Solution phase synthesis of oligonucleotides

• US 7,019,127 B2
• Filed: 11/15/2002
• Issued: 03/28/2006
• Est. Priority Date: 08/13/1997
• Status: Expired due to Term

• Current Assignee: Nitto Denko Avecia, Inc. (Nitto Denko Corporation)
• Original Assignee: Avecia Limited
• Inventors: Song, Quanlai, Reese, Colin Bernard
• Primary Examiner(s): Wilson, James O.
• Assistant Examiner(s): Krishnan, Ganapathy
• Application Number:
  US10/294,794
• Publication Number:
  US 20030083490A1
• Time in Patent Office: 1,229 Days
• Field of Search: 435/91.1, 536/25.33, 536/25.6, 536/26.1, 536/23.1, 536/22.1, 536/25.3, 514/44
• US Class Current: 536/25.3
• CPC Class Codes: C07D 207/48 Sulfur atoms
• CPC Class Codes: C07D 265/33 Two oxygen atoms, in positions 3 and 5
• CPC Class Codes: C07F 9/165 Esters of thiophosphoric acids
• CPC Class Codes: C07F 9/18 with hydroxyaryl compounds
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

A process for the synthesis in solution phase of a phosphorothioate triester is provided. The process comprises the solution phase coupling of an H-phosphonate with an alcohol in the presence of a coupling agent to form an H-phosphonate diester. The H-phosphonate diester is oxidised in situ with a sulfur transfer agent to produce the phosphorothioate triester. Preferably, the H-phosphonate and alcohol are protected nucleosides or oligonucleotides. Oligonucleotide H-phosphonates which can be used in the formation of phosphorothioate triesters are also provided.

Claims

1. In an improved process for the synthesis of a deprotected oligonucleotide, said process comprising

assembling a protected phosphorothioate triester oligonucleotide,

deprotecting the phosphorothioate triester oligonucleotide to form a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide, or a deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages,

the improvement being that said assembling comprises

solution phase coupling, in the presence of a coupling agent,

(i) a protected nucleoside or oligonucleotide H-phosphonate comprising a 3′

- or 5′

H-phosphonate function;

with

(ii) a protected nucleoside or oligonucleotide comprising a free 3′

- or 5′

-hydroxy function;

to form an H-phosphonate diester and, in situ,

reacting the H-phosphonate diester with a sulfur transfer agent to produce said phosphorothioate triester.

2. The process of claim 1, further comprising purifying said deprotected phosphodiester oligonucleotide deprotected phosphorothioate oligonucleotide, or deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages.

3. The process of claim 1, comprising forming said deprotected phosphodiester oligonucleotide.

4. The process of claim 1, comprising forming said deprotected phosphorothioate oligonucleotide.

5. The process of claim 1, comprising forming said deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages.

6. A The process of claim 1, comprising coupling a protected nucleoside or oligonucleotide H-phosphonate comprising a 3′

-H-phosphonate function with a protected nucleoside or oligonucleotide comprising a free 5′

-hydroxy function.

7. The process of claim 1, wherein said coupling agent is a diaryl phosphorochloridate represented by formula (ArO)₂POCl, wherein Ar represents phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl, or 2,4,6-tribromophenyl.

8. The process of claim 1, wherein said sulfur transfer agent is represented by the following formula:

wherein

L represents a leaving group, and

A represents an aryl group, a methyl group, a substituted alkyl group or an alkenyl group.

9. The process or claim 8, wherein

L represents a morpholine-3,5-dione, a phthalimide, a succinimide, a maleimide, or an indazole, and

A represents a 4-halophenyl group, a 4-alkylphenyl group, a methyl group, a benzyl group, an alkylbenzyl group, a halobenzyl group, an ally group, a crotyl group, a 2-cyanoethyl group, or a 2-(4-nitrophenyl)ethyl group.

10. The process of claim 1, wherein said protected nucleoside or oligonucleotide H-phosphonate comprising a 3′

- or 5′

-H-phosphonate function and said protected nucleoside or oligonucleotide comprising a flee 3′

- or 5′

-hydroxy function are selected from the group consisting of deoxyribonucleosides, oligodeoxyribonucleotides, ribonucleosides, 2′

-O-(alkyl, alkoxyalkyl or alkenyl)-ribonucleosides, oligoribonucleotides, and 2′

-O-(alkyl, alkoxyslkyl or alkenyl)-oligoribonucleotides.

11. In an improved process for the synthesis of a deprotected oligonucleotide, said process comprising

assembling a protected phosphorothioate triester oligonucleotide,

deprotecting the phosphorothioate triester oligonucleotide to form a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide, or a deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages,

the improvement being that said assembling comprises

solution phase coupling

(i) a protected nucleoside or oligonucleotide H-phosphonate comprising a 3′

-H-phosphonate function;

with

(ii) a protected nucleoside or oligonucleotide comprising a free 5′

-hydroxy function;

to form an H-phosphonate diester and, in situ,

reacting the H-phosphonate diester with a sulfur transfer agent to produce said phosphorothioate triester

wherein said solution phase coupling is performed in the presence of a diaryl phosphorochloridate represented by formula (ArO)₂POCl, wherein Ar represents phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl, or 2,4,6-tribromophenyl, and

wherein said sulfur transfer agent is represented by the following formula

wherein

L represents a leaving group, and

A represents an aryl group, a methyl group, a substituted alkyl group or an alkenyl group.

12. The process of claim 11, comprising forming said deprotected phosphodiester oligonucleotide.

13. The process of claim 11, comprising forming said deprotected phosphorothioate oligonucleotide.

14. The process of claim 11, comprising forming said deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages.

15. The process of claim 11, wherein

L represents a morpholine-3,5-dione, a phthalimide, a succinimide, a maleimide, or an indazole, and

A represents a 4-halophenyl group, a 4-alkylphenyl group, a methyl group, a benzyl group, an alkylbenzyl group, a halobenzyl group, an ally group, a crotyl group, a 2-cyanoethyl group, or a 2-(4-nitrophenyl)ethyl group.

16. The process of claim 11, wherein said protected nucleoside or oligonucleotide H-phosphonate comprising a 3′

-H-phosphonate function and said protected nucleoside or oligonucleotide comprising a free 5′

-hydroxy function are selected from the group consisting of deoxyribonucleosides, oligodeoxyribonucleotides, ribonucleosides, 2′

-O-(alkyl, alkoxyalkyl or alkenyl)-ribonucleosides, oligoribonucleotides, and 2′

-O-(alkyl, alkoxyalkyl or alkenyl)-oligoribonucleotides.

17. The process of claim 11, further comprising purifying said deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide, or deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages.

20. The process of claim 15, wherein

L represents a morpholine-3,5-dione, a phthalimide, a succinimide, a maleimide, or an indazole, and

A represents a 4-halophenyl group, a 4-alkylphenyl group, a methyl group, a benzyl group, an alkylbenzyl group, a halobenzyl group, an ally group, a crotyl group, a 2-cyanoethyl group, or a 2-(4-nitrophenyl)ethyl group.

18. In an improved process for the synthesis of a purified, deprotected oligonucleotide, said process comprising

assembling a protected phosphorothioate triester oligonucleotide,

deprotecting the phosphorothioate triester oligonucleotide to form a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide, or a deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages,

purifying said deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide, or deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages,

the improvement being that said assembling comprises

solution phase coupling

(i) a protected nucleoside or oligonucleotide H-phosphonate comprising a 3′

-H-phosphonate function;

with

(ii) a protected nucleoside or oligonucleotide comprising a free 5′

-hydroxy function;

to form an H-phosphonate diester and, in situ,

reacting the H-phosphonate diester with a sulfur transfer agent to produce said phosphorothioate triester

wherein said solution phase coupling is performed in the presence of a diaryl phosphorochloridate represented by formula (ArO)₂POCl, wherein Ar represents phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl, or 2,4,6-tribromophenyl, and

wherein said sulfur transfer agent is represented by the following formula

wherein

L represents a leaving group, and

A represents an aryl group, a methyl group, a substituted alkyl group or an alkenyl group.

19. The process of claim 18, wherein said protected nucleoside or oligonucleotide H-phosphonate comprising a 3′

-H-phosphonate function and said protected nucleoside or oligonucleotide comprising a free 5′

-hydroxy function are selected from the group consisting of deoxyribonucleosides, oligodeoxyribonucleotides, ribonucleosides, 2′

-O-alkyl, alkoxyalkyl or alkenyl),ribonucleosides, oligoribonucleotides, and 2′

-O-(alkyl, alkoxyslkyl or alkenyl)-oligoribonucleotides.