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.     

Efficient Synthesis of 2′-Amino-2′-deoxypyrimidine 5′-Triphosphates

Abstract

The synthesis of 2′-amino-2′-deoxypyrimidine 5′-triphosphates is described. The 2′-amino-2′-deoxyuridine 5′-triphosphate is obtained from uridine in four steps with 25% overall yield. The 2′-amino-2′-deoxycytidine 5′-triphosphate is obtained from uridine in seven steps with 13% overall yield.     

Enzymatic Synthesis of Radioactive 5-Methyl-2′-Deoxycytidine 5′-Monophosphate by Using 32P-Postlabeling

Abstract

5-Methyl-2′-deoxycytidine 5′-[³²P]- and deoxycytidine 5-[32 P]-monophosphates were prepared from corresponding nucleotide homopolymers by using a ³² P-postlabeling procedure. The radioactive monophosphates obtained were well suited for biological and biochemical experiments.     

Incorporation of 5′-N-BOC-2′, 5′-Dideoxynucleoside-3′-O-Phosphoramidites Into Oligonucleotides by Automated Synthesis

Abstract

An efficient method for synthesizing 5′-Boc-5′-amino-2′, 5′- dideoxynucleoside phosphoramidites and conditions for their incorporation in solid-phase oligonucleotide synthesis are presented.     

Efficient Synthesis of 5-Carboxy-2′-Deoxypyrimidine Nucleoside 5′-Triphosphates

An efficient P(V)–N activation method for the synthesis of 5-carboxy-2′-deoxyuridine and 5-carboxy-2′-deoxycytidine triphosphates directly from the corresponding phosphoropiperidate precursors has been developed.     

Design and Synthesis of A3 Adenosine Receptor Ligands, 2′-Fluoro Analogues of Cl-IB-MECA

Abstract

Synthesis of 2′-deoxy-2′-fluoro-N ⁶-substituted adenosines as bioisosteres of Cl-IB-MECA and their binding affinities to A₃ adenosine receptor are described.     

Synthesis of 2-Chloro-2′-Deoxyadenosine by Microbiological Transglycosylation

Abstract

The title compound have been synthesized by an enzymatic trans-2′-deoxyribosylation of 2-chloroadenine using the whole cells of E. coli BMT-1D/1A as a biocatalyst and 2′-deoxyguanosine as a donor of glycosyl moiety.     

Synthesis of Guanosine-3′-diphosphate- 5′-diphosphate by Nucleotide Pyrophosphotransferase

An α-glucosidase was purified from sweet corn seeds by fractionation with ammonium sulfate, chromatographies on CM-Sepharose and Sepharose 4B, and gel filtrations on Sephadex G-100. The enzyme was homogeneous in disc electrophoretic analysis. The molecular weight was estimated to be about 9.6 × 10⁴ by SDS-disc electrophoresis.

The enzyme showed high activities toward maltose, nigerose, phenyl-α-maltoside, and maltooligosaccharides. The ratios of maximum velocity for maltose, nigerose, kojibiose, isomaltose, phenyl-α-glucoside, phenyl-α-maltoside, panose, turanose, and soluble starch were estimated to be 100 : 78 : 17 : 11 : 28 : 100 : 31 : 3.4 : 126, and the Km values for these substrates, 1.5 mM, 1.4 mM, 0.48 mM, 14 mM, 4.2 mM, 1.1 mM, 5.0 mM, 0.28 mM and 52mg/ml, respectively. The maximum velocity for soluble starch was high, but this α-glucan was not a favorable substrate because the Km value was also very high. The V_max for maltooligosaccharides were somewhat dependent on the degree of polymerization (n). The Km values for substrates having four or more glucose units increased with the increase in n.     

Facile Synthesis of Base-Labile 2′-Deoxyribonucleosides: An Improved Synthesis of 2′-Deoxy-5-aza-Cytidine

Abstract

The use of the Fmoc group for the protection of the hydroxy functions of the sugar moiety gave an improved overall yield of 2′-deoxy-5-azacytidine (6β), due to the mildly-basic conditions required for its removal from the protected nucleoside.     

Synthesis of 2′-O,4′-C-alkylene-bridged ribonucleosides and their evaluation as inhibitors of HCV NS5B polymerase

The synthesis of 2′-O,4′-C-methylene-bridged bicyclic guanine ribonucleosides bearing 2′-C-methyl or 5′-C-methyl modifications is described. Key to the successful installation of the methyl functionality in both cases was the use of a one-pot oxidation–Grignard procedure to avoid formation of the respective unreactive hydrates prior to alkylation. The 2′-C-methyl- and 5′-C-methyl-modified bicyclic guanosines were evaluated, along with the known uracil-, cytosine-, adenine-, guanine-LNA and guanine-ENA nucleosides, as potential antiviral agents and found to be inactive in the hepatitis C virus (HCV) cell-based replicon assay. Examination of the corresponding nucleoside triphosphates, however, against the purified HCV NS5B polymerase indicated that LNA-G and 2′-C-methyl-LNA-G are potent inhibitors of both 1b wild type and S282T mutant enzymes in vitro. Activity was further demonstrated for the LNA-G-triphosphate against HCV NS5B polymerase genotypes 1a, 2a, 3a and 4a. A phosphorylation by-pass prodrug strategy may be required to promote anti-HCV activity in the replicon assay.     

Synthesis of 2′-deoxythymidine by an enzymatic transdeoyrinosylation

Summary 2-Deoxythymidine was synthesized by an enzymatic transdeoxyribosylation of thymine using either (i) dGuo, dCyd or dAdo, or (ii) the mixture of the same 2-deoxynucleosides resulting from enzymatic hydrolysis of DNA as donors of 2-deoxyribofuranose moiety.     

Stabilization of RNA Bulges by Oligonucleotides Containing 2′-Naphthylmethyl-2′-deoxytubercidine

Abstract

A novel nucleoside analogue, 2′-naphthylmethyl-2′-deoxytubercidine, is synthesized and incorporated in oligonucleotides that stabilize bulges in partially complementary RNA.     

Synthesis,Cytostatic Activity and Inhibition of Ribonucleotide Reductase by 5′-Phosphoramidates and 5′-Diphosphates,of 2′-O-Allyl-arabinofuranosyl Nucleosides

Abstract

The diphosphates of a series of 2′-O-allyl-1-β-D-arabinofuranosyl derivatives, previously obtained by us, have been prepared and tested for their inhibitory activity in an in vitro assay using R1 and R2 subunits of the purified recombinant mouse ribonucleotide reductase (RNR). 2′-O-Allyl-araU diphosphate proved to be inhibitory, with an IC₅₀ of 100 μM. The 5′-phosphoramidate pronucleotide of 2′-O-allyl-araU was also prepared and tested for inhibition of tumor cell proliferation.     

Synthesis of 5′-C-Chain-Extended Uridines by Reaction of 5′-Halonucleosides with Malonic Acid Type Derivatives

Abstract

Reaction of 3-N-benzyl-5′-deoxy-5′-haloderivatives of uridine with the carbanions derived from diethyl malonate, ethyl acetoacetate, ethyl cyanoacetate and malondinitrile afforded the corresponding highly functionalized 5′-C-chain-extended uridines.     

Synthesis of 3′-Amino and 5′-Amino Hydantoin 2′-Deoxynucleosides

Abstract

3′-Amino and 5′-amino derivatives of hydantoin 2′-deoxynucleosides have been prepared from the corresponding 3′-phthalimido and 5′-azido nucleosides, respectively, which in turn were prepared by condensation of appropriate sugars with 5-benzylidenehydantoin. The amino nucleosides were tested for their potential activity against HIV and HSV.     

Synthesis of 2′-Deuterio and 3′-Deuterio Cytidine 5′-Diphosphate

Abstract

2′-²H- and 3′-²H-CDP were synthesized from 5′-MMT-3′-O-TBDMS and 2′,5′- O-diTBDMS cytidine derivatives, respectively, by oxidation followed by acidic removal of 5′-protection, reduction with [NaBD(OAc)₃] and finally displacement of a tosyl group by pyrophosphate.     

Synthesis and Evaluation of an RNA Editing Substrate Bearing 2′-Deoxy-2′-Mercaptoadenosine

The RNA-editing adenosine deaminases (ADARs) catalyze deamination of adenosine to inosine in double stranded structure found in various RNA substrates, including mRNAs. Here we describe the synthesis of a phosphoramidite of 2 ′-deoxy-2 ′-mercaptoadenosine and its incorporation into an ADAR substrate. Surprisingly, no deamination product was observed with this substrate indicating replacing the 2 ′-OH with a 2 ′-SH at the editing site is highly inhibitory. Modeling of nucleotide binding into the active site suggests the side chain of T375 of human ADAR2 to be in proximity of the 2 ′-substituent. Mutation of this residue to cysteine caused a greater that 100-fold reduction in deamination rate with the 2 ′-OH substrate.