The marine environment has recently been described as a source of novel chemical diversity for drug discovery, as many bioactive substances are isolated from marine organisms, including phytoplankton, algae, sponges, tunicates and mollusks.1 Our group has reported the isolation of azaspiracid-222 and JBIR-443 from marine sponges. Microorganisms from marine habitats, especially fungi,4 also constitute a promising untapped resource for discovering novel bioactive substances such as diketopiperazine alkaloids,5 trichodermatides6 and carbonarones.7 We also have reported new sesquiterpenes—JBIR-27 and JBIR-28—from a tunicate-derived fungus Penicillium sp. SS080624SCf1,8 a new aspochracin derivative—JBIR-15—from a marine sponge-derived fungus Aspergillus sclerotiorum Huber Sp080903f04,9 and new glycosyl benzenediols—JBIR-37 and JBIR-38—from a marine sponge-derived fungus, Acremonium sp. SpF080624G1f01.10 Further investigation resulted in the discovery of two new terpeptin analogs, designated as JBIR-81 (1) and JBIR-82 (2), together with terpeptin11 (3), from the culture of a marine-derived Aspergillus sp. SpD081030G1f1 (Figure 1a). This paper describes the fermentation, isolation, structure elucidation, and in brief, the biological activity of 1 and 2.

Figure 1
figure 1

(a) Structures of JBIR-81 (1), JBIR-82 (2) and terpeptin (3). (b) Correlations in DQF-COSY (bold lines) and CT-HMBC (arrows) spectra of 1 and 2.

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The fungus, Aspergillus sp. SpD081030G1f1, was isolated from a seaweed, Sargassum sp., collected at the sea shore of Ishigaki Island, Okinawa Prefecture, Japan. The strain was cultivated in 50-ml test tubes containing 15 ml of the seed medium (24 g l–1 potato dextrose broth, BD Biosciences, San Jose, CA, USA). The test tubes were shaken on a reciprocal shaker (355 r.p.m.) at 27 °C for 3 days. Aliquots (5 ml) of the seed culture were inoculated into 500-ml Erlenmeyer flasks containing 9 g oatmeal (Quaker, Chicago, IL, USA), 30 ml vegetable juice (KAGOME, Nagoya, Japan) and incubated at 27 °C for 14 days in static culture.

The solid culture (20 flasks) was extracted with 80% aqueous acetone (400 ml). The extract was evaporated in vacuo, and the residual aqueous concentrate was partitioned with EtOAc (100 ml × 3). After drying over Na2SO4, the EtOAc layer was evaporated. The dried residue (845 mg) was subjected to normal-phase medium-pressure liquid chromatography (Purif-Pack SI-60, Moritex, Tokyo, Japan) using a CHCl3–MeOH stepwise solvent system (0, 5 and 10% MeOH). The fractions containing 1–3 were collected by HPLC-MS monitoring. The target eluate (5% MeOH, 28 mg) was further purified by preparative reverse-phase HPLC using an L-column 2 ODS column (20 i.d. × 150 mm; Chemical Evaluation and Research Institute, Tokyo, Japan) with a 2996 photodiode array detector (Waters, Milford, MA, USA) and a 3100 mass detector (Waters) developed using 25% aqueous MeOH containing 0.1% formic acid (flow rate, 10 ml min−1) to yield 1 (1.1 mg, retention time (Rt) 9.7 min), 2 (1.4 mg, Rt 21.4 min) and 3 (2.0 mg, Rt 19.2 min).

Compounds 1 and 2 were obtained as colorless amorphous solids ([α]D25 –177.7 for 1 and –145.3 for 2, c 0.1, in MeOH). The molecular formulas of 1 and 2 were determined by HR-electrospray ionization-MS to be C24H34N4O3 (found: 427.2712 [M+H]+, calcd: 427.2709) for 1 and C29H42N4O3 (found: 495.3340 [M+H]+, calcd: 495.3335) for 2. The presence of an amide group was deduced from their IR spectra (νmax (KBr) 3315, 1631 cm−1 for 1; and 3313, 1631 cm−1 for 2). The UV spectra revealed the presence of an indole chromophore (λmax (MeOH) 230 (ɛ=13 880), 280 (ɛ=6780) nm for 1 and 230 (ɛ=15 030), 285 (ɛ=6770) nm for 2] similar to those of 3.11 The structures of 1 and 2 were determined by NMR spectral analyses. The direct connectivity of protons and carbons were established by heteronuclear single quantum coherence spectra, and the tabulated 13C and 1H NMR spectral data for 1 and 2 are shown in Table 1. The structures of 1 and 2 were elucidated in a series of double-quantum-filtered (DQF)-COSY and constant time heteronuclear multiple bond correlation (HMBC)12 as follows.

Table 1 13C (150 MHz) and 1H (600 MHz) NMR data for 1 and 2
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The 1H–1H correlations observed in the DQF-COSY spectrum, together with the spin coupling constants among aromatic protons 4-H (δH 7.57), 5-H (δH 7.03), 6-H (δH 7.12) and 7-H (δH 7.38), revealed a 1,2-disubstituted benzene ring. In the HMBC spectrum, the aromatic proton 4-H was peri coupled to an aromatic quaternary carbon C-3 (δC 109.8), an olefinic proton 2-H (δH 7.56) was coupled to C-3 and an aromatic quaternary carbon C-3a (δC 126.8), and an amino proton 1-NH (δH 11.32) was coupled to an olefinic carbon C-2 (δC 123.7), C-3 and C-3a. Taking into consideration these correlations, the UV absorption and 13C chemical shifts, a 3-substituted indole chromophore was established. The sequence from an olefinic proton 8-H (δH 5.93) to an amide proton 10-NH (δH 8.95) through an olefinic proton 9-H (δH 6.62) in the DQF-COSY spectrum determined an ethenamine moiety. The configuration at C-8 was deduced to be cis by the coupling constants between 8-H and 9-H (J=9.4 Hz). The ethenamine moiety was connected to C-3 of the indole by the 1H-13C long-range couplings from 8-H to C-3 and C-3a, and from 2-H to C-8 (δC 103.3). Therefore, these results indicated (Z)-2-(1H-indol-3-yl)ethenamine.

The remaining peptide sequence, N-methyl leucine-N-acetyl valine, was elucidated as follows. The sequence from an α-methine proton 12-H (δH 5.25) to a methyl proton 24-H (δH 0.88) through methylene protons 22-H (δH 1.68, 1.59) and a methine proton 23-H (δH 1.40), which in turn spin coupled to a methyl proton 25-H (δH 0.79), was observed in the DQF-COSY spectrum. In addition, 1H-13C long-range couplings from the α-methine proton 12-H to an amide carbonyl carbon C-11 (δc 169.3) and an N-methyl carbon 13-CH3 (δc 31.2) established an N-methylleucine residue. Furthermore, the sequence from an amide proton 16-NH (δH 8.11) to a methyl proton 20-H (δH 0.73) through an α-methine proton 15-H (δH 4.47) and a methine proton 19-H (δH 1.91), which was coupled to a methyl proton 21-H (δH 0.81), together with the 1H-13C long-range couplings between the α-methine proton 15-H and an amide carbonyl carbon C-14 (δc 173.3) revealed a valine residue. Finally, 1H-13C long-range couplings from the amide proton 10-NH to the amide carbonyl carbon C-11, from the N-methyl proton 13-CH3 (δH 3.02) to the amide carbonyl carbon C-14, and from the amide proton 16-NH and an acyl methyl proton 18-H (δH 1.81) to an amide carbonyl carbon C-17 (δc 169.2), established the connectivity of these amino-acid derived partial residues. Thus, the planar structure of 1 was determined as shown in Figure 1a.

Compound 2 is structurally related to 1, in which the olefinic proton 2-H is replaced by a dimethylallyl moiety. Correlations interpreted by DQF-COSY and HMBC spectra proved common partial structures in 2 (Figure 1b). Both singlet methyl protons 29-H (δH 1.68) and 30-H (δH 1.67) were 1H-13C long-range coupled to each other, and commonly coupled to an olefinic quaternary carbon C-28 (δC 132.6) and an olefinic methine carbon C-27 (δC 121.1), of which proton was 1H spin-coupled to methylene proton 26-H (δH 3.35). Thus, a dimethylallyl moiety was determined as shown in Figure 1b. 1H-13C long-range couplings from the methylene proton 26-H and the amino proton 1-NH (δH 10.99) to an olefinic quaternary carbon C-2 (δC 136.9) confirmed the structure of 2 as shown in Figure 1a. To our knowledge, this is the first report of these terpeptin analogs.

To evaluate the free radical scavenging activities of 1–3, we tested the protective activity against L-glutamate toxicity in N18-RE-105 cells,13, 14, 15 which can be used to assess free radical scavenging activity. Compounds 1–3 showed stronger protective activity against L-glutamate toxicity in cells with EC50 values of 0.7, 1.5 and 0.9 μM, respectively, compared to that of a representative antioxidant, α-tocopherol (EC50=8.8 μM). Details of the biological activities of 1–3 are now under examination.