Novel acetylenic oxylipins from the moss Dicranum scoparium with antifeeding activity against herbivorous slugs.
Venue: Angewandte Chemie

Mosses are seldom fed upon even in areas where intense herbivore pressure is observed on other plants. But even more than 100 years after the first investigations of Ernst Stahl on the chemical defense of plants and mosses against slugs and snails we know very little about the nature and biosynthesis of metabolites responsible for moss chemical defense. Dicranum scoparium is an example of a well-defended moss; it is globally distributed in temperate and arctic forests and often occurs in dense patches that show no sign of herbivory. In a recent survey of the volatile oxylipins (products of the oxidative transformation of polyunsaturated fatty acids) from mosses, we identified D. scoparium as a prolific producer of such metabolites. In addition, this moss produces unusual acetylenic cyclopentenones from the dominant acetylenic fatty acid dicranin presumably also by means of oxylipin pathways. Motivated by the observation that oxylipins often serve as defense metabolites or regulators of defense reactions in higher plants, 7] we explored the chemistry and chemical ecology of oxylipins of D. scoparium. As observed in higher plants and diatoms, the production of volatile oxylipins is triggered by mechanical wounding in mosses as well. Comparing the ultraperformance liquid chromatography/ mass spectrometry (UPLC/MS) profiles of methanolic extracts obtained from D. scoparium before and after tissue disruption revealed that a complex mixture of metabolites is produced within seconds after wounding . Owing to the complexity of the chromatograms, we aimed to address the metabolic pathways guided by biosynthetic precursors labeled with stable isotopes. Therefore we prepared monoand dideuterated fatty acids and administered them to the moss (Scheme 1). The acetylenic fatty acid dicranin proved to be of special interest as a precursor of unusual novel oxylipins. After a 10 min administration of monoor dideuterated dicranin suspended in water to frozen powdered moss, a complex mix of deuterated metabolites formed. Since not all oxylipins were chromatographically separated a manual evaluation of UPLC/MS data ran the risk of overlooking relevant signals. We therefore used an automated peak extraction routine that delivers mass/retention time pairs for every compound. These data from nontreated controls and mixtures obtained after treatment with monoor dideuterated dicranin were then evaluated using a canonical analysis of principal coordinates that enabled the rapid identification of dicranin-derived peaks (Figure 1; see the Supporting Information for experimental details). In contrast to the established manual evaluation of UPLC/MS runs this analysis allows an automated quick and comprehensive survey of metabolites derived from labeled precursors. This method covers the entire range of polarities and molecular weights recorded in LC/MS runs and picks up even minor or chromatographically not separated metabolites. The use of two precursors with different degrees of labeling in separate experiments supports the automated evaluation of the chromatogram since the second treatment serves as additional independent replicate (Figure 1c). The use of only one labeled precursor is also feasible, but more care has to be taken in the manual verification of the identified signals to avoid false positive hits. Mass/retention time pairs identified in the canonical analysis were used as guides in the purification of dicranin-derived metabolites using preparative HPLC. Structure elucidation of compounds 2–6 and 8 was based on oneand two-dimensional NMR as well as UV, IR, and MS data. The structure of 7 was tentatively assigned based on derivatization and GC/MS data. Scheme 1. Synthesis of monoand dideuterated fatty aicds. a) LDA, quenching with MeOD; b) NaOMe/MeOD; c) KOH/H2O.