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

Article abstract of DOI:10.1016/S0040-4020(02)00435-0

This report describes the synthesis and the conformational analysis of the optically pure dipeptides analogues of aspartame: H-(S)-Asp-(1R,2R)-c₆Phe-OMe and H-(S)-Asp-(1S,2S)-c₆Phe-OMe, in which the Phe residue of aspartame has been replaced by a restricted Phe with a cyclohexane skeleton: 1-amino-2-phenylcyclohexanecarboxylic acid (c₆Phe). Of these, only the dipeptide that incorporates (1R,2R)-c₆Phe is sweet, whereas that incorporates (1S,2S)-c₆Phe is bitter. This relationship between the absolute configuration of the dipeptides and the properties is explained through the different conformational behaviour displayed by each molecule, based on molecular mechanics and molecular dynamics calculations, including solvent effects.

Full text of DOI:10.1016/S0040-4020(02)00435-0

TETRAHEDRON
Pergamon
Tetrahedron 58 .2002) 4899±4905
Aspartame analogues containing
1-amino-2-phenylcyclohexanecarboxylic acids ꢀc6Phe)
a
Alberto Avenoza, Miguel Parõs, Jesus M. Peregrina, Myriam Alõas, Marõa P. Lopez,
a
a
b
b
Â
Jose I. Garcõa and Carlos Cativiela
Â
Â
Â
Â
b
b,p
Â
Â
a
  Â
Departamento de Quõmica, Universidad de La Rioja, Grupo de Sõntesis Quõmica de La Rioja, U.A.-C.S.I.C., 26006 LogronÄo, Spain
b
  Â
Departamento de Quõmica Organica, Facultad de Ciencias, Instituto de Ciencia de Materiales de Aragon,
Universidad de Zaragoza-C.S.I.C., 50009 Zaragoza, Spain
Received 31 August 2001; revised 22 March 2002; accepted 18 April 2002
AbstractÐThis report describes the synthesis and the conformational analysis of the optically pure dipeptides analogues of aspartame:
H-.S)-Asp-.1R,2R)-c6Phe-OMe and H-.S)-Asp-.1S,2S)-c6Phe-OMe, in which the Phe residue of aspartame has been replaced by a restricted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). Of these, only the dipeptide that incorporates
.1R,2R)-c6Phe is sweet, whereas that incorporates .1S,2S)-c6Phe is bitter. This relationship between the absolute con®guration of the
dipeptides and the properties is explained through the different conformational behaviour displayed by each molecule, based on molecular
mechanics and molecular dynamics calculations, including solvent effects. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
tion of those aspartame-like sweeteners. Thus, in order to
develop a three-dimensional receptor model, several confor-
mationally restricted analogues of aspartame have been
synthesised and studied. Since it has been shown that
many changes are well tolerated in the phenylalanine
.Phe) of the dipeptide4±6 and taking into consideration that
one approach involves the use of constrained amino acids,7,8
the Phe of aspartame have been replaced by different
conformationally restricted amino acids and, in particular,
different a-methyl a-amino acids have been used.9±11 More
restriction was observed when the Phe was substituted by
cyclopropane amino acids: 1-aminocyclopropanecarboxylic
acid .Acc)12,13 and 1-amino-2-phenylcyclopropanecar-
boxylic acids .c3Phe)14 but, of these, only H-Asp-Acc-OR
are sweet whereas all four isomers of H-Asp-c3Phe-OMe are
tasteless.
Aspartame .H-.S)-Asp-.S)-Phe-OMe), a dipeptide approxi-
mately 150±200 times sweeter than sucrose,1 was dis-
covered by accident in 1965, when James Schlatter, a
chemist of G. D. Searle Company was testing an anti-
ulcer drug. Due to the explosion of no calorie or low calorie
foods in the marketplace and taking into account that
aspartame is made up primarily of two amino acids, it was
initially regarded as the perfect arti®cial sweetener.
However, since its initial approval by Food and Drug
Administration .FDA) in 1981 for use in United States as
food additive, some questions have arisen about its safety.
Because of this, aspartame may be the most systematically
studied food additive.
In recent years, several efforts have been made in order to
elucidate the stereochemical basis of sweet taste and two
models have emerged as the most appropriates: Temussi2
and Goodman.3 The core of the difference between both
models is the conformational ¯exibility of peptide tastants
like aspartame or its dipeptide analogues and consequently
the dif®culty to elucidate which is the bioactive conforma-
However, to the best of our knowledge, Phe of aspartame
has never been replaced by cyclohexane-a-amino acids, in
which the torsional angle x1 is restricted by tethering Ca to
Cb.15 In this ®eld, we have recently reported the synthesis of
1-amino-2-phenylcyclohexanecarboxylic acids .c6Phe)
using
a
Diels±Alder reaction16 as conformationally
constrained Phe analogues and their incorporation into
some dipeptides to modulate the b-folding mode.17 More-
over, the in¯uence of side chain restriction and NH¼p
interaction on the b-turn folding modes of dipeptides
incorporating c6Phe derivatives have been studied.18
Keywords: Diels±Alder reactions; cyclohexanes; resolution; chromato-
graphy; peptide analogues/mimetics; conformation; computer-assisted
methods.
Corresponding author. Tel./fax: 134-976-761-210;
e-mail: cativiela@posta.unizar.es.
p
Abbreviations: Cbz, carbobenzyloxycarbonyl; Et2O, diethyl ether; EtOAc,
ethyl acetate; EtOH, ethanol; MeOH, methanol; NMM, N-methylmorpho-
line; OtBu, tert-butoxy; OMe, methoxy; OR, alkoxy; TFA, tri¯uoroacetic
acid; Bz, benzoyl.
Therefore, we have considered the replacement of Phe by
c6Phe in the aspartame dipeptide. Thus, in this paper we
report the synthesis and the conformational behaviour of
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020.02)00435-0

4900
A. Avenoza et al. / Tetrahedron 58 52002) 4899±4905
CH2Cl2 at 2158C.21,22 In this way, we synthesised the
diastereoisomers .S,1R,2R)-6 and .S,1S,2S)-6 with an over-
all yield of 93% from amino esters .1R,2R)-4 and .1S,2S)-4,
respectively .Scheme 2). Both optically pure compounds
were characterised by their spectral data.
An alternative way to obtain the protected dipeptides
.S,1R,2R)-6 and .S,1S,2S)-6 as optically pure compounds
involves the same synthetic procedure but now starting
from H-rac-cis-c6Phe-OMe .rac-cis-4) and further chroma-
tographic separation of the corresponding diastereoisomeric
mixture19 .Scheme 2). The synthesis of amino ester rac-cis-
4 was achieved from rac-cis-2, in good yield, by formation
of the methyl ester with thionyl chloride/methanol .Scheme
1). Coupling of rac-cis-4 with .S)-5 provided the mixture of
diastereoisomers in excellent yield. Expected separation of
two stereoisomers could not be achieved by ¯ash chromato-
graphy under any conditions tested. This dif®culty prompted
us to explore the use of semipreparative HPLC for the
resolution of .S,1R,2R)-6 and .S,1S,2S)-6.
Scheme 1. Reagents and conditions: .a) Ref. 16. .b) and .c) Ref. 20.
.d) SOCl2, MeOH.
two stereoisomers of H-.S)-Asp-c6Phe-OMe as conforma-
tionally resctricted aspartame analogues.
2. Results and discussion
2.1. Peptide synthesis
Resolution was ®rstly examined at the analytical scale in
normal phase using silica as stationary phase. Elution and
detection conditions of the best-performed separations are
given in Table 1. Thus, the same amylose-based chiral
column previously used in the separation of rac-cis-3,
proved also to be very ef®cient in other semipreparative
resolutions of phenylalanine surrogates,23,24 was examined.
The mixture of n-hexane/2-propanol/chloroform indicated
in Table 1 was therefore selected as an eluent in an attempt
to optimise the resolution in relation with the column load-
ability.
We have developed two synthesis of the aspartame
analogues. Firstly, our starting material was Bz-rac-cis-
c6Phe-OMe, rac-cis-3, which was previously obtained16
from 1, a precursor of rac-cis-c6Phe´HCl, rac-cis-2 .Scheme
1). Thus, in order to obtain the optically pure amino esters
cis-4, the separation of the enantiomerically pure Bz-
.1R,2R)-c6Phe-OMe and Bz-.1S,2S)-c6Phe-OMe, .1R,2R)-
3 and .1S,2S)-3, from Bz-rac-cis-c6Phe-OMe .rac-cis-3)
was achieved by chiral HPLC using a covalently bonded
amylose-derived CSP on a semipreparative column.19,20
Once enantiomerically pure precursors were available, two
easy steps, hydrolysis followed by formation of the methyl
ester with thionyl chloride/methanol, led respectively to
pure .1R,2R)-4 and .1S,2S)-4 in high yields .Scheme 2).
The extension of the analytical conditions to the semi-
preparative scale proved to be very ef®cient .a and Rs
values are given in Table 1). The sample was dissolved in
chloroform and the separation was achieved by successive
injections using the peak shaving technique. Thus, 2.1 g of
diastereomeric mixture afforded 1.6 g of optically pure
material .ca. 0.8 g of each diastereoisomer) in a single
passage through a 150 mm£20 mm ID column and with a
Starting from .1R,2R)-4 and .1S,2S)-4, we assayed different
methods to couple these amino esters with the conveniently
protected aspartic acid Cbz-.S)-Asp.OtBu)-OH, .S)-5. Of all
coupling conditions tried the best results were obtained
i
when we used BuOCOCl in the presence of NMM in
Scheme 2. Reagents and conditions: .a) Chiral HPLC. .b) .i) 6N HCl; .ii) SOCl2, MeOH. .c) iBuOCOCl, NMM, CH2Cl2. .d) .i) iBuOCOCl, NMM, CH2Cl2;
.ii) HPLC. .e) .i) TFA/CH2Cl2; .ii) H2, Pd/C.

A. Avenoza et al. / Tetrahedron 58 52002) 4899±4905
4901
Table 1. Selected chromatographic data for the HPLC resolution of .S,1R,2R)-6 and .S,1S,2S)-6 on several stationary phases and chromatographic modes
Eluenta A/B/C
Flow .mL/min)
l .nm)
a
Rs
0
k1
Chromatographic mode
Column
Normal phase
Normal phase
Chiral phased
Chiral phased
Chiral phased
Nova-Pakb
98/2/0
98/2/0
98/2/0
96/2/2
96/2/2
0.7
1
1
1
18
210
210
210
225
265
1.15
2.19
2.35
2.81
1.79
1.19
1.18
1.71
1.68
1.55
1.96
2.12
3.09
1.73
0.94
Spherisorbc
Analyticale
Analyticale
Semipreparativef
For the de®nition of k0, a and Rs see Section 4.
A: n-hexane, B: 2-propanol, C: chloroform.
a
b
Radial-pak cartridge, 4 mm Nova-Pakw, 100 mm£8 mm ID .analytical scale).
c
d
e
f
Steel column, 5 mm Spherisorbw, 250 mm£4.6 mm ID .analytical scale).
Mixed 10-undecenoate/3,5-dimethylphenylcarbamate of amylose bonded to 5 mm allylsilica.
Steel column, 150 mm£4.6 mm ID.
Steel column, 150 mm£20 mm ID.
total time of 4 h to complete the process. The optical purity
of the compounds was checked on the analytical column.
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
compound .S,1R,2R)-8 is sweet, although less potent than
aspartame, with a metallic aftertaste; whereas compound .S,
1S,2S)-8 is bitter.
Direct assignment of their absolute con®guration could not
be achieved because none of the diastereoisomers 6 gave
single crystals suitable for X-ray diffraction analysis. Thus,
in order to assign the stereochemistry of both dipeptides, we
compared their spectral data with those corresponding to the
dipeptides previously synthesised from each enantiomer
.1R,2R)-4 and .1S,2S)-4.
Deprotection of each diastereoisomer was carried out in two
steps: ®rstly TFA in CH2Cl2 afforded .S,1R,2R)-7 and
.S,1S,2S)-7, further hydrogenolysis using palladium on
carbon as a catalyst led to the optically pure analogues of
aspartame H-.S)-Asp-.1R,2R)-c6Phe-OMe and H-.S)-Asp-
.1S,2S)-c6Phe-OMe, .S,1R,2R)-8 and .S,1S,2S)-8.
2.3. Molecular modelling studies
Molecular modelling studies carried out for aspartame and
some of its analogues have led to a widely accepted model
which relates the topochemical arrangement of the different
groups of the dipeptide with the taste.3 Following this
Figure 1. Calculated minimum energy conformations of .S,1R,2R)-8 and .S,1S,2S)-8 .hydrogen atoms have been omitted for clarity).

4902
A. Avenoza et al. / Tetrahedron 58 52002) 4899±4905
Table 2. Calculated energies .kcal/mol) and some selected dihedral angles for the most stable conformations of .S,1R,2R)-8 and .S,1S,2S)-8
Compound
Conformer
Dihedral anglesa
EMM2
DGsolv
ETot
DETot
C
F
x1
.S,1R,2R)-8
.S,1S,2S)-8
1x
2z
1x
2z
2156.3
240.9
119.2
272.6
245.8
194.4
293.5
275.2
235.7
254.7
159.4
159.7
256.4
258.9
258.5
263.1
279.8
273.3
281.1
277.3
2136.2
2132.2
2139.6
2140.4
0.0
4.0
0.8
0.0
a
See Fig. 1 for the de®nition of the dihedral angles.
model, the sweet taste is associated to a L-shaped con-
formation, in which the hydrophobic group is pointing to
the arbitrarily chosen 1x coordinate axis .Fig. 1). On
the other hand, the bitter taste is associated to the con-
formation in which the same group points in the 2z
direction, other possible arrangements leading to tasteless
compounds.
3. Conclusion
Two new aspartame derivatives incorporating the
constrained phenylalanine, c6Phe, have been prepared
showing different properties depending on the stereo-
chemistry of the modi®ed phenylalanine residue. The
relations between the absolute con®guration of the dipep-
tides and the properties are explained through the different
conformational behaviour displayed by each molecule. The
use of new constrained phenylalanines is in progress and the
obtained results will be published in due course.
In general, most of molecular modelling studies on this
kinds of systems obviate solvation effects, that however,
can be important for relatively high polar molecules as the
dipeptides considered. In order to account for solvation
effects, at least in an approximate manner, we envisaged a
mixed scheme, combining molecular mechanics and
quantum mechanics calculations. Thus, single point energy
semiempirical AM1 calculations on the MM2 optimised
structures .see Experimental 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 then this energy was added to the MM2 steric
energy to obtain the relative energies of the different con-
formers. Although this procedure does not allow to take into
account solvent effects during the geometry optimisation, it
has the advantage of conjugating the strength of MM2 force
®eld for conformational analysis with that of a successful
quantum mechanical method for calculating the solvation
energy.
4. Experimental
4.1. General
4.1.1. Instrumentation. Solvents were puri®ed 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
¯uorescence or developed by iodine or by ninhydrin chro-
matic reaction as appropriate. Column chromatography was
performed using silica gel 60 .230±400 mesh). Melting
points were determined on a BuÈchi SMP-20 apparatus and
were not corrected. IRspectra were registered on a Mattson
Genesis FTIRspectrophotometer; nmax is given for the main
absorption bands. H and 13C NMRspectra were recorded
1
Table 2 gathers the minimum energy values obtained for
conformations 1x and 2z of compounds .S,1R,2R)-8 and
.S,1S,2S)-8. The corresponding structures are shown in
Fig. 1.
on a Varian Unity-300 or a Bruker ARX-300 instruments.
Spectra were recorded in CDCl3 or DMSO-d6 with 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 polarimeter-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 were in
good agreement with the calculated values. Mass spectra
were obtained in the 1FAB mode on a high resolution
VG-autospectrometer.
As can be seen, based on total energies, compound
.S,1R,2R)-8 should be sweet .since conformer 1x is more
stable than conformer 2z), whereas compound .S,1S,2S)-8
should be bitter .since the reverse relative stability is
obtained). It can be then concluded that molecular
mechanics calculations are in good agreement with the
taste experimentally found for both analogues, which
supports the validity of the model assumed for the
structure±taste relationship.
4.1.2. HPLC. HPLC was carried out using a Waters HPLC
system equipped with a Waters 600-E pump and a Waters
991 photodiode array detector. Radial-Pak cartridge 4 mm
Nova-Pakw 100 mm£8 mm ID, steel column 5 mm
Spherisorbw Silica 250 mm£4.6 mm ID and steel column
5 mm XterraTM RP18 4.6 mm£150 mm ID were purchased
from Waters Chromatogra®a S.A. The chiral stationary
phase consisting of a mixed 10-undecenoate/3,5-dimethyl-
phenylcarbamate of amylose bonded to allylsilica was
prepared according to a previously described procedure25,26
by means of a radical reaction between the alkenyl groups in
From the methodological point of view, it is worth noting
that inclusion of solvation energy is fundamental to
arriving to this result. Thus, in both cases, the gas phase
MM2 energies indicate the 2z conformation as the
most stable; however, the 1x conformation is
differentially more solvated than the 2z conformation,
giving rise to a reversal of stability in solution in the case
of .S,1R,2R)-8.
Ê
the polysaccharide and the modi®ed silica .100 A, 5 mm).

A. Avenoza et al. / Tetrahedron 58 52002) 4899±4905
4903
The chiral stationary phase obtained in this way was packed
into stainless tubes by the slurry method. The analytical
assays were carried out on a 150 mm£4.6 mm ID column
and the semipreparative 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. The solvents used as mobile
phases were chromoscan grade from LabScan.
iBuOCOCl .1.03 g, 7.6 mmol) in dry CH2Cl2 .25mL) was
added. After stirring for 20 min, a precooled solution in
CH2Cl2 .25 mL) of H-rac-cis-c6Phe-OMe, rac-cis-4
.1.73 g, 7.4 mmol), which was used as obtained in Section
4.2 without any further puri®cation, was added dropwise to
the above solution. The reaction was stirred at 2158C for
1 h, then the solution was allowed to warm to room tempera-
ture. The solution was diluted with CH2Cl2 and washed with
5% KHSO4, 5% NaHCO3, brine, dried and evaporated to
dryness. The product was puri®ed by silica gel column chro-
matography using hexane±ethyl acetate .5:2) as eluent
yielding the protected dipeptides in high yield ..90%).
The capacity .k0), selectivity .a) and resolution .Rs) values
were calculated according to the equations k0Rˆ.tR2t0)/t0,
0
aˆk02/k1 , Rsˆ1.18.t22t1)/.w21w1). Subscripts 1 and 2
refer to the ®rst and second eluted diastereoisomer, respec-
tively; tR .Rˆ1,2) are their retention times, and w2 and w1
denote their bandwidths at half-heigth; t0 is the dead time.
Diastereomeric mixture of protected dipeptides .S,1R,2R)-6
and .S,1S,2S)-6 puri®ed by silica gel column chromato-
graphy was resolved by semipreparative HPLC as described
below.
4.1.3. Molecular modelling methodology. All computer
simulations were carried out using the Chem3D program.27
Molecular Mechanics and Molecular Dynamics were
achieved by means of the MM2 force ®eld,28 as implemen-
ted in Chem3D. Molecular Dynamics trajectories were
collected in periods of 1 ns, using a step interval of 1 fs,
and a target temperature of 373 K. It is well-known that
the dihedral angles of the Asp residue remains essentially
unchanged for all minimum energy conformations of aspar-
tame, and that this disposition is very similar to the X-ray
crystal structure.3 Consequently, the geometry of this
residue was kept ®xed in the disposition of the X-ray crystal
structure of aspartame throughout all the simulations.
4.3.2. Procedure B. The reported general synthetic way was
applied separately to the optically pure enantiomers
H-.1R,2R)-c6Phe-OMe, .1R,2R)-4, and H-.1S,2S)-c6Phe-
OMe, .1S,2S)-4. Each protected dipeptide 6 was puri®ed
by silica gel column chromatography using hexane±ethyl
acetate .5:2) as eluent. The optically pure diastereoisomers
.S,1R,2R)-6 and .S,1S,2S)-6 are then obtained separately in
high yield ..90%).
4.3.3. Cbz-ꢀS)-AspꢀOtBu)-ꢀ1R,2R)-c6Phe-OMe, ꢀS,1R,2R)-
20
6. Mp 508C. Rf1ˆ0.35. [a]D ˆ214.9 .c 0.4, CHCl3). IR
.nujol): 3354, 1724, 1708, 1689, 1679 cm21 1H NMR
.
.CDCl3): d 1.10±1.54 .m, 2H), 1.41 .s, 9H), 1.55±2.08
.m, 5H), 2.59 .dd, 1H, Jˆ17.7, 7.8 Hz), 2.72±2.96 .m,
2H), 3.06 .dd, 1H, Jˆ13.2, 3.4 Hz), 3.40 .s, 3H), 4.45±
4.60 .m, 1H), 5.08 .d, 1H, Jˆ12.0 Hz), 5.15 .d, 1H,
Jˆ12.0 Hz), 5.81 .d, 1H, Jˆ6.6 Hz), 6.79 .s, 1H), 7.00±
Solvation effects were taken into account by means of single
point energy semiempirical AM129 calculations on the
MM2 optimised structures, using the COSMO continuum
model,30 with the dielectric constant of water.
7.10 .m, 2H), 7.20±7.40 .m, 8H). 13C NMR.CDCl ): d
4.2. Synthesis of rac-cis-c6Phe-OMe ꢀrac-cis-4)
3
20.7, 25.7, 26.4, 28.0, 30.6, 37.7, 49.7, 51.1, 51.8, 64.1,
67.1, 81.8, 127.5, 127.6, 128.2, 128.3, 128.6, 128.6, 136.1,
139.9, 155.7, 170.0, 171.3, 172.9. MS .m/z, %): 539
[.M11)1, 81]. Anal. Calcd for C30H38N2O7: C, 66.89; H,
7.11; N, 5.20. Found: C, 66.97; H, 7.06; N, 5.14.
The preparation of 1-amino-2-phenylcyclohexane-1-
carboxylic acid hydrochloride, rac-cis-2, from the cyclo-
adduct obtained in the Diels±Alder reaction of 1,3-buta-
diene and .Z)-2-phenyl-4-benzylidene-5.4H)-oxazolone
has already been reported.16,20 In order to obtain the corre-
sponding methyl ester, rac-cis-4, the following procedure
was carried out.
4.3.4. Cbz-ꢀS)-AspꢀOtBu)-ꢀ1S,2S)-c6Phe-OMe, ꢀS,1S,2S)-
20
6. Mp 598C. Rf1ˆ0.35. [a]D ˆ127.8 .c 0.5, CHCl3). IR
.nujol): 3372, 1721, 1703, 1688, 1676 cm21 1H NMR
.
.CDCl3): d 1.25±1.50 .m, 2H), 1.37 .s, 9H), 1.60±2.05
.m, 5H), 2.50 .dd, 1H, Jˆ17.1, 5.4 Hz), 2.84 .dd, 1H,
Jˆ17.1, 5.4 Hz), 2.94 .m, 1H), 3.08 .m, 1H), 3.42 .s, 3H),
4.42 .m, 1H), 5.13 .d, 1H, Jˆ11.7 Hz), 5.20 .d, 1H,
Jˆ11.7 Hz), 6.14 .d, 1H, Jˆ8.7 Hz), 6.68 .s, 1H), 7.05±
7.15 .m, 2H), 7.15±7.25 .m, 3H), 7.25±7.45 .m, 5H). 13C
SOCl2 .0.9 mL, 11.8 mmol) was added dropwise to a stirred
solution of MeOH .35 mL) cooled in an ice-bath. After
15 min of stirring at that temperature, rac-cis-2 .2.13 g,
7.9 mmol) was added to the reaction mixture and re¯ux
for 8 h. After the addition of 3 further portions of SOCl2/
MeOH, prepared as above mentioned, the solvents were
eliminated 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 88%).
NMR.CDCl ): d 20.5, 25.7, 26.6, 27.9, 30.9, 36.4, 49.7,
3
51.7, 51.8, 64.2, 67.2, 81.6, 127.5, 127.7, 127.9, 128.1,
128.3, 128.6, 136.1, 139.9, 156.1, 170.4, 171.4, 173.1. MS
.m/z, %): 1099 [.2M1Na)1, 11], 561 [.M1Na)1, 100], 539
[.M11)1, 32]. Anal. Calcd for C30H38N2O7: C, 66.89; H,
7.11; N, 5.20. Found: C, 67.08; H, 7.03; N, 5.24.
4.3. General procedure for the synthesis of protected
dipeptides ꢀS,1R,2R)-6, ꢀS,1S,2S)-6
4.4. Resolution of two stereoisomers of Cbz-ꢀS)-
AspꢀOtBu)-cis-c6Phe-OMe: isolation of ꢀS,1R,2R)-6,
ꢀS,1S,2S)-6
4.3.1. Procedure A. Cbz-.S)-Asp.OtBu)-OH, .S)-5, .2.46 g,
7.6 mmol) and NMM .768 mg, 7.6 mmol) were dissolved in
dry CH2Cl2 .100 mL) under an inert atmosphere. The
mixture was cooled at 2158C and a precooled solution of
HPLC resolution of a mixture of .S,1R,2R)-6 and .S,1S,2S)-

4904
A. Avenoza et al. / Tetrahedron 58 52002) 4899±4905
6 .2.1 g) dissolved in chloroform .4.2 mL) was carried out
by successive injections of 0.2 mL on a 150 mm£20 mm
column ®lled with mixed 10-undecenoate/3,5-dimethyl-
phenylcarbamate of amylose bonded to allylsilica using a
mixture of n-hexane/2-propanol/chloroform 96/2/2 as the
eluent .¯ow rate 18 mL/min). Each run was collected into
three separated fractions. The combined ®rst fractions,
collected between 4.3 and 5.7 min, contained 804 mg of
optically pure ®rst eluted diastereoisomer .S,1R,2R)-6.
The combined last fractions .collected between 7.1 and
10.5 min) contained 797 mg of optically pure more strongly
retained diastereoisomer .S,1S,2S)-6.
4.6. General procedure for the synthesis of ꢀS,1R,2R)-8,
ꢀS,1S,2S)-8
The corresponding semi-protected dipeptide 7 .1 mmol)
was dissolved in 30 mL EtOAc/MeOH .1:1) and hydro-
genated at room temperature in the presence of 10%
palladium/carbon .45 mg) for 12 h. The solution was
®ltered, evaporated under reduced pressure and further
lyophilised to afford a white solid. Both deprotected
dipeptides were shown to be optically pure by reversed-
phase HPLC .column: 5 mm Xterrae RP18, 4.6 mm£
150 mm ID). The elutions were performed isocratically
with 20% CH3CN/80% 0.0125 M NaH2PO4 .pH 2.5) .v/v)
at a ¯ow rate of 1 mL/min, with UV detection at 210 nm.
4.5. General procedure for the synthesis of ꢀS,1R,2R)-7,
ꢀS,1S,2S)-7
4.6.1. H-ꢀS)-Asp-ꢀ1R,2R)-c6Phe-OMe, ꢀS,1R,2R)-8. This
compound was prepared according to the procedure
described
above:
Cbz-.S)-Asp-.1R,2R)-c6Phe-OMe,
The corresponding protected dipeptide 6 .1 mmol) was
dissolved in CH2Cl2 .15 mL). Then, TFA .7.5 mL) was
added. The solution was stirred at room temperature for
1.5 h. TFA and CH2Cl2 were evaporated under reduced
pressure to afford the ®nal product.
.S,1R,2R)-7 .433 mg, 0.9 mmol); 30 mL EtOAc/MeOH;
10% Pd/C .45 mg). Yield: 95.5%, mp 140±1458C.
20
[a]D ˆ24.9 .c 0.5, MeOH). IR.nujol): 3600±2400, 1729,
1689, 1679 cm21. H NMR.DMSO- d6): d 1.09±1.80 .m,
1
5H), 1.81±2.00 .m, 1H), 2.10±2.30 .m, 1H), 2.50 .m, 1H),
2.62 .dd, 1H, Jˆ17.0, 6.6 Hz), 2.71 .dd, 1H, Jˆ17.0, 6.6 Hz),
2.99 .dd, 1H, Jˆ12.9, 2.7 Hz), 3.29 .s, 3H), 4.17 .t, 1H,
Jˆ6.6 Hz), 7.10±7.40 .m, 5H), 8.09 .br s, 1H). 13C NMR
.DMSO-d6): d 20.3, 25.3, 26.5, 30.5, 37.8, 49.6, 49.9, 51.2,
63.3, 126.9, 128.0, 128.1, 140.7, 168.8, 171.8, 172.1. MS .m/z,
%): 393 [.M2H12Na)1, 100], 371 [.M1Na)1, 89], 349
[.M1H), 23]. Anal. Calcd for C18H24N2O5: C, 62.05; H,
6.94; N, 8.04. Found: C, 62.21; H, 6.97; N, 7.99.
4.5.1. Cbz-ꢀS)-Asp-ꢀ1R,2R)-c6Phe-OMe, ꢀS,1R,2R)-7.
This compound was prepared according to the procedure
described above: Cbz-.S)-Asp.OtBu)-.1R,2R)-c6Phe-OMe,
.S,1R,2R)-6 .646 mg, 1.2 mmol); CH2Cl2 .20 mL); TFA
.10 mL). Yield: quantitative, mp 618C. Rf1ˆ0.07,
20
Rf2ˆ0.77. [a]D ˆ231.6 .c 0.38, CHCl3). IR.nujol):
2300±3600, 1723, 1708, 1688, 1675 cm21 1H NMR
.
.CDCl3): d 1.10±1.58 .m, 2H), 1.58±1.74 .m, 2H), 1.74±
1.90 .m, 2H), 1.98 .ddd, 1H, Jˆ13.5, 13.2, 2.7 Hz), 2.69
.dd, 1H, Jˆ17.1, 6.3 Hz), 2.82±3.02 .m, 2H), 3.08 .dd, 1H,
Jˆ12.0, 3.0 Hz), 3.42 .s, 3H), 4.10 .br s, 1H), 4.59 .m, 1H),
5.09 .d, 1H, Jˆ11.7 Hz), 5.17 .d, 1H, Jˆ11.7 Hz), 5.71 .d,
1H, Jˆ8.7 Hz), 6.56 .s, 1H), 6.95±7.05 .m, 2H), 7.15±7.25
4.6.2. H-ꢀS)-Asp-ꢀ1S,2S)-c6Phe-OMe, ꢀS,1S,2S)-8. This
compound was prepared according to the procedure
described
above:
Cbz-.S)-Asp-.1S,2S)-c6Phe-OMe,
.S,1S,2S)-7 .433 mg, 0.9 mmol); 30 mL EtOAc/MeOH;
10% Pd/C .45 mg). Yield: 98%, mp 145±1508C.
.m, 3H), 7.25±7.40 .m, 5H). 13C NMR.CDCl ): d 20.6,
3
20
[a]D ˆ165.9 .c 0.5, MeOH). IR.nujol): 3600±2400,
25.6, 26.4, 30.7, 35.4, 49.4, 51.3, 52.0, 64.6, 67.6, 127.5,
127.7, 128.4, 128.5, 128.6, 128.7, 135.7, 139.5, 156.2,
170.8, 172.7, 175.4. MS .m/z, %): 506 [.M1H1Na)1,
77], 483 [.M1H)1, 52], 423 [.M2COOCH3)1, 20]. Anal.
Calcd for C26H30N2O7: C, 64.72. H, 6.27; N, 5.81; Found: C,
64.81; H, 6.18; N, 5.86.
1
1728, 1687 cm21. H NMR.DMSO- d6): d 1.25±1.80 .m,
5H), 1.81±2.00 .m, 1H), 2.17 .m, 1H), 2.22 .dd, 1H,
Jˆ16.2, 8.7 Hz), 2.34 .dd, 1H, Jˆ16.2, 4.8 Hz), 2.63 .ªdº,
1H, Jˆ12.9 Hz), 2.94 .dd, 1H, Jˆ12.9, 2.4 Hz), 3.26 .s,
3H), 3.70 .m, 1H), 7.10±7,30 .m, 5H), 8.30 .br s, 1H). 13C
NMR.DMSO- d6): d 20.1, 25.3, 26.5, 30.6, 32.9, 48.7, 49.9,
51.2, 62.9, 126.9, 127.9, 128.0, 129.2, 140.7, 171.5, 172.4,
172.5. MS .m/z, %): 697 [.2M1H)1, 8], 371 [.M1Na)1,
42], 349 [.M1H)1, 100]. Anal. Calcd for C18H24N2O5: C,
62.05; H, 6.94; N, 8.04; Found: C, 62.17; H, 6.98; N, 8.09.
4.5.2. Cbz-ꢀS)-Asp-ꢀ1S,2S)-c6Phe-OMe, ꢀS,1S,2S)-7. This
compound was prepared according to the procedure
described above: Cbz-.S)-Asp.OtBu)-.1S,2S)-c6Phe-OMe,
.S,1S,2S)-6 .646 mg, 1.2 mmol); CH2Cl2 .20 mL); TFA
.10 mL). Yield: quantitative, mp 1838C. Rf1ˆ0.07,
20
Rf2ˆ0.77. [a]D ˆ122.8 .c 0.5, CHCl3). IR.nujol): 3283,
1
1749, 1699, 1684, 1676 cm21. H NMR.CDCl ): d 1.18±
Acknowledgements
3
1.55 .m, 2H), 1.55±1.90 .m, 4H), 1.97 .ddd, 1H, Jˆ14.7,
13.2, 2.0 Hz), 2.64 .dd, 1H, Jˆ17.1, 5.4 Hz), 3.43 .s, 3H),
4.49 .m, 1H), 5.13 .d, 1H, Jˆ12.0 Hz), 5.22 .d, 1H,
Jˆ12.0 Hz), 6.01 .d, 1H, Jˆ8.7 Hz), 6.63 .s, 1H), 7.00±
This work has been supported by the Ministerio de Ciencia y
Â
Tecnologõa .project PPQ2001-1305), Universidad de La
Â
Â
Rioja .project API-01/B02), Diputacion General de Aragon
.project P22/98), PETRI .PTR95/0422-OP) and Productos
Aditivos S. A.
7.20 .m, 2H), 7.21±7.42 .m, 8H). 13C NMR.CDCl ): d
3
20.6, 25.6, 26.5, 29.7, 30.8, 35.0, 49.6, 51.3, 52.0, 64.4,
67.5, 127.6, 128.2, 128.5, 128.7, 139.7, 156.3, 170.5,
172.8, 175.2. MS .m/z, %): 505 [.M1Na)1, 100], 483
[.M1H)1, 93], 451 [.M1H±MeOH)1, 30], 437
[.M2COOH)1, 46], 423 [.M2COOCH3)1, 46]. Anal.
Calcd for C26H30N2O7: C, 64.72; H, 6.27; N, 5.81. Found:
C, 64.83; H, 6.31; N, 5.79.
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