Thiocarbonate linkers for polynucleotides

• US 7,790,387 B2
• Filed: 09/24/2007
• Issued: 09/07/2010
• Est. Priority Date: 09/24/2007
• 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: Myerson, Joel, Dellinger, Geraldine, Timar, Zoltan, Caruthers, Marvin H., Dellinger, Douglas J.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/903,821
• Publication Number:
  US 20090082554A1
• Time in Patent Office: 1,079 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.1
• 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 21/02 with ribosyl as saccharide radical
• 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 polynucleotide bound to a substrate via a cleavable linker with a solution comprising an α

-effect nucleophile to result in cleavage of the polynucleotide from the substrate to produce a free polynucleotide;

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, salts of hydrogen peroxide, and mixtures of hydrogen peroxide and performic acid.

5. 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-, keto-, ester-, phospho- 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-, keto-, ester-, and amido- is present.

6. 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-, keto-, ester-, or amido.

7. The method of claim 1, wherein the 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 polynucleotide from the substrate and deprotection of the polynucleotide.

8. The method according to claim 1, wherein said method further comprises combining said modified free polynucleotide with a pharmaceutically acceptable vehicle.

9. The method according to claim 1, wherein said polynucleotide is a DNA.

10. The method according to claim 1, wherein said polynucleotide is a RNA.

21. A polynucleotide produced according to the method of claim 1.

23. 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.

25. The method of claim 1, wherein:

RPN is a 2′

-, 3′

- or 5′

-position of a terminal nucleotide of the polynucleotide; and

wherein the cleaved polynucleotide comprises a hydroxyl group at the 2′

-, 3′

- or 5′

-position of the terminal nucleotide.

11. A method comprising:

contacting a polynucleotide bound to a substrate via a cleavable linker with a solution comprising an α

-effect nucleophile to result in cleavage of the polynucleotide from the substrate to produce a free polynucleotide;

wherein the cleavable linker has the structure (I);

12. The method of claim 11, wherein the solution is at a pH of about 6 to about 12.

13. The method of claim 11, wherein the α

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

14. The method of claim 11, 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.

15. The method of claim 11, 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-, keto-, phosphor 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-, keto-, ester-, and amido- is present.

16. The method of claim 11, 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-, keto-, ester-, or amido.

17. The method of claim 11, wherein the 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 polynucleotide from the substrate and deprotection of the polynucleotide.

18. The method according to claim 11, wherein said method further comprises chemically modifiying said free polynucleotide to produce a modified free polynucleotide.

19. The method according to claim 11, wherein said polynucleotide is a DNA.

20. The method according to claim 11, wherein said polynucleotide is an RNA.

22. A polynucleotide produced according to the method of claim 11.

24. The method of claim 11, further comprising:

prior to said contacting;

synthesizing said 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.

26. The method of claim 11, wherein:

RPN is a 2′

-, 3′

- or 5′

-position of a terminal nucleotide of the polynucleotide; and

wherein the cleaved polynucleotide comprises a hydroxyl group at the 2′

-, 3′

- or 5′

-position of the terminal nucleotide.