Epigenetic Modulation of Endophytic Eupenicillium sp. LG41 by a

Article pubs.acs.org/jnp

Epigenetic Modulation of Endophytic Eupenicillium sp. LG41 by a Histone Deacetylase Inhibitor for Production of Decalin-Containing Compounds Gang Li,†,⊥ Souvik Kusari,*,† Christopher Golz,‡ Hartmut Laatsch,§ Carsten Strohmann,‡ and Michael Spiteller*,† †

Institute of Environmental Research (INFU), Department of Chemistry and Chemical Biology, Chair of Environmental Chemistry and Analytical Chemistry, TU Dortmund, Otto-Hahn-Straße 6, 44221 Dortmund, Germany ‡ Inorganic Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund, Otto-Hahn-Straße 6, 44221 Dortmund, Germany § Institute for Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, D-37077 Göttingen, Germany S Supporting Information *

ABSTRACT: An endophytic fungus, Eupenicillium sp. LG41, isolated from the Chinese medicinal plant Xanthium sibiricum, was subjected to epigenetic modulation using an NAD+-dependent histone deacetylase (HDAC) inhibitor, nicotinamide. Epigenetic stimulation of the endophyte led to enhanced production of two new decalin-containing compounds, eupenicinicols C and D (3 and 4), along with two biosynthetically related known compounds, eujavanicol A (1) and eupenicinicol A (2). The structures and stereochemistry of the new compounds were elucidated by extensive spectroscopic analysis using LC-HRMS, NMR, optical rotation, and ECD calculations, as well as single-crystal X-ray diffraction. Compounds 3 and 4 exist in chemical equilibrium with two and three cis/trans isomers, respectively, as revealed by LC-MS analysis. Compound 4 was active against Staphylococcus aureus with an MIC of 0.1 μg/mL and demonstrated marked cytotoxicity against the human acute monocytic leukemia cell line (THP-1). We have shown that the HDAC inhibitor, nicotinamide, enhanced the production of compounds 3 and 4 by endophytic Eupenicillium sp. LG41, facilitating their isolation, structure elucidation, and evaluation of their biological activities.

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HDAC inhibitor, in several studies led not only to the increased production of compounds by fungi but also to the biosynthesis of new compounds that were not produced without epigenetic modification.5,7,8 In line with our continuous search for bioactive compounds produced by plant-associated endophytic microbial resources, we recently investigated an endophytic fungus, Eupenicillium sp. LG41, harbored in the roots of the Chinese medicinal plant, Xanthium sibiricum, capable of producing decalin-moietycontaining compounds.9 In order to further explore the metabolome of this endophyte, we evaluated the influence of different culture media and the addition of nicotinamide. With this approach, it was revealed that fermenting the fungus in potato dextrose broth (PDB) triggered the production of further new metabolites 3 and 4 with a decalin motif,10 albeit in

lant-associated endophytic microorganisms, particularly endophytic fungi, are well-known producers of diverse secondary metabolites encompassing many chemical scaffolds with intriguing biological activities.1,2 Whole-genome sequencing data of several fungal endophytes illustrate the diverse biosynthetic pathways in these organisms that remain cryptic or unexpressed under laboratory conditions,2 and only a minor portion of the unexplored repertoire of biosynthetic potential of fungal endophytes is expressed under standard in vitro culture conditions.3 One successful strategy explored in recent years to overcome (or bypass) the aforementioned bottleneck has been modulation of the fungal epigenome or chromatin remodeling in order to activate target and nontarget gene clusters.3,4 In particular, the use of small-molecule epigenetic modulators that inhibit endophytic fungal histone deacetylase (HDAC) and DNA methyl transferase has led to discovery of new and/or formerly unexpressed secondary metabolites.5−7 For example, amending media with nicotinamide, an NAD+-dependent © 2017 American Chemical Society and American Society of Pharmacognosy

Received: November 2, 2016 Published: March 23, 2017 983

DOI: 10.1021/acs.jnatprod.6b00997 J. Nat. Prod. 2017, 80, 983−988

Journal of Natural Products

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assigned by ESI-HRMS, which indicated that a kinetic equilibrium between two isomers was strongly favored. The MS2 spectrum (Figure S17) of 3 displayed the characteristic fragment ion at m/z 217.1949, also found in the co-occurring compounds 1 and 2 (Figures S4 and S6).8 The above MS data indicated a preferred cleavage of the side chain located at C-1 and the loss of two water molecules in 3 to afford a nitrogenfree fragment [M + H − C4H8O4N2]+. Further detailed analysis of the MS2 spectrum (Figure S17) supported the presence of a urea moiety in the above side chain from fragment ions at m/z 348.2168 [M + H − NH3]+, 322.2381 [M + H − CHON]+, and 305.2114 [M + H − CH4ON2]+, and two hydroxy groups from further ions at m/z 287.2006 [M + H − CH4ON2 − H2O]+ and 269.1900 [M + H − CH4ON2 − 2H2O]+. The 1D NMR data of 3 (Table 1 and Figures S10 and S11) exhibited structural features similar to those of 1 and 2, except for the significant chemical shift differences due to the side chain (Figures S1, S2, S7, and S8). The structure of the side chain in 3 was derived from the 1H−1H COSY data and HMBC correlations of 1-Me with C-9, and H-10 and H-11 with C-9, together with key HMBC correlations from H-11 (δH 7.38) to C-12 (δC 157.5) and the chemical shifts of C-11 (δC 140.7) and C-12, locating the urea moiety at C-11 (Figure 1). Specifically, the chemical equilibrium between isomeric forms of 3 as suggested by LC-MS was further confirmed by the 1H NMR spectrum with the aid of 1H−1H COSY data. The coupling constants between H-10 and H-11 supported a mixture of Δ10 cis (J = 9.0 Hz) and trans (J = 13.5 Hz) double bond isomers (Table 1 and Figures S10 and S12). A hydrogen bond between the carbonyl group at C-9 and the urea nitrogen could stabilize the dominant Z-configuration. The relative configuration of 3 should be the same as that of 1 and 2 based on the NOESY correlations of Me-1/H-2, H-4a, H-8 and H-10, H-8a/H-2′b and H-5, and H-6/H-5 and H-7β (Figure S15). Finally, a single-crystal diffraction analysis further confirmed the gross structure and relative configuration of 3 (Figure 2). It has to be noted that the crystallization delivered the pure Z-isomer while a mixture of two diastereomers was present in solution. Compound 3 was named eupenicinicol C. Compound 4 was obtained as a white powder and named as eupenicinicol D. Similar to 3, it exhibited three nonisolated diastereomers on LC-HRMS measurements (Figure S19), which were also in an equilibrium as eupenicinicol C (3). In order to obtain clear 1D NMR data of 4, several NMR solvents were evaluated (for a detailed discussion, see Supporting Information). Deuterated chloroform was finally selected for NMR measurements of 4, after equilibration in pyridine-d5. Under these conditions, the mixture was dominated by only one isomer. The 1D NMR (Table 1) and HSQC data (Figure S25) suggested the presence of four methyls, two methylenes, seven methines (two oxygenated), two cis-disubstituted double bonds, and two quaternary carbons, accounting for 27 carbonconnected protons and 19 13C signals. A dimeric structure for 4 was constructed based on the molecular formula. The above analysis showed structural features very similar to those of 1−3, except for significant differences in the side chain. The latter was assembled by 1H−1H COSY correlations of H-10/H-11 and the HMBC correlations from Me-1 to C-1 and C-9, from H-10 to C-9 (Figure 1). The coupling constant of H-10/H-11 (J = 8.4 Hz) revealed a Z-configured double bond (Table 1). The key 1H−1H COSY correlation between H-11 and NH and MS requirements indicated the connection of two monomers

low amounts. However, PDB media amended with the HDAC inhibitor led to an increased production of the new compounds.

Herein, we report the isolation and structure elucidation of two new decalin-containing compounds, eupenicinicols C and D (3 and 4), along with two biosynthetically related known metabolites, eujavanicol A (1) and eupenicinicol A (2) from Eupenicillium sp. LG41.9,11 The equilibrium between cis/trans isomers found in eupenicinicols C and D (3 and 4) was further investigated. Following up on our previous work on the antibacterial activities of decalin-containing compounds,9 we used Gram-positive and Gram-negative bacteria, both environmental strains as well as risk group 2 (RG2) pathogenic strains, to evaluate the activity of the isolated compounds. The in vitro cytotoxicity of 4 against the human acute monocytic leukemia cell line (THP-1) was evaluated using a resazurin-based test and an ATPlite assay.

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RESULTS AND DISCUSSION Compounds 1 and 2 were isolated as white powder and identified as known decalin-containing compounds with a characteristic fragment ion at m/z 217.19 related to the cleavage of the side chain and the loss of two water molecules in the MS2 spectrum (Figures S1−S8).9−11 Notably, the MS2 pattern exhibited by compounds 1 and 2 should be useful in guiding the isolation of similar decalins and for evaluating their antibacterial efficacies. Preparative HPLC and LC-HRMS of compound 3 displayed peaks of two separated isomers (Figure S9). Each individual isomer changed back to two signals on reinjection, giving a pattern identical to the original chromatographic profile. Both peaks shared the same molecular formula (C20H32O4N2) as

Figure 1. Key HMBC and 1 H− 1 H COSY correlations of eupenicinicols C and D (3 and 4). 984



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Table 1. NMR Data for Eupenicinicols C (3, in CD3OD) and D (4, in CDCl3) 3 position

δC, mult.a

1 2 3 4 4a 5 6 7α 7β 8 8a 9 10

52.8, Cq 54.4, CH 125.5, CH 128.7, CH 40.2, CH 76.7, CH 70.9, CH 42.9, CH2

11

140.7, CHf,g

12 1′ 2′a 2′b 3′ 1-Me 1′-Me 8-Me NH

157.5, Cq 37.7, CH 26.5, CH2

31.2, CH 44.1, CHf 208.2, Cq 99.6, CHf,g

13.0, 20.4, 20.0, 22.2,

CH3 CH3 CH3 CH3

4 δH mult.b (J in Hz) 1.89 5.72 6.05 2.17 3.32 3.94 1.78 1.43 1.76 1.92

me br d (10.0) br d (10.0) br t (10.5) me m m br t (12.0) m me

5.79 6.23 7.38 7.95

d d d d

1.32 0.78 1.58 0.78 1.26 0.90 0.66

m me m me s d (7.0) d (6.5)

(9.0)g (13.5)h (9.0)g (13.5)h

δC, mult.c 51.3, Cq 53.3, CH 125.4, CH 126.4, CH 39.5, CH 75.7, CH 70.0, CH 41.3, CH2 30.5, CH 43.3, CH 203.5, Cq 99.2, CH

δH mult.d (J in Hz) 1.84 5.74 5.99 2.12 3.46 4.03 1.81 1.56 1.70 2.12

me m d (10.2) br t (9.0) dd (3.0, 10.2) m br d (13.8) me m me

5.66 d (8.4)

144.6, CH

6.58 t (8.4)

36.8, CH 25.3, CH2

1.27 me 0.72 me 1.59 me 0.73 me 1.24 s 0.86 d (6.6) 0.65 d (6.0) 13.35 br s

12.9, 20.6, 19.9, 21.7,

CH3 CH3 CH3 CH3

a

Recorded at 125 MHz; 13C multiplicities were determined by HSQC experiment. bRecorded at 500 MHz. cRecorded at 150 MHz; 13C multiplicities were determined by HSQC experiment. dRecorded at 600 MHz. eSignals overlapped. fProposed chemical shifts by HMBC or HSQC correlations. gObserved from the Δ10-Z-configuration (around 80%). hObserved from the Δ10-E-configuration (around 20%).

Figure 3. Key NOESY correlations of eupenicinicol D (4).

2, H-4a, H-8 and H-10, H-8a/H-2′b and H-5, and H-6/H-5, which was consistent with that of compounds 1−3. Similar to 3, a mixture of three isomers of 4 was observed in LC-HRMS measurements and NMR data using several NMR solvents, such as acetone-d6, CD3OD, and DMSO-d6 (Figures S19, S22, and S23). Detailed analysis of NMR data in acetoned6 indicated this phenomenon was derived from the cis- or trans-configured double bonds in the bridge (Figure 4). Two hydrogen bonds between the carbonyl group at C-9 and NH group should play a very important role for this equilibrium, especially for the stability of the cis isomer. To determine the absolute configurations of 3 and 4, CD calculations were performed as previously described for other cases (Figures S36−S51 in the Supporting Information; also see the Experimental Section).9,12

Figure 2. ORTEP drawing of eupenicinicol C (3) with 50% probability ellipsoids.

through a nitrogen linkage, which is further supported by the MS2 data with abundant fragments in the mass range 250−400 Da (Figure S30). The planar structure of 4 was confirmed as depicted. The relative configuration of eupenicinicol D (4) was determined by the NOESY correlations (Figure 3) of Me-1/H985



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Figure 4. Partial 1H−1H COSY spectrum (in acetone-d6) of eupenicinicol D (4). The coupling constants of protons at double bonds are included. Observed 1H−1H COSY correlations (bold) and key HMBC correlations (arrow) are shown in the structures of three isomers (structures A, B, and C in a kinetic equilibrium).

Figure 5. Proposed biosynthetic pathway of eujavanicol A (1) and eupenicinicols A (2), C (3), and D (4).

ketoaldehyde, it could react with urea to 3 (Figure 5)13 or in the same way with ammonia to deliver the respective amine, which adds to a second molecule of 2, thereby affording 4. In fact, in a control experiment, addition of aqueous ammonia to compound 2 yielded compound 4 when incubated for 12 h at room temperature, even though the in vitro conversion was

Compounds 1−4 are decalin-derived polyketides with a double bond between C-3 and C-4.10 An intramolecular Diels− Alder (IMDA) cycloaddition could be responsible for the formation of the decalin motif (Figure 5).10 Eupenicinicol A (2) should be a key intermediate in the formation of further decalins, including 3 and 4. As 2 is the enol form of a 3986



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(D21−D24). Fraction D22 was purified by semipreparative HPLC (MeOH−H2O; 1−20 min 55/45, 20−40 min 75/25; 2.0 mL/min) to afford eupenicinicol A [2, 1.2 mg, tR = 10.0 min; TLC, Rf ∼ 0.6, CH2Cl2−MeOH (20:1); gray on heating with H2SO4/EtOH (1:9, v/ v)]. Fraction D3, following the same procedure as that used for fraction D2, gave eujavanicol A [1, 24.0 mg; HPLC, MeOH−H2O, 40/ 60, 2.0 mL/min, tR = 19.4 min; TLC, Rf = 0.6, CH2Cl2−MeOH (20:1); gray on heating with H2SO4/EtOH (1:9, v/v)]. Fraction E (0.27 g) was subjected to silica gel CC [cyclohexane−EtOAc (100:1 to 1:1) and then CH2Cl2−MeOH (100:0 to 0:100)] to obtain six subfractions (E1−E6). Fractions E3 and E4 were combined and then separated using Sephadex LH-20 CC (MeOH) to give four subfractions (E31−E34). Fraction E32 was purified by HPLC to give eupenicinicol D [4, MeOH−H2O, 80/20, 3.0 mL/min, 3.0 mg (17.0 mg more was obtained under optimized fermentation conditions by using PDB medium spiked with nicotinamide, i.e., 6 times the original amount in PDB medium), tR = 11.4, 14.4, and 17.0 min; TLC, Rf = 0.45 and 0.48, CH2Cl2−MeOH (20:1); two main spots from yellow to gray staining on heating with H2SO4/EtOH (1:9, v/v)]. Fraction E33 was purified by HPLC to yield eupenicinicol C [3, 1.5 mg; HPLC, MeOH−H2O, 50/50, 2.0 mL/min, tR = 13.1 and 19.7 min; TLC, Rf = 0.46, CH2Cl2−MeOH (20:1); from yellow to gray staining on heating with H2SO4/EtOH (1:9, v/v)]. Eupenicinicol C (3): white powder; [α]D20 +87.3 (c 0.15, MeOH); LC-UV [(acetonitrile (aq) in H2O/0.1% formic acid)] λmax 222, 292 nm; IR (film) vmax 3333, 2957, 2918, 2849, 1645, 1568 cm−1; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz), see Table 1; (+)-ESI-HRMS m/z 365.2438 [M + H]+ (calcd for C20H33O4N2, 365.2435, Δ0.7864 ppm). Eupenicinicol D (4): white powder; [α]D20 +37.3 (c 0.60, CHCl3); LC-UV [(acetonitrile (aq) in H2O/0.1% formic acid)] λmax 218, 358 nm; IR (film) vmax 3376, 2956, 2918, 2849, 1644, 1577, 1465, 1061 cm−1; CD (CHCl3) 245 (Δε +0.86), 261 (Δε −0.78), 278 (Δε +0.99), 295 (Δε +0.46), 326 (Δε +1.10), 349 (Δε −0.05), 355 (Δε +0.22), 366 (Δε −0.23), 382 (Δε +0.91) nm; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Table 1; (+)-ESIHRMS m/z 626.4427 [M + H]+ (calcd for C38H60O6N, 626.4415, Δ1.9340 ppm). X-ray Crystallographic Analysis of 3. Colorless crystals were obtained from an acetone solution. The single-crystal X-ray diffraction experiment was performed on an Oxford diffraction Xcalibur Sapphire3 diffractometer at 150 K with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). With the aid of Olex2,14 the crystal structure was solved with the olex2.solve15 structure solution program using charge flipping. The structure was refined with the SHELXL16 refinement program with the least squares minimization. Molecular graphics were accomplished with Ortep-3 (for Windows, version 2014.1).17 Crystal Data of Eupenicinicol C (3): crystal size 0.15 × 0.10 × 0.05 mm3, orthorhombic crystal system, space group P212121, a = 9.0781(4) Å, b = 9.8882(5) Å, c = 26.0188(13) Å, V = 2335.60(19) Å3; Z = 4, T = 150 K, μ(Mo Kα) = 0.084 mm−1, Dcalc = 1.202 g/cm3; 20 307 reflections measured with 2θ ranged from 4.406 to 51.994°; 4579 unique reflections (Rint = 0.0483, Rsigma = 0.0396); R1 = 0.0446, wR2 = 0.1041 (all data). Crystallographic data of eupenicinicol C (3) were deposited in the Cambridge Crystallographic Data Centre with supplementary publication number CCDC 1440259. Copies of the data can be acquired, free of charge, from Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK [fax: + 44(0)1223 336033 or e-mail: [email protected]]. Antibacterial Assay and Cytotoxicity Assay. Compounds were evaluated for antibacterial activities in vitro against five environmental and/or pathogenic strains (DSMZ, Braunschweig, Germany) using the previously described method.9 Streptomycin and gentamicin were used as antibiotic references. The in vitro cytotoxicity of compound 4 against the human acute monocytic leukemia cell line (THP-1) was evaluated using a resazurin-based assay and an ATPlite assay. The experimental details were reported earlier.12,18 Computational Details. The Merck molecular force field program (MMFF, SPARTAN’1419) was used to analyze the conformer

found to be slow. This lends support to our proposed aforementioned biosynthetic pathway and agrees with their different production times, vis-à-vis the growth of the fungus, that is, less than 10 days for compounds 1 and 2 compared to around 20 days for compounds 3 or 4 (Figures S33 and S34). Based on our previous results that altering the substitution at C-11 for this type of decalin-containing secondary metabolites could drastically increase the antibacterial activity against clinically relevant Staphylococcus aureus,9 this Gram-positive bacterium was reused in the present study to further test this hypothesis, and four more bacteria were selected (Table S1, Supporting Information). Compound 4 exhibited pronounced efficacy against S. aureus with an MIC of 0.1 μg/mL, and compound 1 was active against Escherichia coli with an MIC of 5.0 μg/mL (Table S1, Supporting Information). Comparison of 4 with 2 and 39 confirmed that the substitution at C-11 plays an important role in increasing the antibacterial activity against the selected bacterium. Additionally, compound 4 demonstrated cytotoxicity (IC50 = 8.0 μM) in vitro against THP-1 cells (Figure S35).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were performed using an A-Krüss Optronic P8000-T polarimeter. IR data were collected through a Bruker Tensor 27 IR spectrometer. CD spectra were recorded on a Jasco J-715 spectrometer. The NMR spectra were measured on a Bruker Avance DRX 400, Bruker Avance DRX 500, or Varian Unity Inova 600 spectrometer. LC-ESI-HRMSn experiments were carried out on a LTQ-Orbitrap spectrometer (Thermo Fisher, USA) equipped with an Agilent 1200 HPLC system. The semipreparative HPLC system was a Gynkotek pump equipped with a Dionex DG-1210 degasser, a Dionex UVD 340S detector, a Dionex Gina 50 autosampler, and a Gemini column (10 × 250 mm, 10 μm). For column chromatography (CC), silica gel 60 (70−230 mesh; AppliChem, GmbH, Darmstadt, Germany) and Sephadex LH-20 (25− 100 μm; Amersham Biosciences) were used. Thin-layer chromatography (TLC) was performed with glass precoated silica gel 60 plates (0.25 mm; Merck, Darmstadt, Germany). Compounds on TLC were visualized under UV light and by spraying with H2SO4/EtOH (1:9, v/ v) followed by heating. Fungal Material. The isolation, identification, and characterization of the endophytic fungus were described earlier.9 This fungal strain was cultured on PDA medium at 28 ± 2 °C for 1 week. Agar plugs were cut into small pieces, and 40 pieces were selected to inoculate 40 Erlenmeyer flasks (500 mL) each containing 250 mL of PDB. The cultures were incubated at 28 °C on a rotary shaker (150 rpm) for 28 days. The endophytic fungus was further fermented in 35 Erlenmeyer flasks (500 mL) each containing 250 mL of PDB supplemented with 15 mg/100 mL of nicotinamide. The cultures were incubated at 28 °C on a rotary shaker (150 rpm) for 28 days. Extraction and Isolation. The culture medium was filtered to remove mycelia. The filtrate was dried under vacuum to afford a dark brown residue. The residue was suspended in H2O and then partitioned with EtOAc to give an EtOAc layer. The mycelia were extracted with EtOAc (3 × 1 L) with sonication. The above EtOAc layer and extract were combined and concentrated to dryness to yield 6.0 g of crude extract. The extract was subjected to CC on silica gel eluting with a gradient of cyclohexane−EtOAc from 1:0 to 1:1 (v/v) and then CH2Cl2−MeOH from 1:0 to 0:1 (v/v) to obtain nine fractions. All of these nine fractions were subjected to LC-HRMS measurement and combined to give six fractions (A−F). All fractions containing metabolites with a similar MSn pattern as that seen for eupenicinicol A (2) were further separated.9 Fraction D (0.91 g) was chromatographed on a silica gel column eluting with cyclohexane− EtOAc (100:1 to 1:1) and then CH2Cl2−MeOH (100:0 to 0:100) to afford five subfractions (D1−D5). Fraction D2 was further separated using Sephadex LH-20 CC (MeOH) to obtain four subfractions 987



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distribution for compound 3 in a systematic approach, and 15,552 geometries were calculated. With SPARTAN’14,19 a Monte Carlo approach and additionally a semisystematic approach were used for compound 4, as more than 900 million starting geometries were needed for the fully systematic search. After degenerated conformers with identical energies were removed, further geometry optimizations with HF/3-21G delivered the lowest 13 conformers for 3 and 12 for 4 in an energy range of 40 kJ/mol above the global minimum. The results were further optimized within a window of 20 kJ/mol using wB97X-D/6-31G* [= wB97X-D/6-31G(d)]. On basis of the geometries thereof, we obtained the energies, the calculated CD spectra [using the RB3LYP functional and the 6-311G(2d,p) basis set], and for compound 4 the energies also with wB97X-D/6-311+G(2df,2p) for more accurate Boltzmann factors. The experimental CD spectrum fitted nicely with the data calculated for compound 3 (Figure S36, Supporting Information), whereas the CD calculation for compound 4 was much more complicated. Based on the detailed CD calculations and careful analysis (Figures S38−S51, Supporting Information), it follows that (Z,Z)-4 (cis,cis-4) must exist in a (nearly) flat conformation in chloroform, in contrast to the calculations for vacuum.

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(INFU, TU Dortmund) for technical assistance with LC-MS measurements.

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DEDICATION This paper is dedicated to Prof. Dr. Dr. h. c. mult. Gerhard Bringmann on the occasion of his 65th birthday.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00997. Spectral data, proposed biosynthetic pathway of compounds 1−4, biological assay results, a detailed discussion of the CD calculations, and significance of the present work (PDF)

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REFERENCES

(1) Aly, A. H.; Debbab, A.; Proksch, P. Die Pharmazie 2013, 68, 499−505. (2) Kusari, S.; Hertweck, C.; Spiteller, M. Chem. Biol. 2012, 19, 792− 798. (3) Cichewicz, R. H. Nat. Prod. Rep. 2010, 27, 11−22. (4) Felsenfeld, G.; Groudine, M. Nature 2003, 421, 448−453. (5) Mao, X. M.; Xu, W.; Li, D.; Yin, W. B.; Chooi, Y. H.; Li, Y. Q.; Tang, Y.; Hu, Y. Angew. Chem., Int. Ed. 2015, 54, 7592−7596. (6) Albright, J. C.; Henke, M. T.; Soukup, A. A.; McClure, R. A.; Thomson, R. J.; Keller, N. P.; Kelleher, N. L. ACS Chem. Biol. 2015, 10, 1535−1541. (7) Asai, T.; Morita, S.; Taniguchi, T.; Monde, K.; Oshima, Y. Org. Biomol. Chem. 2016, 14, 646−651. (8) Asai, T.; Luo, D.; Yamashita, K.; Oshima, Y. Org. Lett. 2013, 15, 1020−1023. (9) Li, G.; Kusari, S.; Lamshöft, M.; Schüffler, A.; Laatsch, H.; Spiteller, M. J. Nat. Prod. 2014, 77, 2335−2341. (10) Li, G.; Kusari, S.; Spiteller, M. Nat. Prod. Rep. 2014, 31, 1175− 1201. (11) Nakadate, S.; Nozawa, K.; Horie, H.; Fujii, Y.; Nagai, M.; Hosoe, T.; Kawai, K.; Yaguchi, T.; Fukushima, K. J. Nat. Prod. 2007, 70, 1510−1512. (12) Wang, W.-X.; Kusari, S.; Laatsch, H.; Golz, C.; Kusari, P.; Strohmann, C.; Kayser, O.; Spiteller, M. J. Nat. Prod. 2016, 79, 704− 710. (13) Ding, L.; Fotso, S.; Li, F.; Qin, S.; Laatsch, H. J. Nat. Prod. 2008, 71, 1068−1069. (14) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H. J. Appl. Crystallogr. 2009, 42, 339−341. (15) Bourhis, L. J.; Dolomanov, O. V.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H. Acta Crystallogr., Sect. A: Found. Adv. 2015, A71, 59− 75. (16) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, A64, 112−122. (17) Farrugia, L. J. J. Appl. Crystallogr. 2012, 45, 849−854. (18) Kusari, S.; Zühlke, S.; Košuth, J.; Č ellárová, E.; Spiteller, M. J. Nat. Prod. 2009, 72, 1825−1835. (19) SPARTAN’14; Wavefunction, Inc.: Irvine, CA, 2014.

AUTHOR INFORMATION

Corresponding Authors

*Tel: +49-(0)231-755-4086. Fax: +49-(0)231-755-4084. Email: [email protected] (S. Kusari). *Tel: +49-(0)231-755-4080. Fax: +49-(0)231-755-4085. Email: [email protected] (M. Spiteller). ORCID

Souvik Kusari: 0000-0002-4685-0794 Carsten Strohmann: 0000-0002-4787-2135 Present Address ⊥

Department of Medicinal Chemistry, Qingdao University School of Pharmacy, Qingdao 266021, Shandong Province, China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS G.L. gratefully acknowledges the China Scholarship Council (CSC) for a doctoral fellowship. We are grateful to the Ministry of Innovation, Science, Research and Technology of the State of North Rhine-Westphalia, Germany, and the German Research Foundation (DFG) for funding a high-resolution mass spectrometer. We thankfully acknowledge Dr. F.M. Talontsi (formerly at INFU, TU Dortmund) for valuable discussions, Dr. W. Hiller (Department of Chemistry and Chemical Biology, TU Dortmund) for realization of the NMR measurements, Dr. S. Zühlke (INFU, TU Dortmund) for helpful discussions on the mass spectrometric analysis, and Dr. R. Class (Pharmacelsus GmbH, Saarbrücken, Germany) for cytotoxicity tests. We also thank Mr. Dennis Eckelmann 988


Epigenetic Modulation of Endophytic Eupenicillium sp. LG41 by a

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