New Meroterpenoid and Isocoumarins from the Fungus Talaromyces amestolkiae MST1-15 Collected from Coal Area

Three new compounds including a meroterpenoid (1) and two isocoumarins (8 and 9), together with thirteen known compounds (2–7, 10–16) were isolated from the metabolites of Talaromyces amestolkiae MST1-15. Their structures were identified by a combination of spectroscopic analysis. The absolute configuration of compound 1 was elucidated on the basis of experimental and electronic circular dichroism calculation, and compounds 8 and 9 were determined by Mo2(OAc)4-induced circular dichroism experiments. Compounds 7–16 showed weak antibacterial activities against Stenotrophomonas maltophilia with MIC values ranging from 128 to 512 μg/mL (MICs of ceftriaxone sodium and levofloxacin were 128 and 0.25 μg/mL, respectively).


Introduction
The genus Talaromyces was initially established by Benjamin and over 71 species have been reported [1,2]. Secondary metabolites from Talaromyces are rich in structural diversity including anthraquinones, alkaloids, terpenes, and azaphilones, and had a wide range of bioactivities, such as MAO-inhibitory, nematicidal, cytotoxic, antiviral, and antiinflammatory activities [3][4][5]. Talaromyces amestolkiae belongs to the genus Talaromyces, species of which are found to widely inhabit plants, soil, sponges, and foods [6]. This fungus is increasingly attracting attention for its ability to produce high levels of xylanases, cellulases, active compounds, and natural colorants with potential applications in the fields of industry, medicine, and food [7,8]. Recently, a series of different structural types of compounds including meroterpenoids, isocoumarins, and benzofurans were isolated as metabolites of T. amestolkiae for activity screening [5,6,9,10].
Extremophiles have been proven to be a promising source of bioactive compounds with diverse structures since their first discovery in the mid-20th century. Most fungi that inhabit extreme environments are categorized as Ascomycota covering a range of genera, including Acremonium, Alternaria, Aspergillus, Cladosporium, and Penicillium, and have been demonstrated to produce numerous worthwhile compounds with antimicrobial, anticancer, and/or antidiabetic activities [11][12][13]. Coal areas are deemed as multi-extreme environments with high metal contents and/or extreme pH, in which fungi survive through producing extremolytes, extremozymes, as well as small useful molecules to cope with the extreme environments [14]. In our ongoing research on the discovery of bioactive compounds from fungi, a chemical study of the secondary metabolites of T. amestolkiae MST1-15 collected from the Xingren coal area was carried out. Sixteen compounds (1-16, Figure S1) including new meroterpenoid (1) and isocoumarins (8 and 9) were isolated.  To further establish the relative configuration of C-9 and C-11, the chemical shifts of four isomers (1a-1d, Figure S35) were predicted at the B3LYP/6-311+G(d,p) with the PCM solvent model. The calculated chemical shifts of isomer 1a were assigned to be in agreement with the experimental values according to the DP4+ analyses (Table S1). The absolute configuration of compound 1 was finally determined by a comparison of its experimental ECD spectrum with that calculated for the proposed structure by quantum chemical TDDFT. The predicted ECD spectrum of 7R, 9R, 11S, 12S, 22R, and 23R was in good agreement with that of the experimental one that showed a negative Cotton effect around 243 nm ( Figure 3). Therefore, the absolute configuration of 1 was established as (7R, 9R, 11S, 12S, 22R, and 23R) and was named amestolkolide E.   experimental ECD spectrum with that calculated for the proposed structure by quantum chemical TDDFT. The predicted ECD spectrum of 7R, 9R, 11S, 12S, 22R, and 23R was in good agreement with that of the experimental one that showed a negative Cotton effect around 243 nm ( Figure 3). Therefore, the absolute configuration of 1 was established as (7R, 9R, 11S, 12S, 22R, and 23R) and was named amestolkolide E.   experimental ECD spectrum with that calculated for the proposed structure by quantum chemical TDDFT. The predicted ECD spectrum of 7R, 9R, 11S, 12S, 22R, and 23R was in good agreement with that of the experimental one that showed a negative Cotton effect around 243 nm ( Figure 3). Therefore, the absolute configuration of 1 was established as (7R, 9R, 11S, 12S, 22R, and 23R) and was named amestolkolide E.   To further establish the relative configuration of C-9 and C-11, the chemical shifts of four isomers (1a-1d, Figure S35) were predicted at the B3LYP/6-311+G(d,p) with the PCM solvent model. The calculated chemical shifts of isomer 1a were assigned to be in agreement with the experimental values according to the DP4+ analyses (Table S1). The absolute configuration of compound 1 was finally determined by a comparison of its experimental ECD spectrum with that calculated for the proposed structure by quantum chemical TDDFT. The predicted ECD spectrum of 7R, 9R, 11S, 12S, 22R, and 23R was in good agreement with that of the experimental one that showed a negative Cotton effect around 243 nm ( Figure 3). Therefore, the absolute configuration of 1 was established as (7R, 9R, 11S, 12S, 22R, and 23R) and was named amestolkolide E.

General Experimental Procedures
High-resolution electrospray ionization mass spectra (HR ESIMS) were recorded on a Shimadzu LC MS-IT-TOF mass spectrometer equipped with an ESI interface. Optical rotation was determined on an Autopol VI automatic polarimeter. A Chirascan-plus CD spectrometer was employed for the UV and ECD spectra detection. IR spectrum was recorded by the FT-IR-650 spectrometer as KBr disk. Nuclear magnetic resonance (NMR) spectra were measured by a Bruker AVANCE NEO 600M NMR spectrometer ( 1 H: 600 MHz; 13 C: 150 MHz). Reversed-phase semi-preparative high-performance liquid chromatography (RP-HPLC) isolation was performed on an LC 3000 system equipped with a UV detector and a Kromasil C18 column (10 mm × 250 mm, 10 µm) with flow rate of 3 mL/min. Normal phase semi-preparative HPLC (NP-HPLC) isolation was used with an AS20005 system equipped with a UV detector and silica gel column (10 mm × 250 mm, 5 µm, Hanbon Sci & Tech (Suzhou, China), with flow rate of 3 mL/min. Sephadex LH-20 was purchased from GE healthcare company. Silica gel (200-300, 300-400 mesh) for column chromatography (CC) and silica gel GF254 (10-40 µm) for thin layer chromatography (TLC) and preparative TLC were obtained from Qingdao Haiyang Chemical Co., Ltd., Qingdao, China. All solvents were of analytical grade.

Fungal Material
Talaromyces amestolkiae MST1-15 was isolated from the soil collected in the Xingren coal area of Guizhou province in China (25 • 21 54 N, 105 • 9 9 E), in May 2020. The soil sample was suspended in sterile water and stirred for 10 min to obtain the suspension. A serial dilution method was subsequently adopted and the aliquot from each dilution was inoculated on potato dextrose agar (PDA) plates at 28 • C for 72 h. Isolates that appeared morphologically different were selected and purified, and maintained on a PDA slant stored at 4 • C. The strain was finally identified on the basis of the morphology and ITS analysis (see Supplementary Materials S1). The fungus was deposited at the Microbiological Collection Center of Guizhou Medical University (GMU-2020-MST 1-15).

Fermentation, Extraction, and Isolation
The fungus was firstly cultured in potato dextrose broth in aerobic closed conical flask shaking with 140 r/min at 28 • C for 4 days. A 10% volume (vs. rice weight) of the fungi suspension was subsequently inoculated into sterile rice medium with 0.3% peptone and cultured in a 28 • C incubator for 30 days to obtain the fermentation substance.

Antibacterial Assay
The minimum inhibitory concentrations (MICs) were evaluated according to the reported procedure [29,30] with minor modifications. Briefly, Stenotrophomonas maltophilia ATCC 13637 was cultured on 0002 medium (1% peptone, 0.3% beef extract, and 0.5% NaCl in distilled water with pH 7.0) at 30 • C and prepared for its suspension with a final concentration of 5 × 10 5 CFU/mL. Samples (1-16) were severally added into 96-well plates employing serial two-fold dilution from concentrations of 512 to 2 µg/mL. Whereafter, 100 µL bacterial suspension was transferred to the wells to incubate for 16 h at 30 • C. The final concentration of 0.25% TTC (2,3,5-triphenyl tetrazolium chloride, Sigma) in wells as microorganism growth indicator was added and the microorganism was continuously incubated for 3 h, and the concentration in well that showed no red color indicating complete growth inhibition of bacteria was determined as MIC. Equivalent amounts of DMSO (2.5%) and medium were successively used as negative and blank controls. Ceftriaxone sodium and levofloxacin were used as positive controls. Each experiment was performed in triplicate.