Terretonin G, a new sesterterpenoid antibiotic from marine-derived Aspergillus sp. OPMF00272

Marine-derived microorganisms are known as producers of many structurally unique and strong-bioactive compounds, including clinical medicines and research reagents.^{1, 2, 3, 4} During our screening for new metabolites from marine-derived fungi, one new sesterterpenoid, named terretonin G (1), was isolated from the culture broth of Aspergillus sp. OPMF00272 along with eight known natural products, terretonin (2),^{5, 6, 7} LL-S490β (3),⁸ methyl-3,4,5-trimethoxyl-2-[2-(nicotinamide)benzamido]benzoate (4),⁹ territrem B(5),¹⁰ aspernolid A (6),¹¹ butyrolactones I (7)¹² and V (8),¹³ and aspulvinone J (9)¹⁴ (Figure 1). This study describes the fermentation, isolation, structural elucidation and biological activity of 1.

Figure 1

Structures of nine compounds produced by Aspergillus sp. OPMF00272. Terretonin G (1), terretonin (2), LL-S490β (3), methyl-3,4,5-trimethoxyl-2-[2-(nicotinamide)benzamido]benzoate (4), territrem B (5), aspernolid A (6), butyrolactones I (7) and V (8), and aspulvinone J (9).

The fungus Aspergillus sp. OPMF00272 was isolated from poriferan collected on Ishigaki island in Okinawa, Japan, in 2008. The strain was inoculated into a 500-ml Erlenmeyer flask containing 100 ml seed medium (2.0% glucose, 0.2% yeast extract, 0.05% MgSO₄·7H₂O, 0.5% polypeptone, 0.1% KH₂PO₄ and 0.1% agar, pH 6.0). The flask was shaken on a rotary shaker at 27 °C for 3 days. The seed culture (2.0 ml) was transferred in a 1000-ml culture box containing 150 ml production medium (5% oat meal, 0.2% yeast extract, 0.1% Na tartrate, 0.1% KH₂PO₄, 0.8% Daigo authentic seawater, Nihon Pharmaceutical Co., Ltd., Tokyo, Japan). The fermentation was carried out at 27 °C for 7 days under static conditions. The culture broth (150 ml × 3) was extracted with ethanol (450 ml) for 2 h. After this extract had been evaporated to an aqueous solution, the residue was partitioned between water and EtOAc to yield the crude extract (684 mg) after evaporation of the EtOAc fraction. This extract was dissolved in a small volume of chloroform, applied to a silica gel column (40 g, 3.4 × 10 cm, 0.04–0.063 mm; Merck, Darmstadt, Germany), and eluted stepwise with 100% chloroform, 50:1, 10:1, 5:1 and 1:1 (v/v) chloroform–methanol and 100% methanol (300 ml each). Terretonin G was observed in the fraction eluted with 50:1 chloroform–methanol. This fraction was further purified by reversed-phase C-18 HPLC (20 × 250 mm; PEGASIL ODS, Senshu Scientific Co., Tokyo, Japan) under the following conditions: solvent, 53% CH₃CN water isocratic condition; flow rate of 8.0 ml min⁻¹; UV detection at 210 nm. Under these conditions, terretonin G was eluted as a peak with a retention time of 20.1 min. This fraction was collected and concentrated to yield pure terretonin G (1.3 mg) as a colorless solid.

The physico-chemical properties of terretonin G (1) are summarized in Table 1. The molecular formula of 1 was established as C₂₇H₃₈O₉ ([M+Na]⁺ m/z 529.2411) on the basis of HI-ESI-MS measurement, indicating that terretonin G contained nine degrees of unsaturation. The IR spectrum of 1 showed characteristic absorption at 3467, 1738 and 1713 cm⁻¹, suggesting the presence of hydoxy and carbonyl moieties. The structure of terretonin G (1) was mainly elucidated by analysis of NMR spectra including 2D NMR. The ¹³C NMR spectrum (in CD₃OD) showed 27 resolved signals, which were classified into eight sp³ methyl carbons, three sp³ methylene carbons, one sp² methylene carbon, four sp³ methine carbons and 11 quaternary carbons including four carbonyl carbons (C-3, C-6, C-16 and C-18) (Table 2). Analysis of the ¹H–¹H COSY spectra revealed two partial structures C-1 to C-2 and C-9 to C-11 (Figure 2). The ¹³C–¹H long range couplings of 2J and 3J observed in the ¹³C–¹H HMBC experiments (Figure 2) gave the following information: (1) The cross peaks from 1-H₂ (δ 1.75, 2.20) to C-3 (δ 215.9), C-5 (δ 63.8) and C-10 (δ 44.5), from 2-H₂ (δ 2.23, 2.85) to C-3, from 5-H (δ 2.81) to C-4 (δ 47.8), C-6 (δ 210.4), C-9 (δ 54.4), C-10, C-19 (δ 22.6), C-20 (δ 24.5) and C-22 (δ 16.3), from 7-H (δ 4.12) to C-6, C-8 (δ 50.9), C-14 (δ 57.6) and C-21 (δ 12.4), from 9-H (δ 2.05) to C-8 and C-10, from 11-H₂ (δ 2.45, 2.61) to C-8, C-12 (δ 150.7), C-13 (δ 58.4) and C-23 (δ 111.5), from 14-H (δ 3.75) to C-7 (δ 85.9), C-8, C-9, C-12, C-13, C-16 (δ 209.7), C-21 and C-24 (δ 22.6), from 19-H₃ (δ 1.50) to C-3, C-4, C-5 and C-20, from 20-H₃ (δ 1.07) to C-3, C-4, C-5 and C-19, from 21-H₃ (δ 1.17) to C-7, C-8, C-9 and C-14, from 22-H₃ (δ 1.15) to C-1, C-5, C-9 and C-10, from 23-H₂ (δ 4.32, 4.81) to C-11(δ 29.1), C-12 and C-13, and from 24-H₃ (δ 1.52) to C-12, C-13 and C-14 supported the partial structure I. (2) The cross peaks from 25-H₃ (δ 1.58) to C-16, C-17 (δ 83.3) and C-18 (δ 174.5) and from 18-OCH₃ (δ 3.73) to C-18 supported the partial structure II. (3) The cross peak from 15-OCH₃ (δ 3.52) to C-15 (δ 173.0) supported the partial structure III. (4) The cross peaks from 14-H to C-15 and from 24-H₃ to C-16 indicated that the partial structures I, II and III are connected as shown in Figure 2. Taking into consideration the molecular formula, the IR data and chemical shifts of C-7 (δ 85.9) and C-17 (δ 83.3), a hydroxy moiety should be bound to both C-7 and C-17. Thus, the planar structure of terretonin G was elucidated as shown in Figure 1.

Table 1 Physico-chemical properties of terretonin G

Table 2 NMR spectroscopic data for terretonin G in CD₃OD

Figure 2

Key cross peaks observed in ¹H–¹H COSY and ¹³C–¹H HMBC experiments of 1.

The relative configurations of C-5, C-7, C-8, C-9, C-10, C-13 and C-14 were determined by NOE experiments. Observation of NOEs from 5-H to 7-H, 9-H and 20-H₃, from 9-H to 14-H, from 22-H₃ to 19-H₃ and 21-H₃, and from 21-H₃ to 24-H₃ (Figure 3) indicated that they were assigned as 5R*, 7R*, 8R*, 9R*, 10R*, 13R* and 14S*. Stereochemistry of C-17, however, was not determined by NOE experiments.

Figure 3

NOE experiments of 1.

The antimicrobial activity of 1 and 2 was investigated using our routine in-house assay system.¹⁵ From the antimicrobial assay using paper disk, 1 (20 μg per 6 mm disk) showed antimicrobial activity with an inhibitory zone (10, 8 and 8 mm) against Gram-positive bacteria (Staphylococcus aureus FDA209P, Bacillus subtillis PCI219 and Micrococus luteus ATCC9341), but not against Gram-negative bacteria (Pseudomonas aeruginosa IFO12689 and Escherichia coli JM109) and fungi (Candida albicans ATCC64548 and Saccharomyces cerevisiae S288c). Interestingly, 2 (20 μg per 6 mm disk) showed no inhibitory activity against all microorganisms we tested.

Terretonin and its structurally related terretonins A–F have been reported; terretonin was reported as a mycotoxin⁵ and terretonins E and F were reported as inhibitors of the mammalian mitochondrial respiratory chain.⁷ Unfortunately, there has been no report about the biological activity of terretonins A–D.⁶ Accordingly, other biological activities of 1 in our in-house assays such as effect on cell cycle,¹⁶ lipid metabolites¹⁷ and alkaline phosphatase expression in myoblasts¹⁸ were investigated. Unfortunately, 1 showed no significant activity in these assays even at 25 μg ml⁻¹ (data not shown). To our knowledge, this is the first report that terretonins showed antimicrobial activity.