Segment-Type Amidite Oligonucleotide Synthesis Purity
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Solution Overview
Problem
The existing phosphoramidite method for synthesizing oligonucleotides is inefficient in preventing the production of shorter oligonucleotides (N-1)mers, which complicates purification and reduces the purity of the desired oligonucleotides due to similar chromatographic mobility, and the performance of previously suggested activators has not been adequately investigated.
Innovation Solution
A method using a nucleoside phosphoramidite with two or more nucleoside moieties or a linker moiety, in conjunction with specific activators such as 5-mercapto-1-methyltetrazole, 5-mercapto-1-phenyltetrazole, or saccharin 1-methylimidazole, to enhance the efficiency of oligonucleotide synthesis by reducing the formation of (N-1)mers and minimizing degradation during the process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional phosphoramidite method is used for synthesizing oligonucleotides, then the synthesis process is simple and straightforward, but oligonucleotide (N-1)mers with shorter length are inevitably produced which complicates purification and reduces product purity
Solution Approach 1:
The patent divides the oligonucleotide synthesis into segments by using nucleoside phosphoramidites with two or three nucleoside moieties (segment-type amidites) instead of adding single nucleosides sequentially. This segmentation approach allows the formation of longer oligonucleotide chains in fewer coupling steps, thereby reducing the production of (N-1)mer impurities and simplifying the purification process while maintaining high product purity.
2Manufacturing precision
If segment-type amidite is used to suppress (N-1)mer production, then the purity of oligonucleotide product is improved, but the reaction efficiency is insufficient and degradation occurs during synthesis
Solution Approach 1:
The patent optimizes reaction parameters by introducing specific activators (saccharin derivatives or imidazolium salts) and controlling the coupling conditions. These parameter changes enhance the reactivity of segment-type amidites, improve coupling efficiency, and minimize degradation during synthesis, thereby achieving both high purity and high productivity in oligonucleotide production.
3Productivity
If 1H-tetrazole is used as activator in oligonucleotide synthesis, then the coupling reaction proceeds, but safety concerns arise and the performance of alternative activators has not been sufficiently investigated
Solution Approach 1:
The patent replaces the hazardous 1H-tetrazole activator with safer alternatives such as saccharin derivatives or imidazolium salts. These alternative activators maintain sufficient coupling reaction efficiency while eliminating the safety concerns associated with 1H-tetrazole, providing a safer and equally effective solution for oligonucleotide synthesis.
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 significantly reduces the production of impurities like (N-1)mers, improves the purity and yield of oligonucleotides, and minimizes the degradation of segment-type amidites, leading to higher efficiency and purity in oligonucleotide synthesis.
Implementation Method 1
binding a nucleoside phosphoramidite to a 3′ or 5′ hydroxyl or thiol group of a nucleotide or nucleoside in the presence of an activator
Data Source
AI summary
The purpose of the present invention is to provide a method for synthesizing an oligonucleotide by using a segment-type amidite. An oligonucleotide production method comprising at least one coupling step for coupling a nucleoside phosphoramidite with a thiol group or a hydroxyl group at the 3′ or 5′ of a nucleoside or a nucleotide in the presence of an activator, wherein, in the at least one coupling step, said nucleoside phosphoramidite has (a) two or more nucleoside portions or (b) at least one nucleoside portion and a linker portion, and said activator has a structure represented by formula (1) (in formula (1), X represents an organic base) or by formula (2) (in formula (2), R1 and R2 are each independently selected from the group consisting of H, straight chain or branched chain C1-7 alkyl groups and optionally substituted aromatic groups).