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.

中文翻译：

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修饰寡核苷酸合成中羟脯氨酸衍生物中的杂质


本研究探讨了合成修饰寡核苷酸的挑战，特别关注使用羟脯氨酸衍生物时引入的杂质。我们发现，掺入氨基接头，特别是反式-4-羟基-l-脯氨酸骨架，会导致形成难以去除的特定杂质。为了解决这些问题，我们建议将接头预装到固相载体上，以防止产生磷酰胺杂质。此外，建议在切割和脱保护步骤中使用清除剂，以尽量减少不需要的反应。一个重要的发现是三乙胺在洗涤步骤中的关键作用，其中增加其用量可有效防止丙烯腈和乙酰化加合物的形成。通过一系列实验，我们确定了这些杂质的原因并提出了缓解这些杂质的策略。这些策略可确保高效合成纯度更高的寡核苷酸，增强它们在生物医学研究和生物技术中更广泛应用的潜力。