Process

• US 6,768,005 B2
• Filed: 12/20/2000
• Issued: 07/27/2004
• Est. Priority Date: 12/20/2000
• Status: Expired due to Term

• Current Assignee: Nitto Denko Avecia, Inc. (Nitto Denko Corporation)
• Original Assignee: Avecia Limited
• Inventors: Douglas, Mark Edward, Wellings, Donald Alfred, Mellor, Ben James
• Primary Examiner(s): McGarry, Sean
• Assistant Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/740,031
• Publication Number:
  US 20020111479A1
• Time in Patent Office: 1,315 Days
• Field of Search: 536/23.1, 536/25.3, 536/25.34, 435/91.1, 435/72, 435/6
• US Class Current: 536/25.3
• 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 process for the synthesis of phosphorothioate oligonucleotides is provided which comprises assembling an oligonucleotide bound to a solid support in the presence of acetonitrile; prior to cleaving the oligonucleotide from the solid support removing the acetonitrile; and cleaving the oligonucleotide from the solid support. The process is particularly suited to the large scale synthesis of nucleotides.

Claims

1. A process for the preparation of an oligonucleotide which comprises coupling a nucleoside or oligonucleotide phosphoramidite with a nucleoside or oligonucleotide comprising a free hydroxy group supported on a solid support to form an oligonucleotide phosphite triester, wherein the solid support is a poly(acrylamide) microporous support.

2. A process according to claim 1, wherein the solid support is an amine-functionalized support derived from a polymer prepared by copolymerisation of acryloyl-sarcosine methyl ester, N,N-dimethylacrylamide and bis-acryloylethylenediamine.

3. A process according to claim 2, wherein the amine-functionalized support comprises a primary amine functionality derived from reaction of the methyl ester group with an alkyl diamine, preferably ethylene diamine.

4. A process according to claim 1, wherein the oligonucleotide is bound to the solid support via a cleavable linker selected from the group consisting of urethane, oxalyl, succinyl, and amino-derived linkers.

5. A process according to claim 1, which is employed to prepare oligonucleotides having from 1 to 100 internucleoside linkages.

6. A process according to claim 1, which is employed to prepare compounds wherein at least 50% of the internucleoside linkages are phosphorothioated.

7. A process according to claim 6, wherein 90 to 100% of the internucleoside linkages are phosphorothioated.

8. A process according to claim 3, wherein the alkyl diamine is ethylene diamine.

9. A process according to claim 1, wherein the oligonucleotide is cleaved from the solid support by contact with a cleaving reagent.

10. A process according to claim 9 wherein the cleavage reagent comprises methylamine, aqueous methylamine solution, liquefied ammonia, gaseous ammonia or concentrated aqueous ammonia solution.