Analytical Research, Pharmaceutical Science & Technology Unit, Pharmaceutical Profiling & Development Function, Deep Human Biology Learning, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan.
Analytical Research, Pharmaceutical Science & Technology Unit, Pharmaceutical Profiling & Development Function, Deep Human Biology Learning, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan.
J Chromatogr A. 2024 Jun 21;1725:464945. doi: 10.1016/j.chroma.2024.464945. Epub 2024 Apr 25.
In the field of oligonucleotides drug discovery, phosphorothioate (PS) modification has been recognized as an effective tool to overcome the nuclease digestion, and generates 2 of possible diastereomers, where n equals the number of PS linkages. However, it is also well known that differences in drug efficacy and toxicity are caused by differences in stereochemistry of oligonucleotides. Therefore, the development of a high-resolution analytical method that enables stereo discrimination of oligonucleotides is desired. Under this circumstance, capillary electrophoresis (CE) using polyvinylpyrrolidone (PVP) is considered as one of the useful tools for the separation analysis of diastereomers. In this study, we evaluated the several oligonucleotides with the structural diversities in order to understand the separation mechanism of the diastereomers by CE. Especially, five kinds of 2'-moieties were deeply examined by CE with PVP 1,300,000 polymer solution. We found that different trend of the peak shapes and the peak resolution were observed among these oligonucleotides. For example, the better peak resolution was observed in 6 mer PS3-DNA compared to the rigid structure of 6 mer PS3-LNA. As for this reason, the computational simulation revealed that difference of accessible surface area caused by the steric structure of thiophosphate in each oligonucleotide is one of the key attributes to explain the separation of the diastereomers. In addition, we achieved the separation of sixteen peak tops of the diastereomers in 6 mer PS4-DNA, and the complete separation of fifteen diastereomers in 6 mer PS4-RNA. These knowledge for the separation of the diastereomers by CE will be expected to the quality control of the oligonucleotide drugs.
在寡核苷酸药物发现领域，硫代磷酸酯 (PS) 修饰已被认为是克服核酸酶消化的有效工具，并产生 2 种可能的非对映异构体，其中 n 等于 PS 键的数量。然而，众所周知，寡核苷酸的立体化学差异会导致药物疗效和毒性的差异。因此，需要开发一种能够对寡核苷酸进行立体选择性分辨的高分辨率分析方法。在这种情况下，使用聚乙烯吡咯烷酮 (PVP) 的毛细管电泳 (CE) 被认为是分离分析非对映异构体的有用工具之一。在本研究中，我们评估了具有结构多样性的几种寡核苷酸，以了解 CE 中非对映异构体的分离机制。特别是，通过使用 PVP 1,300,000 聚合物溶液对五种 2'-取代基进行了深入的 CE 分析。我们发现，这些寡核苷酸的峰形和峰分辨率呈现出不同的趋势。例如，与刚性结构的 6 聚体 PS3-LNA 相比，3 聚体 PS3-DNA 的峰分辨率更好。由于这个原因，计算模拟表明，每个寡核苷酸中硫代磷酸酯的立体结构引起的可及表面积差异是解释非对映异构体分离的关键属性之一。此外，我们实现了 6 聚体 PS4-DNA 中 16 个非对映异构体峰尖的分离，以及 6 聚体 PS4-RNA 中 15 个非对映异构体的完全分离。这些关于 CE 中非对映异构体分离的知识有望用于寡核苷酸药物的质量控制。
