A novel cyclic dipeptide from deep marine-derived fungus Aspergillus sp. SCSIOW2

A novel cyclic dipeptide, 14-hydroxy-cyclopeptine (1), was purified from a deep sea derived fungal isolate identified as an Aspergillus sp. The structure was elucidated by detailed spectroscopic analyses using 1D and 2D NMR experiments and high resolution mass spectrometry. The absolute configuration of the amino acid was determined by Marfey's method. Two conformational isomers of 1 were established by ROE analyses. 1 inhibited nitric oxide production with IC50 values at 40.3 μg/mL in a lipopolysaccharide and recombinant mouse interferon-γ -activated macrophage-like cell line, RAW 264.7 and showed no cytotoxic effect in the tested dose range up to 100 μg/mL.


Introduction
Macrophages play major roles in the innate and adaptive immune responses by releasing various factors such as pro-inflammatory cytokines, oxygen and nitrogen species. One critical releasing factor of nitric oxide (NO) has been implicated in numerous physiological and pathological processes (Bogdan 2001;Dawn & Bolli 2002). Excessive production of NO appears to associate with many chronic or acute diseases related to inflammation such as rheumatoid arthritis, cancer and even Alzheimer's disease. Therefore, inhibition of activation and NO production may be of therapeutic benefit against various types of diseases (Aktan et al. 2003).
So far, more than 1000 structurally unique and biologically active compounds have been isolated from marine-derived fungi (Rateb & Ebel 2011). Accordingly, marine fungi have attracted increasing attention as a resource for drug discovery (Bhadury et al. 2006). Plinabulin, a synthetic cyclic dipeptide analogue of halimide, which is isolated from a marine fungus species, is now in phase II clinical trial for treatment of non-small cell lung cancer (Yamazaki et al. 2011). In our efforts to identify novel structures and bioactive metabolites from deep sea (over 1000 m) derived fungi, we screened the fermentation extracts of 20 fungi for NO production inhibitory activities using macrophage RAW 264.7 cells. Among them, the EtOAc extracts of Aspergillus sp. SCSIOW2 showed strong potency to inhibit NO production without cytotoxic effects. A bioassay-guided chemical investigation resulted in the isolation of a novel cyclic dipeptide, cyclo-(L-N-MeTyr-anthranilic acid), which we have named 14-hydroxy-cyclopeptine (1).

Results and discussion
14-Hydroxy-cyclopeptine (1) was obtained as a colourless oil, its molecular formula was determined as C 17 H 16 N 2 O 3 , according to its HR-ESI-MS peak at m/z 297.1211 [M þ H] þ and m/ z 593.2375 [2M þ H] þ , requiring 11 degrees of unsaturation. Interestingly, all the proton and carbon signals of 1 recorded in DMSO-d 6 were observed in pairs at room temperature. The temperature dependency of 1 H NMR was then analysed, at þ 508C, the two sets of signals began to merge, at þ 858C, coalesced into single resonances ( Figure S1 -S3), this indicated the paired signals resulted from two different stable conformations in chemical exchange. Analysis of 1 H and 13 C NMR data revealed the presence of four carbonyl signals (d c 171.9, 171.1, 169.6, and 167.2), two amide protons (d H 10.5 and 10.6) and two N-methyl signals (d H 2.94 and 2.87). Comprehensive analysis of 2D (COSY, HSQC and HMBC) NMR spectroscopic data constructed four amino acid residues as two N-MeTyrs and two anthranilic acids (AAs). Two partial sequences of N-MeTyr-AA were established by HMBC correlations of the carbonyl carbons of each AA with the two N-methyl protons, respectively ( Figure 1). However, N-MeTyr- AA only accounted for 10 degrees of unsaturation, which suggested a cyclodipeptide of 1. Finally, HMBC correlations of each amide proton of AA with carbonyl carbon of N-MeTyr confirmed the final planar structures as cyclo-(N-MeTyr-AA). 1 had a specific optical rotation of ½a 20 D 2 93.58, cyclopeptin has a specific optical rotation of ½a 20 D 2 93.58, 4 0 -methoxycyclopeptin had a specific optical rotation of ½a 20 D 2 90.28 (Kusano et al. 2000), indicating the same absolute configuration for the sole chiral centre at C-3 for the three molecules. This result supported stereochemistry at C-3 most probably being S. The absolute configuration of N-MeTyr was finally determined as L-configuration based on hydrolysis and Marfey's analysis ( Figure S12). 1 H NMR integral area shows that the two distinct sets of conformers can be observed in about 4:5 ratio at room temperature. Small cyclic peptides have very different conformational behaviour to acyclic peptides, most significantly with respect to cis/trans isomerisation of the peptide groups. However, cyclic dipeptides usually have both peptide bonds in the cis conformation because this is the only configuration that allows for closure of the ring (Oakley & Johnston 2013). Preferred conformations for 1 were deduced by ROE spectral data. ROE correlations between N-methyl group and H-10 (tyrosine's b proton), H-12/16 in the major conformer (conformer A) indicated that the b-C of tyrosin (C-10) was in a pseudoequatorial direction. ROE correlation between N-methyl and H-3 in the minor conformer (conformer B) supported that the b-C of tyrosin was in a pseudoaxial direction (Figure 1). Up-field shifts of 10a and 10b in B form depend on the shielding effect of the benzene ring. Changing the structure from A to B can be done simply by ring flipping of the seven-membered cyclodipeptide ring with two peptide bonds remaining in cis conformation.
We examined the inhibitory effects of 1 on the production of NO induced by lipopolysaccharide (LPS)/INF-g. 1 showed weak NO production inhibitory activity [IC 50 ¼ 40.3 mg/mL (68.0 mM)]. The cytotoxic effect of 1 was measured with MTT assay. 1 did not show any cytotoxic effect at the tested dose range (30 -100 mg/mL). Quercetin was used as positive control which showed stronger NO inhibitory effect (inhibitory rate 97.4% at 100 mg/ mL).

General experimental procedures
Optical rotations were determined on a Jasco P-1020 polarimeter (Jasco, Hachioji, Tokyo, Japan). UV data were recorded on a Perkin Elmer Lambda 25 UV/vis spectrometer (Perkin Elmer, Boston, MA, USA). IR data were recorded using a Nicolet Avatar 330 FT-IR spectrometer (Thermo Scientific, Waltham, MA, USA). NMR spectra were acquired on a Bruker AVANCE-400 (400 MHz) or a Bruker ASCEND 600 MHz (Bruker, Ettlingen, Germany) NMR magnet system using TMS as internal standard. HR-ESI-MS were recorded on an AB SCIEX TOF/TOFe 5800 system (AB Sciex, Redwood City, CA, USA). Column chromatography (CC) was carried out with silica gel (100 -200 mesh, Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Pharmacia Biotech). TLC was performed on Merck TLC plates (silica gel 60 RP-18 F 254 S and silica gel 60 F254), with compounds were visualised by spraying with 5% (v/v) H 2 SO 4 in EtOH and then heating on a hot plate. HPLC was performed on a Shimadzu LC-20AT pump equipped with a SPD-20A UV -vis detector. A YMC-Pack Pro C18 column (10 £ 250 mm I.D. 5 m) and a YMC-Pack Pro C18 column (4.6 £ 250 mm I.D. 5 m) were used for semi-preparative and analysis purposes, respectively.

Strains
Fungus SCSIOW2 was isolated from deep marine sediment sample collected in the South China Sea (112830.203E, 1881.654N) at a depth of 2439 m. This fungus was characterised as Aspergillus sp. by the analysis of the ITS region sequence with GenebankS1. This fungus was deposited in Marine Microbial Lab., College of Life Science, Shenzhen University (Shenzhen, China).