Monatin, its stereoisomers and derivatives: Modeling the sweet taste chemoreception mechanism

Article abstract of DOI:10.1002/ejoc.200400916

The sweet natural compound monatin 1 has two stereogenic centers, and the 2S,4S absolute configuration has been attributed previously to the natural isomer. Among the four stereoisomers of monatin, three of them, particularly the 2R,4R isomer, tastes intensely sweet. The conformations of the four compounds have been studied by means of molecular modelling techniques. Both the diastereoisomeric forms show strong intramolecular hydrogen bonds which involve different functional groups and give rise to two different minimum energy conformations. The tertiary alcohol group in monatin seems to be indirectly involved in the generation of the taste, acting as an important contraint in generating the active conformation. The most important glucophores have been identified in the terminal -NH₃⁺ and -COO ^- groups and in the indole ring by comparison with known topological models of sweet compounds and through the synthesis of appropriate derivatives in which some of these groups are lacking or modified. The relative affinity of each stereoisomer for its putative sweet taste receptor has been estimated semi-quantitatively with the pseudoreceptor modelling technique. The predicted activity calculated with this technique is in good agreement with the experimental data and explains why the 2R,4R isomer (and not the natural 2S,4S isomer) is the sweetest of the series. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005.

Full text of DOI:10.1002/ejoc.200400916

FULL PAPER
Monatin, Its Stereoisomers and Derivatives: Modeling the Sweet Taste
Chemoreception Mechanism
Angela Bassoli,*[a] Gigliola Borgonovo,[a] Gilberto Busnelli,[a] Gabriella Morini,[a] and
Lucio Merlini[a]
Keywords: Sweet taste / Monatin / Molecular modeling / Sweet taste receptor / Pseudoreceptor modeling
The sweet natural compound monatin 1 has two stereogenic
centers, and the 2S,4S absolute configuration has been at-
tributed previously to the natural isomer. Among the four ste-
reoisomers of monatin, three of them, particularly the 2R,4R
isomer, tastes intensely sweet. The conformations of the four
been identified in the terminal –NH3+ and –COO– groups and
in the indole ring by comparison with known topological
models of sweet compounds and through the synthesis of ap-
propriate derivatives in which some of these groups are lack-
ing or modified. The relative affinity of each stereoisomer for
compounds have been studied by means of molecular model- its putative sweet taste receptor has been estimated semi-
ling techniques. Both the diastereoisomeric forms show
strong intramolecular hydrogen bonds which involve dif-
ferent functional groups and give rise to two different mini-
mum energy conformations. The tertiary alcohol group in
monatin seems to be indirectly involved in the generation of
the taste, acting as an important contraint in generating the
active conformation. The most important glucophores have
quantitatively with the pseudoreceptor modelling technique.
The predicted activity calculated with this technique is in
good agreement with the experimental data and explains
why the 2R,4R isomer (and not the natural 2S,4S isomer) is
the sweetest of the series.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
Introduction
Monatin 1 is a high-intensity sweet natural compound
isolated from the roots of Schlerochiton ilicifolius, a spiny-
leaved hardwood shrub growing in South Africa. Its struc-
ture [4-hydroxy-4-(indol-3-ylmethyl)glutamic acid] has been
elucidated by Ackerman and coworkers.[1] The same au-
thors also assigned the 2S,4S absolute configuration to the
isolated compound. In a previous work,[2] we have de-
scribed the synthesis of all the four stereoisomers of mona-
tin and the results of the tasting trials. In our experiments,
three of the four isomers tasted sweet, and unexpectedly,
the 2R,4R isomer (compound 2) was the sweetest of the
series (Figure 1).
A similar finding has been reported recently in a
patent:[3] all the four monatin isomers tested as sodium salts
elicit a sweet taste, and the 2R,4R isomer is the sweetest.
These results are of remarkable practical importance, since
obtaining a mixture of isomers by synthesis is easier than
the stereoselective preparation of single compounds. Inter-
estingly, we also found a mixture of isomers during the
analysis of an extraction sample of natural origin.[2] From
a theoretical point of view, the low stereoselectivity ob-
served in the binding of monatin isomers to the sweet taste
Figure 1. The four stereoisomers of monatin.
receptor is intriguing, since often the stereoselectivity of
taste chemoreception is very high. For instance, only the l,
l isomer of the dipeptide aspartame is sweet, the others are
tasteless or even bitter. It was particularly surprising that
the sweetest monatin isomer is not the 2S,4S isomer, which
was first isolated and identified as the natural sweet prin-
ciple, but its enantiomer. Therefore, we started to study the
structure–activity relationships for monatin and its stereo-
isomers in order to understand how each of them can bind
to the putative sweet taste receptor with different strengths.
In developing structure–taste relationships of monatin
and derivatives, we followed a three-step procedure: 1) se-
[a] Dipartimento di Scienze Molecolari Agroalimentari – DISMA,
Sezione di Chimica, Università di Milano,
Via Celoria 2, 20133 Milano, Italia
Fax: +39-02-50316801
E-mail: angela.bassoli@unimi.it
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DOI: 10.1002/ejoc.200400916
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Monatin, Its Stereoisomers and Derivatives
FULL PAPER
arch for minimum energy conformation(s); 2) identification terminal amino acid moiety and to the hydroxy acid at the
of important glucophores by use of topological models and C-4 position) and one hydrophobic area (corresponding to
through the synthesis of analogues; 3) modeling of pseudo- the indole ring). In contrast to the peptides, the principal
receptor.
skeleton of this molecule consists of a chain of C–C single
bonds that elongates in space with no particular constraints
beside the steric hindrance of the side substituents. Never-
theless, the presence of polar groups makes the formation
of strong intramolecular hydrogen bonds possible, which
characterize the calculated minimum energy conformations
for the two diastereoisomers, R*R* and S*R* monatin
(Figure 2).
Results and Discussion
The mechanism of action of sweet substances, as well as
that of other flavors, has been under investigation for many
years. From 2001, significant progress has been made in our
understanding of the mechanism of sweet taste chemorecep-
tion, and almost contemporaneously, several independent
groups of researchers[4–9] identified a new family of proteins
of the GPCRs family (T1Rs), which form functional di-
meric receptors that are able to bind several sweet com-
pounds. Since that time, several papers contributed to un-
ravel the biochemistry of the sweet taste receptor.[10,11] Very
recently, the different functional roles of T1Rs subunits in
the heteromeric sweet taste receptor has been proposed;[12]
however, only the primary structure of the proteins is
known, while the three-dimensional structure of the recep-
tor and the binding mode of the active ligands to the recep-
tor active site at a molecular level is not yet known.
In R*R* monatin, a strong hydrogen bond (1.99 Å) in-
+
volves the terminal –NH3 group as the hydrogen donor
and the oxygen atom of the C-4 hydroxyl group as the ac-
ceptor. This conformation is quite different from that ob-
tained by Nakamura and coworkers[23] with the MM3
method, where no intramolecular hydrogen bonds were de-
tected. The other diastereoisomer, R*,S* monatin, has a
different minimum energy conformation with three intra-
molecular hydrogen bonds: one between the H atom of the
hydroxyl group at C-4 and the carboxylate group at C-2,
one between the H atom of COOH at C-4 and the O atom
of the hydroxyl group at C-4; and another between the H
+
atom of NH3 at C-2 and the carboxylate group at C-2.
In the last century, several different topological models
were developed to describe the nature and the spatial ar-
rangement of the glucophores of an ideal sweet compound
and/or the recognition sites of the sweet taste receptor.[13–18]
These kinds of models are quite easy to use for qualita-
tive interpretation of the data, but they do not give any
quantitative information or any clue on the structure of the
receptor active site. In the past, our research group has
studied the (Q)SARs of many sweet tasting compounds by
using topological and statistic models,[19–21] and recently, we
developed a pseudoreceptor model[22] that is able to semi-
quantitatively predict the sweet taste of compounds belong-
ing to many different chemical classes.
Identification of Glucophores
The identification of the glucophores (i.e. the functional
groups involved in direct binding with the receptor) has
often been made by measuring the distances between the
functional groups and comparing them to those of known
topological models. For sweet compounds, the models of
Shallenberger and Acree, and Kier,[14,15] Temussi,[16] Good-
man,[17] and the multipoint attachment model (MPA) of
Tinti and Nofre[18] (which characterizes an ideal sweet com-
pound with eight glucophores, which consists of four high
affinity sites AH, B, G − corresponding to AH, B, X of
Shallenberger–Acree–Kier − and D, and four secondary
sites) are among the most utilized for this purpose. This
Conformational Studies
Monatin has a modified amino acid structure, in which simple method can be useful but suffers from many disad-
it is possible to recognize a glutamic acid fragment and a vantages: in fact, these distances are easily calculated when
tryptophan that has a hydroxyl group instead of an amino an atom in the structure is clearly identified as the “point”
group; the two fragments are partially superimposed. It is glucophore, but the calculation is very approximate when
possible to identify two polar blocks (corresponding to the more undefined areas are considered. In monatin, the AH
Figure 2. Minimum energy conformations: a) 2R*,4R* monatin; b) 2R*,4S* monatin.
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A. Bassoli, G. Borgonovo, G. Busnelli, G. Morini, L. Merlini
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and B glucophores could likely correspond to the amino
The new derivatives were obtained starting from S-pyro-
acid terminal group or to the quaternary hydroxy acid at glutamic acid methylester 5, which is a cheap chiral precur-
C-4, whereas the indole ring is likely to correspond to the sor that is able to undertake stereoselective alkylation reac-
G hydrophobic glucophore. Owing to its magnitude, a great tions at the C-4 position.[24] The general procedure is shown
uncertainty occurs in the measure of the AH–G or B–G in Scheme 1.
distance; depending on which atom is considered as the cen-
ter of the G area, the distances could correspond to the
central or terminal polar groups. Therefore, this method
does not allow the unambiguous identification of the gluco-
phores.
Another method is to identify glucophores by testing the
importance of each functional group in the interaction with
the receptor. Analogues of the target molecule that lack
some functional groups or with modified ones can be syn-
thesized and submitted to biological tests. If the activity is
suppressed or reduced, it is possible to deduce an important
role in activity. We applied this method to monatin and syn-
thesized some analogues with modified functional groups
and some that lacked functional groups.
Synthesis of Monatin Analogues
First we decided to plan the synthesis of some derivatives
that lack the hydroxyl group. This group seems, in fact, to
be important in the molecule, and the deoxy analogues are
Scheme 1. Synthesis of deoxy monatin analogues by alkylation of
pyroglutamate.
easier to access by synthesis. As a second criterion, in some
cases, we substituted the indole ring (which is usually un-
stable under several reaction conditions and therefore needs
The alkylation reaction on N-Boc methylpyroglutamate
special protective groups) with other hydrophobic aromatic 6 was diastereoselective, as expected from analogous alky-
rings which could, theoretically, give a similar interaction lations described in the literature with benzyl bromide, 2-
with the putative receptor. For this purpose, we used bromomethylnaphtalene, and 4-Cl-benzyl bromide, and
phenyl, naphthyl and 4-Cl-phenyl rings: the last one was gave compounds 7, 8, and 9. The stereochemistry of these
chosen since it is known that 4-Cl-tryptophan is sweeter compounds was assigned as 2S,4R on the basis of compari-
than tryptophan itself.
son with literature data and by the NMR spectroscopic
Scheme 2. Synthesis of deoxy derivatives by aldol condensation.
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Monatin, Its Stereoisomers and Derivatives
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Scheme 3. Synthesis of compound 16 by aldol condensation.
spectra which showed a trans relationship between the sub-
stituents at the C-2 and C-4 positions. The acidic hydrolysis
of the lactam ring gave derivatives 10, 11, and 12 in one
step, respectively, without racemization of the chiral centers.
These deoxy derivatives have the same stereochemistry of
natural 2S,4S monatin 1 at both stereocenters (the absolute
configuration in these analogues is 2R,4S as a result of the
changes in the priority of the substituents).
Scheme 4. Synthesis of deamino derivative 23.
A similar approach was used for the synthesis of the in-
dole derivatives 16 and 17 (Scheme 2).
Tasting Trials
In this case the alkylation with bromide 13 was not selec-
tive; the resulting reaction mixture 14 was deprotected in
two steps and chromatographed to give a mixture of dia-
stereoisomers 15 in about a 1:1 ratio as shown by NMR
spectroscopy. A satisfactory separation of the diastereoiso-
mers 16 and 17 was achieved by RP-HPLC on an analytical
scale only. Compound 16 was then obtained as a single en-
antiomer by a different route based on the aldol condensa-
tion of protected pyroglutamic acid 6 and aldehyde 18
(Scheme 3). The resulting mixture of aldols 19 was dehy-
drated to give the unsaturated product 20 as a 3:1 mixture
of E and Z isomers (NMR spectroscopy), which was hydro-
genated stereoselectively to give compound 21. This latter
compound was hydrolyzed and deprotected to give 16 as a
single diastereoisomer. The attribution of the 2S,4S config-
uration to this product was made on the basis of compari-
son with analogous reactions described in the literature,[25]
and confirmed by NMR mono- and bidimensional NOE
experiments on compound 21, that showed a cis relation-
ship between the two hydrogen atoms in the positions C-2
and C-4.
All the new monatin analogues synthesized were submit-
ted to preliminary tasting trials according standard pro-
cedures.[27] The related compound 24 (indole lactic acid)
was also tested and is shown in Figure 3 for comparison.
Figure 3. Tasteless monatin analogues.
A monatin analogue 23 that lacks the terminal amino
None of the derivatives tasted sweet in the tasting trials.
group was prepared as a racemate by hydrolysis of lactone In particular, compound 17 was not sweet: this derivative
22[26] (Scheme 4).
has an identical structure and stereochemistry to that of
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A. Bassoli, G. Borgonovo, G. Busnelli, G. Morini, L. Merlini
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natural monatin, except for the absence of the hydroxyl
group. This functional group seems therefore to be essential
for the sweet taste of monatin, and this is confirmed by the
observation that the biological activity disappeared in all
the new deoxy derivatives. The minimum energy conforma-
tion of deoxymonatin 17 was also calculated and is shown
in Figure 4.
Figure 5. The four stereoisomers of monatin superimposed on the
MPA model. Glucophores are indicated by grey-dot spheres; only
AH, B and G are labeled for clarity.
Figure 4. Minimum energy conformation of deoxyderivative 17.
Qualitatively, all the compounds appear to fit the model.
In particular, when the main glucophores –NH3 and –
The absence of the hydroxyl group changes the minimum
energy conformation; in this case, an intramolecular hydro-
gen bond (1.85 Å) is formed between the terminal amino
group and the oxygen atom of the carboxylic acid at the C-
5 position. As a result, the relative topology of potential
glucophores is completely different from that observed for
S,S monatin. A similar conformation is also observed for
all the other deoxy derivatives (10–12 and 16). It is therefore
possible that the hydroxyl group plays an important role in
the taste of monatin, not as a glucophore itself, but indi-
rectly, by introducing an important constraint which gener-
ates an active conformation. Other evidence that supports
this hypothesis is the fact that indole lactic acid 24 is itself
not sweet, as we would expect if the main AH and B gluco-
phores corresponded to the hydroxy acid moiety. On the
other hand, the terminal amino group is also important for
the taste, since the corresponding derivative 23, which lacks
this group, is completely inactive (tasteless).
+
COO– are set as AH and B sites, the indole ring is always
lying in the large hydrophobic area corresponding to the G
site, so that it is hard to predict any difference in activity
between the compounds. In fact, only a qualitative com-
parison is possible with this methodology as well as with
similar methods, e.g. with the Goodman model,[17] whereas
more refined information is required in order to give a theo-
retical explanation of the observed biological activity.
Therefore, we have applied the so-called pseudoreceptor
modeling technique to obtain some semiquantitative infor-
mation on the binding energy between the ligands and the
putative receptor. For this aim, we used the pseudoreceptor
model developed by our group to be able to explain and
predict the affinity of several sweet-tasting compounds be-
longing to different classes.[22] The four monatin stereoiso-
mers in their minimum energy conformation have been in-
serted in the pseudoreceptor in positions consistent with the
MPA model (utilized as a tool also during the development
of the general pseudoreceptor model to overlap compounds
belonging to different classes for which it was not possible
Pseudoreceptor Modeling
The results seen above seem to indicate that the main to superimpose on the basis of the same structural feature)
glucophores in monatin likely correspond to the indole (G) and then subjected to free ligand relaxation, while the pseu-
and the terminal amino acid fragment (AH and B). We doreceptor was kept rigid. The predicted free energies of
started with this assumption and compared the obtained binding were then compared with the experimental values.
minimum energy conformation of the monatin stereoiso- It is important to underline that the individual conforma-
mers with the Tinti and Nofre multipoint attachment tions of the four isomers after free ligand relaxation are not
model. The four compounds 1–4 have been compared to significantly different from the starting ones. Table 1 shows
the MPA model by overlapping the AH, B, and G sites with the comparison between the predicted free energies of li-
the amino, C-2 carboxylate and indole groups, respectively, gand binding, ΔG°, for isomers 1–4 and the experimental
to find the best possible superimposition (Figure 5).
activity obtained by sensory evaluation.
Table 1. Taste activity of the four stereoisomers by sensory analysis and molecular modeling.
Reference
Monatin stereoisomers
Compound
Absolute configuration
Relative Sweetness (RS)
1
2
3
2S,4R
tasteless
300
4
2S,4S
350
2R,4R
1000
2700
–
2R,4S
250
this paper
[3][a]
50
1200
–10.15
1300
–
–11.32
[1]
–
ΔG°binding (pseudoreceptor)
–13.02
–11.46
+
[a] Tested as Na or Na/NH4 salts.
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Monatin, Its Stereoisomers and Derivatives
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Among the four stereoisomers of monatin the 2R,4R iso-
mer has the highest affinity for the pseudoreceptor. There-
fore, the taste activity predicted by the pseudoreceptor
method is in good agreement with the experimental finding
that this isomer, (and not its natural enantiomer 2S,4S) is
the sweetest of the series. Interestingly, the highest calcu-
lated ΔG° (–10.15) is that of 2S,4S monatin, and this stereo-
isomer is therefore predicted to be the less active of the
series. This result is in contrast with the sensory evaluation
experimental data, where we found compound 3 to be the
least active – it was tasteless at the concentration used. Nev-
ertheless, the result is in agreement with the data indicated
by the more recent sensory analysis experiments made by
the Ajinomoto Co. Inc.[3]
Conclusions
Generally, stereochemistry is a strong discriminating fac-
tor in the chemoreception mechanism of taste recognition.
The sweet natural compound 2S,4S monatin and its three
stereoisomers are a curious exception to this rule, since at
least three of them (or all four, following other authors)
tasted sweet in sensory analysis experiments. In these mole-
cules, the absolute configuration of the two stereogenic cen-
ters is not so important to the binding with the receptor. In
particular, the configuration at C-4 is not crucial; this could
depend on the fact that the substituents on this carbon
atom are not directly involved in the interaction with the
receptor. In fact, in the minimum energy conformation, the
molecule is quite extended and the central groups are steri-
cally hindered; moreover, the tertiary hydroxyl group is en-
gaged in a strong intramolecular hydrogen bond that pre-
vents other interactions. The low stereoselectivity in chemo-
recognition of monatin isomers was not easily foreseen,
since these molecules are structurally related to tryptophan
and this compound, as many amino acids, is sweet in its R-
d form but bitter in the S-l form and shows high stereo-
chemical discrimination in taste chemoreception.
The pseudoreceptor approach also gives some insights
into the binding mode of the active compounds to the pseu-
doreceptor. Figure 6 shows compound 2 inside the pseudo-
receptor and the interactions between the ligand and the
surroundings amino acid residues.
These results therefore suggest that all factors have to be
considered when predicting the chemosensory properties of
new compounds on the basis of the structure and stereo-
chemistry of known leads. The pseudoreceptor modeling
calculations are useful in the prediction of the relative po-
tency of sweet compounds and in the interpretation of ex-
perimental data from sensory analysis, since they give se-
miquantitative information on the binding energy and some
insights into the possible interaction of active ligands with
the putative receptor protein until the detailed three-dimen-
sional structure of the receptor active site(s) is clarified.
Experimental Section
Molecular Modeling
Three-dimensional molecular models were built on a Silicon
Graphics O2, using the programs InsightII and Discover, 97.0 (Ac-
celrys, San Diego, CA). The initial models were energy-refined by
molecular mechanics techniques with conjugate gradients until a
maximum energy derivative value of 0.008 kcalmol–1 Å–1 was ob-
tained using the CVFF force field. Conformational analysis was
performed by molecular dynamics. Monatin and other amino acid
derivatives were minimized in vacuo as zwitterions as they are sup-
posed to be in solution. For atomic partial charges of the ligand
atoms, Mulliken charges calculated on the minimized structures
using the MOPAC program[28] with the MNDO Hamiltonian were
used.
Figure 6. 2R,4R monatin (compound 2) in the pseudoreceptor cav-
ity.
The most important interactions in determining the
binding affinity of the compounds are (in decreasing order
of calculated energy) those involving the charged terminal
amino group (Asp, 33) and C-2 carboxylate (Arg, 19); fol-
lowed by the C-4 carboxylic acid (Pro, 26) and its hydro-
phobic interaction with indole (Phe, 31). In this model, the
hydroxyl group of monatin lies in the center of the cavity
and does not seem to be involved in strong interactions with
any amino acid residue.
The intramolecular hydrogen bond between the –OH and
the –+NH3 groups is retained inside the pseudoreceptor.
This situation confirms that the most important interac-
tions should involve the terminal charged amino acid
groups and indole as glucophores, while the alcohol group
is of key importance in keeping the active conformation.
For pseudoreceptor modeling, the program PrGen 2.1[29] (SIAT
Biographics Laboratory, Basel, CH) was used. Molecules in their
lowest-energy conformation were imported in PrGen and remini-
mized with the Yeti force field.[30]
The equation used in PrGen to correlate free energies of ligand
binding ΔG° with relative sweetness is: ΔG°exp. = RT·lnKd, where
Kd = Kd(sucrose)/RS. Details on PrGen and on the general pseudo-
receptor model for sweet compounds developed by our group can
be found in ref.[22]
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Synthesis of Monatin Derivatives
NMR (DMSO): δ = 1.78 (m, 1 H), 2.10 (m, 1 H), 2.78–3.00 (m, 3
H), 3.78–3.85 (m, 1 H, 2-H), 7.30–7.81 (m, 7 H, ar), 8.20 (broad s,
2 H, NH2) ppm. m/z (%): 269 [M+ – H2O] (40), 224 (10), 181 (10),
152 (20), 141 (100), 115 (50), 84 (40). [α]2D5 = +16.4 (c = 0.53,
MeOH). HRMS (ESI, pos): 288.12256 (M + 1); calcd. 287.11576.
Reagents were of commercial grade purity; solvents were dried with
standard procedures; m.p. are uncorrected. H NMR spectra were
1
recorded with Bruker AMX-300 and AMX-600 instruments, by
using TMS as internal standard; J values are given in Hz. Mass
spectra were recorded with a Finnigan 4021 spectrometer; HPLC
experiments were made with a Varian PROSTAR instrument;
HPLC-MS analysis were made with an Agilent SL 1100 series in-
strument with ESI and ion trap system. HRMS were made with a
Bruker Daltonics APEX II ICR-FTMS instrument, by using the
ESI ionization mode. Optical rotation data were recorded with a
Jasco J 810 spectropolarimeter.
(2S,4R)-2-Amino-4-(4Ј-chlorobenzyl)pentanedioic Acid (12): The
crude product was purified with a cation exchange resin (Amberlist
IR-120) eluting with NH3 solution (1 n⁾. Recrystallization from
ethanol gave the pure product (0.30 g, 89%). 1H NMR
(600 MHz,CDCl3): δ = 2.14 (m, 1 H, 3a-H), 2.29 (m, 1 H, 3b-H),
2.68–2.75 (m, 2 H, 4-H and 5a-H), 3.11 (m, 1 H, 5b-H), 3.98 (dd,
1 H, 2-H), 7.00–7.25 (m, 4 H, ar) ppm. m/z (%): 208 (60), 143 (20),
127 (85), 125 (100). [α]2D5 = –4.0 (c = 0.2, MeOH).
1-tert-Butyl 2-Methyl (2S)-5-Oxopyrrolidine-1,2-dicarboxylate (6):
White solid (7.19 g, 65%). M.p. 67–68. 1H NMR (CDCl3): δ = 1.48
(s, 9 H, Boc), 1.98–2.10 (m, 1 H), 2.25–2.65 (m, 3 H), 3.78 (s, 3 H,
OCH3), 4.63 (dd, 1J = 3.7, 2J = 11.2 Hz, 1 H, 5-H) ppm. IR (nujol):
2990, 1800, 1750, 1700 cm–1.[α]2D5 = –39.61 (c = 1.02, EtOH). m/z
(%): 244 [M+] (5), 188 (40), 84 (80), 59 (100).
3-(Bromomethyl)-1-[(4-methylphenyl)sulfonyl]-1H-indole (13): Com-
pound 13 was obtained from indole-3-carboxyaldehyde in three
steps by protection of the nitrogen atom with p-toluenesulphonyl
chloride, reduction to alcohol with NaBH4, and reaction with bro-
mine and PPh3 in CCl4. White solid (1.30 g, 64%). M.p. 132 °C
(dec.). 1H NMR (CDCl3): δ = 2.35 (s, 3 H, CH3), 4.60 (s, 2 H,
CH2Br), 7.20–7.40 (m, 5 H, ar), 7.65–7.90 (m, 4 H, ar) ppm. m/z
(%): 365 [M+] (2), 284 (100), 155 (20), 129 (25), 102 (20), 91 (50),
65 (20).
General Procedure for the Alkylation (Scheme 1): A 1 m solution of
LiHMDS (13.57 mL, 13.57 mmol) was added to
6 (3.0 g,
12.34 mmol) in dry THF under nitrogen at –78 °C. After 1 h, the
bromide was added dropwise (10% molar excess) and stirred for
2.5 h. The reaction mixture was quenched with saturated NH4Cl
solution and extracted with diethyl ether. The organic phase was
dried on Na2SO4, filtered, and the solvents evaporated to dryness.
The product was purified by flash chromatography.
(2S,4S)- and (2S,4R)-4-[Hydroxy(1-tosylsulfonil-1H-indol-3-yl)-
methyl]-5-oxopyrrolidine-1,2-dicarboxylic Acid (14, Diastereoiso-
meric Mixture): White solid (23%). M.p. 81–82 °C.1H NMR
(CDCl3): δ = 1.51 (s, 18 H, 2 Boc), 1.68 (m, 1 H, 4-H), 1.95 (m, 3
H), 2.34 (s, 6 H, 2 CH3), 2.75 (m, 2 H), 2.94 (m, 2 H), 3.33 (m, 2
H), 3.74 (s, 3 H, OCH3), 3.77 (s, 3 H, OCH3), 4.49 (m, 2 H, two
2-H), 4.1–8.0 (m, 18 H, ar and indole) ppm. m/z (%): 526 [M+]
(22), 426 (80), 284 (100), 272 (90), 211 (30), 168 (22), 155 (60), 130
(45), 91 (90), 65 (18), 57 (30). HRMS (ESI, pos): 549.16416 [M +
Na]; calcd. 526.17737.
1-tert-Butyl 2-Methyl (2S,4R)-4-Benzyl-5-oxopyrrolidine-1,2-dicar-
boxylate (7): White solid (53%, ee Ͼ 98%). M.p. 98–99 °C. 1H
NMR (CDCl3): δ = 1.50 (s, 9 H, Boc), 2.08 (m, 2 H), 2.58 (m, 1
H), 2.85 (m, 1 H), 3.30 (m, 1 H), 3.70 (s, 3 H, OCH3), 4.40 (dd, 1J
= 3.4, 2J = 8.7 Hz, 1 H, 2-H), 7.15–7.30 (m, 6 H, ar) ppm. m/z (%):
334 [M+], 303 (5), 278 (20), 233 (100), 174 (80), 129 (10), 91 (30),
57 (70). [α]2D5 = –38,7 (c = 0.78, CHCl3).
(2S,4R)-2-Amino-4-(1H-indol-3-ylmethyl)pentanedioic Acid (16):
White solid. M.p. 128–130 °C. 1H NMR (aceton-d6): δ = 1.89 (ddd,
1-tert-Butyl 2-Methyl (2S,4R)-4-(2-Naphthylmethyl)-5-oxopyrrolid-
ine-1,2-dicarboxylate (8): White solid (21%, ee Ͼ 98%). M.p. 143–
2
3
1
1J = 12.7, J = 8.0, J = 8.9 Hz, 1 H, 3a-H), 2.64 (ddd, J = 12.7,
1
146 °C. H NMR (CDCl3): δ = 1.48 (s, 9 H, Boc), 2.08 (m, 2 H),
2J = 8.0, J = 8.6, 1 H, 3b-H), 2.87 (ddd, J = 14.4, J = 9.6, J =
1.0, 1 H, 5a-H), 2.92 (m, 1 H, 4-H), 3.29 (ddd, J = 14.4 Hz, J =
3.7, J = 1.3, 1 H, 5b-H) 4.29 (t, J = 8.0 Hz, 1 H, 2-H), 6.9–6.5
(m, 6 H, indole) ppm. m/z (%): (LCMS – ESI): 276 [M+] (35), 258
(55), 130 (100). [α]2D5 = +41.5 (c = 0.27, MeOH).
3
1
2
3
2.88 (m, 1 H), 3.02 (m, 1 H), 3.48 (m, 1 H), 3.75 (s, 3 H, OCH3),
1
2
1
2
4.50 (dd, J = 3.7, J = 7.6 Hz, 1 H, 2-H), 7.30–7.87 (m, 7 H, ar)
ppm. m/z (%): 383 [M+] (15), 327 (10), 283 (60), 224 (20), 167 (20),
141 (100). [α]2D5 = –9.7 (c = 0.89, CHCl3).
3
1
1-tert-Butyl 2-Methyl (2S,4R)-4-(4-Chlorobenzyl)-5-oxopyrrolidine-
1,2-dicarboxylate (9): White solid (32%, ee Ͼ 98%). M.p. 128 °C.
1H NMR (CDCl3): δ = 1.50 (s, 9 H, Boc), 1.94–2.12 (m, 2 H), 2.70
(m, 1 H), 2.92 (m, 1 H), 3.25 (m, 1 H), 3.72 (s, 3 H, OCH3), 4.50
(dd, J = 2.0, J = 9.1 Hz, 1 H, 2-H), 7.10 (d, 2 H), 7.30 (d, 2 H)
ppm. m/z (%): 267 (15), 216 (5), 210 (25), 208 (50), 125 (20), 116
(30). [α]2D5 = –31.7 (c = 0.5, CHCl3).
(2S, 4S)-2-Amino-4-(1H-indol-3-ylmethyl)pentanedioic Acid (17): 1H
NMR (aceton-d6): δ = 1.92 (m, 1 H, 3a-H), 2.27 (m, 1 H, 3b-H),
1
2
2.91 (dd, J = 5.4, J = 14.2 Hz, 1 H, 5a-H), 2.99 (m, 1 H, 4-H),
1
3.14 (m, 1 H, 5b-H) 4.27 (t, J = 8.8, 1 H, 2-H), 7.1–8.1 (m, 6 H,
1
2
indole) ppm. m/z (%): (LCMS-ESI): 276 [M+] (5), 258 (100), 130
(50), 102 (10). [α]2D5 = +35.5 (c = 0.38, MeOH).
Compounds 16 and 17 were separated by RP-HPLC eluting under
gradient conditions from eluent A (H2O + 1% AcOH) to eluent B
(MeOH) in 10 minutes, flow 1 mLmin–1. Retention times (min):
12.92 (compound 16) and 12.26 (compound 17).
General Procedure for Hydrolysis: A solution of 7, 8, or 9 was re-
fluxed in HCl (6 n⁾ for 4 h. The reaction was monitored with TLC
and the spots detected with ninhydrine. The solvent was evaporated
at reduced pressure.
Compound 16 was also obtained as follows: Compound 21
(0.520 g, 0.9 mmol) [see below], a mixture of MeOH/H2O 3:1
(15 mL), and K2CO3 were refluxed for 4 h. After removal of the
solvent, the crude product was purified by flash chromatography
(CH2Cl2/MeOH/NH3/H2O, 8:3:0.2:0.2). White solid (0.12 g, 38%).
Spectral data identical to those reported above.
(2S,4R)-2-Amino-4-benzylpentanedioic Acid (10): The crude oil was
purified through an ion exchange resin (Amberlist IRA-68) eluting
with aqueous NH3 (0.05 n) to give a white solid (0.264 g, 75%). 1H
NMR (DMSO): δ = 1.75 and 2.10 (two m, 2 H), 2.78–3.00 (m, 3
H), 3.80 (m, 1 H, 2-H), 7.15–7.30 (m, 5 H, ar), 8.30 (br. s, 2 H,
-NH2) ppm. m/z (%): 219 (60), 201 (20), 174 (50), 146 (15), 128
(20), 117 (20), 91 (100). HRMS (ESI, pos): 238.10755 (M + 1);
calcd. 237.10011. [α]2D5 = –14 (c = 0.45, MeOH).
1-(Mesitylsulfonyl)-1H-indole-3-carbaldehyde (18): Obtained from
1H-indole-3-carbaldehyde by reaction with mesityl chloride and
NaH in dry DMF at 0 °C. White solid (44%). M.p. 129–131 °C.
(2S,4R)-2-Amino-4-(2-naphthylmethyl)pentanedioic
Acid
(11):
White solid (0.138 g, 87%). M.p. (aq. ethanol) 158–160 °C. 1H 1H NMR (CDCl3): δ = 2.23 (s, 3 H, CH3), 2.49 (s, 6 H, two CH3),
2524
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 2518–2525

Monatin, Its Stereoisomers and Derivatives
FULL PAPER
6.94–7.30 (m, 7 H, indole and ar), 10.50 (s, 1 H, CHO) ppm. m/z
Acknowledgments
(%): 327 [M+] (100), 183 (10), 119 (50), 86 (30), 84 (45).
We thank MIUR (Italian Ministry for Research and the Univer-
sity) and University of Milano for financial support (programs CO-
FIN 2002 and FIRST 2003); Fondazione Fratelli Confalonieri (Mi-
lano) for a PhD fellowship to G. Busnelli; Damien Kelly, National
University of Ireland, Galway, for contribution to experimental
work.
(2S)-4-[Hydroxy(1-mesitylsulfonyl-1H-indol-3-yl)methyl]-5-oxopyr-
rolidine-1,2-dicarboxylic Acid (19): A 1 m solution of LiHMDS in
THF (7.3 mmol) was added to a solution of 6 (1.48 g, 6.09 mmol)
in dry THF whilst stirring at –78 °C under nitrogen. The reaction
mixture was stirred for 1 h at –78 °C prior to addition of a solution
of 18 (2.2 g, 6.75 mmol) in THF and BF3·Et2O (2.16 mL,
6.75 mmol). The mixture was stirred for 2.5 h at –78 °C, quenched
with saturated NH4Cl, and extracted with diethyl ether. The com-
bined organic phases were dried with Na2SO4, filtered, and the
solvents evaporated in vacuo. Flash chromatography (hexane/ethyl
acetate, 45:55) gave mixture of 19 (0.89 g, 26%). 1H NMR (CDCl3):
δ = 1.51 (s, 18 H, 2Boc), 1.85 (m, 1 H, 4a-H), 1.99 (m, 1 H, 4b-H),
2.31 (s, 3 H, CH3), 2.53 (s, 6 H, two CH3), 3.29 (m, 1 H, 3-H), 3.75
(s, 3 H, OCH3), 4.58 (dd, 1,2J = 9.2, 1 H, 5-H), 4.8 (s, 1 H, OH),
5.05 (d, 1J = 9.2, 1 H, 6-H), 6.95 (s, 2 H, ar), 7.2–7.8 (m, 5 H,
indole) ppm. m/z (%): 571 [M + 1] (5), 470 (3), 465 (10), 328 (15),
209 (10), 183 (18), 143 (40), 119 (100), 91 (100).
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(2S)-4-[Hydroxy(1-mesitylsulfonyl-1H-indol-3-yl)methylene]-5-oxo-
pyrrolidine-1,2-dicarboxylic Acid (20): The aldol mixture 19 (0.70 g,
1.23 mmol) was dissolved in dry CH2Cl2 and treated with methane-
sulphonyl chloride (0.1 mL, 1.35 mmol) and TEA (1.46 mL,
13.5 mmol). After 2 d at room temperature, the reaction mixture
was quenched with water and extracted with CH2Cl2. The organic
layer was dried with Na2SO4, and the solvent evaporated under
reduced pressure. After purification of the crude product by flash
chromatography (hexane/ethyl acetate, 6:4), a white solid product
(0.39 g, 57%) was obtained as a mixture of E/Z isomers. M.p. 85–
88 °C. 1H NMR (CDCl3): δ = 1.55 (s, 18 H, Boc E and Z), 2.27 (s,
3 H, pCH3 Z), 2.3 (s, 3 H, pCH3 E), 2.52 (s, 6 H, OCH3 of E), 2.58
(s, 6 H, OCH3 Z), 2.85–2.9 (m, 2 H, 4a-H E and Z), 3.25–3.32 (m,
2 H, 4b-H E and Z), 3.78 (s, 3 H, OCH3 Z), 3.8 (s, 3 H, OCH3 E),
1
2
1
4.68 (dd, J = 4.5, J = 10.0 Hz, 1 H, 5-H Z), 4.78 (dd, J = 3.5,
2J = 10,0 Hz, 1 H, 5-H E), 6.95 (s, 2 H, ar of Z), 6.97 (s, 2 H, ar
of E), 7.1 (br. t, 1 H, CH=C Z), 7.19–7.31 (m, 8 H, indole Z and
1
E), 7.77 (s, 1 H, 8-H E), 7.78 (s, 1 H, 8-H Z), 7.81 (t, J = 3.0, 1
H, CH=C, E) ppm. m/z (%): 552 [M+] (30), 452 (100), 393 (10),
327 (10), 269 (40), 209 (30), 119 (30), 71 (20). [α]2D5 = +23 (c = 1.0,
CHCl3).
(2S,4S)-4-[Hydroxy(1-mesitylsulfonyl-1H-indol-3-yl)methyl]-5-oxo-
pyrrolidine-1,2-dicarboxylic Acid (21): M.p. 58–60 °C. 1H NMR
(CDCl3): δ = 1.50 (s, 9 H, Boc), 1.72 (m, 1 H, 3a-H),2.30 (s, 3 H,
CH3), 2.52 (s, 6 H, two CH3), 2.43 (m, 1 H, 3b-H), 2.84 (m, 1 H,
1
CH2), 2.95 (m, 1 H, 4-H), 3.40 (dd, 1 H, CH2), 4.50 (dd, J = 9.6,
2J = 11.2, 1 H), 6.97 (s, 2 H), 7.20 (m, 2 H), 7.31 (m, 1 H), 7.4 (s,
1 H), 7.5 (m, 1 H) ppm. m/z (%): 554 [M+] (20), 522 (10), 396 (10),
318 (10), 266 (10), 242 (100) 183 (10). [α]2D5 = +18.2 (c = 1.0,
CHCl3). The absolute configuration was assessed by NMR mono-
and bi-dimensional NOE experiments (Supplementary Information
available).
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2-Hydroxy-2-(1H-indol-3-ylmethyl)pentanedioic Acid (23): Lactone
22[26] (71 mg, 0.26 mmol) was stirred in aqueous EtOH and KOH
(0.5 mmol) at room temperature for 2 d. Acidic workup followed
by extraction with ethyl acetate gave compound 23. White solid
(25 mg, 30%). 1H NMR ([D6]acetone): δ = 2.40 (m, 4 H, CH2),
3.45 (m, 2 H, CH2-indole), 7.10–7.70 (m, 5 H, indole), 10.20 (br. s,
1 H, NH) ppm. m/z (%): 287 [M+] (10), 259 (5), 229 (5), 200 (5),
155 (20), 149 (30).
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Received: December 28, 2004
Eur. J. Org. Chem. 2005, 2518–2525
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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