The Analytical Chemistry of Anti-AIDS Agents

使用毛细管电泳（CE）测量和表征合成核苷酸混合物以及细胞内核苷酸代谢物的方法的发展仍在继续。我们之前的工作表明，定量在线样品堆叠对核苷酸的CE分析产生超过100倍的信号增强。这些方法已被用于表征和研究合成核苷酸衍生物混合物的水解和酶稳定性。然而，由于这些样品的高离子强度，生物衍生的核苷酸混合物在样品堆叠和CE分析时显示出信号和分辨率的明显恶化。克服这种影响的样品制备方法和分析策略已经探索并取得了一些成功，导致生物样品的信号增强20至30倍。调查仍在继续。我们还评估了用MALDI质谱分析微量核苷酸代谢物的微制备CE分离的全柱样品堆叠。这将应用于艾滋病治疗中使用的基于核苷的逆转录酶抑制剂的活性细胞内核苷酸代谢物的测定和测量。核苷类逆转录酶抑制剂是一类重要的艾滋病治疗药物。药物化学实验室长期以来一直在研究构象锁定核苷作为潜在的抗艾滋病和抗癌药物。在这些研究过程中，人们已经认识到核苷糖的构象可以深刻地影响其化学和生物学特性。我们已经通过实验证实，在两种构象锁定的双脱氧腺苷（ddA）类似物中确实存在这种情况，其中糖氧孤对轨道的取向被冻结以增强或削弱头型效应的强度。当端粒效应增强时，糖苷键被拉长，核苷更容易被水解。在pH为2时，具有增强的异构效应的构象锁定ddA类似物的去嘌呤率比其结构异构体高6倍以上。然而，这两种ddA类似物都比ddA本身更稳定，在pH为2时，其半衰期仅为2.67 min。

The development of methods using capillary electrophoresis (CE) to measure and characterize synthetic nucleotide mixtures as well as intracellular nucleotide metabolites continues. Our previous work has shown that quantitative on-line sample-stacking produces a more than 100-fold signal enhancement for the CE analysis of nucleotides. These methods have been applied to characterize and study the hydrolytic and enzymatic stability of mixtures of synthetic nucleotide derivatives. Biologically derived mixtures of nucleotides, however, show a marked deterioration in signal and resolution upon sample-stacking and CE analysis because of the high ionic strength of these samples. Sample preparation methods and analysis strategies to overcome this effect have been explored and have provided some success, resulting in a 20- to 30-fold signal enhancement for biological samples. This investigation continues. We are also evaluating whole-column sample stacking for the micropreparative CE isolation of trace nucleotide metabolites for structural analysis by MALDI mass spectrometry. This will have application for the determination and measurement of the active intracellular nucleotide metabolites of the nucleoside-based reverse transcriptase inhibitors that are used in AIDS therapy. Nucleoside-based reverse transcriptase inhibitors comprise an important class of therapeutically useful drugs for AIDS. The Laboratory of Medicinal Chemistry has had a long-standing program studying conformationally locked nucleosides as potential anti-AIDS and anticancer agents. During the course of these studies it has come to be recognized that the conformation of nucleoside sugar can profoundly affect its chemical and biological properties. We have experimentally confirmed that this is indeed the case in two conformationally locked dideoxyadenosine (ddA) analogues where the orientation of the sugar oxygens lone pair orbitals are frozen to either enhance or impair the strength of the anomeric effect. Where the anomeric effect is enhanced, the glycosidic bond is lengthened and the nucleoside becomes more susceptible to hydrolysis. At pH 2, the conformationally locked ddA analogue with an enhanced anomeric effect has a depurination rate more than 6-fold greater than that of its structural isomer. Both ddA analogues, however, are more stable than ddA itself which has a half-life of only 2.67 min at pH 2.