Integrating stereoselective synthesis and molecular-behavioral approaches to characterize aggregation pheromone in...

Aggregation pheromones play a central role in the social behavior of the German cockroach (Blattella germanica L.), yet their precise stereochemical identities and molecular recognition mechanisms remain unclear. Here, we achieved the first stereoselective synthesis of all four stereoisomers of 3,11-dimethylnonacosane via an asymmetric strategy employing Evans' chiral auxiliaries. This concise 11-step route provided ∼30% overall yield and 98% enantiomeric purity. Combined behavioral assays (Y-tube and semi-field assays) and electroantennogram (EAG) recordings revealed that all stereoisomers triggered aggregation responses, with the (3S,11S)-3,11-dimethylnonacosane inducing the strongest attraction. Transcriptional profiling of 48 antennal odorant-binding protein genes identified OBP39 and OBP40 as candidates responsive to (3S,11S)-3,11-dimethylnonacosane. Molecular docking and microscale thermophoresis (MST) assay demonstrated that OBP39 and OBP40 proteins directly interact with (3S,11S)-3,11-dimethylnonacosane, respectively. Furthermore, RNA interference (RNAi)-mediated silencing of either gene abolished antennal EAG responses and reduced aggregation behavior, confirming their essential roles in the perception of (3S,11S)-3,11-dimethylnonacosane. Our work integrates stereoselective synthesis with behavioral ecology, electrophysiology, and molecular biology to uncover a direct mechanistic connection between pheromone stereochemistry and its recognition system in the German cockroach. These findings provide novel insights into insect chemical communication and lay a foundation for developing pheromone-based strategies in sustainable pest management.