Racemic and enantiopure synthesis and physicochemical characterization of the novel taste enhancer N-(1-carboxyethyl)-6-(hydroxymethyl)pyridinium-3-ol inner salt

Article abstract of DOI:10.1021/jf034246+

Convenient syntheses were developed to obtain on a multigram scale the novel taste enhancer N-(1-carboxyethyl)-6-(hydroxymethyl)pyridinium-3-ol 1, called alapyridaine, as a racemic mixture and as pure (+)-(S) and (-)-(R) enantiomers, respectively. 5-(Hydroxymethyl)-2-furaldehyde was used as key intermediate and was reacted with L-alanine under alkaline conditions to obtain racemic 1. Alternatively, reductive amination of 5-(hydroxymethyl)-2-furaldehyde with Raney-Ni/hydrogen and L- or D-alanine followed by mild oxidation led to (+)-(S)-1 and (-)-(R)-1, respectively. Racemization was promoted under alkaline and boiling conditions via a carbanion, the formation of which was facilitated by the electron-withdrawing effect of the iminium cation and the resonance-stabilizing capacity of the pyridinium moiety. Under these conditions, 1 was obtained in a 1:1 mixture of the phenol (1) and phenolate (1-H) forms as shown by X-ray diffraction. Racemic 1 formed monoclinic crystals of high molecular organization in which the phenol-type (RS)-1, the phenolate-type (RS)-1-H, sodium cations, and ethanol molecules are present. The crystal structure of [Na(1)(1-H)·(C₂H₆O)] shows one-dimensional μ₂-bridging-oxygen polymers stabilized by a three-dimensional network of ionic, hydrogen bond, and π-stacking interactions with channels occupied by solvent molecules.

Full text of DOI:10.1021/jf034246+

4040 J. Agric. Food Chem. 2003, 51, 4040−4045
Racemic and Enantiopure Synthesis and Physicochemical
Characterization of the Novel Taste Enhancer
N-(1-Carboxyethyl)-6-(hydroxymethyl)pyridinium-3-ol Inner Salt
RENAUD VILLARD,† FABIEN ROBERT,† IMRE BLANK,*,† GEÄ RALD BERNARDINELLI,‡
#
TOMISLAV SOLDO,§ AND THOMAS HOFMANN
Nestle´ Research Center, Vers-chez-les-Blanc, P.O. Box 44, 1000 Lausanne 26, Switzerland;
Laboratoire de Cristallographie, quai E. Ansermet 24, 1211 Gene`ve 4, Switzerland; Deutsche
Forschungsanstalt fu¨r Lebensmittelchemie, Lichtenbergstrasse 4, 85748 Garching, Germany; and
Institut fu¨r Lebensmittelchemie, Universita¨t Mu¨nster, Corrensstrasse 45, 48149 Mu¨nster, Germany
Convenient syntheses were developed to obtain on a multigram scale the novel taste enhancer N-(1-
carboxyethyl)-6-(hydroxymethyl)pyridinium-3-ol 1, called alapyridaine, as a racemic mixture and as
pure (+)-(S) and (-)-(R) enantiomers, respectively. 5-(Hydroxymethyl)-2-furaldehyde was used as
key intermediate and was reacted with L-alanine under alkaline conditions to obtain racemic 1.
Alternatively, reductive amination of 5-(hydroxymethyl)-2-furaldehyde with Raney-Ni/hydrogen and
L- or D-alanine followed by mild oxidation led to (+)-(S)-1 and (-)-(R)-1, respectively. Racemization
was promoted under alkaline and boiling conditions via a carbanion, the formation of which was
facilitated by the electron-withdrawing effect of the iminium cation and the resonance-stabilizing
capacity of the pyridinium moiety. Under these conditions, 1 was obtained in a 1:1 mixture of the
phenol (1) and phenolate (1-H) forms as shown by X-ray diffraction. Racemic 1 formed monoclinic
crystals of high molecular organization in which the phenol-type (RS)-1, the phenolate-type (RS)-1-
H, sodium cations, and ethanol molecules are present. The crystal structure of [Na(1)(1-H)‚(C2H6O)]
shows one-dimensional µ2-bridging-oxygen polymers stabilized by a three-dimensional network of
ionic, hydrogen bond, and π-stacking interactions with channels occupied by solvent molecules.
KEYWORDS: Maillard reaction; taste enhancer; alapyridaine; crystal structure; pyridinium betaines; X-ray
diffraction
INTRODUCTION
The Maillard reaction of reducing sugars and amino acids
plays an important role in thermal food processing as a source
of color and flavor (1, 2). Although odor-active volatile reaction
products have been studied in great detail (3-5), relatively little
has been published on nonvolatile constituents of the Maillard
reaction such as taste compounds, for example (6-8). Recently,
however, there has been an increasing number of publications
dealing with new structures of taste components generated by
the Maillard reaction. As a result, new chemical structures
having taste properties, such as bitter (9, 10), cooling (11), and
umami-like (12), have been isolated and identified from Maillard
reactions.
Figure 1. Structure of N-(1-carboxyethyl)-6-(hydroxymethyl)pyridinium-3-
ol isolated from a heated aqueous solution of glucose and L-alanine (13).
newly developed comparative taste dilution analysis used as a
screening procedure that combines instrumental analysis and
human taste perception (13). This novel Maillard reaction
product, also called alapyridaine, is tasteless on its own, but
enhances the sweet taste of the sugars glucose and sucrose, the
amino acid L-alanine, and the artificial sweetener aspartame (L-
Asp-L-Phe-OMe). Depending on the pH value, the detection
threshold of glucose was decreased by a factor of 16 in the
presence of an equimolar amount of alapyridaine. The (+)-(S)-1
enantiomer has been reported as the physiologically active
compound, whereas (-)-(R)-1 did not affect sweetness percep-
tion at all (13).
Very recently, N-(1-carboxyethyl)-6-(hydroxymethyl)pyri-
dinium-3-ol inner salt (1, Figure 1) has been identified in heated
aqueous solutions of D-glucose and L-alanine on the basis of a
* Author to whom correspondence should be addressed (telephone +21/
785-8607; fax +21/785-8554; e-mail imre.blank@rdls.nestle.com).
† Nestle´ Research Center.
‡ Laboratoire de Cristallographie.
§ Deutsche Forschungsanstalt fu¨r Lebensmittelchemie.
# Institut fu¨r Lebensmittelchemie.
10.1021/jf034246+ CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/31/2003

Novel Taste Enhancer
J. Agric. Food Chem., Vol. 51, No. 14, 2003 4041
+
Figure 2. Synthesis of racemic and enantiopure 1: a, Amberlite 15 (H resin), NEt3‚HCl, BuOAc/H O, reflux under N , 2.5 h, 52% yield; b, 1.0 equiv
2
2
of L-alanine, 1.9 equiv of 3, EtOH/water (1:1), aq NaOH (32%) f pH 9.4, RT (1.5 h) f reflux (72 h), 65% yield (sodium salt); c, 1.5 equiv of 3, 2 equiv
of L-alanine, water, aq NaOH (32%) f pH 8.5, Ni/H , RT, 5 bar, 48 h, 38% yield (ammonium salt); d, 1 equiv of 4 in water, 0 °C, 1.25 equiv of Br2/MeOH
2
(0.5 h), RT (1 h), 13% yield (ammonium salt). RT, room temperature.
Liquid Chromatography)Mass Spectrometry (LC-MS). An
analytical HPLC column Nucleosil 100-5C18 (Macherey and Nagel,
Du¨ren, Germany) was coupled to an LCQ-MS (Finnigan MAT, Bremen,
Germany) using positive (ESI+) and negative (ESI-) electrospray
ionization. After injection of the sample (2-20 µL), compounds were
chromatographed using the conditions described above.
In the course of the study on 1, we observed racemization of
1 during the synthesis procedure. Also, the target compound
could be obtained in crystalline form, which showed a particular
three-dimensional structure. In addition, racemization resulted
in a lower taste-enhancing activity of the racemate compared
to the (+)-(S) enantiomer, which needed to be studied in more
detail. Therefore, sufficient amounts of highly purified and
structurally well-defined alapyridaine were required in order to
(i) study the taste-enhancing activity of alapyridaine in food
products, (ii) gain an initial insight into the racemization process
as induced by thermal treatment, (iii) investigate the biochemical
mechanisms of alapyridaine on sweet taste enhancement in
human taste buds, and (iv) perform a safety assessment. We
now report on convenient syntheses of racemic 1 and its
enantiomers on a multigram scale, the racemization process,
and the crystal structure observed by X-ray diffraction.
Nuclear Magnetic Resonance (NMR) Spectroscopy. The samples
for NMR spectroscopy were prepared in Wilmad 528-PP 5 mm Pyrex
NMR tubes, using deuterated water (0.7 mL) as solvent. The NMR
spectra were acquired on a Bruker AC-250 or AM-360 spectrometer
(Rheinstetten, Germany), equipped with a quadrinuclear 5 mm probe
head, at 250/360 MHz for 1H and at 41.7/90 MHz for 13C NMR. One-
1
dimensional H NMR, 13C NMR, and distortionless enhancement by
polarization transfer (DEPT-135) spectra were acquired as described
earlier using standard conditions (14). The chemical shifts were
referenced to TSP or ethanol (residual solvent) as an internal standard.
Measurements were performed at room temperature (298 K). The
following abbreviations are used to describe multiplicity: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet. The spectra were interpreted
using MestRe-C 2.3 software.
EXPERIMENTAL PROCEDURES
Infrared (IR) Spectroscopy. IR spectra were recorded on a Perkin-
Elmer 1600 series FTIR apparatus (Norwalk, CT).
Polarimetry. Measurements were performed on a Perkin-Elmer 241
MC polarimeter with a sodium lamp at 589 nm and a slit of 1 mm.
The cuvette length was 100 mm.
Materials. L-Alanine, D-alanine, alumina (neutral), Amberlite 15,
n-butyl acetate (BuOAc), D-fructose, 5-(hydroxymethyl)-2-furaldehyde,
acetic acid, ammonium formate, silica gel 60, Amberlite IRA-400
(OH-), Raney nickel, heavy water (D2O), sodium 3-trimethylsilyl
[2,2,3,3-2H4]-propionate (TSP), dimethyl sulfoxide (DMSO), and tri-
ethylamine hydrochloride (NEt3‚HCl) were from Aldrich/Fluka (Buchs,
Switzerland). Ethanol (EtOH), methanol (MeOH), n-butanol, sodium
hydroxide (NaOH), RP18 reversed phase (LiChroprep, 25-40 µm),
formic acid, petroleum ether, and bromine (Br2) were from Merck
(Darmstadt, Germany). Hydrogen (5.0) was from Messer Griesheim
GmbH (Krefeld, Germany).
High-Resolution Gas chromatography (HRGC). A Carlo Erba
Mega 2 series chromatograph was used equipped with a cold on-column
injector and a flame ionization detector (FID). The column was a 30
× 0.32 mm i.d., 0.25 µm, DB-Wax (J&W Scientific, Folsom, CA).
The temperature was programmed as follows: 35 °C (2 min),
40 °C/min to 50 °C, 6 °C/min to 180 °C, and 10 °C/min to 240 °C
(10 min).
High-Pressure Liquid Chromatography)Diode Array Detection
(HPLC-DAD). HPLC was performed with an apparatus from Jasco
(Gross-Umstadt, Germany) composed of an HPLC pump system (PU
1580) with an in-line degasser (DG-1580-53), a low-pressure gradient
unit (LG-1580-02), and a diode array detector (MD 1515). Analytical
(250 × 4.6 mm) or semipreparative scale (250 × 10 mm) RP-18
columns (ODS-Hypersil, 5 µm) from Phenomenex (Aschaffenburg,
Germany) were used. Operating at a flow rate of 0.8 mL/min (analytical)
or 3.2 mL/min (semipreparative), the mobile phase was a mixture of
aqueous ammonium formate (10 mmol, pH 8.2) and methanol or formic
acid (0.1%, pH 2.5) and methanol. The injection volume was 10-100
µL. Software used included Jasco Borwin v1.50 and Jasco-PDA v1.50.
X-ray Diffraction. Light brown prisms 0.21 × 0.21 × 0.24 mm
were mounted on a quartz fiber with protection oil. Cell dimensions
and intensities were measured at 200 K on a Stoe IPDS diffractometer
with graphite-monochromated Mo[KR] radiation (λ ) 0.71073 Å); θmax
) 25.8°; 19655 measured reflections, 4213 independent reflections (Rint
) 0.072) of which 1819 had |Fo| > 4 σ (Fo). Data were corrected for
Lorentz and polarization effects and for absorption (T min, max )
0.9703, 0.9839). The structure was solved by direct methods using
SIR97 (15); all other calculations were performed with XTAL system
(16) and ORTEP (17) programs. Full-matrix least-squares refinement
based on F using the weight of 1/[σ2(Fo) + 0.00015(Fo2)] gave final
values of R ) 0.039, ωR ) 0.038, and GOF(F) ) 1.46(3) for 352
variables and 2037 contributing reflections. Maximum shift/error )
0.0003, max/min residual electron density ) 0.61/-0.48 eÅ-3. Hy-
drogen atoms were observed and refined with a fixed value of their
isotropic displacement parameter.
Syntheses. Following the synthetic sequence shown in Figure 2,
5-(hydroxymethyl)-2-furaldehyde (3), obtained by acid-catalyzed de-
hydration of D-fructose (2), was reacted with L-alanine under alkaline
conditions, resulting in racemic 1. Enantiopure 1 was prepared by
reductive amination of 3 in the presence of L- or D-alanine, followed
by mild oxidation of the intermediary furfurylamine derivative (4) to
(+)-(S)-1 and (-)-(R)-1, respectively.
5-(Hydroxymethyl)-2-furaldehyde (3). In a flask equipped with a
reflux condenser, D-fructose (10.0 g, 55 mmol), strong acid resin

4042 J. Agric. Food Chem., Vol. 51, No. 14, 2003
Villard et al.
Table 1. Synthesis of 5-(Hydroxymethyl)-2-furaldehyde (3) from
Table 2. 1H NMR Data of Racemic
D-Fructose
N-(1-Carboxyethyl)-6-(hydroxymethyl)pyridinium-3-ol (1) before and
after Refluxing under Alkaline Conditions in D O
2
reaction temp
trial time (h) (°C)
yield of
3a (%) ref
solvent(s)
purification
none
chromatography
none
1 at pD 4.7
1 after 24 h reflux at pD 9.5
1
2
3
16
2
100 DMSO
150 DMSO
32
24
0
nrb
24
nr
proton
δ (ppm)
multa
J (Hz)
δ (ppm)
multa
J (Hz)
CH
1.73
4.68
5.31
7.42
7.52
7.83
d
s
q
dd
d
7.2
1.55
s
3
40
50 H O/EtOAc
2
+
CH OH
2
H , H O
2
CH
H-4
H-5
H-2
7.2
9.0, 2.5
9.0
4
5
1
300
distillation
6
25
7.15
7.30
d
d
9.0
9.0
+
2.5
100 BuOAc, resin H , chromatography
52
nr
H O
2
d
2.5
a Yields of 3 were determined by HR/GC (FID detector). b Not reported.
a Multiplicity of signals: s, singlet; d, doublet; dd, double-doublet; q, quartet.
Amberlite 15 (10.0 g), and NEt3‚HCl (2.0 g) were stirred in a mixture
of n-butyl acetate (150 mL) and water (5 mL) and then heated at 100
°C under a nitrogen atmosphere for 2.5 h. After cooling to room
temperature, the mixture was decanted, and the organic layer was
concentrated under reduced pressure to yield 3 (3.3 g, 26 mmol, yield
) 52%, purity > 80%, GC) as an amorphous solid after filtration over
neutral alumina using a mixture of petroleum ether and ethyl acetate
(3:2, v/v) as the solvent.
water (35 mL) over a period of 30 min. After 1 h of stirring, the
resulting mixture was neutralized by adding a strongly basic ion-
exchange resin [Amberlite IRA-400 (OH-)] and filtered, and the filtrate
was freed from solvent in vacuo. The residue was dissolved in a formic
acid solution (0.1% in water) and applied to a 30 × 3 cm water-cooled
glass column filled with RP-18 (LiChroprep, 25-40 µm) in a formic
acid solution (0.1% in water). Using the same solvent as the mobile
phase and monitoring the effluent at 300 nm, (+)-(S)-1 was eluted
between 150 and 290 mL. Chromatography was repeated using
ammonium formate (10 mmol/L, pH 8.2) as the mobile phase, and the
fraction containing the target compound was freeze-dried twice to obtain
the (+)-(S)-1 inner salt (0.76 mmol) in 13% yield: 1H NMR (360 MHz,
D2O, pD 7.1, TSP) δ 1.86 (d, J ) 6.8 Hz, 3H), 4.84 (s, 2H), 5.42 (q,
J ) 6.8 Hz, 1H), 7.70 (dd, J ) 2.3, 8.6 Hz, 1H), 7.77 (d, J ) 8.6 Hz,
1H), 8.08 (d, J ) 2.3 Hz, 1H); LC-MS (ESI+), m/z (%) 198 (100, [M
+ 1]+), 220 (57, [M + Na]+); UV-vis (Millipore water, 12.5 mmol/
L, pH 7.1), λmax 248, 326 nm; [R]D20 +40.2° (c 0.01, Millipore water,
Racemic 1. A solution of L-alanine (1.0 equiv) and 3 (1.2 equiv) in
EtOH/water (50:50, v/v) was adjusted to pH 9.4 with an aqueous NaOH
solution (32%). After stirring at room temperature for 1.5 h, the mixture
was refluxed for 24 h and then cooled to room temperature. This
procedure was repeated twice after the addition of 0.4 and 0.3 equiv
of 3. The sample was then concentrated under reduced pressure and
purified by chromatography on silica gel 60 with a solvent mixture
composed of butanol, water, and acetic acid (2:2:1, v/v/v). Finally,
chromatography on RP18 reversed phase material with aqueous formic
acid (0.5% in water) and subsequent recrystallization from absolute
ethanol furnished racemic 1 in 65% yield with a purity of >98%
+
pH 7.0, NH4 salt).
(-)-(R)-1. Following the procedure described for the preparation of
(S)-4 and (+)-(S)-1, (-)-(R)-1 was synthesized using D-alanine. LC-
20
(NMR): mp (absolute ethanol), 91 °C; [R]D ) 0° (c 1.0, Millipore
water, pH 4.7, Na+ salt); 13C NMR (90 MHz, D2O, pD 4.7) δ 174.6
20
MS and NMR data were identical to those obtained for (+)-(S)-1: [R]D
(s), 160.9 (s), 142.8 (s), 133.8 (d), 133.1 (d), 128.6 (d), 65.5 (d), 59.7
+
-38.6° (c 0.01, Millipore water, pH 7.0, NH4 salt).
(t), 18.5 (q); IR (KBr) ν ) 3417, 3090, 3080, 3040, 2798, 1616 cm-1
;
UV-vis (ammonium formate, 10 mmol/L, pH 8.2), λmax 248, 326 nm;
UV-vis (formic acid, 0.1% in water, pH 2.5), λmax 300 nm; LC-MS
(ESI+), m/z (%) 198 (21, [M + 1]+), 220 (10, [M + Na]+), 395 (100,
[2M + 1]+), 417 (29, [2M + K]+), 592 (53, [3M + 1]+); crystal data,
light brown monoclinic prism, (C9H11NO4) (C9H10NO4)- Na+ (C2H6O),
Mr ) 462.5; µ ) 0.12 mm-1, dx ) 1.359 g‚cm-3, P 21/c, Z ) 4, a )
7.1705(3), b ) 22.7540(16), c ) 13.8627(7) Å, â ) 91.635(6)°, V )
2260.9(2) Å3.
RESULTS AND DISCUSSION
Syntheses. As shown in Figure 2, the synthesis of racemic
and enantiopure 1 is based on 5-(hydroxymethyl)-2-furanalde-
hyde 3 as the common intermediate. The synthesis of 3 was
studied in more detail, as it is a rather unstable and expensive
material. It has mainly been prepared from the readily available
D-fructose 2 by dehydration using Brønsted and Lewis acids as
catalysts (18-20). In general, the yields were low and strongly
dependent on the reaction conditions such as time and temper-
ature as well as the solvent used. The methods described for its
production so far suffered from the disadvantage that it was
obtained in aqueous or polar media from which isolation was a
challenging task.
To overcome these problems, we applied a phase transfer
procedure with n-butyl acetate/water using the strong acid resin
Amberlite 15 and ammonium salts as catalyst, thus resulting in
∼50% yield after chromatographic workup (Table 1, trial 5).
This procedure could easily be scaled up to 100 g without
decreasing the yield. Another advantage was the feasibility of
recovering the catalyst and solvents. Purification was achieved
by dry chromatography on alumina with petroleum ether/EtOAc
as eluent.
(S)-N-(1-Carboxyethyl)-2-(hydroxymethyl)-5-(methylamino)furan [(S)-
4]. A solution of 3 (30 mmol) and L-alanine (60 mmol) in water (30
mL) was adjusted to pH 8.5 with an NaOH solution (32% in water)
and was stirred in a hydrogenation vessel at room temperature for 30
min. After the addition of Raney nickel (0.75 g), the mixture was stirred
under a hydrogen atmosphere at 5 bar for 48 h. Another aliquot of 3
(10 mmol) was added, and hydrogenation was continued for an
additional 48 h. After filtration of the catalyst and a wash with methanol,
the filtrate was concentrated in vacuo. The residue was dissolved in
ammonium formate (10 mmol/L, pH 8.2) and applied on a water-cooled
30 × 4 cm glass column filled with a slurry of RP-18 (LiChroprep,
25-40 µm) in a mixture (99:1, v/v) of ammonium formate (10 mmol/
L, pH 8.2) and methanol. By using the same solvent mixture as the
mobile phase and monitoring the effluent at 220 nm, the fractions
eluting between 210 and 330 mL were collected to obtain (S)-4 (15.0
mmol) after freeze-drying in 38% yield: 1H NMR (250 MHz, D2O,
TSP) δ 1.43 (d, J ) 7.3 Hz, 3H), 3.57 (q, J ) 7.3 Hz, 1H), 4.18 (d, J
) 2.4 Hz, 2H), 4.56 (s, 2H), 6.39 (d, J ) 3.4 Hz, 1H), 6.51 (d, J ) 3.4
Hz, 1H); 13C NMR (41.7 MHz, D2O, TSP) δ 173.7 (s), 157.7 (s), 148.9
(s), 115.4 (d), 112.1 (d), 59.7 (d), 58.5 (t), 44.7 (t), 18.5 (q); LC-MS
(ESI+), m/z (%) 421 (100, [2 M + Na]+), 399 (95, [2 M + H]+), 222
(42, [M + Na]+), 200 (49, [M + H]+); UV-vis (ammonium formate,
10 mmol/L, pH 8.2), λmax 210 nm.
Alternatively, 3 was obtained at ∼30% yield without puri-
fication using DMSO as solvent (trial 1). However, a much
longer reaction time was required compared to trial 5. Increasing
the reaction temperature to 150 °C led to lower yields (trials 2
and 4). This is most likely due to polymerization reactions,
which we observed in most of the samples shown in Table 1,
known to be favored at high temperatures and in highly
(+)-(S)-1. A solution of bromine in methanol (6.0 mmol in 10 mL)
was added dropwise to a cooled (0 °C) solution of (S)-4 (7.5 mmol) in

Novel Taste Enhancer
J. Agric. Food Chem., Vol. 51, No. 14, 2003 4043
Figure 3. Racemization of 1 in an aqueous alkaline solution under reflux conditions showing the delocalization of the negative charge in the mesomeric
forms 1a, 1b, and 1c.
concentrated samples. Lowering the temperature to 50 °C, on
the other hand, did not yield 3 at all (trial 3). In general,
purification was required to separate the brown polymeric
substances prior to reaction with alanine. Distillation did not
give satisfactory results; total polymerization of 3 occurred at
80 °C and 0.02 mbar pressure.
Reaction of 3 with L-alanine in alkaline aqueous solutions
on a 100 g scale gave rise to racemic 1, as indicated by
polarimetry, in a 65% yield after purification (Figure 2).
Reactant 3 was added in three to five portions and in an overall
excess (alanine to 3, 1:2 equivalent) to improve the yields by
limiting polymerization reactions of 3 and also by entirely
consuming the amino acid. The latter facilitated separation of
the target 1 from residual alanine, which in general is a
challenging task. Purification of 1 was achieved by column
chromatography on silica gel using a ternary eluent composed
of butanol, water, and acetic acid. If very pure material is
required, additional chromatography on an RP18 column using
aqueous formic acid as the eluent is suitable. However,
precipitation from ethanol was found to be a convenient
procedure for purifying multigram amounts of racemic 1. The
overall yield of 1 was 30% on the basis of D-fructose as the
starting material.
Enantiopure 1 was prepared by adapting the procedure
described for N-alkyl pyridinium betaines (23) to alapyridaine
(Figure 2). Reductive amination of 3 and L-alanine with Raney
nickel/hydrogen resulted in the corresponding furfurylamine
derivative (S)-4, (S)-N-(1-carboxyethyl)-2-(hydroxymethyl)-5-
(methylamino)furan. The latter was converted into the target
pyridinium betaine compound by mild oxidation with bromine
in water/methanol to yield (+)-(S)-1. Similarly, the reaction with
D-alanine resulted in (-)-(R)-1. Purification was performed by
column chromatography using an RP18 column and aqueous
formic acid as mobile phase, monitoring the target compound
at 300 nm. Optical rotation of +40.2° and -38.6° indicated
the presence of (S)-1 and (R)-1, respectively.
Figure 4. ORTEP view of the asymmetric unit of racemic [Na(1)(1-H)‚
(C2H O)] (solvent molecule omitted) showing the phenol (labeled a) and
6
phenolate (labeled b) forms. Ellipsoids are represented with 40%
probability. Selected interatomic distances: C4a−O2a ) 1.338(4) Å, C4b−
O2b ) 1.308(4) Å, Na‚‚‚O3a ) 2.376(3) Å, Na‚‚‚O1b ) 2.352(3) Å.
Racemization. Despite the use of enantiopure alanine as the
starting material, 1 was obtained as a racemate under reflux
conditions in alkaline aqueous solutions. To study the racem-
ization process, L-alanine or racemic 1 was refluxed in D2O at
pD 9.5 for 24 h. No proton-deuterium exchange was observed
for L-alanine by 1H NMR (data not shown). This is in agreement
with previously published data, indicating that more severe
conditions are required for racemization of amino acids (24,
25).
However, the data obtained for racemic 1 indicated the
disappearance of the enantiotopic R-proton (Table 2). Compared
to L-alanine, the acidity of the proton at the chiral center is
increased by the pyridinium moiety. Abstraction of the R-proton
results in the formation of a carbanion at the asymmetric carbon,
which can be stabilized by delocalization of the negative charge
through the aromatic pyridinium moiety, thus facilitating the
racemization step via the iminium mesomeric form 1b upon
reprotonation (Figure 3). At present, however, it is not yet clear
if the racemization process is due to the formation mechanism
or the consequence of the long thermal treatment during the
synthesis procedure of 1.
Three-Dimensional Structure Characterization of Racemic
1. Suitable crystals for X-ray diffraction of racemic 1 were
obtained by crystallization in absolute ethanol. An analysis of
the crystal structure shows that the asymmetric unit (Figure 4)
is composed of one molecule of 1 (phenol form), one depro-
tonated molecule at the alcohol function (phenolate form, 1-H)
compensating for the positive charge of one sodium cation, and
one molecule of ethanol. Due to the absence of residual electron
density around O2b and the shorter observed C-O bond
distance, the localization of the negative charge was attributed
to this atom. Moreover, the three other hydroxyl groups are
involved as donors in hydrogen bond interactions with two
distinct carboxylates and O2b as acceptors.

4044 J. Agric. Food Chem., Vol. 51, No. 14, 2003
Villard et al.
nels parallel to [100] in which the ethanol molecules are lo-
cated.
In conclusion, we have developed convenient synthetic
approaches to obtain on a multigram scale the novel, foodborne
taste enhancer 1 as a racemic mixture and also its enantiomers,
required for extended sensorial evaluation of alapyridaine in
food products as well as for safety assessment. Under alkaline
and boiling conditions, racemization occurred via an intermedi-
ary carbanion, the formation of which was facilitated by the
electron-withdrawing effect of the iminium cation and the
resonance-stabilizing capacities of the pyridinium moiety.
Racemic 1 formed monoclinic crystals in which (RS)-1 (phenol),
(RS)-1-H (phenolate), sodium cations, and ethanol molecules
are assembled as [Na(1)(1-H)‚(C2H6O)] in one-dimensional µ2-
bridging-oxygen polymers stabilized by a three-dimensional
network of ionic, hydrogen bond, and π-stacking interactions
with channels occupied by solvent molecules.
ACKNOWLEDGMENT
Figure 5. Perspective view of [Na(1)(1-H)] crystal structure showing the
three phenol and three phenolate forms involved in the distorted octahedral
coordination of Na+.
We are grateful to Dr. Elizabeth Prior for linguistic proofreading
of the manuscript. I.B. and T.H. contributed equally to this work.
Supporting Information Available: Crystallographic data of
racemic 1 and CIF file. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Received for review March 13, 2003. Revised manuscript received May
5, 2003. Accepted May 5, 2003.
JF034246+