Miniolins A–C, novel isomeric furanones induced by epigenetic manipulation of Penicillium minioluteum

Abstract

Cultivation of Penicillium minioluteum with azacitidine, a DNA methyltransferase inhibitor, led to the isolation of a novel type of aspertetronin dimer, named miniolins A–C (1–3), along with their precursor aspertetronin A (4). The structures of 1–3 were elucidated by extensive spectroscopic methods, and the absolute configurations were assigned by the chiral HPLC analysis of chemical degradation products and electronic circular dichroism associated with the TDDFT computational method (CAM-B3LYP/TZVP). The miniolins showed moderate cytotoxic activity against Hela cell lines.

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Introduction

Fungi are unique organisms capable of producing pharmaceutically useful compounds, as exemplified by penicillin, cyclosporine and lovastatin.^1,2 In the current post-genomic era, many microorganisms have been found to harbor significant numbers of biosynthetic pathways to secondary metabolites, such as Aspergillus oryzae³ and A. fumigatus.⁴ However, many natural-product-encoding gene clusters in microorganisms are silent under common culture conditions. Nevertheless, only a few of their small molecule products have been detected in the laboratory.⁵ Thus, in recent years, multiple techniques have been developed to sidestep transcriptional roadblocks and enhance product diversity via silent biosynthetic pathways. To date, chemical approaches targeting histone and DNA posttranslational processes have shown great potential for rationally directing the activation of silent gene clusters.^6,7 The epigenetic manipulation of fungal gene expression by small molecules, DNA methyl transferase and/or histone deacetylase (HDAC) inhibitors influences secondary metabolism in the fungus and is an appropriate method for exploring novel fungal metabolites prepared through cryptic biosynthetic pathways.^8–11

In our ongoing search for novel bioactives from fungi,^12–15 we found that the addition of azacitidine, a DNA methyltransferase inhibitor, to the culture medium of Penicillium minioluteum significantly enhanced the production and diversity of small molecule metabolites (Fig. S1†), leading to three new polyketidefuranones, named miniolins A, B and C (1–3), and their precursor (+)-(E,E)-(S)-aspertetronin A (4),^16,17 which was originally isolated from various Aspergillus sp.^18–20

Results and discussion

Miniolin A (1) was obtained as a colorless oil ([α]²¹_D −85.3 (c0.13, CHCl₃)). Its molecular formula (C₃₂H₄₀O₈) was deduced from HRESIMS at m/z 553.2865 [M + H]⁺ (calcd. 553.2796), indicating 13 degrees of unsaturation. The ¹³C NMR and DEPT spectra (Table 1) revealed 32 carbon signals, including eight methyls (including two oxygenated), three methylenes, ten methines (including nine unsaturated) and eleven quaternary carbons; among these, seven double bonds and four carbonyl groups are found. Apart from the eleven degrees due to the unsaturated bonds, the remaining two degrees implied that 1 possesses two rings in the molecule. Inspection of the signals in the 1D NMR and MS spectra suggested an aspertetronin A-derived dimer skeleton with a characteristic furan-3(2H)-one moiety (δ_C 192.4, 107.6, 198.0, 91.3).¹⁶

Table 1 ¹³C (125 MHz) and ¹H (500 MHz) NMR data of 1–3 in CDCl₃^a

	1		2		3
	δC	δH	δC	δH	δC	δH
a δH multi, (J in Hz).b Overlapped.
1	14.1	1.91, d (7.2)	14.2	1.91, d (7.2)	15.3	1.65, d (6.9)
2	137.8	6.29, q (7.2)	137.3	6.30, q (7.2)	130.6	5.98, m^b
3	132.6		133.0		134.3
4	192.4		192.0		191.1
5	107.6		107.7		109.2
6	198.0		197.9		198.1
7	91.3		91.4		91.4
8	125.5	5.52, d (15.4)	125.3	5.52, d (15.4)	125.3	5.51, d (15.4)
9	131.8	6.32, dd (15.4, 10.4)	132.4	6.26, dd (15.4, 10.4)	132.2	6.27, dd (15.4, 10.4)
10	127.6	5.98, dd (14.8, 10.4)	127.7	5.95, dd (15.7, 10.4)	127.6	5.96, dd (15.1, 10.4)
11	139.5	5.79, dt (14.8, 7.0)	139.7	5.80, m^b	139.7	5.78, m^b
12	25.6	2.10, m^b	25.6	2.10, m^b	25.7	2.09, m^b
13	13.2	1.00, t (7.0)	13.2	0.99, t (7.4)	13.3	0.99, t (7.2)
14	162.8		162.9		162.2
15	22.2	1.53, s	22.4	1.50, s	22.5	1.51, s
16	51.6	3.74, s	51.6	3.76, s	51.7	3.78, s
1′	19.4	1.30, d (6.9)	18.7	1.24, d (6.9)	18.9	1.18, d (6.6)
2′	31.4	3.60, m	31.2	3.56, m	36.7	3.17, m^b
3′	35.7	3.16, dd (14.2, 7.2)	36.5	3.28, dd (13.6, 7.5)	37.1	3.17, m^b
		3.46, dd (14.2, 8.5)		3.36, dd (13.6, 8.2)		3.29, dd (10.7, 7.2)
4′	196.1		196.5		196.5
5′	107.3		107.3		107.3
6′	198.6		198.6		198.4
7′	90.9		90.8		91.3
8′	125.9	5.59, d (15.4)	125.7	5.61, d (15.4)	125.4	5.59, d (15.4)
9′	131.8	6.25, dd (15.4, 10.4)	131.9	6.34, d (15.4, 10.4)	132.1	6.32, dd (15.4, 10.4)
10′	127.8	5.97, dd (15.1, 10.4)	127.9	6.01, dd (15.7, 10.4)	127.7	6.00, dd (14.8, 10.4)
11′	139.4	5.85, dt (15.1, 7.0)	139.3	5.83, m^b	139.6	5.81, m^b
12′	25.6	2.10, m^b	25.6	2.10, m^b	25.7	2.09, m^b
13′	13.2	1.00, t (7.0)	13.2	0.99, t (7.4)	13.2	0.99, t (7.2)
14′	163.2		163.1		163.1
15′	22.5	1.64, s	22.7	1.59, s	22.6	1.61, s
16′	51.5	3.79, s	51.5	3.81, s	51.6	3.80, s

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Extensive analysis of the 2D NMR spectra (HSQC, ¹H–¹H COSY, and HMBC) further established the connectivities among the substituents and the furan-3(2H)-one moieties. Four fragments (a–d, in Fig. 1) were deduced from COSY interaction pairs. Further detailed HMBC analysis confirmed the connectivities of fragments a–d and the two furan-3(2H)-one moieties. The HMBC correlations of CH₃-15/C-6, 7, 8 indicated that moiety a, H₃C-15 and the carbonyl at C-6 were connected by the oxygen-bearing quaternary carbon C-7. The HMBC correlations of H-1/C-3 and H-2/C-4 indicated the connectivity of unit b to C-4 through C-3. The HMBC correlation of CH₃-16/C-14 suggests a methyl ester attached to the central core at C-5. Similar HMBC correlations of the other monomer suggested the same fragments (c, d, and a furan-3(2H)-one moiety, Fig. 1). Furthermore, the HMBC correlations of H-1′/C-3, and H-3′/C-3 revealed that the two monomers were connected between C-2′ and C-3. These data led to the planar structure of miniolin A (1).

Fig. 1 Selected COSY and HMBC correlations of 1.

The geometry of the four double bonds on the two diene side chains was determined by NOESY correlations and coupling constants. The large vicinal coupling constants (15.4 Hz of H-8 and H-9 and H-8′ and 9′, 14.8 of H-10 and H-11, and 15.1 Hz of H-10′ and H-11′) and the NOESY correlations of H-9/H-11 and H-9′/H-11′ (Fig. 2) confirmed the all-trans geometry of the double bonds on C-8, C-10, C-8′ and C-10′. Furthermore, the NOE signals of H-1/H-2′ suggested an E-geometry of the Δ² olefin. However, the relative configurations of the three chiral centers (C-7, 2′ and 7′) in 1 could not be deduced from NOE correlations because of the high flexibility of the compound.

Fig. 2 Key NOESY correlations of 1.

HR ESIMS data of miniolins B (2) and C (3) suggested they possess the same molecular formula as miniolin A (1). The similar NMR data of all the miniolins indicated they are stereoisomeric. The 1D and 2D NMR data of 2 were very similar to those of 1 (Table 1 and Fig. 3), which indicated both compounds share the same planar structure with E-geometry for all double bonds. Compound 3 also had similar ¹H and ¹³C NMR data (Table 1) and COSY and HMBC correlations (Fig. 3) to those of 1, with only one exception, the NOESY correlations of H-2/H-2′, which indicated an Z-geometry of the Δ² olefin in 3. As with compound 1, we were unable to employ NOE data to assign stereochemistry of the chiral centers in 2 and 3 due to their high flexibilities.

Fig. 3 Selected 2D NMR correlations of 2 and 3.

The absolute configurations of the chiral centers (C-7, C-2′, and C-7′) in 1–3 were assigned by chiral HPLC analysis of their chemical degradation products and ECD spectra. To determine the chirality at C-2′ in 1–3, we performed an oxidative cleavage reaction^21,22 to remove unrelated branches as nonchiral mixed organic acids and to obtain products with retention of the stereochemistry of C-2′, as shown in Scheme 1. Unexpectedly, oxidation of 1–3 with RuO₄ (ref. 23) produced only 2′-methylsuccinic acid, which was identified by LC/ESI-MS² analysis (Fig. S2†). The absolute stereochemistry of the 2′-methylsuccinic acid was assigned by chiral HPLC analysis on the basis of comparing the t_R with those of standard S- or R-methylsuccinic acid (Fig. 4). Therefore, the absolute configuration of C-2′ in 1 was assigned as S, while that of both 2 and 3 was R. In addition, the presence of the methylsuccinic acids also supported the linkage between C-2′ and C-3 in 1–3.

Scheme 1 Oxidative cleavages of 1–3 to methylsuccinic acids.

Fig. 4 Chiral analysis of the resulting methylsuccinic acids on a CHIRALPAK AD-H chiral column.

The absolute configurations of the oxygen-bearing quaternary centers C-7 and C-7′ in 1–3 were biogenetically related to those of 4 (Scheme 2). Compound 4 showed the same signs of optical rotation ([α]²¹_D +209.8, c0.13 in CHCl₃) as those reported for both synthetic ([α]²⁰_D +166.1, c0.55 in CHCl₃ (ref. 16)) and natural ([α]_D +133, c0.30 in CHCl₃ (ref. 19)) aspertetronin A, disclosing its 7S-configuration. The configurations of C-7 and C-7′ in 1–3 were also proposed to be S, thus revealing the same chirality.

Scheme 2 Plausible biosynthetic pathway of 1–3.

The stereochemistry of 1 and 4 was further corroborated by their CD spectra. In recent years, TDDFT has become a powerful tool to provide highly precise CD curves.²⁴ Comparison of the theoretical CD curve with the experimental spectrum provides a convenient approach to identify absolute configuration for trace and non-crystalline natural products.^25–27 However, for flexible molecules, it is still difficult to obtain highly accurate results in a limited time period, since the flexibility leads to conformations that are too numerous to be favorable for calculations. In order to balance accuracy and computational time, we utilized an economical molecular force field (MMFF94S) in Conflex 6.7 (ref. 28) to acquire meaningful conformers, followed by DFT optimizations at the B3LYP/6-31G* level for 1 and B3LYP/6-31+G(d,p) for 4 in Gaussian 09.²⁹ Theoretical CD curves were calculated by the TDDFT method at the CAM-B3LYP/TZVP level and simulated using SpecDis³⁰ according to Boltzmann distributions. The resulting calculated CD spectra of (S)-4 matched well with its experimental CD spectra (Fig. 5), which provided another certification of the configuration of C-7 and C-7′ in the monomer and dimers. The similarity in the CD spectra of the dimers (1–3) indicates that the configurations of chiral C-2 do not have a great influence on the CD Cotton effects. Thus, 1 was selected to have CD calculation performed (CAM-B3LYP/TZVP) to check the entire absolute configuration assignments. Accordingly, the calculated CD curve showed a positive Cotton effect for the n–π* transition of the furan-3(2H)-one moiety and conjugated alkene around the crossover point at 265 nm, corresponding to the experimental positive Cotton effects observed around 260 nm. Thus, the entire absolute structures of 1–3 were established as (−)-(7S,2′S,7′S)-1, (−)-(7S,2′R,7′S)-2, and (−)-(7S,2′R,7′S)-3.

Fig. 5 Calculated and experimental CD spectra of 1–4.

Members of the furanone structural class of natural oxygenated heterocycles have been reported from fungi of the genera Cephalosporium (for example, gregatins and graminin A), Aspergillus (aspertetronins, huaspenones A and B),^18–20 Penicillium (penicilliol) and Paraconiothyrium (graminin B),³¹ and they show a wide range of bioactivities, such as phytotoxic³² and DNA polymerase inhibitory effects.³³ To the best of our knowledge, miniolins A–C (1–3) represent the first instances of aspertetronin dimers in nature. Particularly, it should be pointed out that the originally proposed structures of gregatins A–D and aspertetronins A and B have been revised twice by Burghart-Stoll and Brückner through total synthesis.^16,17

Biogenetically, aspertetronin A (4) is the precursor of miniolins A–C (1–3), which were envisioned to be biosynthesized via polyketide pathways. However, they were not artificial products obtained during the purification procedures, but were produced by fermentation (Fig. S1†). A plausible biogenetic pathway for miniolins A–C (1–3) is proposed in Scheme 2. The precursor aspertetronin A (4) is epoxidized by NADP⁺, followed by a ring opening to form a carbonium (ii), which undergoes an addition reaction with another molecule of 4 to generate the 2′-epi-intermediate (iii). Hydriding of iii then yields iv, followed by elimination of the 2-hydroxyl group to afford the title compounds 1–3.

The cytotoxicity of 1–4 was evaluated against Hela cell lines by SRB colorimetric assay, with etoposide as the positive control. Compounds 1–3 displayed moderate inhibition with IC₅₀ values of 33.3, 28.7, and 21.4 μM, respectively, while 4 was non-cytotoxic.

Experimental

General procedures

UV spectra were obtained using a Thermo Scientific Evolution 300 UV-vis spectrophotometer. IR spectra were recorded on a Bruker Tensor 27 spectrophotometer with KBr disks. Optical rotations were measured on a Rudolph Autopol III automatic polarimeter. CD spectra were obtained on a Chirascan CD spectrometer. ESI-MS was performed on a Thermo Fisher LTQ Fleet instrument spectrometer. HR ESIMS was performed on an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer. 1D and 2D NMR spectra were recorded on a Bruker AVANCE III (500 MHz) instrument. Chemical shifts were recorded using the solvent residual peak as the internal standard. High performance liquid chromatography (HPLC) analysis and semi-preparation was performed on a Waters 1525 instrument (Waters Corp.). Chiral separation was performed on a CHIRALPAK AD-H column. Column chromatography was performed on silica gel (90–150 μm; Qingdao Marine Chemical Inc., Qingdao, China), MCI gel (75–150 μm; Mitsubishi Chemical Corp., Tokyo, Japan), Sephadex LH-20 (40–70 μm; Amersham Pharmacia Biotech AB, Uppsala, Sweden), and Lichroprep RP-18 gel (40–63 μm; Merck, Darmstadt, Germany). GF₂₅₄ plates (Qingdao Marine Chemical Inc.) were used for thin-layer chromatography (TLC).

Fungal material, extraction, and isolation

Penicillium minioluteum was purchased from China General Microbiological Culture Collection Center (CGMCC, no. 3.5723). The fungus was incubated in potato dextrose agar (PDA) at 25 °C for 4 days. For the cultural media tests, one piece (approximately 7 mm²) of mycelium was inoculated aseptically into three 500 mL Erlenmeyer flasks containing 150 mL of potato dextrose (PD) liquid medium with 500 μM 5-azacitidine (AZA) as a chemical epigenetic modifier. The flasks were then cultured at 27 ± 0.5 °C for 10 days with shaking at 150 rpm. As a parallel control, another three flasks of medium excluding AZA were cultured under the same conditions. Ethyl acetate (EtOAc) extracts of the cultures and the controls were compared by HPLC with an Agilent TC-C18 column (250 × 4.6 mm). The results (Fig. S1†) showed that 5-azacitidine enriched the diversity of the metabolites. For the scale-up of metabolite production, a seed mycelium of the fungus was inoculated into 150 Erlenmeyer flasks containing 22.5 L of potato dextrose (PD) liquid medium in total, and cultured under the tested conditions.

Extraction, isolation and purification

The cultures of P. minioluteum were extracted with petroleum ether (PE) to yield 2.0 g of residue. The residue was fractionated by silica flash chromatography eluted with a PE-EtOAc gradient to give four fractions (Fr. 1–Fr. 4). Fr. 2 (70 mg) was subjected to PTLC with a developing solvent of CHCl₃–acetone (50 : 1) to yield compound 1 (9 mg) and a mixture (32 mg). The latter was then separated on a semi-preparative HPLC C18 column (Thermo BDS Hypersil 250 × 10 mm) and eluted with 75% MeCN to yield compounds 2 (1.9 mg) and 3 (3.2 mg). Fr. 3 was chromatographed on a Sephadex LH-20 gel column, then purified on a HPLC C18 semi-preparative column with 75% MeOH as eluent to afford 4 (8 mg).

Miniolin A (1). Colorless oil; [α]²¹_D −85.3 (c0.13, CHCl₃); UV (MeCN) λ_max (log ε) 231 nm (4.27); CD (MeCN) λ_max (Δε) 244 (−21.3), 260 (−3.0), 270 (−5.0), 318 (+3.0) nm; IR (KBr) v_max 3444, 2958, 2922, 2852, 1732, 1716, 1703, 1685, 1651, 1635, 1458, 1116 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 1; ESIMS m/z 553.00 [M + H]⁺; HRESIMS m/z 553.2865 [M + H]⁺ (calcd. for C₃₂H₄₀O₈H [M + H]⁺ 553.2796).

Miniolin B (2). Colorless oil; [α]²¹_D −103.7 (c0.1, CHCl₃); UV (MeCN) λ_max (log ε) 233 nm (3.48); CD (MeCN) λ_max (Δε) 244 (−21.3), 260 (−3.0), 270 (−5.0), 318 (+3.0) nm; IR (KBr) v_max 3446, 2956, 2922, 2850, 1716, 1651, 1579, 1560, 1440, 1384 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 1; ESIMS m/z 552.97 [M + H]⁺; HRESIMS m/z 553.2826 [M + H]⁺ (calcd. For C₃₂H₄₀O₈H [M + H]⁺ 553.2796).

Miniolin C (3). Colorless oil; [α]²¹_D −106.2 (c0.05, CHCl₃); UV (MeCN) λ_max (log ε) 195 nm (5.58); CD (MeCN) λ_max (Δε) 244 (−21.3), 260 (−3.0), 270 (−5.0), 318 (+3.0) nm; IR (KBr) v_max 3446, 2954, 2920, 2850, 1733, 1706, 1703, 1651, 1577, 1458, 1377 cm⁻¹; ¹H NMR and ¹³C NMR data, see Table 1; ESIMS m/z 552.64 [M + H]⁺; HRESIMS m/z 553.2810 [M + H]⁺ (calcd. for C₃₂H₄₀O₈H [M + H]⁺ 553.2796).

Aspertetronin A (4). Colorless oil; [α]²¹_D +209.8 (c0.13, CHCl₃); UV (MeCN) λ_max (log ε) 231 (4.09) nm; CD (MeCN) λ_max (Δε) 228 (−18.6), 251 (+7.1), 270 (+7.4), nm; IR (KBr) v_max 3423, 2962, 1743, 1706, 1645, 1438, 1132, 991, 788 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (multi, J in Hz) 2.06 (H₃-1, dd, 6.8, 1.4 Hz), 7.20 (H-2, dq, 15.7, 6.8 Hz), 7.33 (H-3, dq, 15.7, 1.4 Hz), 5.57 (H-8, d, 15.4 Hz), 6.27 (H-9, dd, 15.4, 10.3 Hz), 5.97 (H-10, dd, 15.1, 10.3 Hz), 5.80 (H-11, dt, 15.1, 6.5 Hz), 2.10 (H₂-12, dq, 7.4, 6.5 Hz), 0.99 (H₃-13, t, 7.4 Hz), 1.54 (H₃-15, s), 3.84 (H₃-16, s); ¹³C NMR (100 MHz, CDCl₃) δ (in ppm) 19.3 (C-1), 144.6 (C-2), 120.8 (C-3), 185.2 (C-4), 103.7 (C-5), 198.3 (C-6), 90.4 (C-7), 126.1 (C-8), 131.5 (C-9), 127.8 (C-10), 139.2 (C-11), 25.6 (C-12), 13.2 (C-13), 163.5 (C-14), 22.5 (C-15), 51.5 (C-16); ESIMS m/z 277.00 [M + H]⁺.

Oxidative cleavage reactions and chiral HPLC analysis

Compounds 1 (2.1 mg), 2 (1.9 mg) and 3 (1.5 mg) were, respectively, treated with 50 eq. NaIO₄ and 2.2% mol RuCl₃·xH₂O in the solvent system H₂O/CH₃CN/AcOEt (3 : 2 : 2). The mixture was stirred for 4 h at room temperature. After addition of 0.5 mL 1 N HCl for the suppression of methylsuccinic acid ionization, the mixture was extracted with water saturated n-butyl alcohol (3 × 1.0 mL). The combined organic extracts were dried over MgSO₄. The resulting products and reference methylsuccinic acid were carried forward for comparative analysis on a Thermo LCQ Fleet LC/MS with an Agilent TC-18 column (250 × 4.6 mm) and CHIRALPAK AD-H column (250 × 4.6 mm). t_R and MS/MS data were collected.

Computational method

A preliminary conformational search was performed in Conflex 6.7 using an MMFF94S force field. Conformers with 3 kcal mol⁻¹ were saved and further optimized using the Gaussian 09 software package at the B3LYP/6-31+G(d,p) level for compound 4 and the B3LYP/6-31G* level for 1. The stable conformers with distributions greater than 1% and without imaginary frequencies were submitted for ECD calculation by the TDDFT (CAM-B3LYP/TZVP) method associated with the CPCM solvent model in MeCN. The excitation energies (E), oscillator strength (f), and rotatory strength of the lowest 30 excited states were calculated. The ECD spectra of different conformers were simulated using SpecDis with a half bandwidth of 0.5 eV. The final ECD spectra were generated according to the Boltzmann-calculated distribution of each conformer.

Acknowledgements

We are grateful to Dr Shigeki Matsunaga of the University of Tokyo for kind suggestions about absolute configuration. This work is financially supported from the National Natural Science Foundation of China (no. 31371886, 21102114).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra11712c

‡ H.-Y. Tang and Q. Zhang contributed equally.

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