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) andent-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 fungusEpicoccum nigrum09116viaMS/MS molecular networking guided isolation and chiral separation for the first time. A plausible epicospirocin biosynthetic pathway was elucidated throughin silicogene 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.