Polymorphism-Dependent Bioefficacy of the Prodrug Propionylbrassinolide at Ultra-Low Concentrations

Abstract Polymorphism is widely recognized to alter the bioavailability of active ingredients. Nevertheless, polymorphism-dependent biological behavior at ultra-low concentrations (nM level) has seldom been reported, owing to the intuitive assumption that compounds exist solely as dissolved molecules under these conditions. This assumption is particularly widespread for hormones, which exert potent biological effects and mediate biological regulation at nanomolar to micromolar levels. Consequently, the potential role of solid-state chemistry of these compounds has remained largely unexplored. Herein, we demonstrate that the phytohormone propionylbrassinolide (PB) exhibits pronounced polymorphism-dependent bioefficacy even at 8.5 nM, with Form II consistently displaying higher activity than Form I in rice seed germination assays. The aqueous solubility of PB (<0.05 nM) is far below this application concentration, suggesting that a substantial solid-phase fraction can persist even at nanomolar dosing levels. Differences in solid-state hydrolysis kinetics between the two polymorphs may contribute to their distinct bioefficacy, potentially involving hydrolytic activation of the prodrug. These results uncover a previously unrecognized solid-state dimension of bioactive compounds applied at ultra-low concentrations and highlight the importance of considering the solid-state chemistry of phytohormones.