Antimicrobial and Antioxidant Polyketides from a Deep-Sea-Derived Fungus Aspergillus versicolor SH0105

Fifteen polyketides, including four new compounds, isoversiol F (1), decumbenone D (2), palitantin B (7), and 1,3-di-O-methyl-norsolorinic acid (8), along with 11 known compounds (3–6 and 9–15), were isolated from the deep-sea-derived fungus Aspergillus versicolor SH0105. Their structures and absolute configurations were determined by comprehensive spectroscopic data, including 1D and 2D NMR, HRESIMS, and ECD calculations, and it is the first time to determine the absolute configuration of known decumbenone A (6). All of these compounds were evaluated for their antimicrobial activities against four human pathogenic microbes and five fouling bacterial strains. The results indicated that 3,7-dihydroxy-1,9-dimethyldibenzofuran (14) displayed obvious inhibitory activity against Staphylococcus aureus (ATCC 27154) with the MIC value of 13.7 μM. In addition, the antioxidant assays of the isolated compounds revealed that aspermutarubrol/violaceol-I (15) exhibited significant 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity with the IC50 value of 34.1 μM, and displayed strong reduction of Fe3+ with the ferric reducing antioxidant power (FRAP) value of 9.0 mM under the concentration of 3.1 μg/mL, which were more potent than ascorbic acid.


Introduction
Marine-sourced microbes have been deemed as one of the important resources for the discovery of drug lead compounds, with increasing number of diverse new bioactive natural products reported in recent years [1]. A series of remarkable progress have been made in the exploitation of marine microbial resources using various technical strategies, for instance, epigenetic modification [2,3], coculture [4,5], and genome mining [6,7]. The genus Aspergillus was widely distributed in marine environment and marine-derived Aspergillus species was home to a crucial reservoir for producing new bioactive chemical molecules to promote the development of marine drugs [8,9]. So far, plenty Mar. Drugs 2020, 18, 636; doi:10.3390/md18120636 www.mdpi.com/journal/marinedrugs of novel and active secondary metabolites have been reported from Aspergillus, such as anticancer plinabulin (NPI-2358) [10], α-glucosidase inhibitor aspergillusol A [11], and antiviral ochraceopone A and isoasteltoxin [12]. Inspiringly, plinabulin (NPI-2358) was an inhibitor of tubulin polymerization in third phase of clinical study to treat metastatic advanced nonsmall cell lung cancer (NSCLC) [13]. It was noteworthy that the studies of microbial secondary metabolites from extreme marine environments like deep sea have been gradually brought to the forefront in recent decades [14][15][16]. More and more new bioactive natural products have been discovered from the deep-sea derived Aspergillus, e.g., antifungal versicoloids A and B [17], cytotoxic penicillenols A1 and B1 [18], and anti-inflammatory cyclopenin [19]. As a part of our continuous research for marine bioactive natural products, a variety of bioactive compounds have been obtained from marine-derived Aspergillus genus [20], such as antibacterial (−)-sydonic acid [21], anti-RSV 22-O-(N-Me-L-valyl)-21-epi-aflaquinolone B [22], and antituberculous asperversiamides A-C [23]. Recently, a deep-sea-derived fungus Aspergillus versicolor SH0105 isolated from a Mariana Trench sediment sample (−5455 m) attracted our attention owing to its EtOAc extract of the fungal culture exhibiting antibacterial activity. The further chemical investigation on the EtOAc extract led to the isolation of four new polyketides (1-2 and 7-8), along with 11 known compounds (3-6 and 9-15) ( Figure 1). Herein, we report the isolation, structure elucidation, and biological activities of these compounds.

Results and Discussion
Isoversiol F (1) was obtained as a yellowish oil with a molecular formula of C 16 Figure S9), displaying the same molecular formula with the coisolated 12,13-dedihydroversiol (4), which was first isolated from the marine-derived Aspergillus sp. SCS-KFD66 [24]. The 1 H NMR, 13 C NMR, and HSQC spectra of 1 (Table 1 and Figures S2-S4) revealed the presence of one carbonyl (δ C 200.2), one olefinic quaternary carbon (δ C 130.3), five olefinic methine, two sp 3 quaternary carbons (with one oxygenated (δ C 86.2)), one methylene (δ C 38.8, δ H 1.30 and 2.00), three methines (including one oxymethine δ C 67.5, δ H 5.20), and three methyls. These structural features were also very similar to those of 4. The 1 H-1 H COSY interactions and the key HMBC correlations from H-4 to C-2, C-6, C-10, and C-16, from H-13 to C-8 and C-11, from H 3 -14 to C-7 and C-9, and from H 3 -15 to C-8, C-9, C-10, and C-11 in 1 demonstrated the same planar structure with 4 ( Figure 2 and Figures S5 and S6). The obvious distinctions were the chemical shifts of the oxymethine (δ C 67.5 and δ H 5.20) and methyl (δ C 17.2 and δ H 1.28) in 1 replaced the oxymethine (δ C 66.8 and δ H 3.95) and methyl (δ C 13.5 and δ H 1.17) in 4, respectively, which manifested that compound 1 should be a diastereoisomer of 4.  The relative configuration of 1 was determined by coupling constants, 1D NOE and 2D NOESY spectra. The small coupling constant of JH-1, H-10 = 3.3 Hz reflected the syn-relationship of H-1 and H-10. In the NOE spectrum, the irradiation of H3-15 (δH 1.28) led to the signal increase of H-1 (δH 5.20) and H3-16 (δH 1.04), suggesting that H3-15, H3-16, and H-1 should be positioned at the same planar ( Figure 3 and Figure S8). Besides, in the NOESY spectrum, the correlation was also observed between H-10 and H3-14 indicating the same face of these protons ( Figure 3 and Figure S7). Thus, the relative configuration of 1 was assumed as 1S * ,3S * ,8R * ,9S * ,10S * . The Mosher method was applied to determine the absolute configuration of 1, however, it failed. Fortunately, the absolute configuration of 1 was resolved by ECD calculations. Its experimental ECD spectrum agreed with that of calculated 1S,3S,8R,9S,10S-1, which exhibited negative Cotton effect at around 230 nm and positive Cotton effect at around 260 nm ( Figure 4 and Figure S32). Therefore, the absolute configuration of 1 was assigned as 1S,3S,8R,9S,10S. Compound 1 was a derivative of versiol [25], therefore, we named it as isoversiol F, which followed the reported isoversiols A−E [26].  Figure S8). Besides, in the NOESY spectrum, the correlation was also observed between H-10 and H 3 -14 indicating the same face of these protons ( Figure 3 and Figure S7). Thus, the relative configuration of 1 was assumed as 1S * ,3S * ,8R * ,9S * ,10S * . The Mosher method was applied to determine the absolute configuration of 1, however, it failed. Fortunately, the absolute configuration of 1 was resolved by ECD calculations. Its experimental ECD spectrum agreed with that of calculated 1S,3S,8R,9S,10S-1, which exhibited negative Cotton effect at around 230 nm and positive Cotton effect at around 260 nm ( Figure 4 and Figure S32). Therefore, the absolute configuration of 1 was assigned as 1S,3S,8R,9S,10S. Compound 1 was a derivative of versiol [25], therefore, we named it as isoversiol F, which followed the reported isoversiols A-E [26].   , and four methyl groups. These spectroscopic features suggested the presence of a similar skeleton with those of coisolated decumbenone A (6), which was first discovered from the fungus Penicillium decumbens [27]. The distinct differences were the existence of an additional methyl group (δC 30.8 and δH 2.28) in 2, and the absence of two methylenes (including one oxygenated) of the side chain in 6, indicating an acetyl group [CH3CO−] of the side chain at C-9 in 2 replaced the 3-hydroxypropionyl group [HOCH2CH2CO−] in 6, which was verified by the HMBC correlations from H-12 to C-1 and C-9 ( Figure 2 and Figure S14).
The relative configuration of 2 was also determined by coupling constants, 1D NOE, and 2D NOESY spectra. The small coupling constant of JH-1, H-10 = 3.3 Hz demonstrated the same side of H-1 and H-10. In the 1D NOE experiment measured in CDCl3 ( Figure 3 and Figure S17), the irradiation of H3-14 (δH 1.46) enhanced the signal of H-10 (δH 2.98), and the irradiation of H-10 and H3-15 (δH 1.05) simultaneously resulted the enhancement of H-2b (δH 1.32), implying that H3-14, H3-15, and H-10 should be placed at the same face ( Figure 3). In addition, the NOESY cross-peaks between H-10 and H3-13 indicated that these protons also should be coplanar ( Figure 3 and Figure S15). Herein, the relative configuration of 2 was deduced as 1S * ,3S * ,8R * ,9S * ,10S * . The calculated ECD spectrum of 1S,3S,8R,9S,10S-2 matched the experimental carve of 2 ( Figure 5 and Figure S33). Therefore, the absolute configuration of 2 was assumed as1S,3S,8R,9S,10S. It was worth mentioning that only the relative stereochemistry of the known compound 6 was assumed by Fujii et al. [27]. Herein, we firstly determined the absolute configuration of 6 as 1S,3S,8R,9R,10S by comparing the experimental and calculated ECD spectra ( Figure 5 and Figure S35).  (Table 1 and Figures S10-S12) indicated the presence of one ketone carbonyl (δ C 216.3), four olefinic signals (one quaternary), two sp 3 quaternary carbons (with one oxygenated), three methines (including one oxymethine δ C 67.3, δ H 4.22), one methylene (δ C 40.8, δ H 1.85 and 1.22), and four methyl groups. These spectroscopic features suggested the presence of a similar skeleton with those of coisolated decumbenone A (6), which was first discovered from the fungus Penicillium decumbens [27]. The distinct differences were the existence of an additional methyl group (δ C 30.8 and δ H 2.28) in 2, and the absence of two methylenes (including one oxygenated) of the side chain in 6, indicating an acetyl group [CH 3 CO−] of the side chain at C-9 in 2 replaced the 3-hydroxypropionyl group [HOCH 2 CH 2 CO−] in 6, which was verified by the HMBC correlations from H-12 to C-1 and C-9 ( Figure 2 and Figure S14).
The relative configuration of 2 was also determined by coupling constants, 1D NOE, and 2D NOESY spectra. The small coupling constant of J H-1, H-10 = 3.3 Hz demonstrated the same side of H-1 and H-10. In the 1D NOE experiment measured in CDCl 3 (Figure 3 and Figure S17), the irradiation of H 3 -14 (δ H 1.46) enhanced the signal of H-10 (δ H 2.98), and the irradiation of H-10 and H 3 -15 (δ H 1.05) simultaneously resulted the enhancement of H-2b (δ H 1.32), implying that H 3 -14, H 3 -15, and H-10 should be placed at the same face ( Figure 3). In addition, the NOESY cross-peaks between H-10 and H 3 -13 indicated that these protons also should be coplanar (Figure 3 and Figure S15). Herein, the relative configuration of 2 was deduced as 1S * ,3S * ,8R * ,9S * ,10S * . The calculated ECD spectrum of 1S,3S,8R,9S,10S-2 matched the experimental carve of 2 ( Figure 5 and Figure S33). Therefore, the absolute configuration of 2 was assumed as1S,3S,8R,9S,10S. It was worth mentioning that only the relative stereochemistry of the known compound 6 was assumed by Fujii et al. [27]. Herein, we firstly determined the absolute configuration of 6 as 1S,3S,8R,9R,10S by comparing the experimental and calculated ECD spectra ( Figure 5 and Figure S35). . The observed HMBC correlations from H-8 to C-4, C-5, and C-6 and from H-7 to C-1, C-5, and C-6 suggested the aliphatic chain should be located at C-5 and the hydroxymethyl linked at C-6 ( Figure 2). Because of the demand of five degrees of unsaturation, an additional ring should be proposed, which was confirmed by the HMBC correlations from H-3 to C-1 and C-5, and H-2 to C-1 and C-6 ( Figure 2). Hence, the planar structure of 7 was determined, which was similar with the known palitantin isolated from a plant endophytic A. fumigatiaffinis [28], except that the saturated bond at C-5/C-6 in palitantin was replaced by a double bond in 7.
The relative configuration of 7 was determined by coupling constants and NOESY correlations.  Figure S24). The absolute configuration of 7 was investigated by quantum chemical TDDFT calculations of its ECD spectrum. The experimental ECD spectrum was consistent with the calculated one of 2R,3R-7 ( Figure 6 and Figure S34), suggesting the absolute configuration of 7 as 2R,3R. 1,3-Di-O-methyl-norsolorinic acid (8) was isolated as a red powder and assigned the molecular formula as C22H22O7 based on its HRESIMS data ( Figure S31), including 12 degrees of unsaturation. and an aliphatic spin system C-8 to C-14, respectively, which was also verified by the corresponding HMBC correlations (Figure 2 and Figures  S22 and S23). The observed HMBC correlations from H-8 to C-4, C-5, and C-6 and from H-7 to C-1, C-5, and C-6 suggested the aliphatic chain should be located at C-5 and the hydroxymethyl linked at C-6 ( Figure 2). Because of the demand of five degrees of unsaturation, an additional ring should be proposed, which was confirmed by the HMBC correlations from H-3 to C-1 and C-5, and H-2 to C-1 and C-6 ( Figure 2). Hence, the planar structure of 7 was determined, which was similar with the known palitantin isolated from a plant endophytic A. fumigatiaffinis [28], except that the saturated bond at C-5/C-6 in palitantin was replaced by a double bond in 7.
The relative configuration of 7 was determined by coupling constants and NOESY correlations.  Figure S24). The absolute configuration of 7 was investigated by quantum chemical TDDFT calculations of its ECD spectrum. The experimental ECD spectrum was consistent with the calculated one of 2R,3R-7 ( Figure 6 and Figure  S34), suggesting the absolute configuration of 7 as 2R,3R. Palitantin B (7) was isolated as a yellow solid. Its molecular formula was suggested to be C14H20O4 according to its HRESIMS at m/z 253.1442 [M + H] + (calcd for 253.1434) ( Figure S25), with five degrees of unsaturation. The 1 H NMR, 13 C NMR data of 7 (Table 1 and Figure S19−20) displayed the presence of one carbonyl group, six olefinic carbons, two oxymethines, four methylenes, and one methyl group. In the 1 H- 1

H COSY spectrum, the cross-peaks of H-2/H-3/H-4 and correlations between H-8 to H-14 demonstrated a residue of [-OCHCH(O)CH2-]
and an aliphatic spin system C-8 to C-14, respectively, which was also verified by the corresponding HMBC correlations (Figure 2 and Figure  S22−23). The observed HMBC correlations from H-8 to C-4, C-5, and C-6 and from H-7 to C-1, C-5, and C-6 suggested the aliphatic chain should be located at C-5 and the hydroxymethyl linked at C-6 ( Figure 2). Because of the demand of five degrees of unsaturation, an additional ring should be proposed, which was confirmed by the HMBC correlations from H-3 to C-1 and C-5, and H-2 to C-1 and C-6 ( Figure 2). Hence, the planar structure of 7 was determined, which was similar with the known palitantin isolated from a plant endophytic A. fumigatiaffinis [28], except that the saturated bond at C-5/C-6 in palitantin was replaced by a double bond in 7.
Antimicrobial resistance phenomenon is still a global issue, which is threatening the human's life [37,38], indicating that it is very urgent to discover new antimicrobial molecules or mechanisms. In this study, all the isolated compounds 1-15 were evaluated for their antimicrobial activities against four human pathogenic microbes and five fouling bacterial strains. The results suggested that compound 14 displayed strong inhibitory activity against Staphylococcus aureus (ATCC 27154) with the MIC value of 13.7 µM, which was comparable to the positive control ciprofloxacin (MIC = 9.4 µM), and presented moderate inhibitory activity against Aeromonas salmonicida (ATCC 7965D) with the same MIC value of 13.7 µM (sea nine 211, MIC = 1.4 µM; Table S1). In addition, the antioxidant assays of the isolated compounds were carried out by DPPH radicals scavenging and FRAP models. The results revealed that 15 exhibited significant DPPH radical scavenging activity with the IC 50 value of 34.1 µM and displayed strong reduction of Fe 3+ with the FRAP value of 9.0 mM under the concentration of 3.1 µg/mL; thus, 15 was more potent than the positive control ascorbic acid (DPPH, IC 50 = 115.1 µM; FRAP = 5.6 mM under 3.1 µg/mL; Table S2). However, the radical scavenging effects of 1-14 were less than 50% under the concentration of 50 µg/mL.

General Experimental Procedures
The Optical rotations were measured on a JASCO P-1020 digital polarimeter (Jasco Corp., Tokyo, Japan). UV spectra were recorded by a Milton Roy UV-VIS spectrophotometer (Hitachi, Tokyo, Japan). IR spectra were performed on a Nicolet-Nexus-470 spectrometer using KBr pellets (Thermo Electron, Waltham, MA, USA). NMR spectra were tested by a JEOL JEMECP NMR spectrometer (600 MHz for 1 H NMR, 150 MHz for 13 C NMR and 500 MHz for NOE spectra, JEOL, Tokyo, Japan) using tetramethylsilane (TMS) as an internal standard. ESIMS spectra were measured on a Micromass Q-TOF spectrometer (Waters Corp., Manchester, UK). ECD spectra were obtained on a JASCO J-815 circular dichroism spectrometer (JASCO Electric Co., Ltd., Tokyo, Japan). In the biological assay, the optical densities (OD) were acquired by a multimode reader Spark 10M (Tecan, Männedorf, Switzerland). Semipreparative HPLC was performed on a Hitachi L-2000 HPLC system coupled with a Hitachi L-2455 photodiode array detector and a Kromasil C 18 semipreparative HPLC column (250 mm × 10 mm, 5 µm). Silica gel (Qingdao Haiyang Chemical Group Co., Qingdao, China) and Sephadex LH-20 (Amersham Biosciences Inc., Piscataway, NJ, USA) were used for column chromatography (CC). Precoated silica gel GF254 plates (Yantai Zifu Chemical Group Co., Yantai, China) were used for thin layer chromatography (TLC).

Fungal Material
The fungal strain A. versicolor SH0105 was isolated from a deep-sea sediment sample collected at a depth of 5455 m from the Mariana Trench. The strain was deposited in the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China. The fungal strain was identified as A. versicolor according to its morphological features, amplification and sequencing of the DNA sequences of the ITS region, and construction of phylogenetic tree by MEGA 7.0 (Temple University, Philadelphia, PA, USA; Figure S1). The sequence data was submitted to NCBI with the GeneBank accession number MT620963.

Antimicrobial Assay
The antimicrobial assays were evaluated using a broth microdilution method in 96-well polystyrene microtiter plates Costar 3599 (Corning Inc., New York, NY, USA) according to the standard of Clinical and Laboratory Standards Institute (CLSI) [41]. Three pathogenic bacterial strains, Staphylococcus aureus (ATCC 27154), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 10145); five fouling bacterial strains, P. fulva (ATCC 31418), Aeromonas salmonicida (ATCC 7965D), Photobacterium angustum (ATCC 33975), Enterobacter cloacae (ATCC 39978), and E. hormaechei (ATCC 700323); and one pathogenic fungal strain Candida albicans (ATCC 76485) were used as the test strains. First, the tested pathogenic bacteria, fouling bacteria, and pathogenic fungus were inoculated in 10 mL of LB (yeast extract 5 g/L, peptone 10 g/L, NaCl 10 g/L), 2216E (Hopebio, Qingdao, China), and YM (Hopebio, Qingdao, China) liquid medium, respectively, and cultivated at 37 • C for 12 h to yield the initial microbial liquids. The microbial density was adjusted to 0.5 MacFarland and then diluted 1000 times using the corresponding broth to obtain the tested microbial suspension with an inoculum density of 1 ×10 5 cfu/mL. The tested compounds were dissolved in 100% DMSO to obtain the mother solution with the initial concentration of 1 mg/mL. Following the principle of twofold serial dilution, each well contained 5 µL of tested compounds and 195 µL of the microbial suspension to obtain the final measured concentration of 25-0.098 µg/mL. Finally, the plates were incubated at 37 • C for 24 h and the optical density of each well was recorded by microplate reader (Tecan, Männedorf, Switzerland) at 600 nm. MIC represents the minimal inhibitory concentration of compound without visible microbial growth. The antimicrobial assays were performed in triplicate. Broad-spectrum antimicrobial ciprofloxacin and commercial antifouling sea-nine 211 were used as positive controls for pathogenic and fouling microbial strains, respectively. DMSO was used as a negative control.

Antioxidant Activity
The DPPH radical scavenging assay and ferric reducing antioxidant power assay (FRAP) were used to evaluate the antioxidant activities of the isolated compounds [42]. The samples and positive control ascorbic acid were dissolved in DMSO with final concentrations of 100, 50, 25, 12.5, and 6.25 µg/mL. DPPH was dissolved in anhydrous ethanol (EtOH) with the concentrations of 0.05 mg/mL. Fe 3+ -TPTZ solution consisted of 2 mmol/L FeCl 3 and 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), respectively. Tested samples (100 µL) were added to 100 µL of fresh DPPH or Fe 3+ -TPTZ solution, then reacted in the dark for 30 min. The optical density (OD) was measured by a multimode reader Spark 10 M (Tecan, Männedorf, Switzerland) at 517 and 593 nm, respectively. The EtOH and DMSO were employed as a blank and negative control, respectively. The IC 50 values were calculated on the software of GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA).

Conclusions
Deep-sea derived fungi are potential resources to seek for structural novel and diverse biological natural products. In the present study, chemical investigation of the deep-sea-derived fungus A. versicolor SH0105 led to the isolation of four new polyketides (1-2 and 7-8), along with 11 known compounds (3-6 and 9-15), which enriched the diversity of secondary metabolites from the deep-sea-derived Aspergillus. The structures and absolute configurations of new compounds were elucidated by comprehensive spectroscopic data and ECD calculations, and it is the first time to determine the absolute configuration of known decumbenone A (6). In the bioactive assays, compound 14 displayed obvious inhibitory activity against S. aureus (ATCC 27154) and 15 exhibited significant DPPH radical scavenging activity and displayed strong reduction of Fe 3+ , which were more potent than ascorbic acid, indicating the prospect to discovery of chemical entities with antimicrobial and antioxidant activities from the deep-sea medicinal microbial resources.