Phosphorus protecting groups

• US 7,368,550 B2
• Filed: 03/23/2006
• Issued: 05/06/2008
• Est. Priority Date: 10/31/2005
• 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, Timar, Zoltan
• Primary Examiner(s): CRANE, LAWRENCE E
• Application Number:
  US11/388,339
• Publication Number:
  US 20070099859A1
• Time in Patent Office: 775 Days
• Field of Search: None
• US Class Current: 536/23.1
• 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: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Compounds having a phosphorus group structure of:

wherein:

• • R is alkyl, modified lower alkyl; and
  • Rⁱ and Rⁱ are each independently H, alkyl, modified alkyl, or aryl; are provided. Also provided are polynucleotide compositions that include these compounds and methods of using the compounds in synthesis of the same.

Claims

1. A compound having a structure selected from structure (I) or structure (II):

2. The compound of claim 1, wherein A is a protected 2′

-hydroxyl group, R is methyl, ethyl, n-propyl, or isopropyl;

Rⁱ and Rⁱⁱ are each independently H, methyl, ethyl, n-propyl, or isopropyl;

Rⁱⁱⁱ and R^iv are each isopropyl; and

R^v is a hydroxyl protecting group.

3. The compound of claim 1, wherein R^v is a hydroxyl protecting group.

4. A polynucleotide, the polynucleotide comprising at least one nucleotide subunit having the structure (III):

5. The polynucleotide of claim 4, wherein A is a protected 2′

-hydroxyl group, R is selected from methyl, ethyl, n-propyl, or isopropyl; and

Rⁱ and Rⁱⁱ are each independently H, methyl, ethyl, n-propyl, or isopropyl.

6. A polynucleotide, the polynucleotide comprising a plurality of nucleotide subunits, at least one of said plurality of nucleotide subunits bound to a phosphorus protecting group, the phosphorus protecting group having the structure (V):

7. The polynucleotide of claim 6, wherein R is a methyl, ethyl, n-propyl, or isopropyl;

and R1 and R″

are each independently H, methyl, ethyl, n-propyl, or isopropyl.

8. The polynucleotide of claim 6, wherein each one of at least 50% of the plurality of nucleotide subunits is bound to a respective phosphorus protecting group, each respective phosphorus protecting group having the structure (V).

9. The polynucleotide of claim 6, wherein the polynucleotide is DNA or RNA.

10. A method of deprotecting a protected polynucleotide, the method comprising;

contacting the polynucleotide with a solution comprising an α

-effect nucleophile, wherein the polynucleotide comprises a plurality of nucleotide subunits, at least one of said plurality of nucleotide subunits bound to a phosphorus protecting group, the phosphorus protecting group having the structure (V);

11. The method of claim 10 wherein the solution is at a pH of about 6 to about 11.

12. The method of claim 10 wherein the α

-effect nucleophile has a pK_a in the range of about 4 to 13.

13. The method of claim 10 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 butylhyd roperoxide, a benzylhyd roperoxide, a phenylhydroperoxide, a cumene hydroperoxide, performic acid, peracetic acid, perbenzoic acid, a substituted perbenzoic acid, chloroperbenzoic acid, perbutyric acid, tertiary-butylperoxybenzoic acid, decanediperoxoic acid and corresponding salts of said species.

14. The method of claim 10 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 10 wherein the α

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

16. The method of claim 10 wherein the α

-effect nucleophile has a pK_a of about 4 to 13 and the solution is at a pH in the range from about 6 to about 11.

17. The method of claim 10 wherein the cleavage of the phosphorus protecting group results in a deprotected polynucleotide.

18. The method of claim 10 wherein the cleavage of the phosphorus protecting group results in a solution of deprotected polynucleotide, the method further comprising adding an alcohol to the solution of deprotected polynucleotide to result in precipitation of the deprotected polynucleotide, and recovering the precipitated polynucleotide.

19. The method of claim 10 further comprising wherein the protected polynucleotide is attached to a substrate via a cleavable linker.