Mycousfurans A and B, Antibacterial Usnic Acid Congeners from the Fungus Mycosphaerella sp., Isolated from a Marine Sediment

Mycousfurans (1 and 2), two new usnic acid congeners, along with (−)-mycousnine (3), (−)-placodiolic acid (4), and (+)-usnic acid (5), were isolated using high-performance liquid chromatography-ultraviolet (HPLC-UV)-guided fractionation of extracts of Mycosphaerella sp. isolated from a marine sediment. The planar structures of 1 and 2 were elucidated using 1D and 2D NMR spectra. The relative configurations of the stereogenic carbons of 1 and 2 were established via analysis of their nuclear Overhauser spectroscopy (NOESY) spectra, and their absolute configurations were determined using a comparison of experimental and calculated electronic circular dichroism (ECD) spectra. Compounds 1 and 2 were found to have antibacterial activity, showing moderate activity against Kocuria rhizophila and Staphylococcus aureus.

Mycosphaerella is the largest genus of Ascomycota, with more than 10,000 species. Mycosphaerella species produce secondary metabolites including rosigenin [8], rubellins A and B [9], (−)-mycousnine, and (+)-isomycousnine [10]. Mycousfuranine and mycousnicdiol have also been recently reported to possess antifungal activity [11]. Mycosphaerella species are generally known as foliicolous plant pathogens, isolated from the leaves of plants; however, some species are also found in marine environments. M. ascopliylli and M. pelvetiae are endophytes of the brown algae Ascophyllum nodusum and Pelvetia canaliculate, respectively, while M. apophlaeae is the symbiont of the rhodophyte, Apophlaea lyallii [12][13][14]. In addition, there are recent taxonomy studies demonstrating that Mycosphaerella is not just a terrestrial genus but is spread across marine environments as well. For example, Mycosphaerella sp. was one of the two dominant fungal communities in samples collected from salt marshes in California Bay and the Atlantic east coast of USA [15,16].
Compound 2 was obtained as a yellow amorphous powder. Its molecular formula was determined to be C 18 H 20 O 7 based on a (+)-HRESIMS m/z 349.1305 [M + H] + and 13 C NMR data. Interpretation of the NMR data revealed that the structure of 2 was almost identical to that of 1, except that 2 possessed a methyl group at C-6 and an acetyl group at C-8 ( Figure 1). The HMBC correlations from H 3 -10 (δ H 1.66, s) to C-9a (δ C 106.1), 9-OH (δ H 9.61, s) to C-8 (δ C 107.1) and C-9a (δ C 106.1), H 3 -13 (δ H 2.73, s) to C-8, and H 3 -11 (δ H 2.02, s) to C-5a (δ C 160.7) supported the positions of the acetyl group at C-8, and consequently placed a methyl group at C-6 (δ C 100.4) ( Figures S9 and S10). Thus, 2 was defined as a regioisomer of 1.
The absolute configurations of the stereogenic carbons of 1 were established via the comparison of the experimental electronic circular dichroism (ECD) spectrum with that generated by the computer-assisted ECD calculation. The ECD spectrum of 1 fits well with that of the calculated ECD spectrum of 4aS and 9bR stereoisomers ( Figure 3). Both experimental and calculated ECD spectra of 1 showed the negative absorption in the range from 210 to 235 nm and the positive absorption from 250 to 310 nm ( Figure 3). Therefore, the absolute configurations of C-4a and C-9a in 1 was established as 4aS and 9bR. The experimental ECD spectrum of 2 was almost identical to that of 1, leading to the conclusion that the absolute configurations of C-4a and C-9a in 2 were 4aS and 9bR.

Bioactivity
Compounds 1-5 were tested for their antibacterial activity using three Gram-positive bacteria (Bacillus substilis ATCC 6633, Kocuria rhizophila ATCC 9341, Staphylococcus aureus ATCC 6538) and three Gram-negative bacteria (Escherichia coli ATCC 11775, Salmonella typhimurium ATCC 14208, Klebsiella pneumonia ATCC 4352). Compound 1 exhibited the minimal inhibitory concentration (MIC) values of 8 µg/mL and 32 µg/mL, while 2 exhibited MIC values of 16 µg/mL and 32 µg/mL, against K. rhizophila and S. aureus, respectively (Table 2). Compounds 1 and 2 showed no antibacterial activity against B. substilis and Gram-negative bacteria. Since the MIC values of 3-5 indicated stronger antibacterial activity than that of 1 and 2 against Gram-positive bacteria, it was suggested that the substituents in ring C could play a role in the antibacterial activity. acetyl group at C-8, and consequently placed a methyl group at C-6 (δC 100.4) ( Figure S9, S10). Thus, 2 was defined as a regioisomer of 1.
The absolute configurations of the stereogenic carbons of 1 were established via the comparison of the experimental electronic circular dichroism (ECD) spectrum with that generated by the computer-assisted ECD calculation. The ECD spectrum of 1 fits well with that of the calculated ECD spectrum of 4aS and 9bR stereoisomers (Figure 3). Both experimental and calculated ECD spectra of 1 showed the negative absorption in the range from 210 to 235 nm and the positive absorption from 250 to 310 nm ( Figure 3). Therefore, the absolute configurations of C-4a and C-9a in 1 was established as 4aS and 9bR. The experimental ECD spectrum of 2 was almost identical to that of 1, leading to the conclusion that the absolute configurations of C-4a and C-9a in 2 were 4aS and 9bR.

Bioactivity
Compounds 1-5 were tested for their antibacterial activity using three Gram-positive bacteria (Bacillus substilis ATCC 6633, Kocuria rhizophila ATCC 9341, Staphylococcus aureus ATCC 6538) and three Gram-negative bacteria (Escherichia coli ATCC 11775, Salmonella typhimurium ATCC 14208, Klebsiella pneumonia ATCC 4352). Compound 1 exhibited the minimal inhibitory concentration (MIC) values of 8 μg/mL and 32 μg/mL, while 2 exhibited MIC values of 16 μg/mL and 32 μg/mL, against K. rhizophila and S. aureus, respectively (Table 2). Compounds 1 and 2 showed no antibacterial activity against B. substilis and Gram-negative bacteria. Since the MIC values of 3-5 indicated stronger antibacterial activity than that of 1 and 2 against Gram-positive bacteria, it was suggested that the substituents in ring C could play a role in the antibacterial activity.   acetyl group at C-8, and consequently placed a methyl group at C-6 (δC 100.4) ( Figure S9, S10). Thus, 2 was defined as a regioisomer of 1.
The absolute configurations of the stereogenic carbons of 1 were established via the comparison of the experimental electronic circular dichroism (ECD) spectrum with that generated by the computer-assisted ECD calculation. The ECD spectrum of 1 fits well with that of the calculated ECD spectrum of 4aS and 9bR stereoisomers (Figure 3). Both experimental and calculated ECD spectra of 1 showed the negative absorption in the range from 210 to 235 nm and the positive absorption from 250 to 310 nm ( Figure 3). Therefore, the absolute configurations of C-4a and C-9a in 1 was established as 4aS and 9bR. The experimental ECD spectrum of 2 was almost identical to that of 1, leading to the conclusion that the absolute configurations of C-4a and C-9a in 2 were 4aS and 9bR.

Bioactivity
Compounds 1-5 were tested for their antibacterial activity using three Gram-positive bacteria (Bacillus substilis ATCC 6633, Kocuria rhizophila ATCC 9341, Staphylococcus aureus ATCC 6538) and three Gram-negative bacteria (Escherichia coli ATCC 11775, Salmonella typhimurium ATCC 14208, Klebsiella pneumonia ATCC 4352). Compound 1 exhibited the minimal inhibitory concentration (MIC) values of 8 μg/mL and 32 μg/mL, while 2 exhibited MIC values of 16 μg/mL and 32 μg/mL, against K. rhizophila and S. aureus, respectively (Table 2). Compounds 1 and 2 showed no antibacterial activity against B. substilis and Gram-negative bacteria. Since the MIC values of 3-5 indicated stronger antibacterial activity than that of 1 and 2 against Gram-positive bacteria, it was suggested that the substituents in ring C could play a role in the antibacterial activity.

General Experimental Procedures
Optical rotations were measured using an Autopol III (Rudolph Research Analytical, Hackettstown, NJ, USA) polarimeter with a 5-cm cell. ECD spectra were recorded using a Chirascan™-plus CD Spectrometer (Applied Photophysics Ltd., Surrey, UK) and the UV spectra were recorded on a Scinco UVS-2100 spectrophotometer (Sinco, Daejeon, Korea). IR spectra were obtained using a Scimitar 800 FT-IR spectrometer (Varian Inc., Palo Alto, CA, USA). NMR spectra were recorded on a Bruker Avance 700 MHz spectrometer (Bruker Biospin Group, Karlsruhe, Germany); The residual solvent signals of CDCl 3 (δ H 7.26, δ C 77.0) were referenced for the 1 H and 13 C chemical shift values. HRESIMS spectra were obtained using a JEOL JMS-AX505WA mass spectrometer (JEOL Ltd., Tokyo, Japan). Low-resolution LC-MS data were obtained using an Agilent Technologies 6120 quadrupole LC/MS system (Agilent Technologies, Santa Clara, CA, USA) with a reversed-phase C18 column (Phenomenex Luna C18 (2), 50 mm × 4.6 mm, 5 µm) at a flow rate of 1.0 mL/min. Column chromatography separation was performed using a C18 column (40-63 m, ZEO prep 90), eluting with a gradient of methanol and water. The fractions were purified using a WATERS TM (Milford, MA, USA) 1525 binary HPLC (high-performance liquid chromatography) pump, equipped with a WATERS 2489 UV visible detector using a WATERS reversed-phase HPLC Watchers 120 ODS-BP (250 mm × 10 mm, 5 µm) column, eluting with 80% CH 3 CN in H 2 O at flow rate of 2.5 mL/min.

Fungal Material
The strain F8015-2B was isolated from a marine sediment at a 5-m depth in Donghae-si, Gangwon-do, South Korea. The collected sediment was dried on a clean bench for 24 h and then crushed using a sterile spoon. The powder was stamped onto 1/3 marine agar medium and incubated at 27 • C. After two weeks, fungal spores were observed. The spores were cultured via repeated inoculation on potato dextrose agarplates. F8015-2B was identified as Mycosphaerella sp. based on a 99.6% (496/498) similarity of 18S rRNA genes to the Mycosphaerella nawae strain MY3.

Fermentation, Extraction, and Purifircation
The strain F8015-2B was cultured in 6 × 2.5-L Ultra Yield Flasks (Thomson Instrument Company, Oceanside, CA, USA), each containing 1 L of potato dextrose broth (PDB) dissolved in seawater. The fungus was cultivated on seed agar blocks in 6 × 2.5-L Ultra Yield Flasks, each containing 1 L of PDB dissolved in seawater at 27 • C and 140 rpm in a shaking incubator. After seven days, the mycelia were filtered from the broth using gauze filtration and extracted with acetone and methanol. The broth was extracted with EtOAc and evaporated to obtain the crude extract (4.01 g).

Computer-Assisted Conformational Analyses and ECD Calculations
Preliminary conformational analyses of 1 and 2 were performed with Merck Molecular Force Field (MMFF) by Spartan 10 (Wavefunction, Irvine, CA, USA). The two lowest energy conformers of 1 and 2 were geometrically optimized with the B3LYP/6-31G(d,p) level of density functional theory (DFT) in methanol using Gaussian 16 (Expanding the limits of computational chemistry, Wallingford, CT, USA). The computer-assisted ECD calculation was carried out with the B3LYP/6-31G(d,p) level of time-dependent density functional theory (TDDFT). The calculated ECD spectra of 1 and 2 were obtained via visualization of SpecDis version 1.71 (SpecDis, Berlin, Germany) in combination with the calculated ECD spectra of each conformer on the basis of Boltzmann distribution theory and their relative Gibbs free energy.

Antibacterial Activity
Three Gram-positive (Bacillus substilis ATCC 6633, Kocuria rhizophila ATCC 9341, Staphylococcus aureus ATCC 6538) and three Gram-negative (Escherichia coli ATCC 11775, Salmonella typhimurium ATCC 14208, Klebsiella pneumonia ATCC 4352) strains were used. These bacteria were inoculated onto a Mueller-Hinton agar medium and allowed to grow for 24 h at 37 • C. The bacterial colonies were cultivated in 15-mL round-bottom tubes containing 5 mL of Mueller-Hinton broth (MHB) at 37 • C and 220 rpm for 24 h. One hundred microliter aliquots of test compounds and positive controls (vancomycin and ampicillin) at a concentration of 256 µg/mL in DMSO were added to different wells of a 96-well microtiter plate containing 50 µL of MHB. The samples were serially diluted and 50 µL of bacterial MHB medium was adjusted to a concentration of 1/100 dilution. McFarland 0.5% standard was added to the wells. The 96-well microtiter plate was incubated for 24 h at 37 • C. Subsequently, the minimum inhibitory concentration was determined as the concentration of compounds inhibiting bacterial growth [19].

Conclusions
In conclusion, mycousfurans A and B (1 and 2) and other usnic acid congeners, were isolated from a marine sediment-derived fungus Mycosphaerella sp. The structures of 1 and 2 were established using 1D and 2D NMR spectra. The absolute configurations of the stereogenic carbons of 1 and 2 were determined using NOESY experiments and a comparison between the experimental and calculated ECD spectra. Compounds 1 and 2 exhibited antibacterial activity against K. rhizophila, S. aureus, and E. coli. The present study is the first report of the antibacterial compounds, produced by Mycosphaerella sp., which was isolated from the marine environment.

Conflicts of Interest:
The authors declare no conflict of interest.