Secondary metabolites from the deep-sea derived fungus Aspergillus terreus MCCC M28183

Aspergillus fungi are renowned for producing a diverse range of natural products with promising biological activities. These include lovastatin, itaconic acid, terrin, and geodin, known for their cholesterol-regulating, anti-inflammatory, antitumor, and antibiotic properties. In our current study, we isolated three dimeric nitrophenyl trans-epoxyamides (1–3), along with fifteen known compounds (4–18), from the culture of Aspergillus terreus MCCC M28183, a deep-sea-derived fungus. The structures of compounds 1–3 were elucidated using a combination of NMR, MS, NMR calculation, and ECD calculation. Compound 1 exhibited moderate inhibitory activity against human gastric cancer cells MKN28, while compound 7 showed similar activity against MGC803 cells, with both showing IC50 values below 10 μM. Furthermore, compound 16 exhibited moderate potency against Vibrio parahaemolyticus ATCC 17802, with a minimum inhibitory concentration (MIC) value of 7.8 μg/mL. This promising research suggests potential avenues for developing new pharmaceuticals, particularly in targeting specific cancer cell lines and combating bacterial infections, leveraging the unique properties of these Aspergillus-derived compounds.


Introduction
Aspergillus terreus, a prolific filamentous fungus, synthesizes an array of secondary metabolites with distinct biological functions.Lovastatin, a prominent metabolite, exhibits a unique hexahydronaphthalene ring and a 2-methylbutyric acid moiety, efficaciously inhibiting HMG-CoA reductase for cholesterol regulation (Huang et al., 2021).Itaconic acid, another metabolite, serves as an industrial precursor and exhibits potential anti-inflammatory properties (Diankristanti and Ng, 2023).Terrein, with its 4H-furan-3-one framework, demonstrates multifaceted biological activities, including antitumor and anti-inflammatory effects, and inhibits melanin synthesis, suggesting therapeutic applications in hyperpigmentation (Zhao et al., 2016).The dihydroxyphenylalanine (DOPA) pigments, geodin and epi-geodin, are noted for their antibiotic activity and metal ion chelation capabilities (Boruta et al., 2021).This spectrum of metabolites, each with unique structural and pharmacological properties, underscores the importance of Aspergillus terreus in pharmaceutical and industrial 10.3389/fmicb.2024.1361550Frontiers in Microbiology 02 frontiersin.orgapplications.Ongoing research in this domain is crucial for advancing natural product chemistry and drug discovery, underscoring the significant role of fungi in producing bioactive compounds.
In exploring marine-derived bioactive compounds, a cytotoxic dihydrobenzofuran was extracted from the deep-sea fungus Aspergillus terreus CC-S06-18 (Wang et al., 2020).Following this, extensive large-scale fermentation enabled the identification and characterization of eighteen secondary metabolites.This included three novel dimeric nitrophenyl trans-epoxyamides (1-3) and an additional fifteen compounds (4-18) (Figure 1).Subsequently, each of these compounds underwent evaluation to determine their cytotoxic and antibacterial activities.
2 Materials and methods

Fungal material
The Aspergillus terreus strain, initially isolated from a seawater sample taken at a depth of 5,250 meters in the Pacific Ocean and originally labeled as CC-S06-18, has had its ITS sequence submitted to GenBank, under the sequence number MN463005.Additionally, this strain is currently preserved in the Marine Culture Collection of China (MCCC), with the preservation number M28183.

Fermentation and extraction
The fermentation process was conducted statically using two hundred 1-liter Erlenmeyer flasks, each filled with 30 grams of millet, 70 grams of rice, and 100 milliliters of seawater.Following the inoculation, the flasks were left to incubate at room temperature for 28 days.Subsequently, the product of fermentation was subjected to three rounds of extraction using ethyl acetate.The final step involved filtering and concentrating the extract, resulting in a total yield of 85 grams.

Cytotoxic activity assay
Initially, MGC803 and MKN28 cell lines were dissociated and counted.Following this, 3 × 10 3 cells from each line were seeded into each well of 96-well plates and incubated overnight to promote cell attachment.During the incubation period, the edge wells of the plate were filled with PBS.Post-attachment, the cells were exposed to increasing concentrations of the test compounds for a growth period of 72 h.Subsequently, 10 μL of CCK8 reagent was added to each well, followed by an incubation at 37°C of 1 h.The absorbance was measured at 450 nm using a microplate reader (Tecan, Morrisville, NC, United States) according to the manufacturer's instructions.The CCK8 experimental setup was conducted in triplicate.Data were analyzed with GraphPad Prism software to determine IC 50 of each compound (Wang et al., 2018(Wang et al., , 2019;;Hong et al., 2022;Lv et al., 2022).
Resazurin sodium salt was dissolved in sterile water to give a 2.0 mg/mL solution.A tested bacterial strain, during its mid-logarithmic phase and with an initial inoculum of 1 × 10 5 CFU/ mL, was introduced into plates containing serial dilutions of the test compound and a 10% resazurin solution.These plates were covered Structures of compounds 1-18.(Wang et al., 2018(Wang et al., , 2019;;Hong et al., 2022;Lv et al., 2022).

NMR and ECD calculations
Conformers were initially generated using CREST software (Grimme, 2019;Pracht et al., 2020) and subsequently underwent refinement via DFT optimization at the B3LYP/6-31G(d) level in Gaussian 16 (Frisch et al., 2016).This process targeted conformers within a 10 kcal/mol energy window.Frequency analysis was employed to verify the local minimum status of each conformer.Electronic energies were then recalculated at the more advanced M062X/6-311 + G(2d,p) level.The conformer populations were assessed using Boltzmann distribution, focusing on those that accounted for more than 2% of the total for in-depth analysis.
The GIAO method was used for the calculation of NMR shielding constants at mPW1PW91-SCRF/6-31 + G(d,p) level with tetramethylsilane (TMS) as a reference (Willoughby et al., 2014).ECD calculations were done via TDDFT at the Cam-B3LYP/6-311G(d) level, calculating 36 excited states per conformer (Pescitelli and Bruhn, 2016).Multiwfn 3.6 software was used to generate ECD curves (Lu and Chen, 2012).1).Considering the NMR data and its molecular formula, compound 1 was theorized to be a symmetrically structured dimer.
The planar configuration of compound 1 was established through a comprehensive analysis using both 1D and 2D NMR spectroscopy.The existence of a p-nitrophenyl group was initially inferred from HMBC spectral correlations, including signals from H-9/H-11 to C-7 (δ C 142.7) and C-10 (δ C 148.9), and from H-8/H-12 to C-10.These findings were further corroborated by COSY spectral correlations, specifically between H-9 and H-8, and H-11 and H-12 (Figure 2).Further, the structure was identified to contain a 2-methyl-2,3epoxyamide group, evidenced by HMBC correlations linking H-6 to C-4 and C-5, and H 3 -14 to C-4, C-5, and C-6, with the chemical shifts of C-5 (δ C 65.0) and C-6 (δ C 62.7) confirming the presence of an epoxy group (Figure 2).The isopropyl segment in the structure was pinpointed through COSY correlations from the methyl group H 3 -13 to H-1 and H-2, and its attachment to N-3 was established by HMBC correlations from H-2 to C-4 (Figure 2).As compound 1 was presumed to be a dimer, the molecular formula of its monomer was deduced to be C 14 H 16 N 2 O 5 .This monomer comprised a 2-methyl-2,3epoxyamide group, an isopropyl group, and a 1,4-disubstituted phenyl, along with a methoxy group attached to the C-1 position.Considering the remaining atomic composition of one nitrogen and two oxygens in the monomer, it was concluded that a nitro group was attached at the C-10 position.All these data led to the confirmation of subunit A in the structure, as shown in Figure 2. Additionally, the HMBC correlations between H-1 and C-4′, coupled with the chemical shifts of C-1/1′ (δ C 86.4) (Figure 2), suggested that two subunit A units are linked via two C-N bonds, forming a dimer with a central symmetrical framework, as represented in Figure 2.
Compound 2 has been established to possess the molecular formula C 26 H 28 N 4 O 10 , which was derived from HRESIMS data exhibiting a deprotonated ion peak at a m/z of 555.1711 [M − H] − .This indicated a reduction of 28 atomic mass units (amu) compared to compound 1, along with the presence of 15 degrees of unsaturation.A comparative analysis of NMR data between compounds 1 and 2 revealed that the primary variations are concentrated in the piperazine ring.Notably, the chemical shifts at C-1 and C-2 altered from δ C 86.4 to δ C 91.3, and from δ C 47.6 to δ C 58.9, respectively.The observation of 13 unique carbon resonances in the 13 C NMR spectrum, in conjunction with its molecular weight, indicates that compound 2 shared the same symmetrical framework as compound 1, as detailed in Table 1.In the NOESY spectrum, correlations between H-1,1′ and H-2,2′ in compound 2 suggested that H 3 -13/13′ and H-1/1′ were positioned on opposite sides of the piperazine ring.Furthermore, NOESY correlations between H-8,8′ and H 3 -14,14′ implied that H-6,6′ and H 3 -14,14′ were situated on different faces of the oxirane ring, paralleling the pattern observed in compound 1, as depicted in Figure 2. Considering the biosynthetic pathway, compound 2 is likely the 1,1′-epimer derivative of compound 1, characterized by a hydroxyl group attached to both C-1′ and C-1.Finally, the absolute configuration of compound 2 is designated as 1R,1'R,2S,2'S,5S,5'S,6R,6'R by ECD calculation at CAM-B3LYP/6-311G(d) (Figure 3B).
Compound 3, isolated as a white powder, had a molecular formula of C 27 H 30 N 4 O 10 .This was established from the HRESIMS data, which showed a sodium adduct ion peak at m/z 593.1859 [M + Na] + , indicating a reduction of 14 amu from 1 and 15 degrees of unsaturation.A comparison of the 1 H NMR and 13 C NMR data revealed similarities between 1 and 3.The most significant difference was the substitution of the nitrogenated methine group (δ C 86.4, C-1) in 1 with a formyl group (δ C 198.3) in 3, as detailed in Table 1.This alteration was corroborated by COSY correlations of H 3 -13/H-2/H-1 and HMBC correlations of H-1/C-13, H-2/C-1′, and H-2/C-4, as illustrated in Figure 2. The coupling constant 3 J H1' − H2 of 9.2 Hz indicated that H-1′ and H-2′ were positioned on opposite faces of the octatomic ring, which was formed by a hydrogen bond between the formyl and the amide group.In the NOESY spectrum, correlations between H 3 -13/H-1′, H 3 -13'/H-1′, and H-2/H-2′ indicated that H 3 -13, H-1′, and H 3 -13′ are located on the same side of the octatomic ring.Similarly, the positioning of H-6/6′ and H 3 -14/14′ on opposite faces of the oxirane ring was evidenced by NOESY correlations between H-8/8′ and H 3 -14/14′.Considering the biosynthetic pathway, the absolute configurations of C-2 and C-2′ were supposed to be S. Compound 3 was assigned the absolute configuration as 2S,5S,6R,1'S,2'S,5'S,6'R, which was corroborated by ECD calculations at the CAM-B3LYP/6-311G(d) level, as illustrated in Figure 3B.
The in vitro antibacterial activities of compounds 1-18 were tested against six bacterial strains.Among them, Compound 16 showed potency against Vibrio parahaemolyticus ATCC 17802, exhibiting a MIC of 7.8 μg/mL.This potency was compared to the positive control, chloramphenicol, which had an MIC of 1.1 μg/mL.

Conclusion
This study details the characterization of three new compounds (1-3) and fifteen known compounds (4-18) from the deep-sea fungus Aspergillus terreus.Compound 1 was identified as a symmetrical dimer with a complex structure, including a p-nitrophenyl moiety, a 2-methyl-2,3-epoxyamide group, and an isopropyl fragment.Its absolute configuration was determined using DFT methods and ECD spectrum analysis.2, structurally similar to 1 but differing in the piperazine ring, is considered an epimer of 1. Compound 3 shares a similar structure with 1, but with a formyl group replacing a nitrogenated methine group.The biogenetic pathways of these compounds are proposed, involving precursors like L-phenylalanine and L-alanine.The study also reports the biological activities of these compounds.Specifically, compounds 1 and 7 exhibit moderate growth inhibitory effects on gastric cancer cell lines, while compound 16 shows moderate antibacterial properties.The study emphasizes the need for extensive research on the biological
formation of intermediate a. Acylation of intermediate a with L-Alanine resulted in intermediate b.Two units of b underwent a condensation reaction by removing two molecules of water to give intermediate c, which was reduced to yield 2. Intermediate c underwent reduction and methylation to yield 1. Similarly, two units of b condensed by eliminating one molecule of water, followed by reduction and methylation steps, resulting in 3 (Figure 4).
FIGURE 3 (A) Linear regression analysis between the experimental and calculated NMR data of 1; (B) Experimental and calculated ECD spectra of 1-3.