Design and Synthesis of Fluorophore-Tagged Disparlure Enantiomers to Study Pheromone Enantiomer Discrimination in the Pheromone-Binding Proteins from the Gypsy Moth, Lymantria dispar

Fluorescent analogues of the gypsy moth Sex Pheromone (+)-disparlure (1) and its enantiomer (-)-disparlure (ent-1) were designed, synthesized, and characterized. The fluorescently labelled analogues 6-FAM (+)-disparlure and 1a 6-FAM (-)-disparlure ent-1a were prepared by copper-catalyzed azide-alkyne cycloaddition of disparlure alkyne and 6-FAM azide. These fluorescent disparlure analogues 1a and ent-1a were used to measure disparlure binding to two pheromone-binding proteins from the gypsy moth, LdisPBP1 and LdisPBP2. The fluorescence binding assay showed that LdisPBP1 has a stronger affinity for 6-FAM (-)-disparlure ent-1a, whereas LdisPBP2 has a stronger affinity for 6-FAM (+)-disparlure 1a, consistent with findings from previous studies with disparlure enantiomers. The 6-FAM disparlure enantiomers appeared to be much stronger ligands for LdisPBPs, with binding constants (K_d) in the nanomolar range, compared to the fluorescent reporter 1-NPN (which had K_d values in the micromolar range). Fluorescence competitive binding assays were used to determine the displacement constant (K_i) for the disparlure enantiomers in competition with fluorescent disparlure analogues binding to LdisPBP1 and LdisPBP2. The K_i data show that disparlure enantiomers can effectively displace the fluorescent disparlure from the binding pocket of LdisPBPs and, therefore, occupy the same binding site.

Cis-epoxide; Click reaction; Enantioselective synthesis; Fluorescein; Fluorescence binding assays; Fluorophore-tagged disparlure enantiomers.