Synthesis of 6-(2-thienyl)purine nucleoside derivatives toward the expansion of the genetic code.

Unnatural bases specifically pairing with pyridin-2-one, 2-amino-6-(2-thienyl) purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace 2-amino-6-(N,N-dimethylamino)purine. It was expected that these novel purine analogues, as compared with 2-amino-6-(N,N-dimethylamino)purine, might reduce the interference in the stacking interactions with the neighboring bases in a duplex and improve the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite these unnatural bases. The syntheses of these nucleoside derivatives and the DNA fragments were examined.     

Synthesis of 6-(2-thienyl)purine nucleoside derivatives that form unnatural base pairs with pyridin-2-one nucleosides

Unnatural bases, 2-amino-6-(2-thienyl)purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace the previously developed purine analogue, 2-amino-6-(N,N-dimethylamino)purine, which specifically pairs with pyridin-2-one. These nucleoside derivatives were synthesized via the 6-substitution of 6-iodopurine nucleosides with tributylstannylthiophene or tributylstannylfuran. As compared with 2-amino-6-(N,N-dimethylamino)purine, 2-amino-6-(2-thienyl)purine reduced the interference in the stacking interactions with the neighboring bases in a DNA duplex and improved the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite the unnatural base.     

Enzymatic synthesis of β-D-2', 3'-unsaturated-5-fluorocytidine from β-D-2',3'-unsaturated thymidine

We have used crude preparations of N-deoxyribosyl transferases (NdRT-II) from Lactobacillus helveticus to catalyze the transfer of a glycosyl moiety from a donor nucleoside to an acceptor base. Optimal conditions for the transglycosylation reaction to make D-D4FC starting from D-D4T and 5-FC were determined after the analysis of several experimental parameters including reaction time, concentration of substrate, pH and the type of buffer. For the first time, a practical procedure for enzymatic synthesis of β-D-2',3'-unsaturated-5-fluorocytidine (β-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine, D-D4FC) from β-D-2',3'-unsaturated thymidine (D-D4T) has been established. This method will be useful in the manufacture of important nucleoside analogues for anti-viral therapy.     

Zinc complexes of benzothiazole-derived Schiff bases with antibacterial activity

Reaction of 2-acetamidobenzaldehyde with 2-amino-, 2-amino-4-methyl-, 2-amino-4-methoxy-, 2-amino-4-chloro-, 2-amino-6-nitro- and 2-amino-6-methylsufonylbenzothiazole afforded a series of Schiff bases. These compounds have been used for complexation reactions to obtain Zn(II) chelates having the same metal ion but different anions of the type [Zn(L)2]Xn [L = Schiff base derivative, X = SO4, NO3, C2O4 and CH3CO2 and n = 1 or 2] These complexes (Table I) have been characterized by physical, spectral, and analytical data. The Schiff bases act tridentately and their metal complexes were proposed to possess an octahedral geometry. To evaluate the antibacterial role of the anion, these compounds have been screened for antibacterial properties against pathogenic strains such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.     

Ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all human and Drosphila melanogaster deoxyribonucleoside kinases

Six non-conventional adenosine-2'- and 3'-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP). With dCK (dCyd as acceptor) and dGK (dGuo as acceptor), sharing 45% sequence identity, donor activities ranged from 13 to 119% that for ATP. Products were 5'-phosphates. In some instances, kinetics are dependent on the nature of the acceptor, and donor and acceptors properties are mutually interdependent. Results are highly relevant to studies on the modes of interaction with the enzymes, and to interpretations of reported crystal structures of dCK and dNK with bound ligands.     

Ability of Adenosine-2′(3′)-deoxy-3′(2′)-triphosphates and Related Analogues to Replace ATP as Phosphate Donor for all Human and Drosphila melanogaster Deoxyribonucleoside Kinases

Abstract

Six non-conventional adenosine-2′- and 3′-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3′-deoxyadenosine-2′-triphosphate was an effective donor (5–60% that for ATP). With dCK (dCyd as acceptor) and dGK (dGuo as acceptor), sharing 45% sequence identity, donor activities ranged from 13 to 119% that for ATP. Products were 5′-phosphates. In some instances, kinetics are dependent on the nature of the acceptor, and donor and acceptors properties are mutually interdependent. Results are highly relevant to studies on the modes of interaction with the enzymes, and to interpretations of reported crystal structures of dCK and dNK with bound ligands.     

Identification of 10-methylsphinganine in cerebrosides of the guinea pig harderian gland

A large amount of branched long chain bases was detected in the cerebrosides of guinea pig Harderian gland. The long chain bases of cerebrosides were analyzed by GLC as trimethylsilyl derivatives. The branched long chain bases were separated into four peaks (I, II, III, IV) according to the number of carbon atoms and the position of branching. In the present work, the structures of long chain bases in the four peaks were analyzed by GLC and GC-MS after conversion of them to aldehydes, alcohols, and fatty acids. Furthermore the main component of long chain bases (Peak II) was isolated by HPLC as N-acetyl derivatives and analyzed by NMR. The structures of branched long chain bases in Peaks I, II, III, and IV are as follows. Branched long chain bases of Peak I are 2-amino-10- (main component), 2-amino-9-, and 2-amino-8-methylhexadecane-1,3-diol. Branched long chain bases of Peak II also consist of a mixture of 2-amino-10-, 2-amino-9-, and 2-amino-8-methyl-heptadecane-1,3-diol. The branched long chain base of Peak III is 2-amino-10-methyl-octadecane-1,3-diol, while that of Peak IV is 2-amino-16-methyloctadecane-1,3-diol. Among these branched long chain bases, 10-methylsphinganines are dominant though the chain lengths are different. These branched long chain bases, in which the substituted positions exist in the middle part of aliphatic chain (10-, 9-, or 8-methylsphinganine) are novel long chain bases in mammals.     

Synthesis and characterization of nickel(II) and copper(II) complexes with tetradentate Schiff base ligands

The 1:1 condensation of 1,2-diaminopropane and 1-phenylbutane-1,3-dione at high dilution gives a mixture of two positional isomers of terdentate mono-condensed Schiff bases 6-amino-3-methyl-1-phenyl-4-aza-2-hepten-1-one (HAMPAH) and 6-amino-3,5-dimethyl-1-phenyl-4-aza-2-hexen-1-one (HADPAH). The mixture of the terdentate ligands has been used for further condensation with pyridine-2-carboxaldehyde or 2-acetylpyridine to obtain the unsymmetrical tetradentate Schiff base ligands. The tetradentate Schiff bases are then allowed to react with the methanol solution of copper(II) and nickel(II) perchlorate separately. The X-ray diffraction confirms the structures of two of the complexes and shows that the condensation site of the diamine with 1-phenylbutane-1,3-dione is the same.     

Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis

Purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) is numbered among targets for persistence of the causative agent of tuberculosis. Here, it is shown that MtPNP is more specific to natural 6-oxopurine nucleosides and synthetic compounds, and does not catalyze the phosphorolysis of adenosine. Initial velocity, product inhibition and equilibrium binding data suggest that MtPNP catalyzes 2′-deoxyguanosine (2dGuo) phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate (P_i) binds first followed by 2dGuo, and ribose 1-phosphate dissociates first followed by guanine. pH-rate profiles indicated a general acid as being essential for both catalysis and 2dGuo binding, and that deprotonation of a group abolishes P_i binding. Proton inventory and solvent deuterium isotope effects indicate that a single solvent proton transfer makes a modest contribution to the rate-limiting step. Pre-steady-state kinetic data indicate that product release appears to contribute to the rate-limiting step for MtPNP-catalyzed reaction.     

Chemical and biological consequences of oxidatively damaged guanine in DNA

Of the four native nucleosides, 2'-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)?dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy?dGuo, and the hyperoxidized dGuo products.     

Novel products generated from 2'-deoxyguanosine by hypochlorous acid or a myeloperoxidase-H2O2-Cl- system: identification of diimino-imidazole and amino-imidazolone nucleosides

Hypochlorous acid (HOCl), generated by myeloperoxidase from H2O2 and Cl-, plays an important role in host defense and inflammatory tissue injury. We report here the identification of products generated from 2'-deoxyguanosine (dGuo) with HOCl. When 1 mM dGuo and 1 mM HOCl were reacted at pH 7.4 and 37 degrees C for 15 min and the reaction was terminated with N-acetylcysteine (N-AcCys), two products were generated in addition to 8-chloro-2'-deoxyguanosine (8-Cl-dGuo). One was identified as an amino-imidazolone nucleoside (dIz), a previously reported product of dGuo with other oxidation systems. The other was identified as a novel diimino-imidazole nucleoside, 2,5-diimino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-2H,5H-imidazole (dDiz) by spectrometric measurements. The yields were 1.4% dDiz, 0.6% dIz and 2.4% 8-Cl-dGuo, with 61.5% unreacted dGuo. Precursors of dDiz and dIz containing a chlorine atom were found in the reaction solution in the absence of termination by N-AcCys. dDiz, dIz and 8-Cl-dGuo were also formed from the reaction of dGuo with myeloperoxidase in the presence of H2O2 and Cl- under mildly acidic conditions. These results imply that dDiz and dIz are generated from dGuo via chlorination by electrophilic attack of HOCl and subsequent dechlorination by N-AcCys. These products may play a role in cytotoxic and/or genotoxic effects of HOCl.     

Chemical and biological consequences of oxidatively damaged guanine in DNA

Abstract

Of the four native nucleosides, 2′-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)?dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy?dGuo, and the hyperoxidized dGuo products.     

Structural analysis of two glycosphingolipids from the lipopolysaccharide-lacking bacterium Sphingomonas capsulata.

Two glycosphingolipids, GSL-1 and GSL-3, were isolated from Sphingomonas capsulata and studied by methylation analysis, laser desorption mass spectrometry, and 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY and heteronuclear 13C,1H COSY experiments. GSL-1 and GSL-3 differ in their carbohydrate part, their structures being alpha-D-GlcpA-(1-->1)-Cer and alpha-D-Galp-(1-->6)-alpha-D-GlcpN-(1-->4)-alpha-D-GlcpA(1-- >1)Cer, respectively. Variations occur in the ceramide of GSL-1 and GSL-3, both having the same long-chain bases, erythro-2-amino-1, 3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1, 3-diol and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol, in the ratios 2.6 : 1 : 3.5 in GSL-1 and 1 : 1.2 : 1.5 in GSL-3. All bases are quantitatively substituted by amide-linked (S)-2-hydroxymyristic acid.     

Comparison of inhibitory activity of isomeric triazolopyridine derivatives towards adenosine receptor subtypes or do similar structures reveal similar bioactivities?

The synthesis of an array of 8-amino-2-aryl-[1,2,4]triazolo[1,5-a]pyridine-6-carboxyl amide derivatives is described for the first time. A subset of 20 derivatives were compared to their isomeric 5-amino-2-aryl-[1,2,4]triazolo[1,5-a]pyridine-7-carboxyl amide counterparts with regard to their potential to inhibit the human adenosine 2a (hA2a) receptor and their selectivity against the human adenosine 1 (hA1) receptor. Based on the analysis of H-bond donor/acceptor capabilities of the isomeric triazolopyridine pairs it can be concluded that the H-bond donor strength of the free amino functionality is the main determinant for hA2a inhibitory activity and hA1 selectivity.     

Deoxynucleoside kinases encoded by the yaaG and yaaF genes of Bacillus subtilis. Substrate specificity and kinetic analysis of deoxyguanosine kinase with UTP as the preferred phosphate donor

The overlapping yaaG and yaaF genes from Bacillus subtilis were cloned and overexpressed in Escherichia coli. Purification of the gene products showed that yaaG encoded a homodimeric deoxyguanosine kinase (dGK) and that yaaF encoded a homodimeric deoxynucleoside kinase capable of phosphorylating both deoxyadenosine and deoxycytidine. The latter was identical to a previously characterized dAdo/dCyd kinase (M?llgaard, H. (1980) J. Biol. Chem. 255, 8216-8220). The purified recombinant dGK was highly specific toward 6-oxopurine 2'-deoxyribonucleosides as phosphate acceptors showing only marginal activities with Guo, dAdo, and 2',3'-dideoxyguanosine. UTP was the preferred phosphate donor with a Km value of 6 microm compared with 36 microm for ATP. In addition, the Km for dGuo was 0.6 microm with UTP but 6.5 microm with ATP as phosphate donor. The combination of these two effects makes UTP over 50 times more efficient than ATP. Initial velocity and product inhibition studies indicated that the reaction with dGuo and UTP as substrates followed an Ordered Bi Bi reaction mechanism with UTP as the leading substrate and UDP the last product to leave. dGTP was a potent competitive inhibitor with respect to UTP. Above 30 microm of dGuo, substrate inhibition was observed, but only with UTP as phosphate donor.     

Leukocyte 8-oxo-7,8-dihydro-2'-deoxyguanosine and comet assay in epirubicin-treated patients

Epirubicin fights cancer through topoisomerase II inhibition, hence producing DNA strand breaks that finally lead to cell apoptosis. But anthracyclines produce free radicals that may explain their adverse effects. Dexrazoxane--an iron chelator--was proven to decrease free radical production and anthracycline cardiotoxicity. In this article, we report the concentrations of cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) relative to 2'-deoxyguanosine (dGuo), and comet assay results from a study including 20 cancer patients treated with epirubicin. Plasma concentrations of vitamins A, E, C and carotenoids are also reported. All data were obtained before and immediately after epirubicin infusion. The ratios of 8-Oxo-dGuo to dGuo were measured in leukocyte DNA by HPLC-coulometry after NaI extraction of nucleic acids. Vitamins A and E and carotenoids were measured by HPLC-spectrophotometry. Vitamin C was measured by HPLC-spectrofluorimetry. Median 8-oxo-dGuo/dGuo ratios increased significantly from 0.34 to 0.48 lesions per 100,000 bases while per cent of tail DNA increased from 3.47 to 3.94 after chemotherapy 8-Oxo-dGuo/dGuo and per cent of tail DNA medians remained in the normal range. Only vitamin C decreased significantly from 55.4 to 50.3 microM Decreases in vitamins A, E, lutein and zeaxanthin were not significant, but concentrations were below the lower limit of the normal range both before and after chemotherapy. Only the correlation between comet assay results and vitamin C concentrations was significant (rho =-0.517, p = 0.023). This study shows that cellular DNA is damaged by epirubicin-generated free radicals which produce the mutagenic modified base 8-oxo-dGuo and are responsible for strand breaks. However, strand breaks are created not only by free radicals but also by topoisomerase II inhibition. In a previous study we did not find any significant change in urinary 8-oxo-dGuo excretion after adriamycin treatment. However, 8-oxo-dGuo may have increased at the end of urine collection as DNA repair and subsequent kidney elimination are relatively slow processes. In another study, authors used GC-MS to detect 8-oxo-dGuo in DNA and did not find any change after prolonged adriamycin infusion. Reasons for these apparent discrepancies are discussed.     

Differential effects of 2'-deoxyguanosine on peripheral blood mononuclear cell proliferation in healthy donors and Hashimoto's thyroiditis patients

Abstract. The aim of this study was to determine possible differences in peripheral blood mononuclear cells (PBMC) proliferation of healthy donors and Hashimoto's thyroiditis patients and whether a statistical approach to cell proliferation analysis might be used to discern the differences. The effect of a wide range of 2'-deoxyguanosine (dGuo) concentrations (0–1250 μM) on the mitogen-induced proliferation of PBMC was studied in healthy donors and Hasimoto's thyroiditis patients. Activity levels of purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) in PBMC were also measured. For the first time in a study of these models of dGuo toxicity in vitro , the analysis of polynomial trends of orders from 1 to 7 was applied to evaluate cell proliferation. A dose-dependent inhibition of mitogen-induced PBMC proliferation was observed in both groups. Data for linear trend established that PBMC from Hashimoto's thyroiditis patients were more sensitive to dGuo toxicity than PBMC from healthy donors. A positive quadratic trend at low dGuo doses was found in the cell proliferation of Hashimoto's thyroiditis patients. A decrease in PNP activity ( P < 0.025) and an increase in ADA activity ( P < 0.005) was observed in PBMC of Hashimoto's thyroiditis group. The differences in PBMC proliferation subjected to dGuo toxicity between the two groups could be related with the distinct pattern of purine salvage enzymes observed.     

Structural analysis of a new glycosphingolipid from the lipopolysaccharide-lacking bacterium Sphingomonas adhaesiva.

A new glycosphingolipid, GSL-4B, was isolated from Sphingomonas adhaesiva and found to share the ceramide moiety with GSL-1 and GSL-3 from Sphingomonas capsulata studied earlier [Kawahara, K.; Moll, H.; Knirel, Y. A.; Seydel, U.; Z?hringer, U. Eur. J. Biochem. 2000, 267, 1837-1846]. It is heterogeneous with respect to the long-chain bases erythro-2-amino-1,3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1,3-diol, and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol which in GSL-4B are present in the ratios of 1.1:1.0:1.1, and all bearing amide-linked (S)-2-hydroxymyristic acid. Methylation analysis and MALDI-TOF-MS along with 1H and 13C NMR spectroscopy showed that the carbohydrate part of GSL-4B has the structure of alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-GlcpA-(1-->1)-Cer     

Striking ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all four human,and the Drosophila melanogaster,deoxyribonucleoside kinases

In extension of an earlier report, six non-conventional analogues of ATP, three adenosine-2'-triphosphates (3'-deoxy, 3'-deoxy-3'-fluoro- and 3'-deoxy-3'-fluoroxylo-), and three adenosine-3'-triphosphates (2'-deoxy-, 2'-deoxy-2'-fluoro- and 2'-deoxy-2'-fluoroara-), were compared with ATP as potential phosphate donors for human deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1), mitochondrial TK2, deoxyguanosine kinase (dGK), and the deoxyribonucleoside kinase (dNK) from Drosophila melanogaster. With one group of enzymes, comprising TK1, TK2, dNK and dCK (with dAdo as acceptor), only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP), and the other five analogues much less so, or inactive. With a second set, including dCK (dCyd, but not dAdo, as acceptor) and dGK (dGuo as acceptor), known to share high sequence similarity (approximately 45% sequence identity), all six analogues were good to excellent donors (13-119% that for ATP). With dCK and ATP1, products were shown to be 5'-phosphates. With dCK, donor properties of the analogues were dependent on the nature of the acceptor, as with natural 5'-triphosphate donors. With dCK (dCyd as acceptor), Km and Vmax for the two 2'(3')-deoxyadenosine-3'(2')-triphosphates are similar to those for ATP. With dGK, Km values are higher than for ATP, while Vmax values are comparable. Kinetic studies further demonstrated Michaelis-Menten (non-cooperative) or cooperative kinetics, dependent on the enzyme employed and the nature of the donor. The physiological significance, if any, of the foregoing remains to be elucidated. The overall results are, on the other hand, highly relevant to studies on the modes of interaction of nucleoside kinases with donors and acceptors; and, in particular, to interpretations of the recently reported crystal structures of dGK with bound ATP, of dNK with bound dCyd, and associated modeling studies.     

Measurement of the main photooxidation products of 2'-deoxyguanosine using chromatographic methods coupled to mass spectrometry

Analytical methods were developed for the measurement of the main photooxidation products of 2'-deoxyguanosine (dGuo), arising from either the type I (electron transfer) or the type II (singlet oxygen) photosensitization mechanism. Oxidation of dGuo by a type I mechanism leads to the predominant formation of 2, 2-diamino-5-[2-deoxy-beta-d-erythro-pentofuranosyl)amino]-5(2H)-oxazo lone. On the other hand, the two 4R and 4S diastereomers of 4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine are the main singlet oxygen-mediated dGuo oxidation products. The modified nucleosides were measured by either gas chromatography coupled to mass spectrometry after silylation of the sample or by high-performance liquid chromatography associated to tandem mass spectrometry. In order to improve the accuracy of the assays, isotopically labeled internal standards were synthesized for an isotope dilution mass spectrometry quantitation. The methods were successfully applied to the measurement of methylene blue- and riboflavin-mediated 2'-deoxyguanosine photooxidation reactions. The advantages of the two above-mentioned methods are discussed on the basis of comparative sensitivity and accuracy.