Activators for oligonucleotide synthesis

• US 6,274,725 B1
• Filed: 10/23/1998
• Issued: 08/14/2001
• Est. Priority Date: 06/02/1998
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Sanghvi, Yogesh, Manoharan, Muthiah
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US09/177,953
• Time in Patent Office: 1,026 Days
• Field of Search: 536/22.1, 536/23.1, 536/25.3, 536/25.33, 536/25.34, 536/25.4, 536/25.41, 536/26.26, 536/26.7, 536/26.8, 536/27.6, 536/27.81, 536/28.5, 536/28.53, 536/28.54
• US Class Current: 536/25.34
• 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: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The present invention relates to improved methods for the preparation of nucleoside phosphoramidites and oligonucleotides. In one aspect, the methods of the invention are used to prepare phosphitylating reagents using pyridinium salts as activators. In a further aspect, the methods of the invention are used to prepare internucleoside linkages using activators which include at least one pyridinium salt and at least one substituted imidazole. In a further aspect, methods are provided preparation of internucleoside linkages between nucleosides having 2′-substituents using imidazolium or benzimidazolium salts as an activator. In a further aspect, methods are provided preparation of internucleoside linkages between nucleosides having bioreversible protecting group that confers enhanced chemical and biophysical properties, without exocyclic amine protection, using imidazolium or benzimidazolium salts as an activator.

Claims

1. A method for preparing a phosphitylated compound comprising the steps of:

providing a compound having a hydroxyl group; and

reacting said compound with a phosphitylating reagent in the presence of a pyridinum salt in a solvent under conditions of time, temperature and pressure effective to yield said phosphitylated compound.

2. The method of claim 1 wherein said compound is a nucleoside.

3. The method of claim 2 wherein said compound is a 5′

-O- protected nucleoside having a 3′

hydroxyl group.

4. The method of claim 1 wherein said compound is a nucleoside diner having a 3′

or 5′

hydroxyl group.

5. The method of claim 1 wherein said compound is an oligonucleotide or oligonucleotide analog having a 3′

or 5′

hydroxyl group.

6. The method of claim 2 wherein said nucleoside has a 5′

or a 2′

hydroxyl group.

7. The method of claim 1 wherein said phosphitylating reagent is 2-cyanoethyl-N,N,N′

,N′

-tetraisopropylphosphorodiamidite, bis(N,N-diisopropylamino)-2-methyltrifluoroacetylaminoethoxyphosphine or bis(N,N-diisopropylamino)-2-diphenylmethylsilylethoxyphosphine.

8. The method of claim 1 wherein said pyridinium salt is pyridinium hydrochloride, pyridinium trifluoroacetate or pyridinium dichloroacetate.

9. The method of claim 1 wherein said solvent is dichloromethane, acetonitrile, ethyl acetate, tetrahydrofuran or a mixture thereof.

10. The method of claim 1, wherein said pyridinium salt is bound to a solid support.

11. The method of claim 10 wherein said pyridinium salt is a polyvinyl pyridinium salt.

12. A method for preparing a phosphitylated compound comprising:

providing a compound of the formula;

wherein;

B is an optionally protected nucleobase;

one of R₁₅ and R₁₆ is H and the other of R₁₅ and R₁₆ is a hydroxyl protecting group, or a linker connected to a solid support and R₁₇ is R₄;

or one of R₁₅ and R₁₆ is a hydroxyl protecting group and the other of R₁₅ and R₁₆ is a linker connected to a solid support and R₁₇ is a hydroxyl group;

or both of R₁₅ and R₁₆ are hydroxyl protecting groups and R₁₇ is a hydroxyl group;

W is an optionally protected internucleoside linkage;

q is 0 to about 50;

R₄ is H, F, O-R, S-R, S-H, N-H(R₁₀) or N-R(R₁₀);

R is a protecting group, —

CH₂—

CH₂—

O—

CH₂—

CH₂—

N(R₁₀)(R₁₁), or has one of the formulas;

where

each m is independently from 1 to 10;

y is from 0 to 10;

E is a hydroxyl protecting group, C₁-C₁₀ alkyl, N(R₁₀)(R₁₁) or N═

C(R₁₀)(R₁₁);

substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, wherein the substitutions are selected from one or several halogen, cyano, carboxy, protected hydroxyl, nitro and mercapto residues;

each R₁₀ and R₁₁ is, independently, H, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, wherein the substitutions are selected from one or several halogen, cyano, carboxy, protected hydroxyl, nitro and mercapto residues;

alkylthioalkyl, a nitrogen protecting group, or R₁₀ and R₁₁, together, are a nitrogen protecting group or wherein R₁₀ and R₁₁ are joined in a ring structure that can include at least one heteroatom selected from N and O;

reacting said compound with a phosphitylating reagent in the presence of a pyridinum salt in a solvent under conditions of time, temperature and pressure effective to yield said phosphitylated compound.

13. The method of claim 12 wherein q is 0.

14. The method of claim 13 wherein R₁₅ is said hydroxyl protecting group and R₁₆is H.

15. The method of claim 13 wherein R₁₅ is said hydroxyl protecting group or R₁₇ is H.

16. The method of claim 12 wherein q is 1 and one of said R₁₅ and R₁₆ is H.

17. The method of claim 16 wherein q is from 2 to about 50.

18. The method of claim 12 wherein said phosphitylating reagent is 2-cyanoethyl-N,N,N′

,N-tetraisopropylphosphorodiamidite, bis(N,N-diisopropylamino)-2-methyltrifluoroacetylaminoethoxyphosphine or bis(N,N-diisopropylamino)-2-diphenylmethylsilylethoxyphosphine.

19. The method of claim 12 wherein said pyridinium salt is pyridinium hydrochloride, pyridinium trifluoroacetate or pyridinium dichloroacetate.

20. The method of claim 12 wherein said solvent is dichloromethane, acetonitrile, ethyl acetate, tetrahydrofuran or a mixture thereof.

21. The method of claim 12, wherein said pyridinium salt is bound to a solid support.

22. The method of claim 21 wherein said pyridinium salt is a polyvinyl pyridinium salt.

23. The method of claim 12 wherein B is devoid of exocyclic amine protection.

24. A method for preparing a phosphitylated compound comprising:

providing a compound of the formula;

wherein;

B is an optionally protected nucleobase;

one of R₁₅ and R₁₆ is H and the other of R₁₅ and R₁₆ is a hydroxyl protecting group, or a linker connected to a solid support and R₁₇ is R₄;

or one of R₁₅ and R₁₆ is a hydroxyl protecting group and the other of R₁₅ and R₁₆ is a linker connected to a solid support and R₁₇ is a hydroxyl group;

or both of R₁₅ and R₁₆ are hydroxyl protecting groups and R₁₇ is a hydroxyl group;

W is an optionally protected internucleoside linkage;

q is 0 to about 50;

R₄ is H, F, O-R, S-R, S-H, N-H(R₁₀) or N-R(R₁₀);

R is a protecting group, —

CH₂—

CH₂—

O—

CH₂—

CH₂—

N(R₁₀)(R₁₁), or has one of the formulas;

where

each m is independently from 1 to 10;

y is from 0 to 10;

E is a hydroxyl protecting group, C₁-C₁₀ alkyl, N(R₁₀)(R₁₁) or N═

C(R₁₀)(R₁₁);

substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, wherein the substitutions are selected from one or several halogen, cyano, carboxy, protected hydroxyl, nitro and mercapto residues;

each R₁₀ and R₁₁ is, independently, H, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, wherein the substitutions are selected from one or several halogen, cyano, carboxy, protected hydroxyl, nitro and mercapto residues;

alkylthioalkyl, a nitrogen protecting group, or R₁₀ and R₁₁, together, are a nitrogen protecting group or wherein R₁₀ and R₁₁ are joined in a ring structure that can include at least one heteroatom selected from N and O;

reacting said compound with a phosphitylating reagent in the presence of pyridinium trifluoroacetate in a solvent under conditions of time, temperature and pressure effective to yield said phosphitylated compound.

25. The method of claim 24 wherein q is 0.

26. The method of claim 25 wherein R₁₅ is said hydroxyl protecting group and R₁₆ is H.

27. The method of claim 25 wherein R₁₅ is said hydroxyl protecting group or R₁₇ is H.

28. The method of claim 25 wherein q is 1 and one of said R₁₅ and R₁₆ is H.

29. The method of claim 28 wherein q is from 2 to about 50.

30. The method of claim 25 wherein said phosphitylating reagent is 2-cyanoethyl-N,N,N′

,N′

-tetraisopropylphosphorodiamidite, bis(N,N-diisopropylamino)-2-methyltrifluoroacetylaminoethoxyphosphine or bis(N,N-diisopropylamino)-2-diphenyl-methylsilylethoxyphosphine.

31. The method of claim 25 wherein said solvent is dichloromethane, acetonitrile, ethyl acetate, tetrahydrofuran or a mixture thereof.

32. The method of claim 25 wherein said pyridinium trifluoroacetate is bound to a solid support.

33. The method of claim 25 wherein B is devoid of exocyclic amine protection.