Solution phase synthesis of oligonucleotides

• US 6,506,894 B1
• Filed: 04/26/2000
• Issued: 01/14/2003
• 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): McGarry, Sean
• Assistant Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/485,606
• Time in Patent Office: 993 Days
• Field of Search: 536/25.33, 536/27.21, 536/25.6, 536/26.1, 536/23.1, 536/22.1, 514/44, 435/6, 435/91.1
• 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 oxidized 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. A process for the preparation of a phosphorothioate triester which comprises the solution phase coupling of an H-phosphonate with an alcohol in the presence of a coupling agent thereby to form an H-phosphonate diester and, in situ, reacting the H-phosphonate diester with a sulfur transfer agent to produce a phosphorothioate triester.

2. A process according to claim 1 wherein the H-phosphonate is a protected nucleoside or oligonucleotide comprising a 3′

-H-phosphonate function.

3. A process according to either of claims 1 and 2, wherein the alcohol is a protected nucleoside or oligonucleotide comprising a free 5′

-hydroxy function.

4. A process according to claim 1, wherein the coupling agent is a diaryl phosphorochloridate of formula (ArO)₂POCl, in which Ar represents phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl or 2,4,6-tribromophenyl.

5. A process according to claim 1, wherein the sulfur transfer agent has the general chemical formula:

6. A process according to claim 5, wherein the leaving group is a morpholine-3,5-dione, phthalimide, succinimide, maleimide or indazole, and A represents a 4-halophenyl group, 4-alkylphenyl group, methyl group, benzyl group, alkylbenzyl group, halobenzyl group, allyl group, crotyl group, 2-cyanoethyl group or a 2-(4-nitrophenyl)ethyl group.

7. A process according to claim 1, wherein the H-phosphonate and the alcohol 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.

9. An H-phosphonate according to claim 6, wherein W represents a protecting group, each X represents O, and each R represents a methyl group, a benzyl group, a 2-cyanoethyl group, an unsubstituted phenyl group or a 4-halophenyl group, M+ represents a tri(C_1-4-alkylammonium) ion, and n is 1 to 16.

10. An H-phosphonate according to either of claim 9 or 10, wherein n is 1, 2 or 3.

11. A process according to claim 1, wherein the coupling agent is a diaryl phosphorochloridate of formula (ArO)₂POCl, in which Ar represents phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl or 2,4,6-tribromophenyl;

and the sulfur transfer agent has the general chemical formula;

12. A process according to claim 11, wherein the leaving group is a morpholine-3,5-dione, phthalimide, succinimide, maleimide or indazole, and A represents a 4-halophenyl group, 4-alkylphenyl group, methyl group, benzyl group, alkylbenzyl group, halobenzyl group, allyl group, crotyl group, 2-cyanoethyl group or a 2-(4-nitrophenyl)ethyl group.

13. A process according to claim 11 or 12, wherein the H-phosphonate and the alcohol are each independently a deoxyribonucleoside, an oligodeoxyribonucleotide, a ribonucleoside, a 2′

-O-(alkyl, alkoxyalkyl or alkenyl)-ribonucleoside, an oligoribonucleotide or a 2′

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

22. A process according to claim 2, wherein the H-phosphonate comprising a 3′

H-phosphonate function is a protected oligonucleotide H-phosphonate comprising at least one phosphorothioate internucleotide linkage.

23. A process according to claim 3, wherein the alcohol comprising a free 5′

-hydroxy function is an oligonucleotide comprising a, least one phosphorothioate internucleotide linkage.

24. A process according to claim 23, wherein the H-phosphonate is a protected oligonucleotide H-phosphonate comprising a 3′

H-phosphonate function and also comprises at least one phosphorothioate internucleotide linkage.

8. An H-phosphonate having the general chemical formula:

wherein

each B independently is a base selected from A, G, T, C or U;

each Q independently is H or OR′

wherein R′

is alkyl, substituted alkyl, alkenyl or a protecting group;

each R independently is an aryl, methyl, substituted alkyl or alkenyl group;

W is H, a protecting group or an H-phosphonate group of formula

 

in which

M⁺ is a monovalent cation;

each X independently represent O or S;

each Y represents S;

Z is H, a protecting group or an H-phosphonate group of formula

 

in which

M⁺ is a monovalent cation; and

n is an integer;

provided that when W is H or a protecting group, that Z is a positive H-phosphonate group, and that when Z is H or a protecting group, that W is an H-phosphonate group.

14. A process for the preparation of a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide or a deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages which comprises:

(a) solution phase coupling of a protected nucleoside or oligonucleotide H-phosphonate comprising a 3′

or 5′

-H-phosphonate function with a protected nucleoside or oligonucleotide comprising a free 3′

or 5′

-hydroxy function in the presence of a coupling agent thereby to form an H-phosphonate diester and, in situ, reacting the H-phosphonate diester with a sulfur transfer agent to produce a phosphorothioate triester; and

(b) deprotecting the phosphorothioate triester produced in (a) thereby to form a deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide or a deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages.

15. A process according to claim 14, wherein the protected nucleoside or oligonucleotide H-phosphonate comprises a 3′

H-phosphonate function and the protected nucleoside or oligonucleotide comprises a free 5′

-hydroxy function.

16. A process according to claim 14 or 15, wherein the coupling agent is a diaryl phosphorochloridate of formula (ArO)₂POCl, in which Ar represents phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl or 2,4,6-tribromophenyl.

17. A process according to claim 14 or 15, wherein the sulfur transfer agent has the general chemical formula:

18. A process according to claim 17, wherein the leaving group is a morpholine-3,5-dione, phthalimide, succinimide, maleimide or indazole, and A represents a 4-halophenyl group, 4-alkylphenyl group, methyl group, benzyl group, alkylbenzyl group, halobenzyl group, allyl group, crotyl group, 2-cyanoethyl group or a 2-(4-nitrophenyl)ethyl group.

19. A process according to claim 14 or 15, wherein an oligonucleotide H-phosphonate and/or an oligonucleotide comprising a free 3′

or 5′

-hydroxy function is employed and either or both of the oligonucleotide H-phosphonate and the oligonucleotide comprising a free 3′

or 5′

-hydroxy function comprise one or more phosphorothioate internucleotide linkages.

20. A process according to claim 14 or 15, wherein the deprotected phosphodiester oligonucleotide, deprotected phosphorothioate oligonucleotide or a deprotected oligonucleotide comprising both phosphodiester and phosphorothioate diester internucleotide linkages is subsequently purified.

21. A process according to claim 20, wherein a deprotected phosphorothioate oligonucleotide is produced.