A new cyclopeptide metabolite of marine gut fungus from Ligia oceanica

A new cyclopeptide, together with three known amino acid derivatives, was isolated from marine-derived fungus Aspergillus flavipes, which was found in the gut of isopod Ligia oceanica. The novel peptide contains four amino acid units, proline, 5-methoxyanthranilic acid, isoleucine and 3-aminoacrylic acid. Its structure was determined on the basis of NMR, HR-MS and MSn spectral data analysis. The two unusual amino acid residues, 5-methoxyanthranilic acid and 3-aminoacrylic acid, were first found in natural product. The known compound N-benzoyl-phenylalanine methyl ester was first found as fungal metabolite. This is the first report of natural products isolated from marine gut fungi.


Introduction
In recent years marine-derived fungi have become an important resource of natural products. Hundreds of fungal metabolites were reported one year and these compounds usually showed cytotoxicity, anti-fouling, anti-infective and anti-parasitic activities (Rateb & Ebel 2011). So far the major sources of marine fungi are alga and sponges. Actually various microorganisms including fungi were found in the gut of marine isopods (Cafaro 1999(Cafaro , 2000White 1999;Zimmer et al. 2001;Roa et al. 2009). These gut symbiote showed some ecological roles such as chemical defence (Lindquist et al. 2005). Therefore, gut fungi may be an interesting source of bioactive marine natural products. In the course of our chemical study of marine fungal metabolites (Xu et al. 2006(Xu et al. , 2008, a new cyclopeptide, together with three known ones (Figure 1), was isolated from the broth of Aspergillus flavipes (Z-4), which were found in the gut of marine isopod Ligia oceanica. A. flavipes is an active producer of natural products including cytochalasans (Zhou et al. 2004;Rochfort et al. 2005;Lin et al. 2009), phenols (Kwon et al. 2010(Kwon et al. , 2012, cerebrosides (Jiang et al. 2004), alkaloids (Barrow & Sun 1994), peptide ) and so on. In this work, the structural identification of one new cyclopeptide was described by NMR and mass spectral data analysis.

Results and discussion
HR-ESI-MS data of 1 showed quasi-molecular ion peaks at m/z 429.2136 [M þ H] (170.2, 168.6, 168.4 and 167.7) and indicated that 1 was a tetrapeptide and the interpretation of 1D (see Section 3.3) and 2D NMR ( Figure 2S) data also revealed four amino acid residues (A -D). In the proton NMR spectrum, three signals [(d 8.28,d,J ¼ 8.8 Hz), (d 7.00, dd, J ¼ 8.8, 2.8 Hz), (d 6.95, d, J ¼ 2.8 Hz)] indicated a 1,2,4-trisubstituted benzene group. These substitutes were determined as carbonyl, methoxyl and NH through HMBC (heteronuclear multiple-bond correlation) signals between signals of both benzene cycle and substitutes, from d H 3.82 (CH 3 OZ) to d C 155.7, d H 8.28 to d C 170.2 (carbonyl), and d H 9.72 (NH) to d C 125.8, respectively. Thus substructure A was identified as 5-methoxyanthranilic acid residue. The 1 H -1 H COSY correlations among proton signals of (d 9.44, d, J ¼ 11.6 Hz), (d 5.94, dd, J ¼ 11.6, 9.0 Hz) and (d 5.30, d, J ¼ 9.0 Hz) indicated a ZNHZCHvCHZ group with Z configuration. Additional HMBC signals from d H 5.30 to d C 167.7 confirmed a 3aminoacrylic acid residue (C). Three aliphatic methine groups (d C 58.8, 58.3, 32.3), four aliphatic methylene groups (d C 39.6, 26.6, 24.9, 21.0) and two methyl groups (d C 16.0, 10.3) (DEPT135 and DEPT90, see Supplementary file) were assigned to two independent spin systems of the types CH 2 ZCH 2 ZCH 2 ZCH and CH 3 ZCH 2 ZCH(CH)ZCH 3 by heteronuclear multiple quantum coherence and 1 H -1 H COSY spectra ( Figure S2), suggesting the existence of proline (B) and isoleucine (D) residues. HMBC signals from d H 9.72 to d C 168.4 and d H 9.44 to d C 168.6 indicated the linkages between A and B, C and D, respectively. The result was supported by the major ESI-MS n fragment ion peaks at m/z 181 [M þ H 2 A 2 B] þ and m/z 153 [M þ H 2 A 2 B 2 CO] þ . Thus compound 1 was determined as shown in Figure 1. The tetrapeptide includes two unusual amino acid residues, 5-methoxyanthranilic acid and 3aminoacrylic acid. To the best of our knowledge, this is the first report of cyclopeptide containing these two amino acid residues. Known compounds (2 -4) were also identified as asperphenamate, N-benzoyl-phenylalanine methyl ester and N-benzoylphenylalaninol, respectively, by the 1D and 2D NMR data analysis and the comparison with reported data (Clark & Hufford 1977;Balunas et al. 2009).
The fungus A. flavipes, terrestrial or marine, was reported to produce various secondary metabolites. This is the first report about this strain isolated from the gut environment of marine isopod. Its products included one novel cyclic tetrapeptide containing two unusual amino acid residues, 5-methoxyanthranilic acid and 3-aminoacrylic acid residues. One of the known products, N-benzoyl-phenylalanine methyl ester, was isolated as a natural product from a plant Brassaiopsis glomerulata. This is the first time reported as a fungal metabolite. It is wondered that these rare metabolites were related with gut environment.
3. Experimental 3.1. General experimental procedures NMR spectra were recorded on a Bruker DRX 400 with trimethylsilyl as internal standard. HR-ESI-MS spectra were measured on an Agilent 6224 TOF LC/MS. MS/MS spectra were obtained by Finnigan LTQ linear ion trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA). Medium 2216E (peptone 5.0 g, yeast extract 1.0 g, FePO 4 0.01 g dissolved in 1000 mL seawater, pH adjusted to 7.6 -7.8, added 16 g agar for solid medium).

Fungal isolation and fermentation
Marine isopod L. oceanica was collected in Zhoushan, Zhejiang province of China, in December 2011. Gut fungus (Z-4) was grown on solid 2216E medium (Qingdao Hope Biol-Technology Co., Ltd, Qingdao, China) for a period of 4 weeks at room temperature. Z-4 was identified as A. flavipes by 18S rDNA analysis. The strain Z-4 was cultured in 500-mL Erlenmeyer flasks containing 2216E liquid media (peptone 5.0 g, yeast extract 1.0 g, FePO 4 0.01 g dissolved in 1000 mL seawater, pH adjusted to 7.6-7.8, 200 mL £ 100) for 4 weeks at room temperature.

Extraction and purification
The culture broth was filtered and extracted with ethyl acetate (EtOAc). The extract (10 g) was subjected to silica gel column chromatography eluted in gradient with hexane -EtOAc (

Conclusion
This work reported that four amino acid derivatives, one novel peptide and three known peptides, were isolated from a marine gut fungus. The new peptide concluded two unusual amino acid residues and known compound N-benzoyl-phenylalanine methyl ester was isolated as fungal metabolite for the first time. This report demonstrates that gut fungi are the possible producers of marine natural products.

Supplementary material
Supplementary material relating to this article is available online.