title: Arylacetamide-derived fluorescent probes: synthesis, biological evaluation, and direct fluorescent labeling of kappa opioid receptors in mouse microglial cells.
abstract: Fluorescein isothiocyanate isomer I (FITC-I) conjugates of 2-(3,4-dichlorophenyl)-N-methyl-N-[1-(3- or 4-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide (10 and 14) were prepared either without or with an intervening mono-, di-, or tetraglycyl linker. The 3-substituted fluorescent probes (2-5) were found to retain potent agonist activity in smooth muscle preparations as well as high kappa receptor affinity and selectivity in receptor binding assays. The 4-substituted series (6-9) were substantially less potent than the corresponding 3-substituted compounds. Flow cytometric analysis demonstrated high levels of direct kappa-specific staining of mouse microglial cells by the fluorescent probe 5 containing a tetraglycyl linker, as indicated by a 41% decrease in percent cells positively labeled and a 61% decrease in mean fluorescence intensity in the presence of the kappa-selective antagonist, norbinaltorphimine (norBNI). In similar studies, the probe 2 without a linker exhibited only nonspecific binding. This is the first report of direct, selective staining of kappa opioid receptors by a fluorescent nonpeptide opioid ligand. The results of the present study illustrate the importance of introducing hydrophilic linkers to reduce nonspecific binding of fluorescent probes for opioid receptors.
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, 55455, USA.
authors.forename: A C
authors.initials: AC
authors.lastname: Chang
authors.forename: C C
authors.initials: CC
authors.lastname: Chao
authors.forename: A E
authors.initials: AE
authors.lastname: Takemori
authors.forename: G
authors.initials: G
authors.lastname: Gekker
authors.forename: S
authors.initials: S
authors.lastname: Hu
authors.forename: P K
authors.initials: PK
authors.lastname: Peterson
authors.forename: P S
authors.initials: PS
authors.lastname: Portoghese
mesh_terms: D000081:Acetamides / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D017628:Microglia / Q000737:chemistry*; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D017473:Receptors, Opioid, kappa / Q000032:analysis*
chemical_list: D000081:Acetamides; D005456:Fluorescent Dyes; D017473:Receptors, Opioid, kappa