Method for in vitro oligonucleotide synthesis using H-phosphonates

• US 5,264,566 A
• Filed: 04/26/1990
• Issued: 11/23/1993
• Est. Priority Date: 10/15/1985
• Status: Expired due to Fees

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Matteucci, Mark D., Froehler, Brian C.
• Primary Examiner(s): Brown, Johnnie R.
• Assistant Examiner(s): CRANE, LAWRENCE E
• Application Number:
  US07/515,890
• Time in Patent Office: 1,307 Days
• Field of Search: 536/27-29
• 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

Abstract

A method is provided for the high fidelity, rapid and economical in vitro synthesis of oligonucleotides. Nucleoside H-phosphonates are condensed in seriatim using a dehydrating agent to produce a poly (nucleoside H-phosphonate). The product is oxidized to yield the desired oligonucleotide. A novel reagent is provided for multiple nucleoside additions in single cycles.

Claims

1. A method for preparing a polynucleotide phosphoramidate, said method comprising the steps of:

oxidizing a carrier-bound polynucleoside H-phosphonate having more than two nucleosides with an oxidizing agent capable of oxidizing said carrier-bound polynucleoside H-phosphonate so as to yield carrier-bound polynucleoside phosphoramidate; and

decoupling said polynucleoside phosphoramidate from said carrier so as to yield said polynucleotide phosphoramidate.

2. A method as recited in claim 1 wherein a polymer carrier is bound to a 3'"'"'-hydroxyl of said polynucleoside H-phosphonate.

3. A method as recited in claim 1 wherein said polynucleoside H-phosphonate is bound to a carrier of material selected from the group consisting of controlled pore glass, silica gel and polystyrene.

4. A method as recited in claim 1 wherein said oxidizing agent is selected from the group consisting of iodine, N-chlorosuccinimide, N-bromosuccinimide, and salts of periodic acids.

5. A method as recited in claim 1 wherein said carrier-bound polynucleoside H-phosphonate has at least 40 nucleosides and wherein said oxidizing step occurs:

with a relatively weak basic solution containing an amine with 1-5 S, N, or O atoms and fewer than 10 carbon atoms; and

then

with a relatively strong basic solution containing an amine with 1-5 S, N, or O atoms and fewer than 10 carbon atoms.

6. A method as recited in claim 1 wherein said decoupling step involves incubating a carrier to which said polynucleoside phosphoramidate is bound in concentrated ammonium hydroxide.

7. A method as recited in claim 1 wherein said oxidizing step occurs in a solution containing a base selected from the group comprising ammonia, primary amines and secondary amines, said base containing 1-5 S, N, or O atoms and fewer than 10 carbon atoms.

8. A method as recited in claim 7 wherein said amine includes an alkyl, an aryl, or an heteroaryl.

9. A method as recited in claim 7 wherein said amine is a heterocyclic secondary amine.

10. A method as recited in claim 7 wherein said solvent is selected from the group consisting of tetrahydrofuran and carbon tetrachloride.

11. A method for preparing a polynucleotide phosphoramidate, said method comprising the steps of:

oxidizing a carrier-bound polynucleoside H-phosphonate having more than two nucleosides in a solution of aqueous iodine and a base selected from the group consisting of NH₃, MeNH₂ and n-BuNH₂ in a solvent selected from the group consisting of tetrahydrofuran and carbon tetrachloride so as to yield carrier-bound polynucleoside phosphoramidate; and

incubating a carrier to which said polynucleoside phosphoramidate is bound in concentrated ammonium hydroxide so as to yield said polynucleotide phosphoramidate.