Three new unsaturated fatty acids from marine-derived fungus Aspergillus sp. SCAU150

Abstract Four unsaturated fatty acid derivatives including three new pantheric acids (1–3), together with three known polyketides (5–7), were isolated from a culture broth of the marine-derived fungus Aspergillus sp. SCAU150. Their complete structures were determined by NMR and HRESIMS data analyses. The antifungal activity of the isolated compounds above was evaluated and 2 was found to show moderated activity toward the phytopathogenic fungus Fusarium solani bio-80814 with an inhibition zone diameter of 6 mm under 5 µg/disc. Graphical Abstract


Introduction
Marine fungi are recognized as prolific sources of structurally unique secondary metabolites with promising biological activities (Jin et al. 2016;Carroll et al. 2020). Species of the genus Aspergillus are abundant producers of bioactive natural products for their utility in drug discovery, including antibacterial, antifungal, antiviral, antiparasitic, anticancer, and antidiabetic activities (Lee et al. 2013;Romsdahl and Wang 2019;Xu et al. 2020). Among them, the unsaturated fatty acids (UFAs) are a category of structures consisted of a long-chain hydrocarbon with the presence of at least one double covalent bond and ending in a carboxyl group (-COOH), which are of great interest in the field of drug discovery because of their widespread bioactivities, such as antimalarial, antimicrobial (Calcul et al. 2013;Tang et al. 2019;Lu et al. 2019). During our ongoing search for structurally novel and biologically active compounds from marine-derived microorganisms (Nong et al. 2014;Nong et al. 2016), the secondary metabolites from a coral-associated fungus Aspergillus sp. SCAU150 were chemically investigated. As a result, three new unsaturated fatty acid derivatives pantheric acids D-F (1-3) featuring a terminal double bond, together with four known structures pantheric acid C (4) (Lee et al. 2019), chaetominine (5) (Jiao et al. 2006), monomethylsulochrin (6) (Guo et al. 2020), fumiquinone B (7) (Hayashi et al. 2007), were obtained from the fungal fermentation broth. The structures of these new compounds were elucidated by detailed analysis of NMR and HRESIMS data. And the antifungal activity of compounds 1-7 was also evaluated against a panel of six plant pathogenic fungal strains.

Results and discussion
The molecular formula of pantheric acid D (1) was assigned as C 10 H 16 O 4 by HRESIMS data with 3 degrees of unsaturation (Supplementary material Figure S9). Interpretation of the 1 H NMR spectroscopic data of 1 (Supplementary material Table S1) displayed diagnostic signals for two shielded olefinic protons (d H 6.00, 5.52), one methoxy protons (d H 3.56). Thorough analysis of 13 C NMR spectroscopic data of 1 (Supplementary  material Table S1) revealed resonances for two ester carbonyls (d C 173.4, 168.2), one sp 2 methylene (d C 124.1), one sp 2 none-protonated carbon (d C 141.3), five sp 3 methylene groups (d C 33.3, 31.3, 28.2, 27.8, 24.4) and one methoxy group (d C 51.2). These data provided a speculation that 1 was a fatty acid derivative. Further this was confirmed by observation of the HMBC spectrum of 1, which showed correlations from H-3 to C-1/C-2/C-4/C-5/C-11, from H-7 to C-5/C-6/C-8, from Me-10 to C-8, and from H-11 to C-1/C-2/C-3 (Supplementary material Figure S1). The presence of a carboxylic acid group was determined at C-1 by the molecular formula, whose carbon appeared at d C 168.2. Based on above, the chemical structure of 1 was established as depicted ( Figure 1). Additional analysis of the COSY spectrum of 1 showing cross-peaks between H-3 and both H-4 and H-11, and between H-6 and H-7, also supported the above assignment (Supplementary material Figure S1). Considering the structure of compound 1 was an analogue of pantheric acid C (4) (Lee et al. 2019), it was given a trivial name as pantheric acid D.
The molecular formula of pantheric acid E (2) was assigned as C 11 H 18 O 4 by HRESIMS data with 3 degrees of unsaturation (Supplementary material Figure S16). The 1 H NMR spectroscopic data of 2 (Supplementary material Table S1) exhibited obvious signals for two shielded protons (d H 6.01, 5.55), one oxygenated methylene quartet (d H 4.06), one methyl triplet (d H 1.18). The 13 C NMR spectroscopic data of 2 (Supplementary material Table S1) showed signals for two ester carbonyls (d C 172.9, 168.1), one sp 2 methylene (d C 124.2), one sp 2 none-protonated carbon (d C 141.0), six sp 3 methylene groups including one oxygenated methylene (d C 59.6), and one methyl (d C 14.1). The 1 H and 13 C NMR spectroscopic data of 2 were greatly similar to those of 1 with the only obvious difference of the absence of one methoxy group (d C 51.2 in compound 1) and the additional appearance of one oxygenated methylene and one methyl group (d C 59.6, 14.1) in 2. Careful analysis of the 2 D NMR spectra revealed that 2 was an analogue of 1, with a methoxy group attached to C-8 instead of an oxygenated ethyl group, which was supported by the COSY spectrum showing the presence of a spin system of H 2 -10/Me-11 and the HMBC spectrum exhibiting correlations from Me-11 to C-10, from H 2 -10 to C-8 (Supplementary material Figure S1). Therefore, the structure of 2 was confidently defined as depicted in Figure 1, and given a trivial name as pantheric acid E.
The molecular formula of pantheric acid F (3) was assigned as C 13 H 22 O 4 by HRESIMS data with 3 degrees of unsaturation (Supplementary material Figure S23). The 1 H and 13 C NMR spectroscopic data of 3 (Supplementary material Table S1) was very similar to 2 with the only obvious difference of the addition of two deshielded methylenes (d C 28.3, 28.4). Comprehensive analysis of HMBC spectrum disclosed correlations from H-3 to C-1/C-2/C-4/C-5/C-12, from H-4 to C-2/C-3/C-5/5a, from H-7 to C-6/C-6a/C-8, from H 2 -10 to C-8/C-11 and from Me-11 to C-10 (Supplementary material Figure S1), while the COSY spectrum revealed spin systems of H-3 and H-4/H-12, H-6 and H-6a/H-7, H-10 and H-11 (Supplementary material Figure S1). So the structure of 3 was readily elucidated and given a trivial name of pantheric acid F.
Considering the new compounds above may be artifacts owing to the uses of methanol, ethyl acetate and acetone as elution solvents of column chromatography during the separation process, the initial extract was used to perform a test of LC-HRESIMS experiment. The result indicated the presence of compounds 1-3 in the original crude extract (Supplementary material Figure S24). To the best of our knowledge, the methyl/ethyl ester formation for fatty acids were also naturally occur like ieodomycin A (Mondol et al. 2011), fusarisolin E (Niu et al. 2019), methylester-3-O-a-Lrhamnopyranoside and (À)-9-hydroxyhexylitaconic acid (Antia et al. 2011), hexadecanoic acid ethyl ester and 9,12-octadecadienoic acid ethyl ester (Marante et al. 2012), (10Z,13Z)-ethyl nonadeca-10,13-dienoate and (9Z,12Z)-ethyl nonadeca-9,12-dienoate . And it is reported that the biosynthesis of these fatty acids methyl/ ethyl ester is involved in two different enzymatic mechanisms, esterification or alcoholysis (Saerens et al. 2006;Herrera-Valencia et al. 2012).

General experimental procedures
IR spectra were recorded on a FT-IRNICOLET spectrophotometer. UV spectra were collected on a Beckman DU 640 spectrophotometer. NMR spectra were recorded on a Bruker AV spectrometer (400 MHz for 1 H and 100 MHz for 13 C). TMS was used as a reference. HRESIMS spectra were measured on an amaZon SL ion trap mass spectrometer and MaXis quadrupole-time-of-flight mass spectrometer (Bruker). Semi-preparative HPLC was performed on an Agilent 1260 LC series with a DAD detector using an Agilent Eclipse XDB-C18 column (9.4 Â 250 mm, 5 mm). Silica gel (Qing Dao Hai Yang Chemical Group Co.; were used for open column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin-layer chromatography (TLC).

Biological material
Fungal strain SCAU150 was isolated from the stony coral Acropora digitifera from Subi Reef in the South China Sea (9 39'N, 112 59'E). The strain was identified as an Aspergillus based on BLAST analysis of fungal ribosomal DNA sequence of internal transcribed spacers (GenBank accession MT597427), which was 100% similarity with that of Aspergillus fumigatus strain MEBP0074. Strain SCAU150 was deposited in the Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University.

Fermentation and extraction
Fungal strain Aspergillus sp. SCAU150 was cultured in 125 replicate 1000 mL Erlenmeyer flasks each containing 400 mL of potato dextrose broth (PDB) supplemented with 3% sea salt, under static incubation at 28 C for 28 days. At the end of the fermentation period, the culture broth was separated from the mycelium by filtration. The mycelium was extracted with acetone, while the culture filtrate was extracted with EtOAc solvent. A combined organic extract was afforded. It was noted that the fermentation and isolation work was performed as previous literatures (Liu et al. 2015;Zhao et al. 2015).

Antifungal assay
Hitherto numerous unsaturated fatty acids and related derivatives were reported to show a wide range of antimicrobial activities (Alves et al. 2020). In this work, compounds 1-7 were tested antifungal activity against six plant phytopathogenic fungi of Colletotrichum asianum HNM 408, Colletotrichum acutatum HNMRC 178, Fusarium oxysporum HNM 1003, Pyricularia oryzae HNM 1003, Fusarium solani bio-80814 and Fusarium moniliforme bio-52799 by disc diffusion method as described previously (Huang et al. 2020). The final result revealed compound 2 exhibited moderated activity toward the pathogenic fungus Fusarium solani bio-80814 with an inhibition zone diameter of 6 mm under 5 mg/disc, while carbendazim (Sigma-Aldrich) was used as a positive control showing an inhibition zone diameter of 20 mm under the same sample sizes (Supplementary material Figure S2). Even though there have been several reports regarding the mode of action of unsaturated fatty acids, the precise mechanism for the antimicrobial activity remains unclear. Recently, it was suggested that the antimicrobial activity of unsaturated fatty acids by targeting different cellular functions including protein synthesis and involving in morphogenesis, adhesion and biofilm formation, as well as membrane perturbations (Bhattacharyya et al. 2020).

Conclusion
Three new unsaturated fatty acid derivatives (1-3) and four known compounds (4-7) were isolated from the marine-derived fungus Aspergillus sp. SCAU150. Compounds 1-3 have been characterized by NMR and HRESIMS data. In addition, Compounds 1-7 were evaluated antifungal activity and 2 showed moderated activity toward the pathogenic fungus Fusarium solani bio-80814.