A Rapid and Versatile Method to Label Receptor Ligands Using "Click" Chemistry: Validation with the Muscarinic M1 Antagonist Pirenzepine
详细信息 查看全文
- 作者：Dominique Bonnet ; Brigitte Ilien ; Jean-Luc Galzi ; Sté ; phanie Riché ; Cyril Antheaune ; Marcel Hibert
- 刊名：Bioconjugate Chemistry
- 出版年：2006
- 出版时间：November 2006
- 年：2006
- 卷：17
- 期：6
- 页码：1618 - 1623
- 全文大小：91K
- 年卷期：v.17,no.6(November 2006)
- ISSN：1520-4812
文摘
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 tagshave to be incorporated. The classical method of coupling usually necessitates a tedious final purification step toremove the excess of reagents and to isolate tagged molecules. To overcome this limitation, Cu(I)-catalyzed1,3-dipolar cycloaddition, referred to as "click" chemistry, was evaluated as a tool to facilitate the access tolabeled 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 scaffoldderived 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 reagentswere needed, and the quasi-stoichiometric conditions of the reaction enabled the straightforward isolation of thefinal product by simple precipitation and its use in bioassays. The affinity of the compounds for the human M1muscarinic receptor fused to EGFP was checked under classical radioligand and FRET binding conditions. Thethree pirenzepine constructs display a nanomolar affinity for the M1 receptor. In addition, both dye-labeledderivatives behave as potent acceptors of energy from excited EGFP with a very high quenching efficiency.