Identification of unique highly hetero-substituted benzenes as chemical weapons of springtails by a combination of trace analytical methods with DFT calculations and synthesis

Springtails (Collembola) are important members of the soil mesofauna. They are small, often less than 1-2 mm in length. A typical escape response of most surface-living species is to jump, using their furca. However, some species also use chemical defence against predators. While the defence chemistry of higher insects has been well studied, reports from the basal Collembola are rare, linked to the difficulties in obtaining enough biomass. We herein report on the identification and repellent activity of compounds detected in Ceratophysella denticulata . Extracts with various solvents obtained from only 50 individuals were sufficient for analysis by GC/MS, GC/HR-MS, and GC/IR. The large number of candidate structures of the major components were then prioritised by DFT calculations of IR spectra. Finally, the total synthesis of the top candidates confirmed the structures of the three major compounds to be 4-methoxy-5-(methylthio)benzo-1,3-dioxolane, 5,6,7-trimethoxybenzo-1,3-oxathiolane, and 8-amino-5,6,7-trimethoxybenzo-1,3-oxathiolane, the latter being the first naturally occurring fully hetero-substituted benzene. These highly substituted benzenes have no precedence in nature and carry structural motifs rare in nature, such as the benzo-1,3-oxathiolane ring system or the occurrence of O-, N-, and S-substituents at the same benzene core. Another novel natural compound, 2-methyl-1 H -imidazo[4,5- b ]pyridine, is used by Hypogastrura viatica . 4-Methoxy-5-(methylthio)benzo-1,3-dioxolane showed significant activity in deterrence assays with the ant Lasius niger . The data indicate that the title compounds are used in the chemical defence of these springtails, thus adding a new compound class to the known antipredator defences of arthropods. The results underline the difference in defence chemistry between Collembola and insects. Tiny amounts form tiny springtails reveal highly hetereosubstituted benzenes as a new class of predator repellents.