Use of N-alkyl imidazole for sulfurization of oligonucleotides with an acetyl disulfide

• US 8,309,710 B2
• Filed: 06/29/2009
• Issued: 11/13/2012
• Est. Priority Date: 06/29/2009
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Sierzchala, Agnieszka B., Dellinger, Douglas J, Metz, Paul A., Mokler, Victor R
• Primary Examiner(s): Jiang, Shaojia Anna
• Assistant Examiner(s): Lau, Jonathan S
• Application Number:
  US12/494,118
• Publication Number:
  US 20100331532A1
• Time in Patent Office: 1,233 Days
• Field of Search: 536/124, 536/26.5, 536/25.3, 548/335.1, 568/22, 568/25
• US Class Current: 536/124
• 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, said method comprising:

providing a phosphite or thiophosphite intermediate; and

contacting said phosphite or thiophosphite intermediate with a composition comprising an acetyl disulfide, a solvent, and N-alkyl imidazole, for a time sufficient to convert said phosphite or thiophosphite intermediate to said phosphorothioate or phosphorodithioate

wherein said solvent is a low dielectric constant solvent.

2. A method according to claim 1 wherein said N-alkyl imidazole is N-methyl imidazole.

3. The method of claim 1 wherein said N-alkyl imidazole is at least 1% in volume (v/v) in said solvent.

4. The method of claim 1 wherein said N-alkyl imidazole is at least 5% in volume (v/v) in said solvent.

5. The method of claim 1 wherein said acetyl disulfide is at least at 0.1M concentration in said solvent.

6. The method of claim 2 wherein said acetyl disulfide is at a concentration of at least 0.1 M and said N-methyl imidazole is at least 5% in volume (v/v) in said solvent.

7. The method of claim 1 wherein said oligonucleotide comprises one or more ribonucleotides.

8. The method of claim 1 wherein said oligonucleotide comprises at least one nucleotide analogue.

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

providing a phosphite or thiophosphite intermediate

contacting said phosphite or thiophosphite intermediate with a composition comprising phenylacetyl disulfide, a solvent, and N-methyl imidazole for a time sufficient to convert said phosphite or thiophosphite intermediate to said phosphorothioate or phosphorodithioate

wherein said solvent is a low dielectric constant solvent.

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

11. The method of claim 10 wherein said solvent is toluene.

12. The method of claim 10 wherein said solvent is 2-methyl THF.

13. The method of claim 9 wherein said solvent is toluene, and wherein said phenylacetyl disulfide is at a concentration of at least 0.1 M and said N-methyl imidazole is at least 5% in toluene (v/v).

14. The method of claim 9 wherein said solvent is toluene, said phenylacetyl disulfide is at a concentration of 0.2 M and said N-methyl imidazole is between 10% to 20% in toluene (v/v).

15. The method of claim 9 wherein said solvent is 2-methylTHF, and wherein said phenylacetyl disulfide is in a concentration of at least 0.1 M and said N-methyl imidazole is at least 5% in volume in 2-methyl THF (v/v).

16. The method of claim 9 wherein said solvent is 2-methyl THF, said phenylacetyl disulfide is in a concentration of 0.2 M and said N-methyl imidazole is 5% to 20% in volume in 2-methyl THF (v/v).

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

providing a phosphite or a thiophosphite bound to a solid support,

contacting said phosphite or thiophosphite with a composition comprising N-alkyl imidazole, an acetyl disulfide, and a solvent for a time sufficient to convert said phosphite or thiophosphite to said phosphorothioate or phosphorodithioate linkage

wherein said solvent is a low dielectric constant solvent.

18. The method of claim 17 wherein said solid support is controlled pore glass or polystyrene.

19. The method of claim 18 wherein said solid support is an array or beads.

20. The method of claim 17 wherein said solvent is selected from toluene, xylene, 2-methyl THF and cyclopentyl methyl ether.

21. The method of claim 20 wherein N-alkyl imidazole is at least at 5% in volume and the acetyl disulfide is at least at 0.1 M concentration in said solvent.