Cleavable thiocarbonate linkers for polynucleotide synthesis

• US 7,855,281 B2
• Filed: 03/23/2006
• Issued: 12/21/2010
• Est. Priority Date: 03/23/2006
• 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., The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: Dellinger, Douglas J, Caruthers, Marvin, Myerson, Joel, Dellinger, Geraldine, Timar, Zoltan
• Primary Examiner(s): Staples; Mark
• Application Number:
  US11/389,326
• Publication Number:
  US 20070224602A1
• Time in Patent Office: 1,734 Days
• Field of Search: None
• US Class Current: 536/22.1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

In various embodiments of the invention, novel compositions having a polynucleotide bound to a substrate via a cleavable linker are provided, and methods of cleaving a polynucleotide from a substrate are provided.

Claims

1. A method comprising:

contacting a synthetic polynucleotide that is covalently bound at a terminal nucleotide of the synthetic polynucleotide to a substrate via a cleavable linker with a solution comprising an α

-effect nucleophile to result in cleavage of the synthetic polynucleotide from the substrate;

wherein the cleavable linker has the structure (I);

2. The method of claim 1 wherein the solution is at a pH of about 6 to about 12.

3. The method of claim 1 wherein the α

-effect nucleophile is characterized as having a pKa in the range of about 4 to 13.

4. The method of claim 1 wherein the solution comprising the α

-effect nucleophile is a solution comprising one or more species selected from hydrogen peroxide, a peracid, a perboric acid, an alkylperoxide, a hydroperoxide, a butylhydroperoxide, a benzylhydroperoxide, a phenylhydroperoxide, a cumene hydroperoxide, performic acid, a peracetic acid, perbenzoic acid, a substituted perbenzoic acid, chloroperbenzoic acid, perbutyric acid, tertiary-butylperoxybenzoic acid, decanediperoxoic acid, corresponding salts of said species, and combinations thereof.

5. The method of claim 1 wherein the solution comprising the α

-effect nucleophile is a solution comprising one or more species selected from hydrogen peroxide, salts of hydrogen peroxide, and mixtures of hydrogen peroxide and performic acid.

6. The method of claim 1 wherein the α

-effect nucleophile is formed in situ by a reaction of hydrogen peroxide and a carboxylic acid or carboxylic acid salt.

7. The method of claim 1, wherein the linking group Lnk is selected from:

(1) a lower alkyl group;

(2) a modified lower alkyl group in which one or more linkages selected from ether-, thio-, amino-, oxo-, ester-, and amido- is present;

(3) a modified lower alkyl substituted with one or more groups including lower alkyl;

aryl, aralkyl, alkoxyl, thioalkyl, hydroxyl, amino, amido, sulfonyl, halo;

or

(4) a modified lower alkyl substituted with one or more groups including lower alkyl;

alkoxyl, thioalkyl, hydroxyl, amino, amido, sulfonyl, halo, and in which one or more linkages selected from ether-, thio-, amino-, oxo-, ester-, and amido- is present.

8. The method of claim 1, wherein the linking group Lnk has the structure

—

(CH2)m-Lkg-(CH2)n-

wherein;

m and n are integers independently selected from the range of 1 to about 12, and

Lkg is a linkage selected from ether-, thio-, amino-, oxo-, ester-, or amido-.

9. The method of claim 1, further comprising recovering the polynucleotide after cleavage of the synthetic polynucleotide from the substrate.

10. The method of claim 1, wherein the synthetic polynucleotide comprises at least one protecting group selected from a nucleobase protecting group, a 2′

-hydroxyl protecting group, and a phosphate protecting group, wherein said at least one protecting group is labile under conditions which include an α

-effect nucleophile; and

wherein said contacting results in concurrent cleavage of the synthetic polynucleotide from the substrate and deprotection of the synthetic polynucleotide.

11. The method of claim 1:

wherein the synthetic polynucleotide that is covalently bound at a terminal nucleotide of the synthetic polynucleotide to a substrate via the cleavable linker is an RNA, said RNA has a 2′

-hydroxyl protecting group, said 2′

hydroxyl protecting group is characterized as being peroxyanion-labile;

wherein the α

-effect nucleophile is a peroxyanion; and

wherein said contacting results in concurrent cleavage of the RNA from the substrate and cleavage of the 2′

-hydroxyl protecting group.

12. The method of claim 1:

wherein the synthetic polynucleotide comprises a 2′

-hydroxyl protecting group and at least one additional protecting group selected from a nucleobase protecting group and a phosphorus protecting group;

wherein said 2′

-hydroxyl protecting group is stable under conditions which include an α

-effect nucleophile;

wherein said at least one additional protecting group is labile under conditions which include an α

-effect nucleophile; and

wherein said contacting results in concurrent cleavage of the synthetic polynucleotide from the substrate and cleavage of said at least one additional protecting group.

13. The method of claim 12 further comprising cleaving the 2′

hydroxyl protecting group under conditions sufficient to result in cleavage of the 2′

hydroxyl protecting group, wherein said conditions do not include α

-effect nucleophile.

14. The method of claim 1, further comprising:

prior to said contacting;

synthesizing said synthetic polynucleotide by a sequential addition of nucleotide monomers to a nucleotide or nucleoside monomer that is covalently bound to said substrate via said cleavable linker.