Process for preparing phosphorothioate oligonucleotides

• US 5,644,048 A
• Filed: 12/18/1992
• Issued: 07/01/1997
• Est. Priority Date: 01/10/1992
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Yau, Eric K.
• Primary Examiner(s): Kunz, Gary L.
• Application Number:
  US07/993,115
• Time in Patent Office: 1,656 Days
• Field of Search: 536/25.3, 536/25.31, 536/25.33, 536/25.34
• US Class Current: 536/25.3
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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 preparing phosphorothioate oligonucleotides utilizes S-(alkaryl or aryl) phosphorothioate compounds as intermediates suitable for use in the solution phase. A dinucleotide is prepared from a nucleoside intermediate that has been phosphitylated with an S-(alkaryl or aryl) alkyl phosphorothioate diester salt. The alkyl ester group is removed and the resulting nucleotide diester is reacted with a further nucleoside having a free 5'"'"'-hydroxyl group. Nucleotide units are linked together to yield compounds of the structure: ##STR1## where X is H, a first blocking group, a nucleoside, a nucleotide or an oligonucleotide; Y is H, a second blocking group, a nucleoside, a nucleotide or an oligonucleotide; R is an alkaryl or aryl group; n is an integer greater than 0; and Bx is a heterocyclic base. Deblocking of the S-alkaryl or S-aryl moiety yields a phosphorothioate oligonucleotide.

Claims

1. A process for preparing phosphorothioate oligonucleotides, comprising the steps of:

(a) reacting PSCl₃ with an alkyl alcohol in the presence of base to form a phosphorus-containing alkoxy intermediate, said alkoxy intermediate having an acidic proton located on a carbon atom in a β

-position relative to said phosphorus atom, said alkoxy intermediate having the structure;

##STR9## where R'"'"' is Cl or alkoxy, and R" is alkyl;

(b) hydrolyzing said alkoxy intermediate to form a hydrolyzed alkoxy intermediate of the structure;

##STR10## where R" is as above, and R"'"'"' is OH or alkoxy;

(c) reacting said hydrolyzed alkoxy intermediate with an alkaryl halide or an aryl halide to form a first reaction intermediate of the structure;

##STR11## where;

R is an aryl or alkaryl group;

R" is as above; and

M is a cation;

(d) reacting said first reaction intermediate with a nucleoside having a 3'"'"'-hydroxyl moiety, a nucleotide having a 3'"'"'-hydroxyl moiety, or an oligonucleotide having a 3'"'"'-hydroxyl moiety in the presence of an activating agent to form a second reaction intermediate of the structure;

##STR12## where R and R" are as above, and Y'"'"' is said nucleoside, nucleotide or oligonucleotide; and

(e) reacting said second reaction intermediate with a nucleoside having a 5'"'"'-hydroxyl moiety, a nucleotide having a 5'"'"'-hydroxyl moiety or an oligonucleotide having a 5'"'"'-hydroxyl moiety in the presence of an activating agent to form an ester linkage between an S-(alkaryl or aryl) alkyl phosphorothioate moiety of said second reaction intermediate and said 5'"'"'-hydroxyl moiety.

2. The process of claim 1 further including reacting the product of step (e) with a nucleoside 3'"'"'-S-(alkaryl or aryl) phosphorothioate, a nucleotide 3'"'"'-S-(alkaryl or aryl) phosphorothioate or an oligonucleotide 3'"'"'-S-(alkaryl or aryl) phosphorothioate.

3. The process of claim 1 including selecting α

,2,4-trichlorotoluene as said alkaryl or aryl halide.

4. The process of claim 1 including selecting triisopropylbenzenesulfonylchloride as said activating agent.

5. The process of claim 4 further including adding a nucleophilic catalyst to said activating agent.

6. The process of claim 5 including selecting 1-methylimidazole as said nucleophilic catalyst.

7. The process of claim 1 including selecting β

-cyanoethanol as said alkyl alcohol.

8. The process of claim 1 further including cleaving an S-(alkaryl or aryl) bond of the product of step (e) to form a phosphorothioate diester linkage.

9. A process for preparing a phosphorothioate internucleoside linkage, comprising the steps of:

reacting 5'"'"'-blocked nucleosides with an S-(alkaryl or aryl) alkyl phosphorothioate diester salt of the structure;

##STR13## to form phosphitylated nucleosides of the structure;

##STR14## where;

R is an aryl or alkaryl group;

X is a blocking group;

Y'"'"' is a 5'"'"'-blocked nucleoside; and

M is a cation;

removing the 5'"'"'-blocking group from one of said phosphitylated nucleosides;

removing said X blocking group from a second of said phosphitylated nucleosides; and

reacting said deblocked phosphitylated nucleoside with said 5'"'"'-deblocked phosphitylated nucleoside in the presence of an activating agent to form a dinucleoside having an S-(alkaryl or aryl) phosphorothioate internucleoside linkage.

10. The process of claim 9 further including cleaving an S-(alkaryl or aryl) bond of said S-(alkaryl or aryl) phosphorothioate internucleoside linkage to form a dinucleoside having a phosphorothioate internucleoside linkage.

11. The process of claim 9 wherein said alkaryl or aryl moiety of said S-(alkaryl or aryl) alkyl phosphorothioate diester salt is 2,4-dichlorobenzyl.

12. The process of claim 9 wherein said alkyl moiety of S-(alkaryl or aryl) alkyl phosphorothioate diester salt is β

-cyanoethyl.

13. The process of claim 10 further including cleaving said S-(alkaryl or aryl) bond with thiophenolate.

14. A process for preparing a phosphorothioate oligonucleotide, comprising the steps of:

(a) hydrolyzing PSCl₃ ;

(b) reacting said hydrolyzed PSCl₃ with an alkaryl halide or an aryl halide to form a first reaction intermediate of the structure;

##STR15## where;

R is an aryl or alkaryl group;

X is H or a blocking group; and

M is a cation;

(c) reacting said first reaction intermediate with a compound selected from the group consisting of a nucleoside having a 3'"'"'-hydroxyl moiety, a nucleotide having a 3'"'"'-hydroxyl moiety, and a oligonucleotide having a 3'"'"'-hydroxyl moiety in the presence of an activating agent to form a second reaction intermediate of the structure;

##STR16## where R and X are as above, and Y'"'"' is a nucleoside, nucleotide or oligonucleotide; and

(d) reacting said second reaction intermediate with a compound selected from the group consisting of a nucleoside having a 5'"'"'-hydroxyl moiety, a nucleotide having a 5'"'"'-hydroxyl moiety and an oligonucleotide having a 5'"'"'-hydroxyl moiety in the presence of an activating agent to form an ester linkage between an S-(alkaryl or aryl) phosphorothioate moiety of said second reaction intermediate and said 5'"'"'-hydroxyl moiety.

15. The process of claim 14 wherein said alkaryl or aryl halide is a chloro-substituted benzyl halide.

16. The process of claim 14 wherein said alkaryl or aryl halide is a dichlorobenzyl halide.

17. The process of claim 14 wherein said alkaryl or aryl halide is α

,2,4-trichlorotoluene.

18. The process of claim 14 wherein said activating agent is triisopropylbenzenesulfonylchloride.

19. The process of claim 18 further including adding a nucleophilic catalyst to said activating agent.

20. The process of claim 19 wherein said nucleophilic catalyst is 1-methylimidazole.

21. The process of claim 14 wherein said PSCl₃ is hydrolyzed with aqueous tertiary alkylamine.

22. The process of claim 21 wherein said aqueous tertiary alkylamine is triethylamine or diisopropylethyl amine.

23. A process for preparing a phosphorothioate oligonucleotide, comprising the steps of:

(a) reacting PSCl₃ with an alkaryl halide or an aryl halide to form a first reaction intermediate of the structure;

##STR17## where;

R is an aryl or alkaryl group;

(b) reacting said first reaction intermediate with a compound selected from the group consisting of a 5'"'"'-protected nucleoside having a 3'"'"'-hydroxyl group, a 5'"'"'-protected nucleotide having a 3'"'"'-hydroxyl moiety, and a 5'"'"'-protected oligonucleotide having a 3'"'"'-hydroxyl moiety to form a second reaction intermediate of the structure;

##STR18## where R is as above, and Y'"'"' is said nucleoside, nucleotide or oligonucleotide; and

(c) reacting said second reaction intermediate with a compound selected from the group consisting of a nucleoside having a 5'"'"'-hydroxyl moiety, a nucleotide having a 5'"'"'-hydroxyl moiety and an oligonucleotide having a 5'"'"'-hydroxyl moiety in the presence of an activating agent to form an ester linkage between an S-(alkaryl or aryl) phosphorothioate moiety of said second reaction intermediate and said 5'"'"'-hydroxyl moiety.

24. The process of claim 23 further including reacting the product of step (c) with a nucleoside 3'"'"'-S-(alkaryl or aryl) phosphorothioate, a nucleotide 3'"'"'-S-(alkaryl or aryl) phosphorothioate or a first oligonucleotide 3'"'"'-S-(alkaryl or aryl) phosphorothioate.

25. The process of claim 23 wherein said alkaryl or aryl halide is a chloro-substituted benzyl halide.

26. The process of claim 23 wherein said alkaryl or aryl halide is a dichlorobenzyl halide.

27. The process of claim 23 wherein said alkaryl or aryl halide is α

,2,4-trichlorotoluene.

28. The process of claim 23 wherein said activating agent is triisopropylbenzenesulfonylchloride.

29. The process of claim 28 further including adding a nucleophilic catalyst to said activating agent.

30. The process of claim 29 wherein said nucleophilic catalyst is 1-methylimidazole.

31. The process of claim 23 further including cleaving an S-(alkaryl or aryl) bond of the product of step (c) to form a phosphorothioate diester linkage.