The incorporation of phosphorothioate linkages has recently been extensively employed in therapeutic oligonucleotides. For their separation and quality control, new high-efficient and high-sensitive analytical methods are needed. In this work, a new affinity capillary electrophoresis method has been developed and applied for the separation of a potential anticancer drug, 2′,3′-cyclic diadenosine diphosphorothioate (R_p, R_p) (ADU-S100), and three recently newly synthesized diastereomers of its difluorinated derivative, 3′,3′-cyclic di(2′-fluoro, 2′-deoxyadenosine phosphorothioate). The separation was performed in the various background electrolytes (BGEs) within a pH range 5–9 using several native and derivatized cyclodextrins (CDs) as chiral additives of the BGE. Relatively good separations were obtained with β-, γ-, and 2-hydroxypropyl-γ-CDs in some of the BGEs tested. However, the best separation was achieved using the 2-hydroxypropyl-β-CD chiral selector at 43.5 mM average concentration in the BGE composed of 40 mM Tris, 40 mM tricine, pH 8.1. Under these conditions, all the previous four cyclic dinucleotides (CDNs) were baseline separated within 4 min. Additionally, the average apparent binding constants and the average actual ionic mobilities of the complexes of all four CDNs with 2-hydroxypropyl-β-CD in the above BGE were determined. The formed complexes were found to be relatively weak, with the average apparent binding constants in the range of 12.2–94.1 L mol⁻¹ and with the actual ionic mobilities spanning the interval (−7.8 to −12.7) × 10⁻⁹ m² V⁻¹ s⁻¹. The developed method can be applied for the separation, analysis, and characterization of the above and similar CDNs.

中文翻译：

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亲和毛细管电泳法分离环硫代二腺苷及其二氟衍生物的非对映异构体及其与2-羟丙基-β-环糊精的结合常数和离子淌度

硫代磷酸酯键的掺入最近已广泛应用于治疗性寡核苷酸中。对于它们的分离和质量控制，需要新的高效、高灵敏度的分析方法。在这项工作中，开发了一种新的亲和毛细管电泳方法并应用于分离潜在的抗癌药物2',3'-环二腺苷二磷酸硫代酸酯（R _p，R _p）（ADU-S100）以及最近新推出的三种药物。合成了其二氟化衍生物3',3'-环状二(2'-氟,2'-脱氧腺苷硫代磷酸酯)的非对映体。使用几种天然和衍生环糊精 (CD) 作为 BGE 的手性添加剂，在 pH 范围 5-9 的各种背景电解质 (BGE) 中进行分离。在一些测试的 BGE 中，β-、γ- 和 2-羟丙基-γ-CD 获得了相对较好的分离效果。然而，在由 40 mM Tris、40 mM tricine、pH 8.1 组成的 BGE 中，使用平均浓度为 43.5 mM 的 2-羟丙基-β-CD 手性选择剂实现了最佳分离。在这些条件下，所有先前的四种环状二核苷酸 (CDN) 在 4 分钟内均实现基线分离。此外，还确定了上述 BGE 中所有四种 CDN 与 2-羟丙基-β-CD 的复合物的平均表观结合常数和平均实际离子迁移率。发现形成的复合物相对较弱，平均表观结合常数在12.2–94.1 L mol ^-1范围内，实际离子迁移率跨越区间(-7.8至-12.7) × 10 ^-9  m ²  V ⁻¹  s ⁻¹。所开发的方法可用于上述和类似 CDN 的分离、分析和表征。