Process for preparing oligonucleotides having chiral phosphorus linkages

• US 5,521,302 A
• Filed: 05/05/1993
• Issued: 05/28/1996
• Est. Priority Date: 01/11/1990
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Cook, Phillip D.
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): CARTER, PHILIP
• Application Number:
  US08/058,023
• Time in Patent Office: 1,119 Days
• Field of Search: 536/23.1, 536/24.1, 536/25.3, 536/25.33, 536/25.34, 536/25.31
• US Class Current: 536/25.31
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• 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: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• 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: C12P 19/34 : Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: C12Q 1/6813 : Hybridisation assays
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/701 : Specific hybridization probes
• 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. A method for preparing an oligonucleotide comprising a predetermined sequence of nucleoside units, comprising the steps of:

(a) selecting a first synthon having structure;

##STR10## (b) selecting a second synthon having structure;

##STR11## (c) contacting the second synthon with the first synthon in the presence of a base to effect nucleophilic attack of a 5'"'"'-phosphate of the first synthon at a 3'"'"'-position of the second synthon to yield a coupled synthon via an inversion of configuration at the 3'"'"' position of the second synthon; and

(d) treating the coupled synthon with a reagent to remove blocking group R_F, thereby generating a new first synthon;

wherein;

Q is O or CH₂ ;

R_D is methyl, alkoxy, thioalkoxy, amino or substituted amino;

R_E is O or S;

R_F is a labile blocking group;

R_X is H, OH, or a sugar derivatizing group;

B_X is a naturally occurring or synthetic nucleoside base or blocked nucleoside base;

L is a leaving group or together L and Bx are a 2-3'"'"' or 6-3'"'"' pyrimidine or 8-3'"'"' purine cyclo-nucleoside; and

Y is a stable blocking group, a solid state support, a nucleotide on a solid state support, or an oligonucleotide on a solid state support.

2. The method of claim 1 wherein the new first synthon has the structure:

##STR12##

3. The method of claim 1 wherein L is selected from the group consisting of halogen, alkylsulfonyl, substituted alkylsulfonyl, arylsulfonyl, substituted arylsulfonyl, heterocyclosulfonyl and trichloroacetidimate.

4. The method of claim 1 wherein L is selected from the group consisting of chloro, fluoro, bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl, methylsulfonyl (mesylate), p-methylbenzenesulfonyl (tosylate), p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate), trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl, imidazolesulfonyl and 2,4,6-trichlorophenyl.

5. The method of claim 1 wherein said base is selected from the group consisting of sodium hydride, methylmagnesium chloride, t-butylmagnesium chloride and methylmagnesium fluoride.

6. The method of claim 1 wherein R_F is an acid labile blocking group.

7. The method of claim 1 wherein Y is a hydrogenolysis sensitive blocking group.

8. The method of claim 1 wherein R_F is selected from the group consisting of t-butyl, dimethoxytrityl and tetrahydropyranyl.

9. The method of claim 1 wherein Y is a nucleotide bound to a solid state support, an oligonucleotide bound to a solid state support, or a solid state support.

10. The method of claim 1 further comprising repeating said steps (b), (c), and (d) a plurality of times.

11. The method of claim 1 further comprising contacting said first synthon with said base in a solvent selected from the group consisting of acetonitrile, tetrahydrofuran and dioxane.

12. The method of claim 1 wherein said reagent used to remove blocking group R_F is an acid selected from the group consisting of trichloroacetic acid, acetic acid and trifluoromethane sulfonic acid.