Oligonucleotides having chiral phosphorus linkages

• US 5,852,188 A
• Filed: 04/19/1996
• Issued: 12/22/1998
• Est. Priority Date: 01/11/1990
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Cook, Phillip Dan
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/635,009
• Time in Patent Office: 977 Days
• Field of Search: 536/24.5, 536/25.33, 536/25.34, 536/26.7, 536/26.8, 536/26.1, 536/25.6, 536/25.3
• US Class Current: 536/24.5
• 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: A61P 31/12 Antivirals
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07J 43/003 not condensed
• CPC Class Codes: C07K 14/005 from viruses
• 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 2310/3511 intercalating or cleaving agent
• 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 2740/16322 : New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• 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: C12Q 1/6813 : Hybridisation assays
• CPC Class Codes: C12Y 113/11012 : Linoleate 13S-lipoxygenase (1.13.11.12)

Abstract

Sequence-specific oligonucleotides are provided having substantially pure chiral Sp phosphorothioate, chiral Rp phosphorothioate, chiral Sp alkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate, chiral Rp phosphoamidate, chiral Sp phosphotriester, and chiral Rp phosphotriester linkages. The novel oligonucleotides are prepared via a stereospecific SN₂ nucleophilic attack of a phosphodiester, phosphorothioate, phosphoramidate, phosphotriester or alkylphosphonate anion on the 3'"'"' position of a xylonucleotide. The reaction proceeds via inversion at the 3'"'"' position of the xylo reactant species, resulting in the incorporation of phosphodiester, phosphorothioate, phosphoramidate, phosphotriester or alkylphosphonate linked ribofuranosyl sugar moieties into the oligonucleotide.

Claims

1. An oligonucleotide comprising a plurality of nucleoside units linked together via phosphate linkages, wherein:

at least one of the nucleoside units is a non-naturally occurring nucleoside unit; and

at least two of the nucleoside units are linked via chiral phosphate linkages.

2. The oligonucleotide of claim 1 wherein said chiral phosphate linkages are selected from the group consisting of chiral Sp phosphorothioate, chiral Rp phosphorothioate, chiral Sp alkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate, chiral Rp phosphoamidate, chiral Sp chiral phosphotriester or chiral Rp phosphotriester.

3. The oligonucleotide of claim 1 wherein each of the phosphate linkages is a chiral phosphate linkage.

4. The oligonucleotide of claim 1 wherein:

at least one of the nucleoside units has one of the structures;

##STR10## wherein;

Q is O or CHRG;

R_A and R_B are H, lower alkyl, substituted lower alkyl, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;

R_C is H, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, substituted O-alkyl, S-alkyl, substituted S-alkyl, SOMe, SO₂ Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substituted NH-alkyl, OCH₂ CH═

CH₂, OCH═

CH₂, OCH₂ CCH, OCCH, aralkyl, heteroaralkyl, heterocycloalkyl, polyalkylamino, substituted silyl, an RNA cleaving moiety, a group which improves the pharmacodynamic properties of an oligonucleotide, or a group which improves the pharmacokinetic properties of an oligonucleotide;

R_G is H, lower alkyl, substituted lower alkyl, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide; and

B_X is a naturally occurring or synthetic nucleoside base.

5. The oligonucleotide of claim 4 wherein B_X is a pyrimidinyl-1 or purinyl-9 moiety.

6. The oligonucleotide of claim 4 wherein B_X is adenine, guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine.

7. The oligonucleotide of claim 1 wherein at least one of the modified nucleotides has one of the structures:

##STR11## wherein;

G and K are, independently, C or N;

J is N or CR² R³ ;

R₁ is OH or NH₂ ;

R₂ and R₃ are H, NH, lower alkyl, substituted lower alkyl, lower alkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino, heterocycloalkyl, heterocycloalkylamino, aminoalkylamino, hetrocycloalkylamino, polyalkylamino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;

R₄ and R₅ are, independently, H, OH, NH₂, lower alkyl, substituted lower alkyl, substituted amino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;

R₆ and R₇ are, independently, H, OH, NH₂, SH, halogen, CONH₂, C(NH)NH₂, C(O)O-alkyl, CSNH₂, CN, C(NH)NHOH, lower alkyl, substituted lower alkyl, substituted amino, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide; and

X is a sugar or a sugar analog moiety where said sugar analog moiety is a sugar substituted with at least one substituent comprising an RNA cleaving moiety, a group which improves the pharmacodynamic properties of an oligonucleotide, or a group which improves the pharmacokinetic properties of an oligonucleotide.

8. The oligonucleotide of claim 7 wherein X has one of the structures:

##STR12## wherein;

Q is O or CHRG;

R_A and R_B are H, lower alkyl, substituted lower alkyl, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide;

R_C is H, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, substituted O-alkyl, S-alkyl, substituted S-alkyl, SOMe, SO₂ Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substituted NH-alkyl, OCH₂ CH═

CH₂, OCH═

CH₂, OCH₂ CCH, OCCH, aralkyl, heteroaralkyl, heterocycloalkyl, polyalkylamino, substituted silyl, an RNA cleaving moiety, a group which improves the pharmacodynamic properties of an oligonucleotide, or a group which improves the pharmacokinetic properties of an oligonucleotide; and

R_G is H, lower alkyl, substituted lower alkyl, an RNA cleaving moiety, a group which improves the pharmacokinetic properties of an oligonucleotide, or a group which improves the pharmacodynamic properties of an oligonucleotide.

9. The oligonucleotide of claim 2 wherein the oligonucleotide forms at least a portion of a preselected RNA or DNA sequence.

10. An oligonucleotide comprising a plurality of nucleoside units linked together by all S_p phosphotriester linkages or by all R_p phosphotriester linkages.

11. The oligonucleotide of claim 10 wherein said nucleoside units are linked together in a sequence that is antisense to a preselected RNA or DNA sequence.

12. An oligonucleotide comprising a plurality of linked of nucleoside units linked together by all S_p phosphoramidate linkages or by all R_p phosphoamidate linkages.

13. The oligonucleotide of claim 12 wherein said nucleoside units are linked together in a sequence that is antisense to a preselected RNA or DNA sequence.

14. An oligonucleotide comprising at least 10 nucleoside units linked together by all S_p alkylphosphonate linkages or by all R_p alkylphosphonate linkages.

15. The oligonucleotide of claim 14 wherein said alkylphosphonate linkages are methylphosphonate linkages.

16. The oligonucleotide of claim 14 wherein said nucleoside units are linked together in a sequence that is antisense to a preselected RNA or DNA sequence.