Molecular networking assisted discovery and biosynthesis elucidation of the antimicrobial spiroketals epicospirocins.

Two pairs of dibenzospiroketal racemates, (±)-epicospirocin A (1a/1b) and (±)-1-epi-epicospirocin A (2a/2b), and two (+)-enantiomers of aspermicrones, ent-aspermicrone B (3b) and ent-aspermicrone C (4b), together with two hemiacetal epimeric mixtures, epicospirocin B/1-epi-epicospirocin B (5/6) and epicospirocin C/1-epi-epicospirocin C (7/8), were investigated from the phytopathogenic fungus Epicoccum nigrum 09116 via MS/MS molecular networking guided isolation and chiral separation for the first time. A plausible epicospirocin biosynthetic pathway was elucidated through in silico gene function annotation together with knock-out experiments. This is the first report that has applied MS/MS molecular networking to identify intermediates correlated with a biosynthetic pathway.