Synthesis and Characterization of 4,4-Difluoro-4-bora- 3a,4a-Diaza-s-Indacene (BODIPY)-Labeled Fluorescent Ligands for the Mu Opioid Receptor Paul J. Emmerson,* Sydney Archer,†‡ Wageeh El-Hamouly,† Alfred Mansour,§ Huda Akil§ and Fedor Medzihradsky* ¶ DEPARTMENTS OF *PHARMACOLOGY AND  BIOLOGICAL CHEMISTRY, AND THE §MENTAL HEALTH RESEARCH INSTITUTE,UNIVERSITY OF MICHIGAN MEDICAL SCHOOL,ANN ARBOR, MI 48109; AND †DEPARTMENT OF CHEMISTRY,COGSWELL LABORATORY,RENSSELAER POLYTECHNIC INSTITUTE,TROY, NY 12181, U.S.A. ABSTRACT. 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-phe- nyl-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 [ 3 H]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 ( K i 0.07 and 0.43 nM, respectively) and nanomolar affinity at the delta ( K i 1.4 nM) receptor. Using fluorescence spectroscopy, the binding of 6-BNX in membranes from C 6 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. BIOCHEM PHARMACOL 54;12:1315–1322, 1997. © 1997 Elsevier Science Inc. KEY WORDS. mu opioid receptor; fluorescence; BODIPY; receptor binding The availability of fluorescently labeled ligands for a variety of receptors has permitted the examination of these systems in ways not provided by approaches based on radiolabeled ligands. Utilizing fluorescent ligands, the kinetics of ligand- receptor association and dissociation were examined [1], as well as the interaction of ligand, receptor, and G protein** [2, 3]. Lateral mobility of receptors [4 – 6] and G proteins [7] within the plasma membrane of intact cells was investi- gated by fluorescence recovery after photobleaching. De- termination of the distance between the epidermal growth factor receptor binding site and the cell membrane utilizing resonance energy transfer allowed for the localization of the receptor binding domain [8]. Additionally, resonance en- ergy transfer has also been utilized to determine the distribution of phospholipids surrounding membrane pro- teins [9, 10]. Rhodamine-labeled enkephalins [11] were found to spe- cifically bind to cells expressing delta opioid receptors and resulted in clustering of the receptor on the cell surface, suggesting an agonist-induced sequestration or internaliza- tion of the receptor [12]. Although these ligands main- tained high affinity for the delta receptor, they were found to label mu receptors poorly [13]. Opioid peptides and alkaloids containing different fluorophores including dansyl [14], fluorescein [15], nitrobenzoxadiazole [16], and pyrene [17] have also been synthesized previously. Labeling of receptors with dansyl-enkephalin was hampered by tissue autofluorescence and the destruction of opioid receptors and ligands by light in the ultraviolet spectrum [18]. A derivative of naltrexone with fluorescein (6-FNX) retained high affinity for the mu opioid receptor [15]; however, no fluorescent labeling of opioid receptors with this ligand has been reported. Fluorescent labeling of kappa opioid recep- tors with a fluorescein-labeled arylacetamide has been demonstrated recently [19, 20]. Secondary binding of R- phycoerythrin to the receptor–ligand complex has also ‡ In memoriam: August 22, 1996. ¶ Corresponding author: Fedor Medzihradsky, Ph.D., Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606. Tel. (313) 764-1114; FAX (313) 763-4450. ** Abbreviations: G protein, GTP-binding protein; pCl-DPDPE, [2,5-D- Pen, 4-p-Cl-Phe]enkephalin; DAMGO, Tyr-D-Ala-Gly-(Me)Phe-Gly-ol; BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; 5,7-dimethyl-BODIPY, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s -indacene-3-propionic acid; 5-(4-phenyl-1,3-butadienyl)-BODIPY, 4,4-difluoro-5-(4-phenyl-1,3-buta- dienyl)-4-bora-3a,4a-diaza-s -indacene-3-propionic acid; 6-FNX, 1-N-fluo- resceinyl naltrexone thiosemicarbazone; 6-BNX, WA-III-25; and U69593, 5,7,8-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]benze- neacetamide. Received 16 December 1996; accepted 16 June 1997. Biochemical Pharmacology, Vol. 54, pp. 1315–1322, 1997. ISSN 0006-2952/97/$17.00 + 0.00 © 1997 Elsevier Science Inc. All rights reserved. PII S0006-2952(97)00374-2