Thiol-derivatized oligonucleotides

• US 6,114,513 A
• Filed: 09/05/1997
• Issued: 09/05/2000
• Est. Priority Date: 01/11/1990
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Manoharan, Muthiah, Cook, Phillip Dan
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/924,326
• Time in Patent Office: 1,096 Days
• Field of Search: 536/25.3, 536/25.31, 536/25.32, 536/25.33, 536/25.34
• US Class Current: 536/23.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• 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 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C07J 43/003 not condensed
• CPC Class Codes: C07J 51/00 Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/3125 Methylphosphonates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/32 of the sugar
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/33 of the base
• CPC Class Codes: C12N 2310/333 Modified A
• CPC Class Codes: C12N 2310/336 : Modified G
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3523 : Allyl
• CPC Class Codes: C12N 2310/3527 : Other alkyl chain
• CPC Class Codes: C12N 2310/3531 : Hydrogen
• CPC Class Codes: C12N 2310/3533 : Halogen
• CPC Class Codes: C12N 9/0069 : acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: C12Y 113/11012 : Linoleate 13S-lipoxygenase (1.13.11.12)

Abstract

Nucleosides and linked nucleosides functionalized to include alkylthiol chemical functionality at ribofuranosyl positions, nucleosidic base positions, or on internucleoside linkages. 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 alkylthio group.

Claims

1. A method comprising the step of contacting a biological sample suspected of containing an RNA with a compound comprising a plurality of linked nucleosides, wherein:

each nucleoside comprises 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;

space="preserve" listing-type="equation">--R.sub.S --S--R.sub.1

where;

R_S has formula R_A, R_A --C(O)--Q--R_A, R_A --Q--R_A --Q--R_A ;

each R_A is independently selected from alkyl having from 1 to about 10 carbon atoms;

each Q is independently selected from the group of NH, O, and S;

R₁ is H or a thiol protecting group or has formula S--R₂, CH₂ C(O)--NH--R₂, CH₂ --CH═

CH--C(O)--R₂, --CH₂ --CH₂ --NH--S(O)₂ --R₂, or (maleimido)-R₂ ; and

R₂ comprises a steroid molecule, a reporter molecule, a lipophilic molecule, a reporter enzyme, a peptide, a protein, a reporter group, an alkylator, an intercalator, a cell receptor binding molecule, a crown ether, a crown amine, a porphyrin, a crosslinking agent, a peptide nucleic acid, or a thiol attached to a poly(ethylene glycol).

2. The method of claim 1 wherein more than one of said nucleosides bears said substituent at a 2'"'"'-O-position, a 3'"'"'-O-position, or a 5'"'"'-O-position.

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

4. The method of claim 3 wherein n is 6.

5. The method of claim 1 wherein R₁ is H or trityl.

6. The method of claim 1 wherein R₁ is S--R₂ and R₂ is thiocholesterol.

7. The method of claim 1 wherein R₁ is CH₂ C(O)--NH--R₂ and R₂ is o-phenanthroline.

8. The method of claim 1 wherein R₁ is phospholipid maleimido, fluorescein maleimido, or pyrene maleimido.

24. The method of claim 1 further comprising detecting the presence or absence of hybridization between said RNA and said compound.

9. A method comprising the step of contacting a biological sample suspected of containing an RNA with a compound comprising a plurality of linked nucleosides, wherein:

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

at least one of said nucleosides bears at a 5-pyrimidine position or at a 2-, 6-, or 8-purine position a substituent having formula;

space="preserve" listing-type="equation">--Q--R.sub.A --S--R.sub.1 ;

or

space="preserve" listing-type="equation">--C.tbd.C--R.sub.A --Q--C(O)--R.sub.A --S--R.sub.1 ;

space="preserve" listing-type="equation">--CH═

CH--C(O)--Q--R.sub.A --S--R.sub.1 ;

or

space="preserve" listing-type="equation">--CH═

CH--R.sub.A --Q--C(O)--Q--R.sub.A --S--R.sub.1 ;

wherein;

each R_A is independently alkyl having from 1 to about 10 carbon atoms;

Q is NH, O, or S;

R₁ is H or a thiol protecting group or has formula S--R₂, CH₂ C(O)--NH--R₂, CH₂ --CH═

CH--C(O)--R₂, --CH₂ --CH₂ --NH--S(O)₂ --R₂, or (maleimido)-R₂ ; and

R₂ comprises a steroid molecule, a reporter molecule, a lipophilic molecule, a reporter enzyme, a peptide, a protein, a reporter group, an alkylator, an intercalator, a cell receptor binding molecule, a crown ether, a crown amine, a porphyrin, a crosslinking agent, a peptide nucleic acid, or a thiol attached to a poly(ethylene glycol);

provided that a 6-purine substituent does not have formula --NH--R_A --S--R₁ when R₁ is H or a thiol protecting group.

10. The method of claim 9 wherein more than one of said nucleosides bears said substituent at a 5-pyrimidine position or at a 2-, 6-, or 8-purine position.

11. The method of claim 9 wherein R_A is (CH₂)_n where n is an integer from 1 to about 10.

12. The method of claim 11 wherein n is 6.

13. The method of claim 9 wherein R₁ is H or trityl.

14. The method of claim 11 wherein said nucleoside bears at a 5-pyrimidine position a substituent having formula --C.tbd.C--CH₂ --NH--C(O)--(CH₂)_n --S--R₁.

15. The method of claim 11 wherein said nucleoside bears at a 5-pyrimidine position a substituent having formula --CH═

CH--C(O)--NH--(CH₂)_n --S--R₁.

16. The method of claim 11 wherein said nucleoside bears at a 2- or 8-purine position a substituent having formula --NH--(CH₂)_n --S--R₁.

25. The method of claim 9 further comprising detecting the presence or absence of hybridization between said RNA and said compound.

17. A method comprising the step of contacting a biological sample suspected of containing an RNA with a compound comprising a plurality of linked nucleosides, wherein:

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

at least two of said nucleosides are bound by a linkage having formula;

space="preserve" listing-type="equation">(5'"'"')-O--P[X][Q--R.sub.A --S--R.sub.1 ]--O--(3'"'"')

wherein;

X is O or S;

Q is NH, O, or S;

R_A is alkyl having from 1 to about 10 carbon atoms;

R₁ is H or a thiol protecting group or has formula S--R₂, CH₂ C(O)--NH--R₂, CH₂ --CH═

CH--C(O)--R₂, --CH₂ --CH₂ --NH--S(O)₂ --R₂, or (maleimido)-R₂ ; and

R₂ comprises a steroid molecule, a reporter molecule, a lipophilic molecule, a reporter enzyme, a peptide, a protein, a reporter group, an alkylator, an intercalator, a cell receptor binding molecule, a crown ether, a crown amine, a porphyrin, a crosslinking agent, a peptide nucleic acid, or a thiol attached to a poly(ethylene glycol);

provided that Q is not NH when R₁ is H or a thiol protecting group.

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

19. The method of claim 18 wherein n is 6.

20. The method of claim 18 wherein R₁ is H or trityl.

21. The method of claim 18 wherein said nucleosides are bound by a linkage having formula --O--CH₂ --P[O][S--(CH₂)_n --S--R₁ ]--O--.

26. The method of claim 17 further comprising detecting the presence or absence of hybridization between said RNA and said compound.

22. A method comprising the step of contacting a biological sample suspected of containing an RNA with a compound having formula:

space="preserve" listing-type="equation">L.sub.1 --R.sub.A --S--R.sub.1a

where;

L₁ is a leaving group;

R_A is alkyl having 1 to about 10 carbon atoms; and

R_1a is an acid labile thiol protecting group.

23. The method of claim 22 having formula Br--(CH₂)_n --R_1a.

27. The method of claim 22 further comprising detecting the presence or absence of hybridization between said RNA and said compound.