A Mild and Efficient Preparation of Phytosteryl Amino Acid Ester

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Functional Structure/Activity Relationships

A Mild and Efficient Preparation of Phytosteryl Amino Acid Ester Hydrochlorides and Their Emulsifying Properties Chengsheng Jia, xue xia, Ping Liu, Huiqi Wang, Jiarui Zhang, and Xiaoming Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07153 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 20, 2019

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Journal of Agricultural and Food Chemistry

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A Mild and Efficient Preparation of Phytosteryl Amino Acid Ester

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Hydrochlorides and Their Emulsifying Properties

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Chengsheng Jia *,⊥, Xue Xia⊥, Ping Liu, Huiqi Wang, Jiarui Zhang, Xiaoming Zhang

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State Key Laboratory of Food Science and Technology, School of Food Science and Technology,

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Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, China

1

ACS Paragon Plus Environment


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ABSTRACT

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The aim of this work was to produce a series of phytosteryl amino acid ester hydrochlorides by

11

two-step method, which involved esterification of phytosterols with N-tert-butoxycarbonyl

12

(BOC)-amino acid and deprotection of BOC group. The highest yield of over 95.0% was obtained

13

when the catalysts were the mixtures of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

14

hydrochlide (EDC), 4-dimethylaminopyridine (DMAP) and triethylamine. It was found that batch

15

charging of the reactants and catalysts was conducive to improving yield. In addition, over 99.0% of

16

BOC group deprotection degree was achieved using HCl/ethyl acetate deprotection method. All the

17

compounds were characterized by fourier transform infrared spectroscopy, mass spectroscopy and

18

nuclear magnetic resonance spectroscopy. The emulsifying properties of phytosterols and

19

phytosteryl amino acid ester hydrochlorides were also investigated. The results showed a higher

20

emulsifying properties of phytosteryl amino acid ester hydrochlorides, which could favor its wide

21

application in food systems.

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KEYWORDS: phytosterols, amino acid, esterification, water solubility, emulsification

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INTRODUCTION

25

Phytosterols such as stigmasterol, beta-sitosterol, campesterol and brassicasterol could be found

26

in vegetable oils, nuts, cereals, seeds and other plant sources1. Phytosterols are structurally similar

27

to cholesterol except for some substitutions at the C24 position on the sterol side chain2-4. Many

28

researches have verified that phytosterols, phytostanols as well as phytosteryl and phytostanyl esters

29

have effects on lowering plasma low-density lipoprotein cholesterol (LDL-C) by dietary means4-9.

30

Thus they have been used as food additives in food such as milk, yogurt, meat products, margarine,

31

cream cheese, chocolate, beverage and nondairy minidrink10-12. Besides, phytosterols/phytostanols

32

also have other beneficial properties, including anti-atherogenic, anti-inflammatory, antioxidant,

33

anti-osteoarthritic and anti-cancer activities13-17.

34

However, practical application of phytosterols is greatly restricted by the poor solubility in oil

35

and insolubility in water. Therefore, it is feasible to conjugate some beneficial molecules to

36

phytosterols to improve their solubility and bioavailability. For example, esterification of

37

phytosterols with various fatty acids could significantly enhance their lipid solubility and retain

38

their biological activity18-20, and these phytosteryl esters are hydrolyzed in the intestine to play the

39

role of cholesterol-lowering21. Recently, some researchers have attempted to prepare emulsified

40

water-soluble phytosterols which could potentially increase their absorption, show improved effects

41

on cholesterol and lipid profile and indicate increased dose response in food systems22-26. However,

42

oil-in-water phytosterols microemulsions, nanodispersions and nanoliposomes are limitedly used in

43

food products because of their poor stability which affects the product quality27,28. And improving

44

water-solubility of bioactives can accelerate the speed of going into the tissues and enhance the

45

absorbance rate in the body. For this reason, some other researchers pay attention to the synthesis of

46

water-soluble phytosteryl derivatives which are much more stable, such as phytostanyl sorbitol

47

succinate and phytosteryl L-glutamic esters29,30. However, through repeated trials, it was

48

unsuccessful to adopt the one-step method to synthesize steryl esters from phytosterols and amino

49

acid using sodium bisulfate as catalyst and n-butanol as solvent.30 The reason was probably that 3



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amino acids were poorly soluble or even insoluble in most of organic solvents and high temperature

51

still could not promote the dissolution. As a result, the above-mentioned one-step method was

52

infeasible.

53

The presence of an amino functional group in amino acids and their poor solubility in organic

54

solvents make the amino protection a frequently needed exercise in synthetic chemistry. A large

55

number of researches have proven that tert-butoxycarbonyl (BOC) group has one of the most useful

56

functions for the protection of amines due to the ease of protection and deprotection. Moreover, it

57

has better stability towards catalytic hydrogenolysis and extreme resistance towards basic

58

nucleophilic reactions31-33. In the present study, a novel synthesis of phytosteryl esters with amino

59

acid was developed by a two-step sequence of the esterification of phytosteryl N-BOC-amino acid

60

esters followed by the deprotection of BOC group. Besides, catalysts dose, substrate molar ratio,

61

addition sequence of reactants and catalysts, reaction temperature and time as well as the

62

deprotection methods were also investigated. Finally, phytosteryl glycine ester hydrochloride,

63

phytosteryl aspartic acid ester hydrochloride and phytosteryl glutamic acid ester hydrochloride were

64

obtained. Fourier transform infrared spectroscopy (FT-IR), mass spectroscopy (MS) and nuclear

65

magnetic resonance spectroscopy (NMR) were adopted to confirm the chemical structure of

66

phytosteryl derivatives and the water-solubility and emulsifying properties of the three phytosteryl

67

amino acid ester hydrochlorides were also determined.

68 69

MATERIALS AND METHOD

70

Materials. Phytosterols were a generous gift from Jiangsu Spring Fruit Biological Products Co.,

71

Ltd. (Taixing, P. R. China). The purity of plant sterols was > 97% (63% β-sitosterol and 37%

72

stigmasterol). Stigmasterol (purity > 95%) was purchased from Shanxi Pioneer Biotech Co., Ltd.

73

(Shanxi, China). N-BOC-amino acids (N-BOC-glycine, N-BOC-aspartic acid, N-BOC-glutamic

74

acid, purity > 95%) were obtained from J&K Chemical Technology Co., Ltd. (Shanghai, China).

75

Methylene dichloride, ethyl acetate, petroleum ether, trichloromethane, formic acid, trifluoroacetic 4


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acid (TFA), diphenyl ether, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC),

77

4-dimethylaminopyridine (DMAP), triethylamine and other reagents used were of analytical grades

78

and supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Methanol used for

79

high-performance liquid chromatography (HPLC) analysis was of spectral grade and purchased

80

from Suzhou Kesheng Laboratory Equipment Co., Ltd. (Suzhou, China).

81

Synthesis and Purification of Phytosteryl N-BOC-Amino Acid Ester. The esterification

82

reaction was carried out as follows: EDC (1.0-3.6 mmol) was first dissolved in methylene

83

dichloride in a triangle flask, placed in an ice-water mixture bath with a magnetic stirrer, and then

84

triethylamine (1.5-5.4 mmol) was added and stirred for 5 min followed by the addition of DMAP

85

(1.0-3.6 mmol) and N-BOC-amino acids (1.0-3.0 mmol). After being stirred at 0 oC for 1 h,

86

phytosterols (1.0 mmol) were added into the flask to react at 0 oC for 6-10 h and then at 25 oC for

87

14-18 h. The total reaction time after adding phytosterols was 24 h. Over the time course of the

88

reactions, a portion of the reaction mixture was periodically removed from the flask for thin layer

89

chromatography (TLC) and high performance liquid chromatography (HPLC).

90

A rotary evaporator was used to remove the solvent of the reaction mixtures resulting from

91

esterification of phytosterols and N-BOC-amino acids after the reaction was completed. The solid

92

powders were dissolved in trichloromethane and purified by column chromatography on silica gel

93

(200-300 mesh) and eluted with ethyl acetate/petroleum ether/formic acid (3:2:0.05, v/v/v). The

94

eluent was collected and then detected by TLC. The solvent of the fractions containing phytosteryl

95

N-BOC-amino acid ester was removed with a rotary evaporator. The isolated phytosteryl ester was

96

dried under vacuum at 50 oC for 24 h and used as substrate for the BOC deprotection reaction.

97

BOC Deprotection and Purification of Phytosteryl Amino Acid Ester Hydrochlorides. Four

98

kinds of BOC deprotection methods were investigated as follows and the deprotection degree was

99

detected by HPLC.

100

Thermal Deprotection. Phytosteryl N-BOC-amino acid ester was added into a reaction tube,

101

placed in an oil bath equipped with a magnetic stirrer and then heated to the desirable temperature 5



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(130-180 oC) under constant nitrogen flow (3.0 mL/min). The phytosteryl ester was melted and

103

reacted for a period of time (1-2 h) 34.

104

Diphenyl Ether Deprotection. Phytosteryl N-BOC-amino acid ester was dissolved in diphenyl

105

ether in a three-necked flask with a reflux device, placed in an oil bath and then heated to the

106

desirable temperature (130-180 oC) under constant nitrogen flow (3.0 mL/min). The reaction

107

mixtures were magnetically agitated for 1-2 h35.

108

TFA Deprotection. Phytosteryl N-BOC-amino acid ester was first added to a triangle flask,

109

dissolved in 30% TFA in methylene dichloride solution and then magnetically agitated at 25 oC for

110

0.5-2 h33.

111

HCl Deprotection. Phytosteryl N-BOC-amino acid ester was dissolved in 4 M hydrogen chloride

112

in ethyl acetate solution in a three-necked flask under a magnetic stirring for 0.5-2 h at 25 oC36.

113

After the deprotection reaction was completed, the HCl and ethyl acetate were removed under

114

vacuum with a rotary evaporator. Subsequently, the phytosteryl amino acid ester hydrochloride was

115

redissolved in ethyl acetate and then the solvent was evaporated again by a rotary evaporator. After

116

repeating the dissolution-evaporation step until the hydrogen chloride was entirely removed, the

117

solid powders of phytosteryl amino acid ester hydrochloride were dried under vacuum at 50 oC for

118

24 h.

119

Analysis Methods. Qualitative and quantitative analysis was performed with TLC and HPLC

120

while structure analysis of phytosteryl N-BOC-amino acid esters and phytosteryl amino acid ester

121

hydrochlorides was carried out in accordance with FT-IR, MS and NMR.

122

TLC Analysis. Aliquots (10 µL) were withdrawn from the reaction mixtures by pipette for TLC

123

analysis. Development was carried out in ethyl acetate/petroleum ether/formic acid (3:2:0.05, v/v/v)

124

and then the spots were located by iodine staining for 1 hour. Rf values of different substrates and

125

products were: 0-0.02 (phytosteryl glycine ester hydrochloride, phytosteryl aspartic acid ester

126

hydrochloride and phytosteryl glutamic acid ester hydrochloride), 0.03-0.05 (N-BOC-aspartic acid

127

and N-BOC-glutamic acid), 0.06-0.08 (N-BOC-glycine), 0.68-0.72 (phytosteryl N-BOC-aspartic 6


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128

acid ester and phytosteryl N-BOC-glutamic acid ester), 0.79-0.83 (phytosterols), 0.94-0.98

129

(phytosteryl N-BOC-glycine ester), respectively.

130

HPLC Analysis. Aliquots removed periodically from the reaction mixtures and the purified

131

products were dissolved in methanol for HPLC analysis. The standard curves were prepared using

132

purified phytosteryl N-BOC-amino acid esters and phytosteryl amino acid ester hydrochlorides. The

133

yield (mol %) was defined as the molar ratio of the amount of synthesized phytosteryl

134

N-BOC-amino acid esters to that of phytosterols at the beginning of the reaction. The analysis was

135

carried out with a symmetry-C18 column (5 μm, 4.6×150 mm, Waters) and a evaporative light

136

scattering detector (ELSD) 3300 (Alltech). The mobile phase was the mixture of methanol/formic

137

acid (1000:1, v/v) and the flow rate was 1.0 mL/min. The chromatographic column temperature was

138

35 oC and the ELSD was performed when drift tube temperature was 85 oC and sprayer temperature

139

was 42 oC with nitrogen 0.25 Mpa as carrier gas.

140

FT-IR Analysis. FT-IR spectra were recorded on Nicolet iS10 spectrometer (Nicolet Instrument

141

Corp., USA) with a DTGS detector. Phytosteryl N-BOC-amino acid esters and phytosteryl amino

142

acid ester hydrochlorides samples were diluted in KBr powders and measured with the scanning

143

scope for 400-4000 cm-1 and the number of scans for 16.

144

MS Analysis. Mass spectrum was obtained by mass spectrometry (Waters UPLC-TQD, USA)

145

with electron spray ionization (ESI) mode. The MS parameters were as follows: source block

146

temperature 130 oC, desolvation temperature 350 oC, desolvation gas flow 800 L/h, cone gas flow

147

50 L/h, capillary voltage 3.5 kV, cone voltage 30 V and the mass scan range 50-1000 m/z. Samples

148

were dissolved in methanol and diluted to 1-5 ppm for detection. According to the properties of

149

samples, phytosteryl N-BOC-glycine ester and phytosteryl glycine ester hydrochloride were

150

analyzed with positive ESI while negative ESI was adopted for phytosteryl N-BOC-aspartic acid

151

ester, phytosteryl N-BOC-glutamic acid ester, phytosteryl aspartic acid ester hydrochloride and

152

phytosteryl glutamic acid ester hydrochloride.

153

NMR Analysis. 1H NMR and

13C

NMR spectra of products were recorded with a Bruker NMR 7



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spectrometer (Bruker Avance Ⅲ 400 MHz, Switzerland), operating at 400 MHz and 100 MHz for

155

1H

156

N-BOC-aspartic acid ester, phytosteryl N-BOC-glutamic acid ester and phytosteryl glycine ester

157

hydrochloride while (CD3)2SO was the solvent of phytosteryl aspartic acid ester hydrochloride and

158

phytosteryl glutamic acid ester hydrochloride.

159

and

13C,

respectively. CDCl3 was the solvent of phytosteryl N-BOC-glycine ester, phytosteryl

Determination

of

Thermal

Stability

of

the

Phytosteryl

Ester

Hydrochloride.

160

Thermogravimetric analysis (TGA) was carried out on a thermoanalyzer system Mettler

161

TGA/SDTA 851e under nitrogen atmosphere. The samples were about 5-15 mg and the heating rate

162

was of 20 oC/min in the range of temperature 50-500 oC.

163

Determination of Water-solubility of the Phytosteryl Ester Hydrochloride. To compare the

164

water-solubility of phytosterols and phytosteryl ester hydrochlorides, 0.01 g phytosterols or

165

phytosteryl ester hydrochlorides were dissolved in 1 mL de-ionized water with ultrasonic wave

166

assistance for 6 h and then allowed to stand for 1 h at 25 oC thermostat water bath. The upper phase

167

(50 μL) was withdrawn by pipette and diluted in methanol (10 mL) and each sample (10 μL) was

168

analyzed by HPLC with methanol/formic acid (1000:1, v/v) as the mobile phase and detected by

169

ELSD. The amount of substrate was determined by comparing peak areas of the corresponding

170

standard sample with a known concentration.

171

Determination of Emulsifying Activity of the Phytosteryl Ester Hydrochlorides. The sample

172

was dissolved in phosphate buffered solution (PBS) (0.1 mol/L, pH 7.4) and its concentration was

173

0.2 mg/mL. This sample solution (15 mL) and soybean oil (5 mL) were homogenized at a speed of

174

20,000 rmp, 25 oC for 1 min. Emulsion was pipetted out 50 µL and diluted 100-fold with 0.1% SDS.

175

Then the absorbance of the above emulsion was measured immediately. A500 of the resulting

176

dispersion was measured using a spectrophotometer. Emulsion activity index37 (EAI, m2/g) was

177

calculated as follows:

178

EAI 

2  2.303  A 0  N 1      10000

(1) 8


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179 180


where A0 is the observed absorbance at 0 min, N is the dilution factor, ϕ is oil volume fraction and  is concentration of emulsifier in the solution before emulsification (g/mL).

181

Determination of Emulsifying Stability of the Phytosteryl Ester Hydrochlorides. The

182

emulsifying stability indices of esters were determined using minor modifications of known

183

protocols38. Soybean oil (1.0 g) was mixed into aqueous solutions of the test samples (10.0g) with

184

different concentrations (0.01%, 0.1% and 0.5% w/w). And a solution lacking a test material was

185

used as the blank control. The mixtures were homogenized for 1 min at 12000 rpm using a blender

186

and a 50 µL sample of the resulting emulsion was diluted to 100 times with 1 mg/mL SDS solution

187

before being drawn into a clean spectrometric cuvette. The absorbance of the emulsion at 500 nm

188

was read at 0 min and 10 min. The emulsion stability indices (ESIs) were then calculated using the

189

following equation: A 0 10 A 0  A10

190

ESI 

191

where A0 and A20 were the absorbance obtained at 0 min and 10 min.

192

Statistical Analysis. The statistical analyses were conducted using SPSS 16.0. Statistical

193

(2)

significance was declared at p < 0.05. All data were presented as means ± SEM.

194 195

RESULTS AND DISCUSSION

196

Structural Analysis of Products. The structural analysis of phytosterols, phytosteryl

197

N-BOC-amino acid esters and phytosteryl amino acid ester hydrochlorides is exemplified by

198

stigmasterol, stigmasteryl N-BOC-amino acid esters and stigmasteryl amino acid ester

199

hydrochlorides. HPLC analysis was employed to determine the yield of stigmasteryl esters and the

200

BOC deprotection degree of stigmasteryl ester hydrochlorides. Purified stigmasterol, stigmasteryl

201

N-BOC-amino acid esters and stigmasteryl amino acid ester hydrochlorides were all analyzed by

202

FT-IR, MS and NMR, respectively.

203

Stigmasterol: HPLC retention time (min): 10.757; FT-IR (ν, cm−1): 3431 (vOH, s), 2959 (vCH, s), 9



204

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2937 (vCH, s), 2868 (vCH, s), 1465 (δCH, m), 1382 (δCH, m), 1055 (ring vibration, m).

205

Stigmasteryl N-BOC-glycine ester: HPLC retention time (min): 10.810; ESI-MS, m/z 592

206

[M+Na]+; FT-IR (ν, cm−1): 3369 (s, vNH), 1725 (s, vC=O), 1719 (s, vC=O), 1517 (s, δNH), 1367 (s, δCH),

207

1284 (s, vC-N), 1206 (s, vC-O), 1171 (s, vC-O); 1H NMR (400 MHz, CDCl3, ppm): δ = 0.68 (3H, s,

208

18-H), 0.80 (6H, d, J = 8.0 Hz, 26-27-H), 0.84 (3H, t, J = 6.0 Hz, 29-H), 1.02 (6H, m), 1.03 (3H, s,

209

19-H), 1.10-1.30 (5H, m), 1.45 (9H, s, (CH3)3C-),1.62-1.50 (8H, m), 1.65-1.73 (2H, m), 1.86-2.09

210

(5H, m), 2.33 (2H, d, J = 8.0 Hz), 3.88 (2H, d, J = 4.0 Hz, -CH2CO), 4.64-4.71 (1H, m, 3-H), 5.02

211

(1H, dd, J = 8.0, 16.0 Hz, 22-H or 23-H), 5.15 (1H, dd, J = 8.0, 16.0 Hz, 22-H or 23-H), 5.38 (1H, d,

212

J = 8.0 Hz, 6-H); 13C NMR (100 MHz, CDCl3, ppm): δ = 12.05 (29-CH3), 12.26 (18-CH3), 18.99

213

(21-CH3), 19.29 (19-CH3), 21.02 (26- or 27-CH3), 21.09 (26- or 27-CH3), 21.23 (CH2), 24.36

214

(11-CH2), 25.41 (15-CH2), 27.71 (CH2), 28.33 ((CH3)3C-), 28.91 (CH2), 31.85 (CH2), 31.89 (2C, 8-

215

and 25-CH), 36.58 (quaternary C-10), 36.92 (CH2), 38.03 (CH2), 39.62 (CH2), 40.50 (20-CH),

216

42.21 (quaternary C-13), 42.67 (-CH2CO), 50.04 (9-CH), 51.24 (24-CH), 55.94 (17-CH), 56.79

217

(14-CH), 75.18 (3-CH), 79.91 ((CH3)3C-), 122.92 (6-CH), 129.31 (22-CH), 138.30 (23-CH), 139.36

218

(quaternary C-5), 155.70 (CONH), 169.75 (CH2C=O).

219

Stigmasteryl glycine ester hydrochloride: HPLC retention time (min): 1.479; ESI-MS, m/z 470

220

[M-HCl+H]+; FT-IR (ν, cm−1): 3397 (s, vNH), 1732 (s, vC=O), 1624 (w, δNH), 1217 (s, vC-O); 1H NMR

221

(400 MHz, CDCl3, ppm): δ = 0.70 (3H, s, 18-H), 0.80 (6H, d, J = 8.0 Hz, 26-27-H), 0.84 (3H, t, J =

222

6.0 Hz, 29-H), 1.02 (3H, s, 19-H), 0.93-1.08 (6H, m), 1.12-1.28 (5H, m), 1.38-1.58 (8H, m),

223

1.61-1.73 (2H, m), 1.86-2.09 (5H, m), 2.33 (2H, d, J = 8.0 Hz), 3.40 (2H, s, -CH2CO), 4.63-4.71

224

(1H, m, 3-H), 5.03 (1H, dd, J = 8.0, 16.0 Hz, 22-H or 23-H), 5.16 (1H, dd, J = 8.0, 16.0 Hz, 22-H or

225

23-H), 5.38 (1H, d, J = 8.0 Hz, 6-H); 13C NMR (100 MHz, CDCl3, ppm): δ = 12.05 (29-CH3), 12.26

226

(18-CH3), 18.99 (21-CH3), 19.31 (19-CH3), 21.02 (26- or 27-CH3), 21.09 (26- or 27-CH3), 21.23

227

(CH2), 24.26 (11-CH2), 25.41 (CH2), 27.79 (15-CH2), 28.91 (CH2), 31.86 (CH2), 31.89 (2C, 8- and

228

25-CH), 36.61 (quaternary C-10), 36.96 (CH2), 38.14 (CH2), 39.63 (CH2), 40.50 (20-CH), 42.22

229

(quaternary C-13), 44.25 (-CH2CO), 50.05 (9-CH), 51.24 (24-CH), 55.95 (17-CH), 56.79 (14-CH), 10


Page 11 of 36


230

74.61 (3-CH), 122.83 (6-CH), 129.31 (22-CH), 138.30 (23-CH), 139.49 (quaternary C-5), 173.72

231

(C=O).

232

Stigmasteryl N-BOC-aspartic acid ester: HPLC retention time (min): 9.141; ESI-MS, m/z 626

233

[M-H]+; FT-IR (ν, cm−1): 3600-2500 (s, vOH), 3426 (s, vNH), 1750 (s, vC=O), 1735 (s, vC=O), 1720 (s,

234

vC=O), 1510 (s, δNH), 1368 (s, δCH), 1280 (m, vC-N), 1220 (s, vC-O), 1168 (s, vC-O); 1H NMR (400 MHz,

235

CDCl3, δ, ppm): δ = 0.70 (3H, s, 18-H), 0.80 (6H, d, J = 8.0 Hz, 26-27-H), 0.84 (3H, t, J = 6.0 Hz,

236

29-H), 1.02 (6H, m), 1.03 (3H, s, 19-H), 1.14-1.30 (5H, m), 1.45 (9H, s, (CH3)3C-), 1.50-1.61 (8H,

237

m), 1.65-1.73 (2H, m), 1.85-2.09 (5H, m), 2.29-2.34 (2H, m), 2.78-3.06 (2H, m, -CH2COOH), 4.53

238

(1H, m, -CHCO), 4.63-4.71 (1H, m, 3-H), 5.02 (1H, dd, J = 8.0, 16.0 Hz, 22-H or 23-H), 5.16 (1H,

239

dd, J = 8.0, 16.0 Hz, 22-H or 23-H), 5.37 (1H, d, J = 8.0 Hz, 6-H), 6.22 (1H, m, -NH-); 13C NMR

240

(100 MHz, CDCl3, ppm): δ = 12.08 (29-CH3), 12.24 (18-CH3), 19.02 (21-CH3), 19.32 (19-CH3),

241

21.08 (2C, 26- and 27-CH3), 21.26 (CH2), 24.38 (11-CH2), 25.41 (CH2), 27.68 (15-CH2), 28.33

242

((CH3)3C-), 28.91 (CH2), 31.90 (2C, 8- and 25-CH), 36.60 (quaternary C-10), 36.69 (CH2COOH),

243

36.95 (CH2), 37.91 (CH2), 39.67 (CH2), 40.49 (20-CH), 42.25 (quaternary C-13), 50.08 (2C, 9-CH

244

and -CHCO), 51.27 (24-CH), 56.00 (17-CH), 56.83 (14-CH), 75.16 (3-CH), 80.30 ((CH3)3C-),

245

122.96 (6-CH), 129.37 (22-CH), 138.29 (23-CH), 139.35 (quaternary C-5), 155.56 (CONH), 170.27

246

(CHC=O), 176.35 (-COOH).

247

Stigmasteryl glycine ester hydrochloride: HPLC retention time (min): 2.545; ESI-MS, m/z 526

248

[M-HCl-H]+; FT-IR (ν, cm−1): 3600-2500 (s, vOH), 3427 (s, vNH), 1747 (s, vC=O), 1590 (w, δNH),

249

1225 (s, vC-O); 1H NMR (400 MHz, (CD3)2SO, ppm): δ = 0.68 (3H, s, 18-H), 0.78 (6H, d, J = 8.0 Hz,

250

26-27-H), 0.82 (3H, t, J = 6.0 Hz, 29-H), 0.99 (6H, m), 1.03 (3H, s, 19-H), 1.07-1.24 (5H, m),

251

1.41-1.70 (10H, m), 1.84-2.06 (5H, m), 2.28-2.30 (2H, m), 2.85-2.94 (2H, m, -CH2COOH), 4.22

252

(1H, m, -CHCO), 4.57 (1H, m, 3-H), 5.03 (1H, dd, J = 8.0, 16.0 Hz, 22-H or 23-H), 5.16 (1H, dd, J

253

= 8.0, 16.0 Hz, 22-H or 23-H), 5.36 (1H, d, J = 8.0 Hz, 6-H), 9.43 (1H, s, -COOH); 13C NMR (100

254

MHz, (CD3)2SO, ppm): δ = 11.82 (29-CH3), 12.08 (18-CH3), 18.83 (21-CH3), 18.93 (19-CH3),

255

20.53 (26- or 27-CH3), 20.90 (26- or 27-CH3), 21.09 (CH2), 23.85 (11-CH2), 24.83 (CH2), 27.05 11



Page 12 of 36

256

(15-CH2), 28.43 (CH2), 31.30 (2C, 8- and 25-CH), 34.31 (CH2COOH), 36.07 (quaternary C-10),

257

36.24 (CH2), 37.51 (CH2), 38.93 (CH2), 39.97 (CH2), 40.18 (20-CH), 41.73 (quaternary C-13),

258

48.57 (9-CH), 49.38 (-CHCO), 50.57 (24-CH), 55.35 (17-CH), 56.17 (14-CH), 75.39 (3-CH),

259

122.40 (6-CH), 128.86 (22-CH), 137.97 (23-CH), 139.07 (quaternary C-5), 168.10 (CHC=O),

260

170.73 (-COOH).

261

Stigmasteryl N-BOC-glutamic acid ester: HPLC retention time (min): 10.1381; ESI-MS, m/z 640

262

[M-H]+; FT-IR (ν, cm−1): 3600-2500 (s, vOH), 3427 (s vNH), 1720 (s, vC=O), 1511 (m, δNH), 1368 (s,

263

δCH), 1330 (m, vC-N), 1170 (s, vC-O); 1H NMR (400 MHz, CDCl3, ppm): δ = 0.70 (3H, s, 18-H), 0.80

264

(6H, d, J = 8.0 Hz, 26-27-H), 0.84 (3H, t, J = 6.0 Hz, 29-H), 1.03 (3H, s, 19-H), 1.01 (6H, m),

265

1.14-1.30 (5H, m), 1.45 (9H, s, (CH3)3C-), 1.50-1.57 (8H, m), 1.60-1.72 (2H, m), 1.84-2.05 (5H, m),

266

2.19-2.26 (2H, m, -CH2CH2COOH), 2.31 (2H, d, J = 8.0 Hz), 2.37-2.46 (2H, m, -CH2COOH), 4.33

267

(1H, m, -CHCO), 4.61-4.62 (1H, m, 3-H), 5.02 (1H, dd, J = 8.0, 12.0 Hz, 22-H or 23-H), 5.16 (1H,

268

dd, J = 8.0, 12.0 Hz, 22-H or 23-H), 5.37 (1H, d, J = 8.0 Hz, 6-H), 8.04 (1H, s, -NH-), 10.06 (1H, m,

269

-COOH); 13C NMR (100 MHz, CDCl3, ppm): δ = 12.05 (29-CH3), 12.26 (18-CH3), 19.00 (21-CH3),

270

19.31 (19-CH3), 21.02 (26- or 27-CH3), 21.09 (26- or 27-CH3), 21.24 (CH2), 24.36 (11-CH2), 25.41

271

(CH2), 27.33 (CH2CH2COOH), 27.71 (15-CH2), 28.30 ((CH3)3C-), 28.92 (CH2), 30.85

272

(-CH2COOH), 31.86 (8- or 25-CH), 31.89 (8- or 25-CH), 36.60 (quaternary C-10), 36.96 (CH2),

273

38.06 (CH2), 39.63 (CH2), 40.51 (20-CH), 42.21 (quaternary C-13), 50.04 (9-CH), 51.25 (24-CH),

274

53.01 (-CHCO), 55.94 (17-CH), 56.79 (14-CH), 74.57 (3-CH), 80.45 ((CH3)3C-), 122.76 (6-CH),

275

129.30 (22-CH), 138.31 (23-CH), 139.52 (quaternary C-5), 155.78 (CONH), 172.55 (CHC=O),

276

176.63 (-COOH).

277

Stigmasteryl glutamic acid ester hydrochloride: HPLC retention time (min): 3.336; ESI-MS, m/z

278

540 [M-HCl-H]+; FT-IR (ν, cm−1): 3600-2500 (s, vOH), 3428 (s, vNH), 1732 (s, vC=O), 1600 (w, δNH),

279

1198 (s, vC-O); 1H NMR (400 MHz, (CD3)2SO, ppm): δ = 0.68 (3H, s, 18-H), 0.77 (6H, d, J = 8.0 Hz,

280

26-27-H), 0.82 (3H, t, J = 6.0 Hz, 29-H), 0.98 (6H, m), 1.01 (3H, s, 19-H), 1.10-1.28 (5H, m),

281

1.37-1.65 (10H, m), 1.78-2.00 (5H, m), 2.03-2.12 (2H, m, -CH2COOH), 2.28 (2H, d, J = 8.0 Hz), 12


Page 13 of 36


282

2.40-2.59 (2H, m, CH2CH2COOH), 3.91 (1H, m, -CHCO), 4.44-4.52 (1H, m, 3-H), 5.03 (1H, dd, J

283

= 8.0, 16.0 Hz, 22-H or 23-H), 5.16 (1H, dd, J = 8.0, 16.0 Hz, 22-H or 23-H), 5.34 (1H, d, J = 8.0

284

Hz, 6-H), 8.48 (3H, s, -NH3+), 13.79 (1H, m, -COOH); 13C NMR (100 MHz, (CD3)2SO, ppm): δ =

285

11.82 (29-CH3), 12.07 (18-CH3), 18.83 (21-CH3), 18.94 (19-CH3), 20.72 (26- or 27-CH3), 20.89

286

(26- or 27-CH3), 21.09 (CH2), 23.85 (11-CH2), 24.83 (CH2), 25.17 (CH2CH2COOH), 27.30

287

(15-CH2), 28.45 (CH2), 29.51 (-CH2COOH), 31.35 (8- or 25-CH), 31.35 (8- or 25-CH), 36.09

288

(quaternary C-10), 36.47 (CH2), 37.62 (CH2), 39.13 (CH2), 39.88 (20-CH), 41.73 (quaternary C-13),

289

50.58 (9-CH), 51.17 (24-CH), 55.35 (17-CH), 56.21 (14-CH), 59.70 (-CHCO), 73.54 (3-CH),

290

122.07 (6-CH), 128.85 (22-CH), 137.97 (23-CH), 139.44 (quaternary C-5), 170.48 (CHC=O),

291

171.05 (-COOH).

292

The IR spectra of stigmasterol, stigmasteryl N-BOC-amino acid ester and stigmasteryl amino

293

acid ester hydrochlorides are shown in Figure 1. Compared with stigmasterol, the absorption signal

294

of hydroxyl group in the free carboxyl group at 2400-3300 cm-1 disappeared in the spectra of their

295

corresponding esters, besides, stigmasteryl N-BOC-amino acid ester contained the absorption signal

296

of carbonyl group and BOC group. And the absorption signal of BOC group disappeared in the

297

spectra of stigmasteryl amino acid ester hydrochlorides. In theory, the relative molecular weight of

298

stigmasteryl N-BOC-glycine ester is 569 and stigmasteryl glycine ester hydrochloride is 505. As

299

presented in Figure 2, a sodium adduct molecular ion [M+Na]+ at m/z 592 for stigmasteryl

300

N-BOC-glycine ester and a dehydrochlorinated and protonated molecular ion [M-HCl+H]+ at m/z

301

470 for stigmasteryl glycine ester hydrochloride were observed under positive-ion mode. Other

302

stigmasteryl N-BOC-amino acid ester and stigmasteryl amino acid ester hydrochloride also

303

contained similar molecular ions. The structures of stigmasteryl amino acid esters were also further

304

confirmed by 1H and 13C NMR, and their NMR spectra were displayed in Figs. S1-S6, respectively.

305

Thus, the products were identified to be stigmasteryl N-BOC-amino acid ester and stigmasteryl

306

amino acid ester hydrochlorides.

307

Effect of Addition Sequence of Reactants and Catalysts. It was found that setting an addition 13



Page 14 of 36

308

sequence of reactants and catalysts could increase the conversion of phytosterols to phytosteryl

309

N-BOC-amino acid ester. The reaction conditions were 1:1 molar ratio of N-BOC-aspartic acid to

310

phytosterols, 1:1:1:1.5 molar ratio of N-BOC-aspartic acid to EDC, DMAP and triethylamine, 25 oC

311

for 24 h. When all the reactants and catalysts had no specific addition sequence and were added in

312

the reactor followed by the dissolution in solvent, the yield was 35.4%. However, when the addition

313

sequence was that EDC and triethylamine firstly dissolved in methylene dichloride and then

314

N-BOC-aspartic acid and DMAP were added to react for 1 h, followed by the addition of

315

phytosterols, the yield rose up to 58.9%. The reason why batch charging of reactants and catalysts

316

was conducive to conversion was that EDC was a kind of hydrochloride salt with low catalytic

317

activity which needed triethylamine as the de-acid reagent to transform itself into

318

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). The catalytic mechanism of EDCI was

319

that EDCI firstly combined with the carboxyl of N-BOC-aspartic acid to form the intermediate

320

O-acylurea which then reacted with phytosterols to form phytosteryl N-BOC-aspartic acid ester,

321

accompanied by the production of 1-ethyl-3-(3-dimethylaminopropyl) urea (EDU) which had no

322

catalytic activity39. DMAP was a kind of auxiliary catalyst that was able to stabilize the

323

intermediate O-acylurea and restrain the formation of N-acylurea, usually used to speed up reaction

324

rate and improve conversion when the steric hindrance of reactants was large. Based on the above

325

analyses, batch charging of the reactants and catalysts allowed the completed activation of EDC and

326

carboxyl of acid which increased the conversion, thus it was considered for the synthesis of

327

phytosteryl N-BOC-amino acid ester.

328

Effect of Reaction Temperature and Time. The effect of reaction temperature and time on the

329

esterification is shown in Figure 3 (A). The total time at 0 oC and 25 oC was 24 h. If the reaction

330

was constantly performed at 25 oC, the yield was only 58.9%. However, the yield increased sharply

331

to 69% when the reaction was carried out at 0 oC for 2 h in the early stage and then at 25 oC for 22 h.

332

With the time proportion at 0 oC in the 24 h reaction time increased, the product yield increased

333

gradually and the highest yield 84.1% was obtained at 0 oC for 10 h and 25 oC for 14 h. The reason 14


Page 15 of 36


334

was that low temperature could make the intermediate O-acylurea stable and inhibit the formation

335

of by-product N-acylurea, which contributed to a considerable conversion enhancement39. It had

336

been reported that low temperature was also applied in the synthesis of hydroxycinnamic acid

337

amides of thiazole with coupling reagents EDC, DMAP and triethylamine as catalysts40. As the

338

esterification of acid and alcohol was exothermic reaction, the ice-water mixture bath was necessary

339

to keep the temperature at 0 oC in the early stage of reaction and in order to make the esterification

340

complete, the reaction could continue at 25 oC for a period of time. The effect of reaction time at 0

341

oC

342

difference in the yield between 8 h and 10 h at 0 oC. In consideration of economical cost, the best

343

reaction temperature and time were selected as 0 oC for 8 h in the early stage of reaction and then 25

344

oC

on the esterification is shown in Figure 3 (B). It was also observed that there was no apparent

for 16 h.

345

Effect of Catalysts Dose. As shown in Figure 3 (C), the influence of catalysts dose on

346

esterification was evaluated using different molar ratio of EDC to N-BOC-aspartic acid from 1:1 to

347

1.8:1. In theory, the formation of an equivalent ester accompanied with the production of an

348

equivalent water which required an equivalent catalyst EDC. However, it was showed that an

349

increase in the molar ratio of EDC to N-BOC-aspartic acid from 1:1 to 1.2:1 could lead to a slight

350

increase in the extent of esterification. When the molar ratio increased continually, the conversion

351

of phytosterols to phytosteryl N-BOC-aspartic acid ester had no obvious improvement. In terms of

352

the yield and economical interest of the reaction, the optimum catalysts dose was 1.2:1 molar ratio

353

of EDC to N-BOC-aspartic acid.

354

Effect of Molar Ratio of N-BOC-Amino Acid to Phytosterols. As shown in Figure 3 (D),

355

although an equimolar ratio of both substrates might be ideal for esterification in terms of

356

economical aspect and further purification of products, such a ratio was not advantageous for the

357

synthesis of phytosteryl N-BOC-aspartic acid ester since an excess of N-BOC-aspartic acid could

358

shift the reaction equilibrium to the products. The change of molar ratio of N-BOC-aspartic acid to

359

phytosterols from 1:1 to 2:1 resulted in a gradual rise in the conversion but then a decrease of yield 15



Page 16 of 36

360

was observed when the molar ratio surpassed 2:1. The reason might be that excessive

361

N-BOC-aspartic acid increased the viscosity of the reaction system which reduced the probability of

362

atomic collision and led to the decline of conversion. The yield reached 95.0% when the molar ratio

363

was 1.5:1, just 1.2% lower than the maximum yield when the molar ratio was 2:1. Hence, a 1.5:1

364

molar ratio of N-BOC-aspartic acid to phytosterols was considered to be more suitable.

365

Deprotection Methods Selection. Four kinds of deprotection methods for BOC group of

366

phytosteryl N-BOC-amino acid esters were investigated: Thermal method, Diphenyl ether method,

367

TFA/methylene dichloride method and HCl/ethyl acetate method. And the BOC group deprotection

368

degrees of the four methods were respectively listed as follows: 0%, 0%, 90%, 99%. Using either

369

thermal deprotection method or diphenyl ether deprotection method could not successfully remove

370

BOC group to achieve phytosteryl amino acid ester. Although the reaction was under constant

371

nitrogen flow, a portion of new formed ester bond of phytosteryl N-BOC-amino acid ester was still

372

dissociated and there appeared some side-products. These results were in agreement with those of a

373

previous study, which found that high temperature might favor the dehydration of sterols to

374

dienes41.

375

However, the deprotection degree of TFA/methylene dichloride method was 90% and in order to

376

achieve isolated products, a separation step was still needed. Furthermore, the final products using

377

TFA/methylene dichloride method were phytosteryl amino acid ester trifluoroacetates, which could

378

not be applied in food industry. While using HCl/ethyl acetate method to remove BOC group, the

379

final products were phytosteryl amino acid ester hydrochlorides which had stable properties and

380

wide range of application in food industry. The deprotection degree of HCl/ethyl acetate method

381

reached up to 99% and there were almost no side-products. Therefore, the isolated products were

382

easy to obtain after evaporating all the ethyl acetate and hydrogen chloride. Based on the above

383

analyses, HCl/ethyl acetate method was selected as the optimal method to remove the BOC group

384

of phytosteryl N-BOC-amino acid esters.

385

Thermal Stability of the Phytosteryl Ester Hydrochloride. Thermogravimetric (TG) analysis 16


Page 17 of 36


386

can be used to study the decomposition temperature of the sample. The TG curve usually represents

387

a plot of weight change as a function of temperature or time. The TG curves of phytosterols and

388

three amino acid stigmasteryl ester hydrochlorides were shown in Figure 4. The initial

389

decomposition temperature (Ti) of phytosterols was 240 oC and the final decomposition temperature

390

(Tf) was 360 oC. The TG curve of phytosteryl glycine ester hydrochloride was closer to that of

391

phytosterols. Its Ti and Tf were 240 °C and 370 °C, respectively. The Ti of phytosteryl aspartic acid

392

ester hydrochloride and phytosteryl glutamic acid ester hydrochloride were increased to 260 oC and

393

280 oC, respectively. And their Tf were 400 oC and 370 oC, respectively. Therefore, three

394

phytosteryl amino acid ester hydrochlorides had good thermal stability and their chemical structures

395

will not be easily destroyed in thermal processing.

396

Water-solubility of Phytosteryl Amino Acid Ester Hydrochlorides. As shown in Figure 5, all

397

the synthesized phytosteryl amino acid ester hydrochlorides could dissolve in water. So, the three

398

products can apply in water-based foods. It was observed that the better water-solubility amino

399

acids owned, the greater water-solubility the corresponding phytosteryl amino acid ester

400

hydrochlorides possessed. Theoretically, the water-solubility of amino acids with polar or charged

401

side chains should be greater than those with nonpolar or uncharged side chains. Nevertheless, there

402

were some exceptions according to the polarity and charge. For example, the solubility of glycine

403

which had no side chains reached 249.9 mg/mL and was much better than that of aspartic acid with

404

one more carboxyl in the side chain. Hence, the water-solubility of the final product was decided

405

not only by the polar and charged groups that it owned, but also by the whole character of the

406

molecule structure.

407

Emulsifying Activity of Phytosteryl Amino Acid Ester Hydrochlorides. Phytosterols have a

408

large hydrophilic group and a hydrophilic group -OH at the C-3. So phytosterols have amphiphilic

409

ability and a certain degree of emulsification. The modification of -OH can not only improve the

410

hydrophilicity, but also regulate the emulsifying properties.

411

The emulsifying properties of phytosteryl amino acid ester hydrochlorides were evaluated by EAI 17



Page 18 of 36

412

determination and was compared to that of phytosterols. All samples were analyzed in triplicate.

413

The concentration of different samples was 0.2 mg/mL and their corresponding EAI were shown in

414

Figure 6 (A). Phytosteryl glutamic acid ester hydrochloride showed the highest EAI of 0.392 m2/g.

415

The value of phytosteryl aspartic acid ester hydrochloride was slightly lower than phytosteryl

416

glutamic acid ester hydrochloride and its EAI was 0.375 m2/g. The EAI of phytosterols and

417

phytosteryl glycine ester hydrochloride showed lower values. Phytosteryl glutamic acid ester

418

hydrochloride, phytosteryl aspartic acid ester hydrochloride and phytosteryl glycine ester

419

hydrochloride were more complex than phytosterols in molecular structure, and -COOH and NH3+

420

were present in their molecule, which made the emulsifying activity higher than that of phytosterols.

421

The results indicated that phytosteryl glutamic acid ester hydrochloride and phytosteryl aspartic

422

acid ester hydrochloride could more significantly improve the emulsification of phytosterols, and

423

expand its application in food systems.

424

Emulsifying Stability of Phytosteryl Amino Acid Ester Hydrochlorides. The emulsifying

425

stability indices (ESIs) of stigmasteryl amino acid ester hydrochlorides were determined at 0.01%,

426

0.2% and 0.5% w/w concentrations and the outcomes of such measurements are shown in Figure 6

427

(B). The ESIs of three stigmasteryl amino acid ester hydrochlorides correlate positively with

428

concentration. The ESI of phytosteryl glycine acid ester hydrochloride is the highest. And, the ESI

429

of phytosteryl aspartic acid ester hydrochloride is higher than phytosteryl glutamic ester

430

hydrochloride. The ESI values were found to be affected by the concentration of stigmasteryl amino

431

acid ester hydrochlorides and the length of amino acid side chain, but further experiments should be

432

conducted to understand why the esters with lowest length side chain showed the best emulsion

433

stabilities. The differences between EAIs may be due to the water-solubility, as the better

434

water-solubility is, the higer emulsifying stability is. As an emulsifier in food industry, one must

435

display high stability of the emulsion obtained. According to the results of the current work, they

436

could have greater efficacy in stabilizing emulsions.

437

In conclusion, as an efficient cholesterol-lowering food component, hydrophilic phytosteryl 18


Page 19 of 36


438

amino acid ester hydrochlorides could be successfully synthesized by two-step method. The process

439

includes esterification of phytosterol using N-BOC-amino acid coupling reagents EDC, DMAP and

440

triethylamine as catalysts and deprotection of BOC group. Additionally, compared to phytosterol,

441

the final products phytosteryl amino acid ester hydrochlorides presented greater emulsifying

442

activity, and the water-solubility and emulsifying stability were also improved, suggesting that

443

esterification of phytosterol with amino acids greatly facilitated their incorporation into foods. The

444

protocol reported in this paper could be easily developed into industrial production. Also,

445

phytosteryl amino acid esters could be considered as a potential healthy nutritional ingredient, and

446

the work along this line is underway to evaluate their efficacy, safety and bioaccessibility by further

447

both in vitro and in vivo studies.

448 449

AUTHOR INFORMATION

450

Corresponding Author

451

*(C.J.) Telephone/fax: +86-510-85329057. E-mail: [email protected].

452

Author Contributions

453

⊥C.J.

454

Funding

455

This study was ﬁnancially supported by Natural Science Foundation of Jiangsu Province

456

(BK20161133), and program of “Collaborative innovation center of food safety and quality control

457

in Jiangsu Province”.

458

Notes

459

The authors declare no competing financial interest.

and X.X. contributed equally to this work.

460 461 462

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phytosterol-enriched oil microcapsules for foodstuff application. Food Bioprocess Technol. 2018,

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11,152-163.

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(2) He, W. S.; Li L. L.; Huang, Q. J.; Yin, J.; Cao, X. C. Highly efficient synthesis of phytosterol

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linolenate in the presence of Bronsted acidic ionic liquid. Food Chem. 2018, 263, 1-7.

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straightforward synthesis of novel galloyl phytosterols with excellent antioxidant activity. Food

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Chem. 2014, 163, 171-177.

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(4) He, W. S.; Hu D.; Wang, Y.; Chen, X. Y.; Jia, C. S.; Ma, H. L.; Feng, B. A novel

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chemo-enzymatic synthesis of hydrophilic phytosterol derivatives. Food Chem. 2016, 192, 557-565.

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Sterol and Acetic Anhydride. J. Food Sci. 2016, 81 (7), C1629-C1635.

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(20) Li, R.; Jia, C. S.; Yue, L.; Zhang, X. M.; Xia, Q. Y.; Zhao, S. L.; Feng, B. Lipase-catalyzed

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synthesis of conjugated linoleyl β-sitosterol and its cholesterol-lowering properties in mice. J. Agric.

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Food Chem. 2010, 58, 1898-1902. 21



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(21) Brown, A. W.; Carden, T. J.; Hang, J. L.; Dussault, P. H.; Carr, T. P. Hydrolysis of phytosterol

516

esters in the intestine is required for their cholesterol lowering effects. Faseb J. 2011, 25, 585.

517

(22) Leong, W. F.; Lai, O. M.; Long, K.; Man, Y. B. C.; Misran, M.; Tan, C. P. Preparation and

518

characterisation of water-soluble phytosterol nanodispersions. Food Chem. 2011, 29, 77-83.

519

(23) Sala,A. A.; Llatas, G. G.; Cilla, A.; Barbera, R.; Siles, L. M. S.; Lagarda, M. G. Impact of lipid

520

components and emulsifiers on plant sterols bioaccessibility from milk -based fruit beverages. J.

521

Agric. Food Chem. 2016, 64 (28), 5686–5691.

522

(24) Pennisi Forell, S. C.; Ranalli, N.; Zaritzky, N. E. Effect of type of emulsifiers and antioxidants

523

on oxidative stability, colour and fatty acid profile of low-fat beef burgers enriched with unsaturated

524

fatty acids and phytosterols. Meat Sci. 2010, 86 (2), 364-370.

525

(25) Fisher, S.; Wachtel, E. J.; Aserin, A.; Garti, N. Solubilization of simvastatin and phytosterols

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in a dilutable microemulsion system. Colloids Surf., B 2013, 107, 35-42.

527

(26) Shaghaghi, M. A.; Harding, S. V.; Jones, P. J. H. Water dispersible plant sterol formulation

528

shows improved effect on lipid profile compared to plant sterol esters. J. Funct. Foods 2014, 6,

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280-289.

530

(27) Roberta, T.; Nicola,C.; Aygul, C.; Tchuenbou-Magaia, F. L. Development and

531

characterization of phytosterol-enriched oil microcapsules for foodstuff application. Food

532

Bioprocess Technol. 2018, 11 (1), 152-163.

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(28) Alexander, M.; Lopez, A. A., Fang Y.; Corredig, M. Incorporation of phytosterols in soy

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phospholipids nanoliposomes: encapsulation efficiency and stability. LWT-Food Sci. Technol. 2012,

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47, 427-436.

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(29) He, W. S.; Zhu, H. Y.; Chen, Z. Y. Plant sterols: chemical and enzymatic structural

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modifications and effects on their cholesterol-lowering activity. J. Agric. Food Chem. 2018, 88,

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3047-3062.

539

(30) Pang, M.; Jiang, S. T.; Cao, L. L.; Pan, L. J. Novel synthesis of steryl esters from phytosterols

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and amino acid. J. Agric. Food Chem. 2011, 59, 10732-10736. 22


Page 23 of 36


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(31) Lim, J. C.; Chung, D. W. Study on the synthesis and characterization of surface activities of

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hydrophilic derivatives of β-sitosterol. J. Appl. Polym. Sci. 2011, 125, 888-895.

543

(32) Upadhyaya, D. J.; Barge, A.; Stefania, R.; Cravotto, G. Efficient, solventless N-Boc protection

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of amines carried out at room temperature using sulfamic acid as recyclable catalyst. Tetrahedron

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Lett. 2007, 48, 8318-8322.

546

(33) Chandrudu, S.; Simerska, P.; Toth, I. Chemical methods for peptide and protein production.

547

Molecules 2013, 18, 4373-4388.

548

(34) Yamamoto, T.; Yoshizawa, M.; Mahmut, A.; Abe, M.; Kuroda, S. I.; Imase, T. Preparation of

549

new π-conjugated polypyrroles by organometallic polycondensations. Synthesis of N-BOC

550

(t-butoxycarbonyl) and N-phenylethynyl polymers, thermal deprotection of the BOC group, and

551

packing structure of the N-phenylethynyl polymer. J. Polym. Sci. Pol. Chem. 2005, 43, 6223-6232.

552

(35) Kou, H.; Kazuya, T.; Kiyofumi, I.; Takao, S. Synthesis of 6z-pandanamine by regioselective

553

cyclization reaction of 2-en-4-ynoic acid derivatives promoted by weak base. Heterocycles 2009, 77

554

(1), 493-505.

555

(36) Han, G.; Tamaki, M.; Hruby, V. Fast, efficient and selective deprotection of the

556

tert-butoxycarbonyl (Boc) group using HCl/dioxane (4 M). J. Pept. Res. 2001, 58 (4), 338-341.

557

(37) Cameron, D. R.; Weber, M. E.; Idziak, E. S.; Neufeld, R. J.; Cooper, D. G. Determination of

558

interfacial areas in emulsions using turbidimetric and droplet size data: correction of the formula for

559

emulsifying activity index. J. Agric. Food Chem. 1991, 39(4), 655-659.

560

(38) Pearce, K. N.; Kinsella, J. E. Emulsifying properties of proteins: Evaluation of a turbidimetric

561

technique. J. Agric. Food Chem. 1978, 26, 716–723.

562

(39) Detar, D. F.; Silverstein, R. Reactions of carbodiimides. Ι. The mechanisms of the reactions of

563

acetic acid with dicyclohexylcarbodiimide. J. Am. Chem. Soc. 1966, 88 (5), 1013-1019.

564

(40) Stankova, I.; Chuchkov, K.; Shishkov, S.; Kostova, K.; Mukova, L.; Galabov, A. S. Synthesis,

565

antioxidative and antiviral activity of hydroxycinnamic acid amides of thiazole containing amino

566

acid. Amino Acids 2009, 37, 383-388. 23



Page 24 of 36

567

(41) Meng, X.; Pan, Q.; Yang, T. Synthesis of phytosteryl esters by using alumina-supported zinc

568

oxide (ZnO/Al2O3) from esterification production of phytosterol with fatty acid. J. Am. Oil Chem.

569

Soc. 2010, 88, 143-149.

24


Page 25 of 36

571


Figure captions

572 573

Figure 1. FT-IR spectra of stigmasterol, stigmasteryl N-BOC-amino acid ester and stigmasteryl

574

amino acid ester hydrochlorides. (A: stigmasterol; B: stigmasteryl N-BOC-glycine ester; C:

575

stigmasteryl glycine ester hydrochloride; D: stigmasteryl N-BOC-aspartic acid ester; E: stigmasteryl

576

aspartic acid ester hydrochloride; F: stigmasteryl N-BOC-glutamic acid ester; G: stigmasteryl

577

glutamic acid ester hydrochloride)

578

Figure 2. MS spectra of stigmasteryl N-BOC-amino acid ester and stigmasteryl amino acid ester

579

hydrochlorides. (A: stigmasteryl N-BOC-glycine ester; B: stigmasteryl glycine ester hydrochloride;

580

C: stigmasteryl N-BOC-aspartic acid ester; D: stigmasteryl aspartic acid ester hydrochloride; e:

581

stigmasteryl N-BOC-glutamic acid ester; f: stigmasteryl glutamic acid ester hydrochloride)

582

Figure 3. Effect of reaction time (A), reaction time at 0 oC (B), molar ratio of catalyst EDC to

583

N-BOC-amino acid (C) and molar ratio of N-BOC-amino acid to phytosterols (D) on the conversion

584

of phytosterols to phytosteryl N-BOC- aspartic acid ester.

585

(Reaction condition: (A) 0oC for 8 h and then 25 oC, 1:1 molar ratio of N-BOC-aspartic acid to

586

phytosterols, 1:1:1.5 molar ratio of EDC to DMAP and triethylamine; (B) total time 24 h, 1:1 molar

587

ratio of N-BOC-aspartic acid to phytosterols, 1:1:1:1.5 molar ratio of N-BOC- aspartic acid to EDC,

588

DMAP and triethylamine; (C) 0 oC for 8 h and then 25 oC for 16 h, 1:1 molar ratio of N-BOC-

589

aspartic acid to phytosterols, 1:1:1.5 molar ratio of EDC to DMAP and triethylamine; (D) 0 oC for 8

590

h and then 25 oC for 16 h, 1:1.2:1.2:1.8 molar ratio of N-BOC-aspartic acid to EDC, DMAP and

591

trimethylamine. )

592

Figure 4. The thermal decomposition curves of phytosterols (A), phytosteryl glycine ester

593

hydrochloride (B), phytosteryl aspartic acid ester hydrochloride (C) and phytosteryl glutamic acid

594

ester hydrochloride (D).

595

Figure 5. The water-solubility of phytosteryl amino acid ester hydrochlorides at 25 oC. (p:

596

phytosterols; p-gly: phytosteryl glycine ester hydrochloride; p-asp: phytosteryl aspartic acid ester 25



Page 26 of 36

597

hydrochloride; p-glu: phytosteryl glutamic acid ester hydrochloride)

598

Figure 6. The emulsifying activity (A) and emulsifying stability (B) of phytosterols and phytosteryl

599

amino acid ester hydrochlorides. (p: phytosterols; p-gly: phytosteryl glycine ester hydrochloride;

600

p-asp: phytosteryl aspartic acid ester hydrochloride; p-glu: phytosteryl glutamic acid ester

601

hydrochloride)

26


Page 27 of 36

603


Scheme captions

604 605

Scheme 1. Synthesis of phytosteryl amino acid ester hydrochlorides by two-step approach.

606

Scheme 2. Proposed mechanism of esterification reaction.

27


4000

4000 -0 5

3500

3500 3000

3000 2500 2000

2500

2000

25

35

15

1500

1500 1000

1000 500

4000

F

70

55 65

500

500

4000 4000

-0 5

500 3500

40

35

35

3000

20

15

10

3500

15

10

3500 3500

Wavenumbers(cm-1)

28

ACS Paragon Plus Environment 2500

Wavenumbers (cm-1)

3000

55

Wavenumbers (cm-1)

3000 3000

2000

45

30

25

2500 2000

45

40

30

20

2500

2500 2000

2000

1217.46

1010.91

1500

1500

25

1500

1500

Wavenumbers(cm-1)

50

630.67

801.18

927.60

700.75

1090.09

55

835.87

996.46 961.90

50

971.80

1138.13 1119.58

1623.65

60

1000

1000

1000

1000 525.03

10

925.85 840.28 799.66

20

1138.27 1067.28 1090.15 1010.17 971.97

60

40

1252.46

25

1462.81 1382.74 1367.19

45

1088.80

65

30

1000

1137.08

70

35

1000

972.51

D 1500

996.01

4000

1500

1731.67

2000

1589.56 1504.81 1467.17 1384.38 1368.00 1330.97

-10

1746.65

15

2868.19

90

3397.22

B

2954.27

%Transmittance

50

2000

1198.18

40

464.17

2959.32 2936.66 2868.36

1054.59

1465.08 1381.87

45

1455.51 1383.75

1000

594.63

926.03 866.34 840.05 798.99 780.25

50

1599.79 1504.01

1500

2500

2868.85

40

2500

2955.39

50

597.28

1000

%Transmittance

55

780.76

3431.27

1022.71 959.23

1133.51

1637.95

2361.19

65

1731.92

30

3000

3000

3426.91

45

801.07

1500

843.49

60

1089.72

Wavenumbers (cm-1)

3500

3500

2360.22

50

1055.86 1028.22 1010.72 971.98 960.66

15

2955.82

2000

1170.61

%Transmittance 60

3427.81

35

971.78

20

571.97

2500

1026.21

30

%Transmittance

45

925.75 854.00 779.41

25

1167.79

70

799.20

3000

2000

1366.56 1284.25 1251.48 1206.09

35

1055.01 1027.31 972.35 961.17

3500

2500

1517.39 1456.64

20

1253.60

30

1456.86

4000

1220.20

30

1367.81

80

2359.39

70

1330.17

0

1454.80

10

1752.47 1719.39

2868.22

3369.10

75

1170.39

20

3000

1367.82

3500

1510.47

2956.39

10

1720.05

15

2868.24

0

1510.88

60

2026.15

5

2961.26

%Transmittance 40

1720.25

10

2868.70

4000

2957.37

20

3426.27

%Transmittance 4000

3426.73

%Transmittance

Journal of Agricultural and Food Chemistry Page 28 of 36

Figure 1 A

55

40

25

500

500

Wavenumbers(cm-1)

C 75

70

65

Wavenumbers (cm-1) Wavenumbers (cm-1)

500

E

75

65

60

55

50

-5

Wavenumbers (cm-1) 500

G

60

Wavenumbers (cm-1) 500

500

Page 29 of 36


Figure 2 A

B

Mw: 569

sterol

20130713-13 8 (0.081) Cm (6:11-(1:3+14:33)) 273.9

100

Mw: 505

2: Scan ES+ 6.31e6

273.9

%

[M-Cl+H]+ [M+Na]+ 79.7 245.9

0

318.0

200

C

345.9 465.9 591.9 592.9 748.7 795.1

400

600

800

Mw: 627

1

978.8

m/z 1000

D

[M-H]+

[M-HCl-H]+3: Scan ES-

626.2

100

Mw: 563

3: Scan ES- 2 1.52e7 20131101-28 去 boc-asp 7 (0.076) Cn (Cen,2, 80.00, Ht); Cm (6:10-(1:4+14:31))

20131101-27 boc-asp 7 (0.076) Cn (Cen,2, 80.00, Ht); Cm (5:13-(1:4+14:47))

100

626.5

393.0

134.5 132.8 134.8

%

%

627.7 213.8 213.5 214.2 134.8

0

100

E

214.9 282.7

170.9

200

300

628.2 628.7

113.1

629.3 552.2 393.0408.9 551.5 553.9 638.4

400

500

600

700

800

900

m/z 1000

0

F

Mw: 641

1

116.5

[M-H]+

20131128-1 BOC 7 (0.076) Cn (Cen,2, 80.00, Ht); Cm (6:11-(1:3+14:36))

150

200

285.3

250

300

526.9

392.7 394.8 365.1 437.0

350

400

525.8 475.0

450

527.8

603.0

571.4 583.8

500

550

600

617.8

m/z

Mw: 577

3: Scan ES1.68e7

640.1

100

100

340.8 283.9

218.6 160.6 203.1

526.4

393.3

283.5 282.9 236.9

116.9

627.5

1.31e6

526.1

283.2

626.8

640.6

%

[M-HCl-H]+ 641.9 254.9 116.9 174.8 254.3

0

100

200

642.3 332.9 713.5 332.7 333.3 423.1 566.0 643.0 714.8 826.6 334.2 424.2 491.0 599.8

300

400

500

600

700

800

975.2 976.6

900

m/z 1000

29



Page 30 of 36

Figure 3 70

90 85

A

60

80

40

Yield (%)

Yield (%)

50

30 20 10 0

B

75 70 65 60 55

0

8

16

24

32

40

48

50

56

0

Reaction time (h)

4

6

8

10

o

Reaction time at 0 C (h)

100 95

2

100

C

95

D

Yield (%)

Yield (%)

90 85 80

90

85

75 70 0.8

1.0

1.2

1.4

1.6

1.8

80

2.0

1.0

1.5

2.0

2.5

3.0

Molar ratio of N-BOC-amino acid ester to phytosterols

Molar ratio of EDC to N-BOC-amino acid ester

30


Page 31 of 36


Figure 4 B

A TG

TG

2.5

1 0.5 0

1.5 1 0.5 0

-0.5

-0.5

0

100

200

300

400

500

0

600

100

TG

3

300

400

500

600

500

600

温度 ( C) (oC) Temperature


C

200

o

o

D TG

DTG

DTG

2.5 2

Weight (%) 质量 (mg)

2.5


DTG

2


质量 (mg)(%) Weight

1.5

DTG

2 1.5 1 0.5

1.5 1 0.5 0

0 -0.5

-0.5

0

100

200

300

400

500

600

0

100

200

300

400

o 温度 ( C) (oC) Temperature

o


31



Figure 5

1.5

Solubility (mg/mL)

1.2

0.9

0.6

0.3

0.0

p

p-gly

p-asp

32


p-glu

Page 32 of 36

Page 33 of 36


Figure 6

A

B

0.5

80

0.01% (w/w) 0.10% (w/w) 0.50% (w/w)

Emulsifying Stability

Emulsifying Activity

0.4 0.3 0.2 0.1

60

40

20

0

0.0

p

p-gly

p-asp

p-glu

33


p-gly

p-asp

p-glu


Scheme 1.

HO

DMAP / EDC / Et3N

+

BOC

CH2Cl2

O

H N

BOC OH

O

H N R

O

R

HCl EtOAc

-

O

Cl+H3N R

O

BOC = t-BuOCO; R = H, CH2COOH, CH2CH2COOH

34


Page 34 of 36

Page 35 of 36


Scheme 2. EtN=C=N(CH2)3NMe2.HCl

Et3N

EtN=C=N(CH2)3NMe2

EDC

EDCI R1COOH

O R1

O

O

R2OH

R2

R1

Ester N H

N H

R1

Et

EDU

R1

N H

N H EDU

O R1

N H

R1

DMAP

(CH2)3NMe2

O N Et

O

O

(CH2)3NMe2

O

O

O

R1COOH

O-Acylurea

O Et

HN (CH2)3NMe2 Et O N

O

+

N

O

(CH2)3NMe2

N

R1 R2OH

N-Acylurea

O

O R1

OH

+

R1

O Ester

R1COOH: N-BOC-amino acids; R2OH: Stigmasterol

35


R2 + N

N DMAP


Page 36 of 36

Graphic for table of contents R

R O

+

BOC

+

NH

OH R'

HO

DMAP / EDC / Et3N rt

O

NH BOC

O

R'

Deprotection

Homogenization

R

Oil phase PS dispersion

Phase separation

O

N2H

+

Homogenization

Oil phase PS Ester dispersion

O R'

Emulsion formation

36


A Mild and Efficient Preparation of Phytosteryl Amino Acid Ester

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