Preparation and characterization of four stereoisomers of monatin

Article abstract of DOI:10.1248/cpb.c16-00286

Monatin is a naturally occurring, sweet amino acid comprising four stereoisomers due to its two asymmetric centers at C2 and C4. However, the characteristics of each stereoisomer have not yet been fully investigated. To obtain a sufficient amount of racemic monatin for optical resolution, a synthetic method was developed by modifying a possible biosynthetic pathway, i.e., a cross-aldol reaction and subsequent transamination. The key intermediate, 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid, was obtained via the cross-aldol reaction of pyruvic acid and indole-3-pyruvic acid. Subsequently, the carbonyl group was converted to a hydroxyimino group through reaction with hydroxylamine and then to an amino group via hydrogenation to produce monatin. Next, the racemic monatin was divided into mixtures of two pairs of enantiomers through recrystallization. Finally, both enantiomers of the N-carbobenzoxy-γ-lactone derivatives of monatin were separated by preparative HPLC and deprotected. It was found that all optically pure stereoisomers exhibited a sweet taste. The isomer that displayed the most intense sweetness was the (2R,4R)-isomer, as determined by single crystal X-ray structure analysis of the monatin potassium salt, whereas the least sweet isomer was the (2S,4S)-isomer, which demonstrated a far lower sweetness than was previously reported.

Full text of DOI:10.1248/cpb.c16-00286

Vol. 64, No. 8
Chem. Pharm. Bull. 64, 1161–1171 (2016)
1161
Regular Article
Preparation and Characterization of Four Stereoisomers of Monatin
,a
Yusuke Amino,* Shigeru Kawahara,b Kenichi Mori,c Kazuko Hirasawa,a Hiroyuki Sakata,d and
Tatsuki Kashiwagia
a Institute for Innovation, Ajinomoto Co., Inc.; 1–1 Suzuki-cho, Kawasaki-ku, Kawasaki 210–8681, Japan: b Tokai
c
Plant, Ajinomoto Co., Inc.; 1730 Hinaga, Yokkaichi, Mie 510–0885, Japan: Research Institute for Bioscience
Products & Fine Chemicals, Ajinomoto Co., Inc.; 1–1 Suzuki-cho, Kawasaki-ku, Kawasaki 210–8681, Japan: and
d Institute of Food Research and Technologies, Ajinomoto Co., Inc.; 1–1 Suzuki-cho, Kawasaki-ku, Kawasaki 210–
8681, Japan.
Received March 23, 2016; accepted April 21, 2016
Monatin is a naturally occurring, sweet amino acid comprising four stereoisomers due to its two asym-
metric centers at C2 and C4. However, the characteristics of each stereoisomer have not yet been fully in-
vestigated. To obtain a sufficient amount of racemic monatin for optical resolution, a synthetic method was
developed by modifying a possible biosynthetic pathway, i.e., a cross-aldol reaction and subsequent transami-
nation. The key intermediate, 4-hydroxy-4-(3-indolylmethyl)-2-ketoglutaric acid, was obtained via the cross-
aldol reaction of pyruvic acid and indole-3-pyruvic acid. Subsequently, the carbonyl group was converted
to a hydroxyimino group through reaction with hydroxylamine and then to an amino group via hydrogena-
tion to produce monatin. Next, the racemic monatin was divided into mixtures of two pairs of enantiomers
through recrystallization. Finally, both enantiomers of the N-carbobenzoxy-γ-lactone derivatives of monatin
were separated by preparative HPLC and deprotected. It was found that all optically pure stereoisomers
exhibited a sweet taste. The isomer that displayed the most intense sweetness was the (2R,4R)-isomer, as
determined by single crystal X-ray structure analysis of the monatin potassium salt, whereas the least sweet
isomer was the (2S,4S)-isomer, which demonstrated a far lower sweetness than was previously reported.
Key words monatin; stereoisomer; sweet taste; cross-aldol reaction; natural compound
Monatin is a naturally occurring amino acid derivative 1b exhibited only a slightly sweet taste, presumably due to
isolated from the bark of the roots of Schlerochiton ilicifo- 1a, which is thought to be present as an impurity; however,
lius, which is a plant native to the north western Transvaal the specific intensity of the sweetness was not reported. Ki-
of South Africa. The structure of monatin was reported as tahara et al. reported a selective synthetic method toward each
(2S,4S)-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl)-pentanoic stereoisomer of monatin; however, the degree of sweetness of
acid ((2S,4S)-4-hydroxy-4-(3-indolylmethyl)-glutamic acid each stereoisomer was not sufficiently discussed.7,8) Bassoli et
(refer to structural formula (1a) in Fig. 1) by Vleggaar et al. disclosed the isolation of the four stereoisomers of mona-
al.1) Discrepancies in the numbering system for monatin tin and sensory analysis of each stereoisomer; however, the
have caused some confusion in the case of the (2S,4R)- and chemical and optical purities of each stereoisomer were not
(2R,4S)-diastereomers. In this report, we assigned the α car- sufficiently indicated.12) In their study, the assignment of ste-
bon of glutamic acid as C2. Thus, monatin is numbered in ac- reochemistry of the products was justified by HPLC analysis
cordance with other substituted amino acid derivatives.
using a chiral column and carried out by our laboratories.12)
According to Vleggaar et al., the sweetness of (2S,4S)-
We have been interested in the taste profiles of other ste-
monatin (1a) (natural-type monatin) derived from the natural reoisomers of monatin, particularly that of (2R)-isomers, as
plant is reported to be 1200- to 1400-fold more intense than it is known that (R)-amino acids (D-amino acids) such as
that of sucrose. The (2S,4S) absolute configuration of natural (R)-tryptophan exhibit a sweet taste, while (S)-amino acids
monatin was assigned by nuclear Overhauser effect (NOE) do not, with the exception of (S)-alanine, (S)-proline, and (S)-
NMR spectroscopic experiments based on its cyclic derivative serine.13,14)
and the empirical Clough–Lutz–Jirgenson rule. These results
suggested that monatin is an attractive candidate in the search tensity of sweetness of each monatin stereoisomer in practical
for novel, high-intensity sweeteners.
concentrations corresponding to 5–10% sucrose concentration,
Various methods have been reported regarding the synthesis with the exception of natural monatin.
As mentioned above, there has been no clear data for the in-
of monatin, some of which produce a mixture of stereoiso-
Although the development of a mass production method is
mers,2–5) while others describe stereoselective syntheses.6–11) necessary to thoroughly evaluate monatin stereoisomers, when
However, there have been no reports wherein all four ste- we began our study, to the best of knowledge there were no
reoisomers having the same structural formula as 1a are reports regarding a large-scale synthetic method of monatin.
synthesized, isolated, and characterized as pure substances. Homologs of monatin (γ-hydroxy-glutamic acid) in which
Nakamura et al. synthesized and isolated hydrochlorides of 1a the 3-indolylmethyl at C4 (γ-position) is replaced by various
and (2S,4R)-monatin (1b).6) They reported that, with respect to substituents, including methyl, ethyl, isobutyl, carboxymethyl,
the intensity of sweetness, synthetic 1a exhibited a sweetness or carboxyethyl, are known as natural products.15–19) These
potency equivalent to that of natural monatin, while synthetic compounds, including monatin, must be produced via a com-
*To whom correspondence should be addressed. e-mail: yusuke_amino@ajinomoto.com
© 2016 The Pharmaceutical Society of Japan

1162
Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
Fig. 1. Structures of the Four Stereoisomers of Monatin
mon biosynthetic pathway; i.e., a cross-aldol reaction and
subsequent transamination. The basic structure of monatin
is a substituted ketoglutaric acid, which must be constructed
by a regiospecific cross-aldol reaction between pyruvic acid
and indole-3-pyruvic acid. Early studies on the aldol reaction
of pyruvic acid derivatives included the catalytic asymmetric
homo-aldol reaction of ethyl pyruvate and cross-aldol reaction
of various 2-ketoesters with activated carbonyl compounds
in various organic solvents.20,21) The decarboxylative self-
condensation of oxaloacetic acid and self-aldol condensation
reaction of pyruvic acid in aqueous solution have also been
reported.22–24) However, to the best of our knowledge, the only
example of a cross-aldol reaction of α-keto acids in an aque-
ous solution is the reaction of glyoxylic acid and oxaloacetic
acid.25,26) In this reaction, glyoxylic acid never underwent
self-condensation, allowing for relatively easy production of a
single condensed product. In the same report, transformation
of the obtained γ-hydroxy α-ketoglutaric acid to γ-hydroxy-
L-glutamic acid via an aminotransferase was described as an
example of the biosynthesis of γ-hydroxy-L-glutamic acid. The
development of chemical processes based on this biosynthetic
pathway is an attractive approach toward establishing a large-
scale synthesis of monatin.
Fig. 2. RP-HPLC Chromatogram of Monatin, Lactone, and Lactam
Left chromatogram: (2S,4S)- and (2R,4R)-isomers; eluted in order of lactone,
monatin, lactam. Right chromatogram: (2S,4R)- and (2R,4S)-isomer; eluted in order
of monatin, lactam, and lactone.
In this paper, we report a novel, practical synthetic method
toward racemic monatin, as well as details of the isolation and
characterization of each stereoisomer.
Results and Discussion
Preparation of Stereoisomer Standards Mixtures of the
(2S,4S)- and (2S,4R)-isomers of monatin (1a, b, respectively)
and the (2R,4R)- and (2R,4S)-isomers of monatin (1c, d, re-
spectively) were prepared from (R)-Garner’s aldehyde and (S)-
Garner’s aldehyde, respectively, according to the method de-
scribed by Nakamura et al.6) Each stereoisomer (diastereomer)
was separated by means of reversed-phase (RP)-HPLC using
a C18 column. Under these conditions, 1b and d were eluted
first, followed by 1a and c, as shown in Fig. 2. While studying
the stability of monatin, it was found that strongly acidic con-
ditions were not suitable for characterizing monatin as these
Fig. 3. Resolution of the Four Monatin Stereoisomers via Chiral HPLC
Using a Crownpak CR(+) Column
Synthesis of Racemic Monatin We carried out a study to
conditions induced a structural change to form a mixture develop a chemical synthetic method of monatin through mod-
of monatin, i.e., the lactone and the lactam; therefore, each ification of a biosynthetic pathway in an attempt to establish
isomer of monatin was isolated as the ammonium salt. Using an industrial production method27) (Chart 1). The first step of
these stereoisomers, we established a chiral HPLC analysis in our strategy was to construct a basic substituted ketoglutaric
which the four stereoisomers were clearly resolved into four acid via a regioselective cross-aldol reaction between pyruvic
peaks using a Crownpak CR(+) column. Using this column, acid (3) and indole-3-pyruvic acid (2). Subsequently, the car-
each isomer eluted as a single peak when injected separately bonyl group of the substituted ketoglutaric acid was replaced
with the following order of retention times, 1d>c>b>a, as by an amino group.
shown in Fig. 3. Both the optical purities and amounts of
obtained stereoisomers using the above procedure were not a donor generated the desired 4-hydroxy-4-(3-indolylmethyl)-
sufficient to determine the exact sweetness potency of each.
2-ketoglutaric acid intermediate (5) in moderate yield under
Notably, the reaction of 2 as an acceptor with 5eq of 3 as

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1163
Chart 1. Large-Scale Synthesis of Racemic Monatin
Fig. 4. Separation of Each of the Four Monatin Stereoisomers
basic aqueous conditions; however, there is potential for four lyst under a pressure of 1MPa in aqueous ammonia proceeded
condensation products to be generated. Through the use of ex- smoothly to produce the racemic monatin ammonium salt (1
cess 3, generation of the self-aldol product of 2 and the prod- NH4+ salt) as crystals in good yield. The ratio of the sum of
uct with the reverse arrangement might be suppressed. The 1a and c to the sum of 1b and d was 60:40. Details of this
decarboxylative cross-condensation of oxaloacetic acid (4) and synthesis and application of this strategy to the chiral synthe-
2 also proceeded in moderate yield. Moreover, 5 was easily sis of 1c will be reported in the near future.
transformed to 4-hydroxy-4-(3-indolylmethyl)-2-hydroxyimi-
Isolation of Each Monatin Stereoisomer We obtained
noglutaric acid (6) upon reaction with hydroxylamine in situ, between several tens and one hundred grams of racemic
without the need for further purification. The ammonium salt monatin, which we used to determine the optical resolution of
of 6 (6 NH4+ salt) was easily isolated as a single product via each of the four stereoisomers using the following procedure28)
crystallization from a mixed solvent of aqueous ammonia and (Fig. 4). Crystals of the major enantiomer mixture of 1a and c
2-propanol. The hydrogenation reaction of 6 with a Rh/C cata- were easily obtained in high purity by recrystallization of the

1164
Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
racemic mixture from a mixed solvent of water and ethanol. fold sweetness. It is known that a relationship exists between
Conversely, the crystals of another enantiomer mixture of the taste and stereochemistry of amino acids, in which many
1b and d were obtained from the mother liquor by recrystal- (R)-amino acids (D-amino acids) such as (R)-tryptophan ex-
lization from the same solvent system. Next, each mixture of hibit a sweet taste, while (S)-amino acids (L-amino acids) are
enantiomers was converted to the corresponding N-carboben- not sweet, with the exception of (S)-alanine, (S)-proline, and
zoxy-monatin (Cbz-monatin, 7) upon reaction of monatin with (S)-serine.13,14) This rule can also be broadly applied to 1a and
carbobenzoxy chloride. Cyclization of 7 was carried out by c. It is well-known that the N-terminal α-amino group and
heating in ethyl acetate at 75°C in the presence of a catalytic β-carboxyl group of aspartame (L-α-aspartyl-L-phenylalanine
amount of p-toluenesulfonic acid to give the N-carbobenzoxy- 2-methyl ester) possess the same alignment as the α-amino
γ-lactone of monatin (Cbz-lactone, 8). Each stereoisomer of 8 group and α-carboxyl group of sweet D-amino acids.30) Thus,
was then separated by semi-preparative chiral HPLC. Separa- it is suggested that 1c and aspartame are likely to interact with
tion of (2S,4S)- and (2R,4R)-Cbz-lactone (8a, c, respectively) a common domain of the sweet taste receptor.
was performed using a CHIRALPAK AS column to afford
Isomer 1a, which is expected to be natural monatin and has
optically pure 8a and c. Separation of (2S,4R)- and (2R,4S)- been reported to exhibit a sweetness in the range of >1000
Cbz-lactone (8b, d, respectively) was performed using a times that of sucrose, was found to be only 25–50 times as
CHIRALCEL OJ column to afford optically pure 8b and d. sweet as sucrose. In contrast, 1c exhibited a sweetness poten-
The four stereoisomers of monatin (1a–d) were obtained in cy of 1700–2700 times that of sucrose. However, these results
excellent purities (>99.2%) as crystals of the sodium salt by are difficult to reconcile with the stereochemical assignments
hydrogenation with Pd/C, followed by hydrolysis with aque- made by Vleggaar et al. and Bassoli et al., in which they re-
ous sodium hydroxide. Their optical purities were verified by ported that the natural monatin they obtained was a mixture
chiral HPLC, the results of which are demonstrated in Fig. 3.
of the four stereoisomers.12) We do not have any information
Sweetness of Each Monatin Stereoisomer The four ste- regarding their detailed extraction methodology; thus, it is not
reoisomers were obtained in excellent purities and submitted possible to conclude whether these isomers were already pres-
to taste testing for the first time. The relative sweetness inten- ent in the plant or if they occurred as a result of racemization
sity of the isomers was compared to sucrose, with the results during isolation. Taking the sweetness potency of each isomer
shown in Table 1: 1a: 50 times (Na salt, compared to 5% into account, there is a possibility that the monatin isolated by
sucrose solution), 25 times (K salt, compared to 10% sucrose Vleggaar et al. may be a mixture of 1a and the (2R,4R)-isomer
solution); 1b: 300 times (Na salt, 5% sucrose), 100 times (Na (1c).
salt, 10% sucrose); 1c: 2700 times (Na salt, 5% sucrose), 1700
The specific rotations for (−)1a reported in the literature
times (K salt, 10% sucrose); and 1d: 800 times (Na salt, 5% range from −7.6 to −10.9° (in 1N HCl). However, we learned
sucrose), 300 times (Na salt, 10% sucrose). As seen from these from our stability study that monatin is stable under neutral or
results, as is usual with high-potency sweeteners, the relative weakly acidic conditions, but unstable under strongly acidic
sweetness changed depending on the concentration of the conditions. In aqueous acid, monatin is able to cyclize to the
authentic sucrose solution. Note that the intensity of monatin lactone and subsequently to the lactam, eventually reaching
sweetness was constant regardless of the counter cation. After equilibrium (Fig. 5). As the acidity of the solution is in-
our study was complete, the sweetness potency of 1c was re- creased, the ratio of the lactone form to linear form monatin
ported as approximately 3100 at 5% sucrose equivalent (SE) also increased. Indeed, when a sample of pure monatin was
and 2700 at 8% SE by Fry et al.29)
Surprisingly, all isomers heated at 70°C in pH 3 buffer for an entire day, the RP-HPLC
were found to exhibit a sweet taste; however, 1a was found to chromatogram showed that the single peak had become het-
be the least sweet isomer, while the other three isomers, espe- erogeneous, as shown in Fig. 2. The lactone of 1a and c was
cially 1c, were found to be intensely sweet.28)
then injected and shown to elute much sooner than the linear
When natural monatin was first derived from the plant, form of monatin. Thus, 1N HCl (and acidic solutions in gen-
it was reported that the intensity of sweetness was 1200- to eral) is not a suitable solvent for characterizing monatin, as
1400-fold greater than that of sucrose. Since we have found these conditions induce a structural change to form mixtures
that the sweetness potency of the (2S) form is much weaker of the monatin lactone and lactam; therefore, we measured
than 500-fold, the results suggest that natural monatin should the specific rotation of monatin stereoisomers in aqueous am-
mainly comprise the (2R) form to explain its 1200- to 1400- monia.
Single Crystal X-Ray Structure Analysis of the (2R,4R)-
Monatin (1c) Potassium Salt Dihydrate The 1c potassium
salt was chosen as the subject of this study, due to its relative-
ly high melting point, and was recrystallized from a mixture
of ethanol and water by slow evaporation.
Table 1. Experimental Sensory Analysis Data for the Four Monatin
Stereoisomers
Compound
1a
1b
1c
1d
The crystal structure of the 1c potassium salt dihydrate
was determined by single crystal X-ray structure analysis. A
summary of this crystallographic analysis is shown in Table
2. The asymmetric unit of the crystal was shown to contain
one monatin potassium salt and two water molecules. Figure
6 shows the asymmetric unit of the crystal as an ORTEP
drawing. The absolute structure of monatin in this crystal was
deduced as the (2R,4R)-form, based on the fact that the Flack
parameter31) converged to a value of 0.082(22) when the refine-
Absolute configuration
(2S,4S) (2S,4R) (2R,4R) (2R,4S)
a)
Relative sweetness1),
1200–1400
Relative sweetness (5% sucrose)b)
Relative sweetness (10% sucrose)c)
Optical purityd)
a) Previously reported relative sweetness value.1) b) Relative sweetness values of
monatin sodium salts compared to the 5% sucrose solution. c) Relative sweetness
values of monatin sodium salts [(2S,4R) and (2R,4S)] and potassium salts [(2S,4S) and
(2R,4R)] compared to a 10% sucrose solution. d) Optical purities of the stereoisomers
determined by HPLC.
50
25
300
100
99.4
2700
1700
99.3
1300
800
99.8
99.2

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1165
Fig. 5. Intramolecular Reaction of (2R,4R)-Monatin in Acidic Solution
Stability of (2R,4R)-monatin (1c) in citric acid buffer (0.05M, pH3.0) at 25°C.
Table 2. General Crystallographic Information for the (2R,4R)-Monatin
Potassium Salt Dihydrate Crystal
Formula unit:
C14H13N2O5K·2H2O
P212121
7.762(3) Å
30.883(3) Å
6.745(3) Å
4
Space group:
Unit cell parameters
a:
b:
c:
Z Value:
Calculated density:
No. of unique reflections:
No. of parameters refined:
Goodness of fit:
Residuals
1.349g/cm3
1761
295
1.24
Ra):
0.051
Rwb):
R1c):
0.108
0.036
Max shift error in final cycle:
0.28
Maximum peak in final diff. map:
Minimum peak in final diff. map:
0.17 e−/Å3
−0.24 e−/Å3
a) R=Σ (Fo2−Fc2)/Σ Fo2. b) Rw=(Σ w(Fo2−Fc2)2/Σ w(Fo2)2)1/2, where w=(σ2(Fo2))−1
c) R1=Σ||Fo|−|Fc||/Σ|Fo| for 1422 reflections with I>2 σ(I).
.
ment was performed assuming this absolute structure. The po-
tassium ion was coordinated with five oxygen atoms from four
carboxyl groups of three neighboring monatin molecules and
one water molecule. The potassium ion also interacted with
an indole ring of one of the above three monatin molecules Fig. 6. ORTEP Drawing of the Molecular Structure of the (2R,4R)-
Monatin Potassium Salt Dihydrate with Thermal Ellipsoids at a 50%
Probability Level
through a π–cation interaction. As mentioned above, we suc-
cessfully solved the crystal structure of the sweetest diaste-
reomer of the monatin potassium salt dihydrate and confirmed
that the absolute stereochemistry was (2R,4R).
equilibrium with 1c in water upon standing and regained its
Sweetness of (2R,4R)-Monatin Derivatives Finally, a sweetness.
brief structure–activity relationship study of monatin deriva-
The structure of monatin contains an indole-3-lactic acid
tives was conducted. As described above, we found that mona- fragment, in which the amino group of D-tryptophan, a sweet
tin and its lactone were in equilibrium in strongly acidic solu- amino acid, is replaced by a hydroxyl group. As such, we were
tions, which gradually converted to the lactam (Fig. 5). The interested in the role of the 4-carboxyl group in sweetness ex-
lactone (9) and lactam (10) of 1c were synthesized and tasted, pression. Thus, we synthesized derivatives of 1c in which the
but neither exhibited sweetness. As expected, 9 returned to 4-position carboxyl group was exchanged for other functional

1166
Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
Chart 2. Synthesis of Monatin Derivatives
groups, such as ethylcarbamoyl (12), hydroxymethyl (14), and b) and 711mg of the ammonium salts of (2R)-isomers (a mix-
carbamoyl (relatively unstable), utilizing 8c as the starting ture of 1c, d). Resolution of 660mg of the (2S)-isomers and
material, as shown in Chart 2. All derivatives were found to 711mg of the (2R)-isomers was performed under the following
be as sweet as 1c; thus, the negative charge on the 4-carboxyl preparative conditions. The fractions were neutralized with
group is not required for sweetness expression or for interac- aqueous ammonia and concentrated prior to the preparative
tion with the sweetness receptor.
procedure. The combined fractions were adsorbed on a cation-
exchange resin and eluted with an aqueous ammonia solution.
The eluted fractions were freeze-dried to give 207mg of 1a,
Conclusion
We have developed
a practical racemic synthesis of 233mg of 1b, 261mg of 1c, and 254mg of 1d as amorphous
monatin, in which the key intermediate, 4-hydroxy-4-(3- solids of their ammonium salts.
indolylmethyl)-2-ketoglutaric acid (5), was synthesized by the
cross-aldol reaction of pyruvic acid and indole-3-pyruvic acid. mers:
Subsequently, the carbonyl group was converted to an amino
Preparative conditions for separation of monatin stereoiso-
Guard column: Inertsil ODS-3 30×50mm. Column: Inertsil
group to afford monatin. The four stereoisomers were then ODS-3 30×250mm. Detection: UV 210nm. Eluent: ‹A› aceto-
isolated from the racemic monatin mixture via crystallization, nitrile+0.05% trifluoroacetic acid (TFA), ‹B› H2O+0.05%
separated from their derivatives by HPLC, and deprotected. TFA. Flow rate: 28mL/min. Gradient: 12–18% ‹A› in ‹B› over
It was found that all isomers exhibited a sweet taste, with 25min. Loaded amount: 10–13mg. Temperature: 25°C.
the (2R,4R)-isomer (1c) being intensely sweet. However, the
(2S,4S)-isomer (1a) was the least sweet isomer and had far
Analytical conditions for monatin diastereomers:
Column: Inertsil ODS-80A 6×150mm. Detection: UV
lower sweetness than was previously reported. Based on these 210nm. Eluent: 12% CH3CN(aq)+0.05% TFA. Flow rate:
result, it was concluded that the stereoisomer (steric structure) 1.5mL/min. Temperature: 25°C.
of monatin present in nature is the (2R,4R)-isomer (1c), which
Analytical conditions for determination of optical purity of
has a significantly sweet taste. These findings will assist us monatin stereoisomers:
in our continued investigation of the conformational require-
ments of novel high-potency sweeteners.
Column: Crownpack CR(+) 4×150mm. Detection: UV
210nm. Eluent: aqueous perchloric acid (pH 2.0)–metha-
nol=90:10. Flow rate: 1.2mL/min. Temperature: 25°C.
Optical purity:
Experimental
General 1H-NMR spectra were recorded on a Brucker
Values in parentheses indicate the retention time for each
Avance 400 spectrometer (400MHz), and MS spectra were peak. Results are given for each stereoisomer. (2S,4S)-isomer:
measured using a Thermo Quest TSQ 700 spectrometer. Am- 94% (45.0min); (2S,4R)-isomer: 94% (26.1min); (2R,4R)-iso-
berlite IR 120B H AG was employed as the cation-exchange mer: 94% (20.9min); (2R,4S)-isomer: 96% (16.1min).
resin. Melting point (mp) measurements were performed using
a Micro Melting Point Apparatus from Yanaco (Japan). Optical
rotary power measurements were performed using a DIP-370
Synthesis of Racemic Monatin Ammonium Salts
4-Hydroxy-4-(3-indolylmethyl)-2-ketoglutaric Acid (5)
Compound 2 (12.3g, 58.7mmol, 97% purity by weight)
Digital Polarimeter manufactured by Nippon bunko (Jasco was dissolved in water (209mL) containing sodium hydroxide
Engineerring, Japan). The chiral columns, Crownpak CR(+), (2.45g). Over 2h, a 25% aqueous sodium hydroxide solution
CHIRALPAK AS, and CHIRALCEL OJ were purchased from (47.61g) and a mixture of 3 (25.85g, 293.5mmol) in water
DAICEL CHEMICAL INDUSTRIES, Ltd., Japan.
(25.85g) were added to the resulting solution under a nitrogen
Preparation of Standard Samples (1) Monatin was syn- atmosphere at 35°C, while the reaction system was maintained
thesized using a modified version of the method developed by at pH 11.0. Subsequently, the reaction solution was agitated
Nakamura et al.6) Crude monatin (2S)-isomers (4.30g) and for 14h. This afforded a reaction solution containing 5 in a
(2R)-isomers (1.14g) were adsorbed on a cation-exchange resin 44.1% yield (vs. 2). Hydrochloric acid (1N, 3.60g) was added
(H+-type) and purified by elution with a 3% aqueous ammonia to neutralize the solution (275mL, pH=6.91). A portion of the
solution. Subsequent freeze-drying resulted in 2.92g of the obtained reaction solution (168mL) was passed through a resin
ammonium salts of monatin (2S)-isomers (a mixture of 1a, column (4.8cm diameter) packed with a synthetic adsorbent

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1167
(840mL, DIAION-SP207, Mitsubishi Chemical Corporation, d]=60:40) were dissolved in an aqueous ammonia solution
Japan). Ion-exchanged water was then passed through the (2.5%, 100mL), and the resulting solution was concentrated
column at a flow rate of 23.5mL/min. Fractions eluted from to 20mL. Aqueous ammonia (5%, 3mL) was then added, and
1.7 to 2.9L were collected and concentrated to obtain 5 in a the mixture was homogenized and allowed to stand at room
66.3% yield.
temperature for 30min. After the crystals were separated, a
1H-NMR (D2O) δ: 3.03 (1H, d, J=14.6Hz), 3.11 (1H, d, slurry was formed by adding an aqueous solution comprised
J=14.6Hz), 3.21 (1H, d, J=18.1Hz), 3.40 (1H, d, J=18.1Hz), of an aqueous ammonia solution (5%) and ethanol (80mL,
7.06–7.15 (3H, m), 7.39 (1H, d, J=7.8Hz), 7.66 (1H, d, 25:75, v/v), and the crystals of 1a and c ammonium salts were
J=7.8Hz). 13C-NMR (D2O) δ: 35.43, 47.91, 77.28, 109.49, filtered. The resulting crystals were dissolved in an aqueous
112.05, 119.44, 119.67, 121.91, 125.42, 128.41, 136.21, 169.78, ammonia solution (2.5%, 30mL), concentrated, and recrystal-
181.43, 203.58. Electrospray ionization (ESI)-MS m/z: 290.02 lized from a mixed solution of aqueous ammonia (5%, 0.5mL)
(M−H)−.
4-Hydroxy-4-(3-indolylmethyl)-2-hydroxyiminoglutaric salts (4.80g, 15.52mmol, RP HPLC purity (hereafter referred
and ethanol (30mL) to give crystals of 1a and c ammonium
Acid (6)
Compound 2 (73.8g, 352mmol) was added to an aqueous
to as “HPLC purity”): 98.0%).
1H-NMR (D2O) δ: 1.99 (1H, dd, J=11.8 and 15.3Hz), 2.51
sodium hydroxide solution (16wt%, 917g) and the resulting (1H, dd, J=2.0 and 15.2Hz), 3.03 (1H, d, J=14.6Hz), 3.22
solution was adjusted to 35°C. An aqueous solution of 3 (50%, (1H, d, J=14.9Hz), 3.57 (1H, dd, J=2.0 and 11.8Hz), 7.06–7.11
310.2g, 1761mmol) was added portion-wise to the solution (1H, m), 7.14–7.18 (1H, m), 7.16 (1H, s), 7.42 (1H, d, J=8.1Hz),
over 2h, while the reaction solution was maintained at pH 11.1 7.66 (1H, d, J=7.9Hz). ESI-MS m/z: 291.39 (M−H)−. mp:
using aqueous sodium hydroxide (30%). After an additional 182.0–186.0°C. Degree of sweetness: approximately 1300-fold
4.5h, a reaction solution containing 5 was obtained. An aque- (as compared to a 5% aqueous solution of sucrose).
ous hydroxylamine hydrochloride salt solution (40%, 367.2g,
The mother liquor ([1a and c]:[ 1b and d]=3:10) obtained
2114mmol) was then added to the reaction solution while in the above operation was concentrated to 5mL. An aqueous
maintaining a pH of 7 using aqueous sodium hydroxide (30%). ammonia solution (5%, 3mL) was then added and the mixture
The solution was stirred at 5°C for 17.5h, after which the re- was homogenized and allowed to stand at room temperature
action solution was adjusted to pH 2 using conc. hydrochloric for 10min. After the crystals were separated, a slurry was
acid and extracted with ethyl acetate to remove the organic formed by adding ethanol (80mL), and the crystals of 1b and
material. The organic layer was rinsed with aqueous satu- d ammonium salts were filtered. The resulting crystals were
rated sodium chloride and concentrated to obtain the residue. dissolved in an aqueous ammonia solution (2.5%, 30mL), con-
The residue was recrystallized from aqueous ammonia (28%, centrated, and recrystallized three times from a mixed solu-
60mL) and 2-propanol (1350mL) to obtain the diammonium tion of aqueous ammonia (5%, 0.5mL) and ethanol (30mL) to
salt of 6 (142mmol, 40% yield vs. 2) as crystals.
1H-NMR (D2O) δ: 2.66 (2H, s), 2.89 (1H, d, J=14.4Hz), HPLC purity: 98.2%). The overall recovery rate was 79.0%.
3.04 (1H, d, J=14.4Hz), 6.89–6.94 (1H, m), 6.97–7.03 (1H,
1H-NMR (D2O) δ: 2.14 (1H, dd, J=10.0 and 15.3Hz), 2.41
give crystals of 1b and d ammonium salts (3.10g, 10.02mmol,
m), 7.11 (1H, d, J=2.8Hz), 7.27 (1H, d, J=7.8Hz), 7.53 (1H, d, (1H, dd, J=2.8 and 15.3Hz), 3.16 (2H, s), 3.92 (1H, dd, J=2.8
J=7.8Hz), 10.71 (1H, brs). ESI-MS m/z: 305.17 (M−H)−. FAB- and 10.0Hz), 7.06–7.10 (1H, m), 7.13–7.18 (1H, m), 7.16 (1H,
MS m/z: 307.0956 (M+H) (Calcd for C14H15N2O6: 307.0930).
s), 7.41 (1H, dd, J=0.9 and 7.9Hz), 7.67 (1H, dd, J=1.0 and
Racemic Monatin Ammonium Salts (1 NH4 Salt) The 8.0Hz). ESI-MS m/z: 291.19 (M−H)−. mp: 167.2–168.4°C. De-
ammonium salt of 6 (9.6g, 28.16mmol) was dissolved in aque- gree of sweetness: approximately 800-fold (as compared to a
ous ammonia (28%, 180mL) and Rh/C (5, 50% hydrous, 9.0g) 5% aqueous solution of sucrose).
+
was added to the solution. The solution was stirred under a
Preparation of Racemic Mixture of 2-Benzyloxy-
hydrogen atmosphere (10 atm., 1MPa) at ambient tempera- carbonylamino-4-(3-indolylmethyl)-4-carboxy-γ-buty-
ture for 24h. The catalyst was removed by filtration, and the rolactone (N-Carbobenzoxy-γ-lactone of Monatin, Cbz-
filtrate was concentrated. Aqueous ethanol (90%, 80mL) was Lactone) (2S,4S)- and (2R,4R)-Isomers (8a, c) The am-
added to the residue, and the solution was stirred at 25°C for monium salts of 1a and c (19.51g, 63.07mmol; HPLC purity:
1.5h. The deposited purified crystals were filtered and dried 99.2%) were dissolved in a sodium hydroxide solution (2N,
under reduced pressure to obtain racemic 1 NH4+ salt (7.90g, 94.6mL, 189.2mmol) and water (90mL). Benzyloxycarbonyl
24.17mmol as the diammonium salt).
chloride (12.61mL, 88.30mmol) was added, and the solution
(2S,4S), (2R,4R)-isomers: 1H-NMR (D2O) δ: 1.95–2.02 was stirred for 2h at room temperature. Additional sodium
(1H, m), 2.58–2.62 (1H, m), 3.01–3.05 (1H, m), 3.21–3.24 (1H, hydroxide solution (2N, 15.8mL, 31.54mmol) and benzyloxy-
m), 3.55–3.58 (1H, m), 7.07–7.11 (1H, m), 7.14–7.18 (2H, m), carbonyl chloride (4.50mL, 31.54mmol) were then added and
7.42–7.44 (1H, d), 7.66–7.68 (1H, d).
the solution was stirred overnight at room temperature. The
(2S,4R), (2R,4S)-isomers: 1H-NMR (D2O) δ: 2.11–2.17 resulting aqueous solution was extracted with ether (50mL,
(1H, m), 2.38–2.43 (1H, m), 3.16 (2H, s), 3.90–3.93 (1H, three times) to remove excess benzyloxycarbonyl chloride.
m), 7.06–7.10 (1H, m), 7.13–7.17 (2H, m), 7.41–7.43 (1H, d), The solution was adjusted to pH 3 using hydrochloric acid
7.66–7.68 (1H, d). ESI-MS m/z: 291.28 (M−H)−.
and extracted with ethyl acetate (100mL, three times). The
Resolution of Mixture of Four Monatin Stereoisomer organic layer was dried over anhydrous magnesium sulfate
Ammonium Salts (1) into Racemic Mixture of (2S,4S)- and filtered. The filtrate was concentrated in vacuo to give 7a
Isomer (1a) and (2R,4R)-Isomer (1c) and Racemic Mixture and c (27.93g, 65.5mmol), which were subsequently dissolved
of (2S,4R)-Isomer (1b) and (2R,4S)-Isomer (1d) Am- in ethyl acetate (400mL). After addition of p-toluenesulfonic
monium salts (10.00g, 32.33mmol) of 1 ([1a and c]:[1b and acid (1.25g, 6.65mmol) the solution was heated at 75°C

1168
Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
for 3h. The resulting solution was washed with water and of hydrochloric acid adjusted to pH 3, washed with saturated
saturated saline, dried over anhydrous magnesium sulfate, fil- saline, dried over anhydrous magnesium sulfate, and filtered,
tered, and the filtrate was concentrated in vacuo. Chloroform and the filtrate was concentrated in vacuo. The residue was
(100mL) was then added to the residue, and the crystals were crystallized with n-hexane (100mL) to give a solvate of 8d
separated and collected by filtration to afford 8a and c in an with 0.2 equiv of ethyl acetate (4.88g, 11.45mmol; HPLC pu-
overall yield of 68.5% (17.64g, 43.19mmol; HPLC purity: rity: 97.3%) and a solvate of 8b with 0.2 equiv of ethyl acetate
99.6%).
(5.41g, 12.70mmol; HPLC purity: 96.9%) in an overall recov-
1H-NMR (DMSO-d6) δ: 2.39 (1H, t, J=11.3Hz), 2.67 (1H, ery yield of 99.7%.
1
t, J=12.8Hz), 3.28–3.38 (2H, m), 3.71–3.81 (1H, m), 4.98 (2H,
Solvate of 8b: H-NMR spectrum is virtually identical to
s), 6.98 (1H, t, J=7.0Hz), 7.07 (1H, t, J=7.0Hz), 7.21 (1H, that of the racemic mixture of 8b and d. ESI-MS m/z: 409.58
s), 7.25–7.35 (6H, m), 7.55 (1H, d, J=7.9Hz), 7.67 (1H, d, (M+H)+. mp: 116.1–116.8°C. Solvate of 8d: 1H-NMR spec-
J=8.3Hz), 11.03 (1H, s). ESI-MS m/z: 409.68 (M+H)+. mp: trum is virtually identical to that of the racemic mixture of 8b
195.5–196.9°C.
and d. ESI-MS m/z: 409.58 (M+H)+. mp: 109.1–110.8°C.
Conversion of Cbz-Lactone (2R,4R)-Isomer (8c) into
Preparation of a Racemic Mixture of Cbz-Lactone
(2S,4R)- and (2R,4S)-Isomers (8b, d) The same operation the (2R,4R)-Isomer (1c) Sodium Salt Isomer 8c (14.24g,
described above was performed using a mixture of the 1b and 34.85mmol; HPLC purity: 99.5%) was dissolved in a mixed
d ammonium salts (15.00g, 48.49mmol; HPLC purity: 99.5%). solvent of methanol (400mL) and water (40mL), Pd/C (10%,
Heating was performed at 75°C for 2h after addition of p- 3g) was added, and reduction was performed in a hydrogen
toluenesulfonic acid to afford 8b and d in an overall yield of atmosphere at room temperature for 2h. After reduction,
61.1% (12.10g, 29.64mmol; HPLC purity: 100%).
water (100mL) and aqueous sodium hydroxide (4N, 19.2mL,
1H-NMR (DMSO-d6) δ: 2.20–2.30 (1H, m), 2.60–2.70 76.67mmol) were added, followed by stirring for 10min. The
(1H, m), 3.20 (1H, d, J=15.2Hz), 3.42 (1H, d, J=15.1Hz), catalyst was then removed by filtration and the filtrate was
3.35–3.48 (1H, m), 5.03 (2H, s), 6.98 (1H, t, J=7.8Hz), 7.05 concentrated. In order to remove excess sodium, the residue
(1H, t, J=7.8Hz), 7.18 (1H, s), 7.28–7.40 (6H, m), 7.53 (1H, d, was dissolved in water (160mL) and an ion-exchange resin
J=7.8Hz), 7.80 (1H, d, J=8.5Hz), 10.92 (1H, s). ESI-MS m/z: (Amberlite IR 120B H AG(H+)) was added portion-wise until
409.58 (M+H)+. mp: 156.7–159.1°C.
the solution became weakly acidic. Aqueous ammonia (28%,
Resolution of Cbz-Lactone (2S,4S)-Isomer (8a) and 34.8mL) was added to the solution, and the ion-exchange
Cbz-Lactone (2R,4R)-Isomer (8c) Resolution was per- resin was removed by filtration and washed with a 5% aque-
formed using an optical isomer resolution column for 8a and ous ammonia solution. The filtrate and washing solution were
c (1.17g, 2.86mmol; HPLC purity: 99.7%). CHIRLPAK AS combined, filtered, and concentrated. The residue was dis-
(20×50mm) and CHIRALPAK AS (20×250mm) columns solved in water (100mL), activated charcoal (1g) was added,
were used with an eluent of n-hexane–ethanol–acetic acid and the solution was stirred for 10min. The activated charcoal
(40:60:0.5) and a flow rate of 10mL/min, detection at UV was removed by filtration, the filtrate was concentrated, and
210nm, temperature of 40°C, and a loaded amount of 25mg. the concentrate was crystallized with aqueous ethanol (90%)
The retention time was 13min for 8a and 23min for 8c. Each at room temperature to give a solvate of the 1c sodium salt
preparative fraction was concentrated, dissolved in ethyl with 0.2 equiv of ethanol (6.55g, 20.19mmol; optically active
acetate (50mL), and concentrated again. The residue was column HPLC purity: 99.3%) in an overall yield of 57.9%.
crystallized from chloroform (30mL) to afford 8a (428mg,
1H-NMR (D2O) δ: 2.06 (1H, dd, J=11.6 and 15.2Hz), 2.68
1.05mmol) and 8c (399mg, 0.977mmol) in an overall recov- (1H, dd, J=2.0 and 15.2Hz), 3.10 (1H, d, J=14.8Hz), 3.30 (1H,
ery yield of 70.7%. This operation was scaled up at a contract d, J=14.8Hz), 3.64 (1H, dd, J=2.0 and 11.6Hz), 7.16 (1H, brt,
manufacturing company, providing 8a (16.7g) and 8c (16.1g) J=8.0Hz), 7.23 (1H, brt, J=8.0Hz), 7.24 (1H, s), 7.50 (1H, d,
from a mixture of stereoisomers (35.6g).
J=8.0Hz), 7.74 (1H, d, J=8.0Hz). 13C-NMR (D2O, 100MHz)
Cbz-lactone (2S,4S)-isomer (8a): 1H-NMR spectrum is δ: 38.1, 41.0, 56.5, 83.1, 111.9, 114.5, 121.9, 122.1, 124.5, 127.8,
virtually identical to that of the racemic mixture of 8a and c. 130.7, 138.7, 177.1, 182.9. ESI-MS m/z: 291.49 (M−H)−. FAB-
ESI-MS m/z: 409.68 (M+H)+. mp: 179.8–182.0°C. Cbz-lactone MS m/z: 315.0963 (M+H) (Calcd for C14H16N2NaO5: 315.0957).
(2R,4R)-isomer (8c): 1H-NMR spectrum is virtually identi- mp: 197.1–198.3°C. Specific rotation: [α]D25 +0.64 (c=0.5, 5%
cal to that of the racemic mixture of 8a and c. ESI-MS m/z: NH3). Degree of sweetness: approximately 2700-fold (as com-
409.88 (M+H)+. mp: 179.2–182.8°C.
Resolution of Cbz-Lactone (2S,4R)-Isomer (8b) and
pared to a 5% aqueous solution of sucrose).
Conversion of Cbz-Lactone (2S,4S)-Isomer (8a) into
Cbz-Lactone (2R,4S)-Isomer (8d) Resolution was per- the (2S,4S)-Isomer (1a) Sodium Salt The same operation
formed using an optical isomer resolution column for 8b and described above was performed using 8a (5.00g, 12.25mmol;
d (9.89g, 24.22mmol; HPLC purity: 100%). CHIRALCEL HPLC purity: 99.8%). A solvate of the 1a sodium salt with
OJ (20×50mm) and CHIRALCEL OJ (20×250mm) columns 0.2 equiv of ethanol (3.15g, 9.71mmol; optically active col-
were used as the guard and preparative columns, respec- umn HPLC purity: 99.8%) was obtained in an overall yield of
tively, with an eluent of n-hexane–ethanol–trifluoroacetic 79.3%.
acid (40:60:0.1) and a flow rate of 8mL/min, detection at
1H- and 13C-NMR spectrum are virtually identical to those
UV 210nm, temperature of 40°C, and a loaded amount of of 1c. ESI-MS m/z: 291.59 (M−H)−. mp: 196.1–197.9°C. Spe-
50mg. The retention time was 16min for 8d and 21min for cific rotation: [α]D25 −1.67 (c=0.5, 5% NH3). Degree of sweet-
8b. Each preparative fraction was neutralized with aqueous ness: approximately 50-fold (as compared to a 5% aqueous
ammonia and concentrated. The residue was then dissolved solution of sucrose).
in ethyl acetate (150mL), washed with an aqueous solution
Conversion of Cbz-Lactone (2S,4R)-Isomer (8b) into the

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1169
(2S,4R)-Isomer (1b) Sodium Salt The same operation de- crystals were filtered, affording 12 (244mg, 7.60mmol) as
scribed above was performed using 8b with 0.2 equiv of ethyl crystals.
acetate (5.23g, 12.28mmol; HPLC purity: 96.9%). The 1b
sodium salt (2.57g, 8.14mmol; optically active column HPLC dd, J=9.4 and 15.0Hz), 2.63 (1H, dd, J=4.2 and 15.0Hz),
purity: 99.4%) was obtained in an overall yield of 66.3%.
1H-NMR (CD3OD) δ: 0.88 (3H, t, J=7.2Hz), 2.12 (1H,
3.05–3.17 (2H, m), 3.17 (1H, d, J=14.5Hz), 3.30 (1H, d,
1H-NMR (D2O) δ: 2.21 (1H, dd, J=10.0 and 15.2Hz), J=14.5Hz), 3.68 (1H, dd, J=4.1 and 9.3Hz), 6.90–7.10 (1H,
2.48 (1H, dd, J=2.8 and 15.2Hz), 3.23 (2H, s), 3.99 (1H, dd, m), 7.08 (1H, t, J=7.2Hz), 7.13 (1H, s), 7.32 (1H, d, J=8.4Hz),
J=2.8 and 10.0Hz), 7.17 (1H, brt, J=8.0Hz), 7.23 (1H, brt, 7.62 (1H, d, J=7.8Hz). ESI-MS m/z: 320.21 (M+H)+,
J=8.0Hz), 7.24 (1H, s), 7.49 (1H, d, J=8.0Hz), 7.74 (1H, d, 318.21 (M−H)−. FAB-MS m/z: 320.1617 (M+H) (Calcd for
J=8.0Hz). 13C-NMR (D2O, 100MHz) δ: 36.7, 41.7, 54.8, 81.1, C16H22N3O4: 320.1610).
112.1, 114.5, 121.9, 122.1, 124.4, 127.6, 130.7, 138.6, 177.6,
Synthesis
of
(2R,4R)-2-Amino-4,5-dihydroxy-4-(3-
183.9. ESI-MS m/z: 291.49 (M−H)−. mp: 227.1–229.4°C. Spe- indolylmethyl)pentanoic Acid (14) Isomer 8c (1.02g,
cific rotation: [α]D25 −9.57 (c=0.5, 5% NH3). Degree of sweet- 2.50mmol) and triethylamine (0.52mL, 3.75mmol) were dis-
ness: approximately 300-fold (as compared to a 5% aqueous solved in tetrahydrofuran (7.5mL). The reaction solution was
solution of sucrose).
cooled to between −15 and −40°C, and isobutyl chlorofor-
Conversion of Cbz-Lactone (2R,4S)-Isomer (8d) into mate (0.51mL, 3.75mmol) was added to the solution. After
the (2R,4S)-Isomer (1d) Sodium Salt The same operation stirring at −15 to −40°C for 30min, a solution of sodium bo-
described above was performed using 8d with 0.2 equiv of rohydride (314mg, 8.30mmol) dissolved in water (1.5mL) was
ethyl acetate (3.66g, 8.59mmol; HPLC purity: 97.3%). The 1d added, and the solution was stirred for an additional 20min.
sodium salt (2.23g, 7.07mmol; optically active column HPLC After adding hydrochloric acid (2N, 2.5mL), the solution was
purity: 99.2%) was obtained in an overall yield of 82.3%.
concentrated in vacuo. The residue was dissolved in water
1H- and 13C-NMR spectrum are virtually identical to those (10mL) and extracted with ethyl acetate (25mL, twice). Once
of 1b. ESI-MS m/z: 291.19 (M−H)−. mp: 227.5–229.2°C. Spe- the resulting organic layer was washed with water (10mL) and
cific rotation: [α]D25 +11.08 (c=0.5, 5% NH3). Degree of sweet- a saturated sodium chloride solution (10mL), it was dried over
ness: approximately 1300-fold (as compared to a 5% aqueous anhydrous magnesium sulfate, filtered, and the filtrate was
solution of sucrose).
concentrated in vacuo. The residue was purified by PTLC, af-
Synthesis of (2R,4R)-2-Amino-4-hydroxy-4-ethylcar- fording (2R,4R)-2-benzyloxycarbonylamino-4-hydroxymethyl-
bamoyl-4-(3-indolylmethyl)butyric Acid (12) Isomer 8c 4-(3-indolylmethyl)-γ-butyrolactone (13, 880mg, 2.23mmol)
(817mg, 2.00mmol) was dissolved in a mixed solvent con- as a viscous, oily product.
taining tetrahydrofuran (10mL), dichloromethane (10mL),
Compound 13 (880mg, 2.23mmol) was then dissolved
and dimethylformamide (10mL) maintained at a temperature in methanol (10mL) and Pd/C (10%, containing 50% water,
of 0°C. To this solution, ethylamine hydrochloride (244mg, 410mg) was added. After reduction at room temperature
3.0mmol), triethylamine (0.56mL, 4.0mmol), 1-hydroxy- under a hydrogen atmosphere at atmospheric pressure for 5h,
benzotriazole hydrate (324mg, 2.40mmol), and 1-ethyl-3-(3- sodium hydroxide (1N, 2.15mL) was added. The catalyst was
dimethylaminopropyl)carbodiimide hydrochloride (463mg, removed by filtration, and the filtrate was concentrated in
2.41mmol) were added, and the solution was stirred over- vacuo. The residue was dissolved in water (15mL) and tetra-
night at room temperature. The reaction solution was then hydrofuran (15mL), and the solution was neutralized with
concentrated in vacuo and water (50mL) was added to the a strongly acidic ion-exchange resin (Amberlite IR120B H
residue. The solution was extracted with ethyl acetate (50mL, AG). The resin was removed by filtration, and the filtrate was
twice), and the organic layer was washed with a 5% aque- freeze-dried, affording 14 (228mg, 0.82mmol) as a powder.
ous citric acid solution (30mL, twice), a saturated sodium
1H-NMR (CD3OD) δ: 1.84 (1H, dd, J=10.8 and 15.2Hz),
chloride solution (30mL, once), a 5% aqueous sodium bi- 2.27 (1H, dd, J=2.8 and 15.6Hz), 2.97 (1H, d, J=14.4Hz),
carbonate solution (30mL, twice), and a saturated sodium 3.06 (1H, d, J=1.48Hz), 3.56 (1H, d, J=11.2Hz), 3.67 (1H,
chloride solution (30mL, once). The organic layer was dried d, J=11.6Hz), 3.87 (1H, dd, J=2.8 and 10.8Hz), 7.01 (1H,
over anhydrous magnesium sulfate, filtered, and the filtrate t, J=8.0Hz), 7.10 (1H, t, J=8.0Hz), 7.15 (1H, s), 7.34 (1H,
was concentrated in vacuo. The residue was purified by dd, J=8.0Hz), 7.64 (1H, d, J=8.0Hz). ESI-MS m/z: 265.30
preparative thin-layer chromatography (PTLC), affording (M+H)+, 263.10 (M−H)−. FAB-MS m/z: 279.1358 (M+H)
(2R,4R)-2-benzyloxycarbonylamino-4-ethylcarbamoyl-4-(3- (Calcd for C14H18N2O4: 279.1345).
indolylmethyl)-γ-butyrolactone (11, 688mg, 1.58mmol).
Synthesis of (2R,4R)-2-Amino-2,3-dideoxy-4-C-(1H-in-
Compound 11 (688mg, 1.58mmol) was then dissolved in dol-3-ylmethyl)pentaric Acid 1,4-Lactone (9, Monatin
methanol (10mL), and Pd/C (10%, 317mg) was added to the Lactone) Isomer 8c (2.0g, 4.90mmol) was dissolved in a
solution. Reduction was performed at room temperature in a mixed solvent of tetrahydrofuran (60mL) and water (10mL).
hydrogen atmosphere at atmospheric pressure for 16h. After To this solution was added Pd/C (10%, 1.0g), and the pH was
removing the catalyst by filtration, the reaction solution was adjusted to slightly basic by adding aqueous ammonia (28%).
concentrated in vacuo. The residue was dissolved in tetra- Reduction was performed in a hydrogen atmosphere at room
hydrofuran (15mL) and a sodium hydroxide solution (2N, temperature for 4h. After reduction, the catalyst was removed
1.5mL) was added, followed by stirring for 10min. The reac- by filtration, and the filtrate was concentrated. The residue
tion solution was concentrated in vacuo and the residue was was crystallized with ethyl ether and then the obtained crys-
dissolved in water (5mL). The solution was neutralized with tals were recrystallized from a small amount of water to give
a strongly acidic ion-exchange resin (Amberlite IR120B H 9 (0.76g, 2.76mmol).
AG). Ethanol was then added to the solution and the deposited
1H-NMR (D2O) δ: 2.38 (1H, dd, J=10.8 and 13.6Hz), 2.92

1170
Chem. Pharm. Bull.
Vol. 64, No. 8 (2016)
(1H, dd, J=9.8 and 13.6Hz), 3.17 (1H, t, J=10.6Hz), 3.37 (2H, was prepared without any pH adjustment. Subjects, who were
s), 7.16 (1H, t, J=6.9Hz), 7.18 (1H, t, J=8.2Hz), 7.27 (1H, s), members of our research group, tested each solution main-
7.46 (1H, d, J=8.0Hz), 7.68 (1H, d, J=8.0Hz). ESI-MS m/z: tained at room temperature with a “sip and spit” method.
275.51 (M+H)+.
They diluted each solution with water until they perceived the
Synthesis of (2R,4R)-4-Hydroxy-4-(1H-indol-3-ylmethyl)- same sweetness intensity as a 5% sucrose solution. The sweet-
5-oxo-proline (10, Monatin Lactam) The 1c sodium salt ness potency was calculated by dividing the concentration of
(3.00g, 9.05mmol) was dissolved in aqueous phosphoric acid 5% sucrose by the concentration that was perceived to have
(pH 2.0, 120mL), and the solution was heated at 90°C for 4d. equal sweetness.
The solution was concentrated to 10mL and extracted with
[Method 2] An aqueous solution of each test compound
ethyl acetate (100mL, three times). The organic solution was nearly equal to 10% sucrose was prepared according to the
washed with water (50mL), dried over anhydrous magne- above results (solution A). Solutions of 7.56% (score 1), 8.76%
sium sulfate, filtered, and concentrated in vacuo. The residue (score 2), 10.00% (score 3), 11.50% (score 4), and 13.23%
was recrystallized from chloroform, affording 10 (1.56g, (score 5) sucrose were prepared as external standard solutions
5.70mmol) as crystals.
(solution B). Seven trained panelists scored each test sample
1H-NMR (DMSO-d6) δ: 1.80 (1H, dd, J=6.6 and 13.0Hz), by comparing the sweetness intensity of solutions A and B.
2.41 (1H, dd, J=8.2 and 13.0Hz), 2.89 (1H, d, J=14.1Hz), The point of sucrose equivalency (Y) of solution A was calcu-
2.97 (1H, d, J=14.1Hz), 3.46 (1H, t, J=7.9Hz), 5.45 (1H, lated by an approximate equation (Y=6.575e0.1398X) and the av-
brs), 6.93–6.98 (1H, m), 7.04 (1H, t, J=7.0Hz), 7.16 (1H, d, erage score of each test compound (X). The sweetness potency
J=2.3Hz), 7.32 (1H, d, J=8.0Hz), 7.62 (1H, d, J=7.9Hz), 7.97 was then calculated by dividing Y by the concentration of the
(1H, s), 10.88 (1H, brs). ESI-MS m/z: 275.06 (M+H)+.
Preparation of (2R,4R)-Monatin Potassium Salt Dihy-
drate Crystals for X-Ray Crystal Structure Analysis The
test compound.
Acknowledgments We would like to acknowledge Profes-
1c ammonium salt (1.5g) was dissolved in water (10mL), sor Takeshi Kitahara, Professor Hidenori Watanabe, and late
and the solution was passed through a column filled with a Professor Murray Goodman for their helpful scientific guid-
cation-exchange resin (25mL, Diaion PK 228, potassium-type, ance. We would like to thank Tadashi Takemoto, Akira Otabe,
Mitubishi Chemical). The resulting eluent was concentrated to Kazutami Sakamoto, Takashi Oonuki, and Toshihisa Kato for
11.5g and dissolved in ethanol (60mL) at 60°C. The solution their encouragement and continued support of this work. We
was cooled to 10°C and stirred overnight. Precipitated crystals are grateful to Masakazu Sugiyama, Nao Funakoshi, Eriko
were collected by filtration and dried to give the 1c potassium Ono, and Ryoichiro Nakamura for their helpful discussions
salt (1.1g, mp: 213–214°C).
and assistance.
The 1c potassium salt (200mg) was then dissolved in water
(2.0mL) and heated to 65°C. Ethanol (10mL) was added, and
Conflict of Interest All authors were employees of Aji-
the solution was cooled to room temperature. A small amount nomoto Co., Inc. when this study was conducted and have no
of seed crystal was added to the solution and allowed to stand further conflicts of interest to declare.
until the crystal grew. Approximately 1 month later, the moth-
er liquor was removed and the crystal was dried.
References
1) Vleggaar R., Ackerman L. G. J., Steyn P. S., J. Chem. Soc., Perkin
Trans. 1, 1992, 3095–3098 (1992).
Single Crystal X-Ray Studies X-Ray diffraction data
of a crystal of the 1c potassium salt dihydrate were col-
lected using a Rigaku AFC5S diffractometer operated at 40kV
and 30mA, with graphite monochromated CuKα radiation
(λ=1.54178Å). Cell constants were obtained from a least-
squares refinement using the setting angles of 24 carefully
centered reflections. The data were collected at a temperature
of 296 1K using the ω–2θ scan technique to a maximum 2θ
value of 159.4°. The linear absorption coefficient (μ) for the
CuKα radiation was 31.1cm−1. An empirical absorption cor-
rection based on azimuthal scans of several reflections was
applied, which resulted in transmission factors ranging from
0.86 to 1.00. A correction for the secondary extinction was
applied (coefficient=1.59514e-06). The crystal structure was
solved by direct methods32) and expanded using Fourier tech-
niques.33) In the full-matrix least-squares refinement, non-hy-
drogen atoms and hydrogen atoms were refined anisotropically
and isotropically, respectively. Anomalous dispersion effects
were included in the Fcalc. All calculations were performed
using the teXsan34) crystallographic software package from
Molecular Structure Corporation.
2) Van Wyk P. J., Ackerman L. G., U.S. Patent 4975298 (1990)
3) Van Wyk P. J., Ackerman L. G., U.S. Patent 5128164 (1992).
4) Holzapfel C. W., Bischofberger K., Olivier J., Synth. Commun., 24,
3197–3211 (1994).
5) Abushanab E., Arumugam S., US Patent 5994559 (1999).
6) Nakamura K., Baker T. J., Goodman M., Org. Lett., 2, 2967–2970
(2000).
7) Fujiyasu J., Watanabe H., Kitahara T., Abstract of Papers Presented
at the General Meeting in 2000 of the Agricultural Chemical Soci-
ety of Japan, 3B128β (page 221).
8) Kitahara T., Watanabe H., JP Patent 2002060382A2 (2002).
9) Tamura O., Shiro T., Toyao A., Ishibashi H., Chem. Commun., 2003,
2678–2679 (2003).
10) Oliveira D. J., Coelho F., Tetrahedron Lett., 42, 679–3796 (2001).
11) Tamura O., Shiro T., Ogasawara M., Toyao A., Ishibashi H., J. Org.
Chem., 70, 4569–4577 (2005).
12) Bassoli A., Borgonovo G., Busnelli G., Morini G., Drew M. G. B.,
Eur. J. Org. Chem., 2005, 1652–1658 (2005).
13) Schiffman S. S., Dackis C., Percept. Psychophys., 17, 140–146
(1975).
14) Shallenberger R. S., Acree T. E., Lee C. Y., Nature (London), 221,
555–556 (1969).
Sensory Evaluation The sweetness intensity of each ste-
reoisomer of monatin was determined relative to sucrose in
water by the following two methods.
15) Helaine V., Bolte J., Tetrahedron Asymmetry, 9, 3855–3861 (1998).
16) Kasai T., Nishitoba T., Shiroshita Y., Sakamura S., Agric. Biol.
Chem., 48, 2271–2278 (1984).
[Method 1] An aqueous solution of each test compound

Vol. 64, No. 8 (2016)
Chem. Pharm. Bull.
1171
17) Larsen P. O., Sørensen H., Cochran D. W., Hagaman E. W., Wen- 27) Kawahara S., Amino Y., Mori K., Funakoshi N., Takemoto T., WO
kert E., Phytochemistry, 12, 1713–1719 (1973).
18) Olsen O., Sørensen H., Phytochemistry, 19, 1717–1721 (1980).
19) Rhugenda-Banga N., Welter A., Jadot J., Casimir J., Phytochemistry,
18, 482–484 (1979).
20) Juhl K., Gathergood N., Jørgensen K. A., Chem. Commun., 2000,
2211–2212 (2000).
21) Gathergood N., Juhl K., Poulsen T. B., Thordrup K., Jørgensen K.
A., Org. Biomol. Chem., 2, 1077–1085 (2004).
22) Wiley R. H., Kim K.-S., J. Org. Chem., 38, 3582–3585 (1973).
23) Leussing D., Stanfield C. K., J. Am. Chem. Soc., 86, 2805–2810
(1964).
Patent 2003059865A1 (2003).
28) Amino Y., Yuzawa K., Mori K., Takemoto T., WO Patent
2003045914A1 (2003).
29) Fry J. C., Yurttas N., Biermann K. L., Lindley M. G., Goulson M.
J., J. Food Sci., 77, S362–S364 (2012).
30) Ariyoshi Y., Agric. Biol. Chem., 40, 983–992 (1976).
31) Flack H. D., Acta Crystallogr. A, 39, 876–881 (1983).
32) Fan H.-F., SAPI91: Structure Analysis Programs with Intelligent
Control, Rigaku Corporation, Tokyo, Japan, 1991.
33) Beurskens P. T., Admiraal G., Beurskens G., Bosman W. P., de
Gelder R., Israel R., Smits J. M. M., DIRDIF94: The DIRDIF-94
program system, Technical Report of the Crystallography Labora-
tory, University of Nijmegen, the Netherlands, 1994.
34) teXsan: Crystal Structure Analysis Package, Molecular Structure
Corporation, 1985 & 1999.
24) Margolis S. A., Coxon B., Anal. Chem., 58, 2504–2510 (1986).
25) Passerat N., Bolte J., Tetrahedron Lett., 28, 1277–1280 (1987).
26) Ruffo A., Testa E., Adinolfi A., Pelizza G., Biochem. J., 85, 588–
593 (1962).