Adenine Nucleosides Research Articles

Isolation and substrate specificity of an adenine nucleoside phosphorylase from adult Schistosoma mansoni

An adenine nucleoside phosphorylase (ANP, EC none) activity was identified and partially purified from extracts of Schistosoma mansoni by chromatofocussing column chromatography and molecular sieving. The enzyme is distinct from purine nucleoside phosphorylase (PNP, EC 2.4.2.1). ANP is specific for adenine nucleosides which includes adenosine analogs modified in the aglycone, pentose or both moieties. (e.g. 2′-deoxyadenosine, 5′-deoxy-5′-methylthioadenosine, 5′-deoxy-5′-iodo-2-fluoroadenosine, etc.) The enzyme is also distinct from the mammalian 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAP, EC 2.4.2.28) in that it is able of the phosphorolysis of 2′-deoxyadenosine while mammalian MTAP cannot. Because of ANP unique substrate specificity, the enzyme could play a role as a target for chemotherapy of these parasites. Cytotoxic analogs may be designed as subversive substrates that are selectively activated only by the schistosomal ANP.

Hydroxy fatty acids for the delivery of dideoxynucleosides as anti-HIV agents

A series of α- and β-carboxylated phospholipid prodrugs of dideoxy nucleosides have been synthesized and evaluated against HIV. An increase in biological effect with a factor of 500 has only been observed for the adenine nucleoside, which suggests that this prodrug approach is base specific.

Detection of adenosine triphosphate through polymerization-induced aggregation of actin-conjugated gold/silver nanorods

We have developed a simple and selective nanosensor for the optical detection of adenosine triphosphate (ATP) using globular actin-conjugated gold/silver nanorods (G-actin–Au/Ag NRs). By simply mixing G-actin and Au/Ag NRs (length ∼56 nm and diameter ∼12 nm), G-actin–Au/Ag NRs were prepared which were stable in physiological solutions (25 mM Tris-HCl, 150 mM NaCl, 5.0 mM KCl, 3.0 mM MgCl2 and 1.0 mM CaCl2; pH 7.4). Introduction of ATP into the G-actin–Au/Ag NR solutions in the presence of excess G-actin induced the formation of filamentous actin-conjugated Au/Ag NR aggregates through ATP-induced polymerization of G-actin. When compared to G-actin-modified spherical Au nanoparticles having a size of 13 nm or 56 nm, G-actin–Au/Ag NRs provided better sensitivity for ATP, mainly because the longitudinal surface plasmon absorbance of the Au/Ag NR has a more sensitive response to aggregation. This G-actin–Au/Ag NR probe provided high sensitivity (limit of detection 25 nM) for ATP with remarkable selectivity (>10-fold) over other adenine nucleotides (adenosine, adenosine monophosphate and adenosine diphosphate) and nucleoside triphosphates (guanosine triphosphate, cytidine triphosphate and uridine triphosphate). It also allowed the determination of ATP concentrations in plasma samples without conducting tedious sample pretreatments; the only necessary step was simple dilution. Our experimental results are in good agreement with those obtained from a commercial luciferin–luciferase bioluminescence assay. Our simple, sensitive and selective approach appears to have a practical potential for the clinical diagnosis of diseases (e.g. cystic fibrosis) associated with changes in ATP concentrations.

Structural Phylogenomics Reveals Gradual Evolutionary Replacement of Abiotic Chemistries by Protein Enzymes in Purine Metabolism

The origin of metabolism has been linked to abiotic chemistries that existed in our planet at the beginning of life. While plausible chemical pathways have been proposed, including the synthesis of nucleobases, ribose and ribonucleotides, the cooption of these reactions by modern enzymes remains shrouded in mystery. Here we study the emergence of purine metabolism. The ages of protein domains derived from a census of fold family structure in hundreds of genomes were mapped onto enzymes in metabolic diagrams. We find that the origin of the nucleotide interconversion pathway benefited most parsimoniously from the prebiotic formation of adenine nucleosides. In turn, pathways of nucleotide biosynthesis, catabolism and salvage originated ∼300 million years later by concerted enzymatic recruitments and gradual replacement of abiotic chemistries. Remarkably, this process led to the emergence of the fully enzymatic biosynthetic pathway ∼3 billion years ago, concurrently with the appearance of a functional ribosome. The simultaneous appearance of purine biosynthesis and the ribosome probably fulfilled the expanding matter-energy and processing needs of genomic information.

Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides

Thermostable nucleoside phosphorylases are attractive biocatalysts for the synthesis of modified nucleosides. Hence we report on the recombinant expression of three 'high molecular mass' purine nucleoside phosphorylases (PNPs) derived from the thermophilic bacteria Deinococcus geothermalis, Geobacillus thermoglucosidasius and from the hyperthermophilic archaeon Aeropyrum pernix (5'-methythioadenosine phosphorylase; ApMTAP). Thermostability studies, kinetic analysis and substrate specificities are reported. The PNPs were stable at their optimal temperatures (DgPNP, 55 °C; GtPNP, 70 °C; ApMTAP, activity rising to 99 °C). Substrate properties were investigated for natural purine nucleosides [adenosine, inosine and their C2'-deoxy counterparts (activity within 50-500 U·mg(-1))], analogues with 2'-amino modified 2'-deoxy-adenosine and -inosine (within 0.1-3 U·mg(-1)) as well as 2'-deoxy-2'-fluoroadenosine (9) and its C2'-arabino diastereomer (10, within 0.01-0.03 U·mg(-1)). Our results reveal that the structure of the heterocyclic base (e.g. adenine or hypoxanthine) can play a critical role in the phosphorolysis reaction. The implications of this finding may be helpful for reaction mechanism studies or optimization of reaction conditions. Unexpectedly, the diastereomeric 2'-deoxyfluoro adenine ribo- and arabino-nucleosides displayed similar substrate properties. Moreover, cytidine and 2'-deoxycytidine were found to be moderate substrates of the prepared PNPs, with substrate activities in a range similar to those determined for 2'-deoxyfluoro adenine nucleosides 9 and 10. C2'-modified nucleosides are accepted as substrates by all recombinant enzymes studied, making these enzymes promising biocatalysts for the synthesis of modified nucleosides. Indeed, the prepared PNPs performed well in preliminary transglycosylation reactions resulting in the synthesis of 2'-deoxyfluoro adenine ribo- and arabino- nucleosides in moderate yield (24%).

Stacked base-pair structures of adenine nucleosides stabilized by the formation of hydrogen-bonding network involving the two sugar groups

We have employed a laser desorption technique combined with supersonic-jet cooling for producing base pairs of adenine nucleosides, adenosine (Ado) and N6,N6-dimethyladenosine (DMAdo) under low-temperature conditions. The resulting base pairs are then ionized through resonant two-photon ionization (R2PI) and analyzed by time-of-flight mass spectrometry. It is found that dimers of these adenine nucleosides are stable, especially in the case of DMAdo, with respect to those of the corresponding bases, i.e., adenine and N6,N6-dimethyladenine. Structural analysis of the DMAdo dimer is performed based on the IR–UV double resonance measurements and theoretical calculations. The result demonstrates that the dimer possesses a stacked structure being stabilized by the formation of hydrogen-bonding network involving the two sugar groups. The occurrence of the frequency shift and broadening is explained satisfactorily based on the anharmonic coupling of the OH stretching modes with specific bending modes and low-frequency modes of base and sugar moieties.

1,2,3-Triazoles as leaving groups in purine chemistry: a three-step synthesis of N6-substituted-2-triazolyl-adenine nucleosides and photophysical properties thereof

A novel three-step approach for the synthesis of N6-substituted-2-(1,2,3-triazol-1-yl)-adenine nucleosides is described. 2,6-Bis-(1,2,3-triazol-1-yl)purine nucleosides are obtained, which undergo regioselective nucleophilic aromatic substitution with amines at C(6). Thus, 1,2,3-triazoles are shown to be good leaving groups in purine chemistry. The title compounds exhibit interesting levels of fluorescence with quantum yields of up to 53%.

Reactivity of the Malonaldehyde‐Glyoxal and Malonaldehyde‐Methylglyoxal Adducts of Adenine Nucleosides toward Amino Acid Cross‐Link Formation

AbstractDNA–protein cross‐links are formed upon exposure to a variety of physical and exogenous, as well as endogenous chemical agents. This type of damage poses a threat to genomic integrity. Among the agents capable of inducing DNA–protein cross‐links bifunctional electrophiles arising from cellular processes deserve particular attention. Representatives of such electrophiles include glyoxal, methylglyoxal and malonaldehyde. Herein, we report that glyoxal and methylglyoxal adducts of 2′‐deoxyadenosine formed in the presence of malonaldehyde {M1Gx‐dA [8‐(diformylmethyl)‐3‐(2′‐deoxy‐β‐D‐ribofuranosyl)imidazo[2,1‐i]purine], and M1MGx‐dA [8‐(diformylmethyl)‐7‐methyl‐3‐(2′‐deoxy‐β‐D‐ribofuranosyl)imidazo[2,1‐i]purine], respectively}, display high reactivity towards lysine derivatives. In reactions with Nα‐acetyllysine M1Gx‐dA was found to form three adducts whereas M1MGx‐dA reacted with the lysine congener to form two stable products. All compounds were isolated and their structures determined on the basis of 2D NMR spectroscopy. Formation of the analogous products was shown in the individual reactions of M1Gx‐dA and M1MGx‐dA with Nϵ‐acetyllysine. Mechanisms proposed for formation of all characterised compounds are also discussed. This study reports identification of structures for a potentially new class of DNA–protein cross‐links.

Effects of molecular dynamics and solvation on the electronic structure of molecular probes

Most spectroscopic parameters are influenced by nuclear dynamics and by the chemical environment. However, proper inclusion of these effects still represents a challenge in computational spectroscopy studies. In many cases, a route coupling satisfactory accuracy with reasonable computational costs consists in the integration of DFT-based methods to compute spectroscopic parameters, with ab initio molecular dynamics simulations to sample from the classical phase space of the system. Here, we discuss the application of this approach in two case studies of remarkable practical interest, namely the simulation of the absorption spectrum of 9-methyladenine, an adenine nucleoside model; and the prediction of electron spin resonance parameters for nitroxyl radicals, the prototypical spin probes. In both cases, the accuracy of the results increases significantly when the subtle interplay of intra-molecular dynamics and solvent effects is introduced.

Halogen Bonding in DNA Base Pairs

Halogen bonding (R-X···Y) is a qualitative analogue of hydrogen bonding that may prove useful in the rational design of artificial proteins and nucleotides. We explore halogen-bonded DNA base pairs containing modified guanine, cytosine, adenine and thymine nucleosides. The structures and stabilities of the halogenated systems are compared to the normal hydrogen bonded base pairs. In most cases, energetically stable, coplanar structures are identified. In the most favorable cases, halogenated base pair stabilities are within 2 kcal mol(-1) of the hydrogen bonded analogues. Among the halogens X = Cl, Br, and I, bromine is best suited for inclusion in these biological systems because it possesses the best combination of polarizability and steric suitability. We find that the most stable structures result from a single substitution of a hydrogen bond for a halogen bond in dA:dT and dG:dC base pairs, which allows 1 or 2 hydrogen bonds, respectively, to complement the halogen bond.

Fludarabine combined with radiotherapy in patients with locally advanced NSCLC lung carcinoma: a phase I study

Background and purposeFludarabine is an adenine nucleoside analogue that has significant activity in hematological malignancies and has shown promising activity in combination with radiation in preclinical solid tumor models. We designed a phase I trial exploring concurrent fludarabine and radiotherapy in patients with advanced non-small cell lung cancer (NSCLC) to determine the maximum tolerated dose (MTD) of fludarabine given with concurrent irradiation.Materials and methodsThirteen patients with stage IIIB NSCLC received thoracic irradiation of 60 Gy. Fludarabine was administered during the 5th and 6th week of radiotherapy. Doses started at 10 mg/m2 per day and increased by steps of 3 mg/m2 per day.ResultsAt a daily dose of 16 mg/m2, one out of six patients developed a grade 4 leukopenia, and one a grad 3 pneumonitis. Further grade III toxicity was not observed. The dose of 13 mg/m2 was identified as the MTD. All patients developed a fludarabine dose-dependent lymphocytopenia.ConclusionFludarabine can be safely administered concurrently with radiation at a daily dose of 13 mg/m2 during the final 2 weeks of radiotherapy. Further prospective clinical studies are required to establish the potential role of concurrent fludarabine and radiotherapy in the treatment of locally advanced inoperable NSCLC.

Lung cancer alters the hydrolysis of nucleotides and nucleosides in platelets

BackgroundThe nucleotides and nucleosides of adenine are signaling molecules related to thromboregulation and modulation of immune responses in patients with malignancies. Thus, this study aims to determine NTPDase, 5′-nucleotidase, ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) activities in the platelets of patients with lung cancer. MethodsWe collected blood samples from patients (n=33) previously treated for lung cancer with chemotherapy. Patients were classified as stage IIIb and IV according to the Union for International Cancer Control (UICC). ResultsPatients showed a significant decrease in the hydrolysis of adenosine diphosphate (ADP) and adenosine, whereas the adenosine monophosphate (AMP) hydrolysis and platelet aggregation were significantly increased in this group. Adenosine triphosphate (ATP) hydrolysis did not show significant results between the group of patients and the control group. ConclusionsWe may suggest that ectonucleotidases as well as ADA are enzymes involved in thromboembolic events but especially here we may see that they are also directly involved in the generation of adenosine formation in the cancer patient circulation.

Constant pH Molecular Dynamics Simulations of Nucleic Acids in Explicit Solvent

The nucleosides of adenine and cytosine have pKa values of 3.50 and 4.08, respectively, and are assumed to be unprotonated under physiological conditions. However, evidence from recent NMR and X-Ray crystallography studies has revealed the prevalence of protonated adenine and cytosine in RNA macromolecules. Such nucleotides with elevated pKa values may play a role in stabilizing RNA structure and participate in the mechanism of ribozyme catalysis. With the work presented here, we establish the framework and demonstrate the first constant pH MD simulations (CPHMD) for nucleic acids in explicit solvent in which the protonation state is coupled to the dynamical evolution of the RNA system via λ-dynamics. We adopt the new functional form λ(Nexp) for λ that was recently developed for Multi-Site λ-Dynamics (MSλD) and demonstrate good sampling characteristics in which rapid and frequent transitions between the protonated and unprotonated states at pH = pKa are achieved. Our calculated pKa values of simple nucleotides are in a good agreement with experimentally measured values, with a mean absolute error of 0.24 pKa units. This work demonstrates that CPHMD can be used as a powerful tool to investigate pH-dependent biological properties of RNA macromolecules.

Synthesis of 4′-Ethynyl-2′-deoxy-4′-thioribonucleosides and Discovery of a Highly Potent and Less Toxic NRTI

The synthesis of 4'-ethynyl-2'-deoxy-4'-thioribonucleosides was carried out utilizing an electrophilic glycosidation in which 4-ethynyl-4-thiofuranoid glycal 16 served as a glycosyl donor. Electrophilic glycosidation between 16 and the silylated nucleobases (N4-acetylcytosine, N6-benzoyladenine and N2-acetyl-O6-diphenylcarbamoylguanine) was carried out in the presence of N-iodosuccinimide (NIS) leading to the exclusive formation of the desired β-anomers 29, 33 and 36. Anti-HIV studies demonstrated that these 4'-thio nucleosides were less cytotoxic to T-lymphocyte (i.e. MT-4 cells) than the corresponding 4'-ethynyl derivatives of 2'-deoxycytidine (44), 2'-deoxyadenosine (45) and 2'-deoxyguanosine (46). Comparison of the selectivity indices (SI) was made between 4'-thionucleosides (32, 41 and 43) and the corresponding 4'-oxygen analogues 44-46 by using the reported CC50 and EC50 values. In the case of cytosine and adenine nucleosides, comparable SI values were obtained: 32 (545) and 45 (458); 41 (>230) and 45 (1,630). In contrast, 4'-ethynyl-2'-deoxy-4'-thioguanosine 43 was found to possess a SI value of >18,200, which is twenty times better than that of 46 (933).

Truncated (N)-Methanocarba Nucleosides as A1 Adenosine Receptor Agonists and Partial Agonists: Overcoming Lack of a Recognition Element

A(1) adenosine receptor (AR) agonists are neuroprotective, cardioprotective, and anxiolytic. (N)-Methanocarba adenine nucleosides designed to bind to human A(1)AR were truncated to eliminate 5'-CH(2)OH. This modification previously converted A(3)AR agonists into antagonists, but the comparable effect at A(1)AR is unknown. In comparison to ribosides, affinity at the A(1)AR was less well preserved than at the A(3)AR, although a few derivatives were moderately A(1)AR selective, notably full agonist 21 (N(6)-dicyclopropylmethyl, K(i) 47.9 nM). Thus, at the A(1)AR recognition elements for nucleoside binding depend more on 5'region interactions, and in their absence A(3)AR selectivity predominates. Based on the recently reported agonist-bound AR structure, this difference between subtypes likely correlates with an essential His residue in transmembrane domain 6 of A(1) but not A(3)AR. The derivatives ranged from partial to full agonists in A(1)AR-mediated adenylate cyclase inhibition. Truncated derivatives have more drug-like physical properties than other A(1)AR agonists; this approach is appealing for preclinical development.

Potential influence of the pericardial space on myocardial function: Cardiac interactions of adenosine and endothelin-1

Abstract Adenosine (ADO) and endothelin-1 (ET-1) play an important role in the regulation of myocardial functions. The pericardial fluid accumulates these regulatory agents and reflects well their levels in the myocardial interstitium. The aim was to investigate the interaction and cardiovascular effects of ADO and ET-1 evoked from the pericardial space, determine the pericardial elimination of intrapericardially applied adenosine and its first metabolite, inosine (INO) and observe their effects on the cardiovascular function compared to their intravenous administration. The present studies were performed on in situ dog heart using the closed pericardium model. Significant increase of the pericardial concentrations of adenine nucleosides (adenosine and inosine) after intrapericardial administration of ET-1 has been found. Regarding the adenine nucleosides-ET-1 interaction, we have demonstrated ET-1 liberation after intrapericardial administration of adenine nucleosides. The present study confirmed significantly slower elimination of adenine nucleosides in the pericardial space than in the systemic circulation. In conclusion, characteristic cardiovascular effect and bidirectional interaction of adenine nucleosides and ET-1 could be provoked by and detected in the pericardial space, reflecting parallel changes in the myocardial interstitium. The low pericardial turnover rate of adenine nucleosides may offer the opportunity of utilizing their multiple beneficial effects for local pharmaco-therapeutic interventions.

Synthesis of Cyclobutane Nucleosides

2-(6-Chloropurinyl)-3-benzoyloxymethylcyclobutanone can be prepared by reaction of 6-chloropurine with 3-benzoyloxymethyl-2-bromocyclobutanone. The N-alkylation gave both N-9 and N-7 regioisomers. Both regioisomers upon hydride reduction followed by aminolysis gave the corresponding adenine nucleoside analogues. However, the N-7 series led to the hypoxanthine analogues as byproducts.

Synthesis of an Adenine Nucleoside Containing the (8′R) Epimeric Carbohydrate Core of Amipurimycin and Its Biological Study

The (8'R) epimeric carbohydrate core 2 of amipurimycin was synthesized from D-glucose derived allylic alcohol 3 in 11 steps and 13% overall yield. The key steps involve an acid-catalyzed acetonide ring opening of 9 with concomitant formation of an unprecedented pyranose ring skeleton to give 2,7-dioxabicyclo[3.2.1]octane 10. The α-orientation of the furan ring in 10 readily allows the stereoselective β-glycosylation and opening of the furanose ring that on removal of protecting groups affords the pyranosyl adenine nucleoside 2. The antifungal and anticancer activities of 2 were studied.

Corrigendum: Simple Methodology for Heck Arylation at C‐8 of Adenine Nucleosides

For the full paper by Pallavi Lagisetty, Li Zhang and Mahesh K. Lakshman, in Issue 4, 2008, pp 602–608 (DOI: 10.1002/adsc.200700418), the Supporting Information has been corrected to rectify errors in the HR‐MS analysis, such as the technique used for obtaining the data.

Activities of adenine nucleotide and nucleoside degradation enzymes in platelets of rats infected by Trypanosoma evansi

Nucleotide and nucleoside-degrading enzymes, such as nucleoside triphosphate diphosphohydrose (NTPDase), 5′-nucleotidase and adenosine deaminase (ADA) are present in the surface membranes of platelets, involved in clotting disturbances of Trypanosoma evansi-infected animals. Thus, this study was aimed at evaluating the activities of these enzymes in platelets of rats experimentally infected with T. evansi. Animals were divided into four groups, according to the level of parasitemia. Blood samples were collected on days 3 (group A: at the beginning of parasitemia), 5 (group B: high parasitemia) and 15 (group C: chronic infection), post-infection. Group D (control group) was composed of non-infected animals for platelet count, separation and enzymatic assays. Animals from groups A and B showed marked thrombocytopenia, but platelet count was not affected in chronically infected rats. NTPDase, 5′-nucleotidase and ADA activities decreased (p<0.05) in platelets from rats of groups A and B, when compared to the control group. In group C, only NTPDase and 5′-nucleoside activities decreased (p<0.001). The correlations between platelet count and nucleotide/nucleoside hydrolysis were positive and statistically significant (p<0.05) in groups A and B. Platelet aggregation was decreased in all infected groups, in comparison to the control group (p<0.05). It is concluded that the alterations observed in the activities of NTPDase, 5′-nucleotidase and ADA in platelets of T. evansi-infected animals might be related to thrombocytopenia, that by reducing the number of platelets, there was less release of ATP and ADP. Another possibility being suggested is that changes have occurred in the membrane of these cells, decreasing the expression of these enzymes in the cell membrane.