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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

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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


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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

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

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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

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

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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

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human health.

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

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syringaldehyde.

19 20 21

22


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INTRODUCTION

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

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

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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),

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thioglucosidic multifunction secondary metabolites characterized by a variable side chain that

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

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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

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

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

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

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

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called biofumigation technique. 7-9

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

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

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

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

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

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

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

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GSLs in nature.



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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complex biochemistry of sinalbin metabolism. 24

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

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

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

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

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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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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hydrolysis products in a variety of agriculturally relevant tissues.

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MATERIALS AND METHODS

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

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35

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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



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

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

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

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

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vacuum desiccators.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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analysis was performed in triplicate.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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



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

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

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

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

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

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

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

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

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

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

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Pessina et al.

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

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enzyme able to hydrolyze 1 µmol of sinigrin

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

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

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

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

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

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the ethyl acetate phase.

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

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

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

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

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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


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

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

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

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

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

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

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

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RESULTS AND DISCUSSION

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

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

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

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

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

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

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

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

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

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

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

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

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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

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library.

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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



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

190

described further.

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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-

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

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

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

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

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

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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.

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

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

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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

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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)

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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

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

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

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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


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

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

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

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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.

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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

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other benzylGSL derivatives,

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

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

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

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3,5-dimethoxysinalbin as common name.

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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´

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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).

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

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

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

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

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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



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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

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hydroxyl group.

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

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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

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

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

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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.

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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

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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


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

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

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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

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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.

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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

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

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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

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

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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

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

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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.


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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

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