Oligonucleotide Oxidation Protocol for Selective P═O/P═S Backbone Control

Solution Overview

Problem

Existing oligonucleotide synthesis methods using iodine-based oxidation solutions inadvertently convert phosphorothioate internucleotide linkages to phosphodiester linkages during the oxidation of phosphite triester compounds, leading to higher than expected phosphodiester content.

Innovation Solution

An oxidation protocol involving an aged iodine-based solution, prepared with pyridine and water, is used to selectively oxidize phosphite triester compounds to phosphodiester compounds without affecting phosphorothioate internucleotide linkages, by controlling aging time and temperature.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If a freshly prepared oxidation solution comprising iodine, pyridine, and water is used to oxidize phosphite triester compounds, then the oxidation reaction proceeds efficiently, but phosphorothioate internucleotide linkages are inadvertently converted to phosphodiester linkages

Engineering Contradiction:

Improveoxidation reaction efficiencyVSAvoidselectivity of oxidation

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The oxidation solution is pre-aged for a specific time period (e.g., 16-72 hours) before use to allow selective oxidation of phosphite triesters while deactivating toward phosphorothioates. This preliminary action modifies the solution's reactivity profile to achieve the desired selectivity.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The key parameter changed is the aging time of the oxidation solution. By controlling the aging duration, the solution's oxidizing power is modulated to differentiate between phosphite triester and phosphorothioate substrates, achieving selective oxidation without converting phosphorothioates to phosphodiesters.

Inventive Principle:

Principle #35Parameter changes

2Manufacturing precision

If the oxidation solution is aged for a sufficient time period to selectively oxidize phosphite triesters, then phosphorothioate linkages are preserved, but the process time increases

Engineering Contradiction:

Improveselectivity of oxidationVSAvoidoxidation solution preparation time

Core Design Contradiction:

Manufacturing precisionVSLoss of time

Solution Approach 1:

The oxidation solution is pre-aged for a specific time period (e.g., 16-72 hours) before use to allow selective oxidation of phosphite triesters while deactivating toward phosphorothioates. This preliminary action modifies the solution's reactivity profile to achieve the desired selectivity.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The key parameter changed is the aging time of the oxidation solution. By controlling the aging duration, the solution's oxidizing power is modulated to differentiate between phosphite triester and phosphorothioate substrates, achieving selective oxidation without converting phosphorothioates to phosphodiesters.

Inventive Principle:

Principle #35Parameter changes

3Productivity

If standard oxidation conditions are used, then phosphite triesters are oxidized to phosphodiesters, but the purity of mixed P═O/P═S backbone oligonucleotides decreases due to unwanted P═S to P═O conversion

Engineering Contradiction:

Improveoxidation reaction efficiencyVSAvoidpurity of oligonucleotide product

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The oxidation solution is pre-aged for a specific time period (e.g., 16-72 hours) before use to allow selective oxidation of phosphite triesters while deactivating toward phosphorothioates. This preliminary action modifies the solution's reactivity profile to achieve the desired selectivity.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The key parameter changed is the aging time of the oxidation solution. By controlling the aging duration, the solution's oxidizing power is modulated to differentiate between phosphite triester and phosphorothioate substrates, achieving selective oxidation without converting phosphorothioates to phosphodiesters.

Inventive Principle:

Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This method ensures selective oxidation of phosphite triester compounds to phosphodiester compounds while preserving phosphorothioate internucleotide linkages, improving the purity and yield of mixed P═O/P═S backbone oligonucleotides.

Implementation Method 1

the oxidation of an intermediary phosphite triester compound of formula I into a phosphodiester compound of formula II

Methodology Applied

Scientific EffectOxidation: Oxidation

Data Source

PatentUS12606584B2Process for the preparation of oligonucleotides using modified oxidation protocol

Publication Date: 2026.04.21 F HOFFMANN LA ROCHE INC

AI summary

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 internucleotide linkages.