Furanone, morpholinone and tetrahydrofuran derivatives from the marine-derived fungus Talaromyces sp. PSU-MF07

Abstract Three new compounds including one furanone, one morpholinone and one tetrahydrofuran together with three known compounds were isolated from the broth extract of the marine-derived fungus Talaromyces sp. PSU-MF07. The structures of the isolated compounds were determined on the basis of spectroscopic methods. The relative configuration was assigned using NOEDIFF data whereas the absolute configurations were established by Mosher’s method, specific rotations and electronic circular dichroism (ECD) data. Some isolated compounds were tested for antimicrobial activity. Only known penioxalicin exhibited weak antibacterial activity against methicillin-resistant Staphylococcus aureus SK1 with an MIC value of 200 µg/mL.


Introduction
The genus Talaromyces is an important fungal genus because of the production of diverse bioactive secondary metabolites, for example, anti-monoamine oxidase wortmannolol (Zhi et al. 2016), anti-influenza A viral coculnol (Nonaka et al. 2015), cytotoxic skyrin (Xie et al. 2016) and helicascolide F (Li et al. 2021b), antibacterial pinophol A (Zhao et al. 2021) and diorcinol (Li et al. 2021a), and antifungal and antibacterial penicifuran A (Ma et al. 2022). In addition, fungi in this genus are of interest because this genus is also a producer of a variety of secondary metabolites, which are important in the food products and agriculture (Zhai et al. 2016;Nicoletti and Becchimanzi 2021). The fungus Talaromyces sp. PSU-MF07 was isolated from an unidentified sponge which was collected from Koh Bulon Mai Pai, Satun Province, Thailand. Herein, we report the isolation and structure elucidation as well as biological activity of some isolated compounds.

Results and discussion
Purification of the broth extract of this fungus using chromatographic techniques led to the isolation of six compounds including three new (1-3) and three known compounds including (2 R)-2-hydroxy-3-methyl-N-(2-oxobutyl)butanamide (4), 5-(4-carboxy-3-methyl-3-buten-1-yl)octahydro-4-(methoxycarbonyl)-3-methyl-6-methylene-2-oxo-4benzofuranacetic acid (5) and penioxalicin (6) (Bian et al. 2015) (Figure 1). The structures of all metabolites were elucidated based on spectroscopic analysis. The relative configuration was assigned using NOEDIFF data whereas the absolute configurations were established by Mosher's method (for compounds 3 and 4), specific rotations (for compounds 5 and 6) and electronic circular dichroism (ECD) (for compounds 1-2 and 5-6) data. Based on the SciFinder Database, compounds 4 and 5 were isolated as natural products for the first time and previously reported without any cited references. Accordingly, their NMR spectroscopic data are reported herein. The structure of the known compound 6 was confirmed by comparing the NMR spectroscopic data (Figures S34 and S35), ECD data and specific rotation (Table S6) with those previously reported in the literature (Bian et al. 2015).
The amide 4 was obtained as a colorless gum with the molecular formula C 9 H 17 NO 3 determined by the HRESIMS peak at m/z 210.1109 [M þ Na] þ ( Figure S27). The IR spectrum showed absorption bands for hydroxy, ketone carbonyl and amide carbonyl functional groups at 3358, 1708 and 1658 cm À1 , respectively. The 1 H NMR spectrum (Table S4, Figure S22) Figure S25) of both H-7 and H-8 with C-6 (d C 174.0) as well as the chemical shifts of C-6 and C-7 (d C 76.3) constructed an N-substituted-2-hydroxy-3-methylbutanamide unit. In addition, a 1-substituted-2-butanonyl unit was established according to the 1 H-1 H COSY correlation between H 3 -1 (d H 1.11) and H 2 -2 (d H 2.51) and the HMBC correlations of H 3 -1 and H ab -4 (d H 4.23 and 4.13) with C-3 (d C 206.1). The 1 H-1 H COSY correlations of 5-NH (d H 7.29) with H ab -4 and the HMBC correlations of H ab -4 with C-6 linked 5-NH of the butanamide moiety with C-4 of the 2-butanonyl unit. The absolute configuration at C-7 in 4 was established to be R by Mosher's method ( Figure S1). Accordingly, 4 was identified as (2R)-2-hydroxy-3-methyl-N-(2-oxobutyl)butanamide.
Compound 5 was obtained as a colorless gum with the molecular formula C 20 H 26 O 8 deduced from HRESIMS peak at m/z 417.1522 [M þ Na] þ ( Figure S33). The UV and IR spectra were similar to those of 6. The 1 H ( Figure S28) and 13 C ( Figure S29) NMR spectroscopic data (Table S5) were similar to those of 6 except for the replacement of a signal for one methyl group in 6 with signals of one methyl ester group (d H 3.66, s, 3H and d C 172.1 and 52.0) in 5. The methyl ester group was located at C-4 (d C 50.1) on the basis of the HMBC correlations (Table S5, Figure S31)  .29) to those of 6 as well as a large coupling constant of 11.4 Hz between H-8 and H-9, 5 had the same relative configuration as 6. The absolute configuration of C-3 was assigned to be S as compound 5 displayed the same positive Cotton effect at 213 nm (De ¼ þ0.99, MeOH) as that of 6 (Bian et al. 2015) ( Figure S36). Consequently, the remaining absolute configurations at C-4, C-5, C-8 and C-9 were assigned as 4S, 5S, 8S and 9S. In addition, 5 gave a similar specific rotation, ½a 25 D À13.3 (c 0.32, MeOH), to that of 6 (Table S6), thus supporting their identical absolute configurations.

General experimental procedures
The infrared (IR) spectra were recorded neat using a Perkin-Elmer spectrum BX FT-IR spectrometer. The ultraviolet (UV) absorption spectra were measured in MeOH on a Shimadzu UV-2600 UV-Vis spectrophotometer. ECD spectra were recorded on a JASCO J-815 polarimeter. The 1 H and 13 C NMR spectra were recorded on a 300 or a 500 MHz Bruker FTNMR Ultra Shield TM spectrometer using tetramethylsilane (TMS) as an internal standard. The specific rotations were recorded on a JASCO P-2000 polarimeter. High resolution electrospray ionization mass spectra (HRESIMS) were obtained on a Bruker MicrOTOF mass spectrometer and Liquid chromatograph-mass spectrometer (2090, LCT, water, micromass). Thin-layer chromatography (TLC) was performed on silica gel 60 GF 254 (Merck). Column chromatography (CC) was carried out on Sephadex LH-20, silica gel (Merck) type 60 (70-230 mesh ASTM), or on reversed phase C 18 silica gel.

Fungal material
The marine-derived fungus PSU-MF07 was isolated from an unidentified sponge from Koh Bulon Mai Pai, Satun Province, Thailand. This fungus was deposited as BCC83150 at BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. The fungus PSU-MF07 was identified based on its morphological and molecular characteristics. Colonies on potato dextrose agar (PDA) at 25 C reached 3 cm in 7 days. Colony color was grey-green. Its conidiophores arised from the mycelium singly, bearing brush-like sporulating structures which are the characteristic of the genus Penicillium (Barnett and Hunter 1998). The molecular analysis of the partial large subunit (LSU) (GenBank accession number KY397999) ribosomal RNA gene showed that PSU-MF07 had close relationships with several strains of Talaromyces spp. (MH997857 and KY474346) and T. aurantiacus CBS 314.59 (MH869412) with 99.5-99.62% nucleotide identity. Therefore, this fungus can be identified as Talaromyces sp.

Antimicrobial assays
The activity was conducted using the procedure described by the Clinical and Laboratory Standards Institute (Phongpaichit et al. 2006). Vancomycin was used as a positive control against S. aureus ATCC25923 and methicillin-resistant S. aureus SK1 and displayed the MIC values of 0.5 and 1.0 mg/mL, respectively. Gentamicin, a positive control against P. aeruginosa ATCC27853 and E. coli ATCC25922, showed the MIC values of 0.25 and 0.5 mg/mL, respectively. Colistin was used as a positive control against A. baumannii NPRC005 and A. baubannii NPRC007 and gave the MIC values of 8 and 32 mg/mL, respectively. Finally, amphotericon B, a positive control against C. albicans ATCC90028, C. albicans NCPF3153, C. neoformans ATCC90112 and C. neoformans ATCC90113 flucytosine-resistant, displayed MIC values of 0.25, 0.25, 0.12 and 0.12 mg/ mL, respectively.

Conclusions
One new furanone (1), one new morpholinone (2) and one new tetrahydrofuran (3) together with three known compounds (4-6) were obtained from the marine-derived fungus Talaromyces sp. PSU-MF07. Compound 6 was previously isolated from the fungus Penicillium oxalicum TW01-1 and displayed moderate cytotoxic activity against HL-60 cell line (Bian et al. 2015). Therefore, this is the first report of the antibacterial activity of 6. In addition, the isolation of 4-6 from the genus Talaromyces has never been reported.