Use of thioacetic acid derivatives in the sulfurization of oligonucleotides with phenylacetyl disulfide

• US 8,642,755 B2
• Filed: 06/30/2009
• Issued: 02/04/2014
• Est. Priority Date: 06/30/2009
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Sierzchala, Agnieszka B., Dellinger, Douglas J, Mokler, Victor R, Timar, Zoltan
• Primary Examiner(s): Jiang, Shaojia Anna
• Assistant Examiner(s): Lau, Jonathan S
• Application Number:
  US12/494,493
• Publication Number:
  US 20100331533A1
• Time in Patent Office: 1,680 Days
• Field of Search: 536/25.33, 548/335.1, 568/22, 568/25
• US Class Current: 536/25.33
• 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 methods employ a phenylacetyl disulfide reagent (known as PADS), phenylthioacetic acid (PTAA) in the presence or absence or N-alkyl imidazole in industrially preferred solvents or solvents that are derived from renewable resources. The use of PTAA eliminates the need to “age” the PADS solution prior to its use in sulfurization reactions.

Claims

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

combining a defined amount of phenylacetyl disulfide and a defined amount of phenylthioacetic acid, with a solvent to produce a composition that comprises at least 1% phenylthioacetic acid by volume and at least 0.1 M phenylacetyl disulfide; and

contacting said at least one phosphite or thiophosphite with said composition for a time sufficient to convert said at least one phosphite or thiophosphite to a phosphorothioate or phosphorodithioate internucleotide linkage,

wherein said composition is used in said contacting step immediately after preparing said composition.

2. The method of claim 1 wherein said solvent comprises a high dielectric constant solvent.

3. The method according to claim 1 wherein said combining step further comprises adding a N-alkyl imidazole.

4. The method of claim 3 wherein said N-alkyl imidazole is N-methyl imidazole.

5. The method of claim 3 wherein said solvent comprises toluene, xylene, 2-methyl THF, cyclopentyl methyl ether, acetonitrile or propylene carbonate.

6. The method of claim 3 wherein said solvent comprises toluene.

7. The method of claim 3 wherein said solvent comprises 2-methyl THF.

8. The method of claim 3 wherein said N-alkyl imidazole is at least 5% in volume (v/v) in said composition.

9. The method of claim 8 wherein said solvent comprises toluene, xylene, 2-methyl THF, cyclopentyl methyl ether, acetonitrile or propylene carbonate.

10. The method of claim 3 wherein said phosphite or thiophosphite is bound to a solid support.

11. The method of claim 10 wherein said solid support is controlled pore glass or polystyrene.

12. The method of claim 11 wherein said solid support is an array or beads.

13. The method of claim 10 wherein said N-alkyl imidazole is at least 5% in volume (v/v) in said composition.

14. The method of claim 13 wherein said defined amount of phenylacetyl disulfide is a concentration of 0.2 M, said N-alkylimidazole is at least 10% in volume (v/v) and said defined amount of phenylthioacetic acid is at least 2.5% in volume in said composition.

15. The method of claim 13 wherein said solvent comprises toluene, xylene, 2-methyl THF, cyclopentyl methyl ether, acetonitrile or propylene carbonate.

16. The method according to claim 14 wherein said solvent is toluene or 2-methyl THF.

17. The method of claim 1 wherein said defined amount of phenylacetyl disulfide is a concentration of at least 0.2 M.

18. The method of claim 1 wherein the phosphite or a thiophosphite is bound to a solid support.

19. The method of claim 2 wherein the phosphite or a thiophosphite is bound to a solid support.

20. The method of claim 18 wherein said defined amount of phenylacetyl disulfide is a concentration of at least 0.2 M.

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

22. The method of claim 21 wherein said solid support is an array or beads.

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

24. The method according to claim 19 wherein said defined amount of phenylacetyl disulfide is a concentration of at least 0.2 M.

25. The method of claim 1 wherein said oligonucleotide comprises one or more ribonucleotide.

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