Colletochlorins E and F, New Phytotoxic Tetrasubstituted Pyran-2

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Colletochlorins E and F, New Phytotoxic Tetrasubstituted Pyran-2-one and Dihydrobenzofuran, Isolated from Colletotrichum higginsianum with Potential Herbicidal Activity Marco Masi, Alessio Cimmino, Angela Boari, Angela Tuzi, Maria Chiara Zonno, Riccardo Baroncelli, Maurizio Vurro, and Antonio Evidente J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05193 • Publication Date (Web): 02 Jan 2017 Downloaded from http://pubs.acs.org on January 7, 2017

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Journal of Agricultural and Food Chemistry

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Colletochlorins E and F, New Phytotoxic Tetrasubstituted Pyran-2-one and

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Dihydrobenzofuran, Isolated from Colletotrichum higginsianum with Potential

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Herbicidal Activity

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Marco Masi,† Alessio Cimmino,† Angela Boari,‡ Angela Tuzi,† Maria Chiara Zonno,‡ Riccardo

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Baroncelli,§ Maurizio Vurro‡ and Antonio Evidente*,†

7 8 9

†

Department of Chimical Sciences, University of Naples Federico II, Complesso Universitario

Monte San’Angelo, Via Cintia 4, 80126 Napoli, Italy

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‡

11

Bari, Italy

12

§

13

Western Brittany Technopôle Brest-Iroise, 29280 Plouzané, France

Institute of Food Production Sciences National Research Council, Via Amendola 122/O, 70125

Laboratoire Universitaire de Biodiversité et Ecologie Microbienne (LUBEM), University of

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15

ACS Paragon Plus Environment


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ABSTRACT: A new tetrasubstituted pyran-2-one and a new dihydrobenzofuran, named

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colletochlorins E and F (1 and 2), were isolated from the culture filtrates of the fungus

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Colletotrichum higginsianum together with the already known colletochlorin A, 4-chloroorcinol and

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colletopyrone. Colletochlorins E, the main metabolite, and F were characterized by spectroscopic

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(NMR, HRESIMS) and chemical methods as 3-[7-chloro-4-hydroxy-2-(1-hydroxy-1-methylethyl)-

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6-methyl-2,3-dihydro-benzofuran-5-ylmethyl]-4-hydroxy-5,6-dimethyl-pyran-2-one, and 7-chloro-

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2-(1-hydroxy-1-methylethyl)-6-methyl-2,3-dihydrobenzofuran-4-ol. The absolute configuration 2’S

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of 1 was deduced by X-ray diffractometric analysis while that 2S of 2 was deduced by comparison

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of its NMR and CD data with those of 1. When assayed by leaf puncture on Sonchus arvensis and

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tomato leaves, 2 caused quite large necrosis (wider than 1 cm) while 4-chloroorcinol proved to be

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the most active compound. These results were confirmed to those obtained in the assay on Lemna

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minor and on Phelipanche ramosa seed germination. Furthermore, 1, colletochlorin A and

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colletopyrone were less and modestly active in the last assay, respectively. Interestingly, the

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phytotoxicity was not associated to an antibiotic activity while only 4-chloroorcinol, colletochlorin

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F exhibited zootoxic activity.

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KEYWORDS: Biocontrol; Colletotrichum higginsianum; pyran-2-ones and dihydrobenzofurans; phytotoxins; colletochlorins E and F

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INTRODUCTION

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Fungi belonging to different species as Ascochyta, Botryosphaeria, Botrytis, Fusarium,

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Lasiodiplodia, Mycosphaerella, Neofusicoccum, Pyricularia, Phoma, Phomopsis, Sclerotinia,

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Verticillium etc. are well known plant pathogens of important agrarian cultures being responsible of

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severe diseases with consequent heavy economic losses. Among them Colletotrichum includes

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hundreds of species particularly harmful to cereals, legumes, fruit trees and vegetables. Some

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species also infected ornamental plants. Colletotrichum spp. are worldwide spread and their

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damages affect several food crops including bananas, cassava, coffee and sorghum which are

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critical to the survival of the population and the economy of developing countries. The genus was

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recently considered the eighth most important group of plant pathogenic fungi in the world, based

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on scientific and economic importance.1

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Colletotrichum species are primarily responsible for anthracnose diseases, whose symptoms

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include necrotic spots on leaves, stems, flowers and fruit, although other plant diseases are also

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reported, such as red rot, crown and stem rot, seedling blight and brown blotch.2 Many species may

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be seed-borne and can survive well in soil by growing as saprophytes on dead plant fragments.

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The

phytopathological

importance

of

the

genus,

associated

to

an

uncertain

51

identification/classification of the species have led to extensive studies on their pathogenesis,

52

morphology, genetics, physiology, host range, and disease life cycle. Conversely, due to the same

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reasons, studies on the production of secondary metabolites has been only partially explored.3

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Fungal bioactive metabolites are an excellent source of pharmaceuticals, antibiotics, antifungal and

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herbicidal compounds.4-8 A very wide survey was carried out on 89 strains belonging to many

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species of the genus Colletotrichum, by evaluating their culture filtrates aimed at finding novel

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metabolites with potential phytotoxic, antibiotic, antifungal, and anticancer activities. Among the

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many species considered, the attention was focused on a strain of Colletotrichum higginsianum.

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This latter fungus, belonging to Colletotrichum destructivum species complex,9 causes anthracnose

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leaf spot disease on many cultivated forms of Brassica. Different secondary metabolites were ACS Paragon Plus Environment


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isolated from various Colletotrichum species, whose structural and biosynthetic characteristics were

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reviewed.3 Among these, the phytotoxic metabolites named colletochlorins and colletorins, that can

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be grouped in 3-prenyl or 3-diprenyl orsellinaldeide derivatives having or not a chlorine at C-5,

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were isolated from Colletotrichum tabacum [synonym of Colletotrichum nicotianae] strains which

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causes anthracnose in tobacco.3 Recently, two new diterpenoid α-pyrones, named higginsianins A

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and B, were isolated from the mycelium of a strain of C. higginsianum grown in liquid culture.10

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The same fungus showed to produce in culture phytotoxic metabolites. Thus, this manuscript

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reports on the isolation and chemical and biological characterization of the new tetrasubstituted

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pyran-2-one and dihydrobenzofuran, named colletochlorins E and F (1 and 2, Figure 1), together

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with the chlorinated 3-diprenyl orsellinaldehyde derivative colletochlorin A, 4-chloroorcinol and

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colletopyrone already produced by other species of Colletotrichum.

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MATERIALS AND METHODS

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General Experimental Procedures. Optical rotations were measured in a MeOH solution

74

on a Jasco P-1010 digital polarimeter (Japan, Tokyo); IR spectra were recorded as glassy film on a

75

Perkin-Elmer (Waltham, Massachusetts, USA) Spectrum One FT-IR spectrometer; UV spectra were

76

recorded in CH3OH solution or otherwise noted on a Perkin-Elmer Lambda 25 UV/Vis

77

spectrophotometer. 1H and

78

MHz, respectively, in CDCl3 or CD3OD on Varian (Palo Alto, California, USA) and Bruker

79

(Karlsruhe, Germany) spectrometers. The same solvent was used as an internal standard. Carbon

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multiplicities were determined by DEPT spectra.11 DEPT, COSY-45, HSQC, HMBC and NOESY

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experiments11 were performed using Bruker or Varian microprograms. HRESIMS and ESI spectra

82

were recorded on Shimadzu (Kyoto, Japan) LCMS-IT-TOF Mass Spectrometer and Agilent (Milan,

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Italy) 6230 Accurate-Mass TOF LC/MS instruments. Analytical and preparative TLC were

84

performed on silica gel (Kieselgel 60, F254, 0.25 and 0.5 mm respectively; Merck, Darmstadt,

85

Germany) or on reverse phase (KC18 F254, 0.20 mm; Whatman, Maidstone, UK) plates; the spots

13

C NMR spectra were recorded at 500 and 400, and at 125 and 100


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were visualized by exposure to UV light and/or iodine vapors and/or by spraying first with 10%

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H2SO4 in MeOH, and then with 5% phosphomolybdic acid in EtOH followed by heating at 110 °C

88

for 10 min. CC: silica gel (Merck, Kieselgel 60, 0.063-0.200 mm).

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Fungal Strain. The C. higginsianum strain used in this study is IMI 349063 (CABI Culture

90

Collection). This strain was used as reference as it is the most commonly used in research

91

laboratories for evolutionary and genetic analyses.12 The genome of C. higginsianum IMI 349063

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has also been sequenced and expression resources are available.13

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Extraction and Purification of C. higginsianum Secondary Metabolites. The strain of C.

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higginsianum used in this study was grown on a minimal defined liquid medium named M1-D.14

95

Small plugs of mycelium obtained by colonies actively growing on PDA were used to seed Roux

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bottles containing 200 mL of the sterile medium above mentioned. Bottles were kept in still

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condition at 25° C in the dark in an incubator for 4 weeks, then filtered by filter paper. The

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lyophilized culture filtrate (17.5 L) was dissolved in distilled H2O (1/10 of its original volume, pH

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6.8) and then extracted with EtOAc (3 × 1 L). The organic extracts were combined, dehydrated with

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anhydrous Na2SO4 and evaporated under reduced pressure, yielding a brown oil (3.42 g). This oil,

101

showing high phytotoxic activity, was purified by CC eluted with CHCl3/i-PrOH (95:5, v/v),

102

yielding ten groups of homogeneous fractions. The residues of the third fraction (122.4 mg) was

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further purified by CC eluted with CH2Cl2/i-PrOH (93:7, v/v) yielding six groups of homogeneous

104

fractions. The residue of the fourth fraction (34.6 mg), of this latter column, was purified by TLC

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on silica gel, eluent CHCl3/i-PrOH (95:5, v/v) yielding colletopyrone (5, 18.1 mg, Rf 0.5, Figure 1)

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as a homogeneous amorphous solid. The residue of the fourth fraction (317.3 mg), of the first

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column, was further purified by CC eluted with CH2Cl2/i-PrOH (93:7, v/v) yielding six groups of

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homogeneous fractions. The residue of the second fraction (42.2 mg), of this column, was purified

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by two successive steps on TLC eluted with CHCl3/i-PrOH (97:3, v/v) and then on reverse phase

110

eluted with Me2CO/H2O (8:2, v/v) obtaining an oily homogeneous compound, named colletochlorin



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F (2, 4.52 mg, Rf 0.31, Figure 1). The residue of the fourth fraction (76.7 mg), of the last column,

112

was purified by TLC eluted with CHCl3/i-PrOH (9:1, v/v) obtaining colletochlorin A (3, 29.8 mg, Rf

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0.37, Figure 1) and 4-chloroorcinol (4, 20.1 mg, Rf 0.57, Figure 1) as pure oily compounds. The

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residues of the seventh (474.4 mg) and eighth (282.7 mg) fractions, of the first column, were

115

combined and further purified by CC eluted with Me2CO/n-hexane (6:4, v/v) yielding five groups

116

of homogeneous fractions. The residue of the fourth (60.2 mg) fraction of this last column was

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further purified by TLC eluted CHCl3/i-PrOH/AcOH (9:0.8:0.2, v/v/v) obtaining an amorphous

118

homogeneous solid, named colletochlorin E (1, 35.4 mg, Rf 0.12, Figure 1). This latter was

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crystallized by slow evaporation at -20 °C from EtOAc/MeOH/H2O (1:1:2, v/v/v) solution.

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Colletochlorin E (1), 3-[7-Chloro-4-hydroxy-2(S)-(1-hydroxy-1-methylethyl)-6-methyl-2,3-

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dihydro-benzofuran-5-ylmethyl]-4-hydroxy-5,6-dimethyl-pyran-2-one. White crystals; [α]25D +18.4

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(c 0.64); IR νmax 3482 (OH), 1669, 1625, 1562, 1453, 1264 cm-1; UV λmax nm (log ε) 287 (3.22),

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212 (3.11); 1H and

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calcd 433.0820) 417.1078 [M + Na]+ (C20H23ClNaO6 calcd 417.1081), 395.1265 [M + H]+

125

(C20H24ClO6 calcd 395.1261).

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Colletochlorin

13

C NMR see Table 1 HRESI MS (+) m/z: 433.0824 [M + K]+ (C20H23ClKO6

F

(2),

7-Chloro-2-(1-hydroxy-1-methylethyl)-6-methyl-2,3-dihydro-

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benzofuran-4-ol. Amorphous solid, [α]25D +11.7 (c 0.17); IR νmax 3458, 1581, 1560, 1501, 1447

128

cm-1; UV λmax nm (log ε) 231 (sh), 208 (3.01) nm; 1H and 13C NMR see Table 2; HR ESIMS (+)

129

m/z: 507.1306 [2M + Na]+ (C24H30ClNaO6 calcd 507.1310), 281.0355 [M + K]+ (C12H15ClKO3

130

calcd 281.0347), 256.0600 [M + Na]+ (C12H15ClNaO3 calcd 256.0607), 243.0781 [M + H]+

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(C12H16ClO3 calcd 243.0788).

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Colletochlorin A (3), 3-chloro-5-(6,7-dihydroxy-3,7-dimethyl-oct-2-enyl)-4,6-dihydroxy-2-

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methyl-benzaldehyde. Amorphous solid, [α]25D +6.24 (c 0.20) [lit. 10 [α]25D +11.6 (c 10.0)]; 1H

134

NMR spectrum was very similar to that previously described;15,16 ESIMS (+) m/z: 379 (100) [M +

135

Na]+, 357 (60) [M + H]+.


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4-Chloroorcinol (4), 4-chloro-5-methylbenzene-1,3-diol. Amorphous solid, 1H and

13

C

137

NMR spectra were very similar to those previously described;17 ESIMS (−) m/z: 157 [M− H]-;

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ESIMS (+) m/z: 159 [M + H]+.

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Colletopyrone

(5),

4,4'-dihydroxy-5,5',6,6'-tetramethyl-3,3'-methylene-di-pyr-2-one.

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Amorphous solid, 1H NMR spectrum was very similar to that previously described;18 ESIMS (+)

141

m/z: 315 [M + Na]+, 293 (48) [M + H]+.

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4’-O-Methyl Derivative of Colletochlorin E (6). An ethereal solution of CH2N2 was slowly

143

added to compound 1 (2.6 mg) dissolved in MeOH (0.2 mL), until a yellow colour was persistent.

144

The reaction mixture was left at room temperature for 24 h. The solvent was evaporated under a N2

145

stream giving an oil residue (2.8 mg). This latter was purified by TLC eluted with Me2CO/n-hexane

146

(1:1, v/v) to give 6 as uncoloured oil (2.2 mg, Rf 0.8). Derivative 6: IR νmax 3410, 1669, 1619, 1586,

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1264 cm-1; UV λmax nm (log ε) 287 (3.26), 238 (sh), 209 (4.12); 1H NMR (CDCl3), δ 4.67 (1H, br t,

148

J = 9.3 Hz, H-2'), 4.06 (3H, s, OMe), 3.66 (2H, s, H-8'), 3.25 (1H, dd, J = 15.0 and 9.3 Hz, H-3'A),

149

3.16 (1H, dd, J = 15.0 and 8.3 Hz, H-3'B), 2.38 (3H, s, Me-8), 2.29 (3H, s, Me-4''), 1.93 (3H, s, Me-

150

7), 1.32 and 1.19 (3H each, s, Me-2'' and Me-3''); ESIMS (+) m/z: 839 [2M + Na]+, 447 [M + K]+,

151

431 [M + Na]+, 409 [M + H]+.

152

4’-O-Acetyl Derivative of Colletochlorin E (7). Colletochlorin E (1, 2.2 mg) dissolved in

153

pyridine (20 µL) was acetylated with acetic anhydride (20 µL) at room temperature for 1 h. The

154

reaction was stopped by addition of MeOH, and the azeotrope, obtained by the addition of benzene,

155

was evaporated by an N2 stream. The oily residue (2.4 mg) was purified by preparative TLC eluted

156

with Me2CO/n-hexane (1:1, v/v) yielding derivatives 7 as a homogeneous compound (1.1 mg, Rf

157

0.75). Derivative 7: IR νmax 3454, 1725, 1670, 1624, 1559, 1408, 1263 cm-1; UV λmax nm (log ε)

158

292 (3.40), 203 (4.03); 1H NMR (CDCl3), δ 4.73 (1H, br t, J = 9.5 Hz, H-2'), 3.72 (2H, s, H- 8'),

159

3.18 (1H, dd, J = 16.0 and 8.8 Hz, H-3'A), 3.13 (1H, dd, J = 16.0 and 9.5 Hz, H-3'B), 2.37 (3H, s,



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OAc-4'), 2.25 (3H, s, Me-8), 2.21 (3H, s, Me-4''), 1.83 (3H, s, Me-7), 1.36 and 1.27 (3H each, s,

161

Me-2'' and Me-3''); ESIMS (+) m/z: 437 [M + H]+.

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4'-O-p-Bromobenzoyl Derivative of Colletochlorin E (8). Colletochlorin E (1, 1.5 mg,),

163

was dissolved in CH3CN (0.2 mL), and 4-dimethylaminopyridine (DMAP) (2 mg) and p-

164

bromobenzoyl chloride (2 mg) were added. The reaction mixture was stirred at room temperature

165

for 2 h and then evaporated under reduced pressure. The residue (1.9 mg) was purified by TLC on

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silica gel, eluent Me2CO/n-hexane (1:1, v/v), giving derivatives 8 (1.2 mg, Rf 0.55) as colourless oil.

167

Derivative 8: IR νmax 3410, 1722, 1668 , 1622, 1583, 1260 cm-1; UV λmax nm (log ε) 216 (3.80) nm;

168

1

169

t, J = 8.8 Hz, H-2'), 3.80 (2H, s, H-8'), 3.18 (1H, dd, J = 15.8 and 8.4 Hz, H-3'A), 3.09 (1H, dd, J =

170

15.8 and 8.8 Hz, H-3'B), 2.27 (3H, s, Me-8), 2.19 (3H, s, Me-4''), 1.86 (3H, s, Me-7), 1.38 and 1.27

171

(3H each, s, Me-2'' and Me-3''); ESIMS (+) m/z: 1159 [2M + 2 + H]+, 1157 [2M + H]+, 601 [M + 2

172

+ Na]+, 599 [M + Na]+, 579 [M + 2 + H]+, 577 [M + H]+.

H NMR (CDCl3), δ 8.06 (2H, d, J = 8.3 Hz, H-2',6'), 7.72 (2H, d, J = 8.3 Hz, H-3',5'), 4.76 (1H, br

173

4-O-Methyl Derivative of 2 (9). An ethereal solution of CH2N2 was slowly added to

174

compound 2 (1.0 mg) dissolved in MeOH (0.2 mL), until a yellow colour was persistent. The

175

reaction mixture was left at room temperature for 24 h. The solvent was evaporated under a N2

176

stream giving an oil residue (1.1 mg). This latter was purified by TLC eluted with CHCl3/i-PrOH

177

(95:5, v/v) to give 9 as uncoloured oil (0.9 mg, Rf 0.8). 9 had: IR νmax 3474, 1599, 1491, 1436 cm-1;

178

UV λmax nm (log ε) 230 (sh), 210 (3.14); 1H NMR see Table 2; ESIMS (+) m/z: 279 [M+Na]+, 257

179

[M+H]+.

180

1,3-O,O'-Diacetyl Derivative of 4 (10). 4-Chloroorcinol (4, 1.0 mg) dissolved in pyridine

181

(10 µL) was acetylated with acetic anhydride (10 µL) at room temperature for 24 h. The reaction

182

was stopped by addition of MeOH, and the azeotrope, obtained by the addition of benzene, was

183

evaporated by an N2 stream. The oily residue (1.2 mg) was purified by preparative TLC eluted with

184

CH2Cl2 yielding derivatives 10 (0.7 mg, Rf 0.75). Derivative 10: IR νmax 1745, 1622, 1595, 1260 ACS Paragon Plus Environment

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cm-1; UV λmax nm (log ε) 282 (2.90); 1H NMR (CDCl3), δ 7.03 (1H, s, H-6), 6.83 (1H, s, H-2), 2.30

186

(3H, s, Me-5), 2.17 (3H, s, AcO-C3), 2.09 (3H, s, AcO-C1); ESIMS (+) m/z: 281 [M + K]+, 265 [M

187

+ Na]+.

188

Crystal Structure Determination of Colletochlorin E (1). Colourless single crystals were

189

obtained at −20°C by slow evaporation of a EtOAc/MeOH/H2O (1:1:2, v/v/v) solution. X-ray data

190

collection was performed at ambient temperature on a Bruker-Nonius KappaCCD diffractometer

191

equipped with a graphite monochromated MoKα radiation (λ= 0.71073 Å, CCD rotation images,

192

thick slices, ϕ and ω scans to fill asymmetric unit). Cell parameters were obtained from a least-

193

squares fit of the θ angles of 356 reflections in the range 4.572° ≤ θ ≤ 22.672°. A semiempirical

194

absorption correction (multi-scan, SADABS) was applied. The structure was solved by direct

195

methods using SIR97 program19 and anisotropically refined by the full matrix least-squares method

196

on F2 against all independent measured reflections using SHELX97 program.20 Solvent

197

crystallization water molecules were found. Hydroxy and water H atoms were located in difference

198

Fourier maps and refined according to a riding model. All the other H atoms were placed in

199

calculated positions and allowed to ride on carrier atoms (C-H in the range 0.9-0.98 Å). Absolute

200

configuration was established by anomalous dispersion effects in the experiment. Crystal data and

201

structure refinement details are reported in Supporting information. CCDC-1495800 (compound 1)

202

contains the supplementary crystallographic data for this paper. These data can be obtained free of

203

charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif

204 205 206

Biological assays. Each metabolite was first dissolved in MeOH (except colletopyrone, dissolved in DMSO) and then diluted with distilled water to the desired concentration.

207

Leaf puncture assay. The compounds were tested at 2 µg/µL on two different plant species,

208

i.e. tomato (Lycopersicon esculentum L.) and Sonchus arvensis. A droplet (20 µL) of solution was

209

applied to detached leaves previously punctured with a needle. Five replications were used for each



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metabolite and for each plant species tested. Leaves were kept in a moistened chamber under

211

continuous fluorescent lights. Symptoms were estimated visually between three to five days after

212

droplet application, by using a visual scale from 0 (no symptoms) to 4 (necrosis wider than 1 cm).

213

Control treatments were carried out by applying droplets containing only water containing the same

214

solvents used to dissolve the metabolites.

215

Lemna minor assay. The compounds were tested on Lemna minor by using a protocol

216

previously described21 at a concentration of 2 µg/µL by using 100 µL per each pot. Macroscopic

217

symptoms, consisting in the necrosis of whole leaves, or in chlorosis, were visually observed after

218

one week incubation. Chlorophyll content was also determined as reported,21 and expressed as

219

percentage reduction in comparison to the untreated fronds.

220

Assay on Phelipanche ramosa seeds. An assay to evaluate the effectiveness of the

221

metabolites to inhibit the germination of seeds of the parasitic weed Phelipanche ramosa was

222

carried out, by used the method previously described.22 One week after the treatment, the

223

germination percentage was determined. Suitable control samples were also prepared for

224

comparison.

225

Antibiosis assay. A disk assay on both one Gram+ (Bacillus subtilis) and one Gram-

226

(Escherichia coli) bacterium was carried out, by following the procedure already described.23

227

Compounds were assayed up to 100 µg per diskette.

228

Zootoxic assay. An assay to evaluate the possible zootoxic activity of the metabolites was

229

performed on Artemia salina (brine shrimps) by following the procedure already described.23

230

Concentration of 0.1 µg/µL solution was used. A suitable negative control was also assayed

231

(solvent in water).

232

233

RESULTS AND DISCUSSION


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11 234

The organic extract obtained from the C. higginsianum culture filtrates, was purified by

235

bioassay-guided fractionation as reported in detail in the Experimental section yielding two new

236

bioactive metabolites named colletochlorins E and F (1 and 2, Figure 1).

237

1 and 2 were isolated together with other three well known fungal metabolites, which were

238

identified comparing their physic (OR) and spectroscopic (IR, UV and 1H NMR spectra) data with

239

those previously reported in literature as colletochlorin A, 4-chloroorcinol and colletopyrone (3, 4

240

and 5, respectively, Figure 1).15-18 Their identification was confirmed from the data of their ESIMS

241

spectra. Indeed, those of 3 and 5 showed the sodium cluster [M + Na]+ and the pseudomolecular ion

242

[M + H]+ at m/z 379 and 357 and 315 and 293, respectively, while that of 4 exhibited the

243

pseudomolecular ions [M + H]+ and [M - H]- at m/z 159 and 157. The 1,3-O,O'-diacetyl derivative

244

of 4 (10, Figure 1) was also prepared by usual acetylation with pyridine and acetic anhydride. Its 1H

245

NMR spectrum essentially differed from that of 4 for the presence of the singlets of the two acetyl

246

groups at δ 2.11 and 2.09, while its ESIMS spectrum showed the potassium [M + K]+ and the

247

sodium [M + Na]+ clusters at m/z 281, and 265, respectively.

248

The first inspection of the 1H and

13

C NMR spectra of colletochlorin E and F (1 and 2,

249

Tables 1 and 2) allowed to notice that they were closely related to each other and with

250

colletochlorin A, 4-chloroorcinol and colletopyrone.

251

Colletochlorin E (1) had a molecular formula C24H30ClO6 as deduced from its HRESI MS

252

spectrum and consistent with nine hydrogen deficiencies. Its IR spectrum showed bands typical of

253

hydroxy, lactone and aromatic groups,24 while its UV spectrum exhibited absorption maxima

254

typical of compounds containing an aromatic ring.25 The investigation of 1H NMR spectrum (Table

255

1) of colletochlorin E (1), the main metabolite, showed the signal system of both a tetrasubstituted

256

α-pyrone and pentasubstituted dihydrobenzofuran. Four singlets typical of a vinyl methylene (H2C-

257

8') and three vinyl methyl groups were observed at δ 3.76 and 2.40 (Me-4''), 2.24 (Me-8) and 1.93

258

(Me-7). In addition a triplet (J = 8.7 Hz), due to the proton of oxygenated secondary carbon to CH-



Page 12 of 26

12

259

2', was observed at δ 4.69. This latter coupled in the COSY spectrum11 with the doublet (J = 8.7 Hz)

260

of the adjacent methylene group (H2C-3') resonating at δ 3.20. Finally, the same spectra showed

261

two singlets for two tertiary methyl groups (Me-2'' and Me-3'') resonating at δ 1.27 and 1.25.25 The

262

data of 13C NMR spectrum (Table 1) and the couplings observed in the HSQC spectrum11 allowed

263

us to assign the signals at δ 91.4, 30.2, 25.7, 25.0, 21.6, 17.2, 17.0 and 10.2 to the protonated

264

carbons C-2', C-3', C-3'', C-2'', C-8', C-8, C-4'' and C-7, respectively. The lactone carbonyl as well

265

the quaternary aromatic carbons of both subunits and C-1'' were assigned on the basis of the

266

couplings observed in the HMBC spectrum.11 In particular, the couplings between C-2 and C-3 with

267

H2-8', C-4 with H2-8' and Me-7, C-5 and C-6 both with Me-7 and Me-8, C-2' with Me-2'' and Me-

268

3'', C-3'a with H2-3', C-4' with H2-8', C-5' and C-6' both with H2-8' and Me-4'', C-7' with Me-4'', C-

269

7'a with H2C-3' and C-1' with Me-2'' and Me-3'' allowed us to assign the signal at δ 169.3, 168.3,

270

157.7, 156.9, 149.6, 137.0, 120.0, 113.2, 109.6, 109.1, 102.4 and 76.4 to C-2, C-4, C-6, C-7'a, C-4',

271

C-6', C-5',C-3'a, C-5, C-7', C-3 and C-1''.26 Thus, the chemical shifts were assigned to all the

272

protons and corresponding carbons of 1 as reported in Table 1. All the described long range

273

couplings also allowed to identify the connections between the two subunits and to location of their

274

substituents.

275 276

Thus, 1 was formulated as 3-[7-chloro-4-hydroxy-2-(1-hydroxy-1-methylethyl)-6-methyl2,3-dihydro-benzofuran-5-ylmethyl]-4-hydroxy-5,6-dimethyl-pyran-2-one.

277

The structure assigned to 1 was further supported by the other couplings observed in the

278

HMBC (Table 1) and from the data of its HRESI MS spectrum. This latter showed the potassium

279

[M + K]+ and the sodium [M + Na]+ clusters and the pseudomolecular ion [M + H]+ at m/z

280

433.0824, 417.1078 and 395.1265, respectively.

281

The structure assigned to 1 was confirmed by preparing some key derivatives.

282

Colletochlorin E was converted into the corresponding 4'-O-methyl derivative (6, Figure 1), by

283

reaction with an ethereal solution of diazomethane. Its IR spectrum still showed a band for hydroxy


Page 13 of 26


13 284

groups while its 1H NMR differed from that of 1 only for the singlet of the methoxy group

285

resonating at δ 4.06 and for the appearance of the signal of H-2' and H2-3' as an ABX system

286

resonating as a broad triplet (J = 9.3) and two double doublets (J = 15.0 and 9.3 Hz) and (J = 15.0

287

and 8.3 Hz) at δ 4.67, 3.25 and 3.16, respectively. This result ruled out the alternative metylation of

288

the phenolic group at C-4. The ESIMS spectrum showed the sodiated dimeric form [2M + Na]+, the

289

potassium [M + K]+ and sodium [M + Na]+ clusters and the pseudomolecual ion [M + H]+ at m/z

290

839, 447 and 431, and 409. By usual acetylation carried out with pyridine and acetic anhydride, 1

291

yielded the corresponding 4'-O-acetyl derivative (7, Figure 1). The IR spectrum showed the

292

significant bands of hydroxy groups while its 1H NMR spectrum differed from that of 1 essentially

293

for the presence of the singlet δ 2.37 due to the acetyl group. Furthermore, the same spectrum

294

showed, as above reported for 6, the presence of the signal of an ABX system, due to H-2' and H2-

295

3', resonating as a broad triplet (J = 9.5 Hz) and two double doublets (J = 16.0 and 8.8 Hz) and (J =

296

16.0 and 9.5) at δ 4.73, 3.18 and 3.13. Its ESIMS spectrum showed the pseudomolecular ion [M +

297

H]+ at m/z 437. Finally, by esterification with p-bromobenzoyl chloride, 1 afforded the

298

corresponding 4'-O-benzoate (8, Figure 1). The IR spectrum of 8 showed still bands typical of

299

hydroxy group while its 1H NMR spectrum differed from that of 1 only for the presence of the

300

couple of doublets (J = 8.3 Hz) of the benzoyloxy residue at δ 8.06 and 7.72. Furthermore, the same

301

spectrum showed the ABX system, due to fragment H-2' and H2-3', resonating as a broad triplet (J

302

= 8.8) and two double doublets (J = 15.8 and 8.4 Hz) and (J = 15.8 and 8.8 Hz) at δ 4.76, 3.18 and

303

3.09. Its ESI MS showed the dimeric sodiated form [2M + H]+, the sodium cluster [M + Na]+ and

304

the psuedomolecular ion [M + H]+ at m/z 1157, 599 and 577 as well as their expected corresponding

305

isotopic peaks due to the presence of bromine [2M + 2 + H]+, [M + 2 + Na]+ and [M + 2 + H]+ at

306

m/z 1159, 601 and 579, respectively .

307

The absolute configuration of 1 was determined by X-ray structure analysis of its colorless

308

block shaped crystals obtained from slow evaporation at -20°C from a EtOAc/MeOH/H2O (1:1:2,

309

v/v/v) solution. ACS Paragon Plus Environment


Page 14 of 26

14 310

An ORTEP view of colletochlorin E was reported in Figure 2. Owing to the strong

311

anomalous scattering of chlorine atoms it was possible to assign the absolute configuration from X-

312

ray data and the absolute S configuration was determined at the unique C1 stereogenic center.

313

Compound 1 crystallizes in the monoclinic P21 space group with one molecule and two

314

crystallization water molecules contained in the asymmetric unit. All bond lengths and angles are in

315

the normal range, crystal data and structure refinement details are reported in Supporting

316

information Table 1S. The molecule consists of a substituted α-pyrone ring joined by a methylene

317

unit to a substituted benzofuran ring system. The benzofurane ring system is approximately planar,

318

the largest deviation from the least-squares plane being 0.1096(48) Å for atom C1. The furan ring

319

adopts a twist conformation on C1-C2, the molecule assumes an overall bent V-shape (Supporting

320

information, Figure 1S). The crystal packing is stabilized by strong intermolecular OH····O

321

hydrogen bonds involving all hydroxy groups and crystallization water molecules in a

322

tridimensional H-bonding pattern (Supporting information, Figure 2S). Thus colletochlorin E (1) was formulated as 3-[7-chloro-4-hydroxy-2(S)-(1-hydroxy-1-

323 324

methyl-ethyl)-6-methyl-2,3-dihydro-benzofuran-5-ylmethyl]-4-hydroxy-5,6-dimethyl-pyran-2-one.

325

Colletochlorin F (2), the minor new metabolite, had a molecular formula C12H15ClO3 as

326

deduced from its HRESI MS and consistent with five hydrogen deficiencies. As reported above its

327

1

H and 13C NMR spectra (Table 2) were close to those of 1 and consistent with the bands typical of

328

hydroxy group and aromatic rings observed in its IR spectrum24 as well as the absorption maxima

329

exhibited in the UV spectrum.25 In particular, the inspection of its 1H and 13C NMR spectra showed

330

system signals very close to those of 1 for the dihydrofuran ring while the benzene ring appeared

331

hexa- instead of penta-substituted for the lacking of the α-pyrone residue. In fact, significant signals

332

for a protonated aromatic carbon (HC-5) were observed at δ 6.22 (s)/110.2 (d). The investigation of

333

its COSY, HSQC and HMBC spectra allowed to assign the chemical shifts to all the protons and the


Page 15 of 26


15 334

corresponding carbons, which are reported in Table 2, and to formulate 2 as 7-chloro-2-(1-hydroxy-

335

1-methylethyl)-6-methyl-2,3-dihydro-benzofuran-4-ol.

336

This structure was supported by all the couplings observed in the HMBC spectrum (Table 2)

337

and from the data of its HRESI MS spectrum. In fact, this latter showed the dimeric sodiated form

338

[2M + Na]+, the potassium [M + K]+ and the sodium [M + Na]+ clusters and the pseudomolecular

339

ion [M + H]+ at m/z 507.1306, 281.0355, 256.0600 and 243.0781.

340

The structure assigned to 2 was confirmed by preparing the corresponding 4-O-metyl ether

341

(9, Figure 1) by reaction of colletochlorin F with an ethereal solution of diazomethane. The IR

342

spectrum of 9 still significantly showed bands for hydroxy and aromatic groups.24 Its 1H NMR

343

spectrum (Table 2) differed from that of 2 only for the expected presence of the methoxy group

344

resonating as singlet at δ 3.79. Significant was also the absence of the broad singlet of the phenolic

345

hydroxy group at C-4 present in the 1H NMR spectrum of 2 at δ 4.68, while the tertiary one (HO-1’)

346

was observed at δ 1.93. Its ESIMS spectrum showed the sodium cluster [M+Na]+ and the

347

pseudomolecular ion [M+H]+ at m/z 279 and 257.

348 349

The S absolute stereochemistry at C-2 was deduced from the staking of their CD spectra (Supporting Information, Figure S3).

350

Assayed on punctured Sonchus arvensis leaves, collethochlorin F (2) caused the fast

351

appearance of quite large necrosis (wider than 1 cm) at a lesser extent, on tomato (Lycopersicon

352

esculentum L.) leaves; compound 4, tested on both plants, proved to be the most active among the

353

compounds assayed.

354

These results were confirmed from those obtained in the assay on Lemna minor, both for the

355

visual estimation as well as with regard to the chlorophyll content reduction. Indeed, compound 4

356

caused very clear chlorosis of the plants 1 week after the treatment, corresponding to over 50%

357

reduction in chlorophyll content, whereas 2 caused only a modest chlorosis. Similar results were

358

observed in the bioassay on Phelipanche ramosa seed germination. In this case, however, probably



Page 16 of 26

16 359

due a higher sensitivity of the biological material, both 2 and 4-chloroorcinol caused the complete

360

inhibition of seed germination at the concentration tested. Moreover, also 1 was active, even if less,

361

allowing to only around 40% seeds to germinate, whereas 3 and colletopyrone were very modestly

362

active.

363 364

Interestingly, the phytotoxicity was not associated to an antibiotic activity, as all the compounds were inactive both on the Gram+ and Gram- bacteria used in the antibiosis assay.

365

Opposite to the results on phytotoxicity, in the zootoxic assay the most powerful compound

366

proved to be 4-chloroorcinol, which caused over 80% larval mortality, whereas colletochlorin F

367

caused around 40% mortality. All the other compounds tested were inactive.

368

The results of the leaf-puncture assays carried out on tomato and S. arvensis showed that the

369

phytotoxic activity could be affected by the presence of a suitable substituted orcinol moiety being

370

4-chloroorcinol (4) and colletochlorin F (2), very active. These results were confirmed when the

371

same compounds were tested using the same method on L. minor leaves and the inhibition of seed

372

germination of P. ramosa.

373

In conclusion, this manuscript reports on the isolation of a new tetrasubstituted α-pyrone

374

and a new tetrasubstituted benzofuran, named colletochlorins E and F, together with the well known

375

colletochlorin A, 4-chloroorcinol and colletopyrone, metabolites with potential herbicidal activity

376

produced for the first time by C. higginsianum. The structures of colletochlorins E and F

377

substantially differed form those of both colletochlorins and colletorins isolated from C. tabacum.3

378

Colletochlorin E and F, as well as colletochlorin A belong to the class of naturally occurring 2-

379

prenyl orsellinaldehydes bearing a chlorine attached to the aromatic ring.3 However colletochlorins

380

E and F showed only an isoprenyl unit attached to the cited aromatic ring rearranged into a dihydro

381

furan ring and so that generating its benzofuran skeleton which in colletochlorin E is joined through

382

a methylene bridge to a trisubstituted pyran-2 one. The orcinol moiety appear to be the structural

383

factor that imparts phytotoxicity.


Page 17 of 26


17 384

ASSOCIATED CONTENT

385

Corresponding author.

386

∗

(A.E.) Tel.: +39 081 2539178. E-mail: [email protected]

387

Notes

388

The authors declare no competing financial interest.

389

FUNNDING

390

This research was carried out in part supported by Department of Chemical Science, University of

391

Naples Federico II.

392

ACNOWLEDGMENTS

393

A.E. is associated with Istituto di Chimica Biomolecolare del CNR, Pozzuoli.

394



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18 395

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396

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Dean, R.; Van Kan, J. A. L.; Pretorius, Z. A.; Hammond-Kosack, K. E.; Di Pietro, A.;

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García-Pajón, C. M.; Collado, I. G. Secondary metabolites isolated from Colletotrichum species. Nat. Prod. Rep. 2003, 20, 426-431, and references therein cited.

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Damme, M.; Altomare, C.; Mathieu, V.; Kiss, R.; Evidente, A. Investigations of fungal

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herbicidal activity to control Chenopodium album. Nat. Prod. Comm. 2015, 10, 119-1126.

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diterpenoid α-pyrones produced by Colletotrichum higginsianum, with in vitro cytostatic

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activity. J. Nat. Prod. 2016, 79, 116-125.

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Berger, S.; Braun, S. 200 and More Basic NMR Experiments: a Practical Course; Wiley-

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Dumas, B. A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of

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Bauser, C. A.; Becker, C.; Birren, B. W.; Chen, Z.; Choi, J.; Crouch, J. A.; Duvick, J. P.;

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Farman, M. A.; Gan, P.; Heiman, D.; Henrissat, B.; Howard, R. J.; Kabbage, M.; Koch, C.;

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Kracher, .; Kubo, Y.; Law, A. D.; Lebrun, M.-H.; Lee, Y.-H.; Miyara, I.; Moore, N.;

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Neumann, U.; Nordström, K.; Panaccione, D. G.; Panstruga, R.; Place, M.; Proctor, R. H.;

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Prusky, D.; Rech, G.; Reinhardt, R.; Rollins, J. A.; Rounsley, S.; Schardl, C. L.; Schwartz,

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Takano, Y.; Takahara, H.; Trail, F.: van der Does, H. C.; Voll, L. M., Will, I., Young, S.,

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Zeng, Q., Zhang, J., Zhou, S., Dickman, M. B., Schulze-Lefert, P., Ver Loren van Themaat,

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E., Ma, L.-G., Vaillancourt, L. J. Lifestyle transitions in plant pathogenic Colletotrichum

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Kosuge, Y.; Suzuki, A.; Hirata, S.; Tamura, S. Structure of colletochlorin from Colletotrichum nicotianae. Agr. Biol. Chem. 1973, 37, 455-456.

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Pinkerton, F; Strobel, G. Serinol as an activator of toxin production in attenuated cultures of

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Kosuge, Y.;

Suzuki, A.; Tamura. S. Structures of colletochlorin C, colletorin A and

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Monde, K.; Satoh, H.; Nakamura, M; Tamura, M.; Takasugi, M. Organochlorine compounds

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from a terrestrial higher plant: structures and origin of chlorinated orcinol derivatives from

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diseased bulbs of Lilium maximowiczii. J. Nat. Prod. 1998, 61, 913-921.

450

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colletopyrone from Colletotrichum nicotianae. Agric. Biol. Chem. 1976, 40, 1453-1455.

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Gohbara, M.; Hyeon, S. B.; Suzuki, A.; Tamura, S. Isolation and structure elucidation of

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Altomare, A.; Burla., M. C; Camalli, M.; Cascarano, G.L.; Giacovazzo, C.; Guagliardi, A.;

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Moliterni, A. G. G.; Polidori, G.; R. Spagna. SIR97: a new tool for crystal structure

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determination and refinement. J. Appl. Crystallogr. 1999, 32, 115-119.

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Sheldrick, G. M. A short history of SHELX. Acta Crystallogr., Sect. A, 2008, A64, 112-122.

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Cimmino, A.; Andolfi, A.; Zonno, M. C.; Troise, C.; Santini, A.; Tuzi, A.; Vurro, M.; Ash,

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G.; Evidente, A. Phomentrioloxin: a phytotoxic pentasubstituted geranylcyclohexentriol

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produced by Phomopsis sp., a potential mycoherbicide for Carthamus lanatus biocontrol. J.

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Nat. Prod. 2012, 75, 1130-1137.

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Vurro, M.; Boari, A.; Pilgeram, A. L.; Sands, D. C. Exogenous amino acids inhibit seed

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germination and tubercle formation by Orobanche ramosa (broomrape): potential

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application for management of parasitic weeds. Biol. Control 2006, 36, 258-265.

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Pretsch, E.; Bühlmann, P.; Affolter, C. Structure Determination of Organic Compounds – Tables of Spectral Data; Springer-Verlag: Berlin, 2000; pp 161-243.

468 469

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466 467

Bottalico, A.; Capasso, R.; Evidente, A.; Randazzo, G.; Vurro, M. Cytochalasins: structure-

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471

472 ACS Paragon Plus Environment

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21 473 474

475 476 477 478 479

Table 1. 1H, 13C NMR and HMBC Data of Colletochlorin E (1)a,b in CD3OD. HMBC δCc δH (J in Hz) 2 169.3 C H2-8′ 3 102.4 C H2-8′ 4 168.3 C H2-8′, Me-7 5 109.6 C Me-7, Me-8 6 157.7 C Me-7, Me-8 7 10.2 CH3 1.93 (3H) s 8 17.2 CH3 2.24 (3H) s 2′ 91.4 CH 4.69 (1H) t (8.7) Me-2″, Me-3″ 3′ 30.2 CH2 3.20 (2H) d (8.7) 3′a 113.2 C H2-3′ 4′ 149.6 C H2-8′ 5′ 120.0 C H2-8′, Me-4″ 6′ 137.0 C H2-8′, Me-4″ 7′ 109.1 C Me-4″ 7′a 156.9 C H2-3′ 8′ 21.6 CH2 3.76 (2H) s 1″ 76.4 C Me-2″, Me-3″ 2″ 25.0d CH3 1.27 (3H) sd Me-3″ 3″ 25.7d CH3 1.25 (3H) sd Me-2″ 4″ 17.0 CH3 2.40 (3H) s a The chemical shifts are in δ values (ppm) from TMS. b2D 1H, 1H (COSY) 13C, 1H (HSQC) NMR experiments delineated the correlations of all the protons and the corresponding carbons. cMultiplicities were assigned by DEPT spectrum. dThese signals could be exchangeable.

480



Page 22 of 26

22 481 482

483 484 485 486 487

Table 2. 1H, 13C NMR and HMBC Data of Colletochlorin F (2)a,b and 1H NMR Data of Its 4-O-Methyl Derivative (9) in CDCl3. 2 9 c HMBC δC δH (J in Hz) δH (J in Hz) 2 90.3 CH 4.73 (1H) t (9.2) H2-3, Me-2′, Me-3′ 4.71 (1H) t (8.6) 3 27.4 CH2 3.14 (2H) d (9.2) H-2 3.13 (2H) d (8.6) 3a 111.4 C H2-3 4 157.6 C H2-3 5 110.2 CH 6.22 (1H) s Me-4′ 6.29 (1H) s 6 136.9 C Me-4′ 7 108.6 C H-5 7a 150.3 C H2-3 1′ 71.3 C H2-3, Me-2′, Me-3′ 2′ 22.9 CH3 1.23 (3H) s Me-3′ 1.21 (3H) s 3′ 28.9 CH3 1.37 (3H) s Me-2′ 1.35 (3H) s 2.27 (3H) s H-5 2.33 (3H) s 4′ 18.9 CH3 HO-1′ 1.95 (1H) br s 1.93 (1H) br s HO-4 4.68 (1H) br s OMe 3.79 (3H) s a b 1 1 The chemical shifts are in δ values (ppm) from TMS. 2D H, H (COSY) 13C, 1H (HSQC) NMR experiments delineated the correlations of all the protons and the corresponding carbons. cMultiplicities were assigned by DEPT spectrum.


Page 23 of 26


23 488

Figure Legend

489

Figure 1. Structures of colletochlorins E, F and A (1-3), 4-Chloroorcinol (4), colletopyrone (5),

490

colletochlorin E derivatives (6-8) and 4-O-metyl colletochlorin F (9), and 1,5-O,O'-diacetyl of 4

491

(10).

492

Figure 2. ORTEP view of colletochlorin E (1) with thermal ellipsoids drawn at 30% probability

493

level.

494



Page 24 of 26

24 495

Figure 1 3''

OH

2'

1''

2''

O

Cl

1

O

2

6 5

8

Cl

7' 3'a 4' 6'

4''

OR2

5'

4'

7a

O1

7 6 5

3

OH

2

1'

2'

7'a 1' 3'

O

3'

4

3 3a

OR

8' 4

Colletochlorin F, 2 R=H 4-O-Methyl derivative of 2, 9 R=Me

OR1

7

Colletochlorin E, 1 R1=R2=H 4'-O-Methyl derivative of 1, 6 R1=H, R2=Me 4'-O-Acetyl derivative of 1, 7 R1=H, R2=Ac 4'-O-p-Bromobenzoyl derivative of 1, 8 R1=H, R2=p-BrC6H5CO OH

OR

OH

Cl

Cl OH

OH

OR

CHO

4-Chloroorcinol, 4 R=H 1,3-O,O'-Diacetyl derivative of 4, 10 R=Ac

Colletochlorin A, 3 O

HO

O O OH O

496

Colletopyrone, 5

497


Page 25 of 26


25 498

Figure 2

499

500 501



Page 26 of 26

26 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516

Table of Content Graphic

Colletochlorins E and F, New Phytotoxic Tetrasubstituted Pyran-2one and Dihydrobenzofuran, Isolated from Colletotrichum higginsianum with Potenti al Herbicidal Activity Marco Masi, Alessio Cimmino, Angela Boari, Angela Tuzi, Maria Chiara Zonno, Riccardo Baroncelli, Maurizio Vurro and Antonio Evidente*† OH O OH

Cl OH

O O OH

O Cl OH


Colletochlorins E and F, New Phytotoxic Tetrasubstituted Pyran-2

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