Synthesis and sweetness characteristics of L-aspartyl-D-alanine fenchyl esters

Article abstract of DOI:10.1021/jf010344o

Four isomers of the L-aspartyl-D-alanine fenchyl esters were prepared as potential peptide sweeteners. L-Aspartyl-D-alanine (+)-α-fenchyl ester and L-aspartyl-D-alanine (-)-β-fenchyl ester showed sweetness with potencies 250 and 160 times higher than that of sucrose, respectively. In contrast, L-aspartyl-D-alanine (+)-β-fenchyl ester and L-aspartyl-D-alanine (-)-α-fenchyl ester had the highest sweetness potencies at 5700 and 1100 times that of sucrose, respectively. In particular, L-aspartyl-D-alanine (-)-α-fenchyl ester had an excellent sweetness quality, but L-aspartyl-D-alanine (+)-β-fenchyl ester did not have an excellent quality of sweetness because it displayed an aftertaste caused by the strong sweetness.

Full text of DOI:10.1021/jf010344o

J. Agric. Food Chem. 2001, 49, 5013−5018
5013
Syn th esis a n d Sw eetn ess Ch a r a cter istics of L-Asp a r tyl-D-Ala n in e
F en ch yl Ester s
Yoshifumi Yuasa,* Akira Nagakura, and Haruki Tsuruta
Technical and Engineering Department, Fine Chemical Division, Takasago International Corporation,
1-5-1 Nishiyawata, Hiratsuka, Kanagawa, 254-0073, J apan
Four isomers of the L-aspartyl-D-alanine fenchyl esters were prepared as potential peptide
sweeteners. L-Aspartyl-D-alanine (+)-R-fenchyl ester and L-aspartyl-D-alanine (-)-â-fenchyl ester
showed sweetness with potencies 250 and 160 times higher than that of sucrose, respectively. In
contrast, L-aspartyl-D-alanine (+)-â-fenchyl ester and L-aspartyl-D-alanine (-)-R-fenchyl ester had
the highest sweetness potencies at 5700 and 1100 times that of sucrose, respectively. In particular,
L-aspartyl-D-alanine (-)-R-fenchyl ester had an excellent sweetness quality; but L-aspartyl-D-alanine
(+)-â-fenchyl ester did not have an excellent quality of sweetness because it displayed an aftertaste
caused by the strong sweetness.
Keyw or d s: Sweetener; fenchol; L-aspartyl-D-alanine fenchyl esters; aspartic acid derivatives
INTRODUCTION
MATERIALS AND METHODS
The correlation between sweetness and chemical
structure of a sweetener was proposed by Schellenberger
as the AH-B site theory which is explained by a proton
donor and a proton acceptor (1, 2 and recent references
therein), and by Kier as the dispersion X site theory
which is considered a hydrophobic site (3) (Figure 1).
Since aspartame (1) having AH-B and X sites was
found in 1969 (4, 5), many L-aspartyl dipeptides have
been synthesized (6-8). In particular, the sweetness of
the L-aspartyl-D,L-aminomalonic methyl fenchyl diester
(2), which was found by Fujino and co-workers (9), is
30000 times more potent than sucrose. In 1980, Yin-
Zeng et al. synthesized the four fenchyl alcohol isomers
of (2) and found that the (+)-â-fenchyl ester was 50000
times as sweet as sucrose (10). This (+)-â-fenchyl ester
of (2) is known as the most potent among these L-
aspartyl dipeptides. However, the compound is ex-
tremely unstable for practical use, and also the stability
of aspartame in aqueous solution is not sufficient
because it has a methyl ester unit. To develop good
quality, potent sweetness, and improved chemical sta-
bility of the L-aspartyl dipeptides, we converted the ester
unit into an amino alcohol. As a result, we have recently
reported the new sweetener, L-aspartyl fenchyl amino
alcohol (3) (11, 12), which has a high degree of sweetness
and good stability in solution. For the sweetness of the
L-aspartyl dipeptides, compounds having a fenchyl unit
as a large hydrophobic group (13, 14) have a potent
sweetness. We also described L-aspartyl-D-alanine fenchyl
esters (4) in a previous patent (15, 16). Similar com-
pounds have been reported by Zeng et al. (17); however,
their reported data left us doubtful about the evaluated
sweetness potency.
Ta ste P a n els. The compounds were dissolved in water, and
the threshold value of each compound was determined by a
limiting method using a panel consisting of five skilled
flavorists; and then these sweetness characteristics were
evaluated by 10 panelists.
In str u m en ta tion . Following are the instruments and
specifications used for the analyses. IR: J asco IR-810. 1H NMR
(TMS as internal standard): Bruker AM-400 (400 MHz). 13C
NMR (TMS as internal standard): Bruker AM-400 (100 MHz).
MS: Hitachi M-80A mass spectrometer at 70 eV. Optical
rotations: J asco DIP-4 digital polarimeter. GLC: HP5890 II
with an FID detector; column NB-1 (25 m × 0.25 mm i.d., df
) 0.15 µm); temperature programmed at 100-200 °C, +1.0
°C/min; carrier gas He, 0.1 MPa. HPLC: Hitachi L-6000 with
an L-4000 UV as a detector; column, Inertsil ODS-2 (250 mm
× 4.6 mm); eluent, CH3CN/H2O, 7:3 (pH 2.3; adjusted by using
H3PO4); flow rate 0.5 mL/min; detection, UV (210 or 254 nm).
Column chromatography: Merck Kieselgel 60, Art. Nr. 7734.
Melting points: Yanagimoto micromelting apparatus, uncor-
rected values.
P r ep a r a tion of F en ch ols. Commercial (+)-R-fenchol (10
g, Aldrich, 95.9% purity) was dissolved in 20 mL of n-heptane,
and the solution was cooled to -40 °C. The crystals that
precipitated were separated by filtration to give 3.9 g of 98.3%
purity (+)-R-fenchol. (R/â ) 98.3/1.6), mp. 40-42 °C. [Lit. 42-
43 °C (18)], [R]D26 ) +11.0° (c ) 5, in EtOH). [Lit. [R]D ) +10.5°
(c ) 5, in EtOH) (18)].
(-)-R-Fenchol was prepared by the lithium aluminum
hydride reduction (THF, 0 °C, 16 h) of (+)-fenchone (20 g,
Tokyo Kasei, 98% purity), and the crude crystals obtained (17.6
g) were recrystallized from 35 mL of n-heptane at -40 °C to
afford 3.9 g of 98.1% purity (-)-R-fenchol (R/â ) 98.1/1.9), mp.
42-44.5 °C, [Lit. 43.6-44.5 °C (19)], [R]D26 ) -12.8° (c ) 5, in
27
EtOH), [Lit. [R]D ) -12.7° (c ) 3, in 95% EtOH) (19)].
(+)-â-Fenchol was prepared by the aluminum isopropoxide
reduction of (-)-fenchone (25 g, Aldrich, 98% purity, in
2-propanol, reflux, 420 h), and the crude product (14 g, GC
analysis showed a 22/78 ratio of (+)-R-fenchol/(+)-â-fenchol)
was converted to the p-nitrobenzoate, recrystallized from
n-heptane at -40 °C, and followed by hydrolysis with NaOH
to give 1.02 g of 98.8% purity (+)-â-fenchol (R/â ) 1.2/98.8).
These authors reported that the L-aspartyl-D-alanine
(+)-R-fenchyl ester (4a ) had no sweetness. Therefore,
we independently synthesized four isomers of the L-
aspartyl-D-alanine fenchyl esters (4a -d ) (Table 1) to
reinvestigate the correlation between the fenchyl iso-
mers and the sweetness potency.
26
[R]D ) +23.3° (c ) 5, in EtOH), [Lit. [R]D ) +21.5° (c ) 5, in
* To whom correspondence should be addressed. Tel: 81-
(0)463-21-7150. Fax: 81-(0)463-23-1655. E-mail: TIC00488@
nifty.ne.jp.
EtOH) (20)]. (-)-â-Fenchol was prepared by the procedure
outlined above; (+)-fenchone was converted to crude product
(GC analysis showed a 25/75 ratio of (-)-R-fenchol/(-)-â-
10.1021/jf010344o CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/28/2001

5014 J. Agric. Food Chem., Vol. 49, No. 10, 2001
Yuasa et al.
F igu r e 1. Structures of aspartame (1) and L-aspartylfenchyl derivatives (2-4).
Ta ble 1. Sw eetn ess Ch a r a cter istics of L-Asp a r tyl-D-Ala n in e F en ch yl Ester s
a
b
Sweetness potency vs 5% sucrose. Value reported by Zeng et al. (10). c A, Excellent; B, good; C, poor.
fenchol). The crude fenchol was purified by recrystallization
after conversion to the p-nitrobenzoate and then hydrolyzed
with NaOH to give 1.52 g of 99.3% purity (-)-â-fenchol (R/â )
0.7/99.3). [R]D26 ) -31.60° (c ) 5, in EtOH), [Lit. [R]D ) -21.8°
(c ) 4, in EtOH, 94.4% pure) (18)].
Syn th esis of N-Ben zyloxyca r bon yl-D-a la n in e F en ch yl
Ester s (5a -d ). A 2.23-g (10 mM) aliquot of N-benzyloxycar-
bonyl-D-alanine and 1.7 g (11 mM) of one of the fenchol isomers
were dissolved in dichloromethane (25 mL) and cooled to 0
°C. Dicyclohexylcarbodiimide [2.49 g (12 mM)] and 14 mg (0.1
mM) of 4-(dimethylamino)pyridine were added, and the mix-
ture was stirred for 30 min, then warmed to room temperature
for 6 h. The dicyclohexylurea that formed was filtered off, and
the filtrate was washed with 10% citric acid solution, 5%
sodium bicarbonate solution, and brine, then dried over
anhydrous magnesium sulfate. The solvent was removed under
reduced pressure. The residue was purified by silica gel
chromatography using chloroform as the eluent to give one of
the following N-benzyloxycarbonyl-D-alanine fenchyl esters as
a solid.
N-Ben zyloxyca r b on yl-D-a la n in e (+)-r-F en ch yl E st er
(5a ). Yield 72.5%. Purity by HPLC was 98.5%. mp 90-92 °C.
[R]D ) +26.0° (c ) 2.0, in CHCl3). IR (CHCl3): ν 3430 cm-1
,
26
1
1720, 1510. H NMR (CDCl3): δ 0.77 (3H, s, CH3), 1.04 (3H,
s, CH3), 1.10 (3H, s, CH3), 1.09-1.74 (7H, m, fenchyl), 1.45
(3H, d, J ) 7.1 Hz, CH3CHNH), 4.39 (1H, d, J ) 1.8 Hz), 4.44

Characteristics of L-Aspartyl-D-Alanine Fenchyl Esters
J. Agric. Food Chem., Vol. 49, No. 10, 2001 5015
(1H, q, J ) 7.1 Hz, CH3CHNH), 5.11 (2H, s, PhCH2), 7.29-
7.36 (5H, m, aromH). 13C NMR (CDCl3): δ 19.0 (q), 19.3 (q),
20.2 (q), 25.8 (t), 26.5 (t), 29.6 (q), 39.5 (s), 41.3 (t), 48.3 (d),
48.4 (s), 49.8 (d), 66.9 (t), 87.4 (d), 128.1 (2×d), 128.2 (d), 128.5
(2×d), 136.4 (s), 155.6 (s), 173.3 (s). MS m/z (%): 359 (2) (M+),
223 (17), 206 (7), 178 (26), 153 (20), 137 (34), 107 (36), 91 (100).
Anal. C21H29NO4 (359.5): calcd, C 70.16, H 8.13, N 3.89; found,
C 70.19, H 8.15, N 3.91%.
removed under reduced pressure. The residue was purified by
silica gel column chromatography using chloroform as the
eluent to give one of the following N-benzyloxycarbonyl-â-
benzyl-L-aspartyl-D-alanine fenchyl esters as an oil.
N-Ben zyloxycar bon yl-â-ben zyl-L-aspar tyl-D-alan in e (+)-
r-F en ch yl Ester (6a ). Yield 79%. Purity by HPLC was 99.5%.
[R]D ) +17.6° (c ) 0.6, in CHCl3). IR (CHCl3): ν 3410 cm-1
,
24
1
1725, 1675, 1500. H NMR (CDCl3): δ 0.77 (3H, s, CH3), 1.04
(3H, s, CH3), 1.11 (3H, s, CH3), 1.10-1.77 (7H, m, fenchyl),
1.39 (3H, d, J ) 7.1 Hz, CH3CHNH), 2.75 (1H, dd, J ) 6.2, 17
Hz, CH2CO2Bn), 3.06 (1H, dd, J ) 3.8, 17 Hz, CH2CO2Bn),
4.39 (1H, d, J ) 1.9 Hz, CO2CH-), 4.57 (1H, q, J ) 7.1 Hz,
CH3CHNH). 4.64 (1H, br s, CHCH2CO2Bn), 5.08-5.17 (4H,
m, 2×PhCH2), 7.26-7.37 (10H, m, aromH). 13C NMR
(CDCl3): δ 18.5 (q), 19.3 (q), 20.3 (q), 25.8 (t), 26.5 (t), 29.6
(q), 36.1 (t), 39.5 (s), 41.3 (t), 48.3 (d), 48.4 (s), 48.5 (d), 51.2
(d), 66.9 (t), 67.4 (t), 87.5 (d), 128.1 (d), 128.2 (2×d), 128.3
(2×d), 128.4 (2×d), 128.6 (3×d), 135.4 (s), 135.9 (s), 169.6 (s),
171.4 (s), 172.7 (s), 180.3 (s). MS m/z (%): 565 (19) (M+1), 429
(24), 411 (9), 383 (9), 321 (58), 275 (14), 222 (16), 181 (23), 137
(74), 91 (100).
N-Ben zyloxyca r bon yl-D-a la n in e (+)-â-F en ch yl Ester
(5b). Yield 65%. Purity by HPLC was 98.8%. mp 70-73 °C.
[R]D ) -18.7° (c ) 1.5, in CHCl3). IR (CHCl3): ν 3430 cm-1
,
23
1
1720, 1510. H NMR (CDCl3): δ 0.85 (3H, s, CH3), 1.01 (3H,
s, CH3), 1.06 (3H, s, CH3), 1.08-1.75 (7H, m, fenchyl), 1.44
(3H, d, J ) 7.1 Hz, CH3CHNH), 4.19 (1H, d, J ) 1.3 Hz), 4.40
(1H, q, J ) 7.1 Hz, CH3CHNH), 5.11 (2H, s, PhCH2), 5.35 (1H,
br d, J ) 6.9 Hz, NH), 7.29-7.36 (5H, m, aromH). 13C NMR
(CDCl3): δ 17.0 (q), 18.9 (q), 23.4 (q), 25.4 (t), 25.6 (q), 33.3
(t), 41.7 (t), 43.7 (s), 48.3 (d), 48.4 (d), 49.8 (d), 66.8 (t), 88.6
(s), 128.1 (2×d), 128.2 (d), 128.5 (2×d), 136.3 (s), 155.6 (s),
172.9 (s). MS m/z (%): 360 (2) (M++1), 270 (8), 224 (50), 206
(6), 178 (26), 153 (22), 137 (91), 107 (32), 91 (100). Anal. C21H29
-
NO4 (359.5): calcd, C 70.16, H 8.13, N 3.89; found, C 70.18, H
8.16, N 3.90%.
N-Ben zyloxycar bon yl-â-ben zyl-L-aspar tyl-D-alan in e (+)-
â-F en ch yl Ester (6b). Yield 78%. Purity by HPLC was 99.5%.
[R]D ) -11.0° (c ) 1.0, in CHCl3). IR (CHCl3): ν 3410 cm-1
,
22
N-Ben zyloxyca r b on yl-D-a la n in e (-)-r-F en ch yl E st er
26
1
(5c). Yield 71%. Purity by HPLC was 99%. mp 70-72 °C. [R]D
1725, 1675, 1500. H NMR (CDCl3): δ 0.84 (3H, s, CH3), 1.00
(3H, s, CH3), 1.06 (3H, s, CH3), 1.0-1.76 (7H, m, fenchyl), 1.38
(3H, d, J ) 7.2 Hz, CH3CHNH), 2.75 (1H, dd, J ) 6.3, 17 Hz,
CH2CO2Bn), 3.06 (1H, dd, J ) 3.8, 17 Hz, CH2CO2Bn), 4.21
(1H, d, J ) 1.9 Hz, CO2CH-), 4.54 (1H, q, J ) 7.2 Hz,
CH3CHNH). 4.63 (1H, br s, CHCH2CO2Bn), 5.08-5.17 (4H,
m, 2×PhCH2), 7.26-7.37 (10H, m, aromH). 13C NMR
(CDCl3): δ 17.1 (q), 18.5 (q), 23.5 (q), 25.5 (t), 25.7 (q), 33.3
(t), 36.2 (t), 41.7(t), 41.3 (t), 48.3 (d), 48.4 (s), 48.5 (d), 51.1 (d),
66.9 (t), 67.4 (t), 88.7 (d), 128.1(d), 128.2 (2×d), 128.3 (2×d),
128.4 (2×d), 128.5 (d), 128.6 (2×d), 135.4 (s), 135.9 (s), 169.5
(s), 171.4 (s), 172.2 (s), 180.2 (s). MS m/z (%): 566 (5) (M++2),
519 (4), 429 (27), 411 (22), 383 (12), 312 (18), 268 (50), 222
(16), 181 (40), 137 (72), 91 (100).
) -26.0° (c ) 2.0, in CHCl3). IR (CHCl3): ν 3430 cm-1, 1720,
1510. 1H NMR (CDCl3): δ 0.78 (3H, s, CH3), 1.03 (3H, s, CH3),
1.09 (3H, s, CH3), 1.07-1.74 (7H, m, fenchyl), 1.44 (3H, d, J )
7.1 Hz, CH3CHNH), 4.39 (1H, d, J ) 1.5 Hz), 4.41 (1H, q, J )
7.1 Hz, CH3CHNH), 5.11 (2H, s, PhCH2), 7.29-7.37 (5H, m,
aromH). 13C NMR (CDCl3): δ 19.1 (q), 19.3 (q), 20.1 (q), 25.8
(t), 26.6 (t), 29.6 (q), 39.41 (s), 41.3 (t), 48.3 (d), 48.4 (s), 49.8
(d), 66.9 (t), 87.4 (d), 128.1 (d), 128.2 (d), 128.4 (d), 128.5 (d),
128.6 (d), 136.3 (s), 155.6 (s), 173.3 (s). MS m/z (%): 359 (6)
(M+), 292 (6), 279 (27), 153 (3), 137 (14), 108 (18), 91 (100).
Anal. C21H29NO4 (359.5): calcd, C 70.16, H 8.13, N 3.89; found,
C 70.22, H 8.14, N 3.92%.
N-Ben zyloxyca r bon yl-D-a la n in e (-)-â-F en ch yl Ester
(5d ). Yield 69%. Purity by HPLC was 99.5%. mp 69-71 °C.
N-Ben zyloxycar bon yl-â-ben zyl-L-aspar tyl-D-alan in e (-)-
[R]D ) +15.8° (c ) 2.1, in CHCl3). IR (CHCl3): ν 3430 cm-1
,
r-F en ch yl Ester (6c). Yield 81%. Purity by HPLC was 99%.
26
1
23
1720, 1510. H NMR (CDCl3): δ 0.84 (3H, s, CH3), 1.01 (3H,
s, CH3), 1.06 (3H, s, CH3), 1.07-1.74 (7H, m, fenchyl), 1.44
(3H, d, J ) 7.2 Hz, CH3CHNH), 4.22 (1H, d, J ) 1.6 Hz), 4.41
(1H, q, J ) 7.2 Hz, CH3CHNH), 5.11 (2H, s, PhCH2), 5.35 (1H,
br d, J ) 6.9 Hz, NH), 7.26-7.36 (5H, m, aromH). 13C NMR
(CDCl3): δ 17.0 (q), 18.9 (q), 23.6 (q), 25.5 (t), 25.7 (q), 33.2
(t), 41.7 (t), 43.7 (s), 48.3 (d), 48.4 (d), 49.8 (d), 66.8 (t), 88.5
(s), 128.1 (2×d), 128.2 (d), 128.5 (2×d), 136.4 (s), 155.6 (s),
172.7 (s). MS m/z (%): 359 (6) (M+), 292 (6), 279 (27), 153 (3),
137 (14), 108 (18), 91 (100). Anal. C21H29NO4 (359.5): calcd, C
70.16, H 8.13, N 3.89; found, C 70.17, H 8.13, N 3.90%.
Syn th esis of N-Ben zyloxyca r bon yl-â-ben zyl-L-a sp a r -
tyl-D-a la n in e F en ch yl Ester s (6a -d ). A 1.3-g (3.6 mM)
aliquot of each N-benzyloxycarbonyl-D-alanine fenchyl ester
was dissolved in methanol (25 mL), and 0.4 g of 5% palladium/
carbon was added to the solution and followed by hydrogena-
tion at room temperature for 5 h under atmospheric pressure.
After the catalyst was filtered off, the solvent was removed
under reduced pressure to give the D-alanine fenchyl ester as
an oil.
[R]D ) - 11.9° (c ) 1.0, in CHCl3). IR (CHCl3): ν 3410 cm-1
,
1
1720, 1675, 1500. H NMR (CDCl3): δ 0.77 (3H, s, CH3), 1.03
(3H, s, CH3), 1.09 (3H, s, CH3), 1.07-1.74 (7H, m, fenchyl),
1.38 (3H, d, J ) 7.1 Hz, CH3CHNH), 2.75 (1H, dd, J ) 6.4, 17
Hz, CH2CO2Bn), 3.07 (1H, dd, J ) 3.8, 17 Hz, CH2CO2Bn),
4.04 (1H, d, J ) 1.9 Hz, CO2CH-), 4.56 (1H, q, J ) 7.1 Hz,
CH3CHNH). 4.63 (1H, br s, CHCH2CO2Bn), 5.08-5.17 (4H,
m, 2×PhCH2), 7.26-7.37 (10H, m, aromH). 13C NMR
(CDCl3): δ 18.6 (q), 19.3 (q), 20.1 (q), 25.8 (t), 26.7 (t), 29.7
(q), 36.2 (t), 39.4 (s), 41.3 (t), 48.3 (d), 48.4 (s), 48.6 (d), 51.1
(d), 66.9 (t), 67.4 (t), 87.4 (d), 128.1 (d), 128.2 (2×d), 128.3
(2×d), 128.4 (2×d), 128.6 (3×d), 135.4 (s), 135.9 (s), 169.5 (s),
171.4 (s), 172.7 (s), 180.3 (s). MS m/z (%): 565 (9) (M++1), 429
(4), 411 (9), 383 (5), 321 (12), 275 (30), 222 (17), 181 (23), 137
(72), 91 (100).
N-Ben zyloxyca r b on yl-â-b en zyl-L-a sp a r t yl-D-a la n in e
(-)-â-F en ch yl Ester (6d ). Yield 77%. Purity by HPLC was
99.5%. [R]D23 ) +18.4° (c ) 0.5, in CHCl3). IR (CHCl3): ν 3410
cm-1, 1725, 1680, 1500. 1H NMR (CDCl3): δ 0.84 (3H, s, CH3),
1.00 (3H, s, CH3), 1.07 (3H, s, CH3), 1.09-1.76 (7H, m, fenchyl),
1.39 (3H, d, J ) 7.3 Hz, CH3CHNH), 2.75 (1H, dd, J ) 6.2,
17.1 Hz, CH2CO2Bn), 3.04 (1H, dd, J ) 4.1, 17.1 Hz, CH2CO2-
Bn), 4.21 (1H, d, J ) 1.9 Hz, CO2CH-), 4.55 (1H, q, J ) 7.3
Hz, CH3CHNH). 4.62 (1H, br s, CHCH2CO2Bn), 5.08-5.17 (4H,
m, 2×PhCH2), 7.26-7.37 (10H, m, aromH). 13C NMR
(CDCl3): δ 17.1 (q), 18.4 (q), 23.6 (q), 25.6 (t), 25.7 (q), 33.3
(t), 36.1 (t), 41.7 (t), 43.8 (t), 48.3 (s), 48.4 (d), 48.5 (d), 51.1
(d), 66.9 (t), 67.4 (t), 88.7 (d), 128.1(d), 128.2 (2×d), 128.3 (2×d),
128.4 (2×d), 128.6 (3×d), 135.4 (s), 135.9 (s), 169.5 (s), 171.4
(s), 172.7 (s), 180.3 (s). MS m/z (%): 565 (5) (M++1), 429 (33),
411 (10), 385 (14), 321 (29), 268 (17), 222 (19), 181 (21), 137
(70), 91 (100).
A 1.3-g (3.6 mM) aliquot of N-benzyloxycarbonyl-L-aspartic
acid â-benzyl ester was dissolved in dry dioxane (20 mL), and
0.74 g (4 mM) of N-hydroxy-endo-5-norbornene-2,3-dicarbox-
imide was added to the solution. After the mixture was cooled
on ice, 0.84 g (4.1 mM) of dicyclohexylcarbodiimide was added;
the temperature was then raised to room temperature and
stirring was continued for 4 h. The dicyclohexyl urea that
formed was then filtered off, and the previously prepared
D-alanine fenchyl ester in dry dioxane (10 mL) was added to
the filtrate with ice cooling. The mixture was stirred at room
temperature for 16 h, and the solvent was removed under
reduced pressure. To the residue, ethyl acetate (60 mL) was
added and washed with 10% citric acid solution, 5% sodium
bicarbonate solution, and brine. The organic layer was dried
with anhydrous magnesium sulfate and the solvent was
Syn th esis of L-a sp a r tyl-D-a la n in e F en ch yl Ester s (4a -
d ). 1.1 g (1.95 mM) of N-benzyloxycarbonyl-â-benzyl-L-aspar-
tyl-D-alanine fenchyl ester was dissolved in methanol (20 mL),

5016 J. Agric. Food Chem., Vol. 49, No. 10, 2001
Yuasa et al.
F igu r e 2. Synthesis of 4a -d .
F igu r e 3. 1H NMR spectrum of L-aspartyl-D-alanine (-)-R-fenchyl ester.
and 0.3 g of 5% palladium/carbon was added to the solution
followed by hydrogenation at room temperature for 5 h under
atmospheric pressure. After the catalyst was filtered off, the
solvent was removed under reduced pressure. n-Hexane was
added to the residue, forming a powdery precipitate. The
precipitate was separated by filtration and dried to give one
of following the L-aspartyl-D-alanine fenchyl esters as a
colorless and odorless powder.
(3H, d, J ) 7.3 Hz, CH3CHNH), 2.83 (1H, dd, J ) 8.5, 17.7
Hz, CH2CO2H), 2.94 (1H, dd, J ) 4.6, 17.7 Hz, CH2CO2H), 4.18
(1H, q, J ) 4.6, 8.5 Hz, CHCH2CO2H), 4.17 (1H, d, J ) 1.8
Hz, CO2CH-), 4.47 (1H, q, J ) 7.3 Hz, CH3CHNH). 13C NMR
(CD3OD): δ 17.4 (q), 17.5 (q), 24.0 (q), 26.1 (t), 26.4 (t), 32.7
(s), 34.4 (t), 36.6 (t), 42.6 (t), 44.9 (s), 48.8 (d), 49.9 (d), 51.3
(d), 90.0 (d), 169.1 (s), 173.6 (s), 174.0 (s). MS m/z (%): 341 (4)
(M++1), 187 (25), 169 (4), 159 (38), 141 (8), 137 (15), 113 (4),
102 (8), 88 (100), 81 (15), 70 (4), 44 (31). Anal C17H26N2O5
(340.4): calcd, C 59.98, H 8.29, N 8.23; found, C 60.07, H 8.31,
N 8.19%.
L-Asp a r tyl-D-a la n in e (-)-r-F en ch yl Ester (4c). Purity
by HPLC was 100%. Yield 80%. mp 128-133 °C. [R]D25 ) +9.6°
(c ) 0.35, in MeOH). IR (CHCl3): ν 3350 cm-1, 3200, 1735,
1685. 1H NMR (CD3OD): δ 0.82 (3H, s, CH3), 1.05 (3H, s, CH3),
1.08 (3H, s, CH3), 1.03-1.83 (7H, m, fenchyl), 1.44 (3H, d, J )
7.3 Hz, CH3CHNH), 2.61 (1H, dd, J ) 9.1, 17 Hz, CH2CO2H),
2.74 (1H, dd, J ) 4.8, 17 Hz, CH2CO2H), 4.08 (1H, dd, J )
4.8, 9.1 Hz, CHCH2CO2H), 4.38 (1H, d, J ) 2 Hz, CO2CH-),
4.49 (1H, q, J ) 7.3 Hz, CH3CHNH). 13C NMR (CD3OD): δ
17.7 (q), 19.6 (q), 20.6 (q), 26.7 (t), 27.5 (t), 30.0 (q), 34.4 (s),
38.0 (t), 40.5 (s), 42.2 (t), 48.9 (d), 49.3 (d), 52.2 (d), 88.6 (d),
170.1 (s), 174.5 (s), 175. 7(s). MS m/z (%): 341 (2) (M++1), 187
(27), 169 (8), 159 (42), 141 (13), 137 (13), 113 (6), 102 (27), 88
(100), 81 (12), 70 (4). Anal C17H26N2O5 (340.4): calcd, C 59.98,
H 8.29, N 8.23; found, C 60.22, H 8.42, N 8.03%.
L-Asp a r tyl-D-a la n in e (+)-r-F en ch yl Ester (4a ). Yield
25
83%. Purity by HPLC was 100%. mp 138-141 °C. [R]D
)
+51.6° (c ) 0.55, in MeOH). IR (CHCl3): ν 3400 cm-1, 3220,
1735, 1685. H NMR (CD3OD): δ 0.81 (3H, s, CH3), 1.06 (3H,
1
s, CH3), 1.10 (3H, s, CH3), 1.04-1.84 (7H, m, fenchyl), 1.45
(3H, d, J ) 7.3 Hz, CH3CHNH), 2.57 (1H, dd, J ) 9.2, 17 Hz,
CH2CO2H), 2.71 (1H, dd, J ) 4.7, 17 Hz, CH2CO2H), 4.06 (1H,
dd, J ) 4.7, 9.2 Hz, CHCH2CO2H), 4.38 (1H, d, J ) 1.9 Hz,
CO2CH-), 4.49 (1H, q, J ) 7.3 Hz, CH3CHNH). 13C NMR (CD3-
OD): δ 17.8 (q), 19.6 (q), 20.9 (q), 26.8 (t), 27.5 (t), 30.1 (q),
34.6 (s), 38.3 (t), 40.6 (s), 42.2 (t), 48.6 (d), 49.3 (d), 52.4 (d),
88.6 (d), 170.1 (s), 174.4 (s), 176.0 (s). MS m/z (%): 341 (2)
(M++1), 187 (26), 169 (5), 159 (40), 141 (10), 137 (13), 113 (4),
102 (19), 88 (100), 81 (12), 70 (4), 44 (38). Anal C17H26N2O5
(340.4): calcd, C 59.98, H 8.29, N, 8.23; found, C 60.12, H 8.40,
N 8.09%.
L-Asp a r tyl-D-a la n in e (+)-â-F en ch yl Ester (4b). Yield
80%. Purity by HPLC was 100%. mp 114-117 °C. [R]25
)
D
+10.9° (c ) 0.55, in MeOH). IR (CHCl3): ν 3325 cm-1, 3200,
L-Asp a r tyl-D-a la n in e (-)-â-F en ch yl Ester (4d ). Yield
1
25
1730, 1690. H NMR (CD3OD): δ 0.89 (3H, s, CH3), 1.04 (3H,
79%. Purtity by HPLC was 100%. mp 131-136 °C. [R]D
)
s, CH3), 1.05 (3H, s, CH3), 1.13-1.82 (7H, m, fenchyl), 1.44
+33.3° (c ) 0.33, in MeOH). IR (CHCl3): ν 3350 cm-1, 3200,

Characteristics of L-Aspartyl-D-Alanine Fenchyl Esters
J. Agric. Food Chem., Vol. 49, No. 10, 2001 5017
F igu r e 4. The estimated conformation of L-aspartyl-D-alanine (-)-R-fenchyl ester.
F igu r e 5. Newman projections of sweeteners 1-4.
1
1730, 1690. H NMR (CD3OD): δ 0.89 (3H, s, CH3), 1.04 (3H,
s, CH3), 1.07 (3H, s, CH3), 1.13-1.82 (7H, m, fenchyl), 1.44
(3H, d, J ) 7.3 Hz, CH3CHNH), 2.85 (1H, dd, J ) 8.7, 17.8
Hz, CH2CO2H), 2.97 (1H, dd, J ) 4.4, 17.9 Hz, CH2CO2H), 4.18
(1H, dd, J ) 4.4, 8.7 Hz, CHCH2CO2H), 4.19 (1H, d, J ) 1.6
Hz, CO2CH-), 4.49 (1H, q, J ) 7.3 Hz, CH3CHNH). 13C NMR
(CD3OD): δ 17.4 (q), 17.6 (q), 24.2 (q), 26.0 (t), 26.3 (t), 32.7
(s), 34.3 (t), 36.2 (t), 42.6 (t), 44.8 (s), 49.1 (d), 49.2 (d), 51.0
(d), 89.9 (d), 168.9 (s), 172.9 (s), 173.8 (s). MS m/z (%); 341 (4)
(M++1), 187 (23), 169 (7), 159 (39), 141 (10), 137 (15), 113 (5),
102 (27), 88 (100), 81 (14), 70 (3), 44 (38). Anal C17H26N2O5
(340.4): calcd, C 59.98, H 8.29, N 8.23; found, C 60.02, H 8.38,
N 8.19%.
RESULTS AND DISCUSSION
The syntheses of the L-aspartyl-D-alanine fenchyl
esters (4a -d ) were accomplished as follows: Condensa-
tion of N-benzyloxycarbonyl-D-alanine with fenchol by
dicyclohexylcarbodiimide and a catalytic amount of
4-(dimethylamino)pyridine gave the corresponding N-
benzyloxycarbonyl-D-alanine fenchyl esters (5a -d ) in
65-72.5% yield, which was followed by deprotection by
hydrogenolysis with 5% palladium/carbon. The fenchyl
esters of D-alanine (4a -d ) were condensed with N-ben-
zyloxycarbonyl-â-benzyl-L-aspartic acid by dicyclohexyl-
carbodiimide and a catalytic amount of N-hydroxy-endo-

5018 J. Agric. Food Chem., Vol. 49, No. 10, 2001
Yuasa et al.
(3) Kier, L. B. A molecular theory of sweet taste. J . Pharm.
Sci. 1972, 61, 1394-1397.
(4) Mazur, R. H.; Schlatter, J . M.; Goldkamp, A. H. Struc-
ture-taste relationships of some dipeptides. J . Am.
Chem. Soc. 1969, 91, 2684-2691.
(5) Mazur, R. H.; Goldkamp, A. H.; J ames, P. A.; Schlatter,
J . M. Structure-taste relationships of aspartic acid
amides. J . Med. Chem. 1970, 13, 1217-1221.
(6) Crosby, G. A.; Dubois, G. E.; Wingard, R. E., J r. The
design of synthetic sweeteners. In Drug Design; Aniens,
E. J ., Ed.; Academic Press: New York, 1979; Vol III, pp
215-248.
(7) Pavlova, L. A.; Komarova, T. V.; Davidovich, Yu. A.;
Rogozhin, S. V. Aspartame and its analogues. Russ.
Chem. Rev. 1981, 50, 590-605.
(8) Ariyoshi, Y. Structure-taste relationships of peptides.
Kagaku Kyoiku 1983, 31, 12-17.
(9) Fujino, M.; Wakimasu, M.; Mano, M.; Tanaka, K.;
Nakajima, N.; Aoki, H. Structure-taste relationships
of L-aspartyl aminomalonic acid diesters. Chem. Pharm.
Bull. 1976, 24, 2112-2117.
(10) Ying-Zing, L.; Hiu-qmin, X.; Guohua, J .; Guang-zhi, Z.
Proceedings, The Relationship between the structure
and sweetness of aspartic acid dipeptides. Sino-Amer-
ican Symposium on Chemistry of Natural Products,
1980; Beijing Science Press: Beijing, 1982; pp 254-256.
(11) Yuasa, Y.; Nagakura, A.; Tsuruta, H. The sweetness and
stereochemistry of L-aspartyl-fenchylamino alcohol de-
rivatives. Tetrahedron Lett. 1994, 35, 6891-6895.
(12) Yuasa, Y.; Nagakura, A.; Tsuruta, H. The synthesis of
(2R,3R)-3-(L-aspartylamino)-1-[(2S,3R)-3-methyl-2-nor-
bornyl]-2-butanol, an excellent sweetener. Flavour Fla-
grance J . 1996, 11, 327-332.
(13) Mazur, R. H.; Leuter, J . A.; Swiatek, K. A.; Scllater, J .
M. Synthetic sweeteners. 3. Aspartyl dipeptide esters
from L- and D-alkylglycines. J . Med. Chem. 1973, 16,
1284-1287.
(14) Ariyoshi, Y.; Yasuda, N.; Yamatani, T. The structure-
taste relationships of the dipeptide esters composed of
L-aspartic acid and â-hydroxy amino acids. Bull. Chem.
Soc. J pn. 1974, 47, 326-330.
(15) Nagakura, A.; Yuasa, Y.; Tsuruta, H.; Akutagawa, S.
Pinanyl and fenchyl esters of aspartylalanine. J pn
Patent 61-200999, 1986. Chem. Abstr. 1987, 106, 156864c.
(16) Nagakura, A.; Yuasa, Y.; Tsuruta, H.; Akutagawa, S.
L-Aspartyl-D-alanine (-)-R-fenchyl ester. J pn Patent 61-
291597, 1986. Chem. Abstr. 1987, 107, 23712a.
(17) Zeng, G.-Z.; Chen, J .-T.; He, H.-Z.; Wang, Z.-Q.; Yan,
J .-S. In the pursuit of a better sweetener. J . Agric. Food
Chem. 1991, 39, 782-785.
5-norbornene-2,3-diimide to give the N-benzyloxy-
carbonyl-â-benzyl-L-aspartyl-D-alanine fenchyl esters
(6a -d ) in 77-81% yield, which was followed by depro-
tection by hydrogenolysis with 5% palladium/carbon to
give the L-aspartyl-D-alanine fenchyl ester as a colorless
powder (Figure 2).
We compared the sweetness potency of the L-aspartyl
fenchyl esters (4a -d ) with that of sucrose. As a result
of the evaluation, the (+)-R and (-)-â isomers (4a and
4d ) showed intensities of 250 and 160 times sucrose,
respectively. On the other hand, the sweetness of the
(+)-â- form (4b) was 5700 times and the (-)-R- form (4c)
was 1100 times greater than that of sucrose (Table 1).
From this result, the reported data of Zeng et al. that
showed 4a had no sweetness were corrected. We further
assumed that the configuration of the (+)-â- or (-)-R-
fenchyl unit is a better fitting form for the sweetness
receptor.
Next, we studied the conformation of L-aspartyl-D-
alanine (-)-R-fenchyl ester (4c) by NMR (Figure 3).
Each proton was assigned by 2D NMR, and the nuclear
Overhauser effect (nOe) in L-aspartyl-D-alanine (-)-R-
fenchyl ester (4c) was observed between the bridgehead
methyl group (Me1) of fenchyl and the methyl group
(Me4) of alanine (Figure 4). The coupling constant of NH
and the methine proton (H4) of the alanine unit (J HNR)
was 7 Hz by selective frequency decoupling (SFDEC)
and thus their dihedral angle was calculated to be 145°
(21). The carbonyl oxygen atom of the ester and the
2-position proton of the bicyclo ring is considered to be
eclipsed. Also, the conformation between the carbonyl
of the amide and NH is considered to be antiperiplanar
(Figure 4).
The Newman projections of aspartame (1), the ami-
nomalonic fenchyl ester (2) (9, 10), the fenchyl amino
alcohol (3) (11, 12), and the D-alanine fenchyl ester (4)
prepared by us are shown in Figure 5. From these
Newman projections, we propose the following hypoth-
esis: Sites I and II are present in the sweet taste
receptors for fitting the large and small hydrophobic
groups (22) of these sweeteners, and these sites have a
nonbonding interaction to functional groups; for ex-
ample, the carbonyl of the ester or hydroxy function.
These functional groups act only as an anchor. Sweet-
ener 2 has two anchors to fit both sites I and II, and
therefore, has the highest sweetness potency. On the
other hand, sweeteners 3, 4, and 1 have one anchor to
fit site II or I (Figure 5). We assume that site I has a
higher sweetness potency than site II.
(18) Coulombeay, A.; Rasat, A. Re´duction de la fenchone,
e´pime´risation des fenchol. Bull. Soc. Chim. Fr. 1965,
3338-3345.
In particular, 4c has an excellent quality, potent
sweetness, and is more stable than aspartame in solu-
tion, but not sufficient for use as a sweetener. 4b did
not have an excellent quality of sweetness because it
displayed an aftertaste caused by the strong sweetness.
Unfortunately, 4b and 4c were found to be slowly
hydrolyzed at the pH (3-4) of soft drinks to give fenchol,
a compound that imparts an undesirable off-taste to the
beverage at levels of less than 0.1 ppm (23). We have
previously reported that the D-alanine unit can be
changed to the more stable structure of the optically
active amino alcohol (11, 12).
(19) Doering, W. von E.; Aschner, T. C. Stereochemical
equilibration of alcohols. J . Am. Chem. Soc. 1949, 71,
838-840.
(20) Guenther, E.; Althausen, D. The Essential Oils; D. Van
Nostrand: New York, 1949; Vol. 2, p 254 (rotations for
all four fenchols are given).
(21) Ramachandran, G. N.; Chandrasekarn, R.; Kopple, K.
D. Variation of the NH-CRH coupling constant with
dihedral angle in the NMR spectra of peptides. Biopoly-
mers 1971, 10, 2113-2131.
(22) Ariyoshi, Y. The structure-taste relationships of as-
partyl dipeptide esters. Agric. Biol. Chem. 1976, 40,
983-992.
(23) King, G. A., III; Sweeny, J . G.; Iacobucci, G. A. New high
potency L-aspartyl-3-bicycloalkyl-L-alanine methyl ester
sweeteners. J . Agric. Food Chem. 1991, 39, 52-56.
LITERATURE CITED
(1) Shallenberger, R. S.; Acree, T. E. Molecular theory of
sweet taste. Nature 1967, 216, 480-482.
(2) Shallenberger, R. S. Taste Chemistry; Blackie Academic
& Professional, an imprint of Chapman & Hall: Glas-
gow, UK, 1993.
Received for review March 13, 2001. Revised manuscript
received J uly 26, 2001. Accepted J uly 31, 2001.
J F010344O