Secondary Metabolites from Marine-Derived Fungus Penicillium rubens BTBU20213035

Two new polyketide derivatives, penirubenones A and B (1 and 2), and two naturally rare amino-bis-tetrahydrofuran derivatives, penirubenamides A and B (3 and 4), together with nine known compounds (5–13) were isolated from the marine-derived fungus Penicillium rubens BTBU20213035. The structures were identified by HRESIMS and 1D and 2D NMR analyses, and their absolute configurations were determined by a comparison of experimental and calculated electronic circular dichroism (ECD) spectroscopy and 13C NMR data. We found that 6 exhibited antibacterial activity against Staphylococcus aureus, with an MIC value of 3.125 μg/mL, and 1 and 2 showed synergistic antifungal activity against Candida albicans at 12.5 and 50 μg/mL with 0.0625 μg/mL rapamycin.


Introduction
Over the past several decades, pathogenic fungi have had a devastating impact on human health.As invasive pathogens, Candida species rank as a leading cause of healthcare-associated bloodstream infections in the United States [1][2][3].Infective diseases caused by Candida species result in a high mortality rate (40-60%), leading to an estimated 400,000 deaths globally per year [4].There are five classes of antifungal drugs currently available for treating fungal infections [5].However, with the worldwide spread of drug-resistant strains, pathogenic fungi kill about 1.5 million individuals annually [6,7].Thus, the development of novel antifungal drugs has become an urgent requirement for researchers and clinicians [8].To overcome the challenge of drug resistance in pathogenic microbes, the combination of two compounds, or synergism, has attracted increasing research interest [9,10].
Marine natural products, known for their high chemical biodiversity and potent bioactivity, have served as valuable new resources for the development of antifungal drugs [11][12][13].Nowadays, natural products isolated from marine fungi are predominant in marine natural products, with over 30% of new compounds identified from marinederived fungi [14,15].Penicillium, famed for the discovery of the first antibiotic, penicillin, continues to be an attractive microbial source for new chemical entries.New compounds with antimicrobial, anti-inflammatory, cytotoxic, and other bioactivities with potential applications in drug development have been reported [16][17][18].

Molecular Identification
The strain BTBU20213035 was isolated from a mud sample collected from the intertidal zones of Sanya, Hainan Province, China.The fungus strain was inoculated on a malt extract agar plate and cultured at 28 • C for 7 days, resulting in colonies about 10 mm in diameter.The genomic DNA of BTBU20213035 was extracted using a DNA quick Plant System (Tiangen), and the ITS sequence was amplified using the conventional primer pair of ITS4 (5 ′ -TCCTCCGCTTATTGATATGC-3 ′ ) and ITS5 (5 ′ -GGAAGTAAAAGTCGTAACAAGG-3 ′ ).PCR products were sequenced by Beijing Qingke Biotechnology Co., Ltd.(Beijing, China).The strain BTBU20213035 was identified by comparing ITS sequences with data from the GenBank database using the BLAST program.Alignments and calculations of sequence similarity were carried out using CLUSTAL W [22].The strain was deposited at Beijing Technology and Business University, Beijing, China, with the accession number BTBU20213035.

General Experimental Procedure
HR-ESI MS was measured by an Accurate-Mass-Q-TOF LC/MS 6520 instrument (Santa Clara, CA, USA).NMR spectra were recorded on a Bruker Avance 500 spectrometer (Bruker, Germany) with residual solvent peaks as references.Optical rotations were measured by using an Anton Paar MCP 200 Modular Circular polarimeter (Austria) in a 100 × 2 mm cell at 25 • C. Thin-layer chromatography silica (Qingdao Haiyang Chemical Co. Ltd., Qingdao, China) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used for column chromatography.Semipreparative HPLC was performed on an Agilent 1200 HPLC system equipped with an Agilent DAD UV-vis spectrometric detector, using a reversed-phase column (C8, 5 µm, 9.4 mm × 250 mm, Agilent, Santa Clara, CA, USA).

Fungal Materials, Cultivation, Fermentation, and Isolation
P. rubens BTBU20213035 was inoculated on a malt extract agar plate and incubated at 28 • C for 7 days.A slice of fungal colony (1 cm 2 ) was cut from the plate and put into three conical flasks (250 mL each) containing 50 mL yeast peptone dextrose medium and cultured at 28 • C (180 rpm) for 36 h.Then, 5 mL of the cultured medium was inoculated into fifteen 1 L conical flasks, each containing a sterilized medium of 120 g rice and 120 mL distilled water.The inoculated flasks were incubated in a stationary manner at 28 • C for 28 days.The fermented materials were extracted three times by EtOAc:MeOH (80:20), and the organic solvent was evaporated in vacuo at 45 • C to yield a brown crude extract.The crude extract was resuspended in 500 mL distilled water and extracted with 500 mL EtOAc (three times).Then, EtOAc was evaporated in vacuo at 45

ECD Calculation Methods
A random conformation search of starting geometries in GaussView 6 was used to produce low-energy conformers within 5 kcal/mol of energy, through which 6, 4, and 4 conformers were obtained for 1-1, 1-2, and 2, respectively.The ECD calculation was performed with the software package Gaussian 16 using the DFT method at the B3LYP/6-31G(d) level [23].TDDFT (time-dependent density functional theory) calculations of their low-energy conformations were performed using a solvent model in methanol to simulate their UV-vis spectra at the same level.Origin 9.0 was used to compare the calculated and experimental CD curves. 13C NMR calculations were carried out via the GIAO method at the same level [24].Statistical parameters, including correlation coefficient (R 2 ), mean absolute error (MAE), and maximum error (MaxErr), were used to quantify the agreement between the experimental and calculated data [25].The correlation coefficient (R 2 ) was determined from a plot of δ calc (x axis) against δ exp (y axis) for each particular compound.

Antibacterial Assay
The antibacterial activities against Escherichia coli and Staphylococcus aureus were determined in a 96-well plate using the microdilution method in LB broth medium.The compounds and the positive control were dissolved in dimethyl sulfoxide (DMSO) with nal concentrations ranging from 0.39 to 200 µg/mL using a 2-fold serial dilution method.The test strains were incubated with the diluted compounds at 37 • C for 18 h.DMSO, ciprofloxacin, and vancomycin (ranging from 0.0625 to 4 µg/mL) served as negative and positive controls, respectively.

Antifungal Assay
The assessment of antifungal activity against C. albicans was carried out in 96-well plate format using the microdilution method in RPMI 1640 medium [26].The compounds were dissolved in dimethyl sulfoxide (DMSO) at final concentrations that ranged from 0.39 to 200 µg/mL by using a 2-fold serial dilution method.The test strains were incubated with the diluted compounds at 28 • C for 18 h.DMSO and rapamycin (ranging from 0.125 to 4 µg/mL) were used as negative and positive controls, respectively.

Synergistic Antifungal Assay
Synergistic antifungal activity was determined by combining a positive control, rapamycin, with the isolated compounds at different concentrations.The compounds and rapamycin were dissolved in DMSO.The concentrations of the isolated compounds and rapamycin ranged from 0.39 to 200 µg/mL and from 0.03125 to 4 µg/mL, respectively.In the 96-well plate, the tested compounds were diluted 2-fold across columns 1 to 10, while rapamycin was diluted 2-fold across rows A to G. The fractional inhibitory concentration index (FICI) was defined as the sum of the minimal inhibitory concentration (MIC) of each tested sample used in combination divided by the MIC of the drug used alone.Synergistic activity was defined as an FICI ≤ 0.5 [27].The MIC was defined as the minimum concentration of compound at which no bacterial growth was observed.

Phylogenetic Analysis
A phylogenetic tree based on ITS rDNA sequences was constructed and is shown in Figure 1.The strain BTBU202130355 shared the highest identity (99.64%) with P. rubens CBS 129667.Phylogenetically, the strain BTBU20213035 was identified as belonging to the known strain of P. rubens.

Antifungal Assay
The assessment of antifungal activity against C. albicans was carried out in 96-well plate format using the microdilution method in RPMI 1640 medium [26].The compounds were dissolved in dimethyl sulfoxide (DMSO) at final concentrations that ranged from 0.39 to 200 µg/mL by using a 2-fold serial dilution method.The test strains were incubated with the diluted compounds at 28 °C for 18 h.DMSO and rapamycin (ranging from 0.125 to 4 µg/mL) were used as negative and positive controls, respectively.

Synergistic Antifungal Assay
Synergistic antifungal activity was determined by combining a positive control, rapamycin, with the isolated compounds at different concentrations.The compounds and rapamycin were dissolved in DMSO.The concentrations of the isolated compounds and rapamycin ranged from 0.39 to 200 µg/mL and from 0.03125 to 4 µg/mL, respectively.In the 96-well plate, the tested compounds were diluted 2-fold across columns 1 to 10, while rapamycin was diluted 2-fold across rows A to G. The fractional inhibitory concentration index (FICI) was defined as the sum of the minimal inhibitory concentration (MIC) of each tested sample used in combination divided by the MIC of the drug used alone.Synergistic activity was defined as an FICI ≤ 0.5 [27].The MIC was defined as the minimum concentration of compound at which no bacterial growth was observed.

Antibacterial Activities of the Isolated Compounds
All the isolated compounds were evaluated for their antimicrobial activities against C. albicans, S. aureus, and E. coli, as well as for synergistic antifungal activity against C. albicans.None of the compounds showed inhibitory effects on E. coli.Compound 6 displayed antibacterial activity against S. aureus, with an MIC value of 3.125 µg/mL.All the tested compounds did not show antifungal activity at a concentration of 200 µg/mL (MIC for rapamycin is 0.5 µg/mL).To explore the potential of these compounds, the synergistic antifungal activity in combination with rapamycin was determined.Compounds 1 and 2 exhibited synergistic antifungal activity against C. albicans at 12.5 and 50 µg/mL, respectively, when combined with 0.0625 µg/mL rapamycin.

Conclusions
During the chemical investigation of new chemical entries from marine-derived fungi, two new polyketide derivatives, penirubenones A and B (compounds 1 and 2), together with two naturally rare amino-bis-tetrahydrofuran derivatives, penirubenamides A and B (compounds 3 and 4) (Figure 5), were isolated from the marine-derived Penicillium rubens BTBU20213035.The planar structures and relative configurations were characterized by a detailed HRESIMS and 1 D and 2 D NMR analyses.Furthermore, the absolute configurations were determined by calculations of electronic circular dichroism (ECD) and NMR, as well as by comparison with the reported data.Compounds 1 and 2 share the carbon skeleton of ambuic acid analogues, which have been identified from marine-derived fungus of Penicillium [35].The carbon skeleton of compounds 3 and 4 was reported as a natural product for the first time, to the best of our knowledge.Other classes of compounds were also identified from this marine-derived fungus, such as secalonic acid analogue (6), andrastin-type meroterpenoids (5, 7-9), and ambuic acid analogues (10)(11)(12)(13).These results revealed the high chemical diversity of Penicillium rubens BTBU20213035.Further investigation using the one strain-many compounds (OSMAC) method will lead to the identification of more new secondary metabolites.In the antimicrobial evaluation, compound 6 inhibited the growth of S aureus, with an MIC value of 3.125 µg/mL.Compound 6 has also been reported to have cytotoxic activity against HepG2 cells [31].Our study broadened the application of the secalonic acid analogue.Compounds 1 and 2 The known compounds were identified as penimeroterpenoid A (5) [30], 2,4 ′ -linked secalonic acid (6) [31], dihydrocitreohybridonol (7) [32], 3-deacetylated andrastin A (8) [33], citreohybridonol (9) [34], penicyclone A 10) [35], penicyclone D (11) [35], penicyclone E (12) [35], and peniginsengin A (13) [36].

Antibacterial Activities of the Isolated Compounds
All the isolated compounds were evaluated for their antimicrobial activities against C. albicans, S. aureus, and E. coli, as well as for synergistic antifungal activity against C. albicans.None of the compounds showed inhibitory effects on E. coli.Compound 6 displayed antibacterial activity against S. aureus, with an MIC value of 3.125 µg/mL.All the tested compounds did not show antifungal activity at a concentration of 200 µg/mL (MIC for rapamycin is 0.5 µg/mL).To explore the potential of these compounds, the synergistic antifungal activity in combination with rapamycin was determined.Compounds 1 and 2 exhibited synergistic antifungal activity against C. albicans at 12.5 and 50 µg/mL, respectively, when combined with 0.0625 µg/mL rapamycin.

Conclusions
During the chemical investigation of new chemical entries from marine-derived fungi, two new polyketide derivatives, penirubenones A and B (compounds 1 and 2), together with two naturally rare amino-bis-tetrahydrofuran derivatives, penirubenamides A and B (compounds 3 and 4) (Figure 5), were isolated from the marine-derived Penicillium rubens BTBU20213035.The planar structures and relative configurations were characterized by a detailed HRESIMS and 1 D and 2 D NMR analyses.Furthermore, the absolute configurations were determined by calculations of electronic circular dichroism (ECD) and NMR, as well as by comparison with the reported data.Compounds 1 and 2 share the carbon skeleton of ambuic acid analogues, which have been identified from marine-derived fungus of Penicillium [35].The carbon skeleton of compounds 3 and 4 was reported as a natural product for the first time, to the best of our knowledge.Other classes of compounds were also identified from this marine-derived fungus, such as secalonic acid analogue (6), andrastin-type meroterpenoids (5, 7-9), and ambuic acid analogues (10)(11)(12)(13).These results revealed the high chemical diversity of Penicillium rubens BTBU20213035.Further investigation using the one strain-many compounds (OSMAC) method will lead to the identification of more new secondary metabolites.In the antimicrobial evaluation, compound 6 inhibited the growth of S aureus, with an MIC value of 3.125 µg/mL.Compound 6 has also been reported to have cytotoxic activity against HepG2 cells [31].Our study broadened the application of the secalonic acid analogue.Compounds 1 and 2 showed synergistic antifungal activity when combined with rapamycin.Synergisms can typically delay the development of drug resistance, so, detailed investigations on the drug resistance development of compounds 1 and 2 will be performed in future studies.showed synergistic antifungal activity when combined with rapamycin.Synergisms can typically delay the development of drug resistance, so, detailed investigations on the drug resistance development of compounds 1 and 2 will be performed in future studies.

Figure 1 .
Figure 1.Maximum likelihood analysis based on ITS sequences.Bootstrap values ≥ 75% are indicated at the nodes.The tree was rooted to A. Niger ATCC 16888.

Figure 3 .
Figure 3. Structures and experimental ECD spectra of 1 and 2.

Table 3 .
Comparison of experimental and calculated13C NMR for compound 1.

Table 3 .
Comparison of experimental and calculated 13 C NMR for compound 1.