Aminooxy-modified oligonucleotides and methods for making same

• US 6,172,209 B1
• Filed: 08/07/1998
• Issued: 01/09/2001
• Est. Priority Date: 02/14/1997
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Prakash, Thazha P., Manoharan, Muthiah, Cook, Phillip Dan, Kawasaki, Andrew M.
• Primary Examiner(s): Geist, Gary
• Assistant Examiner(s): CRANE, LAWRENCE E
• Application Number:
  US09/130,973
• Time in Patent Office: 886 Days
• Field of Search: 435/88, 435/91.1, 536/23.1, 536/24.3, 536/24.5, 536/26.7, 536/26.8, 536/27.6, 536/27.8, 536/27.81, 536/28.5, 536/28.53, 536/25.33, 536/25.34, 558/70
• US Class Current: 536/23.1
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 15/203 Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• 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 20/582 Recycling of unreacted starting or intermediate materials

Abstract

Oligonucleotides and other macromolecules are provided which have increased nuclease resistance, substituent groups (such as 2′-aminooxy groups) for increasing binding affinity to complementary strand, and subsequences of 2′-deoxy-erythro-pentofuranosyl nucleotides that activate RNase H. Such oligonucleotides and macromolecules are useful for diagnostics and other research purposes, for modulating the expression of a protein in organisms, and for the diagnosis, detection and treatment of other conditions susceptible to oligonucleotide therapeutics.

Claims

1. An oligonucleotide specifically hybridizable with DNA or RNA comprising a sequence of from about 5 to about 50 nucleotide units, wherein:

said sequence is divided into a first region having 2′

-O-alkyl substituted nucleotide units and a second region being composed of nucleotide units having 2′

-deoxy sugar moieties;

said nucleotide units of at least one of said first or second regions are connected by phosphorothioate linkages; and

at least one of said 2′

-O-alkyl substituted nucleotide units of said first region bears a 2′

-aminooxy group having one of the formulas;

wherein;

m is from 1 to 10;

y is from 1 to 10;

x is 1;

E is N (R₁)(R₂), —

NH—

CH(R₁)(R₂) or N═

C(R₁)(R₂); and

each R₁ and R₂ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protecting group, or R₁ and R₂, together, are a nitrogen protecting group or wherein R₁ and R₂ are joined in a ring structure that can include at least one heteroatom selected from N and O.

2. The oligonucleotide of claim 1 wherein said nucleotide units of said first and second regions are connected by phosphorothioate linkages.

3. The oligonucleotide of claim 1 wherein said nucleotide units of said first region are connected by phosphodiester linkages and said nucleotide units of said second region are connected by phosphorothioate linkages.

4. The oligonucleotide of claim 1 wherein said nucleotide units of said first region are connected by phosphorothioate linkages and said nucleotide units of said second region are connected by phosphodiester linkages.

5. The oligonucleotide of claim 1 wherein said second region has at least three nucleotide units.

6. The oligonucleotide of claim 1 wherein said second region has at least five nucleotide units.

7. The compound of claim 1 wherein said 2′

-aminooxy group is an aminooxyethyl group.

8. The compound of claim 1 wherein R₁ and R₂ are independently H or C₁-C₁₀ alkyl.

9. The compound of claim 1 wherein R₁ and R₂ are joined in a ring structure that can include at least one heteroatom selected from N and O.

10. The compound of claim 9 wherein said ring structure is imidazolyl, pipieridinyl, morpholinyl or substituted piperazinyl.

11. The compound of claim 10 wherein said substituted piperazinyl bears a C₁-C₁₂ alkyl substituent.

12. The oligonucleotide of claim 1 having a third region, said third region having 2′

-O-alkyl substituted nucleotide units, wherein said second region is positioned between said first and third regions.

13. The oligonucleotide of claim 12 wherein said nucleotide units of said first, second and third regions are connected by phosphorothioate linkages.

14. The oligonucleotide of claim 12 wherein said nucleotide units of said first and third regions are connected by phosphodiester linkages and said nucleotide units of said second region are connected by phosphorothioate linkages.

15. The oligonucleotide of claim 12 wherein said nucleotide units of said first and third regions are connected by phosphorothioate linkages and said nucleotide units of said second region are connected by phosphodiester linkages.

16. The oligonucleotide of claim 12 wherein said second region has at least three nucleotide units.

17. The oligonucleotide of claim 12 wherein said second region has at least five nucleotide units.

18. The oligonucleotide of claim 12 wherein at least one of said 2′

-O-alkyl substituted nucleotide units of said third region bears a 2′

-aminooxy group having one of said formulas.

19. A method for preparing a compound of formula:

wherein;

Bx is a heterocyclic base;

T₁ and T₂, independently, are OH, a protected hydroxyl, an activated phosphate group, an activated phosphite group, or a reactive group for forming an internucleotide linkage;

n is from 1 to 10;

E is N(R₁)(R₂), —

NH—

CH(R₁)(R₂)—

or N═

C(R₁)(R₂);

each R₁ and R₂ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protecting group, or R₁ and R₂, together, are a nitrogen protecting group or wherein R₁ and R₂ are joined in a ring structure wherein said ring can include at least one heteroatom selected from N(R₁) and O;

comprising the steps of;

contacting said 2′

-O—

(CH₂)_n—

OH nucleoside with N-hydroxyphthalimide to form a 2′

-O—

(CH₂)_n—

O-phthalimido nucleoside;

contacting said 2′

-O—

(CH₂)_n—

O-phthalimido nucleoside with a basic reagent, followed by a carbonyl-containing compound, to form a 2′

-O—

(CH₂)_n—

O—

N—

C(R₁)(R₂) nucleoside; and

optionally contacting said 2′

-O—

(CH₂)_n—

O—

N═

C(R₁)(R₂) nucleoside with a reducing agent, followed by a carbonyl-containing compound in the presence of a reducing agent, to form a 2′

-O—

(CH₂)_n—

O—

N(R₁)(R₂) nucleoside.

20. The method of claim 19 wherein Bx is a 5-methyluracilyl, uracilyl, cytosinyl, 5-methylcytosinyl, guaninyl, adeninyl, or 2,6-diaminopurinyl group.

21. The method of claim 19 wherein Bx is uracilyl or 5-methyl-uracilyl further comprising the step of contacting said uracilyl or 5-methyl-uracilyl with reagents to convert said uracilyl to cytosinyl or said 5-methyluracilyl to 5-methylcytosinyl.

22. The method of claim 19 further comprising selectively protecting primary hydroxyl and primary amino groups of said nucleosides.

23. The method of claim 19 further comprising contacting said nucleoside with a chemical protecting group to protect a 5′

-hydroxyl group thereof, thereby providing a 5′

-O-protected-2′

-O-substituted nucleoside.

24. The method of claim 23 wherein said chemical protecting group is a trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthine-9-yl or 9-(p-methoxyphenyl)xanthine-9-yl group.

25. The method of claim 23 further comprising contacting said 5′

-O-protected-2′

-O-substituted nucleoside with a phosphitylating reagent to provide a 5′

-O-protected-2′

-O-substituted nucleoside phosphoramidite.

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

,N′

-tetra-isopropylphosphorodiamidite or 2-cyanoethyl diisopropylchlorophosphoramidite.

27. The method of claim 19 wherein said basic reagent comprises hydrazine, methylhydrazine, ammonia, alkylhydrazines, or methylamine.

28. A method for preparing a compound of formula:

wherein;

Bj is a heterocyclic base;

T₁ and T₂, independently, are OH, a protected hydroxyl, an activated phosphate group, an activated phosphite group, or a reactive group for forming an internucleotide linkage;

n is from 1 to 10;

E is N(R₁)(R₂), —

NH—

CH(R₁)(R₂) or N═

C(R₁)(R₂);

each R₁ and R₂ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protecting group, or R₁ and R₂, together, are a nitrogen protecting group or wherein R₁ and R₂ are joined in a ring structure wherein said ring can include at least one heteroatom selected from N(R₁) and O;

comprising the steps of;

alkylating a 2′

-OH nucleoside to provide a 2′

-O—

(CH₂)—

CH═

CH₂nucleoside, wherein z is an integer from 1 to 9;

contacting said 2′

-O—

(CH₂)_z—

CH═

CH₂ nucleoside with at least one oxidizing agent to provide a 2′

-O-(CH₂)_z—

CHO nucleoside;

reducing said 2′

-O—

(CH₂)_z—

CHO nucleoside with a reducing agent to provide a 2′

-O—

(CH₂)_n—

OH nucleoside;

contacting said 2′

-O—

(CH₂)_n—

OH nucleoside with N-hydroxyphthalimide to provide a 2′

-O—

(CH₂)_n—

O-phthalimido substituted nucleoside;

contacting said 2′

-O—

(CH₂)_n—

O-phthalimido nucleoside with a basic reagent, followed by a carbonyl-containing compound, to provide a 2′

-O—

(CH₂)_n—

O—

N═

C(R₁)(R₂) nucleoside; and

optionally contacting said 2′

-O—

(CH₂)_n—

O—

N═

C(R₁)(R₂) nucleoside with a reducing agent, followed by a carbonyl-containing compound in the presence of a reducing agent, to form a 2′

-O—

(CH₂)_n—

O—

N(R₁)(R₂) nucleoside.

29. The method of claim 28 wherein Bj is a 5-methyluracilyl, uracilyl, cytosinyl, 5-methylcytosinyl, adeninyl or 2,6-diaminopurinyl group.

30. The method of claim 29 further comprising selectively chemically protecting primary hydroxyl and primary amino groups of said nucleosides.

31. The method of claim 28 further comprising contacting said 2′

-O—

(CH₂)_n—

CH═

CH₂ nucleoside wherein Bj is 2,6-diaminopurinyl with adenosine deaminase to convert said 2,6-diaminopurinyl to guaninyl.

32. The method of claim 28 further comprising contacting said 2′

-O-substituted nucleoside with a chemical protecting group to provide a 5′

-O-protected-2′

-substituted nucleoside.

33. The method of claim 32 wherein said chemical protecting group is a trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthine-9-yl or 9-(p-methoxyphenyl)xanthine-9-yl group.

34. The method of claim 32 further comprising contacting said 5′

-O-protected-2′

-substituted nucleoside with a phosphitylating reagent to provide a 5′

-O-protected-2′

-substituted nucleoside phosphoramidite.

35. The method of claim 31 wherein said phosphitylating reagent is 2-cyanoethyl-N,N,N′

,N′

-tetra-isopropylphosphorodiamidite or 2-cyanoethyl diisopropyl-chlorophosphoramidite.

36. The method of claim 28 wherein said at least one oxidizing agent is 4-methylmorpholine N-oxide/osmium tetraoxide followed by sodium periodate or ozonolysis.

37. The method of claim 28 wherein said basic reagent comprises hydrazine, methylhydrazine, ammonia, alkylhydrazines, or methylamine.