# US 5,348,868 A · Methods and reagents for cleaving and deprotecting oligonucleotides

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- Number: US 5,348,868 A
- Document ID: US5348868A
- Status: Granted
- Kind: A
- Application number: 07/873,915
- Filed: 1992-04-24
- Granted: 1994-09-20
- Earliest claimed: 1992-04-24
- Assignees: Beckman Instruments, Inc.
- Inventors: Parameswara M. Reddy; Naeem B. Hanna
- Web page: https://patentgrep.com/patent/US5348868A

## CPC

- 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
- 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

Disclosed are reagents of methods for cleaving and deprotecting insolubilized and protected synthetic oligonucleotides. In a particularly preferred embodiment, the reagent comprises methylamine and t-butylamine.

## Claims

### Claim 1

A method for cleaving a support-bound oligonucleotide and/or removing at least one protecting group from a protected oligonucleotide, the method comprising the steps of:
a) introducing an oligonucleotide selected from the group consisting of
i) at least one insoluble, protected oligonucleotide;
ii) at least one soluble, protected oligonucleotide; and
iii) at least one insoluble oligonucleotide,
to a reagent to form a mixture, said reagent comprising methylamine; and
b) incubating said mixture for a sufficient time period at a sufficient temperature to obtain at least one biologically useful oligonucleotide.

### Claim 2 (depends on 1)

The method of claim 1 wherein said reagent further comprises at least one transamination suppression agent selected from the group consisting of straight-chain, branched, cyclic, saturated and unsaturated alkylamines having from between 1 and about 10 carbon atoms; straight-chain, branched, cyclic, saturated and unsaturated alkylamines having from between 1 and about 10 carbon atoms and comprising at least one functional group; ethanol; methanol; isopropylamine; acetylnitrile, dimethylformamide; tetrahydrofuran; and combinations of the foregoing.

### Claim 3 (depends on 2)

The method of claim 2 wherein said transamination suppression reagent is an alkylamine selected from the group consisting of t-butylamine, ethylamine, propylamine, isoprpoylamine, dimethylamine, diethylamine, trimethylamine and secondary butylamine.

### Claim 4 (depends on 1)

The method of claim 1 wherein said reagent further comprises t-butylamine.

### Claim 5 (depends on 1)

The method of claim 1 wherein said reagent further comprises aqueous ammonia.

### Claim 6 (depends on 1)

The method of claim 1 wherein said reagent further comprises aqueous ammonia.

### Claim 7 (depends on 1)

The method of claim 1 wherein said incubation time is less than about 100 minutes.

### Claim 8 (depends on 1)

The method of claim 1 wherein said incubation temperature is less than about 100.degree. C.

### Claim 9

A method for cleaving a support-bound oligonucleotide and/or removing at least one protecting group from a protected oligonucleotide, the method comprising the steps of:
a) introducing an oligonucleotide selected from the group consisting of
i) at least one insoluble, protected oligonucleotide;
ii) at least one soluble, protected oligonucleotide; and
iii) at least one insoluble oligonucleotide to a reagent to form a mixture, said reagent comprising methylamine and aqueous ammonia; and
b) incubating said mixture for a sufficient time period at a sufficient temperature to obtain at least one soluble oligonucleotide.

### Claim 10 (depends on 9)

The method of claim 9 further including transamination suppression reagent is selected from the group consisting of straight-chain, branched, cyclic, saturated and unsaturated alkylamines having from between 1 and about 10 carbon atoms; straight-chain, branched, cyclic, saturated and unsaturated alkylamines having from between 1 and about 10 carbon atoms and comprising at least one functional group; ethanol; methanol; isopropylamine; acetylnitrile, dimethylformamide; tetrahydrofuran; and combinations of the foregoing.

### Claim 11 (depends on 10)

The method of claim 10 wherein said transamination suppression reagent is an alkylamine selected from the group consisting of t-butylamine, ethylamine, propylamine, isoprpoylamine, dimethylamine, diethylamine, trimethylamine and secondary butylamine.

### Claim 12 (depends on 9)

The method of claim 9 wherein said reagent further comprises t-butylamine.

### Claim 13 (depends on 9)

The method of claim 9 wherein said incubation time is less than about 100 minutes.

### Claim 14 (depends on 9)

The method of claim 9 wherein said incubation temperature is less than about 100.degree. C.