Isolation, structure determination, synthesis, and sensory activity of N-phenylpropenoyl-L-amino acids from cocoa (Theobroma cacao)

Article abstract of DOI:10.1021/jf050458q

Application of chromatographic separation and taste dilution analyses recently revealed besides procyanidins a series of N-phenylpropenoyl amino acids as the key contributors to the astringent taste of nonfermented cocoa beans as well as roasted cocoa nib

Full text of DOI:10.1021/jf050458q

J. Agric. Food Chem. 2005, 53, 5419−5428 5419
Isolation, Structure Determination, Synthesis, and Sensory
Activity of N-Phenylpropenoyl- -amino Acids from Cocoa
L
(Theobroma cacao)
†
TIMO STARK AND THOMAS HOFMANN*,#
Deutsche Forschungsanstalt fu¨r Lebensmittelchemie, Lichtenbergstrasse 4, D-85748 Garching,
Germany, and Institut fu¨r Lebensmittelchemie, Universita¨t Mu¨nster, Corrensstrasse 45,
D-48149 Mu¨nster, Germany
Application of chromatographic separation and taste dilution analyses recently revealed besides
procyanidins a series of N-phenylpropenoyl amino acids as the key contributors to the astringent
taste of nonfermented cocoa beans as well as roasted cocoa nibs. Because these amides have as
yet not been reported as key taste compounds, this paper presents the isolation, structure
determination, and sensory activity of these amino acid amides. Besides the previously reported
(-)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine (clovamide), (-)-N-[4′-hydroxy-(E)-cin-
namoyl]-L-tyrosine (deoxyclovamide), and (-)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-tyrosine, seven
additional amides, namely, (+)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[4′-hydroxy-
(E)-cinnamoyl]-L-aspartic acid, (-)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4′-hydroxy-
(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4′-hydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine, (+)-N-[4′-
hydroxy-3′-methoxy-(E)-cinnamoyl]-L-aspartic acid, and (+)-N-[(E)-cinnamoyl]-L-aspartic acid, were
identified for the first time in cocoa products by means of LC-MS/MS, 1D/2D-NMR, UV-vis, CD
spectroscopy, and polarimetry, as well as independent enantiopure synthesis. Using the recently
developed half-tongue test, human recognition thresholds for the astringent and mouth-drying oral
sensation were determined to be between 26 and 220 µmol/L (water) depending on the amino acid
moiety. In addition, exposure to light rapidly converted these [E]-configured N-phenylpropenoyl amino
acids into the corresponding [Z]-isomers, thus indicating that analysis of these compounds in food
and plant materials needs to be performed very carefully in the absence of light to prevent artifact
formation.
KEYWORDS: Cocoa; astringency; taste dilution analysis; (+)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-aspartic
acid; (-)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-glutamic acid; clovamide; deoxyclovamide; half-tongue test
INTRODUCTION
fractionation led to the detection of a series of monomeric and
oligomeric procyanidins, flavonol and flavanon glycosides, and
the N-phenylpropenoyl amino acids 1-10 (Figure 1) as the key
players imparting the astringent oral taste sensation during
consumption of cocoa products (2). Because these N-phenyl-
propenoyl amino acids have as yet not been reported as key
taste compounds in foods, the details on their isolation, structure
determination, stereochemistry, and sensory activity are pre-
sented.
Besides the attractive aroma, the sensory quality of the
fermented and roasted seeds of Theobroma cacoa is driven by
its desirable bitterness and its slight sour taste as well as its
typical astringent mouthfeel, which is perceived as a long-lasting
puckering, shrinking, and drying sensation in the oral cavity
and can enhance the complexity and palate length of cocoa
beverages and chocolate confectionary.
To answer the question as to which nonvolatile, key taste
compounds are responsible for the typical taste of roasted cocoa,
we recently applied the so-called taste dilution analysis (1) on
fractions isolated from ground roasted cocoa nibs by means of
gel permeation chromatography (2). This bioassay-guided
MATERIALS AND METHODS
Chemicals. The following compounds were obtained commer-
cially: Amberlyst 15 (H+, 20-50 mesh) and (dimethylamino)pyridine
(Fluka, Neu-Ulm, Germany); acetic anhydride, (E)-4-hydroxycinnamic
acid, thionyl chloride, (E)-4-hydroxy-3-methoxycinnamic acid, and (E)-
cinnamic acid (Sigma-Aldrich, Steinheim, Germany); and (E)-3,4-
dihydroxycinnamic acid, potassium carbonate, L-aspartic acid, D-aspartic
acid, L-dopa, L-glutamic acid, L-tyrosine, sodium sulfate, pyridine, and
tetrahydrofuran (Merck, Darmstadt, Germany). Solvents were of HPLC
* Author to whom correspondence should be addressed (telephone
+49-251-83-33-391; fax +49-251-83-33-396; e-mail thomas.hofmann@
uni-muenster.de).
† Deutsche Forschungsanstalt fu¨r Lebensmittelchemie.
# Institut fu¨r Lebensmittelchemie.
10.1021/jf050458q CCC: $30.25
© 2005 American Chemical Society
Published on Web 06/02/2005

5420 J. Agric. Food Chem., Vol. 53, No. 13, 2005
Stark and Hofmann
Figure 1. Structures of N-phenylpropenoyl amino acids 1−12.
grade (Merck). Deuterated solvents were obtained from Euriso-Top
(Gif-Sur-Yvette, France). Bottled water (Vittel; low mineralization, 405
mg/L) adjusted to pH 6.0 with aqueous hydrochloric acid (0.1 mol/L)
was used for sensory evaluation. Nonfermented, washed cocoa beans
were collected in Sulawesi.
treated by that procedure are essentially free of the solvents and buffer
compounds used.
Half-Tongue Test. Taste dilution factors as well as human astringency
recognition thresholds were determined by means of the recently
developed half-tongue test (4) using bottled water as the solvent. Serial
1:1 dilutions of the samples were presented in order of increasing
concentrations to a trained panel of 12 persons in three different sessions
using the sip-and-spit method. When the panelist selected correctly,
the same concentration was presented again besides one blank as a
proof for the correctness of the data. The geometric mean of the last
and the second last concentration was calculated and taken as the
individual recognition threshold. The values between individuals and
between five separate sessions differed by not more than plus or minus
one dilution step; that is, a threshold value of 26 µmol/L for (-)-N-
[3′,4′-dihydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine represents a range
from 13 to 52 µmol/L.
Sensory Analyses. Training of the Sensory Panel. Twelve subjects
(five women and seven men, ages 25-38 years) with no history of
known taste disorders were trained to evaluate the taste of aqueous
solutions (3 mL each) of the following standard taste compounds by
using a triangle test as described in the literature (3): sucrose (50 mmol/
L) for sweet taste; lactic acid (20 mmol/L) for sour taste; NaCl (20
mmol/L) for salty taste; caffeine (1 mmol/L) for bitter taste; sodium
glutamate (3 mmol/L) for umami taste; and gallustannic acid (0.05%)
for astringency. The assessors had participated earlier at regular intervals
for at least two years in sensory experiments and were, therefore,
familiar with the techniques applied. Sensory analyses were performed
in a sensory panel room at 22-25 °C in three different sessions.
Pretreatment of Fractions. Prior to sensory analysis, the fractions
or compounds isolated were suspended in water, and, after removal of
the volatiles in high vacuum (<5 mPa), were freeze-dried twice. GC-
MS and ion chromatographic analysis revealed that food fractions
Syntheses of N-Phenylpropenoyl-L-amino Acids, 1-11 (Figure
1). Acetic anhydride (25 mmol) was added dropwise to a solution of
(E)-caffeic acid, (E)-p-coumaric acid, or (E)-ferulic acid (10 mmol
each), respectively, in pyridine (5 mL) and (dimethylamino)pyridine
(0.25 mmol) at 0 °C. The reaction mixture was stirred for 1 h at 0 °C

Taste-Active Phenylpropenoyl Acid Amides in Cocoa
J. Agric. Food Chem., Vol. 53, No. 13, 2005 5421
and, then, poured onto crushed ice. The aqueous phase was acidified
to pH 2.0 with aqueous hydrochloric acid (2 mol/L) and extracted with
a mixture of ethyl acetate and tetrahydrofuran (3:1, v/v; 3 × 50 mL),
and the combined organic extracts were dried over Na2SO4, filtered,
and freed from solvent in a vacuum, affording a colorless, amorphous
powder. An aliquot (1.0 mmol) of the acetylated phenylpropenoic acid
derivative obtained was mixed with thionyl chloride (1.0 mmol) and
was then heated under reflux until no further formation of hydrogen
chloride was observable. After cooling to room temperature, the reaction
mixture was dried under a stream of nitrogen, and either L-aspartic
acid, D-aspartic acid, L-glutamic acid, L-dihydroxyphenylalanine, or
L-tyrosine (each 1.0 mmol) dissolved in dry tetrahydrofuran (30 mL)
was added. After this solution had been stirred for up to 72 h at room
temperature, the solvent was evaporated in a vacuum, and the residue
was dissolved in a solution of K2CO3 (0.25 mol/L; 20 mL) in water/
methanol (1:1; v/v) and stirred at room temperature. After 20 min,
Amberlyst 15 ion-exchange resin was added with stirring until a pH
value of 3.5 was reached. The resin was then filtered off and washed
with water (20 mL), and the filtrate was freed from methanol in a
vacuum and, finally, freeze-dried. The residue was taken up in water
(3 mL) and applied onto the top of a water-cooled glass column (40 ×
140 mm) filled with a slurry of LiChroprep 25-40 µm RP-18 material
(Merck) in aqueous formic acid (0.1% in water; pH 2.5). Chromatog-
raphy was performed using aqueous formic acid (0.1% in water; pH
2.5) as the effluent, followed by aqueous formic acid (0.1% in water;
pH 2.5) containing increasing amounts of methanol. The target
compounds were detected by monitoring the effluent at 300 nm, and
the fractions containing these compounds were confirmed by RP-HPLC-
DAD. After the individual fractions had been freeze-dried three times,
the corresponding N-phenylpropenoyl-L-amino acids were obtained as
white, amorphous powders in high purities of >99%.
(+)-N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-L-aspartic acid 1 (0.33 mmol;
33% yield) (Figure 1): [R]2D0 +9.3° (in H2O); CD (in MeOH), λext(∆ꢀ)
) 237 nm (-2.53); UVmax, 207, 285, 309 nm; LC-MS (ESI+), m/z
613 (100, [2M + Na]+), 296 (95, [M + 1]+), 591 (55, [2M + 1]+),
924 (52, [3M + K]+), 908 (46, [3M + Na]+), 629 (38, [2M + K]+),
318 (25, [M + Na), 163 (21, [M - 132]+); 1H NMR (400 MHz,
DMSO-d6), δ 2.62 [dd, 1H, J ) 7.0, 16.3 Hz, H-C(3a)], 2.73 [dd,
1H, J ) 5.9, 16.3 Hz, H-C(3b)], 4.63 [m, 1H, H-C(2)], 6.44 [d, 1H,
J ) 15.7 Hz, H-C(8′)], 6.75 [d, 1H, J ) 8.2 Hz, H-C(5′)], 6.85 [dd,
1H, J ) 1.8, 8.2 Hz, H-C(6′)], 6.95 [d, 1H, J ) 1.8 Hz, H-C(2′)],
7.25 [d, 1H, J ) 15.7 Hz, H-C(7′)], 8.29 [d, 1H, J ) 8.2 Hz, H-N],
9.16 [s, 1H, HO-C(3′)], 9.41 [s, 1H, HO-C(4′)], 12.55 [s, 2H, HOOC-
(1, 4)]; 13C NMR (100 MHz, DMSO-d6), δ 36.7 [C-3], 48.9 [C-2],
114.1 [C-2′], 116.0 [C-5′], 118.2 [C-8′], 120.8 [C-6′], 126.5 [C-1′],
140.1 [C-7′], 145.7 [C-3′], 147.7 [C-4′], 165.4 [C-9′], 172.0 [C-4], 172.8
[C-1].
HO-C(3, 4,3′,4′)], 12.56 [s, 1H, HOOC(9)]; 13C NMR (100 MHz,
DMSO-d6), δ 35.0 [C-7], 54.3 [C-8], 114.1 [C-2′], 115.5 [C-5], 116.0
[C-5′], 116.7 [C-2], 118.4 [C-8′], 120.0 [C-6], 120.6 [C-6′], 126.5 [C-1′],
128.7 [C-1], 139.7 [C-7′], 144.0 [C-4], 145.1 [C-3], 145.7 [C-3′], 147.6
[C-4′], 165.4 [C-9′], 173.7 [C-9].
(-)-N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-L-tyrosine 4 (0.40 mmol;
40% tield) (Figure 1): [R]D20 -35.6° (in MeOH); UVmax, 225, 255, 297
nm; LC-MS (ESI+), m/z 687 (100, [2M + 1]+), 344 (27, [M + 1]+),
1
163 (10, [M - 180]+); H NMR (400 MHz, DMSO-d6), δ 2.78 [dd,
1H, J ) 9.2, 13.9 Hz, H-C(7a)], 2.98 [dd, 1H, J ) 4.8, 13.9 Hz,
H-C(7b)], 4.48 [m, 1H, H-C(8)], 6.41 [d, 1H, J ) 15.7 Hz, H-C(8′)],
6.65 [d, 2H, J ) 8.5 Hz, H-C(3, 5)], 6.74 [d, 1H, J ) 8.1 Hz,
H-C(5′)], 6.82 [dd, 1H, J ) 2.0, 8.2 Hz, H-C(6′)], 6.94 [d, 1H, J )
2.0 Hz, H-C(2′)], 7.03 [d, 2H, J ) 8.5 Hz, H-C(2, 6)], 7.20 [d, 1H,
J ) 15.7 Hz, H-C(7′)], 7.82 [d, 1H, J ) 8.1 Hz, H-N], 9.10, 9.16,
9.32 [3s, 3 × 1H, HO], 12.60 [s, 1H, HOOC(9)]; 13C NMR (100 MHz,
DMSO-d6), δ 36.4 [C-7], 54.2 [C-8], 114.1 [C-2′], 115.2 [C-3, 5], 115.9
[C-5′], 118.2 [C-8′], 120.7 [C-6′], 126.5 [C-1′], 127.9 [C-1], 130.2 [C-2,
6], 139.8 [C-7′], 145.7 [C-3′], 147.6 [C-4′], 156.1 [C-4], 165.5 [C-9′],
173.5 [C-9].
(+)-N-[4′-Hydroxy-(E)-cinnamoyl]-L-aspartic acid 5 (0.45 mmol;
45% yield) (Figure 1): [R]2D0 +1.2° (in MeOH); UVmax, 207, 285, 309
nm; LC-MS (ESI+), m/z 280 (100, [M + 1]+), 559 (70, [2M + 1]+),
1
597 (50, [2M + K]+), 147 (26, [M - 132]+); H NMR (400 MHz,
DMSO-d6), δ 2.65 [dd, 1H, J ) 7.0, 16.4 Hz, H-C(3a)], 2.73 [dd,
1H, J ) 5.7, 16.4 Hz, H-C(3b)], 4.64 [m, 1H, H-C(2)], 6.52 [d, 1H,
J ) 15.7 Hz, H-C(8′)], 6.79 [d, 2H, J ) 8.6 Hz, H-C(3′, 5′)], 7.34
[d, 1H, J ) 15.7 Hz, H-C(7′)], 7.40 [d, 2H, J ) 8.6 Hz, H-C(2′, 6′)],
8.27 [d, 1H, J ) 8.2 Hz, H-N], 9.85 [s, 1H, HO-C(4′)], 12.62 [s,
2H, HOO-C(1, 4)]; 13C NMR (100 MHz, DMSO-d6), δ 36.5 [C-3],
49.0 [C-2], 116.0 [C-3′, 5′], 118.3 [C-8′], 126.0 [C-1′], 129.5 [C-2′,
6′], 139.7 [C-7′], 159.2 [C-4′], 165.4 [C-9′], 171.9 [C-4], 172.7 [C-1].
(-)-N-[4′-Hydroxy-(E)-cinnamoyl]-L-glutamic acid 6 (0.55 mmol;
55% yield) (Figure 1): [R]2D0 -6.2° (in MeOH); UVmax, 207, 285, 309
nm; LC-MS (ESI+), m/z 918 (100, [3M + K]+), 903 (75, [3M + Na]+),
1
294 (55, [M + 1]+), 587 (35, [2M + 1]+), 147 (19, [M - 146]+); H
NMR (400 MHz, DMSO-d6), δ 1.84 [m, 1H, H-C(3a)], 1.98 [m, 1H,
H-C(3b)], 2.32 [m, 2H, H-C(4)], 4.34 [m, 1H, H-C(2)], 6.50 [d,
1H, J ) 15.7 Hz, H-C(8′)], 6.80 [d, 2H, J ) 8.6 Hz, H-C(3′,5′)],
7.34 [d, 1H, J ) 15.7 Hz, H-C(7′)], 7.40 [d, 2H, J ) 8.6 Hz,
H-C(2′,6′)], 8.23 [s, 1H, H-N], 9.83 [s, 1H, HO-C(4′)], 12.64 [s,
2H, HOO-C(1, 5)]; 13C NMR (100 MHz, DMSO-d6), δ 26.5 [C-3],
30.0 [C-4], 51.2 [C-2], 115.7 [C-3′,5′], 118.0 [C-8′], 125.7 [C-1′], 129.2
[C-2′,6′], 139.3 [C-7′], 158.9 [C-4′], 165.4 [C-9′], 173.3 [C-5], 173.5
[C-1].
(-)-N-[4′-Hydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine 7 (0.30 mmol;
30% yield) (Figure 1): [R]2D0 -24.2° (in MeOH); UVmax, 225, 255,
297 nm; LC-MS (ESI+), m/z 1395 (100, [4M + Na]+), 1068 (85, [3M
+ K]+), 725 (46, [2M + K]+), 1411 (44, [4M + K]+), 147 (23, [M -
(-)-N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-L-glutamic acid 2 (0.50 mmol;
50% yield) (Figure 1): [R]2D0 -5.9° (H2O); UVmax, 207, 285, 309 nm;
LC-MS (ESI+), m/z 619 (100, [2M + 1]+), 310 (35, [M + 1]+), 163
1
196]+), 344 (15, [M + 1]+); H NMR (400 MHz, DMSO-d6), δ 2.73
1
(5, [M - 146]+); H NMR (400 MHz, DMSO-d6), δ 1.82 [m, 1H,
[dd, 1H, J ) 8.8, 13.8 Hz, H-C(7a)], 2.91 [dd, 1H, J ) 4.8, 13.8 Hz,
H-C(7b)], 4.40 [m, 1H, H-C(8)], 6.46 [dd, 1H, J ) 2.0, 8.0 Hz,
H-C(6)], 6.50 [d, 1H, J ) 15.7 Hz, H-C(8′)], 6.59 [d, 1H, J ) 8.0
Hz, H-C(5)], 6.61 [d, 1H, J ) 2.0 Hz, H-C(2)], 6.78 [d, 2H, J ) 8.6
Hz, H-C(3′,5′)], 7.27 [d, 1H, J ) 15.7 Hz, H-C(7′)], 7.38 [d, 2H, J
) 8.6 Hz, H-C(2′,6′)], 8.08 [d, 1H, J ) 8.0 Hz, H-N]; 13C NMR
(100 MHz, DMSO-d6), δ 36.4 [C-7], 54.1 [C-8], 115.2 [C-5], 115.6
[C-3′,5′], 116.4 [C-2], 119.0 [C-8′], 119.7 [C-6], 125.7 [C-1′], 128.5
[C-1], 129.1 [C-2′,6′], 138.9 [C-7′], 143.7 [C-4], 144.8 [C-3], 158.8
[C-4′], 165.0 [C-9′], 173.3 [C-9].
H-C(3a)], 2.01 [m, 1H, H-C(3b)], 2.30 [m, 2H, H-C(4)], 4.33 [m,
1H, H-C(2)], 6.41 [d, 1H, J ) 15.7 Hz, H-C(8′)], 6.75 [d, 1H, J )
8.2 Hz, H-C(5′)], 6.85 [dd, 1H, J ) 2.0, 8.2 Hz, H-C(6′)], 6.95 [d,
1H, J ) 2.0 Hz, H-C(2′)], 7.25 [d, 1H, J ) 15.7 Hz, H-C(7′)], 8.20
[d, 1H, J ) 7.9 Hz, H-N], 9.10 [s, 1H, HO-C(3′)], 9.33 [s, 1H, HO-
C(4′)], 12.37 [s, 2H, HOOC(1, 5)]; 13C NMR (100 MHz, DMSO-d6),
δ 26.7 [C-3], 30.2 [C-4], 51.4 [C-2], 114.1 [C-2′], 115.9 [C-5′], 118.1
[C-8′], 120.6 [C-6′], 126.4 [C-1′], 140.0 [C-7′], 145.7 [C-3′], 147.6
[C-4′], 165.6 [C-9′], 173.2 [C-5], 173.5 [C-1].
(-)-N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine 3 (0.32
mmol; 32% yield) (Figure 1): [R]2D0 -13.0° (in MeOH); UVmax, 225,
255, 297 nm; LC-MS (ESI+), m/z 719 (100, [2M + 1]+), 360 (45, [M
(-)-N-[4′-Hydroxy-(E)-cinnamoyl]-L-tyrosine 8 (0.55 mmol; 55%
yield) (Figure 1): [R]D20 -26.9° (in MeOH); UVmax, 225, 255, 297 nm;
LC-MS (ESI+), m/z 677 (100, [2M + Na]+), 693 (51, [2M + K]+),
1020 (38, [3M + K]+), 1004 (32, [3M + Na]+), 147 (30, [M - 180]+),
328 (25, [M + 1]+), 350 (24, [M + Na]+); 1H NMR (400 MHz, DMSO-
d6), δ 2.81 [dd, 1H, J ) 9.3, 13.9 Hz, H-C(7a)], 3.00 [dd, 1H, J )
4.7, 13.9 Hz, H-C(7b)], 4.49 [m, 1H, H-C(8)], 6.50 [d, 1H, J ) 15.7
Hz, H-C(8′)], 6.66 [d, 2H, J ) 8.3 Hz, H-C(3,5)], 6.79 [d, 2H, J )
8.4 Hz, H-C(3′,5′)], 7.03 [d, 2H, J ) 8.3 Hz, H-C(2, 6)], 7.30 [d,
1H, J ) 15.7 Hz, H-C(7′)], 7.38 [d, 2H, J ) 8.4 Hz, H-C(2′,6′),
8.16 [d, 1H, J ) 8.0 Hz, H-N]; 13C NMR (100 MHz, DMSO-d6), δ
1
+ 1]+), 163 (25, [M - 196]+); H NMR (400 MHz, DMSO-d6), δ
2.72 [dd, 1H, J ) 8.9, 13.6 Hz, H-C(7a)], 2.91 [dd, 1H, J ) 4.6, 13.6
Hz, H-C(7b)], 4.42 [m, 1H, H-C(8)], 6.42 [d, 1H, J ) 15.7 Hz,
H-C(8′)], 6.47 [dd, 1H, J ) 1.8, 8.2 Hz, H-C(6)], 6.60 [d, 1H, J )
8.2 Hz, H-C(5)], 6.62 [d, 1H, J ) 1.8 Hz, H-C(2)], 6.74 [d, 1H, J )
8.2 Hz, H-C(5′)], 6.82 [dd, 1H, J ) 1.8, 8.2 Hz, H-C(6′)], 6.94 [d,
1H, J ) 1.8 Hz, H-C(2′)], 7.19 [d, 1H, J ) 15.7 Hz, H-C(7′)], 8.14
[d, 1H, J ) 7.9 Hz, H-N], 8.63, 8.68, 9.09, 9.32 [4 × s, 4 × 1H,

5422 J. Agric. Food Chem., Vol. 53, No. 13, 2005
Stark and Hofmann
36.4 [C-7], 54.2 [C-8], 115.2 [C-3,5], 116.0 [C-3′,5′], 118.4 [C-8′],
126.0 [C-1′], 127.9 [C-1], 129.5 [C-2′,6′], 130.2 [C-2,6], 139.5 [C-7′],
156.1 [C-4], 159.1 [C-4′], 165.6 [C-9′], 173.5 [C-9].
order of ascending concentrations, and each dilution was evaluated by
means of the half-tongue test. The dilution at which a taste difference
between the diluted extract and one blank (water) could just be detected
was defined as the taste dilution (TD) factor (1). The TD factors
evaluated by four different assessors in three different sessions were
averaged. The TD factors between individuals and separate sessions
did not differ by more than one dilution step.
(+)-N-[4′-Hydroxy-3′-methoxy-(E)-cinnamoyl]-L-aspartic acid 9 (0.4
mmol; 40% yield) (Figure 1): [R]2D0 +10.9° (in H2O); UVmax, 219, 303
nm; LC-MS (ESI+), m/z 310 (100, [M + 1]+), 619 (75, [2M + 1]+),
1
177 (62, [M - 132]+); H NMR (400 MHz, DMSO-d6), δ 2.65 [dd,
1H, J ) 7.0, 16.6 Hz, H-C(3a)], 2.74 [dd, 1H, J ) 5.7, 16.6 Hz,
H-C(3b)], 3.81 [s, 3H, H-C(10′)], 4.65 [m, 1H, H-C(2)], 6.58 [d,
1H, J ) 15.7 Hz, H-C(8′)], 6.79 [d, 1H, J ) 8.1 Hz, H-C(5′)], 7.00
[dd, 1H, J ) 1.8, 8.1 Hz, H-C(6′)], 7.16 [d, 1H, J ) 1.8 Hz, H-C(2′)],
7.34 [d, 1H, J ) 15.7 Hz, H-C(7′)], 8.21 [d, 1H, J ) 7.9 Hz, H-N],
9.44 [s, 1H, HO-C(4′)], 12.65 [s, 2H, HOO-C(1,4)]; 13C NMR (100
MHz, DMSO-d6), δ 36.5 [C-3], 48.9 [C-2], 55.7 [C-10′], 110.9 [C-2′],
115.8 [C-5′], 118.7 [C-8′], 122.0 [C-6′], 126.5 [C-1′], 139.9 [C-7′],
148.0 [C-3′], 148.6 [C-4′], 165.6 [C-9′], 172.0 [C-4], 172.7 [C-1].
(+)-N-(E)-Cinnamoyl-L-aspartic acid 10 (0.55 mmol; 55% yield)
(Figure 1): [R]2D0 +1.7° (in MeOH); UVmax, 207, 261 nm; LC-MS
(ESI+), m/z 264 (100, [M + 1]+), 527 (65, [2M + 1]+), 549 (20, [2M
Isolation of (+)-N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-L-aspartic
Acid (1) and (+)-N-[4′-Hydroxy-(E)-cinnamoyl]-L-aspartic Acid (5)
from RP18 Fractions S1-VIII and S1-IX, Respectively. Aliquots of
fractions S1-VIII and S1-IX were fractionated by semipreparative HPLC
on RP-18, ODS-Hypersil, 5 µm (ThermoHypersil), to afford compounds
1 and 5, respectively, by using methanol/water mixtures as the mobile
phase (flow rate ) 3 mL/min). For the isolation of compound 1,
chromatography was performed with a mixture (20:80, v/v) of methanol
and aqueous formic acid (0.1% in water; pH 2.5) for 10 min, increasing
the methanol content to 60% over 30 min and then to 100% over 0.5
min; thereafter, elution with 100% methanol for 10 min was performed.
For the isolation of compound 5, chromatography was performed with
a mixture (25:75, v/v) of methanol and aqueous formic acid (0.1% in
water; pH 2.5) for 10 min, increasing the methanol content to 60%
over 30 min and then to 100% over 0.5 min; thereafter, elution with
100% methanol for 10 min was performed. The target compounds were
collected, freed from solvent in a vacuum, and freeze-dried three times
to give compounds 1 and 5 as white amorphous powders in high purity
of >99%. Spectroscopic (1H NMR, MS, UV, [R]2D0) and chromato-
graphic data (retention time) as well as sensory activity (taste threshold)
of the isolated materials were identical to the data obtained for the
corresponding synthesized reference materials.
Identification of (-)-N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-L-glutam-
ic Acid (2), (-)-N-[4′-Hydroxy-(E)-cinnamoyl]-L-glutamic Acid (6),
(+)-N-[4′-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-aspartic Acid (9),
and (+)-N-(E)-Cinnamoyl-L-aspartic Acid (10) in RP18 Fraction
S1-X. Aliquots of fraction S1-X were separated by analytical RP-HPLC
(flow rate ) 0.8 mL/min) starting with a mixture (20:80, v/v) of
methanol and aqueous formic acid (0.1% in water; pH 2.5) for 10 min,
then increasing the methanol content to 60% over 30 min and, finally,
to 100% over 0.5 min, thereafter eluting with 100% methanol for 10
min. Comparison of chromatographic data (retention times), spectro-
scopic data (LC-MS, UV), and sensory data with those of the
synthesized reference compounds led to the identification of compounds
2, 6, 9, and 10 (Figure 1) as (-)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-
L-glutamic acid, (-)-N-[4′-hydroxy-(E)-cinnamoyl]-L-glutamic acid,
(+)-N-[4′-hydroxy-3-methoxy-(E)-cinnamoyl]-L-aspartic acid, and (+)-
N-(E)-cinnamoyl-L-aspartic acid, respectively.
Isolation of (-)-N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-3-hydroxy-
L-tyrosine (3), (-)-N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-L-tyrosine (4),
(-)-N-[4′-Hydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine (7), and
(-)-N-[4′-Hydroxy-(E)-cinnamoyl]-L-tyrosine (8) from RP18 Frac-
tions S2-VIII, S2-X, and S2-XII, Respectively. Aliquots of fractions
S2-VIII, S2-X, and S2-XII were separated by semipreparative HPLC
on RP-18, ODS-Hypersil, 5 µm (ThermoHypersil), using the following
methanol/water gradients at a flow rate of 3 mL/min. For the isolation
of compound 3 from fraction S2-VIII, chromatography was performed
starting with a mixture (30:70, v/v) of methanol and aqueous formic
acid (0.1% in water; pH 2.5) for 5 min, then increasing the methanol
content to 50% over 30 min and, finally, to 100% over 0.5 min,
thereafter eluting with 100% methanol for 10 min. For the isolation of
compounds 4 and 7 from fraction S2-X, chromatography was performed
starting with a mixture (25:75, v/v) of methanol and aqueous formic
acid (0.1% in water; pH 2.5) for 10 min, then increasing the methanol
content to 40% over 30 min and, finally, to 100% over 0.5 min,
thereafter eluting with 100% methanol for 10 min. For the isolation of
compound 8 from fraction S2-XII, chromatography was performed
starting with a mixture (30:70, v/v) of methanol and aqueous formic
acid (0.1% in water; pH 2.5) for 10 min, then increasing the methanol
content to 50% over 30 min and, finally, to 100% over 0.5 min,
thereafter eluting with 100% methanol for 10 min. The fractions
containing the target compounds were collected, the solvents were
removed in a vacuum, and the fractions were freeze-dried three times
to give compounds 3, 4, 7, and 8 as white, amorphous powders in
1
+ 23]+), 131 (20, [M - 132]+); H NMR (400 MHz, DMSO-d6), δ
2.67 [dd, 1H, J ) 7.0, 16.5 Hz, H-C(3a)], 2.76 [dd, 1H, J ) 5.5, 16.5
Hz, H-C(3b)], 4.67 [m, 1H, H-C(2)], 6.75 [d, 1H, J ) 15.8 Hz,
H-C(8′)], 7.38-7.44 [m, 3H, H-C(3′,5′,4′)], 7.44 [d, 1H, J ) 15.8
Hz, H-C(7′)], 7.57 [d, 2H, J ) 8.1 Hz, H-C(2′,6′)], 8.39 [d, 1H,
H-N], 12.58 [s, 2H, HOO-C(1, 4)]; 13C NMR (100 MHz, DMSO-
d6), δ 36.3 [C-3], 48.9 [C-2], 121.9 [C-8′], 127.8 [C-2′,6′], 129.1
[C-3′,5′], 129.8 [C-4′], 135.0 [C-1′], 139.5 [C-7′], 165.0 [C-9′], 171.9
[C-4], 172.6 [C-1].
(-)-N-[3′,4′-Dihydroxy-(E)-cinnamoyl]-D-aspartic acid 11 (0.33
mmol; 33% yield) (Figure 1): [R]2D0 -9.3° (in H2O); [R]20 -6.0° (in
D
MeOH); CD (in MeOH), λext(∆ꢀ) ) 238 nm (+1.68); UV, LC-MS,
and NMR data were identical to those obtained for the corresponding
(+)-L-enantiomer 1.
Solvent Extraction of Cocoa Beans. Washed beans (100 g) were
peeled by hand, frozen in liquid nitrogen, crushed in a grinding mill,
and then extracted with n-pentane (5 × 300 mL) at room temperature
for 30 min. After centrifugation, the residual cocoa material was then
extracted five times with a mixture (7:3, v/v; 300 mL each) of acetone
and water for 45 min at room temperature with stirring. After filtration,
the liquid layer was freed from acetone under reduced pressure at 30
°C, and the aqueous solution obtained was extracted with dichlo-
romethane (5 × 150 mL) to remove theobromine and caffeine and then
freeze-dried to give the acetone/water extract.
Fractionation of the Acetone/Water Extract by Solid-Phase
Extraction. Aliquots (1 g each) of the acetone/water extract were
dissolved in Millipore water (10 mL), filtered, and then applied onto
the top of a C18 Sep-Pak cartridge (Waters, Millipore) preconditioned
with methanol, followed by water. Fractionation was performed by
flushing the column with water (50 mL, fraction S1), followed by
methanol/water (20:80, v/v; 40 mL; fraction S2) and methanol (40 mL;
fraction S3). The fractions S1-S3 collected were concentrated in a
vacuum and, after freeze-drying twice, the yields and the taste quality
of the individual fractions were determined. The intensely astringent
fraction S1 was taken up in aqueous formic acid (0.1% in water; pH
2.5; 3 mL), the astringent fraction S2 was dissolved in a mixture (20:
80, v/v; 5 mL) of methanol and aqueous formic acid (0.1% in water;
pH 2.5), and both fractions were separately applied onto the top of a
water-cooled glass column (40 × 140 mm) filled with a slurry of
LiChroprep 25-40 µm RP-18 material (Merck) in the same solvent
mixture. Chromatography was performed by using aqueous formic acid
(0.1% in water; pH 2.5), followed by the same formic acid solution
containing increasing amounts of methanol up to 60%. The effluent
was collected in 13 subfractions, namely, S1-I-XIII and S2-I-XIII,
respectively. These fractions were used for taste dilution analysis as
well as for chemical structure determination.
Taste Dilution Analysis (TDA). Aliquots (250 mg) of fraction S1
were separated by semipreparative HPLC on RP-18, ODS-Hypersil, 5
µm (ThermoHypersil), and the effluent was collected to give seven
subfractions, which were freeze-dried, taken up in water (2 mL), and
then sequentially diluted 1:2 with bottled water. The serial dilutions of
each of these fractions were then presented to the sensory panel in

Taste-Active Phenylpropenoyl Acid Amides in Cocoa
J. Agric. Food Chem., Vol. 53, No. 13, 2005 5423
Figure 2. (A) RP-HPLC chromatogram and (B) TD chromatogram of fraction S1 isolated from cocoa beans by means of solid-phase extraction.
purities of >99%. Spectroscopic data (1H NMR, MS, UV, [R]D20) and
chromatographic data (retention time) as well as sensory activity
(threshold) of the isolated materials were identical to those obtained
for the corresponding synthesized reference materials.
in either an analytical (250 × 4.6 mm i.d.; flow rate ) 0.8 mL/min) or
a semipreparative scale (250 × 10 mm i.d.; flow rate ) 3.0 mL/min).
Liquid Chromatography-Mass Spectrometry (LC-MS). An
analytical ODS-Hypersil, 5 µm, HPLC column (Phenomenex, Aschaffen-
burg, Germany) was coupled to an LCQ mass spectrometer (Finnigan
MAT GmbH, Bremen, Germany) operating in the positive (ESI+) and
negative (ESI-) electrospray ionization mode. After injection of the
sample (20-100 µL), analyses were performed using the solvent
gradient reported above.
Isolation of (+)-N-[3′,4′-Dihydroxy-(Z)-cinnamoyl]-L-aspartic
Acid (12). A solution of compound 1 (0.5 mmol) in methanol/water
(20:80, v/v; pH 3.0; 2 mL) was exposed to UV light (λ ) 365 nm) for
1 h by means of a Spectroline model CM-10 (Spectronics Corp.,
Westbury, NY). Aliquots of the light-exposed mixture were separated
by semipreparative HPLC on RP-18, ODS-Hypersil, 5 µm (Thermo-
Hypersil). Using a flow rate of 3 mL/min, chromatography was
performed starting with a mixture (20:80, v/v) of methanol and aqueous
formic acid (0.1% in water; pH 2.5) for 10 min, then increasing the
methanol content to 60% over 30 min and, finally, to 100% over 0.5
min. The fraction containing the corresponding (Z)-isomer was col-
lected, freed from solvent in a vacuum, and freeze-dried three times to
give (+)-N-[3′,4′-dihydroxy-(Z)-cinnamoyl]-L-aspartic acid (12) as a
Circular Dichroism (CD) Spectroscopy. For CD spectroscopy,
methanolic solutions of the samples were analyzed by means of a Jasco
J600 spectropolarimeter (Hachioji, Japan).
Nuclear Magnetic Resonance Spectroscopy (NMR). 1H, 13C, and
DEPT-135 NMR experiments were performed on an AV-360 spec-
1
trometer (Bruker, Rheinstetten, Germany). H, COSY, HMQC, and
HMBC measurements were performed on an AMX 400-III spectrometer
(Bruker). Evaluation of the experiments was carried out using 1D- and
2D-WIN-NMR (version 6.1) as well as XWin-NMR software (version
3.5; Bruker). Tetramethylsilane was used as the internal standard.
white, amorphous powder (46% yield) with a purity of >99%: UVmax
,
207, 285, 309 nm; LC-MS (ESI+), m/z 613 (100, [2M + Na]+), 296
(95, [M + 1]+), 591 (55, [2M + 1]+), 924 (52, [3M + K]+), 908 (46,
[3M + Na]+), 629 (38, [2M + K]+), 318 (25, [M + Na]+), 163 (21,
[M - 132]+); 1H NMR (400 MHz, CD3OD), δ 2.79 [dd, 1H, J ) 6.9,
16.8 Hz, H-C(3a)], 2.84 [dd, 1H, J ) 5.5, 16.8 Hz, H-C(3b)], 4.80
[m, 1H, H-C(2)], 5.83 [d, 1H, J ) 12.6 Hz, H-C(8′)], 6.59 [d, 1H,
J ) 12.6 Hz, H-C(7′)], 6.70 [d, 1H, J ) 8.1 Hz, H-C(5′)], 6.90 [dd,
1H, J ) 2.0, 8.1 Hz, H-C(6′)], 7.12 [d, 1H, J ) 2.0 Hz, H-C(2′)].
Quantitative Analysis of Light-Induced (E)/(Z)-Isomerization.
Aliquots (50 µL) of solutions of the N-phenylpropenoyl amino acids
(5 µmol/L) in methanol/water (1:1, v/v) were mixed with Millipore
water (2 mL) adjusted to pH 3, 5, or 7 with aqueous hydrochloric acid
(0.1 mmol/L), placed into a crystallization dish (4.6 cm i.d.), and then
radiated for 1 h with UV light at λ ) 254 or 365 nm by means of a
Spectroline model CM-10 at a distance of 13 cm. After light exposure,
the samples were analyzed by means of HPLC-DAD and LC-MS/MS,
and the relative amounts of (E)- and (Z)-isomers were calculated from
the ratio of the corresponding peak areas.
RESULTS AND DISCUSSION
With the aim of isolating the highly polar N-phenylpropenoyl
amino acids, freshly ground, peeled cocoa beans were defatted
by extraction with n-pentane, and the residual cocoa material
was then extracted with acetone/water as reported recently (2).
After removal of the acetone in a vacuum and extraction of the
liquid layer with dichloromethane, the aqueous phase was freeze-
dried to give an acetone/water extract that was further fraction-
ated by means of solid-phase extraction using RP-18 cartridges.
Elution with water, followed by methanol/water (20:80), and
methanol, revealed fractions S1, S2, and S3, respectively, after
freeze-drying. These three fractions, strongly differing in color
and taste, were then analyzed by means of HPLC-DAD, HPLC-
MS, and HPLC-degustation. In particular, fractions S1 and S2
exhibited a pronounced astringent and intense mouth-coating
oral sensation.
Isolation and Identification of N-Phenylpropenoyl-L-
Amino Acids (1, 2, 5, 6, 9, and 10) from Fraction S1. To
locate the key taste compounds in the strongly astringent tasting
fraction S1, this fraction was further separated by HPLC using
RP18 material as the stationary phase (Figure 2A). With the
chromatography monitored at 310 nm, the effluent was collected
in seven fractions S1-1-7, which were individually freeze-dried,
then dissolved in the same amount of water, and, finally, used
Polarimetry. The optical rotation of solutions of N-phenylpropenoyl
amino acids (20 mg) in Millipore water (2 mL) or methanol (2 mL)
was performed on a 241 MC polarimeter (Perkin-Elmer, Rodgau-
Ju¨gesheim, Germany) equipped with a 100 mm cell and operating at
589 nm.
High-Performance Liquid Chromatography (HPLC). The HPLC
apparatus (Jasco, Gross-Umstadt, Germany) consisted of a HPLC pump
system PU 1580 with an in-line degasser (DG-1580-53), a low-pressure
gradient unit (LG-1580-02), and a diode array detector (DAD) type
MD 1515. Chromatography was performed on stainless steel columns
packed with ODS-Hypersil, 5 µm, RP-18 material (ThermoHypersil)

5424 J. Agric. Food Chem., Vol. 53, No. 13, 2005
Stark and Hofmann
Figure 3. 1H NMR (400 MHz; DMSO-d6) of (
+)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-aspartic acid (1) identified in HPLC fraction S1-3.
for the TDA. To achieve this, each solution was stepwise diluted
1:1 with water, and the dilutions were then presented in order
of increasing concentrations to trained sensory panelists, who
were asked to judge the astringent oral sensation by application
of the recently developed half-tongue test (4) until the threshold
was reached. As this so-called TD factor, obtained for each
fraction, is related to its taste activity in water, the seven HPLC
fractions were rated according to their relative astringency
impact (Figure 2B). Due to their high TD factors of 256 and
128, fractions S1-3 and S1-5 were evaluated with by far the
highest taste impacts for astringency, followed by HPLC fraction
S1-2 judged with a TD factor of 64. In comparison, the other
fractions were evaluated with significantly lower astringent taste
impact and should therefore not contribute to cocoa taste.
acid, respectively. To further confirm the structure of the
suggested aspartate moiety, 1D- and 2D-NMR experiments were
performed.
The 1H NMR spectrum of compound 1, displayed in Figure
3, showed 11 resonance signals each integrating for one proton.
Upon the addition of trace amounts of D2O, the proton signals
of two hydroxy groups and one imino group resonating at 8.29,
9.16, and 9.41 ppm disappeared. When these observations were
taken into account and the 13 carbon atoms detected in the 13
C
NMR spectrum considered, the sum formula of C13H13NO7 was
proposed for compound 1. The double doublet of H-C(3a,b)
resonating at 2.68 ppm showed a geminal coupling with a
coupling constant of 16.5 Hz and coupling constants of 7.0 and
5.9 Hz, respectively, assigned as the vicinal couplings with the
proton H-C(2) detected at 4.63 ppm. In addition, a homo-
nuclear, double-quantum filtered δ,δ-correlation experiment
(DQF-COSY) revealed coupling between the proton H-C(2)
and the N-H proton resonating at 8.29 ppm. Furthermore, the
coupling constant of 15.7 Hz for the homonuclear coupling of
H-C(8′) and H-C(7′), resonating at 6.44 and 7.25 ppm, led to
their assignment as two (E)-configured olefinic protons as
expected for cinnamic acid derivatives. Three aromatic protons
showing ortho and meta couplings were detected between 6.74
and 6.95 ppm. Comparison of the 13 carbon signals observed
in the 13C NMR spectrum with the results of a DEPT-135
experiment, which showed 7 signals, identified six carbon
signals as quaternary carbon atoms. Unequivocal assignment
of these quaternary carbon atoms and the hydrogen-substituted
carbon atoms, respectively, could be successfully achieved by
means of heteronuclear multiple-bond correlation spectroscopy
With the aim of isolating the taste compounds exhibiting the
astringent and mouth-coating oral sensation of HPLC fractions
S1-3 and S1-5, fraction S1 was isolated from the acetone/water
extract (31 g) obtained from 222 g of cocoa beans and then
fractionated by preparative column chromatography on RP-18
material. With the effluent monitored at 300 nm, the effluents
of the individual peaks detected were collected and freed from
solvent in a vacuum to give 13 subfractions (S1-I-XIII). Final
purification of the individual subfractions was performed by
semipreparative RP-HPLC, thus giving rise to highly pure
astringent taste compounds, which were analyzed by means of
UV-vis, LC-MS, and NMR spectroscopy.
The astringent compounds detected in subfractions S1-
VIII-X showed similar UV absorption spectra exhibiting
maxima at 207, 285, and 309 nm. LC-MS analysis showed [M
+ H]+ ions with m/z 296 and 280 for the compounds eluting in
fractions S1-VIII and S1-IX, respectively, indicating that one
nitrogen atom is incorporated in both molecules. MS/MS
analysis of the ion m/z 296 or 280 further demonstrated a loss
of 132 amu, most likely corresponding to an aspartate moiety,
to generate the ions m/z 163 and 147 fitting well with the
structure of a 3,4-dihydroxycinnamic acid or 4-hydroxycinnamic
2
3
(HMBC) optimized for JC,H and JC,H coupling constants as
well as heteronuclear single-quantum correlation spectroscopy
1
(HSQC) optimized for JC,H coupling constants.
For an unequivocal confirmation of the proposed structure,
we also measured heteronuclear δ,δ-correlations between the
nitrogen atom and neighboring protons by means of 15N/1H
heteronuclear single-bond (15N-HMQC) and multiple-bond

Taste-Active Phenylpropenoyl Acid Amides in Cocoa
J. Agric. Food Chem., Vol. 53, No. 13, 2005 5425
Figure 4. Synthetic sequence used for the preparation of (+)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-aspartic acid (1).
correlation spectroscopy (15N-HMBC). In the 15N-HMQC
experiment, just one coupling was observed, thus allowing the
conclusion that only one proton, resonating at 8.29 ppm, is
bound to the nitrogen atom. In addition, the 15N-HMBC
experiment showed the expected heteronuclear coupling between
the nitrogen atom and the protons H-C(3a) and H-C(3b) of
N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-aspartic acid 1 (Figure 1)
resonating at 2.62 and 2.73 ppm.
To further confirm the structure of compound 1, N-[3′,4′-
dihydroxy-(E)-cinnamoyl]-L-aspartic acid was synthesized fol-
lowing the reaction sequence outlined in Figure 4. (E)-Caffeic
acid was acetylated with acetic anhydride in pyridine/(dimethyl-
amino)pyridine, converted into the corresponding chloride using
thionyl chloride, and, finally, reacted with L-aspartic acid in dry
tetrahydrofuran. After K2CO3-catalyzed cleavage of the protect-
ing groups and Amberlite treatment, the target compound 1 was
isolated by column chromatography on RP-18 material, followed
by a final HPLC purification yielding N-[3′,4′-dihydroxy-(E)-
cinnamoyl]-L-aspartic acid (Figure 1) as a white, amorphous
powder in a high purity of >99%. The mass spectroscopic and
NMR, as well as the sensory data were well in line with those
determined for the authentic compound isolated from cocoa
beans.
To identify the enantiomer of compound 1 existing naturally
in cocoa beans, the optical rotation of compound 1 isolated from
cocoa beans, synthetic N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-
aspartic acid, and N-[3′,4′-dihydroxy-(E)-cinnamoyl]-D-aspartic
acid synthesized from D-aspartic acid following the procedure
shown in Figure 4 for the L-enantiomer was measured by means
of polarimetry. The synthetic D-enantiomer showed an optical
rotation of [R]2D0 -9.3° (in H2O), whereas the L-enantiomer
revealed a value of +9.3° (in H2O), fitting well with the optical
rotation found for the naturally occurring enantiomer isolated
from cocoa beans. In addition, CD measurements were per-
formed with these samples, showing that the isolated compound
1 and the synthesized (+)-L-enantiomer showed the same
extinction value of λext(∆ꢀ) ) 237 nm (-2.5), whereas the (-)-
D-enantiomer exhibited an extinction value of λext(∆ꢀ) ) 238
nm (+1.68), thus clearly demonstrating that cocoa beans contain
exclusively the (+)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-aspar-
tic acid (1) (Figure 5).
Figure 5. (A) UV spectrum, and (B) CD spectrum of the synthetic (
N-[3 ,4 -dihydroxy-(E)-cinnamoyl]- -aspartic acid (1; ‚ ‚ ‚), the synthetic
)-N-[3 ,4 -dihydroxy-(E)-cinnamoyl]- -aspartic acid (11; - - -), and the
sample of compound 1 isolated from cocoa ( ).
+)-
′
′
L
(
−
′
′
D
s
1). By means of HPLC-degustation, HPLC-MS, and HPLC-
DAD, compound 1 was identified as the taste compound in
HPLC fraction S1-3, which was evaluated with a high TD factor
for its astringent and mouth-coating oral sensation (Figure 2B).
Using the same analytical strategy, LC-MS/MS studies, 1D-
und 2D-NMR experiments, and polarimetry led to the identi-
fication of compound 5 detected in fraction S1-IX and HPLC
fraction S1-5 (Figure 2B) as (+)-N-[4′-hydroxy-(E)-cinnamoyl]-
L-aspartic acid (Figure 1). The structure of that taste compound
Taking all of these data into consideration, LC-MS/MS, 1D-
and 2D-NMR experiments, and CD spectroscopy as well as
enantiopure synthesis led to the unequivocal identification of
the astringent compound 1 isolated from fraction S1-VIII as
(+)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-aspartic acid (Figure

5426 J. Agric. Food Chem., Vol. 53, No. 13, 2005
Stark and Hofmann
was further confirmed by synthesis using the procedure reported
above for compound 1, but starting with 4′-hydroxy-(E)-
cinnamic acid instead of (E)-caffeic acid. Although this
compound has been earlier reported in cell suspension cultures
of Arabidopsis thaliana (5), the taste activity of that amino acid
has not been previously described.
In addition, four astringent compounds were detected in
subfraction S1-X showing similar UV absorption spectra as
shown for compounds 1 and 5 and showing [M + H]+ ions
with m/z 310 (compounds 2 and 9), 294 (compound 6), and
264 (compound 10), thus suggesting that the structures of these
tastants might be (-)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-
glutamic acid (2), (-)-N-[4′-hydroxy-(E)-cinnamoyl]-L-glutamic
acid (6), (+)-N-[4′-hydroxy-3′-methoxy-(E)-cinnamoyl]-L-as-
partic acid (9), and (+)-N-(E)-cinnamoyl-L-aspartic acid (10).
To further strengthen this assumption, compound 2 was
synthesized from 3′,4′-dihydroxy-(E)-cinnamic acid and L-
glutamic acid, compound 6 from 4′-hydroxy-(E)-cinnamic acid
and L-glutamic acid, compound 9 from 4′-hydroxy-3′-methoxy-
(E)-cinnamic acid and L-aspartic acid, and, finally, compound
10 from (E)-cinnamic acid and L-aspartic acid using the synthetic
procedure reported above for compound 1. Comparison of
retention times, UV, and LC-MS/MS as well as sensory data
of the compounds isolated from subfraction S1-X with those
of the synthesized reference compounds led to the unequivocal
identification of these compounds as (-)-N-[3′,4′-dihydroxy-
(E)-cinnamoyl]-L-glutamic acid (2), (-)-N-[4′-hydroxy-(E)-
cinnamoyl]-L-glutamic acid (6), (+)-N-[4′-hydroxy-3′-methoxy-
(E)-cinnamoyl]-L-aspartic acid (9), and (+)-N-(E)-cinnamoyl-
L-aspartic acid (10), respectively. Comparison of retention times
and LC-MS/MS data as well as cochromatography with the
synthetic compounds revealed these taste compounds are present
in HPLC fractions S1-4 (compound 2), S1-6 (compound 6), and
S1-7 (compounds 9 and 10) evaluated with lower taste dilution
factors for astringency (Figure 2B). Although compound 9 has
been earlier reported in cell cultures of Beta Vulgaris (6), the
taste activity of that compound has yet not been reported. To
the best of our knowledge, compounds 2, 6, and 10 have not
been reported previously.
Figure 6. HPLC-MS analysis of an aqueous solution of (
+
)-N-[3
′,4′-
dihydroxy-(E)-cinnamoyl]-L-aspartic acid (1) exposed to UV light with a
wavelength of 365 nm.
3,4-dihydroxycinnamic acid and corresponding to the cleavage
of one molecule of 3-hydroxy-tyrosine and tyrosine, respec-
tively. In addition, MS/MS analysis of the ion m/z 344,
corresponding to the first peak in fraction S2-X, as well as the
ion m/z 328 revealed the loss of 196 and 180 Da, respectively,
to generate the ion m/z 147 fitting well with the structure of a
hydroxycinnamic acid and corresponding to the cleavage of one
molecule of 3-hydroxy-tyrosine and tyrosine. To further confirm
the phenylpropenoic acid group in the molecules and to identify
the amino acid moieties, 1D- and 2D-NMR experiments were
performed.
In brief, LC-MS/MS as well as DQF-COSY-, HMBC- and
HSQC NMR experiments, revealing a complete picture of
chemical shifts, homo- and heteronuclear single- and multiple-
bond correlations, led to the unequivocal identification of the
astringent compounds in fraction S2-VIII, S2-X, and S3-XII
as (-)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine
(3), (-)-N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-tyrosine (4), (-)-
N-[4′-hydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine (7), and
(-)-N-[4′-hydroxy-(E)-cinnamoyl]-L-tyrosine (8), respectively
(Figure 1). Finally, the structures of all of these compounds
were synthesized from 3′,4′-dihydroxy-(E)-cinnamic acid or 4′-
hydroxy-(E)-cinnamic acid and L-tyrosine or 3-hydroxy-L-
tyrosine, respectively, using the synthetic procedure reported
above for compound 1. Although compound 3, named clov-
amide, was earlier identified in red clover (Trifolium pratense)
(7-9), together with compound 8, named deoxyclovamide,
earlier reported as a constituent of the bark of African blackwood
(Dalbergia melanoxylon) (10) and as cocoa antioxidants (11),
the taste activity of these phenolic amino acid derivatives has
not been previously published. In addition, compound 4 has
been earlier identified in cocoa flowers (12) and raw robusta
coffee beans (13), but its taste activity has not been investigated
so far. To the best of our knowledge, compound 7 has not
previously been identified in any food material.
Isolation and Identification of N-Phenylpropenoyl-L-
Amino Acids (3, 4, 7, and 9) from Fraction S2. Aimed at
isolating the taste compounds exhibiting the astringent and
mouth-coating oral sensation in fraction S2, an aliquot of this
fraction obtained from 222 g cocoa beans was fractionated by
preparative column chromatography on RP-18 material. Moni-
toring the effluent at 300 nm, the effluents of the individual
peaks detected were collected and freed from solvent in a
vacuum to give 13 subfractions (S2-I to S2-XIII) which were
evaluated by the sensory panel. Final purification of the taste-
active subfractions was performed by semipreparative RP-
HPLC, thus giving rise to highly pure astringent taste com-
pounds which were analyzed by means of UV/Vis, LC-MS, and
NMR spectroscopy.
(E)/(Z)-Isomerization of N-Phenylpropenoyl Amino Acids.
During isolation of the amino acid amides from cocoa as well
as during purification of the corresponding synthetic compounds,
we observed that the (E)-configured olefinic double bond in
these molecules is partially isomerized in the presence of light.
To study the influence of light on this isomerization process,
an aqueous solution of compound 1 was exposed to UV light
with a wavelength of 365 nm, and, thereafter, the mixture was
analyzed by RP-HPLC. As shown in Figure 6, besides
compound 1, a second compound was formed, which was
isolated by HPLC and unequivocally identified as (+)-N-[3′,4′-
dihydroxy-(Z)-cinnamoyl]-L-aspartic acid (12) by means of LC-
MS/MS and NMR experiments.
LC-MS analysis of the astringent compounds isolated from
these subfractions, all of which exhibiting similar absorption
maxima at 225, 255, and 297 nm, revealed [M+H]+ ions with
m/z 360 for the compound 3 eluting in fraction S2-VIII, m/z
344 for compounds 4 and 7 detected in fraction S2-X, and m/z
328 for compound 8 detected in fraction S2-XII. These data
clearly indicated that one nitrogen atom is incorporated in the
molecules under investigation. MS/MS analysis of the ions m/z
360 and 344, corresponding to the second peak in fraction S2-
X, revealed the loss of 196 and 180 amu, respectively, to
generate the ion m/z 163 fitting well with the structure of a
To investigate the influence of light on this isomerization
process more quantitatively, solutions of N-phenylpropenoyl
amino acids differing in the pH value were exposed to UV light
with the wavelength of 254 or 365 nm, respectively (Table 1).

Taste-Active Phenylpropenoyl Acid Amides in Cocoa
J. Agric. Food Chem., Vol. 53, No. 13, 2005 5427
Table 1. Isomerization of N-Phenylpropenoyl Amino Acids upon UV
Light Exposure
Table 2. Human Recognition Taste Thresholds of N-Phenylpropenoyl
Amino Acids
taste threshold
concn for
ratio of isomers (%) after radiation at
taste compd
astringencya
(µmol/L)
254 nm
365 nm
compd
(Z)-isoa
(E)-isoa
(Z)-isoa
(E)-isoa
(
(
(
(
(
(
(
(
(
(
(
(
+
−
+
+
−
+
−
−
−
+
−
−
)-N-[(E)-cinnamoyl]-
L
-aspartic acid (10)
220b
190b
180b
170b
170b
170b
170b
100
83
)-N-[3
)-N-[4
)-N-[3
)-N-[3
)-N-[3
)-N-[4
)-N-[4
)-N-[3
)-N-[4
)-N-[4
)-N-[3
′
′
′
′
′
′
′
′
′
′
′
,4
′
-dihydroxy-(E)-cinnamoyl]-
L-glutamic acid (2)
pH 3.0
pH 5.0
pH 7.0
1
2
3
4
5
16
12
23
16
21
42
29
84
88
77
84
79
58
71
46
43
62
51
64
70
3
54
57
38
49
36
30
97
-hydroxy-(E)-cinnamoyl]-
L-aspartic acid (5)
,4′
,4′
,4′
-dihydroxy-(E)-cinnamoyl]-
-dihydroxy-(E)-cinnamoyl]-
-dihydroxy-(Z)-cinnamoyl]-
L-aspartic acid (1)
D-aspartic acid (11)
L-aspartic acid (12)
-hydroxy-(E)-cinnamoyl]-
-hydroxy-(E)-cinnamoyl]-
L
-glutamic acid (6)
-tyrosine (8)
9
10
L
,4
-hydroxy-3-methoxy-(E)-cinnamoyl]-
-hydroxy-(E)-cinnamoyl]-3-hydroxy-
,4 -dihydroxy-(E)-cinnamoyl]-3-hydroxy-
′
-dihydroxy-(E)-cinnamoyl]-
L
-tyrosine (4)
-aspartic acid (9)
-tyrosine (7)
-tyrosine (3)
L
57b
1
2
3
4
5
17
14
25
18
22
43
27
83
86
75
82
78
57
73
43
45
69
54
64
70
2
57
55
31
46
36
30
98
L
55
26
′
L
a Taste threshold concentrations were determined by means of the half-tongue
test. b These compounds exhibit sour taste at the recognition taste threshold
concentration of 1500 mol/L, which was determined by means of a triangle test
as reported recently (3).
9
10
µ
1
2
3
4
5
20
16
26
20
22
44
24
80
84
74
80
78
56
76
43
45
66
55
64
70
2
57
55
34
45
36
30
98
imparted by the N-phenylpropenoyl amino acids was described
as mouth-drying and puckering astringent, with threshold
concentrations ranging from 26 to 190 µmol/L. Comparison of
the taste thresholds obtained for the aspartic acid derivatives
showed that the sensory activity was not strongly influenced
by the hydroxyl groups at the cinnamoyl moiety; for example,
the aspartic acid amides containing (E)-cinnamic acid, 4′-
hydroxy-(E)-cinnamic acid, and 3′,4′-dihydroxy-(E)-cinnamic
acid, respectively, showed very similar taste thresholds of 220,
180, and 170 µmol/L. In contrast, compound 9, containing a
4′-hydroxy-3′-methoxy-(E)-cinnamoyl moiety, showed a 3-fold
lower taste threshold when compared to compound 1.
9
10
a The corresponding isomers have been detected by HPLC-DAD as well as
HPLC-MS/MS.
After light exposure, the solutions were subsequently analyzed
by HPLC coupled to a diode array detector or a mass
spectrometer. Compounds 1-5 and 9, containing either a 4′-
hydroxy-(E)-cinnamoyl, a 3′,4′-dihydroxy-(E)-cinnamoyl, or a
4′-hydroxy-3′-methoxy-(E)-cinnamoyl group, were most strongly
isomerized into the corresponding (Z)-configured compound at
pH 7 when exposed to light with a wavelength of 365 nm. In
comparison, exposure to UV light with a wavelength of 254
nm was significantly less active in facilitating the isomerization
process; for example, at pH 3 the (Z)- and (E)-configurations
of 1 were detected in ratios of 16:84 and 46:54 when exposed
to light with the wavelength of 254 or 365 nm, respectively. In
contrast, compound 10, bearing an (E)-cinnamoyl moiety, was
not significantly isomerized upon light exposure at 365 nm, but
was converted by 29% into the (Z)-isomer when exposed to
light at 254 nm (pH 3.0). Independent from the pH value,
compound 9 showed favorably isomerization at 254 nm as well
as at 365 nm; for example, only 30% the (E)-isomer was
recovered after exposure to light with the wavelength of 365
nm. In contrast, compounds 1 and 2 were least susceptible to
isomerization under the conditions used. Due to different UV-
vis absorption maxima, the N-phenylpropenoic acid moiety is
driving the acceptability for (E)/(Z)-isomerization.
As the naturally occurring (E)-configured N-phenylpropenoyl
amino acids were shown to be rapidly converted into the
corresponding (Z)-isomers, quantitative analysis of these com-
pounds in food and plant materials, such as cocoa, has to be
done very carefully in the absence of light to prevent any artifact
formation.
Sensory Activity of N-Phenylpropenoyl Amino Acids. Prior
to sensory analysis, the purity of all compounds was checked
by HPLC-MS as well as H NMR spectroscopy. To study the
sensory activity of these N-phenylpropenoyl amino acids, the
human sensory recognition thresholds were determined in bottled
water using the half-tongue test for the astringent and a triangle
test for the sour impression (Table 2). The oral sensation
Sensory analysis of the L- and D-configured compound 1 as
well as the (Z)-configured isomer 12 revealed the same threshold
concentration of 170 µmol/L for these isomers, thus demonstrat-
ing that the stereochemistry does not play any role for the
sensory activity of these amino acid amides.
In contrast, the amino acid moiety seems to be of major
importance for the sensory impact. For example, the taste
threshold of the N-[3′,4′-dihydroxy-(E)-cinnamoyl]-L-amino
acids decreased from 170 and 190 µmol/L for the aspartic acid
and glutamic acid derivatives 1 and 2, respectively, by a factor
of 2 to 83 µmol/L for the tyrosine derivative 4 and by 7-fold to
26 µmol/L for the dopa derivative 3 (Table 2). Besides the
astringent taste quality, the amides of the aliphatic amino acids
asparatic acid (1, 5, and 9-12) and glutamic acid (2 and 6)
exhibited significant sour taste at concentrations >1.5 mmol/
L, whereas the amides of the aromatic amino acids tyrosine (4
and 8) and 3-hydroxytyrosine (3 and 7) did not induce any sour
taste perception.
Aimed at demonstrating the contribution of the N-phenyl-
propenoyl amino acids to the typical astringent taste of roasted
cocoa, quantitative studies, taste reconstitution, and taste omis-
sion experiments are currently in progress and will be published
elsewhere.
1
ACKNOWLEDGMENT
We thank H.-U. Humpf, University of Muenster, for performing
the CD measurements.

5428 J. Agric. Food Chem., Vol. 53, No. 13, 2005
Stark and Hofmann
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Received for review March 1, 2005. Revised manuscript received April
28, 2005. Accepted April 28, 2005. This research project was supported
by a grant from FEI (Forschungskreis der Erna1hrungsindustrie e.V.,
Bonn, Germany), the AiF (Arbeitsgemeinschaft industrieller Fors-
chung), and the German Ministry of Economics (Project 13235 N).
JF050458Q