Method for polynucleotide synthesis

• US 7,417,139 B2
• Filed: 08/30/2003
• Issued: 08/26/2008
• Est. Priority Date: 08/30/2003
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc., The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Dellinger, Douglas J, Sierzchala, Agnieszka B., Caruthers, Marvin H, Dellinger, Geraldine F
• Primary Examiner(s): Crane; L. E.
• Application Number:
  US10/652,054
• Publication Number:
  US 20050048601A1
• Time in Patent Office: 1,823 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
• 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

Methods of forming an internucleotide bond are disclosed. Such methods find use in synthesis of polynucleotides. The method involves contacting a functionalized support with a precursor having an exocyclic amine triaryl methyl protecting group under conditions and for a time sufficient to result in internucleotide bond formation. The functionalized support includes a solid support, a triaryl methyl linker group, and a nucleoside moiety having a reactive site hydroxyl, the nucleoside moiety attached to the solid support via the triaryl methyl linker group. In particular embodiments, the precursor has the structure:

wherein:

• • O and H represent oxygen and hydrogen, respectively
  • R1 is hydrido, hydroxyl, protected hydroxyl, lower alkyl, modified lower alkyl, or alkoxy,
  • one of R2 or R3 is a hydroxyl protecting group; and the other of R2 or R3 is a reactive group capable of reacting with the reactive site hydroxyl,
  • Base is a heterocyclic base having an exocyclic amine group, and
  • Tram is the exocyclic amine triaryl methyl protecting group.

Claims

1. A method of forming an internucleotide bond, the method comprising:

contacting a functionalized support with a precursor under conditions and for a time sufficient to result in internucleotide bond formation,

said functionalized support comprising a solid support, a triarylmethyl linker group, and a nucleoside moiety having a reactive site hydroxyl, the nucleoside moiety bound to the solid support via the triarylmethyl linker group,

said precursor having the structure (IVc)

2. The method of claim 1, wherein the exocyclic amine triaryl methyl protecting group has the structure (VIc)

3. The method of claim 2, wherein R₄, R₅, and R₆ are independently selected from substituted and unsubstituted phenyl groups.

4. The method of claim 2, wherein R₄, R₅, and R₆ are optionally substituted aryl groups independently selected from phenyl, biphenyl, naphthanyl, indolyl, pyriduiyl, pyrrolyl, thiophenyl, furanyl, annulenyl, quinolinyl, and anthracenyl.

5. The method of claim 2, wherein R₄, R₅, and R₆ are independently selected from phenyl, methoxyphenyl, dimethoxyphenyl, trimethoxyphenyl, and furanyl.

6. The method of claim 1, wherein Ris a hydrido, the hydroxyl protecting group is a carbonate protecting group, and the reactive group has the structure (Vc):

7. The method of claim 6, 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.

8. The method of claim 6, wherein Y is selected from alkyl, lower alkyl, alkenyl, benzyl, substituted benzyl, aryl, aralkyl, cycloalkyl, electron-β

-(electron-withdrawing group) substituted alkyl, electron-β

-(electron-withdrawing group) substituted ethyl;

electron-withdrawing substituted phenyl;

or electron-withdrawing substituted phenylethyl.

9. The method of claim 6, wherein X is a diisopropyl amino group and Y is a cyanoethyl group.

10. The method of claim 6, 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. The method of claim 10, wherein the carbonate protecting group is an optionally substituted aryl carbonate protecting group.