SYNTHESIS AND PROPERTIES OF A NOVEL BRIDGED NUCLEIC ACID ANALOGUE, 5′-AMINO-3′,5′-BNA

An oligonucleotide P3′?N5′ phosphoramidate (5′-amino-DNA) attracts much attention because of its potential for application to DNA sequencing; however, its ability to hybridize with complementary strands is low. To overcome this drawback of the 5′-amino-DNA, we have designed and successfully synthesized a novel nucleic acid analogue having a P3′?N5′ phosphoramidate linkage and a constrained sugar moiety, 5′-amino-3′-C,5′-N-methylene bridged nucleic acid (5′-amino-3′,5′-BNA). The binding affinity of the 5′-amino-3′,5′-BNA towards complementary DNA and RNA strands was investigated by UV melting experiments. The melting temperature (Tm) of the duplex comprising the 5′-amino-3′,5′-BNA and its complementary strand was much higher than that of the duplex containing the corresponding 5′-amino-DNA.     

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).     

3′-AZIDO-3′-DEOXY-5′-O-ISONICOTINOYLTHYMIDINE,A NEW PRODRUG OF ZIDOVUDINE. SYNTHESIS,SOLID STATE CHARACTERIZATION AND ANTI HIV-1 ACTIVITY

ABSTRACT

Synthesis, solid state characterization and anti HIV-1 activity of 3′-azido-3′-deoxy-5′-O-isonicotinoylthymidine (2), a new prodrug of zidovudine (AZT, 1), are described. Two solid forms of 2 prepared by crystallization from ethyl acetate-petroleum ether (form α) and from a melt sample of form α (amorphous form) were characterized by X-ray diffractometry, infrared spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA) techniques. The novel nucleoside exhibited antiviral activity against standard and resistant strain panels of HIV-1 as well as cytotoxicity similar to that of AZT.     

MECHANISM OF INACTIVATION OF S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE BY 5′-DEOXY-5′-S-ALLENYLTHIOADENOSINE AND 5′-DEOXY-5′-S-PROPYNYLTHIOADENOSINE

5′-Deoxy-5′-S-allenylthioadenosine 1 and 5′-deoxy-5′-S-propnylthioadenosine 2, derived from adenosine, were prepared. 1 and 2 caused irreversible inactivation of AdoHcy hydrolase. ESI mass spectra analysis of the inactivated enzyme demonstrated that 1 and 2 were type II “mechanism-based” inhibitors.     

SYNTHESIS OF 3′-THIOAMIDO-MODIFIED 3′-DEOXYTHYMIDINE-5′-TRIPHOSPHATES AND THEIR USE AS CHAIN TERMINATORS IN SANGER-DNA SEQUENCING

The thioamide derivatives 3′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-[(2-methyl-1-thioxo-propyl)amino]thymidine 1 and 3′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-{{6-{[(9H-(fluo-ren-9-ylmethoxy)carbonyl]-amino}-1-thioxohexyl}amino} thymidine 2 were synthesized by regioselective thionation of their corresponding amides 7 and 8 with 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphet-ane-2,4-disulfide (Lawesson's reagent). The thioamides were converted into the corresponding 5′-triphosphates 3 and 4. Compound 3 was chosen for DNA sequencing experiments and 4 was further labelled with fluorescein.     

INACTIVATION OF S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE WITH FLUORINATED ANALOGS OF 2′- AND 3′-DEOXY-5′-METHYLTHIOADENOSINE

Fluorinated analogs of 2′- and 3′-deoxy-5′-methylthioadenosine 1–4 caused irreversible inactivation of AdoHcy hydrolase. Based on the ESI-Mass spectra analysis of the inactivated enzyme with the fluorinated analog 1 a mechanism of inactivation is proposed.     

Synthesis of 3′-Amino-3′-deoxyguanosine 5′-Triphosphate

Abstract

Phosphorylation of 2′-0-acetyl-3′-trifluoroacetamido-3′-deoxy-N²-palmitoylguanosine with N-morpholino-O, O-bis(1-benzotriazolyl)phos-phate gives a 5′-phosphotriester. Removal of the benzotriazolyl group and addition of pyrophosphoric acid gave, after deblocking all protecting groups, GTP(3′NH₂).     

HYDROLYSIS OF 2′-DEOXY AND 2′-FLUORONUCLEOSIDE-3′-PHOSPHODlESTERS

Abstract

Two nucleoside analogs were synthesized to test the ribose conformational and electronic effects on phosphate hydrolysis at the 3′ position. It was found that under alkaline conditions, a 2′-fluoro-nucleoside (C3′-endo) resulted in a phosphate degradation that was ten times faster than the 2′-deoxynucleoside analog (C2′-endo). In addition to kinetic differences, product distributions will be presented.     

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.     

SYNTHESIS AND ANTIVIRAL EVALUATION OF 3′-C-TRIFLUOROMETHYL NUCLEOSIDE DERIVATIVES BEARING ADENINE AS THE BASE

3′-deoxy-3′-C-trifluoromethyl- (3), 2′,3′-dideoxy-3′-C-trifluoromethyl- (5) and 2′,3′-dideoxy-2′,3′-didehydro-3′-C-trifluoromethyladenosine (6) derivatives have been synthesized and their antiviral properties examined. All these derivatives were stereospecifically prepared by glycosylation of adenine with a trifluoromethyl sugar precursor (1), followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV, but they did not show an antiviral effect.     

SYNTHESIS AND ANTIVIRAL ACTIVITY OF D- AND L-2′-AZIDO-2′,3′-DIDEOXY-4′-THIOPYRIMIDINE AND PURINE NUCLEOSIDES

Novel D- and L-2′-azido-2′,3′-dideoxy-4′-thionucleosides were synthesized starting from L- and D-xylose via D- and L-4-thioarabitol derivative as key intermediates and evaluated for antiviral activity, respectively. When the final nucleosides were tested against HIV-1, HSV-1, HSV-2, and HCMV, they were found to be only active against HCMV without cytotoxicity up to 100 μg/ml.     

SYNTHESIS AND PURIFICATION OF FLUORINATED BENZIMIDAZOLE AND BENZENE NUCLEOSIDE-5′-TRIPHOSPHATES

The expression “universal base” is very often used to express hybridization properties and recognition patterns of nucleosides. Their behaviour in biological applications, however, is of great interest regarding, e.g., their incorporation by polymerases. The 4,6-difluorobenzimidazole and the 2,4-difluorobenzene nucleoside analogues have proven to be universal bases that do not discriminate between the four natural nucleobases in RNA duplexes. Therefore, we synthesized the corresponding triphosphates to evaluate their behavior in polymerase catalyzed reactions and to investigate their ability to serve as substrates for the T7 RNA polymerase.     

SYNTHESIS AND BIOLOGICAL EVALUATION OF CONFORMATIONALLY RESTRICTED AND NUCLEOBASE-MODIFIED ANALOGS OF THE ANTICANCER COMPOUND 3′-C-ETHYNYLCYTIDINE (ECYD)

A series of conformationally restricted and nucleobase-modified analogs of the anticancer compound 3′-C-ethynylcytidine (ECyd) and its uracil analog (EUrd) have been synthesized. While none of, the conformationally restricted analogs displayed anticancer activity, 5-iodo-EUrd and 5-bromo-EUrd displayed potent anticancer activity with IC50 values of 35 nM and 0.73 μM.     

SYNTHESIS OF SOME 2′-FLUORO-2′-DEOXY-3′-C-ETHYNYL AND 3′-C-VINYL-β-D-LYXOFURANOSYL NUCLEOSIDES

1-(2-Fluoro-2-deoxy-β-D-arabinofuranosyl)uracil (5) and 1-(2-fluoro-2-deoxy-β-D-arabinofuranosyl)cytosine (6) were synthesized as reported earlier. Both of these compounds were converted into 2′-fluoro-2′-deoxy-3′-C-ethynyl and 3′-C-vinyl-β-D-lyxofuranosyl nucleosides (16–19) by a multistep sequence. All these new nucleosides were evaluated against seven human tumor cell lines in vitro.     

EFFICIENT PRODUCTION OF URIDINE 5′-DIPHOSPHO-N-ACETYLGLUCOSAMINE BY THE COMBINATION OF THREE RECOMBINANT ENZYMES AND YEAST CELLS

Uridine 5′-diphospho N-acetylglucosamine (UDP-GlcNAc) is an important nucleotide sugar in the biochemistry of all living organisms, and it is an important substrate in the synthesis of oligosaccharides. In the present work, three bioactive enzymes, namely, glucokinase (YqgR), GlcNAc-phosphate mutase (Agm1), and N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), were produced effectively as soluble form in recombinant Escherichia coli. These three enzymes and dried yeast together were used to construct a multistep enzymatic system, which could produce UDP-GlcNAc efficiently with N-acetylglucosamine (GlcNAc) as the substrate. After the optimization of various reaction conditions, 31.5 mMUDP-GlcNAc was produced from 50 mMGlcNAc and 50 mMUMP.     

COMPARISON OF THE ANTIVIRAL ACTIVITY OF HYDROPHOBIC AMINO ACID PHOSPHORAMIDATE MONOESTERS OF 2′, 3′-DIDEOXYADENOSINE (DDA) AND 3′-AZIDO-3′-DEOXYTHYMIDINE (AZT)

A series of hydrophobic, water soluble and non-toxic amino acid phosphoramidate monoesters of dideoxyadenosine (ddA) and 3′-azido-3′-deoxythymidine were shown to inhibit the replication of HIV-1 in human peripheral blood mononuclear cells (PBMC) from two donors. The tryptophan methyl ester phosphoramidates of AZT and ddA were equally potent (EC₅₀S = 0.3–0.4 μM), while the phenyl methyl ester of ddA was 40- to 100- fold more potent than the AZT derivatives. The alaninyl methyl ester of AZT was found to be 70- fold more potent than the ddA derivative. The methyl amide derivatives were found to be 5–20 fold less active than the methyl esters for the ddA series, while for AZT the derivatives were found to be of similar potency or 60- to 166- fold more potent than the methylesters.     

Occurrence of Guanosine-5′-diphosphate-3′-diphosphate and Adenosine-5′-triphosphate-3′-diphosphate in Streptomyces galilaeus

Synthetic activity and existence of ppGpp and pppApp in an anthracycline-producing strain Streptomyces galilaeus were determined by radioimmunoassay and ³²P-labeling method during cultivation under both the antibiotic productive and non-productive conditions. The cellular ppGpp(pppGpp)-synthesizing activity was highest at the end of exponential growth, and 3-fold higher in the antibiotic-productive cultivation than in non-productive cultivation. The intracellular level of ppGpp determined by radioimmunoassay was high at the end of exponential growth, and afterwards its level decreased by one fifth. The low level of cellular ppGpp during the period of intense antibiotic production suggests that ppGpp consumption may play an important role in antibiotic production of S. galilaeus. The concentration of pppApp was not determined intracellularly by radioimmunoassay.