Natural pteridines--a chemical hobby.
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Animals↗
Biomedical subjects
W Pfleiderer
Publications and source records attributed to W Pfleiderer.
Laser-sensitized tautomers in dihydrofolate reductase.
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Binding Sites↗
N2-(N,N-Dimethylaminomethylene)-O4-(2-p-nitrophenylethyl)-biopterin: a versatile intermediate for a glycosidation reaction.
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Biopterins↗
(2'-5')Oligoadenylate and intracellular immunity against retrovirus infection.
1. The double-stranded RNA-dependent 2',5'-oligoadenylate (2-5A) synthetase/ribonuclease L (RNase L) system plays an essential role in the establishment of the antiviral state of a cell exposed to virus infection. 2. Until recently, the application of 2-5A derivatives to reinforce this system seemed to be limited mainly due to the low specificity of RNase L for viral RNA. 3. Two new strategies have been developed which yield a selective antiviral effect of 2-5As at least against human immunodeficiency virus-1 (HIV-1) infection: (i) an "intracellular immunization" approach using 2-5A synthetase cDNA linked to HIV trans-acting response element (TAR) and (ii) inhibition of retroviral reverse transcriptase activity by 2-5A analogues.
2',5'-Oligoadenylate Synthetase↗
6-Pyruvoyl tetrahydropterin synthase assay in extracts of cultured human cells using high-performance liquid chromatography with fluorescence detection of biopterin.
An assay for 6-pyruvoyl tetrahydropterin synthase, the second enzyme in the conversion of guanosine triphosphate into tetrahydrobiopterin, has been developed. Cell extracts were incubated with enzymatically prepared dihydroneopterin triphosphate (80 microM) in the presence of Mg2+ (12 mM), excess sepiapterin reductase (EC 1.1.1.153) (2 nmol/min) and NADPH (2 mM). 6-Pyruvoyl tetrahydropterin, the product of the reaction, was thus converted into tetrahydrobiopterin. After oxidation of the reduced biopterin derivatives in acidic iodine solution, biopterin was enriched and separated from the abundant neopterin phosphates by solid-phase extraction on a strong cation exchanger. Biopterin was then directly eluted on a reversed-phase liquid chromatographic column and detected fluorimetrically using excitation at 353 nm and emission at 438 nm. The biopterin concentrations formed by the coupled enzyme reaction increased linearly with incubation times up to 90 min. The assay allows the quantification of 6-pyruvoyl tetrahydropterin synthase in cultured human cells.
Alcohol Oxidoreductases↗
Synthesis and properties of 5,6-dichlorobenzimidazole 2'----5'- and 3'----5'-nucleotide dimers and trimers.
5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (2), synthesised by the fusion method, was used for the synthesis of 2'----5'- and 3'----5'-linked di- and tri-meric oligonucleotides. The protecting groups used were p-methoxytrityl for HO-5', tert-butyldimethylsilyl for HO-2',3', and 2,5-dichlorophenyl and 2-(4-nitrophenyl)ethyl for the phosphate group. The internucleotidic linkages were established by the phosphotriester approach to give the fully protected 2'----5' dimers (15, 17, and 18) and trimers (27 and 28), as well as the 3'----5' dimers (22 and 23) and trimers (31 and 32). Deprotection involved a sequence of steps to afford the corresponding free oligonucleotides 21, 26, 30, and 33 isolated as the triethylammonium salts in good yields. The new compounds were characterised by elemental analysis and by u.v. and 1H-n.m.r. spectroscopy.
Antiviral Agents↗
2,4-diamino-7-hydroxy-pteridines, a new class of catabolites of methotrexate.
Methotrexate remains a commonly used drug in the chemotherapy of various malignancies. The known catabolites are 7-hydroxy-methotrexate, formed in the liver, and diamino-methyl-pteroic acid formed in the gut. We report for the first time evidence that 2,4-diamino-7-hydroxy-pteridine derivatives are present in the biological fluids of patients on high-dose methotrexate protocols. So far, two major derivatives have been identified as 2,4-diamino-6-hydroxymethyl-7-hydroxy-pteridine and 2,4-diamino-6-methyl-7-hydroxy-pteridine. In regard to the actual knowledge of the catabolism of pteridines, these compounds are presumably formed by intestinal bacteria during enterohepatic circulation of the drug. Their slow clearance from the body raises the question of possible interference of these compounds on pteridine-dependent enzymes, which might explain in part some of the toxic effects of methotrexate.
Adolescent↗
Cordycepin analogues of 2',5'-oligoadenylate inhibit human immunodeficiency virus infection via inhibition of reverse transcriptase.
Analogues of 2',5'-oligoadenylates (2-5A), the cordycepin (3'-deoxyadenosine) core trimer (Co3) and its 5'-monophosphate derivative (pCo3), were shown to display pronounced anti-human immunodeficiency virus type 1 (HIV-1) activity in vitro. Treatment of HIV-1 infected H9 cells with 1 microM Co3 or pCo3 resulted in an almost 100% inhibition of virus production. The compounds were encapsulated in liposomes targeted by antibodies specific for the T-cell receptor molecule CD3. Substitution of one or two cordycepin units in Co3 or pCo3 decreased the antiviral activity of the compounds. pCo3 did not stimulate 2-5A-dependent ribonuclease L activity and displayed no effect on the amount of cellular RNA and protein. At a concentration of 10 microM the cellular DNA polymerases alpha, beta, and gamma were almost insensitive toward Co3 or pCo3. In contrast, these compounds reduced the activity of HIV-1 reverse transcriptase (RT) by 90% at a concentration of 10 microM if the viral RNA genome and the cellular tRNALys.3 was used as template/primer system; if the synthetic poly(A).(dT)10 was used as template/primer, no marked inhibition was observed. Dot-blot, gel-retardation, and cross-linking assays showed that Co3 or pCo3 interfere with the binding site of tRNALys.3 to RT. These results indicate that inhibition of RT at the level of initiation of the enzymic reaction is a novel approach to inhibit HIV-1 replication.
Adenine Nucleotides↗
New approaches towards automated oligoribonucleotide synthesis.
Several protecting group combinations have been investigated to achieve an automated oligoribonucleotide synthesis on a solid support. The use of 2'-O-tert-butyldimethylsilyl-5'-O-monomethoxytrityl-ribonucleoside -3'-(p-nitrophenylethyl, N,N-diethyl)-phosphoramidites in conjunction with beta-eliminating blocking groups at the aglycone gave satisfactory results. An inverse protecting group strategy based upon analogously substituted 5'-O-2-dansylethoxycarbonyl-2'-O-4- methoxytetrahydropyran-4-yl-phosphoramidites can be regarded as another promising approach.
Base Sequence↗
Specificities of rabbit anti-(2',5')oligoadenylate antibodies towards phosphorothioate and seco analogs of oligoadenylates.
Rabbit antibodies to 2',5'-linked triadenylate were prepared by immunization with (2',5')A3 conjugated via the 2'3'-levulinic group, (2'5')A3-Lev, to BSA. New radioimmunoassay for (2',5')oligoadenylates was developed using 125I thyrosine labeled derivative of (2',5')A3-Lev. Reactivity of antibodies with phosphorothioate and seco analogs of oligoadenylates was studied. It was found that (i) stereospecific substitution of the diastereotopic oxygens with sulfur in the internucleotide phosphodiester linkages changes the immunoreactivity of such analogs; (ii) the seco analogs of oligoadenylates display in some cases a rather high reactivity.
Adenine Nucleotides↗
Phosphorothioate analogues of (2'-5')(A)4: agonist and antagonist activities in intact cells.
Metabolically stable phosphorothioate tetramer analogues of (2'-5')(A)n with Rp and/or Sp chirality in the 2'-5'-phosphodiester linkages constitute a new class of antiviral agents since they mimic the effects of interferons. Three of the diastereomeric 5'-monophosphates (i.e., pRpRpRp, pSpRpRp, and pRpSpSp) bind to and activate RNase L from extracts of HeLa cells. However, the pSpSpSp (2'-5')-(A)4-phosphorothioate is unique in that it binds to, but cannot activate, RNase L to cleave rRNA. When microinjected into the cytoplasm of HeLa cells followed by virus infection, the pRpRpRp, pSpRpRp, and pRpSpSp (2'-5')(A)4-phosphorothioates demonstrate antiviral activity, as does (2'-5')(A)4ox-red, an active (2'-5')(A)n analogue. When microinjected simultaneously with (2'-5')(A)nox-red, an active the pSpSpSp (2'-5')(A)4-phosphorothioate inhibits activation of RNase L in HeLa cells, thereby blocking direct protection of vesicular stomatitis virus. The agonist and antagonist properties of pRpRpRp and pSpSpSp, respectively, are transient probably as a consequence of the hydrolysis of the 5'-monophosphate and formation of the less active (2'-5')(A)4-phosphorothioate cores. The possible use of these (2'-5')(A)4-phosphorothioates as tools for dissecting the biological significance of the (2'-5')(A)n system or in antiviral chemotherapy is discussed.
Antiviral Agents↗
Tetrahydrobiopterin biosynthetic activities in human macrophages, fibroblasts, THP-1, and T 24 cells. GTP-cyclohydrolase I is stimulated by interferon-gamma, and 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are constitutively present.
Interferon-gamma induces tetrahydrobiopterin biosynthesis in human cells and cell lines. Macrophages are peculiar in the formation of large amounts of neopterin derivatives as compared to tetrahydrobiopterin (Werner, E. R., Werner-Felmayer, G., Fuchs, D., Hausen, A., Reibnegger, G., and Wachter, H. (1989) Biochem J. 262, 861-866). Here we compare the impact of interferon-gamma treatment on activities of GTP-cyclohydrolase I (EC 3.5.4.16), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase (EC 1.1.1.153) in human peripheral blood-derived macrophages, normal dermal fibroblasts, THP-1 myelomonocytic cells, and the T 24 bladder transitional-cell carcinoma line. Upon interferon-gamma treatment, GTP-cyclohydrolase I activity is increased 7- to 40-fold, whereas 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase activities, which are constitutively present in all four investigated cells, remain unchanged. In fibroblasts and T 24 cells GTP cyclohydrolase I activity is the rate-limiting step of tetrahydrobiopterin biosynthesis. In macrophages and in THP-1 cells, however, the induced GTP cyclohydrolase I activity is higher than the 6-pyruvoyl tetrahydropterin synthase activity, leading to the accumulation of neopterin and neopterin phosphates.
Alcohol Oxidoreductases↗
Application of a new electrophoresis technique (2D cryostat section electrophoresis) to synovial tissue of RA patients and comparison with immunohistochemical staining methods.
We describe a new two dimensional electrophoresis technique which is based on the combination of cryostat section technology and IEF- and SDS-gel electrophoresis. The optimal conditions for two dimensional cryostat section electrophoresis are investigated. The application of this technique to synovial membranes of patients suffering from rheumatoid arthritis and osteoarthritis is described.
Arthritis, Rheumatoid↗
Improved synthesis of oligodeoxyribonucleotides.
An improved synthesis of oligodeoxyribonucleotides was achieved with NPE- and NPEOC-protected phosphoramidites leading to easier and time-saving purification and isolation of the gene fragments.
Oligodeoxyribonucleotides↗
Sulfur-containing 1,3-dialkylxanthine derivatives as selective antagonists at A1-adenosine receptors.
Sulfur-containing analogues of 8-substituted xanthines were prepared in an effort to increase selectivity or potency as antagonists at adenosine receptors. Either cyclopentyl or various aryl substituents were utilized at the 8-position, because of the association of these groups with high potency at A1-adenosine receptors. Sulfur was incorporated on the purine ring at positions 2 and/or 6, in the 8-position substituent in the form of 2- or 3-thienyl groups, or via thienyl groups separated from an 8-aryl substituent through an amide-containing chain. The feasibility of using the thienyl group as a prosthetic group for selective iodination via its Hg2+ derivative was explored. Receptor selectivity was determined in binding assays using membrane homogenates from rat cortex [( 3H]-N6-(phenylisopropyl)adenosine as radioligand] or striatum [3H]-5'-(N-ethylcarbamoyl)adenosine as radioligand] for A1- and A2-adenosine receptors, respectively. Generally, 2-thio-8-cycloalkylxanthines were at least as A1 selective as the corresponding oxygen analogue. 2-Thio-8-aryl derivatives tended to be more potent at A2 receptors than the oxygen analogue. 8-[4-[(Carboxy-methyl)oxyl] phenyl]-1,3-dipropyl-2-thioxanthine ethyl ester was greater than 740-fold A1 selective.
Animals↗
Phosphorothioate and cordycepin analogues of 2',5'-oligoadenylate: inhibition of human immunodeficiency virus type 1 reverse transcriptase and infection in vitro.
Natural antiviral activity can be mediated by the interferon-induced synthesis of 2',5'-oligoadenylates (2-5As) and subsequent RNase L activation by these molecules. Analogues of 2-5A that are biologically active and metabolically stable were synthesized and analyzed for antiviral activity against the human immunodeficiency virus type 1 (HIV-1). Replacement of the 3' hydroxyl group of the adenosine moieties of 2-5A with hydrogen atoms (i.e., cordycepin analogues of 2-5A) converted authentic 2-5A trimer into anti-HIV-1 agents in vitro. These cordycepin analogues of 2-5A also inhibited partially purified HIV-1 reverse transcriptase. Introduction of chirality into the 2',5'-phosphodiester internucleotide linkages or 5'-phosphate moieties of the 2-5A molecule (i.e., phosphorothioate analogues of 2-5A) converted authentic 2-5A into more potent inhibitors of HIV-1 reverse transcriptase. However, these phosphorothioate 2-5As demonstrated little or no anti-HIV-1 activity in vitro. Thus, some analogues of 2-5A may form a class of anti-HIV-1 drugs with possible pleiotropic activities that include activation of latent RNase L and inhibition of reverse transcription.
Adenine Nucleotides↗
8-Substituted xanthines as antagonists at A1- and A2-adenosine receptors.
Two classes of 8-substituted analogs of theophylline (1,3-dialkylxanthines), having 8-cycloalkyl, 8-cycloalkenyl or 8-(para-substituted aryl) groups, were shown to be potent and, in some cases, receptor subtype selective antagonists at A1- and A2-adenosine receptors. New analogs based on a functionalized cogener approach and on classical medicinal chemical approaches were prepared. Affinity at A1-adenosine receptors was evaluated by inhibition of binding of [3H]N6-phenylisopropyladenosine to rat brain membranes. Activity at A2-adenosine receptors was measured by the reversal of 5'-N-ethylcarboxamidoadenosine (NECA)-stimulated production of cyclic AMP in membranes from rat pheochromocytoma PC12 cells. Cycloalkenyl analogs containing rigid olefinic bonds differed greatly in potency from the saturated analogs. The selectivity of phenylsulfonamide analogs depended on distal structural features. Novel xanthine analogs include diamino-, thiol-, aldehyde, and halogen-substituted derivatives, peptide conjugates of 8-[4-[2-aminoethylaminocarbonylmethyloxy]phenyl]1,3-dipropylxan thi ne (XAC), and a hydroxyethylamide analog of XAC.
Adenosine↗
A route to 2',5'-oligoadenylates with increased stability towards phosphodiesterases.
The rates of enzymatic hydrolysis of 2',5'-oligoadenylates and their synthetic analogs have been measured. These compounds were treated with either NIH 3T3 cell lysates, mouse liver homogenates or snake venom phosphodiesterase. All analogs with 3'-terminal acyclic nucleoside residues demonstrated greater stability compared with the natural compound adenylyl(2'-5')adenylyl(2'-5')adenosine.
Adenine Nucleotides↗