Rubemamine and Rubescenamine, Two Naturally Occurring N

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Rubemamine and Rubescenamine, Two Naturally Occurring NCinnamoyl Phenethylamines with Umami Taste Modulating Properties Michael Backes, Katja Obst, Juliane Bojahr, Anika Thorhauer, Natacha Roudnitzky, Susanne Paetz, Katharina Verena Reichelt, Gerhard E. Krammer, Wolfgang Meyerhof, and Jakob Ley J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04402 • Publication Date (Web): 16 Sep 2015 Downloaded from http://pubs.acs.org on September 21, 2015

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Journal of Agricultural and Food Chemistry

Rubemamine and Rubescenamine, Two Naturally Occurring N-Cinnamoyl Phenethylamines with Umami Taste Modulating Properties

Michael Backes1, Katja Obst1, Juliane Bojahr2, Anika Thorhauer2, Natacha Roudnitzky2 Susanne Paetz1, Katharina V. Reichelt1, Gerhard E. Krammer1, Wolfgang Meyerhof2, Jakob P. Ley*1 1

Symrise AG, Flavors Division, Research & Technology, P.O. Box 1253, 37603 Holzminden,

Germany 2

German Institute of Human Nutrition, Department of Molecular Genetics, Arthur-

Scheunert-Allee 114-116, 14558 Nuthetal Corresponding Author: Dr. Jakob P. Ley, Symrise, [email protected]

Running Title: Rubemamine and Rubescenamine Act on the Umami Receptor

1 ACS Paragon Plus Environment


1

Abstract

2

Sensory screening of a series of naturally occurring N-cinnamoyl derivatives of substituted

3

phenethylamines r7evealed that rubemamine (9, from Chenopodium album) and

4

rubescenamine (10, from Zanthoxylum rubsecens) elicit strong intrinsic umami taste in water

5

at 50 and 10 ppm, respectively. Sensory tests in glutamate and nucleotides containing bases

6

showed that the compounds influence the whole flavor profile of savory formulations. Both,

7

rubemamine (9) and rubescenamine (10) at 10 - 100 ppm dose dependently positively

8

modulated the umami taste of MSG (0.17 – 0.22 %) up to 3fold. Among the investigated

9

amides, only rubemamine (9) and rubescenamine (10) are able to directly activate the

10

TAS1R1-TAS1R3 umami taste receptor. Moreover, both compounds also synergistically

11

modulated the activation of TAS1R1-TAS1R3 by MSG. Most remarkably, rubemamine (9)

12

was able to further positively modulate the IMP-enhanced TAS1R1-TAS1R3 response to

13

MSG ~1.8fold. Finally, also armatamide (11), zanthosinamide (13), and dioxamine (14),

14

which lack intrinsic umami taste in vivo and direct receptor response in vitro, positively

15

modulated receptor activation by MSG about 2fold and additionally the IMP-enhanced MSG-

16

induced TAS1R1-TAS1R3 responses approximately by 50%. In sensory experiments,

17

dioxamine (14) at 25 ppm in combination with 0.17 % MSG exhibited a sensory equivalent to

18

0.37 % MSG.

19

Keywords: umami, cinnamic acid phenethylamides, human sensory, flavor modulators,

20

umami receptor

21


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22

Introduction

23

Umami, is one of our five basic tastes, which is believed to signal the presence of calories

24

derived from protein and is correlated to the occurrence of monosodium glutamate (MSG) in

25

food.1 And rightly so, since it represents the most important umami taste eliciting compound.

26

Interestingly, some ribonucleotides, especially disodium 5’-guanylate (GMP) and disodium

27

5’-inosinate (IMP) which often co-occur with MSG in food are well known enhancers of

28

MSG-evoked umami taste.2

29

Umami taste is mainly mediated by the TAS1R1-TAS1R3 receptor heteromer,3 a G protein-

30

coupled receptor belonging to the family of glutamate receptors. The TAS1R1-TAS1R3

31

receptor is expressed in specialized epithelial cells of taste buds which are located on the soft

32

palate and in vallate, fungiform and foliate papilla of the tongue.4, 5 The mechanisms for MSG

33

reception and also the positive allosteric modulation by nucleotides is achieved through

34

binding of MSG and ribonucleotides to adjacent sites in the cleft of the extra-cellular Venus-

35

fly-trap motive of the TAS1R1 subunit.6 Moreover interaction sites for taste modulators have

36

been found within the transmembrane regions of the TAS1R1 and TAS1R3 subunit.6-8

37

In order to expand the set of applicable flavor molecules with umami taste characteristics

38

previous studies investigated mainly savory flavor constituents such as phthalides9, and

39

especially amino acid or nucleotide type compounds from foodstuff rich in umami taste.1, 10, 11

40

Such studies led to the discovery of several new artificial modulators for umami taste, e.g. N-

41

(heptan-4-yl)benzo[d][1.3]dioxole-5-carboxamide, 2E,6Z-nonadienoic acid N-ethylamide,

42

2E,6Z-nonadienoic acid N-cyclopropylamide, cyclopropanoic acid N-neomenthylamide or

43

some substituted N-benzyl-N-(2-pyridylethyl)oxalic acid amides.12,

44

effective modulators from natural sources were not available.


13

Until lately, such


45

For the efficacy of the majority of known synthetic umami taste and umami taste modulating

46

compounds, generally a central amide group or diamide in combination with two nonpolar,

47

medium sized substituents seems to be relevant.12 Among natural substances, the group of N-

48

cinnamoyl derivatives of aromatic amines exhibits a similar structural motif and was

49

investigated further. Important sub-groups are the N-cinnamoyl phenethylamines14 found in

50

various plants, N-cinnamoyl phenylalanins (clovamide type)15

51

cinnamoyl serotonins16 found in safflower seeds, and N-cinnamoyl anthranilic acids

52

(avenanthramides)17 described in oat. Indeed, several of these compounds were found to be

53

taste active. For example, the clovamides have been attributed astringent and bitter taste,15

54

the feruloyl-3-methoxytyramines have been described as weakly pungent substances18 and

55

some avenanthramides show cooling properties.19 Until recently, no data about the umami

56

effect of these secondary metabolites where reported, But during the reviewing process of this

57

paper a study was published showing the umami effects of rubemamine (9) and its homolog

58

N-3,4-dimethoxycinnamoyl-4-methoxyphenethylamine (16).20

59

In order to identify potential umami modulating compounds, a series of naturally occurring N-

60

cinnamoyl derivatives of substituted phenethylamines and serotonin was prepared (Figure 1,

61

natural occurrence Table 1) and tested by a sensory panel. In addition, advanced sensory

62

studies investigated the potential of these compounds to function as general taste modifiers

63

and synergistic umami taste modulators. Furthermore, in order to identify a biological basis

64

for the observed sensory effects, the compounds were tested by means of an in vitro TAS1R1-

65

TAS1R3 receptor assay for their intrinsic and synergistic activity together with MSG.

66

Materials and Methods

67

General. Reagents, solvents and consumables were purchased from commercial suppliers

68

(Acros Organics, Geel, Belgium; Sigma-Aldrich, Steinheim, Germany,; Alfa Aesar GmbH &

69

Co KG, Karlsruhe, Germany) or produced by Symrise and used without further purification or 4 ACS Paragon Plus Environment

found e.g. or cocoa, N-

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drying. Monosodium glutamate (MSG) was obtained from Oskar Berg GmbH, Germany.

71

Monitoring of the reaction was accomplished by thin-layer chromatography on silica gel

72

GF254 plates with either UV detection or by use of a staining reagent consisting of

73

ammonium molybdate, cerium sulfate and sulfuric acid. Purity was determined via GC

74

chromatography using either an Agilent 6890N or Agilent 7890N (Agilent Technologies,

75

Santa Clara, USA) system equipped with DB-WAX and DB-1 columns (length, 20 m; inside

76

diameter, 0.18 mm; film, 0.18 µm); flow rate, 0.5 – 3.0 ml min–1; injector, split ratio 1/70,

77

80°C – 12°/s – 250°C; carrier gas, H2; detector, FID, 275°C (Agilent Technologies, Santa

78

Clara, USA) or a high temperature MXT column (length, 15 m; inside diameter, 0.25 mm;

79

film, 0.10 µm); flow rate, 1.0 – 5.0 ml min–1; injector, split ratio 1/100, 60°C – 10°/s – 380°C;

80

carrier gas, H2; detector, FID, 420°C (Restek, Bad Homburg, Germany). If the purity could

81

not be determined via GC due to decomposition, below mentioned NMR and UPLC

82

instruments were used to determine the purity. The purity of all reported compounds was ≥

83

95%. 1H and

84

Varian Mercury Plus or Varian Unity Innova spectrometer (Varian, Darmstadt, Germany)

85

using tetramethylsilane as internal reference. GC-MS spectra (EI, 70 eV, detector:

86

quadrupole) were obtained by using either a Shimadzu GC2010/QP2010 (Shimadzu

87

Corporation, Kyoto, Japan) or an Agilent 6890N (Agilent Technologies, Santa Clara, USA)

88

system. HR-MS spectra (EI, 70 eV, detector: TOF) were recorded on a coupled system

89

consisting of a Bruker micrOTOF Q-II (Bruker Daltonik GmbH, Bremen, Germany) and a

90

Waters Acquity UPLC (Waters, Eschborn, Germany).

91

General procedure A for the synthesis of amides (1) – (7), (11), (12), (15)

92

1.0 eq. of the corresponding cinnamic acid derivative and 1.0 eq. of the N-hydroxysuccin-

93

imide were dissolved in 2.5 – 5.0 ml/mmol dioxane at 35°C. Subsequently 1.0 eq. of DCC in

94

1.5 – 2.5 ml/mmol dioxane was added and the resulting mixture was stirred overnight at room

13

C nuclear magnetic resonance spectra were recorded on a Varian Gemini,



95

temperature. The formed solid was removed by filtration and washed with dioxane. This

96

mixture was added slowly to a suspension of 1.1 – 1.5 eq. of the corresponding amine in 2.0 –

97

3.0 ml/mmol water containing 1.0 – 2.5 eq. of NaHCO3 keeping the temperature below 40°C,

98

after completion of the addition the mixture is stirred at room temperature and monitored by

99

TLC. After the reaction is finished, 10 % aqueous HCl is added to adjust the pH to 1.5 and the

100

reaction mixture is extracted two times with ethyl acetate. The combined organic layers are

101

consecutively washed with saturated aqueous NaHCO3 solution and brine before being dried

102

over NaSO4 (if necessary activated charcoal can be added during the drying process). After

103

removal of the solvent, the products were subjected to crystallization, flash chromatography

104

or preparative HPLC for final purification.

105

General procedure B for the synthesis of amides (9), (10), (13), (14)

106

1.05 – 1.2 eq. of oxalyl chloride were slowly added to a mixture of 1.0 eq. of the

107

corresponding cinnamic acid derivative in 2.0 – 4.0 ml/mmol of dichloromethane. The

108

mixture was allowed to stir overnight before refluxing for additional 30 minutes. After

109

evaporation of the solvent, the crude acid chloride was dissolved in 1.0 – 2.0 ml/mmol of

110

acetone and added to a mixture of the amine (or the corresponding hydrochloride) in 1.0 –

111

2.0 ml/mmol acetone and 1.0 – 2.0 ml/mmol water containing 1.0 eq. (or 2.0 eq. if the

112

hydrochlorides are used) of sodium hydroxide. The mixture is allowed to stir for additional

113

two hours before the product is filtered of and washed several times with water. Further

114

purification is possible – where needed – by recrystallization. Spectral data of all compounds

115

were in accordance with those reported in the literature as listed in Table S.1 of the

116

Supplementary Material.

117

N-E-Coumaroyltyramine (1) was prepared following A starting from p-coumaric acid and

118

tyramine hydrochloride. Final purification was possible via crystallization from ethyl acetate.


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119

1

120

3.64 (s, 1H), 6.40 (d, J = 15.7 Hz, 1H), 6.73 (m, 2H), 6.80 (m, 2H), 7.07 (m, 2H), 7.41 (m,

121

2H), 7.49 (d, J = 15.8, 1H) ppm. HR-MS (ESI+): 284.1281 (M+● + H, C17H19NO3+; calc.

122

284.1281).

123

N-(E)-Coumaryldopamine (2) was prepared following A starting from p-coumaric acid and

124

dopamine hydrochloride. Final purification was possible via two consecutive flash

125

chromatography steps (1st chromatography employing ethyl acetate as eluent; 2nd

126

chromatography employing chloroform/methanol 10:1 as eluent).

127

1

128

Hz, 1H), 6.54 (dd, J = 7.9, 2.2 Hz, 1H), 6.67 (d, J = 2.1 Hz, 1H), 6.71 (d, J = 8.0, 1H), 6.78

129

(m, 2H), 7.39 (m, 2H), 7.48 (d, J = 15.7, 1H) ppm. HR-MS (ESI+): 300.1235 (M+● + H,

130

C17H18NO4+; calc. 300.1230).

131

N-(E)-Caffeoyltyramine (3) was prepared following A starting from caffeic acid and

132

tyramine. Final purification was possible via crystallization from chloroform/methanol 10:1.

133

1

134

Hz, 1H), 6.71 (m, 2H), 6.74 (d, J = 8.2 Hz, 1H), 6.88 (ddd, J = 8.2, 2.1, ~ 0.5 Hz, 1H), 6.98(d,

135

J = 2.1 Hz, 1H), 7.05 (m, 2H), 7.37 (d, J = 15.6 Hz, 1H) ppm. HR-MS (ESI+): 300.1235 (M+●

136

+ H, C17H18NO4+; calc. 300.1230).

137

N-(E)-Feruloyltyramine (4) was prepared following A starting from ferulic acid and tyramine.

138

Final purification was possible via flash chromatography employing ethyl acetate as eluent.

139

1

140

3H), 6.38 (dd, J = 15.5, 1.1 Hz, 1H), 6.72 (m, 2H), 6.77 (dd, J = 8.3, 1.1 Hz, 1H), 7.01 (dd, J

141

= 8.3, 1.9 Hz, 1H), 7.05 (m, 2H), 7.10 (d, J = 1.9 Hz, 1H), 7.43 (d, J = 15.7 Hz, 1H) ppm.

H-NMR (200 MHz, CD3OD): 2.74 (dd, J = 8.1, 6.6 Hz, 2H), 3.44 (dd, J = 8.2, 6.5 Hz, 2H),

H-NMR (200 MHz, CD3OD): 2.69 (dd, J = 8.1, 6.5 Hz, 2H), 3.44 (m, 2H), 6.38 (d, J = 15.7

H-NMR (400 MHz, CD3OD): 2.74 (dd, J = 8.0, 6.9 Hz, 2H), 3.45 (m, 2H), 6.32 (d, J =15.7

H-NMR (400 MHz, CD3OD): 2.75 (t, J = 7.4 Hz, 2H), 3.35 (s, 1H), 3.46 (m, 2H), 3.88 (s,



142

GC/EI-MS: 343 (6, M+●), 329 (20), 194(12), 193(10), 192(10), 177(40), 151(10), 150(100),

143

149(9), 145(14), 137(11), 89(8).

144

N-(E)-Feruloyloctopamine (5) was prepared following AAV1 starting from ferulic acid and

145

octopamine hydrochloride. Final purification was possible via flash chromatography

146

employing ethyl acetate as eluent.

147

1

148

1H), 3.88 (s, 3H), 4.72 (m, 1H), 6.45 (d, J = 15.7, 1H), 6.76 (m, 2H), 6.79 (d, J = 8.2, 1H),

149

7.02 (dd, J = 8.1, 2.0 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.22 (m, 2H), 7.44 (d, J = 15.6 Hz,

150

1H) ppm. HR-MS (ESI+): 330.1335 (M+● + H, C18H20NO5+; calc. 330.1336).

151

N-(E)-Feruloyldopamine (6) was prepared following AAV1 starting from ferulic acid and

152

dopamine hydrochloride. Final purification was possible via two consecutive flash

153

chromatography steps (1st chromatography employing ethyl acetate as eluent; 2nd

154


155

1

156

3H), 6.40 (dd, J = 15.7, 0.4 Hz, 1H), 6.55 (dd, J = 8.0, 2.1 Hz, 1H), 6.67 (d, J = 2.1 Hz, 1H),

157

6.69 (d, J = 8.0 Hz, 1H), 6.79 (d, J = 8.2 Hz, 1H), 7.02 (dd, J = 8.2, 2.0, 1H), 7.11 (d, J = 2.0

158

Hz, 1H), 7.43 (d, J = 15.7 Hz, 1H) ppm. GC/EI-MS: 329 (20, M+●), 194 (64), 193 (13), 192

159

(29), 177 (100), 145 (30), 136 (38), 123 (13), 117 (14), 89 (15).

160

N-(E)-Feruloyl-3-methoxytyramine (7) was prepared following A starting from ferulic acid

161

and 3-methoxytyramine hydrochloride. Final purification was possible via two consecutive

162

flash chromatography steps (1st chromatography employing ethyl acetate as eluent; 2nd

163


H-NMR (400 MHz, CD3OD): 3.43 (dd, J = 13.6, 7.9 Hz, 1H), 3.53 (dd, J = 13.6, 5.0 Hz,

H-NMR (400 MHz, CD3OD): 2.70 (t, J = 7.4 Hz, 2H), 3.45 (dd, J = 8.0, 6.7 Hz, 2H), 3.87 (s,


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164

1

165

3H), 3.81 (s, 3H), 6.48 (d, J = 15.5 Hz, 1H), 6.73 (dd, J = 8.0, 1.8 Hz, 1H), 6.79 (d, J = 8.1

166

Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 1.9 Hz, 1H), 7.09 (m,1H), 7.17 (d, J = 1.9 Hz,

167

1H), 7.49 (d, J = 15.6 Hz, 1H) ppm. GC/EI-MS: 343 (6, M+●), 194 (12), 193 (10), 192 (10),

168

177 (40), 151 (10), 150 (100), 149 (9), 145 (14), 137 (11), 89 (8).

169

N-(Z)-Feruloyl-3-methoxytyramine (8) was prepared by dissolving N-E-Feruloyl-3-

170

methoxytyramine (7) in 100 ml/g ethanol and subsequent UV-irradiation (LW 366) for 100 h.

171

The resulting E/Z mixture of isomers was purified by HPLC chromatography.

172

1

173

3H), 3.82 (s, 3H), 5.81 (d, J = 12.7, 1H), 6.60 (dd, J = 8.0, 2.0, 1H), 6.62 (d, J = 12.6 Hz, 1H),

174

6.69 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H), 6.77 (d, J = 2.0 Hz, 1H), 6.92 (dd, J = 8.2,

175

2.0 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H) ppm. GC/EI-MS: 343 (5, M+● ), 194 (12), 193 (8), 177

176

(39), 151 (11), 150 (100), 145 (19), 137 (10), 135 (8), 117 (9), 89 (10).

177

Rubemamine (9) was prepared following B starting from (E)-3,4-dimethoxy cinnamic acid

178

and 3,4-dimethoxyphenylethylamine. If necessary, further purification is possible via

179

crystallization from n-propanol/n-hexane 2:3. Spectral data were in accordance with those

180

reported in the literature.21

181

1

182

3H), 3.87 (s, 3H), 3.90 (s, 3H), 3.90 (s, 3H), 5.58 (t, J = 5.9 Hz, 1H), 6.20 (d, J = 15.5 Hz,

183

1H), 6.75 (d, J = 2.0 Hz, 1H), 6.77 (dd, J = 8.0, 1.9 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.85 (d,

184

J = 8.3 Hz, 1H), 7.00 (d, J = 2.0 Hz, 1H), 7.07 (ddd, J = 8.2, 2.0, ~ 0.5 Hz, 1H), 7.56 (d, J =

185

15.5 Hz, 1H) ppm. GC/EI-MS: 371 (6, M+●), 207 (3), 206 (10), 192 (5), 191 (29), 165 (11),

186

164 (100), 163 (5), 151 (7), 149 (6).

H-NMR (200 MHz, CD3OD): 2.74 (t, J = 7.2 Hz, 2H), 3.44 (dd, J =7.9, 6.5 Hz, 2H), 3.76 (s,

H-NMR (400 MHz, CD3OD): 2.70 (t, J = 7.3 Hz, 2H), 3.42 (dd, J = 7.9, 6.7 Hz, 2H), 3.78 (s,

H-NMR (400 MHz, CDCl3): 2.84 (t, J = 6.9 Hz, 2H), 3.64 (td, J = 6.9, 5.9 Hz, 2H), 3.87 (s,



187

Rubescenamine (10) was prepared following B starting from (E)-3,4-dimethoxy cinnamic

188

acid and 2-(1,3-benzodioxol-5-yl)ethanamine hydrochloride. If necessary, further purification

189

is possible via crystallization from n-propanol/n-hexane 2:3. Spectral data were in accordance

190

with those reported in the literature.21

191

1

192

6H), 5.56 (t, J = 6.1 Hz, 1H), 5.94 (s, 2H), 6.20 (d, J = 15.5 Hz, 1H), 6.67 (dd, J = 7.8, 1.7 Hz,

193

1H), 6.72 (d, J = 1.7 Hz, 1H), 6.77 (d, J = 7.9 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 7.01 (d, J =

194

2.0 Hz, 1H), 7.07 (dd, J = 8.3, 2.0 Hz, 1H), 7.56 (d, J = 15.5 Hz, 1H) ppm. GC/EI-MS: 355

195

(17, M+●), 207 (58), 206 (79), 192 (12), 191 (69), 163 (11), 149 (11), 148 (100), 147 (13), 77

196

(11).

197

Armatamide (11) was prepared following A starting from (E)-3,4-methylendioxy cinnamic

198

acid and 2-(4-methoxyphenyl)ethanamine . Final purification was possible via crystallization

199

from ethyl acetate.

200

1

201

(s, 2H), 6.42 (d, J = 15.6 Hz, 1H), 6.83 (d, J = 8.1, 1H), 6.86 (d, J = 8.7 Hz, 2H), 7.04 (ddd, J

202

= 8.0, 1.7, ~0.5 Hz, 1H), 7.11 (m, 1H), 7.16 (m, 2H), 7.44 (d, J = 15.6, 1H) ppm. HR-MS

203

(ESI+): 326.1387 (M+● + H, C19H20NO4+; calc. 326.1387).

204

Zanthosine (12) was prepared following A staring from (E)-3,4-methylendioxy cinnamic acid

205

and 3,4-dimethoxyphenylethylamine. For final purification the product was dissolved in ethyl

206

acetate under reflux. After cooling down to room temperature, the product was precipitated by

207

the addition of diethylether.

208

1

209

3H), 3.87 (s, 3H), 5.66 (m, 1H), 5.99 (s, 2H), 6.15 (d, J = 15.5 Hz, 1H), 6.70 – 6.88 (m, 4H),

H-NMR (400 MHz, CDCl3): 2.80 (t, J = 6.8 Hz, 2H), 3.61(td, J = 6.8, 5.9 Hz, 2H), 3.90 (s,

H-NMR (200 MHz, CD3OD): 2.77 (dd, J = 8.0, 6.6 Hz, 2H), 3.46 (m, 2H), 3.73 (s, 3H), 6.02

H-NMR (200 MHz, CDCl3): 2.83 (t, J = 6.9 Hz, 2H), 3.63 (td, J = 6.9, 5.9 Hz, 2H), 3.87 (s,


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210

6.92 – 7.02 (m, 2H), 7.53 (d, J = 15.5 Hz, 1H) ppm. HR-MS (ESI+): 356.1490 (M+● + H,

211

C20H22NO5+; calc. 356.1492).

212

Zanthosinamide (13) was prepared following B staring from (E)-3,4-methylendioxy cinnamic

213

acid and 2-(3,4-dimethoxyphenyl)-N-methyl-ethanamine. As the expected product did not

214

precipitate from the reaction mixture, the product is extracted with ethyl acetate. After

215

evaporation of the solvent no further purification was necessary.

216

1

217

3.80 (s, 3H), 5.94 (s, 2H), 6.50 (d, J = 15.3 Hz, 1H), 6.70 (s, 2H), 6.72 (d, J = 2.0 Hz, 1H),

218

6.78 (d, J = 8.3 Hz, 1H), 6.80 (d, J = 8.1 Hz, 1H), 6.92 (d, J = 6.5 Hz, 2H), 7.46 (d, J = 15.3

219

Hz, 1H) ppm. GC/EI-MS: 369 (7, M+● ), 176 (9), 175 (78), 165 (11), 164 (100), 151 (7), 149

220

(4), 145 (25), 117 (12), 89 (15). HR-MS (ESI+): 353.1369 (M+● + H, C20H21N2O4+, calc.

221

353.1496).

222

Dioxamine (14) was prepared following B starting from (E)-3,4-methylendioxy cinnamic

223

acid and 2-(1,3-benzodioxol-5-yl)ethanamine hydrochloride.

224

1

225

1H), 5.94 (s, 2H), 5.99 (s, 2H), 6.14 (d, J = 15.5 Hz, 1H), 6.66 (dd, J = 7.8, 1.6 Hz, 1H), 6.71

226

(d, J = 1.5 Hz, 1H), 6.76 (d, J = 7.8, 1H), 6.79 (dd, J = 7.6, ~0.8 Hz, 1H), 6.96 (dd, J = 7.6,

227

1.7, 1H), 6.98 (s, 1H), 7.53 (d, J = 15.5 Hz, 1H) ppm. HR-MS: 340.1172 (M+● + H,

228

C19H18NO5, calc. 340.1179).

229

Moschamine (15) was prepared following AAV1 starting from ferulic acid and serotonine

230

hydrochloride. Final purification was possible via flash chromatography employing ethyl

231

acetate as eluent with subsequent recrystallization from ethylacetate.

H-NMR (100 °C, 400 MHz, CDCl3): 2.81 (m, 2H), 2.99 (s, 3H), 3.62 (m, 2H), 3.77 (s, 3H),

H-NMR (200 MHz, CD3OD): 2.80 (t, J = 6.8 Hz, 2H), 3.60 (td, J = 6.8, 5.9 Hz, 2H), 5.54 (s,



232

1

233

(d, J = 15.7 Hz, 1H), 6.59 (dd, J = 8.6, 2.3 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 6.85 (d, J = 2.4

234

Hz, 1H), 6.98 (dd, J = 8.1, 1.9 Hz, 1H), 7.05 (d, J = 2.5 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H),

235

7.13 (dd, J = 8.8, ~ 0.6 Hz, 1H), 7.33 (d, J = 15.7 Hz, 1H), 8.03 (t, J = 5.7 Hz, 1H), 8.59 (s,

236

1H), 10.48 (d, J = 2.4 Hz, 1H) ppm. HR-MS: 353.1374 (M+● + H, C20H21N2O4, calc.

237

353.1496).

H-NMR (400 MHz, CD3S=OCD3): 2.78 (t, J = 7.4 Hz, 2H), 3.43 (m, 2H), 3.80 (s, 3H), 6.45

238 239

Sensory tests

240

Sensory tests were carried out with healthy and trained panelists without known taste

241

disorders. Panelists were fully informed about procedure and intention of the project and had

242

given written consent. Due to the FEMA/GRAS status of rubemamine (9)22 and

243

rubescenamine (10)23 and positive evaluation EFSA for rubemamine (9),24 the other

244

compounds were considered to be a low risk for the screening phase. In addition, the panelists

245

were advised not to swallow the samples (max. 10 mL solution), but to use the sip and spit

246

method. The tests were conducted in sensory panel rooms under standardized conditions, and

247

they were given blind and randomized. For the preparation of the test solutions Vittel® water

248

was used. The panelists had participated earlier at regular intervals in sensory work and were,

249

therefore, familiar with the techniques applied.

250

Pre-Evaluation. A number of 5–8 trained panelists received each compound in a dosage of 10,

251

50, and 100 ppm in a 0.5% salt and a 5.0% sugar solution, and were asked to describe the

252

sample and give a qualitative statement for the umami intensity. No additional reference (e.g.,

253

MSG) was presented during this session.

254

An American beef extract (Beef Meat Extract Stock Type, Symrise) as the base, a

255

corresponding base mixed with MSG and a corresponding base mixed with MSG and the test 12 ACS Paragon Plus Environment

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compound were in each case administered blind individually to 15-25 trained panelists at

257

slightly elevated temperatures (30-35°C) for profiling. Using the descriptors previously

258

decided upon by discussion (mouth-feel, salty, metallic, meaty, bitter, mouth-watering,

259

barbecued-roasted, sweet, sour, lingering) the strength of these was assessed on a scale from

260

0 (imperceptible) through 9 (very strong). The individual results of the panelists were

261

averaged.

262

The taste detection threshold for rubemamine (9) in water (Vittel®) was determined by a

263

forced-choice ascending concentration series methods of limits according to the established

264

ASTM protocol. 25 Threshold for rubescenamine (10) was determined at elevated temperature

265

(~ 30°C) according to the same method.

266

Quantification of taste modulation effects of rubemamine (9) and rubescenamine (10) on

267

umami taste of MSG (Yamaguchi protocol)

268

For the quantification of the synergistic effects, the intrinsic and positive umami modulating

269

activity of the test compound in comparison to MSG was determined by means of a paired

270

choice comparison test as proposed by Yamaguchi.2 For the determination of the intrinsic

271

activity a binary solution (pH 6.0) containing the test compound in water (fixed sample) was

272

compared to a series of aqueous solutions containing logarithmically (30% intervals, 0.17%,

273

0.22%, 0.28%, 0.37%, 0.49%, 0.63%, 0.82%, 1.07%, 1.4 %, 1.82 w/w) increasing

274

concentrations of MSG (reference samples). For the determination of the modulating activity

275

a binary solution (pH 6.0) containing the umami compound and MSG (0.17% or 0.22 %) in

276

water (fixed sample) was compared to a series of aqueous solutions containing logarithmically

277

(30% intervals) increasing concentrations of MSG (reference samples). In each sensory

278

session, the assessors were asked to evaluate four sample pairs, presented in randomly coded

279

cups, and to identify the sample exhibiting the stronger umami taste using a forced choice

280

methodology (2-AFC). The panelists wore nose clips during tasting. Each test was repeated 3 13 ACS Paragon Plus Environment


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times for each panelist. To control significance, Student’s t-test was conducted and the level

282

of significance was set to p < 0.1 due to the relative low panelist number (n = 14-18).

283

Activation of TAS1R1-TAS1R3 umami receptor in transfected HEK 293 cells

284

To elucidate umami receptor activation by the test compounds a cell based assay was used as

285

described elsewhere.26 In

286

HEK293PEAKrapid cells (American Type Culture Collection), modified for stable expression

287

of human TAS1R1 (hTAS1R1) subunit and the promiscuous G protein subunit Gα15. For

288

umami receptor expression cells were transiently transfected with vector encoding for the rat

289

Tas1r3 (rTas1r3) subunit using Lipofectamine2000 (Invitrogen). Transfection was carried out

290

24 h prior to calcium imaging experiments.

291

To enable functional calcium imaging, transfected cells were loaded with 2 µM Fluo4-AM

292

(Molecular Probes) in serum-free DMEM low glucose Glutamax (Gibco) medium containing

293

2.5 mM probenecide. After 1 h of incubation at 37 °C cells were washed 3-times with bath

294

solution (130 mM NaCl, 5 mM KCl, 10 mM Hepes, 2 mM CaCl2, pH 7.4) and incubated for

295

20 min at room temperature in the dark between washing steps to allow complete de-

296

esterification of the dye. Calcium imaging experiments were carried out using fluorimetric

297

imaging plate reader (FLIPRtetra, Molecular Devices). This allows automated substance

298

application and simultaneous fluorescence measurements. Compounds were solved and

299

applied in bath solution or low concentrations of DMSO (0.03% - 0.3%) for better solubility.

300

Subsequent to application of test compounds, isoproterenol was applied onto the cells to

301

control for cell number and vitality. Empty vector transfected cells were used to control for

302

unspecific responses of the cellular background. To control for responses of the diluent cells

303

were treated with bath solution or the respective concentration of DMSO.

brief, functional experiments were carried out


using

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Raw fluorescence changes were corrected for unspecific fluorescence changes of empty

305

vector expressing cells using ScreenWorks 2.0. Relative fluorescence change was then

306

calculated (∆F=Fmax-Fmin) and normalized to baseline fluorescence (∆F/F). To calculate

307

concentration-response curves ∆F/F values were plotted half-logarithmically against

308

concentration of the test compound. Half-maximal effective agonist concentrations (EC50)

309

were calculated using non-linear regression to the sigmoidal function f(x) = min + (max – min

310

/ 1 + [x / EC50]Hillslope) (SigmaPlot 9.01, Systat Software). To control for significance one-way

311

analysis of variance was performed using SPSS.

312 313

Results and Discussions

314

In order to investigate their possible potential as modulators of umami taste, a subset of the

315

known naturally occurring N-cinnamoyl phenethylamines similar to rubemamine (9) and

316

rubescenamine (10) with various substitution patterns were prepared. In Figure 1 the

317

synthesized structures are shown; their natural occurrence is listed in Table 1. Depending on

318

the substitution pattern there are several methods known to obtain these N-cinnamoyl amines.

319

In case of free hydroxyl groups, the cinammic acid has to be activated as the corresponding N-

320

hydroxysuccinimide ester employing N,N-dicyclohexylcarbodiimide as coupling reagent27

321

These intermediates were reacted with an appropriate amine to obtain the desired compounds.

322

The target compounds could be purified by column chromatography or crystallization.

323

Alternatively – if the cinnamic acid moiety contains no hydroxyl groups – it may be more

324

convenient to transfer the cinnamic acid into the corresponding acid chloride by using oxalyl

325

chloride and subsequent reaction with a suitable amine under Schotten-Baumann conditions.

326

Usually, no further purification step is necessary; in the supplementary material, the 1H-NMR

327

and GC-MS or LC-MS spectra for rubemamine (9) and rubescenamine (10) are shown to

328

demonstrate purity. The best method to obtain (Z) isomers of the parent (E) configured 15 ACS Paragon Plus Environment


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cinnamic acid amides is an isomerization reaction under UV28 as described for N-(Z)-feruloyl-

330

3-methoxytyramine (8).

331

All synthesized compounds were evaluated at 10, 50 and 100 ppm in pure water by a panel of

332

8 experts (Table 1). Whereas many of these N-cinnamoyl derivatives mostly evoked

333

bitterness, mouth drying or were associated with trigeminal sensations at 100 ppm, only

334

rubemamine (9) and rubescenamine (10) showed an intrinsic umami taste. Zanthosinamide

335

(13) was found to elicit umami taste at 100 ppm as well but in addition it was rated very bitter

336

at the same concentration. Just recently, the umami taste of 9 was reported20 but from the

337

remaining umami active cinnamoyl amines, only the homolog N-3,4-dimethoxycinnamoyl-4-

338

methoxyphenethylamine (16) could be identified in natural sources. According to the study, a

339

3,4-dimethoxycinnamic acid moiety seems to be necessary for a strong umami activity which

340

is very well reflected by the results shown in Table 1 for the series of naturally occurring N-

341

cinnamoyl phenethylamines.

342

Besides the well-known umami taste of foodstuffs or raw materials rich in amino acids,

343

ribonucleotides, peptides, their derivatives, or their reaction products generated during food

344

processing1, 13, 29 there are only singular reports about plant secondary metabolites exhibiting

345

or modulating umami taste. E.g., the malic acid derivative morelid was found in morel

346

mushrooms and weakly modulates umami taste evoked by MSG30 and the polyphenol

347

theogallin was identified to be one component of the umami character of green tea.31 The

348

reason for this paucity may be the very low concentration of such substances in the original

349

botanical sources; e.g. rubemamine (9) was found in white goosefoot (Chenopodium album)32

350

at a level of 6 mg kg-1 , N-3,4-dimethoxycinnamoyl-4-methoxyphenethylamine (16) as trace

351

compound in Zanthoxylum piperitum20 and rubescenamine (10) at a level of 10 ppm in the

352

bark of Zanthoxylum rubescens.21 The detection threshold for rubemamine (9) in water was

353

determined at a level of 2263 ppb. Therefore, even for white goosefoot which is at least 16 ACS Paragon Plus Environment

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sporadically consumed in Western European countries as salad or green, the effective amount

355

in food prepared from goosefoot is near or even below the threshold for the pure compound.

356

Due to the limited solubility of rubescenamine (10), the threshold of the neat compound was

357

determined at elevated temperatures (30 °C) at 903 ppb. Zanthoxylum piperitum bark is

358

reported to be used solely for pharmaceutical treatments and therefore only limited sensorial

359

data are available.

360

The ability of the investigated N-cinnamoyl amines (1) – (15) to activate the heterodimeric

361

umami receptor in a heterologous cell system was investigated via calcium imaging. Of the

362

synthesized compounds, only rubemamine (9) and rubescenamine (10) showed activation of

363

the heterologously expressed umami receptor hTAS1R1-rTas1r3 (Figure 2, 3). Since

364

glutamate and nucleotides were shown to interact with the TAS1R1 subunit of the receptor6 it

365

can be assumed that the functionally expressed umami receptor resembles the native human

366

umami receptor in taste cells. Moreover the EC50 value we found for the prototypical umami

367

substance glutamate (MSG) is in the range of other in vitro data and also correlated well with

368

in vivo threshold for glutamate.

369

identified umami compounds concentration-response curves were established. Figure 3

370

demonstrates that rubemamine (9) is a powerful umami receptor agonist which is ~40fold

371

more potent than MSG and equally efficient. In contrast, rubescenamine (10) has a much

372

lower efficacy. However, this compound (10) is able to activate the receptor at even lower

373

concentrations than rubemamine (9) and has therefore a 200fold higher potency than MSG.

374

This is can be seen by the lower EC50 value of rubescenamine (10) (7.5 ± 0.4 µM) compared

375

to rubemamine (9) (44 ± 8.0 µM) and MSG (1704 ± 335 µM). Although the difference in the

376

EC50 value of rubemamine and rubescenamine is statistically not significant by one-way

377

analysis of variance, the higher potency of rubescenamine (10) relative to rubemamine (9) to

3, 33-36

To compare the potency and efficacy of these newly



378

act on the umami receptor is also reflected by their detection thresholds in water (903 ppb vs.

379

2263).

380

Furthermore the potential of the two naturally occurring compounds to modulate umami

381

receptor activation by MSG and MSG + IMP was evaluated. Rubemamine (9) und

382

rubescenamine (10) were both able to positively modulate the receptor response to MSG, yet

383

the effect mediated by 9 was much larger than that mediated by (10). For rubescenamine (10)

384

no further effect was observed in the presence of IMP (Figure 4). Most remarkably,

385

rubemamine (9) further positively influenced the IMP-enhanced receptor responses to MSG,

386

whereas, vice versa, IMP did not further enhance the receptor response to MSG and 9. Thus,

387

the data do not allow to decide if IMP and 9 bind to the same site or different sites in

388

hTAS1R1-rTas1r3. Yet the data demonstrate that 9 positively modulates MSG-mediated

389

receptor responses to an extent that cannot be further enlarged by IMP. The substances 8, 11,

390

13 and 14, which showed no receptor activation when applied alone onto the cells, were also

391

able to positively modulate receptor activation by MSG even though their effects were smaller

392

than that of 9. Additionally, like compound 9, compounds 11, 13 and 14 enlarged the IMP-

393

enhanced TAS1R1-Tas1r3 responses to MSG. However, there are differences between the

394

effects mediated by 9 or 11, 13 and 14. First of all the positive effect of rubemamine (9) on

395

IMP-enhanced MSG-mediated receptor responses is smaller than that contributed by 11, 13

396

and 14. Secondly and more importantly and unlike the case of 9, the effect of the latter

397

compounds on MSG-mediated receptor response can be further enhanced by IMP. These

398

findings suggest that the binding sites for 11, 13 and 14 differ from that of IMP. Positive

399

modulation of a ribonucleotide-enhanced MSG-mediated umami receptor responses was also

400

shown for another amide.37 However, the reported effect was much smaller than effects

401

elicited by 9, 11, 13 and 14.


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402

Although binding and sensory studies need to confirm these results, the data show that little

403

differences in molecular structure can have large impact on receptor responses. For the related

404

sweet taste receptor TAS1R2-TAS1R3 which has the TAS1R3 subunit in common with the

405

umami receptor binding of structurally related substances has been described to occur in

406

similar regions.7, 8, 38 While umami receptor positive modulation by nucleotides is described to

407

occur via the TAS1R1 subunit,6 the known sweet and umami taste inhibitor lactisole

408

negatively modulates the umami receptor via the transmembrane regions of the TAS1R3

409

subunit.7,

410

yl)benzo[d][1,3]dioxole-5-carboxamide) has been demonstrated to interact with the

411

transmembrane region of the TAS1R1 subunit.6 If the binding occured via the rTas1r3 subunit

412

the modulatory effect were not seen in human sensory studies. But the regions of the receptor

413

which are targets for interaction with the N-cinnamoyl amides are currently unknown and

414

need to be determined exactly in further investigations.

415

To quantify the modulating effects for the naturally occurring N-cinnamoyl amines 9, 10, 11,

416

13 and 14 in vivo, further sensory studies according to the method described by YAMAGUCHI2

417

were performed; amide 8 was not evaluated due to its disturbing dry off-taste. Generally it is

418

very difficult to quantify umami effects due to the strong lingering character of this taste

419

quality. Especially a single experimental comparison of a test solution against a full

420

concentration series of MSG within a single sensory session fails due to carry-over effects.

421

Therefore, Yamaguchi proposed a series of forced choice paired tests which compare

422

mixtures of a standard MSG concentration (e.g. 0.17 %) and the test compound with each

423

sample of a MSG concentration series (here 0.17 up to 1.82 % MSG). Multiple sessions for

424

each panelist are necessary to get data on the full series of dilutions. In a single test, the

425

panelist has only to decide which solution is stronger in umami. At the end all experiments are

426

ranked according to the answers of the panelists. The same experiment is done with the test

427

compound alone and finally the MSG equivalent concentration is calculated as that last

8

Also the positively umami taste modulating compound S807 (N-(heptan-4-



Page 20 of 40

428

concentration where the mixture of MSG and test compound is non-significantly weaker than

429

the MSG reference solution. The known synergistic effects for GMP

430

validation of the panel; 40 ppm GMP (corresponds to 0.37 % MSG) and 0.17 % MSG were

431

described to elicit the same intensity of umami taste as a MSG solution of 0.8 %. Therefore,

432

GMP exhibited a clear and expected synergistic effect on umami taste for the panelists.

433

Amides 11 (10 ppm) and 13 (50 ppm) showed no significant modulating effect in the

434

Yamaguchi sensory protocol (data not shown). Dioxamine (14) at 25 ppm in combination

435

with 0.17 % MSG exhibited a sensory equivalent to 0.37 % MSG. Due to the limited

436

solubility of 14 the latter test was done at increased temperature (35 - 40 °C). Unfortunately,

437

some bitterness was detected which could potentially mislead the panelists and therefore the

438

tests with 14 were not extended in depth. For rubemamine (9) positive modulating effects

439

were found and are summarized in Figure 6. For all tested combinations of rubemamine (9)

440

with MSG (columns 1-9) the compound shows a positive synergistic effect. In contrast,

441

rubescenamine (10) (50 ppm column 10, 0.17 % MSG) showed no effect at room temperature

442

(20-22°C, see Figure 5, column 10) but the experiment was repeated with a warmed solution

443

also in comparison with rubemamine (9) (Figure 6). At increased temperature, for 9 the effect

444

is still more pronounced compared to ambient conditions and also rubescenamine (10) shows

445

positive modulating properties on the umami taste of MSG. The limited solubility of 10 at

446

ambient conditions noticed in this experiment might be also the reason for the lower efficacy

447

of 10 compared to rubemamine (9) seen in the dose-response studies on the hTAS1R1-

448

rTas1r3 receptor.

449

Although the interaction sites of the cinnamoyl amides at the umami receptor remain to be

450

elucidated, the quantitative sensory results support the studies using the receptor assays and

451

the proposal of two different binding sites: only 9 and 10 are able to induce strong intrinsic

452

umami effects and cause a positive modulation of the MSG umami taste in vitro and in vivo. 20 ACS Paragon Plus Environment

2

was used for the

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In contrast, the potential modulators 11, 13, and 14 putatively binding to an alternative

454

receptor site exhibited only weak or no intrinsic umami effects. Only 14 showed some

455

modulating effect on MSG in vivo which was slightly disturbed by limited solubility and

456

bitterness. For the observed differences between the umami modulation in vitro and in vivo by

457

the compounds 11 and 13 it must be considered that we used the rat Tas1r3 in the

458

heterologous expression system which could be responsible for this effect.. In order to

459

evaluate the potential of rubemamine (9) and rubescenamine (10) as flavor substances

460

showing also general taste modifying properties, advanced profiling studies in a model broth

461

were performed with a larger panel (n = 16 – 20) on an American beef extract already

462

containing the typical umami tastants such as glutamate and nucleotides. In this case, the

463

attribute “umami” itself was excluded due to some uncertainty of its description in a complex

464

flavor environment. Instead umami-related attributes such as “mouth watering” or “meaty”

465

were used. In Figure 7 the differences of these descriptors in relation to the ratings for the

466

base are shown: 0.05 % MSG increases most of the attributes significantly. Rubemamine (9)

467

at 50 ppm significantly raises the important descriptors mouth feel, meaty, and mouth

468

watering but with the exception for the parameter mouth watering, this amount is not able to

469

match the intensity of the positive control (base with 0.05% added MSG) (Figure 7A).

470

Rubescenamine (10) at 5 and especially at 10 ppm is able to boost significantly mouth feel,

471

meaty, mouth watering, sour, and long lasting (Figure 7A) and shows at 10 ppm no

472

sifgnificant difference to the positive control for mouth feel, saltiness, and mouth watering,

473

reflecting the higher potential as flavor modifier. In Figure 7B, the potential of 9 and 10 to

474

boost the flavour profile of a low added MSG concentration in the model broth is shown. The

475

effect of a mixture of low MSG and rubemamine (9) matches most ratings of the attributes of

476

the positive control. Rubescenamine (10) at 5 ppm in combination with 0.0025 % MSG is

477

also able to boost the profile of the base showing mostly significant changes compared to the

478

base but non-significant differences to the positive control. 21 ACS Paragon Plus Environment


479

As result of the study, besides the known umami tastant rubemamine (9) also the naturally

480

occurring rubescenamine (10) was found to modulate complex flavor systems. Both are able

481

to synergistically modulate the umami sensation of MSG and are acting on the umami

482

receptor as shown in heterologous cell models. New insights for the binding mode can be

483

deduced from the combined sensory and in vitro experiments which lead to further studies to

484

validate the binding mode and binding site within the umami receptor.

485

Acknowledgments

486

The authors thank Maria Blings, Petra Hoffmann-Lücke, Carsten Strempel, Bernd Wiedwald

487

and Susanne Mundt for technical support. We also gratefully acknowledge Dr. Anne

488

Brockhoff (Nuthetal) for her continuous advice how to run the umami receptors assays.

489

Funding Sources

490

The study was partly funded by BMBF (Federal Ministry of Education and Research,

491

Germany), FKZ 01EA1324C.

492


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References

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1.

Behrens, M.; Meyerhof, W.; Hellfritsch, C.; Hofmann, T., Sweet and Umami Taste:

495

Natural Products, Their Chemosensory Targets, and Beyond. Angew. Chem. Int. Ed.

496

2011, 10, 2220-2242.

497

2.

498 499

5′-Inosinate. J. Food Sci. 1967, 32, 473-478. 3.

500 501

Nelson, G.; Chandrashekar, J.; Hoon, M. A.; Feng, L.; Zhao, G.; Ryba, N. J. P.; Zuker, C. S., An amino-acid taste receptor. Nature 2002, 416, 199-202.

4.

502 503

Yamaguchi, S., The Synergistic Taste Effect of Monosodium Glutamate and Disodium

Nelson, G.; Hoon, M. A.; Chandrashekar, J.; Zhang, Y.; Ryba, N. J.; Zuker, C. S., Mammalian sweet taste receptors. Cell 2001, 106, 381-390.

5.

Voigt, A.; Hubner, S.; Lossow, K.; Hermans-Borgmeyer, I.; Boehm, U.; Meyerhof,

504

W., Genetic labeling of Tas1r1 and Tas2r131 taste receptor cells in mice. Chem. Sens.

505

2012, 37, 897-911.

506

6.

Zhang, F.; Klebansky, B.; Fine, R. M.; Xu, H.; Pronin, A.; Liu, H.; Tachdjian, C.; Li,

507

X., Molecular mechanism for the umami taste synergism. Proc. Natl. Acad. Sci. U.S.A.

508

2008, 105, 20930-20934.

509

7.

Xu, H.; Staszewski, L.; Tang, H.; Adler, E.; Zoller, M.; Li, X., Different functional

510

roles of T1R subunits in the heteromeric taste receptors. Proc. Natl. Acad. Sci. U.S.A.

511

2004, 101, 14258-14263.

512

8.

Winnig, M.; Bufe, B.; Meyerhof, W., Valine 738 and lysine 735 in the fifth

513

transmembrane domain of rTas1r3 mediate insensitivity towards lactisole of the rat

514

sweet taste receptor. BMC Neurosci. 2005, 6, 22-22.

515

9.

Kurobayashi, Y.; Katsumi, Y.; Fujita, A.; Morimitsu, Y.; Kubota, K., Flavour

516

Enhancement of Chicken Broth from Boiled Celery Constituents. J. Agric. Food

517

Chem. 2008, 56, 512-516. 23 ACS Paragon Plus Environment


518

10.

519 520

Taste Sensations. Chimia 2011, 65, 407-410. 11.

521 522

Kuroda, M., Proteins and peptides with taste. CRC Press: Boca Raton, Fla., 2012; p 277-296.

12.

523 524

Starkenmann, C.; Cayeux, I.; Birkbeck, A. A., Exploring Natural Products for New

Backes, M.; Paetz, S.; Vössing, T.; Ley, J. P., Synthesis and Sensory Studies of Umami-Active Scaffolds. Chem. & Biodiv. 2014, 11, 1782-1797.

13.

Winkel, C.; Klerk, A. d.; Visser, J.; de Rijke, E.; Bakker, J.; Koenig, T.; Renes, H.,

525

New Developments in Umami (Enhancing) Molecules. Chem. & Biodiv. 2008, 5,

526

1195-1203.

527

14.

528 529

Bassard, J.-E.; Ullmann, P.; Bernier, F.; Werck-Reichhart, D., Phenolamides: Bridging polyamines to the phenolic metabolism. Phytochemistry 2010, 71, 1808-1824.

15.

Stark, T.; Hofmann, T., Isolation, Structure Determination, Synthesis, and Sensory

530

Activity of N-Phenylpropenoyl-L-amino Acids from Cocoa (Theobroma cacao). J.

531

Agric. Food Chem. 2005, 53, 5419-5428.

532

16.

Koyama, N.; Kuribayashi, K.; Seki, T.; Kobayashi, K.; Furuhata, Y.; Suzuki, K.;

533

Arisaka, H.; Nakano, T.; Amino, Y.; Ishii, K., Serotonin Derivatives, Major Safflower

534

(Carthamus tinctorius L.) Seed Antioxidants, Inhibit Low-Density Lipoprotein (LDL)

535

Oxidation and Atherosclerosis in Apolipoprotein E-Deficient Mice. J. Agric. Food

536

Chem. 2006, 54, 4970-4976.

537

17.

Bratt, K.; Sunnerheim, K.; Bryngelsson, S.; Fagerlund, A.; Engman, L.; Andersson, R.

538

E.; Dimberg, L. H., Avenanthramides in oats (Avena sativa L.) and Structure-

539

Antioxidant activity relationships. J. Agric. Food Chem. 2003, 51, 594–600.

540

18.

Ley, J. P.; Krammer, G.; Kindel, G.; Bertram, H.-J., Pungency of Various Substituted

541

Hydroxycinnamic Amides of Hydroxyphenethylamines. In Polyphenol

542

Communications 2004, Hoikkala, A.; Soidinsalo, O.; Wähälä, K., Eds. University of

543

Helsinki: Helsinki, Finland, 2004; pp 269-270. 24 ACS Paragon Plus Environment

Page 24 of 40

Page 25 of 40

544


19.

Bassoli, A.; Borgonovo, G.; Busnelli, G.; Morini, G. Use of amide derivatives as taste-

545

modifying agents, flavouring compositions and products containing them. WO 2006

546

117,602.

547

20.

Frerot, E.; Neyrinck, N.; Cayeux, I.; Yuan, Y.; Yuan, Y.-M., New Umami Amides:

548

Structure-Taste Relationship Studies of Cinnamic Acid-Derived Amides and Natural

549

Occurrence of an Intense Umami Amide in Zanthoxylum piperitum. J. Agric. Food

550

Chem. 2015, 63, 7161-7168.

551

21.

552 553

Adesina, S. K., Three new amides from Zanthoxylum rubescens. Planta Med. 1989, 55, 324-326.

22.

Waddell, W. J.; Cohen, S. M.; Feron, V. J.; Goodman, J. I.; Marnett, L. J.; Portoghese,

554

P. S.; Rietjens, I. M. C. M.; Smith, R. L.; Adams, T. B.; Gavin, C. L.; McGowen, M.

555

M.; Williams, M. C., GRAS flavoring substances 23. Food Tech. 2007, 61, 22-24, 26-

556

28, 30-4.

557

23.

Marnett, L. J.; Cohen, S. M.; Fukushima, S.; Gooderham, N. J.; Hecht, S. S.; Rietjens,

558

I. M. C. M.; Smith, R. L.; Adams, T. B.; Hallagan, J. B.; Harman, C.; McGowen, M.

559

M.; Taylor, S. V., GRAS Flavoring Substances 26. Food Tech. 2013, August, 38-56.

560

24.

Sundh, U. B.; Binderup, M.-L.; Bolognesi, C.; Brimer, L.; Castle, L.; Di Domenico,

561

A.; Engel, K.-H.; Franz, R.; Gontard, N.; Gurtler, R.; Husoy, T.; Jany, K.-D.; Kolf-

562

Clauw, M.; Mennes, W.; Milana, M. R.; Pratt, I.; Svensson, K.; de Fatima Tavares

563

Pocas, M.; Toldra, F.; Wolfle, D.; Carere, A.; Frandsen, H.; Hill, F.; Mulder, G.;

564

Wallin, H.; Beltoft, V.; Lund, P.; Norby, K., Scientific opinion on flavouring group

565

evaluation 94, revision 2 (FGE.94Rev2): consideration of aliphatic amines and amides

566

evaluated in an addendum to the group of aliphatic and aromatic amines and amides

567

evaluated by the JECFA (68th meeting). EFSA Journal 2015, 12, 3622/1-3622/34.



568

25.

Standard Practice for Determination of Odor and Taste Thresholds By a Forced-

569

Choice Ascending Concentration Series Method of Limits. ASTM 2011, Practice E679

570

– 04.

571

26.

Suess, B.; Brockhoff, A.; Degenhardt, A.; Billmayer, S.; Meyerhof, W.; Hofmann, T.,

572

Human Taste and Umami Receptor Responses to Chemosensorica Generated by

573

Maillard-type N2-Alkyl- and N2-Arylthiomethylation of Guanosine 5'-

574

Monophosphates. J. Agric. Food Chem. 2014, 62, 11429-11440.

575

27.

576 577

Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M., N-Hydroxysuccinimide Esters in Peptide Synthesis. J. Am. Chem. Soc. 1963, 85, 3039-3039.

28.

Yoshihara, T.; Yamaguchi, K.; Takamatsu, S.; Sakamura, S., A New Lignan Amide,

578

Grossamide, from Bell Pepper Capsicum annuum var. grossum. Agric. Biol. Chem.

579

1981, 45, 2593-2598.

580

29.

Hofmann, T.; Suess, B.; Brockhoff, A.; Meyerhof, W., Structures, Orosensory

581

Activity, and T1R1/T1R3-Receptor Activation of Amides Generated by Lactone

582

Aminolysis Using Food-Related Processing Conditions. Eur. Food Res. Technol.

583

2013, 237, 57-70.

584

30.

Rotzoll, N.; Dunkel, A.; Hofmann, T., Activity-Guided Identification of (S)-Malic

585

Acid 1-O-D-Glucopyranoside (Morelid) and -Aminobutyric Acid as Contributors to

586

Umami Taste and Mouth-Drying Oral Sensation of Morel Mushrooms (Morchella

587

deliciosa Fr.). J. Agric. Food Chem. 2005, 53, 4149-4156.

588

31.

Kaneko, S.; Kumazawa, K.; Masuda, H.; Henze, A.; Hofmann, T., Molecular and

589

Sensory Studies on the Umami Taste of Japanese Green Tea. J. Agric. Food Chem.

590

2006, 54, 2688-2694.

591

32.

Cutillo, F.; D'Abrosca, B.; DellaGreca, M.; Di Marino, C.; Golino, A.; Previtera, L.;

592

Zarrelli, A., Cinnamic acid amide from Chenopodium album: effects on seeds

593

germination and plant growth. Phytochemistry 2003, 64, 1381-1387. 26 ACS Paragon Plus Environment

Page 26 of 40

Page 27 of 40

594


33.

Raliou, M.; Grauso, M.; Hoffmann, B.; Schlegel–Le-Poupon, C.; Nespoulous, C.;

595

Débat, H.; Belloir, C.; Wiencis, A.; Sigoillot, M.; Preet Bano, S.; Trotier, D.;

596

Pernollet, J.-C.; Montmayeur, J.-P.; Faurion, A.; Briand, L., Human Genetic

597

Polymorphisms in T1R1 and T1R3 Taste Receptor Subunits Affect Their Function.

598

Chem. Sens. 2011, 36, 527-537.

599

34.

600 601

Li, X.; Staszewski, L.; Xu, H.; Durick, K.; Zoller, M.; Adler, E., Human receptors for sweet and umami taste. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4692-4696.

35.

Xu, H.; Staszewski, L.; Tang, H.; Adler, E.; Zoller, M.; Li, X., Different functional

602

roles of T1R subunits in the heteromeric taste receptors. Proc. Natl. Acad. Sci. U.S.A.

603

2004, 101, 14258-14263.

604

36.

Shigemura, N.; Shirosaki, S.; Sanematsu, K.; Yoshida, R.; Ninomiya, Y., Genetic and

605

Molecular Basis of Individual Differences in Human Umami Taste Perception. PLoS

606

ONE 2009, 4, e6717-e6717.

607

37.

Suess, B.; Brockhoff, A.; Meyerhof, W.; Hofmann, T., Structures, orosensory activity,

608

and T1R1/T1R3 receptor activation of amides generated by lactone aminolysis using

609

food-related processing conditions. Eur. Food Res. Technol. 2013, 237, 57-70.

610

38.

Masuda, K.; Koizumi, A.; Nakajima, K.; Tanaka, T.; Abe, K.; Misaka, T.; Ishiguro,

611

M., Characterization of the modes of binding between human sweet taste receptor and

612

low-molecular-weight sweet compounds. PLoS One 2012, 7, e35380-e35380.

613

39.

614 615

Wu, P.-L.; Su, G.-C.; Wu, T.-S., Constituents from the Stems of Aristolochia manshuriensis. J. Nat. Prod. 2003, 66, 996-998.

40.

El-Gamal, A. A.; Takeya, K.; Itokawa, H.; Halim, A. F.; Amer, M. M.; Saad, H. E. A.;

616

Awad, S. A., Studies on the chemical constituents of Atraphaxis spinosa L., var.

617

sinaica Boiss. Nat. Med. 1994, 48, 304-306.

618 619

41.

Chen, C.-Y.; Yeh, Y.-T.; Yang, W.-L., Amides from the Stem of Capsicum annuum. Nat. Prod. Com. 2011, 6, 227-229. 27 ACS Paragon Plus Environment


620

42.

Kujala, T. S.; Vienola, M. S.; Klika, K. D.; Loponen, J. M.; Pihlaja, K., Betalain and

621

phenolic compositions of four beetroot (Beta vulgaris) cultivars. Eur. Food Res.

622

Technol. 2002, 214, 505-510.

623

43.

624 625

Oueslati, M. H.; Jannet, H. B.; Mighri, Z.; Chriaa, J.; Abreu, P. M., Phytochemical constituents from Salsolia tetranda. J. Nat. Prod. 2006, 69, 1366-1369.

44.

Pedersen, H. A.; Steffensen, S. K.; Christophersen, C.; Mortensen, A. G.; Jørgensen,

626

L. N.; Niveyro, S.; Troiani, R. M. d.; Rodrguez-Enrquez, R. J.; Rosa, A. P. B.-d. l.;

627

Fomsgaard, I. S., Synthesis and Quantitation of Six Phenolic Amides in Amaranthus

628

spp. J. Agric. Food Chem. 2010, 58, 6306-6311.

629

45.

630 631

Hay Nat. Prod. Res. 2011, 25, 26-30. 46.

632 633

Adesina, S. K.; Reisch, J., Amides from Zanthoxylum rubescens. Phytochemistry 1989, 28, (3), 839-842.

47.

634 635

Chen, C.-Y.; Yeh, H.-C., A new amide from the stems of Cinnamomum reticulatum

Kalia, N. K.; Singh, B.; Sood, R. P., A New Amide from Zanthoxylum armatum. J. Nat. Prod. 1999, 62, 311-312.

48.

Adesina, S. K.; Olatunji, O. A.; Bergenthal, D.; Reisch, J., Natural product chemistry.

636

Part 122. Transcinnamoylamides from Zanthoxylum rubescens pericarbs. Pharmazie

637

1988, 43, 517-518.

638

49.

Kang, G.-H.; Chang, E.-J.; Choi, S.-W., Antioxidative activity of phenolic compounds

639

in roasted safflower (Carthamus tinctorius L.) seeds. J. Food Sci.Nutr. 1999, 4, 221-

640

225.

641


Page 28 of 40

Page 29 of 40


642

Figure Captions

643

Figure 1: Structures of naturally occurring N-cinnamoyl amines 1 – 16; for natural

644

occurrence see Table 1.

645

Figure 2: Test for umami receptor activation hTAS1R1-rTas1r3 in HEK293 cell model by N-

646

cinnamoyl amines 1 – 15 and MSG measured in calcium imaging experiments.

647

Mean ± Standard deviation of 3 independent experiments, each measured in triplicates. One-

648

way variance analysis was performed to test differences between substances, including

649

vehicle. Contrast test, with a multiple comparison Bonferroni adjustment, were performed

650

between substances and their respective vehicle; *p

Rubemamine and Rubescenamine, Two Naturally Occurring N

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