Mechanistic insight into deuterium loss from C-2 in hydroxycoumarins during postharvest deterioration in cassava

Abstract Background : Postharvest physiological deterioration (PPD) involves complex biochemical transformations, including the biosynthesis of hydroxycoumarin derivatives in cassava. An interesting observation was made when tracer studies were done on cassava roots using E -cinnamic- d 7 acid. Instead of the anticipated d 4 -labelled hydroxycoumarins, d 3 -labelled products were predominantly observed, indicating an in vivo isotopic substitution event. Two potential mechanisms were proposed to explain the unexpected result: (i) nucleophilic addition of glutathione (GSH) across the alkene bond followed by bond rotation and elimination, and (ii) nucleophilic addition of water followed by a similar elimination process. There are currently no published scientific reports that have evaluated the proposed hypotheses in cassava. Study aim : This study investigated the unexpected isotopic exchange from carbon 2 (C-2) of hydroxycoumarins following tracer studies using E -cinnamic- d 7 acid in cassava roots. The study focused on the second proposed mechanism. Method : Owing to the instability of GSH adducts in planta, the water-mediated mechanism was experimentally explored via labelled substrate feeding experiments. Extracts were analysed via liquid chromatography-mass spectrometry (LC-MS). Results : LC-MS analysis showed possible d 4 -labelled products when deuterium oxide (D 2 O) was introduced with labelled substrate feeding while d 3 -labelled hydroxycoumarin products were diminished. Conclusion : LC-MS analysis and isotopic tracing support a Michael-type nucleophilic addition of endogenous water to the carbon 2-carbon 3 (C2-C3)-unsaturated system, leading to deuterium loss at C-2. This novel mechanism provides insight into isotope exchange processes during hydroxycoumarin biosynthesis in cassava PPD. These findings enhance the understanding of plant secondary metabolism under stress conditions and may inform strategies to mitigate postharvest deterioration in cassava and related crops.