Phomanones A-C From Phoma sp. HDN16-618: A Mariana Trench Fungus

Abstract

Three new compounds, phomanones A-C (1-3), and a known compound, 2-hydroxymethyl-3-methylcyclopent-2-enone (4), were isolated from Phoma sp. HDN16-618, a fungus derived from a sea water sample collected from Mariana Trench. Their structures, including absolute configurations, were elucidated by extensive Nuclear Magnetic Resonance (NMR), Mass Spectrometry (MS), and electronic circular dichroism analyses. None of the compounds showed significant cytotoxic activity against a panel of cancer cell lines.

Mariana Trench, located in the western Pacific Ocean, is known as the deepest trench in the world, extending to a depth of approximately 10 920 m, corresponding to about 110 MPa of hydrostatic pressure. ^{1 -3} Due to the unique geographical location and environment, it is regarded to be a unique ecosystem, which is the habitat for deep-sea microorganisms with the ability to produce structurally diversified secondary metabolites. ⁴

Within our ongoing work of searching for new metabolites from deep-sea derived fungi, ^{5 -7} a Phoma sp. strain (HDN16-618) was obtained from a seawater sample collected from the Mariana Trench at a depth of 4000 m. Further chemical studies of this strain led to the isolation of 4 polyketides (1-4), including 3 new compounds, namely phomanones A-C (1-3) (Figure 1), among which 1 is an unusual sulfur-containing cyclopentenone. Herein, we report the isolation, structure elucidation, and bioactivity of the new compounds.

Figure 1

Structures of compounds 1 to 4.

The Phoma sp. was incubated for 60 days in a rice medium in Erlenmeyer flasks under static conditions. The whole (about 40 L) culture was then extracted with MeOH and the organic extract (28.0 g) fractionated by repeated column chromatography (CC) using silica gel and High Performance Liquid Chromatography (HPLC) octadecyl silane (ODS) columns leading to the isolation of compounds 1 (13.3 mg), 2 (14.1 mg), 3 (12.0 mg), and 4 (30.0 mg).

Phomanone A (1) was obtained as a colorless oil. Its molecular formula was established as C₈H₁₂O₂S on the basis of the protonated molecular peak at m/z 173.0628 [M+H]⁺ in its High Resolution Electrospray Ionization Mass Spectroscopy (HRESIMS), requiring 3 degrees of unsaturation. The 1D and 2D NMR data (Table 1) of 1 were similar to the known compound 4, ^8,9 revealing the existence of a 2-hydroxymethyl-3-methylcyclopent-2-enone moiety, which was further confirmed by the 1H-1H Correlation Spectroscopy (COSY) and 1H Detected Heteronuclear Multiple Bond Correlation (HMBC) correlations (Figure 2). The additional signals were assigned to a thiomethyl group moiety attached to C-4 on the basis of the key COSY correlations of H-4 (δ_H 3.88)/H₂-5(δ_H 2.81, 2.32) and the HMBC correlations from H₃-8 (δ_H 1.81) to C-4 (δ_C 45.9). Consequently, the planar structure of compound 1 was established as 2-hydroxymethyl-3-methyl-4-(methylthio)cyclopent-2-enone.

Table 1

¹H (500 MHz) and ¹³C (125 MHz) NMR Data for Compound 1 in DMSO-d ₆ .

Position	δC, type	δH (J in Hz)
1	205.1, C
2	171.8, C
3	141.8, C
4	45.9, CH	3.88, d (6.8)
5	42.3, CH2	2.32, dd (2.0,19.0)2.81, dd (6.8,19.0)
6	52.3, CH2	4.02, s
7	15.4, CH3	2.13, s
8	10.4, CH3	1.81, s

Table 2

¹H (500 MHz) and ¹³C (125 MHz) NMR Data for Compounds 2 to 3 in DMSO-d₆ .

Position	2		3
	δC, type	δH (J in Hz)	δC, type	δH (J in Hz)
1	166.5, C		166.5, C
2	90.3, CH	5.14, d (1.4)	90.2, CH	5.14, d (1.7)
3	173.6, C		173.3, C
4	32.4, CH2	2.36, m2.48, m	32.3, CH2	2.39, m2.51, m
5	75.0, CH	4.35, m	74.9, CH	4.35, m
6	29.7, CH2	2.34, m	29.6, CH2	2.42, m
7	29.6, CH2	1.86, dd (1.4, 7.7)	29.4, CH2	1.88, dd (1.4, 6.4)
8	174.3, C		173.6, C
9	56.7, CH3	3.71, s	56.7, CH3	3.70, s
10			51.9, CH3	3.58, s

Figure 2

Key ¹H-¹H COSY and HMBC correlation of compounds 1 to 3.

To determine the absolute configuration, the solution conformers and electronic circular dichroism (ECD) spectra of (4R)-1a/(4S)-1b were calculated and compared with the experimental solution ECD spectrum of 1. The Merck molecular force field (MMFF) conformational search of truncated model (4R)-1a/(4S)-1b, followed by density functional theory (DFT) optimization at the B3LYP/6-31G(d) level afforded 4 slightly different conformers above 2.0% population with 45.0%, 35.6%, 12.0%, and 5.4% populations. The Boltzmann-weighted ECD spectrum of (4R)-1a gave the best agreement (Figure 3), which led to the determination of the 4R absolute configuration of 1.

Figure 3

Experimental electronic circular dichroism spectrum of 1 (black curve), and calculated electronic circular dichroism spectrum of truncated models 1a (blue curve) and 1b (red curve).

Phomanone B (2) was obtained as a colorless oil. Its molecular formula was established as C₉H₁₂O₅ on the basis of the protonated molecular peak at m/z 201.0756 [M+H]⁺ in its HRESIMS, requiring 4 degrees of unsaturation. Careful comparison of its ¹H and ¹³C NMR data (Table 1) with those of 3-(4-methoxy-2-oxo-2H-pyran-6-yl) propanoic acid ¹⁰ indicated that they shared the same skeleton with the only difference being the absence of the C-4 double bond, which was further confirmed by the contiguous correlations of H₂-4/H-5/H₂-6 in the COSY spectrum (Figure 2). Therefore, the planar structure of 2 was established. The absolute configuration of compound 2 was also determined as 5R by ECD calculation (Figure 4).

Figure 4

Experimental electronic circular dichroism spectra of 2 (black curve) and 3 (green curve), and calculated electronic circular dichroism spectrum of truncated models 2a (red curve) and 2b (blue curve).

Phomanone C (3) was assigned with the molecular formula of C₁₀H₁₄O₅ on the basis of the HRESIMS ion at m/z 215.0911[M+H]⁺, which is 14 Da bigger than that of 2. Comparison of the 1D NMR (Table 2) with 2 revealed high similarity in the structure, except that the carboxyl group was methylated in 3, which was confirmed by the HMBC correlation from CH₃-10 (δ_H 3.58) to C-8 (δ_C 173.6) (Figure 2). The similar ECD spectra of 2 and 3 indicated that they shared the same absolute configuration.

All new compounds (1 -3) were tested for their cytotoxicity against the HL-60, K562, BEL-7402, HCT-116, A549, Hela, L-02, MGC-803, HO8910, SH-SY5Y, PC-3, U87, and MDA-MB-231 cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Sulforhodamine B assays ^11,12 with adriamycin as a positive control. However, these compounds showed no significant cytotoxicities against the tested cell lines (IC₅₀ > 30 µM).

In conclusion, 3 new compounds phomanones A-C (1-3) were isolated and identified from Phoma sp. HDN16-618, a Mariana Trench derived fungus. Notably, compound 1 possesses an unusual sulfur-containing cyclopentenone segment which has never been found in a natural product before.

Experimental

General

Specific rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a HITACHI 5430, CD spectra on a JASCO J-715 spectropolarimeter, and NMR spectra on an Agilent 500 MHz DD2 spectrometer (Agilent, Beijing, China) using tetramethylsilane as an internal standard; chemical shifts were recorded as δ values. HRESIMS were obtained using a Thermo Scientific LTQ Orbitrap XL mass spectrometer. Semipreparative HPLC was performed using an ODS column (HPLC [YMC-Pack ODS-A, 10 × 250 mm, 5 µm, 3 mL/min]).

Medium-pressure preparative liquid chromatography was conducted on a Bona-Agela CHEETAH HP100 (Beijing Agela Technologies Co., Ltd.). For CC, silica gel (100-200 mesh, 200-300 mesh, Qingdao Marine Chemical Inc.) and Sephadex LH-20 (Amersham Biosciences) columns were used.

Fungal Material

The strain HDN16-618 was isolated from a seawater sample (142°25.823′E, 11°21.136′N) collected at a depth of 4000 m from the Mariana Trench. The strain was identified by Internal Transcribed Spacers sequence and the sequence data have been submitted to GenBank (accession number: HQ914816). A voucher specimen is deposited in our laboratory at −20°C and its working stocks were prepared on potato dextrose agar slants stored at 4°C.

Fermentation, Extraction, and Isolation

Strain HDN16-618 was cultured on a PDA plate at 28°C for 5 days. The fresh mycelia and spores were inoculated into 1000 mL Erlenmeyer flasks (×10) each containing 80 g of rice and 120 mL of sea water to perform large-scale fermentation. The cultures were incubated statically at 28°C for 60 days.

The fermented broth was then extracted first with MeOH 3 times, and then with EtOAc 3 times. The organic solvent was evaporated under reduced pressure to afford an organic extract (32.0 g), which was partitioned with 90% MeOH in H₂O and then extracted with light petroleum (PE) 4 times. The MeOH layer was concentrated to provide a defatted extract (28.0 g), which was chromatographed over silica gel using gradient elution with PE-DCM-MeOH (1:1:0→ 0:5:1) to obtain 8 fractions (Fr.1-Fr.8). Fr.4 (2.1 g) was subjected to CC on ODS H₂O-MeOH (20%→100%) to obtain 8 fractions (Fr.4-1-Fr.4-8). Fr.4-5, followed by repeated CC over silica gel using a gradient of DCM-MeOH (50:0 → 5:1), yielded subfraction Fr.4-5-6. Final purification by preparative HPLC (PHPLC, H₂O-MeOH, 35%) yielded 1 (13.3 mg) and 2 (14.1 mg). Fr.5 (1.1 g) was subjected to CC on ODS, H₂O-MeOH (20%→100%), to give 8 fractions (Fr.5-1-Fr.5-8). Fr.5-1, after final purification by PHPLC (H₂O-MeOH, 20%), yielded 4 (30 mg), Fr.5-4, after repeated CC over silica gel using a gradient of DCM-MeOH (50:0 → 5:1), gave subfractions 5-4-6. Final purification by PHPLC (H₂O-MeOH, 40%) yielded 3 (12.0 mg).

Compound 1

Colorless oil.

${\displaystyle {[\alpha ]}_D^{25}}$ : 41.8 (c 0.10, MeOH).

IR (MeOH) ν _max: 3397, 2917, 1695, 1639, 1429, 1384, 1205, 1176, 1003, 676, cm⁻¹;

¹H and ¹³C NMR: Table 1.

HRESIMS: m/z 173.0628 [M+H]⁺ (calcd for C₈H₁₂O₂S, 173.0631).

Compound 2

Colorless oil.

${\displaystyle {[\alpha ]}_D^{25}}$ : −8.4 (c 0.23, MeOH).

IR (MeOH) ν _max: 2956, 2357, 1728, 1632, 1211, 841, 776 cm⁻¹.

¹H and ¹³C NMR: Table 1.

HRESIMS: m/z 201.0756 [M+H]⁺ (calcd for C₉H₁₃O₅, 201.0757).

Compound 3

Colorless crystals.

${\displaystyle {[\alpha ]}_D^{25}}$ : −25.4 (c 0.18, MeOH).

IR (MeOH) ν _max: 3096, 2933, 1715, 1621, 1470, 1380, 1090, 868, 784, 638 cm⁻¹;

¹H and ¹³C NMR: Table 1.

HRESIMS: m/z 215.0911 [M+H]⁺ (calcd for C₁₀H₁₅O₅, 215.0914).

Computation Section

Conformational searches were run by employing the “systematic” procedure implemented in Spartan’14 using the MMFF. All MMFF minima were reoptimized with DFT calculations at the B3LYP/6-31+G(d) level using the Gaussian09 program. The geometry was optimized starting from various initial conformations, with vibrational frequency calculations confirming the presence of minima. ^13,14 Time-dependent DFT calculations were performed on the 8 lowest-energy conformations for (4R)-1a and (4S)-1b and the 4 lowest-energy conformations for (5R)-2a and (5S)-2b (>5% population) using 30 excited states and using a polarizable continuum model for MeOH. Electronic circular dichroism spectra were generated using the program SpecDis by applying a Gaussian band shape with 0.30 eV width for 1a/1b and 0.35 eV for 2a/2b from dipole length rotational strengths. The dipole velocity forms yielded negligible differences. The spectra of the conformers were combined using Boltzmann weighting, with the lowest-energy conformations accounting for about 98.0% and 99.6% of the weights, respectively. The calculated spectra were shifted by 10 nm for 1a/1b and 15 nm for 2a/2b to facilitate comparison with the experimental data.