Methods of synthesizing oligonucleotides using carbonate protecting groups and alpha-effect nucleophile deprotection

• US 7,101,986 B2
• Filed: 08/25/2003
• Issued: 09/05/2006
• Est. Priority Date: 08/03/1998
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc., The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Caruthers, Marvin H., Dellinger, Douglas J., Betley, Jason R.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/648,740
• Publication Number:
  US 20040230052A1
• Time in Patent Office: 1,107 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 536/25.3, 536/26.6
• US Class Current: 536/23.1
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention provides methods for synthesizing oligonucleotides using nucleoside monomers having carbonate protected hydroxyl groups that are deprotected with α-effect nucleophiles. The α-effect nucleophile irreversibly cleave the carbonate protecting groups while simultaneously oxidizing the internucleotide phosphite triester linkage to a phosphodiester linkage. The procedure may be carried out in aqueous solution at neutral to mildly basic pH. The method eliminates the need for separate deprotection and oxidation steps, and, since the use of acid to remove protecting groups is unnecessary, acid-induced depurination is avoided. Fluorescent or other readily detectable carbonate protecting groups can be used, enabling monitoring of individual reaction steps during oligonucleotide synthesis. The invention is particularly useful in the highly parallel, microscale synthesis of oligonucleotides.

Claims

1. A method of synthesizing an oligonucleotide on a solid support comprising:

(a) coupling a nucleoside monomer having a protected hydroxyl group to a free hydroxyl group on a support-bound nucleoside monomer, wherein the hydroxyl group on the coupled nucleoside monomer is protected with a carbonate protecting group and the coupling reaction gives rise to a phosphite triester bond between the support-bound nucleoside monomer and the coupled nucleoside monomer; and

(b) contacting the coupled nucleoside monomer with an alpha-effect nucleophile to simultaneously (i) irreversibly remove the carbonate protecting group, and (ii) oxidize the phosphite triester linkage to a phosphotriester linkage.

2. The method of claim 1, wherein steps (a) and (b) are conducted in aqueous solution at neutral or mild pH.

3. The method of claim 2, wherein the α

-effect nucleophile is a peroxide.

4. The method of claim 3, wherein the peroxide is an inorganic peroxide of the formula M⁺OOH⁻

, wherein M⁺ is a counterion selected from the group consisting of H⁺, Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺.

5. The method of claim 3, wherein the peroxide is an organic peroxide of the formula ROOH, wherein R is hydrocarbyl optionally substituted with one or more nonhydrocarbyl substituents and optionally containing one or more nonhydrocarbyl linkages.

6. The method of claim 3, wherein the peroxide is activated by increasing the pH of the aqueous solution above the pKa of the peroxide such that the peroxide is converted to peroxy anion.

7. The method of claim 6, wherein the peroxy anion also removes exocyclic amine-protecting groups from the support-bound nucleoside monomer.

8. The method of claim 7, wherein the exocyclic amine-protecting groups that are removed from the support-bound nucleoside monomer are acetyl, trifluoroacetyl, difluoroacetyl, or trifluoroacetyl moieties.

9. The method of claim 1, wherein the oligonucleotide synthesis is in the 3′

to 5′

direction.

10. The method of claim 9, wherein support-bound nucleoside monomer is bound to the support through its 3′

-hydroxyl group.

11. The method of claim 10, wherein the coupled nucleoside monomer has a phosphorous derivative at the 3′

position and a carbonate protecting group at the 5′

position.

12. The method of claim 11, wherein the coupling reaction yields the structure of formula (IV)

13. The method of claim 12, wherein when R is hydrido, the support-bound nucleoside is a deoxyribonucleoside and when R is hydroxyl, the support-bound nucleoside is a ribonucleoside.

14. The method of claim 12, wherein R¹ is aryl carbonate selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenycarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′

-(-methyl-2-nitropiperonyl)oxycarbonyl, and 9-fluoroenylmethylcarbonyl.

15. The method of claim 12, wherein the purine base or the pyrimidine base may be protected with a protecting group selected from the group consisting of acetyl, difluoroacetyl, trifluoroacetyl, isobutyrul, and benzoyl.

16. The method of claim 12, wherein when Y is hydrocarbyl, Y is selected from the group consisting of lower alkyl, electron-withdrawing substituted aliphatic, electron-withdrawing substituted phenyl, or electron-withdrawing substituted phenylethyl.

17. The method of claim 12, wherein the simultaneous deprotection and oxidation of the structure of formula (IV) with the α

-effect nucleophile yields the structure of formula (VIII)

18. The method of claim 1, wherein the oligonucleotide synthesis is in the 5′

to 3′

direction.

19. The method of claim 18, wherein the support-bound nucleoside is bound to the support through its 5′

-hydroxyl group.

20. The method of claim 19, wherein the coupled nucleoside monomer has a phosphorous derivative at the 5′

position and a carbonate protecting group at the 3′

position.

21. The method of claim 20, wherein the coupling reaction yields the structure of formula (XI)

22. The method of claim 21, wherein when R is hydrido, the support-bound nucleoside is a deoxyribonucleoside and when R is hydroxyl, the support-bound nucleoside is a ribonucleoside.

23. The method of claim 21, wherein R¹ is aryl carbonate selected from the group consisting of o-nitrophenylcarbonyl, p-phenylazophenycarbonyl, phenylcarbonyl, p-chlorophenylcarbonyl, 5′

-(-methyl-2-nitropiperonyl)oxycarbonyl, and 9-fluoroenylmethylcarbonyl.

24. The method of claim 21, wherein the purine base or the pyrimidine base may be protected with a protecting group selected from the group consisting of acetyl, difluoroacetyl, trifluoroacetyl, isobutyrul, and benzoyl.

25. The method of claim 21, wherein when Y is hydrocarbyl, Y is selected from the group consisting of lower alkyl, electron-withdrawing substituted aliphatic, electron-withdrawing substituted phenyl, or electron-withdrawing substituted phenylethyl.

26. The method of claim 21, wherein the simultaneous deprotection and oxidation of the structure of formula (XI) with the α

-effect nucleophile results in cleavage of the carbonate linkage such that the moiety —

OR¹ is converted to —

OH and the phosphate triester linkage —

O—

P(OY)—

O—

is oxidized to a phosphotriester linkage.

27. The method of claim 12, wherein R³ comprises a moiety that fluoresces or becomes colored upon cleavage of the carbonate protecting group.

28. The method of claim 21, wherein R³ comprises a moiety that fluoresces or becomes colored upon cleavage of the carbonate protecting group.

29. The method of claim 1, wherein steps (a) and (b) are repeated until the oligonucleotide is of a desired length and sequence.

30. The method of claim 29, wherein upon completion of the oligonucleotide synthesis, the oligonucleotide is cleaved from the solid support.