Methods for preparing oligonucleotides having chiral phosphorothioate linkages

• US 20060041115A1
• Filed: 03/15/2005
• Published: 02/23/2006
• Est. Priority Date: 06/14/2001
• Status: Abandoned Application

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Ravikumar, Vasulinga
• Application Number:
  US11/081,880
• Publication Number:
  US 20060041115A1
• Time in Patent Office: Days
• US Class Current: 536/25.300
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Abstract

Methods are provided for preparing internucleotide phosphorothioate linkages that are enriched in the Sp or Rp enantiomer comprising coupling a synthon with a 2′-substituted nucleoside in the presence of coupling agent that is selected to enhance either the Rp or Spenantiomer according to its pKa.

Claims

1. A method for preparing an internucleotide phosphorothioate linkage enriched in the Rp enantiomer between a synthon having a hydroxyl moiety at the 5′

position and a 2′

-substituted nucleoside having an activated phosphate moiety at the 3′

-position comprising selecting a coupling agent having a pKa ranging from about 3.3 to about 4.5 and coupling said synthon to said 2′

-substituted nucleoside in the presence of said coupling agent.

2. The method of claim 1 wherein said synthon is bound to a support.

3. The method of claim 1 wherein the synthon having a hydroxyl moiety at the 5′

position is a deoxyribonucleotide, a ribonucleotide, a 2′

-protected ribonucleotide or a 2′

-substitued ribonucleotide.

8. The method of claim 1 wherein said coupling agent is 5-(ethylthio)-1H-tetrazole.

10. The method of claim 1 wherein said 2′

-substituted nucleoside comprises a 2′

-substituent, said 2′

-substituent is O-alkyl, O-PG, O(CH₂)_nOCH₃, or O[(CH₂)_nO]_mCH₃, wherein PG is a hydroxyl protecting group and n and m are from about 1 to about 10.

11. The method of claim 10 wherein said 2′

substituent is 2′

-O—

C₁ to C₁₂ alkyl.

13. The method of claim 11 wherein said C₁ to C₁₂ alkyl group is methyl.

15. The method of claim 10 wherein said 2′

-substituent is O(CH₂)₂OCH₃.

16. The method of claim 1 wherein said activated phosphate moiety comprises either a B-cyanoethyl protecting group or an acetoxy phenoxy ethyl group.

4. (canceled)

5. (canceled)

6. (canceled)

7. (canceled)

9. (canceled)

12. (canceled)

14. (canceled)

17. (canceled)

18. A method for preparing an internucleotide phosphorothioate linkage enriched in the Sp enantiomer between a synthon having a hydroxyl moiety at the 5′

position and a 2′

-substituted nucleoside having an activated phosphate moiety at the 3′

-position comprising selecting a coupling agent having a pKa ranging from about 6.0 to about 8.0 and coupling said synthon to said 2′

-substituted nucleoside in the presence of said coupling agent.

19. The method of claim 18 wherein said synthon is bound to a support.

20. The method of claim 18 wherein the synthon having a hydroxyl moiety at the 5′

position is a deoxyribonucleotide, a ribonucleotide, a 2′

-protected ribonucleotide or a 2′

-substitued ribonucleotide.

26. The method of claim 18 wherein said coupling agent is an imidazolium or pyridinium derivative or salt thereof.

28. The method of claim 26 wherein said coupling agent is imidazolium trifluoroacetate, imidazolium triflate, imidazolium perchlorate, imidazolium acetate, imidazolium tosylate or imidazolium nitrate.

29. The method of claim 26 wherein said coupling agent is pyridinium trifluoroacetate, pyridinium triflate, pyridinium perchlorate, pyridinium acetate, pyridinium tosylate or pyridinium nitrate.

31. The method of claim 18 wherein said 2′

-substituted nucleoside comprises a 2′

-substituent said 2′

-substituent is O-alkyl, O-PG, O(CH₂)_nOCH₃, or O[(CH₂)_nO]_mCH₃, wherein PG is a hydroxyl protecting group and n and m are from about 1 to about 10.

32. The method of claim 31 wherein said 2′

substituent is 2′

-O—

C₁ to C₁₂ alkyl.

34. The method of claim 32 wherein said C₁ to C₁₂ alkyl group is methyl.

36. The method of claim 31 wherein said 2′

-substituent is O(CH₂)₂OCH₃.

37. The method of claim 18 wherein said activated phosphate moiety comprises either a B-cyanoethyl protecting group or an acetoxy phenoxy ethyl group.

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

27. (canceled)

30. (canceled)

33. (canceled)

35. (canceled)

38-47. -47. (canceled)