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

• US 8,309,706 B2
• Filed: 09/06/2007
• Issued: 11/13/2012
• Est. Priority Date: 08/03/1998
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Dellinger, Douglas J., Caruthers, Marvin H., Betley, Jason R.
• Primary Examiner(s): OLSON, ERIC
• Application Number:
  US11/899,828
• Publication Number:
  US 20080076913A1
• Time in Patent Office: 1,895 Days
• Field of Search: None
• US Class Current: 536/25.31
• 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 coupling a nucleoside monomer to a nucleoside, said method comprising:

(a) coupling a nucleoside monomer having a carbonate protected hydroxyl group and a phosphoramidite group to a free hydroxyl group on the nucleoside, wherein the coupling reaction gives rise to a phosphite triester bond between the nucleoside and the coupled nucleoside monomer; and

(b) removing the carbonate protecting group from the coupled nucleoside monomer with an α

-effect nucleophile reagent through a nucleophilic attack.

2. The method of claim 1, wherein step (b) is conducted in an aqueous solution at a pH less than 10.

3. The method of claim 1, 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 selected from the group consisting of an alkyl, an aryl, a substituted alkyl, and a substituted aryl.

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 1, wherein the nucleoside monomer has an exocyclic amine-protecting group.

8. The method of claim 7, wherein the exocyclic amine-protecting group is acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl or benzoyl.

9. The method of claim 1, wherein the coupling of the nucleoside monomer and the nucleoside allows the synthesis of an oligonucleotide in the 3′

to 5′

direction.

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

-hydroxyl group.

11. The method of claim 10, wherein the carbonate protected hydroxyl group of the nucleoside monomer is at the 5′

position and the phosphoramidite group of the nucleoside monomer is at the 3′

position, wherein the phosphoramidite group has the structure;

12. The method of claim 1, wherein the coupling of the nucleoside monomer and the nucleoside allows the synthesis of an oligonucleotide in the 5′

to 3′

direction.

13. The method of claim 12, wherein the nucleoside is bound to a support through its 5′

-hydroxyl group.

14. The method of claim 13, wherein the phosphoramidite group of the nucleoside monomer is at the 5′

position and the carbonate protected hydroxyl group of the nucleoside monomer is at the 3′

position.

15. The method of claim 1, further comprising:

oxidizing said phosphite triester bond between steps (a) and (b), wherein steps (a) and (b) and said oxidizing step are repeated until an oligonucleotide is of a desired length and sequence is synthesized.

16. The method of claim 15, wherein the nucleoside is bound to a support, and wherein upon completion of the oligonucleotide synthesis, the oligonucleotide is cleaved from the solid support.

17. The method of claim 1, further comprising:

oxidizing said phosphite triester bond simultaneous with step (b), wherein steps (a) and (b) and said oxidizing step are repeated until an oligonucleotide of a desired length and sequence is synthesized.

18. The method of claim 17, wherein the nucleoside is bound to a support, and wherein upon completion of the oligonucleotide synthesis, the oligonucleotide is cleaved from the solid support.

19. The method of claim 1, wherein the nucleoside is bound to a support.