New Antimicrobial Cyclopentenones from Nigrospora sphaerica

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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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†

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510642, China

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‡

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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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ACS Paragon Plus Environment


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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

108

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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13

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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)

9



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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

215

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

217

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

218

5R.

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

220

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

228

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

229

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

230

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



232

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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

248

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

249

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

250

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

251

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

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

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

262

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

263

moderate antibacterial activities against S. aureus belonging to Gram positive

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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

272

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

273

activity to F. graminearum .16 13


exhibited

antifungal


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

276

(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

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

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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

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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.

18


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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.

19



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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.

20


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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

21



Figure 1

22


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Figure 2

23



Figure 3

24


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TABLE OF CONTENTS GRAPHICS

25


New Antimicrobial Cyclopentenones from Nigrospora sphaerica

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