Aspartame analogues containing 1-amino-2-phenylcyclohexanecarboxylic acids (c6Phe). Part 2

Article abstract of DOI:10.1016/j.tet.2005.01.033

This report describes the synthesis and conformational analysis of optically pure dipeptide analogues of aspartame, namely H-(S)-Asp-(1R,2S)-c ₆Phe-OMe and H-(S)-Asp-(1S,2R)-c₆Phe-OMe, in which the Phe residue of aspartame has been r

Full text of DOI:10.1016/j.tet.2005.01.033

Tetrahedron 61 (2005) 2913–2919
Aspartame analogues containing
1-amino-2-phenylcyclohexanecarboxylic acids (c6Phe). Part 2
*
´ ´ ´ ´
Miriam Alıas, Marta Lasa, Pilar Lopez, Jose I. Garcıa and Carlos Cativiela
´ ´
Departamento de Quımica Organica, ICMA, Facultad de Ciencias, Universidad de Zaragoza-CSIC, 50009 Zaragoza, Spain
Received 22 November 2004; revised 7 January 2005; accepted 12 January 2005
Abstract—This report describes the synthesis and conformational analysis of optically pure dipeptide analogues of aspartame, namely
H-(S)-Asp-(1R,2S)-c6Phe-OMe and H-(S)-Asp-(1S,2R)-c6Phe-OMe, in which the Phe residue of aspartame has been replaced by a restricted
Phe with a cyclohexane skeleton: 1-amino-2-phenylcyclohexanecarboxylic acid (c6Phe). The dipeptide that incorporates (1R,2S)-c6Phe is
sweet whereas the compound that incorporates (1S,2R)-c6Phe is bitter. This relationship between the absolute configuration of the dipeptides
and the properties is explained in terms of the different conformational behaviour displayed by each molecule, as determined by molecular
mechanics and molecular dynamics calculations that take into account solvent effects.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
respect to unmodified residues. Thus, the incorporation of
amino acids with well-defined c1 tendencies into bioactive
peptides may provide fundamental insights into the precise
conformational requirements of the side-chain groups to fit
the receptor binding site.
The discovery of the dipeptide sweetener Aspartame1
[H-(S)-Asp-(S)-Phe-OMe] has led to the synthesis of a
large number of dipeptide analogues in an attempt to find a
better taste profile and to elucidate the mechanism for the
sweet response.
Several conformationally restricted analogues of aspartame
in which the Phe residue has been replaced by different
conformationally restricted amino acids have been synthe-
sised and studied. Particular examples have incorporated
different 1-aminocycloalkanecarboxylic acids4,5 and
1-amino 2-phenylcyclopropanecarboxylic acids (c3Phe).6
Some of the resulting compounds were found to be sweet
whereas others are bitter or tasteless.
Analysis of the preferred conformations of aspartame using
1
a combination of X-ray crystallography, H NMR spectro-
scopy and molecular mechanics calculations has led several
authors to propose different models for the tastant–receptor
interaction. Two such models have emerged as the most
appropriate: Temussi2 and Goodman.3 The difference
between the two models concerns the conformational
flexibility of aspartame dipeptides. As a result, the
availability of conformationally restricted analogues is
very important to establish the molecular arrays required
for sweet and bitter tastes. The incorporation of side-chain
constrained amino acids constitutes a powerful tool to
explore this aspect. Restrictions on the c1 torsion angles will
affect the conformational preferences of the side substitu-
ents and have an impact on the rotameric distribution with
The presence in the phenylalanine analogue of a cyclo-
hexane structure, which tethers Ca to Cb, (c6Phe) appears to
be very efficient for the restriction of the side-chain
flexibility and is far superior to other restrictions such as
a- and/or b-methylation. For each enantiomer only two c1
arrangements are accessible, with one of these being
strongly discriminated by the propensity of the bulky
phenyl group to occupy equatorial positions in a chair
conformation. As a result, (1S,2R)-c6Phe and (1S,2S)-c6Phe
can be viewed as frozen gauche (C) side-chain analogues
of (S)-Phe and, conversely, (1R,2S)-c6Phe as gauche (K)
analogues of (R)-Phe.7
Keywords: Strecker; Cyclohexanes; Constrained phenylalanines; Aspar-
tame analogues; Computer-assisted methods; Chiral stationary phase;
HPLC resolution.
Abbreviations: TEA, triethylamine; Cbz, benzyloxycarbonyl; EtOAc, ethyl
acetate; MeOH, methanol; NMM, N-Methylmorpholine; OtBu, tert-butoxy;
OMe, methoxy; TFA, trifluoroacetic acid; Bz, benzoyl.
* Corresponding author. Tel./fax: C34 976 761 210;
e-mail: cativiela@unizar.es
Due to this interesting restriction, and as an extension of our
studies on constrained analogues of aspartame, we pre-
viously described the replacement of the Phe residue with
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.01.033

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M. Alıas et al. / Tetrahedron 61 (2005) 2913–2919
2914
Scheme 1. Synthetic route to racemic trans-c6Phe. Reagents and conditions: (a) NaCN, NH4Cl, iPrOH, NH4OH. (b) CH3COCl, TEA, CH2Cl2. (c) 12 N HCl,
reflux.
two enantiomers of cis-c6Phe, namely (1R,2R)-c6Phe and
(1S,2S)-c6Phe, which resulted in a sweet and a bitter
compound, respectively.8 The different organoleptical
properties of the two molecules are related to the different
conformational behaviour of each dipeptide, as exemplified
by molecular calculations. These conformational prefer-
ences depend on the absolute configuration of each
enantiomer of the Phe analogue.
indispensable to complete the synthesis and conformational
studies of aspartame analogues incorporating all four
isomers of restricted phenylalanine c6Phe.
We report here the synthesis and conformational behaviour
of H-(S)-Asp-(1R,2S)-c6Phe-OMe and H-(S)-Asp-(1S,2R)-
c6Phe-OMe as conformationally restricted aspartame
analogues.
2. Results and discussion
2.1. Peptide synthesis
In the course of our work on the synthesis of conformation-
ally restricted phenylalanines, we recently developed9
The first step in the synthesis of the aspartame analogues
involved the preparation of rac-trans-3. As previously
described by our group,9 a convenient route for the
preparation of trans-c6Phe in its racemic form involves a
Strecker reaction of 2-phenylcyclohexanone and subsequent
hydrolysis of the product (Scheme 1). The synthesis of
amino ester rac-trans-4 was achieved in moderate yield
from rac-trans-3 by formation of the methyl ester with
thionyl chloride/methanol (Scheme 2). Once this starting
material had been obtained, we attempted to couple these
amino esters, rac-trans-4, with conveniently protected
aspartic acid Cbz-(S)-Asp(OtBu)-OH, (S)-5. Coupling by
the mixed-anhydride procedure,10,11 using iBuOCOCl in the
presence of NMM in CH2Cl2 at K15 8C, afforded a mixture
of the desired diastereoisomers (S,1R,2S)-6 and (S,1S,2R)-6
in very good yield.
a
convenient route for the preparation of trans-c6Phe in
racemic form by a Strecker reaction on 2-phenylcyclo-
hexanone. The importance of the absolute configuration of
the Phe analogue in the sweet properties of the aspartame
dipeptide makes the incorporation of the two enantiomers of
trans-c6Phe, i.e. (1S,2R)-c6Phe and (1R,2S)-c6Phe,
However, all attempts to separate the diastereoisomers by
flash chromatography were unsuccessful. We therefore
decided to explore the use of semi-preparative HPLC for
the resolution of (S,1R,2S)-6 and (S,1S,2R)-6. Analytical
separation was initially examined in normal phase using
silica as the stationary phase, but separation was not
observed under any of the conditions tested. Better results
were obtained on an amylose-derived chiral stationary
phase, which has proved to be very efficient in other semi-
preparative resolutions of phenylalanine surrogates.8,12–16
Elution and detection conditions for the best separations are
given in Table 1 and the analytical resolution is shown in
Figure 1.
Scheme 2. Reagents and conditions: (a) SOCl2, MeOH. (b) (i) iBuOCOCl,
NMM, CH2Cl2; (ii) HPLC. (c) TFA/CH2Cl2. (d) H2, Pd/C.
Table 1. Selected chromatographic data for the HPLC resolution of (S,1R,2S)-6 and (S,1S,2R)-6 on the amylose-derived chiral stationary phase
Eluenta A/B/C
Flow (mL/min)
l (nm)
a
Rs
0
Column
k1
Analyticalb
97/3/0
96/3/1
0.8
15
210
254
1.97
1.79
1.25
1.22
1.67
0.90
Semi-preparativec
For the definition of k0, a and Rs see Section 4.
a A: n-hexane, B: 2-propanol, C: chloroform.
b Steel column, 150 mm!4.6 mm ID, toZ2.64 min. cZ5 mg/mL. Temperature: 25 8C.
c Steel column, 150 mm!20 mm ID, toZ2.52 min. cZ200 mg/mL. Temperature: 25 8C.

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M. Alıas et al. / Tetrahedron 61 (2005) 2913–2919
2915
Figure 1. HPLC analytical resolution of Cbz-Asp(OtBu)-rac-trans-c6Phe-OMe and resolved diastereoisomers (S,1R,2S)-6 and (S,1S,2R)-6.
For the extension of the analytical conditions to the semi-
preparative scale we selected the mixture n-hexane/
2-propanol/chloroform indicated in Table 1. This solvent
system was found to be the most appropriate eluent to
optimise the resolution in relation to the column loadability.
The final steps leading to deprotected dipeptides were
carried out with each diastereoisomer as follows. Firstly,
treatment with TFA in CH2Cl2 afforded (S,1R,2S)-7 and
(S,1S,2R)-7. Secondly, hydrogenolysis using palladium on
carbon as a catalyst and further purification by RP-HPLC
gave the optically pure analogues of aspartame: H-(S)-Asp-
(1R,2S)-c6Phe-OMe and H-(S)-Asp-(1S,2R)-c6Phe-OMe,
(S,1R,2S)-8 and (S, 1S,2R)-8.
Direct assignment of the stereochemistry of the dipeptides
(S,1R,2S)-6 and (S,1S,2R)-6 was made by comparing their
spectral data with those corresponding to the dipeptides
synthesised by coupling the two optically pure enantiomers
(1R,2S)-4 and (1S,2R)-4, prepared from optically pure
amino acids (1R,2S)-3 and (1S,2R)-3,9 with protected
aspartic acid (S)-5.
2.2. Peptide taste determination
Taste tests were carried out by a ‘sip and spit’ qualitative
assessment of solutions of the compounds using a three-
volunteer taste panel. The analogues were tasted in water at
room temperature without any pH adjustment at 0.5% (w/v)
concentration. These taste determinations show that com-
pound (S,1R,2S)-8 is sweet, albeit less potent than
aspartame, whereas compound (S,1S,2R)-8 is bitter.
2.3. Molecular modelling studies
The research carried out on the elucidation of detailed
structure-taste relationships of aspartame and its analogues
has led to a widely accepted model that has considerable
predictive power for related molecules.3 According to this
model, the sweet taste is associated with an L-shaped
conformation, in which the hydrophobic group projects
along the Cx axis (Fig. 2). A reverse L-shaped structure, in
which the hydrophobic group points to the Kz axis, is
associated with a bitter taste. Other possible topochemical
arrays would lead to tasteless compounds.
In order to account for solvation effects, we decided to carry
out the same mixed scheme as reported previously,8
combining molecular mechanics and quantum mechanics
calculations. Thus, single point energy semi-empirical AM1
calculations on the MM2 optimised structures (see Section 4
for details) were carried out using the COSMO continuum
model with the dielectric constant of water. The solvation
energy was obtained as the energy difference between the
solvated and the isolated structures, and this energy was
then added to the MM2 steric energy to obtain the relative
energies of the different conformers. Although this pro-
cedure does not take into account solvent effects during the
geometry optimisation, it has the advantage of combining
Figure 2. Calculated minimum energy conformations of (S,1R,2S)-8 and
(S,1S,2R)-8 (hydrogen atoms have been omitted for clarity).

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M. Alıas et al. / Tetrahedron 61 (2005) 2913–2919
2916
Table 2. Calculated energies (kcal molK1) and some selected dihedral angles for the most stable conformations of (S,1R,2S)-8 and (S,1S,2R)-8
Compound
Conformer
Dihedral anglesa
EMM2
DGsolv
ETot
DETot
J
F
c1
(S,1R,2S)-8
(S,1S,2R)-8
Cx
Kz
K56.3
K55.7
175.5
90.4
K58.2
K60.3
K58.9
K54.3
K77.7
K81.9
K136.6
K136.2
0
0.4
Cx
Kz
K89.5
K166.6
39.2
45.7
57.2
60.8
K51.2
K55.4
K80.0
K79.0
K131.2
K134.4
3.2
0
a See Figure 2 for the definition of the dihedral angles.
the strength of the MM2 force field for conformational
analysis with that of a successful quantum mechanical
method for calculating the solvation energy.
relationship in all four aspartame analogues that incorporate
c6Phe.
The minimum energy values obtained for conformations
Cx and Kz for compounds (S,1R,2S)-8 are gathered in
Table 2 and the corresponding structures are shown in
Figure 2. As can be seen, based on total energies, compound
(S,1R,2S)-8 should be sweet (since conformer Cx is more
stable than conformer Kz), whereas compound (S,1S,2R)-8
should be bitter (since the reverse relative stability is
obtained).
3. Conclusion
Two new aspartame derivatives that incorporate the
constrained phenylalanines trans-c6Phe have been synthe-
sised. The isomer H-(S)-Asp-(1R,2S)-c6Phe-OMe is sweet
whereas isomer H-(S)-Asp-(1S,2R)-c6Phe-OMe is bitter.
The relationship between the absolute configurations of the
dipeptides and the taste response can be explained in terms
of the different conformational behaviour displayed by each
molecule.
Analysis of the results obtained for the four new analogues
of aspartame reveals that the conformational preferences of
the c6Phe residues have been shifted with respect to their
(S)-Phe and (R)-Phe counterparts.17 Thus, in (1S,2R)-c6Phe
and (1S,2S)-c6Phe, analogues of (S)-Phe, a gauche (C) side-
chain conformation is shown, while this rotamer proves to
be the least favoured for the (S)-Phe residue. As for (1R,2R)-
c6Phe and (1R,2S)-c6Phe, analogues of (R)-Phe, a gauche
(K) disposition is exhibited, whereas this rotamer is the
least favoured for the (R)-Phe residue.
4. Experimental
4.1. General
4.1.1. Instrumentation. Solvents were purified according to
standard procedures. Analytical TLC was performed using
Merck 60 SI F254 precoated silica gel polyester plates using
the following solvent systems: 1 (hexane/EtOAc, 5:2); 2
(CH2Cl2/MeOH, 8:2). The products were examined by UV
fluorescence or developed by iodine or ninhydrin chromatic
reaction as appropriate. Column chromatography was
performed using silica gel 60 (230–400 mesh). Melting
points were determined on a Bu¨chi SMP-20 apparatus and
were not corrected. IR spectra were registered on a Mattson
Genesis FTIR spectrophotometer; nmax is given for the main
It has been reported3,18 that in the minimum energy
conformations calculated for sweet H-(S)-Asp-(S)-Phe-
OMe, the side chain in (S)-Phe can assume any of the
three possible staggered Ca-Cb rotamers: gauche (C),
gauche (K) and anti; but the conformation responsible for
sweet taste demands a value of c1ZK608 in the C-terminal
residue.
absorption bands. H and 13C NMR spectra were recorded
1
In addition, it should be noted that the incorporation into
aspartame of c6Phe residues with a gauche (K) preference,
i.e. (1R,2R)-c6Phe and (1R,2S)-c6Phe, leads to sweet
derivatives whereas replacement by c6Phe residues where
a gauche (C) preference is exhibited, i.e. (1S,2S)-c6Phe and
(1S,2R)-c6Phe, leads to bitter analogues. This fact suggests
that, for a sweet response, the stereochemistry at Ca in the
Phe residue is not as crucial as the value of the dihedral
angle c1, with the latter being necessary but not sufficient on
its own.
on Varian Unity-300 or Bruker ARX-300 instruments, using
TMS as the internal standard; chemical shifts are reported in
ppm on the d scale, coupling constants in Hz. Optical
rotations were measured on a Perkin–Elmer 241 polari-
meter-C in a 1 dm cell of 1 mL capacity. Microanalyses
were carried out on a Perkin–Elmer 200 C, H, N, S analyser
and are in good agreement with the calculated values.
4.1.2. High performance liquid chromatography. HPLC
was carried out using a Waters HPLC system equipped with
a Waters 600-E pump and a Waters 991 photodiode array
detector. The chiral stationary phase, which consisted of
mixed 10-undecenoate/3,5-dimethylphenylcarbamate of
amylose bonded to allylsilica, was prepared according to a
previously described procedure.19,20 The analytical assays
were carried out on a 150 mm!4.6 mm ID column and the
semi-preparative resolution was achieved on a 150 mm!
20 mm ID column. All analytical assays and semi-
preparative chromatography were performed under the
conditions given in Table 1. Final products were purified
It can therefore be concluded that the results of molecular
mechanics calculations are in good agreement with the taste
experimentally found. Once again, the procedure followed
to describe the solvation effects has proved to be both
convenient and very useful, as the results obtained in vacuo
would have led to incorrect taste predictions for some of the
new analogues; e.g. the case of (S,1R,2R)-8.8
These results support the model reported by Goodman et al.3
which has been assumed to explain the structure-taste

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M. Alıas et al. / Tetrahedron 61 (2005) 2913–2919
2917
on a Xterrae MS C8 250 mm!4.6 mm ID column. The
solvents used as mobile phases were of spectral grade.
temperature. The solution was diluted with CH2Cl2 and
washed with 5% KHSO4, 5% NaHCO3, brine, dried and
evaporated to dryness. The product was purified by silica gel
column chromatography using hexane/ethyl acetate (5:2)
as eluent to give the protected dipeptides in high yield
(O99%). The diastereomeric mixture of protected dipep-
tides was purified by silica gel column chromatography and
then resolved by semi-preparative HPLC as described
below.
The capacity (k0), selectivity (a) and resolution (Rs) values
0
were calculated according to the equations kR Z(tRKt0)/t0,
0
0
aZk2 /k1 , RsZ1.18(t2Kt1)/(w2Cw1). Subscripts 1 and 2
refer to the first and second eluted diastereoisomers,
respectively; tR (RZ1, 2) are their retention times, and w2
and w1 denote their bandwidths at half height; t0 is the dead
time.
4.3.1. Resolution of two stereoisomers of Cbz-(S)-
Asp(OtBu)-rac-trans-c6Phe-OMe: isolation of (S,1R,2S)-
6 and (S,1S,2R)-6. HPLC resolution of a mixture of
(S,1R,2S)-6 and (S,1S,2R)-6 (235 mg) dissolved in chloro-
form (1.2 mL) was carried out by successive injections of
0.10 mL onto a 150 mm!20 mm column filled with mixed
10-undecenoate-3,5-dimethyl phenylcarbamate of amylose
bonded to allylsilica. A mixture of n-hexane/2-propanol/
chloroform (96/3/1) was used as the eluent (flow rate
15 mL/min). Each run was collected into three separate
fractions: 6.5–7.2, 7.2–8.2 and 8.2–9.6 min. In this way
85 mg and 53 mg of the less and more strongly retained
diastereoisomers were obtained, respectively. The com-
bined second fractions, containing 95 mg of diastereomeric
mixture, were reinjected to afford 23 mg and 32 mg of the
less and more strongly retained diastereoisomers, respect-
ively. As a result, 108 mg of optically pure first eluted
diastereoisomer and 85 mg of optically pure last eluted
diastereoisomer were obtained.
4.1.3. Molecular modelling methodology. All computer
simulations were carried out using the Chem3D program.21
Molecular Dynamics were obtained by means of the MM2
force field,22 as implemented in Chem3D. Molecular
dynamics trajectories were collected in periods of 1 ns,
using a step interval of 1 fs and a target temperature of
473 K. It is well known that the dihedral angles of the Asp
residue remain essentially unchanged for all minimum
energy conformations of aspartame, and that this disposition
is very similar to the X-ray crystal structure.3 Consequently,
the geometry of this residue was kept fixed in the position of
the X-ray crystal structure of aspartame throughout all the
simulations.
Solvation effects were taken into account by means of single
point energy semi-empirical AM123 calculations on the
MM2 optimised structures, using the COSMO continuum
model24,25 with the dielectric constant of water.
4.2. Synthesis of rac-trans-c6Phe-OMe, rac-trans-4
4.3.2. CBz-(S)-Asp(OtBu)-(1R,2S)-c6Phe-OMe, (S,1R,
2S)-6. Mp: oil. Rf1Z0.40. [a]2D0ZK5.45 (cZ1, CHCl3).
IR: 3336.94; 1727.73; 1711.31; 1693.51; 1678.15 cmK1. 1H
NMR (CDCl3 300 MHz) d 1.2–2.0 (m, 5H); 1.38 (m, 9H);
2.1–2.5 (m, 3H); 2.54 (dd, 1H, JZ6 Hz, JZ16.5 Hz); 2.9
(m, 1H); 3.56 (m, 1H); 3.62 (s, 3H); 4.49 (m, 1H); 5.09 (m,
2H); 5.84 (d, 1H, JZ7.8 Hz); 7.0–7.4 (m, 11H). 13C NMR
(CDCl3 75 MHz) d 172.34; 170.96 169.38; 155.79; 140.97;
136.30; 128.54; 128.33; 128.23; 128.18; 128.06; 127.20;
81.67; 67.01; 64.26; 51.95; 51.59; 47.07; 37.54; 32.35;
28.39; 27.97; 25.02; 22.54. Anal. Calcd for C30H38N2O7: C,
66.89; H, 7.11; N, 5.20. Found: C, 66.99; H, 7.11; N, 5.17.
The synthesis of 1-amino-2-phenylcyclohexane-1-carb-
oxylic acid hydrochloride, rac-trans-3, was performed as
described previously.9 The corresponding methyl ester,
rac-trans-4, was prepared using the following procedure.
SOCl2 (1.8 mL, 24.67 mmol) was added dropwise to a
stirred solution of MeOH (20 mL) cooled in an ice-bath.
After 15 min stirring at that temperature, rac-trans-3
(316 mg, 1.24 mmol) was added to the reaction mixture
and this was heated under reflux for 8 h. A further 3 portions
of SOCl2/MeOH, prepared as described above, were added
and the solvents were removed in vacuo. The residue was
partitioned between 5% NaHCO3/EtOAc. The organic layer
was washed with an additional portion of 5% NaHCO3,
dried and concentrated in vacuo to afford the racemic
a-amino ester (yield 42%).
4.3.3. CBz-(S)-Asp(OtBu)-(1S,2R)-c6Phe-OMe, (S,1S,
2R)-6. Mp: oil. Rf1Z0.40. [a]2D0ZC30.34 (cZ0.92,
1
CHCl3). IR: 3336.58; 1729.23; 1678.47 cmK1. H NMR
(CDCl3 300 MHz) d 1.2–2.5 (m, 8H); 1.45 (s, 9H); 2.58 (dd,
1H, JZ6 Hz, JZ18.3 Hz); 2.87 (dd, 1H, JZ17.1 Hz, JZ
3 Hz); 3.57 (m, 1H); 3.63 (s, 3H); 4.45 (m, 1H); 5.07 (s, 2H);
5.93 (d, 1H, JZ8.4 Hz); 7.1–7.4 (m, 11H). 13C NMR
(CDCl3 75 MHz) d 172.93; 171.49; 169.85; 156.51; 141.35;
136.68; 129.01; 128.91; 128.88; 128.60; 128.46; 127.59;
82.09; 67.56; 64.88; 52.47; 47.28; 37.33; 32.39; 29.82;
28.77; 28.50; 25.32; 22.97. Anal. Calcd for C30H38N2O7: C,
66.89; H, 7.11; N, 5.20. Found: C, 66.75; H, 7.15; N, 5.27.
4.3. General procedure for the synthesis of protected
dipeptides (S,1R,2S)-6 and (S,1S,2R)-6
Cbz-(S)-Asp(OtBu)-OH, (S)-5, (198 mg, 0.61 mmol) and
NMM (62 mg, 0.61 mmol) were dissolved in dry CH2Cl2
(15 mL) under an inert atmosphere. The mixture was cooled
i
to K15 8C and a precooled solution of BuOCOCl (84 mg,
0.61 mmol) in dry CH2Cl2 (3 mL) was added. The mixture
was stirred for 20 min and a precooled solution of H-rac-
trans-c6Phe-OMe, rac-trans-4 (137 g, 0.51 mmol)—
obtained as described in Section 4.2 and used without
further purification—in CH2Cl2 (3 mL) was added drop-
wise. The reaction mixture was stirred at K15 8C for 1 h
and the solution was allowed to warm up to room
4.4. General procedure for the synthesis of (S,1R,2S)-7
and (S,1S,2R)-7
The corresponding protected dipeptide (1 mmol) was
dissolved in CH2Cl2 (15 mL) and TFA (7.5 mL) was
added. The solution was stirred at room temperature for

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2918
1.5 h. The TFA and CH2Cl2 were evaporated under reduced
pressure to afford the final product.
21.25. Anal. Calcd for C18H24N2O5: C, 62.05; H, 6.94; N,
8.04. Found: C, 62.12; H, 6.95; N, 8.07.
4.4.1. CBz-(S)-Asp-(1R,2S)-c6Phe-OMe, (S,1R,2S)-7.
This compound was prepared according to the procedure
described above. Cbz-(S)-Asp(OtBu)-(1R,2S)-c6Phe-OMe,
(S,1R,2S)-6, (100 mg, 0.19 mmol); CH2Cl2 (3 mL); TFA
(1.55 mL). Yield: quantitative. Mp: oil. Rf1Z0.04, Rf2Z
0.79. [a]2D0ZK19.76 (cZ0.47, CHCl3). IR (nujol): 2500–
3600; 1725.02 cmK1. 1H NMR (CDCl3 300 MHz) d 1.2–2.0
(m, 5H); 2.0–2.4 (m, 3H); 2.72 (dd, 1H, JZ6.1 Hz JZ
17.3 Hz); 2.92 (m, 1H); 3.55 (m, 1H); 3.64 (s, 3H); 4.54 (m,
1H); 5.09 (s, 2H); 5.92 (d, 1H, JZ8 Hz); 6.8–7.4 (m, 11H);
8.50 (s, 1H). 13C NMR (CDCl3 75 MHz) d 175.58; 172.49;
169.68; 159.76; 156.13; 140.60; 135.78; 128.58; 128.32;
128.15; 127.29; 67.50; 64.61; 52.24; 51.41; 47.26; 35.87;
31.93; 29.37; 24.93; 22.68. Anal. Calcd for C26H30N2O7: C,
64.72; H, 6.27; N, 5.81. Found: C, 64.77; H, 6.20; N, 5.74.
4.5.2. H-(S)-Asp-(1S,2R)-c6Phe-OMe, (S,1S,2R)-8. This
compound was prepared according to the procedure
described above. Cbz-(S)-Asp-(S,1S,2R)-c6Phe-OMe,
(S,1S,2R)-7, (47 mg, 0.1 mmol); EtOAc/MeOH (4 mL);
10% Pd/C (5 mg). Yield: 84%. Mp: 165 8C. Rf2Z0.51.
[a]2D0ZC36.94 (cZ0.71, MeOH). IR (nujol): 2400–3600;
1
1730.81; 1684.52 cmK1. H NMR (D2O 300 MHz) d 1.4–
1.6 (m, 1H); 1.6–1.8 (m, 2H); 1.8–2.0 (m, 3H); 2.15 (m,
1H); 2.45 (m, 1H); 2.53 (dd, 1H, JZ6 Hz, JZ13.2 Hz);
2.71 (dd, 1H, JZ4.2 Hz, JZ12.9 Hz); 3.03 (dd, 1H, JZ
3 Hz, JZ6.9 Hz); 3.53 (s, 3H); 4.22 (t, 1H, JZ4.2 Hz);
7.2–7.4 (m, 5H). 13C NMR (D2O/MeOH-d4 75 MHz) d
175.79; 173.61; 167.86; 140.98; 129.13; 128.18; 127.25;
63.28; 52.06; 50.44; 49.52; 37.14; 32.35; 27.78; 23.38;
21.35. Anal. Calcd for C18H24N2O5: C, 62.05; H, 6.94; N,
8.04. Found: C, 62.25; H, 6.91; N, 8.09.
4.4.2. CBz-(S)-Asp-(1S,2R)-c6Phe-OMe, (S,1S,2R)-7.
This compound was prepared according to the procedure
described above. Cbz-(S)-Asp(OtBu)-(1S,2R)-c6Phe-OMe,
(S,1S,2R)-6, (65 mg, 0.12 mmol); CH2Cl2 (2 mL); TFA
(1 mL). Yield: quantitative. Mp: oil. Rf1Z0.04, Rf2Z0.79.
[a]2D0ZC16.63 (cZ0.19, CHCl3). IR (nujol): 2500–3600;
Acknowledgements
This work was supported by FEDER, Ministerio de Ciencia
´
y Tecnologıa (PPQ2001-1834) and Productos Aditivos.
1723.26; 1686.86; 1671.37; 1660.74 cmK1 1H NMR
.
(CDCl3 300 MHz) d 1.1–2.0 (m, 5H); 2.05 (m, 1H); 2.15
(m, 1H); 2.30 (m, 1H); 2.63 (dd, 1H, JZ4.2 Hz, JZ
12.9 Hz); 2.84 (dd, 1H, JZ3 Hz, JZ12.6 Hz); 3.47 (m,
1H); 3.56 (s, 3H); 4.46 (m, 1H); 4.98 (m, 2H); 5.93 (d, 1H,
JZ6.6 Hz); 6.87 (s, 1H); 6.9–7.4 (m, 11H). 13C NMR
(CDCl3 75 MHz) d 175.74; 172.47; 169.42; 156.17; 140.72;
135.98; 128.54; 128.30; 128.24; 128.15; 128.02; 127.19;
67.32; 64.48; 52.11; 51.54; 46.95; 35.44; 31.92; 28.30;
24.87; 22.45. Anal. Calcd for C26H30N2O7: C, 64.72; H,
6.27; N, 5.81. Found: C, 64.79; H, 6.30; N, 5.77.
References and notes
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4.5.1. H-(S)-Asp-(1R,2S)-c6Phe-OMe, (S,1R,2S)-8. This
compound was prepared according to the procedure
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(S,1R,2S)-7, (78 mg, 0.16 mmol); EtOAc/MeOH (6 mL);
10% Pd/C (15 mg). Yield: 97%. Mp: 144 8C. Rf2Z0.51.
[a]2D0ZK14.76 (cZ0.49, MeOH). IR (nujol): 2400–3600;
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1
1720.20; 1690.31 cmK1. H NMR (D2O 300 MHz) d 1.5–
´
´
´
1.7 (m, 2H); 1.8–2.2 (m, 5H); 2.37 (m, 1H); 2.90 (m, 2H);
3.19 (dd, 1H, JZ3 Hz, JZ6 Hz); 3.58 (s, 3H); 4.31 (t, 1H,
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´