A novel cyclic dipeptide, named penicimutide (1), and four known cyclic dipeptides, cyclo(L-Val-L-Pro) (2), cyclo(L-Ile-L-Pro) (3), cyclo(L-Leu-L-Pro) (4) and cyclo(L-Phe-L-Pro) (5), were isolated from a neomycin-resistant mutant of the marine-derived fungus Penicillium purpurogenum G59. The structure of 1, including the absolute configuration, was determined by spectroscopic and chemical methods, especially NMR and Marfey's analysis. An unusual amino acid in 1, 4,5-didehydro-L-leucine, was found for the first time occurring in nature. HPLC-ESI-MS analysis evidenced that 1-3 were produced only in the mutant strain, but 4 and 5 were produced in both the mutant and parental strains, indicating that the introduction of neomycin-resistance in the mutant activated pathways of 1-3 biosynthesis that were silent in the parental strain. Compound 1 selectively inhibited HeLa cells (among five tested human cancer cell lines) with an inhibition rate (IR %) of 39.4 % at 100 µg/mL, a similar inhibition intensity to that of the positive control 5-fluorouracil (IR % of 41.4 % at 100 µg/mL against HeLa cells). The present work exemplifies the effectiveness of our previous DMSO-mediated method for introducing drug-resistance in fungi to activate silent biosynthetic pathways to obtain new bioactive compounds.
Keywords: Cyclic dipeptide; Fungal metabolite; Marine-derived Penicillium purpurogenum; Neomycin-resistant mutant; Penicimutide; Structure determination.