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

Govardhana R Pinnelli,Erika Plettner

doi:10.1007/s10886-021-01318-2

Govardhana R Pinnelli, Erika Plettner

Open Access

https://doi.org/10.1007/s10886-021-01318-2

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Abstract

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 (Kd) in the nanomolar range, compared to the fluorescent reporter 1-NPN (which had Kd values in the micromolar range). Fluorescence competitive binding assays were used to determine the displacement constant (Ki) for the disparlure enantiomers in competition with fluorescent disparlure analogues binding to LdisPBP1 and LdisPBP2. The Ki data show that disparlure enantiomers can effectively displace the fluorescent disparlure from the binding pocket of LdisPBPs and, therefore, occupy the same binding site.

Highlights

Pheromones are detected by moths using a very organized system, located on the antennae (Krieger and Breer 1999)
Pores in the cuticle of the hairs allow the diffusion of pheromones to the interior, where they first interact with pheromonebinding proteins (PBPs) in the lymph and with pheromone receptors (PRs)
The target molecule has been divided into three parts: (+)-disparlure (1) for the selective binding towards its pheromone binding protein, 6-FAM moiety for the fluorescence detection, and between the two parts, a diethylene glycol linker to increase the hydrophilicity of the final target molecule and provide some separation between the recognition part and the fluorophore (Scheme. 1)

Summary

Introduction

Pheromones are detected by moths using a very organized system, located on the antennae (Krieger and Breer 1999). Pheromones are signals that are transformed into neuronal signals by pheromone sensory neurons found in the antennae of male moths. Antennae of male moths are covered in sensory hairs (sensilla), which are innervated with dendrites of olfactory sensory neurons. Pores in the cuticle of the hairs allow the diffusion of pheromones to the interior, where they first interact with pheromonebinding proteins (PBPs) in the lymph and with pheromone receptors (PRs). The latter are embedded in the dendritic end of pheromone sensory neurons and are bathed in sensillum lymph (an aqueous solution rich in PBPs, fatty acids and ions). The PBPs bind pheromones and other compounds reversibly and selectively, in order to keep them in the lymph

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