Advantages of Liquid-phase Chemical Synthesis Method

The liquid-phase technique provides an alternative to the extensively employed solid-phase oligonucleotide synthesis method. In this strategy, a soluble polymeric carrier integrated into the reaction medium acts as the scaffold for elongating the chain. Afterward, the polymer is separated from unused reagents and soluble by-products using filtration or crystallization techniques. This approach eliminates any inconsistency resulting from support insolubility, thus enhancing the potential for process upscaling. This is particularly pertinent now as synthetic oligonucleotides are transitioning from research settings to practical biomedical applications. Consequently, the scientific community is actively exploring the development and evaluation of a more user-friendly liquid-phase chemical synthesis method for oligonucleotides. An innovative strategy known as high-performance liquid phase (HELP), utilizing soluble polyethylene glycol (PEG) as a polymeric support and employing phosphotriester chemistry for the coupling reaction, has demonstrated promising outcomes.

Fig.1 General scheme of liquid-phase DNA synthesis. (Creusen, et al., 2020)

Liquid-phase Chemical Synthesis Method for Oligonucleotide at CD BioGlyco

CD BioGlyco commits to providing global clients with industry-leading Custom Carbohydrate Synthesis services, such as Custom Sugar-nucleotides Synthesis services. The HELP-based Synthesis of Oligonucleotides we provide mainly includes 3 key steps: functionalization of PEG, oligonucleotide assembly, deprotection & purification.

• PEG Functionalization
  The initial nucleoside is attached through PEG, which is linked to a symmetrical anhydride derivative of four 5'-O-dimethoxytrityl (DMT)-2'-deoxynucleoside-3'-O-succinates. The PEG-nucleosides are then subjected to analysis using ¹H-NMR spectroscopy. To confirm the complete removal of reagents, TLC is initially employed, followed by product characterization. Any residual unreacted PEG molecules are treated with acetic anhydride.
• Oligonucleotide assembly
  The process of oligonucleotide assembly is executed through detritylation, condensation, capping, and oxidation steps, adhering to established procedures.
• Deprotection & purification
  Upon the synthesis's completion, the oligomers are separated from the polymer using standard deprotection techniques. Most of the product is detached from PEG through oximation, succeeded by comprehensive hydrolysis using concentrated ammonia. Partially deprotected oligonucleotides are subjected to analysis via HPLC. Finally, after full deprotection with acetic acid, the product undergoes purification through ion exchange chromatography.

Fig.2 Process of HELP for oligonucleotide synthesis. (CD BioGlyco)

Applications of Liquid-phase Chemical Synthesis Method

• Biomedical research: The liquid-phase synthesis method is extensively used to produce custom oligonucleotides for various research purposes, such as investigating gene expression, studying genetic variations, and exploring nucleic acid-protein interactions.
• Diagnostic assays: Oligonucleotides synthesized using the liquid-phase method are crucial components of diagnostic assays, including polymerase chain reactions (PCR), reverse transcription PCR (RT-PCR), and nucleic acid hybridization techniques.
• Next-generation sequencing (NGS): Oligonucleotides produced through the liquid-phase approach serve as primers and adapters in NGS technologies. They facilitate the high-throughput sequencing of DNA and RNA, enabling comprehensive genomic and transcriptomic analyses.

Advantages

• Liquid-phase oligonucleotide synthesis employs a uniform reaction mixture, enabling potentially boundless scalability.
• Oligonucleotides are synthesized using the liquid-phase technique and hold the potential for advancing extensive drug development efforts.
• The liquid-phase approach to oligonucleotide synthesis is expected to contribute to the progression of large-scale fabrication of DNA materials.

At CD BioGlyco, we have established the high-quality Glyco™ Synthesis Platform that is dedicated to glycobiology research. Our commitment to excellence is evident in every aspect of the Chemical-based Oligonucleotide Synthesis Service. Our team is dedicated to maintaining the highest standards and ensuring client satisfaction, particularly in the realm of liquid-phase chemical synthesis methods for oligonucleotides. If you are interested in our services, please do not hesitate to contact us for more details.