Hydroxyl and Methoxyl Derivatives of Benzylglucosinolate in Lepidium

Subscriber access provided by University of Newcastle, Australia

Article

Hydroxyl and Methoxyl Derivatives of Benzylglucosinolate in Lepidium densiflorum with Hydrolysis to Isothiocyanates and non-Isothiocyanate Products: Substitution Governs Product Type and Mass Spectral Fragmentation ELEONORA PAGNOTTA, Niels Agerbirk, Carl Erik Olsen, Luisa Ugolini, Susanna Cinti, and Luca Lazzeri J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00529 • Publication Date (Web): 25 Mar 2017 Downloaded from http://pubs.acs.org on April 2, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42

Journal of Agricultural and Food Chemistry

Hydroxyl and Methoxyl Derivatives of Benzylglucosinolate in Lepidium densiflorum with Hydrolysis to Isothiocyanates and non-Isothiocyanate Products: Substitution Governs Product Type and Mass Spectral Fragmentation

Eleonora Pagnottaa*, Niels Agerbirkb, Carl E. Olsenb, Luisa Ugolinia, Susanna Cintia, Luca Lazzeria a

Council for Agricultural Research and Economics. Research Centre for Industrial Crops, CREA-

CIN, via di Corticella,133. 40128, Bologna (Italy). b

Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University

of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.

*Corresponding Author: Eleonora Pagnotta, Council for Agricultural Research and Economics. Research Centre for Industrial Crops (CREA-CIN), via di Corticella,133. 40128, Bologna (Italy), tel +39-051-6316852, fax + 39-051-374857, e-mail: [email protected]

1 ACS Paragon Plus Environment


Page 2 of 42

1

Abstract A system of benzylic glucosinolates was found and characterized in common

2

pepperweed, Lepidium densiflorum Schrad. The major glucosinolate was the novel 4-hydroxy-3,5-

3

dimethoxybenzylglucosinolate (3,5-dimethoxysinalbin), present at high levels in seeds, leaves and

4

roots. Medium level glucosinolates were 3,4-dimethoxybenzylglucosinolate and 3,4,5-

5

trimethoxybenzylglucosinolate. Minor glucosinolates included benzylglucosinolate, 3-hydroxy and

6

3-methoxybenzylglucosinolate, 4-hydroxybenzylglucosinolate (sinalbin), the novel 4-hydroxy-3-

7

methoxybenzylglucosinolate (3-methoxysinalbin) and indole-type glucosinolates. A biosynthetic

8

connection is suggested. NMR, UV and ion trap MS/MS spectral data are reported, showing

9

contrasting MS-fragmentation of para-hydroxyls and para-methoxyls. Additional investigations by

10

GC-MS focused on glucosinolate hydrolysis products. While glucosinolates generally yielded

11

isothiocyanates, the dominating 3,5-dimethoxysinalbin with a free para-hydroxyl group produced

12

the corresponding alcohol and syringaldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde). After

13

thermal deactivation of the endogenous myrosinase enzyme, massive accumulation of the

14

corresponding nitrile was detected. This case study points out how non-isothiocyanate glucosinolate

15

hydrolysis products are prevalent in nature and of interest both in plant-pathogen interactions and

16

human health.

17

Keywords: Brassicaceae; novel glucosinolate; methoxylation; hydroxylation; phenolics;

18

syringaldehyde.

19 20 21

22


Page 3 of 42


23

INTRODUCTION

24

The Brassicaceae are one of the most important families in the Brassicales, containing about 350

25

plant genera and about 3700 species. 1 Only few genera of this wide variety of germplasm are

26

exploited agronomically. Nevertheless their production in Europe alone has recently been estimated

27

to be approximately 70 million tons/year. 2 All these species contain glucosinolates (GSLs),

28

thioglucosidic multifunction secondary metabolites characterized by a variable side chain that

29

confers the chemical and biological properties of the around 132 GSLs that were documented by

30

2011. 3-5 GSLs naturally coexist with the endogenous enzyme myrosinase (MYR) (thioglucoside

31

glucohydrolase, EC 3.2.1.147), which, in the presence of water, catalyzes their hydrolysis to a series

32

of breakdown products. The chemical structures of these products depend on the structure of the

33

GSL and on the reaction conditions, in particular pH and the presence of specifier proteins. 6 A

34

well-characterized function of the GSL–MYR system is the release of isothiocyanates (ITCs) that

35

play a defense role against pathogens, insects and generalist herbivores. That is why the

36

Brassicaceae have been applied agronomically against some soil borne pests and diseases in the so

37

called biofumigation technique. 7-9

38

Another function relates to the health properties of the GSL-MYR system. Indeed, GSLs and ITCs

39

have been found to be central in a variety of beneficial effects of cruciferous vegetables. 10-12A rich

40

literature reports on in vitro and in vivo effects of ITCs both as anticancer, anti-inflammatory and

41

antioxidant molecules, and as bactericidal, antiviral, and antidiabetic agents, protective for the

42

neurological, gastrointestinal, cardiovascular and skin systems. 13 Consequently, a major goal of

43

GSL research is to understand the relationship between GSL chemical structure and biological

44

activity, with the aim of finding new applications for the entire palette of known and yet unknown

45

GSLs in nature.



Page 4 of 42

46

An intriguing aspect of this diversity is the recurrence of derivatives of benzylGSL in wild and

47

cultivated plants such as several species of Lepidium, Sinapis, Tropaeolum, and Moringa, which are

48

all characterized by quite different biological activities. It is relevant to classify benzylic GSLs in

49

two groups with respect to the stability of their corresponding ITCs released from endogenous

50

MYR hydrolysis. The first group consists of benzylic GSLs able to release stable ITCs. These are

51

characterized by high biological activities, (e.g., benzyl glucosinolate and the p-rhamnosyloxy

52

derivative glucomoringin) tested both in agriculture, ecology and human health maintenance. 14-17

53

The second group consists of substituted derivatives activated in the ortho or para position by

54

hydroxylation. This benzylic activation results in quick hydrolysis of the resulting ITC in aqueous

55

solutions to benzylic alcohols and thiocyanate ion because p-activation stabilizes a carbocation

56

intermediate. 3 A paradigm of this chemistry is p-hydroxybenzylGSL (sinalbin). Although this GSL

57

seems to be involved in defense in vivo, 18 several trials showed how the breakdown products of

58

isolated sinalbin were less harmful against several pathogens than other aromatic ITCs such as

59

benzyl ITC and phenethyl ITC. 19-22 On the other hand, with regard to food safety, it has recently

60

been proposed that sinalbin derivatives may be natural precursors of bisphenol F, a chemical used in

61

the production of plastic and resins, and of considerable concern with respect to the balance of

62

sexual hormones. 23 Excretion of a peculiar sinalbin metabolite in pig urine is another indication of

63

complex biochemistry of sinalbin metabolism. 24

64

Unfortunately, the taxonomic distribution of sinalbin is poorly known because old published

65

screenings of plant species employed gas chromatography at conditions that did not include sinalbin

66

ITC and its products. 25 Nevertheless, sinalbin has been detected in several genera when analyzed by

67

suitable methods, often at high levels. 26, 27 Recently, open field experiments with multiple

68

Arabidopsis thaliana GSL types revealed how all chemotypes can take advantage in specific

69

conditions. 28 From these results it is conceivable to presume that the GSL profile of any plant is


Page 5 of 42


70

optimized for some ecological situation. Hence, if GSLs help the plant to defend itself against

71

attacks of herbivores or pathogens, the lack of known toxic properties of sinalbin makes it a

72

mystery how the accumulation of this and related GSLs would be of advantage for the plant.

73

We call this conflict between low detectable toxicities of sinalbin products and expected defensive

74

properties of GSL ”the sinalbin paradox”. A hypothetic advantage of a species containing sinalbin

75

could be linked to the enzymatic conversion of thiocyanate ion to methyl thiocyanate. 29 Otherwise

76

plants could possibly benefit from the benzyl alcohol derived from hydrolysis of sinalbin, although

77

toxicity in standard bioassays is low. 19-24 Alternatively, a more indirect advantage related to plant-

78

defense response signaling or secondary reaction products could exist. 30-32

79

Among Brassicaceae, L. densiflorum (common pepperweed) is an annual/biennial plant growing to

80

0.5m of height, which is diffused in all continents except for Antarctica. The leaves are considered

81

edible and seeds used as mustard. It is particularly resistant to arid climates, salt stress and ionizing

82

radiation. 33 It is considered an alien species for Eastern Europe, where it spread at the middle of

83

last century and where it is considered invasive. Despite its high seed GSL content, defatted seed

84

meals were remarkably less toxic to the root nematode Meloidogyne incognita than other GSL

85

containing defatted seed meals. 34 The aim of the present study was the elucidation of the structures

86

of the GSLs in L. densiflorum, recording and interpretation of desulfoGSL ion trap mass spectra for

87

future routine GSL identification, as well as an investigation of GSL profiles and natural GSL

88

hydrolysis products in a variety of agriculturally relevant tissues.

89

MATERIALS AND METHODS

90

Plant material. L. densiflorum seeds were from the seed collection of CREA-CIN (Bologna, Italy).

91

35

92

sowing on a surface of around 10 square meters (Figure 1). All cultivation phases, from sowing to

They were reproduced at the experimental farm of Budrio (Bologna, Italy) in 2013 autumnal



Page 6 of 42

93

harvesting, were performed by low impact cultivation techniques, applying neither irrigation nor

94

chemical treatments. After harvesting, seeds were cleaned, dried to maximum 8% of moisture and

95

stored in darkness at 19°C and 40% relative humidity. The samples of leaves and roots were

96

collected from three different plants, immediately frozen and finally freeze-dried for storage in glass

97

vacuum desiccators.

98

Chemicals. Methanol (MeOH) and acetonitrile (ACN) (HPLC-grade, Lichrosolv®) were purchased

99

from Merck (Darmstadt, Germany). Formic acid (98% purity) and ethanol (99.8% purity) were

100

from Fluka (Buchs, Switzerland), hexane (≥ 97% purity, Chromasolv®), ethyl acetate (LC-MS

101

Chromasolv®) and syringaldehyde were from Sigma-Aldrich (Milan, Italy), 4-hydroxy-3,5-

102

dimethoxyphenylacetonitrile was from Fluorochem Ltd (Hadfield, UK). Water of HPLC grade

103

(Millipore) was produced using a Milli-Q Synthesis A 10 (Molsheim, France) system. All the

104

solvents were of analytical grade. The standard of sinigrin was isolated as previously reported. 36

105

Glucosinolate analysis by HPLC-UV. GSLs were extracted from seeds (250 mg), and from finely

106

powdered freeze-dried leaves (350 mg) and roots (500 mg), using hot solvents as previously

107

reported. 37 From the entire crude extract (10 mL), aliquots of each aqueous ethanolic extract (1 mL)

108

were loaded onto a mini-column filled with DEAE Sephadex A-25 anion-exchange resin (GE

109

Healthcare, Freiburg, Germany), conditioned with 25 mM sodium acetate buffer (pH 5.6). After

110

washing with the same buffer (3 mL), purified sulfatase (200 µl, 0.35 U/mL) was loaded onto the

111

mini-column which was then left overnight at 20 °C. DesulfoGSLs were eluted with tree 1 mL

112

portions of ultrapure water, allowing the water to drain after each addiction, and their analysis was

113

performed using an HPLC (Hewlett-Packard chromatograph 1100) equipped with a diode array

114

detector, a ChromSep HPLC column SS (250 × 3.0 mm, 5 µm) and a ChromSep guard column

115

(Intersil 5 ODS-3) (Varian). The column was eluted at a flow rate of 0.8 mL min-1 with aqueous

116

ACN (solvent A: water; solvent B: ACN) at 30°C following the program: 0-1 min, isocratic 1% B; 6 ACS Paragon Plus Environment

Page 7 of 42


117

1-22 min linear gradient 1-22% B; 22-30 min, linear gradient 22-1% B; 30-33 min, isocratic 1% B.

118

The desulfoGSLs were detected monitoring their absorbance at 229 nm and their amounts were

119

estimated using a calibration curve of pure desulfo-sinigrin (range from 0.1 to 3 mM, y=5768.1 x +

120

205.77, r2=0.9997). In lack of available relative response factors for several L. densiflorum GSLs,

121

the factor 0.5 measured for sinalbin (relative to sinigrin) was arbitrarily used. 38 Each extraction and

122

analysis was performed in triplicate.

123

HPLC-MS, isolation and spectroscopy of desulfoglucosinolates. L. densiflorum seeds were

124

crushed in a mortar and the entire resulting meal (2.5 g) was extracted 3 times in boiling 70% aq.

125

MeOH. The extract (50 mL) was divided among five DEAE Sephadex mini-columns and subjected

126

to a wash, on-column enzymatic desulfation and elution with water.

127

investigated by ion trap HPLC-MS/MS of 1 mL of this fraction as previously reported.

128

remaining desulfoGSL fraction was concentrated by freeze drying and dissolved in 2.5 mL water.

129

For preparative HPLC of d1-d5 we used a Supelcosil LC-ABZ ‘amide C-16’ column (250 mm x

130

4.6 mm, 5 µm) (Supelco, Bellefonte, PA) for optimal separation of closely eluting desulfoGSLs d1

131

and d2. This resulted in the reverse elution order of d1 and d2 as shown in Figure 2. Five

132

desulfoGSLs were separated, collected and pooled from 7 runs (inj. vol 300 µL). These fractions

133

were freeze dried (after removal of methanol under gentle air stream) and subjected to NMR and

134

HPLC-MS/MS for structural identification. The instrumentation for NMR spectroscopy (1H: 400

135

MHz, 13C: 100 MHz, D2O solvent, internal standard dioxane) was exactly as previously reported.

136

All fractions, representing desulfoGSLs d1-d5, were subjected to 1H-NMR and d3-d5 to 13C NMR,

137

supplemented by COSY, HSQC, NOESY and HMBC as relevant.

39

The seed GSL profile was 39

The

3

138 139

GC−MS analysis of glucosinolate hydrolysis products. GSL hydrolysis products were measured

140

after a variety of treatments as follows: For the effect of pH on endogenous MYR activity, hexane-



Page 8 of 42

141

defatted seed meal from L. densiflorum (100 mg) was mixed with 250 µL (sufficient liquid to form

142

a paste) of either: 1) 0.1 M potassium phosphate buffer, pH 6.5; 2) 0.1 M sodium acetate buffer, pH

143

4.5; or 3) 0.1 M HCl, pH 1.5. As a control, hexane-defatted seed meal, not incubated with any

144

buffer, was also extracted and analyzed. For the effect of heat-deactivation of endogenous

145

myrosinase in hexane-defatted seed meal, MYR activity was totally deactivated in defatted seed

146

meal by incubation in an autoclave (120°C for 20 min) followed by incubation in a stove (100°C for

147

30 min). No residual MYR activity was detected by the pH-stat technique in the heat-treated seed

148

meal after subsequent addition of 0.1 M potassium phosphate buffer, pH 6.5. 40 Deactivated defatted

149

seed meal was mixed as described before with 0.1 M potassium phosphate buffer, pH 6.5, both in

150

presence or absence of exogenous 9 U/g MYR isolated from Sinapis alba seeds as reported in

151

Pessina et al.

152

activity (36 U/mL) was tested before each use. One MYR unit (U) was defined as the amount of

153

enzyme able to hydrolyze 1 µmol of sinigrin

154

investigating GSL products from roots and leaves, 100 mg of freeze-dried plant material, was mixed

155

with 250 µL of 0.05 M potassium phosphate buffer, pH 6.5. In all experiments, endogenous or

156

exogenous MYR hydrolysis was allowed for 15 min at 37°C, then 500 µL ethyl acetate was added,

157

and the mixture was vortexed for extraction of hydrolysis products. The control was extracted

158

similarly. The samples were finally centrifuged at 10,000 rpm and 4 °C before GC-MS analysis of

159

the ethyl acetate phase.

160

Extracted compounds were separated by a Bruker GC 451 gas chromatograph equipped with a HP-5

161

fused silica capillary column (30 m by 0.25 mm inside diameter; 0.25 µm film thickness, J&W

162

Scientific Inc, Folsom, CA) connected to a quadrupole mass detector (Bruker Scion SQ Premium,

163

Bruker Daltonics, Macerata, Italy). The oven temperature was set at 60 °C, and maintained for 4

164

min, then it was programmed to rise from 60 to 220 °C at 10 °C min-1, and finally held at 220 °C for

41

The enzyme solution was stored at 4 °C in sterile distilled H2O until use and its

per minute at pH 6.5 and 37 °C. Finally for


Page 9 of 42


165

4 min. The transfer line was maintained at 280 °C and the ion source at 220 °C. Split injection

166

(1:20) was applied and the carrier gas flow (helium) was 1 mL min-1. The mass spectrometer was

167

operated in electron impact mode at 70 eV, scanning the range of 10-250 m/z, in a full scan

168

acquisition mode. Mass spectra were identified by matching the recorded mass spectra with the

169

NIST/EPA/NIH Mass Spectral Database (NIST 11, Gaithersburg, MD), or compared with those of

170

ethyl acetate solutions of authentic standards if they were absent in the database (4-hydroxy-3,5-

171

dimethoxyphenylacetonitrile). Data were expressed as % of the total peak area.

172

RESULTS AND DISCUSSION

173

Glucosinolate profiles of Lepidium densiflorum seeds; roots; leaves. Seeds; roots; leaves of L.

174

densiflorum showed several differences in their GSL profiles when analyzed by the desulfoGSL

175

method (Figure 2). High levels were found in seeds, leaves had around one third of that level, while

176

root levels were very low. In seeds, five major benzylic GSLs (numbered from 1 to 5 in order of

177

elution) were detected. Corresponding desulfoGSLs are designated d1 - d5, respectively. Among

178

them, a prevalent desulfoGSL eluting at 12.6 min (d3), as well as another one present in low

179

amount and eluting at 11.7 min (d1), had unknown UV spectra when compared to our in-house

180

libraries of desulfoGSLs, and all five were identified as described below. The prevalent 3, at an

181

estimated level of 90 µmol/g, represented around 75% of total seed GSLs. Another major seed GSL

182

(5) was very low in leaves and roots. Overall, the GSL pattern of L. densiflorum appeared in all

183

analyzed plant tissues particularly rich in derivatives of benzylGSL. In addition, traces of indole

184

GSLs, indol-3-ylmethylGSL (glucobrassicin) and its 4-hydroxy derivative, were apparent by

185

HPLC-UV in leaves and roots, while traces of 3-methoxybenzylGSL (glucolimnanthin) were found

186

in roots (S. I. 1). They were identified by UV spectra and HPLC retention times based on our

187

library.

188

structure elucidation began with large scale desulfation of the native seed GSLs followed by HPLC

38, 42

Since seeds were a good source of all major GSLs in the investigated plant parts, the



Page 10 of 42

189

isolation and spectroscopy. Further analysis of seeds by LC-MS revealed additional minor GSLs as

190

described further.

191

Structure elucidation of desulfoglucosinolates. The last eluting desulfoGSL (d5) proved by 1H

192

and 13C NMR including 2D spectra and by ion trap MS/MS to be the desulfo derivative of a well-

193

known L. densiflorum GSL: 3,4,5-trimethoxybenzylGSL (5)

194

spectrum was quite simple owing to the symmetry of the side chain, and it was interesting to notice

195

a long range coupling in COSY between the aromatic 2´ and 6´ hydrogens (s at 6.69 ppm) and the

196

hydrogens of the methylene group (s at 4.00 ppm). This long range (four bonds) interaction proved

197

to be useful in the subsequent interpretation of the more complex spectra of d4 and the novel d3.

198

From HPLC-MS and proton NMR, the remaining four desulfoGSLs seemed of similar structure but

199

with fewer methyl or methoxyl groups, and were identified as follows.

200

The third eluting peak (d3) was available in considerable amounts. The proton NMR spectrum (S.I.

201

2) showed expectable signals from the thioglucose moiety as well as two aromatic protons in one

202

singlet (2H), two methyl groups in one singlet signal (6H) with chemical shift suitable for methoxyl

203

groups, and a double doublet at a chemical shift suitable for a benzylic methylene group, suggesting

204

a trisubstituted benzyl group with symmetrical substitution including two methoxyl groups. The m/z

205

value from ion trap MS agreed with the substituents being a hydroxyl group and two methoxyl

206

groups. The exact structure was now elucidated, aided by 2D NMR, as follows. Strong (3-bond)

207

HMBC correlations between the methoxyl protons and an aromatic carbon (C3´/C5´) confirmed the

208

presence of the deduced methoxyl groups on the aromatic ring. The carbon NMR supported a

209

symmetrically substituted benzyl group (four chemical shifts with intensities reflecting six carbons),

210

and HMBC confirmed connectivity between the methylene group, the thiohydroximate carbon (0)

211

and the anomeric hydrogen (g1) (Figure 3), confirming the hypothesis of a symmetrically

212

substituted desulfo-4-hydroxy-x,y-dimethoxybenzylGSL. Hence, the two hydrogens on the phenyl

43

( Tables 1, 2 and 3, S.I. 2). This


Page 11 of 42


213

group could either be ortho or meta to the methylene group, and solving their position would enable

214

to choose among the two possible symmetrical structures. These alternatives were distinguished by

215

HMBC: strong HMBC correlation of the aromatic hydrogens to all aromatic carbons as well as to

216

the methylene group was observed. This result was only compatible with the hydrogens being ortho

217

to the methylene group, meaning that the two methoxy groups were each in meta position.

218

Supporting this deduction, weak (4-bond) coupling from the aromatic hydrogens to the methylene

219

group was detected by COSY (Figure 3, Table 1). The general chemical shifts agreed with those of

220

other benzylGSL derivatives,

221

isolated desulfo derivative d3 was desulfo 4-hydroxy-3,5-dimethoxybenzylGSL. As the

222

corresponding native glucosinolate 3 bound to an anion exchange column and was released by

223

sulfatase treatment, we infer the presence of this novel GSL in L. densiflorum seeds and propose

224

3,5-dimethoxysinalbin as common name.

225

The fourth eluting peak, d4, was also available in sufficient amounts for 13C NMR, and its structure

226

elucidated in a similar way. Three aromatic hydrogens were found to be situated at the 2´, 5´ and 6´

227

positions from coupling patterns, long range COSY with the methylene hydrogens, and HMBC

228

interactions. Two methoxyl groups were apparent from the area (6H) in proton NMR. Their proton

229

chemical shifts were identical, but their carbon chemical shifts were not quite identical, in

230

accordance with the 3,4-disubstitution deduced above (Figure 3). Hence, d4 was the desulfo

231

derivative of a known GSL, 3,4-dimethoxybenzylGSL (4).

232

The early eluting d1 and d2 were only available at very low amounts, and d1 seemed to represent a

233

second novel GSL. Three aromatic protons, their coupling pattern, a 3H singlet at 3.86 ppm and the

234

general characteristics clearly demonstrated a 3,4-disubstituted hydroxy-methoxybenzyl side chain

235

for d1. The remaining question was the position of the methoxyl group, and this was elucidated by

236

NOESY (with presaturation of the water signal) (S.I. 2). There was a clear NOE interaction between

26

except for expectable effects of the substitution pattern. Hence, the



Page 12 of 42

237

the methoxyl hydrogens at 3.86 ppm and the aromatic H2 at 6.95 ppm, allowing us to conclude that

238

the corresponding novel GSL was 4-hydroxy-3-methoxybenzylGSL (1, 3-methoxysinalbin) (Figure

239

3). In contrast, the second eluting peak d2 proved to be the known 3-hydroxybenzylGSL

240

(glucolepigramin) 44 from ion trap MS/MS, 1H NMR and HSQC analysis (tables 1 and 2). A well

241

separated aromatic triplet as well as a singlet was unequivocal evidence for the position of the

242

hydroxyl group.

243

Analytical parameters of desulfoglucosinolates. The UV spectra of the desulfoGSLs were

244

characteristic and useful for peak recognition (Table 3, S.I. 3). Three bands, at around 205 nm, 230

245

nm and 275 nm (“B-band”) showed very variable relative intensities. The B-band was relatively

246

weak for the symmetrically substituted d3 and d5, and relatively prominent for d1, d2, and d4.

247

Prediction of relative UV response factors for quantitative analysis was not straightforward, leading

248

us to the tentative use of the response factor of a structurally related GSL, 4-hydroxybenzylGSL

249

(sinalbin), for all five desulfoGSL peaks.

250

All five investigated desulfoGSLs exhibited the general MS2 fragmentation of these analytes. The

251

resulting common fragment types have elsewhere been rationalized and named as types a, b, c and d

252

39,45

253

analysis, because this would allow partial structure elucidation from MS2 spectra.

254

exciting to observe unexpected minor and major fragment ions in the spectra of d1, d3, d4 and d5

255

(but not d2).

256

Both para-hydroxylated analytes d1 and d3 exhibited an additional fragment ion 77 amu smaller

257

than the type c fragment ion, while both para-methoxylated analytes d4 and d5 exhibited a

258

fragment 99 amu smaller than the type c fragment ion (Table 3, Figure 4). It can be hypothesized

259

that the type c minus 77 fragment from d1 and d3 was due to loss of HON=CH-SH as a neutral

(Table 3, Figure 4). There is currently much focus on side chain specific fragmentation in GSL 45

Hence, it was


Page 13 of 42


260

fragment, leaving the sodium adduct of a keto benzyl group with either one or two methoxyl

261

groups. Still, the interpretation allowed a meaningful interpretation of the fragmentation of the four

262

L. densiflorum desulfoGSLs with para-hydroxyl and para-methoxyl derivatives, resulting in “keto

263

type” and “oxonium ion type” fragment ions. The type c minus 99 fragment from d4 and d5 could

264

be due to the loss of a neutral ion pair, HON=CH-S-, Na+, leaving a positively charged oxonium ion

265

of the general formula para H3C-O+=benzyl. This interpretation was compatible with the lack of a

266

similar fragment from d2 due to the lack of any para-oxygen functionality.

267

The above interpretation of MS2 fragmentation would predict the same types of fragments from

268

desulfo

269

(glucoaubrietin): m/z 129 (c-77) and 121 (c-99), respectively. However, inspection of published ion

270

trap MS2 spectra of those desulfoGSLs from our laboratory (including the low mass range not

271

previously illustrated) 15 did not reveal any such fragments, meaning that the rule was not of general

272

applicability even at our specific MS2 conditions. Apparently, the presence of at least one meta-

273

methoxyl group was needed for the formation of a minor fragment ion of either type (c-77 or c-99),

274

and two meta-methoxyl groups were needed for the formation of a major fragment ion of either

275

type. Still, the interpretation allowed a meaningful interpretation of the fragmentation of the four L.

276

densiflorum desulfoGSLs with para-hydroxyl and para-methoxyl derivatives, resulting in “keto

277

type”” and “oxonium ion type” fragment ions. However, major loss of 77 amu from desulfo indole

278

GSL type-c fragments was observed for d7 (Figure 4) and indol-3-ylmethylGSL (results not

279

shown). This observation is in agreement with our interpretation, as also the indol-3-ylmethyl

280

moiety, with or without a 4-methoxy substituent, can form a conjugated fragment similar to the

281

fragments envisioned from d1 and d3 along with loss of H+ from the indole N-H. The fragment

282

from 7 was detected as both proton and sodium adduct (Figure 4B).

para-hydroxybenzylGSL

(sinalbin,

6)

and

desulfo

para-methoxybenzylGSL



Page 14 of 42

283

Glucosinolate products from seed meal at different pH. GC-MS qualitative analysis confirmed

284

the presence of an active endogenous MYR in defatted L. densiflorum seed meal. The products

285

after incubation in buffers included benzaldehydes, a benzyl alcohol and benzyl ITCs (Table 4). As

286

no volatile compounds were detected from direct analysis of obtained meals (data not shown), it

287

appears evident that endogenous MYR catalyzed the hydrolysis of 4 and 5 to the corresponding

288

ITCs at all the tested conditions, even at pH 1.5. The wide pH range is in agreement with reports for

289

other Brassicaceae.

290

ITCs (Table 4, Figure 5B). Regarding the dominating 3, the formation of both alcohol (4-hydroxy-

291

3,5-dimethoxybenzyl

292

syringaldehyde) products were evident both at pH 4.5 and pH 6.5, with the sum of these products

293

being about 20% of total GC area. The mechanism of formation of an alcohol starting from the ITC

294

from 3 would be expected to be hydrolysis facilitated by para-activation at the benzylic position

295

(Figure 5B).

296

relatively rapid oxidation to take place in our plant extracts. The carboxylic acid (4-hydroxy-3-

297

methoxybenzoic acid or vanillic acid) corresponding to 1 could be formed in a similar way.

298

Aldehydes were even detected corresponding to GSLs without para-hydroxyl groups, suggesting

299

some hydrolysis and additional oxidation even of the para-methoxyl derivatives 4 and 5 (Table 4).

300

The presence of benzaldehydes corresponding to benzylic GSLs or ITCs would seem to be a

301

common phenomenon, having also been reported for 4-rhamnosyloxybenzyl ITC,

302

3,4,5- trimethoxybenzyl ITC,

303

GLSs (3, 4, and 5) characterizing the profile of L. densiflorum seeds, corresponding aldehydes were

304

produced at all pH conditions. The relative content of these aldehydes increased with acidity during

305

the hydrolysis reaction, and the aldehydes from 4 and 5 were of very low relative peak area in

306

weakly acidic and near neutral solution (pH 4.5-6.5). However, for 3 the aldehyde was of

18

46

In contrast, the para-hydroxylated 3 and 1 apparently did not form stable

alcohol)

and

aldehyde

(4-hydroxy-3,5-dimethoxybenzaldehyde

or

The detection of an aldehyde (syringaldehyde) in addition to the alcohol suggests

43

47

benzyl ITC,

and 3,4-dimethoxybenzylGSL. 42 In conclusion, for the three main


Page 15 of 42


307

appreciable relative peak area even at these physiologically realistic pH values. Likewise, formation

308

of alcohol was only detected for the para-hydroxylated GSL (3). This pattern suggested an

309

alternative mechanism of formation of the aldehyde from 3: by oxidation via the alcohol, as

310

discussed further in the following section.

311

Exogenous myrosinase treatment of seed meal confirms the glucosinolate origin of products.

312

Thorough heat-inactivation of native MYR was followed by addition of MYR from S. alba, in order

313

to test the GSL origin of hydrolysis products. The addition of S. alba enzyme allowed this

314

conclusion because of its well understood specific substrate recognition of GSLs.

315

treatment resulted in a partial conversion of main GSLs into nitriles, revealed by detection of

316

nitriles in experiments without added MYR (Table 5), confirming the high susceptibility of

317

benzylglucosinolates to thermal degradation to nitriles.

318

methoxyphenylacetonitrile was detectable also in absence of active MYR, while 3-

319

hydroxyphenylacetonitrile was detected only in presence of MYR, probably for its low content.

320

Comparison of mass spectra and retention time with an authentic standard allowed detection of 4-

321

hydroxy-3,5-dimethoxyphenylacetonitrile, as it was not listed in NIST11 library (S.I. 4). The

322

addition of exogenous MYR produced ITCs derived from 4 and 5 as known GSL hydrolysis

323

products. Syringaldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde) was detected both in absence or

324

presence of exogenous MYR, at about equal relative peak areas, suggesting that at least some

325

syringaldehyde was formed during heat treatment. We confirmed the identification of

326

syringaldehyde by comparison with the authentic standard, and quantified it before and after

327

exogenous MYR addition. We

328

r2=0.9549) and found an increase from 10 µmol/g in heat deactivated seed meal to 25 µmol/g after

329

15 min of hydrolysis by added S. alba MYR at 37 °C. This quantification demonstrated that the

330

majority of the detected syringaldehyde was formed by MYR hydrolysis of 3. The syringaldehyde

49, 50

48

Thermic

For minor GSLs, 1-2, 4-hydroxy-3-

obtained a calibration curve of syringaldehyde (0.1-1 mM,



Page 16 of 42

331

formation may occur by hydrolysis of the GSL to the unstable ITC followed by spontaneous

332

hydrolysis to the benzylic alcohol and subsequent oxidation to the benzaldehyde (Figure 5B and C).

333

Surprisingly, the benzylic alcohol, observed in the seed autolysis experiment (Table 4), was not

334

observed in this experiment. The full conversion to syringaldehyde in this experiment could not be

335

enzymatic, considering the preceding heat treatment of the seed meal. Possibly, the oxidative

336

capacity of the seed meal had increased due to the heat treatment (e.g. by oxidation of

337

biochemically bound Fe2+ to Fe3+), resulting in full conversion of the alcohol product of 3 to

338

syringaldehyde.

339

Glucosinolate breakdown products in leaves and roots. In table 6, a list of all apparent GSL

340

breakdown products in roots and leaves is reported. In leaves, only products derived from 3 and 4

341

were detectable, in agreement with the leaf GSL profile being dominated by 3 and 4 (Figure 2). The

342

main hydrolysis product of 4 is 3,4-dimethoxybenzyl ITC. Major peaks of 4-hydroxy-3,5-

343

dimethoxybenzyl alcohol and syringaldehyde were observed also in this experiment, in agreement

344

with their suggested formation from the dominating leaf GSL, 3. In roots, major products were

345

always 3,4-dimethoxybenzyl ITC, 3,4,5-trimethoxybenzyl ITC

346

(limnanthin), the latter in agreement with detected low levels of glucolimnanthin in lyophilized

347

roots.

and 3-methoxybenzyl ITC

348 Searching for biosynthetic intermediates and related glucosinolates in seeds. A comparison of 349 the elucidated structures 1-5 immediately suggested the presence of a new benzylic GSL system with 350 an apparently simple biosynthetic relationship (Figure 5A). The putative parent GSL would appear to 351 be benzylGSL, converted to monosubstituted intermediates such as the meta hydroxyl derivative 2 352 and further to more complex derivatives. This biosynthesis could be similar to the known 353 biosynthesis of substituted indole GSLs by consecutive oxidation and methyl transfer.

51, 52

As a

354 preliminary test of the apparent biosynthesis, additional intermediates were therefore sought. The


Page 17 of 42


355 putative parent GSL, benzylGSL, was detected at trace level (0.5% of the level of 2) by HPLC-MS. 356 The 4-hydroxyderivative (sinalbin, 6) was conclusively detected, albeit at very low levels (2% of the 357 level of 2, assuming equal ionization efficiency) (Figure 4A, Figure 6B+D). A hypothetic 358 intermediate

54

(3,4-dihydroxybenzylGSL, ‘glucomatronalin’) was sought as the sodium adduct in

359 HPLC-MS data but not detected. As another possible intermediate, 3-methoxybenzylGSL (8), was 360 tentatively detected in roots and confirmed by detection of the corresponding ITC (Table 6), it was 361 included in the biosynthetic model (Figure 5A). 362 Indole GSLs were also searched for in seeds, and 4-methoxyindole-3-ylmethylGSL (7) was identified 363 with certainty in seeds at 3% of the level of 2 (based on retention time, m/z value and MS2 spectrum 364 in agreement with an authentic standard

45

) (Figure 4, Figure 6). The known biosynthetic precursors

365 indole-3-ylmethylGSL and 4-hydroxyindole-3-ylmethylGSL and the related 1-hydroxyindole-3366 ylmethylGSL

52

and 1-methoxyindole-3-ylmethylGSL were not detected at all in seeds. No aliphatic

367 GSL peaks were observed. 368 In conclusion, the illustrated biosynthesis from benzylGSL, starting with hydroxylation in either meta 369 or para position and proceeding with additional oxidation and methyl transfer, was supported by 370 detected putative intermediates (Figure 5A). This would be a parallel to the known biosynthesis of 371 hydroxyl and methoxyl derivatives of indol-3-ylmethylGSL.

51, 52

From the co-occurrence of the

372 indole GSL 7, it seems likely that the biosynthesis of 1-5 described here is catalyzed by similar 373 enzymes, possibly evolutionarily related to the Arabidopsis thaliana indole GSL biosynthesis 374 enzymes (cytochromes P450 for hydroxylation and specific indole glucosinolate methyl transferases 375 (IGMTs) for methylation). Another attractive hypothesis is the involvement of other CYPs and 376 methyl transferases related to the enzymes responsible for hydroxylation and methylation of aromatic 53

377 acids such as ferulic acid, the precursor of vanillin.

The dominance of the novel 3 lacking a methyl

378 group at the para position could possibly be due to steric hindrance of a particular methyl transfer,



Page 18 of 42

379 caused by a second meta methoxylation before the para methylation (Figure 5A). This lack of 380 conversion of 3 to 5 is the feature that allows formation of 4-hydroxy-3,5-dimethoxybenzyl alcohol 381 and syringaldehyde. It is tempting to speculate that the structurally related vanillin

53

could be formed

382 if the biosynthesis was blocked after 1. 383

Biological perspectives. The general dominance of the novel phenolic GSL 3 in L. densiflorum and

384

the detection of the corresponding alcohol and aldehyde products extends the “sinalbin paradox”

385

represented by dominance of non-ITC forming sinalbin accumulated by both wild and cultivated

386

forms of the common crop S. alba. 26 These plants are able to biosynthesize GSLs that form stable,

387

toxic ITC (benzyl ITC in S. alba leaves, and 3,4-dimethoxy and 3,4,5-trimethoxybenzyl ITC in all

388

tested L. densiflorum parts), nevertheless they mainly accumulate p-hydroxylated benzylic GSLs

389

that are precursors of apparently harmless alcohols (Figure 5B). If Brassicaceae are generally

390

adapted to be optimally defended by their own arsenal of GSLs,

391

3,5 dimethoxysinalbin (3) should also exist. The observed conversion to phenolics such as

392

syringaldehyde (Figure 5C) might indicate a new biological function. Indeed, a phenolic indole

393

GSL in A. thaliana mutants was associated with increased resistance to nematodes.

394

densiflorum would also appear to be a good source of genes and enzymes involved in hydroxylation

395

and subsequent methylation of benzylic GSLs. Syringaldehyde is naturally present in fruits, nuts,

396

grains, and other edible plants with antimicrobial, antifungal, antiparasite activities, and medicinally

397

interesting properties for inflammatory metabolic syndrome.

398

peculiar fusion in L. densiflorum between two of the most important systems of plant defense: the

399

GSL-MYR 2-9 and the phenolic systems. 54

54

28

a relevant function of the novel

52

L.

So, the new GSL 3 looks like a

400 Prolonged heating resulted in formation of nitriles in seed meal. To date there are no known specific 401 biological

activities

for

3,4,5-trimethoxyphenylacetonitrile

or

4-hydroxy-3,5-

402 dimethoxyphenylacetonitrile. The most interesting use of these compounds, that for the first time we


Page 19 of 42


403 describe as natural derivatives of GSLs, is their utilization as building blocks for the development of 404 new anticancer agents such as combretastatin analogues, or diarylacrylonitriles.

55

The minor novel

405 GSL identified here may also occur elsewhere: traces of 4-hydroxy-3-methoxyphenylacetonitrile 406 were also detected in the fruit of Bretschneidera sinensis Hemsl, suggesting the presence of traces of 407 this new GSL in at least one additional species, although the GSL itself was not detected. 56 408 The present discovery adds two novel natural GSLs (1, 3) to the documented GSL structures. A key 409 feature of this variation is variable substitution of aromatic rings with profound effect on degradation 410 chemistry. Future investigations will hopefully demonstrate still further agronomic and nutritional 411 advantages of this astounding biodiversity. 412 Abbreviations and Nomenclature: 413 GSL: glucosinolate; MYR: myrosinase; ITC: isothiocyanate; 414 Acknowledgments: 415 We thank Nerio Casadei (CREA-CIN) for supporting L. densiflorum reproduction in Bologna, 416 Lorena Malaguti (CREA-CIN) for her technical support and preliminary analysis of L. densiflorum, 417 Dr. Carla Boga (University of Bologna) for valuable discussions concerning GC-MS analysis, and 418 Susanne Bidstrup (University of Copenhagen) for desulfoGSL isolation for NMR. 419 Funding 420 This work was carried out partially within the activities of the Project “SUSCACE”, research activity 421 “Axbb” financed by the Italian Ministry of Agricultural, Food and Forestry Policies (Mipaaf), 422 CREA-CIN, Bologna, Italy. Glucosinolate identification by LC-MS and NMR was supported by 423 Torben og Alice Frimodts Fond, Denmark.



Page 20 of 42

424 Supporting Information description: 425

S.I.1. HPLC chromatograms of Lepidium densiflorum desulfated glucosinolates from leaf and root

426

extracts and UV spectra of detected desulfo indole glucosinolates and desulfo 3-

427

methoxybenzylglucosinolate (desulfo glucolimnanthin).

428

S.I.2. NMR spectra of isolated desulfoglucosinolates d1-d5.

429

S.I.3. HPLC chromatograms of isolated desulfoglucosinolates from Lepidium densiflorum seeds and

430

their corresponding UV spectra.

431

S.I.4. Comparison of GC-MS chromatograms and MS spectra from the analysis of Lepidium

432

densiflorum defatted seed meal extract, after exogenous myrosinase hydrolysis, and an ethyl acetate

433

solution of authentic 4-hydroxy-3,5-dimethoxyphenylacetonitrile.

434

435

436


Page 21 of 42


437

References

438

1. Warwick, S.; Francis, A.; Al-Shehbaz, I.A. Brassicaceae: species checklist and database on CD-

439

Rom. Plant Syst. Evol. 2006, 259, 249-258 .

440

2. Avato, P.; Argentieri, M. P. Brassicaceae: a rich source of health improving phytochemicals.

441

Phytochemistry 2015, 14, 6, 1019-1033.

442

3. Agerbirk, N.; Olsen, C. E. Glucosinolate structures in evolution. Phytochemistry 2012, 77, 16-45.

443

4. Fahey, J. W; Zalcmann, A. T.; Talalay, P. The chemical diversity and distribution of

444

glucosinolates and isothiocyanates among plants. Phytochemistry 2001, 56, 5-51.

445

5. Ettlinger, M.G.; Kjaer, A. Sulfur compounds in plants. Recent Adv. Phytochem., 1968, 1, 59-144.

446

6. Hanschen, F. S.; Lamy, E.; Schreiner, M.; Rohn, S. Reactivity and stability of glucosinolates and

447

their breakdown products in foods. Angew. Chem. Int. Ed. 2014, 53, 11430-11450.

448

7. Lazzeri, L.; Malaguti, L.; Cinti, S.; Ugolini, L; De Nicola, G. R.; Bagatta, M.; .Matteo, R.;

449

Casadei, N.; Patalano, G.; D’Avino, L. The Brassicaceae biofumigation system for plant cultivation

450

and defence. An Italian twenty-year experience of study and application. Acta Horticulturae. 2013,

451

1005, 331-338.

452

8. Matthiessen, J. N.; Kirkegaard, J. A. Biofumigation and enhanced biodegradation: opportunity

453

and challenge in soilborne pest and disease management. Crit. Rev. Plant Sciences. 2006, 25, 235-

454

265.

455

9. Lazzeri, L.; Curto, G.; Dallavalle, E.; D’Avino, L.; Malaguti, L.; Santi, R.; Patalano, G.

456

Nematicidal efficacy of biofumigation by defatted Brassicaceae meal for control of Meloidogyne

457

incognita (Kofoid et White) Chitw. on zucchini crop. J. Sustain. Agric. 2009, 33, 349-358.

458

10. Zhang Y.; Talalay P.; Cho C. G.; Posner G. H. A major inducer of anticarcinogenic protective

459

enzymes from broccoli: isolation and elucidation of structure. Proc. Natl. Acad. Sci. USA 1992, 89,

460

2399-2403.



Page 22 of 42

461

11. Abdull Razis, A. F.; Bagatta, M.; De Nicola, G. R.; Iori, R.; Ioannides, C. Intact glucosinolates

462

modulate hepatic cytochrome P450 and phase II conjugation activities and may contribute directly

463

to the chemopreventive activity of cruciferous vegetables. Toxicology 2010, 277, 74-85.

464

12. Jiang, Y.; Wu, S. H.; Shu, X. O.; Xiang, Y. B.; Ji B. T.; Milne, G. L.; Cai, Q.; Zhang, X.; Gao,

465

Y. T.; Zheng, W.; Yang, G. Cruciferous vegetable intake is inversely correlated with circulating

466

levels of proinflammatory markers in women. J. Acad. Nutr. Diet. 2014, 114, 700-708.

467

13. Frankel, F.; Priven, M.; Richard, E.; Schweinshault,; C.; Tongo, O.; Webster, A.; Barth, E.;

468

Slejzer, K.; Edelstein, S. Health functionality of organosulfides: A Review. Int. J. Food Prop. 2016,

469

19, 537-548.

470

14. Fourie, H.; Ahuja, P.; Lammers, J.; Daneel, M. Brassicacea-based management strategies as an

471

alternative to combat nematode pests: A synopsis, Crop Protec. 2016, 80, 21-41.

472

15. Morris, M. E.; Dave, R. A. Pharmacokinetics and pharmacodynamics of phenethyl

473

isothiocyanate: implications in breast cancer prevention, AAPS J. 2014, 16, 704-708.

474

16. Stohs, S. J.; Hartman, M. J. Review of the safety and efficacy of Moringa oleifera. Phytother.

475

Res. 2015, 29, 796–804.

476

17. Müller, C.; van Loon, J.; Ruschioni, S.; De Nicola, G. R.; Olsen, C. E.; Iori, R.; Agerbirk, N.

477

Taste detection of the non-volatile isothiocyanate moringin results in deterrence to glucosinolate-

478

adapted insect larvae. Phytochemistry 2015, 118, 139–148.

479

18. Borek, V.; Morra, M. J. Ionic Thiocyanate (SCN-) Production from 4-Hydroxybenzyl

480

glucosinolate contained in Sinapis alba seed meal. J. Agric. Food Chem. 2005, 53, 8650-8654.

481

19. Lazzeri, L.; Tacconi, R.; Palmieri, S. In vitro activity of some glucosinolates and their reaction

482

products toward a population of the nematode Heterodera schachtii. J. Agric. Food Chem. 1993,

483

41, 825-829.


Page 23 of 42


484

20. Nastruzzi, C.; Cortesi, R.; Esposito, E.; Menegatti, E.; Leoni, O.; Iori, R.; Palmieri, S. In vitro

485

antiproliferative activity of isothiocyanates and nitriles generated by myrosinase-mediated

486

hydrolysis of glucosinolates from seeds of cruciferous vegetables. J. Agric. Food Chem. 2000, 48,

487

3572-3575.

488

21. Manici, L. M.; Lazzeri, L.; Palmieri, S. In vitro antifungal activity of glucosinolates and their

489

enzyme derived products towards plant pathogenic fungi. J. Agric. Food Chem. 1997, 45, 2768-

490

2773.

491

22. Lazzeri, L.; Curto, G.; Leoni, O.; Dellavalle, E. Effects of glucosinolates and their enzymatic

492

hydrolysis products via myrosinase on the root knot nematode Meloidogyne incognita (Kofoid et

493

White) Chitw. J. Agric. Food Chem. 2004, 52, 6703-6707.

494

23. Zoller, O.; Bruschweiler, B. J.; Magnin, R.; Rehinard, H.; Rhyn, P.; Rupp, H., Zeltner, S.;

495

Felleisen, R. Natural occurrence of bisphenol F in mustard. Food Addit. Contam. A 2016, 33, 137-

496

146.

497

24. Sørensen, J.C.; Frandsen, H.B.; Jensen, S.K.; Kristensen, N.B.; Sørensen, S.; Sørensen, H.

498

Bioavailability and in vivo metabolism of intact glucosinolates. J. Functional Foods 2016, 24, 450-

499

460

500

25. Daxenbichler, M. E.; Spencer, G. F.; Carlson, D. G.; Rose, G. B.; Brinker, A. M.; Powell, R. G.

501

Glucosinolate composition of seeds from 297 species of wild plants. Phytochemistry 1991, 30,

502

2623-2638.

503

26. Agerbirk, N.; Warwick, S.; Hansen, P.R.; Olsen, C.E. Sinapis phylogeny and evolution of

504

glucosinolates and specific nitrile degrading enzymes. Phytochemistry 2008, 69, 2937-2949.

505

27. Bennett, R. N.; Mellon, F. A.; Kroon, P. A. Screening crucifer seeds as sources of specific intact

506

glucosinolates using ion-pair high-performance liquid chromatography negative ion electro-spray

507

mass spectrometry. J. Agric. Food Chem. 2004, 52, 428-438.



Page 24 of 42

508

28. Kerwin, R.; Feusier, J.; Corwin, J.; Rubin, M.; Lin, C.; Muok, A.; Larson, B.; Li, B.; Joseph, B.;

509

Francisco, M.; Copeland, D.; Weinig, C.; Kliebenstein, D. J. Natural genetic variation in

510

Arabidopsis thaliana defense metabolism genes modulates field fitness. eLife Sciences 2015, 4,

511

e05604.

512

29. Nakatoshi, Y.; Nakamura, T. Arabidopsis harmless to ozone layer protein methylates a

513

glucosinolate breakdown product and functions in resistance to Pseudomonas syringae pv.

514

maculicola. J. Biol. Chem. 2009, 284, 19301-19309.

515

30. Brader, C.; Mikkelsen, M. D.; Halkier, B. A.; Palva, E. T. Altering glucosinolate profiles

516

modulates disease resistance in plants. Plant J. 2006, 46, 758-767.

517

31. Bednarek, P.; Pislewska-Bednarek, M.; Svatoš, A.; Schneider, B.; Doubský, J.; Mansurova, M.;

518

et al. A glucosinolate metabolism pathway in living plant cells mediates broad-spectrum antifungal

519

defense. Science 2009, 323, 101–106.

520

32. Clay, N. K.; Adio, A. M.; Denoux, C.; Jander, G.; Ausubel, F. M. Glucosinolate metabolites

521

required for an Arabidopsis innate immune response. Science 2009, 323, 95–101.

522

33. Orsini, F.; D'Urzo, M.P.; Inan, G.; Serra, S.; Oh, D. H.; Mickelbart, M.V.; Consiglio, F.; Li, X.;

523

Jeong, J.C.; Yun, D.-J.; Bohnert, H.J.; Bressan, R.A.; Maggio, A. A comparative study of salt

524

tolerance parameters in 11 wild relatives of Arabidopsis thaliana. J. Exp. Bot., 2010, 61, 3787-

525

3798.

526

34. Curto, G.; Dallavalle, E.; Matteo, R.; Lazzeri, L. Biofumigant effect of new defatted seed meals

527

against the southern 1 root-knot nematode Meloidogyne incognita. Ann. Appl. Biol. 2016, 169, 17-

528

26.

529

35. Lazzeri, L.; Malaguti, L.; Bagatta, M.; D'Avino, L.; Ugolini, L.; De Nicola, G. R.; Casadei, N.;

530

Cinti, S.; Matteo, R.; Iori, R. Characterization of the main glucosinolate content and fatty acid

531

composition in non-food Brassicaceae seeds. Acta Horticulturae 2013, 1005, 331-338.


Page 25 of 42


532

36. Leoni, O.; Iori, R.; Palmieri, S. Hydrolysis of glucosinolates using nylon-immobilized

533

myrosinase to produce pure bioactive molecules. Biotech. Bioeng. 2000, 68, 660-664.

534

37. Franco, P.; Spinozzi, S.; Pagnotta, E.; Lazzeri, L.; Ugolini, L.; Camborata, C.; Roda, A.

535

Development of a liquid chromatography-electrospray ionization-tandem mass spectrometry

536

method for the simultaneous analysis of intact glucosinolates and isothiocyanates in Brassicaceae

537

seeds and functional foods. J. Chromatogr. A 2016, 1428, 154-161.

538

38. Wathelet, J. P.; Iori, R.; Leoni, O.; Rollin, P.; Quinsac, A.; Palmieri, S. Guidelines for

539

glucosinolates analysis in green tissues used for biofumigation. Agroindustria, 2004, 257-266.

540

39. Agerbirk, N.; Olsen, C.E.; Cipollini D.; Orgaard M.; Linde-Laursen I.; Chew, F.S. Specific

541

glucosinolate analysis reveals variable levels of epimeric glucobarbarins, dietary precursors of 5-

542

phenyloxazolidine-2-thiones, in watercress types with contrasting chromosome numbers. J. Agric.

543

Food Chem. 2014, 62, 9586-9596.

544

40. Finiguerra, M.G.; Iori, R.; Palmieri, S. Soluble and total myrosinase activity in defatted Crambe

545

abyssinica meal, J. Agric. Food Chem. 2001, 49, 840–845.

546

41. Pessina, A.; Thomas, R.M.; Palmieri, S.; Luisi, P.L. An improved method for the purification of

547

myrosinase and its physicochemical characterization. Arch. Biochem. Biophys. 1990, 280, 383–389.

548

42. De Nicola, G. R.; Nyegue, M.; Montaut, S.; Iori, R.; Menut, C.; Tatibouët, A.: Rollin, P.;

549

Ndoyé, C.; Amvam Zollo, P. H. Profile and quantification of glucosinolates in Pentadiplandra

550

brazzeana Baillon. Phytochemistry 2012, 73, 51–56.

551

43. Kjaer, A.; Wagnieres, M. 3,4,5-Trimethoxybenzylglucosinolate: a constituent of Lepidium

552

sordidum. Phytochemistry 1971, 10, 2195-2198.

553

44. Friis, P.; Kjær, A. Glucolepigramin, a new thioglucoside, present in Lepidium graminifolium L.

554

Acta Chem. Scand. 1963, 17, 1515-1520.



Page 26 of 42

555

45. Olsen, C.E.; Huang, X.-C.; Hansen, C.I.C.; Cipollini, D.; Ørgaard, M.; Matthes, A.; Geu-Flores,

556

F.; Koch, M.A.; Agerbirk, N. Glucosinolate diversity within a phylogenetic framework of the tribe

557

Cardamineae (Brassicaceae) unraveled with HPLC-MS/MS and NMR based analytical distinction

558

of 70 desulfoglucosinolates. Phytochemistry 2016, 132, 33-56.

559

46. Vaughn, S. F.; Berhow, M. A. Glucosinolate hydrolysis products from various plant sources: pH

560

effects, isolation, and purification. Ind. Crop Prod. 2005, 21, 193-202.

561

47. Alemayehu, M.; Tarekegn, G. Chemical investigation of the leaves of Moringa stenopetala.

562

Bull. Chem. Soc. Ethiop. 2000, 14, 51-56.

563

48. Burmeister, W. P.; Cottaz, S.; Driguez, H.; Iori, R,; Palmieri, S.; Henrissat, B. The crystal

564

structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights

565

into the substrate recognition and active-site machinery of an S-glycosidase. Structure, 1997, 5,

566

663-675.

567

49. Bones, A. M.; Rossiter, J, T. The enzymic and chemically induced decomposition of

568

glucosinolates. Phytochemistry, 2006, 67, 1053–1067.

569

50. Williams, D. J.; Critchley C.; Pun, S.; Chaliha, M.; O’Hare, T. J. Differing mechanisms of

570

simple nitrile formation on glucosinolate degradation in Lepidium sativum and Nasturtium

571

officinale seeds. Phytochemistry, 2009, 70, 1401–1409.

572

51. Pfalz, M.; Mikkelsen, M.D.; Bednarek, P.; Olsen, C.E.; Halkier, B.A.; Kroymann, J. Metabolic

573

engineering in Nicotiana benthamiana revelas key enzyme functions in Arabidopsis thaliana indole

574

glucosinolate modification. Plant Cell 2011, 23, 716-729.

575

52. Pfalz, M.; Mukhaimar, M., Perreau, F.; Kirk, J.; Hansen, C.I.C.; Olsen, C.E.; Agerbirk, N.;

576

Kroymann, J. Methyl transfer in glucosinolate biosynthesis mediated by indole glucosinolate O-

577

methyltransferase 51. Plant Phys. 2016, 172, 2190-2203.


Page 27 of 42


578

53. Gallage, N.J.; Hansen, E.H.; Kannangara, R.; Olsen, C.E.; Motawia, M.S.; Jørgensen, K.;

579

Holme, I.; Hebelstrup, K.; Grisoni, M.; Møller, B.L. Vanillin formation from ferulic acid in Vanilla

580

planifolia is catalyzed by a single enzyme. Nature Comm. 2014, 5, 4037.

581

54. Yancheva, D.; Velcheva, E.; Glavcheva, Z.; Stamboliyska , B.; Smelcerovic, A. Insights in the

582

radical scavenging mechanism of syringaldehyde and generation of its anion, J. Mol. Struct. 2016,

583

1108, 552-559.

584

55. Penthala, N.R.; Sonar, V.N.; Horn, J.; Leggas, M; Yadlapalli, J.S.; Crooks, P.A. Synthesis and

585

evaluation of a series of benzothiophene acrylonitrile analogs as anticancer agents.

586

MedChemComm. 2013, 4, 1073–1078.

587

56. Montaut, S.; Zhang, W.D.; Nuzillard, J.M.; De Nicola, G.R.; Rollin, P. Glucosinolate diversity

588

in Bretschneidera sinensis of Chinese origin, J. Nat. Prod. 2015, 78, 2001−2006.

589 590

591

592

593

594

595

596

597



Page 28 of 42

598

Figure captions

599

Figure 1. Detail of a portion of the field cultivated with Lepidium densiflorum at flowering time in

600

2013 in Bologna.

601

Figure 2. Content of the major benzylic glucosinolates in Lepidium densiflorum seeds, leaves and

602

roots. The results represent the mean of three independent analyses and are reported on a dry weight

603

basis. The insert shows a typical chromatogram of Lepidium densiflorum seeds after desulfation,

604

with the indication of the characterized desulfoglucosinolates d1-d5.

605

Figure 3. Selected long range NMR interactions critical for the structure elucidation of desulfo

606

derivatives (d1, d3, d4) of the corresponding glucosinolates (1, 3 and 4). Straight arrows represent

607

HMBC interactions, curved arrows represent either NOESY or COSY correlations as indicated.

608

Dashed lines signify weaker interactions.

609

Figure 4. Ion trap MS2 spectra of of desulfoglucosinolates (A) showing characteristic fragmentation

610

(B). Fragment codes: a, [anhydroGlc+Na]+; b, [thioGlc+Na]+; c, loss of ahGlc (162); c-77 and c-99,

611

characteristic fragmentations as illustrated. GSL; Glucosinolate; d1, desulfo 3-methoxysinalbin; d2,

612

desulfo 3-hydroxybenzylGSL; d3, desulfo 3,5-dimethoxysinalbin; d4, desulfo 3,4-

613

dimethoxybenzylGSL; d5, desulfo 3,4,5-trimethoxybenzylGSL; d6, desulfo sinalbin; d7, desulfo 4-

614

methoxyindol-3-ylmethylGSL.

615

Figure 5. A. Detected Phe-derived glucosinolates (GSLs) in Lepidium densiflorum with suggested

616

biosynthetic relationships. 1, 3-methoxysinalbin; 2, 3-hydroxybenzylGSL; 3, 3,5-

617

dimethoxysinalbin; 4, 3-4-dimethoxybenzylGSL; 5, 3,4,5-trimethoxybenzylGSL; 6, sinalbin; 8, 3-

618

methoxybenzylGSL. B. The hydrolysis of the activated isothiocyanates from 3 to a benzylic

619

alcohol. C Structure of syringaldehyde for comparison with alcohol product from 3.


Page 29 of 42


620

Figure 6. Conclusive detection of minor glucosinolates (GSLs) in L. densiflorum seeds by ion trap

621

HPLC-MS of Na+ adducts of desulfoGSL derivatives. A. Total ion chromatogram showing major

622

peaks, over-loaded in order to detect trace-level GSLs. B. Extracted ion chromatogram (m/z 368)

623

corresponding to [M+Na]+ of d2 at 5.0 min and the isomer desulfo sinalbin (d6) at 3.8 min. C.

624

Extracted ion chromatogram (m/z 421) corresponding to [M+Na]+ of desulfo 4-methoxyindol-3-

625

ylmethylGSL (d7). D. Enlargement of peak of desulfo sinalbin (d6) at 3.8 min. E. Structure of the

626

Trp-derived 4-methoxyindol-3-ylmethylGSL (7).



Page 30 of 42

Table 1. Data from 1H NMR of desulfoglucosinolates d1-d5 prepared from the native glucosinolates 1-5 in Lepidium densiflorum. d1 Aglucone 1a 3.97 br s 1b 2´ 6.96 d 2 Hz 3´ 4´ 5´ 6.91 d 8 Hz 6´ 6.96 dd 8/2 Hz OMe (m) 3.86 s OMe (p) Thioglucoside moiety g1 Ca 4,75 g2 Ca 3.35 (m) g3 Ca 3.35 (m) g4 3.40 (m) g5 Ca. 3.2 (m) g6a 4.65 (m)

d2

d3

d4

d5

3.98 s 6.82 ‘s’ 6.83 m 7.30 ‘tr’ 8 Hz 6.90 ‘d’ 8 Hz -

3.98 d 15 Hz 3.95 d 15 Hz 6.66 s 6.66 s 3.85 s -

4.00 d 15 Hz 3.96 d 15 Hz 6.97 d 2 Hz 7.04 d 8 Hz 6.93 dd 8/2 Hz 3.85 s 3.85 s

4.02 d 15 Hz 3.98 d 15 Hz 6.69 s 6.69 s 3.86 s 3.77 s

Ca 4.75 Ca 3.35 m Ca 3.35 m Ca 3.4 m 3.22 m 3.65 m

4.73 ‘d’ 7 Hz Ca 4.75 Ca 4.75 Ca 3.35 m Ca 3.30 Ca 3.35 Ca 3.35 m Ca 3.30 Ca 3.35 3.41 ‘tr’ 6 Hz 3.40 ‘tr’ 7 Hz 3.42 ‘tr’ 7 Hz 3.12 m 3.16 m 3.11 m 3.64 dd 13/5 Ca 3.6 m 3.63 dd 13/5 Hz Hz g6b 3.57 dd 13/2 Ca 3.6 m 3.54 dd 13/2 Hz Hz NMR conditions were: 400 MHz, solvent D2O, chemical shifts (δ) relative to that of dioxane set to 3.750 ppm. Chemical shifts with only one decimal or preceded by ‘ca’ are approximate values as extracted from 2D spectra. Multiplicity in quotation marks (‘s’, ‘d’, ‘tr’) indicates additional complexity assigned to long range coupling. Multiple coupling constants are separated by a dash (/). A broad singlet attributed to a slight difference in chemical shift is abbreviated br s.


Page 31 of 42


Table 2. Data from 13C NMR of desulfoglucosinolates d1-d5 prepared from the native glucosinolates 1-5 in Lepidium densiflorum.

Aglucone 0 1 1´ 2´ 3´ 4´ 5´ 6´ OMe (m) OMe (p)

d1

d2

d3

d4

d5

n.d. 38 n.d. 112 n.d. n.d. 115 121 57 -

n.d. 39 n.d. 116 n.d. 116 132 121 -

155.4 38.8 128.6 106.3 148.9 133.8 148.9 106.3 57.2 -

155.5 38.4 129.9 112.4 149.2* 148.1* 113.1 121.5 56.61** 56.56**

155.1 38.9 133.8 106.2 153.6 136.5 153.6 106.2 56.9 61.8

Thioglucoside moiety g1 82 82 82.0 82.0 82.1 g2 73 73 72.8 72.8 72.8 g3 78 78 78.0 77.9 78.0 g4 70 70 69.5 69.6 69.5 g5 81 81 80.7 80.6 80.7 g6 61 61 60.9 61.0 61.0 NMR conditions were: 100 MHz, solvent D2O, chemical shifts (δ) relative to that of dioxane set to 67.400 ppm. Chemical shifts without decimals are approximate as extracted from 2D spectra. For chemical shifts labelled with * or **, respectively, the assignments are uncertain within each label group.



Page 32 of 42

Table 3. Analytical parameters for peak identification of the main Lepidium densiflorum glucosinolates, after analytical desulfation, by HPLC with UV and MS/MS detection.

UV (λmax/nm)

d1 HPLC-DAD (aq. ACN): 203 224 (sh) 282 HPLC-DAD (aq. MeOH): 226 (sh) 281

m/z [M+Na]+

d2

d3

d4

d5

204 220 (sh) 274 280

208 230 (sh) 274 (w)

206 228 280

208 228 (sh) 270 (w)

215 (sh) 273 278

230 (sh) 271 (w)

227 278

230 (sh) 270 (w)

428

412

428

185 203 219 (base) 266

185 203 219 (base) 250

185 203 219 (base) 280

189 (major) -

151 (minor)

181 (major)

HPLC-MS/MS (ion trap) 398 368 MS2 (intermediate or major fragments) “Type a” 185 185 “Type d” 203 203 “Type b” 219 (base) 219 (base) “Type c” 236 206

[ahGlc+Na]+ [Glc+Na]+ [thioGlc+Na]+ [M+NaahGlc]+ Type c – 77 “quinone” Type c – 99 “oxonium” (sh): shoulder, (w): weak

159 (minor) -

-


Page 33 of 42


Table 4. Volatile products from native Lepidium densiflorum defatted seed meal subjected to hydrolysis by endogenous enzymes with aqueous buffers (pH 4.5 and 6.5) or dilute HCl (pH 1.5). GC tR (min) 15.0 15.4

16.8

17.6

18.7

19.3

20.7

18.2

pH

Area %

Component

MS, 70 eV, m/z (rel.int.)

6.5 4.5 6.5 4.5 1.5 6.5 4.5 1.5 6.5

0.2 2.0 1.0 100° C

HO H3CO

For Table of Contents Only


Hydroxyl and Methoxyl Derivatives of Benzylglucosinolate in Lepidium

Recommend Documents