GNPS‐Guided Discovery of Madurastatin Siderophores from the Termite‐Associated Actinomadura sp. RB99

In this study, we analyzed if Actinomadura sp. RB99 produces siderophores that that could be responsible for the antimicrobial activity observed in co‐cultivation studies. Dereplication of high‐resolution tandem mass spectrometry (HRMS/MS) and global natural product social molecular networking platform (GNPS) analysis of fungus‐bacterium co‐cultures resulted in the identification of five madurastatin derivatives (A1, A2, E1, F, and G1), of which were four new derivatives. Chemical structures were unambiguously confirmed by HR‐ESI‐MS, 1D and 2D NMR experiments, as well as MS/MS data and their absolute structures were elucidated based on Marfey's analysis, DP4+ probability calculation and total synthesis. Structure analysis revealed that madurastatin E1 (2) contained a rare 4‐imidazolidinone cyclic moiety and madurastatin A1 (5) was characterized as a Ga(3+)‐complex. The function of madurastatins as siderophores was evaluated using the fungal pathogen Cryptococcus neoformans as model organism. Based on homology models, we identified the putative NRPS‐based gene cluster region of the siderophores in Actinomadura sp. RB99.