Anhydrous amine cleavage of oligonucleotides

• US 5,750,672 A
• Filed: 11/22/1996
• Issued: 05/12/1998
• Est. Priority Date: 11/22/1996
• Status: Expired due to Fees

• Current Assignee: Barrskogen, Inc.
• Original Assignee: Barrskogen, Inc.
• Inventors: Kempe, Tomas
• Primary Examiner(s): Kunz, Gary L.
• Application Number:
  US08/755,398
• Time in Patent Office: 536 Days
• Field of Search: 536/25.4, 536/23.1, 536/25.31, 536/24.3
• US Class Current: 536/25.31
• CPC Class Codes: C07H 1/06 Separation; Purification

Abstract

A method for recovering synthesized oligonucleotides from a solid support that includes the step of incubating the solid support with an anhydrous amine reagent under conditions suitable to cleave and deprotect the oligonucleotide. The cleaved and deprotected oligonucleotide will be substantially insoluble in the reagent and/or will exhibit preferential affinity for the support. Reagent kits for use in such a method, and cleaved, deprotected oligonucleotides prepared by means of such a method are provided.

Claims

1. A method for recovering synthesized oligonucleotides from a solid support, the method comprising the steps of;

(1) providing a solid synthetic support having synthesized oligonucleotides bound thereto, and (2) incubating the solid support with an anhydrous amine reagent in an organic solvent solution under conditions suitable to cleave and deprotect the oligonucleotide, and (3) removing the solution phase reagent in a manner that substantially removes free residues of cleaved protecting groups and permits the cleaved and deprotected oligonucleotide to be preferentially retained on the support.

2. A method according to claim 1 comprising the further step of washing the support under conditions suitable to remove the reagent with the oligonucleotide retained on the support.

3. A method according to claim 1 comprising the further step of recovering the oligonucleotide from the support.

4. A method according to claim 1 comprising the further step of using the recovered oligonucleotide in situ.

5. A method according to claim 1 wherein the reagent is selected from the group consisting of primary alkylamines, secondary amines, and cyclic amines, as well as combinations thereof.

6. A method according to claim 5 wherein the reagent comprises a primary alkylamine selected from the group consisting of methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, and isomers thereof.

7. A method according to claim 5 wherein the reagent comprises a secondary amines selected from the group consisting of dimethylamine, ethylmethylamine, diethylamine, methylisopropylamine, and diisopropylamine.

8. A method according to claim 5 wherein the reagent comprises a cyclic amine selected from the group consisting of ethyleneimine, pyrrole, pyrrolidine, cyclohexylamine, benzylamine, and substituted derivatives thereof.

9. A method according to claim 5 wherein the reagent further comprises a nonpolar organic solvent.

10. A method according to claim 9 wherein the solvent is selected from the group consisting of tetrahydrofuran and acetonitrile.

18. A method according to claim 1 wherein the support is selected from the group consisting of controlled pore glass and polystyrene.

11. A composition comprising a solid support and a cleaved, deprotected synthetic oligonucleotide thereon, wherein the oligonucleotide has been cleaved and deprotected by incubating the solid support with an anhydrous amine reagent in an organic solvent solution under conditions suitable to cleave and deprotect the oligonucleotide, and wherein the solution phase reagent has been removed in a manner that substantially removes free residues of cleaved protecting groups and permits the cleaved and deprotected oligonucleotide to be preferentially retained on the support.

12. A composition according to claim 11 wherein the amine reagent is selected from the group consisting of primary alkylamines, secondary amines, and cyclic amines, as well as combinations thereof.

13. A composition according to claim 12 wherein the amine reagent comprises a primary alkylamine selected from the group consisting of methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, and isomers thereof.

14. A composition according to claim 12 wherein the reagent comprises a secondary amines selected from the group consisting of dimethylamine, ethylmethylamine, diethylamine, methylisopropylamine, and diisopropylamine.

15. A composition according to claim 12 wherein the reagent comprises a cyclic amine selected from the group consisting of ethyleneimine, pyrrole, pyrrolidine, cyclohexylamine, benzylamine, and substituted derivatives thereof.

16. A composition according to claim 12 wherein the reagent further comprises a nonpolar organic solvent.

17. A composition according to claim 16 wherein the solvent is selected from the group consisting of tetrahydrofuran and acetonitrile.

19. A composition according to claim 11 wherein the support is selected from the group consisting of controlled pore glass and polystyrene.