Biochemical synthesis of uniformly (13)C-labeled diterpene hydrocarbons and their bioconversion to diterpenoid phytoalexins in planta.

Phytocassanes and momilactones are the major diterpenoid phytoalexins inductively produced in rice as bioactive substances. Regardless of extensive studies on the biosynthetic pathways of these phytoalexins, bioconversion of diterpene hydrocarbons is not shown in planta. To elucidate the entire biosynthetic pathways of these phytoalexins, uniformly (13)C-labeled ent-cassadiene and syn-pimaradiene were enzymatically synthesized with structural verification by GC-MS and (13)C-NMR. Application of the (13)C-labeled substrates on rice leaves led to the detection of (13)C-labeled metabolites using LC-MS/MS. Further application of this method in the moss Hypnum plumaeforme and the nearest out-group of Oryza species Leersia perrieri, respectively, resulted in successful bioconversion of these labeled substrates into phytoalexins in these plants. These results demonstrate that genuine biosynthetic pathways from these diterpene hydrocarbons to the end product phytoalexins occur in these plants and that enzymatically synthesized [U-(13)C20] diterpene substrates are a powerful tool for chasing endogenous metabolites without dilution with naturally abundant unlabeled compounds.