Method of synthesizing polynucleotides using ionic liquids

• US 7,411,061 B2
• Filed: 12/22/2004
• Issued: 08/12/2008
• Est. Priority Date: 10/31/2001
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Dellinger, Douglas J., Myerson, Joel, Perbost, Michel G. M., Dellinger, Geraldine F
• Primary Examiner(s): Jiang; Shaojia Anna
• Assistant Examiner(s): McIntosh, III; Traviss C
• Application Number:
  US11/020,408
• Publication Number:
  US 20050106685A1
• Time in Patent Office: 1,329 Days
• Field of Search: None
• US Class Current: 536/25.3
• CPC Class Codes: C07D 213/20 Quaternary compounds thereof
• CPC Class Codes: C07D 231/12 with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
• CPC Class Codes: C07D 233/54 having two double bonds between ring members or between ring members and non-ring members
• CPC Class Codes: C07D 233/56 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
• CPC Class Codes: C07D 249/08 1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
• 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 method of synthesizing polynucleotides is disclosed. The method involves contacting a first nucleotide with a selected reactive group in the presence of an ionic liquid. The selected reactive group may be on a second nucleotide, a polynucleotide, or on a moiety on an insoluble substrate, for example in an oligonucleotide synthesizer.

Claims

1. A method of forming an internucleotide bond comprising contacting a free hydroxyl of a growing polynucleotide with a solution comprising a nucleotide monomer and at least 50% by weight of an ionic liquid.

2. The method of claim 1 wherein the solution includes at least 75% by weight of an ionic liquid.

3. The method of claim 1 wherein the solution includes at least 90% by weight of an ionic liquid.

4. The method of claim 1 wherein the solution includes at least 98% by weight of an ionic liquid.

5. The method of claim 1 wherein the ionic liquid is an organic salt comprising a substituted heterocyclic organic cation.

6. The method of claim 5 wherein the organic salt further comprises an anion selected from chloride (Cl⁻

), bromide (Br⁻

), tetrafluoroborate ([BF₄]⁻

), hexafluorophosphate ([PF₆]⁻

), [SbF₆]⁻

, [CuCl₂]⁻

, [AlCl₄]⁻

, [Al₂Cl₇]⁻

, [Al₃Cl₁₀]⁻

, methylsulfate (CH₃SO₄⁻

), trifluoroacetate (CF₃CO₂⁻

), heptafluorobutanoate (CF₃(CF₂)₂CO₂⁻

), triflate (CF₃SO₂⁻

), nonaflate (C₂F₅SO₂⁻

), bis(trifluoromethylsulfonyl)imide ((CF₃SO₂)₂N⁻

), bis(perfluoroethylsulfonyl)imide ((C₂F₅SO₂)₂N⁻

), and tris(trifluoromethylsulfonyl)methide ((CF₃SO₂)₃C⁻

).

7. The method of claim 5 wherein the organic salt is characterized as being a liquid when being >

98% pure and at standard temperature and pressure.

8. The method of claim 1 wherein the organic salt is characterized as being liquid below 80°

C. at standard atmospheric pressure.

9. The method of claim 5 wherein the cation is an N-substituted pyridine having the formula

10. The method of claim 5 wherein the cation has the formula

11. The method of claim 1 wherein the ionic liquid is an organic salt comprising a cation selected from an N-substituted pyridine and a 1,3-disubstituted imidazole.

12. A method of forming an intemucleotide bond between a first nucleoside moiety and a second nucleoside moiety, the method comprising

(a) dissolving the first nucleoside moiety in a solution comprising at least 50% by weight of an ionic liquid, and

(b) contacting the second nucleoside moiety with the product of (a) to form the internucleotide bond.

13. The method of claim 12 wherein the solution includes at least 75% by weight of an ionic liquid.

14. The method of claim 12 wherein the solution includes at least 90% by weight of an ionic liquid.

15. The method of claim 12 wherein the solution includes at least 98% by weight of an ionic liquid.

16. The method of claim 12, further comprising

(c) contacting the internucleotide bond with an oxidizing reagent to oxidize the internucleotide bond.

17. The method of claim 12 wherein the second nucleoside moiety is immobilized on a solid support.

18. The method of claim 12 wherein the ionic liquid is an organic salt comprising a substituted heterocyclic organic cation.

19. The method of claim 18 wherein the organic salt further comprises an anion selected from chloride (Cl⁻

), bromide (Br⁻

), tetrafluoroborate ([BF₄]⁻

), hexafluorophosphate ([PF₆]⁻

), [SbF₆]⁻

, [CuCl₂]⁻

, [AlCl₄]⁻

, [Al₂Cl₇]⁻

, [Al₃Cl₁₀]⁻

, methylsulfate (CH₃SO₄⁻

), trifluoroacetate (CF₃CO₂⁻

), heptafluorobutanoate (CF₃(CF₂)₂CO₂⁻

), triflate (CF₃SO₂⁻

), nonaflate (C₂F₅SO₂⁻

), bis(trifluoromethylsulfonyl)imide ((CF₃SO₂)₂N⁻

), bis(perfluoroethylsulfonyl)imide ((C₂F₅SO₂)₂N⁻

), and tris(trifluoromethylsulfonyl)methide ((CF₃SO₂)₃C⁻

).

20. The method of claim 18 wherein the organic salt is characterized as being a liquid when being >

98% pure and at standard temperature and pressure.

21. The method of claim 18 wherein the cation is an N-substituted pyridine having the formula

22. The method of claim 18 wherein the cation has the formula

23. The method of claim 12 wherein the ionic liquid is an organic salt comprising a cation selected from an N-substituted pyridine and a 1,3-disubstituted imidazole.

24. The method of claim 12 wherein the organic salt is characterized as being liquid below 80°

C. at standard atmospheric pressure.