Process for the preparation of phosphorothioate oligonucleotides

• US 7,227,017 B2
• Filed: 06/05/2003
• Issued: 06/05/2007
• Est. Priority Date: 12/05/2000
• Status: Expired due to Term

• Current Assignee: Nitto Denko Avecia, Inc. (Nitto Denko Corporation)
• Original Assignee: Avecia Limited
• Inventors: Wellings, Donald Alfred, Douglas, Mark Edward, Mellor, Ben James
• Primary Examiner(s): Jiang; S. Anna
• Assistant Examiner(s): CRANE, LAWRENCE E
• Application Number:
  US10/454,841
• Publication Number:
  US 20030229221A1
• Time in Patent Office: 1,461 Days
• Field of Search: 536/25.3, 536/25.33, 536/25.34
• US Class Current: 536/25.34
• 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

A process for the synthesis of phosphorothioate oligonucleotides is provided which comprises assembling an oligonucleotide bound to a solid support in the presence of acetonitrile; prior to cleaving the oligonucleotide from the solid support removing the acetonitrile; and cleaving the oligonucleotide from the solid support. The process is particularly suited to the large scale synthesis of nucleotides. The acetonitrile may be removed from the solid support by one or both of drying and by washing with solvents. Preferred washing solvents comprise trialkylamines.

Claims

1. In an improved process for the large-scale synthesis of phosphorothioate oligonucleotides comprising:

a) assembling by phosphoramidite chemistry a phosphorothioate oligonucleotide bound to a solid support in the presence of acetonitrile;

b) reduction of the content of acetonitrile in the solid support; and

c) cleaving the oligonucleotide from the solid support;

wherein the improved process reduces the concentration of acetonitrile to less than 10% by weight of the oligonucleotide plus solid support.

2. The improved process according to claim 1, wherein the process reduces the concentration of acetonitrile to less than 5% by weight of the oligonucleotide plus solid support.

3. The improved process according to claim 2, wherein the process reduces the concentration of acetonitrile to less than 1% by weight of the oligonucleotide plus solid support.

4. The improved process according to claim 1, which is operated at or above a batch size of 10 mmol of oligonucleotide.

5. The improved process according to claim 1, wherein the oligonucleotide is bound to the solid support via a cleavable linker selected from the group consisting of an urethanyl linker an oxalyl linker and a succinyl linkes

6. The improved process according to claim 1 wherein the oligonucleotide is cleaved from the solid support by contact with a cleaving reagent, said cleavage reagent comprising methylamine, aqueous methylamine solution, liquified ammonia, gaseous ammonia or concentrated aqueous ammonia solution.

7. The improved process according to claim 1 which is employed to prepare oligonucleotides having from 1 to 100 internucleoside linkages.

8. The improved process according to claim 7, which is employed to prepare compounds wherein at least 50% of the internucleoside linkages are phosphorothioate linkages.

9. The improved process according to claim 8, wherein 90 to 100% of the internucleoside linkages are phosphorothioate linkages.

28. The process according to claim 9, wherein the process reduces the concentration of acetonitrile to less than 1% by weight of the oligonucleotide plus solid support, and which is operated at or above a batch size of 10 mmol of oligonucleotide.

31. The improved process according to claim 1, wherein the phosphoramidite chemistry comprises the reaction of betacyanoethyloxy phosphoramidites.

34. The process according to claim 28, wherein the phosphoramidite chemistry comprises the reaction of betacyanoethyloxy phosphoramidites.

10. An improved process for the synthesis of phosphorothioate nucleotides comprising:

a) assembling by phosphoramidite chemistry a phosphorothoate oligonucleotide bound to a solid support in the presence of acetonitrile;

b) prior to cleaving the oligonucleotide from the solid support, washing the oligonucleotide bound to the solid support with a washing regime employing one or more solvent washes to reduce the acetonitrile concentration, wherein the final wash of the washing regime employs a solvent selected from the group consisting of aromatic hydrocarbons, aliphatic hydrocarbons, haloalkanes, esters, alcohols, amides, pyridine, alkylamines, polar ethers, sulphoxides, water, aqueous buffer solutions and mixtures of water and water miscible esters, alcohols, amides, pyridine, alkylamines, polar ethers or sulphoxides; and

c) cleaving the oligonucleotide from the solid support.

11. The process according to claim 10, wherein the solvent employed in the final wash of the washing regime is selected from the group consisting of toluene, cyclohexane, dichloromethane, ethyl acetate, methyl propionate, ethyl propionate, C_1-4 alkyl alcohols, dimethylformamide N-methylpyrrolidinone, tri(C_1-4-alkyl)amines, tetrahydrofuran, dimethylsulphoxide and aqueous sodium acetate solution.

12. The process according to claim 11, wherein the solvent is triethylamine.

13. The process according to claim 10, wherein the oligonucleotide is bound to the solid support via a cleavable linker selected from the group consisting of a urethanyl linker an oxalyl linker, and a succinyl linker.

14. The process according to claim 13, wherein the oligonucleotide is cleaved from the solid support by contact with a cleaving reagent, said cleaving reagent comprising methylamine, aqueous methylamine solution, liquified ammonia, gaseous ammonia or concentrated aqueous ammonia solution.

15. The process according to claim 10, which is employed to prepare oligonucleotides having from 1 to 100 internucleoside linkages.

16. The process according to claim 15, which is employed to prepare compounds wherein at least 50% of the internucleoside linkages are phosphorothioate linkages.

17. The process according to claim 16, wherein 90 to 100% of the internucleoside linkages are phosphorothioate linkages.

29. The process according to claim 17, wherein the process reduces the concentration of acetonitrile to less than 1% by weight of the oligonucleotide plus solid support, and which is operated at or above a batch size of 10 mmol of oligonucleotide.

32. The process according to claim 10, wherein the phosphoramidite chemistry comprises the reaction of betacyanoethyloxy phosphoramidites.

35. The process according to claim 29, wherein the phosphoramidite chemistry comprises the use of betacyanoethyloxy phosphoramidites.

18. An improved process for the synthesis of phosphorothioate nucleotides comprising:

a) assembling by phosphoramidite chemistry a phosphorothoate oligonucleotide bound to a solid support in the presence of acetonitrile;

b) prior to cleaving the oligonucleotide from the solid support, washing the oligonucleotide bound to the solid support with a washing regime employing one or more solvent washes to reduce the acetonitrile concentration, wherein the final wash of the washing regime employs as solvent a solution comprising a trialkylamine; and

c) cleaving the oligonucleotide from the solid support.

19. The process according to claim 18, wherein the solvent is triethylamine.

33. The process according to claim 18, wherein the phosphoramidite chemistry comprises the reaction of betacyanoethyloxy phosphoramidites.

20. An improved process for the synthesis of phosphorothioate nucleotides comprising assembling by phosphoramidite chemistry a phosphorothoate oligonucleotide bound to a solid support in the presence of acetonitrile;

air drying the supported oligonucleotide for sufficient time to reduce the concentration of acetonitrile;

contacting the dried supported oligonucleotide with a trialkylamine for sufficient time to deprotect the the oligonucleotide, and subsequently cleaving the oligonucleotide from the solid support.

21. The process according to claim 20, wherein the process reduces the concentration of acetonitrile to less than 5% by weight of the oligonucleotide plus solid support.

22. The process according to claim 21, wherein the process reduces the concentration of acetonitrile to less than 1% by weight of the oligonucleotide plus solid support.

23. The process according to claim 20, wherein the oligonucleotide is bound to the solid support via a cleavable linker selected from the group consisting of a urethanyl linker an oxalyl linker, and a succinyl linker.

24. The process according to claim 23, wherein the oligonucleotide is cleaved from the solid support by contact with acleaving reagent, said cleavage reagent comprising methylamine, aqueous methylamine solution, liquified ammonia, gaseous ammonia or concentrated aqueous ammonia solution.

25. The process according to claim 20, which is employed to prepare oligonucleotides having from 1 to 100 internucleoside linkages.

26. The process according to claim 25, which is employed to prepare compounds wherein at least 50% of the internucleoside linkages are phosphorothioate linkages.

27. The process according to claim 26, wherein 90 to 100% of the intemucleoside linkages are phosphorothioate linkages.

30. The process according to claim 27, wherein the process reduces the concentration of acetonitrile to less than 1% by weight of the oligonucleotide plus solid support, and which is operated at or above a batch size of 10 mmol of oligonucleotide.

36. The process according to claim 30, wherein the phosphoramidite chemistry comprises the reaction of betacyanoethyloxy phosphoramidites.

37. The process according to claim 20, wherein the trialkylamine is triethylamine.