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New Fusaric Acid Derivatives from the Endophytic Fungus Fusarium oxysporum and Their Phytotoxicity to Barley Leaves Shuai Liu, hao-fu Dai, Raha Orfali, Wenhan Lin, Zhen Liu, and Peter Proksch J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00219 • Publication Date (Web): 06 Apr 2016 Downloaded from http://pubs.acs.org on April 6, 2016

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Journal of Agricultural and Food Chemistry

1

New Fusaric Acid Derivatives from the Endophytic Fungus

2

Fusarium oxysporum and Their Phytotoxicity to Barley Leaves

3 4

Shuai Liu,† Haofu Dai,‡ Raha S. Orfali,§ Wenhan Lin,# Zhen Liu,*,† Peter

5

Proksch*,†

6 7

†

8

Universitätsstrasse 1, 40225 Düsseldorf, Germany

9

‡

Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-Universität Düsseldorf,

Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture,

10

Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural

11

Sciences, Haikou 571101, China

12

§

13

Arabia

14

#

15

China

Department of Pharmacognosy, Faculty of Pharmacy, King Saud University, Riyadh, Saudi

State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191,

16 17

*

18

(Tel: +49 211 81 15979; Fax: +49 211 81 11923; E-mail: [email protected]).

19

(Tel: +49 211 81 14163; Fax: +49 211 81 11923; E-mail: [email protected]).

Corresponding authors

1

ACS Paragon Plus Environment


20

ABSTRACT:

21

Chemical investigation of the endophytic fungus Fusarium oxysporum isolated

22

from fruits of Drepanocarpus lunatus afforded eight new fusaric acid derivatives

23

fusaricates A-G, 1−7, and 10-hydroxy-11-chlorofusaric acid, 8, along with four

24

known compounds. Their structures were elucidated by one- and two-dimensional

25

NMR as well as MS data, and by comparison with the literature. The absolute

26

configurations of fusaricates C-E, 3-5, were determined using chiral GC-MS.

27

Fusaricates A-G, 1-7, represent the first examples of fusaric acid linked to a

28

polyalcohol moiety via an ester bond. All isolated fusaric acid derivatives 1−8

29

showed significant phytotoxicity to leaves of barley.

30 31

KEYWORDS: Fusarium oxysporum, fusaricates A-G, structural elucidation,

32

phytotoxicity

2


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INTRODUCTION

34

Endophytes are defined as microorganisms, including bacteria and fungi that

35

live within plant tissues for all or part of their life cycle without causing any visible

36

symptoms of their presence such as diseases.1-2 The relationships between plants and

37

their endophytes are complex and not well understood in all aspects. It is assumed

38

that plants survive and flourish in stressed ecosystems due to their endophytes which

39

have co-evolved and are essential for their adaptation to changing environments.3 It

40

has also been shown that endophytes influence the growth of their host plants by

41

manipulating phytohormones such as indole-3-acetic acid.4 As an important source

42

of natural products, endophytic fungi provide a wide range of bioactive compounds

43

with anticancer, antibacterial, antiviral and immunosuppressive activities.5

44

Fusarium is a genus of filamentous fungi that produce diverse bioactive

45

secondary metabolites, including beauvericin which possesses antibacterial and

46

insecticidal activities, trichothecenes which are strongly associated with chronic and

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fatal toxic effects in animals or humans, and gibberellins which are plant hormones.6

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Fusaric acid (5-butylpyridinecarboxylic acid) is a typical secondary metabolite of

49

members of this genus showing various bioactivities such as inhibition of

50

dopamine-β-hydroxylase in vitro at 10-8 M.7-8 A recent evaluation of the

51

phytotoxicity of fusaric acid analogues underlined their potential as bioherbicides for

52

organic farming.9-10

53

As part of our ongoing studies on bioactive natural products from endophytic

54

fungi,11-14 Fusarium oxysporum which was isolated from fresh and healthy fruits of 3



55

the mangrove plant Drepanocarpus lunatus (Fabaceae) growing in Cameroon was

56

investigated. Eight new fusaric acid derivatives and four known compounds were

57

isolated. Herein, we report isolation and structural elucidation of the new compounds,

58

as well as their phytotoxic and cytotoxic activities.

59

MATERIALS AND METHODS

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General Method. Optical rotation values were measured by a P-1020

61

polarimeter (JASCO, Tokyo, Japan). NMR spectra were recorded on a Bruker

62

Avance III 600 spectrometer (Bruker, Karlsruhe, Germany). HRESIMS data were

63

obtained from an UHR-QTOF maxis 4G mass spectrometer (Bruker Daltonics,

64

Bremen, Germany). HPLC analysis was conducted by a Dionex P580 system

65

(Dionex Softron, Germering, Germany) with a 125 mm × 4 mm i.d., 5 µm,

66

Eurospher C18 (Knauer, Berlin, Germany) column. Silica gel 60 F254 pre-coated

67

aluminium plates (Merck, Darmstadt, Germany) were used for thin layer

68

chromatography under detection at 254 and 365 nm. Silica 60M (Macherey-Nagel,

69

Dueren, Germany) was applied to column chromatography. The final purification of

70

compounds was performed on a Merck-Hitachi Lachrom semi-preparative RP-HPLC

71

system (Merck, Darmstadt, Germany) with a 300 mm × 8 mm i.d., 10 µm, Eurospher

72

C18 column (Knauer, Berlin, Germany). The analyses of chiral polyalcohols were

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carried out on a GC-MS QP-2010 Plus instrument (Shimadzu, Tokyo, Japan),

74

equipped with a 50 m × 0.25 mm i.d., 0.25 µm, FS-CYCLODEX beta-I/P column

75

(Chromatographie Service GmbH, Langerwehe, Germany). The chiral polyalcohol

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standards were acquired as follows: erythritol and xylitol (Alfa Aesar GmbH, 4


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Karlsruhe, Germany), L-threitol (TCI GmbH, Eschborn, Germany), adonitol and

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D-threitol

79

(Acros Organics, Geel, Belgium).

(Sigma-Aldrich Chemie GmbH, Steinheim, Germany), D- and L-arabitol

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Fungal Material and Identification. This fungus was isolated from fresh fruits

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of Drepanocarpus lunatus (Fabaceae) collected in August 2013 from Douala in

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Cameroon. Isolation of fungal strain was achieved according to standard procedure

83

as described before.15 Based on DNA amplification and sequencing of the ITS

84

region,16 the fungus was identified as F. oxysporum (GenBank accession number

85

KT373853). A voucher strain (Code DL-F-3-1) was deposited in one of the authors’

86

lab (P. P.).

87

Fermentation, Extraction, and Isolation. The fermentation was performed in

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Erlenmeyer flasks (2 × 1L) on solid rice medium containing 100 g rice, 3.5 g sea salt

89

and 110 mL demineralized water. After autoclaving at 121 °C for 20 min and then

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cooling to room temperature, each flask was inoculated and then incubated at 20 °C

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under static conditions. After 30 days, the fermentation was stopped by adding 500

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mL of EtOAc to each flask. The extraction was completed after shaking the flasks on

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a laboratory shaker at 150 r/min for 8 h.

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Evaporation of the EtOAc solution gave a brown extract (1.5 g), which was

95

subjected to vacuum liquid chromatography upon a 15 cm × 8 cm i.d. silica gel

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column using gradient solvent system (n-hexane/EtOAc, 10:0, 9:1, 7:3, 1:1, 3:7;

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CH2Cl2/MeOH, 9:1, 7:3, 1:1, 0:10; 500 mL each gradient) to obtain 9 fractions (Fr.

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A-I). Fr. D (82.5 mg) was subjected to a Sephadex LH-20 column using 100% 5



99

MeOH as mobile phase to remove pigments and further purified by semi-preparative

100

HPLC with MeOH-H2O as eluting system (0-5 min 55% MeOH, 5-15 min from 55%

101

to 64% MeOH, 15-18 min 100% MeOH) to yield pestalotiollide A (1.6 mg),

102

pestalotiollide B (8.8 mg) and dehydroisopenicillide (1.4 mg). Following a similar

103

purification process, Fr. E (116.1 mg) yielded beauvericin (53.5 mg) (HPLC

104

sequence: 0-3 min 80% MeOH, 3-11 min from 80% to 95% MeOH, 11-13 min

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100% MeOH), Fr. F (197.0 mg) afforded 1 (3.9 mg), 3 (2.0 mg), a mixture of 4 and 5

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(12.4 mg) and a mixture of 6 and 7 (5.4 mg) (HPLC sequence: 0-5 min 40% MeOH,

107

5-18 min from 40% to 57% MeOH, 18-22 min 100% MeOH), while Fr. G (152.9 mg)

108

gave 2 (1.9 mg) and 8 (3.2 mg) (HPLC sequence: 0-3 min 20% MeOH, 3-17min

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from 20% to 60% MeOH, 17-21 min 100% MeOH).

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Determination of the Absolute Configuration of Polyalcohols in 3-5. 0.7 mg

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of each sample was hydrolyzed with 2N HCl (1.0 ml) at 90 °C for 2 h. After

112

evaporation in a freeze dryer overnight, the hydrolysis products were treated with

113

trifluoroacetic anhydride (200 µL) and DMAP (0.1 mg) in dichloromethane (200 µL).

114

Trifluoroacetylation of polyalcohols was performed by heating the mixture at 80 °C

115

for 10 minutes and the product was subsequently analyzed by gas chromatography

116

under the following conditions. The column temperature was set at 80 °C and

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ramped to 200 °C at a rate of 2 °C/min. In co-chromatography experiments with D-

118

or L- arabitol, the column temperature were set at 80 °C, ramped to 120 °C at a rate

119

of 2 °C/min and then to 200 °C at a rate of 1 °C/min. The temperature of the injector

120

and detector were set at 200°C. Helium was used as carrier gas with a flow rate of 30 6


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cm/min.

122

Fusaricate A, 1: white amorphous powder; [α]20D +7.0 (c 0.55, EtOH); UV

123

(MeOH) λmax (log ε) 233 (4.11), 270 (3.83) nm; 1H and 13C NMR data, see Table 1;

124

HRESIMS m/z 254.1384 [M + H]+ (calcd for C13H20NO4, 254.1387).

125

Fusaricate B, 2: light yellow solid; [α]20D +7.1 (c 0.48, MeOH); UV (MeOH)

126

λmax (log ε) 233 (4.13), 270 (3.86) nm; 1H and

13

127


C NMR data, see Table 1;

128

Fusaricate C, 3: white amorphous powder; [α]20D -26.9 (c 0.10, acetone); UV

129


130


131

Mixture of fusaricate D, 4, and fusaricate E, 5: white amorphous powder; UV

132


133


134

Mixture of fusaricate F, 6, and fusaricate G, 7: light yellow solid; UV (MeOH)

135

λmax (log ε) 232 (4.21), 271 (3.92) nm; 1H and

13

136


C NMR data, see Table 2;

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10-Hydroxy-11-chlorofusaric acid, 8: white amorphous powder; [α]20D +30.7 (c

138

0.80, MeOH); UV (MeOH) λmax (log ε) 222 (3.78), 272 (3.88) nm; 1H and 13C NMR

139

data, see Table 3; HRESIMS m/z 230.0577 [M + H]+ (calcd for C10H13ClNO3,

140

230.0578).

141

Phytotoxicity Assay. Phytotoxicity was investigated according to the cotton

142

cotyledonary leaf bioassay described by Stipanovic et al.9 Barley plants ('Bonus', a 7



143

Swedish cultivar) were inoculated 15 days after planting and scored 48 h after

144

inoculation. A pipette tip was used to scratch the surface of barley leaf, and then the

145

test solution (20 µL) was placed at three locations on each leaf. Different

146

concentrations (0.5−8.0 mM) of test compounds were prepared by dissolving the

147

compounds with distilled water. Commercial fusaric acid and distilled water were

148

used as positive and negative control, respectively.

149

Cell Proliferation Assay. MTT method was performed to test cytotoxicity

150

against L5178Y mouse lymphoma cell line.17 Experiments were carried out for three

151

times with Kahalalide F as positive control and media with 0.1 % EGMME-DMSO

152

as negative control.

153

RESULTS AND DISSCUSSION

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Compound 1 was obtained as a white amorphous powder. Its molecular formula

155

was established as C13H19NO4 by HRESIMS, indicating five degrees of unsaturation.

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Its UV spectrum showed characteristic peaks of fusaric acid analogs at λmax 233 and

157

270 nm.18 The 1H NMR spectrum (Table 1) exhibited three aromatic protons typical

158

for an ABX coupling system at δH 8.54 (H-6, d, J = 2.2 Hz), δH 8.13 (H-3, d, J = 8.0

159

Hz) and δH 7.86 (H-4, dd, J = 8.0, 2.2 Hz), suggesting the presence of a

160

2,5-substituted pyridine ring, which was confirmed by the HMBC correlations from

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H-3 to C-5 (δC 144.6), from H-4 to C-2 (δC 146.3) and C-6 (δC 150.7), and from H-6

162

to C-2 and C-4 (δC 138.9). A butyl group at C-5 was deduced from the COSY

163

correlations between H2-8 (δH 2.76, t)/H2-9 (δH 1.66, qui), H2-9/H2-10 (δH 1.40, sext),

164

and H2-10/H3-11 (δH 0.97, t), together with the HMBC correlations from H2-8 to C-4, 8


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C-5 and C-6. According to the HSQC spectrum, five proton signals at δH 4.46 (Ha-1',

166

dd, J = 11.3, 3.8 Hz), 4.33 (Hb-1', dd, J = 11.3, 6.6 Hz), 3.99 (H-2', ddt, J = 6.6, 3.8,

167

5.7 Hz), and 3.65 (2H, H2-3', d, J = 5.7 Hz) were assigned to three oxygenated

168

carbons at δC 68.1 (C-1'), 71.1 (C-2'), and 63.9 (C-3'), which were attributed to a

169

glycerol unit, as further supported by the COSY correlations between Ha-1'/H-2',

170

Hb-1'/H-2', and H-2'/H2-3'. An ester linkage between C-2 of the pyridine ring and

171

C-1' of the glycerol unit was confirmed by key HMBC correlations from H-3 and

172

H2-1' to C-7 (δC 166.0). Thus, the structure of 1 was identified as a new fusaric acid

173

derivative named fusaricate A (Figure 2). By comparing its optical rotation value

174

{[α]20D +7.0, (c 0.55, EtOH)} with those of similar known compounds such as

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(R)-1-benzoyloxypropane-2,3-diol {[α]20D -15.9, (c 1, EtOH)} and (S)-1-

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benzoyloxypropane-2,3-diol

177

configuration of fusaricate A was proposed to be S.

{[α]20D

+15.8,

(c

1,

EtOH)}19,

the

absolute

178

The HRESIMS spectrum of fusaricate B, 2, indicated the molecular formula

179

C13H19NO5, which contains an additional oxygen atom compared to 1. The NMR

180

data (Table 1) of 2 were closely related to those of 1, except for the presence of

181

signals of a hydroxymethine group (δC 67.6, δH 3.75) in 2. The proton of this

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hydroxymethine exhibited correlations with H2-9 (δH 1.77, m) and H3-11 (δH 1.21, d)

183

in the COSY spectrum, indicating fusaricate B, 2, to be a 10-hydroxy derivative of

184

fusaricate A, 1. Attempts to determine the absolute configuration of C-10 were

185

unsuccessful due to the insufficient amount of this sample.

186

Fusaricate C, 3, was obtained as a white amorphous powder and its UV 9



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spectrum was almost identical to that of fusaricate A (1). The molecular formula of 3

188

was determined to be C14H21NO5 by HRESIMS, implying additional 30 Da

189

compared to 1. Comparison of the NMR data (Table 1) indicated the gross structure

190

of 3 to be similar to that of 1, except for the replacement of the glycerol unit of 1 by

191

a tetritol unit. This was confirmed by the COSY correlations between H2-1' (δH 4.60,

192

dd; 4.41, dd)/H-2' (δH 3.90, ddd), H-2'/H2-3' (δH 3.67, m) and H-3'/H2-4' (δH 3.81, m;

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3.66, m), as well as by the HMBC correlations from H2-1' to C-7 (δC 166.0).

194

Moreover, the tetritol unit was determined to be erythritol using chiral GC-MS after

195

hydrolysis of the compound.20 By comparing the optical rotation of 3 {[α]20D -26.9,

196

(c

197

(2R,3S)-montagnetol {[α]20D +11, (c 0.4, acetone)}21 and (2S,3R)-montagnetol

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{[α]20D -10.1, (c 0.5, acetone)},22 the absolute configuration of 3 is suggested to be

199

2'S, 3'R.

0.10, acetone)} with

those

of

the

two

related

known

compounds

200

Fusaricates D and E, 4 and 5, were obtained as a pair of inseparable

201

diastereomers with a ratio of 1:1 as deduced by 1H NMR. Both compounds shared

202

the molecular formula C15H23NO6 as determined by HRESIMS data. By comparing

203

their 1H and 13C NMR data (Table 2) with those of 1 and 3, the fusaric acid partial

204

structure of N-1 to C-11 could be easily identified. The additional fourteen proton

205

signals detected between δH 4.70−3.50 were assigned to ten oxygenated carbons

206

between δC 73.0−64.0 according to the HSQC spectrum. A close inspection of the

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COSY and HMBC spectra revealed the presence of two pentitol units and their

208

attachments to C-7 through ester linkages. The chiral GC-MS method20 was used to 10


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determine the absolute configurations of pentitol following hydrolysis of 4 and 5.

210

Both 4 and 5 contained D-arabitol, which is also found in the fungi Gilocladium

211

roseum,20 Gliocladium catenulatum23 and Clonostachys candelabrum.24 The

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coupling constants of 4 and 5 were 1.7 and 8.6 Hz between H-2'/H-3', and 8.5 and

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1.5 Hz between H-3'/H2-4', respectively. Thus, fusaricates D and E, 4 and 5, were

214

determined to have the absolute configurations 2'R, 3'R, 4'R and 2'R, 3'S, 4'R,

215

respectively. It could be assumed that esterification of the fusaric acid moiety with

216

the different terminal hydroxy group of D-arabitol during biosynthesis led to

217

3'-epimers 4 and 5.

218

Fusaricates F and G, 6 and 7, were isolated as a mixture of stereoisomers with a

219

ratio of 1:1. Attempts to separate both compounds were unsuccessful. The

220

HRESIMS spectrum of 6 and 7 showed the pseudomolecular ion peak indicating the

221

molecular formula C15H21NO6. The 1H NMR data were very similar to those of 4 and

222

5. The disappearance of the signals for a methyl and a methylene group, together

223

with the detection of three additional olefinic protons at δH 5.85 (H-10, m), 5.01

224

(Ha-11, d, J = 17.1 Hz) and 4.99 (Hb-11, d, J = 9.8 Hz) in the 1H NMR spectrum of 6

225

and 7 suggested the presence of a double bond at C-10/C-11 in the latter two

226

compounds. This was confirmed by the COSY correlations between H2-11/H-10,

227

H-10/H2-9 (δH 2.44, m), and H2-9/H2-8 (δH 2.86, m). Except for the presence of a

228

double bond the structures of 6 and 7 including the pyridine ring, a D-arabitol unit

229

and an ester linkage were identical to those of 4 and 5 as evident from detailed

230

examination of 1D and 2D NMR. Combined with analysis of the coupling constants, 11



231

the structures of 6 and 7 were elucidated as shown. The molecular formula of 8 was established as C10H12ClNO3 by HRESIMS. Its

232 233

1

234

The obvious difference between both compounds was that C-11 (δC 50.0, CH2) of 8

235

was shifted upfield by approximately 17 ppm compared to 10,11-dihydroxyfusaric

236

acid. These findings indicated the attachment of the chlorine atom to C-11 in 8

237

instead of a hydroxy group as present in 10,11-dihydroxyfusaric acid. Thus,

238

compound 8 was elucidated as 10-hydroxy-11-chlorofusaric acid. Attempts to

239

determine its absolute stereochemistry using Mosher’s method were unsuccessful

240

because it was unstable under Mosher’s reaction condition. It should be noted that

241

compound 8 could not be detected in HPLC-MS analysis of the crude extract when

242

growth medium without sea salt was used for fermentation. Therefore, sea salt added

243

in the growth medium may be the source of chlorine in 8.

H and

13

C NMR data (Table 3) resembled those of 10,11-dihydroxyfusaric acid.25

244

The known compounds were identified as pestalotiollides A and B26,

245

dehydroisopenicillide27, and beauvericin28 by comparison of their spectroscopic data

246

with those in the literature.

247

The cytotoxicity of compounds 1−8 against L5178Y mouse lymphoma cells

248

was evaluated using the MTT assay. However, only 2 showed weak cytotoxicity with

249

an IC50 value of 37.7 µM. Compounds 1−8 were further evaluated for their

250

phytotoxicity against barley leaves ('Bonus', a Swedish cultivar) (Table 4). The

251

phytotoxicity of compounds 1, 4, 5 and 8 were found to be almost equal to that of

252

commercial fusaric acid, suggesting their promising potential in organic farming. 12


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Comparison of the phytotoxicity of fusaricate A, 1, vs. B, 2, suggested that the

254

presence of the hydroxyl group at C-10 led to decreased phytotoxicity. The presence

255

of a double bond between C-10 and C-11 also decreased phytotoxicity as evident

256

from comparision of 4 and 5 vs. 6 and 7.

257

Phytotoxicity of fusaric acid derivatives is non-specific and harmful to both

258

crops and weeds.29 Their potential use as herbicides and application in agriculture

259

have been discussed.10 We described this strain F. oxysporum as symptomless

260

endophyte because it did not cause any visible symptoms to the host plant, which is

261

similar to the relationship between F. verticillioides and maize.30-31 The ecological

262

role of fusaric acid derivatives could lie in the maintainance of an equilibrium

263

between host and endophyte. For the host plant, the production of fusaric acid

264

derivatives by endophyte is probably harmful whereas the major fungal component

265

beauvericin (around 3.6% of the EtOAc extract) may be beneficial due to its

266

insecticidal activity.32 The true nature of interaction between this endophytic fungus

267

and its host plant remains to be elucidated.

268

13

C and 14C isotopic tracer studies revealed that C-5, C-6, C-8, C-9, C-10, and

269

C-11 of fusaric acid were derived from 3 units of acetate, while C-2, C-3, C-4, C-7

270

were derived from oxaloacetate.33-34 It has been proved that the nitrogen atom

271

present in the pyridine ring originates mainly from glutamine.35 According to a

272

recent report, a 12-gene biosynthetic gene cluster was involved in the biosynthesis of

273

fusaric acid.36 The esterification of fusaric acid and the polyalcohols could be

274

catalysed by lipases similar to Novozyme 435 from Candida antarctica.37 In 13



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275

summary, a proposed biosynthetic pathway for the fusaric acid derivatives as found

276

in this study is presented in Figure 3. Fusaricates A-G, 1-7, represent the first

277

examples of fusaric acid linked to a polyalcohol moiety via an ester bond.

278

ASSOCIATED CONTENT

279

Supporting Information

280

GC-MS analyses of polyalcohol standards and degraded samples from compounds

281

3−5, UV, HRESIMS and NMR spectra of compounds 1−8, and phytotoxicity assay

282

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

283

AUTHOR INFORMATION

284

Corresponding Authors

285

*

286

*

287

ACKNOWLEDGMENTS

288

Financial support by China Scholarship Council to S. L. is gratefully acknowledged.

289

P. P. wishes to thank the Manchot Foundation for support. The authors acknowledge

290

the assistance and collaboration of Ansgar Rühlmann (Institute of Biochemistry,

291

Heinrich-Heine-Universität Düsseldorf, Germany) for carrying out GC-MS

292

experiments.

293

(Heinrich-Heine-Universität Düsseldorf, Germany) for collection of the plant

294

material.

Tel: +49 211 81 15979; Fax: +49 211 81 11923; E-mail: [email protected]. Tel: +49 211 81 14163; Fax: +49 211 81 11923; E-mail: [email protected].

We

furthermore

wish

to

thank

14


Sergi

Herve

Akone

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(3) Chadha, N.; Mishra, M.; Rajpal, K.; Bajaj, R.; Choudhary, D. K.; Varma, A. An

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ecological role of fungal endophytes to ameliorate plants under biotic stress.

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Arch. Microbiol. 2015, 197, 869−881.

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20


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Figure Captions Figure 1. Structures of new fusaric acid derivatives isolated from F. oxysporum. Figure 2. COSY (—) and key HMBC (

) correlations of 1.

Figure 3. Proposed biosynthetic pathway of fusaric acid derivatives.

21



Page 22 of 29

Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data of Fusaricates A-C, 1-3 (CD3OD). position

1 δH (J in Hz)

2

2 δC

δH (J in Hz)

146.3, C

3 δC

δH (J in Hz)

146.4, C

δC 146.4, C

3

8.13, d (8.0)

126.4, CH

8.14, d (8.1)

126.5, CH

8.15, d (8.0)

126.5, CH

4

7.86, dd (8.0, 2.2)

138.9, CH

7.88, dd (8.1, 2.2)

138.9, CH

7.87, dd (8.0, 2.2)

138.9, CH

8.57, d (2.2)

150.8, CH

5 6

144.6, C 8.54, d (2.2)

7 8

150.7, CH

144.4, C

166.0, C 2.76, t (7.5)

33.5, CH2

144.6, C 8.54, d (2.2)

166.0, C 2.89, ddd (13.9, 9.1, 6.4)

150.7, CH 166.0, C

30.2, CH2

2.76, t (7.5)

33.5, CH2

2.80, ddd (13.9, 9.1, 7.4) 9

1.66, tt (7.5, 7.5)

34.2, CH2

1.77, m

41.2, CH2

1.67, tt (7.5, 7.5)

34.2, CH2

10

1.40, tq (7.5, 7.5)

23.3, CH2

3.75, tq (6.2, 6.2)

67.6, CH

1.40, tq (7.5, 7.5)

23.3, CH2

11

0.97, t (7.5)

14.1, CH3

1.21, d (6.2)

23.6, CH3

0.97, t (7.5)

14.1, CH3

1'

4.46, dd (11.3, 3.8)

68.1, CH2

4.46, dd (11.3, 3.8)

68.2, CH2

4.60, dd (11.4, 2.8)

68.9, CH2

4.33, dd (11.3, 6.6)

4.34, dd (11.3, 6.6)

4.41, dd (11.4, 6.6)

2'

3.99, ddt (6.6, 3.8, 5.7)

71.1, CH

3.99, ddt (6.6, 3.8, 5.6)

71.2, CH

3'

3.65, d (5.7)

63.9, CH2

3.65, d (5.6)

63.9, CH2

4'

3.90, ddd (7.5, 6.6, 2.8)

71.5, CH

3.67, m

73.5, CH

3.81, m

63.9, CH2

3.66, m

22


Page 23 of 29


Table 2. 1H (600 MHz) and 13C (150 MHz) NMR Data of Fusaricates D-G, 4-7 (CD3OD). position

4 δH (J in Hz)

2

5 δC

δH (J in Hz)

146.3, C

6 δC

δH (J in Hz)

146.4, C

7 δC

δH (J in Hz)

146.5, C

δC 146.5, C

3

8.13, br d (8.1)

126.4, CH

8.15, br d (8.1)

126.5, CH

8.13, br d (8.1)

126.4, CH

8.15, br d (8.1)

126.4, CH

4

7.85, br d (8.1)

138.8, CH

7.86, br d (8.1)

138.9, CH

7.86, br d (8.1)

139.1, CH

7.87, br d (8.1)

139.1, CH

5 6

144.5, C 8.53, br s

7

150.6,CH

144.6, C 8.53, br s

143.6, C

150.7, CH

166.0, C

8.54, br s

150.8,CH

166.1, C

143.6, C 8.54, br s

150.8, CH

166.0, C

166.1, C

8

2.76, t (7.5)

33.5, CH2

2.76, t (7.5)

33.5, CH2

2.86, m

33.2, CH2

2.86, m

33.2, CH2

9

1.66, tt (7.5, 7.5)

34.2, CH2

1.66, tt (7.5, 7.5)

34.2, CH2

2.44, m

35.8, CH2

2.44, m

35.8, CH2

10

1.39, tq (7.5, 7.5)

23.3, CH2

1.39, tq (7.5, 7.5)

23.3, CH2

5.85, m

138.1, CH

5.85, m

138.1, CH

11

0.96, t (7.5)

14.1, CH3

0.96, t (7.5)

14.1, CH3

5.01, d (17.1)

116.5, CH3

5.01, d (17.1)

116.5, CH3

4.99, d (9.8) 1'

4.45, m

68.6, CH2

4.64, dd (11.3, 2.5)

69.1, CH2

4.45, m

4.99, d (9.8) 68.6, CH2

4.45, dd (11.3, 6.2) 2'

4.27, ddd (7.3, 5.4, 1.7)

69.2, CH

4.64, dd (11.3, 2.5)

69.1, CH2

4.45, m

4.04, ddd (8.6, 6.2, 2.5)

70.4, CH

4.27, ddd (7.3, 5.4, 1.7)

69.2, CH

4.03, ddd (8.6, 6.2, 2.5)

70.4, CH

3'

3.59, dd (8.5, 1.7)

72.2, CH

3.64, dd (8.6, 1.5)

71.8, CH

3.59, dd (8.5, 1.7)

72.2, CH

3.65, m

71.8, CH

4'

3.76, ddd (8.5, 5.5, 3.4)

72.7, CH

3.96, ddd (6.3, 6.3, 1.5)

71.5, CH

3.76, ddd (8.5, 5.5, 3.4)

72.7, CH

3.96, ddd (6.3, 6.3, 1.5)

71.5, CH

3.83, dd (11.3, 3.4)

65.0, CH2

3.65, m

64.8, CH2

3.82, dd (11.3, 3.4)

65.0, CH2

3.66, m

64.8, CH2

5'

3.66, dd (11.3, 5.5)

3.66, dd (11.3, 5.5)

23



Table 3. 1H (600 MHz) and 13C (150 MHz) NMR Data of 8 (CD3OD). 8 position δH (J in Hz) 2 3

8.03, d (7.7)

4

7.84, d (7.7)

5 6

125.7 CH 139.6 CH 142.7 C

8.59, s

7 8

δC 145.8 C

149.9 CH not detected

2.88, m

29.7 CH2

2.77, m 9

1.89, m

36.2 CH2

1.77, m 10 11

3.68, m

71.3 CH

3.55, dd (11.2, 4.6)

50.0 CH2

3.50, dd (11.2, 5.8)

24


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Table 4. Relative Phytotoxicity [0 to 5 (±S.D.)] of Fusaric Acid Derivatives 1−8b. Compound

Concentration (mM) 0.5

1.0

2.0

4.0

8.0

1

1.3 (0.6)

1.3 (0.6)

1.7 (0.6)

2.3 (0.6)

5.0 (0.0)

2

0.3 (0.6)

0.0 (0.0)

1.0 (0.0)

1.7 (0.6)

2.7 (0.6)

1.3 (0.6)

0.7 (0.6)

1.3 (0.6)

1.7 (0.6)

2.7 (0.6)

a

4 and 5

1.3 (0.6)

1.3 (0.6)

2.7 (0.6)

3.0 (0.6)

3.7 (0.6)

6 and 7a

1.0 (0.0)

0.7 (0.6)

1.7 (0.6)

1.7 (0.6)

3.0 (1.0)

8

1.3 (0.6)

1.3 (0.6)

2.0 (0.0)

3.0 (0.0)

3.7 (0.6)

Fusaric acid

1.7 (0.6)

1.7 (0.6)

3.0 (0.0)

3.0 (0.0)

4.0 (0.0)

3

a b

4 and 5, 6 and 7 were tested as mixtures. Visual rating: 0 = no necrosis; 5 = severe necrosis; fusaric acid was used as positive control.

25



Page 26 of 29

R 8 10

6

N

5

11

4

N

OH 7

OH OH O

O

2

2'

3

4'

1'

1'

O

3' 2'

3'

O

OH

1R=H 2 R = OH

OH

3 10

N

OH OH

N

OH OH

11

O

3' 2'

4'

O

5'

4'

5'

1'

O

OH OH

O

4 3'R 5 3'S

6 3'R 7 3'S

OH Cl

3' 2'

1'

N OH O 8

26


OH OH

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27



28


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Table of Contents Graphic

29


New Fusaric Acid Derivatives from the Endophytic ... - ACS Publications

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