Nonenzymatic Transglycosylation Reactions Induced by Roasting

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Non-enzymatic Transglycosylation Reactions Induced by Roasting: New Insights from Models Mimicking Coffee Bean Regions with Distinct Polysaccharide Composition Ana SP Moreira, Joana Simões, Fernando Milheiro Nunes, Dmitry Victorovitch Evtuguin, Pedro Domingues, Manuel A. Coimbra, and Maria Rosario M. Domingues J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00342 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

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Journal of Agricultural and Food Chemistry

Non-enzymatic Transglycosylation Reactions Induced by Roasting: New Insights from Models Mimicking Coffee Bean Regions with Distinct Polysaccharide Composition

Ana S. P. Moreira,† Joana Simões,† Fernando M. Nunes,‡ Dmitry V. Evtuguin,§ Pedro Domingues,† Manuel A. Coimbra,† M. Rosário M. Domingues†*

†

QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

‡

CQ-VR, Chemistry Research Centre, Department of Chemistry, University of Trás-os-

Montes e Alto Douro, 5001- 801 Vila Real, Portugal §

CICECO, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

*Corresponding author (Tel: +351 234 370 698; Fax: +351 234 370 084; E-mail: [email protected])

1 ACS Paragon Plus Environment


1

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Abstract

2

Three mixtures containing different molar proportions of (β1→4)-D-mannotriose

3

and

(α1→5)-L-arabinotriose,

oligosaccharides

structurally

related

to

coffee

4

polysaccharides (galactomannans and arabinogalactans), were roasted at 200 °C for

5

different periods. Electrospray ionization mass spectrometry (ESI-MS) and tandem

6

mass spectrometry (ESI-MSn) analyses of labeled (18O) and unlabeled samples allowed

7

identification of non-hybrid oligosaccharides, but also hybrid oligosaccharides

8

composed by both hexose and pentose units. The identification of hybrid

9

oligosaccharides allowed us to infer the occurrence of non-enzymatic transglycosylation

10

reactions involving both oligosaccharides in the starting mixtures. Also, it was observed

11

that using different proportion of the oligosaccharides in the starting mixtures and

12

extent of the thermal treatment lead to a variation in the composition of the compounds

13

formed. These results have led to the conclusion that, depending on the distribution of

14

the polysaccharides in the bean cell walls and the roasting conditions, different non-

15

hybrid and hybrid structures can be formed during coffee roasting.

16 17

Keywords:

thermal

processing;

18

transglycosylation; isobars; isomers

coffee;

galactomannans;

19


arabinogalactans;

Page 3 of 34


20

INTRODUCTION

21

During the roasting process, galactomannans (GM) and type II arabinogalactans

22

(AG), the most abundant polysaccharides of green coffee beans,1 undergo structural

23

modifications, which are far from being completely elucidated due to the complexity

24

and diversity of the structures formed. However, it is well known that they are

25

depolymerized and debranched.2-5 Together with proteins, chlorogenic acids and

26

sucrose, GM and AG are involved in the formation of melanoidins, which are defined as

27

high molecular weight nitrogenous brown-colored compounds.6, 7 However, their exact

28

structures remain unclear. Also, GM are modified at the reducing end during coffee

29

roasting by the occurrence of dehydration, isomerization, decarboxylation, oxidation,

30

caramelization, and Maillard reactions.8

31

The dry thermal processing of oligosaccharides structurally related to coffee GM,9

32

namely (β1→4)-D-mannotriose (Man3), and (α1→5)-L-arabinotriose (Ara3), which is

33

structurally related to the arabinose side chains of coffee AG,10 promoted the formation

34

of new oligosaccharides. These oligosaccharides have a higher number of

35

monosaccharide units, referred to as degree of polymerization, and new types of

36

glycosidic linkages, showing the occurrence of non-enzymatic transglycosylation

37

reactions (TGR), i.e. the transfer of glycosyl units to the hydroxyl groups of other

38

glycosides, induced by roasting.11, 12 When mixtures of Man3 with Ara3 were roasted,

39

hybrid structures composed by arabinose and mannose residues derived from different

40

origins, Ara3 and Man3, respectively, were formed. The identification of the same type

41

of hybrid structures in roasted coffee polysaccharide-rich fractions supports the

42

hypothesis of the occurrence of non-enzymatic TGR during coffee roasting involving

43

GM and AG side chains.13



44

Considering that the distribution of GM and AG in coffee bean cell walls is

45

heterogeneous,14 and they have different vulnerability to roasting-induced degradation,15

46

it is hypothesized that different non-hybrid and hybrid structures can be formed during

47

coffee roasting depending on the distribution of the polysaccharides in the bean cell

48

walls and the roasting conditions. In this work, untreated and thermally treated mixtures

49

with different proportions of Man3 and Ara3, mimicking possible regions within the cell

50

walls with distinct polysaccharide composition, were analyzed. Electrospray ionization

51

mass spectrometry (ESI-MS) and electrospray ionization collision-induced dissociation

52

tandem mass spectrometry (ESI-CID-MSn) analyses were performed by infusing labeled

53

(18O) and unlabeled samples. To verify if the structures formed during the thermal

54

processing of the model mixtures have the same structural features as those formed

55

during coffee roasting, or additional roasting treatments beyond the roasting of the

56

green coffee beans, a GM-rich fraction isolated from spent coffee grounds (SCG),

57

submitted to additional roasting treatments, was treated with an endo-(β1→4)-D-

58

mannanase. The hydrolyzed material was further fractioned by ligand exchange/size-

59

exclusion chromatography (LEX-SEC) and analyzed by ESI-MS and ESI-CID-MSn.

60

The elemental composition of the ions identified in the ESI-MS spectra of both

61

oligosaccharide mixtures and SCG sample was obtained by high resolution and high

62

mass accuracy measurements using an Orbitrap-based mass spectrometer.

63 64

MATERIAL AND METHODS

65 66

Oligosaccharide samples and preparation of the mixtures. Oligosaccharides,

67

Ara3 and Man3, with a purity ≥ 95%, were purchased from Megazyme (County

68

Wicklow, Ireland). As previously described,13 solutions of each oligosaccharide (0.129


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69

mmol in 200 mL of ultrapure water) were prepared, and then used to prepare three

70

mixtures containing different molar proportions of Ara3 and Man3 as follows: 25%

71

Ara3:75% Man3 (A25M75); 50% Ara3:50% Man3 (A50M50); and 75% Ara3:25% Man3

72

(A75M25). Each mixture (in solution) was freeze-dried, and the resulting solid material

73

was powdered with an agate mortar and pestle, and then stored in a desiccator under a

74

phosphorous pentoxide environment.

75 76

Dry thermal treatments. The thermal treatments were performed on a TGA-50

77

thermogravimetric analyzer (Shimadzu, Kyoto, Japan), using the conditions previously

78

described.11-13 Briefly, three samples of each solid mixture (3-8 mg) were heated from

79

room temperature up to 200 °C, maintained at 200 °C for different periods of time: 0

80

(T1), 30 (T2), and 60 (T3) min. For each condition, the thermal treatment was repeated

81

at least twice in different days. The thermally treated mixtures were recuperated,

82

weighed, and suspended in ultrapure water in a concentration of 5 mg/mL. To facilitate

83

their dissolution, they were then stirred at 37 ºC for 3 h. The water-soluble fractions

84

were separated and kept frozen at -20 °C until analysis by MS. Solutions (1 mg/mL) of

85

each untreated mixture (T0) were also prepared and stored under the same conditions.

86 87

Labeling with oxygen-18. To label with oxygen-18 (18O) the carbonyl oxygen of

88

non-modified reducing sugar residues, or new carbonyl groups formed by dry thermal

89

processing, 50 µL of untreated and thermally treated oligosaccharide mixtures,

90

previously dissolved in water, were dried and redissolved in 50 µL of

91

water (H218O, 97%) (Sigma-Aldrich, St. Louis, MO). Each solution was then kept under

92

stirring at 500 rpm for 4 h at 37 °C in a sealed vial, after which time it was frozen at -20

93

°C until analysis by ESI-MS (4 days). To check the 18O-labeling of a keto group under


18

O-enriched


94

the conditions used because they had previously been used only to label the carbonyl

95

group of aldehydes, 2 µL of 3-octanone (99%) (Aldrich-Chemie, Steinheim, Germany)

96

were diluted in 50 µL of H218O, and then submitted to the same conditions of stirring

97

and freezing before ESI-MS analysis.

98 99

SCG sample, enzymatic hydrolysis and fractionation by LEX-SEC. SCG,

100

obtained after a commercial espresso coffee preparation, were submitted to a roasting

101

pre-treatment at 160 or 220 °C, and then sequentially extracted with water at 90 °C and

102

4 M NaOH solutions at 20, 60 and 120 °C. A GM-rich fraction, containing 89% of

103

mannose, was recovered from SCG roasted at 160 °C upon extraction with 4 M NaOH

104

at 120 °C, and became water insoluble upon neutralization.16 In order to convert this

105

fraction into cold water-soluble material, it was submitted to sequential roasting

106

treatments of 1 h at 200 ºC in a pre-heated oven (Binder). After each roasting procedure,

107

the material was suspended in water at room temperature with stirring during 1h. The

108

suspension was then centrifuged and the cold water-soluble material was recovered and

109

freeze-dried.15 A sample (2.4 mg) of the cold water-soluble material recovered after the

110

second roasting procedure, referred as R2W20sn,15 was hydrolyzed with 0.3 U of a pure

111

endo-(β1→4)-D-mannanase preparation (Aspergillus niger, EC 3.2.1.78) (Megazyme,

112

County Wicklow, Ireland) during 60 h at 37 ºC with continuous stirring in 600 µL of

113

100 mM Na-acetate buffer, pH 5.5, containing 0.02% sodium azide. The hydrolyzed

114

sample was filtrated using a Cronus nylon syringe filter (0.2 µm of pore size and 13 mm

115

of diameter) and then fractioned by LEX-SEC on a high-performance liquid

116

chromatograph equipped with a 300 mm x 20 mm i.d. Shodex sugar KS2002 column

117

from Showa Denko K. K. (Tokyo, Japan). The column was maintained at 30 °C, the

118

injected sample volume was 500 µL, and ultrapure water was used as eluent at a flow


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119

rate of 2.80 mL/min. A K-2401 refractive index detector (Knauer, Berlin, Germany)

120

was used. All collected fractions were dried, redissolved in 100 µL of ultrapure water,

121

and kept frozen at -20 °C until analysis by MS. To check its LEX-SEC elution profile,

122

the enzyme (0.3 U in 600 µL of 100 mM Na-acetate buffer) was injected and eluted

123

using the same conditions used for the hydrolyzed SCG sample.

124 125

ESI-MS conditions. Immediately prior to ESI-MS analysis, each sample,

126

previously dissolved in water, was diluted in methanol/water (1:1, v/v) containing

127

formic acid (0.1%, v/v).

128

Linear ion trap (LIT) conditions. ESI-MS and ESI-CID-MSn spectra were

129

acquired from all the mixtures and the LEX-SEC fraction obtained from the hydrolyzed

130

SCG sample in positive mode on a LXQ linear ion trap (LIT) mass spectrometer

131

(Thermo Fisher Scientific Inc., Waltham, MA), using the following operating

132

conditions: electrospray voltage, 5 kV; capillary temperature, 275 °C; capillary voltage,

133

1 V; and tube lens voltage, 40 V. Samples were introduced at a flow rate of 8 µL/min

134

into the ESI source. Nitrogen was used as nebulizing and drying gas. ESI-MS spectra

135

were acquired over the range m/z 100-1500. ESI-CID-MSn spectra were acquired with

136

the energy collision set between 19 and 29 (arbitrary units). Data were acquired and

137

analyzed using Xcalibur software.

138

Q Exactive Orbitrap conditions. The Q Exactive hybrid quadrupole-Orbitrap mass

139

spectrometer (Thermo Firsher Scientific, Germany), interfaced with H-ESI II ion

140

source, was employed for accurate mass measurements of the mixtures submitted to the

141

T3 treatment and the A50M50 mixture submitted to the T1 treatment, as well as the

142

LEX-SEC fraction obtained from the hydrolyzed SCG sample. The acquisition method

143

was set with a full scan and 140,000 resolution (relative to m/z 200) in positive mode.



144

The method parameters were: AGC, 3e6; IT, 100 ms; scan range, 100-1000; spray

145

voltage, 3.0 kV; sheath gas, 5; aux gas, 1; capillary temperature, 250 °C; S-lens RF

146

level, 50; probe heater temperature, 50 °C; and flow rate, 5 µL/min. The Q Exactive

147

system was tuned and calibrated in positive mode using peaks of known mass from a

148

calibration solution (Thermo Scientific) to achieve a mass accuracy of < 0.5 ppm RMS.

149

The data were processed with Xcalibur 3.0.63 software.

150 151

RESULTS AND DISCUSSION

152 153

Color and water-solubility of each oligosaccharide mixture upon dry heating.

154

The possible occurrence of non-enzymatic TGR involving GM and AG side chains

155

during coffee roasting was investigated using as model solid mixtures of Man3 and

156

Ara3. Three mixtures containing different molar proportions of each oligosaccharide

157

(A25M75, A50M50 and A75M25) were used to mimic possible regions within the cell

158

walls with distinct polysaccharide composition. In order to mimic coffee roasting

159

conditions, the oligosaccharide mixtures were submitted to dry thermal treatments at

160

200 °C. Three different samples of each mixture were heated up to 200 °C and

161

maintained at 200 ºC for different periods: T1 (0 min), T2 (30 min) and T3 (60 min).

162

After dry thermal processing, the mixtures (white or off-white powders) acquired a

163

brown coloration, possibly due to caramelization reactions. In general, the brown

164

coloration was more intense for the longer treatments. However, the coloration of the

165

compounds resulting from the longer treatments (T2 and T3) of the mixtures with

166

higher proportion of Ara3 (A50M50 and A75M25) was darker than that observed when

167

A25M75 mixture was submitted to the same treatments. These observations are in line

168

with the lower thermal stability of Ara3 when compared with Man3,11, 12 which is also


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169

corroborated by the percentages of mass loss in each treatment from 150 °C, excluding

170

the initial mass loss due to the loss of adsorbed water molecules. Considering the

171

different mixtures, the mass loss percentages from 150 °C were 0.7-3.5% for T1, 11.0-

172

17.8% for T2, and 15.0-19.1% for T3. Also, independently of the starting mixture, the

173

compounds resulting from T1 were completely dissolved in water. However, as

174

observed for Ara3 (but not for Man3), the compounds resulting from T2 treatment of the

175

A75M25 mixture and T3 treatment of all mixtures were only partially dissolved in

176

water. The percentage of water-soluble material was 65.2% for T2 treatment of the

177

A75M25 mixture. For materials resulting from T3, the percentages of water-soluble

178

material were 68.8% for A25M75, 40.7% for A50M50, and 44.4% for A75M25. These

179

values are higher than those obtained with materials resulting from T2 and T3

180

treatments of Ara3 (35.3 and 24.6%, respectively),12 which shows the formation of a

181

higher amount of hydrophobic compounds when the amount of Ara3 is higher.

182

In summary, the observed differences in the coloration and water-solubility of the

183

thermally treated mixtures suggest that the structural modifications occurred in higher

184

extent with increasing of the molar proportion of Ara3 in the starting mixture, and the

185

time at 200 °C. This is in agreement with the higher diversity of ions observed in the

186

matrix-assisted laser desorption/ionization mass spectrometry spectra of the

187

oligosaccharide mixtures subjected to the longer treatments (T2 and T3).13 In order to

188

obtain a deeper insight into the structural modifications induced by dry thermal

189

processing, both untreated samples and water-soluble fractions recovered from thermal

190

treated samples were analyzed by ESI-MS and ESI-MSn.

191 192

Identification of hybrid and non-hybrid compounds upon dry heating. In

193

preliminary testing, we have found that, under ESI-MS conditions, neutral



194

oligosaccharides ionize better in positive than in negative mode. For this reason,

195

positive mode was preferred for ESI-MS analysis of both untreated and thermally

196

treated mixtures.

197

As typical of neutral oligosaccharides,11, 12, 17 the oligosaccharides in the starting

198

mixtures (Man3 and Ara3), but also the roasting-induced compounds, were mainly

199

detected as sodium adduct ions ([M+Na]+). Accordingly, the most abundant ions in the

200

ESI-MS spectra of the untreated mixtures were observed at m/z 437 ([Ara3+Na]+) and

201

527 ([Man3+Na]+).

202

After thermal processing, new ions, not observed in the ESI-MS spectra of the

203

untreated mixtures, were identified. Independently of the starting mixture (A25M75,

204

A50M50, or A75M25), the diversity of ions was higher in the ESI-MS spectra obtained

205

after the longer treatments (T2 and T3). Due to the complexity of these ESI-MS spectra,

206

only the ions observed with a relative abundance equal or higher than 15% in at least

207

two ESI-MS spectra acquired from the thermally treated mixtures were considered

208

(Table 1). The assignment of these ions was supported based on their fragmentation

209

pattern under ESI-CID-MSn conditions, as will be later described, and corroborated by

210

the elemental composition obtained from high resolution and high mass accuracy

211

measurements using a hybrid quadrupole-Orbitrap mass spectrometer. Accordingly, the

212

ions identified in the ESI-MS spectra of the thermally treated mixtures were attributed

213

to [M+Na]+ ions of non-hybrid compounds composed by hexose (Hex) or pentose

214

(Pent) residues and derivatives, and hybrid compounds composed by both Hex and Pent

215

residues and derivatives (Table 1). As supported by sugar and glycosidic linkage

216

analyses,13 Pent and Hex were mainly arabinose (Ara) and mannose (Man),

217

respectively. However, new sugar residues, although in minor amounts, were formed

218

during thermal processing, namely ribose (Rib), xylose (Xyl) and lyxose (Lyx) that are


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219

isomers, i.e. have the same elemental composition, of Ara, and thus they are not

220

distinguishable by MS. Therefore, in Table 1, Pent represents mainly Ara, but also Rib,

221

Xyl and Lyx that are present in minor amounts. Similarly, Hex represents mainly Man,

222

but also isomeric sugars (glucose and galactose) that are present in minor amounts.

223

As observed for Ara3,12 the non-hybrid compounds composed by Pent residues

224

and derivatives identified after thermal treatment of the oligosaccharide mixtures

225

include Pent oligosaccharides (Pentn) with a lower (n=2) and higher (n=4-5) degree of

226

polymerization than that of the Pent oligosaccharide in the starting mixtures (Ara3). The

227

respective monosaccharide was also observed at m/z 173 ([Pent+Na]+). Also, Pentn

228

(n=2-6) derivatives resulting from the formation of a keto group (-2 Da), and

229

dehydration with loss of one, two and three water molecules, as well as resulting from

230

the oxidative scission of a furanose ring with loss of C2H4O2, C3H4O2, C2H6O3, C3H4O3,

231

C3H6O3 and C2H8O5 were identified.

232

As observed for Man3,11 the non-hybrid compounds composed by Hex residues

233

and derivatives identified after thermal treatment of the model mixtures include Hex

234

mono- and oligosaccharides (Hexn, n=1-3). The corresponding dehydrated derivatives

235

formed by loss of one and three up to six water molecules were also observed ([Hexn-

236

xH2O+Na]+; n=1-5; x=1, 3-6). Revisiting the ESI-MS spectra previously acquired from

237

Man3 subjected to T2 or T3 treatment, beyond the derivatives resulting from loss of one

238

and three water molecules previously reported,11 it was possible to observe the ions

239

attributed to dehydrated Hex oligosaccharides resulting from loss of four, five, and six

240

water molecules.

241

In

agreement

with

previous

observation

by

MALDI-MS,13

hybrid

242

oligosaccharides composed by both Hex and Pent units were also identified as [M+Na]+

243

ions in the ESI-MS spectra of the thermally treated mixtures, namely PentHex2 (m/z



244

497), PentHex3 (m/z 659) and Pent2Hex2 (m/z 629). The observation of these hybrids

245

corroborates the hypothesis of the occurrence of non-enzymatic TGR involving GM and

246

Ara side chains of AG during coffee roasting. Also, several ions (Table 1) were

247

attributed to dehydrated derivatives formed by loss of one and three up to six water

248

molecules from hybrid oligosaccharides ([PentmHexn-xH2O+Na]+; m, n=1-4; x=1, 3-6).

249

As highlighted in Table 1, among the compounds identified in the ESI-MS spectra

250

of the thermally treated mixtures as [M+Na]+ ions, there are compounds with the same

251

nominal mass (calculated by adding the mass of the predominant isotope of each

252

element contributing to the molecule rounded to the nearest integer value). Some of

253

these compounds are isobaric compounds, i.e. have the same nominal mass but different

254

elemental composition, and thus, different accurate mass; other are isomers, i.e. have

255

the same elemental composition, and thus, the same exact mass. Isobaric and isomeric

256

compounds were differentiated based on specific fragmentation seen in the ESI-CID-

257

MSn spectra.

258 259

Diagnostic neutral losses and product ions observed under ESI-CID-MSn The fragmentation of [M+Na]+ ions of neutral and reducing

260

conditions.

261

oligosaccharides under ESI-CID-MSn conditions results of glycosidic linkage cleavages,

262

cross-ring cleavages, and loss of water. As inferred by

263

oxygen of standard oligosaccharides,12,

264

mainly occur between the anomeric carbon and the glycosidic linkage oxygen, whereas

265

cross-ring cleavages and loss of water occur mainly at the reducing end residue with

266

loss of the carbonyl oxygen. The cross-ring cleavages at the reducing end residue

267

depend on the oligosaccharide structure, giving rise to neutral losses of CH2O (-30 Da),

268

C2H4O2 (-60 Da), C3H6O3 (-90 Da), and C4H8O4 (-120 Da). Based on the knowledge of

18, 19

18

O-labeling of the carbonyl

including Ara3,12 glycosidic cleavages


Page 12 of 34

Page 13 of 34


269

the typical fragmentation pathways of non-modified oligosaccharides, but also of

270

derivatives formed when Man3 and Ara3 were individually submitted to the thermal

271

treatments at 200 °C,11,

272

diagnostic of different ion series observed in the ESI-MS spectra of the thermally

273

treated mixtures, which are summarized in Table 2.

12

it was possible to identify neutral losses and product ions

274

The ESI-MSn fragmentation of [M+Na]+ ions of Hex oligosaccharides produces

275

product ions resulting from glycosidic cleavages with loss of a Hex residue (Hexres)

276

(162 Da) and a Hex (180 Da), and diagnostic product ions at m/z 203 ([Hex+Na]+) and

277

185

278

oligosaccharides show product ions resulting from glycosidic cleavages with loss a Pent

279

residue (Pentres) (132 Da) and a Pent (150 Da), and product ions at m/z 173 ([Pent+Na]+)

280

and 155 ([Pentres+Na]+) (Figure 2A). Product ions resulting from the neutral loss of 162

281

Da observed with relative abundance ≤10% in the ESI-MSn spectra of Pent

282

oligosaccharides, as Pent5 and derivatives (Table 3), is not due to the loss of Hexres, but

283

due to the combined loss of Pentres and CH2O (30 Da) from cross-ring cleavage. Neutral

284

losses and product ions observed in the ESI-MSn spectra of non-hybrid oligosaccharides

285

are also observed in the ESI-MSn spectra of Pent-Hex hybrid oligosaccharides.

286

However, the neutral loss of 294 Da, due to the combined loss of Pentres and Hexres, is

287

specific of Pent-Hex hybrid oligosaccharides, as well as the product ion at m/z 335

288

([PentHex+Na]+) (Figure 3A).

([Hexres+Na]+) (Figure

1A).

Similarly,

the

ESI-MSn

spectra

of

Pent

289

When sugar residues are modified, they yield new diagnostic neutral losses and

290

product ions, which can be specific or not of one type of modification. The ESI-MSn

291

fragmentation of an oligosaccharide bearing a (Hex-3H2O) produces a product ion

292

resulting from the neutral loss of 126 Da, corresponding to the loss of (Hex-3H2O), and

293

a product ion at m/z 149 ([Hex-3H2O+Na]+). The loss of 144 Da is observed either in



294

the ESI-MSn spectra of (Hex-H2O) derivatives, resulting from the loss of (Hex-H2O)res,

295

and of (Hex-2H2O) derivatives, resulting from the loss of (Hex-2H2O). The product ion

296

at m/z 167 is also observed in the ESI-MSn spectra of both (Hex-H2O) and (Hex-2H2O)

297

derivatives, corresponding to [(Hex-H2O)res+Na]+ and [Hex-2H2O+Na]+, respectively.

298

In the case of (Hex-H2O) derivatives, the product ion at m/z 185 can also be due to

299

[Hex-H2O+Na]+, and not exclusively to [Hexres+Na]+. Similarly, the neutral loss of 114

300

Da is observed either in the ESI-MSn spectra of (Pent-H2O) derivatives, resulting from

301

the loss of (Pent-H2O)res, and in the ESI-MSn spectra of (Pent-2H2O) derivatives,

302

resulting from the loss of (Pent-2H2O). In the case of (Pent-H2O) derivatives, the

303

product ion at m/z 155 can also be due to [Pent-H2O+Na]+, and not exclusively to

304

[Pentres+Na]+. The neutral loss of 132 and 162 Da can also be due to the presence a

305

mono-dehydrated sugar, (Pent-H2O) and (Hex-H2O), respectively. Also, the neutral loss

306

of 96 Da can be due either to the loss of (Pent-2H2O)res, or (Pent-3H2O). In the case of

307

oligosaccharides bearing a (Pent-2H), their fragmentation produces a product ion

308

resulting from neutral loss of 148 Da, due to the loss of (Pent-2H), and the product ion

309

at m/z 171 ([Pent-2H+Na]+). The identification of neutral losses and product ions

310

diagnostic of different ion series (Table 2) was essential to disclose the presence of

311

isobaric and isomeric compounds.

312 313

Differentiation of isobaric and isomeric compounds by ESI-CID-MSn. The

314

presence of isobaric/isomeric compounds was inferred by the observation of specific

315

product ions in the ESI-MS2 spectra acquired on the LIT mass spectrometer (Table 3).

316

In most of the cases, the presence of isobaric/isomeric compounds was confirmed by

317

ESI-MSn, n=3-4. As example, it is shown the tandem MS-based strategy to discriminate

318

between the pairs Hex3/(Pent3Hex-4H2O) (m/z 527) and Pent3/(Hex3-5H2O) (m/z 437).


Page 14 of 34

Page 15 of 34


319

In the MS analysis of carbohydrate-rich samples, the ion (or product ion) at m/z

320

527 is usually indicative of Hex3. The ESI-MS2 spectrum acquired from the A50M50

321

mixture submitted to the T1 treatment (Figure 1B) is similar to that acquired from the

322

untreated mixture (Figure 1A), suggesting the exclusive presence of the precursor ion

323

[Hex3+Na]+, as corroborated by high resolution and high mass accuracy MS data.

324

However, new product ions, absent in the ESI-MS2 spectrum acquired after T1 (Figure

325

1B), namely the product ions at m/z 401 (-126 Da) and 395 (-132 Da), identified as

326

resulting from the loss of (Hex-3H2O) and Pentres, and the product ions at m/z 287 and

327

263, attributed to [PentPentres+Na]+ and [HexPent-4H2O+Na]+, respectively, were

328

observed in the ESI-MS2 spectra acquired after the longer treatments, T2 (C) and T3

329

(D). These new ions suggest the presence of the precursor ion [Pent3Hex-4H2O+Na]+

330

beyond [Hex3+Na]+, as corroborated by high resolution and high mass accuracy MS

331

data. The relative abundance of the new product ions at m/z 401 and 395 increases with

332

increasing of the treatment time, whereas that at m/z 365 (-162 Da, -Hexres) decreases.

333

These changes suggest the increase of the proportion of [Pent3Hex-4H2O+Na]+ and the

334

decrease of [Hex3+Na]+ ions with increasing of the treatment time.

335

Figure 2 (C-E) shows the ESI-MS2 spectra of the ion at m/z 437 acquired from the

336

A75M25, A50M50, and A25M75 mixtures subjected to the T2 treatment. The product

337

ions at m/z 305 (-Pentres) and 173 (-2 Pentres), also observed in the ESI-MS2 spectrum of

338

the Ara3 (Figure 2A), corroborate the presence of [Pent3+Na]+ as precursor ion. The

339

coexistence of other precursor ion, [Hex3-5H2O+Na]+, is supported by the product ions

340

at m/z 311 (-126 Da), 293 (-144 Da), and 275 (-162 Da), formed respectively by loss of

341

(Hex-3H2O), (Hex-2H2O), and Hexres, as well as those at m/z 203 ([Hex+Na]+), 185

342

([Hexres+Na]+), and 149 ([Hex-3H2O+Na]+). These product ions were also observed in

343

the ESI-MS2 spectrum of (Hex3-5H2O) formed when Man3 was individually subjected



344

to the treatment T2 (Figure 2B).11 The relative abundance of the product ions at m/z

345

311, 293, and 275 suggests the increase of the proportion of [Hex3-5H2O+Na]+ ions and

346

the decrease of [Pent3+Na]+ ions when the proportion of Man3 increases (Figure 2C-E).

347

As illustrated by the ESI-MS2 spectra of the ions at m/z 527 and 437 in Figures 1

348

and 2, the formation of each isobaric compound, but also of each isomeric compound,

349

was dependent on the thermal treatment and the starting mixture. This suggests that a

350

variety of structures may be formed during the green coffee roasting, depending on the

351

roasting conditions and distribution of the polysaccharides in the coffee beans.

352 353

ESI-MSn analysis after labeling with oxygen-18. Aiming to gain more

354

information about the structure of the compounds formed during thermal processing,

355

thermally treated mixtures were dissolved in H218O before MS analysis. Based on the

356

principles of nucleophilic addition reactions of aldehydes and ketones, they react with

357

water to yield geminal diols (hydrates). The hydration reaction is reversible, and a

358

geminal diol can eliminate water to regenerate an aldehyde or ketone.20 Accordingly,

359

the

360

observed, but also new carbonyl groups, either of ketones or aldehydes, formed by dry

361

thermal processing.

18

O-labeling of the carbonyl oxygen of non-modified reducing sugar residues was

362 363

ESI-MSn of

18

O-labeled Pent-Hex hybrid oligosaccharides. The ESI-MS2

364

spectrum of the ion at m/z 659 acquired from the A50M50 mixture subjected to the T1

365

treatment, and that acquired after

366

accordance to what was previously presented, this species could have the contribution

367

of two isobaric compounds, PentHex3 and (Pent4Hex-4H2O). As corroborated by the

368

ESI-MS2 spectrum before

18

18

O-labeling are shown in Figures 3A and B. In

O-labeling (Figure 3A), namely by the absence of the


Page 16 of 34

Page 17 of 34


369

product ion at m/z 533, resulting from the neutral loss of (Hex-3H2O), only PentHex3

370

was present.

371

The ESI-MS2 spectrum of

18

O-labeled PentHex3 (Figure 3B) shows the product

372

ion at m/z 529, resulting from the loss of unlabeled Pentres, and the product ion at m/z

373

205, attributed to [M+Na]+ of a 18O-labeled Hex. These product ions indicate that one of

374

the three Hex units, and not the Pent, was 18O-labeled. This means that a Hex is located

375

at the reducing end of PentHex3. The observation of the product ion at m/z 529 also

376

indicates that the three Hex units are linked together, having several possible binding

377

sites for the Pent. The product ion at m/z 337 (-2 Hexres) suggests that the Pent is linked

378

to the Hex located at the reducing end (Figure 3C). However, the product ion at m/z

379

479, formed by loss of the

380

with the Pent not linked to the Hex located at the reducing end (Figure 3D).

18

O-labeled Hex, supports the presence of other structures

381

In any of the possible structures, it is of note that the Pent-Hex glycosidic linkage

382

involves the anomeric carbon of the Pent. The formation of the Pent-Hex glycosidic

383

linkage involving the anomeric carbon of the Pent, and not of the Hex, can be favored

384

by the higher reactivity of pentoses compared to hexoses. In the case of the thermally

385

treated mixtures, the most abundant pentoses and hexoses are Ara and Man,

386

respectively. As also previously observed by glycosidic linkage analysis,13 new types of

387

Man glycosidic linkages, absent in the untreated mixtures, namely (1→2), (1→6),

388

(1→2,3), (1→2,6), (1→3,6), and (1→2,3,6) linkages, were formed during thermal

389

processing of the Ara3-Man3 mixtures. Accordingly, these new Man linkages were

390

identified when a coffee GM-rich fraction was submitted to different dry thermal

391

treatments.15 In both cases, the formation of (1→6) Man linkages was favored over that

392

of other types of Man linkages. As reported for British gums produced by dry heating of

393

starch,21 the formation of (1→6) linkages can be favored over others in terms of



Page 18 of 34

394

stereochemical and thermodynamic aspects. This can be due to the primary hydroxyl

395

group located at the C6-position, while the other hydroxyl groups of Man are secondary.

396

According to Tomasik et al.,21 anhydrosugars are possible intermediates in the

397

formation of new glycosidic linkages, but the mechanisms involved in the non-

398

enzymatic TGR induced by dry heating are far from being elucidated.

399 ESI-MSn of

400

18

O-labeled oligosaccharides containing a tri-dehydrated hexose.

401

Both non-hybrid and hybrid oligosaccharides containing a (Hex-3H2O) were labeled by

402

dissolving selected thermally treated mixtures in H218O. In Figure 4 are shown the ESI-

403

MS2 spectrum of the ion at m/z 413 ([Pent2Hex-3H2O+Na]+) acquired from the A50M50

404

mixture subjected to the T2 treatment (A), and that acquired after 18O-labeling (B). The

405

product ion observed after 18O-labeling (B) at m/z 287 (-128 Da), resulting from the loss

406

of

407

unlabeled Pentres, attributed to a

408

(Pent2Hex-3H2O) structures with the (Hex-3H2O) located at the reducing end of the

409

corresponding non-modified oligosaccharide.

18

O-labeled (Hex-3H2O), and that observed at m/z 151, formed by loss of two 18

O-labeled (Hex-3H2O), confirm the presence of

410

The occurrence of 18O-labeling at the (Hex-3H2O) suggests that this moiety has a

411

carbonyl group. As previously demonstrated by deuterium-labeling of oligosaccharides

412

bearing a (Hex-3H2O) formed from Man3,11 (Hex-3H2O) may be isomaltol, which has a

413

keto group. Due to the reversibility of the hydration reaction of ketones,20 the

414

labeling of the keto group of isomaltol moieties present in the thermally treated

415

mixtures dissolved in H218O could be expected. However, since the conditions used in

416

this work for 18O-labeling had previously been used only to label the carbonyl group of

417

aldehydes, the ketone 3-octanone was used as standard, confirming the 18O-labeling of a

418

keto group under the conditions used.


18

O-

Page 19 of 34


419

Analysis of a coffee galactomannan-rich fraction. In order to check if the

420

structures formed during thermal processing of the model mixtures have the same

421

structural features as those formed during coffee roasting, or additional roasting

422

treatments beyond the roasting of the green coffee beans, a GM-rich fraction isolated

423

from SCG and submitted to two additional roasting treatments of 1 h at 200 °C was

424

analyzed. First, this GM-rich fraction was treated with an endo-(β1→4)-D-mannanase to

425

selectively cleave the (β1→4)-D-mannan backbone between adjacent (β1→4)-linked

426

Man residues. According to the mechanism of action of this enzyme, the hydrolysis of

427

the (β1→4)-D-mannan backbone is hindered by the presence of substituted Man

428

residues, non-Man residues interspersed in the mannan backbone, modified Man

429

residues, or other Man glycosidic linkages that are not (β1→4), yielding

430

oligosaccharides that contain these structural details. Thus, the hydrolyzed material was

431

further fractionated by LEX-SEC and then analyzed by ESI-MS and ESI-MSn.

432

In accord with previous reports,22, 23 the LEX-SEC fraction that eluted between

433

16-17 min was assigned to the neutral oligosaccharides with the higher molecular

434

weight that resulted from the enzymatic hydrolysis. Due to the enzyme action, these

435

oligosaccharides contain structural details of the coffee GM under study. In the ESI-MS

436

spectrum of this fraction, it was possible to identify the ions at m/z 497, 509, 527, 641

437

and 659, also identified in the mixtures, and other ions at m/z 671, 689, 803, 833, and

438

851. Based on their ESI-CID-MSn fragmentation patterns, and the elemental

439

composition obtained from high resolution and high mass accuracy measurements using

440

a hybrid quadrupole-Orbitrap mass spectrometer, the identification of these ions is as

441

follows: 497, [PentHex2+Na]+; 509, [Hex3-H2O+Na]+; 527, [Hex3+Na]+; 641,

442

[PentHex3-H2O+Na]+; 659, [PentHex3+Na]+; 671, [Hex4-H2O+Na]+; 689, [Hex4+Na]+;

443

803, [PentHex4-H2O+Na]+; 833, [Hex5-H2O+Na]+; 851, [Hex5+Na]+.



444

According to the glycosidic linkage composition of the coffee GM-rich fraction

445

under study,15 the observation of Hex3-5 after enzymatic hydrolysis can be related to the

446

presence of substituted Man residues, namely by single Gal residues as occur in green

447

coffee GM; non-mannose residues (Glc and Gal) interspersed in the mannan backbone;

448

and new glycosidic linkages resistant to the enzyme action, such as (1→6) Man

449

linkages, formed during roasting. Since single Ara residues occur as side chains in green

450

coffee GMs,9 PentHex2 and PentHex3 can result from cleavage of the original mannan

451

backbone, without any modification promoted by roasting. Contrarily, the dehydrated

452

derivatives are the result of dehydration reactions occurring during roasting. The ESI-

453

MSn fragmentation of (PentHex3-H2O) and (PentHex4-H2O) suggests that loss of a

454

water molecule occurred at a Hex unit, as inferred by the loss of an intact Pentres (132

455

Da) and (Hex-H2O)res (144 Da). This is in accordance to what was observed with the

456

Ara3-Man3 mixtures, reinforcing the validity of the models used. The absence of hybrid

457

domains formed by non-enzymatic TGR between GM and AG during roasting in the

458

GM-rich fraction analyzed in this study can be related to its original location in the

459

green coffee beans, and the roasting conditions used.

460

In summary, the analysis of the model mixtures containing different molar

461

proportions of Ara3 and Man3, maintained at 200 °C for different periods, showed that

462

different structures can be formed during coffee roasting, depending on the distribution

463

of the polysaccharides in the beans and the roasting conditions. Furthermore, the

464

diversity of isobaric and isomeric compounds formed highlights the importance of a

465

detailed structural characterization when analyzing real roasted carbohydrate-rich

466

matrices such as coffee.

467 468


Page 20 of 34

Page 21 of 34


469

ABBREVIATIONS USED

470

A25M75 – 25% Ara3:75% Man3

471

A50M50 –50% Ara3:50% Man3

472

A75M25 – 75% Ara3:25% Man3

473

AG – Arabinogalactan

474

Ara3 – (α1→5)-L-arabinotriose

475

GM – Galactomannan

476

Hex – Hexose

477

LEX-SEC – Ligand exchange/size-exclusion chromatography

478

Lyx – Lyxose

479

Man3 – (β1→4)-D-mannotriose

480

Pent – Pentose

481

Rib – Ribose

482

SCG – Spent coffee grounds

483

TGR – Transglycosylation reactions

484

Xyl – Xylose

485 486

ACKNOWLEDGMENT

487

Thanks are due to Fundação para a Ciência e a Tecnologia (FCT, Portugal),

488

European Union, QREN, FEDER, and COMPETE for funding the QOPNA research

489

unit (project PEst-C/QUI/UI0062/2013; FCOMP-01-0124-FEDER-037296), and the

490

Portuguese Mass Spectrometry Network (REDE/1504/REM/2005). Thanks are also due

491

to FCT for the grants of Ana Moreira (SFRH/BD/80553/2011) and Joana Simões

492

(SFRH/BPD/90447/2012).

493



Page 22 of 34

494

ASSOCIATED CONTENT

495

Supporting Information

496

The Supporting Information is available free of charge on the ACS Publications

497

website.

498

ESI-MS spectra acquired from the A75M25 mixture, before and after thermal 18

499

treatments (Figure S1), ESI-MS spectra of 3-octanone, before and after

500

procedure (Figure S2), ESI-MS2 spectra of [M+H]+ ions of unlabeled and 18O-labeled 3-

501

octanone (Figure S3), LEX-SEC chromatograms (Figure S4), ESI-MS spectrum of F16

502

obtained by LEX-SEC (Figure S5), total mass loss and mass loss from 150 °C during

503

each thermal treatment and water-solubility of the resulting compounds (Table S1),

504

accurate masses found by Q Exactive Orbitrap for the ions identified after heating of the

505

A50M50 mixture submitted to the T3 treatment (Table S2).

O-labeling

506 507

REFERENCES

508

(1)

Bradbury, A. G. W.; Halliday, D. J. Chemical structures of green coffee bean

509

polysaccharides. J. Agric. Food Chem. 1990, 38, 389-392.

510

(2)

511

arabinogalactans from two arabica coffee infusions as affected by the degree of roast. J.

512

Agric. Food Chem. 2002, 50, 1429-1434.

513

(3)

514

characterization of polysaccharides from green and roasted Coffea arabica beans.

515

Carbohydr. Polym. 2003, 52, 285-296.

516

(4)

517

carbohydrate composition of Coffea arabica beans. Carbohydr. Polym. 2003, 54, 183-

518

192.

Nunes, F. M.; Coimbra, M. A. Chemical characterization of galactomannans and

Oosterveld, A.; Harmsen, J. S.; Voragen, A. G. J.; Schols, H. A. Extraction and

Oosterveld, A.; Voragen, A. G. J.; Schols, H. A. Effect of roasting on the


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519

(5)

Redgwell, R. J.; Trovato, V.; Curti, D.; Fischer, M. Effect of roasting on

520

degradation and structural features of polysaccharides in Arabica coffee beans.

521

Carbohydr. Res. 2002, 337, 421-431.

522

(6)

523

melanoidins: structures, mechanisms of formation and potential health impacts. Food

524

Funct. 2012, 3, 903-915.

525

(7)

526

coffee melanoidin formation using modified “in bean” models. J. Agric. Food Chem.

527

2012, 60, 8710-8719.

528

(8)

529

of galactomannan derivatives in roasted coffee beverages. J. Agric. Food Chem. 2006,

530

54, 3428-3439

531

(9)

532

residues as structural features of acetylated galactomannans from green and roasted

533

coffee infusions. Carbohydr. Res. 2005, 340, 1689-1698.

534

(10)

535

Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of

536

coffee arabinogalactans. Phytochemistry 2008, 69, 1573-1585.

537

(11)

538

M. Evaluation of the effect of roasting on the structure of coffee galactomannans using

539

model oligosaccharides. J. Agric. Food Chem. 2011, 59, 10078-10087.

540

(12)

541

Roasting-induced changes in arabinotriose, a model of coffee arabinogalactan side

542

chains. Food Chem. 2013, 138, 2291-2299.

Moreira, A. S. P.; Nunes, F. M.; Domingues, M. R.; Coimbra, M. A. Coffee

Nunes, F. M.; Cruz, A. C. S.; Coimbra, M. A. Insight into the mechanism of

Nunes, F. M.; Reis, A.; Domingues, M. R. M.; Coimbra, M. A. Characterization

Nunes, F. M.; Domingues, M. R.; Coimbra, M. A. Arabinosyl and glucosyl

Nunes, F. M.; Reis, A.; Silva, A. M. S.; Domingues, M. R. M.; Coimbra, M. A.

Moreira, A. S. P.; Coimbra, M. A.; Nunes, F. M.; Simões, J.; Domingues, M. R.

Moreira, A. S. P.; Coimbra, M. A.; Nunes, F. M.; Domingues, M. R. M.



543

(13)

Moreira, A. S. P.; Simões, J.; Pereira, A. T.; Passos, C. P.; Nunes, F. M.;

544

Domingues, M. R. M.; Coimbra, M. A. Transglycosylation reactions between

545

galactomannans and arabinogalactans during dry thermal treatment. Carbohydr. Polym.

546

2014, 112, 48-55.

547

(14)

548

Cytochemistry and immunolocalisation of polysaccharides and proteoglycans in the

549

endosperm of green Arabica coffee beans. Protoplasma 2004, 223, 203-211.

550

(15)

551

Thermal stability of spent coffee ground polysaccharides: Galactomannans and

552

arabinogalactans. Carbohydr. Polym. 2014, 101, 256-264.

553

(16)

554

structure of spent coffee ground polysaccharides by roasting pre-treatments. Carbohydr.

555

Polym. 2013, 97, 81-89.

556

(17)

557

R. M. Fragmentation pattern of underivatised xylo-oligosaccharides and their alditol

558

derivatives by electrospray tandem mass spectrometry. Carbohydr. Polym. 2004, 55,

559

401-409.

560

(18)

561

V.; Domingues, M. R. M. Differentiation of isomeric pentose disaccharides by

562

electrospray ionization tandem mass spectrometry and discriminant analysis. Rapid

563

Commun. Mass Spectrom. 2012, 26, 2897-2904.

564

(19)

565

lithium-cationized disaccharides: tandem mass spectrometry and semiempirical

566

calculations. J. Am. Chem. Soc. 1991, 113, 5964-5970.

Sutherland, P. W.; Hallett, I. C.; MacRae, E.; Fischer, M.; Redgwell, R. J.

Simões, J.; Maricato, É.; Nunes, F. M.; Domingues, M. R.; Coimbra, M. A.

Simões, J.; Nunes, F. M.; Domingues, M. R.; Coimbra, M. A. Extractability and

Reis, A.; Coimbra, M. A.; Domingues, P.; Ferrer-Correia, A. J.; Domingues, M.

da Costa, E. V.; Moreira, A. S. P.; Nunes, F. M.; Coimbra, M. A.; Evtuguin, D.

Hofmeister, G. E.; Zhou, Z.; Leary, J. A. Linkage position determination in


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McMurry, J., Organic chemistry - International student edition. 7th ed.;

567

(20)

568

Thomson Brooks/Cole: Belmont, CA, 2008.

569

(21)

570

carbohydrates. Part II.* The decomposition of starch. In Adv. Carbohydr. Chem.

571

Biochem., Tipson, R. S.; Derek, H., Eds. Academic Press: 1989; Vol. Volume 47, pp

572

279-343.

573

(22)

574

F.; Coimbra, M. A.; Barros, A. I. R. N. A.; Domingues, M. R. M. Oxidation of

575

mannosyl oligosaccharides by hydroxyl radicals as assessed by electrospray mass

576

spectrometry. Carbohydr. Res. 2011, 346, 2603-2611.

577

(23)

578

M.; Domingues, M. R. M. Neutral and acidic products derived from hydroxyl radical-

579

induced oxidation of arabinotriose assessed by electrospray ionisation mass

580

spectrometry. J. Mass Spectrom. 2014, 49, 280-290.

Tomasik, P.; Wiejak, S.; Pałasiński, M., The thermal decomposition of

Tudella, J.; Nunes, F. M.; Paradela, R.; Evtuguin, D. V.; Domingues, P.; Amado,

Moreira, A. S. P.; da Costa, E. V.; Evtuguin, D. V.; Coimbra, M. A.; Nunes, F.



FIGURE CAPTIONS

Figure 1. ESI-MS2 spectra of the ion at m/z 527 acquired from the A. untreated A50M50 mixture, and samples of the A50M50 mixture subjected to the B. T1, C. T2, and D. T3 treatments.

Figure 2. ESI-MS2 spectra of the ion at m/z 437 acquired from the A. untreated A75M25 mixture, B. Man3 sample subjected to the T2 treatment, and mixtures subjected to the T2 treatment: C. A75M25, D. A50M50, and E. A25M75.

Figure 3. A. ESI-MS2 spectrum of the ion at m/z 659 ([PentHex3+Na]+) acquired from the A50M50 mixture subjected to T1 treatment, and B. the corresponding ESI-MS2 spectrum acquired after labeling with oxygen-18. The different proposed structures are represented in C and D.

Figure 4. A. ESI-MS2 spectrum of the ion at m/z 413 ([Pent2Hex-3H2O+Na]+) acquired from the A50M50 mixture subjected to the T2 treatment, and B. the corresponding ESIMS2 spectrum acquired after labeling with oxygen-18.


Page 26 of 34

Page 27 of 34


Table 1. Summary of the [M+Na]+ Ions Identified in the ESI-LIT-MS Spectra Acquired from the Thermally Treated Mixtures. Number (n) of pentose (Pent) units 1 2 3 4 5 6 Non-hybrid compounds composed by pentose residues and derivatives [Pentn+Na]+ 173a 305 †437b †569 †701 + c [Pentn-2H+Na] (-2) 435 [Pentn-H2O+Na]+ (-18) 287 †419 †551 †683 [Pentn-2H2O+Na]+ (-36) 401 [Pentn-3H2O+Na]+ (-54) 383 [Pentn-C2H4O2+Na]+ (-60) 377 †509 †641 + [Pentn-C3H4O2+Na] (-72) †365 †497 †629 (-78) 227 [Pentn-C2H6O3+Na]+ [Pentn-C3H4O3+Na]+ (-88) 217 [Pentn-C3H6O3+Na]+ (-90) †347 †479 †611 ‡743 (-112) 193 325 [Pentn-C2H8O5+Na]+ Proposed assignments

Non-hybrid compounds composed by hexose residues and derivatives 203 †365 †527 [Hexn+Na]+ [Hexn-H2O+Na]+ (-18) 185 †347 †509 (-54) 149 311 473 [Hexn-3H2O+Na]+ [Hexn-4H2O+Na]+ (-72) 293 455 [Hexn-5H2O+Na]+ (-90) †437 [Hexn-6H2O+Na]+ (-108) 257 †419 †581 ‡743 Pentose-hexose hybrid oligosaccharides and derivatives [PentHexn+Na]+ †497 †659 [PentHexn-H2O+Na]+ (-18) 317 †479 †641 [PentHexn-3H2O+Na]+ (-54) 281 443 605 (-72) 425 587 [PentHexn-4H2O+Na]+ [PentHexn-5H2O+Na]+ (-90) †569 (-108) 389 †551 †713 [PentHexn-6H2O+Na]+ [Pent2Hexn+Na]+ †629 [Pent2Hexn-H2O+Na]+ (-18) 449 †611 [Pent2Hexn-3H2O+Na]+ (-54) 413 575 [Pent2Hexn-4H2O+Na]+ (-72) 395 [Pent2Hexn-5H2O+Na]+ (-90) †701 [Pent2Hexn-6H2O+Na]+ (-108) 521 †683 [Pent3Hexn-H2O+Na]+ (-18) †581 ‡743 [Pent3Hexn-3H2O+Na]+ (-54) 545 [Pent3Hexn-4H2O+Na]+ (-72) †527 [Pent4Hexn-H2O+Na]+ (-18) †713 [Pent4Hexn-3H2O+Na]+ (-54) 677 [Pent4Hexn-4H2O+Na]+ (-72) †659 Only the ions observed with a relative abundance equal or higher than 15% in at least two of the ESI-LIT-MS spectra were considered. am/z values. bIons attributed to different compounds having the same nominal mass are marked with a symbol: † for two and ‡ for three compounds. cValues in brackets are the m/z value differences compared to the [M+Na]+ ions of the corresponding non-modified oligosaccharide.



Table 2. Diagnostic Neutral Losses and Product Ions Observed Under ESI-CID-Msn Conditions. Neutral losses and product ions Neutral loss (Da) 96 114 126 132 144 148 150 162 180 294 Product ion (m/z) 149 155 167 171 173 185 203 335

Assignment(s)

(Pent-2H2O)res; (Pent-3H2O) (Pent-H2O)res; (Pent-2H2O) (Hex-3H2O); (Hex-2H2O)res Pentres; (Pent-H2O) (Hex-H2O)res; (Hex-2H2O) (Pent-2H) Pent Hexres; (Hex-H2O); Combined loss of Pentres and CH2O (30 Da) from cross-ring cleavage Hex Combined loss of Pentres and Hexres

[Hex-3H2O+Na]+ [Pentres+Na]+; [Pent-H2O+Na]+ [(Hex-H2O)res+Na]+; [Hex-2H2O+Na]+ [Pent-2H+Na]+ [Pent+Na]+ [Hexres+Na]+; [Hex-H2O+Na]+ [Hex+Na]+ [PentHex+Na]+


Page 28 of 34

Page 29 of 34


Table 3. Summary of the Pairs of Isobaric/Isomeric Compounds and One Set of Three Compounds having the Same Nominal Mass Identified as [M+Na]+ Ions in the ESI-MS Spectra of the Thermally Treated Mixtures. Ion (m/z)

Proposed assignments

347

[Pent3-C3H6O3+Na]+ [Hex2-H2O+Na]+

Formula C12H20NaO10

+

Characteristic neutral losses 162 Da 144 Da

132 Da Pentres

Hexres/(Hex-H2O)

365

[Pent3-C3H4O2+Na] [Hex2+Na]+

C12H22NaO11

419

[Pent3-H2O+Na]+ [Hex3-6H2O+Na]+

C15H24NaO12 C18H20NaO10

Pentres/(Pent-H2O)

C15H26NaO13

Pentres

[Pent3+Na] 437

+

[Hex3-5H2O+Na]+

C18H22NaO11

479

[Pent4-C3H6O3+Na]+ [PentHex2-H2O+Na]+

C17H28NaO14

497

[Pent4-C3H4O2+Na]+ [PentHex2+Na]+

C17H30NaO15

509

[Pent4-C2H4O2+Na]+ [Hex3-H2O+Na]+

C18H30NaO15

+

Pentres

(Pentres+CH2O) Hexres

(Hex-H2O)res

a

Hexres

(Hex-3H2O)

Hexres

(Hex-2H2O)

Pentres Pentres

(Pentres+CH2O)a Hexres/(Hex-H2O)

(Hex-H2O)res

Pentres Pentres

(Pentres+CH2O)a Hexres

Pentres


[Hex3+Na] [Pent3Hex-4H2O+Na]+


Pentres

551

[Pent4-H2O+Na]+ [PentHex3-6H2O+Na]+


Pentres/(Pent-H2O) Pentres

+

C20H34NaO17

Pentres

C23H30NaO15

Pentres

Hexres

(Hex-2H2O)

Pentres

Hexres (Hex-H2O)

(Hex-H2O)res

Pentres Pentres


[Pent4+Na]

[PentHex3-5H2O+Na]

+

581

[Hex4-6H2O+Na]+ [Pent3Hex-H2O+Na]+


611

[Pent5-C3H6O3+Na]+ [Pent2Hex2-H2O+Na]+

C22H36NaO18

+

Hexres (Hex-3H2O) Hexres

(Hex-3H2O)

C22H38NaO19

Pentres Pentres

(Pentres+CH2O) Hexres

641

[Pent5-C2H4O2+Na]+ [PentHex3-H2O+Na]+

C23H38NaO19

Pentres Pentres

(Pentres+CH2O)a Hexres/(Hex-H2O) Hexres

[PentHex3+Na] [Pent4Hex-4H2O+Na]+


Pentres Pentres

683

[Pent5-H2O+Na]+ [Pent2Hex3-6H2O+Na]+


Pentres/(Pent-H2O) Pentres

(Hex-H2O)res

a

[Pent5-C3H4O2+Na] [Pent2Hex2+Na]+

659

(Hex-H2O)res (Hex-3H2O)

Hexres

(Hex-3H2O)

+

C25H42NaO21

Pentres

(Pentres+CH2O)

[Pent2Hex3-5H2O+Na]+

C28H38NaO19

Pentres

Hexres

(Hex-2H2O)

713

[PentHex4-6H2O+Na]+ [Pent4Hex-H2O+Na]+


Pentres Pentres

Hexres (Hex-H2O)

(Hex-H2O)res

[Hex5-6H2O+Na]+ [Pent6-C3H6O3+Na]+ [Pent3Hex2-H2O+Na]+

C30H40NaO20

743

[Pent5+Na] 701

a

C27H44NaO22

(Hex-3H2O)/ (Hex-2H2O)res (Hex-3H2O)

629

+

(Hex-3H2O)/ (Hex-2H2O)res

(Hex-H2O)res

527

569

126 Da

a

(Hex-3H2O)

Hexres Pentres Pentres

Hexres/(Hex-H2O)

The corresponding product ion was observed with a relative abundance ≤10%.


(Hex-3H2O)/ (Hex-2H2O)res

(Hex-3H2O) (Hex-H2O)res


Man3

203

365

Na+

437 185

100

A50M50-T0

60 40 [Hexres+Na]+ 20 0

100

-60 Da

[Hex+Na]+ 185.0 203.0

250

-(Hexres+60 Da) 305.1

300

[M+Na]+ 527.2

-120 Da -90 Da 467.2 407.2 437.1

350 m/z

400

450

500

-Hexres -Hex 347.2 365.2

A50M50 - T1

[Hex3+Na]+

80 60

-H2O 509.2 -60 Da [M+Na]+ -120 Da -90 Da 467.2 527.2 479.2 407.2 437.2

20 0

[Hex+Na]+ -(Hexres+60 Da) 185.0 203.0 305.1

200

C 100

-48 Da

40 [Hexres+Na]+

250

300

350 m/z

-Hex 347.1

[HexPent-4H2O+Na]+

40 20 0

D 100

203.1

[PentPentres+Na]+ 263.0 287.1 305.1

200

250

[Hex+Na]+

300

-Pentres 395.1

400

-Pentres 395.1

80 60

[HexPent-4H2O+Na]+ [PentPentres+Na]+

40 20 0

[Hex+Na]+ 203.1

200

287.1 263.1 305.2 250

300

500

-H2O 509.2

-60 Da -(Hex-3H2O) 467.1 [M+Na]+ 401.1 461.1 -32 Da 527.3 495.1

350 m/z

A50M50 - T3

450

[Hex3+Na]+ and [Pent3Hex-4H2O+Na]+

-Hexres 365.1

A50M50 - T2

80 60

400

-Hexres 365.1 -Hex 347.1

350 m/z

450

401.1 437.2 400

500

[Hex3+Na]+ and [Pent3Hex-4H2O+Na]+

-90 Da

Relative Abundance

B

Relative Abundance

[Man3+Na]+

80

200

Relative Abundance

467

-Hexres -Hex 365.2 347.2

-(Hex-3H2O)

Relative Abundance

A

347

305

[M+Na]+ -H2O 527.2 -60 Da 467.2 509.2

450

Figure 1


500

Page 30 of 34


C

347 215

Ara3

173

Na+

305

407

377 245 155

100

A75M25 - T0

-90 Da 347.1

80

-60 Da 377.1

20 0

-Pentres -(Pentres+60 Da) 305.1 -Pent [Pent+Na]+ 287.0 215.0 245.1 155.0 173.0 343.1 -(Pentres+90 Da)

[Pentres+Na]+

150

200

250

300 m/z

350

-30 Da

407.1 -H2O [M+Na]+ 419.1 437.1 400

450

60 40 20 0

-(Pentres+90 Da) -(Pentres+60 Da) -90 Da -Hexres -102 Da 347.1 -60 Da [Pent+Na]+ 377.1 275.0 335.1 155.1 173.0 215.0 245.0

150

100

[Hex-3H2O+Na]+ [Hex+Na]+

40 20 0

-H2O

+Na]+

[Hexres 149.1 185.1 203.1

150

200

-(Hex-2H2O) -90 Da 293.1 -Hex 347.1 257.1 317.2

250

300 m/z

350

419.3 -32 Da + 405.3 [M+Na] 437.4 400

250

300 m/z

350

400

450

[Pent3+Na]+

and [Hex3-5H2O+Na]+

-Pentres 305.1

A50M50 - T2

60 40 20 0

-(Pentres+60 Da) [Hex-3H2O+Na]+ [M+Na]+ -90 Da -60 Da -Hexres -(Pentres+90 Da) 347.1 377.1 -32 Da 437.2 275.1 -(Hex-3H2O) [Pent+Na]+ 405.1 311.1 215.1 245.1 149.0 173.1

100

200

A25M75 - T2

0

300 m/z

350

400

450

[Pent3+Na]+ and [Hex3-5H2O+Na]+

-Hexres 275.1

-(Hex-3H2O) -60 Da -H2O 311.1 -90 Da 377.1 419.1 347.1 -(Hex-2H2O) -32 Da [M+Na]+ 405.1 437.3 293.1 -102 Da -Hex 257.1 335.1

305.1

60 [Hex-3H O+Na]+ 2 [Hex+Na]+ 40 20

250

-Pentres

80

[Hexres+Na]+ 149.1 185.0 203.1

150

450

200

[M+Na]+ 437.2

80

E Relative Abundance

-(Hex-3H2O) 311.2

80 60

[Hex3-5H2O+Na]+

-120 Da

Relative Abundance

100

-Hexres 275.1

Man3 - T2

[Pent3+Na]+ and [Hex3-5H2O+Na]+

[Pentres+Na]+

150

B

-Pentres 305.0

A75M25 - T2

80

D

60 40

100

[Ara3+Na]+

Relative Abundance

A Relative Abundance

287

Relative Abundance

Page 31 of 34

Figure 2

31

ACS Paragon Plus Environment

200

250

300 m/z

350

400

450

20 0

[HexHexres+Na]+ [PentHex+Na]+ [Hex2+Na]+ 335.1

347.1

203.1

275.1 317.1

200

407.1 467.1

300

400

-H2O

641.2 [M+Na]+ 659.2

-60Da 539.2 599.2 -90Da 569.2

365.1

500

m/z 185

D

600

m/z

20 0

[HexHexres+Na]+ [Pent(18Hex)+Na]+

205.1 200

317.1 275.2

[Hex(18Hex)+Na]+

347.1 337.1

300

―O―

m/z 205

m/z 529

m/z 479

479.2 -62Da

367.1 -122Da

437.2 400

539.2 500

-H218O

40

[18Hex+Na]+

Relative Abundance

60

m/z 185

-(18Hex)

80

Na+

Pent

Hex―O―Hex―O―18Hex

-Hexres 529.2 499.2

100

m/z 347

m/z 499 -Pentres

B

529

Hex―O―Hex―O―18Hex

Pent

[M+Na]+

661.5

599.2 641.2 600

m/z

Figure 3


m/z 529

Na+

m/z 155

40

Na+

Pent

m/z 499 m/z 337

―O―

60

C

―O―

80

-Pentres 527.1 -Hexres 497.1 -Hex 479.1 -120Da

m/z 155

100

[Hex+Na]+

Relative Abundance

A

Page 32 of 34

m/z 155


m/z 367

m/z 205

Hex―O―Hex―O―18Hex m/z 317

m/z 479

Page 33 of 34


A

m/z 281

m/z 149

Pent―O―Pent―O―(Hex-3H2O) m/z 287

-Pentres 281.0

100 80 60 40 20 0

[Hex-3H2O+Na]+

Relative Abundance

m/z 155

Na+

-(Hex-3H2O) 287.0 [Pentres+Na]+

149.0

155.0

150

+ -90 Da -60 Da -H2O [M+Na] 413.1 323.0 353.0 395.1

-(Pentres+30 Da) 251.0

200

250

300

350

400

m/z

B

m/z 283

m/z 151 Na+

Pent―O―Pent―O―18(Hex-3H2O)

80 60 40 20 0

[18(Hex-3H2O)+Na]+

Relative Abundance

m/z 155

100

151.1 150

m/z 287

-Pentres 283.1

-18(Hex-3H2O) 287.1 [Pentres+Na]+

155.0 200

-90 Da -60 Da 325.1 355.1

-(Pentres+30 Da) 253.1

250

300

350

m/z

Figure 4


[M+Na]+ 415.2

400


Table of Contents Graphic


Page 34 of 34

Nonenzymatic Transglycosylation Reactions Induced by Roasting

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