A rapid and versatile method to label receptor ligands using "click" chemistry: Validation with the muscarinic M1 antagonist pirenzepine.
Fiche publication
Date publication
novembre 2006
Auteurs
Membres identifiés du Cancéropôle Est :
Dr GALZI Jean-Luc, Pr HIBERT Marcel
Tous les auteurs :
Bonnet D, Ilien B, Galzi JL, Riche S, Antheaune C, Hibert M
Lien Pubmed
Résumé
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.
Référence
Bioconjug Chem. 2006 Nov-Dec;17(6):1618-23.