Synthesis and incorporation of pyrrole carboxamide nucleoside triphosphates by DNA polymerases

We have synthesised and examined the enzymatic incorporation properties of the 5'-triphosphates of 2'-deoxyribosyl pyrrole 3-monocarboxamide (dMTP) and 2'-deoxyribosyl pyrrole 3,4-dicarboxamide (dDTP). These analogues we had hoped would behave as ambivalent base analogues in that they can present two alternative hydrogen-bonding faces either by rotation about the carboxamide group or about the glycosidic bond. The two pyrrole derivatives, dMTP and dDTP, exhibit a preference for incorporation with Klenow polymerase. They are preferentially incorporated as either A or C.     

Synthesis of Analogues of Ribosylbarbituric Acid

Abstract

Syntheses of 5-alkyl (5 b, c), 2′-deoxyribosyl (9, 10) and arabinofuranosyl (13) analogues of ribosylbarbituric acid (5a) are described. The compounds were prepared by condensation of persilylated barbituric acids with pentofuranosyl moieties. The 2′-tolylthio analogue (16) was obtained by an alternative procedure involving ring-opening of 6,2′-O-cyclouridine (15).     

Oligonucleotide Labeling Methods 4. Direct Labeling Reagents with a Novel,Non-Nucleosidic,Chirally Defined 2-Deoxy-β-D-Ribosyl Backbone.

Abstract

Novel solid supports and CE-phosphoramidite reagents have been prepared featuring a unique 2′-deoxyribosyl backbone. These chirally pure reagents form the basis of an oligonucleotide labeling system which provides diastereomerically pure oligonucleotides.     

Acetylation of Nucleosides by Acetylsalicylic Acid (Aspirin)1

Abstract

Acetylsalicylic acid (aspirin) reacted with adenosine, cytidine, guanosine and their 2′-deoxynucleosides to give acetylated nucleosides. Cytidine and 2′-deoxycytidine gave N⁴-acetylated nucleosides in nitromethane while in pyridine fully acetylated products were obtained. Adenosine and 2′-deoxyadenosine also gave fully acetylated products. However, guanosine and 2′-deoxyguanosine gave 2′,3′,5′-tri-O-acetylribosyl and 3′,5′-di-O-acetyl-2′-deoxyribosyl nucleosides, respectively. The corresponding aglycons also gave acetylated heterocycles under various conditions.     

FORMATION OF 2′-DEOXY-2-NITROADENOSINES BY REACTION OF 2′-DEOXYADENOSINES WITH COPPER(II) NITRATE/ACETIC ANHYDRIDE

ABSTRACT

Nitration of 9-substituted [ethyl, (Ac)₂-2′-deoxyribosyl, (Ac)₃-ribosyl] N ⁶-acetyladenine derivatives with Cu(NO₃)₂·3H₂O/Ac₂O was examined. Nitration proceeded at the 2-position, although the yield was low. Removal of the acetyl groups gave 2′-deoxy-2-nitroadenosine derivatives.     

Mechanism-Based Inhibition of Ribonucleotide Reductases: New Mechanistic Considerations and Promising Biological Applications

Abstract

Ribonucleotide reductases (RNRs) perform the de novo biosynthesis of 2′-deoxynucleoside 5′-(di or tri)phosphates. Inhibition of RNRs removes a crucial source of genetic components and enhances the probability of salvage incorporation of analogues into DNA. Several laboratories have clarified aspects of the reaction cascades initiated by generation of substrate nucleotide C3′ free radicals by RNRs. New considerations for radical-mediated mechanism-based inhibition and biological applications are discussed.     

Conformation Analysis of Two Anti-HIV Nucleoside Analogues 2′,3′-Dideoxy-3′-fluorocytidine and Its N4-Dimethylaminomethylene Prodrug Derivative

Abstract

Crystal structure analysis of 2′,3′-dideoxy-3′-fluorocytidine (1) and its prodrug derivative, N⁴-dimethylaminomethylene-2′,3′-dideoxy-3′-fluorocytidine (2), active anti-HIV nucleoside analogues, reveals that both structures adopt an anti conformation about the glycosyl bond. The furanose ring is C2′-endo for (2) and C2′-endo/C1′-exo and C2′-endo/C3′-exo for the two independent molecules of (1), respectively.     

Preparation and Properties of a New Type of Acyclic,Achiral Nucleoside Analogue

Abstract

Preparation of the nucleoside analogues 1 and incorporation of 1, B = T, in deoxyribooligonucleotides by the phosphoramidite method is described. A two-step deprotection procedure was developed to reduce cleavage of the modified allylic unit. The binding properties of the modified oligonucleotides towards complementary DNA and RNA has been evaluated by T_m measurements showing a ΔT_m of ?2 to ?6.5°C per modification. An oligonucleotide with two modifications at the 3′-end showed considerable resistance towards cleavage by a 3′-exonuclease. No antiviral activity against HIV-1 or HSV-1 was found for 1, B = G or T, or for any of the trihydroxy derivatives 5.     

Synthesis and Evaluation of a Series of 2′-Deoxy Analogues of The Antiviral Agent 5,6-Dichloro-2-Isopropylamino-1-(β-L-Ribofuranosyl)-1H-Benzimidazole (1263W94)

Abstract

A series of 2′-deoxy analogues of the antiviral agent 5,6-dichloro-2-isopropylamino-1-(β-L-ribofuranosyl)-1H-benzimidazole (1263W94) were synthesized and evaluated for activity against human cytomegalovirus (HCMV) and for cytotoxicity. The 2-substituents in the benzimidazole moiety correspond to those that were used in the 1263W94 series. In general, as was found in the 1263W94 series, cyclic and branched alkylamino groups were needed for potent activity against HCMV. Three analogues 3a, 3b and 3d were as potent as 1263W94. Further evaluation of two analogues, 3a and 3b, suggested that these 2′-deoxy analogues may act via a novel mechanism of action similar to that of 1263W94. These 2′-deoxy analogues generally lacked cytotoxicity in vitro. Pharmacokinetic parameters in mice and protein binding properties of 3a were quite similar to 1263W94. However, the oral bioavailability of 3a was only half of that observed for 1263W94.     

Synthesis of Some 2′,3′-Dideoxy-2′-C-Methyl-Substituted Nucleosides

Abstract

Nucleoside analogues analogues1-(2′,3′-dideoxy-2′-C-hydroxymethyl-β-D-erythro-pentofuranos-yl)thymine (1), 2′,3′-dideoxy-2′-C-hydroxymethylcytidine (2), 2′,3′-dideoxy-2′-C-hydroxymethyladenosine (3), 1-(2′-C-azidomethyl-2′,3′-dideoxy-β-D-erythro-pento-furanosyl)thymine (4), 2′-C-azidomethyl-2′,3′-dideoxycytidine (5), and 2′3′-dideoxy-2′-C-methylcytidine (6) have been synthesized from (S)-4-hydroxymethyl-y-butyro-lactone (7)     

An Improved Synthesis of 2′-Deoxy-9-deazaadenosine and an N-7 Blocked Derivative Useful for the Synthesis of Modified Oligonucleotides Containing 2′-Deoxy-9-deazaadenosine

Abstract

As an epimerization resistant synthon in the synthesis of oligo-nucleotides consisting of C-nucleoside analogues, hitherto unknown 5-benzyloxy-methyl-3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrrolo[3,2-d]pyrimpyrimidine (7-benzyloxymethyl-2′-deoxy-9-deazaadenosine) was prepared in seven steps from the known 3-amino-2-cyano-4-(2,3-O-isopropylidene-5-O-trityl-β-D-ribofuranosyl)-pyrrolpyrrole (1). Treatment of 1 with benzyl chloromethyl ether in the presence of potassium t-butoxide and 18-crown-6 afforded the N-protected pyrrole 2, which was converted into the 9-deazapurine derivative 3 in high yield by heating in EtOH. 7-Benzyloxymethyl-9-deazaadenosine 4 was obtained from 3 by acid hydrolysis in 2.5% methanolic hydrogen chloride. After protection of the hydroxyl groups of 4 with Markievicz's reagent, the product 5 was converted into the 2′-O-phenoxythiocarbonyl derivative 6. Reduction of 6 with butyltin hydride in the presence of 2,2′-azobis(2-methylpropionitrile), followed by desilylation with triethylammonium fluoride, afforded the desired 7-benzyloxymethyl-2′-deoxy-9-deazaadenosine (8) in high overall yield. The benzyloxymethyl group of 8 was removed by hydrogenolysis over palladium hydroxide (Degussa type) to give 2′-deoxy-9-deazaadenosine (9) in quantitative yield. The structure of 9 is discussed.     

The Synthesis and Preliminary Biological Evaluations of Selected Analogues of the Potent Broad-Spectrum Antiviral Agent Ganciclovir 1′,3′-Cyclic Monophosphate1 (2′-Nor-cGMP)

Abstract

The syntheses and antiviral activity of a series of analogues of the potent antiherpetic 2′-nor-cGMP are described. These derivatives contain systematic changes in either the heterocycle, the acyclic moiety, or the phosphate ring.     

Synthesis and Antiviral Activity of C-5 Substituted Analogues of D4T Bearing Methylamino- or Methyldiamino-Linker Arms

Abstract

A general strategy is reported for the preparation of C-5-methylamino- or methyldiamino-d4T analogues of “different sizes”. Reactions of the 2′,3′-didehydro-2′,3′-dideoxy-C-5 hydroxymethyl precursor (7) with either polymethylene diamines (n = 6, 8, 10 and 12) or propargylamine proceed regioselectively via subtitution reactions at the C-5 position of uracil. The compounds were evaluated for antiviral activity and cytotoxicity. No significant activity was observed for compounds 9, 11, and 13, but 10 and 12 exhibited a weak activity against HIV-1.     

Chemistry and Anti-HIV Activity of 2′-β-Fluoro-2′,3′-dideoxyguanosine

Abstract

The 2′-β-fluoro analogue of 2′,3′-dideoxyguanosine has been prepared by two synthetic routes. This compound and two analogues have anti-HIV activity in at least two of three host cell systems used (ATH8, CEM, PBL). These compounds, as well as their ddGuo parents, have been characterized with regard to their acid-stabilities, octanol-water partition coefficients, and enzyme substrate properties for adenosine deaminase and purine nucleoside phosphorylase. F-ddGuo analogues are less potent but more stable than their non-fluorinated parent compounds.     

2-Bromoadenosine-Substituted 2′,5′-Oligoadenylates Modulate Binding and Activation Abilities of Human Recombinant RNase L

Abstract

2-Bromoadenosine-substituted analogues of 2–5A, p5′A2′p-5′A2′p5′(br²A), p5′(br²A)2′p5′A2′p5′A, and p5′(br²A)2′p5′(br²A)2′p-S′(br²A), were prepared via a modification of a lead ion-catalyzed ligation reaction and were subsequently converted into the corresponding 5′-triphosphates. Both binding and activation of human recombinant RNase L by various 2-bromoadenosine-substituted 2–5A analogues were examined. Among the 2-bromoadenosine-substituted 2–5A analogues, the analogue with 2-bromoadenosine residing in the 2′-terminal position, p5′A2′p5′A2′p-5′(br²A), showed the strongest binding affinity and was as effective as 2–5A itself as an activator of RNase L. The CD spectrum of p5′A2′p-5′A2′p5′(br²A) was superimposable on that of p5′A2′p5′A2′p5′A, indicative of an anti orientation about the base-glycoside bonds as in naturally occurring 2–5A.     

Synthesis of Novel 2′-Fluoro-3′-Hydroxymethyl-5′-Deoxythreosyl Phosphonic Acid Nucleoside Analogues As Antiviral Agents

A series of purine 5′-deoxyphosphonate analogues were designed and synthesized to mimic naturally occurring purine monophosphate from 1,3-dihydroxyacetone as starting material. The discovery of threosyl phosphonate nucleoside (PMDTA, EC₅₀ = 2.53 μM) as a potent anti-HIV agent has led to the synthesis and biological evaluation of 2′,3′-modified 5′-deoxyversions of the threosyl phosphonate nucleosides. The synthesized 2′-fluoro-3′-hydroxymethyl 5′-deoxythreosyl phosphonic acid nucleoside analogues 14, 18, 23, and 27 were tested for anti-HIV activity as well as cytotoxicity. The adenine analogue 18 exhibits weak in vitro anti-HIV-1 activity (EC₅₀ = 19.2 μM).     

Light-induced formation of NO in endothelial cells by photoactivatable NADPH analogues targeting nitric-oxide synthase

BackgroundNitric-oxide synthases (NOS) catalyze the formation of NO using NADPH as electron donor. We have recently designed and synthesized a new series of two-photon absorbing and photoactivatable NADPH analogues (NT). These compounds bear one or two carboxymethyl group(s) on the 2′- or/and 3′-position(s) of the ribose in the adenosine moiety, instead of a 2′-phosphate group, and differ by the nature of the electron donor in their photoactivatable chromophore (replacing the nicotinamide moiety). Here, we addressed the ability of NTs to photoinduce eNOS-dependent NO production in endothelial cells.MethodsThe cellular fate of NTs and their photoinduced effects were studied using multiphoton fluorescence imaging, cell viability assays and a BODIPY-derived NO probe for NO measurements. The eNOS dependence of photoinduced NO production was addressed using two NOS inhibitors (NS1 and L-NAME) targeting the reductase and the oxygenase domains, respectively.ResultsWe found that, two compounds, those bearing a single carboxymethyl group on the 3′-position of the ribose, colocalize with the Golgi apparatus (the main intracellular location of eNOS) and display high intracellular two-photon brightness. Furthermore, a eNOS-dependent photooxidation was observed for these two compounds only, which is accompanied by a substantial intracellular NO production accounting for specific photocytotoxic effects.ConclusionsWe show for the first time that NT photoactivation efficiently triggers electron flow at the eNOS level and increases the basal production of NO by endothelial cells.General significanceEfficient photoactivatable NADPH analogues targeting NOS could have important implications for generating apoptosis in tumor cells or modulating NO-dependent physiological processes.     

A Solid-Phase Synthesis of 2′,5′-Linked Oligoadenylates (2-5A)

Abstract

2′,5′-Oligoadenylate 5′-triphosphates (2-5A) as products of 2-5A synthetase and activators of ribonuclease L (RNase L), are mediators in one of the mechanisms of interferon′s antiviral action. Upon activation, RNase L inhibits protein synthesis due to the degradation of RNAs. This activity of 2-5A could possibly find an application in virus or cancer chemotherapy, but two major barriers prevent the use of 2′,5′-linked oligoadenylates as therapeutic agents. The 2-5A is readily degraded by a 2′,5′ phosphodiesterase and as a highly negatively charged molecule, is not readily taken up by cells. One possible solution to this latter limitation might be found in chemical modifications of the 2-5A structure. Many analogues of 2-5A have been already obtained with modified base, ribose or phosphate moieties. While these have provided some important information about the enzyme- activator interactions, the cell permeability problem still remains unsolved. One of the major obstacles in this study is lack of a convenient method of synthesis of 2′,5′ ribonucleotides of widely varying structure.     

Synthesis and Anti-HIV Activity of Novel 2′-Deoxy-2′-β-Fluoro-Threosyl Nucleoside Phosphonic Acid Analogues

Novel 2′-deoxy-2′-β-fluoro-threose purine phosphonic acid analogues were designed and racemically synthesized from 2-propanone-1,3-diacetate. Condensation successfully proceeded from a glycosyl donor 9 under Vorbrüggen conditions. Cross-metathesis of vinyl analogues 13 and 23 with diethyl vinylphosphonate yielded the desired nucleoside phosphonate analogues 14 and 24, respectively. Ammonolysis and hydrolysis of phosphonates yielded the nucleoside phosphonic acid analogues 16, 19, 26, and 29. The synthesized nucleoside analogues were subjected to antiviral screening against human immunodeficiency virus (HIV)-1. Adenine analogue 18 exhibited weak in vitro activities against human immunodeficiency virus (HIV)-1.     

Phosphate Modified Analogues of 5′-O-Phosphorylated 2′,3′-Dideoxynucleosides: Synthesis and Anti-HIV Activity

Abstract

Phosphonate analogues of 5′-O-phosphoryl-2′,3′-dideoxyribofuranosyl adenine, cytosine, hypoxanthine and thymine were synthesized. The hypoxanthine and thymine analogues were inactive against HIV induced cyopathy in the CEM-4 T-cell lines. The 2′,3′-ddC and 2′,3′-ddA analogues were marginally active.