Synthesis and characterization of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled fluorescent ligands for the mu opioid receptor.

A series of opioid ligands utilizing the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid or 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza- s-indacene-3-propionic acid were synthesized and characterized for their ability to act as a suitable fluorescent label for the mu opioid receptor. All compounds displaced the mu opioid receptor binding of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in monkey brain membranes with high affinity. The binding of fluorescent ligands to delta and kappa receptors was highly variable. 5,7-Dimethyl-BODIPY naltrexamine, "6-BNX," displayed subnanomolar affinities for the mu and kappa opioid receptors (Ki 0.07 and 0.43 nM, respectively) and nanomolar affinity at the delta (Ki 1.4 nM) receptor. Using fluorescence spectroscopy, the binding of 6-BNX in membranes from C6 glioma cells transfected with the cloned mu opioid receptor was investigated. In these membranes containing a high receptor density (10-80 pmol/mg protein), 6-BNX labeling was saturable, mu opioid specific, stereoselective (as determined with the isomers dextrorphan and levorphanol), and more than 90% specific. The results describe a series of newly developed fluorescent ligands for the mu opioid receptor and the use of one of these ligands as a label for the cloned mu receptor. These ligands provide a new approach for studying the structural and biophysical nature of opioid receptors.