Generation of cytidine 5′-triphosphate using adenylate kinase

A membrane-enclosed enzyme reactor containing adenylate kinase (E.C. 2.7.4.3, rabbit muscle) and pyruvate kinase (E.C. 2.7.1.40) converts cytidine 5′-monophosphate (CMP) and phosphoenolpyruvate (PEP) to cytidine 5′-triphosphate (CTP) and pyruvate on a gram scale.     

The first chemical synthesis of pyrazofurin 5′-triphosphate

As an archetype C-nucleoside, pyrazofurin possesses broad-spectrum antiviral and antitumor activities. However, the presence of the acidic enol in the nucleobase of pyrazofurin poses a huge challenge to the conventional NTP synthetic methods. On the basis of a selective phosphorylation method and the P(V)-N activation strategy, the first chemical synthesis of pyrazofurin 5′-triphosphate (PTP) was accomplished, which will greatly facilitate the investigation on the interactions of viral RNA polymerases and PTP, an important cellular metabolite of pyrazofurin.     

Recognition and catalytic hydrolysis of adenosine 5′-triphosphate by cadmium(II) and L-glutamic acid

Interactions among Cd²⁺, glutamic acid (Glu), and adenosine 5′-triphosphate (ATP) have been studied by potentiometric pH titration, IR, Raman, fluorescence, and NMR methods. In the Cd²⁺–ATP binary system, the main interaction sites are the α-, β-, and γ-phosphate groups, N-1, and/or N-7. Cd²⁺ binds to the N-1 site at relatively low pH and binds to the N-7 site of adenosine ring of ATP with increasing pH. In the Cd²⁺–Glu–ATP ternary system, ATP mainly binds to Cd²⁺ by the triphosphate chain. Oxygens of Glu coordinate with Cd²⁺ to form a complex to catalyze ATP hydrolysis. Hydrolysis of ATP catalyzed by the CdGlu complex was studied at pH 7.0 and 80°C by ³¹P-NMR spectrometry. Kinetics studies showed that the rate constant of ATP hydrolysis was 0.0199?min^?1 in the ternary system, which is 9.9-fold faster than that in the ATP solution (2.01?×?10^?3?min^?1). Hydrolysis occurs through an addition–elimination reaction mechanism with Cd²⁺ regulating the recognition and catalytic hydrolysis of ATP; water participates in the hydrolysis reaction of ATP at different steps with different functions in the ternary system.     

Efficient method for the synthesis of dideoxyuridine-5′-triphosphate fluorescent derivative

An approach to the synthesis of a fluorescent labeled dideoxyuridine triphosphate derivative using the pentafluorophenyl ester of a dicarbocyanine dye was suggested. The use of the dye pentafluorophenyl ester instead of N-oxysuccinimidyl one has the advantage of giving significantly higher yield of the fluorescent labeled dideoxyuridine-5??-triphosphate derivative.     

Separation of Cytidine 5′-triphosphate Biosynthesized from Cytidine 5′-monophosphate on Ion-exchange Resin and HPLC Analysis of Cytidine Compounds

Conditions were studied in the biosynthesis of cytidine 5′-triphosphate (CTP) from cytidine 5′-monophosphate (CMP). A 201 × 7 anion ion-exchange resin was applied for the separation of CTP from CMP. Adsorption isotherm and elution conditions (eluant, eluant concentration, flow rate, sample volume loaded) were investigated. At the same time, a new high-performance liquid chromatography on an anion ion-exchange column WAX-1 with UV detector at 260 nm was developed to measure CMP, cytidine 5′-diphosphate (CDP), and CTP. The retention time for CMP, CDP, and CTP are 0.723, 1.448, and 4.432 min, respectively. This new rapid high-performance liquid chromatography (HPLC) method for the analysis of cytidine compounds in biological sample has a wide linear range with high precision and repeatability.     

Synthesis of 5′-deoxy-5′-[18F]fluoro-adenosine by Radiofluorination of 5′-deoxy-5′-haloadenosine Derivatives

5-Deoxy-5-[¹⁸F]fluoro-adenosine was synthesised by nucleophilic radiofluorination reactions of 5-deoxy-5-haloadenosines. The homogeneous isotope exchange in 5-deoxy-5-fluoro-adenosine was also investigated. The conversion of these reactions was found to be rather low and depends on the strength of the halogen-carbon bond: 0.248% for chloride-, 0.488% for bromide- and 1.070% for iodide-derivative; there was no reaction observed in the case of fluoro-compound.     

The adenine ring influences the adenosine 5′-triphosphate hydrolysis

DFT calculations (ωB97X-D) of the adenosine 5′-triphosphate (ATP) hydrolysis were carried out. Four reactions (A), (B), (C), and (D) were investigated. Reaction (A) is of the acid catalysis and (B) is of the Mg²⁺ one with (H₂O) _n, n = 5, 11, and 17. (C) is of the myosin-model catalysis, and (D) is of the F₁-ATPase one. Transition states were determined precisely. For reaction (A), the experimental activation energy was reproduced well by the n = 11 and 17 models. For the Mg²⁺-containing hydrolysis, (B), 2 reactions (a) [without the Mg²⁺-adenine linkage] and (b) [with the connection] were examined and were compared. The reaction (b) was found to be more likely than (a) in both total and activation energies. The adenine ring works to enhance the polarity of transition states. For the myosin catalyzed (C) and in F₁-ATPase (D) hydrolyses, again, the reaction (b) is much more likely than (a). The proton transfer from the lytic water molecule leads to the protonation of the carboxylate of Glu459 in (C). The adenine ring was suggested to influence and promote the ATP hydrolysis.     

Investigations into the electrooxidation of guanosine-5′-triphosphate at the pyrolytic graphite electrode

The electrochemical oxidation of guanosine-5-triphosphate has been investigated in phosphate-containing electrolytes in the pH range 1.5–10.9 at a pyrolytic graphite electrode by cyclic sweep voltammetry, spectral studies, bulk electrolysis and related techniques. In this pH range, the oxidation occurred in a single well-defined peak (I_a). The peak potential of oxidation peaks (E_p) was found to be dependent on pH, concentration and sweep rate. The kinetics of the UV-absorbing intermediates was followed spectrophotometrically and the decay of the intermediate occurred in a pseudo-first-order reaction. The first-order rate constants for the disappearance of the UV-absorbing intermediate have also been calculated. The products of the electrode reaction were characterized by HPLC and GC/MS. A tentative mechanism for the formation of the products has also been suggested.     

L-Arginine and zinc ion effect on recognition and hydrolysis rate of adenosine 5′-triphosphate

Zn²⁺ can interact with adenosine 5′-triphosphate (ATP) by electrostatic and coordination interactions, and the interaction sites between Zn²⁺ and ATP vary at different pH in the ATP–Zn²⁺ binary system. Non-covalent interactions exist between the carboxyl of arginine (Arg) and Zn²⁺, which led to competition between ATP and Arg to interact with Zn²⁺ in the ATP–Zn²⁺–Arg ternary system. Kinetics studies show that the hydrolysis rate constant of ATP in the ATP–Zn²⁺ binary system was 2.44?×?10^?2?min^?1, about 11-fold faster than that (2.27?×?10^?3?min^?1) in the ATP–Zn²⁺–Arg ternary system. This may be attributed to coordination interactions between the carboxyl of Arg and Zn²⁺ and the decreased activity of zinc ion toward the phosphate groups via nucleophilic attack. A mechanism that the hydrolysis occurred through an addition–elimination mechanism is proposed.     

In vitro digestibility of soybean β-conglycinin hydrolysates

The in vitro digestibility of alcalase enzymatic hydrolysates ofβ-conglycinin was studied.The results showed that the zeta potentials ofβ-conglycinin hydrolysates decreased and their electronegativity increased when digested with pepsin and trypsin.Furthermore,the content of peptides with molecular weight from 10 kDa to 20 kDa remained stable,while those with higher molecular weight(>20 kDa) decreased,and those with lower molecular weight(<10 kDa) increased.The proportion of highly hydrophobic peptides decreased in the process of the in vitro digestion,but no significant change in the surface hydropliobicity indices of digestion products was observed(P<0.05).These results indicate that theβ-congiycinin hydrolysates were degraded through in vitro digestion,but the degree of degradation was relatively low.Peptides with molecular weight from 10 kDa to 20 kDa in theβ-conglycinin hydrolysates resisted the digestion by pepsin and trypsin and they remained stable during the in vitro digestion processes.     

A multinuclear NMR study on the microscopic ionization constants of adenosine-5′-triphosphate in aqueous solution

A ¹H, ¹³C and ³¹P NMR study of adenosine-5′-triphosphate has been performed as a function of pH. The examination of the chemical shifts allowed the proposal of an ionization scheme in which the third deprotonation takes place both on the adenine ring and on the phosphate group in a ratio of about 2:1. Furthermore, an analysis of carbon relaxation times supports this mechanism of ionization and confirms that the adenine ring binds the proton both on the NH₂ group and the N₇ nitrogen atom.     

Thermodynamic parameters for the interaction of adenosine 5′-diphosphate,and adenosine 5′-triphosphate with Mg2+ from 323.15 to 398.15 K

The interaction of adenosine 5-diphosphate (ADP) and adenosine 5-triphosphate (ATP) with Mg²⁺ in water has been studied calorimetrically at 323.15, 348.15, 373.15, and 398.15 K for ATP and at 348.15 and 373.15 K for ADP. The enthalpies of reaction of Mg²⁺ with ADP and ATP were obtained from the heats of mixing of aqueous solutions of tetramethylammonium salts of ADP and ATP with MgCl₂ solutions in an isothermal flow calorimeter. Equilibrium constant (K), enthalpy change (H°), entropy change (S°), and heat capacity change (Cp°) values were calculated for the interaction: Mg²⁺+L^n–=MgL^2–n and Mg²⁺+MgL^2–n=Mg₂L^4–n, where n=4 for L=ATP and n=3 for L=ADP. The results are consistent with those at lower temperatures. For the two nucleotides studied, the above two reactions are endothermic and entropy-driven in the temperature range studied. Large Cp° values for the interaction of Mg²⁺ with ADP with ATP indicate the involvement of phosphate groups of nucleotides in the coordination of Mg²⁺. The coordination of the first and second Mg²⁺ ions involves the phosphate chain in both ADP and ATP. No evidence was found for the involvement of the adenine ring or the ribose moiety in the coordination of Mg²⁺ with these nucleotides. Approximate values of logK, H°, and S°, and Cp° for the self-association of ADP and ATP in the presence of Mg²⁺ are also given.     

Ternary complexes in solution. Comparison of the coordination tendency of some biologically important zwitterionic buffers toward the binary complexes of Cu(II) and adenosine 5′-mono-, 5′-di-, and 5′-triphosphate

Summary Potentiometric equilibrium measurements have been made at 25.0±0.1 °C and ionic strengthI=0.1 mol dm^–3 KNO₃ for the interaction of adenosine 5-mono-, 5-di-, and 5-triphosphate (AMP,ADP andATP) and Cu(II) with biologically important secondary ligand zwitterionic buffers (N,N-bis-(2-hydroxyethyl)-2-aminoethanesulphonic acid (BES), N-tris-(hydroxymethyl)-methyl-2-aminoethanesulphonic acid (TES), N,N-bis-(2-hydroxyethyl)-glycine (Bicine) andtris-(hydroxymethyl)-methylaminopropane sulphonic acid (TAPS)) in a 1:1:1 ratio and the formation of various 1:1:1 mixed ligand complex species inferred from the potentiometricpH titration curves. Initial estimates of the formation constants of the resulting species and the acid dissociation constants ofAMP,ADP,ATP, and secondary ligands have been refined with the SUPERQUAD computer program. Negative and positive logK values were obtained for the ternary systems studied. In some Cu(II) ternary systems studied the interligand interactions or some cooperativity between the coordinate ligands, possibly H bond formation, has been found to be most effective in deciding the stability of the ternary complexes formed in solution. Stabilities of mixed ligand complexes increase in the orderAMP<ADP<ATP. The trend in stability constants of the mixed-ligand complexes of the title zwitterionic buffer ligands is found to beTAPS>Bicine>TES>BES.

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Ternäre Komplexe in Lösung. Vergleich der Koordinationstendenz einiger biologisch wichtiger zwitterionischer Puffer an binäre Komplexe von Cu(II) und Adenosin-5-mono-, -5-di- und-5-triphosphat

Zusammenfassung Die Wechselwirkungen zwischen Adenosin-5-mono, -5-di- und-5-triphosphat (AMP,ADP undATP), Cu(II) und biologisch wichtigen zwitterionischen Puffern mit Sekundärligandeigenschaften (N,N-bis-(2-Hydroxyethyl)-2-aminoethansulfonsäure (BES), N-tris-(Hydroxymethyl)-methyl-2-aminoethansulfonsäure (TES), N,N-bis-(2-Hydroxyethyl)-glycin (Bicin) undtris-(Hydroxymethyl)-methylaminopropansulfonsäure (TAPS) im Verhältnis 1:1:1 wurden mittels potentiometrischer Gleichgewichtsmessungen bei 25.0±0.1 °C undI=0.1 mol·dm^–3 KNO₃ untersucht. Die Titrationskurven lassen auf verschiedene Komplexe mit gemischten Liganden im Verhältnis von 1:1:1 schließen. Erste Abschätzungen der Bildungskonstanten der entstehenden Produkte und der Dissoziationskonstanten vonAMP,ADP,ATP und der Sekundärliganden wurden mit HIlfe des Programms SUPERQUAD verfeinert. Für die untersuchten ternären Systeme wurden negative und positive Werte für logK erhalten. In einigen der Cu(II)-Komplexe wird die Stabilität des ternären Systems in Lösung hauptsächlich durch Wechselwirkungen zwischen den Liganden, möglicherweise durch die Ausbildung von Wasserstoffbrückenbindungen, bestimmt. Die Stabilität der Komplexe steigt in der ReihenfolgeAMP<ADP<ATP. Der entsprechende Trend der Stabilitätskonstanten bei den gemischten Komplexen mit den im Titel genannten zwitterionischen Puffern lautetTAPS>Bicin>TES>BES.

     

Syntheses of 5′-O-glycosylnucleosides

A general synthetic approach to 2,3-unsaturated glycosides connecting with nucleosides involving Ferrier rearrangements of glycals is discussed. The new compounds were identified by NMR and MS (HRFAB⁺). The hydroxylation of the resulting 2,3-unsaturated glycosides was completed using OsO₄ to give 5′-O-glycosylnucleosides in good yield.     

In vitro metabolism of 2′-ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry

Antisense oligonucleotides (ASOs) have been touted as an emerging therapeutic class to treat genetic disorders and infections. The evaluation of metabolic stability of ASOs during biotransformation is critical due to concerns regarding drug safety. Because the effects of the modifications in ASOs on their metabolic stabilities are different from unmodified ASOs, studies that afford an understanding of these effects as well as propose proper methods to determine modified and unmodified ASO metabolites are imperative. An LC–tandem mass spectrometry method offering good selectivity with a high-quality separation using 30 mm N,N-dimethylcyclohexylamine and 100 mm 1,1,1,3,3,3-hexafluoro-2-propanol was utilized to identify each oligonucleotide metabolite. Subsequently, the method was successfully applied to a variety of in vitro systems including endo/exonuclease digestion, mouse liver homogenates, and then liver microsomes, after which the metabolic stability of unmodified versus modified ASOs was compared. Typical patterns of chain-shortened metabolites generated by mainly 3′-exonucleases were observed in phosphodiester and phosphorothioate ASOs, and endonuclease activity was identically observed in gapmers that showed relatively more resistance to nuclease degradation. Overall, the degradation of each ASO occurred more slowly corresponding to the degree of chemical modifications, while 5′-exonuclease activities were only observed in gapmers incubated in mouse liver homogenates. Our findings provide further understanding of the impact of modifications on the metabolic stability of ASOs, which facilitates the development of future ASO therapeutics.     

Thermal analysis of 5′-deoxy-5′-iodo-2′,3′-O-isopropylidene-5-fluorouridine

The melting temperature, melting enthalpy, and specific heat capacities (C _p) of 5′-deoxy-5′-iodo-2′,3′-O-isopropylidene-5-fluorouridine (DIOIPF) were measured using DSC-60 Differential Scanning Calorimetry. The melting temperature and melting enthalpy were obtained to be 453.80 K and 33.22 J g^?1, respectively. The relationship between the specific heat capacity and temperature was obtained to be C _p/J g^?1 K^?1 = 2.0261 – 0.0096T + 2 × 10^?5 T ² at the temperature range from 320.15 to 430.15 K. The thermal decomposition process was studied by the TG–DTA analyzer. The results showed that the thermal decomposition temperature of DIOIPF was above 487.84 K, and the decomposition process can be divided into three stages: the first stage is the decomposition of impurities, the mass loss in the second stage may be the sublimation of iodine and thermal decomposition process of the side-group C₄H₂O₂N₂F, and the third stage may be the thermal decomposition process of both the groups –CH₃ and –CH₂OCH₂–. The obtained thermodynamic basic data are helpful for exploiting new synthetic method, engineering design, and commercial process of DIOIPF.     

The structural modification of DNA nucleosides by nonenzymatic glycation: an <Emphasis Type="Italic">in vitro</Emphasis> study based on the reactions of glyoxal and methylglyoxal with 2′-deoxyguanosine

Methylglyoxal and glyoxal are generated from the oxidation of carbohydrates and lipids, and like d-glucose have been shown to nonenzymatically react with proteins to form advanced glycation end products (AGEs). AGEs can occur both in vitro and in vivo, and these compounds have been shown to exacerbate many of the long-term complications of diabetes. Earlier studies in our laboratory reported d-glucose, d-galactose, and d/l-glyceraldehyde formed AGEs with nucleosides. The objective of this study was to focus on purines and pyrimidines and to analyze these DNA nucleoside derived AGE adducts with glyoxal or methylglyoxal using a combination of analytical techniques. Studies using UV and fluorescence spectroscopy along with mass spectrometry provided for a thorough analysis of the nucleoside AGEs and demonstrated that methylglyoxal and glyoxal reacted with 2′-deoxyguanosine via the classic Amadori pathway, and did not react appreciably with 2′-deoxyadenosine, 2′-deoxythymidine, and 2′-deoxycytidine. Additional findings revealed that methylglyoxal was more reactive than glyoxal. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In vitro and in vivo evaluation of β-cyclodextrin-based nanosponges of telmisartan

Telmisartan (TEL) is a BCS Class II drug having dissolution rate limited bioavailability. The aim of work was to enhance the solubility of TEL so that bioavailability problems are solved. β-Cyclodextrin (β-CD) based nanosponges (NSs) were formed by cross-linking β-CD with carbonate bonds, which were porous as well as nanosized. Drug was incorporated by solvent evaporation method. The effect of ternary component alkalizer (NaHCO₃) on solubility of TEL was studied. In order to find out the solubilization efficiency of NS, phase solubility study was carried out. Saturation solubility and in vitro dissolution study of β-CD complex of TEL was compared with plain TEL and NS complexes of TEL. The NS and NS complexes of TEL were characterized by differential scanning calorimetry, powder X-ray diffraction, Fourier transform infrared spectroscopy, nuclear magnetic resonance and scanning electron microscope. It was found that solubility of TEL was increased by 8.53-fold in distilled water; 3.35-fold in 0.1 N HCl and 4.66-fold in phosphate buffer pH 6.8 by incorporating NaHCO₃ in drug–NS complex than TEL. It was found that the NaHCO₃ in NS based complex synergistically enhanced dissolution of TEL by modulating microenvironmental pH and by changing amorphization of the drug. The highest solubility and in vitro drug release was observed in inclusion complex prepared from NS and NaHCO₃. An increase of 54.4 % in AUC was seen in case the ternary NS complex whereas β-CD ternary complex exhibited an increase of 79.65 %.     

Stepwise conformational transition of crystalline disodium adenosine 5′-triphosphate with relative humidity as studied by high resolution solid state 13C and 31P NMR

Stepwise conformational transition of disodium adenosine 5′-triphosphate (Na₂ATP) crystals as a function of relative humidity (RH), was examined by means of high resolution solid state NMR spectroscopy. The presence of two types of molecules, A and B, with altered conformations in an asymmetric unit, is evident from both ³¹P and ¹³C NMR spectra, irrespective of the three different crystalline forms, mono-, di- and trihydrates, in which, cell parameters changed linearly with RH. Hydration-dependent conformational transition from the monohydrate (RH 0–10%) through dihydrate (20–40%) to trihydrate (60–90%), was well monitored by stepwise upfield displacements of the C1′ ¹³C signals of ribose moiety (molecule B), although, the corresponding peak for the molecule A, is almost unchanged. This means that, adaptation of stepwise structural transitions to the linear expansion of cell parameters is almost entirely ascribed to the conformational readjustment of the molecule B, together with varying proportion of two types of hydrate forms, at the RH range of 20–40%. We consider that, small clusters or thin layers are formed in the beginning of the transition and subsequently, the number of them, rather than the size or thickness increases, because the phosphorous spin–lattice relaxation times, T₁^P, for the three phosphorous nuclei, including those of mixed state, of the two hydrate forms, turned out to be similar due to sufficient fast spin diffusion rates among them. Since only one direction of the cell parameters is changed, as determined by the previous X-ray diffraction study, we conclude that thin layer type micro crystals might be produced during the stepwise transition. Further, it was found that, these molecules acquired motional flexibility mainly at the bound water molecules, together with increased relative humidity, as determined by a variety of relaxation parameters.