Analysis of the Binding Sites of Opioid Receptor by Photoaffinity Labeling

研究目的：最近成功构建的嵌合阿片受体表明，存在几个细胞外区域，这些区域可能导致每种类型的阿片受体具有不同的配体结合特征。然而，通常不可能排除导致配体结合改变的潜在构象变化。本研究的目的是开发和应用基于二氮杂环的光探针分析阿片类药物的结合部位。结果：合成了一种简便的间和对-CF3-二氮杂环苯甲酸的合成方法。利用这些光反应基团，合成了一对新型的光探针，它们只是在卡宾的生成位置上有所不同。探针的这种结构特征可能有助于绘制配体结合口袋内的不同位置。为了避免使用放射性探针，还开发了一种新的生物素化的光反应纳曲酮类似物。所有新的含二氮杂环的探针都与Mu受体、Delta受体和Kappa受体可逆结合，亲和力很高。用生物素探针研究了重组Delta受体蛋白的光亲和标记。通过光化学生物素化蛋白质的化学发光检测，成功地实现了靶分子的可视化。

Research Purpose : Recent success in the construction of chimeric opioid receptors demonstrated the presence of several extra cellular regions which could be responsible for the distinct ligand-binding profiles of the each type of opioid receptors. However, it is generally impossible to exclude potential conformational changes which result in alterations of the ligand binding. The purpose of this research is the development and application of diazirine based photoprobes for the analysis of opioid binding sites.Results : A convenient synthesis of m- and p-CF_3-diazirinylbenzoic acid was developed. Using these photoreactive moieties, a novel pair of photoprobes which only differ in the carbene generating sites were synthesized. This structural feature of the probes are potentially useful to map the different sites within the ligand binding pocket. For avoiding the use of radioactive probes, a novel biotinylated photoreactive naltrexone analog was also developed. All new probes bearing diazirine bind reversibly with high affinity at mu-, delta-, and kappa- receptors. The photoaffinity labeling of recombinant delta-receptor protein was investigated with the biotinyl probe. The visualization of target molecule was successfully accomplished by the chemiluminescent detection of photochemically biotinylated proteins.