title: A rapid and versatile method to label receptor ligands using "click" chemistry: Validation with the muscarinic M1 antagonist pirenzepine.
abstract: 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.
authors.affiliation: Département de Pharmacochimie de la Communication Cellulaire, Département Récepteurs et Protéines Membranaires, and IFR85, Service commun de RMN, Institut Gilbert Laustriat, Faculté de Pharmacie de Strasbourg, France. dominique.bonnet@pharma.u-strasbg.fr
authors.forename: Dominique
authors.initials: D
authors.lastname: Bonnet
authors.forename: Brigitte
authors.initials: B
authors.lastname: Ilien
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Galzi
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Riché
authors.forename: Cyril
authors.initials: C
authors.lastname: Antheaune
authors.forename: Marcel
authors.initials: M
authors.lastname: Hibert
mesh_terms: D002384:Catalysis; D002460:Cell Line; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D018727:Muscarinic Antagonists / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D010890:Pirenzepine / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D043643:Receptor, Muscarinic M1 / Q000378:metabolism*; D013997:Time Factors
chemical_list: D008024:Ligands; D018727:Muscarinic Antagonists; D043643:Receptor, Muscarinic M1; D010890:Pirenzepine