Biochemical synthesis of uniformly ¹³C-labeled diterpene hydrocarbons and their bioconversion to diterpenoid phytoalexins <i>in planta</i>

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 <i>in planta</i>. To elucidate the entire biosynthetic pathways of these phytoalexins, uniformly ¹³C-labeled <i>ent</i>-cassadiene and <i>syn</i>-pimaradiene were enzymatically synthesized with structural verification by GC–MS and ¹³C-NMR. Application of the ¹³C-labeled substrates on rice leaves led to the detection of ¹³C-labeled metabolites using LC-MS/MS. Further application of this method in the moss <i>Hypnum plumaeforme</i> and the nearest out-group of <i>Oryza</i> species <i>Leersia perrieri</i>, 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-¹³C₂₀] diterpene substrates are a powerful tool for chasing endogenous metabolites without dilution with naturally abundant unlabeled compounds. Uniformly labeled ¹³C-diterpene: A powerful tool for tracing endogenous metabolites without dilution with naturally abundant unlabeled compounds

植卡桑类（Phytocassanes）与稻壳酮类（momilactones）是水稻中诱导产生的主要二萜类植保素，属于生物活性物质。尽管针对这类植保素的生物合成途径已有大量研究，但二萜烃类的植物体内生物转化尚未见报道。为阐明这类植保素的完整生物合成途径，研究人员通过酶法合成了全碳¹³C标记的对映-卡桑二烯（ent-cassadiene）和顺式-海松二烯（syn-pimaradiene），并通过气相色谱-质谱联用（GC–MS）与碳-13核磁共振波谱（¹³C-NMR）完成结构确证。将该¹³C标记底物施加于水稻叶片后，借助液相色谱-串联质谱联用（LC-MS/MS）可检测到¹³C标记的代谢产物。进一步将该方法分别应用于苔藓植物灰藓（Hypnum plumaeforme）以及稻属（Oryza）物种的最近外类群Perrier李氏禾（Leersia perrieri），成功实现了标记底物在这两种植物体内向植保素的生物转化。上述结果证实，从这类二萜烃类至终产物植保素的真实生物合成途径确实存在于这些植物中，且酶法合成的[U-¹³C₂₀]二萜底物可作为高效工具，用于示踪内源性代谢物而不受天然丰度未标记化合物的稀释干扰。全碳¹³C标记二萜：一种无需被天然丰度未标记化合物稀释即可示踪内源性代谢物的高效工具

搜集汇总

背景与挑战

背景概述

该数据集聚焦于通过酶法合成均匀13C标记的二萜烯烃（如ent-松香二烯和syn-海松二烯），并利用这些标记底物在水稻、苔藓等植物中追踪其生物转化为植物抗毒素（如稻壳酮和茉莉内酯）的完整途径。研究证实了标记底物可作为有效工具，避免未标记化合物的稀释干扰，从而阐明植物内源性代谢过程。