The synthesis of oligonucleotides containing 2′-deoxy-2′-fluoro-4′-thioarabinonucleotides is described. 2′-Deoxy-2′-fluoro-5-methyl-4′-thioarabinouridine (4′S-FMAU) was incorporated into 18-mer antisense oligonucleotides (AONs). 4′S-FMAU adopts a predominantly northern sugar conformation. Oligonucleotides containing 4′S-FMAU, unlike those containing FMAU, were unable to elicit E. coli or human RNase H activity, thus corroborating the hypothesis that RNase H prefers duplexes containing oligonucleotides that can adopt eastern conformations in the antisense strand. The duplex structure and stability of these oligonucleotides was also investigated via circular dichroism (CD)- and UV- binding studies. Replacement of the 4′-oxygen by a sulfur atom resulted in a marked decrease in melting temperature of AON:RNA as well as AON:DNA duplexes. 2′-Deoxy-2′-fluoro-4′-thioarabinouridine (4′S-FAU) was incorporated into 21-mer small interfering RNA (siRNA) and the resulting siRNA molecules were able to trigger RNA interference with good efficiency. Positional effects were explored, and synergy with 2′F-ANA, which has been previously established as a functional siRNA modification, was demonstrated.
An improved synthesis of 2'-deoxy-2'-fluoro-5-methyl-4'-thioarabinouridine (4'S-FMAU) is described. Participation of the 3'-O-benzoyl protecting group in the thiosugar precursor influenced the stereochemistry of the N-glycosylation reaction in nonpolar solvents, permitting a higher beta/alpha ratio than previously observed for similar Lewis acid catalyzed glycosylations. Conformational analysis of the nucleoside using 3JHH and 3JHF NMR coupling constants together with the PSEUROT program showed that it adopted a predominantly northern conformation in contrast to 2'-deoxy-2'-fluoro-5-methylarabinouridine (FMAU), whose PSEUROT conformational analysis is presented here for the first time, which showed a dominantly southeast conformation. The sharp conformational switch attained by replacing the ring heteroatom is attributed to a decrease in relevant steric and stereoelectronic effects.
During the work 4-hydroxybenzyl isothiocyanate (PHBITC) was measured during the hydrolysis of sinalbin with myrosinase while imposing the temperature and pH regimen used for the fermentation phase of dry fermented sausage production. The results on PHBITC stability in maltodextrin presented provide precautionary information on the limited ability of this material to afford stability of PHBITC expected.
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