Process for the preparation of phosphorothioate triesters

1. In process for the preparation of a phosphorothioate triester which comprises coupling an H-phosphonate with an alcohol in the presence of a coupling agent thereby to form an H-phosphonate diester, and subsequently, reacting the H-phosphonate diester with a sulfur transfer agent thereby to form a phosphorothioate triester, the improvement wherein the coupling reaction between the H-phosphonate and the alcohol is carried out in the presence of the sulfur transfer agent which is later used to form the triester.

2. A process according to claim 1 wherein the H-phosphonate is a protected nucleoside or oligonucleotide comprising a 3′-H-phosphonate function.

3. A process according to either of claims 1 and 2 , wherein the alcohol is a protected nucleoside or oligonucleotide comprising a free 5′-hydroxy function.

4. A process according to claim 1 , wherein the coupling agent is a diaryl phosphorochloridate of formula (ArO) 2 POCl, in which Ar represents phenyl, 2-chiorophenyl, 2,4,6-trichlorophenyl or 2,4,6-tribromophenyl.

5. A process according to claim 1 , wherein the sulfur transfer agent has the general chemical formula:

L-------S--------D

wherein L represents a leaving group, and D represents an aryl group, a methyl group, a substituted alkyl group or an alkenyl group.

6. A process according to claim 5 , wherein the leaving group is a morpholine-3,5-dione, phthalimide, succinimide, maleimide or indazole, and D represents a 4-halophenyl group, 4-alkylphenyl group, methyl group, benzyl group, alkylbenzyl group, halobenzyl group, allyl group, crotyl group, 2-cyanoethyl group or a 2-(4-nitrophenyl)ethyl group.

7. A process according to claim 1 , wherein the H-phosphonate and the alcohol are independently selected from the group consisting of deoxyribonucleosides, oligodeoxyribonucleosides, ribonucleosides, 2′-O-(alkyl, alkoxyalkyl or alkenyl)-ribonucleosides, oligoribonucleotides and 2′-O-(alkyl, alkoxyalkyl or alkenyl)-oligoribonucleotides.

8. A process according to claim 1 , wherein the H-phosphonate or the alcohol is linked to a solid support.

9. A process according to claim 8 , wherein an alcohol is linked to a solid support, the alcohol is a nucleoside or nucleotide having a free hydroxy group at the 5′-position, and is linked to the solid support via the 3′-position.

10. A process according to claim 8 or 9 , wherein an alcohol linked to a solid support is contacted with a solution comprising an H-phosphonate, a coupling agent and a sulfur transfer agent.

11. A process according to claim 1 , wherein the process is carried out in the solution phase.

12. A process according to claim 11 , wherein a solution comprising the coupling agent and sulfur transfer agent is added to a solution comprising the H-phosphonate and the alcohol.

13. A process according to claim 1 , wherein an oligonucleotide H-phosphonate and/or an oligonucleotide comprising a free 3′ or 5′-hydroxy function is employed and either or both of the oligonucleotide H-phosphonate and the oligonucleotide comprising a free 3′ or 5′-hydroxy function comprise one or more phosphorothioate internucleotide linkages.

14. A process according to claim 1 , wherein the phosphorothioate triester comprises from 2 to 8 nucleotide residues.

15. A process for the preparation of a deprotected oligonucleotide, oligonucleotide phosphorothioate or mixed oligonucleotide/oligonucleotide phosphorothioate which comprises:

a) coupling a protected nucleoside or oligonucleotide H-phosphonate comprising a 3′ or 5′-H-phosphonate function with a protected nucleoside or oligonucleotide comprising a free 3′ or 5′-hydroxy function in the presence of a coupling agent thereby to form an H-phosphonate diester and, in situ, reacting the H-phosphonate diester with a sulfur transfer agent to produce a phosphorothioate triester, wherein the coupling reaction between the H-phosphonate and the alcohol, occurs in the presence of the sulfur transfer agent; and

b) deprotecting the phosphorothioate triester produced in a) thereby to form a deprotected oligonucleotide, oligonucleotide phosphorothioate or mixed oligonucleotide, oligonucleotide/phosphorothioate.

16. A process according to claim 15 , wherein the deprotected oligonucleotide, oligonucleotide phosphorothioate or mixed oligonucleotide/oligonucleotide phosphorothioate is subsequently purified.

17. A process according to claim 15 , wherein the protected nucleoside or oligonucleotide H-phosphonate comprises a 3′-H-phosphonate function and the protected nucleoside or oligonucleotide comprising a free hydroxy function comprises a free 5′-hydroxy function.

18. A process according to claim 15 , wherein the coupling agent is a diaryl phosphorochloridate of formula (ArO) 2 POCl, in which Ar represents phenyl, 2-chlorophenyl, 2,4,6-trichiorophenyl or 2,4,6-tribromophenyl.

19. A process according to either of claims 15 or 16 , wherein the sulfur transfer agent has the general chemical formula:

L--------S--------D

wherein L represents a leaving group, and D represents an aryl group, a methyl group, a substituted alkyl group or an alkenyl group.

20. A process according to claim 19 , wherein the leaving group is a morpholine-3,5-dione, phthalimide, succinimide, maleimide or indazole, and D represents a 4-halophenyl group, 4-alkylphenyl group, methyl group, benzyl group, alkylbenzyl group, halobenzyl group, allyl group, crotyl group, 2-cyanoethyl group or a 2-(4-nitrophenyl)ethyl group.

21. A process according to claim 15 , wherein an oligonucleotide H-phosphonate and/or an oligonucleotide comprising a free 3′- or 5′-hydroxy function is employed and either or both of the oligonucleotide H-phosphonate and the oligonucleotide comprising a free 3′- or 5′-hydroxy function comprise one or more phosphorothioate internucleotide linkages.

22. A process for the synthesis of a deprotected oligonucleotide, said process comprising an assembly process in which a protected oligonucleotide is assembled, and a deprotection process in which the deprotected oligonucleotide is produced, the assembly process comprising coupling of a protected nucleoside or oligonucleotide H-phosphonate with a protected nucleoside or oligonucleotide comprising a free hydroxy function in the presence of a coupling agent thereby to form an H-phosphonate diester, characterised in that the coupling of the protected nucleoside or oligonucleotide H-phosphonate with the protected nucleoside or oligonucleotide comprising a free hydroxy function occurs in the presence of the sulfur transfer agent.

23. A process according to claim 22 , wherein the deprotected oligonucleotide is a phosphodiester oligonucleotide, phosphorothioate oligonucleotide or an oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages.

24. A process according to claim 22 , wherein the protected nucleoside or oligonucleotide H-phosphonate comprises a 3′- or 5′-H-phosphonate group.

25. A process according to claim 22 , wherein the protected nucleoside or oligonucleotide comprising a free hydroxy function comprises a free 3′- or 5′-hydroxy function.

26. A process according to claim 22 , wherein the oligonucleotide is subsequently purified.

27. A process for the preparation of an H-phosphonate diester, which comprises coupling an H-phosphonate with an alcohol in the presence of a coupling agent, characterised in that the coupling reaction between the H-phosphonate and the alcohol occurs in the presence of the sulfur transfer agent.

28. A process according to claim 27 , wherein the H-phosphonate is a protected nucleoside or oligonucleotide comprising a 3′-H-phosphonate function and the alcohol is a protected nucleoside or oligonucleotide comprising a free 5′-hydroxy function.

29. A process according to claim 8 , wherein the alcohol is linked to the solid support.

30. A process according to claim 14 , wherein the phosphorothioate triester comprises from 2 to 4 nucleotide residues.