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 cocultures
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 Ga3+-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.