USE OF N-ALKYL IMIDAZOLE FOR SULFURIZATION OF OLIGONUCLEOTIDES WITH AN ACETYL DISULFIDE

• US 20130035261A1
• Filed: 10/09/2012
• Published: 02/07/2013
• Est. Priority Date: 06/29/2009
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Dellinger, Douglas J., Mokler, Victor R., Sierzchala, Agnieszka B., Metz, Paul A.
• Granted Patent: US 9,169,286 B2
• Time in Patent Office: Days
• US Class Current: 506/30
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical

Abstract

A method and compositions for sulfurizing at least one phosphite or thiophosphite linkage in an oligonucleotide. The addition of N-alkyl imidazole to the acetyldisulfide sulfurization solution enables the use of industrially preferred solvents or solvents that are derived from renewable resources.

Claims

1. A method of sulfurizing at least one phosphite or thiophosphite linkage in an oligonucleotide, the method comprising:

contacting an oligonucleotide comprising a phosphite or thiophosphite intermediate with a composition comprising an acetyl disulfide, a solvent, and a nucleophilic acylation catalyst, for a time sufficient to convert the phosphite or thiophosphite intermediate to a phosphorothioate or phosphorodithioate;

wherein the nucleophilic acylation catalyst is at up to 25% (v/v) in the solvent.

2. The method of claim 1, wherein the nucleophilic acylation catalyst is an N-alkyl imidazole.

3. The method of claim 2, wherein the N-alkyl imidazole is N-methyl imidazole.

4. The method of claim 1, wherein the solvent is at least 80% (v/v) of the composition.

5. The method of claim 2, wherein the N-alkyl imidazole is at 10% (v/v) or less in the solvent.

6. The method of claim 1, wherein the solvent is a high dielectric constant solvent.

7. The method of claim 5, wherein said solvent comprises acetonitrile or propylene carbonate.

8. The method of claim 7, wherein the solvent is acetonitrile.

9. The method of claim 1, wherein the solvent is a low dielectric constant solvent.

10. The method of claim 9, wherein said solvent comprises toluene, xylene, 2-methyl THF, cyclopentyl methyl ether or 1-methylpyrrolidinone.

11. The method of claim 10, wherein the solvent is toluene.

12. The method of claim 2, wherein the N-alkyl imidazole is at least 5% in volume (v/v) in the solvent.

13. The method of claim 12, wherein the acetyl disulfide is at least at 0.1M concentration in said solvent.

14. The method of claim 1, wherein the oligonucleotide is bound to a solid support.

15. The method of claim 14, wherein said solid support is an array or beads.

16. The method of claim 1, wherein the oligonucleotide comprises one or more ribonucleotides.

17. The method of claim 1, wherein the oligonucleotide comprises at least one nucleotide analogue.

18. A method of sulfurizing at least one phosphite or thiophosphite linkage in an oligonucleotide, the method comprising:

contacting an oligonucleotide comprising a phosphite or thiophosphite intermediate with a composition comprising an acetyl disulfide, a high dielectric solvent, and N-methyl imidazole, for a time sufficient to convert the phosphite or thiophosphite intermediate to a phosphorothioate or phosphorodithioate;

wherein the high dielectric solvent is at least 80% (v/v) of the composition, and the N-methyl imidazole is at 10% (v/v) or less in the high dielectric solvent.

19. The method of claim 18, wherein the solvent is acetonitrile.