Synthesis of polynucleotides using combined oxidation/deprotection chemistry

• US 7,135,565 B2
• Filed: 07/27/2001
• Issued: 11/14/2006
• Est. Priority Date: 07/28/2000
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc., The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: Agilent Technologies, Inc., University of Colorado System
• Inventors: Perbost, Michael G. M., Caruthers, Marvin H., Dellinger, Douglas J., Betley, Jason R.
• Primary Examiner(s): Barts, Samuel
• Assistant Examiner(s): LEWIS, PATRICK T
• Application Number:
  US09/916,369
• Publication Number:
  US 20020058802A1
• Time in Patent Office: 1,936 Days
• Field of Search: 536/25.3, 536/22.1, 536/23.1, 536/24.3, 536/25.31, 536/25.33, 536/25.34, 435/6
• US Class Current: 536/25.3
• 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

A method of synthesizing a polynucleotide which can, for example, be used during fabrication of an array. A second nucleoside is coupled to a first nucleoside through a phosphite linkage, with the second nucleoside having a hydroxyl protecting group that is a non-carbonate protecting group. The product of the foregoing step is exposed to a composition which both oxidizes the formed phosphite to a phosphate and deprotects the protected hydroxyl of the coupled nucleoside. The method has particular application to fabricating an addressable array of polynucleotides on a substrate which carries substrate bound moieties each with a hydroxyl group.

Claims

1. A method of synthesizing a polynucleotide, comprising:

(a) coupling a second nucleoside to a first nucleoside through a phosphite linkage,

wherein the second nucleoside has a non-carbonate protecting group protecting a hydroxyl; and

(b) exposing the product of step (a) to a composition which concurrently oxidizes the phosphite formed in step (a) to a phosphate and deprotects the protected hydroxyl of the second nucleoside.

2. A method according to claim 1 wherein the second nucleoside is a phosphoramidite and wherein steps (a) and (b) are repeated and the hydroxyl deprotected in a first iteration of step (b) reacts to form the phosphite linkage with the second nucleoside in the next iteration of step (a).

3. A method according to claim 1 wherein the non-carbonate protecting group is an acid labile protecting group and the composition comprises an acid to remove the non-carbonate protecting group.

4. A method according to claim 1 wherein the composition comprises a solution with a solvent which is primarily non-aqueous.

5. A method according to claim 4 wherein the solution is anhydrous.

6. A method according to claim 2 wherein the solution comprises iodine, an oxaziridine or a peroxide as an oxidizing agent.

7. A method according to claim 2 wherein the composition comprises an acetic acid and iodine, an oxaziridine, or an organic peroxide.

8. A method according to claim 1 wherein the non-carbonate protecting group is labile under nucleophilic attack under neutral or mildly basic conditions and the composition comprises a nucleophile that exhibits an alpha effect at neutral to mildly basic pH.

9. The method of claim 8 wherein the nucleophile is an inorganic peroxide of the formula M+OOH−

, wherein M+ is a counterion selected from the group consisting of H+, Li+, Na+, K+, Rb+ and Cs+.

10. The method of claim 8, wherein the nucleophile is an organic peroxide of the formula (V), (VI) or (VII),

in which R4 through R10 are hydrocarbyl optionally substituted with one or more nonhydrocarbyl substituents and optionally containing one or more nonhydrocarbyl linkages.

11. The method of claim 8 wherein the nucleophile is one of t-butyl hydroperoxide or m-chloroperoxybenzoic acid, or mixtures thereof.