Cleavable linker for polynucleotide synthesis

• US 7,385,050 B2
• Filed: 08/30/2003
• Issued: 06/10/2008
• Est. Priority Date: 08/30/2003
• Status: Active Grant

• 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, Dellinger, Geraldine F
• Primary Examiner(s): CRANE, LAWRENCE E
• Application Number:
  US10/652,063
• Publication Number:
  US 20050048497A1
• Time in Patent Office: 1,746 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: 536/25.3
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

Functionalized supports for polynucleotide synthesis are disclosed. The supports have linker moieties that are stable to conditions used in polynucleotide synthesis, but may be cleaved to release synthesized polynucleotides from the support. Methods of making the functionalized supports and methods of using are also disclosed. In particular embodiments of methods of making the functionalized supports, a solid support, on which an available reactive group is bound, is contacted with a reagent having the structure (I)
Phos-Cgp-Trl-Cgp′Nucl   (I)
wherein the groups are defined as follows:

• • Phos is a reactive phosphorus group capable of specifically reacting with an available reactive group on the support,
  • Trl is a triaryl methyl linker group having three aryl groups, each bound to a central methyl carbon, at least one of said three aryl groups having one or more substituents,
  • Cgp is a linking group linking the reactive phosphorus group and the triaryl methyl linker group, or is a bond linking the reactive phosphorus group and the triaryl methyl linker group,
  • Nucl is a nucleoside moiety, wherein the nucleoside moiety is optionally part of a polynucleotide moiety, and
  • Cgp′ is a linking group linking the nucleoside moiety and the triaryl methyl linker group, or is a bond linking the nucleoside moiety and the triaryl methyl linker group.
    In typical embodiments, the solid support is contacted with the reagent having the structure (I) under conditions and for a time sufficient to result in a functionalized support having a nucleoside moiety bound to the solid support via a triaryl methyl linker group.

Claims

1. A compound having the structure (I)

Phos-Cgp-Trl-Cgp′

-Nucl 

 

(I)

wherein;

Phos is a reactive phosphorus group which specifically reacts with a reactive group on a solid support to produce a phosphorous containing linkage group,

Trl is a triaryl methyl linker group having three aryl groups, wherein each of the three aryl groups are bound to a central methyl carbon, and at least one of said three aryl groups has one or more substituents, wherein one of said substituents is bound to Cgp and the central methyl carbon is bound to Cgp′

,

Cgp is a linking group linking the reactive phosphorus group and the triaryl methyl linker group, or is a bond linking the reactive phosphorus group and the triaryl methyl linker group,

Nucl is a nucleoside moiety, and

Cgp′

is a linking group linking the nucleoside moiety at the 3′

O or the 5′

O and the triaryl methyl linker group, or is a bond linking the nucleoside moiety at the 3′

O or the 5′

O and the central methyl carbon of the triaryl methyl linker group; and

wherein the reactive phosphorous group has the structure (VI)

2. The compound of claim 1, wherein the triaryl methyl linker group has the structure (IV)

3. The compound of claim 2, wherein R₁₂, R₁₃, and R₁₄ are independently selected from substituted phenyl and unsubstituted phenyl, provided that one of R₁₂, R₁₃, and R₁₄ is substituted by being bound to the reactive phosphorus group via the Cgp group.

4. The compound of claim 2, wherein R₁₂, R₁₃, and R₁₄ are independently selected from unsubstituted or substituted aryl groups selected from phenyl, biphenyl, naphthanyl, indolyl, pyridinyl, pyrrolyl, 2-thienyl, 3-thienyl, furanyl, annulenyl, quinolinyl, and anthracenyl, provided that one of R₁₂, R₁₃, and R₁₄ is substituted by being bound to the reactive phosphorous group via the Cgp group.

5. The compound of claim 4, wherein at least one of R₁₂, R₁₃, and R₁₄ is selected from naphthanyl, indolyl, pyridinyl, pyrrolyl, 2-thienyl, 3-thienyl, furanyl, annulenyl, quinolinyl, and anthracenyl, provided that one of R₁₂, R₁₃, and R₁₄ is substituted by being bound to the reactive phosphorous group via the Cgp group.

6. The compound of claim 2, wherein R₁₂, R₁₃, and R₁₄ are independently selected from phenyl, methoxyphenyl, dimethoxyphenyl, trimethoxyphenyl, and furanyl, provided that one of R₁₂, R₁₃, and R₁₄ is substituted by being bound to the reactive phosphorous group via the Cgp group.

7. The compound of claim 1, wherein the linking group denoted Cgp′

comprises a polynucleotide moiety.

8. The compound of claim 1, wherein X is a secondary amino group having the structure —

NQ₁Q₂;

in which Q₁ and Q₂ are independently selected from the group consisting of alkyl, aryl, aralkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, and cycloalkynyl.

9. The compound of claim 1, wherein Y is selected from alkyl, lower alkyl, alkenyl, benzyl, substituted benzyl, aryl, aralkyl, cycloalkyl, electron-withdrawing β

-substituted alkyl, electron-withdrawing β

-substituted ethyl;

electron-withdrawing substituted phenyl; and

electron-withdrawing substituted phenylethyl.

10. The compound of claim 1, wherein X is a diisopropyl amino group and Y is selected from methyl, benzyl, substituted benzyl, β

-cyanoethyl, methyl-β

-cyanoethyl, dimethyl-β

-cyanoethyl, phenylsulfonylethyl, methyl-sulfonylethyl, p-nitrophenylsulfonylethyl, 2,2,2-trichloro-1,1-dimethylethyl, 2-(4-pyridyl)ethyl, 2-(2-pyridyl)ethyl, allyl, 4-methylene-1-acetylphenol, β

-thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl, 2,2,2-trichloroethyl,p-nitrophenylethyl, p-cyanophenyl-ethyl, 9-fluorenylmethyl, 1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl, 2-methylthioethyl, 2-(diphenylphosphino)-ethyl, 1-methyl-1-phenylethyl, 3-buten-1-yl, 4-(trimethylsilyl)-2-buten-1-yl, cinnamyl, α

-methylcinnamyl, and 8-quinolyl.

11. A method comprising:

(a) providing a solid support having an available hydroxyl, amino, or thio reactive group bound thereto;

(b) contacting said solid support with a compound having the structure (I)

Phos-Cgp-Trl-Cgp′

-Nucl 

 

(I)

wherein;

Phos is a reactive phosphorus group which specifically reacts with a reactive group on a solid support to produce a phosphorous containing linkage group,

Trl is a triaryl methyl linker group having three aryl groups, wherein each of the three aryl groups are bound to a central methyl carbon, and at least one of said three aryl groups has one or more substituents, wherein one of said substituents is bound to Cgp and the central methyl carbon is bound to Cgp′

,

Cgp is a linking group linking the reactive phosphorus group and the triaryl methyl linker group, or is a bond linking the reactive phosphorus group and the triaryl methyl linker group,

Nucl is a nucleoside moiety, and

Cgp′

is a linking group linking the nucleoside moiety at the 3′

O or the 5′

O to the triaryl methyl linker group, or is a bond linking the nucleoside moiety at the 3′

O or the 5′

O to the central methyl carbon of the triaryl methyl linker group; and

wherein the reactive phosphorous group has the structure (VI)

12. The method of claim 11, wherein the available reactive group is selected from hydroxyl, and thio.

13. The method of claim 12, wherein X is a secondary amino group having the structure —

NQ₁Q₂;

in which Q₁ and Q₂ are independently selected from the group consisting of alkyl, aryl, aralkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, and cycloalkynyl.

14. The method of claim 12, wherein Y is selected from alkyl, lower alkyl, alkenyl, benzyl, substituted benzyl, aryl, aralkyl, cycloalkyl, electron-withdrawing β

-substituted alkyl, electron-withdrawing β

-substituted ethyl;

electron-withdrawing substituted phenyl; and

electron-withdrawing substituted phenylethyl.

15. The method of claim 11, wherein the nucleoside moiety has a hydroxyl protecting group bound thereto.

16. The method of claim 15, said method further comprising contacting the functionalized solid support with a combined deprotection/oxidation agent.

17. The method of claim 16, wherein the combined deprotection/oxidation agent comprises an alpha effect nucleophile.

18. The method of claim 11, wherein the triaryl methyl linker group has the structure (IV)

19. The method of claim 18, wherein R₁₂, R₁₃, R₁₄ are independently selected from substituted phenyl and unsubstituted phenyl, provided that one of R₁₂, R₁₃, and R₁₄ is substituted by being bound to the reactive phosphorus group.

20. The method of claim 18, wherein R₁₂, R₁₃, and R₁₄ are independently selected from unsubstituted or substituted aryl groups selected from phenyl, biphenyl, naphthanyl, indolyl, pyridinyl, pyrrolyl, 2-thienyl, 3-thienyl, furanyl, annulenyl, quinolinyl, and anthracenyl.

21. The method of claim 18, wherein at least one of R₁₂, R₁₃, and R₁₄ is selected from naphthanyl, indolyl, pyridinyl, pyrrolyl, 2-thienyl, 3-thienyl, furanyl, annulenyl, quinolinyl, and anthracenyl.