Oligonucleotide synthesis with alternative solvents

• US 7,276,599 B2
• Filed: 06/02/2004
• Issued: 10/02/2007
• Est. Priority Date: 06/02/2003
• Status: Active Grant

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Krotz, Achim, Andrade, Mark, Moore, Max N., Scozzari, Anthony, Carty, Recaldo
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/858,917
• Publication Number:
  US 20050026192A1
• Time in Patent Office: 1,217 Days
• Field of Search: 536/23.1, 536/25.3, 536/25.34, 435/6
• US Class Current: 536/25.3
• 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
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

The invention provides for methods of manufacturing an oligonucleotide comprising a pentavalent phosphate triester. In particular, the method comprises providing a 5′ blocked-nucleoside, deblocking the 5′ blocked-nucleoside to form a 5′ OH-nucleoside, coupling the 5′ OH-nucleoside with a phosphoramidite to form and oligonucleotide comprising a trivalent phosphite triester; and oxidizing the oligonucleotide comprising a trivalent phosphite triester to the oligonucleotide comprising a pentavalent phosphate triester. In some embodiments, the wash between any of the steps above is with at least one solvent wash comprising a toluene.

Claims

1. A method of manufacturing an oligonucleotide comprising a pentavalent phosphate triester, said method comprising:

(a) providing a 5′

blocked-nucleoside;

(b) deblocking the 5′

blocked-nucleoside to form a 5′

OH-nucleoside;

(c) coupling the 5′

OH-nucleoside with a phosphoramidite of formula (IV)

2. The method of claim 1, wherein a wash between any of the steps above is with at least one solvent wash comprising a toluene.

3. The method of claim 1, wherein the 5′

blocked-nucleoside is linked to another nucleoside.

4. The method of claim 1, wherein the 5′

blocked-nucleoside is linked to a solid support.

5. The method of claim 1 wherein the 5′

blocked-nucleoside comprises a sugar moiety and a base.

6. The method of claim 1, wherein the solvent wash comprises a toluene and an acetonitrile.

7. The method of claim 1, wherein the solvent wash is substantially free of an acetonitrile.

8. A method of manufacturing a compound of Formula I:

9. The method of claim 8, wherein the first solvent wash comprises the toluene.

10. The method of claim 8, wherein the first solvent wash comprises the toluene and an acetonitrile.

11. The method of claim 8, wherein the first solvent wash is substantially free of an acetonitrile.

12. The process according to claim 8, wherein the second solvent wash comprises a toluene.

13. The method of claim 8, wherein the second solvent wash comprises the toluene and an acetonitrile.

14. The method of claim 8, wherein the second solvent wash is substantially free of an acetonitrile.

15. The method of claim 8, after (e), further comprising:

(f) contacting the compound of Formula I with a third solvent wash.

16. The method of claim 15, after (f), further comprising:

(g) capping any compound of Formula III that has not reacted with the compound of Formula IV.

17. The method of claim 15, wherein the third solvent wash comprises at least one of a toluene, an acetonitrile, or mixture thereof.

18. The method of claim 15, wherein the third solvent wash is substantially free of an acetonitrile.

19. The method of claim 8, after (e), further comprising capping any compound of Formula IV that has not reacted with the compound of Formula IV.

20. The process according to claim 8, wherein each Y is 0.

21. The process of claim 8, wherein n is about 7 to about 79.

22. The process of claim 8, wherein each R′

₂ is H or a 2′

-substituent.

23. The process of claim 8, wherein each R′

₂ is H or a 2′

-substituent, said 2′

-substituent being a member of the group consisting of 2′

-O—

CH₃, 2′

-O—

CH₂CH₂OCH₃, or 2′

-O—

(CH₂)₃NH₂.

24. The process of claim 8, wherein said 2′

-substituent is 2′

-OCH₂CH₂OCH₃.

25. The process of claim 8, wherein each q′

is 1.

26. The process of claim 8, wherein each G is S.

27. A process of claim 8, further comprising cleaving the compound of Formula I from the solid support to form a compound of Formula X: