Process for separating and deprotecting oligonucleotides

• US 20040044195A1
• Filed: 08/28/2002
• Published: 03/04/2004
• Est. Priority Date: 08/28/2002
• Status: Active Grant

• Current Assignee: Quiatech AB (Quiapeg Pharmaceuticals Holding AB)
• Original Assignee: Quiatech AB (Quiapeg Pharmaceuticals Holding AB)
• Inventors: Kwiatkowski, Marek
• Granted Patent: US 7,345,163 B2
• Time in Patent Office: Days
• US Class Current: 536/25.3
• 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

Abstract

Methods for deprotecting and derivatizing oligonucleotides that are non-covalently bound to a solid support are described.

Claims

1. A method for deprotecting oligonucleotides, said method comprising:

a) providing a plurality of oligonucleotides, said plurality comprising protected oligonucleotides, wherein (i) the 5′

end of each said protected oligonucleotide is linked to a hydrophobic separation function or (ii) each said protected oligonucleotide is linked to a pair of hydrophobic separation functions, wherein one member of said pair of hydrophobic separation functions is less hydrophobic than the other member of said pair;

b) precipitating said plurality of oligonucleotides on a hydrophobic solid support using an organic solvent to produce non-covalently immobilized oligonucleotides; and

c) deprotecting said immobilized oligonucleotides by (i) selectively removing said 5′

separation function from said immobilized oligonucleotides or (ii) sequentially removing said pair of hydrophobic separation functions from said immobilized oligonucleotides, using a reagent dissolved in an organic solvent.

2. The method of claim 1, wherein said method further comprises, before step b), adsorbing said plurality of oligonucleotides on a hydrophobic support;

and eluting (i) oligonucleotides lacking said 5′

hydrophobic separation function or (ii) oligonucleotides lacking any protecting group and oligonucleotides containing only the less hydrophobic separation function of said pair of hydrophobic separation functions.

3. The method of claim 1, said method further comprising eluting said deprotected oligonucleotides.

4. The method of claim 1, wherein said organic solvent is dichloromethane, chloroform, acetonitrile, tetrahydrofuran, ethyl acetate, acetone, or isopropanol.

5. The method of claim 1, wherein said precipitating step comprises pre-drying said plurality of oligonucleotides on said hydrophobic solid support then washing said hydrophobic solid support with said organic solvent.

6. The method of claim 1, wherein said 5′

hydrophobic separation function is a trityl moiety, a hydrophobic acetal, or a thioacetal group.

7. The method of claim 6, wherein said trityl moiety is selectively removed using a non-aqueous solution of acid.

8. The method of claim 7, wherein said non-aqueous solution of acid is 2% trichloroacetic acid in dichloromethane.

9. The method of claim 1, wherein said hydrophobic solid support is provided in the form of a cartridge or a manifold, said manifold containing a plurality of cartridges.

10. The method of claim 1, wherein said hydrophobic solid support is polystyrene, charcoal, graphite, or silica based.

11. The method of claim 6, wherein said trityl moiety is a dimethoxytrityl, trimethoxytrityl, pixyl, or monomethoxytrityl moiety.

12. The method of claim 6, wherein said trityl moiety is selected from the group consisting of 4-hexyloxy methoxytrityl, 4-decyloxymethoxytrityl, 4-hexadecyloxymethoxy trityl, 4-octadecyloxyphenylxanthyl, 4-4′

-bis-hexyloxymethoxytrityl, 4-4′

-bis-decyloxymethoxytrityl, 4-4′

-bis-hexadecyloxymethoxytrityl, 4-octadecyloxytrityl, 4-hexadecyloxytrityl, 4-decyloxytrityl, and 4-hexyloxy trityl moiety.

13. The method of claim 1, wherein one member of said pair of hydrophobic separation functions is a straight chain alkyl, branched alkyl, arylalkyl, or an aryl group linked to the oligonucleotide by a linker that is removable under conditions that do not remove the other member of said pair of hydrophobic separation functions.

15. The method of claim 1, wherein the oligonucleotides within said plurality contain deoxyribonucleotides or ribonucleotides.

16. The method of claim 15, wherein the oligonucleotides contain a non-standard backbone.

17. The method of claim 16, wherein said non-standard backbone is phosphorothioate, phosphorodithioate, or phosphoramido.

18. The method of claim 15, wherein said oligonucleotide is a Locked Nucleic Acid (LNA).

19. The method of claim 15, wherein the oligonucleotides contain one or more nucleotides linked to a functional group.

20. The method of claim 19, wherein said functional group is an amino, thiol, phosphate, aldehyde, intercalating reagent, quencher, or a fluorophore.

21. The method of claim 19, wherein said functional group is placed on either end of said oligonucleotide.

22. The method of claim 19, wherein said functional group is placed on both ends of said oligonucleotide.

23. The method of claim 19, wherein said functional group is different on each end.

24. The method of claim 19, wherein said 5′

separation function or one member of said pair of hydrophobic separation functions is attached to said functional group.

25. The method of claim 19, wherein one member of said pair of hydrophobic separation functions is attached to a linker and said linker is attached to said functional group.

26. The method of claim 1, said method further comprising derivatizing said immobilized oligonucleotides using non-aqueous conditions.

27. The method of claim 26, wherein said derivatizing step comprises incorporating a fluorophore into said purified oligonucleotides.

28. The method of claim 26, said method further comprising eluting said derivatized oligonucleotides.

14. The method of claim 14, wherein said linker is a siloxyl or disiloxyl function.