Synthesis and Properties of Oligonucleotide Chimeras Containing 5′-Amino-2′-deoxy-2′-fluoroarabinonucleosides

Abstract

Oligonucleotide analogues comprised of 2′-deoxy-2′-fluoro-β-D-arabinose units joined via P3′-N5′ phosphoramidate linkages (2′F-ANA^5′N) were prepared for the first time. Among the compounds prepared were a series of 2′OMe-RNA-[GAP]-2′OMe-RNA ‘chimeras’, whereby the “GAP” consisted of DNA, DNA^5′N, 2′F-ANA or 2′F-ANA^5′N segments. The chimeras with the 2′F-ANA and DNA gaps exhibited the highest affinity towards a complementary RNA target, followed by the 5′-amino derivatives, i.e., 2′F-ANA > DNA > 2′F-ANA^5′N > DNA^5′N. Importantly, hybrids between these chimeras and target RNA were all substrates of both human RNase HII and E.coli RNase HI. In terms of efficiency of the chimera in recruiting the bacterial enzyme, the following order was observed: gap DNA > 2′F-ANA > 2′F-ANA^5′N > DNA^5′N. The corresponding relative rates observed with the human enzyme were: gap DNA > 2′F-ANA^5′N > 2′F-ANA > DNA^5′N.     

Synthesis and anti-HIV activity of 2′-deoxy-2′-fluoro-4′-C-ethynyl nucleoside analogs

Based on the favorable antiviral profiles of 4′-substituted nucleosides, novel 1-(2′-deoxy-2′-fluoro-4′-C-ethynyl-β-d-arabinofuranosyl)-uracil (1a), -thymine (1b), and -cytosine (2) analogs were synthesized. Compounds 1b and 2 exhibited potent anti-HIV-1 activity with IC₅₀ values of 86 and 1.34 nM, respectively, without significant cytotoxicity. Compound 2 was 35-fold more potent than AZT against wild-type virus, and also retained nanomolar antiviral activity against resistant strains, NL4-3 (K101E) and RTMDR. Thus, 2 merits further development as a novel NRTI drug.     

2′-Deoxy-1-methylpseudocytidine,a stable analog of 2′-deoxy-5-methylisocytidine

2′-Deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 2′-deoxy-5-methylisocytidine are expensive, because of its intrinsic chemical instability. We report here a C-glycoside analog that is more stable and, in oligonucleotides, pairs with 2′-deoxyisoguanosine, contributing to duplex stability about as much as a standard 2′-deoxycytidine and 2′-deoxyguanosine pair.     

Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

DNA polymerase α₁, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α₁ and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α₁ activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.     

A New Synthesis of 2′,3′-Didehydro-3′-deoxy-3-Alkylthymidine

Abstract

The preparation of 3-alkyl D4T derivatives has been carried out starting from the corresponding 5′-O-t-butyldimethylsilyl-3′-O-methanesulfonylthymidine 2 by way of deprotection-elimination and succesive alkylation reactions.     

Syn- and anti-conformations of 5′-deoxy- and 5′-O-methyl-uridine 2′,3′-cyclic monophosphate

Two uridine 2′,3′-cyclic monophosphate (cUMP) derivatives, 5′-deoxy (DcUMP) and 5′-O-methyl (McUMP), were studied by means of quantum chemical methods. Aqueous solvent effects were estimated based on the isodensity-surface polarized-continuum model (IPCM). Gas phase calculations revealed only slight energy differences between the syn- and anti-conformers of both compounds: the relative energies of the syn-structure are −0.9 and 0.2 kcal mol^-1 for DcUMP and McUMP, respectively. According to the results from the IPCM calculations, however, both syn-conformers become about 14 kcal mol^-1 more stable in aqueous solution than their corresponding anti-structures. Additionally, the effects of a countercation and protonation on DcUMP were studied, revealing that the syn-structure is also favored over the anti-one for these systems.     

Polynucleotides. LII1 Synthesis and properties of poly(2′-deoxy-2′-fluoroadenylic acid)

2′-Deoxy-2′-fluoroadenosine was chemically transformed to its 5′-diphosphate and polymerized with polynucleotide phosphorylase to give poly(2′-deoxy-2′-fluoroadenylic acid) [poly(Af)]. Polymerization proceeded smoothly as in the case of poly(A) and the yield of the polymerization was 55%. The UV absorption spectra of poly(Af) closely resembled those of poly(A) and the hypochromicity was 32% at pH 7.0. The CD profile at 25° and neutrality showed similar pattern to that of other poly(2′-deoxy-2′-halogenoadenylic acids) with somewhat larger [θ] values both in the positive and negative maxima. Acid titration of poly(Af) showed a transition point at pH 5.2 and the Tm of the acid form was 37° which was significantly lower than that of poly(A), but similar to that of poly(2′-azido-2′-deoxyadenylic acid). Poly(Af) formed 1:1 and 1:2 complexes with poly-(U) having Tm of 49° and 62° at 0.04M and 0.15M Na⁺ concentration, respectively. Poly(Af) also formed a 1:2 complex with poly(I) and its Tm was 36° at 0.05M Na⁺ concentration. These data showed that poly(Af) has rather similar properties to those of poly(A), but not to poly(dA).     

A homopolymer of the potent antitumor drug 2′-deoxy-5-fluorouridylate

Poly(5-fluoro-2′-deoxyuridylic acid) was synthesized and its properties were compared with those of poly(dT) and poly(dU). It readily complexed with poly(dA). The 1:1 complex melted at about 20°C lower than poly(dA) · poly(dT). A triple-stranded helix, poly(dA)·2 poly(dF⁵U) was formed only in high salt (2.0 M NaCl).     

Template-directed oligomerization of 5′-deoxy-5′-nucleosideacetic acid derivatives

5-Deoxy-5-nucleosideacetic acids II–V are isostructural analogues of nucleotides with a carboxylate group in the place of the 5-phosphate group. We have studied their oligomerization in aqueous solution using a water-soluble carbodiimide as the condensing agent in the presence or absence of an appropriate polynucleotide template. Condensation of adenylic acid analogues IIa, IIIa, and Va in the presence of polyuridylic acid were found to be the most efficient reactions. Cyclization of the activated monomers to lactones and the insolubility of the oligomers in aqueous solution were found to be obstacles to the efficient formation of long oligomers.     

Conversion of 1′-deoxy-2H-ABA to2H-ABA in cell-free extracts fromCercospora rosicola

Abscisic acid (ABA) biosynthesis in the fungusCercospora rosicola has been studied previously by feeding potential intermediates to growing cultures and examining the products. The present work describes the isolation and partial characterization of an enzyme system which catalyzes the last step in this pathway, the conversion of 1′-deoxy-²H-ABA to²H-ABA. Enzyme extracts were prepared from cold-pressed mycelia of actively growingC. rosicola in tricine buffer containing sucrose and β-mercaptoethanol. Low-speed supernatants and resuspended microsomal preparations were active in converting 1′-deoxy-²H-ABA to²H-ABA. Optimum conditions for the reactions were established at 10 μM substrate, 300 μl enzyme extract in a total volume of 1 ml. Reaction products were chromatographed by reverse-phase high-performance liquid chromatography (HPLC). The presumptive ABA fractions were collected and the²H-ABA was measured by combined gas chromatography-mass spectrometry (GC-MS) with an²H-ABA standard. Most of the enzyme activity was found in the microsomal fraction. Typical reaction rates were on the order of 1.5 pmol²H-ABA formed/min/mg protein. The reaction required NADPH and was enhanced by FAD. Triarimol, a substituted pyrimidine, which inhibits ABA synthesis inC. rosicola, had no effect on the conversion of 1′-deoxy-²H-ABA to²H-ABA.     

Discovery of β-d-2′-deoxy-2′-dichlorouridine nucleotide prodrugs as potent inhibitors of hepatitis C virus replication

Discovery of sofosbuvir has radically changed hepatitis C treatment and nucleoside/tide NS5B inhibitors are now viewed as one of the key components in combination therapies with other direct-acting antiviral agents. As part of our program to identify new nucleoside inhibitors of HCV replication, we now wish to report on the discovery of β-d-2′-deoxy-2′-dichlorouridine nucleotide prodrugs as potent inhibitors of HCV replication. Although, cytidine analogues have long been recognized to be metabolized to both cytidine and uridine triphosphates through the action of cytidine deaminase, uridine analogues are generally believed to produce exclusively uridine triphosphate. Detailed investigation of the intracellular metabolism of our newly discovered uridine prodrugs, as well as of sofosbuvir, has now revealed the formation of both uridine and cytidine triphosphates. This occurs, not only in vitro in cell lines, but also in vivo upon oral dosing to dogs.     

Synthesis and anti-HCV activity of β-d-2′-deoxy-2′-α-chloro-2′-β-fluoro and β-d-2′-deoxy-2′-α-bromo-2′-β-fluoro nucleosides and their phosphoramidate prodrugs

We report herein the synthesis and evaluation of a series of β-d-2′-deoxy-2′-α-chloro-2′-β-fluoro and β-d-2′-deoxy-2′-α-bromo-2′-β-fluoro nucleosides along with their corresponding phosphoramidate prodrugs. Key intermediates, lactols 11 and 12, were obtained by a diastereoselective fluorination of protected 2-deoxy-2-chloro/bromo-ribonolactones 7 and 8. All synthesized nucleosides and prodrugs were evaluated with a hepatitis C virus (HCV) subgenomic replicon system.     

Biocatalytic synthesis of base-modified 2′-deoxy-β-D-ribonucleosides with bacterial whole cells

Whole-cell syntheses of representative modified purine and pyrimidine 2-deoxy--D-ribonucleosides are described. The transglycosylation reactions were carried out at 55¡C using the thermostable bacteria, Bacillus stearothermophilus. These transformations were efficient and gave yields close to or greater than 50% (conversions >70%).     

Facile synthesis of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N 6-ethenoadenosine nucleosides

Facile synthetic methods of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N ⁶-ethenoadenosine nucleosides by either an enzymatic dideoxyribosyl transfer reaction or a simple chemical reaction were proposed. The synthetic products were isolated and purified by preparative HPLC and their structures were confirmed by¹H NMR (500 MHz) and FAB-MS including high resolution mass measurement. These modified nucleoside analogs have not been reported yet. Therefore, these modified nucleoside analogs are of potential value to be studied further for biological activity such as anticancer or antiviral.     

Synthesis and anti-HCV activity of a series of β-d-2′-deoxy-2′-dibromo nucleosides and their corresponding phosphoramidate prodrugs

Several β-d-2′-deoxy-2′-substituted nucleoside analogs have displayed potent and selective anti-HCV activities and some of them have reached human clinical trials. In that regard, we report herein the synthesis of a series of 2′-deoxy,2′-dibromo substituted U, C, G and A nucleosides 10a–d and their corresponding phosphoramidate prodrugs 13a–d. The synthesized nucleosides 10a–d and prodrugs 13a–d were evaluated for their inhibitory activity against HCV as well as cellular toxicity. The results showed that the most potent compound was prodrug 13a, which exhibited micromolar inhibitory activity (EC₅₀?=?1.5?±?0.8?µM) with no observed toxicity. In addition, molecular modeling and free energy perturbation calculations for the 5′-triphosphate formed from 13a and related 2′-modified nucleotides are discussed.     

Selective targeting of 2′-deoxy-5-fluorouridine to urokinase positive malignant cells in vitro

A urokinase targeting conjugate of 2′-deoxy-5-fluorouridine (5-FUdr) was synthesized and tested for tumor-cell selective cytotoxicity in vitro. The 5-FUdr prodrug 2′-deoxy-5-fluoro-3′-O-(3-carboxypropanoyl)uridine (5-FUdrsuccOH) containing an ester-labile succinate linker was attached to the specific urokinase inhibitor plasminogen activator inhibitor type II (PAI-2) and was found to preferentially kill urokinase-over expressing cancer cells. Up to 7 molecules of 5-FUdr were incorporated per PAI-2 molecule without affecting protein activity. This is the first time a small organic cytotoxin has been conjugated to PAI-2.     

Cobalt-requiring 5′-deoxy-5′-methylthioadenosine nucleosidase from soybean (Glycine max) cotyledon

5??-Deoxy-5??-methylthioadenosine nucleosidase (MTA nucleosidase, EC 3.2.2.9) was purified from soybean (Glycine max) cotyledon. The nucleosidase was a trimer consisting of three identical subunits with a molecular mass of 59.5?kDa. The nucleosidase was a cobalt-requiring enzyme for its catalytic function. The enzymatic activity increased in a dose-dependent manner in the presence of cobalt. Cobalt was bound to the nucleosidase with a stoichiometry of 1 equivalent of cobalt/subunit. A thiol group-specific reagent reduced the enzymatic activity. Four cysteinyl residues of each subunit are considered to play an important role in binding cobalt.