New Secondary Metabolites from Marine-Derived Fungus Talaromyces minnesotensis BTBU20220184

Six new compounds, talamitones A and B (1 and 2), demethyltalamitone B (3), talamiisocoumaringlycosides A and B (4 and 5), and talaminaphtholglycoside (6), together with six known compounds (7–12), were isolated from the marine-derived fungus Talaromyces minnesotensis BTBU20220184. The new structures were characterized by using HRESIMS and NMR. This is the first report of isocoumaringlycoside derivatives from a fungus of the Talaromyces genus. Compounds 5, 6, and 9 showed synergistic antibacterial activity against Staphylococcus aureus.


Introduction
The marine ecosystem is a rich pool for filamentous fungi.With the quick development in sampling using marine and molecular taxonomy technology, the fungal diversity of the marine environment has been greatly improved [1].Over 1800 species of marinederived fungi, belonging to 769 genera, have been studied, such as Aspergillus, Penicillium, Trichoderma, Eurotium, Talaromyces, and so on [2,3].The section of Talaromyces was first introduced by Stolk and Samson in 1972 [4], and over 200 species have been reported in this genus [5,6].Species of Talaromyces are commonly distributed in various environments, such as soil [6,7], marine [8], food products [9], leaf litter [10], and atmospheric environments [11].A chemical investigation of Talaromyces species revealed the potential for new bioactive chemical entries [12].A panel of new structural classes have been reported from this fungus, including ergosteroids [13,14], meroterpenoids [15], and pyridone derivatives [16,17].Nicoletti reviewed the chemical diversity of Talaromyces species from marine environments.Over 500 natural products have been identified from fungi of the Talaromyces genus, and 45% of the compounds were new structures.These compounds displayed a wide spectrum of biological activities and chemical diversity, attracting researchers to screening new pharmaceutical entries [18].
In our ongoing chemical investigation of marine-derived microorganisms, new antibacterial compounds have been identified from marine-derived Talaromyces strains [19,20].
The HMBC correlations from H 2 -8, H 2 -9, and H 3 -OMe to C-10 confirmed the presence of a C-8/C-9/C-10/OMe moiety.The HMBC correlations from H 2 -8 to C-1, C-2, and C-3; from H 2 -9 to C-2; and from H-3 ′ and H-5 ′ to C-1 ′ and C-3 revealed the connection from C-2 to C-8 and from C-3 to C-1 ′ , respectively.Therefore, the structure of 1 was assigned as shown in Figure 1 and named talamitone A.
Table 1. 1 H (500 MHz) and 13 C NMR (125 MHz) data of 1-3 (CD 3 OD).Compound 2 was isolated as a light-yellow powder.The molecular formula of 2 was determined to be C20H18O9 based on the HRESIMS spectrum (m/z [M + H] + 403.1011, calcd for C20H19O9, 403.1024), indicating twelve degrees of unsaturation (Figure S7).The analysis of the 1 H, 13 C, HSQC, and 1 H-1 H COSY spectra (Table 1, Figures S8  Compound 2 was isolated as a light-yellow powder.The molecular formula of 2 was determined to be C 20 H 18 O 9 based on the HRESIMS spectrum (m/z [M + H] + 403.1011, calcd for C 20 H 19 O 9 , 403.1024), indicating twelve degrees of unsaturation (Figure S7).The analysis of the 1 H, 13 C, HSQC, and 1 H- ; two carbonyls at δ C 201.0 (C-1) and 202.8 (C-3); as well as two carboxyls at δ C 175.5 (C-10) and δ C 168.6 (C-11).All of the above NMR data indicated that the structure of 2 is similar to that of 1.A detailed comparison of the NMR data of compounds 1 and 2 showed that the double bond C-2/C-3 was modified.The double bonds of C-2/C-3 in 1 were replaced by one sp 3 methylene (δ H 3.06, 2H, t, J = 7.0 Hz; δ C 38.8, C-2) and one carbonyl (δ C 202.8, C-3).In the HMBC spectrum (Figure 2 and Figure S12), the long-range correlation from H-5 to C-3 confirmed the carbonyl (C-3)'s bearing on C-3a.The HMBC correlations from H-3 ′ and H-5 ′ to C-3 and from H 2 -2 to C-1 and C-7a indicated that the α,β-unsaturated moiety of C-1/C-2/C-3 in 1 changed to the moiety of C-1 (carbonyl)/C-2 (methylene) and the other carbonyl of C-3 in 2. Therefore, the structure of 2 was assigned as shown in Figure 1 S13).Compound 3 showed similar NMR data to those of 2. By comparing the molecular formula and NMR data (Table 1, Figures S14 and S15), 3 was deduced to be the product of the demethylation of 2. The structure of 3 was confirmed by detailed analysis of the HSQC, 1 H-1 H COSY, and HMBC spectra (Figures S16-S18).The long-range correlation from H-5 to C-3 confirmed the carbonyl (C-3)'s bearing on C-3a.The presence of HMBC correlations from H 2 -8 and H 2 -9 to C-10 indicated the carboxyl of C-10.Therefore, the structure of 3 was assigned as shown in Figure 1 S19).The 1 H and 13 C NMR data of 4 (Table 2, Figures S20 and S21) showed signals for one isocoumarin substructure at δ C 167.9 (C-1, carboxyl), 157.9 (C-3, oxygenated), δ C 101.2 (C-4)/δ H 6.94 (s, H-4), 133.5 (C-4a), 132.9 (C-5), 161.

Biological Activity
The biological evaluations were conducted to assess the antibacterial activities against Candida albicans ATCC 10231, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25923.None of the compounds showed markable antimicrobial activities, except for 8 inhabiting the growth of S. aureus at 200 µg/mL.When combing them with 0.125 µg/mL of methicillin, 5, 6, and 9 showed synergistic antibacterial activity against S. aureus at concentrations of 25, 50, and 12.5 µg/mL, respectively.

General Experimental Procedures
Optical rotations [α] 25  D were recorded on an Anton Paar MCP 200 Modular Circular Polarimeter (Austria) in a 100 × 2 mm cell.NMR spectra were measured on a Bruker Avance 500 spectrometer with residual solvent peaks as references (CD 3 OD: δ H 3.31, δ C 49.0).High-resolution HRESIMS measurements were obtained on a Xevo G2 XS QToF system (Manchester, UK) in positive-ion mode.HPLC was performed using an Agilent 1200 Series separation module equipped with an Agilent 1200 Series diode array and Agilent 1260 Series fraction collector and an Agilent ZORBAX RX-C8 column (250 × 9.4 mm, 5 µm).

Microbial Material, Fermentation, Extraction, and Purification
The strain T. minnesotensis BTBU20220184 was isolated from a sediment sample collected from Qinzhou Bay, Guangxi Province, China.It was cultured on a malt extract agar plate at 26 • C for 4 days.The genomic DNA of T. minnesotensis BTBU20220184 were extracted using a Fungi Genomic DNA Extraction Kit (Solarbio Life Sciences, Beijing, China).The β-tubulin gene sequence region was amplified using the conventional primer pair of Bt2a (5 ′ -GGTAACCAAATCGGTGCTGCTTTC-3 ′ ) and Bt2b (5 ′ -ACCCTCAGTGTAGTGAC CCTTGGC-3 ′ ).PCR products were sent to Beijing Qingke Biotechnology Co., Ltd.(Beijing, China) for DNA sequencing.BTBU20220184 was identified as T. minnesotensis by comparing the β-tubulin gene sequence with the GenBank database using the BLAST program.A neighbor-joining (NJ) tree (Figure S38) was constructed using a software package, Mega version 5 [28].The fungus was assigned the accession number BTBU20220184 in the culture collection at Beijing Technology and Business University, Beijing.

Biological Activity
Compounds 1-12 were evaluated against S. aureus, E. coli, and C. albicans for their antimicrobial activities in 96-well plates according to the Antimicrobial Susceptibility Testing Standards outlined by the Clinical and Laboratory Standards Institute document M07-A7 (CLSI) [29] and our previous report [30,31].The MIC was defined as the minimum concentration of the compound that prevented visible growth of the microbes.The synergistic antimicrobial activities were determined by combing one-fourth of the minimum inhibitory concentration of the positive control (MIC of methicillin was 0.5 µg/mL) for S. aureus with two-fold diluted tested compounds.The synergistic antimicrobial activities were calculated based on a previous report [32].Briefly, final concentrations were preprepared from 1.5625 to 100 µg/mL of isolated compounds.Methicillin was added by a column, while the tested compounds were diluted 2-fold by row in a 96-well microtiter plate.The fractional inhibitory concentration index (FICI) was calculated as the sum of the MIC of each drug when used in combination, divided by the MIC of the drug used alone.Synergistic antibacterial activity was defined by FICI ≤ 0.5.

Table 2 .
1H (500 MHz) and13C NMR (125 MHz) data of 4-6 (CD 3 OD).Compound 5 was isolated as a light-yellow powder.The molecular formula of 5 was determined to be C 19 H 23 ClO 11 based on the HRESIMS spectrum (m/z [M + Na] + 485.0830, calcd for C 19 H 23 ClO 11 Na, 485.0821), indicating eight degrees of unsaturation (Figure and named talamiisocoumaringlycoside B. Compound 6 was isolated as a light-yellow powder.The molecular formula of 6 was determined to be C 19 H 22 O 9 based on the HRESIMS spectrum (m/z [M + H] + 395.1338, calcd for C 19 H 23 O 9 , 395.1337), indicating nine degrees of unsaturation (Figure