New Antimicrobial Cyclopentenones from Nigrospora sphaerica

May 10, 2018 - College of Materials and Energy, South China Agricultural University ... School of Chemistry and Chemical Engineering, Sun Yat-sen Univ...
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Bioactive Constituents, Metabolites, and Functions

New Antimicrobial Cyclopentenones from Nigrospora sphaerica ZMT05, a Fungus Derived from Oxya chinensis Thunber Zhihui Wu, Zihui Xie, Manlin Wu, Xiaoqi Li, Weilin Li, Weijia Ding, Zhigang She, and Chunyuan Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01376 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 11, 2018

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Journal of Agricultural and Food Chemistry

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New Antimicrobial Cyclopentenones from Nigrospora sphaerica

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ZMT05, a Fungus Derived from Oxya chinensis Thunber

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Zhihui Wu,† Zihui Xie,† Manlin Wu,† Xiaoqi Li,† Weilin Li,† Weijia Ding,† Zhigang

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She,‡ and Chunyuan Li*, †

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7

510642, China

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9

510275, China

College of Materials and Energy, South China Agricultural University, Guangzhou

School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou

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*Corresponding Author (Tel: +86-20-85280319; Fax: +86-20-85282366; E-mail:

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[email protected])

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ABSTRACT: Six new cyclopentenone derivatives (+)-nigrosporione A, (+)-1, (‒)-

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nigrosporione A, (‒)-1, nigrosporione B (2), nigrosporione C (3), (+)-nigrosporione D,

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(+)-4, and (‒)-nigrosporione D, (‒)-4 were isolated from an endophytic fungus

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Nigrospora sphaerica ZMT05, collected from the rice grasshopper (Oxya chinensis

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Thunberg) which is an insect pest in rice and also used as a food for people in some

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countries. Their planar and spatial structures were determined by spectroscopic

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analyses and ECD calculations. Compounds (+)-1, (‒)-1 and 2 inhibited the plant

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pathogens Fusarium oxysporum, Colletotrichum musae, Penicillium italicum and

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Fusarium graminearum, compounds 3 and (‒)-4 inhibited F. oxysporum, C. musae and

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P. italicum, and compound (+)-4 inhibited F. oxysporum, C. musae and F.

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graminearum, showing antifungal activities stronger than triadimefon. Additionally,

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compounds (+)-1, (‒)-1, 2 and 3 displayed moderate antibacterial activities against

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Staphyloccocus aureus and Escherichia coli.

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KEYWORDS:

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Antifungal activity; Oxya chinensis Thunberg

Insect-derived fungi; Nigrospora sphaerica; Cyclopentenone;

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INTRODUCTION

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In recent years, fungi inhabiting insect organs have been recognized as abundant

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sources of biologically active natural products with novel structures.1-3 Nigrospora

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sphaerica, a filamentous fungus belonging to the phylum Ascomycota,4 has been

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proven to produce different types of secondary metabolites with specific agricultural

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and pharmaceutical values.5-8 Oxya chinensis Thunberg (Orthoptera: Acrididae) is a

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main insect pest that threaten Oryza sativa L. It is distributed in rice growing zones all

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over China.9 A recent report shows that the pest feeds on various plants including Oryza

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sativa L., Saccharum officinarum L., Zea mays L., Sorghum vulgare Pers., and others.10

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Since this insect contains rich nutrients, such as protein, fatty acids, vitamins, it is also

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widely used for food among people in some areas in China, Japan and Thailand.11 As

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part of our program to search for leads of new fungicides used in agriculture from

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microorganisms,12, 13 a fungus Nigrospora sphaerica (collection No. ZMT05) isolated

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from Oxya chinensis Thunberg collected from Guangzhou, China attracted our interests

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because of its antifungal activities against several plant pathogens in vitro. Our

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investigation on the metabolites of this fungus, led to the separation of six new

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cyclopentenone derivatives, whose structures were identified by spectroscopic and

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spectrometric method, and the antifungal and antibacterial activities of which were

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determined.

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MATERIALS AND METHODS

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General Experimental Procedures. 3

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NMR data were recorded using an AVIII 600MHz NMR spectrometer (Bruker

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BioSpin GmbH company, Rheinstetten, Germany), adopting the residual solvent

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signals of (CD3)2SO or CDCl3 as references. Optical rotation was measured on a P-

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1020 digital polarimeter (Jasco International Co., Ltd., Tokyo, Japan). UV, IR, CD and

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HRESIMS spectra were obtained using UV-2550 spectrophotometer (Shimadzu

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Corporation, Tokyo, Japan), Nicolet iS10 Fourier transform infrared spectrophotometer

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(Thermo Electron Corporation, Madison, WI), Chirascan CD spectrometer (Applied

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Photophysics Ltd., London, UK), and Q-TOF mass spectrometer (Thermo Fisher

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Scientific Inc., Frankfurt, Germany), respectively. Column and thin layer

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chromatography (TLC) were performed using 200–300 mesh silica gel and G60, F-254

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silica gel plates (Qingdao Haiyang Chemicals Co., Ltd., Qingdao, China), respectively.

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Chiral purification was carried out using a 1260 Infinity Series HPLC system (Agilent

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Corporation, Santa Clara, CA). The HPLC column used was a 250 mm × 4.6 mm i.d.,

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5 μm, Lux Cellulose-2, without a guard column. The HPLC solvents and other

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chemicals adopted were of chromatographic and analytical pure grades, respectively.

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Fungal Material.

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Nigrospora sphaerica ZMT05 was isolated from Oxya chinensis Thunberg and

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stored in the College of Materials and Energy, South China Agricultural University.

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This fungus was identified through molecular analyses. The use of BLAST disclosed

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that its ITS sequence (No. MG171196 in GenBank) was identical to those of two

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Nigrospora sphaerica strains KM893076.1 and KM111472.1. 4

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Fermentation, Extraction and Isolation.

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A scraping of the agar with mycelium of the fungus growing on potato dextrose agar

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medium at 28 °C for 3‒4 d was put into the liquid medium (2% glucose, 2% peptone,

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1% sea salt), and cultured at 28 °C, 180 rpm for about 4 d as seed culture. Then 6 mL

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of the culture was transferred to an Erlenmeyer flask (1 L) with the autoclaved rice

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medium (80 mL H2O, 60 g rice, and 0.1 g sea salt) and incubated for 30 d at room

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temperature. One hundred Erlenmeyer flasks containing fermented solid rice media and

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mycelia were continuously extracted three times using 95% ethanol. The solvent was

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evaporated in vacuo to 2 L and extracted three times with ethyl acetate to afford 28.0 g

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of a yellow extract. The extract was divided into seven fractions (Fr. 1−Fr. 7) by column

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(40 × 6 cm) chromatography, using gradient of petroleum ether/ethyl acetate (92:8,

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1000 mL; 82:18, 510 mL; 76:24, 750 mL; 50:50, 510 mL; 24:76, 510 mL; 20:80, 500

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mL; 0: 100, 1000 mL; v/v) as eluents. Fr. 3 was purified on column chromatography

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(40 × 1.5 cm) and eluted with petroleum ether/ethyl acetate (92:8, 200 mL; 83:17, 200

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mL; 80:20, 200 mL; 76:24, 200 mL; v/v), affording eighty subfractions (Fr. 3.1− Fr.

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3.80, 10 mL per subfraction). Fr.3. 31 and Fr. 36 were slowly recrystallized in the

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solvent acetone at room temperature to give compounds (+)-4 (2.2 mg) and (‒)-4 (1.5

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mg), respectively. Fr. 4 was fractioned by column chromatography (40 × 1.5 cm) using

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petroleum ether/ethyl acetate (92:8, 200 mL; 82:18, 200 mL; 76:24, 250 mL; 50:50,

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300 mL; v/v) as eluents to afford Fr. 4.1–Fr. 4.4. Fr. 4.2 was separated by preparative

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TLC (petroleum ether/ethyl acetate, 3:1, v/v) to give compound 2 (5 mg, Rf = 0.62). Fr. 5

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4.3 was separated in the same way to afford compounds (±)-1 (3.8 mg, Rf = 0.36) and

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3 (4.3 mg, Rf = 0.51). Racemic (±)-1 was separated into a pair of enantiomers (+)-1 (1.2

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mg, tR = 7.6 min) and (‒)-1 (1.1 mg, tR = 9.0 min) through chiral HPLC (CH3CN/H2O,

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70:30; 25 °C; 1.0 mL/min).

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(+)-Nigrosporione A, (+)-1: yellow oil; UV (CH3CN) λmax (log ε) 239 (1.94) nm; IR

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(KBr) νmax 3374, 2944, 2872, 1684, 1588, 1357, 1264, 1142, 990 cm‒1; [α]25 D 25.29 (c

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0.28, MeOH); HRESIMS m/z 185.0803 ([M + H]+, calcd. for C9H13O4 185.0808); 13C

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NMR and 1H NMR (Tables 1 and 2).

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(‒)-Nigrosporione A, (‒)-1: yellow oil; UV (CH3CN) λmax (log ε) 239 (1.94) nm; IR

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(KBr) νmax 3374, 2944, 2872, 1684, 1588, 1357, 1264, 1142, 990 cm‒1; [α]25 D −25.18 (c

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0.28, MeOH); HRESIMS m/z 185.0803( [M + H]+, calcd. for C9H13O4 185.0808); 13C

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NMR and 1H NMR (Tables 1 and 2).

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Nigrosporione B (2): yellow oil; UV (CH3CN) λmax (log ε) 238 (1.98) nm; IR (KBr)

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νmax 3364, 2916, 1679, 1596, 1355, 1038, 829 cm‒1; [α]25 D −36.63 (c 0.17, MeOH);

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HRESIMS m/z 209.0789 ([M + Na]+, calcd. for C9H15O4 209.0784); 13C NMR and 1H

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NMR (Tables 1 and 2).

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Nigrosporione C (3): yellow oil; UV (CH3CN) λmax (log ε) =239 (1.88) nm; IR (KBr)

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νmax 3342, 2931, 1679, 1594, 1447, 1347, 1367, 1239, 1038, 829, 551 cm‒1; [α]25 D 20.56

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(c 0.16, MeOH); HRESIMS m/z 187.0967 ([M + H]+, calcd. for C9H15O4 107.0964);

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C NMR and 1H NMR (Tables 1 and 2). (+)-Nigrosporione D, (+)-4: yellow oil; UV (CH3CN) λmax (log ε) 265 (1.94) nm; IR 6

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(KBr) νmax 3414, 2920, 1708, 1572, 1357, 1230, 1038, 823 cm‒1; [α]25 D 12.35 (c 0.17,

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MeOH); HRESIMS m/z 169.0860 ([M + H]+, calcd. for C9H13O3 169.0859); 13C NMR

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and 1H NMR (Tables 1 and 2).

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(‒)-Nigrosporione D, (‒)-4: yellow oil; UV (CH3CN) λmax (log ε) 265 (1.94) nm; IR

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(KBr) νmax 3414, 2920, 1708, 1572, 1357, 1230, 1038, 823 cm‒1; [α]25 D −13.50 (c 0.17,

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MeOH); HRESIMS m/z 169.0860 ([M + H]+, calcd. for C9H13O3 169.0859); 13C NMR

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and 1H NMR (Tables 1 and 2).

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ECD Calculations.

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The theoretical ECD spectra of the isolated compounds were calculated based on the

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relative configurations determined by their NOESY spectra. Conformational analyses

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were accomplished by the MMFF94 force field calculation through the software

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Spartan'10 (Wavefunction Inc., Irvine, CA). DFT calculations were used to generate

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and optimize the conformers with energy ≤ 10 kcal/mol at the 6-31 G (d, p) level. The

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ECD calculations were performed for the stable conformers by the method of DFT (TD-

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DFT) using Gaussian 09 (Gaussian Inc., Wallingford, CT) software at the B3LYP/6-

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311+G (d, p) level. The rotary strengths of 30 excited states were calculated. MeOH

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was used as the IEF-PCM solvent. A half-bandwidth of 0.3 eV was applied to Gaussian

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according to dipole-length rotational strengths. Then the softwares SpecDis 1.64

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(University of Wurzburg, Wurzburg, Germany) and OriginPro 8.5 (OriginLab, Ltd.,

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Northampton, MA) were used to generate the ECD curves.

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Antimicrobial Activity Assay. 7

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Four fungi including Fusarium oxysporum (F. oxysporum), Colletotrichum musae

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(C. musae), Fusarium graminearum (F. graminearum) and Penicillium italicum (P.

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italicum), along with two bacteria Escherichia coli (E. coli) and Staphylococcus aureus

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(S. aureus) used for bioassay were acquired from College of Agriculture, South China

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Agricultural University. The antimicrobial effects were examined as the minimum

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inhibitory concentration (MIC) values.16 In brief, a stock solution of each test sample

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was prepared in 5% aqueous DMSO (v/v), following which 0.5 mL of the solution was

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serially diluted with 0.5 mL of potato dextrose broth (PDB) to final concentrations of

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200, 100, 50, 25, 12.5, 6.25 and 3.13 μg/mL in a set of capped test tubes. Ten microliters

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of an inoculum suspension of the test microorganism (105 colony-forming units/mL in

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PDB) was added to each test tube. Then the fungi and bacteria were cultured at 28 °C

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for 48 h, and at 37 °C for 24 h, respectively. The MIC value for each sample was

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determined to be the lowest concentration with invisible microbial growth. The solvent

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(0.5 mL of 5% DMSO/H2O + 0.5 mL PDB) was used as negative control, whereas

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triadimefon (Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China) and

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kanamycin (Yuanye Bio-Technology Co., Ltd., Shanghai, China) were adopted as

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positive controls with respect to fungi and bacteria, respectively.

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RESULTS AND DISCUSSION

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(±) Nigrosporione A, (±)-1 was a yellow oil, and its molecular formula was

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elucidated as C9H12O4, based on high-resolution ESIMS data, suggesting that (±)-1 had

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four degrees of unsaturation. The IR spectrum displayed the functional groups of 8

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hydroxyl (3374 cm−1), carbonyl (1684 cm−1), and double bond (1588 cm−1). The 1H

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NMR spectrum (Table 2) exhibited one hydroxyl at δH 6.52 (8-OH, exchangeable), one

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methyl at δH 1.17 (H-10), two methines at δH 2.82 (H-4) and 5.26 (H-8), one

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oxymethylene at δH 3.76 (H-6a) and 3.59 (H-6b), one oxymethyl at δH 3.84 (H-9) and

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one olefinic methine at δH 5.37 (H-2). The

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exhibited nine signals consisting of one carbonyl group at δC 207.4 (C-1), one double

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bond at δC 104.1 (C-2) and 188.2 (C-3), one methyl at δC 18.8(C-10), one oxymethyl at

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δC 59.8(C-9), one oxymethylene at δC 72.7 (C-6), two methines at δC 98.2 (C-8) and

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61.0 (C-4), and one quaternary carbon at δC 54.8(C-5), accounting for two of the four

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degrees of unsaturation in (±)-1. This result also implied that (±)-1 contained two rings.

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HMBC correlations from H-10 to C-1, C-5, and C-6; H-6 to C-1, C-5, and C-10; and

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H-4 to C-1, C-5, and C-10 unequivocally established the direct connections of C-5 with

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C-1, C-4, C-6 and C-10. Similarly, those from H-2 to C-1, C-3, C-4, and C-5; and H-9

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to C-3 established the five-membered enone ring and placed the oxymethyl group at C-

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3. Furthermore, the correlations from H-8 to C-4, C-5 and C-6; and 8-OH to C-4, C-8,

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indicated a tetrahydrofuran ring and placed 8-OH at C-8. Accordingly, the planar

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structure of (±)-1 was determined (Figure 1). NOE correlations (Figure 2) between H-

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4 and H-10 indicated that H-4 was on the same side relative to H-10. No detectable

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coupling constant between H-4 and H-8 in the 1H NMR spectrum of (±)-1 placed the

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two protons on the same side, which was supported by no visible NOE cross peak

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signals between H-4 and 8-OH. Notably, the optical rotation and CD maximum of (±)-

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C NMR and HSQC spectra (Table 1)

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1 were both close to zero (Figure 3), suggesting that (±)-1 was a raceme. A subsequent

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chiral HPLC analysis of (±)-1 displayed two different chromatographic peaks in the

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proportion of 1:1, which further confirmed this deduction. Then the racemate was

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separated into a pair of enantiomers, (+) 1 and (‒) 1. The theoretical ECD spectra of

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(+)-1 and (‒)-1 were further calculated and compared with the experimental ones to

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determine the absolute configurations. They (Figure 3) were very similar (Figure 3), so

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that (+)-1 and (‒)-1 were 4S, 5S, 8S and 4R, 5R, 8R, respectively.

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Nigrosporione B (2) was obtained as yellow oil and showed IR bands for hydroxyl

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(3364 cm-1), carbonyl (1679 cm-1) and double bond groups. The molecular formula was

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determined as C9H14O4 according to HRESIMS (three degrees of unsaturation). The

200

13

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resonances, including one carbonyl group at δC 208.5 (C-1), one double bond at δC

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102.6 (C-2) and 189.2 (C-3), one methyl at δC 15.9(C-9), one oxymethyl at δC 58.9(C-

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9), two oxymethylenes at δC 67.6 (C-6) and δC 59.4 (C-7), one methine at δC 49.8 (C-

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4), and one quaternary carbon at δC 52.7(C-5). The carbonyl and double bond groups

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accounted for two of the three elements of unsaturation, indicating that the molecule

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only possessed one cyclic ring. These data together with the molecular formula revealed

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by HRESIMS, indicated that compound 2 was a cyclopentenone with the substitutes of

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one methyl, one methoxyl, and two hydroxymethyls. Furthermore, the HMBC spectrum

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showed correlations from H-9 at δH 1.17 to C-1, C-4, C-5, and C-6; H-6 at δH 3.57 and

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3.63 to C-1, C-4, C-5, and C-9; H-4 at δH 3.04 to C-3, C-5, and C-9; H-2 at δH 5.24 to

C NMR (Table 1) and HSQC spectroscopic data for 2, indicated nine carbon

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C-1, C-3, C-4, and C-5; H-8 at δH 3.84 to C-3; and H-7 at δH 3.75 and 3.95 to C-3, C-4,

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and C-5, which established the planar structure of 2 (Figure 1). NOE correlations

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(Figure 2) between H-7 and H-9, and the absence of correlations between H-7 and H-

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6, revealed that H-7 was cis to H-9 and trans to H-6. Additionally, the lack of

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correlations between H-4 and H-9 revealed that H-4 and H-9 faced the opposite

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direction. The trend of the experimental ECD curve was almost the same as that of the

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theoretical one for (4R, 5R)-2 (Figure 3), establishing the chiral carbons of 2 to be 4R,

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5R.

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Nigrosporione C (3) had the same molecular formula C9H14O4 as 2 deduced from its

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HRESIMS spectrum. The carbon types of 3 (Table 1) were the same as those of 2.

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Moreover, they shared similar chemical shifts at most carbons. However, the carbon

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chemical shifts of compound 3 at C-4 and C-9 showed obvious differences with the

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deviations of 4.6 and 6.3 ppm with those of 2, respectively. Subsequently, detailed

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analysis of the HMBC correlations of 3 revealed that 3 had the same planar structure as

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2. These results implied that 3 might be a stereoisomer of 2 (Figure 1). NOE correlations

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between H-4 and H-9, and the lack of correlations between H-4 and H-6, indicated that

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H-4 was cis to H-9 and trans to H-6 (Figure 2). Moreover, NOE correlations of H-7a

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and H-7b to H-6a and H-6b, indicated that H-6 was cis to H-7. The experimental ECD

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spectra of 3 was quite similar to the calculated one of compound 4S, 5R-3 (Figure 3),

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establishing the absolute configuration of 3 as 4S, 5R.

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Nigrosporione D, (+)-4 was purified as yellow oil, and its molecular formula 11

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C9H12O3 was determined based on the HRESIMS, indicating one fewer degrees of

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unsaturation than 3. There were great resemblances between the NMR (Tables 1 and 2)

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spectra of (+)-4 and those of 3. However, compared with 3, the proton signals of H-4

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at δH 2.70, H-7 at δH 4.01 and 3.92 were missing, and changing to exocyclic olefinic

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methylene signals at δH 5.22 and 5.69 in (+)-4. Together, the signals at δC 54.4 (CH, C-

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4) for the methine group and at δC 59.1 (CH2, C-7) for the oxymethylene group were

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replaced by two olefinic signals at δC 145.9 (C, C-4) and δC 109.5 (CH2, C-7),

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respectively, in (+)-4. It could be concluded that the hydroxymethyl group (CH2, C-7)

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at C-4 in 3 was changed to the exocyclic olefin Δ4(7) substituent at C-4 in (+)-4. The

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clear HMBC correlations from H-7 to C-3, 4 and 5 supported the deduction (Figure 2).

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Finally, all NMR data for (+)-4 were readily assigned by HMBC analysis. Compound

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(+)-4 only has one chiral carbon, and its experimental ECD was similar to the

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calculative one of (S)-4 (Figure 3), with the absolute configuration of (+)-4 being 5S.

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(‒)-Nigrosporione D, (‒)-4 shared almost the same 1D and 2D NMR spectra with

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compound (+)-4, suggesting that their planar structures were identical. However, the

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specific rotation ([α]25 D ‒13.50) and the Cotton effects in the CD spectrum of (‒)-4 were

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completely opposite to those of (+)-4, indicating that compound (‒)-4 was the

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enantiomer of the latter. Moreover, the experimental ECD spectra of (‒)-4 was quite

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similar to the calculated one of the 5R-4 (Figure 3), which also confirmed the 5R

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configuration of (‒)-4.

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All the compounds were examined for inhibory activity toward the plant pathogenic 12

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fungi F. oxysporum, C. musae, P. italicum and F. graminearum, and the bacteria E. coli

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and S. aureus (Table 3). In particular, compounds (+)-1, (‒)-1 and 2 exhibited antifungal

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activities against all the tested plant pathogens (MIC values, 3.13‒25 μg/mL), higher

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than triadimefon (the positive control). Compounds 3 and (‒)-4 showed stronger

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antifungal activities toward F. oxysporum, C. musae and P. italicum (MIC values, 3.13‒

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25 μg/mL), than the control. Additionally, compound (+)-4 displayed antifungal

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activities against F. oxysporum, C. musae and F. graminearum with MIC values of 50,

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25, and 100 μg/mL, respectively, which were stronger than triadimefon. The results

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indicated the potential values of these new cyclopentenones as fungicides used in

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agriculture. As shown in Table 3, compounds (+)-1, (‒)-1, 2 and 3 also displayed

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moderate antibacterial activities against S. aureus belonging to Gram positive

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bacterium and E. coli belonging to Gram negative bacterium (MIC values, 3.13‒12.5

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μg/mL). It is interesting that the configuration seems to have no impact on the

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antimicrobial activities for compounds (+)-1 and (‒)-1.

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In previous investigations, only one example, nosporin A, with the same bicyclic ring

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system as compounds (+)-1 and (‒)-1 had been found as fungal metabolites.14 It was

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reported to have moderate antibacterial activity toward Bacillus subtilis ATCC 6633.

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In addition, cyclopentenones, hygrophorones A–G were reported to have inhibitory

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activities toward Cladosporium cucumerinum.15 (±)-(4S*, 5S*)-2, 4, 5-trihydroxy-3-

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methoxy-4-methoxycarbonyl-5-methyl-2-cyclopenten-1-one

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activity to F. graminearum .16 13

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antifungal

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FUNDING SOURCES This work was supported by the National Natural Science Foundation of China

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(21102049),

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(2015A030313405), the Science and Technology Project of Guangdong Province

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(2016A020222019), and the Science and Technology Project of Guangzhou City

279

(201707010342).

280

SUPPORTING INFORMATION

281

This material is available free of charge via the Internet at http://pubs.acs.org.

282

1

the

Natural

Science

Foundation

of

Guangdong

Province

H NMR, 13C NMR, HSQC, HMBC, 1H-1HCOSY, NOESY, HRESIMS, UV and IR

283

spectra of the new compounds (Figures S1‒S41), and the Chiral-HPLC separation profile

284

of the racemate (±) 1 (Figure S42).

285

Author Contributions: Chunyuan Li and Weijia Ding conceived and designed the

286

experiments; Zhihui Wu, Zihui Xie, Manlin Wu, Xiaoqi Li, Weilin Li and Chunyuan Li

287

performed the experiments; Zhigang She, Chunyuan Li and Weijia Ding analyzed the

288

data; Zhihui Wu and Weijia Ding wrote the paper; Chunyuan Li and Weijia Ding revised

289

and edited the manuscript.

290 291

REFERENCES

292

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2. Chen, C.; Wang, J.; Liu, J.; Zhu, H.; Sun, B.; Wang, J.; Zhang, J.; Luo, Z.; Yao, G.;

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Xue, Y.; Zhang, Y. Armochaetoglobins A−J: cytochalasan alkaloids from Chaetomium

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globosum TW1-1, a fungus derived from the terrestrial arthropod Armadillidium

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3. Ge, H.; Tan, R. Symbionts, an important source of new bioactive natural products.

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fungus Nigrospora sphaerica. Phytochem. Lett. 2014, 7, 1‒5.

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6. Kim, J.-C.; Choi, G. J.; Park, J.-H.; Kim, H. T.; Cho, K. Y. Activity against plant

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7. Cutler, H. G.; Hoogsteen, K.; Littrell, R. H.; Arison, B. H. Epoxyexserohilone, a

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novel metabolite from Nigrospora sphaerica. Agric. Biol. Chem. 1991, 55, 2037–2042.

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8. Harwooda, J. S.; Cutler, H. G.; Jacyno, J. M. Nigrosporolide, a plant growth-

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9. Wu, H.; Zhang, R.; Liu, J.; Guo, Y.; Ma, E. Effects of malathion and chlorpyrifos on 15

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antifungal activity of compounds from the mangrove endophytic fungus Aspergillus

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Nosporins A and B, new metabolites from a filamentous fungus, VKM-3750.

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bioactivity of compounds from the mangrove endophytic fungus Alternaria sp. Mar.

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FIGURE CAPTIONS Figure 1. Chemical structures of the isolated compounds. Figure 2. Key HMBC and NOESY of the isolated compounds. Figure 3 The calculated and experimental ECD spectra of the isolated compounds.

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Table 1. 13C NMR Data of the Isolated Compounds (±)-1a

2b

3b

(+)-4b

(-)-4 b

position δc/ppm 1

207.4

208.5

209.0

206.8

206.3

2

104.1

102.6

103.9

104.4

104.4

3

188.2

189.2

189.7

180.8

180.5

4

61.0

49.8

54.4

145.9

146.2

5

54.8

52.7

52.8

51.3

51.2

6

72.7

67.6

64.5

109.5

109.2

59.4

59.1

67.3

67.4

7

a

8

98.2

58.9

59.0

58.5

58.4

9

59.8

15.9

22.8

18.9

18.9

10

18.8

Measured in (CD3)2SO at 150 MHz ; b Measured in CDCl3 at 150MHz.

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Table 2. 1H NMR Data of the Isolated Compounds

(±)1a

2b

3b

(+)-4b

(-)-4b

position δH/ppm, multi (J/Hz)

2

5.37,s

5.24,d(1.2)

5.34,s(3.0)

4

2.82,s

3.04,ddd(1.8,4.8,4.8)

2.70,d(4.8)

6

3.76,d(9.0)

3.57,d(10.2)

3.59,d(9.0) 7

5.43,d(1.2)

3.67,d(12.0)

5.22,d(1.2)

5.22,d(1.2)

3.63,d(10.2)

3.84,dd(3.0,12.0)

5.69,d(1.2)

5.69,d(1.2)

3.75,dd(4.8,11.4)

4.01,dd(2.4,12.0)

3.60,d(10.8)

3.60,d(10.8)

3.95,dd(4.8,11.4)

3.92,dd(6.0,12.0)

3.71,d(10.8)

3.70,d(10.8)

8

5.26,d(3.6)

3.84,s

3.86,s

3.92,s

3.92,s

9

3.84,s

1.17,s

1.10,s

1.20,s

1.22,s

10

1.17,s

6-OH

unobservable

4.51-4.66c

7-OH

unobservable

4.51-4.66c

8-OH a

5.44,d(1.2)

6.52,s

Measured in (CD3)2SO at 600MHz; b Measured in CDCl3 at 600MHz; c The chemical

shifts of 6-OH and 7-OH of compound 3 were not differentiated due to the fact that no HMBC correlations for them were observed.

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Table 3. The Antifungal and Antibacterial Activity of the Isolated Compounds Measured as MIC values Compds

F. oxysporum

C. musae

P. italicum

F. graminearum

E. coli

S. aureus

MIC, μg/mL (+)-1

12.5

6.25

12.5

25

3.13

3.13

(‒)-1

12.5

6.25

12.5

25

3.13

3.13

2

12.5

25

12.5

3.13

3.13

6.25

3

25

12.5

12.5

>200

12.5

12.5

(+)-4

50

25

200

100

>200

50

(‒)-4

3.13

3.13

6.25

>200

>200

>200

Triadimefona

100

80

50

150

NT

NT

Kanamycinb

NT

NT

NT

NT

1.0

1.0

a

positive control towards fungi; b positive control towards bacteria

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Figure 1

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Figure 2

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Figure 3

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TABLE OF CONTENTS GRAPHICS

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