Food Chemistry 141 (2013) 1078–1086

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Food Chemistry

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Formation of cysteine-S-conjugates in the Maillard reaction of cysteineand xylose

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.04.043

⇑ Corresponding author. Tel.: +41 22 780 2211.E-mail address: christoph.cerny@firmenich.com (C. Cerny).

Christoph Cerny ⇑, Renée Guntz-DubiniCorporate Research, Firmenich SA, routes des Jeunes 1, 1211 Geneva 8, Switzerland

a r t i c l e i n f o

Article history:Received 4 March 2013Received in revised form 15 April 2013Accepted 17 April 2013Available online 24 April 2013

Keywords:Maillard reactionCysteine-S-conjugateCysteineXyloseFurfuryl alcohol2-Furfurylthiol2-Methyl-3-furanthiol

a b s t r a c t

Cysteine-S-conjugates (CS-conjugates) occur in foods derived from plant sources like grape, passion fruit,onion, garlic, bell pepper and hops. During eating CS-conjugates are degraded into aroma-active thiols byb-lyases that originate from oral microflora. The present study provides evidence for the formation of theCS-conjugates S-furfuryl-L-cysteine (FFT-S-Cys) and S-(2-methyl-3-furyl)-L-cysteine (MFT-S-Cys) in theMaillard reaction of xylose with cysteine at 100 �C for 2 h. The CS-conjugates were isolated using cationicexchange and reversed-phase chromatography and identified by 1H NMR, 13C NMR and LC–MS2. Spectraand LC retention times matched those of authentic standards. To the best of our knowledge, this is thefirst time that CS-conjugates are described as Maillard reaction products. Furfuryl alcohol (FFA) is pro-posed as an intermediate which undergoes a nucleophilic substitution with cysteine. Both FFT-S-Cysand MFT-S-Cys are odourless but produce strong aroma when tasted in aqueous solutions, supposedlyinduced by b -lyases from the oral microflora. The perceived aromas resemble those of the correspondingaroma-active thiols 2-furfurylthiol (FFT) and 2-methyl-3-furanthiol (MFT) which smell coffee-like andmeaty, respectively.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Cysteine-S-conjugates (CS-conjugates) are thioether derivativesof cysteine. They play multiple roles in physiology, for example asintermediates in the detoxification of xenobiotics and endogenoustoxic compounds. After the formation of a glutathione–S-conjugatefrom glutathione and a toxic substance, the glycine and glutamatemoieties are enzymatically cleaved to give the corresponding CS-conjugate. After N-acetylation of the CS-conjugate it is excretedby the urinary system (Hayden, Schaeffer, Larsen, & Stevens, 1987).

CS-conjugates also occur in foods, such as onion, garlic, bellpepper, passion fruit, grapes and hops (Gros, Peeters, & Collin,2012; Kimura et al., 1990; Lawson & Gardener, 2005; Pena-Gallego,Hernandez-Orte, Cacho, & Ferreira, 2012; Starkenmann & Niclass,2011; Starkenmann, Niclass, & Troccaz, 2011; Tominaga & Dubour-dieu, 2000; Tominaga, Masneuf, & Dubourdieu, 1995). They play animportant role in aroma generation during wine fermentation(Tominaga, Peyrot des Gachons, & Dubourdieu, 1998). CS-conju-gates are formed by catabolism of glutathione–S-conjugates thatare present in grape juice (Capone, Sefton, Hayasaka, & Jeffery,2010; Kobayashi et al., 2010; Peyrot des Gachons, Tominaga, &Dubourdieu, 2002; Roland, Vialaret, Razungles, Rigou, & Schneider,2010; Thibon, Cluzet, Merillon, Darriet, & Dubourdieu, 2011). Then

b-lyases from the wine yeast cleave the C–S bond and convert theCS-conjugate into pyruvic acid, ammonia and the aroma-activethiol (Roncoroni et al., 2010; Tominaga et al., 1995). For example,the conjugate S-[4-(2-hydroxy-4-methylpentyl)]-L-cysteine istransformed into the aroma compound 4-sulfanyl-4-methyl-2-pentanol which exhibits a fruity and grapefruit-like odour. BothCS-conjugate and its thiol are typical for wines made from thegrape variety Vitis vinifera L. cv. Sauvignon blanc, known for theirexotic fruit-like and grapefruit character (Peyrot des Gachons,Tominaga, & Dubourdieu, 2000; Tominaga, Furrer, Henry, &Dubourdieu, 1998).

Recent experiments shed light on certain CS-conjugates whichact as powerful in-mouth flavour precursors. Starkenmann et al.(2008) demonstrated that S-(1-hydroxy-3-hexyl)]-L-cysteine, S-propyl-L-cysteine and S-(2-heptyl)-L-cysteine, respectively, whichnaturally occur in wine, onion and bell pepper, respectively, aretransformed in the mouth into their corresponding aroma-activethiols 3-sulfanyl-1-hexanol, 1-propanethiol and 2-heptanethiol.They could show that Fusobacterium nucleatum, an oral bacterium,is able to transform the odourless CS-conjugate S-(1-hydroxy-3-hexyl)-L-cysteine into the aroma-active thiol.

The Maillard reaction is one of the central pathways to aroma-active volatiles during heating of food (e.g., boiling, roasting, grill-ing and frying). The Maillard reaction of cysteine with reducingsugars in particular plays a key role in the formation of sulphur-containing odorants. For instance, the reaction of L-cysteine with

C. Cerny, R. Guntz-Dubini / Food Chemistry 141 (2013) 1078–1086 1079

pentoses (ribose, xylose or arabinose) generates furfurylthiol (FFT),2-methyl-3-furanthiol (MFT) and other compounds which areessential for the aroma of cooked meat (Cerny & Davidek, 2003;Grosch, Sen, Guth, & Zeiler-Hilgart, 1990; Hofmann & Schieberle,1995; Mottram & Nobrega, 2002). Although volatiles from Maillardreaction represent a minuscule mass fraction in the reaction prod-uct, they have been intensively studied because of their importantaroma contribution to heated food. On the contrary, non-volatileMaillard compounds have been largely neglected in flavour re-search and only a few studies have looked at flavour-active non-volatile Maillard products (Frank, Jezussek, & Hofmann, 2003,2005; Schlichtherle-Cerny, Affolter, & Cerny, 2003). Hence, theobjective of the present study was to gain more knowledge onthe formation of non-volatiles in the Maillard reaction betweencysteine and xylose with focus on the possible formation of cys-teine derivatives. The present paper reports on the isolation andidentification of two new CS-conjugates in the Maillard reactionproduct (MRP) from xylose and cysteine: S-furfuryl-L-cysteine(FFT-S-Cys) and S-(2-methyl-3-furyl)-L-cysteine (MFT-S-Cys).

2. Materials and methods

2.1. Materials

Thionyl chloride was obtained from Acros (Geel, Belgium),hydrogen chloride in dioxane (4.0 M), hydrogen chloride in ethanol(1.25 M) and formic acid (puriss. p.a. for MS) from Fluka (Buchs,Switzerland), and lithium hydroxide and sodium hydroxide fromMerck (Darmstadt, Germany). L-Cysteine hydrochloride monohy-drate, furfuryl alcohol (FFA), 2-methyl-3-furanthiol (MFT), L-serineand D-xylose were Firmenich raw materials. Solvents were of ana-lytical grade. Acetonitrile for HPLC (hypergrade for LC–MS) wasfrom Merck. Water for HPLC was prepared using a MilliporeMilli-Q Plus PF water purification system (Molsheim, France).Dowex 50WX8 was purchased from Alfa Aesar (Karlsruhe, Ger-many) and LiChroprep RP-18 (40–63 lm) from Merck.

2.2. LC–MS/ELSD and LC-DAD

HPLC was done on a Waters 2790 Alliance instrument (Baden-Dättwil, Switzerland) using a Phenomenex Luna C18(2) column(4.6 mm internal diameter, 150 mm length, 3 lm particle size)from Brechbühler (Schlieren, Switzerland). Freeze-dried sampleswere dissolved in mobile phase (10% aqueous acetonitrile with0.1% formic acid) at approximately 0.05%. The injection volumewas 10 ll. Elution solvents were 0.1% aqueous formic acid (solventA) and acetonitrile containing 0.1% formic acid (solvent B). The gra-dient was 90% A for 2 min, decreasing gradually to 30% A over6 min, keeping 30% A for 4 min and increasing again to 90% A with-in 3 min. The flow was 0.5 ml/min. At the end of the column theflow was split (approximately 1:1), one part of the effluent goingto the mass spectrometer, the other to the evaporative light scat-tering detector (ELSD) from Polymer Laboratories (now part of Agi-lent, Santa Clara, CA), model PL-ELS 2100 (neb = 45 �C, evap = 40 �C,gas = 1.0 SLM). The mass spectrometer was a Bruker Esquire 3000plus ion trap (Bruker Daltonics, Bremen, Germany) with the elec-trospray ion source in positive mode (ESI+). Nitrogen was used asnebulising and drying gas. Drying temperature was 300 �C. Thecapillary voltage was set to 4.2 kV. The scan range was m/z 50–1000. LC-DAD was performed on an Agilent series 1100 HPLC(Basel, Switzerland), equipped with a DAD (G1315B) using thesame column and conditions as for LC–MS/ELSD.

2.3. Synthesis of reference compounds

2.3.1. Synthesis of S-furfuryl-L-cysteine (FFT-S-Cys)Cysteine hydrochloride monohydrate (7.90 g, 45 mmol) and FFA

(3.95 g, 40 mmol) were heated in an ethanolic solution of hydrogenchloride (1.25 M, 60 ml) at 80 �C for 60 min. The resulting darkbrown solution was purified on a Dowex 50WX8 column, whichhad been pre-treated with hydrochloric acid (0.1 M) and rinsedwith water until neutral. Elution was first with water until the elu-ate became clear, then with aqueous ammonium hydroxide (2.0 M,200 ml). Fractions with FFT-S-Cys were freeze-dried (VirTis Freeze-mobile 25EL, SP Scientific, Ipswich, UK) and then purified on an RP-18 column yielding 0.62 g of FFT-S-Cys (13% molar yield). LC–MSESI+ (rt 6.5 min). MS2 fragments from the ESI-MS+ parent ion m/z202 (M+H)+: 185, 184, 156, 113, 81. NMR spectra were measuredon a Bruker Avance-500 spectrometer (Bruker, Fällanden, Switzer-land). 1H NMR (500 MHz, D2O) d 7.51 (dd, J = 1.9, 0.7, 1H); 6.45 (dd,J = 3.2, 1.9, 1H); 6.39 (dd, J = 3.2, 0.7, 1H); 3.86 (d, J = 1.9, 2H); 3.82(dd, J = 7.8, 4.2, 1H); 3.09 (dd, J = 15.0, 4.2, 1H); 2.99 (dd, J = 15.0,7.8, 1H). 13C NMR (125 MHz, D2O) d 173.1 (s), 150.9 (s), 143.4(d), 111.2 (d), 109.0 (d), 53.8 (d), 32.0 (t), 28.8 (t).

2.3.2. Synthesis of S-(2-methyl-3-furyl)-L-cysteine (MFT-S-Cys)2.3.1.1. 3-Chloro-L-alanine. The synthesis is based on the procedureof Yamashita, Inoue, Kinoshita, Ueda, and Murao (2000). L-Serine(30.0 g, 283 mmol) was added to a stirred solution of hydrogenchloride in dioxane (4.0 M, 600 ml). Then thionyl chloride (41.8 g,351 mmol) was added dropwise at room temperature over40 min. The temperature was raised to 40 �C and held for 20 h.Then approximately 300 ml of volatiles were evaporated using arotary evaporator (20 �C). The residue was cooled to 8 �C followedby addition of 200 ml water while keeping the temperature con-stant and then concentrated to approximately 200 g using a rotaryevaporator (20 �C). After the addition of active carbon (3.0 g), filter-ing through a suction filter and rinsing with water, the filtrate wasconcentrated at 20 �C in vacuo to 120 ml. The cooled (0–10 �C) con-centrate was added to a saturated aqueous solution of lithiumhydroxide (75 ml), followed by the addition of acetone (600 ml)and left to stand for 80 min at 0–10 �C. The formed precipitatewas recovered by filtration, washed with acetone (100 ml) anddried in a desiccator (31.1 g 3-chloro-L-alanine; 88% molar yield).MS ESI+: m/z 126, 124, 80, 78. 1H NMR (500 MHz, D2O) d 4.79 (s,2H); 4.18 (dd, J = 4.9, 3.2, 1H); 4.10 (dd, J = 12.5, 4.9, 1H); 4.03(dd, J = 12.5, 3.2, 1H). 13C NMR (125 MHz, D2O) d 173.8 (s), 58.2(d), 46.1 (t).

2.3.1.2. MFT-S-Cys. 3-Chloro-L-alanine (3.71 g, 27 mmol) wasadded to a stirred aqueous sodium hydroxide solution (4% w/w,80 ml). A solution of 2-methyl-3-furanthiol (3.48 g, 31 mmol) inmethanol (80 ml) was rapidly added, causing the temperature ofthe yellow solution to increase to 30 �C. After 90 min, MFT-S-Cyshad started to form, as indicated by an ESI-MS peak of m/z 202in the LC–MS chromatogram. After 26 h – the peak had substan-tially increased – the reaction mixture was adjusted to pH 5 withhydrochloric acid (12%, 20 ml) and then washed with diethyl etherto remove any unreacted thiol. An aliquot of the freeze-dried aque-ous phase (2.3 g) was fractionated by column chromatography onLiChroprep RP-18 (40–63 lm) and an ethanol gradient from 0%to 20% ethanol. MFT-S-Cys eluted with the 15% ethanol fraction.LC–MS ESI+ (rt 8.0 min). MS2 fragments from the ESI-MS+ parention m/z 202 (M+H): 185, 156, 113. 1H NMR (500 MHz, D2O) d7.44 (d, J = 2.1, 1H); 6.51 (d, J = 2.1, 1H); 3.76 (dd, J = 8.2, 4.0,1H); 3.27 (dd, J = 14.7, 4.0, 1H); 3.09 (dd, J = 14.7, 8.2, 1H); 2.34(s, 3H). 13C NMR (125 MHz, D2O) d 175.2 (s), 158.9 (s), 144.4 (d),117.3 (d), 110.5 (s), 56.8 (d), 39.2 (t), 14.0 (q).

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2.4. Preparation of the Maillard reaction product (MRP)

D-Xylose (90.0 g, 600 mmol) and L-cysteine hydrochloridemonohydrate (95.0 g, 541 mmol) were dissolved in water (50 ml)at 40 �C. Sodium hydroxide solution (50%, 45.5 g) was added andthe pH was recorded. After 15 min, the pH had equilibrated at a va-lue of 4.0. Then the mixture was heated in a stirred glass reactor at100 �C for 120 min under reflux.

2.5. Model reaction of FFA with cysteine

L-Cysteine hydrochloride monohydrate (105.0 g, 600 mmol)was dissolved in water (100 ml), neutralised with aqueous sodiumhydroxide (50%, 48.0 g) and the pH adjusted to 4.0 with hydrochlo-ric acid (12%). Then furfuryl alcohol (5.95 g, 61 mmol) was addedand the mixture heated at 100 �C for 120 min. After addition ofwater (150 ml) the reacted mixture was filtered. The filtrate wasdiluted 1:1 (w/w) with water for LC–MS and 1:19 for LC-DADanalyses.

2.6. Isolation of cysteine-S-conjugates

2.6.1. Isolation of FFT-S-CysWater was added to the MRP to obtain a total mass of 320 g. An

aliquot (200 g) was fractionated by ion exchange chromatographyon Dowex 50WX8 (80 g) which had been pre-treated with hydro-chloric acid (7%) and rinsed with water until neutral. Elution wasfirst with water (750 ml) then with aqueous ammonium hydroxideof increasing molarity (0.3 M, 200 ml; 0.6 M, 200 ml; 0.9 M,200 ml). All fractions were freeze-dried and analysed by LC–MS(as 0.05% solutions in the mobile phase). The fraction eluting withaqueous ammonium hydroxide (0.3 M; 5.5 g freeze-dried solids)was selected for further fractionation on RP-18 (LiChroprep RP18,70 g) using a water/ethanol (v/v) gradient (water, 300 ml; 10% eth-anol, 75 ml; 20% ethanol, 75 ml; 30% ethanol, 75 ml; 40% ethanol,150 ml). The RP-18 fraction obtained with 10% ethanol (v/v) wasfurther purified on a smaller RP-18 column (LiChroprep, 3.0 g) withwater (20 � 3 ml) as eluent. The 6 ml of the last two fractions werepooled and freeze-dried. The freeze-dried fraction (fraction A) wassufficiently pure for characterisation by NMR.

2.6.2. Isolation of MFT-S-CysThe RP-18 fraction eluting with 20% ethanol (cf. 2.5.1.) was

purified on a smaller RP-18 column (LiChroprep, 2.0 g) with wateras mobile phase (9 � 3 ml). The last two fractions were pooled,freeze-dried and measured by NMR and LC–MS (fraction B).

3. Results

A concentrated aqueous solution of cysteine and xylose washeated at an initial pH of 4 for 2 h at 100 �C. The resulting Maillardreaction product (MRP) was first fractionated by cation exchangechromatography followed by sub-fractionation on RP-18 using awater/ethanol gradient.

3.1. Identification of FFT-S-Cys

The RP-18 fraction which eluted with 10% ethanol, showed anLC–MS peak with a pseudomolecular ion at m/z 202. It was furtherpurified on a smaller RP-18 column. The resulting fraction A gave asingle major peak in the LC–MS/ELSD chromatogram (cf. Fig. 1).The corresponding molecular weight of 201 is in agreement withFFT-S-Cys structure. A FFT-S-Cys reference was synthesised andcompared to fraction A. Both had the same retention time. TheMS2 spectra (from parent ion m/z 202) likewise showed identical

fragmentation patterns. Table 1 lists mass-to-charge ratios ofMS2 fragments and lost neutral particles for FFT-S-Cys: Loss ofammonia and water, respectively, from the pseudomolecular ionm/z 202 led to m/z 185 and 184. The fragment m/z 156 stems fromloss of formic acid. Splitting-off of the alanine moiety results in thefurfurylthio cation (m/z 113) while scission of cysteine producesfragment m/z 81.

The NMR data strongly support the MS based identification.Fig. 2 compares 1H and 13C NMR spectra of the reference com-pound with FFT-S-Cys isolated from fraction A. The 1H NMR furanring signals appear at 7.51, 6.45 and 6.39 ppm while the methylenegroup hydrogen atoms between the furan ring and the sulphuratom show up as a doublet at 3.86 ppm. The three hydrogen atomsfrom the cysteine moiety correspond to one double doublet at 3.82and two others around 3.04 ppm. The signals due to the amino andcarboxylic hydrogen atoms are not observed due to rapid H–D ex-change in deuterium oxide. It is noteworthy that all signals of thereference spectrum (cf. Fig 2a) clearly show up in the spectrumfrom fraction A (cf. Fig. 2b). Additional smaller signals in the spec-trum suggest that it contains minor, unidentified impurities. Theseimpurities correlate presumably to the small peaks at 5.6 and6.1 min in the LC-ELSD chromatogram (cf. Fig. 1b). The 13C NMRspectra confirms furthermore the identification of FFT-S-Cys infraction A (cf. Fig. 2c and d). The signals from the carbonyl group(173.1 ppm), the furan ring (150.9, 143.4, 111.2 and 109.0 ppm),the carbon atom adjacent to the amino group (53.8 ppm) and themethylene carbons (32.0 and 28.8 ppm) appear both in the spectraof fraction A and the reference compound. As in the 1H NMR spec-trum, marginal signals from impurities appear in the 13C NMRspectrum.

Taken together, FFT-S-Cys was positively identified in the MRPfrom xylose and cysteine by means of 13C NMR and 1H NMR as wellas LC–MS2.

3.2. Identification of MFT-S-Cys

MFT-S-Cys eluted in the RP-18 chromatography fraction with20% ethanol, as indicated by a peak with m/z 202 peak. The elutionwith 20% ethanol points to a lower polarity than FFT-S-Cys whicheluted already with 10% ethanol. Purification of this fraction on asmaller RP-18 column yielded fraction B which consisted essen-tially of a single compound.

Fraction B showed the same pseudomolecular ion signal (m/z202) as FFT-S-Cys in the ESI-MS spectrum (cf. Fig. 3f) suggestingthat it is an isomer. Moreover the MS2 fragments m/z 185, 156and 113 are in common, underlining a similar structure encom-passing amino, carboxy and alanyl moieties. Synthesis of theauthentic reference compound and comparison with fraction B re-vealed MFT-S-Cys as the compound of fraction B on the basis ofidentical MS spectra and retention times.

Comparison of 1H NMR and 13C NMR spectra of MFT-S-Cys andfraction B confirmed the identification (cf. Fig. 4). The furan ringprotons show doublets at 6.54 and 7.44 ppm and the methyl groupa singlet at 2.34 ppm. The cysteine hydrogen atoms correspond tothree doublets of doublets (3.09, 3.27 and 3.76 ppm). The 13C NMRspectrum shows a simple pattern with furan carbons at 110.5,117.3, 144.4 and 158.9 ppm, the methyl group at 14.0 ppm andcysteine signals at 39.2, 56.8 and 175.2 ppm. 1H NMR and 13CNMR spectra match precisely the ones of fraction B.

In summary, MFT-S-Cys was clearly identified in the MRP fromxylose and cysteine, based on NMR spectra, MS data and LC reten-tion times. It is equally noteworthy that the UV spectra from LC-DAD analyses of fractions A and B match those of the synthesisedreferences. The absorption maxima kmax relate to the furan struc-ture of the molecules and were found at 224 nm for FFT-S-Cysand 220 nm for MFT-S-Cys, respectively.

Fig. 1. (a) LC-ELSD chromatogram of FFT-S-Cys. (b) LC-ELSD chromatogram of fraction A. (c) LC–MS trace of FFT-S-Cys (ESI+, m/z 202 ± 0.3). (d) LC–MS trace of fraction A (ESI+,m/z 202 ± 0.3). (e) MS (ESI+) (top) and MS2 spectra (parent ion m/z 202.0 ± 0.3) (bottom) of FFT-S-Cys (6.7 min). (f) MS (ESI+) (top) and MS2 spectra (parent ion m/z 202.0 ± 0.3)(bottom) of fraction A (6.7 min).

Table 1Fragmentation (MS2) of pseudomolecular ions (ESI+) m/z 202 of FFT-S-Cys and MFT-S-Cys.

MS2 of FFT-S-Cys(m/z 202 ± 0.3)

Neutral particle loss Fragment

m/z 185 �17 [(M+H)�NH3]+

m/z 184 �18 [(M+H)�H2O]+

m/z 156 �46 [(M+H)�H2O�CO]+

m/z 113 �89 [(M+H)�alanine]+

m/z 81 �121 [(M+H)�cysteine]+

MS2 of MFT-S-Cys(m/z 202 ± 0.3)

m/z 185 �17 [(M+H)�NH3]+

m/z 184 �18 [(M+H)�H2O]+

m/z 113 �89 [(M+H)�alanine]+

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3.3. Model reaction of FFA with cysteine

A model reaction with FFA and cysteine (molar ratio 1:10) wascarried out using the same reaction conditions as for the reactionbetween cysteine and xylose. Both LC-DAD and LC–MS chromato-grams of the reaction product showed peaks with retention timesmatching those of FFT-S-Cys and MFT-S-Cys. The area of the MFT-S-Cys peak was approximately 40-times smaller than the FFT-S-Cys peak in the LC-DAD chromatogram (chromatograms notshown).

4. Discussion

The results demonstrate that both FFT-S-Cys and MFT-S-Cys areformed in the Maillard reaction of xylose and cysteine. Naturally,the question of a plausible formation pathway to both productsarises. It is known that the corresponding thiols FFT and MFT areMRPs from xylose and cysteine (Cerny & Davidek, 2003; Farmer,Mottram, & Whitfield, 1989; Hofmann & Schieberle, 1995) and itis conceivable that both could be intermediates for the observedCS-conjugates. However the substitution of FFT and MFT by cys-teine i.e. of one thiol by another thiol is not straightforward.

A more probable mechanism is the nucleophilic substitution ofan alcohol by cysteine. Fig. 5 illustrates the pathway that involvesformation of FFA as an intermediate which then undergoes nucle-ophilic substitution by cysteine. Under strongly acidic conditions(hydrochloric ethanol) FFA and cysteine gave FFT-S-Cys in 13% mo-lar yield (cf. Section 2) suggesting a nucleophilic substitution. Weassume that the same mechanism could still be valid at a lessacidic pH of 4. Both cysteine and FFA are present in the reaction,cysteine as reactant, and FFA was identified in the solvent extractof the present MRP (data not shown). FFA is also known to occurin Maillard reaction products as well as in heated foods (De Rijke,Van Dort, & Boelens, 1981; Rizzi, 1995; Tressl, Helak, Kersten, &Nittka, 1993).

Consequently, we investigated the reaction of FFA with cysteineunder the same reaction conditions as for the MRP (i.e. 100 �C, 2 h,

Fig. 2. 1H NMR spectra: (a) FFT-S-Cys; (b) Fraction A; 13C NMR spectra: (c) FFT-S-Cys; (d) Fraction A;

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Fig. 3. (a) LC-ELSD trace of MFT-S-Cys. (b) LC-ELSD trace of fraction B. (c) LC–MS trace of MFT-S-Cys (ESI+, m/z 202 ± 0.3). (d) LC–MS trace of fraction B (ESI+, m/z 202 ± 0.3).(e) MS (ESI+) (top) and MS2 spectra (parent ion m/z 202.0 ± 0.3) (bottom) of MFT-S-Cys (7.8 min). (f) MS (ESI+) (top) and MS2 spectra (parent ion m/z 202.0 ± 0.3) (bottom) offraction B (7.9 min).

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and pH 4) in order to test our assumption. Peaks with retentiontimes of FFT-S-Cys (6.5 min) and MFT-S-Cys (7.9 min), respectively,in the LC–MS trace m/z 202 (M+H) suggest that both CS-conjugateshad formed and FFA is an effective precursor. It should be notedthat MFT-S-Cys apparently forms to a much smaller extent thanFFT-S-Cys. Its LC-DAD peak is only 2.5% of the FFT-S-Cys area.

Rizzi (1995) investigated already the reaction of FFA with cys-teine at pH 4 and 100 �C in 1995. He focused on the gas chromatog-raphy analysis of volatiles and found traces of FFT in thedichloromethane extract of the reaction product, but did not ana-lyse non-volatiles. Interestingly, Rizzi suggests a furfuryl cationas an intermediate for FFT, difurfuryl ether and polymers, howeverwithout speculating on the possible reaction of this intermediatewith cysteine. The same furfuryl cation was proposed by Nikolovand Yaylayan (2012) as intermediate in the co-pyrolysis of FFAwith furfurylamine to give difurfurylamine.

We postulate that the nucleophilic substitution of FFA is accom-panied by a rearrangement, similar to an allylic shift that occurs inthe nucleophilic substitution of allylic compounds (De Wolfe &Young, 1956). When cysteine attacks the methylene group ofFFA, the protonated hydroxyl group is replaced, yielding FFT-S-Cys. On the other hand, the reaction of cysteine at the C-3 atomof the furan ring leads to rearrangement of the double bond, lossof water and formation of the postulated intermediate S-(2-meth-ylene-2,3-dihydro-3-furyl)-L-cysteine (cf. Fig. 5, in square brackets)

and subsequently MFT-S-Cys. Whether this proceeds via SN1 or SN2mechanism was not investigated. It seems likely that other mech-anisms are involved in the formation of MFT-S-Cys, since it isformed much less than FFT-S-Cys in the model reaction of cysteinewith FFA. A third theoretical isomer, S-(2-methyl-5-furyl)-L-cys-teine, was not observed. Possibly the nucleophilic attack at the C-5 atom of the furan ring is disfavoured, the concentration is belowthe analytical detection threshold or the peak hidden by FFT-S-Cys.

Maillard reactions of CS-conjugates are known; after all they areamino acids. For example, Roessner, Velisek, Kubec, and Davidek(2000) have studied the Strecker degradation (which is commonlylinked to Maillard reaction) of S-methyl-, S-allyl-L-cysteine andother CS-conjugates and identified the corresponding aldehydes2-methylthioacetaldehyde and 2-allylthioacetaldehyde. The pres-ent study showed that the Maillard reactions can also be at thesource of CS-conjugates. However, it remains unknown to whichextent formed MFT-S-Cys and FFT-S-Cys are subsequently de-graded further in the Maillard reaction, and what ratio exists be-tween formation and reduction.

As far as we know, MFT-S-Cys has never been reported in theliterature and is a new compound. FFT-S-Cys has been mentionedfor oral treatment of seborrhoea (Kalopissis & Manoussos, 1976),but no details about the compound are mentioned. Neither of themhas been reported in grape must, oak used for wine barrels, inplants or elsewhere in nature.

Fig. 4. 1H NMR spectra: (a) MFT-S-Cys; (b) Fraction B; 13C NMR spectra: (c) MFT-S-Cys; (d) Fraction B;

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Meat contains the relevant precursor compounds cysteine andribose (Macy, Naumann, & Bailey, 1964; Schlichtherle-Cerny &Grosch, 1998; Warendorf & Belitz, 1992). Ribose has similar

reactivity to xylose. Hence, the occurrence of FFT-S-Cys andMFT-S-Cys is likely in cooked meat. Similarly, FFA and protein-bound cysteine are found in coffee (Arnold & Ludwig, 2012;

Fig. 5. Hypothetical formation of FFT-S-Cys and MFT-S-Cys from D-xylose and L-cysteine via FFA.

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Meichelbeck & Zahn, 2012; Moon & Shibamoto, 2009; Vitzthum,Werkhoff, & Ablanque, 1975). Wine contains both FFA and free cys-teine (Baumes, Cordonnier, Nitz, & Drawert, 1986; Valero, Millan,Ortega, & Mauricio, 2003). It seems plausible that FFT-S-Cys andMFT-S-Cys could form from their precursors both in coffee andwine. More in-depth analytical research is needed to verify theoccurrence of both CS-conjugates in foodstuffs.

We were curious to assay the flavour properties of both FFT-S-Cys and MFT-S-Cys. In a preliminary flavour evaluation (panel of 7tasters, data not shown) both compounds were evaluated at1.0 mg/L in water. The solutions smelled absolutely neutral. How-ever when tasted, in the mouth both exhibited a distinct and long-lasting retronasal aroma. FFT-S-Cys produced a coffee-like, sulfuryaroma, strongly reminiscent of FFT, whereas MFT-S-Cys tastedmeaty and sulfury, similar to MFT. This indicates strongly that bothnew CS-conjugates release the corresponding thiols in the mouth,just as reported by Starkenmann et al. (2008) for the CS-conjugatesS-[3-(1-hexanol)]-L-cysteine, S-(1-propyl)-L-cysteine, and S-(2-heptyl)-L-cysteine. Presumably the same release mechanism,mediated by CS-lyases from oral microflora, applies.

This is to our knowledge the first report on the formation of CS-conjugates in the Maillard reaction. Previously, several CS-conju-gates have been identified in foods derived from plant sources likegrape, trifoliate oranges (Citrus trifoliate L.), passion fruit, onion,garlic, asparagus and bell pepper (Kimura et al., 1990; Lawson &Gardener, 2005; Starkenmann, Niclass, & Escher, 2007;Starkenmann, Troccaz, & Howell, 2008; Starkenmann et al., 2011;Tominaga & Dubourdieu, 2000), and as precursors for thiols, enzy-matically released by Saccharomyces cerevisiae (i.e. by b-lyaseactivity), during wine fermentation (Tominaga et al., 1995; Tomi-naga, Peyrot des Gachons et al., 1998). All these CS-conjugates formbiochemically through plant and microbial metabolism. Contrarily,FFT-S-Cys and MFT-S-Cys are Maillard-derived and the first re-ported prototypes of their kind. More in-depth research is neededto elucidate the formation of CS-conjugates in Maillard reactionproducts with different sugars and their potential as in-mouth fla-vour precursors. Additionally, they could exhibit taste or taste-enhancing properties (sweet, bitter, umami, cooling and others)like other amino acid derivatives (Frerot & Benzi, 2004; Grigorov,Schlichtherle-Cerny, Affolter, Kochhar, & Juillerat, 2003) or protein-ogenic amino acids (Solms, 1969).

Acknowledgements

We thank Wolfgang Fieber, Robert Brauchli, Sandy Frank, HorstSommer and Lu Yang for NMR measurements and interpretation ofthe spectra, Christian Starkenmann for critical discussions andKushrav Crawford and Andreas Taglieber for critically reading themanuscript.

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