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.     

Hydrogen Bond Equilibrium Constants of Some Unusual Nucleotide Base Pairs

Abstract

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.     

Facile 5′-Halogenation of Unprotected Nucleosides

Abstract

Facile and efficient 5′-bromination and 5′-iodination of unprotected nucleosides, such as uridine, thymidine, 5-ethyluridine, inosine, cytidine and adenosine, were achieved by the use of carbon tetrahalide and triphenylphosphine in N,N-dimethylacetamide or hexamethyl-phosphoramide.     

An Efficient Method for the Synthesis of [4–15N]Cytidine, 2′-Deoxy[4–15N]Cytidine, [6–15N]adenosine,and 2′-Deoxy [6–15N]adenosine Derivatives

Abstract

Nucleophilic substitution reactions of 4-azolyl-1 β-P-D-ribofuranosylpyrimidin-2(1H)-one and 6-azolyl-9-β-D-ribofuranosyl-9H-purine derivatives, which were converted from uridine and inosine, with [¹⁵N]phthalimide in the presence of triethylamine or DBU gave N ⁴-phthaloyl[4-¹⁵N]cytidine and N ⁶-phthaloyl[6-¹⁵N]- adenosine derivatives, respectively, in high yields. Similar reactions of those azolyl derivatives with succinimide afforded N ⁴-succinylcytidine and N ⁶-succinyladenosine derivatives in high yields. The corresponding 2′-deoxyribonucleosides were also synthesized efficiently through the same procedure.

     

Nucleic Acid Related Compounds. 82. Conversions of Adenosine to Inosine 5′-Thioether Derivatives with Aspergillus oryzae Adenosine Deaminase or Alkyl Nitrites. Substrate and Inhibitory Activities of Inosine 5′-Thioether Derivatives with Purine Nucleoside Phosphorylase

Abstract

Adenosine derivatives lacking a 5′-hydroxyl group seldom act as alternative substrates of adenosine deaminases from calf intestine and other mammalian sources. A deaminase from Aspergillus oryzae deaminated adenosine 5′-thioether derivatives cleanly and more efficiently than alkyl nitrites. The inosine derivatives were very poor alternative substrates and weak inhibitors of purine nucleoside phosphorylase.     

Comparison of the diastereoisomeric excess of uridine,inosine and adenosine cyanohydrins determined by HPLC-DAD and 1H NMR.

The separation of the diastereoisomers of the nucleoside derivatives of uridine, inosine and adenosine was performed by HPLC using chiral and no chiral columns, it was observed with the no chiral columns the resolution was good enough to determine diastereoisomeric excess. These methods were compared with ¹H NMR, and no significant differences were observed between the three techniques. Diastereoisomeric uridine (3a), inosine (3b) and adenosine (4c) cyanohydrins were resolved by ¹H nuclear magnetic resonance (¹H NMR), chiral normal phase-high-performance liquid chromatography-diode array detector (NP-HPLC-DAD) and reversed phase (RP-HPLC-DAD); these methods allowed the assesment of the percent diastereoisomeric excess (% de) of the nucleosidic cyanohydrins of 3a (4, 6 and 4), 3b (10, 8 and 6) and 4c (4, 4 and 4). To the best of our knowledge, there are no reports using analytical techniques for the separation of the epimers of 3a, 3b and 4c.     

Structure-Activity Relationships of Adenosine Deaminase Inhibitors

Abstract

Adenosine deaminase (ADA) is an important catabolic enzyme which converts adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. ADA exists in two different isoenzymes, namely ADA1 and ADA2, whose balance in monocytes-macrophages seems to guarantee the homeostasis of adenine nucleosides. Modifications of the purine moiety or/and substitution of the sugar moiety of adenosine with aliphatic chains led to derivatives which are good ADA inhibitors.     

Ribonucleoside Diphosphate Adducts with Elliptinium Acetate,an Antitumor Agent

Abstract

The oxidized form of the antitumor agent elliptinium acetate is able to arylate adenosine and uridine 5′-diphosphate by attack of the 2′-0 position of the ribose and cyclisation, leading to spiro derivatives. Ring opening occurs under reducing conditions and leads to the formation of adducts at 2′ or 3′ positions. Spiro and uncyclised adducts showed low cytotoxicity against L1210 cells in vitro.     

Daunorubicin Derivatives Obtained from Daunorubicin and Nucleoside Dialdehydes

Abstract

Nucleoside dialdehydes were obtained by periodate oxidation of adenosine, cytidine, guanosine, uridine or 6-azauridine in the presence of Dowex (1×8; CH₃COO). Reductive alkylation of daunorubicin with these dialdehydes in the presence of NaBH₃CN produced a series of 3′-deamino-3′-(4-morpholino)daunorubicin or 13-(R, S)-dihydrodaunorubicin derivatives, the latter being mixtures of two diastereomers at 13-C atom. The morpholino-daunorubicin derivatives containing nucleic base moieties are less cytotoxic than cyanomorpholino-daunorubicin, morpholino-daunorubicin and even than the parent antibiotic.     

Glycosylations of Inosine and Uridine Nucleoside Bases and Synthesis of the New 1-(β-D-Glucopyranosyl)-Inosine-5′, 6″-diphosphate

Abstract

Gluco- and ribosylation of the bases of sugar protected inosine and uridine were investigated, obtaining only adducts with β-configuration at the new glycosidic carbon; stereospecific insertion of a sugar moiety at the 1-N of inosine was achieved either using a Mitsunobu approach (for ribosylation) or by direct coupling of 1-δ-bromoglucose 13 with 2′,3′,5′-tri-O-acetylinosine for glucosylation. 1-(β-D-glucosyl)-inosine, chosen as starting substrate for glucosylated analogs of cyclic IDP-ribose, was phosphorylated at the primary hydroxyls and tested in intramolecular pyrophosphate bond formation.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. I. Kinetics of incorporation of nucleosides into nucleotide pools and pool sizes during growth cycle

Results from kinetic studies on the incorporation of ³H-5-uridine and ³H-8-adenosine into the acid-soluble nucleotide poor and nucleic acids by Novikoff hepatoma cells (subline N1S1-67) in suspension culture indicate that the uridine transport reaction is saturated at about 100 μM and that for adenosine at about 10 μM nucleoside in the medium, and that above 100 μM simple diffusion becomes the predominant mode of entry of both nucleosides into the cell. The Km of the transport reactions is approximately 1.3 × 10^?5 M for uridine and 6 × 10^?6 M for adenosine. The incorporation of these nucleosides into both the nucleotide pool and into nucleic acids seems to be limited by the rate of entry of the nucleic acid synthesis from the rate of incorporation of nucleosides. Other complicating factors are a change with time of labeling in the relative proporation of nucleoside incorporated into DNA and into the individual nucleotides of RNA, the splitting of uridine to uracil by th ecells, the deamination of adenosine kto inosine and the subsequent cleavage of inosine to hypoxanthine. Various lines of evidence are presented which indicate that the overall nucleotide pools of the cells are very small under normal growth conditions. During growth in the presence of 200 μM uridine or adenosine, however, the cells continue to convert the nucleosides into intracellular nucleotides much more rapidly than required for nucleic acid synthesis. This results in an accumulation of free uridine and adenosine nucleotides in the cells, the maximum amounts of which are at least equivalent to the amount of these nucleotides in total cellular RNA.     

Determination of the activation energy for pseudorotation of the furanose ring in nucleosides by 13-C nuclear-magnetic-resonance relaxation.

The activation energies for the pseudorotation of the furanose ring in adenosine, guanosine, inosine and xanthosine dissolved in liquid deuteroammonia have been determined by analysis of the longitudinal relaxation rates of the single tertiary carbons between +40 degrees C and minus 60 degrees C. For the purine ribosides the average activation energy was found to be 4.7 plus or minus 0.5 kcal x mol-1 (20 plus or minus 2 kJ x mol-1). For the pyrimidine nucleosides cytidine and uridine the respective activation energy should be higher since it could not be determined by 13-C relaxation measurements. This result can be explained by the formation of a hydrogen bond between the 5'-hydroxymethyl group and the base. In adenosine, guanosine, inosine and xanthosine the relaxation rates of C(5') are smaller than all others thus excluding the formation of a hydrogen bond between the purine base and the 5'-hydroxymethyl group of a strength comparable to the one suggested for cytidine and uridine.     

Synthesis And Biological Activity Op Hydpdxymetflyl Analogs op 5-Benzylacyclouridine and 5-Benzyloxybenzylacycloaridine

Abstract

Hydroxymethyl analogs of 5-benzylacyclouridine (BAU) and 5-benzyloxybenzylacyclouridine (BBAU) were synthesized by the condensation of appropriately blocked 2-(chloromethyl)glycerols with substituted 2, 4-dimethoxypyrimidines. The HM derivatives were found to be potent inhibitors of the enzyme uridine phosphorylase and to potentiate significantly the growth-inhibiting action of FdUrd in cell culture.     

Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of 14C-labeled precursors and activity of key enzymes

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐¹⁴C]adenine and [8‐¹⁴C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐¹⁴C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐¹⁴C]adenosine, [8‐¹⁴C]adenine, and [8‐¹⁴C]inosine was also detected in ureide, especially allantoic acid, and CO₂. Among these 3 precursors, the radioactivity from [8‐¹⁴C]inosine was predominantly incorporated into CO₂. These results suggest the operation of a conventional degradation pathway. Both [2‐¹⁴C]uracil and [2‐¹⁴C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐¹⁴C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐¹⁴C]uracil was recovered as CO₂ and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO₂.     

Characterization of inosine–uridine nucleoside hydrolase (RihC) from Escherichia coli

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480 μM. No activity was exhibited against 2′-OH and 3′-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5′-OH group or with the 2′-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.     

Purine catabolism in plants : purification and some properties of inosine nucleosidase from yellow lupin (lupinus luteus L.) seeds

Inosine nucleosidase (EC 3.2.2.2), the enzyme which hydrolyzes inosine to hypoxanthine and ribose, has been partially purified from Lupinus luteus L. cv. Topaz seeds by extraction of the seed meal with low ionic strength buffer, ammonium sulfate fractionation, and chromatography on aminohexyl-Sepharose, Sephadex G-100, and hydroxyapatite.

Molecular weight of the native enzyme is 62,000 as judged by gel filtration. The inosine nucleosidase exhibits optimum activity around pH 8. Energy of activation for inosine hydrolysis estimated from Arrhenius plot is 14.2 kilocalories per mole. The K_m value computed for inosine is 65 micromolar.

Among the inosine analogs tested, the following nucleosides are substrates for the lupin inosine nucleosidase: xanthosine, purine riboside (nebularine), 6-mercaptopurine riboside, 8-azainosine, adenosine, and guanosine. The ratio of the velocities measured at 500 micromolar concentration of inosine, adenosine, and guanosine was 100:11:1, respectively. Specificity (V_max/K_m) towards adenosine is 48 times lower than that towards inosine.

In contrast to the adenosine nucleosidase activity which is absent from lupin seeds and appears in the cotyledons during germination (Guranowski, Pawełkiewicz 1978 Planta 139: 245-247), the inosine nucleosidase is present in both lupin seeds and seedlings.

     

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

The transport of [${}^3\text{H}$]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 mm for hypoxanthine was derived from linear, monophasic plots of $\text{v}\text{S}$ against v. Nucleosides are competitive inhibitors of this process; adenosine and thymidine give respective K_i values of 86 and 300 μm. The corresponding bases give much higher inhibition constants with adenine and thymine yielding values of 3100 and 1700 μm, 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.     

O6-(4-Nitrophenyl)inosine and -Guanosine as Chromogenic Substrates for Adenosine Deaminase

Abstract

O⁶-(4-Nitrophenyl)inosine (la), O⁶ -(4-nitrophenyl)guanosine (1c) and O⁶ -(4-methylumbelliferonyl)inosine (2) were obtained by reaction of 6-chloro-9-(β-D-ribofuranosyl)purine (3a) or 2-amino-6-chloro-9-(β-D-ribofuranosyl)purine (3c) with sodium salts of 4-nitrophenol or 4-methylumbelliferone in N,N-dimethylformamide. Similarly, 6-chloro-9-(β-D-2,3-isopropylideneribofuranosyl)purine (3b) was transformed to 2′,3′-O-isopropylidene-O⁶-(4-nitrophenyl)inosine (1b). Deprotection of 1b with CF₃COOH gave compound la and O⁶ -(4-nitrophenyl)hypoxanthine (4). Compounds 1a and 1c are substrates for adenosine deaminase releasing 4-nitrophenol which is readily detected visually or spectrophotomemcally. Rate and extent of hydrolysis of la are significantly increased in the presence of purine nucleoside phosphorylase but xanthine oxidase has no influence. A potential fluorogenic analogue 2 is not a substrate for adenosine deaminase.

     

Stereocontrolled Synthesis of Diene and Enyne Sugar-Modified Nucleosides and Their Interaction with S-Adenosyl-L-homocysteine Hydrolase

Abstract

Conjugated diene 5–7 and enyne 8 analogs derived from adenosine and uridine were synthesized employing Pd-catalyzed cross-coupling reactions.     

Independent blood-brain barrier transport systems for nucleic acid precursors

The blood-brain barrier permeability to certain ¹⁴C-labelled purine and pyrimidine compounds was studied by simultaneous injection in conjunction with two reference isotopes into the rat common carotid artery and decapitation 15 s later. The amount of ¹⁴C-labelled base or nucleoside remaining in brain was expressed in relation to ³H₂O (a highly diffusible internal standard) and ^113mIn-labelled EDTA (an essentially non-diffusible internal standard).Of the 17 compounds tested, measurable, saturable uptakes were established for adenine, adenosine, guanosine, inosine and uridine.Two independent transport systems in the rat blood-brain barrier were defined. One transported adenine (K_m = 0.027 mM) and could be inhibited with hypoxanthine. Adenosine (K_m = 0.018 mM), guanosine, inosine and uridine all cross-inhibit, defining a second independent nucleoside carrier system. Adenosine inhibited [¹⁴C]uridine uptake more effectively than did uridine, suggesting a weaker affinity of uridine for this nucleoside carrier.