(II) adeninates and adenosinates in DMSO, but does not react with Zn(II), Co(II), and Cd(II) adeninates and adenosinates (with both bridging and monodentate type of the ligand coordination). H₂(t-Bu)₄Pc enters the complex formation reaction with adeninates and adenosinates of all studied metals in DMSO at almost equal rates. The states of adenine and adenosine complexes of different d-metals in DMSO and ethanol are proposed on the basis of kinetic data obtained.     

Regioselective alkylation of the exocyclic nitrogen of adenine and adenosine by the Mitsunobu reaction

A novel synthetic route to N⁶-substitution of adenine is presented, employing the Mitsunobu reaction as the key step. A range of primary and secondary alcohols all coupled in very good to excellent yields within 30 min at 45 °C, offering a milder alternative to the traditional nucleophilic aromatic substitution of 6-chloropurine. The utility of this protocol is further demonstrated by its application to the syntheses of N⁶,N⁹-di-substituted adenines, including the potent and selective A₁ adenosine receptor agonist N⁶-cyclopentyladenosine.     

Cathodic reduction of nicotinamide adenine dinucleotide and other adenine-containing compounds in acidic media

Both nicotinamide adenine dinucleotide (NAD⁺) and acid-hydrated NADH, as well as adenine, adenosine, adenosine mono-, di-, and tri-phosphate and adenosine diphosphoribose, undergo four-electron reductions of the protonated adenine ring in acidic media. The values of αn_a (transfer coefficient times the number of electrons involved in the rate-determining step), n (total number of electron transferred), and p (number of protons involved in the rate-determining step) agree well with values previously reported for adenine. Cathodic stripping voltammetry of an adsorbed film can be applied to these compounds. Rapid scan rates are required to eliminate the slow desorption step at ?1.1 V vs. SCE for some of these compounds. Hydration of the nicotinamide ring of NADH appears to inhibit this desorption step, but does not appear to be related directly to the electroactivity of the hydration product.     

Nafion-CNT coated carbon-fiber microelectrodes for enhanced detection of adenosine

Adenosine is a neuromodulator that regulates neurotransmission. Adenosine can be monitored using fast-scan cyclic voltammetry at carbon-fiber microelectrodes and ATP is a possible interferent in vivo because the electroactive moiety, adenine, is the same for both molecules. In this study, we investigated carbon-fiber microelectrodes coated with Nafion and carbon nanotubes (CNTs) to enhance the sensitivity of adenosine and decrease interference by ATP. Electrodes coated in 0.05 mg mL(-1) CNTs in Nafion had a 4.2 ± 0.2 fold increase in current for adenosine, twice as large as for Nafion alone. Nafion-CNT electrodes were 6 times more sensitive to adenosine than ATP. The Nafion-CNT coating did not slow the temporal response of the electrode. Comparing different purine bases shows that the presence of an amine group enhances sensitivity and that purines with carbonyl groups, such as guanine and hypoxanthine, do not have as great an enhancement after Nafion-CNT coating. The ribose group provides additional sensitivity enhancement for adenosine over adenine. The Nafion-CNT modified electrodes exhibited significantly more current for adenosine than ATP in brain slices. Therefore, Nafion-CNT modified electrodes are useful for sensitive, selective detection of adenosine in biological samples.     

Measurement of adenine nucleotide levels with an adenine analyser as an index of freshness of porgy

In a prototype of an adenine analyser, adenosine and adenine nucleotides were derivatized with a fluorescent reagent, bromoacetaldehyde, after separation on a Hitachi gel No. 3012-N column by high-performance liquid chromatography. The previous analyser was improved by using a shorter reaction coil and by introduction of a Hitachi gel No. 3013-N with 5-microns particles of porous polystyrene-divinylbenzene, and applied to estimate the freshness of porgy. Total amounts of ATP, ADP and AMP in an isolated muscle just after death gradually decreased to 60% of the original amount after 5 h, and the ATP content rapidly decreased to 20% after 1 h. A good correlation was found between the levels of total adenine compounds and the energy charge values obtained from nineteen porgies frozen at a prerigour state. On the other hand, there existed no relationship between total adenine levels and the K values, which were indices for estimating freshness of fish. The analyser will be useful to evaluate the freshness of tissues and cells based on the higher contents of total adenine compounds, especially ATP.     

Site-selective direct arylation of unprotected adenine nucleosides mediated by palladium and copper: insights into the reaction mechanism

Reaction conditions facilitating the site-selective direct aryl functionalisation at the C-8 position of adenine nucleosides have been identified. Many different aromatic components may be effectively cross-coupled to provide a diverse array of arylated adenine nucleoside products without the need for ribose or adenine protecting groups. The optimal palladium catalyst loading lies between 0.5 and 5 mol %. Addition of excess mercury to the reaction had a negligible affect on catalysis, suggesting the involvement of a homogeneous catalytic species. A study by transmission electron microscopy (TEM) shows that metal containing nanoparticles, ca. 3 nm with good uniformity, are formed during the latter stages of the reaction. Stabilised PVP palladium colloids (PVP=N-polyvinylpyrrolidone) are catalytically active in the direct arylation process, releasing homogenous palladium into solution. The effect of various substituted 2-pyridine ligand additives has been investigated. A mechanism for the site-selective arylation of adenosine is proposed.     

Theoretical study on the hydrolytic deamination mechanism of adenosine

The hydrolytic deamination mechanism of adenosine to produce inosine was studied using density functional method on two models. One is adenine and the other is adenosine. Optimized geometries of reactants, intermediates, transition states, and products were determined at B3LYP/6-311G(d,p) level. IRC calculations were performed on the transition states to verify whether it is the real transition state that connects the corresponding intermediates. Single point calculations were carried out on the previous optimized geometries obtained during IRC calculations. Four pathways have been determined for the hydrolytic deamination of adenosine. Pathway d is the most favorable pathway. In this pathway a tetra-coordinated intermediate is formed through hydrolysis reaction, then the deamination reaction takes place, which causes the cleavage of C6–N10 bond and the creation of C=O bond. Unlike the deamination of adenine, the attacking side of water molecule has effect on the deamination of adenosine. The energy barriers of adenosine deamination are a little higher than those of adenine deamination. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ionization potentials of adenine along the internal conversion pathways

Ionization potentials of adenine in the vertical spectrum and along the main internal conversion pathways are computed with several high-level methods and an assessment of the quality of these calculations is provided. A long-standing divergence between experimental and theoretical results, concerning the assignment of the surface on which adenine relaxes, is reviewed based on these calculations. Ionization energy variations up to 4.5 eV between the Franck-Condon region and the conical intersections were found, with general implications for pump-probe experiments with organic molecules. The ionization potentials computed along the reaction pathways can be used as a general guide for aiding the setup and analysis of further experiments with adenine and other heterocycles.     

Highly selective recognition of adenine nucleobases by synthetic hosts with a linked five-six-five-membered triheteroaromatic structure and the application to potentiometric sensing of the adenine nucleotide

A new structure for an adenine-selective host molecule, featuring the pertinent link of five-six-five-membered heteroaromatic rings and two carbamoyl NH sites, was developed. This structure provides a correctly oriented array of complementary hydrogen bonding sites for the adenine nucleobase, which exploits both Watson-Crick and Hoogsteen-type interactions. The complexation with adenine nucleobases by multiple hydrogen bonding was supported by (1)H NMR spectroscopy. This type of host displayed high selectivity in complexation, with an accompanying fluorescent response to lipophilized adenosine in CHCl(3). Furthermore, a remarkably selective potentiometric response was attained for adenosine 5'-monophosphate over 5'-GMP, 5'-CMP, and 5'-UMP by using an ion-selective electrode with a PVC-supported solvent polymeric membrane. This indicates recognition of water-soluble nucleotide guests through the membrane-water interface. These findings are expected to form a reliable basis for the development of artificial sensing systems for mononucleotides in biological systems.     

Elimination voltammetry of adenine and cytosine mixtures.

The elimination voltammetry with linear scan (EVLS) was used to study adenine and cytosine reduction signals at the mercury electrode. In comparison with the linear scan voltammetry (which provides only one unresolved peak), two elimination functions provide good resolution of individual peaks and significant increase of sensitivity. The first elimination function eliminates the kinetic current (I(k)) and conserves the diffusion current (I(d)). The second elimination function eliminates kinetic and charging currents (I(k) and I(c)) simultaneously and conserves the diffusion current (I(d)). Both functions give two well-resolved peaks of adenine and cytosine in a wide concentration range, while the linear sweep voltammetry gives badly resolved peaks due to hydrogen evolution. The best resolution of peaks is observed in acetate buffer at pH 3.8 and the detection limit for both substances is 500 nM. The concentration dependence of EVLS peak heights for one substance at the constant concentration of the other substance is linear. The peak potentials differ in these elimination functions. The difference in EVLS peak potentials gives the possibility to evaluate alpha n(a). Elimination voltammetry with linear scan contributes to the resolution of cathodic signals of purine and pyrimidine bases at very negative potentials near supporting electrolyte discharge.     

Interaction of adenine and thymine with aqueous sugar solutions

The solubility of the nucleic acid bases, adenine and thymine, in aqueous erythritol, xylose, glucose, and sucrose solutions has been studied. The solubility of adenine increases linearly with glucose and sucrose concentration, whereas with the other reagents a nonlinear increase is observed. Below 1.5M reagent concentration, the solubility of adenine increases in the order erythritol < robose, xylose < glucose < sucrose. The solubility of thymine in these solutions, on the other hand, decreases, increases, or does not change depending upon the reagent. The effect of temperature on the solubility of adenine and thymine in sugar solution indicates that the transfer of these molecules from water to sugar solution is exothermic.Presented in part at the VIIth All-India Symposium in Biophysics held at Visva Bharati University during October 1976.     

Ultra‐fast adenosine 5′‐triphosphate,adenosine 5′‐diphosphate and adenosine 5′‐monophosphate detection by pressure‐assisted capillary electrophoresis UV detection

Herein, we report a new CE method to measure adenine nucleotides adenosine 5′‐triphosphate, adenosine 5′‐diphosphate, and adenosine 5′‐monophosphate in red blood cells. For this purpose, 20 mmol/L sodium acetate buffer at pH 3.80 was used as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of 25 kV and an overimposed pressure of 0.2 psi from inlet to outlet. A rapid separation of these analytes in less than 1.5 min was obtained with a good reproducibility for intra‐ and inter‐assay (CV<4 and 8%, respectively) and an excellent analytical recovery (from 98.3 to 99%). The applicability of our method was proved by measuring adenine nucleotides in red blood cells.     

腺嘌呤、腺苷和5'-腺苷酸电化学氧化行为的比较研究

本文使用微分脉冲伏安法, 循环伏安法, 计时电流法, 和线性扫描伏安法对这三种生物分子在碳糊电极上的氧化行为进行比较研究, 并对典电极反应动力学参数(βnb)、扩散系数(D)及反应速度常数(Kb)进行测量比较.     

Application and validation of an ion-exchange high-performance liquid chromatographic method for measuring adenine nucleotides, creatine and creatine phosphate in mouse brain

We have modified and applied an ion-exchange high-performance liquid chromatographic method for measuring adenine nucleotides (adenosine monophosphate, adenosine diphosphate and adenosine triphosphate) as well as creatine and creatine phosphate in brain tissue. There was a linear relationship between the area of each peak and the amount of standard injected onto the column in the concentration range 0.5-25 nmol per 50 microliters. The concentrations of creatine phosphate and creatine were not stable in a standard mixture for 20 h at 4 degrees C unless the pH of the standard mixture was adjusted to neutral. We therefore strongly recommended the neutralization of all standard mixtures and samples before storage. The measurements of adenine nucleotides, creatine and phosphate in control mouse brain determined by this method agreed well with an enzymic method of nucleotide measurement. Furthermore, both methods detected similar decreases in the concentrations of adenosine triphosphate and creatine phosphate, together with concomitant increases in the concentrations of adenosine diphosphate, adenosine monophosphate and creatine when mice were placed under anoxic conditions (either 30 s or 2 min); these changes were greater after 2 min of anoxia than after 30 s of anoxia.     

Differential pulse-polarographic determination of adenine, adenosine and mono-, di- and tri-phosphate of adenosine

Summary The application of differential pulse polarography for the quantitative trace determination of adenine, adenosine, adenosine-3-monophosphate, adenosine-5-monophosphate, adenosine-5-diphosphate and adenosine-5-triphosphate has been investigated. Optimum conditions were found (pulse amplitude 100 mV, scan rate 2 mV · s^–1, drop time 2s). The effect of pH was studied, and the optimum pH was determined to give the highest sensitivity. The detection limit for adenine, adenosine and A-5-MP is ca. 2.2 ×10^–6M, 1.5×10^–6M and 2.99×10^–6M, respectively; for the other adenosine nucleotides it is 4.45×10^–6M. The validity of this method is supported by the constancy of the i _DPP/C values.

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Bestimmung von Adenin, Adenosin sowie Adenosinmono-, -di- und -triphosphat durch Differential-Puls-Polarographie

Zusammenfassung Die Anwendbarkeit der Differential-Puls-Polarographie zur quantitativen Spurenbestimmung von Adenin, Adenosin, Adenosin-3-monophosphat, Adenosin-5-monophosphat, Adenosin-5-diphosphat und Adenosin-5-triphosphat wurde geprüft und optimale Bedingungen ausgearbeitet (Pulsamplitude 100mV, Abtastgeschwindigkeit 2 mV/s, Tropfzeit 2s). Der Einfluß des pH-Wertes wurde untersucht und der optimale pH für maximale Empfindlichkeit bestimmt. Die Nachweisgrenzen für Adenin, Adenosin und A-5-MP betragen etwa 2,2 · 10^–6M, 1,5 · 10^–6M bzw. 2,99 · 10^–6M, für die übrigen Adenosinnucleotide 4,45 · 10^–6M. Die Brauchbarkeit der Methode ergibt sich aus der Konstanz der i _DPP/C-Werte.