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A number of pesticides that inhibit the function of mitochondria have been found recently. Several commercial insecticidal and acaricidal compounds show inhibitory effects against the electron transport system complexes I and III, for example, fenpyroximate and strobilurin derivatives.1, 2 However, commercial pesticides targeting mitochondria are currently limited to those compounds inhibiting the electron transmission system.

ATP is produced in the mitochondrial inner membrane and transported by an ADP/ATP carrier protein (AAC). If compounds inhibit this crucial AAC, the mitochondria will not be able to produce ATP. Thus, the AAC would appear to be a good pesticidal target. Consequently, a screening system using insect AAC-expressing S. cerevisiae was developed by our group to find the compounds, especially those with a novel mechanism of action.3 Two new mitochondrial inhibitors, ascosteroside C (1) and trichopolyn VІ, have been found by this system to date.4, 5

Further investigation of a cultured broth of Aspergillus sp. FKI-6682, producer of 1, led us to discover a new mitochondrial inhibitor, named ascosteroside D (2) (Figure 1). Aspergillus sp. FKI-6682, isolated from a soil sample collected in Haha-jima, Japan, was cultured on an agar slant consisting of 0.1% glycerol, 0.08% KH2PO4, 0.02% K2HPO4, 0.02% MgSO4·7H2O, 0.02% KCl, 0.2% NaNO3, 0.02% yeast extract and 1.5% agar (adjusted to pH 6.0 before sterilization).4 A loopful of spores was inoculated into seed medium (100 ml), consisting of 2.0% glucose, 0.5% Polypepton (Nihon Pharmaceutical Co., Tokyo, Japan), 0.2% yeast extract, 0.1% KH2PO4, 0.05% MgSO4·7H2O and 0.1% agar (adjusted to pH 6.0 before sterilization), in each of five 500 ml Erlenmeyer flasks. The flasks were incubated on a rotary shaker (210 r.p.m.) at 27 °C for 2 days. The seed culture (25 ml) was inoculated into each of 20 culture bags (Ulpack 47, HOKKEN Co. Ltd, Tochigi, Japan) containing a production medium (500 g of wet rice). Static fermentation was continued at 27 °C for 14 days.

Figure 1
figure 1

Structures of ascosterosides C (1) and D (2).

The stationary culture (10 kg) was extracted with acetone (16 l). After filtration, the filtrate was concentrated in vacuo to remove acetone. The obtained aqueous solution was applied to an HP20 chromatography column (600 ml resin, Mitsubishi Chemical Co., Tokyo, Japan). After washing with H2O (2.0 l) and 50% MeOH aq. (2.0 l), the active fraction eluted with MeOH was concentrated in vacuo to afford crude material. The active fraction was applied to an ODS chromatography column (300 ml resin, YMC Co., Kyoto, Japan). After being washed with H2O (1.0 l), the column was eluted stepwise with 20, 40, 60, 80, 90 and 100% MeOH (each 1.0 l). The active material, eluted with 80% and 90% MeOH, was concentrated in vacuo. The remaining aqueous solution (200 ml) was extracted three times with ethyl acetate (200 ml) and the organic layer was concentrated in vacuo to dryness. The extract (1.42 g) was applied to a silica gel chromatography column (125 ml resin, Merck KGaA, Darmstadt, Germany) and eluted stepwise with a solvent mixture of CHCl3:MeOH (100:0, 100:1, 100:2 100:5, 100:10, 35:8, 1:1, 0:100 and 0:100 with 0.1% trifluoroacetic acid). The active compound was eluted with CHCl3:MeOH (35:8) and dried in vacuo. Finally, the active material (171 mg) was purified by high-performance liquid chromatography (Pegasil ODS SP 100, 10 i.d. × 250 mm, Senshu Scientific Co., Tokyo, Japan) with an isocratic solvent system of 50% aqueous acetonitrile at a flow rate of 4.0 ml min−1. The peaks with the retention time of 16–19 min and 23–27 min were collected and freeze-dried to afford 2 (24.0 mg) and 1 (76.0 mg), respectively.

Compound 2 was obtained as a white powder; soluble in MeOH, CHCl3, acetonitrile and dimethyl sulfoxide; [α]D23 +31.4° (c=0.1, MeOH); IR (KBr) λmax 3434, 2956, 2871, 1691, 1452, 1378 and 1027 cm−1; and UV (MeOH) λmax nm (ɛ) 203 (13680) and 231 (sh, 2530). The similarity of these physico-chemical characteristics with those of 1 strongly suggested that these congeners are analogs. The molecular formula of 2 was elucidated as C35H54O9 from the [M+Na]+ ion at m/z 641.3662 (calcd. for [M+Na]+, m/z 641.3660) in high-resolution electrospray ionization mass spectrometry analysis, indicating that the structure of 2 differs from that of 1 by a CH2.

The 1D NMR spectral data of 2 in CD3OD are shown in Table 1. The 1H and 13C NMR and HSQC spectra of 2 indicated the presence of five methyls, 10 methylenes including one oxymethylene and one sp2 exomethylene, 10 sp3 methines including five oxymethines, one anomeric methine and one olefinic methine, three sp3 quaternary carbons, four fully substituted olefinic carbons and one carbonyl carbon.

Table 1 NMR spectroscopic data for ascosterosides D (1) and C (2) in CD3OD at 400 MHz for 1H and 100 MHz 13C

The 1H and 13C NMR spectra of 2 resembled those of 1. However, the sp2 carbon at C-24, sp3methine at C-25 and exomethylene at C-28, as well as two doublet methyls at C-26 and C-27 of 1, were not observed. Instead of the above signals in 1, sp2 olefinic methine at C-24, sp2 carbon at C-25 and two singlet olefinic methyls at C-26 and C-27 appeared in 2. These chemical shifts strongly suggested that the structure of 2 is almost the same as that of 1 except for a side chain. The 1H-13C HMBC correlations from H2-23 (δH 1.91, 2.01) to C-24 (δC 126.0); from H-24 (δH, 5.10) to C-26 (δC 17.7) and C-27 (δC 25.9); from H3-26 (δH 1.60) to C-24, C-25 (δC 131.9) and C-27; and from H3-27 (δH 1.68) to C-24, C-25 and C-26 showed that 2 has 6-methyl-hep-5-en-2-yl group instead of the 6-methyl-5-methylidene-heptan-2-yl group in 1. Therefore, the structure of 2 was elucidated as shown in Figure 2 and 2 was named ascosteroside D.

Figure 2
figure 2

Key correlation of 1H-1H COSY and 1H-13C HMBC in ascosteroside D (2).

The inhibitory effect of 2 against mitochondrial function was evaluated using the paper disc method on agar plates with endogenous AAC-disrupted and Acyrthosiphon pisum AAC-expressing S. cerevisiae and AAC-disrupted empty vector-carrying S. cerevisiae (Δaac S. cerevisiae).3, 6 The A. pisum aac-transformed S. cerevisiae grown in glycerol-containing medium can only produce ATP in its mitochondria. The Δaac S. cerevisiae grown in glucose-containing medium cannot produce ATP in its mitochondria but it can produce ATP in the cytosol. If any compound inhibits the growth of insect AAC-expressing S. cerevisiae more than Δ aac S. cerevisiae, this suggests that it inhibits mitochondrial function.4, 5

Sterile filter discs impregnated with each compound solution (10 μl) were placed on the agar plate and the plates incubated at 30 °C for 48 h. After incubation, the inhibition zones were measured. Compound 2 inhibited the growth of insect AAC-expressing S. cerevisiae in glycerol-containing medium (Table 2). However, it did not inhibit Δaac S. cerevisiae in glucose-containing medium. Compound 1 showed similar results.4 This suggests that 2 inhibited the mitochondrial function as did 1. Cell growth inhibition assays were performed against several tumor cell lines using the method employed for evaluating 1.4 The IC50 values of 2 were 47, 46, 119, 142 and 67 μM against HeLa S3, HT29, A549, H1299 and Panc1 cells, respectively. Conversely, 1 did not inhibit cell growth, even at a concentration of 100 μM. These results suggested that the difference in structure between 1 and 2 might be important for influencing biological activity.

Table 2 Selective growth inhibition activity of ascosterosides D (1) and C (2) against recombinant S. cerevisiae

Antimicrobial activities of 2 against 11 microorganisms, S. cerevisiae ATCC9763, Candida albicans ATCC64548, Mucor racemosus IFO4581, Aspergillus niger ATCC6275, Staphylococcus aureus ATCC6538p, Bacillus subtilis ATCC6633, Escherichia coli NIHJ, Pseudomonas aeruginosa IFO3080, Xanthomonas campestris pv. oryzae KB88, Acholeplasma laidlawii PG8 and Kocuria rhizophila ATCC9341, were evaluated using the paper disc method.7 Compound 2 inhibited the growth of S. cerevisiae and C. albicans at 0.1 μg per disc, whereas it did not show antimicrobial activity against the other microorganisms.

In conclusion, 2 was isolated from the fungus Aspergillus sp. FKI-6682 as a new mitochondrial inhibitor, which could inhibit insect AAC-expressing S. cerevisiae in glycerol-containing medium. However, 2 did not inhibit Δaac S. cerevisiae in glucose-containing medium. These results suggest that 2 inhibited ATP production in mitochondria in the same manner as ascosteroside C (1).4

Dedication

We dedicate this article to the pioneering work of Professor Dr. Hamao Umezawa.