PEG-Based Liquid Phase Synthesis

Polyethylene glycol (PEG) is a highly popular polymer support in liquid phase oligonucleotide synthesis (LPOS), gaining significant attention and advancements in various research circles. Because the synthesis is carried out in a homogeneous solution, the PEG-based liquid phase method uses fewer excess reagents than the solid phase method. In addition, the growing intermediates can be isolated through precipitation and filtration, enabling the production of large amounts of oligonucleotides in one run. More importantly, the liquid phase method also allows easy monitoring of the synthesis process by non-destructive spectrophotometry.

Advantages of Using PEG as Soluble Support

The macromolecular PEG carrier is highly favored for several key reasons. First, it allows acetonitrile to be used as an optimal solvent for the coupling reaction, streamlining the purification process in oligonucleotide synthesis. Additionally, the PEG support accommodates various coupling strategies, including the H-phosphonate and phosphotriester methods.

The low cost of the PEG process also favors scalability due to the use of conventional batch reactors. For example, the lower consumption of monomeric building blocks compared to solid-support oligonucleotide synthesis significantly reduces the overall manufacturing price. In addition, the high coupling efficiency and the possibilities of using a convergent synthesis makes the PEG-based platform ideal for large-scale production. PEG-based liquid phase synthesis would benefit further if the number of separations in the chain elongation cycle could be reduced.

Research Progress

The first oligonucleotide syntheses using the PEG-based liquid phase method utilized the well-known phosphotriester chemistry. It was in 1991 when Bonora's team^[1] introduced a new technique called HELP (high efficiency liquid phase) for the rapid production of large quantities of short oligodeoxynucleotides. This method, utilizing PEG as a soluble polymer support, enables the generation of hundreds of milligrams of final product in a single synthesis run, while avoiding the time-consuming steps of traditional solution technique. Additionally, the process is easily scalable for larger production.

Phosphoramidite chemistry^[2] and H-phosphonate chemistry^[3] can also be used in conjunction with PEG-based liquid phase method. The phosphoramidite HELP method ("HELP Plus") represented a significant improvement over the original HELP method, delivering faster reaction times, higher yields, and longer oligomer chains. Although phosphoramidite synthons are commonly used in solid phase synthesis, HELP Plus is the first liquid phase method to adopt them. The use of H-phosphonate nucleotides makes the HELP Plus method more cost-effective than other liquid phase methods because they are equally reactive, more stable, and less expensive than phosphoramidite nucleotides. Moreover, only a single oxidation step was needed at the end of the process, rather than after each coupling step.

Currently, the most important PEG-based liquid phase synthesis may be the large-scale synthesis of phosphorothioate oligonucleotides using the blockmer strategy. This approach minimizes the level of n−1 deletion sequences and enhances yield by reducing the number of precipitation and filtration steps. For instance, Bonora's research team^[4] employs PEG as a soluble and inert support throughout the synthesis process. The use of pre-formed dimeric phosphoramidite as synthons improves the quality and amount of the desired oligonucleotides.

Using PEG as a soluble support in liquid phase synthesis has become a common strategy for the commercial large-scale production of oligonucleotides. Alfa Chemistry is at the forefront of this innovative strategy. If you have any needs, please feel free to contact us.

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