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Antibacterial Secondary Metabolites from an Endophytic Fungus, Eupenicillium sp. LG41 Gang Li,† Souvik Kusari,*,† Marc Lamshöft,† Anja Schüffler,‡ Hartmut Laatsch,§ 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 of Biotechnology and Drug Research, Erwin-Schrödinger-Straße 56, D-67663 Kaiserslautern, Germany § Institute for Organic and Biomolecular Chemistry, Georg-August University, Tammannstraße 2, D-37077 Göttingen, Germany S Supporting Information *

ABSTRACT: Two new compounds containing the decalin moiety, eupenicinicols A and B (1 and 2), two new sirenin derivatives, eupenicisirenins A and B (3 and 4), and four known compounds, (2S)-butylitaconic acid (5), (2S)-hexylitaconic acid (6), xanthomegnin (7), and viridicatumtoxin (8), were isolated from an endophytic fungus, Eupenicillium sp. LG41, harbored in the roots of the Chinese medicinal plant Xanthium sibiricum. Their structures were confirmed through combined spectroscopic analysis (NMR and HRMSn), and their absolute configurations were deduced by ECD calculations or optical rotation data. Since the endophytic fungus was isolated from the roots, the antibacterial efficacies of the compounds 1−6 were investigated against Bacillus subtilis and Acinetobacter sp. BD4, which typically inhabit soil, as well as the clinically important Staphylococcus aureus and Escherichia coli. (2S)-Butylitaconic acid (5) and (2S)-hexylitaconic acid (6) exhibited pronounced efficacy against Acinetobacter sp., corroborating the notion that root-endophytes provide chemical defense to the host plants. Compound 2 was highly active against the clinically relevant S. aureus. By comparing 1 with 2, it was revealed that altering the substitution at C-11 could drastically increase the antibacterial efficacy of 1. Our study reveals plausible ecological roles of the endophyte and its potential pharmaceutical use as a source of antibacterial compounds.

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treating several ailments including fever and dysentery.13,14 Earlier investigations of this plant have led to the isolation of sesquiterpene lactones, which are designated as the chemical markers for Xanthium species.14 We recently isolated an endophytic fungus, Eupenicillium sp. LG41, from the roots of X. sibiricum collected from Taian, People’s Republic of China. Here we report the isolation, characterization, and antimicrobial activities of four new compounds, including two compounds containing the decalin moiety, eupenicinicols A and B (1 and 2), and two sirenin derivatives, eupenicisirenins A and B (3 and 4), together with four known compounds,15−19 (2S)-butylitaconic acid (5), (2S)hexylitaconic acid (6), xanthomegnin (7), and viridicatumtoxin (8), from the isolated endophytic fungus Eupenicillium sp. LG41. In order to evaluate the ecological role of the endophyte and its further use as a source of antimicrobial compounds, the antimicrobial efficacies of the isolated new compounds have been investigated. In this study, clinically important bacteria (such as the Gram-positive bacterium Staphylococcus aureus subsp. aureus and the Gram-negative bacterium Escherichia coli) and bacteria inhabiting soil and the rhizosphere (such as the Gram-positive bacterium Bacillus subtilis and the Gram-negative

ndophytic fungi, a distinct group of microorganisms that asymptomatically colonize living tissues of healthy plants, have attracted considerable attention owing to their ecological and biotechnological potential.1,2 Their complex association with host plants and other organisms contributes toward their adaptation to both biotic and abiotic selection pressures in their ecological niches.3,4 Many reports have shown that plants and associated microorganisms have a substantial influence on the metabolic processes of endophytes, leading to their enormous biological diversity and a variety of biosynthetic capabilities.5−9 Such biosynthetic blueprints enable endophytes to produce bioactive secondary metabolites that can serve as an invaluable source of lead compounds against a plethora of diseases. Traditional Chinese Medicine (TCM) is of great importance to health maintenance not only for the people of Asia but also for many countries in the West.10 Many of these trace their origins back thousands of years and have been investigated worldwide for their chemical and pharmacological properties.10,11 Endophytic fungi from traditional medical plants have already proven to be promising sources of pharmaceutical leads, and as such, several reports demonstrate the importance of these organisms.7,12 Xanthium sibiricum (Asteraceae) is an important medicinal plant that has been used in TCM for decades.13 X. sibiricum is commonly distributed in China and its fruit (Cang Er Zi), leaves, and roots have been used in TCM for © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 5, 2014

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dx.doi.org/10.1021/np500111w | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Structures of compounds 1−8.

bacterium Acinetobacter sp. BD4) were used. Furthermore, the fungi Aspergillus aculeatus and Colletotrichum circinans were used to evaluate the antifungal efficacy of the compounds produced by the endophyte.

Table 1. NMR Spectral Data for Compound 1 position

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RESULTS AND DISCUSSION Compound 1 (Figure 1) was isolated as a white powder, with the molecular formula C19H30O4 (five double-bond equivalents) as derived from ESI-HRMS ([M + H]+ at m/z 323.2219, calcd 323.2217) and its fragment information ([M + H − H2O]+ at m/z 305.2113, [M + H-2 × H2O]+ at m/z 287.2007, and [M + H-3 × H2O]+ at m/z 269.1899, Figure S7, Supporting Information). Furthermore, the MS2 spectrum shown in Figure S7 (Supporting Information) denotes the successive loss of up to four water molecules and/or a CO group ([M + H-2 × H2O]+ at m/z 287.2005, [M + H-2 × H2O − CO]+ at m/z 259.2055, [M + H-3 × H2O − CO]+ at m/z 241.1949; or [M + H-2 × H2O]+ at m/z 287.2005, [M + H-3 × H2O]+ at m/z 269.1898, [M + H-4 × H2O]+ at m/z 251.1790), revealing that there should be at least three hydroxy groups and/or a carbonyl moiety in 1. Moreover, a characteristic fragment ion at m/z 217.1951 [M + H − C3H6O4]+ was found. The 1H NMR data (Table 1) suggested the presence of four methyls, two oxygenated methines, and four protons associated with two double bonds. The 1D NMR data in combination with the HSQC spectrum revealed four methyls, two methylenes, seven methines (two oxygenated), two cisdisubstituted double bonds (one oxygenated carbon), and the remaining two quaternary carbons (Figures S1−S3, Supporting Information). Interpretation of 1H−1H COSY NMR data (Table 1 and Figure 2) suggested the proton spin systems from C-2 to C-7, C-2 to C-3′, 8-Me to C-4a, and C-10 to C-11. The HMBC correlations of Me-8/C-7, C-8, and C-8a and Me-1/C1, C-2, and C-8a established the connection of C-7 to C-8 and of C-1 to C-2 and C-8a and assigned Me-1 and Me-8 at C-1 and C-8, respectively, indicating a decalin ring system.20 The remaining side chain was verified by the HMBC correlations of Me-1 and a pair of protons of the Δ10-double bond to C-9 (Figure 2). Three hydroxy groups were attached to C-5 (δH 3.47, δC 75.4), C-6 (δH 4.06, δC 69.7), and C-11 (δH 7.97, δC 175.4), respectively, from their chemical shifts, which was also consistent with its MS data. Accordingly, the planar structure of compound 1 was unambiguously established as depicted (Figure 1). The NOESY correlations (Figure S6, Supporting Information) of Me-1/H-2, H-4a, H-8, and H-10, H-8a/H-2′b

δC, mult.a

1 2

49.4, Cq 54.0, CH

3

124.9, CH

4 4a 5 6 7α 7β 8 8a 9 10 11 1′ 2′a 2′b 3′ 1-Me 1′-Me 8-Me

126.3, 39.1, 75.4, 69.7, 41.4,

CH CH CH CH CH2

30.3, 43.3, 205.7, 99.7, 175.4, 36.8, 25.5,

CH CH Cq CH CH CH CH2

12.6, 19.8, 19.6, 20.8,

CH3 CH3 CH3 CH3

δH mult.b (J in Hz)

COSYb

1.84 mc

1′, 3, 4

5.75 dd (10.0, 3.0) 6.02 d (10.0) 2.18 m 3.47 m 4.06 br s 1.88 m 1.52 m 1.78 m 1.94 t (10.0)

2, 4

5.83 7.97 1.33 0.79 1.56 0.80 1.21 0.92 0.76

d (5.0) d (5.0) m mc m mc s d (7.0) d (7.0)

HMBCb 1′, 2′, 1, 3, 4, 8a, 1Me 1, 2, 4a

2, 3, 4a 4, 5, 6, 8a 4a, 6 5, 7 6

8 5, 6, 8, 8a

8a, 8-Me 4a, 8

1, 4a, 5, 7, 8, 1-Me

11 10 2, 1′-Me 1′, 3′

9, 11 9, 10 2′, 3′ 2, 1′, 3′, 1′-Me

2′

1′, 2′ 1, 2, 8a, 9 2, 1′, 2′ 7, 8, 8a

1′ 8

2, 4a, 5, 8a 3, 4, 5

a

Recorded in CDCl3 at 125 MHz; 13C multiplicities were determined by HSQC experiment. bRecorded in CDCl3 at 500 MHz. cSignals overlapped.

Figure 2. Key HMBC and 1H−1H COSY correlations of 1 and 3.

and H-5, and H-6/H-5 and H-7β determined the relative configuration of 1. The above conclusions support that compound 1 shares the same relative configuration with its analogue, eujavanicol A (isolated from the same genus).20,21 Compound 1 showed an optical rotation of [α]22D +47.8 (c B



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0.23, MeOH) compared to [α]22D +49.9 (c 1.33, MeOH) for eujavanicol A and [α]D +30.3 (c 0.11, MeOH) for tandyukisin (Figure S39, Supporting Information).21,22 Considering the same biosynthetic origin, the absolute configuration assigned to compound 1 is the same as that of eujavanicol A21 and tandyukisin.22 The minor component 2 (Figure 1) was also obtained as a white powder. A molecular formula of C20H32O4 was assigned from its quasimolecular ion at m/z 337.2373 [M + H]+ and its fragments ([M + H − H2O]+ at m/z 319.2268 and [M + H-2 × H2O]+ at m/z 301.2162, Figure S14, Supporting Information). The MS2 pattern of 2 was similar to that of compound 1, especially in the abundant fragment ion at m/z 217.1948 [M + H − C4H8O4]+, except that a lost CH3OH molecule was evident ([M + H − H2O]+ at m/z 319.2262, [M + H − H2O − CH3OH]+ at m/z 287.2002, or [M + H − CH3OH]+ at m/z 305.2115, Figure S14, Supporting Information). The fragmentation pattern indicated that compound 2 was similar to 1, except for an additional methoxy group. Unlike compound 1, the 1H NMR spectrum of 2 (Figure S10, Supporting Information) showed a large coupling constant (J = 12.0 Hz in 2; J = 5.0 Hz in 1) between H-10 and H-11 and a significant methoxy singlet (δH 3.77), revealing a trans-disubstituted double bond and suggesting the location of the methoxy group at C-11. Unfortunately, owing to the limited amount of 2 available for NMR measurement, clear 13C, HSQC, and HMBC data were not obtained (Figures S11 and S12, Supporting Information). However, for the moiety modified by the methoxy group, it was still possible to assign the NMR data to 2 (for OMe-11, δH 3.77, δC 58.0; for C-11, δH, 7.59, δC 163.5) from the HMBC correlations of OMe-11 to C-11 and of H-11 to OMe-11. The whole structure was also supported by 1 H−1H COSY NMR data (Figure S13, Supporting Information). From a biosynthetic standpoint, 2 (Figure 1) should have the same absolute configuration as 1. The molecular formula of compound 3 was deduced as C15H22O3 as determined by ESI-HRMS (m/z 251.1638, [M + H]+, calcd 251.1642; m/z 233.1536, [M + H − H2O]+, calcd 233.1536, Figure S22, Supporting Information), revealing five double-bond equivalents. The ESI-HRMS2 spectrum of 3 (Figure S22, Supporting Information) revealed the presence of a carboxyl or lactone group from fragment ions at m/z 215.1426 [M + H-2 × H2O]+, 197.1320 [M + H-3 × H2O]+, and 187.1477 [M + H − H2O − CH2O2]+ and of a 2(or 3)methylpent-2-ene unit corresponding to other product ions at m/z 177.0907 [M + H − H2O − C4H8]+, 163.0751 [M + H − H2O − C5H10]+, and 149.0598 [M + H − H2O − C6H12]+. Detailed analysis of 1H, 13C, and HSQC NMR data of 3 (Table 2 and Supporting Information) showed the presence of three methyls, three methylenes, three methines (one oxygenated), one quaternary carbon, and four olefinic carbons. The α,βunsaturated carboxylic acid or lactone, suggested partly by MS2 analysis, was constructed by the detailed HMBC correlations of H-4β to C-2, C-3, and C-15 and of H-2 to C-15. Furthermore, the six-membered ring system in 3 was readily confirmed on the basis of the 1H−1H COSY correlations of H-1/H-2, H-1/ H-6, H-5/H2-4, and H-5/H-6 (Figure 2). Another spin system of CH2(8)−CH2(9)−CH(10) together with the HMBC correlations of H3-12/C-10, C-11, and C-13 and H3-13/C-10, C-11, and C-12 confirmed a 2-methylpent-2-ene moiety as indicated by MS2 data (Figure 2). The HMBC correlations from H3-14 (δH 1.04, s) to C-1, C-6, C-7, and C-8 revealed the connection of C-7 to C-1, C-6, C-8, and C-14. The oxygenated

Table 2. 1H and 13C NMR Data for Compounds 3 (in CDCl3) and 4 (in CD3OD) 3 position

δC, mult.a

1

28.1, CH

2 3 4α

141.4, CH 124.6, Cq 32.1, CH2

4β 5

65.6, CH

6 7 8

32.8, CH 34.7, Cq 43.5, CH2

9 10 11 12 13 14 15

25.7, 123.9, 131.9, 25.6, 17.7, 13.4, 169.5,

CH2 CH Cq CH3 CH3 CH3 Cq

4

δH mult.b (J in Hz) 1.66 mc 7.37 dd (3.0, 5.5) 1.83 ddd (3.0, 8.5, 17.5) 3.01 dd (8.5, 17.5) 4.28 ddd (5.5, 8.5) 1.54 mc 1.54 1.28 2.16 5.14

mc m m t (7.0)

δC, mult.a 26.1, CH 136.5, CH 132.5, Cqd 22.3, CH2

17.2, CH2 25.7, CH 34.0, Cqd 178.2, Cqd 10.1, CH3 170.5, Cqd

δH mult.b (J in Hz) 2.18 dd (5.5, 8.5) 7.10 m 2.05 m 2.39 2.01 1.88 1.97

m mc α, m mc

1.15 s

1.72 s 1.65 s 1.04 s

a

Recorded at 125 MHz; 13C multiplicities were determined by HSQC experiment. bRecorded at 500 MHz. cSignals overlapped. dProposed by HMBC correlations.

carbon C-5 at δC 65.6 and the MS requirement suggested a hydroxy and a carboxyl group located at C-5 and C-3, respectively. The key NOESY correlations of H-4α (δH 1.83)/H3-14 and H-10/H3-12 and the strong NOESY correlation between H-4β (δH 3.01) and H-5 allowed the determination of its relative configuration as shown (Figure 3).

Figure 3. Key NOESY correlations of 3.

The absolute configuration of 3 was calculated using semiempirical and ab initio methods. With SPARTAN’14,23 using PM3 and the Monte Carlo technique, 648 starting geometries were tested, which resulted in 100 conformers within 6.9 kcal above the global minimum. A further geometry optimization with Gaussian g0924 using DFT calculations with the B3LYP functional and the 6-311G(2d,p) basis set delivered 53 conformers with Boltzmann factors > 0.001 (corresponding to an energy difference of ΔE < 2.5 kcal/mol). The ECD C



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calculated optical rotation ([α]D −43), as found for the natural product ([α]22D −27.3). With respect to their NOE data, the chirality centers in eupenicisirenins A (3) and B (4) have the same relative configuration. As the hydroxy group in 3 did not influence the chromophoric system, the close similarity of the ECD spectra of 3 and 4 was expected. However, their optical rotations had opposite signs and also differed strongly in their absolute values. Out of the 53 conformers populated at room temperature in 3, 26 had a negative sign and contributed −1.54 to the optical rotation. This was compensated by 27 conformers with a positive rotation value of in total +2.60, resulting in a positive optical rotation of [α]D +1.06. The conformation of the prenoid side chain in 3 is responsible for this effect (Figure 6). Obviously, the optical rotation of 3 is

spectra (Figure 4) of all conformers were calculated at the same level of theory; the individual conformer values were summed

Figure 4. Experimental (red) and calculated (blue) ECD curves of eupenicisirenin A (3).

with respect to their Boltzmann factors. The calculations reflected the experimental CD data correctly (Figure 4), so that the absolute configuration of 3 is that depicted in Figure 1. ORD calculations with WB97XD/6-311G(d,p) resulted in a specific optical rotation of [α]D +1.06 (measured [α]D +2.7) and confirmed this configuration. Compound 4 (Figure 1) was obtained as a white solid. ESIMS, MS2, and MS3 in the negative mode showed product ions at m/z 195.06, 151.07, and 107.03 (Figure S31, Supporting Information), corresponding to [M − H]−, [M − H − COO]−, and [M − H-2 × COO]−, respectively, and suggesting two carboxyl groups in 4. Furthermore, considering the 1H NMR data (Table 2), its molecular formula was deduced to be C10H12O4. Compound 4 also had a similar ring system to 3, which was verified by the 1H−1H COSY correlations of H-1/H2, H-1/H-6, and H-4β/H-5α and the HMBC correlations from H-1 to C-3 and C-7, from H-2 to C-1 and C-4, from H2-5 to C3 and C-6, and from H3-9 to C-6 and C-7 (Figures S28 and S29, Supporting Information). Two carboxylic acids (δC 170.5 and 178.2) were located at C-3 and C-7, respectively, supported by the HMBC correlations of H-1 to C-8, H-2 to C-10, and H39 to C-8 and MS analysis. The relative configuration of 4 was the same as that of 3 on the basis of NOESY correlations of H39/H-4α (δH 2.05), H3-9/H-5α (δH 1.88), and H-1/H-6 (Figure S30, Supporting Information). As compound 4 has fewer degrees of freedom for internal rotations than 3, SPARTAN’1423 calculated only 12 conformers; all were within a range of 2.1 kcal above the global minimum. A further minimization of the individual geometries with Gaussian g0924 reduced the energy range to 10.0 5.0 >10.0 10

1.0 5.0 >10.0 >10.0

>10.0 5.0 >10.0 5.0

>10.0 10.0 >10.0 5.0

10.0 10.0 >10.0 1.0

10.0 10.0 >10.0 1.0

5.0 1.0 5.0 10.0

1.0 1.0 1.0 5.0

All values are in μg/mL and derived from experiments in triplicate. E



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of 99.8% (in 547 bp) to Eupenicillium brefeldianum CBS 233.81 (GenBank Accession No.: GU981615) and 99.8% (in 547 bp) to Eupenicillium meridianum CBS 443.75 (GenBank Accession No.: GU981616.1). The fungus was assigned the strain designation LG41 and was deposited in the internal culture collection at INFU, TU Dortmund, Germany. For the purpose of large-scale fermentation, the fungal strain was first cultured in two flasks (250 mL) each containing 100 mL of PDB to obtain seed broth. This culture was incubated at 30 °C on a shaker (INFORS HT Multitron 2, Einsbach, Germany) at 150 rpm for 7 days. Further, the seed broth (5 mL) was added to 20 flasks (500 mL) each containing 80 g of rice, 120 mL of water, and 0.3% peptone. The cultures were incubated at room temperature for 7 weeks. Extraction and Isolation. At the end of the fermentation, the rice culture was extracted with ethyl acetate (EtOAc). The organic solvent was evaporated to dryness under reduced pressure to give 13.4 g of a crude extract. The extract was separated by column chromatography (CC) on silica gel eluting with a gradient of CH2Cl2−MeOH from 100:0 to 0:100 (v/v). Eight fractions (A−H) obtained on the basis of TLC were subjected to LC-HRMS analysis. Fraction C, with poor solubility in MeOH, from 50:1 (CH2Cl2−MeOH) elution, was chromatographed over a silica gel column with a gradient of cyclohexane−EtOAc (100:1 to 0:100) to afford 7 (55.5 mg) and 8 (219.8 mg). Fraction D from the same 50:1 (CH2Cl2−MeOH) elution was subjected to a Sephadex LH-20 column (MeOH) to obtain four subfractions (D1−D4). Fraction D1 was further separated by silica gel column chromatography with cyclohexane−EtOAc into five subfractions (D11−D15). Fraction D13 was purified by semipreparative HPLC (MeOH−H2O, 45/55, 2.0 mL/min) to afford 1 (2.3 mg, tR = 19.0 min) and 2 (0.5 mg, tR = 37.0 min). Fraction E, also from the CH2Cl2−MeOH (50:1) elution, following a similar procedure to that used for fraction D yielded four subfractions (E11−E14). Further separation of fraction E11 by silica gel CC using CH2Cl2−MeOH mixtures (100:0 to 1:1) gave four subfractions (E111−E114). Fraction E111 was purified by HPLC (MeOH−H2O, 50/50, 2.0 mL/min) to afford 5 (10.4 mg, tR = 9.5 min) and 6 (105.6 mg, tR = 29.5 min). Fraction E113 was subjected to preparative TLC, followed by preparative HPLC (MeOH−H2O, 70/30, 2.0 mL/min), yielding 3 (1.1 mg, tR = 15.5 min) and 4 (1.5 mg, tR = 17.2 min). Eupenicinicol A (1): white powder; [α]22D +47.8 (c 0.23, MeOH); LC-UV [(acetonitrile(aq) in H2O−0.1% FA)] λmax 226, 274 nm; IR (film) νmax 3452, 1632, 1114 cm−1; CD (MeCN) 194 (Δε +4.21), 290 (Δε −0.09), 343 (Δε +0.11) nm; 1H NMR (CDCl3, 500 MHz) and 13 C NMR (CDCl3, 125 MHz), see Table 1; positive ESI-HRMS m/z 323.2219 [M + H]+ (calcd for C19H31O4, 323.2217). Eupenicinicol B (2): white powder; [α]22D +16.0 (c 0.05, MeOH); LC-UV [(acetonitrile(aq) in H2O−0.1% FA)] λmax 226, 258 nm; IR (film) νmax 3444, 1640, 1109 cm−1; 1H NMR (CDCl3, 500 MHz) see Figure S10; positive ESI-HRMS m/z 337.2373 [M + H]+ (calcd for C20H33O4, 337.2379). Eupenicisirenin A (3): white powder; [α]22D +2.7 (c 0.11, MeOH); LC-UV [(acetonitrile(aq) in H2O−0.1% FA)] λmax 250 nm; IR (film) νmax 3415, 1643, 1375, 1321, 1101 cm−1; CD (MeOH) 209 (Δε −1.11), 260 (Δε +1.54) nm; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Table 2; positive ESI-HRMS m/z 251.1638, [M + H]+ (calcd for C15H23O3, 251.1642); 233.1536 [M + H − H2O]+ (calcd for C15H21O2, 233.1536). Eupenicisirenin B (4): white solid; [α]22D −27.3 (c 0.15, MeOH); LC-UV [(acetonitrile(aq) in H2O−0.1% FA)] λmax 246 nm; IR (film) νmax 3452, 1627, 1380, 1112, 1031 cm−1; CD (MeOH) 207 (Δε −4.80), 253 (Δε +1.19) nm; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz), see Table 2; negative ESIMS m/z 195.06 [M − H]−, 151.07 [M − H − CO2]−, and 107.03 [M − H-2 × CO2]−. (2S)-Butylitaconic acid (5): white solid; [α]22D +10.3 (c 1.04, MeOH); LC-UV [(acetonitrile(aq) in H2O−0.1% FA)] λmax 214 nm; IR (film) νmax 3401, 1641, 1112 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Table 3; positive APCI-HRMS m/z 187.0967 [M + H]+ (calcd for C9H15O4, 187.0965). Compounds and Microorganisms Used for Antimicrobial Assay. The in vitro antibacterial activities of the compounds 1−6 were

tested against a panel of standard pathogenic control strains (LeibnizInstitut DSMZ, Braunschweig, Germany). The Gram-positive bacteria Staphylococcus aureus subsp. aureus (DSM 799) and Bacillus subtilis (DSM 1088) and Gram-negative bacteria Acinetobacter sp. BD4 (DSM 586) and Escherichia coli (DSM 1116) were used. The activation, maintenance, and preparation of working culture suspensions were carried out in accordance with established procedures.28 The compounds tested (1−6) were dissolved in HPLC-grade MeOH at a concentration range of 0.1 to 10 μg/mL. Additionally, streptomycin and gentamicin (both from Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were used simultaneously as reference standards. The standards were also prepared at the same concentration range as the tested compounds in sterile double-distilled H2O. Furthermore, the fungi Colletotrichum circinans (DSM 62125) and Aspergillus aculeatus (DSM 2344) were used to test the antifungal activities of compounds 1−6. Antibacterial and Antifungal Assays. The disk diffusion method, according to the Clinical and Laboratory Standards Institute (CLSI),29 was employed for the determination of the antibacterial and antifungal activities of the samples. All agar plates were prepared in 90 mm sterile Petri dishes (TPP, Trasadingen, Switzerland) with 22 mL of agar, giving a final depth of 4 mm (nutrient agar for bacteria and potato dextrose agar for fungi). The inoculum suspension (200 μL) was spread on the solid media plates using the standard spread-plate technique. For fungi, the mycelia were first thoroughly macerated in a sterile mortar and pestle to yield a homogeneous inoculum. Sterile assay paper disks (Schleicher & Schuell GmbH, Dassel, Germany; 6.0 mm in diameter) were impregnated with 40 μL of the samples, airdried under the laminar airflow hood, and placed on inoculated plates. After standing at 4 °C for 2 h, the plates were incubated at 37 °C for 24 h for bacteria or at 28 °C for 48 h for fungi. Three control sets were included. The first control was the organism control and consisted of a seeded Petri dish with no sample. In the second control, samples were applied to unseeded Petri dishes to check for sterility. Finally, the solvent effect was controlled by a disk treated with 40 μL of HPLCgrade MeOH or with 40 μL of sterile double-distilled H2O. We used the standard antibiotics streptomycin and gentamicin as reference in parallel to reveal the comparative antimicrobial efficacy of compounds 1−6 against the tested organisms. The minimum inhibitory concentrations of each tested compound as well as the reference antibiotics against each bacterium were calculated according to CLSI.29 Each test was performed in triplicate. Computational Details. Computational methods were applied as described previously.30

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ASSOCIATED CONTENT

S Supporting Information *

Spectral data of compounds 1−5 and ITS sequence of the endophytic fungus. This material is available free of charge via the Internet at http://pubs.acs.org.

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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.

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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. G.L. gratefully acknowledges the China Scholarship Council (CSC) for a doctoral fellowship. S.K. was a Visiting Researcher at the F



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Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J., Fox, D. J. Gaussian 09W, Version 7.0; Gaussian: Wallingford, CT, 2009. (25) Kondru, R. K.; Beratan, D. N.; Friestad, G. K.; Smith, A. B.; Wipf, P. Org. Lett. 2000, 2, 1509−1512. (26) Talontsi, F. M.; Lamshöft, M.; Bauer, J. O.; Razakarivony, A. A.; Andriamihaja, B.; Strohmann, C.; Spiteller, M. J. Nat. Prod. 2013, 76, 97−102. (27) Kusari, S.; Lamshöft, M.; Zühlke, S.; Spiteller, M. J. Nat. Prod. 2008, 71, 159−162. (28) Kusari, S.; Lamshöft, M.; Spiteller, M. J. Appl. Microbiol. 2009, 107, 1019−1030. (29) Wikler, M. A. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard, ninth ed. (M2-A9); CLSI: Wayne, PA, 2006. (30) Kouam, S. F.; Ngouonpe, A. W.; Lamshöft, M.; Talontsi, F. M.; Bauer, J. O.; Strohmann, C.; Ngadjui, B. T.; Laatsch, H.; Spiteller, M. Phytochemistry 2014, 105, 52−59.

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 15 3RB, United Kingdom, during part of the work and preparation of the manuscript (Apr 2013 to Mar 2014). S.K. gratefully acknowledges M.S. for approving and authorizing, Dr. G. M. Preston for hosting, and TU Dortmund for supporting his stay at the University of Oxford. We thank Dr. F. M. Talontsi (INFU, TU Dortmund) for valuable discussions and technical assistance.

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