Oligonucleotides having chiral phosphorus linkages

• US 6,239,265 B1
• Filed: 12/09/1998
• Issued: 05/29/2001
• Est. Priority Date: 01/11/1990
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Cook, Phillip Dan
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US09/208,533
• Time in Patent Office: 902 Days
• Field of Search: 536/22.1, 536/23.1, 536/25.3, 536/25.31, 536/25.33, 536/25.34, 536/25.4, 536/25.6
• 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: 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 two of the nucleoside units are linked via a chiral phosphate linkage;

at least one of said chiral phosphate linkages being between the 3′

-terminal nucleosicle and its adjoining nucleoside; and

with the proviso that said oligonucleotide has less than all of said nucleoside units linked together by either all Rp or all Sp linkages.

2. The oligonucleotide of claim 1 wherein two of the nucleoside units are linked via a chiral phosphate linkage.

3. The oligonucleotide of claim 1 wherein the chiral phosphate linkages are Rp linkages.

4. The oligonucleotide of claim 2 wherein the chiral phosphate linkage is an Rp linkage.

5. The oligonucleotide of claim 1 wherein the chiral phosphate linkages are Sp linkages.

6. The oligonucleotide of claim 2 wherein the chiral phosphate linkage is an Sp linkage.

7. 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.

8. The oligonuzleotide of claim 2 or 5 wherein said chiral phosphate linkage is 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.

9. The oligonucleotide of claim 4 or 6 wherein said chiral phosphate linkage is selected from the group consisting of chiral Sp phosphorothioate, chiral Sp alkylphosphonate, chiral Sp phosphoamidate, and chiral Sp chiral phosphotriester.

10. The oligonicleotide of claim 1, 4 or 6 wherein:

at least one of the nucleoside units is selected from the group consisting of

wherein;

Q is O or CHR_G;

R_A and R_B are H, lower alkyl, substituted lower alkyl;

R_C is H, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN, CF₃, OCF₃, SOMe, SO₂Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substituted NH-alkyl, OCH₂CH═

CH₂, OCH═

CH₂, OCH₂CCH, OCCH, aralkyl, heteroaralkyl, heterocycloaryl, polyalkylamino and substituted silyl;

R_G is H, lower alkyl and substituted lower alkyl; and

B_X is a naturally occurring or synthetic nucleoside base.