Oligonucleotide Synthesis Segment for High-Yield Coupling
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Solution Overview
Problem
Conventional oligonucleotide synthesis methods face challenges such as low yield in coupling reactions, inefficiencies in oxidation/sulfurization and deprotection steps, and a heavy burden in purification due to the generation of similar by-products, which complicates the synthesis of oligonucleotides with longer lengths.
Innovation Solution
The use of a segment represented by formula (I) for oligonucleotide synthesis, which involves a series of reactions with phosphitylating compounds and nucleosides to produce a segment that can be used to synthesize oligonucleotides with improved efficiency and reduced by-product generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional monomer amidite is used for step-by-step oligonucleotide synthesis, then the synthesis process can be performed, but the yield of coupling reactions does not reach 100% and the synthesis efficiency decreases as the oligonucleotide length increases
Solution Approach 1:
The invention divides the oligonucleotide synthesis process into two stages: first synthesizing a pre-formed segment (oligonucleotide with 2-23 nucleotides) with high purity, then using this segment as a building block for further extension. This segmentation allows the difficult high-yield coupling reactions to be performed during segment formation, while subsequent extensions benefit from the high-purity starting material.
Solution Approach 2:
The invention performs preliminary synthesis of a high-purity oligonucleotide segment before using it as a substrate for further elongation. This preliminary action ensures that the starting material for subsequent coupling reactions has minimal impurities, thereby maintaining high reaction yields throughout the synthesis process.
2Ease of manufacture
If step-by-step extension method is used, then oligonucleotide synthesis can be achieved, but by-products such as (N-1)-mer and (N-2)-mer are generated which complicate purification
Solution Approach 1:
The invention performs preliminary synthesis of a high-purity oligonucleotide segment before using it as a substrate for further elongation. This preliminary action ensures that the starting material for subsequent coupling reactions has minimal impurities, thereby maintaining high reaction yields throughout the synthesis process.
Solution Approach 2:
The invention changes the parameters of the synthesis process by using a pre-formed segment with specific length (2-23 nucleotides) and high purity as the starting material, rather than beginning with a single nucleotide. This parameter change fundamentally alters the by-product profile, eliminating the generation of (N-1)-mer and (N-2)-mer by-products that plague conventional step-by-step synthesis.
3Productivity
If commercially available monomer amidite is used, then oligonucleotide synthesis can be performed, but the cost increases and large-scale synthesis is obstructed
Solution Approach 1:
The invention divides the oligonucleotide synthesis process into two stages: first synthesizing a pre-formed segment (oligonucleotide with 2-23 nucleotides) with high purity, then using this segment as a building block for further extension. This segmentation allows the difficult high-yield coupling reactions to be performed during segment formation, while subsequent extensions benefit from the high-purity starting material.
Solution Approach 2:
The invention changes the parameters of the synthesis process by using a pre-formed segment with specific length (2-23 nucleotides) and high purity as the starting material, rather than beginning with a single nucleotide. This parameter change fundamentally alters the by-product profile, eliminating the generation of (N-1)-mer and (N-2)-mer by-products that plague conventional step-by-step 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 reduces the number of synthesis steps, improves the yield of oligonucleotides with target lengths, simplifies purification by minimizing by-product formation, and allows for the synthesis of larger quantities of oligonucleotides at lower costs.
Implementation Method 1
a method of extending the length of a nucleotide through a coupling reaction for each base step by step
Implementation Method 2
a step of oxidizing or sulfurizing phosphite
Data Source
AI summary
A segment for use in synthesis of an oligonucleotide, represented by the following formula (I), a method for producing the same, and a method for synthesizing an oligonucleotide therefrom are provided. In formula (I), B is a protected/unprotected nucleoside base; R1 is a protecting group; R2, R3 and R4 are OCH2CH2CN, OCH2CH═CH2, etc.; R5 is a substituted/unsubstituted aliphatic group/aromatic group; X is a lone pair, O or S; Y is NHR6, a halogen, CN, etc., or a hydroxyl group protected with an acyl, ether or silyl protecting group; R6 is H, an aliphatic group or an aromatic group; Z is H, an alkyl, an O— or N-alkyl or a halogen, or forms a Z—Y bond with Y; and (m+n) is an integer of 2 or more and 23 or less.