Antibacterial Azaphilones from an Endophytic Fungus, Colletotrichum


Feb 24, 2016 - Institute of Environmental Research (INFU), Department of Chemistry and Chemical Biology, Chair of Environmental Chemistry and Analytic...
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Antibacterial Azaphilones from an Endophytic Fungus, Colletotrichum sp. BS4 Wen-Xuan Wang,† Souvik Kusari,*,† Hartmut Laatsch,‡ Christopher Golz,§ Parijat Kusari,⊥ Carsten Strohmann,§ Oliver Kayser,⊥ 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, D-44221 Dortmund, Germany ‡ Institute for Organic and Biomolecular Chemistry, Georg-August University, Tammannstrasse 2, D-37077 Göttingen, Germany § Inorganic Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund, Otto-Hahn-Straße 6, D-44221 Dortmund, Germany ⊥ Department of Biochemical and Chemical Engineering, Chair of Technical Biochemistry, TU Dortmund, Emil-Figge-Straße 66, D-44227 Dortmund, Germany S Supporting Information *

ABSTRACT: Three new compounds, colletotrichones A−C (1−3), and one known compound, chermesinone B (4a), were isolated from an endophytic fungus, Colletotrichum sp. BS4, harbored in the leaves of Buxus sinica, a well-known boxwood plant used in traditional Chinese medicine (TCM). Their structures were determined by extensive spectroscopic analyses including 1D and 2D NMR, HRMS, ECD spectra, UV, and IR, as well as single-crystal X-ray diffraction, and shown to be azaphilones sharing a 3,6a-dimethyl-9-(2methylbutanoyl)-9H-furo[2,3-h]isochromene-6,8-dione scaffold. Owing to the remarkable antibacterial potency of known azaphilones coupled to the usage of the host plant in TCM, we evaluated the antibacterial efficacy of the isolated compounds against two commonly dispersed environmental strains of Escherichia coli and Bacillus subtilis, as well as against two human pathogenic clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa. Compound 1 exhibited marked antibacterial potencies against the environmental strains that were comparable to the standard antibiotics. Compound 3 was also active against E. coli. Finally, compound 2a exhibited the same efficacy as streptomycin against the clinically relevant bacterium S. aureus. The in vitro cytotoxicity of these compounds on a human acute monocytic leukemia cell line (THP-1) was also assessed. Our results provide a scientific rationale for further investigations into endophyte-mediated host chemical defense against specialist and generalist pathogens.

E

and investigated cultivable endophytic fungi harbored in leaves and stems of B. sinica, particularly aiming to discover fungi capable of producing antibacterial compounds. Herein we report the isolation of an endophytic fungus, Colletotrichum sp. BS4, and the structural characterization and antibacterial efficacies of compounds produced on rice medium. Earlier investigations have revealed a shifting lifestyle of this fungal genus, varying from being endophytes to dangerous plant pathogens causing anthracnose.12 Moreover, a plethora of secondary metabolites including alkaloids, peptides, polyketides, and steroids were identified from this genus.12 On screening different media for fermenting this strain using the OSMAC (One Strain MAny Compounds) approach,13 we noticed the production of a series of interesting secondary metabolites on rice medium. By successive chromatographic and extensive spectroscopic methods as well as X-ray

ndophytic fungi, which are distributed in tissues of every plant investigated to date, establish remarkable mutualistic associations with their host plants.1 The pursuit of discovering novel bioactive compounds and revealing the relationship(s) between plants and their endophytic microflora has attracted the attention of chemists and biologists worldwide.1−5 Buxus sinica (Buxaceae), commonly known as the boxwood plant, is used not only in traditional Chinese medicine (TCM) for treating diseases, including malaria, syphilis, rheumatism, dermatitis, and rabies,6 but also as a topiary hedge in People’s Republic of China.7 It shows strong tolerance toward pruning and shearing and attracted our interest toward the potential endophytes harbored in leaves and stems. Given the utility of this plant both in TCM and for its tolerant morphology, it is conceivable that endophytes associated with this plant in a balanced mutualistic manner2 might aid in fitness of the host against biotic and abiotic stress.8 As a part of the continuation of our investigations on endophytic fungi from various medicinal plants including those used in TCM,9−11 we isolated © 2016 American Chemical Society and American Society of Pharmacognosy

Received: May 15, 2015 Published: February 24, 2016 704

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Table 1. 1H NMR Spectroscopic Data of 1−3 at 500 MHz

diffraction, three new compounds were isolated and identified, namely, colletotrichones A−C (1−3), along with one known compound, chermesinone B (4a)14 (Figure 1). We evaluated

1a

position

3b

1

5.90, 1H, s

7.30, 1H, s

3 4 7 8

5.82, 1H, s 5.82, 1H, s

6.00, 1H, s 5.36, 1H, br s 3.89, 1H, br s

10 12 13

3.39, 1H, m 1.41, 2H, m

14

0.69, 3H, (7.5) 2.09, 3H, 1.65, 3H, 1.15, 3H, (7.0)

15 16 17 a

2ab

t s s d

3.89, 1H, 3.10, 1H, 1.60, 1H, 1.37, 1H, 0.81, 3H, (7.4) 2.14, 3H, 1.57, 3H, 1.09, 3H, (6.5)

br s m m m t

3.95, 1H, dd (11.5, 5.4) 3.84, 1H, dd (13.5, 11.5) 5.48, 1H, br s 5.62, 1H, br s 3.10, 1H, dd (12.5, 11.5) 2.74, 1H, dddd (13.5, 11.5, 5.4, 1.5) 3.93, 1H, d (12.5) 3.07, 1H, m 1.79, 1H, m 1.42, 1H, m 0.90, 3H, t (7.5)

s s d

1.95, 3H, s 1.50, 3H, s 1.19, 3H, d (7.2)

Measured in CD3OD. bMeasured in CDCl3.

Table 2. 13C NMR Spectroscopic Data of 1−3 at 125 MHz

Figure 1. Structures of compounds 1−4.

the antibacterial efficacies of these compounds against the clinically important risk-group 2 (RG2) pathogenic bacteria Staphylococcus aureus and Pseudomonas aeruginosa, as well as two environmental strains of Escherichia coli and Bacillus subtilis. Furthermore, we evaluated their cytotoxicity against human acute monocytic leukemia cells (THP-1) in vitro using a resazurin-based assay to measure the THP-1 mitochondrial metabolic inhibition as well as an ATPlite assay to measure the THP-1 cytoplasmic ATP depletion.



RESULTS AND DISCUSSION Compound 1 was isolated as a colorless, amorphous powder with the formula C18H20O7 determined by ESI-HRMS, which showed nine double-bond equivalents (DBEs). The 1H NMR data showed four methyl groups, δH 0.69 (3H, t, J = 7.5, Me14), 2.09 (3H, s, Me-15), 1.65 (3H, s, Me-16), and 1.15 (3H, d, J = 7.0, Me-17), two olefinic protons, δH 5.82 (2H, s, H-3 and H-4), one acetal proton, δH 5.90, (1H, s, H-1) (identified by HSQC), and three further alkyl protons at δH 3.39 (1H, m, H12) and 1.41 (2H, m, CH2-13) (Table 1), which suggested two acidic protons exchanged by deuterium. Four methyl carbons, four downfield oxygen-connected sp3 carbons, eight sp2 carbons including two carbonyl groups, and one sp3 methylene and one sp3 methine carbon were observed in the 13C NMR data (Table 2) and HSQC experiments. 1H,1H COSY showed a spin−spin coupling system consisting of Me-14/CH2-13/CH-12/Me-17 (Figure 2). The key heteronuclear correlations between carbons and protons (J2,3) in HMBC confirmed a dimethyl isochromene structure and a sec-butyl group (Figure 2). Since the five double bonds and two identified rings contribute only seven DBEs, the remaining two DBEs belong to two further rings, as no additional sp2 signals were visible. However, the information above was not sufficient to connect these moieties, because of the lack of protons at positions 6 to 11 for 2D NMR

a

position

1a

2ab

3b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

97.2 159.5 101.2 118.9 194.9 85.8 73.7 63.4 149.2 103.2 168.2 35.1 27.0 11.0 19.4 17.1 17.0 168.4

147.2 159.4 107.3 105.8c 191.5 83.2c 43.0 114.6 144.5 55.7 206.3 46.6 26.2 11.1 19.5 23.2 14.1 168.6

69.0 163.9c 101.1 115.0 192.5 83.4 44.5 35.6 153.0 52.6 206.2 47.8 24.5 11.5 20.6 18.8 16.7 168.8c

Measured in CD3OD. bMeasured in CDCl3. cAssigned by HMBC.

Figure 2. 1H,1H COSY and key HMBC correlations and X-ray diffraction crystal structure (ORTEP drawing) of colletotrichone A (1).

interpretations. To determine the structure of compound 1, a single crystal from ethyl acetate and methanol (1:1) was subjected to X-ray crystallography to analyze the structure 705

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(Figure 2). The crystal structure showed an isochromene group cis-fused with a furan ring and a pyran ring, which formed a unique stereostructure with a rigid and nearly rectangular bent core skeleton. As the relative configuration was unequivocally defined by Xray diffraction, the absolute configuration must be identical with that depicted in the structure of 1 or with the mirror image thereof. The determination was made by comparing the calculated ECD and ORD data with the experimental values. Molecular mechanics calculations (SPARTAN’14, MMFF15) for 1 in a systematic approach delivered the lowest 18 conformers in an energy range of 45 kJ/mol above the global minimum. By ab initio calculations using SPARTAN’1415 and Gaussian 0916 [finally with the wB97X-D functional and the 6311++G(2df,2p) basis set], 11 conformers within a range of 16 kJ/mol (Gibbs free energy) above the global minimum were selected for further DFT calculations of the ECD and ORD data as described previously.11 As all conformers delivered positive OR values, the Boltzmann-weighted optical rotation was positive (calcd molar rotation = +396°/specific rotation +11.4°), and an experimental value of 340.4° was found for 1. As the enantiomer ent-1 would give a negative optical rotation and a mirror-imaged ECD spectrum with respect to Figure 3, the high similarity of experimental and calculated ECD and OR data unambiguously confirmed the absolute (1S,6R,7S,8S,12S)configuration of 1, as depicted in the structure.

Figure 4. 1H,1H COSY, key HMBC, and NOESY correlations of colletotrichone B (2a). The 3D structure is the conformer with the lowest energy.

a 1D NOESY experiment with adequate scan times (Figure S19, Supporting Information). Therefore, the configuration of H-10 related to Me-16 and H-7 was not ascertained by the interpretation of NOESY spectra. For the relative configuration of H-12/Me-17, the NOE correlation was observed only between H-10 and H-12, which suggested that the average distance between H-10 and Me-17 is considerably larger than between H-10 and H-12 and below the detection limit. It is worth mentioning here that both H-10 and Me-17 as well as H10 and H-12 gave cross-peaks in the NOESY spectrum of rel(7S,10S,12S)-chermesinone C reported earlier.14 On the basis of the H-10/H-12 and H-10/Me-17 distances of calculated preferential conformers (Boltzmann factor >0.01; see Table S2 in the Supporting Information), the possible configurations were proposed to be rel-(6R,6R,10R,12S) or rel-(6R,6R,10S,12R), the latter being less likely. To distinguish between both diastereomers, we compared ab initio-calculated and experimental NMR data. In accordance with the comparison of the experimental and calculated NMR data (see Tables S3 and S4 in the Supporting Information), the chemical shift of Me-17 [δC17 = 14.1, δH17 = 1.09; calcd 15.4 (err. 1.06) for (6R,6R,10R,12S)-2a] suggested that H-10 and Me-17 have the same relative orientations as in monochaetin (4b) (Me-17, δC17 = 14.4, δH17 = 1.11).18 For the other 6R,7R,10ξ,12ξ diastereomers, 2c and 2d, higher shifts were obtained (see Table S4 in the Supporting Information), as also found experimentally for chermesinone B (4a: Me-17, δC17 = 17.1, δH17 = 1.24).14 To differentiate between the structures 2a and 2b, ab initio ECD calculations were performed. The calculated ECD curve of 2a showed a Cotton effect coinciding with the experimental ECD spectrum (Figure 5). In contrast to this, the major negative Cotton effect at 290 nm in the calculated ECD value of 2b could not be explained by the experimental data. Therefore, the structure of colletotrichone B was elucidated as 2a, and the absolute configuration was determined to be 6R,7R,10R,12S. The assignment of the 6R,12S configuration could also be explained in view of its proposed biosynthetic pathway. Colletotrichone C (3) was obtained as a colorless, amorphous powder showing a pseudomolecular [M + H]+ ion, with the formula C18H22O5. In comparison with 2a and 4a, the 1H NMR data (Table 1) of 3 displayed three further peaks at δH 2.74, 3.95 and 3.84 and the absence of the sp2 CH signal

Figure 3. Experimental (black continuous line) and calculated (red, dashed curve) ECD spectra (in MeOH) of colletotrichone A (1).

Compound 2a was isolated as a yellowish oil with the formula C18H20O5 determined by ESI-HRMS. The 1H NMR, 13 C NMR, and HSQC data of 2a indicated the same pattern of sp2 and sp3 carbons as the known compound 4a isolated here, while the signals of CH-1, CH-10, Me-16, and Me-17 were quite different. HMBC and 1H,1H COSY experiments (Figure 4) revealed the same planar structure as for 4a and 4b. In 2D NOESY experiments (in acetone-d6, see Figure S18 in the Supporting Information), nuclear Overhauser effects (NOE) were clearly observed between Me-16 and H-7 (H-7 and H-10 have different chemical shifts in acetone-d6, Table S1 in the Supporting Information), which indicated that the lactone ring is cis fused (Figure 4). Although there was an NOE cross-peak between H-7 and H-10, this did not confirm that H-7 and H-10 are on the same side of the lactone ring, because they are too close and the cross-peak between H-7 and H-10 also appeared in the NOESY spectrum of chermesinone B (in which H-7 and H-10 have different orientations).14 Moreover, no unambiguous NOE could be observed between H-10 and Me-16 even in 706

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Figure 7. Experimental (black continuous line) and calculated (red, dashed curve) ECD spectra (in MeOH) of colletotrichone C (3). Figure 5. Experimental CD data in MeOH (black continuous line) and calculated ECD spectra of colletotrichone B (2a; red, dashed curve) and (10S,12R)-epi-colletotrichone B (2b; blue dashed curve).

hydrolysis.21 However, considering the steric effects or SN2 reaction mechanism (H2O would preferentially attack C-8 from the opposite direction of the epoxy group as shown in the proposed biosynthetic pathway of chaetoviridin I in ref 21), the hydrolysis of an epoxy group is more likely to generate two trans-hydroxy groups. Therefore, we propose our aforementioned biosynthetic pathway to explain the ring system and configuration of compound 1 (Figure 8). Azaphilones are well-known as having a broad range of biological activities such as antimicrobial, cytotoxic, anticancer, antiviral, and anti-inflammatory properties.19 In an earlier investigation on azaphilones from stromata of Xylariaceae, Hellwig et al. suggested that they are maintained in the course of their coevolution with angiosperm hosts and involved in a kind of chemical defense reaction to keep the fitness of their producers.22 Moreover, some azaphilones with similar lactone substructures were reported to have antibacterial activities. For instance, deflectin B-2a showed a minimum inhibitory concentration (MIC) of 5 μg/mL against Bacillus brevis and Bacillus subtilis ATCC 6633 on a complex medium.23 Sassafrins A−C showed moderate antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, and Escherichia coli.24 In another study, multiformins A−D were shown to have moderate to strong activity against S. aureus, P. aeruginosa, K. pneumonia, E. coli, and Salmonella enteritidis.25 Therefore, given the potent antibacterial efficacies of azaphilones, we evaluated the antibacterial effectiveness of our isolated compounds against two widely distributed environmental strains of E. coli (Gram-negative) and B. subtilis (Grampositive), as well as against two human pathogenic bacterial strains of S. aureus (Gram-positive) and P. aeruginosa (Gramnegative).11 Compound 1 showed pronounced efficacies particularly against both the tested environmental strains comparable to the standard antibiotics (Table 3). Additionally, compound 3 was quite active against the environmental strain of E. coli. Furthermore, compound 2a exhibited the same efficacy as streptomycin against the clinically relevant, RG2 bacterium S. aureus. Since the endophytic fungus was isolated from the leaves of B. sinica, it is compelling that it provides some form of azaphilone-mediated chemical defense to the host plant against invading specialist and generalist bacteria. Interestingly, the tested compounds (1−4) did not demonstrate significant cytotoxic efficacies in vitro against THP-1 cells (Figure S31, Supporting Information).

of H-1 in compounds 2a (δH 7.30) and 4a (δH 6.83). 13C NMR (Table 2), HMBC, and 1H,1H COSY data (Figure 6) indicated

Figure 6. 1H,1H COSY, key HMBC, and NOESY correlations of colletotrichone C (3).

that the pyran ring was hydrogenated at positions 1 and 8. The coupling constants of H-7 (δH 3.10, 1H, dd, 12.5, 11.5), H-8 (δH 2.74, 1H, dddd, 13.5, 11.5, 5.4, 1.5), and H-10 (δH 3.93, 1H, d, 12.5) suggested that these three methine protons at positions 7, 8, and 10 are at axial positions, which was further confirmed by NOESY experiments (Figure 6). To determine the absolute configuration, the ECD spectrum of 3 was calculated as described above, and the spectrum of expected (6R,7R,8S,10S)-configuration was in agreement with the experimental spectrum (Figure 7). The configuration of C-12 was assigned as S, based on the Me-17 shift (δC 16.7, δH 1.19) of 3, in comparison with the NMR data of compounds 2a and 4a. The compounds 1−4 are typical polyketides, whose biosynthesis has been extensively investigated in numerous studies.19 Interestingly, for the sec-butyl substructure, a condensation product originating from two different polyketides, but not from a single octaketide chain, was proposed.18,19 With respect to their structures, the biosynthesis of compounds 1−4 might proceed from a pentaketide via an isochromene analogue, which is first acylated and then condensed with a 4methyl-3-oxohexanoic acid, and further modified by oxidation or hydrogenation, according to the proposed biosynthesis of other azaphilones (Figure 8).19,20 Borges et al. proposed a pathway of cyclization of chaetoviridin I, whose essential steps were an epoxidation at double bond Δ7 and subsequent



EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on an A. Krüss P8000-T Optronic polarimeter, and ECD measurements were performed using a Jasco J-715 spectrometer. IR spectra were obtained using a Bruker TENSOR27 IR spectrometer.

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Figure 8. Proposed biosynthetic pathway of compounds 1−4.

Table 3. Minimum Inhibitory Concentrations (MIC) of Compounds 1−4 against Gram-Positive and Gram-Negative Bacteria Compared to Standard References (Streptomycin and Gentamicin)a

a

organism (DSMZ no.)

1

2a

3

4a

streptomycin

gentamicin

Staphylococcus aureus (DSM 799) Escherichia coli (DSM 1116) Bacillus subtilis (DSM 1088) Pseudomonas aeruginosa (DSM 22644)

>10 1.0 0.1 >10

5.0 >10 >10 >10

>10 5.0 >10 >10

>10 >10 >10 >10

5.0 1.0 5.0 10.0

1.0 1.0 1.0 1.0

All values are in μg/mL and derived from experiments in triplicate. each 1000 mL flask) at 25 °C with PDB culture as seed broth. After 30 days, the culture (1.6 kg) was extracted with 4 L of ethyl acetate (EtOAc) at room temperature three times to yield 33 g of brown residue after removing the solvent under vacuum. This extract was subjected to a silica gel column (2 × 10 cm) and eluted by a stepwise cyclohexane−EtOAc gradient from ratio 1:0 to 0:1 into four fractions (Fr. 1−Fr. 4). Fr. 3 was separated on a silica gel column with CH2Cl2− EtOAc (3:1) to obtain two subfractions (Fr. 3a, 3b). Successive purification of Fr. 3a by semipreparative HPLC (UV detection at 355 nm, flow rate 3 mL/min, mobile phase MeCN and H2O (0.1% formic acid)) yielded 5 mg of colletotrichone A (1) (33% MeCN, tR: 31.8 min), 10 mg of colletotrichone B (2a) (45% MeCN, tR: 27.6 min), 2 mg of colletotrichone C (3) (55% MeCN, tR: 25.0 min), and 8 mg of chermesinone B (4a) (45% MeCN, tR: 32 min). Colletotrichone A (1): colorless, amorphous powder; [α]20D +340.4 (c 0.05, MeOH); LC-UV [(acetonitrile in H2O−0.1% FA)] λmax 222, 340 nm; IR (film) νmax 3368, 2917, 2849, 1741, 1653, 1612, 1081, 1060 cm−1; CD spectrum (0.1 mg/mL, MeOH), 200 (Δε −1.85), 213 (Δε +0.66), 256 (Δε −8.23), 326 nm (Δε +10.21); 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz), see Tables 1 and 2; ESI-HRMS m/z 349.1280, [M + H]+ (calcd for C18H21O7, 349.1282, Δ −0.5108 ppm), 366.1547, [M + NH4]+ (calcd for C18H24O7N, 366.1547, Δ −0.0934 ppm). X-ray Crystallographic Analysis of 1. The diffraction experiment was performed on an Oxford Diffraction Xcalibur S diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.710 73 Å). The crystal structures were solved with direct methods (SHELXS97) and refined against F2 with the full-matrix least-squares method (SHELXL97).27 A multiscan absorption correction using the CrysAlis RED program (Oxford Diffraction, 2006) was employed. The

NMR spectra were recorded on a Bruker DRX-500 spectrometer operating at 500 (1H) and 125 (13C) MHz with tetramethylsilane as internal standard. HRMS and HRMS/MS experiments were carried out on an LTQ-Orbitrap spectrometer (Thermo Fisher, USA) equipped with an HESI-II source. The spectrometer was equipped with an Agilent 1200 HPLC system including pump, PDA detector, column oven (30 °C), and autosampler (injection volume 5 μL). The detailed MS parameters have been reported earlier.11 HPLC for separations and purifications was performed on a Venusil XBP (2) C18 column (10 × 250 mm) using a Gynkotek pump equipped with a Dionex DG-1210 degasser, a Dionex UVD 340S detector, and a Dionex Gina 50 autosampler. All solvents used were of analytical grade. Silica gel 60 (70−230 mesh; AppliChem, GmbH, Darmstadt, Germany) was used for column chromatography. Thin-layer chromatography was carried out with glass precoated silica gel 60 plates (0.25 mm; Merck, Darmstadt, Germany). Spots were visualized under UV light and by spraying with H2SO4−EtOH (1:9, v/v) followed by heating. Fungal Material. The endophytic fungus Colletotrichum sp. BS4 was isolated from the leaves of Buxus sinica (Buxaceae) collected from Guangzhou, Guangdong Province, People’s Republic of China. For identification, the fungal strain was cultured on potato dextrose agar (PDA) at 28 ± 2 °C for 1 week in an incubator. The fungus was identified by ITS sequencing following our previously established method.26 The ITS sequence of the identified endophytic fungus has been deposited at the EMBL-Bank (accession number LN552210). The fungus was assigned the strain designation BS4 and deposited in the internal culture collection at INFU, TU Dortmund, Germany. Extraction and Isolation. The large-scale fermentation was performed on solid rice medium (80 g of rice, 100 mL of water, in 708

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non-hydrogen atoms were placed in geometrically calculated positions, and each was assigned a fixed isotropic displacement parameter based on a riding model.17 A colorless crystal was obtained from a solution of EtOAc−MeOH (1:1, v/v): orthorhombic crystal system; space group P21212; a = 15.0896(7) Å, b = 15.6838(7) Å, c = 7.1843(3) Å, α = β = γ = 90°, V = 1700.25(13) Å3; Z = 4, d = 1.361 g/cm3; crystal dimensions 0.12 × 0.09 × 0.04 mm; the final indices were R1 = 0.0346, wR2 = 0.0796. Crystallographic data of colletotrichone A (1) were deposited in the Cambridge Crystallographic Data Centre with supplementary publication number CCDC 1013027. Copies of the data can be obtained, free of charge, on application to the Director, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44(0)1223 336033 or by email: [email protected]]. Colletotrichone B (2a): yellowish oil; [α]20D +81.0 (c 1.29, CHCl3); LC-UV [(acetonitrile in H2O−0.1% FA)] λmax 222, 366 nm; IR (film) νmax 3453, 2968, 2929, 1773, 1623, 1550, 1088 cm−1; CD spectrum (0.1 mg/mL, MeOH), 209 nm (Δε −1.63), 254 (Δε +1.89), 327 (Δε +8.19), 361 (Δε −4.81), 373 nm (−4.86); 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Tables 1 and 2; ESIHRMS m/z 317.1385, [M + H]+ (calcd for C18H21O5, 317.1384, Δ 0.3609 ppm). Colletotrichone C (3): colorless, amorphous powder; [α]20D +124.2 (c 0.07, CHCl3); LC-UV [(acetonitrile in H2O−0.1% FA)] λmax 214, 332 nm; IR (film) νmax 2971, 2933, 1779, 1711, 1612, 1219, 1105, 1029 cm−1; CD spectrum (0.1 mg/mL, MeOH), 202 (Δε +2.45), 224 (Δε −0.26), 250 (Δε +2.28), 286 (Δε −0.67), 344 nm (Δε +3.64); 1 H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Tables 1 and 2; ESI-HRMS m/z 319.1542, [M + H]+ (calcd for C18H23O5, 319.1540, Δ 0.7083 ppm), 341.1361, [M + Na]+ (calcd for C18H22O5Na, 341.1359, Δ −0.5143 ppm), 659.2836, [2M + Na]+ (calcd for C36H44O10Na, 659.2827, Δ 1.4080 ppm). Antibacterial Assays. The in vitro antibacterial activities of compounds 1, 2a, 3, and 4a were tested against a panel of standard pathogenic control strains (obtained from Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) using our previously described method.11



Chemical Biology, TU Dortmund) for realization of the NMR measurements, Dr. S. Zühlke (INFU, TU Dortmund) for valuable discussions and realization of mass spectrometric analyses, and Dr. R. Class (Pharmacelsus GmbH, Saarbrücken, Germany) for realization of the cytotoxic assays.



(1) Kusari, S.; Spiteller, M. Nat. Prod. Rep. 2011, 28, 1203−1207. (2) Kusari, S.; Hertweck, C.; Spiteller, M. Chem. Biol. 2012, 19, 792− 798. (3) Kusari, S.; Pandey, S. P.; Spiteller, M. Phytochemistry 2013, 91, 81−87. (4) Kusari, S.; Singh, S.; Jayabaskaran, C. Trends Biotechnol. 2014, 32, 304−311. (5) Mousa, W. K.; Raizada, M. N. Front. Microbiol. 2013, 27, 65. (6) Lin, Y.-L.; Qiu, M.-H.; Li, Z.-R.; Zhou, L.; Liu, J.-Q. Acta Bot. Yunnan. 2006, 28, 429−432. (7) Musselwhite, S.; Harris, R.; Latimer, J.; Wright, R. J. Environ. Hort. 2004, 22, 124−128. (8) Savatin, D. V.; Gramegna, G.; Modesti, V.; Cervone, F. Front. Plant Sci. 2014, 5, 470. (9) Kusari, P.; Kusari, S.; Spiteller, M.; Kayser, O. Fungal Divers. 2013, 60, 137−151. (10) Kusari, S.; Zühlke, S.; Spiteller, M. J. Nat. Prod. 2011, 74, 764− 775. (11) Li, G.; Kusari, S.; Lamshöft, M.; Schüffler, A.; Laatsch, H.; Spiteller, M. J. Nat. Prod. 2014, 77, 2335−2341. (12) García-Pajón, C. M.; Collado, I. G. Nat. Prod. Rep. 2003, 20, 426−431. (13) Bode, H. B.; Bethe, B.; Höfs, R.; Zeeck, A. ChemBioChem 2002, 3, 619−627. (14) Huang, H.; Feng, X.; Xiao, Z.; Liu, L.; Li, H.; Ma, L.; Lu, Y.; Ju, J.; She, Z.; Lin, Y. J. Nat. Prod. 2011, 74, 997−1002. (15) Shao, Y.; Molnar, L. F.; Jung, Y.; Kussmann, J.; Ochsenfeld, C.; Brown, S. T.; Gilbert, A. T. B.; Slipchenko, L. V.; Levchenko, S. V.; O’Neill, D. P.; DiStasio, R. A., Jr.; Lochan, R. C.; Wang, T.; Beran, G. J. O.; Besley, N. A.; Herbert, J. M.; Lin, C. Y.; Van Voorhis, T.; Chien, S. H.; Sodt, A.; Steele, R. P.; Rassolov, V. A.; Maslen, P. E.; Korambath, P. P.; Adamson, R. D.; Austin, B.; Baker, J.; Byrd, E. F. C.; Dachsel, H.; Doerksen, R. J.; Dreuw, A.; Dunietz, B. D.; Dutoi, A. D.; Furlani, T. R.; Gwaltney, S. R.; Heyden, A.; Hirata, S.; Hsu, C.-P.; Kedziora, G.; Khalliulin, R. Z.; Klunzinger, P.; Lee, A. M.; Lee, M. S.; Liang, W. Z.; Lotan, I.; Nair, N.; Peters, B.; Proynov, E. I.; Pieniazek, P. A.; Rhee, Y. M.; Ritchie, J.; Rosta, E.; Sherrill, C. D.; Simmonett, A. C.; Subotnik, J. E.; Woodcock, H. L., III; Zhang, W.; Bell, A. T.; Chakraborty, A. K.; Chipman, D. M.; Keil, F. J.; Warshel, A.; Hehre, W. J.; Schaefer, H. F.; Kong, J.; Krylov, A. I.; Gill, P. M. W.; Head-Gordon, M. Phys. Chem. Chem. Phys. 2006, 8, 3172. (16) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09W, Version 7.0; Gaussian: Wallingford, CT, 2009. (17) Talontsi, F. M.; Lamshöft, M.; Bauer, J. O.; Razakarivony, A. A.; Andriamihaja, B.; Strohmann, C.; Spiteller, M. J. Nat. Prod. 2013, 76, 97−102.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00436. Spectroscopic data of compounds 1−3 and cytotoxicity of compounds 1, 2a, 3, and 4a (PDF)



REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Tel: +49-(0)231-755-4086. Fax: +49-(0)231-755-4084. Email: [email protected] *Tel: +49-(0)231-755-4080. Fax: +49-(0)231-755-4085. Email: [email protected] Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We are grateful to the Ministry of Innovation, Science, Research and Technology of the State of North RhineWestphalia, Germany, and the German Research Foundation (DFG) for funding a high-resolution mass spectrometer. W.X.W. gratefully acknowledges the China Scholarship Council (CSC) for a doctoral fellowship. We are very much indebted to Mr. W. Hehre (Wavefunction, Inc., Irvine, CA, USA) for valuable discussions about NMR calculations. We gratefully acknowledge Dr. W. Hiller (Department of Chemistry and 709

DOI: 10.1021/acs.jnatprod.5b00436 J. Nat. Prod. 2016, 79, 704−710

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(18) Steyn, P. S.; Vleggaar, R. J. Chem. Soc., Perkin Trans. 1 1986, 1975−1976. (19) Gao, J.-M.; Yang, S.-X.; Qin, J.-C. Chem. Rev. 2013, 113, 4755− 4811. (20) Balakrishnan, B.; Chen, C.-C.; Pan, T.-M.; Kwon, H.-J. Tetrahedron Lett. 2014, 55, 1640−1643. (21) Borges, W. S.; Mancilla, G.; Guimarães, D. O.; Durán-Patrón, R.; Collado, I. G.; Pupo, M. T. J. Nat. Prod. 2011, 74, 1182−1187. (22) Hellwig, V.; Ju, Y.-M.; Rogers, J. D.; Fournier, J.; Stadler, M. Mycol. Prog. 2005, 4, 39−54. (23) Anke, H.; Kemmer, T.; Höfle, G. J. Antibiot. 1981, 34, 923−928. (24) Quang, D. N.; Hashimoto, T.; Fournier, J.; Stadler, M.; Radulović, N.; Asakawa, Y. Tetrahedron 2005, 61, 1743−1748. (25) Quang, D. N.; Hashimoto, T.; Stadler, M.; Radulović, N.; Asakawa, Y. Planta Med. 2005, 71, 1058−1062. (26) Wang, W.-X.; Kusari, S.; Sezgin, S.; Lamshöft, M.; Kusari, P.; Kayser, O.; Spiteller, M. Appl. Microbiol. Biotechnol. 2015, 99, 7651− 7662. (27) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, A64, 112−122.

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