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Synthesis, Identification, and Structure Elucidation of Adducts Formed by Reactions of Hydroxycinnamic Acids with Glutathione or Cysteinylglycine Nayla Ferreira-Lima,*,†,‡ Anna Vallverdú-Queralt,*,†,§ Emmanuelle Meudec,† Jean-Paul Mazauric,† Nicolas Sommerer,† Marilde T. Bordignon-Luiz,‡ Véronique Cheynier,† and Christine Le Guernevé† †

INRA, UMR1083 Sciences Pour l’œnologie, Plateforme Polyphénols, 2, Place Viala, Montpellier Cedex, 34060, France Departamento de Ciência e Tecnologia de Alimentos CAL/CCA, Universidade Federal de Santa Catarina, Rod. Admar Gonzaga, 1346, Itacorubi, Florianópolis, SC, Brazil § Centro de Investigación Biomédica en Red de la Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid 28029, Spain ‡

S Supporting Information *

ABSTRACT: Grape polyphenols, especially hydroxycinnamic acids such as caftaric and caffeic acid, are prone to enzymatic oxidation reactions during the winemaking process, forming o-quinones and leading to color darkening. Glutathione is capable of trapping these o-quinones and thus limiting juice browning. In this study, the addition of glutathione or cysteinylglycine onto caftaric or caffeic acid o-quinones formed by polyphenoloxidase-catalyzed reactions was investigated by UPLC-DAD-ESIMS and NMR data analyses. Complete identification of adducts has been achieved via NMR data. The results confirmed that the favored reaction is the substitution of the sulfanyl group of cysteine at C-2 of the aromatic ring. Several minor isomers, namely, the cis-isomer of the 2-S adduct and trans-isomers of the 5-S and 6-S adducts, and the 2,5-di-S-glutathionyl adducts were also identified and quantified by qNMR. With the exception of 2-(S-glutathionyl)- and 2,5-di(S-glutathionyl)-trans-caftaric acid, these products had never been formally identified. In particular, the 5-S and 6-S derivatives are reported here for the first time. The first formal identification of 2-S cisderivatives is also provided. Moreover, NMR and UPLC-DAD-ESIMS analysis showed that signature UV and MS spectra can serve as markers of the conformation and substitution position in the aromatic ring for each of the isomers.

H

It readily oxidizes reducing compounds such as sulfur dioxide or ascorbic acid but also other o-diphenols with lower redox potential, including GRP.5 In this coupled oxidation reaction, GRP is oxidized to GRP-o-quinone, while caftaric acid-o-quinone is reduced to caftaric acid. Addition of another GSH molecule on GRP-o-quinone yields 2,5-di(S-glutathionyl)-trans-caftaric acid, or GRP2 (2, Figure 1).6 Juice browning susceptibility depends to a large extent on the molar ratio of GSH to caftaric (and coutaric) acid.7 However, monitoring of caftaric acid, GRP, GRP2, and caftaric acid- and GRP-o-quinones during oxidation indicated that other juice components compete with GSH for trapping of caftaric acid-o-quinones. A GRP isomer has recently been detected and tentatively attributed to the cis-isomer (3, Figure 1) on the basis of its UV−visible spectrum and further evidence based on MS fragmentation pattern and modeling.8 A mixture of GRP isomers, attributed to trans- and cis-GRP (95:5 based on peak area %), was also obtained by

ydroxycinnamic acids, especially caftaric acid (i.e., caffeoyltartaric acid), are major phenolic compounds in white grapes and wines made with minimal pomace contact.1,2 Caftaric acid is also a good substrate for enzymatic oxidation catalyzed by polyphenoloxidase (PPO). Enzymatic oxidation is an important concern in the wine industry, as it leads to excessive juice browning and loss of varietal aroma compounds and subsequently affects the main characteristics of white wines such as color and flavor.3 Enzymatic oxidation reactions in juice convert caftaric acid into caftaric acid-o-quinone, which is highly reactive. Owing to its electrophilic character, caftaric acid-o-quinone can react with nucleophiles to form adducts. A well-known example of such reactions is the addition of glutathione (γ-L-glutamyl-Lcysteinyl-L-glycine; GSH), a natural tripeptide found in grapes and wines, yielding grape reaction product (GRP),2 which has been identified as 2-(S-glutathionyl)-trans-caftaric acid (1, Figure 1).4 As GRP is not a substrate for grape PPO, its formation could be an interesting way of limiting juice browning.2,4 Caftaric acid-o-quinone is also a powerful oxidant. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 30, 2016

A

DOI: 10.1021/acs.jnatprod.6b00279 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Chemical structures of the synthesized compounds.

enzymatic oxidation of caftaric acid in the presence of GSH.9 In addition, cysteinyl, cysteinylglycyl, and glutamylcysteinyl derivatives of caftaric or caffeic acid have been detected in wines.4,8

These molecules accumulate with aging, suggesting that they result from hydrolysis of GRP. Moreover, preliminary experiments performed in our laboratory have detected B



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Figure 2. UPLC profiles at 280 nm of methanol fractions obtained after SPE of reaction media resulting from (A) glutathione with caftaric acid-oquinone (compounds 1−3); (B) glutathione with caffeic acid-o-quinone (compounds 5−8); and (C) cysteinylglycine with caftaric acid-o-quinone (compounds 9−13) and UV spectra of the major products.

Table 1. Summary of LC-DAD-MS Data of the Synthesized Products

a

compound

no.

tR (min)

[M − H]− (fragments)b (m/z)

[M + H]+ (fragments)b (m/z)

2-(S-glutathionyl)-trans-caftaric acid 2,5-di(S-glutathionyl)- trans-caftaric acid 2-(S-glutathionyl)-cis-caftaric acid 5-(S-glutathionyl)-trans-caftaric acid 2-(S-glutathionyl)-trans-caffeic acid 5-(S-glutathionyl)-trans-caffeic acid 6-(S-glutathionyl)-trans-caffeic acid 2-(S-glutathionyl)-cis-caffeic acid 2,5-di(S-glutathionyl)-trans-caffeic acid 2-(S-cysteinylglycyl)-trans-caftaric acid 5-(S-cysteinylglycyl)-trans-caftaric acid 6-(S-cysteinylglycyl)-trans-caftaric acid 2-(S-cysteinylglycyl)-cis-caftaric acid

1 2 3 4 5 6 7 8 9 10 11 12 13

8.4 9.0 9.8 9.3 11.3 12.5 12.7 13.7 11.9 7.5 7.8 8.0 10.5

616 (484; 440; 272) 921/460a (792; 771; 648; 394;a 372;a 272) 616 (484; 440; 272) 616 (484; 440; 272) 484 (440, 272) 484 (440, 272) 484 (440, 272) 484 (440, 272) 789/394a (745; 660; 616; 472) 487 (355; 311) 487 (355; 311) 487 (355; 311) 487 (355; 311)

618 (264; 489) 462a (665, 397.5,a 314) 618 (489; 322; 264) 618 (489, 393, 321) 486 (264) 486 (393; 357; 321) 486 (357, 264) 486 (357, 264) 791 (773; 662; 644; 553) 489 (264) 489 (264; 321) 489 (264) 489 (322; 264)

UV λmax 252; 275; 254; 260; 252; 260; 270; 250; 271; 252; 260; 269; 250;

329 327 315 327 321 320 301; 333 315 320 328 328 306; 342 316

Doubly charged ion [M − 2H]2− or [M + 2H]2+. bThe main fragments are highlighted in bold.

characteristic of the different isomers (position of the thioether bond on the phenolic ring and cis- or trans- configuration of the vinyl chain double bond).

cysteinylglycylcaftaric acid in juice, consistent with the presence of cysteinylglycine, also reported as a conjugate of 3-mercaptohexan-1-ol.9,10 However, only tentative identification based on UV and mass spectra has been provided for GRP isomers and their hydrolysis products.8 The aim of the present study was to synthesize and identify glutathionyl and cysteinylglycyl adducts of caftaric acid, as well as glutathionyl adducts of caffeic acid. Synthesis was achieved by PPO-catalyzed oxidation of caftaric or caffeic acid in grape juice-like model systems containing GSH or cysteinylglycine. Products were identified by UPLC-DAD-ESIMS and NMR data analyses. Formal identification of the minor adducts is provided for the first time, as well as accurate determination of the proportions of the different isomers using quantitative NMR (qNMR). Comparison of both approaches also permitted identification of UV and MS fragmentation spectra

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RESULTS AND DISCUSSION LC-DAD-MS Analysis. UPLC-DAD-MS/MS analysis of the MeOH fractions obtained from each reaction medium after solid-phase extraction allowed detection of several adducts (Figure 2 and Table 1). Glutathionyl Adducts of Caftaric Acid. Analysis of the GSH and caftaric acid reaction products (Figure 2A) showed the presence of a major peak (1) detected at m/z 616, in the negative ion mode, with fragment ions at m/z 484 (loss of the tartaric acid moiety: −132 amu), 440 (loss of the tartaric acid moiety and of a carboxylic group: −132−44 amu), and 272 (loss of caftaric acid thiol: −344 amu), as described by C



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Figure 3. Fragmentation spectra in the positive ion mode of the major products formed.

Boselli et al.11 Compound 1 was also detected at m/z 618 in the positive ion mode, with fragment ions at m/z 489 (loss of glutamic acid through cleavage of the peptide bond: −129 amu)12 and m/z 264, that has been attributed to cleavage of the ether bond and loss of a carboxylic group (−310 to −44 amu).8,11 It was tentatively identified as the well-known 2-(S-glutathionyl)-trans-caftaric acid (GRP; 1).4 Minor peaks

(2, 3, 4) were also observed. Two of these (3 and 4) showed the same mass spectrum as GRP in the negative ion mode, indicating that they were GRP isomers. Slight differences were observed in their UV spectra (Figure 2A). GRP (1) showed a characteristic UV spectrum with absorption bands at 252 and 329 nm, and its two isomers showed absorption bands at 254 and 315 nm (3) and 260 and 327 nm (4), respectively (Figure 2A). D



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Table 2. 1H and 13C NMR Chemical Shifts of Glutathionyl Caftaric Acid Adducts in Methanol-d4 at 25 °C 2-(S-glutathionyl)-trans-caftaric acid position Caffeic 1 2 3 4 5 6 α β COO Tartaric 1 2 3 4 Cysteine α ββ′ CO Glutamic Acid α ββ′ γγ′ CO COOH Glycine αα′ COOH a

δ 1H (ppm), m, JHH (Hz)

6.85, 7.23, 8.44, 6.39,

d d d d

(8.6) (8.6) (16) (16)

5.54, d (2.4) 4.73, d (2.4)

4.28, dd (9.6; 4.3) 3.41, dd (13.8; 4.4) 3.01, dd (13.8; 9.6)

δ 13C

HMBC

130.8 121.5 148.8 149.0 117.4 120.5 146.3 116.8 168.1

6, 5, α, β 6,5, α, β, Cys ββ′ 6, 5, α 6, 5, α 6, α 5, α 6, β 6, α α, β

171.5 75.7 72.4 174.8

2tart, 3tart, 4tart β 2tart, 4tart 1tart, 2tart, 3tart

54.8 37.5

5-(S-glutathionyl)-trans-caftaric acid δ 1H (ppm), m, JHH (Hz)

6.99, d (1.7)

7.24, d (1.7) 7.62, d (16) 6.43, d (16)

δ 13C

HMBC

127.8 115.5 149.8 149.8 nda 126.8 147.6 115.7

β 6,α 2,6 2,6 2, α 2,6

2,5-di(S- glutathionyl)trans-caftaric acid δ 1H (ppm), m, JHH (Hz)

δ 13C

7.45, s 8.36, d (16) 6.61, d (16)

131.9 112.7 149.2 149.7 nda 124.9 145.7 118.1

2-(S-glutathionyl)-ciscaftaric acid δ 1H (ppm), m, JHH (Hz)

6.76, 7.02, 7.43, 6.03,

d d d d

(8.3) (8.3) (12.4) (12.4)

δ 13C 132.1 120 146.5 147.5 116.8 124.1 145.8 119.3

α, β

Cys ββ′ Cys α

173.2

Cys α, Cys ββ′

3.76, m 2.10, m 2.50, m

55.1 27.5 32.9 175.1 173.3

Glu ββ′, Glu γγ′ Glu α, Glu γγ′ Glu α, Glu ββ′ Glu ββ′, Glu γγ′ Glu α, Glu ββ′, Glu γγ′

3.83, s

42.2 173.0

Gly NH Gly, Gly NH

nd = undetermined. Due to overlaps or too low signal intensity, only some aromatic and vinyl protons and carbons of minor species could be attributed.

Glutathionyl Adducts of Caffeic Acid. The synthesis of the glutathionylcaffeic acid adducts yielded five peaks (Figure 2B). The major one and three of the minor compounds (5−8) were detected at m/z 484 in the negative ion mode, with the same fragment ions at 440 and 272 as GRP isomers, in agreement with the expected glutathionylcaffeic acid structures (Figure 1). These compounds showed different UV−vis spectra with absorption bands at 252 and 321 nm for the major peak [5, presumably 2-(S-glutathionyl)-trans-caffeic acid] and 260 and 320 nm (6), 270, 301, and 333 nm (7), and 250 and 315 nm (8), respectively, for the other isomers (Figure 2B). Furthermore, they showed distinctive fragmentation spectra in the positive ion mode (Figure 3B). Compound 6 showed the same fragment ion at m/z 321 as compound 4 and also lacked the fragment ion at m/z 264, suggesting that it was the equivalent caffeic acid derivative (i.e., presumably a 5-substituted transproduct). The intensity of the fragment ion at m/z 264 was much lower in the spectrum of compound 8 than in those of compounds 5 and 7 and was indicative of a cis-isomer. The last compound (9) was detected at m/z 789 and m/z 394 ([M − H]− and [M − 2H]2−, respectively), with a fragment ion at 745 corresponding to the loss of the carboxylic group (data not shown), and thus could be attributed to 2,5-di(S-glutathionyl)caffeic acid.

Compound 3 showed a characteristic MS fragmentation spectrum in the positive ion mode, with lower intensity of the fragment ion at m/z 264 than in the trans-GRP fragmentation spectrum (Figure 3A). A minor form of GRP showing the same UV and mass spectra as compound 3 has been observed earlier in white wines and tentatively attributed to cis-GRP.8 The other GRP isomer (4), which likely corresponds to the 5-substituted trans-isomer, has never been reported. It yielded a specific fragmentation pattern in the positive ion mode, with low intensity of the fragment ion at m/z 264 and a major fragment ion at m/z 321 that was not detected in the fragmentation spectra of the two other isomers (Figure 3A). The last glutathionylcaftaric acid adduct was detected both as the singly charged ion at m/z 921 and as the doubly charged ion at m/z 460, in the negative ion mode (462 in the positive ion mode). It yielded the expected fragment ions corresponding to the loss of tartrate and the loss of one or two glutamate(s) in both positive and negative ion modes and as monocharged and/or doubly charged ions (data not shown). It was thus attributed to 2,5di(S-glutathionyl)-trans-caftaric acid (2), or GRP2 identified earlier.6 Formation of this compound by coupled oxidation of GRP and subsequent addition of a second GSH molecule to the GRP-o-quinone has been reported in model solutions and juices containing excess GSH.7 E


F

a

d d d d

(8.5) (8.5) (16) (16)

52.9 26.1 31.2 171.7 170.7

40.8 170.7

3.64, m

170.6

52.3 36.1

128.6 120.7 147.3 147.2 115.8 118.0 142.5 117.2 167.9

δ 13C

3.38, m 1.90, m 2.36, m

4.11, m 2.24, dd (13.2; 4.3) 2.88, dd (13.2; 4.3)

6.64, 7.17, 8.15, 6.19,


Gly NH Gly, Gly NH

Glu ββ′, Glu γγ′ Glu α, Glu γγ′ Glu α, Glu ββ′ Glu ββ′, Glu γγ′ Glu α, Glu ββ′, Glu γγ′

Cys α, Cys ββ′

Cys ββ′ Cys α

6, 5, α, β 6,5, α, β, Cys ββ′ 6, 5, α 6, 5, α 6, α 5, α 6, β 6, α α, β

HMBC

4.38, m 3.31, m 2.98, m

7.09, d (1.8) 7.38, d (16) 6.30, d (16)

6.94, d (1.8)


51.1 33.8

125.7 113.2 146.0 146.7 122.1 121.1 143.7 115.9 167.7

δ 13C

α, β

2, α 2,6

β 6, α 2,6 2,6

HMBC

5-(S-glutathionyl)-trans-caffeic acid

7.22, s 8.11, d (16) 6.40, d (16)

δ 1H (ppm), m, JHH (Hz) nda nda nda nda nda 118 142.4 117.7 nda

δ 13C

2,5-di(S-glutathionyl)-transcaffeic acid

8.02, d (16) 6.12, d (16)

7.03, s*

7.12, s*

δ 1H (ppm), m, JHH (Hz) nda nda nda nda nda nda

δ 13C



CO Glutamic acid α ββ′ γγ′ CO COOH Glycine αα′ COOH

Caffeic 1 2 3 4 5 6 α β COO Cysteine α ββ′

position


Table 3. 1H and 13C NMR Chemical Shifts of Glutathionyl Caffeic Acid Adducts in DMSO-d6 at 25 °C

6.73 6.90 7.16, d (12) 5.84, d (12)


nda nda nda nda nda nda nda nda

δ 13C

2-(S-glutathionyl)-cis-caffeic acid

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Table 4. 1H and 13C NMR Chemical Shifts of Cysteinylglycyl Caftaric Acid Adducts in Methanol-d4 at 25°C 2-(S-cysteinylglycyl)-trans-caftaric acid position Caffeic 1 2 3 4 5 6 α β COO Tartaric 1 2 3 4 Cysteine α ββ′ COOH Glycine αα′ COOH a


6.89, 7.26, 8.43, 6.36,

d d d d

(8.5) (8.5) (16) (16)

5.48, d (2.4) 4.66, d (2.4)

3.97, m 3.26, dd (14.3; 5.5) 3.18, dd (14.3; 7.8)

δ 13C

HMBC

130.9 120.7 148.9 149.1 117.7 120.7 145.5 117.9 168.1

6, 5, α, β 6,5, α, β, Cys ββ′ 6, 5, α 6,5 6, α 5, α 6, β 6, α α, β

172.6 76.4 72.9 175.9

2tart, 3tart, 4tart β 2tart, 4tart 1tart, 2tart, 3tart

54.2 37.9 169.0

3.87, d (17.8) 3.77, d (17.8)

42.8 173.6

5-(S-cysteinylglycyl)-trans-caftaric acid δ 1H (ppm), m, JHH (Hz)

7.08, d (1.8)

7.25, d (1.8) 7.6, d (16) 6.36, d (16)

δ 13C

HMBC

128.4 115.5 nda nda nda 128.7 146.8 117.9

2, β, α 6, α

2, α 2 α, β

2-(S-cysteinylglycyl)-cis-caftaric acid δ 1H (ppm), m, JHH (Hz)

6.82, 7.01, 7.46, 5.99,

d d d d

(8.3) (8.3) (12) (12)

δ 13C 133.9 117.7 148.4 147.4 117.4 124.8 145.7 120.7

HMBC

6 6 6

α, β

Cys ββ′ Cy sα Cys α, Cys ββ′ Gly NH Gly, Gly NH


Figure 4. DOSY spectrum of the main glutathionyl caftaric acid adduct. Top: 1D 1H NMR spectrum.

Cysteinylglycyl Adducts of Caftaric Acid. The reaction of cysteinylglycine with caftaric acid-o-quinone was also investigated. It yielded four compounds (10−13; Figure 2C) detected at m/z 487 in the negative ion mode, with fragment ions at m/z 355 (−132 amu, loss of tartaric acid) and m/z 311

(−44 amu, loss of a carboxylic group), corresponding to isomers of (S-cysteinylglycyl)caftaric acid, but the formation of the disubstituted compound was not observed. The UV spectra of 10−13 (Figure 2C) were similar to those observed for the series of glutathionyl derivatives of caftaric acid and caffeic acid, G



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and the elution order was retained. Fragmentation of all compounds in the positive ion mode yielded an ion at m/z 264 (Figure 3C), which indicated that it does not result from cleavage of the thioether bond and loss of a carboxylic group, as previously proposed for the GSH adducts,8,11 but from the rearrangement of the cysteinylglycylcaffeic acid part. In addition, compound 11 showed the same ion at m/z 321 as compounds 4 and 6 (Figure 3C), again suggesting a 5-substituted trans-product. NMR Analysis. 2D NMR spectra permitted the assignment of all protons and carbons of the main synthetic adducts (Figures S1−S15, Supporting Information). The assignment of some aromatic and vinylic protons and carbons of minor adducts was also performed (Tables 2, 3, and 4). Glutathionyl Adducts of Caftaric Acid. The 1H and 13C NMR chemical shifts of all glutathionylcaftaric acid adducts detected in the reaction medium are shown in Table 2. The main reaction product was readily identified as 2-(S-glutathionyl)-trans-caftaric acid (GRP, 1; Figure 1) from the NMR data. Aside from MeOD and HOD peaks, the 2D DOSY spectrum (Figure 4) showed that the most intense signals have the same diffusion coefficient value (D). In the aromatic-vinyl region, two ortho-coupled protons appearing as doublets (J = 8.6 Hz) at δH 6.85 and 7.23 are attributed to H-5 and H-6 of the aromatic ring of caftaric acid substituted at C-2, whereas the vinylic H-α and H-β gave two doublets (J = 16 Hz) at δH 8.44 and 6.39, characteristic of caftaric acid in a trans-configuration. The two aliphatic protons of caftaric acid appear as doublets (J = 2.5 Hz) at δH 4.73 and 5.54. GSH protons give signals in a more shielded region of the spectrum (δH 2.1−4.3), and their identification is straightforward (Table 2). The bond between the sulfanyl group of the glutathionyl moiety and C-2 on the caffeoyl unit is formally confirmed by long-range correlations between H-β and H-β′ of the cysteine residue and C-2 of the aromatic ring, and ROEs involving protons of both caftaric acid and the glutathionyl moiety. Especially, the ROE of H-α of caftaric acid with the cysteinyl protons and H-γ and H-γ′ of the glutamyl groups indicated their close proximity (Figure 5). A zoom-in view of the DOSY spectrum in the aromatic-vinyl region allows detection of minor components. Indeed, sets of less intense signals having D values either similar to that of GRP (1) or lower can be observed (Figure 6). Since D values are related to the molecular weight, the former correspond to isomers of GRP (1), whereas the latter are due to caftaric acid substituted with two glutathionyl moieties (2). The coupling constants of the aromatic and vinyl protons, respectively, enable determination of the substitution position on the aromatic ring and of the double-bond configuration. Thus, three minor products were identified. The 5-(S-glutathionyl)caftaric acid in trans-configuration (4, Figure 1) gives two doublets (J = 1.7 Hz) characteristic of meta-coupled ring protons at δH 6.99 and 7.24 and a pair of doublets at δH 6.43 and 7.62 (J = 16 Hz). ROE correlations of H-6 with the cysteine H-α, H-β, and H-β′ were consistent with the 5-S substitution. The 2-(S-glutathionyl)caftaric acid in cis-configuration (3, Figure 1) was characterized by two aromatic proton doublets (J = 8.3 Hz) at δH 6.76 and 7.02 and two vinylic proton doublets (J = 12.4 Hz) at δH 6.03 and 7.43. The set of signals at lower D values is composed of the H-α and H-β vinylic doublets (J = 16 Hz) at δH 6.61 and 8.36 characteristic of caftaric acid in trans-configuration and a singlet at δH 7.45 corresponding to one residual aromatic ring proton, proving a disubstitution of the caftaric acid. This proton was definitively assigned to H-6 through its strong ROE

Figure 5. ROESY spectrum showing correlations implying protons of caftaric acid and glutathionyl moieties of the main glutathionyl caftaric acid.

correlations with vinylic H-α and H-β, cysteine H-α, H-β, and H-β′, and glutamic acid H-γ and H-γ′ (Figures S16 and S17, Supporting Information), confirming this adduct to be the 2,5-di(S-glutathionyl)-trans-caftaric acid (GRP2, 2; Figure 1). The absolute molar concentration of all products formed was determined by integration of proton signal area after calibration of the probe with an external traceable standard, allowing assessment of the purity of the final product. The relative molar percentage of each adduct was calculated: GRP (1) represents ∼92% of all adducts formed, 5-(S-glutathionyl)-trans-caftaric acid (4) ∼5%, 2,5-di(S-glutathionyl)-trans-caftaric acid (2) ∼2.4%, and 2-(S-glutathionyl)-cis-caftaric acid (3) ∼1.6%. Glutathionyl Adducts of Caffeic Acid. Similar experiments were performed to analyze the reaction products obtained for the synthesis of glutathionylcaffeic acid. All adducts were identified using the typical patterns described above (Table 3 and Figures S7−S10, Supporting Information). The 1D and DOSY NMR spectra (Figure S18, Supporting Information) showed the presence of one main product, 2-(S-glutathionyl)-transcaffeic acid (5; Figure 1), which represented about 92% of all adducts formed. The other minor adducts detected were 5-(S-glutathionyl)-trans-caffeic acid, 6 (∼4.5%), 2,5-di(Sglutathionyl)-trans-caffeic acid, 9 (∼2.6%), 2-(S-glutathionyl)cis-caffeic acid, 8 (∼0.6%), and 6-(S-glutathionyl)-trans-caffeic acid, 7 (∼0.6%). This is the first report of a 6-(S-glutathionyl) derivative, which was characterized by the presence of two singlets at δH 7.03 and 7.12, corresponding to the residual H-2 and H-5, and of two vinylic proton doublets (J = 16 Hz) at δH 6.12 and 8.02, indicating that it is in the trans-configuration. Cysteinylglycyl Adducts of Caftaric Acid. Identification of the cysteinylglycylcaftaric acid adducts and assignments of their 1H and 13C NMR signals (Table 4 and Figures S11−S15, Supporting Information) were performed in the same way. All spectra were similar to those of the GSH derivatives, except H



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Figure 6. DOSY spectrum showing aromatic and vinyl signals of the main and minor glutathionyl caftaric acid adducts. Apparent diffusion coefficients of monosubstituted (−) and disubstituted (---) products. Top: 1D 1H NMR spectrum. The following symbols are used: ○ 2-(S-glutathionyl)-trans-caftaric acid, * 2,5-di(S-glutathionyl)-trans-caftaric acid, ● 5-(S-glutathionyl)-trans-caftaric acid, ◊ 2-(S-glutathionyl)cis-caftaric acid, FA = formic acid.

the trans-isomers of the 5-S adducts (4, 6, and 11) and cisisomers of the 2-S adducts (3, 8, and 13). The 5-(S-glutathionyl) adducts were more abundant than the 2-S-cis-isomers, while the reverse was true for cysteinylglycylcaftaric acid adducts. Although relative concentrations cannot be determined from relative peak areas in UV or MS profiles, comparison of the molar proportions determined by NMR spectroscopy with UPLC-DAD-MS data (retention times, UV spectra, MS spectra) enables identification of all peaks detected in the HPLC profiles. Indeed, each profile shows that peaks eluted in the same order that have similar UV spectra and yield the same fragment ions in the positive ion mode, indicating that they share the same structural features. The 6-S adduct in the trans-configuration (7) was formally identified only in the GSH-caffeic acid solutions and could thus be attributed to the peak eluted at 12.7 min (Figure 1B). The compound eluted at 8 min can consequently be attributed to the 6-(S-cysteinylglycyl)-trans-caftaric acid adduct (12) on the basis of its retention time and UV−visible spectrum (Figure 1C). The presence of additional signals corresponding to H-α and H-β of trans-configured vinylic bonds reinforces this attribution, although the NMR signals were too weak to allow complete assignment of this minor species. The 6-S adduct was not detected in the caftaric acid-GSH reaction medium. The hypsochromically shifted UV spectra of compounds eluted at 9.8, 13.7, and 10.5 min suggest that they may be cisisomers of the 2-S adducts (3, 8, and 13), as postulated earlier.8 The peak area of the GRP isomer increased after exposure of the solution to UV light (results not shown), confirming that it corresponds to cis-GRP (3). Compounds 3, 8, and 13 were formed in the course of the reaction since the cis-isomers were

for the lack of the glutamic acid signals. Pairs of doublets (J = 8.5 Hz) at δH 6.89 and 7.26, characteristic of ortho-coupled ring protons, and at δH 6.36 and 8.43 (J = 16 Hz), characteristic of the vinyl bond in the trans-configuration, permitted identification of the major product as 2-(S-cysteinylglycyl)-transcaftaric acid (10; Figure 1), representing about 95% of all adducts formed. Two other adducts were detected: the 2-(Scysteinylglycyl)-cis-caftaric acid, 13 (3%), and the 5-(S-cysteinylglycyl)-trans-caftaric acid, 11 (2%). The former showed two doublets (J = 8.3 Hz) characteristic of ortho-coupled ring protons at δH 6.82 and 7.01 and a pair of doublets at δH 5.99 and 7.46 (J = 12 Hz), indicating that the vinylic bond was cis-configured. The latter showed two pairs of doublets at δH 7.08 and 7.25 (J = 1.8 Hz), characteristic of meta-coupled ring protons, and at δH 6.36 and 7.6 (J = 16 Hz), characteristic of a vinyl trans-configured bond, respectively. NMR analysis allowed structural elucidation of most adducts detected by LC-DAD-MS and accurate determination of their molarities. The major adducts formed in all cases were confirmed to be 2-S adducts in the trans-configuration (1, 5, and 10), as shown earlier.4 The disubstituted adducts were shown to be 2,5-di-S adducts in the trans-configuration (2 and 9). These compounds arise from oxidation of the 2-S-glutathionyl adduct followed by nucleophilic addition of a second GSH in the 5 position of the aromatic ring, as described earlier.7 No evidence of disubstitution was observed when GSH was replaced with cysteinylglycine, suggesting that the latter reacts faster than the former with caftaric acid-o-quinone. The 6-S adduct in the trans-configuration was identified only in the GSH-caffeic acid solution (7). The other minor compounds were identified as I



Article

amino group on the carbonyl of the terminal acid group of cysteine with loss of water, to form a diketopiperazine, as illustrated for compound 11 (Figure 7). The ion at m/z 321 is specific to 5-S adducts, while the ion at m/z 264 is more intense for other isomers [except for cis-2-(S glutathionyl) adducts as discussed above], suggesting that their formation is affected by molecular conformation. ROESY NMR spectra showed correlations of different intensities between the caffeic and peptide protons for trans-2-S, 5-S, and 2,5-di(Sglutathionyl)caftaric acid. Stronger correlations were observed between the H-6 and the cysteine H-α, H-β, and H-β′ in the case of the 5-S adduct (not shown), indicating a greater proximity between this amino acid and the aromatic H-6. Moreover, ROE correlations between the H-α of caftaric acid and the cysteinyl protons and glutamyl H-γ and H-γ′ in the trans-2-S adduct (Figure 5) indicate proximity between the peptide group and the caffeic vinyl chain. This may favor the formation of the fragment ion at m/z 264, resulting from reaction between the cysteinyl amino and the tartrate carboxylic groups. In the 5-S adducts, the amino acid, being further away from the tartrate ester, is more likely to form the diketopiperazine through loss of water from cysteinylglycine cyclization, leading to m/z 321 after loss of tartaric acid. Except for GRP and GRP2, glutathionylcaftaric acid, glutathionylcaffeic acid, and cysteinylglycylcaftaric acid adducts had never been formally identified. In particular, the 5-S and 6-S adducts are reported here for the first time, demonstrating that sulfanyl substitution on caffeic acid adducts is not exclusive to C-2. The first formal identification of 2-S cis-adducts is provided, confirming the structural hypothesis proposed by other authors. The elution order under the chromatographic conditions is 1:2-S-trans-, 2:2,5-di-S-trans-, 3:5-S-trans-, 4:6-S-trans-, and 5:2-S-cis-. Moreover, signature UV and MS spectra that can serve for identification of all these molecules in HPLC profiles are proposed. This work also leads to revision of the fragmentation schemes in the positive ion mode. Full description of the reaction media, including accurate quantification of products using qNMR, is an important step toward the development of methods to analyze these compounds, paving the way for a better understanding of reactions taking place during winemaking and aging and their impact on wine quality. Indeed, cysteinylglycylcaftaric acid and glutathionylcaffeic acid have been reported earlier in juices and wines and could be formed in rather large amounts as a result of hydrolytic activities arising from microbial contamination or enzyme treatment. Moreover, although the 2-S-trans-forms are largely predominant, they can be partly converted to the cisisomers upon light exposure. Finally, there has been some controversy about the contribution of caftaric acid and GRP to wine taste and in particular bitterness. Further studies are needed to determine whether the different derivatives including the isomers elicit different taste properties.

not detected by NMR or UPLC-DAD-MS in the caftaric and caffeic acid samples used as precursors. The cis−trans isomerization may have been catalyzed by light exposure, as no special care was taken to avoid it.13 The fragment ion at m/z 264 was less intense in the fragmentation spectra of cis-configured compounds 3 and 8, as described earlier.8 Moreover, it is almost absent in the spectra of compounds 4 and 6. This fragment has been attributed to cleavage of the thioether bond and loss of a carboxylic group on the resulting GSH fragment.8 However, it is present in all cysteinylglycyl adducts, indicating that it does not correspond to the glutathionyl part of the molecule. It probably arises from a rearrangement between the peptide group and the caffeic acid carboxylic group, through an attack of the terminal amino group on the carbonyl of the ester linkage of tartaric group, with loss of tartaric acid (or loss of water in the case of glutathionylcaffeic acid) and cyclization and loss of glycine by the b-y pathway, classically described for peptides,14 as shown in Figure 7. The lower abundance of the ion at m/z 264 for the

Figure 7. Scheme of the fragmentation pathway leading to fragment ions at m/z 264 and m/z 321, respectively, for 2-(S-cysteinylglycyl)trans-caftaric acid (10) and 5-(S-cysteinylglycyl)-trans-caftaric acid (11).

cis-isomer is likely due to steric effects. Depending on the position of the double bond, the amino acid is too far away from the carboxylic group of the caffeic acid or is too close and causes an impedant effect, preventing the formation of the fragment at m/z 264. In the case of compounds 3 and 8, the ion at m/z 264 is less intense than fragment ions at m/z 489 and 357, respectively, corresponding to the loss of glutamic acid (129 amu). It is the major ion in the spectrum of compound 13, which lacks the glutamic acid group and cannot yield the −129 amu fragment. The last group of compounds, identified as the 5-S adducts in the trans-configuration (4, 6, and 11), show a characteristic fragment ion at m/z 321 that can serve as a signature of their particular structures. This fragment is tentatively attributed to the loss of glutamic acid (−129 amu), tartaric acid (−150 amu), if present, and water and probably due to a rearrangement inside the peptide group, through an attack of the terminal

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EXPERIMENTAL SECTION

General Experimental Procedures. All the experiments were carried out by Plateforme Polyphenols at the Institut National de la Recherche Agronomique (INRA, Montpellier, France). NMR spectra were acquired using an Agilent 500 MHz DD2 NMR spectrometer operating at 500.05 MHz for 1H and 125.75 MHz for 13C, equipped with a 5 mm indirect detection Z-gradient probe. UPLC-DAD-MS/ MS data were acquired using a Waters Acquity UPLC-DAD system coupled to a Bruker Daltonics Amazon mass spectrometer equipped with an electrospray source and an ion trap mass analyzer. All solvents utilized in this study were of HPLC grade, and all chemicals of J



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After probe tuning, a single-scan 1D 1H NMR spectrum of DMS was collected using a 90 deg pulse. The surface area of the DMS signal was used for probe calibration using VNMRJ absolute intensity qNMR utility software. The probe calibration was then verified using the gallic acid sample. The calibration probe was checked on a regular basis. The absolute concentration of products formed during the synthesis was determined from 1D 1H NMR spectra using a 90 deg pulse and an interscan delay equal to 20 s (>5T1). Well-separated signals were used for the surface signal integration.

analytical reagent grade. Methanol, formic acid, and acetone were purchased from VWR Prolabo (Fontenay sous Bois, France). Caffeic and caftaric acids, GSH, and cysteinylglycine were purchased from Sigma (St. Louis, MO, USA). Solid-phase extraction cartridges were purchased from Waters (Waters Sep-Pak Vac 20 mL tC18 − 5g) (Milford, MA, USA). All deuterated solvents were purchased from Euriso-top (France). Purified deionized water (Milli-Q purification system, Millipore, France) was used for the preparation of all solutions. Preparation of Crude Grape Polyphenol Oxidase. The grape polyphenol oxidase extract was prepared from Macabeu grapes harvested at oenological maturity from the INRA experimental vineyard at Pech Rouge (Gruissan, France) according to the method previously described by Cheynier and Ricardo da Silva.15 Grape must was obtained by pressing, and the pressed material allowed to settle overnight at 4 °C. A volume of 4 mL of the deposit (5 L/100 L juice) thus obtained was washed twice with 10 mL of acetone (80%) at 4 °C, and the fraction containing PPO recovered by filtration on Whatman #1 paper and resuspended in 3 mL of water acidified with formic acid at pH 2.5. Enzymatic Reactions. The enzymatic reactions were carried out in 20 mL of acidified H2O (pH 2.5 with HCOOH) containing caffeic or caftaric acid (1 mM) and GSH or cysteinylglycine in the molar ratio 1:3. The solutions were incubated with the PPO extract (10% v/v) for 1 h under stirring at room temperature. After reaction, the solution was filtered through a Whatman filter (0.45 μm), and the solid-phase extraction was carried out. Solid-Phase Extraction (SPE). The glutathionyl and cysteinylglycyl adducts of caftaric and caffeic acid were isolated by solid-phase extraction performed with tC18 SPE cartridges (Waters Sep-Pak Vac 20 cm3 tC18 − 5g) using a glass vacuum manifold. The cartridges were conditioned with 10 mL of MeOH and 20 mL of ultrapure H2O adjusted to pH 2.5 with HCOOH. After conditioning, the cartridges were loaded with the samples (50 mL). In order to eliminate the excess of peptide and the residual PPO, a washing step was carried out with 80 mL of ultrapure H2O (pH 2.5). Adducts were eluted and recovered with 20 mL of MeOH/H2O (70:30 v/v). The MeOH was then evaporated in a rotatory evaporator at 30 °C, and the final solution was freeze-dried and kept under vacuum prior to UPLCDAD-MS/MS and NMR analysis. UPLC-DAD-MS/MS Analysis. UPLC analyses were performed using a reversed-phase Acquity BEH C18 column (150 mm length; 1 mm i.d.; 1.7 μm particle size; Waters) maintained at 35 °C. The samples were dissolved in MeOH/H2O (50:50 v/v) (20 mg/L), filtered, and directly injected. The mobile phase was a binary solvent system of A (ultrapure H2O/HCOOH, 99:1 v/v) and B (MeOH/ HCOOH, 99:1, v/v). The elution program was as follows: isocratic with 2% B (1 min), 2−30% B (1−10 min), isocratic with 30% B (10−12 min), 30−75% B (12−25 min), 75−90% B (25−30 min), and isocratic with 90% B (30−35 min), followed by reconditioning of the column. The injection volume was 0.5 μL, and the flow rate was 0.08 mL min−1. UV−visible spectra were recorded from 200 to 650 nm. ESIMS analyses were performed with a Bruker Daltonics Amazon (Bruker, Darmstadt, Germany) mass spectrometer, coupled to the UPLC-DAD system and equipped with an electrospray source and an ion trap mass analyzer. The spectrometer was operated in the negative ion mode (capillary voltage, 4.5 kV; end plate offset, −500 V; temperature, 200 °C; nebulizer gas, 10 psi; and dry gas, 5 L/min) or in the positive ion mode (capillary voltage, 2.5 kV; end plate offset: −500 V; temperature, 200 °C; nebulizer gas, 10 psi; and dry gas, 5 L/min). The fragmentation amplitude used for MS2 experiments was set at 1 V. NMR Spectroscopy. NMR analysis was carried out following the method described by Vallverdú-Queralt et al.16 The chemical shifts were reported relative to that of the internal organic solvent methanold4 (3.31 and 49.1 ppm for 1H and 13C, respectively) or DMSO-d6 (2.5 and 39.5 ppm for 1H and 13C, respectively). Data were processed and analyzed using both VNMRJ and ACD/Laboratories software. Absolute concentration measurements were carried out by qNMR using an external calibration method.17 Dimethyl sulfide (DMS) (20 mg) and gallic acid (20 mg) were accurately weighed and dissolved in a precise volume of D2O (5 mL) and DMSO-d6 (5 mL), respectively.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00279. Additional information relating to the NMR data of all synthesized compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel (N. Ferreira-Lima): +55 (0) 48 3721 5376. Fax: +55 (0) 48 3334 3726. E-mail: [email protected]; [email protected]. *Tel (A. Vallverdú-Queralt):+33 (0) 4 99 61 25 84. Fax: +33 (0) 4 99 61 28 57. E-mail: [email protected], [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge CAPES (Coordençaõ ́ Superior) for the de Aperfeiçoamento de Pessoal de Nivel financial support of a doctoral sandwich program at Plateforme Polyphénols, UMR SPO, INRA, for N.F.-L. A.V.-Q. is grateful to the Alfonso Martiń Escudero Foundation for the postdoctoral fellowship for carrying out research abroad. Funding for this work was provided by the Instituto de Salud Carlos III, ISCIII (CIBEROBN). Financial support from GIS IBiSA (Infrastructures en Biologie Santé et Agronomie), Région Languedoc Roussillon, and INRA CNOC for funding of the UPLC-MS and NMR equipment is also acknowledged.

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REFERENCES

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