We analyzed the abundance of bacterial sulfonosphingolipids, including rosette-inducing (RIFs) factors, in seven bacterial prey strains using high-resolution tandem mass spectrometry (HRMS 2 ) and molecular networking (MN) within the Global Natural Product Social Molecular Networking (GNPS) web platform. Isolation and structure elucidation of six sulfonosphingolipids resembling RIFs was accomplished based on comparative MS and NMR studies. Here, we also report the first total synthesis of two RIF-2 diastereomers and one congener in 15 and eight synthetic steps, respectively. For the total synthesis of RIF-2 congeners, we employed a decarboxylative cross-coupling reaction to synthesize the necessary branched a-hydroxy fatty acids, and the Garner-aldehyde approach to generate the capnine base carrying three stereogenic centers. Bioactivity studies in the choanoflagellate Salpingoeca rosetta revealed that rosette inducing activity of RIFs is inhibited dose-dependently by the co-occuring sulfonosphingolipid sulfobacin D and F and that activity of RIFs is specific for isolates obtained from Algoriphagus .