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Aug 22, 2016 - ABSTRACT: Glutathione (GSH) conjugates of the mycotoxin 4-deoxynivalenol (DON), 1, have been detected in plants by. LC-MS, but their ...
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Characterization of Deoxynivalenol−Glutathione Conjugates Using Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography−High-Resolution Mass Spectrometry Ana Stanic,†,§ Silvio Uhlig,† Morten Sandvik,† Frode Rise,§ Alistair L. Wilkins,†,⊥ and Christopher O. Miles*,† †

Norwegian Veterinary Institute, P.O. Box 750, Sentrum, NO-0106 Oslo, Norway Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway ⊥ Chemistry Department, University of Waikato, Private Bag 3105, 3240 Hamilton, New Zealand §

S Supporting Information *

ABSTRACT: Glutathione (GSH) conjugates of the mycotoxin 4-deoxynivalenol (DON), 1, have been detected in plants by LC-MS, but their identities were not confirmed due to a lack of standards. We have synthesized DON−GSH conjugates in alkaline solution. The major products 2 and 5 were isolated and their structures determined by mass spectrometry and NMR spectroscopy as GSH adducts at C-13 and C-10 (via epoxide and Michael addition, respectively) of 1. Other Michael addition products were also tentatively identified by LC-MS. Concentrations of 2 and 5 were determined by quantitative NMR and are suitable for use as quantitative standards for LC-MS studies of plant and animal metabolism of 1. LC-MS showed that in the presence of human glutathione S-transferases of the alpha and mu classes, the reaction of DON and GSH proceeded with a halflife of 17 h, identical with the rate of the uncatalyzed reaction rate, indicating an absence of catalysis. KEYWORDS: thiol, mycotoxin, HRMS, LC-MS, NMR, ERETIC, conjugation, GSH, GST

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INTRODUCTION Fungi of the genus Fusarium infect grain and other crops worldwide and are responsible for contamination of grain products with a range of mycotoxins, including trichothecenes, zearalenone, and fumonisins.1 Among the trichothecene mycotoxins, 4-deoxynivalenol (DON), 1 (Figure 1), is one of the most widespread and economically important, causing feed refusal, reduced weight gain, and emesis in livestock consuming heavily contaminated grain products.2 Several detoxification mechanisms for 1 have been identified in plants infected with Fusarium, including sulfation,3 glycosylation,4 and conjugation with the tripeptide L-glutathione (GSH).5 The presence of DON−GSH, as well as its presumed degradation products DON−cysteinylglycine and DON−cysteine (DON−Cys), was recently demonstrated by LC-MS in DON-treated wheat lines,6 although the actual structures of the conjugates were not determined. This, together with the observed up-regulation of glutathione transferase (GST) and related pathways in contaminated plants,7 suggests a role for GST-catalyzed conjugation in this detoxification process. GSTs are a widely distributed superfamily of multifunctional enzymes found in invertebrates and vertebrates, plants, yeasts, and bacteria.8 Their main role is catalysis of the nucleophilic conjugation of GSH to electrophilic substrates, but they also have less well characterized roles in endogenous metabolism including functioning as GSH-dependent peroxidases or GSH-dependent isomerases or have noncatalytic functions including binding of nonsubstrate ligands and modulation of signaling processes.9 Thus, a wide array of glutathione conjugates can be formed with endogenous and xenobiotic electrophilic species.10,11 © 2016 American Chemical Society

Characteristic features of glutathione are its high cellular concentration relative to other thiols, with glutathione accumulating to millimolar concentrations in tissues, well in excess of the concentration of free cysteine, and the high reduction state of the cellular glutathione pool.12 Recent studies with 1 suggested that it reacts with a wide range of thiols, including Cys and GSH, and that the reactive species is the thiolate.13 The reaction of 1 with Cys leads to reversible Michael addition of the thiolate to C-10 to form multiple diastereoisomers and to irreversible nucleophilic addition of the thiolate to C-13 of the epoxy group.14 Thus, the Cys and GSH conjugates of 1 formed naturally in plants could plausibly have a number of structures. Here we report the base-catalyzed synthesis and isolation of GSH adducts of 1 and their identification by LC-MS and NMR. The adducts were then used as standards to study the possible role of GST catalysis in the conjugation of 1 with GSH in vitro.

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MATERIALS AND METHODS

Chemicals. DON, 1 (≥98%), GSH (≥98%), 5.0 mm Norell Standard Series NMR tubes, D2O (99.9 atom % D), glutathione Stransferase (GST) assay kit (CS0410, human GSTA4 GSTM1), and sodium carbonate (pro analysis) were from Sigma-Aldrich (Steinheim, Germany). LC-MS grade water and acetonitrile were from Fisher Scientific (Oslo, Norway), whereas ammonium formate (puriss p.a. for HPLC) and dioxane were from Fluka (Steinheim, Germany). Sodium Received: Revised: Accepted: Published: 6903

June 24, 2016 August 18, 2016 August 22, 2016 August 22, 2016 DOI: 10.1021/acs.jafc.6b02853 J. Agric. Food Chem. 2016, 64, 6903−6910

Article

Journal of Agricultural and Food Chemistry

mode (m/z 200−400) for detection of 1 (tR 15.9 min, [M + HCO2]− for 1 at m/z 341.1). This method was used to follow the incubation of 1 with glutathione in the enzymatic assay. LC-HRMS. Method 2A. An alternative HPLC method was applied to better separate the isomeric reaction products using a 150 × 2.1 mm i.d., 2.6 μm, Kinetex F5 column (Phenomenex, Torrance, CA, USA). Injection volumes were 2 μL. The mobile phase (250 μL/min) consisted of 5 mM ammonium formate (A) and 5 mM ammonium formate in 95:5 methanol/water (B), in a linear gradient from 3 to 40% B over 14.5 min, then to 100% B at 14.7 min (2 min hold), followed by return to 3% B at 16.9 min and equilibration with 3% B for 3.1 min using a Dionex UltiMate 3000 UHPLC pump (ThermoFisher Scientific). The detector was a Q-Exactive Fourier-transform highresolution mass spectrometer (ThermoFisher Scientific) equipped with a heated electrospray ionization interface (HESI). The HRMS was run in positive and negative ion full-scan mode using fast polarity switching (i.e., alternating positive and negative ion scans) in the mass range m/z 150−700. The mass resolution was set to 70,000 at m/z 200. The spray voltage was 4 kV, the transfer capillary temperature was 250 °C, and the sheath and auxiliary gas flow rates were 35 and 10 units, respectively. Method 2B. LC-HRMS2 was used to acquire high-resolution MS2 spectra for the reaction products, using the same chromatographic conditions and detector as for method 2A. Analyses were performed in positive or negative mode, using alternating full scan (performed as for method 2A) and targeted MS2 scans. The precursor ion mass for the MS2 spectra was set to m/z 604.2 ([M + H]+ of 2−5) in positive mode and to m/z 602.2 ([M − H]− of 2−5) in negative mode, with an isolation window of m/z 2.0. The normalized collision energy for higher energy collisional dissociation was set to 30, and the resolution during product ion scanning was set to 17,500. Analytical Scale Reaction between 1 and GSH. DON, 1 (100 μg), was dissolved in 1 mL of a freshly prepared solution of glutathione (50 mM) in 0.2 M carbonate buffer (pH 10.7) and held at ambient temperature (ca. 20 °C). Aliquots (20 μL) were periodically (ca. hourly on the first day and daily thereafter for 20 days) transferred to chromatography vials and diluted to 1 mL with water; 1.5 μL of acetic acid was added to stop the reaction, and the mixture was analyzed by LC-MS (methods 2A and 2B). Preparation of DON 13−GSH (2). DON, 1 (1 mg, 3 μmol), was dissolved in a freshly prepared solution of GSH (30.7 mg, 100 μmol) in carbonate buffer (pH 10.7, 1 mL) and placed in an incubator at 37 °C for 15 days. At this time, epoxide adduct 2 was the most prominent component in the reaction mixture as assessed by LC-MS (method 1A). An aliquot of the reaction mixture (800 μL) was applied to a Strata-X SPE column. The column was eluted with water (5 × 1 mL) followed by 0.5% methanol (5 × 1 mL), and the 10 fractions were analyzed by LC-MS (method 1A). The third water fraction contained most of epoxide adduct 2 and was freeze-dried. Preparation of DON 10−GSH (5). DON, 1 (1 mg, 3 μmol), was dissolved in a freshly prepared solution of GSH (30.7 mg, 100 μmol) in carbonate buffer (pH 10.7, 1 mL) and placed in the LC-MS autosampler tray at 20 °C. The reaction mixture was followed approximately hourly on the first day and daily thereafter by LC-MS (method 1A). After 9 days, an aliquot (400 μL) of the reaction mixture was diluted with water to 600 μL and applied to a Strata-X SPE column. The column was eluted with water (4 × 1 mL), 0.5% methanol (4 × 1 mL), and 5% methanol (3 × 1 mL). To each fraction was added 5 μL of acetic acid, and the fractions were analyzed with LC-MS method 1A. Fractions 4 and 5 (the fourth water fraction and first fraction containing 0.5% methanol) contained most of Michael adduct 5. The two fractions were combined and freeze-dried. Specific Activity of GST. Phosphate-buffered saline (PBS) (pH 7.3, 2940 μL), GSH (200 mM, 30 μL), and 1-chloro-2,4dinitrobenzene (100 mM, 30 μL) as substrate were prepared in two 3 mL quartz cuvettes, and GST (6 μL; 0.25 mg/mL) was added to one of the cuvettes. Absorbance was measured relative to the control cuvette on a UV-1800 Shimadzu UV spectrophotometer (Bergman AS, Rælingen, Norway) at 340 nm for 300 s at 1 s intervals. The absorbance increased linearly with time and indicated the GST

Figure 1. Chemical structures of 4-deoxynivalenol (DON (1)) and its Michael (DON 10−GSH (2)) and epoxy (DON 13−GSH (5)) adducts with glutathione (GSH). In solution these compounds exist as equilibrium mixtures of their ketone (1a, 2a, and 5a) and hemiketal (1b, 2b, and 5b) forms. Atoms in the amino acids Gly, Cys, and Glu in the GSH side chains are numbered from their carboxylic acid termini as shown for 2a. Compounds 3 and 4 (Figures 2 and 5−7) are believed to be 9,10-diastereoisomers of 5. bicarbonate (pro analysis) (Merck, Darmstadt, Germany) and sodium carbonate were used to prepare 0.2 M carbonate buffer (pH 10.7). Acetic acid-d4 (99.5 atom % D) was from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Strata-X SPE columns (500 mg) from Phenomenex (Torrance, CA, USA) were conditioned with 5 mL of methanol and 5 mL of water before use. Methanol was from Romil (Cambridge, UK). Values for m/z of conjugates were calculated with Fusarium Toxin Mass calculator (version 9 in Excel),15 whereas m/z values for fragment ions and mass deviations were calculated with Xcalibur 2.3 (Thermo Scientific). LC-MS. Method 1A. Chromatography was performed on a 150 × 2.1 mm, i.d., 3 μm, Atlantis T3 column (Waters, Milford, MA, USA). The flow rate was 300 μL/min, and the injection volume was 2 μL. The mobile phase consisted of 5 mM aqueous ammonium formate (A) and 5 mM ammonium formate in 95:5 acetonitrile/water (B). A Surveyor HPLC system was interfaced to a Finnigan LTQ linear ion trap mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) operated in full scan mode. The mass spectrometer was equipped with an electrospray ionization (ESI) interface operated in positive or negative mode. Important source parameters included a heated capillary temperature of 275 °C, a sheath gas flow rate of 45 units, and an auxiliary gas flow rate of 8 units. The source voltages were 5 and −4 kV in positive and negative modes, respectively. This method was used to analyze fractions from SPE of the reaction mixtures, using a linear gradient from 0.5 to 15% B over 15 min, then to 100% B over 5 min, with a 3 min hold, followed by a return to 0.5% B with a 3 min hold for equilibration. The mass spectrometer was sequentially run in either positive or negative ion mode in the mass range m/z 100−1000. Method 1B. This was identical to method 1A, with the following exceptions. A linear gradient was used from 0.5 to 15% B over 15 min, then to 100% B over 1 min, with a 1 min hold, followed by a return to 0.5% B with a 3 min hold for equilibration. The first segment (0−14.5 min) was full scan in positive mode (m/z 100−1000) for the detection of DON−GSH adducts ([M + H]+ for 2−5 at m/z 604.2). In the second segment (14.5−20 min), the polarity was switched to negative 6904

DOI: 10.1021/acs.jafc.6b02853 J. Agric. Food Chem. 2016, 64, 6903−6910

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Journal of Agricultural and Food Chemistry solution had a specific activity of 4.13 μmol/min/mg at ambient temperature. Reaction of GSH with 1, with and without GST. DON, 1 (100 μg), was dissolved in PBS (1 mL) containing GSH (200 mM) in two LC-MS vials, which were equilibrated to the LC-MS tray temperature (36 °C) prior to commencement of the experiment. To one vial was added GST (30 μL; 0.25 mg/mL); the other vial was used as a control. Reactions were followed by regular injections with the LC-MS (method 1B) for 20 h and then reanalyzed after 25 days. The reaction was performed a second time, but in addition to analysis by injection with the LC-MS, aliquots (20 μL) were transferred after 1, 4.5, and 24 h to chromatography vials and diluted to 1 mL with water, and 2 μL of acetic acid added. The aliquots were analyzed by LC-HRMS2 (method 2B) for 4 and 5. NMR Spectroscopy. Purified 2 and 5 were dissolved in 545 μL of D2O to which 5 μL of acetic acid-d4 had been added. 1H, SELTOCSY, COSY, TOCSY, HSQC, HMBC, band selective HMBC, NOESY, and ROESY NMR experiments on 2 and 5 and 13C and DEPT135 NMR experiments on 2 were conducted on an Avance AVIII HD 800 MHz NMR spectrometer (BioSpin GmbH, Rheinstetten, Germany) equipped with a 5 mm TCI cryoprobe (1H, 13C, 15N) with automatic tuning and matching and Z-gradient accessories. 13C and DEPT135 NMR experiments on 5 were conducted on an AVII 600 MHz instrument. Data were recorded and processed using Bruker TopSpin (versions 3.0 and 3.2), and chemical shifts are reported relative to internal CD2HCO2D, 2.08 ppm, and CD3CO2D, 21.03 ppm.16 Quantitative NMR (qNMR). qNMR was performed using the Electronic REference To access In vivo Concentrations (ERETIC 2) procedure,17 using a 527 ± 7 μM dioxane solution as the external standard as described previously.14 Quantitation was performed on an Avance AVII 600 MHz (Bruker) NMR spectrometer equipped with a 5 mm CP-TCI (1H/13C, 15N−2H) triple-resonance inverse cryoprobe with Z-gradient coils. The number of scans was set to 16, the flip angle 90°, the relaxation delay (d1) 10 s, the pulse width (p1) 7.43 μs, the acquisition time 3.635 s, and the receiver gain (RG) 32.0, with lowpower continuous wave presaturation on the water peak. The areas from the H-15 protons in 2a and H-10 in 2b, as well as the H-14 methyl protons of 5a and 5b, were manually integrated, and concentrations were calculated with reference to external dioxane. The calculated concentrations were obtained from the average of 11 independent measurements.

Figure 2. Extracted ion LC-HRMS chromatograms (method 2A; positive ion mode, m/z 604.2171 (corresponding to [M + H]+ for 2− 5), 5 ppm window) from the reaction between DON and GSH followed for 20 days. The same vertical scale is used for all chromatograms. An essentially identical series of chromatograms was obtained in negative ion mode at the mass corresponding to [M − H]− of 2−5.

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peaks corresponding to 3 and 4/5 gradually decreased in signal height, whereas the intensity of another peak, 2, with the same mass increased. Furthermore, the 4/5 peak moved to slightly later retention time, indicating that the amount of 5 relative to 4 increased with time. Similar reaction dynamics were observed for the reaction of 1 with mercaptoethanol and cysteine,13,14 where later-eluting peaks, due to Michael addition of thiols to the 9,10-double bond of 1, gradually disappeared and were replaced by the earlier-eluting epoxide adduct that formed more slowly. The identities of 2 and 5 were determined by NMR analysis after purification. NMR Analysis of DON−GSH Conjugates 2 and 5. LCMS data established that 2 and 5 both contained 1 conjugated to one molecule of GSH. NMR spectroscopy was used to verify this and to establish the point of attachment between 1 and GSH in 2 and 5 and the stereochemistry of the attachment for 5. Compounds 2 and 5 were dissolved in D2O containing 0.9% v/v CD3CO2D, as this was shown to stabilize thia-Michael adducts of Cys with 1.14 No signs of deconjugation or isomerization of 5 were observed under these conditions by NMR or LC-MS over a period of >6 months, and the corresponding Cys adduct14 of 1 was similarly found to be stable for at least 9 months (Stanic, Uhlig, Rise, Wilkins, and Miles, unpublished observations). The presence of acid also promoted equilibration between the ketone and hemiketal isomers of the adducts (i.e., 2a/2b and 5a/5b), leading to weak correlations between pairs of resonances in some of the

RESULTS AND DISCUSSION LC-HRMS Monitoring of DON−GSH Reaction. Previously optimized conditions for reaction of DON with thiols13,14 were applied to study the reaction of 1 with GSH on an analytical scale. Because lowering the pH was recently shown to stop the reaction of 1 with Cys and to stabilize the resulting conjugates,14 the reaction was monitored by taking aliquots at appropriate times and adding acetic acid, followed by analysis with a variety of LC-MS methods. LC-HRMS in positive (Figure 2) and negative ion modes showed fast formation of 3 and closely eluting 4/5 and confirmed that the GSH adducts of 1 were stable under weakly acidic conditions. The three products afforded positively charged ions at m/z 604.2167−604.2169, consistent with C25H38O12N3S+ (Δ −0.7 to −0.2 ppm), and negatively charged ions at m/z 602.2040− 602.2041, consistent with C25H36O12N3S− (Δ 2.4−2.7 ppm), indicating attachment of a single GSH moiety to DON. Despite the improved separation of DON−GSH isomers on the pentafluorophenyl propyl column (Kinetex F5) compared to the ODS column, 4 and 5 could not be completely separated. The chromatographic peak from 4/5 was unusually broad, and the product ion spectra from targeted-HRMS2 in positive ion mode were different at the peak front compared to its tail. Thus, we concluded that the peak contained a mixture of at least two isomers (Figure 2). Over a period of 20 days, the 6905

DOI: 10.1021/acs.jafc.6b02853 J. Agric. Food Chem. 2016, 64, 6903−6910

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Journal of Agricultural and Food Chemistry

Table 1. 1H and 13C NMR Assignments (δ) for the Hemiketal Form of DON 13−GSH (2b) (Epoxide Adduct) and DON 10− GSH (5) (Michael Adduct) in D2O Containing CD3CO2D (1% v/v)a DON 10−GSH (5) (Michael adduct) 5a (ketone form) 1

atom DON

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Cys

Gly

Glu

1 2 3

1 2 1 2 3 4 5

3.75 4.47 2.17 2.06

H

(d, 4.4) (dt, 11.0, 4.4) (m) (dd, 14.4, 11.0)

4.79 (br s) 2.83 (dq, 12.4, 6.5) 3.43 (dd, 12.4, 3.3) 4.73 (d, 3.3) 3.32 3.22 1.03 3.67 3.91 1.24

(d, 3.5) (d, 3.5) (3H, s) (d, 12.2)* (d, 12.2)* (3H, d, 6.5)

4.65 (dd, 8.2, 5.7) 3.16 (dd, 14.1, 5.7)* 3.13 (dd, 14.1, 8.2)*

3.99 (2H, m)

3.83 (t, 6.4) 2.19 (2H, m) 2.56 (2H, m)

DON 13−GSH (2) (epoxide adduct)

5b (hemiketal form) 13

1

C

81.6 69.5 44.1 47.0 56.6 75.9 214.2 44.6 52.5 77.0 68.2 49.5 14.4 60.2 13.0 173.8 54.5 34.2

174.8 42.9 174.9 55.1 27.3 32.5 176.0

3.71 4.47 1.87 2.06

H

(d, 4.4) (dt, 11.0, 4.4) (dd, 14.4, 4.4) (dd, 14.4, 11.0)

4.00 (br s) 1.86 (dq, 12.0, 6.5) 2.96 (dd, 12.0, 5.8) 4.56 (d, 5.8) 3.35 3.27 1.09 4.25 3.72 1.14

(d, 3.8) (d, 3.8) (3H, s) (d, 8.7) (d, 8.7) (3H, d, 6.5)

4.66 (dd, 7.5, 5.8) 3.22 (dd, 14.0, 7.5) 3.11 (dd, 14.0, 5.8)

3.99 (2H, m)

3.83 (t, 6.4) 2.19 (2H, m) 2.56 (2H, m)

2b (hemiketal form) 13

1

C

82.4 69.6 45.0 44.6 56.7 77.1 107.5 43.3 49.9 75.6 68.7 49.3 15.3 68.7 13.2 173.8 54.6 32.7

174.8 42.9 174.9 55.1 27.3 32.5 176.0

H

4.02 4.63 1.54 2.11

(d, 4.6) (ddd, 10.8, 4.6, 3.8) (dd, 14.4, 3.8) (dd, 14.4, 10.8)

4.08 (s)

5.55 (dq 3.8, 1.6) 4.85 (m) 3.18 3.47 1.32 4.28 3.48 1.81

(d, 13.1) (d, 13.1) (3H, s) (d, 9.0) (d, 9.0) (3H, t, 1.6)

4.62 (dd, 8.1, 5.5) 3.00 (dd, 14.1, 8.1)* 3.14 (dd, 14.1, 5.5)*

3.96 (2H, bs)

3.82 (t, 6.5) 2.18 (2H, m) 2.56 (2H, m)

13

C

80.7 70.40 44.8 51.8 52.1 77.2 105.8 143.0 123.0 77.5 83.1 36.1 16.3 70.41 16.7 173.8 54.6 35.4

175.2 43.1 174.9 55.1 27.3 32.6 176.0

a

Format: chemical shift in ppm (multiplicity, coupling constants in Hz). Atom numbering is as shown in Figure 1. Except where marked with an asterisk, pairs of distinguishable methylene protons are in the order Hpro‑R, Hpro‑S (for stereochemically significant NOE correlations for 5b, see Figure 4). Carbon chemical shifts assigned to two decimal places are to differentiate close but resolved resonances. NMR data for DON, 1a, and 1b, acquired in the same solvent at 600 MHz, have been presented by Stanic et al.14

homonuclear 2D NMR spectra, as previously observed14 for Cys adducts of 1, but this did not prevent structural analysis. Examination of the COSY and TOCSY NMR spectra of 2 identified resonances attributable to spin systems for 2b of H2/H-3/H-4a/H-4b, H-14, H-7, H-15a/H-15b, H-16/H-10/H11, Cys H-2/H-3a/H-3b, Gly H-2a/H-2b, and Glu H-2/H-3a/ H-3b/H-4a/H-4b, whereas only a few resonances were unambiguously identifiable for 2a due to its low abundance (2a:2b ca. 9:91) and overlap with resonances from 2b. Thus, full assignments of resonances were obtained only for 2b (Table 1). Chemical shifts of the protonated carbons were assigned from the HSQC NMR spectrum, whereas an HMBC spectrum and a band-selective HMBC spectrum were used to assign nonprotonated carbon resonances and to connect the spin systems. Correlations observed in the HMBC spectrum of 2b from H-16 (1.81 ppm) to 13C resonances at 105.8 (C-8), 123.0 (C-10), and 143.0 ppm (C-9) and from H-7 (4.08) to 13 C resonances at 70.41 (C-15) and 105.8 ppm (C-8) identified 2b as the hemiketal isomer. Additional HMBC correlations (Figure 3) were observed between the Cys H-3a and H-3b

(3.00 and 3.14 ppm) protons and 13C resonances at 36.1 (C13), 54.6 (Cys C-2), and 173.8 ppm (Cys C-1) and between the H-13pro‑R and H-13pro‑S (3.18 and 3.47 ppm) protons and the 13C resonances at 35.4 (Cys C-3), 51.8 (C-5), and 80.7 ppm (C-2). This, together with the presence of an intact 9,10olefinic bond (H-10, 5.55 ppm; C-9 and C-10 at 143.0 and 123.0 ppm) and NOE correlations observed in the ROESY NMR spectrum between Cys H-3a (3.00 ppm) and the trichothecene resonances at 3.47 (H-13pro-S) and 4.63 ppm (H3), unambiguously identified 2b as being formed by attachment of the cysteinyl thiol group of GSH to C-13, via nucleophilic attack of the thiol group of GSH on the epoxide group of 1. The 1H NMR spectrum indicated 5 to be a 32:68 mixture of two isomers (5a and 5b). Notable features included doublet methyl signals at 1.24 and 1.14 ppm (H-16 of 5a and 5b, respectively), indicating a proton on C-9, and the apparent absence of an identifiable olefinic proton on C-10, consistent with a Michael adduct of GSH at C-10. COSY and TOCSY NMR spectra of 5 were used to identify resonances attributable to the H-2/H-3/H-4a/H-4b, H-14, H-7, H-15a/H-15b, H-16/ 6906

DOI: 10.1021/acs.jafc.6b02853 J. Agric. Food Chem. 2016, 64, 6903−6910

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Journal of Agricultural and Food Chemistry

between H-9 and H-10 (ca. 12 Hz) of 5a and 5b were consistent with these protons both being axially oriented, and examination of the NOE correlations observed in the ROESY NMR spectrum of 5b (Figure 4) revealed the stereochemistry

Figure 4. Structurally significant NOE correlations observed for 5b in the ROESY NMR spectrum of 5 that established the stereochemistry at C-9 and C-10 shown in Figure 1. For simplicity, the majority of the glutathione side chain is not shown.

Figure 3. Selected region of the HMBC NMR spectrum of 2, showing the H-13 and Cys H-3 1H NMR resonances for 2b and their corresponding correlated 13C NMR resonances. Correlations marked with asterisks originate from other sources. Note that the resonance of H-13pro‑S (3.47 ppm, d, J = 13.1 Hz) partially overlaps with that of H15pro‑S (3.48 ppm, d, J = 9.0 Hz).

of 5 at C-9 and C-10 to be as shown in Figure 1. A corresponding examination of ROESY NMR correlations for minor isomer 5a was similarly consistent with the structure shown in Figure 1. The 9,10-stereochemistry of the Cys Michael adduct of 1 formed at long reaction time14 was identical to that found for 5, indicating that this is the thermodynamically most favorable Michael addition product. However, for both Cys and GSH, other diastereoisomers of 5 were formed more rapidly but presumably isomerized to 5 over time. Both GSH adducts 2 and 5 in this study, and their corresponding Cys adducts in an earlier study,14 existed predominantly (ca. 90 and 70%, respectively) in the hemiketal form in solution, whereas DON exists largely (ca. 90%) in the ketone form.14,18 Because only the ketone forms can be expected to undergo Michael addition and retro-Michael addition,13 the position and rate of the ketone−hemiketal equilibrium is likely to influence both the reaction kinetics of thiol addition and the mass spectrometric fragmentations of the adducts. ERETIC2. Compounds 2 and 5 were purified on a submilligram scale and quantitated with the ERETIC2 1H qNMR experiment. The peak area in 1H NMR spectra is directly proportional to the number of nuclei, making NMR a method that is well suited not only for quantitation of compounds in solution but also for the quantitative determination of mixtures, for example, of isomers. The ERETIC signal was calibrated using a solution of dioxane in D2O as the external reference.14 Because DON−GSH conjugates were equilibrating mixtures of a hemiketal and ketone form, the masses of both isomers were calculated and added together. Calculated masses of purified 2 and 5 were 572 and 144 μg, respectively, with relative standard deviations of 0.5 and 0.4%, respectively. Mass Spectrometric Fragmentation. The MS fragmentation of DON−GSH conjugates was studied by targetedHRMS2 analysis of their positively charged ([M + H]+) and negatively charged (M − H]−) ions (Figure 5). Fragmentation of the [M − H]− ions of DON−GSH epoxide adduct 2

H-9/H-10/H-11, and Cys H-2/H-3a/H-3b spin systems of both 5a and 5b, whereas only a single set of superimposed resonances was observed for the Gly H-2a/H-2b and Glu H-2/ H-3a/H-3b/H-4a/H-4b spin systems of 5a and 5b. Chemical shifts of the protonated carbons were assigned from the HSQC NMR spectrum, whereas an HMBC spectrum and a bandselective HMBC spectrum were used to assign nonprotonated carbon resonances and to connect the spin systems. The chemical shifts of C-12 and C-13 (ca. 69 and 49 ppm, compared to ca. 83 and 36 ppm, respectively, for 2b) were consistent with the presence of an intact epoxy group in 5a and 5b, as were chemical shifts and coupling constants (ca. 3.5 Hz, compared to 13.1 Hz for 2b) of the H-13 methylene signals. H16 (1.24 ppm) of 5a showed HMBC correlations to 13C resonances at 44.6 (C-9), 52.5 (C-10), and 214.2 ppm (C-8), and H-7 (4.79 ppm) showed HMBC correlations to 13C signals at 60.2 (C-15) and 214.2 ppm (C-8), establishing 5a as a ketone isomer without a 9,10-olefinic bond. For 5b, HMBC correlations from H-16 (1.14 ppm) to 13C signals at 43.3 (C-9), 49.9 (C-10), and 107.5 ppm (C-8); from H-7 (4.00 ppm) to 13 C resonances at 68.7 (C-15) and 107.5 ppm (C-8); from H15pro‑R (4.25 ppm) to 13C signals at 44.6 (C-5), 56.7 (C-6), and 75.6 ppm (C-11); and from H-15pro‑S (3.72 ppm) to 13C signals at 56.7 (C-6), 75.6 (C-11) and 107.5 ppm (C-8) established the absence of a 9,10-olefinic bond and the presence of an intramolecular 15-hemiketal at C-8. Furthermore, Cys H-3pro-R and Cys H-3pro-S of 5b showed HMBC correlations to 13C signals at 49.9 (C-10), 54.6 (Cys C-2), and 173.8 ppm (Cys C1), whereas H-10 (2.96 ppm) showed HMBC correlations to 13 C resonances at 32.7 (Cys C-3) and 43.3 ppm (C-9), establishing the point of attachment of the thiol group of GSH at C-10 of 5, produced via Michael addition of GSH to 1. NOE correlations observed between the Cys H-2 and the Cys H-3 protons and H-10 of the trichothecene nucleus were consistent with this as the point of attachment. The coupling constants 6907

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Figure 5. LC-HRMS2 (method 2B) product ion spectra from higher energy collisional dissociation of DON−GSH isomers 2−5 obtained during monitoring of the reaction of DON with GSH (Figure 2). (A−D) Positive ion spectra (at reaction times of 10 days for 2, 3, and 5 and 46.5 h for 4) of [M + H]+ (m/z 604.2); (E−H) corresponding negative ion spectra of [M − H]− (m/z 602.2) at the same reaction times. Note that spectra of 4 and 5 were obtained using subtraction due to partial overlap.

afforded a product ion with m/z 299.0958 corresponding to C14H19O5S− (Δ −0.3 ppm, ca. 15% relative intensity). This product ion is attributable to cleavage of the side chain attached at C-6 and loss of the GSH moiety, leaving the sulfhydryl group on the trichothecene. The same product ion was also observed when the [M − H]− ions of the DON−Cys epoxide adduct were fragmented.14 However, the most prominent product ion in the HRMS2 spectrum of 2 was m/z 272.0893 (corresponding to C10H14N3O6−, Δ 3.1 ppm). This product ion appears to be related to deprotonated GSH, lacking the sulfhydryl group. The negatively charged DON−GSH Michael adducts 3−5 fragmented differently from epoxide adduct 2. However, there was no obvious difference in the fragmentation patterns of the different isomers 3, 4, and 5 (Figure 5). The most prominent product ion from fragmentation of [M − H]− of 3, 4, and 5 was m/z 306.0772 (C10H16N3O6S−, Δ 0.1 ppm), corresponding to [M − H]− of GSH, indicating a facile elimination of GSH from Michael adducts 3−5 in negative ion mode. None of the remaining major product ions were related to the trichothecene moiety (Figure 5). Fragmentation of the [M + H]+ ions of the four DON−GSH adducts also resulted in marked differences in the relative abundances of product ions for the epoxide adduct 2, compared to Michael adducts 3−5 (Figure 5). Fragmentation of [M + H]+ of 2 gave product ions at m/z 529.1852 (C23H33O10N2S+, Δ 0.3 ppm), 281.0836 (C14H17O4S+, Δ −2.2 ppm), 263.0733 (C14H15O3S+, Δ −1.3 ppm), and 231.1013 (C14H15O3+, Δ −1.2 ppm) (Figure 5). [M + H]+ of DON−GSH Michael adducts

3−5 afforded largely the same product ions, but their fragmentation patterns differed significantly from that of 2. As for 2, 3−5 afforded product ions at m/z 529.1837−529.1846 (C23H33O10N2S+, Δ −2.5 to −0.8 ppm) attributable to loss of glycine. In addition to this, 3−5 gave product ions at m/z 458.1470 (C20H28O9NS+, Δ −2.0 ppm), attributable to loss of glutamic acid from their GSH moieties. These two fragments were among the most prominent in the HRMS2 spectra reported for a putative DON−GSH adduct in wheat extracted 96 h after treatment with DON,5 suggesting that the adducts detected in the wheat were probably Michael rather than epoxide adducts of DON and GSH. Other common fragments in the HRMS2 spectra of 3−5 were m/z 475.1733−475.1740 (C20H31O9N2S+, Δ −2.5 to −1.0 ppm) and m/z 329.1047 (C15H21O6S+, Δ −1.9 ppm). A product ion at m/z 475.172 was the base peak in the HRMS2 spectra of the putative DON− GSH adduct in the study of Kluger at al.,5 again indicating that the putative GSH adduct detected in the DON-treated wheat was the product of Michael addition of GSH to 1. The three product ions at m/z 179.0482 (C5H11O3N2S+, Δ −1.6 ppm), m/z 130.0498 (C5H8O3N+, Δ −0.5 ppm), and m/z 162.0217 (C5H8O3NS+, Δ −1.5 ppm) were all related to the GSH moiety and were especially useful in distinguishing between the closely eluting peaks of 3, 4, and 5. The product ion at m/z 130.0499 was present in the HRMS2 spectrum of 3, the base peak in the spectrum of 4, and nearly absent in the spectrum of 5, and the product ion at m/z 162.0217 was the base peak in the HRMS2 spectrum of 5 (Figure 5), but was much weaker in the spectra 6908

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Journal of Agricultural and Food Chemistry of 3 and 4. Targeted-HRMS2 LC chromatograms, extracted at masses for product ions that were selective for the various isomers, permitted identification of 2−5 in reaction mixtures including the presence of overlapping peaks due to 4 and 5 (Figure 6).

Figure 7. LC-MS analysis (method 1B) of the reaction of DON with GSH with (circles, solid lines) and without (triangles, dashed lines) addition of GST. The graph shows the change in peak areas for DON (solid symbols) and DON−GSH (sum of 3−5) (open symbols) with time, and the lines show three-parameter exponential curves (decay, or rise, to a maximum) fitted with SigmaPlot 13.0.

glutathione, “DON−2H”−S-glutathione, DON−S-cysteinylglycine, and DON−S-Cys) have also been detected in DONtreated wheat.6 Whether this conjugation is the result of enzymatic action or merely due to chemical reaction is unknown. Even though the human alpha and mu GST we tested had no effect on the GSH conjugation of DON, this does not exclude the possibility that the formation of DON−GSH conjugates might be catalyzed by other mammalian or plant GSTs. The theta and zeta GSTs in plants have counterparts in animals, but other classes of GSTs are specific to plants.10 For example, both phi and tau GSTs occur only in plants and have a broad range of conjugating activity toward xenobiotics.19 In this study we have characterized the structures of two DON−GSH conjugates, each of which exists in equilibrium between ketone and hemiketal forms. The conjugates, 5 and 2, were obtained from nucleophilic addition of the thiol to C-10 or C-13, respectively, of DON at basic pH. Although the toxicities of these GSH adducts were not tested, they would likely be low given that the corresponding Cys, mercaptoethanol, and methanethiol adducts of DON were found to be much less toxic than DON.13,14,20 However, it should be noted that the thia-Michael addition reaction with DON is reversible,13,14 which could lead to slow release of DON from GSH adducts 3−5. The epoxide groups of trichothecenes such as DON have generally been regarded as unreactive,21,22 although the inactivation of some thiol-containing enzymes is reported to be associated with the presence of the 12,13-epoxy group.23 This suggests that the epoxide group of trichothecenes may be more reactive than suspected, as is indicated by recent results presented here and elsewhere, and may be associated with some of the biological activities displayed by this group of toxins. Knowledge of the reactivity of trichothecenes, and the structures and chemical characteristics of their thiol adducts, will assist in the identification of the isomers that are found in natural samples and may help deepen our understanding of their mechanisms of action.

Figure 6. LC-HRMS2 analysis (5 ppm mass windows) in positive ion mode (method 2B) of the reaction of 1 with GSH at pH 10.7 after 46.5 h at ambient temperature (see Figure 2). (A) Full scan extracted ion chromatogram at m/z 604.2171 (exact mass of [M + H]+ for 2− 5); (B−D) extracted ion chromatograms at the indicated isomerspecific (Figure 5A−D) masses obtained from LC-HRMS2 analysis (positive ion mode) at m/z 604.2 (Figure 5), with the proposed structures of the product ions shown. Chromatograms C and D clearly show that the broadened peak eluting at ca. 5.5 min at intermediate reaction times in Figure 2 is composed of at least two isomers (4 and 5).

Enzymatic Conjugation with GSH. The formation of DON−GSH conjugate 4, together with small amounts of 3 and 5, was observed during the incubation of 1 and GSH both with and without GST for 18 h. Furthermore, the conjugation rates (t1/2 = 17 h) and product ratios were essentially identical in both incubations, indicating primarily nonenzymatic formation of DON−GSH conjugates under the conditions used (Figure 7). No 2 was detected but, on the basis of the reaction progress at higher pH (Figure 2), this product would be expected in significant amounts only at long reaction times. However, analysis of the reactions after 25 days indicated that the conjugation reaction had stopped or partially reversed. This is attributable to the reversibility of the Michael addition to 1 and autoxidation of the thiol (GSH) to its disulfide (GSSG).13,14 Nevertheless, our finding that conjugation of DON with GSH occurs at physiological pH in the absence of catalysis by GST is consistent with the results of Gardiner et al.,7 who detected DON−GSH adducts in experiments with both living and heatinactivated yeast. GSH conjugation of DON (DON−S6909

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(7) Gardiner, S. A.; Boddu, J.; Berthiller, F.; Hametner, C.; Stupar, R. M.; Adam, G.; Muehlbauer, G. J. Transcriptome analysis of the barley−deoxynivalenol interaction: evidence for a role of glutathione in deoxynivalenol detoxification. Mol. Plant-Microbe Interact. 2010, 23, 962−976. (8) Liu, Y.-J.; Han, X.-M.; Ren, L.-L.; Yang, H.-L.; Zeng, Q.-Y. Functional divergence of the glutathione S-transferase supergene family in Physcomitrella patens reveals complex patterns of large gene family evolution in land plants. Plant Physiol. 2013, 161, 773−786. (9) Frova, C. Glutathione transferases in the genomics era: new insights and perspectives. Biomol. Eng. 2006, 23, 149−169. (10) Dixon, D. P.; Edwards, R. Glutathione transferases. In The Arabidopsis Book; American Society of Plant Biologists: 2010; Vol. 8, p e0131. (11) Wang, W.; Ballatori, N. Endogenous glutathione conjugates: occurrence and biological functions. Pharmacol. Rev. 1998, 50, 335− 356. (12) Noctor, G.; Mhamdi, A.; Chaouch, S.; Han, Y.; Neukermans, J.; Marquez-Garcia, B.; Queval, G.; Foyer, C. H. Glutathione in plants: an integrated overview. Plant, Cell Environ. 2012, 35, 454−484. (13) Stanic, A.; Uhlig, S.; Solhaug, A.; Rise, F.; Wilkins, A. L.; Miles, C. O. Nucleophilic addition of thiols to deoxynivalenol. J. Agric. Food Chem. 2015, 63, 7556−7566. (14) Stanic, A.; Uhlig, S.; Solhaug, A.; Rise, F.; Wilkins, A. L.; Miles, C. O. Preparation and characterization of cysteine adducts of deoxynivalenol. J. Agric. Food Chem. 2016, 64, 4777−4785. (15) Miles, C. O. Fusarium toxin mass calculator (version 9 in Excel), 2016; DOI: 10.13140/RG.2.1.3694.0401, https://www.researchgate. net/publication/304249256_Fusarium_toxin_mass_calculator_ version_9_in_Excel (accessed June 23, 2016). (16) Fulmer, G. R.; Miller, A. J. M.; Sherden, N. H.; Gottlieb, H. E.; Nudelman, A.; Stoltz, B. M.; Bercaw, J. E.; Goldberg, K. I. NMR chemical shifts of trace impurities: common laboratory solvents, organics, and gases in deuterated solvents relevant to the organometallic chemist. Organometallics 2010, 29, 2176−2179. (17) Wider, G.; Dreier, L. Measuring protein concentrations by NMR spectroscopy. J. Am. Chem. Soc. 2006, 128, 2571−2576. (18) Jarvis, B. B.; Mazzocchi, D. B.; Ammon, H. L.; Mazzola, E. P.; Flippen-Anderson, J. L.; Gilardi, R. D.; George, C. F. Conformational effects in trichothecenes: structures of 15-hydroxy C4 and C8 ketones. J. Org. Chem. 1990, 55, 3660−3662. (19) Edwards, R.; Dixon, D. P. Plant glutathione transferases. Methods Enzymol. 2005, 401, 169−186. (20) Fruhmann, P.; Weigl-Pollack, T.; Mikula, H.; Wiesenberger, G.; Adam, G.; Varga, E.; Berthiller, F.; Krska, R.; Hametner, C.; Frohlich, J. Methylthiodeoxynivalenol (MTD): insight into the chemistry, structure and toxicity of thia-Michael adducts of trichothecenes. Org. Biomol. Chem. 2014, 12, 5144−5150. (21) Nakamura, Y.; Ohta, M.; Ueno, Y. Reactivity of 12,13epoxytrichothecenes with epoxide hydrolase, glutathione-S-transferase and glutathione. Chem. Pharm. Bull. 1977, 25, 3410−3414. (22) Karlovsky, P. Biological detoxification of the mycotoxin deoxynivalenol and its use in genetically engineered crops and feed additives. Appl. Microbiol. Biotechnol. 2011, 91, 491−504. (23) Ueno, Y.; Matsumoto, H. Inactivation of some thiol-enzymes by trichothecene mycotoxins from Fusarium species. Chem. Pharm. Bull. 1975, 23, 2439−2442.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02853. Tabulated HRMS data for 1−5; LC-MS chromatograms from conjugation using GST; NMR spectra of 2 and 5, and partial NMR assignments for 2a; molecular model of 5b labeled with chemical shifts, stereochemistry, and NOE correlations; positive and negative ion chromatograms of 2−5 extracted from LC-HRMS2 analysis (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(C.O.M.) Phone: +47 2321-6228. Fax: +47 2321-6201. Email: [email protected]. Funding

This work was part of the project “Mycotoxins and toxigenic fungi in Norwegian pig farming: consequences for animal health and possible intervention strategies” funded by the Research Council of Norway (Grant 225332) and cofinanced by Animalia, Lantmännen Research Foundation and Felleskjøpet Fôrutvikling. The Research Council of Norway is acknowledged for support through the Norwegian NMR Platform, NNP (226244/F50). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED COSY, correlation spectroscopy; DEPT, distortionless enhancement of polarization transfer; DON, deoxynivalenol; GSH, glutathione; ERETIC, electronic reference to access in vivo concentrations; HMBC, heteronuclear multiple-bond correlation; HRMS, high resolution mass spectrometry; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser spectroscopy; ROESY, rotating frame Overhauser spectroscopy; SELTOCSY, selective total correlation spectroscopy; TOCSY, total correlation spectroscopy

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REFERENCES

(1) Nesic, K.; Ivanovic, S.; Nesic, V. Fusarial toxins: secondary metabolites of Fusarium fungi. Rev. Environ. Contam. Toxicol. 2014, 228, 101−120. (2) Rotter, B. A.; Prelusky, D. B.; Pestka, J. J. Toxicology of deoxynivalenol (vomitoxin). J. Toxicol. Environ. Health 1996, 48, 1−34. (3) Warth, B.; Fruhmann, P.; Wiesenberger, G.; Kluger, B.; Sarkanj, B.; Lemmens, M.; Hametner, C.; Fröhlich, J.; Adam, G.; Krska, R.; Schuhmacher, R. Deoxynivalenol-sulfates: identification and quantification of novel conjugated (masked) mycotoxins in wheat. Anal. Bioanal. Chem. 2015, 407, 1033−1039. (4) Sewald, N.; von Gleissenthall, J. L.; Schuster, M.; Müller, G.; Aplin, R. T. Structure elucidation of a plant metabolite of 4desoxynivalenol. Tetrahedron: Asymmetry 1992, 3, 953−960. (5) Kluger, B.; Bueschl, C.; Lemmens, M.; Berthiller, F.; Häubl, G.; Jaunecker, G.; Adam, G.; Krska, R.; Schuhmacher, R. Stable isotopic labelling-assisted untargeted metabolic profiling reveals novel conjugates of the mycotoxin deoxynivalenol in wheat. Anal. Bioanal. Chem. 2013, 405, 5031−5036. (6) Kluger, B.; Bueschl, C.; Lemmens, M.; Michlmayr, H.; Malachova, A.; Koutnik, A.; Maloku, I.; Berthiller, F.; Adam, G.; Krska, R.; Schuhmacher, R. Biotransformation of the mycotoxin deoxynivalenol in Fusarium resistant and susceptible near isogenic wheat lines. PLoS One 2015, 10, e0119656. 6910

DOI: 10.1021/acs.jafc.6b02853 J. Agric. Food Chem. 2016, 64, 6903−6910