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

• US 20080076913A1
• Filed: 09/06/2007
• Published: 03/27/2008
• Est. Priority Date: 08/03/1998
• Status: Active Grant

• Current Assignee: Douglas Dellinger, Jason Betley, Marvin Caruthers
• Original Assignee: Douglas Dellinger, Jason Betley, Marvin Caruthers
• Inventors: Betley, Jason, Dellinger, Douglas, Caruthers, Marvin
• Granted Patent: US 8,309,706 B2
• Time in Patent Office: Days
• 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

1-30. -30. (canceled)

31. A method of synthesizing an oligonucleotide on a solid support, said method 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 bond between the support-bound nucleoside monomer and the coupled nucleoside monomer; and

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

-effect nucleophile.

32. The method of claim 31, wherein steps (a) and (b) are conducted in aqueous solution at neutral or mild pH.

33. The method of claim 32, wherein the α

-effect nucleophile is a peroxide.

34. The method of claim 33, 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⁺.

35. The method of claim 33, 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.

36. The method of claim 33, 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.

37. The method of claim 36, wherein the peroxy anion also removes exocyclic amine-protecting groups from the support-bound nucleoside monomer.

38. The method of claim 37, wherein the exocyclic amine-protecting groups that are removed from the support-bound nucleoside monomer are acetyl, trifluoroacetyl, difluoroacetyl, or trifluoroacetyl moieties.

39. The method of claim 31, wherein the oligonucleotide synthesis is in the 3′

to 5′

direction.

40. The method of claim 39, wherein support-bound-nucleoside monomer is bound to the support through its 3′

-hydroxyl group.

41. The method of claim 40, wherein the coupled nucleoside monomer has a phosphorous derivative at the 3′

position and a carbonate protecting group at the 5′

position.