The Analytical Chemistry of Anti-AIDS Agents

使用毛细管电泳法(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。我们以前的工作表明，定量在线样品堆积为核苷酸的CE分析产生了100多倍的信号增强。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性和酶稳定性。然而，生物衍生的核苷酸混合物在样品堆积和CE分析中显示出明显的信号和分辨率恶化，因为这些样品的离子强度很高。已经探索了克服这种影响的样品制备方法和分析策略，并取得了一些成功，导致生物样品的信号增强了20至30倍。这项调查仍在继续。我们还评估了用于微制备毛细管电泳法分离微量核苷酸代谢物的全柱样品堆积，以用于MALDI质谱仪的结构分析。这将在艾滋病治疗中使用的核苷类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中得到应用。核苷类逆转录酶抑制剂是一类重要的艾滋病治疗药物。药物化学实验室有一个长期的项目，研究构象锁定的核苷作为潜在的抗艾滋病和抗癌药物。在这些研究过程中，人们逐渐认识到核苷糖的构象可以深刻地影响其化学和生物学性质。我们已经在实验上证实，在两个构象锁定的双脱氧腺苷(DDA)类似物中确实存在这种情况，其中糖氧基孤对轨道的取向被冻结，以增强或削弱反常效应的强度。当异构体效应增强时，糖苷键被延长，核苷变得更容易被水解。在pH=2时，构象锁定的DDA类似物具有增强的异构体效应，其脱嘌率是其结构异构体的6倍以上。然而，这两种DDA类似物都比DDA本身更稳定，后者在pH值为2时的半衰期仅为2.67分钟。

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.