Studies on the Preparation,Isolation, and Reactions of Adenosine Schiff Bases

Abstract

A systematic study of the preparation, isolation, and reactions of adenosine Schiff bases is presented. Schiff bases of nucleosides can be prepared and isolated, but the reaction appears to be specific for 2′, 3′-0-isopropylidene adenosines. The use of nucleoside Schiff bases as synthetic intermediates is not yet a viable process.     

Development of a FIA system with immobilized enzymes for specific post-column detection of purine bases and their nucleosides separated by HPLC column.

A sensitive and highly selective method for the simultaneous determination of purine bases and their nucleosides is proposed. An amperometric flow-injection system with the two immobilized enzyme reactors (guanase immobilized reactor and purine nucleoside phosphorylase/xanthine oxidase co-immobilized reactor) is used as the specific post-column detection system of HPLC, to convert compounds separated by a reversed-phase. HPLC column to electroactive species (hydrogen peroxide and uric acid) which can be detected at a flow-through platinum electrode. The proposed detection system is specific for a group of purine bases and purine nucleosides and does not respond for purine nucleotides and pyrimidine bases. The linear determination ranges are from 10 pmol to 5 nmol for four purine bases (hypoxanthine, xanthine, guanine, and adenine) and four purine nucleosides (inosine, xanthosine, guanosine, and adenosine). The detection limits are 1.2-5.5 pmol.     

Hydration of nucleosides in dilute aqueous solutions. Ultrasonic velocity and density measurements

The values of the concentration increments of the ultrasound velocity and their temperature slopes, apparent molar volumes, apparent molar expansibilities, apparent molar adiabatic compressibilities and their temperature gradients for 12 nucleosides and their analogs, as well as for ribose and deoxyribose, have been obtained using precision measurements of ultrasound velocity and density. The following hydration parameters for the atomic groups of the nucleosides, reflecting the state of water in the hydration shells of these groups, have been analyzed: (1) the contribution of ribose to the values of the concentration increment of ultrasound velocity A, the apparent molar volumes phi v and apparent molar adiabatic compressibilities phi ks of nucleosides; (2) contributions of the CH3, NH2 and O = ... -H groups of nucleic bases to the A, phi v and phi ks values of nucleosides and free nucleic bases; (3) contributions of the 2'-OH group of ribose to the values of A, phi v and phi ks nucleosides; (4) changes in the A values of nucleosides and free nucleic bases upon their protonation and deprotonation. Data have been obtained on the mutual influence of the atomic groups of nucleosides on their hydration. It is shown that the GC pairs of free deoxynucleosides undergo hydration more vigorously than the AT pairs, which contrasts with the relation of the degree of hydration of the GC and AT pairs of the double helix.     

Measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography

A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in conjunction with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 10³ concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed.     

Interconversion and uptake of nucleotides, nucleosides, and purine bases by the marine bacterium MB22.

Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases. Nucleosides and purine bases formed were taken up by distinct transport systems. We found a high-affinity common transport system for adenine, guanine, and hypoxanthine, with a Km of 40 nM. This system was inhibited noncompetitively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 microM and another one for inosine and adenosine with a Km of 1.4 microM. The nucleoside transport systems exhibited both mixed and noncompetitive inhibition by different nucleosides other than those translocated; purine and pyrimidine bases had no effect. The transport of nucleosides and purine bases was inhibited by dinitrophenol or azide, thus suggesting that transport is energy dependent. Inside the cell all of the substrates were converted mainly into guanosine, xanthine, and uric acid, but also anabolic products, such as nucleotides and nucleic acids, could be found.     

High performance liquid chromatographic profiles of nucleosides, bases and tryptophan in the plasma of the Tasmanian devil and four other marsupial species

Plasma profiles of nucleosides, bases and trytophan of five marsupial species were established using the reversed-phase mode of high performance liquid chromatography (RHPLC). Within each species, the profiles were highly reproducible and between species there were distinct differences. In the Tasmanian devil, the circulating levels of constituents examined with one exception, were generally lower than in the other marsupials. The exception was a constituent present in large amounts and having the characteristics of a purine nucleoside derivative which was found only in the plasma of the devil.     

Capillary electrophoretic profiling and pattern recognition analysis of urinary nucleosides from uterine myoma and cervical cancer patients

Capillary electrophoretic (CE) profiling analysis combined with pattern recognition methods is described for the correlation between urinary nucleoside profiles and uterine cervical cancer. Nucleosides were extracted from urine specimens by solid-phase extraction in affinity mode using phenylboronic acid gel. CE separation was carried out with an uncoated fused-silica capillary (570 mm×50 μm I.D.) maintained at 20°C, using 25 mM borate–42.5 mM phosphate buffer (pH 6.7) containing 200 mM sodium dodecyl sulfate as the run buffer under the applied voltage of 20 kV. A total of 15 nucleosides were positively identified in urine samples (2 ml) from eight uterine myoma (benign tumor group), 10 uterine cervical cancer (malignant tumor group) patients and 10 healthy females (normal group) studied. The star symbol plots drawn based on each mean concentration of nucleosides normalized to that in normal group enabled one to discriminate malignant and benign groups from normal group. In addition, canonical discriminant analysis performed on the nucleoside data of 28 individual urine specimens correctly classified into three separate clusters according to groups in the canonical plot.     

Quantitative changes in the content of nucleic acid bases and their derivatives in a culture ofPenicillium sizowi

Acid-soluble (“free”) nucleotides, nucleosides and bases were analyzed in the mycelium and in the culture filtrate of the fungusPenicillium sizowi, using micro-thin-layer chromatography on alumina and densitometry of the zones of the individual components. It was found that the levels of the various components underwent complicated changes, the corresponding curves exhibiting from one to three maxima which occur at different periods of cultivation. It was observed that a substantial amount of nucleotides, nucleosides and bases occurs in the medium as early as at the beginning of the exponential phase of growth. An attempt was made to elucidate some peculiarities of the nucleotide pool ofPenicillium species, using enzymes responsible for the individual transformations of nucleotides, nucleosides and bases.     

Cyclohexenyl Nucleosides and Related Compounds

Abstract

Cis and trans-1-(4-hydroxy-2-cyclohexenyl)- and 1-(2-hydroxy-5-cyclohexenyl) thymines were obtained by stereospecific routes. Oxidation of the 1, 4-products afforded 1-(4-oxo-2-cyclohexenyl)thymine, the carbocyclic analog of a reportedly antiviral ketopyranosyl nucleoside. Exclusive 1, 6-conjugate addition occurred with heterocyclic bases and methyl 1, 3-cyclohexadiene-1-carboxylate. Reduction of the thymine adduct gave 1-(4-hydroxymethyl1-3-cyclohexenyl)thymine. Michael-type addition provided a direct route to 3-oxocycloalkyl nucleosides, and lactone nucleosides resulted from addition of bases to α-methylene-γ-butyrolactone. Anti-HIV screening revealed no activity for the new compounds.     

Metabolisms of Nucleosides in Bacteria

In this study, the authors developed a simplified method for the separation and the quantitative determination of nucleosides and bases, using paper electraophoretic technique. By this method, nucleosides and bases were well separated and determined in a fairly short time. Thus, this method was expected to be as accurate as the published methods and was believed to be a better method for both quantitative determination and detection of the nucleosides and bases in a large number of samples.     

Quantification of 8-oxo-guanine and guanine as the nucleobase, nucleoside and deoxynucleoside forms in human urine by high-performance liquid chromatography–electrospray tandem mass spectrometry

Oxidative DNA damage, linked pathogenically to a variety of diseases such as cancer and ageing, can be investigated by measuring specific DNA repair products in urine. Within the last decade, since it was established that such products were excreted into urine, progress in their analysis in urine has been limited. Guanine is the DNA base most prone to oxidation. We present a method for determination of the urinary 8-hydroxylated species of guanine, based on direct injection of urine onto a high-performance liquid chromatography (HPLC)–tandem mass spectrometry system. The analysis covers the 8-hydroxylated base, ribonucleoside and deoxynucleoside, and the corresponding non-oxidised species. Without pre-treatment of urine the detection limits for the nucleobases are ~2 nM (50 fmol injected) and for the nucleosides ~0.5 nM (12.5 fmol injected). Previously, liquid chromatography of the nucleobases has been problematic but is made possible by low-temperature reverse-phase C18 chromatography, a method that increases retention on the column. In the case of the nucleosides, retention was almost total and provides a means for on-column concentration of larger urine samples and controlled high peak gradient elution. The total excretion of 8-hydroylated guanine species was 212 nmol/24 h. The oxidised base accounted for 64%, the ribonucleoside for 23% and the deoxynucleoside for 13%, indicating substantial oxidation of RNA in humans. In rat urine, excretion of the oxidised base was more dominant, the percentages of the oxidised base, ribonucleoside and deoxynucleosides being 89, 8 and 3%. This finding is at odds with previous reports using immunoaffinity pre-purification and HPLC–electrochemical detection analysis. The developed method now makes it possible to measure oxidative nucleic acid stress to both RNA and DNA in epidemiological and intervention settings, and our findings indicate a substantial RNA oxidation in addition to DNA oxidation. The small volume needed also makes the method applicable to small experimental animals.     

Simultaneous determination of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in biological samples using solid-phase extraction and high-performance liquid chromatography

A sensitive and rapid method for measuring simultaneously adenosine, S-adenosylhomocysteine and S-adenosylmethionine in renal tissue, and for the analysis of adenosine and S-adenosylhomocysteine concentrations in the urine is presented. Separation and quantification of the nucleosides are performed following solid-phase extraction by reversed-phase ion-pair high-performance liquid chromatography with a binary gradient system. N⁶-Methyladenosine is used as the internal standard. This method is characterized by an absolute recovery of over 90% of the nucleosides plus the following limits of quantification: 0.25–1.0 nmol/g wet weight for renal tissue and 0.25–0.5 μM for urine. The relative recovery (corrected for internal standard) of the three nucleosides ranges between 98.1±2.6% and 102.5±4.0% for renal tissue and urine, respectively (mean±S.D., n=3). Since the adenosine content in kidney tissue increases instantly after the onset of ischemia, a stop freezing technique is mandatory to observe the tissue levels of the nucleosides under normoxic conditions. The resulting tissue contents of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in normoxic rat kidney are 5.64±2.2, 0.67±0.18 and 46.2±1.9 nmol/g wet weight, respectively (mean±S.D., n=6). Urine concentrations of adenosine and S-adenosylhomocysteine of man and rat are in the low μM range and are negatively correlated with urine flow-rate.     

Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels

1. The following methods for hydrolysis of methyl-(14)C-labelled RNA, and for chromatographic isolation and determination of the products, were investigated: enzymic digestion to nucleosides at pH6 or 8; alkaline hydrolysis and conversion into nucleosides; hydrolysis by acid to pyrimidine nucleotides and purine bases, or completely to bases; chromatography on Dowex 50 (NH(4) (+) form) at pH6 or 8.9, or on Dowex 50 (H(+) form), or on Sephadex G-10. 2. The suitability of the various methods for determination of methylation products was assessed. The principal product, 7-methylguanosine, was unstable under the conditions used for determinations of nucleosides. 3- and 7-Methyladenine and 3- and 7-methylguanine are best determined as bases; 1-methyladenine and 3-methylcytosine can be isolated as either nucleosides or bases; O(6)-methylguanine is unstable under the acid hydrolysis conditions used and can be determined as the nucleoside; 3-methyluracil was detected, but may be derived from methylation of the ionized form of uracil. 3. Differences between the patterns of methylation of RNA and homopolyribonucleotides by the N-methyl-N-nitroso compounds and dimethyl sulphate were found: the nitroso compounds were able to methylate O-6 of guanine, were relatively more reactive at N-7 of adenine and probably at N-3 of guanine, but less reactive at N-1 of adenine, N-3 of cytosine and probably at N-3 of uridine. They probably reacted more with the ribose-phosphate chain, but no products from this were identified. 4. The possible influences of these differences on biological action of the methylating agents is discussed. Nitroso compounds may differ principally in their ability to induce miscoding in the Watson-Crick sense by reaction at O-6 of guanine. Both types of agent may induce miscoding to a lesser extent through methylation at N-3 of guanine; both can methylate N atoms, presumably preventing Watson-Crick hydrogen-bonding. N-Methyl-N-nitrosourea can degrade RNA, possibly through phosphotriester formation, but this mechanism is not proven.     

An isotopic assay of cyclic 3',5'-nucleotide phosphodiesterase with aluminum oxide columns

A method is described for separating purine bases and nucleosides from cyclic 3′,5′-nucleotides on aluminum oxide columns. Purine bases and nucleosides were found to pass through columns equilibrated with ammonium acetate buffer at pH 4.0 while the cyclic nucleotides were retarded enough to permit separation. Optimal conditions and factors affecting separation are described. The method was shown to be applicable in the isotopic assay of cyclic 3′,5′-nucleotide phosphodiesterase activity over a broad range of substrate concentrations. The advantages of this method and its possible use in a simultaneous cyclase-phosphodiesterase assay are described.     

Synthesis and antiviral activities of enantiomeric 1-[2-(hydroxymethyl) cyclopropyl] methyl nucleosides

Cyclopropyl carbocyclic nucleosides have been synthesized from the key intermediate 2 which was converted to the mesylated cyclopropyl methyl alcohol 3. Condensation of compound 3 with various purine and pyrimidine bases gave the desired nucleosides. All synthesized nucleosides were evaluated for antiviral activity and cellular toxicity. Among them adenine 22 and guanine 23 derivatives showed moderate antiviral activity against HIV-1 and HBV. None of the other compounds showed any significant antiviral activities against HIV-1, HBV, HSV-1 and HSV-2 in vitro up to 100 microM.     

Synthesis and Antiviral Activities of Enantiomeric 1-[2-(Hydroxymethyl) Cyclopropyl] Methyl Nucleosides

Abstract

Cyclopropyl carbocyclic nucleosides have been synthesized from the key intermediate 2 which was converted to the mesylated cyclopropyl methyl alcohol 3. Condensation of compound 3 with various purine and pyrimidine bases gave the desired nucleosides. All synthesized nucleosides were evaluated for antiviral activity and cellular toxicity. Among them adenine 22 and guanine 23 derivatives showed moderate antiviral activity against HIV-1 and HBV. None of the other compounds showed any significant antiviral activities against HIV-1, HBV, HSV-1 and HSV-2 in vitro up to 100μM.     

Urinary excretion of modified purines and nucleosides in immunodeficient children

Studies have been carried out using an XAD-4 resin and ion-exchange chromatography for determination of urinary purines and nucleosides in seven children with severe combined immunodeficiency and in six normal children. These studies have included analyses for five methylated purines or nucleosides produced by catabolism of nucleic acids. The following compounds have been quantitatively determined: 1-methyladenosine, 1-methylinosine, 1-methylguanosine, 1-methylguanine, 3-methylcytidine, adenosine, methylthioadenosine sulfoxide, cytidine, and deoxycytidine. 1-Methyladenosine and 1-methylinosine were most consistently elevated in the urine of immunodeficient children. Methylthioadenosine sulfoxide was very markedly increased in urine of two of the immunodeficient children while more moderate increases were noted with a number of other nucleosides. The germ-free child with severe combined immunodeficiency showed consistently lower excretion levels of these compounds when compared to normal children.     

Synthesis of 2′-Deoxy-2′ -Fluoro-D-Arabinopyranopyranosyl Nucleosides and Their 3′,4′-Seco analogues

Abstract

2′-Deoxy-2′-fluoro-D-arabinopyranosyl nucleosides were synthesized by condensation of 1,3,4-tri-O-benzoyl-2-deoxy-2-fluoro-D-arabinopyranose with the appropriate silylated bases in the presence of trimethylsilyl triflate. Scission of the 3′,4′-bond by periodate oxidation followed by sodium borohydride reduction resulted in the formation of the 3′,4′-seco analogues of the 2′-deoxy-2′-fluoro-D-arabinofuranosyl nucleosides.     

Purine Nucleoside Phosphorylase Activity in Rat Cerebrospinal Fluid

The sequential hydrolysis of purines is present in rat CSF and generates nucleosides as inosine and guanosine that are usual substrates for purine nucleoside phosphorylase (PNP). PNP catalyzes phosphorolysis of the purine nucleosides and deoxynucleosides releasing purine bases. Here we investigated the presence of PNP in CSF of rats using: i) a specific chromophoric analogue of nucleosides, 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), and ii) an inhibitor of PNP activity, immucillin-H. Additionally, we performed a preliminary kinetic characterization (K(M): Henry-Michaelis-Menten constant; V: maximal velocity) for MESG and inorganic phosphate (Pi). The values of K(M) and V for MESG (n = 3, mean+/-SD) were 142.5+/-29.5 microM and 0.0102+/-0.0006 U mg(-1), respectively. For Pi (n=3, mean+/-SD), the K(M) values and V were 186.8+/-43.7 microM and 0.0104+/-0.0016 U mg(-1), respectively. The results indicated that PNP is present in rat CSF and provided a preliminary kinetic characterization.     

Propeller Twisting in Single Crystals of Nucleosides

Abstract

Propeller twists were measured for base-paired nucleosides and nucleic acid bases. The occurrence of propeller twist outside a double helical framework is shown, and conclusions drawn on the relative magnitude of the effect in single nucleosides.