Amine-derivatized nucleosides and oligonucleosides

• US 7,037,646 B1
• Filed: 09/02/1994
• Issued: 05/02/2006
• Est. Priority Date: 01/11/1990
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Cook, Phillip Dan, Guinosso, Charles J.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US08/602,862
• Time in Patent Office: 4,260 Days
• Field of Search: 435/6, 514/44, 536/24.5, 536/23.1
• US Class Current: 435/6.12
• 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 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

Nucleosides and oligonucleosides functionalized to include alkylamino functionality, and derivatives thereof. In certain embodiments, the compounds of the invention further include steroids, reporter molecules, reporter enzymes, lipophilic molecules, peptides or proteins attached to the nucleosides through the alkylamino group.

Claims

1. A method for detecting the presence or absence of an RNA in a biological sample suspected of containing said RNA comprising selecting a compound capable of hybridizing with said RNA, contacting said sample with said compound, and identifying any hybrid of said compound and an RNA wherein said compound comprises a plurality of linked nucleosides, wherein:

each nucleoside includes a ribofuranosyl sugar portion and a base portion; and

at least one of said nucleosides bears at a 2′

-O-position, a 3′

-O-position, or a 5′

-O-position a substituent having formula;

—

R_A—

N(R_1a)(R_1b)

where;

each R_A, independently, is a straight chain or branched hydrocarbon alkylene group including from 1 to about 10 carbon atoms or (CH₂—

CH₂—

Q)_x;

R_1a and R_1b, independently, are H, R₂, or have formula C(X)—

R₂, C(X)—

R_A—

R₂, C(X)—

Q—

R_A—

R₂, or C(X)—

Q—

R₂, provided R_1a and R_1b are not both H;

R₂ is a folate, a steroid molecule, a reporter molecule, a lipophilic molecule, a reporter enzyme, a peptide, a protein, or has formula —

Q—

(CH₂CH₂—

Q—

)_x—

R₃;

X is O or S;

each Q is, independently, is NH, O, or S;

x is 1 to about 200;

R₃ is H, R_A, C(O)OH, C(O)OR_A, C(O)R₄, R_A—

N3, or R_A—

NH₂;

R₄ is Cl, Br, I, SO₂R₅ or has structure;

m is 2 to 7; and

R₅ is alkyl having 1 to about 10 carbon atoms.

3. The method of claim 1 wherein more than one of said nucleosides bear said substituent at a 2′

-O-position, a 3′

-O-position, or a 5′

-O-position.

4. The method of claim 1 wherein R_A is (CH₂)_n where n is an integer from 1 to about 10.

5. The method of claim 1 wherein R_1a is H and R_1b is C(O)—

(CH₂)_n—

R₂ where n is an integer from 1 to about 10.

6. The method of claim 1 wherein R_1a is H and R_1b is R₂.

7. The method of claim 1 wherein R_1a is H and R_1b is C(O)—

O—

R₂.

8. The method of claim 1 wherein R_1a and R_1b both are a straight chain or branched hydrocarbon alkylene group including from 1 to about 10 carbon atoms.

9. The method of claim 1 wherein R_1a is H and R_1b is C(O)—

(CH₂)_n—

R₂ where n is an integer from 1 to about 10.

10. The method of claim 1 wherein R_1a is H and R_A is (CH₂)₅.

11. The method of claim 1 wherein R_1a is H and R_1b is C(S)—

NH—

R₂.

12. The method of claim 1 wherein R₂ includes pyrene, fluorescein, dinitrophenyl, cholesterol, or acridine.

13. The method of claim 1 wherein R_1a is H and R_1b is C(O)—

R₂.

14. The method of claim 1 wherein R₂ has formula —

O—

(CH₂CH₂—

O—

)_x—

R₃.

15. The method of claim 1 wherein a 3′

-position of at least one of said nucleosides is linked to a 5′

-position of an adjacent nucleoside.

16. The method of claim 1 wherein a 2′

-position of at least one of said nucleosides is linked to a 5′

-position of an adjacent nucleoside.

2. A method for detecting the presence or absence of an RNA in a biological sample suspected of containing said RNA comprising selecting an oligonucleoside capable of hybridizing with said RNA, contacting said sample with said oligonucleoside, and identifying any hybrid of said oligonucleoside and an RNA wherein said oligonucleoside comprises a ribofuranosyl sugar portion and a base portion, wherein said oligonucleoside bears at a 3′

-O-position or a 5′

-O-position a substituent having formula;

—

R_A—

N(R_1a)(R_1b)

where;

each R_A, independently, is a straight chain or branched hydrocarbon alkylene group including from 1 to about 10 carbon atoms;

R_1a and R_1b, independently, are H, R₂, or have formula C(X)—

R₂, C(X)—

R_A—

R₂, C(X)—

Q—

R_A—

R₂, or C(X)—

Q—

R₂, provided R_1a and R_1b are not both H;

R₂ is a folate, a steroid molecule, a reporter molecule, a lipophilic molecule, a reporter enzyme, a peptide, a protein, or has formula —

Q—

(CH₂CH₂—

Q—

)_x—

R₃;

X is O or S;

each Q is, independently, is NH, O, or S;

x is 1 to about 200;

R₃ is H, R_A, C(O)OH, C(O)OR_A, C(O)R₄, R_A—

N3, or R_A—

NH₂;

R₄ is Cl, Br, I, SO₂R₅ or has structure;

m is 2 to 7; and

R₅ is alkyl having 1 to about 10 carbon atoms.

17. The method of claim 2 wherein R_A is (CH₂)_n where n is an integer from 1 to about 10.

18. The method of claim 2 wherein R_1a is H and R_1b is C(O)—

(CH₂)_n—

R₂ where n is an integer from 1 to about 10.

19. The method of claim 2 wherein R_1a is H and R_1b is R₂.

20. The method of claim 2 wherein R_1a is H and R_1b is C(O)—

O—

R₂.

21. The method of claim 2 wherein R_1a and R_1b both are a straight chain or branched hydrocarbon alkylene group including from 1 to about 10 carbon atoms.

22. The method of claim 2 wherein R_1a is H and R_1b is C(O)—

(CH₂)_n—

R₂ where n is an integer from 1 to about 10.

23. The method of claim 22 wherein R_A is (CH₂)₅.

24. The method of claim 2 wherein R_1a is H and R_1b is C(S)—

NH—

R₂.

25. The method of claim 2 wherein R₂ includes pyrene, fluorescein, dinitrophenyl, cholesterol, or acridine.

26. The method of claim 2 wherein R_1a is H and R_1b is C(O)—

R₂.

27. The method of claim 26 wherein R₂ has formula —

O—

(CH₂CH₂—

O—

)_n—

R₃.