Stereodefined Phosphorothioate Oligonucleotide Synthesis via Periodic Coupling
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Solution Overview
Problem
The synthesis of stereodefined phosphorothioate oligonucleotides is inefficient compared to non-stereodefined phosphorothioate oligonucleotides, requiring improved methods to enhance yield and purity.
Innovation Solution
A method involving multiple coupling and oxidation cycles within a single elongation cycle using oxazaphospholidine phosphoramidite monomers, with deprotection, coupling, oxidation, and optional washing steps repeated to stabilize phosphite-triester intermediates, enhancing the coupling efficacy and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If multiple coupling and oxidation cycles are performed within a single elongation cycle, then the yield and purity of stereodefined phosphorothioate oligonucleotides is improved, but the synthesis time and process complexity increase
Solution Approach 1:
The patent applies periodic action by implementing multiple alternating coupling and oxidation cycles within a single elongation cycle. Instead of performing a single coupling step followed by a single oxidation step, the method repeatedly alternates between coupling oxazaphospholidine phosphoramidite monomers and oxidizing the resulting phosphite triester intermediates. This periodic repetition of coupling-oxidation sequences ensures complete conversion and stabilizes intermediates, thereby improving yield and purity of stereodefined phosphorothioate oligonucleotides despite the increased number of steps.
2Productivity
If multiple coupling and oxidation cycles are performed within a single elongation cycle, then the yield of stereodefined phosphorothioate oligonucleotides is improved, but the process complexity increases
Solution Approach 1:
The patent merges multiple coupling and oxidation operations into a single integrated elongation cycle. By combining several coupling steps and several oxidation steps within one elongation cycle rather than treating them as separate cycles, the method achieves higher yield through repeated stabilization of phosphite triester intermediates while managing process complexity through systematic integration of steps.
Solution Approach 2:
The patent applies preliminary action by performing oxidation steps immediately after coupling steps to stabilize phosphite triester intermediates before they can decompose or undergo side reactions. This preliminary stabilization through repeated oxidation prevents loss of intermediate compounds and ensures high yield of the final stereodefined phosphorothioate product.
3Reliability
If prolonged coupling steps are used to achieve efficient coupling, then the coupling efficacy is improved, but the stability of phosphite-triester intermediates decreases
Solution Approach 1:
The patent resolves the contradiction between coupling efficacy and intermediate stability by implementing periodic alternating cycles of coupling and oxidation. Instead of using prolonged continuous coupling steps that lead to intermediate decomposition, the method performs brief coupling steps followed by immediate oxidation steps. This periodic interruption and stabilization prevents phosphite triester intermediate decomposition while ensuring complete coupling through repetition of the cycle.
Solution Approach 2:
The patent applies preliminary action by performing oxidation immediately after each coupling step to stabilize the phosphite triester intermediate before it can decompose. This preliminary stabilization action prevents the loss of intermediates that would occur during prolonged coupling steps, thereby maintaining both high coupling efficacy and intermediate stability.
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 significantly improves the yield and purity of stereodefined phosphorothioate oligonucleotides by stabilizing intermediate phosphite-triester compounds, addressing inefficiencies in existing synthesis methods.
Implementation Method 1
oxidizing the phosphite triester intermediate with a sulfurizing reagent
Data Source
AI summary
The present invention relates to methods of synthesis of stereodefined oligonucleotides wherein the protected 5′-hydroxy terminus of a nucleoside or oligonucleotide attached to a solid support is first unprotected, followed by coupling an oxazaphospholidine phosphoramidite monomer to the now deprotected 5′-hydroxy terminus, oxidizing the resultant phosphite triester intermediate with a sulfurizing reagent, and optionally washing the product. The method of synthesis of the present invention described above may be repeated to optionally elongate the stereodefined oligonucleotide being synthesized until the elongation cycle is intentionally terminated via deprotecting and cleavaging the resulting stereodefined oligonucleotide from the solid support. The oxazaphospholidine phosphoramidite monomer to be coupled in the present invention may optionally be chosen from Formula 1a and 1b. Generally, the present invention relates to enhanced synthesis methods wherein a single elongation cycle, optionally comprising repeated coupling and oxidation steps, results in an enhanced yield and higher purity of stereodefined phosphorothioate oligonucleotides.