Expanding the chemical diversity of an endophytic fungus Bulgaria inquinans, an ascomycete associated with mistletoe, through an OSMAC approach

Abstract

An endophytic fungus Bulgaria inquinans (isolate MSp3-1), isolated from mistletoe (Viscum album), was subjected to fermentation on solid Czapek medium. Chromatographic workup of the crude EtOAc extract yielded five new natural products (1-5). Subsequent application of the "One Strain, MAny Compounds" (OSMAC) strategy on this strain by the addition of a mixture of salts (MgSO₄, NaNO₃ and NaCl) to solid Czapek medium induced the accumulation of nine additional new secondary metabolites (6-13, 16), with most of them (8, 10-12) not detectable in cultures lacking the salt mixture. The structures of the new compounds were established on the basis of the 1D/2D NMR and HRESIMS data. The TDDFT-ECD method was applied to determine the absolute configurations of the new compounds 1, 4 and 6 as well as of the previously reported bulgarialactone B (14), for which the absolute configuration was unknown so far. The modified Mosher's method was performed to assign the absolute configurations of 12 and 13. TDDFT-ECD analysis also allowed determining the absolute configuration of (+)-epicocconone, which had an enantiomeric absolute configuration in the tricyclic moiety compared to that of bulgarialactone B (14). All the isolated metabolites were evaluated for their cytotoxic activity. Compound 2 was found to possess strong cytotoxic activity against the murine lymphoma cell line L5178Y with an IC₅₀ value of 1.8 μM, while the remaining metabolites were shown to be inactive.

Fig. 1. Structures of compounds 1–18 isolated from B. inquinans.

Fig. 2. COSY and selected HMBC correlations of 1, 7 and 9.

Fig. 3. Experimental ECD spectrum (black) of 1 in MeCN compared with the Boltzmann-weighted PBE0/TZVP PCM/MeCN ECD spectrum (purple) of (S)-1 computed for the eight low-energy CAM-B3LYP/TZVP PCM/MeCN conformers. The bars represent the rotational strength of the lowest-energy conformer.

Fig. 4. Experimental ECD spectrum (black) of 4 in MeCN compared with the Boltzmann-weighted PBE0/TZVP PCM/MeCN ECD spectrum (purple) of (8R,9S)-4 computed for the 22 low-energy CAM-B3LYP/TZVP PCM/MeCN conformers. The bars represent the rotational strength of the lowest-energy conformer.

Fig. 5. Δδ_(S)–(R) values in ppm for the MTPA esters of 12.

Fig. 6. Experimental ECD spectrum of 14 in MeCN compared with the Boltzmann-weighted BH&HLYP/TZVP PCM/MeCN spectra of (3S,11S,23R)-14 and (3S,11S,23S)-14 computed for the low-energy (≥1%) CAM-B3LYP/TZVP PCM/MeCN conformers (26 and 26 conformers, respectively).

Fig. 7. Classification of the 26 low-energy (≥1%) CAM-B3LYP/TZVP PCM/MeCN conformers of (3S,11S,23R)-14 into conformer groups. Group A (70.3%) contains conformers A, B, C, D, E, F, H, I, J, L, M, N, Q, R, S, T, U, V, W, Y, Z; group B (5.6%) contains conformers G, K, X; group C (3.2%) contains conformers O and P.