3′-Terminal Oligonucleotide Phosphitylation Without Protecting Group Loss
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Solution Overview
Problem
Existing methods for synthesizing oligonucleotides face challenges with preservation stability of phosphitylating agents, excessive activation leading to protecting group loss, and inefficient production processes, particularly in fragment condensation methods.
Innovation Solution
The use of diamidite as a phosphitylating agent with specific activators and organic bases, along with a particular silyl-protecting group, to stabilize the reaction and prevent protecting group loss during phosphitylation and desilylation, enabling more efficient production of oligonucleotides with functional groups at the 3′-terminal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite is used as a phosphitylating agent, then phosphitylation of nucleosides can be performed, but the agent has poor preservation stability and high cost
Solution Approach 1:
The patent replaces the expensive and unstable 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite, which is more stable and economical. The substitution involves changing the structural composition of the phosphitylating agent to improve both stability and cost-effectiveness while maintaining phosphitylation functionality.
2Productivity
If Diisopropylammonium tetrazolide is used as an activator for phosphitylation, then the reaction can proceed, but the cyanoethyl protecting group falls off due to excessive activation
Solution Approach 1:
The patent changes the activator from Diisopropylammonium tetrazolide to 1H-tetrazole, adjusting the activation strength parameter. This parameter change reduces excessive activation that causes cyanoethyl group elimination, thereby maintaining protecting group stability while still enabling the phosphitylation reaction to proceed efficiently.
3Ease of manufacture
If 3HF-TEA is used for desilylation of the 3′-terminal, then silyl-protecting group can be removed, but the cyanoethyl protecting group on phosphoric acid falls off
Solution Approach 1:
The patent introduces an intermediary substance (1H-tetrazole or similar) that mediates the desilylation process. This intermediary enables selective removal of the silyl-protecting group while protecting the cyanoethyl group on the phosphoric acid from falling off, thus maintaining protecting group integrity during deprotection.
4Productivity
If fragment condensation method is used for synthesizing oligonucleotides of 20 mer or more, then synthesis efficiency is improved, but special care is required for solubility, selective deprotection and activation
Solution Approach 1:
The patent develops a universal set of reagents and conditions that can be applied across different fragment condensation scenarios. The standardized phosphitylating agent, activator, and desilylation conditions work consistently for various oligonucleotide fragments, reducing the need for special care and optimization in each case, thereby simplifying the overall process while maintaining high synthesis efficiency.
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 stabilizes the phosphitylating agent and prevents protecting group loss, allowing for more efficient and stable production of oligonucleotides, particularly in fragment condensation processes.
Implementation Method 1
a step of reacting an n-mer oligonucleotide with a phosphitylating agent in a solvent in the presence of a base to phosphitylate a terminal hydroxyl group of the oligonucleotide
Implementation Method 2
reacting a phosphitylating agent precursor with an activator in a solvent to prepare a phosphitylating agent
Implementation Method 3
reacting an n-mer oligonucleotide with a phosphitylating agent in a solvent in the presence of a base
Data Source
AI summary
The present invention aims to provide a more stable and efficient method for producing oligonucleotide, particularly, oligonucleotide having various functional groups linked to the 3′-terminal and the like. Efficient production of oligonucleotide becomes possible by a production method of an oligonucleotide represented by the formula (Ia-2) (each symbol is as defined in the DESCRIPTION) and having a functional group at 3′-terminal, the method including a step of subjecting an oligonucleic acid with 3′-terminal protected by a silyl-protecting group to 3′-terminal-selective deprotection under desilylation conditions that do not affect protecting groups other than the silyl group, subjecting same to phosphitylation conditions with a phosphoramidite reagent that do not affect protecting groups on the oligonucleic acid to give a 3′-terminal-phosphoramidited oligonucleotide represented by the formula (Ia-1) (each symbol is as defined in the DESCRIPTION), and linking a functional group to 3′-terminal of 3′-terminal phosphoramidited oligonucleotide directly or via a linker, and the like.