Oligonucleotides having chiral phosphorus linkages

• US 20020137921A1
• Filed: 05/17/2002
• Published: 09/26/2002
• Est. Priority Date: 10/15/1991
• Status: Active Grant

• Current Assignee: Phillip Dan Cook
• Original Assignee: Phillip Dan Cook
• Inventors: Cook, Phillip Dan
• Granted Patent: US 6,699,979 B2
• Time in Patent Office: Days
• US Class Current: 536/25.33
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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 21/04 with deoxyribosyl as saccharide radical
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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

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;

(b) selecting a second synthon having structure;

(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 new first synthon via a stereospecific inversion of configuration at the 3′

position of the second synthon; and

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

wherein;

Q is O or CH₂;

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

R_E is O or S;

R_F is H or 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 L is selected from the group consisting of halogen, alkylsulfonyl, substituted alkylsulfonyl, arylsulfonyl, substituted arylsulfonyl, hetercyclcosulfonyl or trichloroacetimidate

3. 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.

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

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

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

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

8. 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.

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

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 f irst 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 RF is an acid selected from the group consisting of trichloroacetic acid, acetic acid or trifluoromethane sulfonic acid.

13. The product of the process of claim 1.

15. The oligonucleotide of claim 14 wherein said chiral phosphate linkages are selected from the group consisting of chiral Sp phosphorothioate, chiral Rp phosphorothioiate, chiral Sp alkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate, chiral Rp phosphoamidate, chiral Sp chiral phosphotriester or chiral Rp phosphotriester.

16. The oligonucleotide of claim 14 wherein each of the phosphate linkages is a chiral phosphate linkage.

17. The oligonucleotide of claim 14 wherein:

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

wherein;

Q is O or CHR_G;

R_Aand 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, SO₂Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substituted NH-alkyl, OCH₂CH═

CH₂, OCH═

CH₂, OCH₂CCH, 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.

18. The oligonucleotide of claim 17 wherein B_X is a pyrimidinyl-1 or purinyl-9 moiety.

19. The oligonucleotide of claim 17 wherein B_X is adenine, guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine.

20. The oligonucleotide of claim 14 wherein at least one of the modified nucleotides has one of the structures:

21. The oligonucleotide of claim 20 wherein X has one of the structures:

22. The oligonucleotide of claim 14 wherein the oligonucleotide forms at least a portion of a RNA or DNA sequence.

24. The oligonucleotide of claim 23 wherein said nucleoside units are linked together in a sequence that is antisense to an RNA or DNA sequence.

26. The oligonucleotide of claim 25 wherein said nucleoside units are linked together in a sequence that is antisense to an RNA or DNA sequence.

28. The oligonucleotide of claim 27 wherein said alkylphosphonate linkages are methylphosphonate linkages.

29. The oligonucleotide of claim 27 wherein said nucleoside units are linked together in a sequence that is antisense to an RNA or DNA sequence.

14. 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.

23. An oligonucleotide comprising a plurality of nucleoside units linked together by all S_P phosphotriester linkages or by all R_P phosphotriester linkages.

25. 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.

27. An oligonucleotide comprising at least 10 nucleoside units linked together by all S_P alkylphosphonate linkages or by all R_P alkylphosphonate linkages.

30. A composition of matter having the structure:

31. The composition of matter of claim 30 wherein L is selected from the group consisting of halogen, alkylsulfonyl, substituted alkylsulfonyl, arylsulfonyl, substituted arylsulfonyl, hetercyclcosulfonyl or trichloroacetimidate

32. The composition of matter of claim 30 wherein L is selected from the group consisting of chloro, fluoro, bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl, benzene-sulfonyl, methylsulfonyl (mesylate), p-methylbenzenesulfonyl (tosylate), p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate), trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl, imidazolesulfonyl and 2,4,6-trichlorophenyl.

33. The composition of matter of claim 30 wherein R_X 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, SO₂Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substituted NH-alkyl, OCH₂CH═

CH₂, OCH═

CH₂, OCH₂CCH, OCCH, aralkyl, heteroaralkyl, heterocycloalkyl, polyalkylamino, substituted silyl, an RNA cleaving moiety, a group for improving the pharmacodynamic properties of an oligonucleotide or a group for improving the pharmacokinetic properties of an oligonucleotide.

34. The composition of matter of claim 30 wherein R_Fis selected from the group consisting of H, t-butyl, di-methoxytrityl or tetrahydropyranyl.

35. The composition of matter of claim 30 wherein B_X is a pyrimidinyl-1 or purinyl-9 moiety.

36. The composition of claim 30 wherein B_X is adenine, guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or 5-methylcytosine.

37. The composition of claim 30 wherein Q is O.

38. The composition of claim 30 having Sp stereochemistry.

39. The composition of claim 30 having Rp stereochemistry.