Derivatized oligonucleotides having improved uptake and other properties

• US 6,153,737 A
• Filed: 04/22/1994
• Issued: 11/28/2000
• Est. Priority Date: 01/11/1990
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc., Northern Telecom Limited (Nortel Networks Corporation)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Manoharan, Muthiah, Cook, Phillip Dan, Bennett, Clarence Frank
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US08/211,882
• Time in Patent Office: 2,412 Days
• Field of Search: 536/22.1, 536/23.1, 536/25.3, 536/25.32, 536/26.6, 435/6, 514/1, 514/44, 935/77
• US Class Current: 536/22.1
• CPC Class Codes: A01N 37/46 N-acyl derivatives
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• 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

Linked nucleosides having at least one functionalized nucleoside that bears a substituent such as a steroid molecule, a reporter molecule, a non-aromatic lipophilic molecule, a reporter enzyme, a peptide, a protein, a water soluble vitamin, a lipid soluble vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a pyrene, a hybrid photonuclease/intercalator, or an aryl azide photo-crosslinking agent exhibit increased cellular uptake and other properties. The substituent can be attached at the 2'"'"'-position of the functionalized nucleoside via a linking group. If at least a portion of the remaining liked nucleosides are 2'"'"'-deoxy-2'"'"'-fluoro, 2'"'"'-O-methoxy, 2'"'"'-O-ethoxy, 2'"'"'-O-propoxy, 2'"'"'-O-aminoalkoxy or 2'"'"'-O-allyloxy nucleosides, the substituent can be attached via a linking group at any of the 3'"'"' or the 5'"'"' positions of the nucleoside or on the heterocyclic base of the nucleoside or on the inter-nucleotide linkage linking the nucleoside to an adjacent nucleoside.

Claims

1. A compound comprising a plurality of linked nucleosides, wherein at least one of said nucleosides is functionalized at a 2'"'"'-O-position or a 2'"'"'-S-position thereof by attachment of a steroid molecule, an RNA cleaving complex, a porphyrin, or an aryl azide photo-crosslinking agent.

2. The compound of claim 1 wherein said functionalized nucleoside comprises a steroid molecule attached to the 2'"'"'-O-position or the 2'"'"'-S-position of said nucleoside, said steroid molecule being selected from the group consisting of cholic acid, deoxycholic acid, dehydrocholic acid, cortisone, digoxigenin, testosterone, cholesterol and digoxigenin.

3. The compound of claim 2 wherein said steroid molecule is cholesterol.

4. The compound of claim 1 wherein said functionalized nucleoside comprises an RNA cleaving complex attached to the 2'"'"'-O-position or the 2'"'"'-S-position of said nucleoside, said RNA cleaving complex being selected from the group consisting of a phenanthroline/metal complex and a bipyridine/metal complex.

5. The compound of claim 1 wherein said functionalized nucleoside comprises an aryl azide photo-crosslinking agent attached to the 2'"'"'-O-position or the 2'"'"'-S-position of said nucleoside.

6. The compound of claim 1 wherein said functionalized nucleoside comprises a porphyrin attached to the 2'"'"'-O-position or the 2'"'"'-S-position of said nucleoside.

7. The compound of claim 1 wherein folic acid is attached at the 2'"'"'-O-position or the 2'"'"'-S-position of said nucleoside.

8. The compound of claim 1 wherein cholic acid is attached at the 2'"'"'-O-position or the 2'"'"'-S-position of said nucleoside.

9. The compound of claim 8 wherein said attachment is through a linking moiety comprises an Ω

-aminoalkoxy or Ω

-aminoalkylamino moiety.

10. The compound of claim 9 wherein said linking moiety comprises an Ω

-aminoalkoxy moiety.

11. The compound of claim 9 wherein said linking moiety comprises a 5-aminopentoxy group.

12. The compound of claim 1 wherein at least two of the linked nucleosides are linked by phosphorothioate linking groups.

13. A compound comprising a plurality of linked nucleosides, wherein at least one of the nucleosides is a

2'"'"' functionalized nucleoside having cholic acid linked to a 2'"'"'-O-position or a 2'"'"'-S-position of the nucleoside.

14. The compound of claim 13 wherein a linking group links said cholic acid and the functionalized nucleoside.

15. The compound of claim 13 wherein at least one of the linked nucleosides is a 2'"'"'-deoxy'"'"'-2'"'"'-fluoro, 2'"'"'-methoxy, 2'"'"'-ethoxy, 2'"'"'-propoxy, 2'"'"'-aminoalkoxy or 2'"'"'-allyloxy nucleoside.

16. The compound of claim 13 wherein at least two of the linked nucleosides are linked by phosphorothioate linking groups.

17. The compound of claim 13 wherein said functionalized nucleoside is a 3'"'"' terminal nucleoside having cholesterol linked to the 3'"'"'-position of said 3'"'"' terminal nucleoside.

18. The compound of claim 13 wherein said functionalized nucleoside is a 5'"'"' terminal nucleoside having cholesterol linked to the 5'"'"'-position of said 5'"'"' terminal nucleoside.

19. The compound of claim 13 wherein said functionalized nucleoside is a heterocyclic base functionalized nucleoside having cholesterol linked to the heterocyclic base of said nucleoside.

20. The compound of claim 13 wherein said functionalized nucleoside is an inter-strand nucleoside having cholesterol linked to an inter-nucleotide linkage linking said inter-strand nucleoside to an adjacent nucleoside.

21. A compound comprising a plurality of linked nucleosides, wherein:

at least one of the nucleosides is a 2'"'"'-deoxy'"'"'-2'"'"'-fluoro, 2'"'"'-O-C₁ -C₂₀ -alkyl, 2'"'"'-O-C₂ -C₂₀ -alkenyl, 2'"'"'-O-C₂ -C₂₀ -alkynyl, 2'"'"'-S-C₁ -C₂₀ -alkyl, 2'"'"'-S-C₂ -C₂₀ -alkenyl, or 2'"'"'-S-C₂ -C₂₀ -alkynyl nucleoside; and

at least one of the nucleosides is a functionalized nucleoside selected from the group consisting of;

a 2'"'"'-O-functionalized or a 2'"'"'-S-functionalized nucleoside having a steroid molecule, an RNA cleaving complex, a porphyrin, or an aryl azide photo-crosslinking agent linked to the 2'"'"'-position of the nucleoside;

a heterocyclic base functionalized nucleoside having a steroid molecule, a reporter molecule, an enzyme, a peptide, a protein, a vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a hybrid photonuclease/intercalator, or an aryl azide photo-crosslinking agent linked to the heterocyclic base of the nucleoside;

a 5'"'"' terminal nucleoside having a steroid molecule, a reporter molecule, an enzyme, a peptide, a protein, a vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a hybrid photonuclease/inter-calator, or an aryl azide photo-crosslinking agent linked to a 5'"'"'-O-position or a 5'"'"'-S-position of the nucleoside;

a 3'"'"' terminal nucleoside having a steroid molecule, a reporter molecule, an enzyme, a peptide, a protein, a vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a hybrid photonuclease/intercalator, or an aryl azide photo-crosslinking agent linked to the 3'"'"'-position of the nucleoside; and

an inter-strand nucleoside having a steroid molecule, a reporter molecule, an enzyme, a peptide, a protein, a vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a hybrid photo-nuclease/intercalator, or an aryl azide photo-crosslinking agent linked to an inter-stand linkage linking said inter-strand nucleoside to an adjacent nucleoside.

22. The compound of claim 21 wherein:

said functionalized nucleoside is a nucleoside having a steroid molecule, a vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a hybrid photonuclease/intercalator, or an aryl azide photo-crosslinking agent linked to the 2'"'"'-position of said nucleoside; and

the remainder of said linked nucleosides are 2'"'"'-deoxy-2'"'"'-fluoro, 2'"'"'-methoxy, 2'"'"'-ethoxy, 2'"'"'-propoxy, 2'"'"'-aminoalkoxy or 2'"'"'-allyloxy nucleosides.

24. The compound of claim 21 wherein said functionalized nucleoside is a 5'"'"' nucleoside having a steroid molecule, a reporter molecule, an enzyme, a peptide, a protein, a vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a hybrid photonuclease/intercalator, or an aryl azide photo-crosslinking agent linked to the 5'"'"'-O-position or the 5'"'"'-S-position of the nucleoside.

25. The compound of claim 21 wherein said functionalized nucleoside is a heterocyclic base functionalized nucleoside having a steroid molecule, a reporter molecule, an enzyme, a peptide, a protein, a vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a hybrid photo-nuclease/intercalator, or an aryl azide photo-crosslinking agent linked to the heterocyclic base of the nucleoside.

26. The compound of claim 21 wherein said functionalized nucleoside is an inter-strand nucleoside having a steroid molecule, a reporter molecule, an enzyme, a peptide, a protein, a vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a hybrid photonuclease/intercalator, or an aryl azide photo-crosslinking agent linked to an inter-strand linkage linking said nucleoside to an adjacent nucleoside.

27. The compound of claim 21 wherein at least two of the linked nucleosides are linked by phosphorothioate linking groups.

23. A compound comprising a plurality of linked nucleosides, wherein:

at least one of the nucleosides is a 2'"'"'-deoxy'"'"'-2'"'"'-fluoro, 2'"'"'-O-C₁ -C₂₀ -alkyl, 2'"'"'-O-C₂ -C₂₀ -alkenyl, 2'"'"'-O-C₂ -C₂₀ -alkynyl, 2'"'"'-S-C₁ -C₂₀ -alkyl, 2'"'"'-S-C₂ -C₂₀ -alkenyl, 2'"'"'-S-C₂ -C₂₀ -alkynyl, 2'"'"'-NH-C₁ -C₂₀ -alkyl, 2'"'"'-NH-C₂ -C₂₀ -alkenyl, 2'"'"'-NH-C₂ -C₂₀ -alkynyl nucleoside; and

at least one of the nucleosides is a 3'"'"' terminal nucleoside having a steroid molecule, a reporter molecule, an enzyme, a peptide, a protein, a vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a hybrid photonuclease/intercalator, or an aryl azide photo-crosslinking agent linked to the 3'"'"'-position of the nucleoside.

28. A method of effecting cellular uptake of a compound having a plurality of linked nucleosides, comprising contacting an organism with said compound wherein said compound includes at least one nucleoside functionalized at a 2'"'"'-O-position or a 2'"'"'-S-position by attachment of a steroid molecule, an enzyme, a peptide, a protein, or a vitamin to the 2'"'"'-position with a linking moiety.

29. The method of claim 28 further including selecting said steroid molecule from the group consisting of cholic acid, deoxycholic acid, dehydrocholic acid, cortisone, digoxigenin, testosterone, cholesterol and digoxigenin.

30. The method of claim 28 further including selecting cholic acid as said steroid molecule.

31. The method of claim 28 further including selecting an Ω

-amino alkylamino moiety as said linking molecule.

32. A compound comprising a plurality of linked nucleosides, wherein at least one of the nucleosides is a

2'"'"'-O-functionalized or 2'"'"'-S-functionalized nucleoside having folic acid linked to the 2'"'"' position of the nucleoside.

33. A compound of claim 32 wherein said functionalized nucleoside is a 2'"'"' functionalized nucleoside having folic acid linked to the 2'"'"' position of the nucleoside.

34. A compound comprising a plurality of linked nucleosides, wherein at least one of the nucleosides is a functionalized nucleoside selected from the group consisting of:

a 5'"'"' terminal nucleoside having pyrene linked to the 5'"'"'-position of the nucleoside;

a 3'"'"' terminal nucleoside having pyrene linked to the 3'"'"'-position of the nucleoside; and

an inter-strand nucleoside having pyrene linked to an inter-nucleotide linkage linking said inter-strand nucleoside to an adjacent nucleoside.