Synthesis of sulfurized oligonucleotides
View Patent ↗Methods for the formation of sulfurized oligonucleotides are provided. The methods allow for the formation of phosphorothioate linkages in the oligonucleotides or derivatives, without the need for complex solvent mixtures and repeated washing or solvent changes. Oligonucleotides having from about 8, and up to about 50, nucleotides can be sulfurized according to the methods of the invention with higher yields than have been previously reported.
1. A method for the preparation of a phosphorothioate internucleoside linkage comprising:
contacting the 5′ hydroxyl group of a solid support bound nucleic acid moiety with a phosphite moiety in acetonitrile to form a phosphite diester intermediate; said phosphite moiety being bonded to a nucleoside, a nucleotide, an oligonucleoside or an oligonucleotide; and
oxidizing said phosphite diester intermediate with an arylacetyl disulfide in a solvent mixture of acetonitrile and a lutidine, acetonitrile and a collidine and/or acetonitrile and a picoline in a ratio of 1.5:1 to 1:1.5, for a time sufficient to effect conversion of said phosphite diester intermediate to said phosphorothioate.
2. The method of claim 1 wherein said solvent mixture is either acetonitrile and a lutidine or acetonitrile and a picoline in a ratio of 1.5:1 to 1:1.5.
3. The method of claim 1 wherein said solvent mixture is acetonitrile and a picoline in a ratio of 1.5:1 to 1:1.5.
4. The method of claim 3 , wherein said picoline is 3-picoline.
5. The method of claim 4 wherein said ratio is 1:1.
6. The method of claim 1 wherein said arylacetyl disulfide is phenylacetyl disulfide.
7. The method of claim 1 wherein said phosphite moiety is a phosphoramidite group that is bonded to a nucleoside.
8. The method of claim 1 wherein said phosphite diester intermediate is a phosphite linked dinucleotide.
9. The method of claim 8 wherein said dinucleotide is formed by reaction of a phosphoramidite group.
10. The method of claim 1 wherein said phosphite moiety is attached to an oligonucleotide.
11. The method of claim 10 wherein said phosphite moiety is a phosphoramidite group.
12. A method for the preparing an oligonucleotide wherein at least one of the internucleoside linkages is a phosphorothioate internucleoside linkage prepared according to the method of claim 1 and wherein said oligonucleotide comprises from about 8 to about 30 nucleotides.
13. The method of claim 12 wherein said oligonucleotide comprises about 20 nucleotides.