Oligodeoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus.

Abstract

Modified oligodeoxynucleotides complementary to RNA of human immunodeficiency virus 1 (HIV-1) were tested for their ability to inhibit virally induced syncytium formation and expression of viral p24 protein. The Fig. 1. All the modified oligonucleotides were found to be resistant to nucleases such as snake venom or spleen phosphodiesterases. amounts of material for toxicity studies were made by a manual solid-support method on an Omnifit assembly (13). Unmodified oligomers were made and purified by methods described previously (2). Phosphorothioates and phosphoramidates were synthesized by a modification of the Hphosphonate procedure (14,15). To generate phosphorothioate linkages, oxidation following the final coupling and detritylation was replaced by treatment with 0.1 M sulfur in carbon disulfide/triethylamine (9:1, vol/vol) at room temperature for up to 2 hr, depending on chain length (16). For the phosphoramidates, this step was replaced by treatment with a 10% solution of the appropriate amine in carbon tetrachloride for up to 1.5 hr, depending on chain length (16).After deblocking in concentrated ammonium hydroxide (550C for 51/2 hr or room temperature for >2 days), products were purified on 2-mm-thick preparative layer plates (Merck silica gel 60) in propanol/water/concentrated ammonium hydroxide (55:35:10, vol/vol). Phosphorothioates were further purified on DEAE-cellulose and C18 silica (details will be published elsewhere). Phosphoramidates from preparative layer plates were passed through Sephadex G-25 in aqueous 30% (vol/vol) ethanol and then through C18 silica and dialyzed. In all cases, small amounts of oligomers attached to the solid support were taken before the final treatment with sulfur or amine and were oxidized with 2% iodine in pyridine/water (98:2, vol/vol) (15) to phosphodiesters. These were used for determination of base composition by enzymatic degradation to nucleosides followed by HPLC. The phosphorothioates were also characterized by polyacrylamide gel electrophoresis, where they had similar mobility to the corresponding diesters.Phosphoramidates were further characterized by hydrolysis with formic acid to the phosphodiesters (16) for comparison with authentic samples on HPLC and degradation to nucleosides for determination of nucleoside composition and integrity by HPLC. A useful indicator of oligonucleotide purity and integrity is the melting curve of its duplex with a complementary DNA Abbreviations: HIV, human immunodeficiency virus; AIDS, acquired immunodeficiency syndrome. 7079The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues.

“…Oligonucleotides were synthesized on an automated synthesizer (model 8700, Milligen Biosearch, Bedford, MA). Both normal phosphodiester oligonucleotides and their phosphorothioate analogues were assembled by using H-phosphonate chemistry (11,16,17).…”

“…However, relatively short half-lives of normal oligonucleotides (9) in serum and in cells due to the presence of nucleases, and possibly the low permeability of these charged molecules into normal cells, limit their potential usefulness in vivo. To overcome this limitation, we and others (10-15) have studied internucleotide phosphate backbone-modified oligonucleotides, such as methylphosphonates (3, 10) as well as phosphorothioates and various phosphoramidates (11,12), for their antiviral activities against HIV.Here we report both the inhibition of HIV replication and the effect on cell growth of MOLT-3 cells that have been infected just 24 or 48 hr before addition of oligomers and also the effect on MOLT-3 cells chronically infected with HIV that were mixed with uninfected cells in the presence of oligodeoxynucleotides and their phosphorothioate analogues. …”

“…However, relatively short half-lives of normal oligonucleotides (9) in serum and in cells due to the presence of nucleases, and possibly the low permeability of these charged molecules into normal cells, limit their potential usefulness in vivo. To overcome this limitation, we and others (10)(11)(12)(13)(14)(15) have studied internucleotide phosphate backbone-modified oligonucleotides, such as methylphosphonates (3,10) as well as phosphorothioates and various phosphoramidates (11,12), for their antiviral activities against HIV.…”

“…Oligonucleotides were synthesized on an automated synthesizer (model 8700, Milligen Biosearch, Bedford, MA). Both normal phosphodiester oligonucleotides and their phosphorothioate analogues were assembled by using H-phosphonate chemistry (11,16,17).Synthesis was carried out on a 15-to 25-,umol scale on controlled pore glass support (loading of nucleoside = 35-40 ,umol/g). For each coupling reaction, a 2-to 3-fold molar excess of nucleoside H-phosphonate and a 20-to 30-fold molar excess of adamantanecarbonyl chloride in acetonitrile/ pyridine (1:1, vol/vol) were used for 60-80 sec, and 3% dichloroacetic acid in dichloroethane for 90-120 sec was used for each detritylation step.…”

Morpholino antisense oligomer targeting human midkine: Its application for cancer therapy

“…Modifications that impart resistance to nuclease degradation include phosphorothioate (PS, Fig. 1c), 6,7 methylphosphonate, 8 and 2Ј-O-methyl-modified oligonucleotides. 9 PS-modified oligonucleotides are used commonly and effectively inhibit synthesis of proteins in various cell types.…”