5-thio-D-ribopyranose : Part III. Conformational equilibria in the methyl D-ribopyranosides,their 1-thio, 5-thio,and 1,5-dithio analogues,and the related triacetates

Conformational equilibria have been estimated by n.m.r. spectroscopy for the methyl 2,3,4-tri-O-acetyl-α- and -β-D-ribopyranosides (1′a and 1′b), their 1-thio (2′a, 2′b), their 5-thio (3′a, 3′b), and their 1,5-dithio (4′a, 4′b) analogues. Only 1′b shows a preference for the 1C conformation; the others favour the C1 form to various extents. These results are discussed in terms of polar and steric effects. Similar estimations have been made on the unacetylated D-ribopyranosides (1–4) and, where a definite conformational assignment is possible, these follow the same trend as the triacetates (1′–4′). These results are compared, where possible, with the results of X-ray crystallographic studies.     

A simple, specific, radioisotopic assay for 5'-nucleotidase.

A method is presented using [¹⁴C]5′-AMP as a substrate for measuring 5′-nucleotidase activity in the presence of interfering phosphatases. An inhibitor of 5′-nucleotidase, α,β-methyleneadenosine diphosphate is utilized, and the enzyme activity is measured as the difference between total phosphatase activity and inhibitor-insensitive activity.     

THE SYNTHESIS OF,AND STUDIES ON, 2′-C-MODIFIED NUCLEOTIDES

The synthesis of uridine monomers containing either a 2′-deoxy-2′-C-methy- lcyano or ethylcyano group is described. These monomers are intended for incorporation into oligonucleotides to investigate a proposed duplex-stabilising effect exerted by 2′-tethered amide groups.     

The comparative effects of 5′-methylthioadenosine and some of its analogs on cells containing,and deficient in, 5′-methylthioadenosine phosphorylase

The antiproliferative effects of 5′-methylthioadenosine and the 5′-methylthioadenosine analogs, 5′-isobutylthioadenosine, 5′-deoxyadenosine and 5′-methylthiotubercidin were examined using two mouse cell lines, one 5′-methylthioadenosine phosphorylase-deficient the other containing 5′-methylthioadenosine phosphorylase. All of the compounds were found to be growth inhibitory to both cell lines, demonstrating that these compounds need not be degraded to exert their inhibitory effects. A correlation was observed between the potency of the growth inhibitory effect and the ability of the cells to degrade these compounds. 5′-Methylthioadenosine, 5′-deoxyadenosine and 5′-isobutylthioadenosine, all of which are substrates for the 5′-methylthioadenosine phosphorylase in vitro, were more growth inhibitory to the 5′-methylthioadenosine phosphorylase-deficient cells than to the 5′-methylthioadenosine phosphorylase-containing cells, whereas, the 7-deaza analog, 5′-methylthiotubercidin, a nondegradable inhibitor of the 5′-methylthioadenosine phosphorylase, was a more potent inhibitor of the 5′-methylthioadenosine phosphorylase-containing cell line. Due to the inhibition by 5′-methylthiotubercidin on 5′-methylthioadenosine phosphorylase in vitro the disposition of cellularly-synthesized 5′-methylthioadenosine was explored using both cell types. 5′-Methylthiotubercidin inhibited the accumulation of exogenous 5′-methylthioadenosine from 5′-methylthioadenosine phosphorylase-deficient cells with no effect on intracellular 5′-methylthioadenosine. In contrast, 5′-methylthiotubercidin caused a large accumulation of extracellular 5′-methylthioadenosine with a concomitant smaller increase intracellularly in 5′-methylthioadenosine phosphorylase-containing cells. That cellularly-synthesized 5′-methylthioadenosine as well as the cellular excretion of this nucleoside are altered in response to treatment with 5′-methylthiotubercidin suggests two possible sites at which 5′-methylthiotubercidin may exert its effect.     

Proton magnetic resonance study of nucleosides,nucleotides, and dideoxynucleoside monophosphates containing a syn pyrimidine base

Proton magnetic resonance data have been obtained for 6-methyl-2′-deoxyuridine (dT*), its 3′- and 5′-monophosphates, and its 3′,5′-diphosphate, as well as for the corresponding thymine derivatives. The synthesis of the dideoxynucleoside monophosphates—d(TpT), d(T*pT), d(TpT*), and d(T*pT*)—was accomplished, and spectral data were obtained for these four dimers. The data show that the 6-methyluracil base prefers the syn conformation about the N-glycosyl bond at the monomer and dimer levels. The presence of the syn base leads to increases in the cis couplings of the sugar ring, J_1′2″ and J_2′3′, which indicate a trend towards eclipsing of the substituents on the C1′-C2′ and C2′-C3′ fragments. This trend is discussed in terms of changes in the pseudorotational parameters which describe the pucker of the ring. The syn base destabilizes the g⁺ conformer about the C4′-C5′ bond, leading to a preference for the t conformer in all dT* residues at the monomer and dimer levels. Preliminary work on the formation of cyclobutane-type photodimers in d(T*pT) and d(T*pT*) is discussed and presented as evidence for the capability of the syn 6-methyluracil base to form base-stacked complexes.     

Hydrolysis of 2',3'-cyclic adenosine monophosphate and 3',5'-cyclic adenosine monophosphate in subcellular fractions of normal and neoplastic mouse spleen

A comparison has been made between the capacity to hydrolyse 2′,3′-cyclic adenosine monophosphate and 3′,5′-cyclic adenosine monophosphate in subcellular fractions of normal and neoplastic (lymphosarcoma) spleen of C₅₇BL mice. The effect of X-irradiation on these activities was tested. Subcellular fractionation of normal and lymphosarcoma spleen points to a different overall localization of the enzymes. The 2′,3′-cyclic nucleotide phosphodiesterase (2′,3′-cAMPase) has its highest specific activity in the particulate fractions of the cell, while the data on 3′,5′-cyclic nucleotide phosphodiesterase (3′,5′-cAMPase) show the highest activity in the soluble fraction. The 2′,3′-cAMPase activity is higher in the tumor as compared to the normal tissue, while the opposite holds for 3′,5′-cAMPase. Total body irradiation of normal mice with a dose of 600 rads of X-rays, results in a clear drop in 2′,3′-cAMPase 48 hours after the exposure. The 3′,5′-cAMPase is hardly affected at this time. Neither imidazol nor Mg⁺⁺ has any influence on the 2′,3′-cAMPase. The pH optimum for 3′,5′-cAMPase and 2′,3′-cAMPase appears to be 7.7 and 6.2 respectively. This report suggests a no-identity of the two enzymes in mouse spleen, a situation different from that found in certain plants.     

Synthesis of a fluorescent 2′3′-dideoxycytosine analog,tCdd

The pathway leading to the preparation of a novel tricyclic 2′3′-dideoxycytosine analog, tCdd (1) is reported. A protected 2′3′-dideoxyribose prepared from l-glutamic acid was coupled to a silylated fluorescent base to yield a mixture of the α- and β-anomers of the 2′3′-dideoxyribonucleoside of 1,3-diaza-2-oxophenothiazine, tCdd (1). The fluorescent base analog retains a high fluorescence emission over a large pH range and should be useful in a variety of probe applications.     

Terminally modified,short phosphorothioate oligonucleotides as inhibitors of gene expression in cells

Phosphorothioates are excellent antisense inhibitors, which are active both in cells and in vivo. Since their affinity to complementary ribonucleic acids is rather low, long strands (⩾20-mers) are typically required to achieve the desired biological activity. However, mismatch discrimination of long inhibitors is reduced. In contrast, shorter phosphorothioates exhibit better sequence specificity, but have in most cases too low affinity for practical applications in cells. We screened a range of terminal modifiers of a 14-mer phosphorothioate sequence, which is complementary to mRNA of a representative gene, whose protein product is fluorescent (DsRed2) and easy to monitor in cells. We found that optimal combinations of 5′- and 3′-modifications include 5′-trimethoxystilbene with 3′-uracil(anthraquinone)-cap, 5′-chloic acid derivative with 3′-uracyl(anthraquinone)-cap and 5′-cholic acid derivative with three 3′-LNA moieties. In contrast to the LNA, stabilizing and activity-enhancing effects of other mentioned modifiers for PTO/RNA duplexes have not been previously reported. We observed that the 14-mer inhibitor carrying 5′-cholic acid derivative with three 3′-LNA moieties inhibits expression of DsRed2 in cells stronger than the unmodified 21-mer. Mismatch discrimination of this inhibitor was found to be comparable to that of the unmodified 14-mer.     

Oligonucleotide conformations. III. Comparative optical and thermodynamic studies of uridylyl-3'-5'-nucleosides containing ribose, deoxyribose, or 2'-deoxy-2'-fluororibose in the uridine moiety.

The synthesis of uridylyl-3′-5′-nucleosides containing ribose, deoxyribose, or 2′-fluoro-2′-deoxyribose in the uridine-3′-bound moiety and adenosine, guanosine, cytidine or uridine in the 5′-nucleoside is reported. The temperature dependence of the circular dichroism of these dinucleoside phosphates in 0.06 M phosphate buffer at pH 7 was analyzed by the two-state model and the oscillating dimer model. From the former, apparent thermodynamic parameters were determined by means of an iterative computer method. The comparison between the three different dinucleoside phosphates in each series indicated that the fluororiboside and the riboside resembled each other and were more stacked than the analogue containing deoxyribose. It further appeared that the similarity between the fluororiboside and the riboside is influenced by the nature of the neighboring 5′-bound base. The interaction between the 3′-bound sugar moiety and the 5′-bound base is evoked as a possible stabilization mechanism.     

A simple, specific radiometric assay for 5'-nucleotidase.

A radiometric assay for 5′-nucleotidese (EC 3.1.3.5) has been developed, which is applicable for all 5′-nucleotide substrates. Various column materials and eluants were evaluated for their suitability in the separation of purine and pyrimidine bases and nucleosides produced in the reaction. Neutral alumina columns were found to be the best. The unadsorbed nucleosides and their bases could be quantitatively eluted with 0.1 m Tris-HCl, pH 7.4; subsequent elution of the 5′-nucleotide was then accomplished with 0.2 m sodium phosphate, pH 7.4. Differential measurement of 5′-nucleotidase can be accomplished in the presence of acid or alkaline phosphatases by inclusion of concanavalin A into the reaction mixture. It completely inhibits 5′-nucleotidase without effecting the phosphatases. The applicability of this assay has been demonstrated by studying the properties of 5′-nucleotidase present in a purified plasma membrane preparation from a rat tumor which is enriched with both 5′-nucleotidase and alkaline phosphatase.     

Design,synthesis, and cytotoxicity of stabilized mycolactone analogs

On exposure to visible light, mycolactone A/B, the causative toxin of Buruli ulcer, rearranges to a mixture of four photo-mycolactones apparently via a rare photochemically-induced [₄π_s + ₂π_a] cycloaddition. In order to prevent the rearrangement, two C6′-C7′ dihydromycolactone analogs 6′α-15 and 6′β-15 were designed and synthesized. 6′α-15 and 6′β-15 were shown to be stable under not only photochemical, but also acidic and basic conditions. Cytotoxicity was tested against arbitrarily chosen four cell lines (human Hek-293, human lung carcinoma A-549, human melanoma LOX-IMVI, and mouse L-929), thereby revealing that: (1) both analogs maintain potent cytotoxicity; (2) 6′β-15 exhibits significantly higher potency against human cell lines than 6′α-15; (3) in comparison with parent mycolactone A/B, 6′β-15 exhibits equal potency against human Hek-293, whereas significantly lower potency against human lung carcinoma A-549 and human melanoma LOX-IMVI.     

Potentiometric and colorimetric methods for the assay of 2',3'-cyclic nucleotide 3'-phosphohydrolase

Abstract— A potentiometric titration method for the assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase is presented. Progress curves of the reaction were recorded automatically by pH-stat. 2-Mercaptoethanol was added to the reaction mixture to maintain a linear rate of reaction. The method is suitable for obtaining kinetic parameters and can be used for the rapid assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in nervous tissues. An improved colorimetric method for estimation of 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity at the optimum pH is described. This method employs the two-step procedure in which decyclization by 2′,3′-cyclic nucleotide 3′-phosphohydrolase and dephosphorylation by Escherichia coli alkaline phosphatase (EC 3.1.3.1) are carried out separately under the optimum conditions for each enzyme. The method is sensitive and most convenient for routine assays.     

ANTI-HIV ACTIVITY OF NOVEL PHOSPHONATE DERIVATIVES OF AZT,d4T,AND ddA

Anti-HIV activity and cytotoxicity were tested for novel phosphonate derivatives of AZT, d4T and ddA. For d4T phosphonate derivatives the most active was 2′,3′-Dideoxy-2′,3′-didehydrothymidine 5′-isopropylphosphite and among the AZT phosphonate derivatives highest activity was shown by 2′,3′-Dideoxy-3′-azidothymidine 5′-cyclohexylphosphite.     

Enzymic analysis of cyclic 3', 5'-AMP in mammalian tissues and urine

The details are presented for the analysis of 3′,5′ cyclic adenosine monophosphate (3′5′CAMP) in milligram amounts of mammalian tissues (muscles, liver, brain, and kidney) and in microliter samples of urine. An examination of the sources of difficulty and how they are effectively handled is also included. In the determination of tissue 3′5′CAMP the cyclic nucleotide is first separated from 5′-nucleoside mono-, di-, and triphosphates by cellulose thin-layer chromatography following Ba(OH)₂-ZnSO₄ precipitation of extracts. After quantitative recovery 3′,5′CAMP is converted to 5′ AMP and subsequently to ATP by the actions of phosphodiesterase, myokinase, and pyruvate kinase. Enzymic cycling with the hexokinase-pyruvate kinase system is then used to produce a proportional concentration of G-6-P equivalent to several thousand fold the ATP concentration and the G-6-P measured fluorometrically. Cyclic adenylate in urine samples is determined directly without prior separation from any urinary components. Examples are presented of the analytical procedures applied to the measurement of 3′5′CAMP levels in tissues and urine after various experimental treatments. These include the effects of epinephrine in skeletal muscle in vitro and in vivo, of adrenalectomy and hydrocortisone in liver, of ischemia in brain, and of hypertonic infusion on urinary excretion of 3′5′CAMP.     

Metabolism of naturally occurring propenylbenzene derivatives III. Allybenzene,propenyl benzene,and related metabolic products

The acidic and neutral urinary metabolites of allylbenzene, propenylbenzene, 1′-hydroxyallylbenzene, and cinnamyl alcohol were identified and related through a common metabolic scheme. The rat metabolizes allylbenzene to 1′-hydroxyallylbenzene which can rearrange to yield cinnamyl alcohol which is further metabolized. This mechanism is proposed to account for the appearance of “propenyl type” metabolites from allylbenzene compounds. Propenylbenzene is oxidized to cinnamyl alcohol. Both 1′-hydroxyallylbenzene and cinnamyl alcohol are excreted unchanged in the neutral extract when given to rats. Allylbenzene and 1′-hydroxyallylbenzene yield basic ninhydrin-positive metabolites. Allylbenzene is first oxidized on the benzylic carbon to form 1′-hydroxyallylbenzene, which is further oxidized to form phenyl vinyl ketone, which condenses with the secondary amines piperidine and dimethylamine to form tertiary aminopropiophenones (Mannich bases). Analogous compounds, cinnamyl alcohol and propenylbenzene, do not yield Mannich base metabolites. This proposed metabolic scheme is consistent with the chemical mechanism operative in the synthesis of Mannich base from allylbenzene via chromic acid oxidation followed by amine addition.     

Index to Subjects,Vol. 27

Abstract

A constrained model building procedure is used to generate nucleic acid structures of the familiar A-, B-, and Z-DNA duplexes. Attention is focused upon the multiple structural solutions associated with the arrangements of nucleic acid base pairs rather than the optimum sugar-phosphate structure. The glycosyl (χ) and sugar torsions (both the ring puckering and the exocyclic C5′-C4′ (ψ) torsion) are treated as independent variables and the resulting O3′…O5′ distances are used as closure determinants. When such distances conform to the known geometry of phosphate chemical bonding, an intervening phosphorus atom with correct C-O-P valence angles can be located. Four sequential torsion angles- φ,ω,ω,ω and φ about the C3′-O3′-P-O5′-C5′ bonds are then obtained as dependent variables. The resulting structures are categorized in terms of conformation, ranked in potential energy, and analyzed for torsional correlations. The numerical results are quite interesting with implications regarding nucleic acid models constructed to fit less than ideal experimental data. The multiple solutions to the problem are useful for comprehending the conformational complexities of thelocal sugar-phosphate backbone and for understanding the transitions between different helical forms. According to these studies, unique characterization of a nucleic acid duplex involves more than the determination of its base pair morphology, its sugar puckering preferences, or its groove binding features.     

Conformational study of ribonucleotides, 2′-deoxyribonucleotides,and arabinonucleotides by carbon-13 nuclear magnetic resonance

The geminal and vicinal ¹³C-³¹P coupling constants have been monitored, as a function of pH, for a series of uracil and cytosine 3′- and 5′-nucleotides with a ribose, arabinose, or 2′-deoxyribose sugar. Data were also obtained for two 3′,5′-diphosphates in the ribose and arabinose series. The geminal J(C5′-P5′) and J(C3′-P3′) couplings show only a small dependence on the ionization state of the phosphate, decreasing by < 0.5 Hz in the pH 5–7 range. For the ribose and arabinose 3′-nucleotides, the vicinal J(C4′-P3′) increase (up to 1.5 Hz) on secondary phosphate ionization in the pH 5–7 range, whereas their J(C2′-P3′) couplings decrease (up to 1.5 Hz) over the same pH range. In contrast for the 2′-deoxyribose molecules, both couplings decrease (～0.5 Hz) on phosphate ionization. The titration curves provide information about the influence of the sugar on the conformation about the C3′? O3′ bond. Some conformational trends could be rationalized by consideration of the sugar-puckerdependent contact interactions between the 3′-phosphate and the substituents on the furanose ring.     

2′-MODIFIED OLIGONUCLEOTIDES FROM METHOXYOXALAMIDO AND SUCCINIMIDO PRECURSORS: SYNTHESIS,PROPERTIES, AND APPLICATIONS

Synthesis of 2′-modified oligonucleotides from 2′-methoxyoxalamido (MOX) and 2′-succinimido (SUC) precursors is described. Their physical and biochemical properties were assessed. Synthesized oligonucleotides were used as primers in advanced DNA sequencing protocols. An example of sequencing directly off genomic DNA template without prior cloning or PCR amplification is presented.     

Zeyherol,a dilignol from Zeyhera digitalis

The stem wood of Zeyhera digitalis (Bignoniaceae) contains, besides d-glucose, vanillic acid, veratric acid and lapachol, a natural dilignol type compound for which the structure of 2,2′-dihydroxy-3,3′-dimethoxy-5-ω-oxo-E-propenyl)-5′-(ω-hydroxy-Z-propenyl)-biphenyl is proposed.