# US 7,411,061 B2 · Method of synthesizing polynucleotides using ionic liquids

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- Number: US 7,411,061 B2
- Document ID: US7411061B2
- Status: Granted
- Kind: B2
- Application number: 11/020,408
- Filed: 2004-12-22
- Published: 2005-05-19
- Granted: 2008-08-12
- Earliest claimed: 2001-10-31
- Assignees: Agilent Technologies, Inc.
- Inventors: Joel Myerson; Michel G. M. Perbost; Douglas J. Dellinger; Geraldine F Dellinger
- Web page: https://patentgrep.com/patent/US7411061B2

## CPC

- C07D 233/56: with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
- C07D 213/20: Quaternary compounds thereof
- C07D 231/12: with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
- C07D 233/54: having two double bonds between ring members or between ring members and non-ring members
- C07D 249/08: 1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
- 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
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)

## 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

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 5)

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−).

### Claim 7 (depends on 5)

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.

### Claim 8 (depends on 1)

The method of claim 1 wherein the organic salt is characterized as being liquid below 80° C. at standard atmospheric pressure.

### Claim 9 (depends on 5)

The method of claim 5 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.

### Claim 10 (depends on 5)

The method of claim 5 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.

### Claim 11 (depends on 1)

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.

### Claim 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.

### Claim 13 (depends on 12)

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

### Claim 14 (depends on 12)

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

### Claim 15 (depends on 12)

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

### Claim 16 (depends on 12)

The method of claim 12, further comprising
(c) contacting the internucleotide bond with an oxidizing reagent to oxidize the internucleotide bond.

### Claim 17 (depends on 12)

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

### Claim 18 (depends on 12)

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

### Claim 19 (depends on 18)

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−).

### Claim 20 (depends on 18)

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.

### Claim 21 (depends on 18)

The method of claim 18 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.

### Claim 22 (depends on 18)

The method of claim 18 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.

### Claim 23 (depends on 12)

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.

### Claim 24 (depends on 12)

The method of claim 12 wherein the organic salt is characterized as being liquid below 80° C. at standard atmospheric pressure.