Synthesis and RNase L Binding and Activation of a 2-5A-(5′)-DNA-(3′)-PNA Chimera,a Novel Potential Antisense Molecule

Abstract

Fully automated solid-phase synthesis gave access to a hybrid in which 5′-phosphorylated-2′-5′-linked oligoadenylate (2–5A) is connected to the 5′-terminus of DNA which, in turn, is linked at the 3′-end to PNA [2–5A-(5′)-DNA-(3′)-PNA chimera]. This novel antisense molecule retains full RNase L activation potency while suffering only a slight reduction in binding affinity.     

Synthesis of P-Thioadenylyl (2′-5′) Adenosine and P-Thioadenylyl (2′-5′)-P-Thioadenylyl-(2′-5′)-Adenosine

Abstract

Dimer and trimer adenylates with 2′-5′ phosphorothioate linkages were synthesized via the phosphoramidite method using p-nitrophenyl-ethyl group for phosphate protection and followed by sulfur oxidation. The various diastereoisomers were separated and characterized.     

5′-O-Dephosphorylated 2′,5′-oligoadenylate (2-5A) with 8-methyladenosine at the 2′-terminus activates human RNase L

Human ribonuclease L (RNase L), an interferon-induced endoribonuclease, becomes enzymatically active after binding to 2-5A. The 5′-phosphoryl group of 2-5A is reportedly necessary for the conformational change leading to RNase L activation. However, we found that 5′-O-dephosphorylated 2-5A tetramer analogs with 8-methyladenosine at the 2′-terminus were more effective as an activator of RNase L than the parent 2-5A tetramer. Introduction of 8-methyladenosine is thought to induce a dramatic shift of 2-5A in the binding site of RNase L.     

Stereospecific Synthesis and Anti-HIV Activity of (Z)2′- and (E)3′-Deoxy-2′(3′)-C-(chloromethylene) Pyrimidine Nucleosides

Abstract

Reaction of 1-[2,5(and 3,5)-di-O-trityl-β-D-erythro-pentofuran-3 (and 2)-ulosyl]uracil derivatives 5 and 6 with (chloromethyl)triphenylphosphorane resulted in the stereoselective formation of (E)-3′- and (Z)-2′-chloromethylene derivatives 7 (69%) and 8 (53%), respectively, deprotection of which gave 9 and 10. Transformation of the uracil nucleoside 7 into cytosine one followed by deprotection yielded 12. The latter was converted into the arabinoside 14. The fully deprotected chloromethylene nucleosides were tested for their activity against HIV-1 and HIV-2.     

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 Cytidylyl(3′→5′)cytidylyl(3′→5′)-(2′)3′-O-[Lα-alanyl]adenosine

Abstract

A new synthesis of protected C-C-A-[L^α-Ala] 14 is reported using a new set of complementary groups such as 2-phenylsulfonylethoxycarbonyl (PSEC) for the protection of exocyclic amino functions, o-chlorophenyl (o-CIPh) for the internucleotide phosphotriester, 3-methoxy-1,5-dicarbomethoxypentan-3-yl (MDMP) and the 4-monomethoxytrityl (MMTr) for the protection of the ã-amino fuction of the amino acid. 14 could be deprotected in two steps by treatments with 1,1,3,3-tetramethylguanidinium oximate under a dry condition and then by neat trifluoroacetic acid. Treatment with neat trifluoroacetic acid produced a stable salt: [C-C-A-Ala-N^ãH₃+ CF₃CO₂-] and did not promote any internucleotide phosphate migration or degradation of the oligomeric molecule. This salt was considerably more stable than C-C-A-Ala conjugate with a free ã-amino group, and, therefore, it could be easily purified on a silica gel column and was isolated in 82 % yield. This strategy should be useful for the synthesis of longer oligonucleotide-aminoacyl conjugate.     

3′-3′,3′-5′ and 5′-5′ TpT-Amides: Synthesis,Characterization and Alkaline Hydrolysis

Abstract

A simple procedure is described for the preparation of the title compounds 1, 8 and 9. 3′-3′ or 3′-5′ or 5′-5′ TpT was reacted with a twofold molar excess of TPS in anhydrous DMF, at room temperature, for 5 min, followed by a 1 min in situ treatment of the reaction mixture with excess 7.0 N NH₄OH, at 0°C. The alkaline hydrolysis of 1, 8 and 9 proceeds without the assistance of 3′- and 5′-hydroxyl groups resulting in equimolar mixtures of thymidine (4) and thymidine 3′-phosphoramidate (6) (for the 3′-3′ isomer) or thymidine 5′-phosphoramidate (7) (for the 5′-5′ isomer) or 6 and 7 in equal quantities (for the 3′-5′ isomer).     

Synthesis of the 2′-,3′-Didehydro-2′-,3′-dideoxy and 2′-,3′-Dideoxy Derivatives of 6-Azauridine and a New Route to 2′-,3′-Didehydro-2′-,3′-dideoxy-5-chlorouridine

Abstract

The 5′-O-(4,4′-dimethoxytrityl) and 5′-O-(tert-butyldimethylsilyl) derivatives of 2′-,3′-O-thiocarbonyl-6-azauridine and 2′,3′-O-thiocarbonyl-5-chlorouridine were synthesized from the parent nucleosides by reaction with 4, 4′-dimethoxytrityl chloride and tert-butyldimethylsilyl chloride, respectively, followed by treatment with 1,1′-thiocarbonyldiimidazole. Introduction of a 2′-,3′-double bond into the sugar ring by reaction of the 5′-protected 2′-,3′-O-thionocarbonates with 1, 3-dimethyl-2-phenyl-1, 3, 2-diazaphospholidiine was unsuccessful, but could be accomplished satisfactorily with trimethyl phosphite. Reactions were generally more successful with the 5′-silylated than with the 5′-tritylated nucleosides. Formation of 2′-,3′-O-thiocarbonyl derivatives proceeded in higher yield with 5′-protected 6-azauridines than with the corresponding 5-chlorouridines because of the propensity of the latter to form 2,2′-anhydro derivatives. In the reaction of 5′-O-(tert-butyldimethylsilyl)-2′-,3′-O-thiocarbonyl-6-azauridine with trimethyl phosphite, introduction of the double bond was accompanied by N³-methylation. However this side reaction was not a problem with 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-O-thioarbonyl-5-chlorouridine. Treatment of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-6-azauridine with tetrabutylammonium fluoride followed by hydrogenation afforded 2′-,3′-dideoxy-6-azauridine. Deprotection of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-5-chlorouridine yielded 2′-,3′-didehydro-2′-,3′-dide-oxy-5-chlorouridine.     

A Direct and Efficient Synthesis of 5′-Deoxy-2′, 3′-

Abstract

A direct and efficient synthesis of 5′-deoxy-2′,3′-O-isopropylideneinosine, 7, from readily available inosine is described. An example of a potentially general synthesis of N -substituted-5′-deoxyadenosines from 7 is also described.     

A Convergent Regiospecific Synthesis of the Lariat-Trinucleotides and A2′p 5′G and A2′p 5′G 3′p 5′C from A O4-2-Nitrophenyl) -Uridine Building Block

Abstract

Total synthesis of title compounds 1_ and 2_ from a common intermediate 7 is reported using the phosphotriester-phosphiteamidite approach. Appropriate NMR evidence has been presented in support of the regiospecific synthesis of target molecules in addition to enzymatic analysis. Present work clearly shows that the NMR evidence is mandatory to establish the isomeric purity of branched RNA molecules; enzymatic or/and electrophoretic analysis alone as tools for confirmation of branched RNA structures can be misleading.     

Synthesis and Properties of 2′4′- and 3′-5′-Linked Ribodinucleoside Monophosphates Containing 2-Aminoadenosine and Uridine

Abstract

Self complementary diribonucleoside monophosphates containing 2-aminoadenosine (n²A) and uridine (U) residues, (2′-5′) n²ApU (1), (3′-5′) n²ApU (2), (2′-5′) Upn²A (3) and (3′-5′) Upn²A (4), were synthesized by condensation of suitably protected nucleoside and nucleotide units using dicyclohexylcarbodiimide (DCC). The dimers, (3) and (41, were also obtained from uridine 2′,3′-cyclic phosphate and unprotected 2-aminoadenosine using 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl) as the condensing agent. The conformational properties of these dimers were examined by UV, CD and NMR spectroscopy. The results reveal that the 2′-5′ isomers take a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′ isomers. The n²ApU isomers have more stacked structure than the Upn²A isomers.     

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract

The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g⁺ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.     

Synthesis,Solution Conformation and Biological properties of 2′,3′-Dideoxy-3′-fluoro-D-erythro-pentofuranosides of 2-Thiouracil and 2-Thiothymine

Abstract

The synthesis of the α- and β-anomers of 2′,3′-dideoxy-3′-fluoro-2-thiouridine and 2′,3′-dideoxy-3′-fluoro-2-thiothymidine via Lewis acid catalysed nucleoside condensation is described. High resolution ¹H NMR data, solution conformations and biological properties are also presented.     

Synthesis,Molecular Conformation and Activity Against Herpes Simplex Virus of (E)-5-(2-Bromovinyl)-2′-Deoxycytidine Analogs

Analogs of (E)-5-(2-bromovinyl)-2 ′-deoxycytidine (BrVdCyd) (1) by substitution at N⁴ were synthesized to impart resistance against deamination. The anti-HSV-1 activity and solution conformation of these analogs were determined. N⁴-Acetyl-BrVdCyd (2) was a potent inhibitor of HSV-1 replication whereas N⁴-propanoyl-BrVdCyd (3) had good activity and N⁴-Butanoyl-BrVdCyd (4) had only low activity against HSV-1 replication. N⁴-Methyl-BrVdCyd (5) was devoid of activity against HSV-1.     

Solid-phase synthesis of 5′-triphosphate 2′-5′-oligoadenylates analogs with 3′-O-biolabile groups and their evaluation as RNase L activators and antiviral drugs

5′-Triphosphate 2′-5′-oligoadenylate (2–5A) is the central player in the 2–5A system that is an innate immunity pathway in response to the presence of infectious agents. Intracellular endoribonuclease RNase L activated by 2–5A cleaves viral and cellular RNA resulting in apoptosis. The major limitations of 2–5A for therapeutic applications is the short biological half-life and poor cellular uptake. Modification of 2–5A with biolabile and lipophilic groups that facilitate its uptake, increase its in vivo stability and release the parent 2–5A drug in an intact form offer an alternative approach to therapeutic use of 2–5A. Here we have synthesized the trimeric and tetrameric 2–5A species bearing hydrophobic and enzymolabile pivaloyloxymethyl groups at 3′-positions and a triphosphate at the 5′-end. Both analogs were able to activate RNase L and the production of the trimer 2–5A (the most active) was scaled up to the milligram scale for antiviral evaluation in cells infected by influenza virus or respiratory syncytial virus. The trimer analog demonstrated some significant antiviral activity.     

A Convenient Synthesis of N4,O3′, O5′-Triacetyl-2′-Ketocytidine

Abstract

A convenient synthesis of the title compound in four steps from cytidine is reported. Key transformations include differentiation of the 2′ position as N⁴,O^3′,O^5′-triacetyl-2,2′-anhydrocytidine, opening to the arabino derivative, and oxidation of the 2′ position with the Dess-Martin reagent.