Determination of the absolute configurations of the stereogenic centers of ustilaginoidins by studying the biosynthetic monomers from a gene knockout mutant of Villosiclava virens.

Ustilaginoidins are a kind of mycotoxins with 9,9'-linked bis-naphtho-γ-pyrones structures produced by the rice false smut pathogen Villosiclava virens. These metabolites displayed a wide range of bioactivities, such as teratogenic, cytotoxic, phytotoxic, and antibacterial activities. So far 26 ustilaginoidins have been isolated from V. virens, among which 18 compounds contained stereogenic center(s), however, most of them were unknown for the absolute configurations, except that of ustilaginoidin D. In this study, the absolute structures of these ustilaginoidins were constructed for the first time by analysis of the biosynthetic monomers obtained from a gene knockout mutant (ΔUV_2091) of V. virens. The gene UV_2091 was predicted to encode an enzyme that dimerized the monomeric naphtho-γ-pyrones in V. virens. Knockout of this gene led to the accumulation of three monomers, namely hemiustilaginoidin F (1), epihemiustilaginoidin D (2), and hemiustilaginoidin D (3), but the production of ustilaginoidins was completely blocked. The structures of the monomers were deduced by spectroscopic analysis, in combination with TDDFT ECD calculations for determining the absolute configurations. These compounds were tested for their phytotoxic, cytotoxic, antibacterial, and antifungal activities. Compounds 1 and 3 showed inhibition against the radicle and plumule elongation of rice and lettuce seeds at the tested concentrations. Compound 1 was active against the tested five human cancer cells, with half maximal inhibitory concentrations (IC(50)s) of 13.2~37.3 μM. Compounds 1~3 inhibited the growth of the tested pathogenic bacteria with minimum inhibitory concentrations of 8~32 µg/mL, while compound 3 exhibited antifungal activity against Magnaporthe oryzae (IC(50), 5.21 µg/mL). A comparison of these data with those of the ustilaginoidins provided insights into the structure-bioactivity relationships.