Oligonucleotide Synthesis via Liquid Phase Condensation and Purification
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Solution Overview
Problem
Current methods for producing oligonucleotides, such as the solid phase and liquid phase methods, face limitations in scalability and efficiency, particularly in large-scale synthesis of long oligonucleotides, with impurities present during condensation steps.
Innovation Solution
A novel production method involving the steps of condensing a nucleoside or oligonucleotide with a protected 3′-hydroxy group and a nucleoside or oligonucleotide with a phosphoramidited 3′-hydroxy group, followed by oxidation or sulfurization, and then removing the 5′-hydroxy protecting group to purify the oligonucleotide, which avoids impurities during condensation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If solid phase method is used for oligonucleotide production, then synthesis speed is improved and automation progresses, but scalability is limited due to facility restriction
Solution Approach 1:
The patent extracts the oligonucleotide synthesis process from the solid phase support into liquid phase solution chemistry, allowing the reaction to proceed in homogeneous solution rather than being constrained by solid phase facility limitations. This enables scalable production while maintaining synthesis efficiency
Solution Approach 2:
The patent changes the physical state parameter from solid phase to liquid phase, fundamentally altering the reaction environment from heterogeneous to homogeneous. This parameter change enables both rapid synthesis and scalable production by eliminating the facility restrictions inherent to solid phase methods
2Productivity
If liquid phase method is used for oligonucleotide production, then scalability is improved, but operation complexity increases and yield decreases
Solution Approach 1:
The patent applies preliminary protection to the 3'-hydroxy group of the nucleoside before condensation, and uses pre-formed phosphoramidite derivatives. This preliminary preparation simplifies the actual condensation step and reduces operational complexity during the synthesis process, enabling scalable production with manageable procedure complexity
Solution Approach 2:
The patent uses hydrophobic protecting groups as intermediaries that facilitate both the condensation reaction and subsequent purification. These protecting groups act as mediators that enable the reaction to proceed efficiently and allow for simple extraction-based purification, reducing operational complexity
3Ease of operation
If deprotection is performed before condensation in conventional methods, then 5'-hydroxy group is activated, but impurities are present during condensation step
Solution Approach 1:
The patent inverts the conventional sequence by performing condensation before deprotection. The 5'-hydroxy group remains protected during condensation, preventing impurity formation, and is only deprotected after the condensation step is complete. This inverted sequence eliminates impurities during condensation while maintaining ease of operation
Solution Approach 2:
The patent uses preliminary protection of the 5'-hydroxy group with acid-labile protecting groups that remain intact during condensation. This preliminary protection prevents unwanted side reactions and impurity formation during the condensation step, ensuring high manufacturing precision
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 enables efficient condensation and purification of oligonucleotides, improving the scalability and yield of oligonucleotide synthesis by eliminating impurities and enhancing the efficiency of the process.
Implementation Method 1
condensing a nucleoside, nucleotide or oligonucleotide (a) and a nucleoside, nucleotide or oligonucleotide (b) to give a phosphite triester product (c)
Implementation Method 2
oxidizing or sulfurizing a phosphite triester product (c) obtained by the condensation
Implementation Method 3
oxidizing or sulfurizing a phosphite triester product (c) obtained by the condensation
Implementation Method 4
removing the 5′-hydroxy-protecting group from an oligonucleotide (d) obtained by the oxidation or sulfurization (deprotection)
Implementation Method 5
a nucleoside, nucleotide or oligonucleotide (b) wherein a 3′-hydroxy group or 3′-amino group is phosphoramidited and a 5′-hydroxy group is protected by a protecting group
Implementation Method 6
purifying by solid-liquid separating or extracting an oligonucleotide (e) wherein the 5′-hydroxy group is not protected
Data Source
AI summary
Oligonucleotides may be produced by a process, including (1) condensing a nucleoside, nucleotide or oligonucleotide (b), and a nucleoside, nucleotide or oligonucleotide (a), or a substituted nucleotide or oligonucleotide (α) in a non-polar solvent to give a reaction solution containing a phosphite triester product (c); (3) oxidizing or sulfurizing the phosphite triester product (c) to give a reaction solution containing an oligonucleotide (d) wherein the 5′-hydroxy group is protected; (4) deprotecting the oligonucleotide (d) to give a reaction solution containing an oligonucleotide (e) wherein the 5′-hydroxy group is not protected; and (6) adding a polar solvent to the reaction solution containing the oligonucleotide (e) and purifying the oligonucleotide (e) by solid-liquid separation, wherein said nucleoside, nucleotide or oligonucleotide (a) or said substituted nucleotide or oligonucleotide (α) is a compound represented by formula (a-i):wherein Base, Rp1, R10, m, L, Y, and Z are defined herein.