New Alkylitaconic Acid Derivatives from Nodulisporium sp. A21 and

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Bioactive Constituents, Metabolites, and Functions

New alkylitaconic acid derivatives from Nodulisporium sp. A21 and their auxin herbicidal activities on weed seeds Lingling Cao, Wei Yan, Chenguang Gu, Zhiyang Wang, Shuangshuang Zhao, Shuang Kang, Babar Khan, Hai-Liang Zhu, Jun Li, and Yonghao Ye J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04996 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 24, 2019

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Journal of Agricultural and Food Chemistry

New alkylitaconic acid derivatives from Nodulisporium sp. A21 and their auxin herbicidal activities on weed seeds

Lingling Cao,†,‡,§ Wei Yan,†,‡,§ Chenguang Gu,†,‡ Zhiyang Wang,†,‡ Shuangshuang Zhao,†,‡ Shuang Kang†,‡, Babar Khan,†,‡ Hailiang Zhu,§ Jun Li,†,‡ Yonghao Ye†,‡,*

†

College of Plant Protection, State & Local Joint Engineering Research Center of

Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing 210095, P. R. China. ‡

Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of

Education, Nanjing 210095, P. R. China §State

Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,

Nanjing University, Nanjing 210023, P. R. China

* Corresponding Author: Email: [email protected]; Phone: +86-25-8439-9753; Fax: +86-25-8439-9753. § L.C. and W.Y. contributed equally to this paper.

1

ACS Paragon Plus Environment


1

Abstract

2

Five alkylitaconic acid (AA) derivatives, including two novel compounds,

3

epideoxysporothric acid (2) and sporochartine F (5), and three known compounds,

4

deoxysporothric acid (1), deoxyisosporothric acid (3) and 1-undecen-2,3-dicarboxylic

5

acid (4), were obtained from the fermentation culture of the endophytic fungus

6

Nodulisporium sp. A21. The auxin herbicidal activities of compounds 1-4 against

7

weed seeds were investigated under laboratory conditions. In general, the tested

8

compounds displayed radicle growth promoting activity at low doses, and inhibitory

9

activity at higher doses. Compounds 1 and 2 could significantly inhibit the radicle

10

growth of dicotyledon weeds, Eclipta prostrata and Veronica persica, at a

11

concentration range from 50 to 200 μg mL-1, while 3 notably stimulated radicle

12

growth at the same concentration range. The results suggested that these AA

13

derivatives have the potential to be used as the lead scaffold for novel auxin herbicide

14

development. In addition, the biosynthetic pathways of 1-4 were deduced based on

15

13C

labeling experiment.

16 17

Keywords: Nodulisporium sp., alkylitaconic acid derivatives, auxin herbicidal

18

activity, fatty acids

19

2


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20

Introduction

21

Weeds have been the major biotic cause of crop yield losses since the origins of

22

agriculture and result in 30% to 90% losses of crop yield, varying across different

23

crops and locations [1]. Although there are many approaches to reduce the damage of

24

weeds, chemical prevention is still the most wildly used method, in which the

25

compounds possessing phytohormonal auxin activity have been among the most

26

successful herbicides and bioregulators [2-4]. Since the natural plant hormone

27

indole-3-acetic acid (IAA) was discovered, a number of structurally similar auxin

28

herbicides were synthesized and sold commercially, and they were classified into four

29

categories according to their chemical scaffolds, viz. benzoic acid, pyridine carboxylic

30

acid, phenoxycarboxylic acid and quinolinecarboxylic acid [5-9]. However, since

31

cases of resistance have constantly emerged in recent years due to unrestricted usage

32

[10], novel auxin herbicides with new modes of action are still in demand.

33

Natural products have a high structural diversity and environmentally friendly

34

attributes, which were considered to be the most promising source of the lead

35

compounds in pesticide discovery [11-16]. It is reported that natural alkylitaconic acid

36

(AA) derivatives possess various bioactivities. For example, (2S)-butylitaconic acid

37

and (2S)-hexylitaconic acid from Eupenicillium sp. LG41 exhibited antibacterial

38

activity [17], and the latter compound displayed plant growth regulation activity [18];

39

decumbic acid from the stem of Dendrobium nobile exhibited antifungal activity [19];

40

striatisporolide A from rhizomes of Athyrium multidentatum (Doell.) Ching displayed

41

cytoproliferative activity [20]; deoxysporothric acid and lichesterinic acid from the 3



42

fungus Hypoxylon monticulosum CLL-205 and the lichen Cetraria islandica,

43

respectively, showed cytotoxicity against tumor cell lines [21,22].

44

In our continuous work on the characterization of structurally novel and/or

45

biologically active metabolites from endophytic fungi [23,24], we found that the

46

endophyte Nodulisporium sp. A21 showed potent antifungal and auxin herbicidal

47

activity. Previous bioassay-guide fractionation obtained sporothriolide, an AA

48

metabolite with significant anti-phytopathogenic fungal activity [25]. In this study,

49

five AA derivatives, including two new compounds, epideoxysporothric acid (2) and

50

sporochartine F (5), were isolated from the A21 culture. Their auxin herbicidal

51

activities against monocotyledon and dicotyledon weeds were evaluated. In addition,

52

the biosynthetic pathway of these compounds was proposed and verified using the

53

method of feeding [1-13C] acetate and [2-13C] acetate during the liquid fermentation.

54

MATERIALS AND METHODS

55

Instruments and Chemicals.

56

Optical rotations were measured in a CDCl3 solution on a Rudolph II Autopol

57

automatic polarimeter (Rudolph, NJ, USA). Circular dichroism (CD) spectra were

58

recorded on a JASCO J-810 CD spectrometer (JASCO, Tokyo, Japan). NMR spectra

59

were recorded on Bruker Avance III 400MHz and 600MHz NMR spectrometer

60

(Bruker, Rheinstetten, Germany) in CDCl3 at room temperature. Chemical shifts ()

61

were calculated in ppm with reference to internal TMS, and coupling constants (J) are

62

given in Hz. HR-ESI-MS spectra were recorded on a Agilent 6210 TOF LC-MS

63

spectrometer (Agilent, CA, USA). The X-ray single crystal diffraction analysis were 4


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64

accomplished on a Bruker APEX-II CCD diffractometer (Bruker, MA, USA). High

65

performance liquid chromatography (HPLC) analysis was performed on a 1200

66

Infinity LC system (Agilent, CA, USA), and the columns used were 250 mm × 4.6

67

mm i.d., 5 μm, ZORBAX Eclipse XDB, (Agilent, CA, USA). Silica gel (200-300

68

mesh, Qingdao Marine Chemical Inc., China), Sephadex LH-20 (Pharmacia Biotech

69

AB, Uppsala, Sweden) and ODS-A-HG 50 μm (YMC Kyoto, Japan) were used for

70

column chromatography. (R)-(-)-α-Methoxy-α-(trifluoromethyl) phenylacetyl chloride

71

((R)-(-)-MTPA chloride, CAS: 39637-99-5), (S)-(+)-α-Methoxy-α-(trifluoromethyl)

72

phenylacetyl

73

acetate-1-13C (CAS: 23424-28-4) and sodium acetate-2-13C (CAS: 13291-89-9) were

74

purchased from Sigma-Aldrich (MA, USA). The positive pesticide indole-3-acetic

75

acid (99%, CAS: 87-51-4) was purchased from J&K Chemicals (Beijing, China). All

76

chemicals used in the study were of analytical grade.

77

Fungal Strain and Weed Seeds.

78

The endophytic fungus Nodulisporium sp. A21 was previously isolated from Ginkgo

79

biloba in 2012, which has been deposited in China General Microbiological Culture

80

Collection Center (CGMCC) with an accession number 15377. The weed seeds of E.

81

crusgalli, E. prostrata, A. japonicus and V. persica were provided by the Lab of

82

Herbicide Toxicology and Resistance, Nanjing Agricultural University, China.

83

Extraction and Purification.

84

The fungus A21 was transferred to PDA medium and cultured at 25 °C in the dark for

85

5 days. Five millimeter plugs of mycelia obtained by colony growth were transferred

chloride

((S)-(+)-MTPA

chloride,

CAS:

5


20445-33-4),

sodium


86

into a 1000 mL conical flask containing 400 mL of the sterile PDB medium, with

87

periodical shaking at 130 rpm for 12 days at 25 °C on a rotary shaker afterwards. The

88

fermentation broth was filtered through three layers of muslin cloth and extracted with

89

EtOAc three times, and the organic extract was dried (40 g). The organic extract was

90

subjected to silica gel chromatography (1000 mm × 50 mm i.d.) eluted stepwise with

91

CH2Cl2-MeOH (100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32, and 0:100) as the

92

mobile phase to produce eight fractions, F1-F8. F2 (3.2 g) was separated by ODS

93

reversed-phase column chromatography using MeOH-H2O (2:8, 3:7, 4:6, 5:5, 6:4, 7:3,

94

8:2, 9:1, 10:0) as the mobile phase to obtain nine fractions, I-IX. Fractions V and VI

95

of F2 were subjected to gel chromatography on a Sephadex LH-20 eluted with MeOH

96

to yield a mixture, respectively. The mixture was purified by HPLC applying 57% of

97

CH3CN (with 0.1% trifluoroacetic acid added to water) with the flow rate of 2 mL/min.

98

Pure compounds 1 (41.8 mg), 2 (16.4 mg) and 3 (23.4 mg) were obtained at 36.6 min,

99

39 min and 56.8 min, respectively. The UV detection was at 220 nm. One colorless oil

100

compound 4 (24 mg) and white powder 5 (4 mg) of fraction VI of F3 were obtained at

101

42.3 min and 60.9 min applying the same purification condition of HPLC (Fig. 1).

102

Formation of the (S)- and (R)-MTPA Esters of 5

103

Compound 5 (1.0 mg) was dissolved in 500 μL of anhydrous pyridine and 10 μL of

104

(R)-(-)-MTPA chloride were added in the reaction mixture. After 12h, the solvent was

105

evaporated under reduced pressure, and the residue was purified by HPLC

106

(MeOH/H2O, 85:15) to give (S)-MTPA ester 5a (0.7 mg). 5 (1.0 mg) and

107

(S)-(+)-MTPA chloride were treated with the same procedure to afford (R)-MTPA 6


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108

ester 5b (0.7 mg).

109

X-ray Crystallographic Analysis of Compounds 1, 2, 3 and 5.

110

The colorless co-crystal of 1 and 2, as well as the colorless single-crystal of 3 and 5,

111

were all obtained from slow evaporation of CH3OH solution, respectively. Diffraction

112

data of the crystals were collected on a Bruker Apex-II CCD X-ray diffractometer

113

using Cu Kα radiation (λ = 1.5418 Å). The structures were solved by direct methods

114

and refined by full-matrix least-squares calculations on F2 using SHELXL via OLEX2

115

[26-28]. All non-hydrogen atoms were refined anisotropically. The hydrogen atom

116

positions were geometrically idealized and allowed to ride on their parent atoms.

117

Crystallographic data for 1, 2, 3, and 5 have been deposited in the Cambridge

118

Crystallographic Data Centre (CCDC). Copies of these data can be obtained free of

119

charge from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44 (0)

120

1223-336033 or e-mail: [email protected]].

121

Crystallographic data for 1 and 2: C13H20O4, M = 240.29, monoclinic, a = 14.7076(13)

122

Å, b = 5.1175(5) Å, c = 17.6085(14) Å, α = 90°, β = 91.741(6)°, γ = 90°, V

123

=1324.7(2) Å3, T =130 K, space group P21, Z = 4, μ(Cu Kα) = 0.724 mm−1, 10290

124

reflections measured, 3612 independent reflections (Rint = 0.0526). The final R1 values

125

were 0.0501 [I > 2σ(I)]. The final wR(F2) values were 0.1345 [I > 2σ(I)]. The final R1

126

values were 0.0517 (all data). The final wR(F2) values were 0.1369 (all data). The

127

goodness of fit on F2 was 1.060. Flack parameter: -0.1(2). CCDC numbers: 1551389.

128

Crystallographic data for 3: C13H20O4, M = 240.29, monoclinic, a = 7.0633(15) Å, b =

129

7.9184(16) Å, c= 24.437(5) Å, α = 90°, β = 95.613(15)°, γ = 90°, V =1360.2(5) Å3, T 7



130

=153 K, space group C2, Z = 4, μ(Cu Kα) = 0.705 mm−1, 4048 reflections measured,

131

1780 independent reflections (Rint = 0.0445). The final R1 values were 0.0673 [I >

132

2σ(I)]. The final wR(F2) values were 0.1714 [I > 2σ(I)]. The final R1 values were

133

0.0692 (all data). The final wR(F2) values were 0.1725 (all data). The goodness of fit

134

on F2 was 1.106. Flack parameter: 0.15(17). CCDC numbers: 1834131.

135

Crystallographic data for 5: C27H40O9, M = 508.58, monoclinic, a = 14.054(3) Å, b =

136

24.833(4) Å, c= 16.920(3) Å, α = 90°, β = 109.321(4)°, γ = 90°, V =5572.5(17) Å3, T

137

= 153 K, space group P21, Z = 2, μ(Cu Kα) = 0.745 mm−1, 24383 reflections measured,

138

14930 independent reflections (Rint = 0.0684). The final R1 values were 0.0947 [I >

139

2σ(I)]. The final wR(F2) values were 0.2524 [I > 2σ(I)]. The final R1 values were

140

0.1249 (all data). The final wR(F2) values were 0.2791 (all data). The goodness of fit

141

on F2 was 1.014. Flack parameter: 0.13(19). CCDC numbers: 1847592.

142

Bioassays with Weed Species.

143

Auxin herbicidal assays of the natural compounds were tested in 6 cm diameter Petri

144

dishes against four weeds, including two monocotyledon species E. crusgalli and A.

145

japonicus, and two dicotyledon species E. prostrata and V. persica. The seeds were

146

sterilized with 1.0% (v/v) sodium hypochlorite for 10 min. The seeds were washed 2 -

147

3 times with tap water [29]. For these assays, stock solutions of test compounds were

148

prepared in dimethyl sulfoxide (DMSO), and the final concentration of 6.25 μg mL-1,

149

12.5 μg mL-1, 25.0 μg mL-1, 50 μg mL-1, 100 μg mL-1, 200 μg mL-1 and 400 μg mL-1

150

were prepared using distilled water containing 0.25% DMSO [30]. A total of 0.25%

151

DMSO in distilled water was used as the negative control. The positive control used 8


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152

natural auxin IAA, and a series of concentrations was set from 0.78 to 400 μg mL-1.

153

The test solutions (2 mL) were transferred into the Petri dishes, and 20 seeds were

154

placed in every dish. The Petri dishes were sealed with polyethylene wrapping film

155

with several small holes for ventilation and placed in a growth chamber calibrated to

156

provide 12 h light / 12 h darkness at 25 ± 1 °C or 20 ± 1 °C. Three replicates were

157

used for each treatment. The primary radicle and germ lengths were measured after 7

158

or 10 days. The inhibition rate was calculated using the following formula: inhibition

159

rate (%) = ((Lc - Lt) / Lc) × 100, where Lc is the length of the control and Lt is the

160

length of the treatment [31].

161

Administration of 13C-Labeled Sodium Acetate Assay.

162

In an effort to verify the biosynthesis pathway of AA derivatives, a

163

experiment was carried out. The fungus A21 was fermented used the methods above

164

and supplemented with [1-13C] - and [2-13C] - sodium acetate at a final concentration

165

of 7.2 μM after 5, 6, 7 and 8 days of incubation, respectively. Then the incubation was

166

continued till 12 days [32]. The fermentation broth was harvested and extracted with

167

ethyl acetate three times. Then the extract was purified used the same approach as that

168

of unlabeled metabolites described above.

169

Statistical Analysis.

170

The inhibition rates of radicle lengths and germ lengths of tested weeds were analyzed

171

using SPSS 19.0 with the probit analysis. All quantitative data were presented as the

172

mean ± SE of at least three independent experiments using the Duncan statistical test

173

for group differences. The value p ≤ 0.05 was considered as statistically significant. 9


13C-labeling


Page 10 of 31

174

RESULTS AND DISCUSSION.

175

Structure Elucidation.

176

Five secondary metabolites were isolated from the culture broth of Nodulisporium sp.

177

A21. Their structures were elucidated by 1D-NMR, 2D-NMR and X-ray

178

crystallography analyses.

179

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

180

C13H20O4, was deduced by HR-ESI-MS. The 1H and

181

consistent with those of deoxysporothric acid isolated from H. monticulosum

182

CLL-205 [21]. The structure of 1 was confirmed by single-crystal X-ray diffraction

183

(Cu Kα) analysis, which also determined its absolute configuration [a Flack parameter

184

of -0.1(2)] (Fig. 2).

185

Compound 2 was obtained as a white amorphous powder; [α] 𝐷 +29.3 (c 0.20, CHCl3);

186

HR-ESI-MS (+) m/z 263.1242 [M+Na]+ (calcd for C13H20NaO4, 263.1260); for 1H

187

NMR and

188

were similar to those of 1 expect for a NOESY correlation. The NOESY correlation

189

between H-2 and H-6 was observed in 1 but disappeared in 2, indicating a

190

trans-configuration of 2. The structure of 2 as the co-crystal former of compound 1

191

was also confirmed by single-crystal X-ray diffraction (Cu Kα) analysis (Fig. 2).

192

Thus, compound 2 was defined as 2-[(2S,6R)-6-hexyl-2-oxotetrahydrofuran-3-yl]

193

acrylic acid and designated epideoxysporothric acid.

194

Compound

195

(S)-2-heptyl-4-methyl-5-oxo-2,5-dihydrofuran-3-carboxylic acid. It was previously

13C

NMR spectra of 1 were

25

13C

NMR data (CDCl3), see Table 1. The 1D and 2D NMR spectra of 2

3,

obtained

as

a

colorless

crystal,

10


was

identified

as

Page 11 of 31


196

synthesized by Sweidan et al. as B-7 to explore the antibacterial activity, but it didn’t

197

show any activity in vitro [33]. It is the first report of 3 as a natural product, as well as

198

its crystal data (Fig. 3). This compound was designated as deoxyisosporothric acid

199

according to its homologues of natural products.

200

The known compound 4 was obtained as a colorless oil. Its molecular formula,

201

C13H22O4, was deduced by HR-ESI-MS. The 1H-NMR spectra were fully consistent

202

with 1-undecen-2, 3-dicarboxylic acid, which was first isolated as a natural product

203

from the endophytic fungus Pestalotiopsis theae [34]. The absolute configuration was

204

determined to be 2R by an ECD comparison with the literature [34] (Fig. S16).

205

Compound 5 was isolated as a colorless crystal and assigned the molecular formula

206

C27H40O9 in accordance with its HR-ESI-MS data, indicating 8 degrees of

207

unsaturation. The 13C NMR revealed the presence of 27 carbons attributed by HSQC

208

to three methyl carbons (δC 14.0, 14.0 and 11.4), one oxygen-bond methyl carbon (δC

209

52.9), eleven aliphatic methylene groups (δC 34.7, 31.6, 31.5, 31.3, 28.9, 28.8, 28.8,

210

25.7, 25.3, 22,5 and 22.5), five methine groups including three oxygen-bond methines

211

at δC 82.4 (C-6′), 78.6 (C-5′) and 74.5 (C-6), four lactonic ester groups at δC 175.6

212

(C-1′), 174.9 (C-4′), 171.4 (C-4) and 163.4 (C-1), two methines forming a double

213

bond at δC 146.5 (C-2) and 139.5 (C-3), and a quaternary carbon at δC 90.5 (C-5). The

214

1H

215

(H-5′) and 4.54 (H-6′) and two CH group at δH 3.38 (H-2′) and 2.97 (H-3′). The

216

COSY correlations between H-3′ and H-13′, H-2′ and H-3′, H-2′ and H-5′, H-5′ and

217

H-6′ and H-6′ and H-7′ together with the HMBC correlations between H-2′, H-3′, H-5′

NMR spectrum of compound 5 revealed two oxygenated CH groups at δH 5.04

11



218

and C-1′ and between H-2′, H-3′, H13′ and C-4′ enabled us to form the bis-γ-lactone

219

ring. All of these structural components indicated that a sporothriolide moiety was

220

involved in compound 5. In addition, the HMBC correlations from H-13 (δH 2.21) to

221

C-1, C-2, C-3, C-4 and C-5, and those from H-6 (δH 3.98) to C-3 showed that 5

222

contained an isosporothric acid scaffold [35]. The connection of the two moieties was

223

deduced based on the HMBC correlations between H-13′ (δH 2.97, 2.51) and C-2 and

224

C-5 (Fig. 4). The relative configuration between C2′, C5′ and C6′, C2′ and C13′, C6

225

and C13′ were determined by NOESY. The structure of compound 5 was further

226

confirmed by X-ray diffraction using Cu Kα radiation (Fig. 5, the selected rotamer). It

227

should be noted that the solid state structure of 5 possesses four conformation isomers

228

(Fig. S25). Alkyl steric hindrance and hydrogen bonding between the rotamer

229

molecules were the prime factors controlling the structures of these rotamers with the

230

same configuration of 5.

231

The absolute configuration of C-6 in 5 was determined by modified Mosher’s method.

232

Compound 5 was esterified with (R)-(-)-MTPA chloride and (S)-(+)-MTPA chloride,

233

respectively, to give the (S)- and (R)-MTPA esters 5a and 5b. The Δδ values between

234

5a and 5b demonstrated that C-6 possessed a R configuration (Fig. 6, Fig. S26-S28)

235

[36]. Therefore, the absolute configuration of all chiral carbons in 5 was assigned as

236

5S, 6R, 2′S, 3′S, 5′R, and 6′R. This compound, named sporochartine F, had an unusual

237

skeleton with a carbon-carbon bond between sporothriolide and isosporothric acid

238

derivative.

239

Bioassays with Weed Species. 12


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240

An assay to evaluate the effectiveness of the plant auxin activities on the radicle and

241

germ elongation of four weed species were conducted using the Petri dish assay

242

method. The results showed that the compounds 1-4 exhibited remarkable radicle and

243

germ growth regulation activities against the four weed species. As shown in Fig. 7,

244

Table S1 and S2, the compounds could promote radicle growth at low doses, while

245

inhibiting plant growth at higher doses. Compounds 1 and 2 almost completely

246

inhibited the radicle and germ growth of the two dicotyledon species (E. prostrate and

247

V. persica) at 400 μg mL-1 and had low toxicity against the two monocotyledon

248

species (E. crusgalli and A. japonicus). Simultaneously, 3 had a good growth

249

regulation effect of the radicle against E. prostrate with a promotion ratio of 44.5% at

250

50μg mL-1 and against V. persica with the promote ratio of 20.3%. The bioassay

251

results of compound 4 were consistent with those of the homologous compound

252

hexylitaconic acid, which was a plant growth regulator [18]. The natural auxin IAA

253

was assayed as a positive control, and the results are showed in Table S3.

254

Biosynthetic pathway.

255

The biosynthetic pathways of structurally related metabolites such as canadensolide

256

and dihydrocanadensolide had been studied [37,38]. They indicated that these

257

compounds resulted from the condensation of fatty acids with the tricarboxylic acid

258

cycle intermediate, commonly oxaloacetate. Given these findings, we proposed a

259

similar pathway for the biosynthesis of compounds 1-4. First, a C10 fatty acid unit

260

condensed

261

decarboxylation and dehydration to compound 4. Cyclization between C-1 and C-6 in

with

oxaloacetate

to

form

intermediate

13


A,

which

underwent


262

4 produced 1 and 2. Lactonization between C-4 and C-5 could produce intermediate

263

B, which isomerized to 3. Labeling studies with [1-13C] and [2-13C]-acetate revealed

264

that the incorporation patterns of compounds 1 and 3 were consistent with the

265

predicted manner [39] (Fig. 8, Table S4). Compound 5, possessing a novel skeleton,

266

might derive from the condensation of two AA derivatives, sporothriolide and

267

isosporothric acid, via Michael addition reaction.

268

In conclusion, we characterized two new compounds, epideoxysporothric acid (2) and

269

sporochartine F (5), together with three known compounds, deoxysporothric acid (1),

270

deoxyisoporothric acid (3) and 1-undecen-2,3-dicarboxylic acid (4), by NMR and

271

X-ray analysis. The bioassay of the regular radicle and germ elongation of the four

272

weed species on compounds 1-4 was conducted. These AA derivatives not only

273

showed a potent phytotoxin but could also cause substantial growth stimulation at

274

subtoxic doses, a phenomenon known as phytohormone activity. The fatty acid

275

biosynthetic pathway was deduced and verified using 13C labeling experiments. Given

276

the importance of natural products in pesticides, the chemical scaffold of these AA

277

derivatives could be considered to be the lead framework to develop novel auxin

278

herbicides.

279 280

ASSOCIATED CONTENT

281

Supporting Information

282

Spectroscopic information of compounds 1-5 (Figure S1 - Figure S24). Four rotamers

283

in the solid-state structure of compound 5 (Figure S25). The 1H-NMR and 1H-1H 14


Page 14 of 31

Page 15 of 31


284

COSY spectrum about Mosher’s analysis of compound 5 (Figure S26 - Figure S28).

285

The

286

Figure S32 and Table S4). Herbicidal activity data of compounds 1-4 (Table S1 - S4).

13C-NMR

data of

13C-labelled

sodium acetate feeding experiment (Figure S29-

287 288

AUTHOR INFORMATION

289

Corresponding Author

290

Tel.: +86-25-84399753. Fax: +86-25-84399753.

291

E-mail address: [email protected]

292

Funding

293

This work was supported by the National Key Research and Development Program of

294

China (2017YFD0201300), the National Natural Science Foundation of China

295

(31572043 & 21602109), Qing Lan Project of Jiangsu Province, and Research

296

Innovation Program for College Graduates of Jiangsu Province (KYLX16_1067).

297

Notes

298

The authors declare no competing financial interest.

299 300

References

301

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414

20


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Page 21 of 31


Table 1. 1H (600 MHz) and 13C NMR (150 MHz) data of compound 2 in CDCl3. position

δC, type

δH, mult (J in Hz)

1

176.6, C

2

43.1, CH

3

136.2, C

4

170.3, C

5

33.8, CH2

2.40, m; 2.21, m

6

79.1, CH

4.62, m

7

35.6, CH2

1.73, m; 1.59, m

8

25.2, CH2

1.46-1.29, m

3.68, t (9.3)

9

29.0, CH2

1.46-1.29, m

10

31.6, CH2

1.46-1.29, m

11

22.5, CH2

1.46-1.29, m

12

14.1, CH3

0.89, t (7.0)

13

131.2, CH2

6.53, s; 5.92, s

21



Page 22 of 31

Table 2. 1H (600 MHz) and 13C NMR (150 MHz) data of compound 5 in CDCl3. position

δC, type

1

δH, mult (J in Hz)

position

δC, type

163.4, C

1′

175.6, C

2

146.5, C

2′

47.2, CH

3.38, dd (6.45)

3

139.5, C

3′

39.3, CH

2.97, m

4

171.4, C

4′

174.9, C

5

90.5, C

5′

78.6, CH

5.04, dd (6.6, 4.3)

6

74.5, CH

3.98, dd (7.1)

6′

82.4, CH

4.54, ddd (8.1, 6.2, 4.5)

7

28.9, CH2

1.46, m

7′

28.8, CH2

1.84, m; 1.77, m

8

31.3, CH2

1.30-1.21, m

8′

25.3, CH2

1.30-1.21, m

9

25.7, CH2

1.30-1.21, m

9′

28.8, CH2

1.30-1.21, m

10

31.6, CH2

1.30-1.21, m

10′

31.5, CH2

1.30-1.21, m

11

22.5, CH2

1.30-1.21, m

11′

22.5, CH2

1.30-1.21, m

12

14.0, CH3

0.87, t (7.0)

12′

14.0, CH3

0.87, t (7.1)

13

11.4, CH3

2.21, s

13′

34.8, CH2

2.97, m; 2.51, m

14

52.9, CH3

3.92, s

22


δH, mult (J in Hz)

Page 23 of 31


O

1

O

13

O

O

OH

5

6

1

O

4

2

11

9

7

2

OH

5

6

O

3 4

2

1

O 11'

9 '

7'

O

5'

O O 12

10

5 8

6

HO

2'

13'

1'

O 3

1

13

HOOC 1

2

3

4 5

6

3 2

13

1

OH O

O 14

8

COOH 4

O

3

O

3'

6'

O

O

4'

10 12

5

4

Figure 1. The structures of compounds 1-5 from the endophytic fungus Nodulisporium sp. A21.

23



Figure 2. X-ray crystallographic structures of compound 1 and 2.

24


Page 24 of 31

Page 25 of 31


Figure 3. X-ray crystallographic structure of compound 3.

25



Page 26 of 31

O O

O

O

O

O

O O

O O O

COSY

O

O

OH O

OH O

O

NOESY

HMBC

Figure 4. Key COSY, HMBC and NOESY correlations of compound 5.

26


Page 27 of 31


Figure 5. X-ray crystallographic structure of compound 5.

27



Page 28 of 31

O -0.080

O

O

-0.058

-0.001

O

-0.027

O

+0.013 -0.080 -0.162

O +0.025

+0.118

O

0.000

OR O

+0.109

overlapped +0.021

5a R=(S)-MTPA 5b R=(R)-MTPA

Figure 6. Selected ΔδH (ΔδH = δS − δR) values for MTPA esters 5a and 5b.

28


Page 29 of 31


Figure 7. Effects of compounds 1-4 on radicle growth of the four weeds.

29



COSCoA

O

O HO

O O

A O

H2O+CO2

O

O isomerization

O 4,5-cyclization

O

O

HO 1

O

HO

3

[2-13C] sodium acetate

O

OH OH O

O

[1-13C] sodium acetate

OH OH OH

O

+

SCoA

Page 30 of 31

B

HO

4

6

13 2

3 O 4 OH

OH

1

1,6-cyclization +

O

5

O

O

2

Figure 8. The hypothetical biosynthetic pathways for 1-4.

30


O

O

OH

Page 31 of 31


For Table of Contents Only

31


New Alkylitaconic Acid Derivatives from Nodulisporium sp. A21 and

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