An efficient stereoselective synthesis of 4′-β-thioribonucleosides 14, 15, 27, and 30 using the Pummerer reaction as the key step is described. The Pummerer reaction of 1,4-anhydro-2-O-(2,4dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-sulfinyl-D-ribitol (R-10:S-10 ) 2.7:1) in the presence of silylated uracil afforded the desired β-anomer of the 4′-thiouridine derivative 11 in 66% yield without formation of its R-anomer. The reaction with R-10 gave 11 in 87% yield, while the one with S-10 resulted in a 27% decrease of the desired product 11 along with a 22% yield of 3,6-O-(1,1,3,3tetraisopropyldisiloxane-1,3-diyl)-3-hydroxy-2-hydroxymethylthiophene (12). A likely explanation for the observed difference in the reaction of R-10 and S-10 is that the reaction proceeds via an E2 type pathway, which prefers anti elimination. Thus, R-10 would preferentially afford the R-thiocarbocation intermediate 21 via an E2 anti elimination under the reaction conditions. The resulting 21 would be expected to react with silylated uracil stereoselectively to give 11 in good yield. However, formation of the more stable tertiary R-thiocarbocation intermediate 23, which would prefer to give 12 and/or to decompose, would compete with the formation of the desired 21 in the reaction with S-10. Consequently, this argument would explain the low yields of the desired product 11 and the poor mass balance in the reaction with S-10. When the sulfoxide 10 (R-10:S-10 ) >16:1) prepared by oxidation of 9 with ozone was used for the Pummerer reaction, the desired 11 was obtained in 80% yield. Compound 11 was converted to 4′-β-thiouridine ( 14) by treatment of 11 with ammonium fluoride, followed by methanolic ammonia. Similarly, 4′-β-thiocytidine (15) was prepared when silylated N 4 -acetylcytosine was used in the Pummerer reaction. For the Pummerer reactions with purine bases, 6-chloropurine and 2-amino-6-chloropurine were found to be the most suitable. When the reactions were conducted in a mixture of acetonitrile and 1,2-dichloroethane at room temperature, followed by reflux, the desired products 25 and 28 were obtained in 65% and 56% yields, respectively. These compounds were then converted to 4′-β-thioadenosine (27) and 4′-β-thioguanosine (30) under the usual conditions. This is therefore the first time that the stereoselective synthesis of 4′-β-thioribonucleosides has been performed using the neighboring group participation of the Pummerer reaction.

The synthesis and thermal stability of oligodeoxynucleotides (ODNs) containing imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleosides 1-4 (N(N), O(O), N(O), and O(N), respectively) with the aim of developing two sets of new base pairing motifs consisting of four hydrogen bonds (H-bonds) is described. The proposed four tricyclic nucleosides 1-4 were synthesized through the Stille coupling reaction of a 5-iodoimidazole nucleoside with an appropriate 5-stannylpyrimidine derivative, followed by an intramolecular cyclization. These nucleosides were incorporated into ODNs to investigate the H-bonding ability. When one molecule of the tricyclic nucleosides was incorporated into the center of each ODN (ODN I and II, each 17mer), no apparent specificity of base pairing was observed, and all duplexes were less stable than the duplexes containing natural G:C and A:T pairs. On the other hand, when three molecules of the tricyclic nucleosides were consecutively incorporated into the center of each ODN (ODN III and IV, each 17mer), thermal and thermodynamic stabilization of the duplexes due to the specific base pairings was observed. The melting temperature (T(m)) of the duplex containing the N(O):O(N) pairs showed the highest T(m) of 84.0 degrees C, which was 18.2 and 23.5 degrees C higher than that of the duplexes containing G:C and A:T pairs, respectively. This result implies that N(O)and O(N) form base pairs with four H-bonds when they are incorporated into ODNs. The duplex containing N(O):O(N) pairs was markedly stabilized by the assistance of the stacking ability of the imidazopyridopyrimidine bases. Thus, we developed a thermally stable new base pairing motif, which should be useful for the stabilization and regulation of a variety of DNA structures.

A series of novel compounds, 5-alkynyl-1-P-D-ribofuranosylimidazole-4-carboxamides, have been identified as broad-spectrum antiviral agents. 5-Ethynyl-1-o-D-ribofuranosylimidazole-4-carboxamide (EICAR), the most potent congener of the group, showed antiviral potency about 10-to 100-fold greater than that of ribavirin. Similar in spectrum to ribavirin, EICAR was particularly active (50% inhibitory concentration, 0.2 to 4 ,ig/ml) against poxviruses (vaccinia virus), togaviruses (Sindbis and Semliki forest viruses), arenaviruses (Junin and Tacaribe viruses), reoviruses (reovirus type 1), orthomyxoviruses (influenza A and B viruses), and paramyxoviruses (parainfluenza virus type 3, measles virus, subacute sclerosing panencephalitis virus, and respiratory syncytial virus). EICAR was also cytostatic for rapidly growing cells (50% inhibitory concentration, 0.2 to 0.9 ug/mml). EICAR inhibited vaccinia virus tail lesion formation at doses that were not toxic to the host. EICAR is a candidate antiviral drug for the treatment of pox-, toga-, arena-, reo-, orthomyxo-, and paramyxovirus infections.

Short interfering RNAs (siRNAs) variously modified with 4 0 -thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4 0 -thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4 0 -thioribonucleosides. Since modifications of siRNAs with 4 0 -thioribonucleosides were well tolerated in RNAi activity compared with that of 2 0 -O-methyl nucleosides, 4 0 -thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.