Enzymatic Synthesis of a Novel Neuroprotective Hydroxytyrosyl

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ENZYMATIC SYNTHESIS OF A NOVEL NEUROPROTECTIVE HYDROXYTYROSYL GLYCOSIDE Manuel Nieto Dominguez, Laura I. de Eugenio, Pablo Peñalver, Efres Belmonte-Reche, Juan C. Morales, Ana Poveda, Jesús Jiménez-Barbero, Alicia Prieto, Francisco J. Plou, and María Jesús Martínez J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04176 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 12, 2017

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Journal of Agricultural and Food Chemistry

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ENZYMATIC SYNTHESIS OF A NOVEL NEUROPROTECTIVE

2

HYDROXYTYROSYL GLYCOSIDE

3

Manuel Nieto-Domíngueza, Laura I. de Eugenioa, Pablo Peñalverb, Efres Belmonte-

4

Recheb, Juan Carlos Moralesb, Ana Povedac, Jesus Jimenez-Barberoc, Alicia Prietoa,

5

Francisco J. Ploud,*, María Jesús Martíneza,*.

6

a

7

Spain.

8

b

9

de Ciencias de la Salud, Avenida del Conocimiento, s/n, 18016, Armilla, Granada,

Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040, Madrid,

Instituto de Parasitología y Biomedicina "López - Neyra", CSIC, Parque Tecnológico

10

Spain.

11

c

12

Bizkaia building 801A, 48160 Derio, Biscay, Spain.

13

d

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*Tel: +34-91-837-31-12; e-mail: [email protected] (M.J. Martínez)

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*Tel: +34-91-585-4869; e-mail: [email protected] (F.J. Plou)

Center for Cooperative Research in Biosciences, Parque Científico Tecnológico de

Instituto de Catálisis y Petroleoquímica, CSIC, Marie Curie, 2, 28049, Madrid, Spain.

16

1 ACS Paragon Plus Environment


17

ABSTRACT

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The eco-friendly synthesis of non-natural glycosides from different phenolic

19

antioxidants was carried out using a fungal β-xylosidase in order to evaluate changes in

20

their bioactivities. Xylosides from hydroquinone and catechol were successfully formed

21

although the best results were obtained for hydroxytyrosol, the main antioxidant from

22

olive oil. The formation of the new products was followed by thin layer

23

chromatography, liquid chromatography and mass spectrometry. The hydroxytyrosyl

24

xyloside was analyzed in more detail, in order to maximize its production and evaluate

25

the effect of glycosylation on some hydroxytyrosol properties. The synthesis was

26

optimized up to the highest production reported for a hydroxytyrosyl glycoside. The

27

structure of this compound was solved by 2D-NMR and identified as 3,4-

28

dihydroxyphenyl-ethyl-O-β-D-xylopyranoside. Evaluation of its biological effect

29

showed an enhancement of both its neuroprotective capacity and its ability to ameliorate

30

intracellular levels of reactive oxygen species.

31 32 33

KEYWORDS Glycosylation; xyloside, regioselectivity, plant phenols, olive oil

34 35

INTRODUCTION

36

Olive oil is one of the main commodities of the Mediterranean agro-industry and, as a

37

consequence, olive production in Southern Europe amounted to 9 million tons in 2014

38

(FAOSTAT). Both olive oil and its by-products contain high amounts of phenols, which

39

implies an invaluable source of bioactive compounds1. Among them, the most potent

40

antioxidant is 3,4-dihydroxyphenylethanol, a phenylethanoid commonly named

41

hydroxytyrosol (HT), known for its strong anti-inflammatory and neuroprotective


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properties2;3. In recent years several studies have demonstrated the effect of HT on the

43

prevention of cardiovascular problems, cancer and other chronic diseases4-6.

44

HT is well absorbed in a dose-dependent manner, but it displays a poor

45

bioavailability for being quickly converted into its sulphate and glucuronide metabolites

46

by a classic phase I/II biotransformation7. In this sense, glycosylation has demonstrated

47

to be a useful approach not only for enhancing bioavailability, but also other specific

48

properties of phenolic antioxidants, such as solubility, stability or biosafety8-10.

49

Glycosylation can be accomplished by synthetic chemical approaches, but they imply

50

many steps of protection and de-protection11;12, low specificity and toxic side-products.

51

On the contrary, the use of glycosidases or glycosyltransferases for the same purpose is

52

an eco-friendly and highly specific alternative9;13. Several reports deal with in vitro

53

synthesis or isolation of physiological metabolites of HT14;15, but there is little

54

information about the production of non-natural glycosides, which may possess

55

enhanced properties or simplify in vivo studies16 to determine its role.

56

The aim of this work was double. On the one hand, to develop a one-step method to

57

enzymatically synthesize non-natural glycosides of polyphenolic antioxidants with

58

commercial interest and, on the other hand, to assess the effect of xylosylation on some

59

of the bioactivities reported for these antioxidants. The GH3 β-xylosidase BxTW1 from

60

Talaromyces amestolkiae, heterologously expressed in Pichia pastoris (rBxTW1), was

61

used as catalyst. This enzyme, previously characterized in our group, catalyzed the

62

regioselective transxylosylation of substrates and showed to have remarkable acceptor

63

promiscuity17;18. The current work reports the screening of several antioxidants, the

64

regioselective and efficient xylosylation of HT, and positive results for catechol and

65

hydroquinone (HQ). In addition, as an approach for improving the properties of

66

bioactive compounds, the ability to reduce the level of intracellular reactive oxygen



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species (ROS) and the neuroprotective effect of this novel HT xyloside have been

68

evaluated.

69 70

MATERIALS AND METHODS

71

Materials

72

Hydroxytyrosol was provided by Seprox Biotech (Madrid, Spain). Quercetin, ferulic

73

acid,

74

lipopolysaccharides from Escherichia coli 055:B5 (LPS), MTT 3-(4,5-dimethyl-2-

75

thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay kit, 2´,7´-dichlorofluorescein

76

diacetate (H2DCFDA) and 4-nitrophenyl-β-D-xylopyranoside (pNPX) were purchased

77

from Sigma-Aldrich (St. Louis, MO, USA). Xylobiose was from TCI Europe

78

(Zwijndrecht, Belgium). Hydroquinone was from Acros Organics. Catechol was

79

purchased from Fisher Scientific (Loughborough, UK). Resveratrol (RES) was from

80

Seebio Biotechnology, Inc. (Shanghai, China). Epigallocatechin gallate (EGCG) was

81

provided from Zhejiang Yixin Pharmaceutical Co., Ltd. (Jinhua Shi, China). Silica gel

82

G/UV254 polyester sheets (0.2 mm thickness and 40 x 80 mm plate size) for thin layer

83

chromatography (TLC) were provided by Macherey-Nagel (Düren, Germany). BxTW1

84

from T. amestolkiae, heterologously expressed in P. pastoris, was produced and purified

85

as previously reported18.

gallic

acid,

(±)-α-tocopherol,

L-ascorbic

acid,

hesperidin,

hesperetin,

86 87

Enzyme activity and acceptor screening

88

Enzyme

89

Transxylosylation reactions were carried out using 0.5 and 5 U/mL of rBxTW1 in the

90

presence of 0.1% BSA and 50 mM sodium formate buffer (pH 3.0). 40 mM xylobiose

91

(11.3 g/L) was used as donor and a series of plant phenolic antioxidants were tested as

activity

was

defined

and

determined

as


previously

reported17.

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acceptors. Negative control reactions were carried out with no enzyme added.

93

Concentration of acceptors and selected co-solvents are listed in Table 1. Acceptor

94

stocks of gallic, ferulic and ascorbic acids were neutralized to pH 3 with sodium

95

hydroxide to prevent pH changes of the reactions. Reactions were carried out at 50 °C

96

and 1200 rpm with a final volume of 200 µL.

97

Samples (2 µL) were collected at different time intervals from 10 min to 72 h and

98

reactions were followed by TLC developed with ethyl acetate/methanol/H2O 10:2:1

99

(v/v). Detection was performed under 254 nm UV light, and the pattern of spots in each

100

reaction and its no-enzyme control were compared.

101 102

Mass Spectrometry

103

Conventional mass spectrometry analyses were performed on a hybrid QTOF analyzer,

104

model QSTAR, Pulsar I, from AB Sciex (Framingham, MA, USA). Reaction samples

105

were analyzed by direct infusion and ionized by electrospray (ESI-MS) with methanol

106

as mobile phase in positive reflector mode.

107

HR-MS analysis was carried out by FIA-ESI-MS on a QTOF Agilent G6530A

108

Accurate Mass Q-TOF LCMS system (Agilent Technologies, Santa Clara CA, USA).

109

The sample was directly infused and ionized by ESI in negative reflector mode.

110

Ionization was enhanced by JetStream technology and the mobile phase was

111

H2O/Formic acid 99.9:0.1 (v/v). Data were processed with Masshunter Data Acquisition

112

B.05.01 and Masshunter Qualitative Analysis B.07.00 software (Agilent Technologies).

113 114

Optimization of transxylosylation

115

The reaction with HT or HQ as acceptors and xylobiose as donor was optimized using a

116

range of donor and acceptor concentrations as well as different reaction times. The



117

reactions were carried out in 50 mM sodium formate buffer (pH 3.0), at 50 °C and 1200

118

rpm (see Supporting Information for the assayed conditions).

119

At the selected time intervals, reactions were stopped by enzyme denaturation at 100 °C

120

for 5 min and stored at 4 °C. The concentration of the new xylosides was determined by

121

HPLC as described below. Concentration data, in terms of molarity, were used to

122

calculate reaction yields according to the following equation:

123

Yield (%) = [Product]/[Acceptor]0 ×100

124 125

Reactions were performed in triplicate. Standard deviations were in all cases lower than 5%.

126

In order to identify the HT-xyloside (HTX) produced and to assay its biological

127

activity, the reaction was scaled-up to 1 mL in the conditions of highest production: 350

128

mM xylobiose, 300 mM HT, 50 mM sodium formate buffer (pH 3.0) at 50 °C and 1200

129

rpm for 2 h. The reaction was stopped by incubation at 100 °C for 5 min without loss of

130

product by physicochemical hydrolysis (see Supporting Information). The reaction

131

mixture was analyzed by HPLC as described below. Fractions corresponding to HTX

132

were collected and the solvent was evaporated. The product was stored at 4 °C.

133 134

HPLC analysis

135

HPLC analysis was performed in an instrument equipped with a semipreparative HPLC

136

quaternary pump 600E (from Waters Corporation, Milford, MA, USA), an autosampler

137

ProStar (Agilent Technologies) and a photodiode-array detector controlled by software

138

Varian LC Workstation 6.41 (Agilent Technologies). The reaction products were

139

detected monitoring their absorbance at 241 and 268 nm, using the chromatogram

140

recorded at 268 nm for quantitation. The sample was loaded in an ACE 3 C18-PFP

141

column (15 x 4.6 mm, 5 µm, Advanced Chromatography Technologies Ltd., Aberdeen,


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United Kingdom) that was kept constant at 40 °C. The mobile phase was methanol/H2O

143

20:80 v/v (both containing 0.1% acetic acid) at 0.7 mL/min for 12 min in the case of

144

samples with HT as acceptor. Methanol/H2O 5:95 v/v (both containing 0.1% acetic

145

acid) at 0.7 mL/min for 15 min was selected for HQ assays. The quantitation was

146

carried out using the Varian Star LC Workstation 6.41. Calibration curves of HT and

147

HQ standards were used for the quantification of HT- and HQ-xylosides, respectively.

148

HTX was purified by semipreparative HPLC. The 1 mL reaction mixture was

149

loaded onto the semipreparative column ACE 5 C18-PFP 15 x 10 mm, 5 µm (Advanced

150

Chromatography Technologies Ltd.). The mobile phase was methanol/H2O 20:80 v/v

151

(both containing 0.1% acetic acid) at 5.5 mL/min for 12 min. A three-way flow splitter

152

(model Accurate, Dionex, Sunnyvale, CA, USA) was used. The fractions containing the

153

product were pooled and the solvent evaporated.

154 155

Nuclear Magnetic Resonance (NMR)

156

The structure of the xyloside was determined using a combination of 1D (1H, 1D-

157

selective NOESY experiments) and 2D (COSY, DEPT-HSQC, NOESY) NMR

158

techniques. The sample was dissolved in deuterated water (ca. 10 mM), recording

159

spectra on a Bruker AV-III 800 spectrometer (Bruker, Billerica, MA, USA) equipped

160

with a TCI cryoprobe with gradients in the Z axis, at a temperature of 298 K. Chemical

161

shifts were expressed in parts per million with respect to the 0 ppm point of DSS (4-

162

dimethyl-4-silapentane-1-sulfonic acid), used as internal standard. All pulse sequences

163

used were provided by Bruker. The values set for the DEPT-HSQC experiment were of

164

7 ppm and 2K points, for the 1H dimension, and 160 ppm and 256 points for the

165

dimension. For the homonuclear experiments COSY and NOESY, 7 ppm windows were


13

C


166

used with a 2K x 256 points matrix. For the NOESY and 1D-selective NOESY

167

experiments, mixing times of 500-600 ms were applied.

168 169

Evaluating the biological properties of HTX

170

The potential of the HTX as neuroprotective compound and its capacity to ameliorate

171

intracellular ROS levels were assayed and compared with those of HT and RES, a well-

172

characterized antioxidant. The tests were carried out in vitro, in cell cultures. SH-S5Y5

173

neurons were cultured in collagen-pretreated Petri-dishes with Dulbecco's Modified

174

Eagle’s Medium (DMEM): Nutrient Mixture F-12 medium supplemented with

175

Penicillin/Streptomycin and 10% inactivated fetal bovine serum (iFBS). RAW 264.7

176

macrophages were cultured in DMEM high glucose medium supplemented as above.

177

To carry out all the following assays, the tested compounds were dissolved in

178

dimethyl sulfoxide (DMSO) and added at different concentrations (1, 10 and 100 µM)

179

to the cell cultures. The final DMSO percentage in each cell culture was adjusted to 1%.

180

Averages and standard deviations of at least eight different readings from various

181

experiments were calculated.

182

The cytotoxicity of the tested compounds to macrophages and neurons was

183

evaluated before conducting the ROS and neuroprotection assays. Neuron assays were

184

done in collagen-pretreated 96-well plates by seeding 2 x 104 neurons per well in a 100-

185

µL volume, incubating for 24 h before compound addition. Macrophage assays were

186

done in 96-well plates by seeding 2.5 x 104 macrophages per well in a 100-µL volume

187

and incubating for 4 h before compound addition. Cell viability was evaluated 24 h after

188

compound addition by the mitochondrial MTT assay, according to the manufacturer.

189

The intracellular ROS level was determined on macrophages that were cultured and

190

plated as described for the cell viability assay. After 2-h incubation of the cells with


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solutions of the tested compounds in DMSO, 100 ng/mL of LPS were added in order to

192

induce ROS production. ROS levels were evaluated 24 h after compound addition by

193

using the ROS-sensitive H2DCFDA staining method. Following treatments, the medium

194

was removed and cells were incubated with 25 µM H2DCFDA for 2 h at 37 °C in the

195

dark. H2DCFDA, a cell-permeable non-fluorescent compound, is intracellularly de-

196

esterified

197

dichlorofluorescein (DCF) in the presence of intracellular ROS upon oxidation.

198

Fluorescence intensity was measured at an excitation wavelength of 485 nm and an

199

emission wavelength of 530 nm using a multimode microplate reader (TECAN,

200

Männedorf, Switzerland). The direct effect of the assayed compounds on the

201

fluorescence intensity was evaluated in cell-free controls, in order to discard potential

202

artifacts (see Supporting Information).

and

turns

into

the

highly

fluorescent

permeant

molecule

2,7-

203

To determine the neuroprotective effect, neurons were also cultured and plated as

204

described in the cell viability assay. The compounds were added at three different

205

concentrations and incubated for 10 min before adding 100 µM hydrogen peroxide. Cell

206

viability was evaluated 24 h after compound addition by mitochondrial MTT assay.

207

Neuron viability was normalized to the H2O2 negative control.

208 209

RESULTS AND DISCUSSION

210

Screening of phenolic plant antioxidants as transxylosylation acceptors

211

A previous report on rBxTW1 described the transxylosylation of xylobiose with 2,6-

212

hydroxynaphthalene, obtaining the anti-tumorigenic 2-(6-hydroxynaphthyl) β-D-

213

xylopyranoside with a modest yield of 0.47 g/L. In the same work the wide acceptor

214

versatility of rBxTW1 was demonstrated by a high-throughput analysis18.



215

Because of its acceptor promiscuity, the enzyme was considered a potential

216

candidate for glycosylating polyphenolic antioxidants, although the presence of bulky

217

aromatic rings and their high insolubility were supposed to decrease transxylosylation.

218

In this sense, the bioactive compound hydroxytyrosol was a good candidate for this

219

assay for containing an aliphatic chain with a primary alcohol group, which was

220

expected to be a suitable target for efficient glycosylation by rBxTW1. Besides this

221

compound, a panel of phenols from biomass were further explored because of their

222

commercial interest: resveratrol, quercetin, (±)-α-tocopherol, hesperetin, hesperidin,

223

gallic acid, L-ascorbic acid, epigallocatechin gallate (EGCG), ferulic acid, HQ and

224

catechol.

225

The screening was carried out at pH 3, using 40 mM xylobiose as the donor and 10

226

g/L of each phenolic antioxidant. This low pH value is optimum for rBxTW1 action,

227

enhances the stability of the acceptors, preventing self-oxidation, and allowed long

228

reaction times and long-term storage of the potential xylosides. The reactions were

229

followed by TLC, and spots likely corresponding to newly formed xylosides, were

230

detected under UV-light in reactions involving HT, HQ and catechol as the acceptors

231

(Fig. 1 and Supporting Information). No sign of glycoconjugates was observed for other

232

screened acceptor candidates.

233

The reaction mixtures obtained with HT, HQ and catechol were analyzed by ESI-

234

MS. The existence of mono-xylosylated derivatives was confirmed for each of the three

235

acceptors (Fig. 2 and Supporting Information). In addition, xylotriose was also detected

236

in the reactions with HQ and catechol, indicating that xylobiose itself could also serve

237

as acceptor17.

238

Hydroquinone and catechol are well-known phenolic antioxidants. HQ is the most

239

used skin-whitening agent for its role as tyrosinase inhibitor, but there is a rising


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concern about its safety on human health and industry is looking for safer and active

241

hydroquinone derivatives19. The xyloside from hydroquinone has been reported as an

242

alternative for inhibiting tyrosinase20, so it constitutes an interesting compound to be

243

synthesized by rBxTW1.

244

Regarding catechol, it is the core of many compounds used as drugs in the

245

treatment of bronchial asthma, hypertension, Parkinson's disease, myocardial infarction

246

or even HIV21;22. Catechyl glycosides, as the catechyl xyloside reported in this work,

247

may lead to novel drugs with improved functionalities23. The enzymatic synthesis of

248

catechyl xyloside was previously reported by Chiku et al.24 using a bacterial xylanase,

249

but the process produced a mixture of glycoconjugates with one to four units of xylose.

250 251

Reaction monitoring by HPLC and evaluation of catalysis specificity

252

Upon preliminary analysis of the reactions by TLC, the positive hits were analyzed by

253

HPLC for a deeper insight into the regio- and stereo-specificity in transxylosylation of

254

these compounds catalyzed by rBxTW1. Different mixtures of methanol and water were

255

employed as mobile phases in order to get the best separation. Surprisingly, peaks

256

corresponding to the catechyl-xylosides were not detected, which could be probably

257

attributed to the low concentration of the xyloside formed.

258

The number of HT- and HQ-xylosides was determined by HPLC. Compounds

259

containing an aromatic ring (derived from HQ or HT) were detected by measuring the

260

absorbance at 241 and 268 nm, and hence the remaining xylobiose, xylose or other side

261

products did not interfere with the analysis. The chromatograms displayed a single new

262

peak for both acceptors, thus confirming monoglycosylation. These results suggest that

263

rBxTW1-catalyzed transxylosylation was highly selective, although the possibility of

264

producing other secondary products as xylobiosides or, in the case of HT, also of



265

different mono-xylosides in amounts below the detection limits of the techniques used,

266

cannot be ruled out.

267 268

Determination of the acceptor/donor balance for maximizing the synthesis yield

269

Trying to find optimal conditions, we tested the influence of donor and acceptor

270

concentration in the reaction mixture, as well as the degree of hydrolytic activity of the

271

enzyme on the synthesized products. HPLC was used to determine the concentration of

272

the xyloside under the different experimental conditions (see Supporting Information).

273

All assays using HT as acceptor contained 18 U/mL of rBxTW1. The effect of HT

274

concentration was analyzed in the range of 75-600 mM, setting xylobiose at 40 mM and

275

following HTX production along the time (Fig. 3A). Then, the HT concentration that

276

yielded the highest amount of xyloside was selected for assessing a range of

277

concentrations of xylobiose (Fig. 3B).

278

Figure 3A shows that the maximal production of the xyloside was quickly achieved

279

at all the HT concentrations tested. However, with 75 and 150 mM HT a dramatic

280

decrease of the product occurred beyond the first 10 min. This suggests that the HT-

281

xyloside is also a substrate for rBxTW1, causing the kinetic equilibrium

282

transglycosylation-hydrolysis to shift towards hydrolysis when the transglycosylation

283

product is available at similar concentrations to the donor. However, when the reaction

284

medium contained 300 and 600 mM HT, the amount of xyloside remained stable at least

285

for 4 hours after reaching the maximum. Then, at high HT concentrations, the

286

hydrolysis-transxylosylation balance shifts towards the synthesis. HTX can act as the

287

donor of a new transxylosylation reaction, attaching the xylosyl moiety to another HT

288

molecule, maintaining the overall concentration of the product. The lower production


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reached with 600 mM HT in comparison to 300 mM could be due to substrate

290

inhibition.

291

Figure 3B illustrates how the production of xyloside increases in parallel to the

292

donor concentration, with 300 mM HT as acceptor according to the previous

293

experiment. The amount of xyloside produced in reactions with 40 mM xylobiose was

294

virtually twice of that obtained with 20 mM of the donor. Nevertheless, this trend was

295

less significant at xylobiose concentrations over 80 mM, with HTX yields around 30%

296

higher for each donor increment (2-fold). Taking into account that the disaccharide is

297

also an efficient acceptor for rBxTW118, as demonstrated by the synthesis of xylotriose

298

as co-product (Fig. 2), this effect may be due to the competition between xylobiose,

299

when present in high excess, and HT.

300

As defined before, the reaction yield describes the molar ratio between product and

301

acceptor. Among the conditions assayed, the best result (26.6% yield) was attained with

302

300 mM HT and 350 mM xylobiose, yielding 77.7 mM of HT-xyloside (22.2 g/L) in 2

303

h. This production is the highest reported for a non-natural hydroxytyrosyl glycoside

304

synthesized by transglycosylation. Trincone et al.16 reported 45% conversion for the

305

glucosylation of HT catalyzed by the α-glucosidase from Aplysia fasciata, but they used

306

a low concentration of HT (5 mM), which led to a scarce net production, and a high

307

excess (30:1) of maltose. In addition, the process was not chemo- or regioselective,

308

giving rise to the three possible glucosides: one from the aliphatic alcohol and one from

309

each of the phenolic hydroxyls. On the contrary, rBxTW1 catalyzes the synthesis of

310

high concentrations of a single xyloside (or at least of a very predominant one), which

311

simplifies product purification and reaction control.

312

The same experimental design was followed to evaluate the effect of HQ and

313

xylobiose concentration on the production of HQ-xyloside (HQX). The preliminary



314

assay, with xylobiose at 40 mM, revealed that synthesis and hydrolysis of the product

315

occurred very quickly (data not shown). Then, the enzyme concentration was lowered to

316

0.9 U/mL, and the formation of HQX was analyzed along a range of concentrations of

317

HQ (Fig. 4A) and xylobiose (Fig. 4B) over time. The highest yield (4.7%) was obtained

318

using 110 mM HQ and 160 mM xylobiose in 2 h, producing 5.2 mM (1.2 g/L) of HQ-

319

xyloside. This value is similar to those reported for other transglycosylation products of

320

HQ8;25 but far away from the high levels reported by Seo et al.26 and remarkably low in

321

comparison to the results displayed for HT. The HPLC profiles of the reaction mixtures

322

for HTX and HQX production, in optimum conditions, are presented in the Supporting

323

Information.

324 325

Product characterization by NMR and MS

326

The synthesis of HTX, the xyloside produced at higher yields, was scaled-up to 1-mL in

327

order to confirm its structure by NMR and to assay its biological properties. The HTX

328

was purified by semipreparative HPLC as described above, obtaining 21.4 mg of a

329

mixture of xyloside and acetic acid. The molar ratio of the mixture was determined to be

330

1:0.5 by NMR, corresponding to a purity of 90.5% of the xyloside. The reaction yield

331

was 23.4%, close to the theoretical value determined by HPLC (26.6%), and the isolated

332

product had the appearance of highly viscous oil with a pale yellow color.

333

The NMR signals were assigned using standard 1H, COSY and HSQC-edited

334

experiments. The observed chemical shifts (C1 and CA, see Fig. 5 and Table 2) as well

335

as the existence of a strong NOE between the anomeric Xyl H1 and the methylene HA

336

protons (see Supporting Information for the NMR spectra) permitted to unequivocally

337

identify the glycosylation product as 3,4-dihydroxyphenyl-ethyl-O-β-D-xylopyranoside.

338

To the best of our knowledge, the glycoside synthesized by rBxTW1 is a novel


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compound. The purified product was also analyzed by HR-MS obtaining the signals

340

corresponding to the (M−H)− anion of the xyloside (see Supporting Information for the

341

HR-MS spectrum). The theoretical molar mass and the exact mass of 3,4-

342

dihydroxyphenyl-ethyl-O-β-D-xylopyranoside were calculated to be 286.28 g/mol and

343

286.1053 Da (85.0090% abundance) respectively. The theoretical exact mass of the

344

(M−H)− anion was determined to be 285.0980 Da and the experimental value obtained

345

by HR-MS was 285.0985, validating the identity of the new xyloside.

346

These results confirmed the high chemo- and regioselectivivity of rBxTW1 for this

347

reaction and the preference of the enzyme for primary-alcohols as acceptors, proposed

348

in a previous paper17, as only this functional group of HT was confirmed to be modified

349

by transglycosylation. This preference may also serve to explain the higher yields

350

obtained for HT in comparison with HQ, with no primary alcohols.

351 352

Biological activity of 3,4-dihydroxyphenyl-ethyl-O-β-D-xylopyranoside (HTX)

353

Neuroprotection and oxidative stress amelioration are well-known effects of HT

354

itself3;27, therefore the assays focused on comparing its effects with those produced by

355

HTX and resveratrol, a well-known antioxidant with proven neuroprotective and ROS

356

inhibition capacities28.

357

First, cell viability in the presence of the tested substances was evaluated by adding

358

different concentrations of HTX, HT and RES to RAW 264.7 macrophage and SH-

359

SY5Y neuroblastoma cultures. None of these compounds but RES at 100 µM was toxic

360

to macrophages (Fig. 6A) or neurons (Fig. 6C). Treatment with 100 µM RES induced a

361

remarkable viability decrease, reducing it up to 44% in RAW macrophages, which

362

indicated certain cytotoxicity of this compound. This effect has been previously

363

reported in the literature29. Interestingly, the cell viability of neurons in the presence of



364

100 µM HTX increases markedly, to values above those of the controls, suggesting its

365

beneficial effect even in the absence of an external stress.

366

Once established the safety of HTX, its potential to alleviate intracellular ROS

367

levels and its neuroprotective activity were assayed and compared to those of the other

368

compounds tested. The former assays were carried out in the presence of LPS as

369

intracellular ROS trigger. ROS levels (Fig. 6B) were measured from the fluorescence

370

intensity of DCF as explained in the Experimental section. As a general rule, all

371

compounds produced a decrease in ROS regardless of the dose added to the cell culture,

372

but this effect was greater for HTX and RES, comparing to HT. It should be noted that

373

treatment with 100 µM HTX reduced ROS levels below 40% of the value detected in

374

the negative control. A similar trend was found upon treatment with 100 µM RES, but

375

considering the results from the cell viability tests, this effect can be attributed to RES

376

toxicity.

377

Regarding neuroprotection, the activity of the compounds evaluated was very

378

different (Fig. 6D). While for RES and HT non-significant neuroprotection from

379

oxidative damage was observed in the concentration range assayed, HTX is

380

neuroprotective at 10 and 100 µM. In fact 100 µM of HTX allow restoration of cell

381

viability to values equal or above those of non-challenged cells.

382

The absence of a protective effect of HT may seem surprising, since it has been

383

reported as a strong neuroprotective agent. However, the methodological differences

384

among different studies make it difficult a precise comparison of the results, and may

385

explain the lack of neuroprotection found in the current study3;30-32.

386

The results presented here confirm that, in the tested conditions, HTX is non-toxic

387

and lowers intracellular ROS levels to a greater degree than HT itself, displaying also

388

considerably higher neuroprotective effect. Thus, this xyloside might be a stronger


Page 16 of 33

Page 17 of 33


389

activator of the Nrf2 signaling pathway33. These preliminary analyses on the biological

390

activity of HTX suggest that uptake of the xyloside by the cell and its potential to cross

391

the blood-brain barrier may merit attention in further research.

392

Beyond the specific bioactivities evaluated, this work demonstrates that

393

xylosylation is a valid approach for the valorization of biomass’ compounds with

394

pharmacological interest. Given the abundance of xylan, a natural source of xylobiose,

395

the synthesis of novel bioactive xylosides by β-xylosidases may be an expanding field

396

in the near future.

397 398

ABBREVIATIONS

399

DCF: 2,7-dichlorofluorescein; DMEM: Dulbecco's Modified Eagle’s Medium; EGCG:

400

Epigallocatechin

401

Hydroquinone;

402

dihydroxyphenyl-ethyl-O-β-D-xylopyranoside;

403

xylopyranoside; RES: Resveratrol.

gallate; HT:

H2DCFDA:

hydroxytyrosol;

2´,7´-dichlorofluorescein HQX:

Xyloside pNPX:

of

diacetate

HQ;

HTX:

HQ: 3,4-

4-nitrophenyl-β-D-

404 405 406

ACKNOWLEGDMENTS The authors thank Inmaculada Alvarez for the HR-MS analysis.

407 408

FUNDING

409

This work was carried out with funding from projects BIO2013-48779-C4-1-R,

410

BIO2015-68387-R and RTC-2014-1777-3 from MINECO, FQM-7316 from Junta de

411

Andalucía and S2013/MAE-2972 from Comunidad de Madrid. M. Nieto-Domínguez

412

thanks the MINECO for an FPU fellowship.

413



414

SUPPORTING INFORMATION

415

List of tested conditions for the transxylosylation of hydroxytyrosol and hydroquinone;

416

stability assay of HTX; cell free controls for the amelioration of ROS levels; TLCs from

417

screening reactions with HT, HQ and catechol; HPLC profiles from transxylosylation

418

reactions with HT and HQ; complete NMR data for HTX identification; complete data

419

of mass spectrometry from transxylosylation reactions and pure HTX.

420


Page 18 of 33

Page 19 of 33


421 422 423

(1) Romero-Garcia, J.; Nino, L.; Martinez-Patino, C.; Alvarez, C.; Castro, E.; Negro,

424

M. Biorefinery based on olive biomass. State of the art and future trends. Bioresour.

425

Technol. 2014, 159, 421-432.

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Hydroxytyrosol is the major anti-inflammatory compound in aqueous olive extracts and

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impairs cytokine and chemokine production in macrophages. Plant Med. 2011, 77,

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1890-1897.

430

(3) De La Cruz, J.P.; Ruiz-Moreno, M.I.; Guerrero, A.; Reyes, J.J.; Benitez-Guerrero,

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A.; Espartero, J.L.; González-Correa, J.A. Differences in the neuroprotective effect of

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orally administered virgin olive oil (Olea europaea) polyphenols tyrosol and

433

hydroxytyrosol in rats. J. Agric. Food Chem. 2015, 63, 5957-5963.

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(4) Hu, T.; He, X.W.; Jiang, J.G.; Xu, X.L. Hydroxytyrosol and its potential therapeutic effects. J. Agric. Food Chem. 2014, 62, 1449-1455.

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(5) López de las Hazas, M.-C.; Piñol, C.; Maciá, A.; Motilva, M.J. Hydroxytyrosol

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and the colonic metabolites derived from virgin olive oil intake induce cell cycle arrest

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and apoptosis in colon cancer cells. J. Agric. Food Chem. 2017, 65, 6467-6476.

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(6) Jemai, H.; El Feki, A.; Sayadi, S. Antidiabetic and antioxidant effects of

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hydroxytyrosol and oleuropein from olive leaves in alloxan-diabetic rats. J. Agric. Food

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Chem. 2009, 57, 8798-8804.

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Ballesteros, A.O.; Plou, F.J. Enzymatic synthesis of α-glucosides of resveratrol with

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surfactant activity. Adv. Synth. Catal. 2011, 353, 1077-1086.

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F.J.; Morales, J.C. Antioxidant activity of resveratrol in several fish lipid matrices:

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Effect of acylation and glucosylation. J. Agric. Food Chem. 2010, 58, 9778-9786.

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(12) Alluis, B.; Dangles, O. Quercetin (=2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-

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4H-1-benzopyran-4-one) glycosides and sulfates: Chemical synthesis, complexation,

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and antioxidant properties. Helv. Chim. Acta 2001, 84, 1133-1156.

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(13) Mathew, S.; Adlercreutz, P. Regioselective glycosylation of hydroquinone to α-

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arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp. Biochem.

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(14) Lucas, R.; Alcantara, D.; Morales, J.C. A concise synthesis of glucuronide

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metabolites of urolithin-B, resveratrol, and hydroxytyrosol. Carbohydr. Res. 2009, 344,

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1340-1346.

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(15) Gomes, V.P.M.; Torres, C.; Rodríguez-Borges, J.E.; Paiva-Martins, F. A

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convenient synthesis of hydroxytyrosol monosulfate metabolites. J. Agric. Food Chem.

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2015, 63, 9565-9571.

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(16) Trincone, A.; Pagnotta, E.; Tramice, A. Enzymatic routes for the production of

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mono- and di-glucosylated derivatives of hydroxytyrosol. Bioresour. Technol. 2012,

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(17) Nieto-Dominguez, M.; de Eugenio, L.I.; Barriuso, J.; Prieto, A.; Fernandez de

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Toro, B.; Canales-Mayordomo, A.; Martinez, M.J. Novel pH-stable glycoside hydrolase

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family 3 β-xylosidase from Talaromyces amestolkiae: an enzyme displaying

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regioselective transxylosylation. Appl. Environ. Microbiol. 2015, 81, 6380-6392.

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(18) Nieto-Domínguez, M.; Prieto, A.; Fernández de Toro, B.; Cañada, F.J.; Barriuso,

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J.; Armstrong, Z.; Withers, S.G.; de Eugenio, L.I.; Marínez, M.J. Enzymatic fine-tuning

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for 2-(6-hydroxynaphthyl) β-D-xylopyranoside synthesis catalyzed by the recombinant

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β-xylosidase BxTW1 from Talaromyces amestolkiae. Microb. Cell Fact. 2016, 15, 1-

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15.

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(19) Draelos, Z.D. Skin lightening preparations and the hydroquinone controversy. Dermatol. Ther. 2007, 20, 308-313.

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(20) Chiku, K.; Dohi, H.; Saito, A.; Ebise, H.; Kouzai, Y.; Shinoyama, H.; Nishida,

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Y.; Ando, A. Enzymatic synthesis of 4-hydroxyphenyl β-D-oligoxylosides and their

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notable tyrosinase inhibitory activity. Biosci. Biotechnol. Biochem. 2009, 73, 1123-

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1128.

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(21) Bollini, M.; Domaoal, R.A.; Thakur, V.V.; Gallardo-Macias, R.; Spasov, K.A.;

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Anderson, K.S.; Jorgensen, W.L. Computationally-guided optimization of a docking hit

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to yield catechol diethers as potent anti-HIV agents. J. Med. Chem. 2011, 54, 8582-

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8591.

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spectrophotometric estimation of catechol derivatives in pharmaceutical preparations.

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Talanta 2001, 55, 1039-1046.

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(23) Kumar, M.; Rawat, P.; Rahuja, N.; Srivastava, A.K.; Maurya, R.

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Antihyperglycemic activity of phenylpropanoyl esters of catechol glycoside and its

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dimers from Dodecadenia grandiflora. Phytochemistry 2009, 70, 1448-1455. 21 ACS Paragon Plus Environment


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Characterization of a novel polyphenol-specific oligoxyloside transfer reaction by a

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family 11 xylanase from Bacillus sp KT12. Biosci. Biotechnol. Biochem. 2008, 72,

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2285-2293.

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(25) Kang, J.; Kim, Y.M.; Kim, N.; Kim, D.W.; Nam, S.H.; Kim, D. Synthesis and

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characterization of hydroquinone fructoside using Leuconostoc mesenteroides

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levansucrase. Appl. Microbiol. Biotechnol. 2009, 83, 1009-1016.

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(26) Seo, D.H.; Jung, J.H.; Ha, S.J.; Cho, H.K.; Jung, D.H.; Kim, T.J.; Baek, N.I.;

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Yoo, S.H.; Park, C.S. High-yield enzymatic bioconversion of hydroquinone to α-

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arbutin, a powerful skin lightening agent, by amylosucrase. Appl. Microbiol.

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Biotechnol. 2012, 94, 1189-1197.

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(27) Di Benedetto, R.; Vari, R.; Scazzocchio, B.; Filesi, C.; Santangelo, C.;

509

Giovannini, C.; Matarrese, P.; D'Archivio, M.; Masella, R. Tyrosol, the major extra

510

virgin olive oil compound, restored intracellular antioxidant defences in spite of its

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weak antioxidative effectiveness. Nutr. Metab. Cardiovasc. Dis. 2007, 17, 535-545.

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(28) Pangeni, R.; Sahni, J.K.; Ali, J.; Sharma, S.; Baboota, S. Resveratrol: Review on

513

therapeutic potential and recent advances in drug delivery. Expert Opin. Drug Deliv.

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2014, 11, 1285-1298.

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(29) Billack, B.; Radkar, V.; Adiabouah, C. In vitro evaluation of the cytotoxic and

516

anti-proliferative properties of resveratrol and several of its analogs. Cell Mol. Biol.

517

Lett. 2008, 13, 553-569.

518

(30) Cabrerizo, S.; Pedro De La Cruz, J.; Antonio Lopez-Villodres, J.; Munoz-Marin,

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J.; Guerrero, A.; Julio Reyes, J.; Teresa Labajos, M.; Antonio Gonzalez-Correa, J. Role

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of the inhibition of oxidative stress and inflammatory mediators in the neuroprotective


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521

effects of hydroxytyrosol in rat brain slices subjected to hypoxia reoxygenationo. J.

522

Nutr. Biochem. 2013, 24, 2152-2157.

523

(31) Schaffer, S.; Podstawa, M.; Visioli, F.; Bogani, P.; Mueller, W.E.; Eckert, G.P.

524

Hydroxytyrosol-rich olive mill wastewater extract protects brain cells in vitro and ex

525

vivo. J. Agric. Food Chem. 2007, 55, 5043-5049.

526

(32) Peng, S.; Zhang, B.; Yao, J.; Duan, D.; Fang, J. Dual protection of

527

hydroxytyrosol, an olive oil polyphenol, against oxidative damage in PC12 cells. Food

528

Funct. 2015, 6, 2091-2100.

529

(33) Forman, H.J.; Davies, K.J.A.; Ursini, F. How do nutritional antioxidants really

530

work: Nucleophilic tone and para-hormesis versus free radical scavenging in vivo. Free

531

Radic. Biol. Med. 2014, 66, 24-35.

532 533



534

FIGURE CAPTIONS

535

Figure 1. Positive acceptors for transxylosylation with rBxTW1. Hydroxytyrosol (A),

536

hydroquinone (B) and catechol (C).

537

Figure 2. Mass spectra of a transxylosylation reaction mixture with hydroxytyrosol (A),

538

hydroquinone (B) and catechol (C). The identified adducts are indicated, a:

539

Xylobiose+Na+; b: HT-Xyloside+Na+; c: 2HT+Na+; d1: HQ-xyloside+Na+; d2:

540

Catechyl-xyloside+Na+; e: Xylotriose+Na+.

541

Figure 3. Optimization of acceptor (A) and donor (B) concentration for the synthesis of

542

HTX. Reactions were performed at pH 3.0 and 50 °C. (A) Time course of the HT-

543

xyloside production varying the concentration of HT. Initial concentration of xylobiose

544

was fixed as 40 mM. (B) Relation between xyloside production and initial xylobiose.

545

Reactions contained 300 mM HT as acceptor.

546

Figure 4. Optimization of acceptor (A) and donor (B) concentration for the synthesis of

547

HQX. Reactions were performed at pH 3.0 and 50 °C. (A) Time course of the HQ-

548

xyloside production using 40 mM xylobiose and varying concentration of HQ. (B)

549

Relation between xyloside production and initial xylobiose by using the optimum

550

concentration of HQ (110 mM).

551

Figure 5. Structure of 3,4-dihydroxyphenyl-ethyl-O-β-D-xylopyranoside. Atoms are

552

labeled in agreement with Table 2.

553

Figure 6. Biological activity of 3,4-dihydroxyphenyl-ethyl-O-β-D-xylopyranoside:

554

Cells viability (A, C), intracellular ROS levels (B) and neuroprotection (D) were

555

evaluated in the presence of HTX and three commercial antioxidants. Assays were

556

performed on macrophage (A, B) and neuron (C, D) cell cultures. Controls with (+) and

557

without (-) the stress agent were included in every case.

558


Page 24 of 33

Page 25 of 33


TABLES Table 1. Phenolic antioxidants and co-solvents selected for the acceptor screening. Acceptor α-tocopherol Catechol EGCG Ferulic acid Gallic acid Hesperetin Hesperidin Hydroquinone Hydroxytyrosol L-ascorbic acid Quercetin Resveratrol

Concentration (g/L) 10 40 10 10 40 10 10 10 40 10 40 10 10 10 10 40 10 40

Co-solvent 20% Acetonitrile 50% Ethyl acetate 20% Acetonitrile 50% Ethyl acetate 20% Acetonitrile 50% Ethyl acetate 20% Acetonitrile 50% Ethyl acetate 20% Acetonitrile 50% Ethyl acetate



Table 2. Chemical shifts data (ppm) from 3,4-dihydroxyphenyl-ethyl-O-β-Dxylopyranoside.

Aryl-H6' Aryl-H2' Aryl-H5' H1-Xylose H3-Xylose H2-Xylose CH2(A)α CH2(A)β H4-Xylose H5-Xyloseα H5-Xyloseβ CH2(B)

δH (Multiplicity/J/nH) 6.57 (dd/8.1,2.1 Hz/1H) 6.67 (d/2.0 Hz/1H) 6.70 (d/8.1 Hz/1H) 4.21 (d/7.9 Hz/1H) 3.25 (t/9.2 Hz/1H) 3.07 (dd/9.3,7.9 Hz/1H) 3.65 (dt/10.2,7.1 Hz/1H) 3.84 (dt/10.1,7.0 Hz/1H) 3.43 (ddd/10.5,9.1,5.5 Hz/1H) 3.11 (dd/11.6,10.5 Hz/1H) 3.76 (dd/11.6,5.5 Hz/1H) 2.64 (t/7.0 Hz/2H)

δC 123.6 119.0 119.0 105.7 78.3 75.6 73.6 71.8 67.8 67.8 37.1

δ: chemical shift; d: doublet; dd: doublet of doublets; ddd: doublet of doublet of doublets; dt: doublet of triplets; t: triplet; J: coupling constant; nH: number of protons.


Page 26 of 33

Page 27 of 33


FIGURES

A)

B)

C)

Figure 1



A

a

8000

60000

b

332.13

309.1

6000

45000

4000 2000

30000 15000

360.34

301.15 251.08

0

Intensity (counts)

75000

331.13

0 250

B

300

d1 265.07

350

400

m/z

450

a 360.33

305.09

200000

60000

160000

45000

120000

30000

80000

15000

306.09

e

361.33

266.07

40000

437.14

0

Intensity (counts)

Intensity (counts)

c

305.09

10000

Intensity (counts)

Page 28 of 33

0 250

300

350

400

450

m/z

Intensity (counts)

a

d2 120000

305.08

265.07

600000

100000

500000

80000

400000

60000

300000 306.09

40000 20000

200000

360.33

e 437.13

266.07

100000

0

0 250

300

350

400

450

m/z

Figure 2


Intensity (counts)

C


A

75 mM HT

150 mM HT

300 mM HT

600 mM HT

[HT-X] (mM)

20 16 12 8 4 0 0

50

100

150

200

time (min)

B

90 80

1 hour

2 hour

70

[HT-Xyl] (mM)

Page 29 of 33

60 50 40 30 20 10 0 20

40

80

160

[Xb] (mM)

Figure 3


350


A [HQ-X] (mM)

4 1 hour 2 hour

3 2 1 0 50

150

250

350

450

[HQ] (mM)

B [HQ-Xyl] (mM)

5

1 hour 2 hour

4 3 2 1 0 10

20

40

80

160

[Xb] (mM)

Figure 4


350

Page 30 of 33

Page 31 of 33


Figure 5



140 120 100 80 60 40 20 0 HTX

HT

RES

Fluorescence Intensity (a.u.)

Compound

B

DMSO (-) DMSO (+)

100 µM 10 µM 1 µM

100 µM 10 µM 1 µM

140 120 100 80 60 40 20 0 HTX

HT

RES

Compound

LPS (-) LPS (+)

Viability (%)

A

C 100 µM 10 µM 1 µM

160 120 80

0 HTX

HT

RES

Compound

D 100 µM 10 µM 1 µM

120

Viability (%)

DMSO (-) DMSO (+)

40

100 80 60 40 20 0 HTX

HT

Compound

RES

H2O2 (-) H2O2 (+)

Viability (%)

200

Figure 6


Page 32 of 33

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+

+

β-xylosidase

23.4% yield

↑↑Neuroprotection ↓ROS

TOC graphic


Enzymatic Synthesis of a Novel Neuroprotective Hydroxytyrosyl

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