1 Condensed tannin reacts with SO2 during wine aging, yielding

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Food and Beverage Chemistry/Biochemistry

Condensed tannin reacts with SO2 during wine aging, yielding flavan-3-ol sulfonates Lingjun Ma, Aude Annie Watrelot, Bennett Addison, and Andrew L. Waterhouse J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01996 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

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Journal of Agricultural and Food Chemistry

Condensed tannin reacts with SO2 during wine aging, yielding flavan-3-ol sulfonates Lingjun Maa,b, Aude A. Watrelotb, Bennett Addisonc, Andrew L.Waterhouse*b

a

Agricultural and Environmental Chemistry, University of California, Davis, CA 95616

b

c

Department of Viticulture and Enology, University of California, Davis, CA 95616

Nuclear Magnetic Resonance Facility, University of California, Davis, CA 95616,

current address: Department of Chemistry, San Diego State University, San Diego, CA 92182 *Email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT

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Numerous monomeric and oligomeric flavanol sulfonation products were observed in 10

3

wines. Levels of 0.85-20.06 mg/L and 0-14.72 mg/L were quantified for two monomeric

4

sulfonated flavan-3-ols, and surprisingly, were generally higher than the well-known

5

native flavan-3-ol monomers. Increasing SO2 levels during wine aging increased the

6

sulfonate modified flavan-3-ol monomers and dimers along with higher concentrations of

7

native monomers. The results indicate that >10% of SO2 is reacting with the C4

8

carbocation, formed from acid cleavage of the interflavan bond, perhaps by a bimolecular

9

SN2 type reaction, and as a reducing agent. In addition, the high SO2 wine had the lowest

10

protein-binding tannin levels, tannin activity, and mean degree of polymerization (mDP),

11

and acidic SO2 treatment of condensed tannin abolishes protein binding. Thus, SO2

12

changes tannin composition during wine aging and the substantial formation of sulfonate-

13

modified flavan-3-ols may provide an additional explanation for the reduction in

14

astringency of aged red wines.

15 16

KEYWORDS: flavan-3-ol sulfonate, tannin sulfonation, astringency, wine


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INTRODUCTION Astringency has been recognized as a major sensory property and plays a very

19

important role in the overall quality of red wine. It is a tactile sensation combining drying

20

of the mouth, roughing of oral tissues and puckering of the cheeks and muscles of the

21

face1. A major contributor to astringency are condensed tannins (flavan-3-ol oligomers

22

and polymers; proanthocyanidins), a wine component extracted from grapes skins and

23

seeds during wine making2 . They are oligomers and polymers of flavan-3-ols, and arise

24

from the condensation of flavan-3-ol units, where the flavan-3-ol units are commonly

25

bonded through a C4-C8 interflavan bonds (Figure 1) 3. Red wine contains condensed

26

tannins extracted from grapes, especially skins and seeds. Seed tannins are mainly made

27

up of (+)-catechin, (−)-epicatechin and (−)-epicatechin-3-gallate 4, whereas skin tannin

28

also have (−)-epigallocatechin. On average, the major extension unit in condensed

29

tannins from grape skins is (-)-epicatechin. And (+)-catechin is the next most abundant

30

unit usually found in the terminal units 3. Tannins are able to interact with salivary glyco-

31

proteins through non-polar interactions either by binding directly to them or by linking

32

two or more proteins together5, 6, impairing saliva lubrication in the mouth7.

33

The tannin profile of wine changes during aging, and the perception of

34

astringency changes as well. In young red wine, tannin perception is described as harsh,

35

green, rough, and hard. However, it becomes silky, smooth and well integrated after

36

aging 8, 9. Therefore, tannins are gradually softened and modifications of the sensory

37

properties occur during wine aging10.

38 39

To date, the decline of tannin mean degree of polymerization (mDP), which is the average number of constitutive units, has been described as the principle reason for


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decreasing tannin reactivity toward human salivary proteins and low astringency intensity

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observed during wine aging11. The explanation is based on the fact that larger tannins

42

precipitate with salivary glycoproteins, leading to a degraded lubrication and enhanced

43

friction elicited by the precipitated particles, while monomers/dimers of flavan-3-ols

44

increase the stability of salivary proteins and possibly also increase bitterness 12, 13.

45

Therefore, the age-related reduction of average proanthocyanidin molecular weight may

46

thus be responsible for the reduction of red wine astringency, but bitterness is not

47

reported to rise, a property associated with monomers. Reactions of tannins with

48

anthocyanins and in particular replacement of part of the tannin chain by an anthocyanin

49

(addition of anthocyanin onto the carbocation generated by acid catalyzed cleavage of a

50

proanthocyanidin) yield tannin-anthocyanin adducts during ageing of red wines, another

51

route to astringency reduction 14. Differences in astringency sensation are also believed to

52

be correlated with tannin concentration, tannin: protein ratio 15, and wine matrix

53

components such as alcohol, pH, polysaccharides, etc. 16.

54

Under acidic conditions, the interflavan bond of proanthocyanidins is labile,

55

leading to bond-cleavage, releasing a C-4 carbocation on one subunit. The expected

56

reaction is a rearrangement where the C-4 carbocation that is released, and then reacts

57

with a different flavan-3-ol subunit on the A-ring, restoring an interflavan bond17, 18.

58

Tannin-anthocyanin adducts are also reported to form through this acid catalyzed

59

hydrolysis to generate a carbocation, followed by reaction with a carbinol form of an

60

anthocyanin as the nucleophile19, 20. In addition, the electrophilic intermediate could be

61

trapped by other nucleophiles, such as thiols, and that is the basis of the “thiolysis”

62

method for analyzing the components of proanthocyanidins 21.


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Bisulfite is a well known nucleophile, and it is the dominant form of SO2 at wine

64

pH; SO2 is widely used in winemaking and wine preservation22. Therefore, these C-4

65

carbocations could form sulfonic acid functionalized flavan-3-ols by reaction of bisulfite

66

with the C-4 carbocation. This reaction would effectively reduce the size of the

67

condensed tannins and also possibly alter the sensation of astringency by adding an

68

ionized functional group to the molecule. Foo et al. 23 first investigated the reaction of

69

loblolly pine (Pinus taeda L.) bark tannins with sodium hydrogen sulphite and revealed

70

that the major products are epicatechin-(4β)-sulfonate and sodium epicatechin-(4β → 8)-

71

epicatechin-(4β)- sulfonate. This tannin sulfonation in the presence of SO2 was further

72

studied by others in the leather industry 24-26. In the adhesive industry, sulfonation of

73

condensed tannins are commonly used to enhance their solubility and reduce their

74

viscosity in water 27.

75

However, only a limited number of reports have suggested the presence of flavan-

76

3-ol sulfonates in wine 28, 29. Mattivi et al tentatively identified these products based on

77

high resolution MS and isotopic distributions28. They later reported NMR information for

78

four flavanol-sulfonate monomers and dimers from the thermal reaction of apple tannin

79

with bisulfite under acidic conditions29. Tao et.al suggested that SO2 changes tannin

80

structure and could have an influence on wine astringency 24, but did not directly

81

investigate the question or offer any possible chemical reactions or structures to explain

82

the idea. To date, these obscure products have not been quantified in wine and no effect

83

of tannin sulfonation on astringency has been proposed.

84

Here we quantify flavan-3-ol sulfonates in aged red wines, and investigate their

85

ability to precipitate protein for the first time. We also analyze the relationship between


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86

the levels of SO2 at bottling and formation of sulfonates in red wine as well as the

87

condensed tannin profiles.

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MATERIALS AND METHODS Reagent and Chemicals. (+)-Catechin hydrate, (-)-epicatechin, (-)-gallocatechin,

91

(-)-epigallocatechin, sinapic acid, glacial acetic acid, bovine serum albumin, sodium

92

dodecyl sulfate (SDS), triethanolamine (TEA) and ferric chloride hexahydrate were

93

purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Sodium bisulfite and formic acid

94

97% were purchased from Acros Organics (Morris Plains, NJ). Methanol and L-tartaric

95

acid were purchased from Fisher Bioreagents, Fisher Scientific (Fair Lawn, NJ). Water

96

was purified using a Milli-Q system filtered at 0.20 µm (Millipore, Billerica, MA). All

97

chemicals were of analytical grade or of the highest available purity.

98 99 100

Grape skin extracts and wine samples. Cabernet Sauvignon grape skins were extracted with acetone:water (66:34) under N2 atmosphere and stored under -20 ℃. Cabernet Sauvignon wines (10 red wines) from the UC Davis winery, made from

101

the UC Davis Oakville station vineyard (Oakville, CA) were obtained from years of 1985,

102

1991, 1994, 1997, 2001, 2004, 2007, 2010, 2012 and 2014.

103

Cabernet Sauvignon wines from Oakville, Napa, 1999 were bottled in 2000 with

104

three different levels: 30, 60 and 120 mg/L SO2 and labeled wine A, B and C

105

respectively, to create three aging conditions during aging at 12 ℃ for 17 years. Ethanol

106

was analyzed using an alcolyzer (Anton-Paar, Ashland, VA). The pH was measured

107

using an Orion 5 Star (Thermo Scientific, Boston, MA). Titratable acidity, expressed as

108

tartaric acid, was determined by titration with a sodium hydroxide solution to pH 7.030. A


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109

photometric measurement based on the formation or consumption of coenzyme

110

nicotinamide adenine dinucleotide (NAD) or its reduced form of NADH was used to

111

determine malic acid. For these analyses, a photometric analyzer Thermo Scientific

112

Gallery (manufactured by Thermo Fisher Scientific Oy, Finland) was used. Free and total

113

SO2 were determined using the aspiration method 30. Parameters of the different wine

114

samples were shown in Table 1.

115

Identification of flavan-3-ol sulfonate compounds by LC-QToF. Wine samples

116

were analyzed on an Agilent 1290 UHPLC coupled to an Agilent 6530 QTOF MS.

117

Filtered wine samples (10 µL) were injected onto a reversed phase C18 Phenomenex (100

118

mm× 4.60 mm, 2.6 µm) column with a guard column filled with the same phase. The

119

mobile phases were (a) 0.1% formic acid in water and (B) 0.1% formic acid in

120

acetonitrile. The flow rate was 0.5 mL/min and column temperature was 25 ℃. The

121

gradient was as follows: 0min, 3% B; 2 min, 3% B; 10 min, 6% B; 25 min, 42% B; 30

122

min, 100% B; 32 min, 100% B; 33 min, 3% B; 36 min, 3% B. Samples in the

123

autosampler tray were held at 10 ℃.

124

Samples were run in negative mode using an Agilent Dual ESI Jet Stream source.

125

Nitrogen was used for both the drying gas and sheath gas in the source. The source

126

parameters were as follows: drying gas flow, 10.0 L/min; drying gas temperature, 325 ℃;

127

sheath gas temperature, 350 ℃; sheath gas flow, 11.0 L/min; nebulizer pressure, 35 psi;

128

capillary voltage, 3000 V; fragmentor voltage, 130 V; nozzle, 500 V. Samples were

129

analysed in full MS scan mode m/z 100-2000. Prior to running each sequence, the

130

instrument was calibrated according to the manufacturer's specifications. Reference mass

131

ions were run continuously into the source at a rate of 3 µL/min during the entirety of the


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132

run to insure accurate mass calibration. Reference ions were proton abstracted CF3 (m/z

133

68.9958), pruine (m/z 119.0363) and HP-0921 (+ formate, m/z 966.0007). All analyses

134

were performed in triplicate. Catechin was used as a standard to determine the average

135

relative response of tentatively identified flavan-3-ol sulfonate.

136

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MS/MS spectra were acquired using the Auto MS/MS mode of the instrument.

137

Source conditions were the same as for single MS mode acquisition. Precursor ions were

138

acquired in narrow isolation width (~1.3 amu) to ensure ion selectivity with a minimum

139

threshold of 200 counts with a collision energy of 35 eV. MS data were acquired over a

140

range of m/z 100-1000 at a rate of 3 spectra/s. A maximum of 2 precursors were allowed

141

per cycle. The MS/MS acquisition range was m/z 50-1000 at a rate of 3 spectra s-1.

142

Synthesis, isolation and characterization of major flavan-3-ol sulfonates. A

143

1.0 g/L grape skin extract was made up in 100% MeOH. The solution was treated 1:1

144

with 10 g/L SO2 solution in H2O (from sodium metabisulfite) and pH adjusted to 2.0 with

145

HCl. Vessels were sealed and placed in a 50 ℃ water bath overnight. The mixture was

146

then evaporated under vacuum. Part of the residue was dissolved in water, filtered, and

147

was purified using an Agilent 1100 series Prep-LC and a LiChrospher 100 RP-18 end

148

capped column (250 mm × 10 mm, 10 μm) with 3.0 mL min-1 flow rate of mobile phase

149

A, 0.1% formic acid in water and B, methanol. The injection volume was set to 100 L of

150

each crude reaction mixture. Acquisitions were performed using Chemstation software

151

and, data was collected at 280 nm. The gradient was as follows: 0min, 3.0% B; 2min,

152

3.0% B; 22min, 5.1% B; 24min, 100% B, followed by washing and reconditioning of the

153

column. Collected fractions were concentrated and then evaporated under vacuum

154

overnight to furnish pure epicatechin-(4)-sulfonate and epigallocatechin-(4)-sulfonate


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155

products. Epicatechin-(4)-sulfonate: ESI-QTOF (m/z) [M-H]-, calcd for C15H13O9S,

156

369.0280; found 369.0281. Epigallocatechin-(4)-sulfonate: ESI-QTOF (m/z) [M-H]-,

157

calcd for C15H13O10S, 385.0229; found 385.0234. Purity of epicatechin-(4)-sulfonate

158

and epigallocatechin-(4)-sulfonate were determined by LC-QTof as 95% and 97%

159

respectively, based on total ion data.

160

NMR analysis of major flavan-3-ol sulfonates. All NMR experiments were

161

performed using a Bruker Avance-III 600 MHz NMR spectrometer equipped with a 5

162

mm CPTCI H/C/N/D Z-gradient cryogenic probe, with the temperature regulated at 298

163

K. 1H data were collected using a Bruker zg30 pulse program, 32 scan averages and 4

164

dummy scans, a 20 ppm spectral width, and 64k complex points. 2D 1H-1H COSY data

165

were collected using 2 scans per increment, 2048 and 128 acquired points in F2 and F1, a

166

8.6 ppm spectral width, and a 1 second recycle delay. 2D 1H-13C HSQC data were

167

collected using the HSQCETGPSISP2.2 Bruker parameter set with 4 or 8 scans per

168

increment, 4096 and 256 acquired points in F2 and F1, a 16 ppm spectral width, and a 1

169

second recycle delay. NMR data were processed and analyzed in Mestrenova version 11,

170

and all chemical shifts (1H and 13C) are referenced to the Methanol methyl peak (1H and

171

13C shifts at 3.31 ppm and 49.0 ppm, respectively).

172

Measurement of epicatechin-(4 )-sulfonate, epigallocatechin-(4 )-sulfonate,

173

(+)-catechin, (-)-epicatechin, (+)-gallocatechin, (-)-epigallocatechin and dimer

174

sulfonates in wines. External standards of epicatechin-(4)-sulfonate, epigallocatechin-

175

(4)-sulfonate, (+)-catechin, (-)-epicatechin, (+)-gallocatechin and (-)-epigallocatechin

176

were used for quantitation by LC-MS and the calibration curve ranged from 0.4 mg/L to

177

100 mg/L, respectively. Sinapic acid (50 mg/L) was added to each sample before analysis


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as the internal standard. Method validation was determined by spike recovery, precision

179

of the analysis, and limit of quantification (LOQ) for the standard compounds. The low

180

concentration standard (0.4 mg/L) were injected 7 times to determine the LOQ. All

181

analyses were performed in triplicate. The spike recoveries are between 80% and 120%.

182

LOQ is 0.07 mg/L, which is determined by 10 times standard deviation of low

183

concentration standard (0.4 mg/L). All calibration curves have a correlation coefficient

184

(r2) more than 0.90. Dimer flavan-3-ol sulfonates were determined by peak area at

185

expected accurate mass by LC-QTof. Analyses were performed on an Agilent 1290

186

UHPLC coupled to an Agilent 6530 QTOF MS. LC-QTof methods and parameters were

187

same as the flavan-3-ol sulfonate identification described in 3.

188

Preparation of sulfonated tannins, acid treated tannins and native tannins.

189

Three grape seed proanthocyanidin extracts (1.0 g/L, 10 mL) were made up in 100%

190

methanol, one was treated 1:1 with 10 g/L (10 mL) sodium bisulfite and pH adjusted to

191

2.0 with HCl; one underwent 10 mL water addition and pH adjusted to 2.0 with HCl; the

192

other one was treated with 10 ml water as a control. Each treatment was prepared in

193

triplicate. All samples were placed at 50 ℃ overnight and then evaporated to 7 mL under

194

vacuum.

195

Total Sulfur Analysis. Total sulfur was determined by a nitric acid/hydrogen

196

peroxide microwave digestion and Inductively Coupled Plasma Atomic Emission

197

Spectrometry (ICP-AES)31. The microwave oven was a CEM model MARS 5 and the

198

ICP Thermo iCAP 6500. Sulfur percentage was calculated by the weight of sulfur

199

component divided by the weight of tannin residue.


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Measurement of tannin concentrations. The determination of tannin

201

concentration was performed by protein precipitation (Bovine Serum Albumin-BSA)

202

method 32 to determine the precipitable tannin levels.

203

Tannin activity determination by HPLC-DAD. Tannin activity was determined

204

based upon a recently developed HPLC method 33-35, slightly modified by Watrelot et al.

205

16

206

temperatures, from 25 ℃ to 40 ℃ in 5 ℃ increments instead of eight temperatures as

207

previously conducted by Barak and Kennedy et al 33. In order to reduce the analysis run

208

time, a grape skin fraction known to highly interact with the hydrophobic surface was

209

used as a control. Tannin activity (specific enthalpy ΔH0) was calculated as described.

210

The specific entropy of interaction was always negative and negligible in the calculation

211

of the tannin activity.

212

as follows. For elucidation of tannin activity, samples were run at four different

Tannin characterization by acid-catalyzed degradation with an excess of

213

phloroglucinol. Condensed tannins from red wines (A, B and C) were extracted in

214

triplicate using SPE C18 cartridge (Hypersep, 1g, 6mL, Thermo Fisher scientific, USA)

215

as follows. Cartridges were activated using 3 volumes of 5 mL methanol followed by 3

216

volumes of 5 mL deionized water. Then 1 mL of wine was loaded and the cartridge was

217

washed by 3 volumes of 5 mL water to remove anthocyanins, sugars and organic acids.

218

Tannins were then eluted using 9 mL methanol. The methanol was evaporated using a

219

Centrivap Cold Trap (Labconco, Kansas city, MO, USA), then 1 mL methanol was added

220

in each tannin fractions. Constitutive subunit composition (extension and terminal

221

subunits) and the mean degree of polymerization (mDP) of tannins were determined by

222

HPLC-DAD (Agilent 1100 Series) after acid-catalysis with an excess of phloroglucinol,


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according to the procedure first described by Kennedy and Jone 36 and following applied

224

by Watrelot et.al 16. Briefly, tannins dissolved in methanol were added to the

225

phloroglucinol reagent solution (0.1 N hydrochloric acid in methanol, containing 50 g/L

226

phloroglucinol and 10 g/L ascorbic acid) (1:1, v/v) and maintained at 50 °C for 20 min.

227

The reaction was stopped by addition of 500 µL of 40 mM aqueous sodium acetate to

228

100 µL of samples, prior to injection of 20 µL by HPLC. The HPLC system consisted of

229

two Chromolith RP-18e (100×4.6 mm) columns connected in series and protected by a

230

guard column containing the same material. The binary gradient run consisted of mobile

231

phase A, 1% v/v aqueous acetic acid and B, 1% v/v acetic acid in acetonitrile. The

232

column flow rate was 1.5 mL/min and with a column temperature of 30 °C. The gradient

233

was as follows: 0min, 3% B; 8.0min, 3% B; 28.0min, 18% B; 28.02 min, 80% B; 32.0

234

min, 80% B; 32.02min, 3% B; 40.0 min, 3% B. Detection of eluting peaks was made

235

using a diode array detector (DAD) at 280 nm. For the calculations of the subunit

236

composition, calibration curves of terminal subunits were carried out on (+)-catechin, (-)-

237

epicatechin and (-)-epicatechin-3-O-gallate standards and procyanidin B1 standard after

238

acid-catalysis reaction was used for the calibration curve of (-)-epicatechin as extension

239

unit. The mDP was calculated by the sum of all constitutive subunit (in moles) divided by

240

the sum of all terminal subunits (in moles).

241

Statistical analysis. Statistical calculations were performed with R Studio

242

(Version 0.99.903). Pairwise comparisons were conducted by T-test and global p values

243

were obtained by ANOVA. The level of significance was less than 0.05 (p < 0.05) if not

244

stated otherwise. Statistics were carried out on analytical replicates. Tannin levels, tannin

245

activity, degree of polymerization of tannins, flavan-3-ol sulfonates and flavan-3-ols were


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246

analyzed in experimental triplicate for each wine, and statistics of these parameters were

247

carried out on these experimental triplicates.

248 249

RESULTS AND DISCUSSION

250

Quantification of flavan-3-ol sulfonates in 10 aged red wines. A putative

251

compound list was compiled according to the masses of monomeric and oligomeric

252

flavan-3-ol sulfonates. Molecular features related to these sulfonate compounds were

253

observed in wines by LC-QToF (Table 2), and 11 sulfonate compounds (flavan-3-ol

254

sulfonate monomers and dimers) were tentatively identified in over 80% of the wines.

255

Isomers with the same molecular formula but with different retention times could initially

256

not be distinguished. Molecular formulas were supported by appropriate isotope

257

distribution 28, 37 and expected accurate mass values (mass error < 2 ppm). Fragmentation

258

spectra were reviewed to partially determine the structures as well as confirm the

259

molecular features. For example, the detection of m/z 369.0281 (M-H)- and the isotope

260

distribution suggested the sulfonate addition to catechin and/or epicatechin of compound

261

1 and 2 with a formula of C15H14O9S; MS/MS fragmentation with A1 (m/z 216.9816), B1

262

(m/z 241.9890) and C1 (m/z 287.0567) indicated their partial structures as shown in

263

Figure S1 (a) (R1=H), revealing that sulfonation was at the C4 position.

264

The MS signal response of candidate compounds versus catechin in 10 different

265

wines was measured. As the signals for compounds 1 and 3 were much stronger than the

266

others, a route to prepare compounds 1 and 3 was pursued for complete structure

267

determination and quantification.


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Compounds 1 and 3 were isolated by prep-LC, using a C-18 adsorbent and

269

acidified (1% formic acid) aqueous mobile phase with a methanol gradient. Sulfonate

270

products were not detected in a control sample. The structures of compounds 1 and 3

271

were determined by 1H and 2D 1H-1H COSY and 1H-13C HSQC NMR data (Table 3 and

272

Figures S2-8). Functionalization at the C-4 position was confirmed since the

273

unfunctionalized diastereotopic CH2 protons H4' and H4'' near 2.7 ppm disappeared after

274

sulfonation, giving rise to a new CH proton downfield near 5.4 ppm; significant

275

deshielding occured for both C4 and H4 due to the electron-withdrawing sulfonyl group.

276

Both C-2 and H-2 were shifted upfield significantly after SO2 functionalization at the C-4

277

position, due to the gamma-gauche effect, indicating that the SO3H group was diaxial

278

with H-2. Additionally, the peak shape and J-coupling constants between vicinal protons

279

H2, H3 and H4 indicated a cis-trans relationship of the C-2, C-3, C-4 substitutions. The

280

small J-coupling values between the vicinal protons can be explained by two primary

281

causes: first, electronegative substituents, which tended to decrease 3JHH coupling values,

282

were present on each of the three sites C-2, C-3 and C-438; and second, a cis-trans

283

relationship between neighboring protons would result in a dihedral angle near 90

284

degrees, thus small J-values would be expected according to the Karplus relationship.

285

Based on these observations, compounds 1 and 3 are epicatechin-(4)-sulfonate and

286

epigallocatechin-(4)-sulfonate respectively as drawn in Figure 2. These results are in

287

agreement with the structure elucidation of Foo et.al.23 that sodium epicatechin-(4β)-

288

sulfonate formed from the reaction of condensed tannin with bisulfite at pH 5.5 and

289

100 °C, and Mattivi et.al. that epicatechin-(4β)-sulfonate and procyanidin B2-4β-

290

sulfonate were generated from the reaction of apple tannins with bisulfite in model wine29.


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The levels of epicatechin-(4)-sulfonate (1) and epigallocatechin-(4)-sulfonate

292

(3) respectively ranged from 0.85-20.06 mg/L and 0-14.72 mg/L in aged wines, and

293

surprisingly, were much higher than the well-known native flavan-3-ol monomers in

294

most of the samples (Figure 3). These results demonstrate that sulfonation of tannins

295

readily occurs under wine aging conditions, releasing flavan-3-ol sulfonates in significant

296

quantities. If the amounts of sulfonated dimers, trimers and larger oligomers are also

297

greater than the native proanthocyanidins, such a large fraction of modified condensed

298

tannin would affect taste and astringency. We admit that it is most surprising that such

299

major components have not been previously quantified, but their high polarity might

300

make them difficult to distinguish from salts on typical chromatographic separations. In

301

an acid-SO2 treated grape-tannin sample prepared below for the protein binding assay,

302

sulfur content (by ICP-AES) increased by 0.4%, supporting our hypothesis that the

303

condensed tannin was modified by sulfite addition.

304

Effects on protein binding. Since protein precipitation has been correlated with

305

the perception of astringency39, the effect of tannin sulfonation on the protein

306

precipitation was studied. Grape seed proanthocyanidin extracts (GSE) underwent three

307

treatments at 50 °C for 10 hours: H2SO3 at pH 2, with water at pH 2, and neutral water as

308

controls. The precipitable tannin concentration of each sample was determined by

309

interaction with protein (Bovine Serum Albumin) 32 and significant differences between

310

samples were observed. The precipitable tannin of GSE treated with just acid at pH=2

311

(47.1±6.4 mg/L) was lower than the water control (77.8±5.0 mg/L), indicating that some

312

of the high molecular tannin was broken down into smaller oligomers that have less

313

binding to proteins 11. However, it was surprising to find that the precipitable tannin


15


Page 16 of 39

314

concentration of the GSE sample treated with H2SO3 was negligible (0.6±1.5 mg/L)

315

compared to the controls. This suggests that the sulfonate derivatives, which have ionic

316

properties, have greatly reduced affinity for protein, and thus are likely to have reduced

317

astringency5. These results are analogous to tannin-anthocyanin adducts, which are likely

318

to involve acid-catalyzed processes, and are generally reported to reduce wine

319

astringency20, 40.

320

The effects of SO2 levels on flavan-3-ol sulfonate formation during aging.

321

Wine A, B and C were created from one new Cabernet Sauvignon wine, but bottled with

322

30 mg/L, 60 mg/L and 120 mg/L SO2, respectively. After aging for 18 years, free SO2

323

was only detected in wine C at 12.50±0.71 mg/L, while total SO2 was measured at

324

50.66±2.38, 65.70±6.96 and 112.2±1.10 mg/L for Wine A, B and C, respectively.

325

The levels of flavan-3-ol sulfonates and flavan-3-ols were significantly different

326

between wine samples, except when concentrations were very low and difficult to

327

quantify. A dose-response relationship was observed, in that the levels of the flavan-3-ol

328

sulfonates increased with higher levels of SO2 added at bottling (Figure 4A), suggesting

329

that the sulfonation of tannins occurs through a bond cleavage, generating the flavan-3-ol

330

sulfonates. However, the levels of the flavan-3-ol monomers also increased as the levels

331

of the added SO2 goes up (Figure 4. B), suggesting in addition that the flavan-3-ol C-4

332

carbocations might also react with SO2 through an electron transfer reaction generating

333

the corresponding flavan-3-ols and sulfuric acid. Such a reaction would not be totally

334

unexpected in light of the similar two pathways reported for sulfites reacting with

335

quinones, one by addition to make sulfonates, and one by reduction of the quinone to

336

catechol 41.


16

Page 17 of 39

337


It was surprising to note that the flavan-3-ol sulfonate concentrations were higher

338

than the flavan-3-ol monomer contents in the three wines. Epicatechin-(4)-sulfonate

339

was detected at ~2.4 times, ~3 times and ~4.1 times the (+)-catechin concentration in

340

wines bottled with 30 ppm, 60 ppm and 120 ppm SO2 addition, respectively (Figure 4).

341

This was consistent with the above finding that the concentration of flavan-3-ol

342

sulfonates were generally higher than well-known flavan-3-ol monomers in most of the

343

wines (Figure 3), suggesting the importance of the tannin sulfonation in wines.

344

Since the standards of flavan-3-ol sulfonate dimer products were not available, the

345

peak area responses of the dimer flavan-3-ol sulfonates are compared among the wines to

346

get an idea of the effect of SO2 on the dimer sulfonate formation. Table 4 showed that

347

these dimer flavan-3-ol sulfonates were significantly different among wine samples with

348

different SO2 additions. Compounds “1-3” were Catechin2-SO3H (C30H26O15S, m/z=

349

657.0913 [M-H]-) at the retention time 12.22, 14.70 and 16.09 min, respectively;

350

Compound “4” was Gallocatechin2-SO3H (C30H26O17S, m/z= 689.0813 [M-H]-, RT=7.69

351

min); Compounds “5-8” were Gallocatechin-catechin-SO3H (C30H26O16S, m/z=

352

673.0864[M-H]-) at the retention time 8.09, 11.39, 14.52 and 15.10 min, respectively.

353

These molecular formulas were matched for the expected accurate mass with a mass error

354

less than 2 ppm. Compounds which have the same molecular formula are not

355

distinguishable by the stereochemistry (e.g. catechin vs epicatechin), and so only the term

356

“catechin” and “gallocatechin” are used. In addition, the order of subunits (e.g. catechin-

357

gallocatechin vs gallocatechin-catechin) cannot be distinguished. It was revealed that the

358

peak area responses of the dimer sulfonates were higher in the wines with higher SO2

359

levels. This is consistent with the monomer sulfonate results, revealing that the higher the


17


Page 18 of 39

360

SO2 levels, the higher the flavan-3-ol sulfonate compounds, confirming the role of added

361

SO2 in the formation of these compounds during aging.

362

By comparing the amount of free SO2 available at bottling, and the measured

363

amounts of the monomer sulfonates, it is apparent that of that free SO2, a sizable fraction

364

went into forming the two major monomers, epicatechin-(4β)-sulfonate and

365

epigallocatechin-(4)-sulfonate. These are the only products with standards available,

366

and in Wines A, B and C, the molar ratio fraction of sulfur dioxide appearing in the

367

monomers was 15%, 11% and 12% respectively. The total amount is actually higher

368

since other sulfonate products, i.e. dimers trimers and higher are also formed. This

369

demonstrates that an important pathway for SO2 consumption during red wine aging is

370

loss to sulfonate production, of particular interest because it is a reaction pathway that

371

would consume SO2 in the absence of oxygen.

372

The effects of SO2 levels on tannin profile changes during aging. Tannin

373

profiles, including the tannin concentration, tannin activity and polymerization of tannins

374

were analyzed in wine A, B and C. The properties were significantly different among

375

wine samples (Figure 5). The higher the SO2 addition, the lower the precipitable tannin

376

levels (Figure 5. A). One possibility is that SO2 reacts with tannins and breaks them down

377

into smaller molecules thus reducing the protein binding capacity. In fact protein

378

precipitation decreased, in response to bottling with increased SO2 levels.

379

To provide further insight of the SO2 effect on the strength of tannin-protein

380

interactions, an analysis of tannin activity was undertaken, where the thermodynamic

381

parameters of interaction between tannins and the hydrophobic surface using HPLC was

382

measured, allowing the determination of the enthalpy of the tannin interactions by


18

Page 19 of 39


383

adsorptive binding. The result of tannin activity (Figure 5. B) followed the same trend as

384

tannin concentrations—the higher the SO2 levels at bottling, the lower the tannin activity.

385

This shows that increased SO2 levels would not only decrease precipitable tannins, but

386

also decrease the enthalpy of interaction with protein on a molar basis.

387

The chain length of tannins has been well documented as a major contributor to

388

wine astringency 11. It would be interesting to know whether SO2 reaction might also

389

reduce the molecular weight of condensed tannins. Measurements revealed that the

390

apparent mDP of tannins was small (~3) in the three measured wines (Figure 5. C), which

391

was in agreement with Mc Rae et al42 who observed that the mDP decreased during aging

392

due to tannin structural changes. Although the measured mDP was small, and in wine the

393

irregular modifications that occur during aging invalidate the absolute value of the

394

measurement, the observed negative correlation with the level of SO2 addition shows an

395

effect of SO2. In particular the observed differences could support the hypothesis that the

396

reaction of SO2 with condensed tannin would reduce the chain length of tannins. It is

397

possible that the electron transfer reaction mentioned above, where sulfites reduce the C4

398

carbocation, could be playing a role here.

399

In summary, the combination of these three results strongly suggest that the

400

changes induced by SO2 during aging would decrease the binding strength to proteins. As

401

astringency is caused by the tannin-induced aggregation and the precipitation of salivary

402

proteins 8, the results in this study would suggest that the changes to tannin by SO2 during

403

aging could contribute to astringency reduction.

404


19


405

Page 20 of 39

Proposed mechanism. Epicatechin and epigallocatechin are not the dominant

406

flavan-3-ol monomers in wine but are the major extension units of proanthocyanidin

407

oligomers that constitute grape tannin (Figure 1) 2, 43. The formation of the these specific

408

flavan-3-ol sulfonates, 1 and 3, as the major observed forms suggest their origin as

409

arising from cleavage of the interflavan bond in the wine’s condensed tannin, and the

410

addition of sulfite to the electrophilic C4 site. The substantial formation of flavan-3-ol

411

sulfonates in aged red wines and their higher levels in wines bottled with higher SO2

412

levels can now be interpreted satisfactorily in terms of the proposed mechanism (Figure

413

6). Under acidic conditions, protonation occurs at the A-ring on an interflavan linkage.

414

The interflavan linkage is liable at 20 ℃ over a pH of 3.6 to 5.4, with an estimated half-

415

life of 200-400 hours44. This protonation is thought to be primarily dependent on pH and

416

temperature 45, and cleavage of the C4-C8 linkage has been described as the rate-

417

determining step, because the effect of any sterochemical factor of the interflavan bond is

418

small 17. However, this prior investigation was conducted without a nucleophile, and the

419

limited data presented here, comparing Wines A-C, suggests that the SO2 concentration

420

affects the reaction rate. That implicates a SN2 type mechanism where the bisulfite ion is

421

involved in the rate limiting step. This involvement would also help explain why the

422

major products observed have one isomer at C4. Kinetic studies of these reactions are

423

needed to fully explain the dependence of the reaction on the concentration of hydrogen

424

bisulfite as well as precursor stereochemistry to clarify the details of the reaction

425

mechanism.

426 427

This mechanism is parallel to the well-known pathway for the formation of C4 modified flavan-3-ol units observed when proanthocyanidins are treated with acid,


20

Page 21 of 39


428

breaking the interflavan bond and a thiol or phloroglucinol36 nucleophile react with the

429

C4 carbocations formed by the acid 18, as well as reactions of proanthocyanidins with

430

anthocyanins under acid conditions 19, 20.

431

Instead of adding as a nucleophile, SO2, may also reduce the C4 carbocation to a

432

methylene (Figure 6, reduction). If this occurred, this would lead to a smaller degree of

433

polymerization, but this process is a new proposal and merits further investigation.

434

Figure 7 shows examples of the sulfonation pathway of a proanthocyanidin

435

trimer, where a sulfonate monomer or dimer are generated from the acid catalyzed

436

cleavage at bond A or bond B, accompanied by formation of dimer or monomer

437

sulfonates and dimer or monomer proanthocyanindins. This is an example of how

438

condensed tannin substrates could react with SO2 under acid conditions to give rise to a

439

number of different sulfonated products. The cumulative amount of these products

440

would be important to measure as well as the sensory effect of these products.

441

In conclusion, this work shows that there are higher levels of the sulfonated

442

flavan-3-ol monomers than the well-known native compounds such as epicatechin in

443

aged wines. The data shows that the source of these sulfonated flavanols arise from the

444

cleavage of the proanthocyanidins via cleavage of the interflavan bond and nucleophilic

445

attack by SO2 on the C4 carbocation. Since the amount of flavan-3-ol sulfonates in aged

446

wines was increased with higher levels of SO2 at bottling, the mechanism for the reaction

447

between the flavan-3-ols and SO2 appears to involve a bimolecular (SN2 type) mechanism,

448

which could also explain the specific stereoselectivity of the major products. In the small

449

set of wines observed, it appears that a sizable fraction >15% of the SO2 lost during aging

450

was consumed by these reactions, altering the perception that SO2 is solely consumed by


21


Page 22 of 39

451

oxygen derived reactions. In addition, since the high-SO2 aged wine had the lowest

452

tannin levels, tannin activity and mDP, (and as noted, SO2 treatment of grape tannin

453

abolishes protein binding) this indicates that SO2 alters the tannin profile during aging

454

and this process may influence the perception of astringency in aged wines. In summary,

455

these observations suggest that tannin sulfonation could contribute to the decline of

456

astringency observed in aged red wines. Future work could focus on the sulfonation

457

mechanism and sensory analysis of the sulfonated tannins to clarify the impact of these

458

aging reactions.


22

Page 23 of 39


459

Acknowledgements

460

The authors thank the UC Davis winery for providing the red wines. We would like to

461

thank Dr. Patricia Howe for helpful suggestions and Mauri Anderson for helping prepare

462

the sulfonated grape seed extract.

463

Supporting Information

464

LC-QTof Complementary Result and NMR Assignments

465

Conflict of interest

466

The authors declare no conflict of interest.


23


467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511

Page 24 of 39

References 1. Lee, C. B.; Lawless, H. T., Time-Course of Astringent Sensations. Chem Senses 1991, 16, 225-238. 2. Waterhouse, A. L., Wine Phenolics. Annals of the New York Academy of Sciences 2002, 957, 21-36. 3. Souquet, J. M.; Cheynier, V.; Brossaud, F.; Moutounet, M., Polymeric proanthocyanidins from grape skins. Phytochemistry 1996, 43, 509-512. 4. Ricardo-Da-Silva, J. M.; Rigaud, J.; Cheynier, V.; Cheminat, A.; Moutounet, M., Procyanidin Dimers and Trimers from Grape Seeds. Phytochemistry 1991, 30, 12591264. 5. de Freitas, V.; Mateus, N., Protein/Polyphenol Interactions: Past and Present Contributions. Mechanisms of Astringency Perception. Curr Org Chem 2012, 16, 724-746. 6. Waterhouse, A. L.; Sacks, G. L.; Jeffery, D. W., Understanding Wine Chemistry. John Wiley & Sons, Ltd: Chichester, West Sussex, United Kingdom, 2016; p 443. 7. Mcmanus, J. P.; Davis, K. G.; Lilley, T. H.; Haslam, E., The Association of Proteins with Polyphenols. J Chem Soc Chem Comm 1981, 309-311. 8. Soares, S.; Sousa, A.; Mateus, N.; de Freitas, V., Effect of Condensed Tannins Addition on the Astringency of Red Wines. Chem Senses 2012, 37, 191-198. 9. Vidal, L.; Antunez, L.; Gimenez, A.; Medina, K.; Boido, E.; Ares, G., Dynamic characterization of red wine astringency: Case study with Uruguayan Tannat wines. Food Research International 2016, 82, 128-135. 10. Gawel, R.; Iland, P. G.; Francis, I. L., Characterizing the astringency of red wine: a case study. Food Qual Prefer 2001, 12, 83-94. 11. Sun, B. S.; de Sa, M.; Leandro, C.; Caldeira, I.; Duarte, F. L.; Spranger, I., Reactivity of Polymeric Proanthocyanidins toward Salivary Proteins and Their Contribution to Young Red Wine Astringency. Journal of Agricultural and Food Chemistry 2013, 61, 939-946. 12. Zanchi, D.; Poulain, C.; Konarev, P.; Tribet, C.; Svergun, D. I., Colloidal stability of tannins: astringency, wine tasting and beyond. J Phys-Condens Mat 2008, 20. 13. Brossaud, F.; Cheynier, V.; Noble, A. C., Bitterness and astringency of grape and wine polyphenols. Austr. J. Grape Wine Res. 2001, 7, 33-39. 14. Weber, F.; Greve, K.; Durner, D.; Fischer, U.; Winterhalter, P., Sensory and Chemical Characterization of Phenolic Polymers from Red Wine Obtained by Gel Permeation Chromatography. Am J Enol Viticult 2013, 64, 15-25. 15. Poncet-Legrand, C.; Cabane, B.; Bautista-Ortin, A. B.; Carrillo, S.; Fulcrand, H.; Perez, J.; Vernhet, A., Tannin Oxidation: Intra- versus Intermolecular Reactions. Biomacromolecules 2010, 11, 2376-2386. 16. Watrelot, A. A.; Schulz, D. L.; Kennedy, J. A., Wine polysaccharides influence tannin-protein interactions. Food Hydrocolloid 2017, 63, 571-579. 17. Beart, J. E.; Lilley, T. H.; Haslam, E., Polyphenol Interactions .2. Covalent Binding of Procyanidins to Proteins during Acid-Catalyzed Decomposition Observations on Some Polymeric Proanthocyanidins. J Chem Soc Perk T 2 1985, 1439-1443.


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18. Vidal, S.; Cartalade, D.; Souquet, J. M.; Fulcrand, H.; Cheynier, V., Changes in proanthocyanidin chain length in winelike model solutions. Journal of Agricultural and Food Chemistry 2002, 50, 2261-2266. 19. Cheynier, V., Polyphenols in foods are more complex than often thought. Am J Clin Nutr 2005, 81, 223s-229s. 20. Haslam, E., INVINO VERITAS - OLIGOMERIC PROCYANIDINS AND THE AGING OF RED WINES. Phytochemistry 1980, 19, 2577-2582. 21. Thompson, R. S.; Jacques, D.; Haslam, E., Plant Proanthocyanidins .1. Introduction - Isolation, Structure, and Distribution in Nature of Plant Procyanidins. J Chem Soc Perk T 1 1972, 1387-&. 22. Nikolantonaki, M.; Waterhouse, A. L., A Method To Quantify Quinone Reaction Rates with Wine Relevant Nucleophiles: A Key to the Understanding of Oxidative Loss of Varietal Thiols. J.Agric. Food Chem. 2012, 60, 8484-8491. 23. Foo, L. Y.; Mcgraw, G. W.; Hemingway, R. W., Condensed Tannins Preferential Substitution at the Interflavanoid Bond by Sulfite Ion. J Chem Soc Chem Comm 1983, 672-673. 24. Tao, J.; Dykes, S. I.; Kilmartin, P. A., Effect of SO2 Concentration on Polyphenol Development during Red Wine Micro-oxygenation. Journal of Agricultural and Food Chemistry 2007, 55, 6104-6109. 25. Bae, Y. S.; Malan, J. C. S.; Karchesy, J. J., Sulfonation of Procyanidin Polymers Evidence of Intramolecular Rearrangement and Aromatic Ring Substitution. Holzforschung 1994, 48, 119-123. 26. Karchesy, J. J.; Foo, L. Y.; Hemingway, R. W.; Barofsky, E.; Barofsky, D. F., Fast Atom Bombardment Mass-Spectrometry of Condensed Tannin Sulfonate Derivatives. Wood Fiber Sci 1989, 21, 155-162. 27. Jorge, F. C. P., Lina; Portugal, António; Gil, Maria Helena; Irle, Mark A.; Costa, Rui Pereira da Improved extraction of pine bark for wood adhesives. 3rd European Panel Products Symposium 1999, 301-307. 28. Arapitsas, P.; Speri, G.; Angeli, A.; Perenzoni, D.; Mattivi, F., The influence of storage on the "chemical age" of red wines. Metabolomics 2014, 10, 816-832. 29. Mattivi, F.; Arapitsas, P.; Perenzoni, D.; Guella, G., Influence of Storage Conditions on the Composition of Red Wines. Acs Sym Ser 2015, 1203, 29-49. 30. Patrick Iland, N. B., Greg Edwards, Sue Caloghiris, Eric Wilkes, Chemical analysis of grapes and wines: Techniques and concepts. Patrick Iland, Campbelltown, Australia. 2004. 31. Soon, Y. K.; Kalra, Y. P.; Abboud, S. A., Comparison of some methods for the determination of total sulfur in plant tissues. Commun Soil Sci Plan 1996, 27, 809818. 32. Harbertson, J. F.; Picciotto, E. A.; Adams, D. O., Measurement of polymeric pigments in grape berry extracts and wines using a protein precipitation assay combined with bisulfite bleaching. Am. J. Enol. Vitic. 2003, 54, 301-306. 33. Barak, J. A.; Kennedy, J. A., HPLC Retention Thermodynamics of Grape and Wine Tannins. Journal of Agricultural and Food Chemistry 2013, 61, 4270-4277. 34. Revelette, M. R.; Barak, J. A.; Kennedy, J. A., High-Performance Liquid Chromatography Determination of Red Wine Tannin Stickiness. J. Agric. Food Chem. 2014, 62, 6626-6631.


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558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589

35. Yacco, R. S.; Watrelot, A. A.; Kennedy, J. A., Red Wine Tannin StructureActivity Relationships during Fermentation and Maceration. Journal of Agricultural and Food Chemistry 2016, 64, 860-869. 36. Kennedy, J. A.; Jones, G. P., Analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. J. Agric. Food Chem. 2001, 49, 1740-1746. 37. Bae, Y., Douglas-fir inner bark procyanidins : sulfonation, isolation and characterization. Oregon State University Ph.D. thesis 1989. 38. Bothner-By, A., Advances in Magnetic resonance. New York-London 1965, 1, 195. 39. Kennedy, J. A.; Ferrier, J.; Harbertson, J. F.; Gachons, C. P. D., Analysis of tannins in red wine using multiple methods: Correlation with perceived astringency. Am. J. Enol. Vitic. 2006, 57, 481-485. 40. Singleton, V. L.; Trousdale, E. K., Anthocyanin-Tannin Interactions Explaining Differences in Polymeric Phenols between White and Red Wines. American Journal of Enology and Viticulture 1992, 43, 63-70. 41. Danilewicz, J. C.; Seccombe, J. T.; Whelan, J., Mechanism of interaction of polyphenols, oxygen, and sulfur dioxide in model wine and wine. Am. J. Enol. Vitic. 2008, 59, 128-136. 42. McRae, J. M.; Dambergs, R. G.; Kassara, S.; Parker, M.; Jeffery, D. W.; Herderich, M. J.; Smith, P. A., Phenolic Compositions of 50 and 30 Year Sequences of Australian Red Wines: The Impact of Wine Age. Journal of Agricultural and Food Chemistry 2012, 60, 10093-10102. 43. Quijada-Morin, N.; Williams, P.; Rivas-Gonzalo, J. C.; Doco, T.; EscribanoBailon, M. T., Polyphenolic, polysaccharide and oligosaccharide composition of Tempranillo red wines and their relationship with the perceived astringency. Food Chem. 2014, 154, 44-51. 44. Hemingway, R. W.; McGraw, G. W., Kinetics of acid-catalyzed cleavage of procyanidins. Journal of Wood Chemistry and Technology 1983, 3, 421-435. 45. Mattivi, F.; Arapitsas, P.; Perenzoni, D., Influence of storage conditions on the composition of red wines. Abstr Pap Am Chem S 2014, 248.

590 591 592

Funding This research is supported by the American Vineyard Foundation (No. 2016-1820)

593


26

Page 27 of 39


594

FIGURE CAPTIONS

595

Figure 1. Condensed Tannin Example. Tetrameric Proanthocyanidin composed, from the

596

top, of epicatechin, epigallocatechin and epicatechin extension subunits and a catechin

597

terminal unit.

598

Figure 2. The structure of Compounds 1 and 3.

599

Figure 3. Flavan-3-ol-(4)-sulfonate and flavan-3-ol levels in wines (n=10, Cabernet

600

Sauvignon, Oakville, Napa, California, USA, 1985-2014).

601

Figure 4. Quantitation of A) major flavan-3-ol sulfonates (epicatechin-(4)-sulfonate

602

and epigallocatechin-(4)-sulfonate) and B) major flavan-3-ols ((-)-epicatechin, (-)-

603

epigallocatechin, (+)-catechin and (+)-gallocatechin) in three wines (Wine A, Wine B and

604

Wine C were spiked with 30, 60 and 120 mg/L SO2 levels after bottling)

605

Figure 5. Measurement of A) tannin levels precipitated by BSA, B) tannin activity and C)

606

mean degree of polymerization of tannins in three wines (Wine A, Wine B and Wine C

607

were spiked with 30, 60 and 120 mg/L SO2 levels after bottling, respectively).

608

Figure 6. Hypothetical mechanism of flavan-3-ol sulfonates formation from tannin

609

sulfonylation. Acid catalyzed interflavan bond cleavage allows for nucleophilic attack at

610

the C4 carbocation intermediate by bisulfite18, 36.

611

Figure 7. Pathway for the formation of sulfonate monomer or dimer from the acid

612

catalyzed cleavage of a proanthocyanidin trimer, followed by nucleophilic trapping by

613

SO2.


27


Page 28 of 39

Table 1. Wine Parameters Measured in 2017. Wine

Free SO2 at

Alcohol (%)

pH

Titratable Acidity (g/L)

Residual Sugar (g/L)

Malic acid (mg/L)

Free SO2 (mg/L)

Total SO2 (mg/L)

bottling, 2000 (mg/L) A

30

13.85±0.08

3.69±0.00

5.52±0.02

0.66±0.01

1658±0.00

-

50.66±2.38

B

60

13.75±0.01

3.66±0.01

5.59±0.01

0.64±0.00

1669±0.00

-

65.70±6.96

C

120

13.72±0.01

3.64±0.01

5.72±0.01

0.65±0.00

1661±0.70

12.50±0.71

112.2±1.10


28

Page 29 of 39


Table 2. Monomer and Dimer Flavan-3-ol Sulfonates Detected in Aged Wines (n=10, Cabernet Sauvignon, Oakville, Napa, California, USA, 1985-2014). No

Compoundsa

Formula

Relative Responseb

RT (min)

Isotope Intensity of Parent Ion[H-]

Incidencec

369.0281(100%), 370.0322(18.64%), 371.0297((7.88%), 372.0313(1.14%), 373.0316(0.19%) 369.0281(95.23%), 370.0316(15.16%), 371.0299(6.51%), 372.0339(1.09%) 385.0231(100%), 386.0276(17.11%), 387.0243(7.18%), 388.0254(1.16%), 389.0256(0.24%)

100%

657.0915(100%), 658.0962(34.11%), 659.0948(12.57%), 660.0957(2.89%), 661.0970(0.56%) 657.0913(100%), 658.0918(33.41%), 659.0910(12.68%), 660.0938(3.32%) 657.0914(100%), 658.095(33.39%), 659.094(12.13%), 660.0956(3.02%), 661.1006(0.83%) 689.0813(100%), 690.085(33.95%), 691.0836(12.95%), 692.0854(2.84%), 693.0866(0.71%) 673.0863(100%), 674.0905(32%), 675.0897(10.88%), 676.0916(2.36%), 677.0933(0.55%) 673.0863(100%), 674.0896(33.06%), 675. 088(12.38%), 676.089(2.95%), 677.0919(0.66%) 673.0863(100%), 674.0903(31.94%), 675.0894(12.84%), 676.0906(3.3%) 673.086(100%), 674.0889(33.02%), 675.0907(13.7%), 676.0945(4.12%)

100%

Flavan-3-ol monomer sulfonates 1

Catechin- SO3H 1

C15H14O9S

2.84

8.77

2

Catechin- SO3H 2

C15H14O9S

0.23

14.17

3

Gallocatechin- SO3H

C15H14O10S

1.36

6.25

100% 100%

Flavan-3-ol dimer sulfonates 4

2Catechin-SO3H 1

C30H26O15S

0.54

12.22

5

2Catechin-SO3H 2

C30H26O15S

0.04

14.70

6

2Catechin-SO3H 3

C30H26O15S

0.06

16.09

7

2Gallocatechin-SO3H

C30H26O17S

0.32

7.69

8

Gallocatechin-catechin- SO3H 1

C30H26O16S

0.29

8.09

9


C30H26O16S

0.32

11.39

10


C30H26O16S

0.05

14.52

11


C30H26O16S

0.04

15.10

80% 100% 100% 100% 100% 90% 80%

a. Compounds which have the same molecular formula were initially not distinguished by the stereochemistry, sulfonate bonding position or the order of bonding unit. b. Average relative MS response of potential compound versus catechin in 10 different wines. c. Percentage of wines (out of 10 tested) in which this signal was detected.


29


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Table 3. Selected 1H and 13C NMR Data for Epi(gallo)catechin and Compound 1 and 3. 1 13 Atom H (ppm) C (ppm) J(Hz) Compound 2 4.83 79.55 app s 3 4.2 67.16 m dd 16.7, Epicatechin 4' 2.73 28.94 2.9 dd 16.7, 4'' 2.86 28.94 4.5 d 1.4 2 4.15 61.38 b Compound 1 3 4.53 67.32 t 1.4 a 4 5.44 76.24 d 1.0 2 4.76 79.56 d 1.5 td 4.7, 3.2, 3 4.17 67.18 1.5 Epigallocatechin dd 16.7, 4' 2.73 28.82 3.2 dd 16.7, 4'' 2.84 28.82 4.7 b d 1.4 2 4.14 61.13 Compound 3 3 4.52 67.28 t 1.4 a 4 5.38 76.13 app s a. Indicates downfield shift at the C-4 position due to sulfonyl group functionalization. b. Indicates upfield shift at C-2 due to the gamma-gauche effect after C-4 functionalization.


30

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Table 4. Peak Area Response (×106) of Dimers of Flavan-3-ol Sulfonates in Three Wines (Wine A, Wine B and Wine C Were Spiked with 30, 60 and 120 mg/L SO2 Levels After Bottling; Compounds “1-3” were Catechin2-SO3H at the Retention Time 12.22, 14.70 and 16.09 min, respectively; Compounds “4” was Gallocatechin2-SO3H; Compounds “5-8” were Gallocatechin-catechin-SO3H at the Retention Time 8.09, 11.39, 14.52 and 15.10 min, respectively). 1

2

3

4

5

6

7

8

0.96±0.05

2.94±0.15

3.11±0.23

3.43±0.16

0.58±0.03

0.45±0.03

Wine A

8.21±0.67

Wine B

15.31±2.03

0.49 ±0.06

1.67±0.18

5.50±0.65

5.89±0.56

6.66±0.80

0.93±0.06

0.72±0.06

Wine C

39.51±2.99

1.17±0.09

4.10±0.08

13.94±0.24

14.23±1.01

16.84±0.47

2.20±0.07

1.66±0.03

P value

1.6e-08

2.0e-08

8.2e-11

8.1e-11

6.4e-09

1.7e-10

5.2e-11

5.3e-11

0.21 ± 0.01


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Figure 1


32

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epicatechin-(4)-sulfonate (1)

epigallocatechin-(4)-sulfonate (3)

Figure 2


33


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Figure 3


34

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A)

B)

Figure 4


35


A)

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C)

B)

Figure 5


36

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Figure 6


37


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Figure 7


38

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Graphic for table of contents


39

1 Condensed tannin reacts with SO2 during wine aging, yielding

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