Inosine Derivatives Research Articles

Hydrogen bond equilibrium constants for the complexes of benzotriazole and some nucleoside derivatives. A near infrared study.

Approximate hydrogen bond association constants were determined by near infrared spectroscopy for pairs formed by benzotriazole and a number of nucleoside derivatives. The molecule of benzotriazole forms, in chloroform, hydrogen bonds with inosine, uridine and adenosine derivatives. The order of decreasing association constants for complexes formed by benzotriazole and uridine or inosine derivatives is the opposite of the one observed for pairs formed by adenosine and uridine or inosine derivatives.

Hypotensive compounds isolated from the dried body of Naja naja Kaouthia Lesson. I. Isolation of inosine as a hypotensive principle and structure-activity study of related compounds.

From the dried body of Naja naja Kaouthia from which the internal organs had been removed, three hypotensive compounds were isolated and one of them was identified as inosine.Inosine produced a transient increase in blood pressure followed by a prolonged fall lasting for 30 min in spontaneously hypertensive rats. Next, the relationship between the chemical structure and the hypotensive effect was examined by studying ribose-modified derivatives of inosine. 2'-Deoxyinosine and 3'-deoxyinosine completely lacked hypotensive effect. Phosphorylation at the 3' or 5'-position except for inosine 5'-diphosphoribose and 5'-chloro-5'-deoxyinosine slightly decreased the hypotensive effect. The results indicate that the ribose configuration is important in relation to the hypotensive effect of inosine.

Spectroscopic studies on the copper(II)—Inosine system

Interactions of inosine derivatives with copper(II) were studied in the pH range 1.4–13 in 50% H 2O-50% DMSO solution. The distinct pH dependence of the optical spectra observed in copper(II)-inosine complexes are correlated to their respective EPR changes as a function of pH. It was concluded that a simple 1:1 complex of copper(II)-inosine is formed in the pH range 1.4–5.0 and bis complexes are present in the pH 5.0–6.2 region solutions of inosine and Cu(II). From pH 6.2 to 7.8 a diamagnetic, hydroxybridged complex dominates. At pH 7.8–9.2 an insoluble, oxybridged species is formed in addition to the soluble paramagnetic Cu(NI) 4 complex. Starting from pH 9.1 the N-polymeric complex is formed which is stable up to pH 12.5, and above pH 12.5 the only species is the Cu(ribose) 2 complex.

Effect of the dialdehyde derivative of 5′-deoxyinosine on pyrimidine deoxyribonucleoside metabolism in L1210 cells

The effects of the dialdehyde derivatives of inosine (Inox) and 5′-deoxyinosine (5′-dInox) on L1210 cells were compared. The growth of L1210 cells was inhibited to a greater extent by 5′-dInox than by Inox. The increased inhibition of L1210 cell growth by 5′-dInox was also reflected by the increased inhibition of the incorporation of precursors into RNA, DNA and proteins. Even though 5′-dInox was a more potent inhibitor, Inox accumulated in the L1210 cells to levels 4- to 5-fold greater than 5′-dInox. The metabolism of [5- 3H]deoxycytidine and [5- 3H]deoxyuridine by L1210 cells in culture, in the presence of Inox or 5′-dInox, indicated that dCMP deaminase was an intracellular site of action for 5′-dInox. The dCMP deaminase activity in cell-free extracts prepared from 5′-dInox-treated cells was reduced markedly. This decrease in activity was not reversed by increased substrate concentrations nor was the activity subject to allosteric activation by dCTP. Deoxyuridine and deoxycytidine were able to reverse the effects of 5′-dInox on the inhibition of L1210 cell growth.

Hydrogen bond equilibrium constants of some unusual nucleotide base pairs.

Approximate hydrogen bond association constants were determined for base pairs formed by an adenine derivative and a number of unusual pyrimidine bases. A series is found in which the H-bond strength in the base-pairs varies. In certain cases the H-bond equilibrium constant is larger than in the adenine-thymine pair. Inosine derivatives seem to have a non-negligible chance of replacing guanosine in the guanosine-cytosine pair. Infrared, near-infrared (overtone) and NMR spectra were used to determine the equilibrium constants.

Effects of adenosine transport inhibitors on adenosine- and nerve-mediated relaxations of guinea pig taenia caeci

Dipyridamole and 6-thiobenzyl derivatives of guanosine and inosine were studied in isolated guinea pig taenia caeci with respect to their direct relaxing effects and their influence on relaxations caused by adenosine and nonadrenergic inhibitory nerve stimulation. Direct inhibitory effects were noted with all drugs at 10 μM and these were usually reversible. All inhibitors potentiated the relaxant effects of a submaximal concentration of adenosine in a concentration dependent manner and the potentiating effects persisted for at least 2–3 h after washout of inhibitors. Among the nucleoside-type inhibitors 6-[4-nitrobenzyl-thio]-purine riboside (NBMPR) was slightly more potent and longer lasting than the others and showed less direct depressant actions. Adenosine concentration-response curves were shifted to the left by all inhibitors but relaxations caused by 2-chloroadenosine remained unaffected. Frequency response curves (0.2–5 −1), obtained under conditions of selective stimulation of nonadrenergic inhibitory nerves, were not affected by any of the nucleoside-type inhibitors while dipyridamole caused small but significant enhancement of relaxations at low frequencies only. All inhibitors shifted concentration-response curves of adenosine, but not of 2-chloroadenosine, to the left.

Synthesis and characterisation of 2:1 guanosine and inosine derivatives of aromatic diamine platinum(II) complexes

The six platinum complexes of formula [Pt(LL)(NuO) 2]Cl 2, where LL is o-phenylenediamine, 4,5-dimethyl- o-phenylenediamine or 2,2′-bipyridyl and NuO is a nucleoside such as guanosine or inosine, have been prepared by interacting an appropriate dichlorodiamine-platinum(II) with an appropriate nucleoside in excess. The resulting complexes have been characterized by chemical analysis and by ultraviolet-visible, infrared and 1H NMR spectroscopy. The 1H NMR study has established the binding of guanosine and inosine to platinum through N 7. The dicationic nature of the complexes has been established by conductivity measurements which show them to be 1:2 electrolytes.

Activation mechanisms of human lymphocytes by the inosine derivative, isoprinosine (R)(IP)

Activation mechanisms of human lymphocytes by the inosine derivative, isoprinosine (R)(IP)

Nucleosides containing chemically reactive groups.

5'-Amino-5'-deoxyinosine (1) and 1-(6-amino-2,5,6-trideoxy-beta-D-erythro-hexofuranosyl)thymine (9) were prepared and substituted on the amino group with chemically reactive functions in an effort to find inhibitors of enzymes that metabolize the corresponding nucleotides. The resulting 5'-substituted methynitrosoureas 3, 11a, and 11b, bromoacetamides 4 and 13, phenyl carbamates 5 and 14, and 4-(fluorosulfonyl)benzamides 6 and 15 were tested for cytotoxicity to H.Ep-2 cells in culture and as inhibitors of incorporation of precursors into nucleic acids of L1210 cells. The inosine derivatives were also evaluated as inhibitors of hypoxanthine phosphoribosyltransferase. Compounds 4, 6 and 13 showed moderate inhibition of formation of nucleic acids, and compound 4 demonstrated significant cytotoxicity (ED50 less than 5 microgram/mL).

Preparation of derivatives of inosine 5'-phosphorothioate for use in affinity chromatography

In the paper the known methods of preparation of ribonucleoside 5'-phosphorothioates II are evaluated and an improved method of their synthesis is described, based on the reaction of 2',3'-O-ethoxymethyleneribonucleosides III with thiophosphoryl chloride in the presence of one equivalent of pyridine, and subsequent hydrolysis. Inosine 5'-phosphate and inosine 5'-phosphorothioate(IIc) were converted to 2',3'-O-cyclic ketals of levulinic acid Vb, VIb on reaction with ethyl levulinate and ethyl orthoformate and subsequent alkaline hydrolysis. O-(4-Aminophenyl)inosine 5'-phosphorothioate (IX) was prepared from 2',3'-O-isopropylideneinosine (VII) with O-(4-nitrophenyl)thiophosphoryldiimidazolide and subsequent catalytic hydrogenation of the O-(4-nitrophenyl)ester VIII formed.

Hypoxanthine transport by Chinese hamster lung fibroblasts: Kinetics and inhibition by nucleosides

The transport of [ 3H ]hypoxanthine was studied in monolayer cultures of mutant Chinese hamster lung fibroblasts lacking hypoxanthine-guanine phosphoribosyltransferase. Initial rates of transport were determined by rapid uptake experiments (8 to 20 s); a Michaelis constant of 0.68 ± 0.09 m m for hypoxanthine was derived from linear, monophasic plots of v S against v. Nucleosides are competitive inhibitors of this process; adenosine and thymidine give respective K i values of 86 and 300 μ m. The corresponding bases give much higher inhibition constants with adenine and thymine yielding values of 3100 and 1700 μ m, respectively. A similar pattern was observed for competitive inhibition of hypoxanthine transport by inosine, adenine arabinoside, uridine, cytidine, and two ribofuranosylimidazo derivatives of pyrimidin-4-one; in every case the nucleoside exhibited a lower K i value than the corresponding homologous base. The inhibition constants observed for nucleosides are remarkably similar to their K m values for nucleoside transport by cultured cells recently reported by others. Hypoxanthine transport was also blocked by the 6-(2-hydroxy-5-nitrobenzylthio) derivatives of inosine and guanosine and by dipyridamole; these agents are also inhibitors of nucleoside transport. These results indicate a closer relationship between base and nucleoside transport than previously recognized and suggest that these two transport processes may involve identical or very similar transport proteins.

Immobilisierung von Adenosinacetalen mit variablem Alkylidenrest — die Funktion des Spacers bei enzymatischer Desaminierung / Immobilization of Adenosine-Acetals with Variable Alkylidene Residues — the Function of the Spacer during Enzymatic Deamination —

Abstract Acetalation of the cis-diol moiety of adenosine or inosine with aliphatic ketoesters of different chain lengths leads to alkylidene derivatives of the nucleosides, which differ in the number of methylene groups in the hydrocarbon chain. By alkaline hydrolysis of the ester group in 1a - c or 3a - c the corresponding acids 2a - c and 4a - c have been prepared. The configuration of the new chiral centres has been determined as R. Enzymatic deamination of the alkylidene derivatives of adenosine leads to the inosine compounds. The rate of deamination reaction is raised by an increasing number of methylene groups in the alkylidene residues or by use of the esters instead of the acids. The alkylidene derivatives of adenosine were coupled with 6-aminohexylagarose yielding polymers with adenosine as ligands. No enzymatic deamination of the polymer with the shortest spacer was observed. The polymers with the longer spacers were converted to the corresponding inosine derivatives. The velocity of the deamination reaction was raised by an increasing spacer length.

Proton transfer from heterocyclic compounds. Part 7. Methylated guanosine and inosine derivatives and the question of zwitterionic involvement

The rates of detritiation of 1-methyl[8-3H]guanosine, 7-methyl[8-3H]guanosine, and 1-methyl[8-3H]inosine have been measured over a pH range at 85 °C and the results compared with those already available for guanosine and inosine. At low pH where the mechanism involves hydroxide ion attack on the protonated substrate methyl substitution has only a marginal effect on the rate. By contrast the results at high pH show that methyl substitution in the 1-position brings about a large rate retardation (ca. 103) and leads to the conclusion that in the case of both guanosine and inosine the hydroxide ion reacts with both the neutral and zwitterionic forms.

米のα‐グルコシダーゼ粗標品によるイノシンα‐グルコシドの生成

Two inosine derivatives were found to be formed when soluble starch and inosine were incubated with the crude preparation of α-glucosidase from rice seeds. One of the derivatives was purified from the incubation mixture by paper chromatography and Sephadex G-15 gel filtration, and obtained in a crystalline form as its hexaacetate. The derivative was identified as 5'-(α-glucosyl) inosine, on the basis of the various experimental results., viz., elemental analyses, UV and NMR absorption spectra, electrophoretic mobilities on paper, and products by hydrolysis with acid and Aspergillus niger α-glucosidase, and oxidation with sodium metaperiodate.

A relation between inhibition of protein synthesis and conformation of 5′-phosphorylated 7-methylguanosine derivatives

The inhibition of protein synthesis by 5′-phosphorylated derivatives of 7-methylguanosine and 7-methylinosine has been studied in a wheat germ cell-free system programmed by exogenous messenger RNA. The di- and triphosphate derivatives were found to inhibit protein synthesis at lower concentrations than 7-methylguanosine-5′-monophosphate. Inosine derivatives were consistently poor inhibitors when tested with different eucaryotic and viral RNAs. Dinucleotides derived from G5′ppp5′A by methylation of guanosine in the 7 position and/or of the adenosine in the 2′-O position were also tested as inhibitors of protein synthesis. A dinucleotide without the 7-methyl group is not inhibitory. Dinucleotides containing m7G‡ inhibit protein synthesis at about the same concentration as m7G5′ppp. The presence of the 2′-O methyl group has no effect on the inhibition of protein synthesis. A correlation between inhibition of protein synthesis and conformation in solution of the phosphorylated derivatives of m7G has been sought by nuclear magnetic resonance analysis. In aqueous solutions m7G exhibits considerable conformational freedom and various conformers are present. In the 5′-phosphorylated derivatives of m7G a very dramatic effect on the conformational freedom of the backbone is observed. This results in an increase in conformational purity and loss of the flexibility about the C-4′−C-5′ bond and a significant increase in the population of 3E conformers of the ribofuranose ring. Little conformational difference is observed between the di- and triphosphate derivatives of m7G. It seems likely that the electrostatic interaction between the positively charged N-7 and the negatively charged phosphate groups plays the dominant role in imparting conformational “rigidity” to the backbone of the 5′-phosphorylated derivatives of m7G.

Control of ribonucleotide reductase in mammalian cells

Studies of the ribonucleotide reductase system from Ehrlich ascites tumor cells have shown that there are several criteria which indicate that more than one enzyme may be responsible for CDP and ADP reduction. The ratio of CDP to ADP reductase activities did not remain constant during various purification steps. The ADP reductase activity was more sensitive to DMF and DMSO than was CDP reductase activity. The CDP reductase activity was more sensitive to inhibition by the dialdehyde derivatives of adenosine, inosine, purine riboside, 6-N-methyladenosine and AMP than was ADP reduction. Pyridoxal phosphate/borohydride treatment caused a marked alteration in the ratio of CDP/ADP reductase activities. The optimal Mg +2 ion concentration for CDP, UDP and ADP reductase activities were 3–4 m m, 0.4 m m and 0.1 m m, respectively. The modulator effects of the various nucleoside triphosphates were compared for CDP and ADP reductase activities. ATP was the prime activator of CDP reduction. However, the ATP concentration required for maximum CDP reduction caused a 90% decrease in ADP reduction. dGTP was the prime activator of ADP reduction. GTP was only one-sixth as effective as dGTP in terms of maximum ADP reduction. Analysis of the GTP and dGTP levels in the tumor cells showed that the levels of dGTP were probably too low to modulate the ADP reductase in the intact cell. Further, GTP and dGTP were negative effectors of CDP reduction. dTTP was much more inhibitory to CDP reductase than to ADP reductase. The level of CDP reductase was shown to increase upon incubation of the Ehrlich tumor cells in serum-free media in culture. The increase in activity was not blocked by cycloheximide or actinomycin D. There was a corresponding increase in the level of thymidine incorporation into DNA. These data suggest that there are other factors not related to protein or RNA synthesis which are involved in initiating DNA synthesis.

A study of 3':5'-cyclic mononucleotide-dependent protein kinase from canine prostate glands.

1. An adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase, located predominantly in the cytosol, was studied in canine prostate. 2. The enzyme exhibited cyclic AMP-binding activity, and could be isolated by chromatography on diethylaminoethyl cellulose. 3. The enzyme was maximally stimulated (fourfold) by 1mum-cyclic AMP, and half-maximal activation of the enzyme was observed in presence of 50nm-cyclic AMP. 4. Equilibrium studies at pH5.0 indicated the presence of one major class of binding site for cyclic AMP, with an association constant of approx. 10(8)m(-1). 5. Stimulation of the enzyme was also observed with the 3':5'-cyclic monophosphate derivatives of cytidine, inosine, guanosine and uridine as well as with dibutyryl cyclic AMP, but higher concentrations of these cyclic nucleotides were required to provide the same degree of activation as that seen with cyclic AMP. 6. Comparing alpha-casein, protamine and different histone subfractions as substrates, highest cyclic AMP stimulation was demonstrated with histones. 7. Although maximum velocity of the enzyme was enhanced approximately fivefold in presence of cyclic AMP, kinetic studies indicated that the apparent K(m) for histone (0.5mg/ml) remained the same whether determined in the presence or absence of the cyclic nucleotide. 8. In addition, cyclic AMP did not significantly change the apparent K(m) for ATP (1.2x10(-5)m). 9. The purified enzyme showed an absolute requirement for bivalent metal ion. Substitution of Mn(2+) for Mg(2+) decreased basal protein kinase activity as well as the stimulation noted with cyclic AMP. Similarly, the basal activity was lowered when Mg(2+) was replaced by Ca(2+) and cyclic AMP produced only little stimulation of the prostatic enzyme.

Activation and phosphorylation of carbonic anhydrase by adenosine 3′,5′-monophosphate-dependent protein kinases

Two isozymes of carbonic anhydrase (carbonate dehydratase, EC 4.2.1.1) from bovine erythrocyte were obtained on DE 52 cellulose column chromatography, one of which was activated in the presence of ATP and Mg 2+ by adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase from hog muscle, or bovine brain, and the other one was not. The activation of the enzyme by the protein kinase was dependent on preincubation time and concentration of protein kinase, cyclic AMP and ATP. The concentrations of cyclic AMP and ATP required to give half-maximal activation of carbonic anhydrase by protein kinase were about 1.6·10 −6 and 1.5·10 −5 M, respectively. The activation of carbonic anhydrase was also observed in the presence of the cyclic 3′,5′-monophosphate derivatives of inosine, guanosine, uridine and cytidine as well as with N 6-2′-O- dibutyryladenosine 3′,5′-monophosphate by using a much higher concentration. Protein kinase modulator inhibited the activation of carbonic anhydrase by protein kinase. The protein kinase was no longer capable of activating carbonic anhydrase when it was boiled previously. Carbonic anhydrase was phosphorylated by the protein kinase, and its reaction was cyclic AMP dependent. Phosphorylation of carbonic anhydrase was dependent on incubation time and protein concentration. The results indicate that carbonic anhydrase is activated by cyclic AMP-dependent protein kinase, and that its activation is associated with phosphorylation of the enzyme protein.

Raman studies of nucleic acids IV: Vibrational spectra and associative interactions of aqueous inosine derivatives

Laser-excited Raman spectra of H 2O and 2H 2O solutions of inosine, inosine-5′-monophosphate and I-methylinosine are reported. The spectra are discussed in terms of the changes in molecular structure produced by changes in pH and p 2H. The temperature dependence of Raman scattering intensities from aqueous inosine and inosine-5′-monophosphate are also reported. Raman hypochromism occurs in several lines in the spectra and is attributed to stacking of the hypoxanthine rings at low temperature. The association of the nucleotide is accompanied by enhanced Raman scattering near 820 cm −1, similar to that observed previously for ordered polynucleotide structures and RNA. Effects of solute concentration and Mg 2+ concentration on spectra of the nucleotide are also discussed. The Raman data suggest that the nucleotide bases are stacked singly and that the phosphate substituents are largely unaffected in structure and environment by the base stacking.

Cyclic nucleotide-dependent protein kinases. V. Preparation and properties of adenosine 3′,5′-monophosphate-dependent protein kinase from various bovine tissues

Adenosine 3′,5′-monophosphate (cyclic AMP)_dependent protein kinases, which catalyze the phosphorylation of proteins by ATP, have been found in each of fifteen bovine tissues examined. The enzymes were partially purified from these tissues, and their properties studied. Cyclic AMP increased by 5–15-fold the ability of each of these enzymes to phosphorylate histones. The concentration of cyclic AMP required to give half-maximal activation ranged from 30 to 160 nM. The 3′,5′-monophosphate derivatives of inosine, guanosine, uridine, and cytidine also activated the enzymes, but only at concentrations considerably higher than those required for cyclic AMP. 2′-Deoxythymidine 3′,5′-monophosphate was incapable of stimulating enzyme activity. Of the proteins tested as substrates, histone was the most effective for all of the enzymes studied. Protamine and casein were also phosphorylated by the enzymes. All of the enzymes had an absolute requirement for a divalent metal. Cyclic AMP stimulated enzyme activity in the presence of Mg 2+, Mn 2+, or Co 2+, whereas it inhibited enzyme activity in the presence of Ca 2+. The concentration of ATP required to give half-maximal velocity was determined for several of the enzyme preparations in the absence and presence of cyclic AMP; in each case, the apparent K m for ATP was significantly lower in the presence than in the absence of the activator. It was found that adenine, adenosine, AMP, ADP, GDP, riboflavin, FMN, and FAD inhibited enzyme activity to varying degrees. The fifteen enzyme preparations were found to be generally similar, but some important differences were observed.