Enzymatic alcoholysis of 3′,5′-di-O-acetyl-2′-deoxynucleosides

Candida antarctica-B (CAL-B) lipase-catalysed alcoholysis of a set of 3′,5′-di-O-acetyl-2′-deoxynucleosides (1a–e) gave the corresponding 3′-O-acetyl-2′-deoxy-nucleosides (2a–e) in yields ranging from 50 to 96%. The alcohol employed in the biotransformation affected the rate of the enzymatic reaction and the yield of the 3′-O-acetylated product, but in all cases only this regioisomer was formed. The obtained results are in agreement with the regioselectivity displayed by CAL-B lipase in previously reported biotransformations of nucleosides. CAL-B catalysed alcoholysis of 2′,3′,5′-tri-O-acetyl-cytidine and 4-N-acetyl-2′,3′,5′-tri-O-acetylcytidine was also studied, affording with the same regioselectivity the corresponding free 5′-hydroxyl nucleosides.     

Synthesis and serotonin receptor binding properties of 5-substituted 3-(1′,2′,5′,6′-tetrahydropyridin-3′-yl) indoles

A semi-rigid 5-hydroxytryptamine (5-HT) analogue, RU28253 [5-methoxy-3-(1′,2′,5′,6′-tetrahydropyridin-3′-yl) indole], is a potent 5-HT₁ and 5-HT₂ agonist. It is isomeric to RU24969 [5-methoxy-3-(1′,2′,5′,6′-tetrahydropyridin-4′-yl) indole], a conformationally restricted 5-HT homologue, which has been extensively used in the study and classification of 5-HT receptors. A series of RU28253 derivatives with diverse substituents on indole 5-position were synthesized and their dissociation constants determined at the 5-HT₁ and 5-HT₂ receptors.     

Regulation of DNA synthesis by guanosine-5′-diphosphate, cyclic guanosine-3′,5′-monophosphate, and cyclic adenosine-3′,5′-monophosphate in mouse lymphoid cells

The effect of various adenine and guanine nucleotides and nucleosides on DNA synthesis was studied in various types of mouse lymphoid cells. Two out of the ten compounds tested, namely guanosine-5′-diphosphate (GDP) and cyclic guanosine-3′,5′-monophosphate (cGMP) increased the thymidine incorporation into the DNA of the spleen cells and counteracted completely or partially the inhibitory action of cyclic adenosine-3′,5′-monophosphate (cAMP) on spleen cells stimulated by various B or T cell mitogens. GDP seems to act preferentially on thymus cells while cGMP acts better on bone marrow cells. The possible significance of the results for the mechanism of the mitogenic signal is discussed.     

Synthesis and evaluation of 4′-5′-dehydro-5′-fluoroaristeromycins as S-adenosyl-L-homocysteine (AdoHcy) hydrolase inhibitors

4′,5′-Dehydro-5′-fluoro analogs of aristeromycin were synthesized and shown to be potent inhibitors of recombinant rat liver AdoHcy hydrolase.     

3′-Fluoro-3′-deoxyribonucleoside 5′-triphosphates: Synthesis and use as terminators of RNA biosynthesis

3′-Fluoro-3′-deoxy-uridine, -cytidine, -adenosine and -guanosine have been synthesized by glycosylation of the corresponding silylated bases with 1-O-acetyl-2,5-di-O-benzoyl-3-fluoro-3-deoxy-D-ribofuranose in the presence of Friedel-Crafts catalysts and were converted to the 5′- triphosphates, NTP(3′-F). It was shown that NTP(3′-F) are terminators of RNA synthesis catalyzed by DNA-dependent RNA polymerase from E. coli and may thus serve as tools for DNA sequencing.     

The synthesis of 2,1′-anhydro-,2,1′:3,6-dianhydro-, and 2,1′:3,6: 3′,6′-trianhydro-sucrose

1′-O-Mesyl-6,6′-di-O-tritylsucrose and the corresponding 1′-O-tosyl derivative were prepared from 6,6′-di-O-tritylsucrose by selective sulphonylation. Both sulphonates underwent intramolecular cyclisation reactions, to give 2,1′-anhydrosucrose in high yields rather than the isomeric 1′,4′-anhydride. Sequential benzoylation, detritylation, and mesylation of the 2,1′-anhydride afforded 2,1′-anhydro-6,6′-di-O-mesylsucrose tetrabenzoate which, in the presence of base, gave 2,1′:3,6:3′,6′-trianhydrosucrose that was not identical with the product previously claimed to have this structure. Several derivatives of 2,1′-anhydrosucrose were prepared possessing different functional groups at either the 6,6′- or 4,6′-positions. Dimolar mesitylene-sulphonylation of 3,3′,4′6′-tetra-O-acetylsucrose gave the 6,1′-disulphonate, which, in the presence of alkali, gave 2,1′:3,6-dianhydrosucrose, which was transformed into the 2,1′:3,6:3′,6′-trianhydride by sequential bromination at C-6′ (carbon tetrabromide-triphenylphosphine) and base-catalysed cyclisation. Treatment of 3,3′,4′,6′-tetra-O-benzoylsucrose with sulphuryl chloride furnished the 4,6,1′-trichloro derivative, which, on alkaline hydrolysis, was converted into 2,1′:3,6-dianhydro-4-chloro-4-deoxy-galacto-sucrose.     

Synthesis of 4′-methoxyadenosine and related compounds

Addition of iodine and methanol to N⁶,N⁶-dibenzoyl-9(2,3-O-carbonyl-5-deoxy-β-d-erythro-pent-4-enofuranosyl)adenine (4) selectively gives N⁶,N⁶-dibenzoyl-2′,3′-O-carbonyl-5′-deoxy-5′-iodo-4′-methoxyadenosine (5). Compound 5 can be converted into 4′-methoxyadenosine via hydrolysis of the carbonate followed by benzoylation, displacement of the 5′-iodo function by benzoate ion, and hydrolysis with ammonia. Configurational assignments are based upon comparisons of ¹H- and ¹³C-n.m.r. spectra with those of previously characterised analogues in the uracil series and by borate electrophoresis. Intermediates in the above scheme have also been converted into 5′-amino-5′-deoxy-4′-methoxyadenosine, 4′-methoxy-5′-O-sulfamoyladenosine, and ethyl 4′-methoxyadenosine-5′-carboxylate, each of which is a 4′-methoxy analogue of biologically active derivatives of adenosine.     

Two simple high-performance liquid chromatographic methods for simultaneous determination of 2′-deoxycytidine 5′-triphosphate and cytosine arabinoside 5′-triphosphate concentrations in biological samples

Cytosine arabinoside (ara-C) has been used in the treatment of leukemia, but its exact mechanism of cytotoxicity is not yet known. One of the proposed mechanisms for the effectiveness of this drug in treating leukemias suggests that a metabolite of ara-C, i.e., 2′-deoxycytidine 5′-triphosphate (araCTP), competes with cytosine arabinoside 5′-triphosphate (dCTP) for binding to DNA polymerase. The ratio of the drug metabolite to the endogenous nucleotide (araCTP/dCTP) may, therefore, be important in determining the effectiveness of ara-C therapy. This ratio may also play a role in drug resistance. Previously published methods have focused on either araCTP or dCTP, along with metabolites and analogues of one of these compounds. The methods presented here provide two simple, sensitive ways to measure dCTP and araCTP in the same biological sample.     

3,5,3′,5′-Tetrachlorobiphenyl is a weak oestrogen agonist in vitro and in vivo

Polychlorinated biphenyls (PCBs) are widespread, persistent environmental contaminants of which some congeners can act as endocrine disrupters. Previous work has shown that 3,4,3′,4′-tetrachlorobiphenyl (PCB77) can act as an oestrogen with actions mediated through the oestrogen receptor. Here, oestrogenic actions have been assessed for two further tetrachlorobiphenyl isomers. Assays of oestrogenic action have involved (1) ligand regulation of oestrogen-sensitive gene expression; (2) ligand regulation of cell growth in oestrogen-dependent human breast cancer cell lines MCF7 McGrath and ZR-75-1; and (3) ligand activity in the immature mouse uterine weight bioassay in vivo. These results demonstrate that 3,5,3′,5′-tetrachlorobiphenyl (PCB 80) can be considered to be a weak oestrogen agonist, but the 2,5,2′,5′-congener (PCB 52) revealed no oestrogenic properties in any of these assays. Implications of these results are discussed in relation to structure-activity predictions for environmental oestogens.     

Guanosine 5′-triphosphate inhibits growth and stimulates differentiated functions in B16 melanoma cells

Exogenous guanosine 5′-triphosphate (GTP) at a concentration of 0.5 mM causes in vitro growth inhibition and induces morphological and biochemical differentiation of B16 melanoma cells. After two days in the presence of GTP, cell proliferation is markedly reduced. Cessation of cell proliferation is followed by the extension of numerous dendrite-like processes and marked increase in melanin production. Other nucleotides such as guanosine 5′-diphosphate (GDP), guanosine 5′-monophosphate (GMP), guanosine 3′:5′-cyclic-monophosphoric acid (cGMP) or adenosine 5′-triphosphate (ATP) have little or no effect on cell morphology or melanin production in B16 melanoma cells, although these compounds retard cell proliferation similar to GTP. These findings are discussed in light of a possible relationship between cell proliferation and differentiation.     

Inhibition of 5′-nucleotidase activity after growth of Dictyostelium discoideum

The specific activity of 5′-nucleotidase activity in cell-free extracts of Dictyostelium discoideum at both exponential and stationary growth phases was determined. The 5′-nucleotidase activity of both membrane and soluble fractions was determined. The results show that at exponential growth more activity is found in the soluble fraction. Furthermore, the results show that stationary phase cells contain about 10-fold less activity than cells at exponential growth. To determine if stationary phase cells contained an inhibitor of 5′-nucleotidase, purified membranes were incubated with a high speed supernatant (S-100) prepared from cells at this stage. The results showed not only a time and concentration dependent loss of membrane bound activity, but also that most of the lost activity could be recovered in a soluble form. This result suggested that the 5′-nucleotidase was being released by a factor in the S-100. Additional studies showed inactivation of the releasing factor by a protease and further, that this inactivation could be prevented by serine protease inhibitors. The specificity of releasing factor with respect to two other membrane bound activities was determined. The results indicated no loss of either 3′5′-cyclic phosphodiesterase or adenylate cyclase. In addition, the results of a comparison of the activity of the releasing factor at two stages of growth showed similar values at both exponential and stationary growth phase. This latter finding suggests that the loss of 5′-nucleotidase activity at stationary phase is not due to modulation of the releasing factor activity. An alternative mechanism is proposed.     

Synthesis of (+/−)–1′-Aza-carbocyclic-pyrimidine-2′,3′-dideoxynucleoside analogues as potential anti-HIV agents

1′-Aza-carbocyclic-2′, 3′-dideoxyuridine, 3′-deoxythymidine and 2′, 3′-dideoxycytidine were synthesized from 1-aminopyrrolidine intermediate 13 and evaluated as anti-HIV agents in MT-4 cells.     

Stereoselective synthesis of 3′-C-methylene- and 2′-methyl-3′-C-methylene-3′-deoxythymidine

Stereoselective synthesis of 3′-C-methylene- and 2′-methyl-3′-C-methylene-3′-deoxythymidine is described, the key reaction being the formation of 3-C-methylene function by catalytic isomerization of a chiral epoxyalcohol, prepared from commercially available 3-methyl-2-butenal and 3-methyl-2-pentenal.     

Synthesis of 3-methylpseudouridine and 2′-deoxy-3-methyl-pseudouridine

The first chemical synthesis of 3-methyl-ψ-uridine (5) and its 2′-deoxy analogue (9) has been achieved. ψ-Uridine was trimethylsilylated and the crude product was treated with acetyl chloride, to give the 1-acetyl derivative (3). Crude 3 was methylated with dimethoxymethyldimethylamine and then saponified, to give crystalline 5 in 82% overall yield. Treatment of 5 with 1,3-dichloro-1,1,3,3-tetraiso-propyldisiloxane afforded the 3′,5′-protected product, which was converted into the 2′-O-[(imidazol-1-yl)thiocarbonyl] derivative 7. Reduction of 7 with tributyltin hydride followed by deblocking of the product gave crystalline 2′-deoxy-3-methyl-ψ-uridine (9) in 35% yield from 5.     

Synthesis and fungicidal activity of 2,2′-bipyridine derivatives

A series of substituted 2,2′-bipyridine derivatives was prepared using the Kröhnke reaction and alkylation of 4,4′-dimethyl-2,2′-bipyridine. These compounds were screened for fungicidal activity against 9 plant diseases. 5-Phenyl-2,2′-bipyridine exhibited strong preventative and curative fungicidal activity against wheat powdery mildew (Erisyphe graminis) and wheat leaf rust (Puccinia recondita).     

Pyridoxal 5′-phosphate related changes in retention of 1,25-dihydroxy vitamin D-receptor ligands in rat intestinal mucosa cell nuclei

After feeding rats a vitamin B-6-deficient diet, we observed a decrease in pyridoxal 5′-phosphate concentrations in intestinal mucosa cells to 32 and 48% of control in cytoplasm and cell nuclei, respectively. Correlation analysis suggested that there were two pyridoxal 5′-phosphate pools in the nuclei: a “mobile” pool (equivalent to about 5% the concentration of the cytoplasmic pyridoxal 5′-phosphate), and a “stable” pool, which was independent of cytoplasmic fluctuations of pyridoxal 5′-phosphate (about 9 pmol pyridoxal 5′-phosphate/mg DNA). Reduction in pyridoxal 5′-phosphate content in the cells of vitamin B-6-deficient animals was accompanied by a substantial increase in 1,25-dihydroxyvitamin D-receptor ligand concentration in the cell nuclei (76.6 ± 19.7 vs 762 ± 291 fmol/mg DNA, mean ± SEM). The degree of 1,25-dihydrovitamin D accumulation in the nuclei appeared to be an exponential function of the “mobile” nuclear pyridoxal 5′-phosphate concentration. Semilogarithmic transformation of the data yielded a straight line, representing an inverse correlation between the cytoplasm-related nuclear pool of pyridoxal 5′-phosphate and the logarithm of the 1,25-dihydroxyvitamin D concentration in the nuclei (r=−0.95). These data suggest that pyridoxal 5′-phosphate may be related to 1,25-dihydroxyvitamin D retention in the nuclei, possibly through interaction of the pyridoxal 5′-phosphate with the vitamin D receptor protein in the nuclei.     

Syntheses and molecular structures of ruthenium(II) complexes of the atropisomeric ligands 1,1′-biphenyl-2,2′-diamine and 3,3′-diamino-2,2′-bipyridine

The unique ligands of [Ru(bipy)₂(bpda)](PF₆)₂ (1, BPDA=1,1′-biphenyl-2,2′-diamine) and [Ru(bipy)₂(dabipy)](PF₆)₂ (2, DABIPY=3,3′-diamino-2,2′-bipyridine) are atropisomeric (exhibit hindered rotation about the sigma bonds that connect the two aromatic groups), so the complexes are diasteromeric with conformation isomers possible for the atropisomeric ligands and configurational isomers possible at the metal centers. Only one diastereomer is observed in the solid-state in both cases. The seven- (1) and five-membered (2) chelate ring of dabipy and bpda (the ligand is bound through its pyridyl groups) ligands are δ when the configuration at the metal is Δ. No evidence for atropisomerization is found in solution. For 1, we conclude bpda binds stereospecifically; however, the atropisomerization barrier of dabipy may be sufficiently low for 2 to preclude the observation of diastereomers by low-temperature NMR spectroscopy.     

The role of 5′-methylthioadenosine phosphorylase in 5′-methylthioadenosine-mediated inhibition of lymphocyte transformation

To determine if increased 5′-methylthioadenosine phosphorylase activity in activated lymphocytes may be responsible for the decreased inhibitory effect noted when 5′-methylthioadenosine is added after stimulation, the activity of this enzyme was monitored during lymphocyte transformation. A direct correlation existed between the transformation process and 5′-methylthioadenosine phosphorylase activity; the longer the stimulation process progressed, the greater the enzyme activity. The 7-deaza analog of 5′-methylthioadenosine, 5′-methylthiotubercidin, was utilized to explore further the role that the phosphorylase may play in the reversal process. 5′-Methylthioadenosine acted as a potent inhibitor, but not a substrate, of the 5′-methylthioadenosine phosphorylase, and was an even more potent inhibitor of lymphocyte transformation than 5′-methylthioadenosine. However, in direct contrast to the 5′-methylthioadenosine effect, inhibition by 5′-methylthiotubercidin could not be completely reversed. These data suggest the 5′-methylthioadenosine phosphorylase plays an important role in reversing 5′-methylthioadenosine-mediated inhibition and that the potent, nonreversible inhibitory effects of 5′-methylthiotubercidin are due to its resistance to 5′-methylthioadenosine phosphorylase degradation.     

Selection of the 3′-splice site in group I introns

A model for selection of 3′-splice sites in splicing of RNA precursors containing group I introns is presented. The key feature of this model is a newly identified tertiary interaction between the catalytic core of the intron and the 3′-splice site. This tertiary pairing would bring the 3′-splice site into the core of the intron, which is known to contain RNA sequences and structures essential for catalyzing the splicing reactions. The proposed tertiary interaction can coexist with P10, a pairing between 3′-exon sequences and the ‘internal guide sequence’ near the 5′-end of the intron. The model predicts that three RNA-RNA interactions are important in selection of 3′-splice sites: (i) binding of intron sequences with the core; (ii) pairing of exon sequences with the internal guide sequence; and (iii) binding of the terminal guanosine to an unknown site within the core.     

Purification of chemically synthesised dinucleoside(5′,5′) polyphosphates by displacement chromatography

Dinucleoside(5′,5′) polyphosphates (Ap_nA, Ap_nG, Gp_nG, n=3–6) are new group of hormones controlling important biological processes. Because some of the dinucleoside(5′,5′) polyphosphates are commercially not available purification of chemical synthesised dinucleoside(5′,5′) polyphosphates became necessary in order to test their physiological and pharmacological properties. It was the aim of this study to find a method which allows purification of 0.1–0.2 g quantities of dinucleoside polyphosphates by analytical HPLC columns yielding products with impurities lower than 1.0%. Adenosine(5′)-polyphospho-(5′)guanosines were synthesised by mixing the corresponding mononucleotides. The reaction results in a complex mixture of Ap_nA, Ap_nG and Gp_nG (with n=3–6 in all cases). The reaction mixture was concentrated on a preparative C₁₈ reversed-phase column. The concentrate was displaced on a reversed-phase stationary. As a result of displacement chromatography, anion-exchange chromatography in gradient modus yielded baseline separated dinucleoside polyphosphates (homogeneity of the fractions>99%). The identity of the substances were determined by matrix assisted laser desorption ionisation mass spectrometry.