Abstract：
In the field of oligonucleotides drug discovery, phosphorothioate (PS) modification has been recognized as an effective tool to overcome the nuclease digestion, and generates 2n 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.
摘要：
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2n个可能的非对映异构体，其中n等于PS连接的数量。然而,众所周知，药物功效和毒性的差异是由寡核苷酸的立体化学差异引起的。因此,需要开发能够立体区分寡核苷酸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）被认为是分离非对映异构体的有用工具之一。在这项研究中,我们评估了几种具有结构多样性的寡核苷酸，以了解CE非对映异构体的分离机理。尤其是,用PVP1,300,000聚合物溶液对五种2'-基团进行了深入的CE检查。我们发现在这些寡核苷酸中观察到峰形状和峰分辨率的不同趋势。例如,与6merPS3-LNA的刚性结构相比,在6merPS3-DNA中观察到更好的峰分辨率。因为这个原因,计算模拟显示，每个寡核苷酸中硫代磷酸酯的空间结构引起的可接近表面积的差异是解释非对映体分离的关键属性之一。此外,我们实现了6聚体PS4-DNA中非对映异构体的16个峰顶的分离，6聚体PS4-RNA中15种非对映体的完全分离。通过CE分离非对映异构体的这些知识将被预期用于寡核苷酸药物的质量控制。
