Impurities from Hydroxyproline Derivatives in the Synthesis of Modified Oligonucleotides

This study explores the challenges of synthesizing modified oligonucleotides, particularly focusing on the impurities introduced when using hydroxyproline derivatives. We found that incorporating amino linkers, specifically the trans-4-hydroxy-l-prolinol backbone, leads to the formation of specific difficult-to-remove impurities. To address these issues, we recommend preloading the linker onto the solid-phase carrier to prevent the generation of phosphoramide impurities. Additionally, using a scavenger during the cleavage and deprotection steps is suggested to minimize unwanted reactions. A significant finding is the crucial role of triethylamine in the washing steps, where increasing its amount effectively prevents the formation of acrylonitrile and acetylation adducts. Through a series of experiments, we have identified the causes of these impurities and proposed strategies for their mitigation. These strategies ensure the efficient synthesis of oligonucleotides with improved purity, enhancing their potential for broader applications in biomedical research and biotechnology.