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′-DEOXY-3′-C-HYDROXYMETHYL ANALOGUES OF TIAZOFURIN AND RIBAVIRIN

On the basis of potent biological activity of 3′-branched-3′-deoxynucleoside analogues, novel ribavirin and tiazofurin derivatives with 3′-C-hydroxymethyl substituent were synthesized, starting from D-xylose.     

CHEMICAL STABILITY OF 2′-DEOXY-5-METHYLISOCYTIDINE DURING OLIGODEOXYNUCLEOTIDE SYNTHESIS AND DEPROTECTION

ABSTRACT

Previous studies have encountered difficulties with degradation of some isocytidine derivatives during solid-phase synthesis and deprotection of oligonucleotides. Here we investigate the degradation of a commonly used derivative, 2′-deoxy-5-methylisocytidine, during oligodeoxynucleotide synthesis and deprotection. A small, but detectable amount of hydrolytic deamination occurred at ca. 0.5% of 2′-deoxy-5-methyliso-cytidine residues using routine synthesis and deprotection conditions. Depyrimidination, or cleavage of the glycosylic bond, occurred to a far lesser extent during alkaline deprotection than previously suggested. In contrast to model studies of nucleoside monomers, significant depyrimidination was not observed, even at extended incubation times.     

SYNTHESIS AND STRUCTURAL STUDY OF QUADRUPLEX STRUCTURES CONTAINING 2′-DEOXY-8-METHYLADENOSINE

The synthesis and preliminary structural studies of ODNs A^8MeGGGT and TA^8MeGGGT, where A^8Me represents 2′-deoxy-8-methyladenosine, are reported.     

6-AZAPYRIMIDINE AND 7-DEAZAPURINE 2′-DEOXY-2′-FLUOROARABINONUCLEOSIDES: SYNTHESIS,CONFORMATION AND PROPERTIES OF OLIGONUCLEOTIDES

The synthesis of 2′-deoxy-2′-fluoro-β-d-arabinofuranosyl nucleosides (1b, 2b, and 3b) were described and their conformation in solution as well as in the solid state was determined. In addition to this, building blocks 10a,b and 13a,b were prepared and employed in solid-phase oligonucleotide synthesis. For compounds 1a and 1b the lactime proton is protected to avoid unresolved degradation of its phosphoramidites 10a,b. UV-melting studies have been carried out to assess the thermal stability of oligonucleotides containing compounds 1a,b, and 3a,b.     

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.     

STRUCTURE-ACTIVITY RELATIONSHIP OF 5′-SUBSTITUTED FLUORO-NEPLANOCIN A ANALOGUES AS POTENT INHIBITORS OF S-ADENOSYLHOMOCYSTEINE HYDROLASE

Four 5′-substituted fluoro-neplanocin A analogues 1a–d were designed and synthesized, and the inhibitory activity against SAH was in the following order: NH₂ > SH > F, N₃, indicating a hydrogen bonding donor is essential for inhibitory activity.     

SYNTHESIS AND STUDY OF CONFORMATIONALLY RESTRICTED 3′-DEOXY-3′,4′-EXO-METHYLENE NUCLEOSIDE ANALOGUES

Hitherto unknown restricted 3′-deoxy-3′,4′-exo-methylene nucleoside derivatives bearing the nucleic acid naturally occurring pyrimidine bases have been synthesized. The compounds were tested for their activity against HIV, HBV, and several RNA viruses, but they did not show significant antiviral effect.     

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.     

SYNTHESIS AND IN VITRO ANTI-HCV ACTIVITY OF β-D- and L-2′-DEOXY-2′-FLUORORIBONUCLEOSIDES

Based on the discovery of β-D-2′-deoxy-2′-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of β-D- and l-2′-deoxy-2′-fluororibonucleosides with modifications at 5 and/or 4 positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The introduction of the 2′-fluoro group was achieved by either fluorination of 2,2′-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds, namely β-D-2′-deoxy-2′,5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon cell line, and inhibitory activity against ribosomal RNA. As β-D-N4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, the two functionalities of the N4-hydroxyl and the 2′-fluoro were combined into one molecule, yielding β-D-2′-deoxy-2′-fluoro-N4-hydroxycytidine (12). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot reliably predict anti-HCV activity in vitro.     

SYNTHESIS AND BIOLOGICAL ACTIVITY OF 2′-DEOXY-4′-THIO-PYRAZOLO[3,4-d]PYRIMIDINE NUCLEOSIDES

The coupling of 4-aminopyrazolo [3, 4-d]pyrimidine with the appropriate thio sugar gave a 3:1 ratio of α,β blocked 4-amino-1-(2-deoxy-4-thio-D-erythropentofuranosyl)- 1H pyrazolo[3,4-d]pyrimidine nucleosides. The mixture was deblocked, both the anomers were separated, and the β-anomer was readily deaminated by adenosine deaminase. The nucleosides have been characterized, and their anomeric configurations have been determined by proton NMR. All three nucleosides were evaluated against a panel of human tumor cell lines for cytotoxicity in vitro. The details of a convenient and high yielding synthesis of these nucleosides are described.     

INHIBITION OF HUMAN TELOMERASE BY L-ENANTIOMERS OF NATURAL 2′-DEOXYRIBONUCLEOSIDE 5′-TRIPHOSPHATES

In order to clarify whether L-enantiomers of natural 2′-deoxyribonucleoside 5′-triphosphates (dNTPs) are recognized by human telomerase, a quantitative telomerase assay based on the ‘stretch PCR’ method was developed and used for kinetic analysis. Among the four L-dNTPs, L-dTTP and L-dGTP inhibited telomerase activity and the others showed slight or no inhibitory effect. Lineweaver-Burk plot analysis showed that the inhibition mode L-dGTP was competitive with dGTP.     

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.     

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.     

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 OF NOVEL D- AND L-3′-DEOXY-3′-C-HYDROXYMETHYL NUCLEOSIDE WITH EXOCYCLIC METHYLENE AS POTENTIAL RIBONUCLEOTIDE REDUCTASE INHIBITOR

D- and L-3′-Deoxy-3′-C-hydroxymethyl thymidine substituted with exocyclic methylene at 2′-position were synthesized, starting from D- and L-xylose as potential ribonucleotide reductase inhibitor, respectively, but they were found to be inactive against several tumor cell lines.     

SYNTHESIS AND EVALUATION OF ANTI-HSV ACTIVITY OF NEW 5-ALKYNYL-2′-DEOXYURIDINES

Eight 5-alkynyl-2′deoxyuridines containing different bulky substituents have been prepared and tested against HSV-1 in Vero cells. The compounds show positive antiviral activity. There is no obvious correlation between activity and substituent size. The nature of the linker between uracil and a substituent appears to be more important for antiviral properties: nucleosides containing arylethynyl groups show higher activity.     

DEAGGREGATION OF eIF4E INDUCED BY mRNA 5′ CAP BINDING

All eukaryotic mRNAs contain a 5′ terminal cap structure, which consists of 7-methylguanosine linked by a 5′-5′ triphosphate bridge to the first transcribed nucleoside (m⁷GpppN). Specific recognition of the cap by the eukaryotic initiation factor eIF4E plays a key role in regulation of translation initiation as a rate-limiting step. Using dynamic light scattering (DLS), the apo-form of murine eIF4E (33–217) was shown to aggregate. After addition of m⁷GTP, progressive deaggregation with the time of incubation in the presence of the cap analogue has been observed.     

HYDROLYSIS OF BULGED NUCLEOTIDES IN HYBRIDS FORMED BY RNA AND IMIDAZOLE-DERIVATIZED OLIGO-2′-O-METHYLRIBONUCLEOTIDES

In order to enhance the efficacy of small antisense molecules, we examined a series of antisense oligonucleotides derivatized with functional groups designed to enable them to hydrolyze their RNA target. Solid phase synthetic methods were used to prepare imidazole-derivatized antisense oligo-2′-O-methylribonucleotides. Upon binding, these oligonucleotides create internal bulged bases in the target RNA that serve as sites for hydrolysis. We observed that an oligonucleotide derivatized with a side chain containing two imidazole groups was capable of hydrolyzing 58% of its RNA target when incubated with the target for 48 hours at 37°C and physiological pH.