Oligonucleotide Protected Base for Liquid-Phase Extraction
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Solution Overview
Problem
Current oligonucleotide synthesis methods, such as the phosphate triester and phosphoramidite methods, face challenges in scaling up, efficiency, and purification, particularly in liquid phase synthesis, due to limitations in reagent usage, reaction monitoring, and the need for complex purification processes.
Innovation Solution
A phosphoramidite method using an oligonucleotide with a protected base, featuring a C5-30 straight chain or branched chain alkyl group or alkenyl group for nucleic acid base protection, allowing for efficient purification through liquid-liquid extraction, eliminating the need for solidification isolation and enabling continuous oligonucleotide production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If solid phase synthesis method is used, then synthesis speed is improved, but scaling-up is limited due to facility restriction and reagents are used in excess
Solution Approach 1:
The patent uses a soluble polymer as an intermediary support for oligonucleotide synthesis. This polymer acts as a mediator that provides the benefits of solid phase synthesis (ease of handling, automated synthesis) while allowing for liquid phase reactions and simplified purification through filtration and solvent removal, thereby enabling scaling-up without facility restrictions
Solution Approach 2:
The patent changes the physical state parameter of the support from solid (traditional solid phase synthesis) to soluble polymer in liquid phase. This parameter change allows the system to maintain the advantages of solid phase synthesis while enabling liquid phase reactions, improved mass transfer, and simplified purification processes that facilitate scaling-up
2Productivity
If liquid phase synthesis method is used, then scaling-up is improved, but purification operation is complicated and yield is low
Solution Approach 1:
The soluble polymer serves as an intermediary that attaches to the oligonucleotide during synthesis, allowing the product to remain associated with a handle that facilitates simple filtration and purification. This intermediary approach maintains ease of manufacture by enabling straightforward separation through filtration and solvent removal rather than complex chromatographic procedures
3Quantity of substance
If MPEG soluble polymer is used as protecting group, then synthesis of up to 20 mer DNA is achieved, but crystallization isolation operation is essential and progress status confirmation is difficult
Solution Approach 1:
The patent employs protecting groups with distinctive UV-absorbing chromophores that enable monitoring of the synthesis progress through UV spectroscopy. The chromophores provide color changes or UV absorption characteristics that allow real-time monitoring of coupling efficiency and reaction progress without requiring removal of the product for analysis
4Quantity of substance
If ionic liquid protecting group is used, then oligonucleotide synthesis up to pentamers is achieved, but crystallization isolation operation is essential and operation time is increased
Solution Approach 1:
The patent modifies the protecting group parameters by using soluble polymers with optimized hydrophobicity and molecular weight. These parameter changes enable the protecting groups to remain soluble throughout the synthesis process without requiring crystallization for isolation, thereby reducing operation time while maintaining the capability to synthesize longer oligonucleotides
5Quantity of substance
If hydrophobic group-linked nucleoside method is used, then 21 mer oligonucleotide synthesis is achieved, but the method is markedly complicated with solidification isolation required at every step
Solution Approach 1:
The patent implements continuous useful action by using soluble polymer protecting groups that remain in solution throughout the synthesis process. This eliminates the need for repeated solidification and isolation steps, allowing the synthesis to proceed continuously with simple filtration and solvent removal between cycles, thereby reducing process complexity while maintaining the capability to synthesize long oligonucleotides
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 approach enhances the solubility and isolability of intermediate oligonucleotides, improving synthesis speed and efficiency by allowing for extraction-based purification, enabling continuous production without the need for intermediate removal from the reaction apparatus.
Implementation Method 1
Base2 in the number of q+1 are each independently a nucleic acid base protected by a group having a C5-30 straight chain or branched chain alkyl group and/or a C5-30 straight chain or branched chain alkenyl group
Implementation Method 2
efficient purification through liquid-liquid extraction, eliminating the need for solidification isolation
Data Source
AI summary
The present invention provides a protected nucleotide for elongation, which can be purified efficiently and in a high yield by a liquid-liquid extraction operation, and can achieve an oligonucleotide production method by a phosphoramidite method.It has been found that the above-mentioned problem can be solved by a particular oligonucleotide comprising a protected base and/or particular oligonucleotide protected by a branched chain-containing aromatic group at 3′-position.