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 preparing an oligonucleotide having at least one phosphorothioate internucleoside linkage, said method comprising:
phosphitylating the 5′-hydroxyl of a nucleic acid moiety in an acetonitrile-containing solvent system to form a phosphite intermediate, said nucleic acid moiety being bound to a solid support; and
oxidizing said phosphite intermediate with phenylacetyl disulfide in an aprotic solvent mixture including said acetonitrile-containing solvent system and a basic solvent for a time sufficient to effect said conversion of said phosphite intermediate to said phosphorothioate.
2. The method of claim 1 , wherein the oligonucleotide is between 8 nucleotides and 30 nucleotides in length.
3. The method of claim 1 , wherein the aprotic solvent mixture includes a basic solvent selected from pyridine and hindered pyridines.
4. The method of claim 1 , wherein said oligonucleotide comprises at least one 2′-modified nucleoside.
5. The method of claim 1 , wherein said oligonucleotide comprises at least one deoxynucleoside.
6. The method of claim 3 , wherein the hindered pyridine is a collidine, a picoline or a lutidine.
7. The method of claim 6 , wherein said picoline is 3-picoline.
8. The method of claim 1 , wherein said phosphite intermediate is a dinucleotide phosphite.
9. The method of claim 1 , wherein said phosphitylating is performed with a phosphoramidite.