title: Fluorescent glucagon derivatives. I. Synthesis and characterisation of fluorescent glucagon derivatives.
abstract: The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC50 values of 6.8.10(-9), 1.7.10(-8), 1.8.10(-8) and 5.4.10(-9) M were measured in rat liver membranes for NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. From the IC50 values Kd values of 2.16.10(-9), 4.10(-9), 2.10(-9) and 1.72.10(-9) M were calculated for the binding of NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-Trp25-glucagon in phosphate-buffered saline (pH 7.4). Difluorescein-glucagon was also prepared by reacting the amino groups of histidine-1 and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiolTrp25-glucagon. Difluorescein-glucagon bound only weakly to glucagon receptors and displayed antagonist properties. The dimer derivative formed from two difluorescein-2-thiolTrp25-glucagon molecules had similar poor binding qualities, whereas the dimer formed from difluorescein-2-thiolTrp25-glucagon and 2-thiolTrp25-glucagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp25-glucagon and difluorescein-glucagon to rat hepatocytes was followed using flow cytometry.
authors.affiliation: Department of Physiological Chemistry, University of Würzburg, F.R.G.
authors.forename: H
authors.initials: H
authors.lastname: Heithier
authors.forename: L D
authors.initials: LD
authors.lastname: Ward
authors.forename: R C
authors.initials: RC
authors.lastname: Cantrill
authors.forename: H W
authors.initials: HW
authors.lastname: Klein
authors.forename: M J
authors.initials: MJ
authors.lastname: Im
authors.forename: G
authors.initials: G
authors.lastname: Pollak
authors.forename: B
authors.initials: B
authors.lastname: Freeman
authors.forename: E
authors.initials: E
authors.lastname: Schiltz
authors.forename: R
authors.initials: R
authors.lastname: Peters
authors.forename: E J
authors.initials: EJ
authors.lastname: Helmreich
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000494:pharmacology; D005934:Glucagon / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000187:drug effects / Q000378:metabolism; D018027:Receptors, Glucagon; D013329:Structure-Activity Relationship
chemical_list: D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D005934:Glucagon; D000262:Adenylyl Cyclases