Identification and Antifungal Activity of Compounds from the Mangrove Endophytic Fungus Aspergillus clavatus R7

Two new coumarin derivatives, 4,4′-dimethoxy-5,5′-dimethyl-7,7′-oxydicoumarin (1), 7-(γ,γ-dimethylallyloxy)-5-methoxy-4-methylcoumarin (2), a new chromone derivative, (S)-5-hydroxy-2,6-dimethyl-4H-furo[3,4-g]benzopyran-4,8(6H)-dione (5), and a new sterone derivative, 24-hydroxylergosta-4,6,8(14),22-tetraen-3-one (6), along with two known bicoumarins, kotanin (3) and orlandin (4), were isolated from an endophytic fungus Aspergillus clavatus (collection No. R7), isolated from the root of Myoporum bontioides collected from Leizhou Peninsula, China. Their structures were elucidated using 1D- and 2D- NMR spectroscopy, and HRESIMS. The absolute configuration of compound 5 was determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Compound 6 significantly inhibited the plant pathogenic fungi Fusarium oxysporum, Colletotrichum musae and Penicillium italicum, compound 5 significantly inhibited Colletotrichum musae, and compounds 1, 3 and 4 greatly inhibited Fusarium oxysporum, showing the antifungal activities higher than those of the positive control, triadimefon.

Compound 5 was obtained as colorless powders, and its molecular formula was established as C 13 H 10 O 5 with nine degrees of unsaturation by positive HR-ESI-MS (m/z 269.0423, [M + Na] + , calcd. 269.0420). The characteristic UV absorption maxima at 229, 242, 263, 345 nm suggested the presence of a chromone pattern in 5 [16,17]. The 1 H and 13 C NMR spectral data of 5 are listed in Table 2. The 1 H NMR spectrum exhibited signals of one olefinic methyl at δ H 2.52 (s, 1H), one secondary methyl at δ H 1.67 (d, 6.6 Hz 3H) connected to one oxomethine at δ H 5.73 (q, 6.6 Hz, 1H), one hydroxyl at δ H 13.43 (s, 1H), and two aromatic proton singlets at δ H 6.37 and 7.83. The olefinic methyl was revealed to be attached at C-2 due to HMBC correlations ( Figure 2) from the 2-CH 3 proton at δ H 2.52 to C-2 (δ C 170.3) and C-3 (δ C 108.9), and from the aromatic H-3 proton (δ H 6.37) to C-2 and C-4a (δ C 112.7). The hydroxyl was proved to be substituted at C-5 based on HMBC correlations from 5-OH (δ H 13.43) to C-4a, C-5 (δ C 155.8) and C-5a (δ C 130.7). These results, combined with the HMBC correlations, including H-9 (δ H 7.37) to C-4a, C-5a, and the oxygen-bearing C-9a (δ C 157.2), ambiguously established the chromone substructure, indicating that the positions of C-8a and C-9 of the aromatic ring were substituted by the remaining moiety. Subsequently, HMBC correlations from H-9 to C-8 (δ C 168.2), from H-6 (δ H 5.73) to C-5a, C-8, 6-CH 3 (δ H 1.67), from 6-CH 3 to C-5a, C-6 (δ C 76.5), together with the remaining 2 degrees of unsaturation revealed by the molecular formula, suggested a γ-valerolactone ring system attached to C-8a and C-9 through C-8 and C-6, respectively. Thus, the planar structure of 5 was completely established. The absolute configuration of 5 was determined by comparing the theoretical calculation of ECD (electronic circular dichroism) with the experimental ECD [18,19]. The experimental ECD of 5 is similar to the ECD of the (S)-model compound (Figure 3), so as to determine that the absolute configuration of 5 was 6S. Therefore, the structure of 5 was as shown in Figure 1.    The configuration of C-24 could not be assigned based on the obtained NOE data. Therefore, compound 6 was elucidated as 24-hydroxylergosta-4, 6,8(14),22-tetraen-3-one, as shown in Figure 1. In addition, the structures of the known compounds 3 and 4 [11] were identified by comparison of their spectroscopic data with those reported in the literature. HRESIMS, 1 H, 13 Table 4, all of the compounds showed broad-spectrum inhibitory activities against these fungi except compound 2, which is inactive towards P. italicm with MIC value >729.66 µM. Moreover, compound 6 exhibited the strongest broad-spectrum inhibitory activities against all the three pathogenic fungi F. oxysporum, C. musae

General Experimental Procetures
Melting points were determined using a JH30 melting point detector (Jia Hang Instrument Co., Ltd., Shanghai, China). Optical rotations were measured using a Horiba SEPA-300 polarimeter at 25 • C. The UV spectra were obtained on a Shimadzu UV-2550 spectrophotometer (Shimadzu, Tokyo, Japan), and IR spectra were run on a Nicolet 5DX-Fourier transform infrared spectrophotometer (Thermo Electron Corporation, Madison, WI, USA). NMR spectra data were recorded at Bruker AV-600 MHz NMR spectrometers (Bruker Biospin AG, Fällanden, Switzerland), with tetramethylsilane (TMS) as internal standard, and the chemical shifts were reported in δ values (ppm). The HRESIMS spectra were recorded on an Q-TOF mass spectrometer (Thermo Fisher, Frankfurt, Germany). CD spectra were recorded with a Chirascan™ CD spectrometer (Applied Photophysics, Leatherhead, UK). Silica gel (200-300 mesh) for column chromatography was purchased from Qingdao Haiyang Chemical Co., Ltd., Qingdao, China. Sephadex LH-20 was purchased from Amersham Pharmacia Biotech. Buckinghamshire, UK. All other chemicals were of analytical grade.

Fungal Material and Fermentation
The fungal strain R7 was isolated from the root of M. bontioides, collected from the mangrove in Leizhou peninsula, China, in May 2014, and deposited at the College of Materials and Energy, South China Agricultural University, Guangdong Province, China. The strain has been identified as A. clavatus, according to morphologic traits and molecular identification [10]. Its 599 base pair ITS sequence had 99% sequence identity to those of several A. clavatus strains (AY373847.1, NR121482.1, KF669481.1) by a NCBI BLAST search. The sequence data has been submitted to GenBank with accession number KY765893.
A small agar scrap with mycelium of the fungal isolate which was grown on potato dextrose agar medium for 5 days at 28 • C was added into 250 mL GYT medium (1% glucose, 0.1% yeast extract, 0.2% peptone, 0.2% crude sea salt), and incubated at 28 • C, 180 rpm for 6 days as seed culture. Then the seed culture was grown on a solid autoclaved rice substrate medium (one hundred 1000 mL Erlenmeyer flasks, each containing 100 mL water, 100 g rice and 0.3 g crude sea salt) for 30 days at 25 • C under static stations.

Computational Analyses
Conformational analyses for compound 5 were performed via Spartan'10 software (Wavefunction, Inc., Irvine, CA, USA) using the MMFF94 molecular mechanics force field calculation. Conformers within a 10 kcal/mol energy window were generated and optimized using DFT calculations at the B3LYP/6-31G (d) level. Conformers for R or S were chosen for ECD calculations in MeOH at the B3LYP/6-311+G (2d, p) level. Rotary strengths for a total of 50 excited states were calculated. The IEF-PCM solvent model for MeOH was used. The calculated ECD spectra were obtained by density functional theory (DFT) and time-dependent DFT (TD-DFT) using Gaussian 09 (Gaussian Inc., Wallingford, CT, USA) program package. The calculated ECD curve was generated using SpecDis 1.6 software package (University of Wurzburg, Wurzburg, Germany) with a half-bandwidth of 0.2 eV.

Antifungal Activity Assay
The following four phytopathogenic fungi were used for bioassay: F. oxysporum, C. musae, and P. italicm. They were obtained from the College of Agriculture, South China Agricultural University. The antifungal activities of the isolated compounds were determined by the broth dilution method as described in the previous report to get the minimum inhibitory concentration (MIC) [21]. Triadimefon and the solvent were used as positive and negative control, respectively.