PROCESS FOR THE PREPARATION OF OLIGONUCLEOTIDES USING MODIFIED OXIDATION PROTOCOL

Abstract

The invention relates to a process for the production of a mixed P═O/P═S backbone oligonucleotide comprising a selective oxidation of an intermediary phosphite triester compound of formula I into a phosphodiester compound of formula II according to the scheme with an oxidation solution obtained by mixing iodine, an organic solvent and water, characterized in that the oxidation solution has been aged for a time period that is sufficient to selectively oxidize the phosphite triester compound of formula I into the phosphodiester compound of formula II without oxidizing the phosphorothioate intenucleotide linkages.

Description

Claims

1. A process for the production of a mixed P═O/P═S backbone oligonucleotide comprising the oxidation of an intermediary phosphite triester compound of formula I into a phosphodiester compound of formula II according to the scheme with an oxidation solution obtained by mixing iodine, an organic solvent and water, characterized in that the oxidation solution has been aged for a time period that is sufficient to selectively oxidize the phosphite triester compound of formula I into the phosphodiester compound of formula II without oxidizing the phosphorothioate internucleotide linkages.

2. The process of claim 1, wherein the organic solvent is pyridine or a C1-6 alkyl-substituted pyridine.

3. The process of claim 2, wherein the volume ratio of pyridine or C1-6 alkyl-substituted pyridine to water is from 1:1 to 20:1.

4. The process of claim 1, wherein the iodine concentration in the oxidation solution is 10 mM to 100 mM.

5. The process of claim 1, wherein the aging of the oxidation solution takes place at a temperature of 20° C. to 100° C.

6. The process of claim 1, wherein the oxidation solution has been aged for a time period of at least 1 day, 3 days, 5 days, 10 days, 15 days or at least 20 days.

7. The process of claim 1, wherein the process comprises the monitoring of the pH and the conductivity to determine the time period that is sufficient to selectively oxidize the phosphite triester compound of formula I into the phosphodiester compound of formula II without oxidizing the phosphorothioate internucleotide linkages.

8. The process of claim 1, wherein the amount of oxidant used in the oxidation reaction is selected between 1.1 equivalents and 15 equivalents.

9. The process of claim 1, wherein the reaction temperature for the oxidation reaction is selected between 15° C. and 27° C.

10. The process of claim 1, wherein the oligonucleotide consists of optionally modified DNA or RNA nucleoside monomers or combinations thereof and is 10 to 40 nucleotides in length.

11. The process of claim 3, wherein the volume ratio of pyridine or C1-6 alkyl-substituted pyridine to water is from 5:1 to 15:1.

12. The process of claim 3, wherein the volume ratio of pyridine or C1-6 alkyl-substituted pyridine to water is 9:1.

13. The process of claim 4, wherein the iodine concentration in the oxidation solution is 20 mM to 50 mM.

14. The process of claim 5, wherein the aging of the oxidation solution takes place at a temperature of 30° C. to 60° C.

15. The process of claim 8, wherein the amount of oxidant used in the oxidation reaction is selected between 1.5 equivalents and 4.5 equivalents.

16. The process of claim 8, wherein the amount of oxidant used in the oxidation reaction is selected between 2 equivalents and 4 equivalents.

17. The process of claim 9, wherein the reaction temperature for the oxidation reaction is selected between 18° C. and 24° C.

18. The process of claim 10, wherein the oligonucleotide consists of optionally modified DNA or RNA nucleoside monomers or combinations thereof and is 10 to 25 nucleotides in length.