Methods and reagents for cleaving and deprotecting oligonucleotides

• US 5,348,868 A
• Filed: 04/24/1992
• Issued: 09/20/1994
• Est. Priority Date: 04/24/1992
• Status: Expired due to Term

• Current Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Original Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Inventors: Reddy, Parameswara M., Hanna, Naeem B.
• Primary Examiner(s): Lilling, Herbert J.
• Application Number:
  US07/873,915
• Time in Patent Office: 879 Days
• Field of Search: 536/26.71, 536/25.31, 536/25.3, 435/91.1
• US Class Current: 435/91.1
• 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

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

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.

2. 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.

3. 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.

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

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

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

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

8. The method of claim 1 wherein said incubation temperature is less than about 100°

C.

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.

10. 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.

11. 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.

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

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

14. The method of claim 9 wherein said incubation temperature is less than about 100°

C.