Method of polynucleotide synthesis using modified support

• US 7,585,970 B2
• Filed: 08/30/2003
• Issued: 09/08/2009
• Est. Priority Date: 08/30/2003
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Hargreaves, John S, Dellinger, Geraldine F, Dellinger, Douglas J
• Primary Examiner(s): Crane; Lawrence E.
• Application Number:
  US10/652,049
• Publication Number:
  US 20050048496A1
• Time in Patent Office: 2,201 Days
• Field of Search: 536/25.3, 536/25.31, 536/25.33, 536/26.7, 536/26.8, 536/27.6, 536/27.81, 536/28.5, 536/25.34, 536/28.53
• US Class Current: 536/25.34
• 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: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

A method for synthesizing a polynucleotide is disclosed. The method comprises providing a functionalized support comprising a triaryl methyl linker bound to the solid support through a substitution on one of the aryl groups, a nucleotide chain bound to the central methyl carbon of the triaryl methyl group and a nucleoside with a reactive site hydroxyl. The method further provides contacting that functionalized support with a precursor having a hydroxyl protecting group and a phosphorous derivative reactive group to produce internucleotide linkage. The resulting compound is then treated to both remove the hydroxyl protecting group on the precursor, and to oxidize the internucleotide bond.

Claims

1. A method for synthesizing a solid-support-attached oligonucleotide or polynucleotide, the method comprising:

(a) providing a functionalized support having the structure (Id)

2. The method of claim 1, wherein steps (b) and (c) are repeated until an oligonucleotide or a polynucleotide having a desired sequence and length is provided.

3. The method of claim 2, further comprising wherein, after the oligonucleotide or polynucleotide having a desired sequence and length is provided, said oligonucleotide or polynucleotide is cleaved from the solid support to yield the oligonucleotide or polynucleotide free in solution, wherein the cleaving is performed by exposure of the oligonucleotide or polynucleotide attached to the solid support to mild acid.

4. The method of claim 1, wherein the triaryl methyl linker group has the structure (VId)

5. The method of claim 4, wherein R₁₂, R₁₃, and R₁₄ are independently selected from substituted and unsubstituted phenyl groups.

6. The method of claim 4, wherein R₁₂, R₁₃, and R₁₄ are optionally substituted aryl groups independently selected from phenyl, biphenyl, naphthanyl, 2-thienyl, 3-thienyl, furanyl, annulenyl, and anthracenyl.

7. The method of claim 4, wherein R₁₂, R₁₃, and R₁₄ are independently selected from phenyl, methoxyphenyl, dimethoxyphenyl, trimethoxyphenyl, and furanyl.

8. The method of claim 4, wherein one of R₁₂, R₁₃, and R₁₄ is a p-methoxyphenyl, and the other two of R₁₂, R₁₃, and R₁₄ are phenyl, provided that one of the two phenyl is substituted by being bound to the solid support via Lnk′

.

9. The method of claim 8 wherein Lnk′

has the structure —

O—

R₂₈—

N—

C(O)N—

wherein R₂₈ is a substituted or unsubstituted hydrocarbyl;

wherein substituted hydrocarbyl is substituted with a group selected from ether, oxo-, ester-, and amido-, and/or being substituted with one or more additional groups including lower alkyl, aryl, alkoxy, thioalkyl, halo, cyano, azido, sulfide, and boronyl.

10. The method of claim 4 wherein the phosphorus derivative capable of reacting with the reactive site hydroxyl is a phosphoramidite group.

11. The method of claim 1, wherein the step (c) is conducted at neutral to mildly basic pH.

12. The method of claim 1, wherein step (c) is conducted at a pH that is greater than the PK_a of said nucleophile.

13. The method of claim 12, wherein the peroxide is an inorganic peroxide of the formula M⁺OOH⁻

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

14. The method of claim 12, wherein the peroxide is an organic peroxide of the formula ROOH, wherein R is selected from alkyl, aryl, substituted alkyl, substituted aryl, and substituted lower alkyl, wherein

substituted aryl is substituted with a group selected from lower alkyl, aryl, aralkyl, lower alkoxy, thioalkyl, halo, cyano, azido, sulfide, and boronyl;

and lower alkyl, substituted with one or more groups selected from lower alkyl, alkoxy, thioalkyl, halo, cyano, azido, carboxy, sulfide, boronyl, and fused aromatic rings;

substituted alkyl and substituted lower alkyl are substituted with a group selected from ether, oxo, ester, and amido, and/or being substituted with one or more additional groups including lower alkyl, aryl, alkoxy, thioalkyl, halo, cyano, azido, sulfide, and boronyl.

15. The method of claim 12, wherein the peroxide is selected from t-butyl hydroperoxide, m-chloroperoxybenzoic acid, and mixtures thereof.

16. The method of claim 12, wherein the peroxide has the structure of formula (Xd), (XId) or (XIId)

17. The method of claim 1, wherein the synthesis is conducted in the 3′

-to-5′

direction.

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

-to-3′

direction.

19. The method of claim 1, wherein the heterocyclic base is a naturally occurring purine or pyrimidine base.

20. A method for synthesizing an oligonucleotide or a polynucleotide, the method comprising:

(a) providing a functionalized support having the structure (Id)