Phosphoramidite coupling to oligonucleotides bearing unprotected internucleosidic phosphate moieties.
The coupling of 2-cyanoethyl thymidine phosphoramidite to solid-support-bound, phosphate-unprotected oligothymidylates and their phosphorothioate analogues was studied. The yield of the coupling reaction depended on the pK(BH)()+ values of protonated nitrogen bases that served as counterions to the phosphodiester functions of oligonucleotides. To maximize the coupling efficiency, the oligonucleotides were detritylated and washed with a mixture of 0.1 M DMAP and 0.1 M 1H-tetrazole, which resulted in a 98+% coupling efficiency. The utility of the results was demonstrated in the preparation of oligonucleotides with a mixed backbone that required the successive use of H-phosphonate and phosphoramidite methods of synthesis. Using this approach, 20-mer antisense oligonucleotides containing 2'-O-(2-methoxyethyl) ribonucleoside residues and phosphorothioate and phosphoramidate internucleosidic linkages were synthesized in high yield.
Department of Medicinal Chemistry, Isis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA 92008, USA. aguzaev@isisph.com
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Guzaev
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Manoharan
D002851:Chromatography, High Pressure Liquid; D009682:Magnetic Resonance Spectroscopy; D009705:Nucleosides / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D010759:Phosphorus Isotopes; D013873:Thionucleotides / Q000737:chemistry*
D009705:Nucleosides; D009943:Organophosphorus Compounds; D010759:Phosphorus Isotopes; D013873:Thionucleotides