A rapid and versatile method to label receptor ligands using "click" chemistry:: Validation with the muscarinic M1 antagonist pirenzepine
A rapid and versatile method to label receptor ligands using "click" chemistry:: Validation with the muscarinic M1 antagonist pirenzepine
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DOI：
10.1021/bc060140j
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发表时间：
2006-11-15
影响因子：
4.7
通讯作者：
Hibert, Marcel
中科院分区：
文献类型：
--
作者：
Bonnet, Dominique;Ilien, Brigitte;Hibert, Marcel
标记的生物活性分子代表了研究和表征配体-受体相互作用的强大药理学工具。然而，这种分子的标记并不是微不足道的，特别是当必须掺入难溶性标签时。经典的偶联方法通常需要繁琐的最终纯化步骤来去除过量的试剂并分离标记的分子。为了克服这一限制，评价了Cu（I）催化的1，3-偶极环加成（称为“点击”化学）作为促进接近标记分子的工具。为了验证该方法，我们将注意力集中在将荧光团（丽丝胺罗丹明B）、非荧光染料（专利蓝VF）或生物素掺入到来自哌仑西平的毒蕈碱拮抗剂支架中。反应在乙腈/水中进行，在CuSO 4和Cu线的存在下，使我们能够获得三个新的哌仑西平衍生物，具有高纯度和良好的产率。不需要偶联试剂，并且反应的准化学计量条件使得能够通过简单沉淀直接分离最终产物并将其用于生物测定。在经典的放射性配体和FRET结合条件下检查化合物对与EGFP融合的人M1毒蕈碱受体的亲和力。三种哌仑西平构建体显示出对M1受体的纳摩尔亲和力。此外，这两种染料标记的衍生物表现为来自激发的EGFP的能量的有效受体，具有非常高的猝灭效率。
Tagged biologically active molecules represent powerful pharmacological tools to study and characterize ligand-receptor interactions. However, the labeling of such molecules is not trivial, especially when poorly soluble tags have to be incorporated. The classical method of coupling usually necessitates a tedious final purification step to remove the excess of reagents and to isolate tagged molecules. To overcome this limitation, Cu(I)- catalyzed 1,3- dipolar cycloaddition, referred to as "click" chemistry, was evaluated as a tool to facilitate the access to labeled molecules. In order to validate the approach, we focused our attention on the incorporation of a fluorophore ( Lissamine Rhodamine B), a nonfluorescent dye ( Patent Blue VF), or biotin into a muscarinic antagonist scaffold derived from pirenzepine. The reaction performed in acetonitrile/ water, in the presence of CuSO4 and Cu wire, allowed us to obtain three novel pirenzepine derivatives with high purity and in good yield. No coupling reagents were needed, and the quasi- stoichiometric conditions of the reaction enabled the straightforward isolation of the final product by simple precipitation and its use in bioassays. The affinity of the compounds for the human M1 muscarinic receptor fused to EGFP was checked under classical radioligand and FRET binding conditions. The three pirenzepine constructs display a nanomolar affinity for the M1 receptor. In addition, both dye- labeled derivatives behave as potent acceptors of energy from excited EGFP with a very high quenching efficiency.