Method of synthesizing polynucleotides using ionic liquids

• US 6,858,720 B2
• Filed: 10/31/2001
• Issued: 02/22/2005
• Est. Priority Date: 10/31/2001
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Perobost, Michel G. M., Dellinger, Douglas J., Myerson, Joel, Dellinger, Geraldine F
• Primary Examiner(s): Wilson, James O.
• Assistant Examiner(s): McIntosh, III, Traviss C.
• Application Number:
  US09/999,623
• Publication Number:
  US 20030083489A1
• Time in Patent Office: 1,210 Days
• Field of Search: 536/25.3, 536/25.33, 536/25.34, 536/22.1, 536/23.1, 536/25.4, 536/25.41
• 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 25% by weight of an ionic liquid.

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

3. The method of claim 2 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⁻

).

4. The method of claim 2 wherein the organic salt is characterized as being a liquid when being >

98% pure and at standard temperature and pressure.

5. The method of claim 2 wherein the cation is an N-substituted pyridine having the

wherein R is alkyl and each R′

is independently selected from hyrido, alkyl, or halogen group.

6. The method of claim 2 wherein the cation has the formula

wherein each R is independently selected from alkyl, each R′

is independently selected from hydrido, alkyl, or halogen, and R″

is selected from hydrido or methyl.

7. 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.

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

(a) obtaining a solution comprising the first nucleoside moiety and at least 25 percent by weight of an ionic liquid, and

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

9. The method of claim 8, further comprising

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

10. The method of claim 8 wherein the second nucleoside moiety is immobilized on a solid support.

11. The method of claim 8 wherein the ionic liquid is an organic salt comprising a substituted heterocyclic organic cation.

12. The method of claim 11 wherein the organic salt further comprises an anion selected from chloride (Cl⁻

), bromide (Br⁻

), tetrafluoroborate ([BF₄]⁻

), hexafluorophosphate ([PF_6]⁻

), [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⁻

).

13. The method of claim 11 wherein the organic salt is characterized as being a liquid when being >

98% pure and at standard temperature and pressure.

14. The method of claim 11 wherein the cation is an N-substituted pyridine having the formula

wherein R is alkyl and each R′

is independently selected from hyrido, alkyl, or halogen group.

15. The method of claim 11 wherein the cation has the formula

wherein each R is independently selected from alkyl, each R′

is independently selected from hydrido, alkyl, or halogen, and R″

is selected from hydrido or methyl.

16. The method of claim 8 wherein the ionic liquid is an organic salt comprising a cation selected from an N-substituted pyridine and a 1,3-disubstituted imidazole.