A Simple Synthesis of Alliin and allo-Alliin: X-ray Diffraction Analysis and Determination of Their Absolute Configurations

Article abstract of DOI:10.1021/acs.jafc.5b05501

A simple method for the isolation of the bioactive compound alliin from garlic, as well as a method for the synthesis of diastereomerically pure alliin and allo-alliin on a preparative laboratory scale, was developed. The absolute configuration of the sulfur atom in alliin and allo-alliin was assigned on the basis of enzyme reactivity, optical rotatory dispersion, and circular dichroism analyses. A comparison of the results from these analyses, in combination with an X-ray diffraction study on a protected allo-alliin derivative, revealed S and R configurations of the sulfur atoms in alliin and allo-alliin, respectively. In addition, the same ¹H NMR spectrum was observed for synthetic and natural alliin. The absolute configuration of natural alliin was assigned for the first time on the basis of the NMR spectrum and X-ray coordinates.

Full text of DOI:10.1021/acs.jafc.5b05501

Article
pubs.acs.org/JAFC
A Simple Synthesis of Alliin and allo-Alliin: X‑ray Diffraction Analysis
and Determination of Their Absolute Configurations
Wataru Hakamata,*,† Ryosuke Koyama,† Mizuki Tanida,† Tomomi Haga,† Takako Hirano,†
Makoto Akao,† Hitomi Kumagai,† and Toshiyuki Nishio†
†Department of Chemistry and Life Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi,
Kanagawa 252-0880, Japan
S
* Supporting Information
ABSTRACT: A simple method for the isolation of the bioactive compound alliin from garlic, as well as a method for the
synthesis of diastereomerically pure alliin and allo-alliin on a preparative laboratory scale, was developed. The absolute
configuration of the sulfur atom in alliin and allo-alliin was assigned on the basis of enzyme reactivity, optical rotatory dispersion,
and circular dichroism analyses. A comparison of the results from these analyses, in combination with an X-ray diffraction study
on a protected allo-alliin derivative, revealed S and R configurations of the sulfur atoms in alliin and allo-alliin, respectively. In
1
addition, the same H NMR spectrum was observed for synthetic and natural alliin. The absolute configuration of natural alliin
was assigned for the first time on the basis of the NMR spectrum and X-ray coordinates.
KEYWORDS: absolute configuration, alliin, allo-alliin, diastereoselective, garlic, synthesis, X-ray crystallography
the absolute configuration at the sulfur atom of the sulfoxide
moiety could not be determined.
INTRODUCTION
■
The beneficial dietary properties of Allium species have been
known for centuries and extensively exploited in food, spices,
and herbal remedies.1−5 Their major component, the sulfur-
containing amino acid alliin, can be converted into other
biologically active compounds such as allicin and ajoene via the
enzyme alliinase.6−10 It seems that alliin as a glycoside
precursor of allicin appeared for the first time in the literature
in 1909,11 but in 1948, another amino acid precursor of allicin
was isolated from Allium sativum and Allium ursinum and also
called alliin.12 Alliin exhibits attractive biological activities, for
example, an antioxidant activity, an affinity to N-methyl-D-
aspartate receptors, an inhibitory effect on platelet aggregation,
an antimicrobial activity, a modification the membrane fluidity
of tumor cells, and an effect on human tumor cell
proliferation.13−19 However, to gain a deeper understanding
of these biological properties, a close examination of the
chemistry of alliin and its diastereomer allo-alliin, the presence
of which in garlic was first reported in 2005,20 is required. Even
though the structures of alliin, 1, and allo-alliin, 2, are shown in
Figure 1, the absolute configurations of alliin and allo-alliin
remain so far uncertain.
Since the late 19th century the active components of Allium
species have attracted the attention of researchers, and many
studies have been devoted to the understanding of their
composition and biological activities.21−24 In the 1940s, the
presence of alliin in garlic and its role as a precursor in the
alliinase-mediated formation of allicin was established.12 In the
same study, discrepancies between the optical rotation of
natural and synthesized alliin were observed, which were
attributed to a difference in the absolute configuration of
cysteine derivatives 1−4 (Figure 1). Later, the same authors
were the first to propose a relationship between the absolute
configurations of 1−4 and the reactivity of alliinase.24 However,
Other attempts to determine the absolute configuration of
alliin unequivocally, for example, based on X-ray crystallo-
graphic analysis of structural analogues such as 5 or 6 (Figure
2),25−27 or based on ORD and CD analyses of propyl
derivatives of alliin such as 7 and 8,28−30 also failed to provide
a definitive result. To the best of our best knowledge, no report
of the absolute configuration of the sulfur atom in alliin has
appeared in the literature since 1971. Accordingly, the absolute
configuration of alliin has so far been deduced only on the basis
of the reactivity of alliin toward alliinase and its ORD and CD
curves compared to those of 5. Hence, an unambiguous
determination of the absolute configuration of the sulfur atom
of alliin and allo-alliin for further chemical and biological
studies remains to be carried out.
Moreover, the preparation of diastereomerically pure alliin
and allo-alliin also remains challenging. Previous reported
attempts to obtain diastereomerically pure alliin and allo-alliin
by recrystallization failed to achieve a satisfactory diastereo-
meric separation of allo-alliin.23,31,32 However, the diastereose-
lective synthesis of alliin via asymmetric oxidation of the sulfur
atom using [Ti(O-iPr)4],33 and the enzymatic syntheses of
alliin via asymmetric oxidation of the sulfur atom using Fe(II)/
α-ketoglutarate-dependent dioxygenase from Bacillus thurin-
giensis and dried cell powder of α-ketoglutarate-dependent
dioxygenase-expressing Escherichia coli were reported.34 Never-
theless, the diastereomeric excess of alliin did not exceed 83%
in any of the cases and thus remains to be improved.
Received: August 21, 2015
Revised: December 3, 2015
Accepted: December 5, 2015
Published: December 5, 2015
© 2015 American Chemical Society
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Figure 1. Structures of the S-allyl-cysteine sulfoxide stereoisomers.
Figure 2. Structures of the S-methyl-L-cysteine sulfoxide and S-propyl-L-cysteine sulfoxide diastereoisomers.
Japan) was dissolved in EtOH/H2O (2:1, v/v, 10 mL), before
triethylamine (1.3 mL, 17.5 mmol) and allyl bromide (0.9 mL, 5.83
mmol) were added. After for 20 h of stirring, the solvent was removed
under reduced pressure, and the obtained residue was purified by
column chromatography on silica gel (MeOH/2-propanol, 1:2, v/v) to
afford 0.73 g (71.5%) of S-allyl-L-cysteine methyl ester, 9.
In this study, we developed a simple method for the isolation
of pure alliin from garlic and a synthetic method for the
preparation of diastereomerically pure alliin and allo-alliin and
also determined the absolute configuration of the sulfur atoms
in alliin and allo-alliin based on NMR spectroscopy and X-ray
diffraction analysis.
Synthesis and Separation of Alliin Methyl Ester, 10, and
allo-Alliin Methyl Ester, 11. A solution of S-allyl-L-cysteine methyl
ester, 9 (501.2 mg, 2.85 mmol), in MeCN (5 mL) was treated with a
30% w/w solution of H2O2 in water (450 μL). After 26 h of stirring at
room temperature, 30 drops of a saturated aqueous solution of sodium
thiosulfate were added. A hydrogen peroxide concentration in the
mixture of <0.5 mg/mL was established by using a Quantofix peroxide
25 test stick (Sigma-Aldrich, St. Louis, MO, USA). Subsequently, all
solvents were removed to afford a residue containing 294.4 mg of a
mixture of alliin methyl ester, 9, and allo-alliin methyl ester, 10, which
were separated by column chromatography on silica gel (MeOH/2-
propanol, 1:2, v/v). After three purification cycles under these
conditions, 107.9 mg (32.4%) of allo-alliin methyl ester, 10, and 162.6
mg (21.5%) of alliin methyl ester, 9, were obtained with a good purity
(>99%).
TLC Analysis of the Diastereomer Mixture of Alliin Methyl
Ester, 10, and allo-Alliin Methyl Ester, 11. The diastereomeric
mixture was analyzed by TLC on silica gel using MeOH/2-propanol
(1:2, v/v), followed by spray-staining with a 2% ninhydrin solution
(Ninhydrin Spray II) (Wako Pure Chemical Industries, Tokyo, Japan).
The diastereomeric mixture displayed two spots that represent allo-
alliin methyl ester, 10 (Rf = 0.55), and alliin methyl ester, 11 (Rf =
0.47). The mixture of diastereomers was also analyzed by TLC on
MATERIALS AND METHODS
■
Synthesis of Alliin Derivatives. All new compounds were
1
1
characterized by H NMR, H−1H COSY, HMQC, and 13C NMR
spectrometry, mass spectrometry, and elemental analysis. The NMR
spectra were recorded on an ECA500 spectrometer (JEOL, Tokyo,
1
Japan) (500 MHz for H and 125 MHz for 13C). Chemical shifts are
expressed in parts per million relative to Me4Si (0 ppm). Low-
resolution mass spectra were obtained on a Quattro premier XE
instrument under positive and negative ion ESI conditions (Waters,
Milford, MA, USA). The single-crystal structure analysis was
performed using X-ray diffraction on a D8 Venture diffractometer
(Bruker AXS, Madison, WI, USA). Melting points were determined on
an MP-21 capillary apparatus (Yamato Scientific, Tokyo, Japan) and
are uncorrected. Optical rotation was measured on a P-1020 digital
polarimeter (JASCO, Tokyo, Japan). Column chromatography was
carried out using silica gel 60N (spherical neutral, particle size = 100−
210 μm) (Kanto Chemical, Tokyo, Japan). The progress of all
reactions was monitored by TLC on silica gel 60 F254 (0.25 mm)
(Merck Millipore, Darmstadt, Germany).
Synthesis of S-Allyl-L-cysteine Methyl Ester, 9. L-Cysteine
methyl ester hydrochloride (1.0 g, 5.83 mmol) (TCI Japan, Tokyo,
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Figure 3. (A) Synthetic route to 10 and 11: (a) allyl bromide, EtOH/H2O (2:1, v/v), 71.5%; (b) 30% H2O2, MeCN, 54.0%. (B) Formation of
intramolecular hydrogen bonds in 10 and 11.
silica gel using two other solvent systems, (A) CH2Cl2/MeOH/25%
aqueous NH4OH (7:1:0.1) and (B) EtOH/2-propanol/H2O (1:2:0.5),
followed by spray-staining with a 2% ninhydrin solution. Both solvent
systems exhibited two isomer spots (A, Rf = 0.53/0.47; B, Rf = 0.50/
0.45).
3.35 (dd, 1H, J = 5.5 Hz, J = 13.5 Hz, −S(O)CH2−), 3.49 (dd, 1H,
J = 7.5 Hz, J = 13.0 Hz, −CH2CHCH2), 3.61 (dd, 1H, J = 7.5 Hz, J
= 13.0 Hz, −CH2CHCH2), 3.80 (s, 3H, −COOCH3), 4.66 (dd, 1H,
J = 7.5 Hz, J = 11.0 Hz, −CHCOOCH3), 5.42 (dd, 1H, J = 1.0 Hz, J =
17.0 Hz, −CHCH2), 5.47 (d, 1H, J = 10.0 Hz, −CHCH2), 5.61
(d, 1H, J = 7.0 Hz, −CONH−), 5.90 (dddd, 1H, J = 7.5 Hz, J = 10.5
Hz, J = 15.0 Hz, −CHCH2); 13C NMR (125 MHz, CDCl3) δ 28.25
(−OC(CH3)3), 49.71 (−CH(NH)−), 52.82 (−CH(NHBoc)
CH2−), 53.06 (−OCH3), 56.12 (−CH2CHCH2), 80.64
(−OC(CH3)3), 124.08 (−CHCH2), 125.29 (−CHCH2),
155.15 (−CONH−), 170.51 (−COOCH3); ESI-MS (positive) m/z
314 [M + Na]+. Anal. Calcd for C12H21NO5S: C, 49.47; H, 7.27; N,
Alliin Methyl Ester, 9: 1H NMR (500 MHz, DMSO-d6) δ 2.05 (s,
2H, −NH2), 2.80 (t, 1H, J = 10.5 Hz, −CH2CH(NH2)−), 2.91 (dd,
1H, J = 3.5 Hz, J = 13.0 Hz, −CH2CH(NH2)−), 3.45 (dd, 1H, J = 8.0
Hz, J = 13.0 Hz, −CH2CHCH2), 3.62 (dd, 1H, J = 7.0 Hz, J = 13.0
Hz, −CH2CHCH2), 3.66 (s, 3H, −COOCH3), 3.67 (dd, 1H, J = 2.5
Hz, J = 10.5 Hz, −CH(NH2)−), 5.35 (dd, 1H, J = 2.0 Hz, J = 9.5 Hz,
−CHCH2), 5.37 (d, 1H, J = 1.0 Hz, −CHCH2), 5.87 (dddd, 1H,
J = 7.5 Hz, J = 10.0 Hz, J = 14.5 Hz, −CHCH2); 13C NMR (125
MHz, DMSO-d6) δ 49.15 (−CH(NH2)−), 51.88 (−OCH3),
55.07 (−CH2CH(NH2)− and −CH2CHCH2), 122.63 (−CH
CH2), 127.38 (−CHCH2), 174.85 (−COOCH3); ESI-MS (pos-
itive) m/z 192 [M + H]+. Anal. Calcd for C7H13NO3S: C, 43.96; H,
6.85; N, 7.32. Found: C, 43.94; H, 6.61; N, 7.10. [α]D23 −50.3° (c 1.0,
MeOH).
23
4.81; S, 11.00. Found: C, 49.19; H, 7.16; N, 4.81; S, 10.69. [α]D
+57.1° (c 1.0, CHCl3).
Synthesis of N-(tert-Butoxycarbonyl) allo-Alliin Methyl Ester,
13. A solution of alliin methyl ester, 10 (206.2 mg, 1.1 mmol), in
DMF (5 mL) was treated with di-tert-butyl dicarbonate (495.4 mg, 2.2
mmol). After 24 h of stirring, the solvent was removed in a centrifugal
evaporator at room temperature for 30 min. The obtained residue was
purified by column chromatography on silica gel (hexane/EtOAc, 1:3,
v/v) to afford 218.5 mg (70.0%) of N-(tert-butoxycarbonyl) allo-alliin
methyl ester, 13. Compound 13 was recrystallized from a hexane/
EtOAc solution to afford a single crystal suitable for X-ray
crystallography: 1H NMR (500 MHz, CDCl3) δ 1.45 (s, 9H,
−C(CH3)3), 3.15 (dd, 1H, J = 4.0 Hz, J = 13.0 Hz, −S(O)
CH2−), 3.24 (dd, 1H, J = 8.0 Hz, J = 13.0 Hz, −S(O)CH2−), 3.49
(dd, 1H, J = 7.5 Hz, J = 13.0 Hz, −CH2CHCH2), 3.59 (dd, 1H, J =
7.5 Hz, J = 13.0 Hz, −CH2CHCH2), 3.80 (s, 3H, −COOCH3), 4.72
(dd, 1H, J = 7.5 Hz, J = 11.0 Hz, −CHCOOCH3), 5.42 (dd, 1H, J =
1.0 Hz, J = 17.0 Hz, −CHCH2), 5.48 (d, 1H, J = 10.0 Hz, −CH
CH2), 5.73 (d, 1H, J = 7.0 Hz, −CONH−), 5.89 (dddd, 1H, J = 7.5
Hz, J = 10.5 Hz, J = 15.0 Hz, −CHCH2); 13C NMR (125 MHz,
CDCl3) δ 28.25 (−OC(CH3)3), 50.19 (−CH(NH)−), 51.79
(−CH(NHBoc)CH2−), 52.94 (−OCH3), 56.61 (−CH2−CH
CH2), 80.51 (−OC(CH3)3), 124.28 (−CHCH2), 125.12
(−CHCH2), 155.31 (−CONH−), 170.86 (−COOCH3); ESI-MS
(positive) m/z 314 [M + Na]+. Anal. Calcd for C12H21NO5S: C, 49.47;
H, 7.27; N, 4.81; S, 11.00. Found: C, 49.27; H, 7.19; N, 4.82; S, 10.72.
1
allo-Alliin Methyl Ester, 10: H NMR (500 MHz, DMSO-d6) δ
2.10 (s, 2H, −NH2), 2.87 (dd, 1H, J = 7.0 Hz, J = 13.5 Hz,
−CH2CH(NH2)−), 3.06 (dd, 1H, J = 6.5 Hz, J = 13.5 Hz,
−CH2CH(NH2)−), 3.51 (dd, 1H, J = 8.0 Hz, J = 13.0 Hz,
−CH2CHCH2), 3.64 (s, 3H, −COOCH3), 3.69 (dd, 1H, J = 7.5
Hz, J = 13.5 Hz, −CH2CHCH2), 3.82 (t, 1H, J = 7.0 Hz,
−CH(NH2)−), 5.36 (dd, 1H, J = 2.5 Hz, J = 10.0 Hz, −CHCH2),
5.39 (d, 1H, J = 1.0 Hz, −CHCH2), 5.87 (dddd, 1H, J = 7.0 Hz, J =
10.0 Hz, J = 14.5 Hz, −CHCH2); 13C NMR (125 MHz, DMSO-d6)
δ 49.30 (−CH(NH2)−), 51.76 (−OCH3), 54.19 (−CH2CH-
(NH2)−), 54.72 (−CH2CHCH2), 122.72 (−CHCH2), 127.28
(−CHCH2), 174.00 (−COOCH3); ESI-MS (positive) m/z 214 [M
+ Na]+. Anal. Calcd for C7H13NO3S: C, 43.96; H, 6.85; N, 7.32.
23
Found: C, 43.95; H, 6.80; N, 6.97. [α]D +52.9° (c 1.0, MeOH).
Synthesis of N-(tert-Butoxycarbonyl) Alliin Methyl Ester, 12.
A solution of alliin methyl ester, 9 (348.0 mg, 1.8 mmol), in DMF (5
mL) was treated with di-tert-butyl dicarbonate (836.2 mg, 3.6 mmol).
After 24 h of stirring, the solvent was removed in a centrifugal
evaporator at room temperature for 30 min. The obtained residue was
purified by column chromatography on silica gel (hexane/EtOAc, 1:3,
v/v) to afford 169.5 mg (31.9%) of N-(tert-butoxycarbonyl) alliin
methyl ester 12: 1H NMR (500 MHz, CDCl3) δ 1.45 (s, 9H,
-C(CH3)3), 3.18 (dd, 1H, J = 5.5 Hz, J = 13.5 Hz, −S(O)CH2−),
23
[α]D −101.1° (c 1.0, CHCl3). mp, 104.0−104.8 °C.
X-ray Diffraction Study of N-(tert-Butoxycarbonyl) allo-Alliin
Methyl Ester, 13. Colorless pieces of 13 were mounted on a quartz
fiber. Cell dimensions and intensities were measured using a D8
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Venture diffractometer using Cu Kα radiation (λ = 1.5418 Å). The
structure was resolved with X-Presso and APEX2. Crystallographic
data in CIF format are in the Supporting Information.35
Synthesis of Alliin, 1. Sodium methoxide (28% methanolic
solution, 50 μL) was added to a solution of alliin methyl ester, 9 (99.6
mg, 0.52 mmol) in MeOH/H2O (4:1, v/v, 10 mL) cooled to −20 °C.
After 2 h of stirring at −20 °C, dry Dowex 50W-X8, 200−400 mesh
(H+ form, 100 mg), was added, before the resin was removed by
filtration and the solvent was evaporated. The obtained residue was
purified by column chromatography on silica gel (12:3:1:0.5:0.5,
CH2Cl2/EtOH/MeOH/H2O/CF3COOH) to afford 26.1 mg (28.2%)
of alliin, 1.
Synthesis of allo-Alliin, 2. Sodium methoxide (28% methanolic
solution, 50 μL) was added to a solution of allo-alliin methyl ester, 10
(103.8 mg, 0.59 mmol) in MeOH/H2O (4:1, v/v, 10 mL) cooled to
−20 °C. After stirring at −20 °C for 2 h, dry Dowex 50W-X8, 200−
400 mesh (H+ form, 100 mg), was added, before the resin was
removed by filtration and the solvent evaporated. The obtained residue
was purified by column chromatography on silica gel (CH2Cl2/EtOH/
MeOH/H2O/CF3COOH, 12:3:1:0.5:0.5) to afford 15.2 mg (16.4%)
of allo-alliin, 2.
Figure 5. Molecular structure of 13 (thermal ellipsoids set at 50%
probability; red, oxygen; yellow, sulfur; blue, nitrogen).
Preparation of Garlic Lyophilization Powder. Peeled fresh
garlic was heated using a 500 W microwave oven (30 min/3 g). The
thus obtained garlic was homogenized with the corresponding weight
of water, before the homogenate was filtered using four layers of gauze.
The obtained solution was centrifuged at 15000g for 20 min at 4 °C.
The resulting supernatant was lyophilized.
Extraction and Purification of Alliin from Garlic Lyophiliza-
tion Powder. Garlic lyophilization powder (1.0 g) was ground in a
mortar and placed in an ice bath before CH2Cl2/EtOH/MeOH/H2O
(12:3:1:0.5, v/v, 10 mL) was added according to a modified reported
method.36 After 30 min of extraction, the precipitate was removed by
filtration and the solvent was evaporated to afford 30.9 mg of a crude
residue, which was purified by column chromatography on silica gel
(CH2Cl2/EtOH/MeOH/H2O/CF3COOH, 12:3:1:0.5:0.5, v/v) to
afford 14.7 mg of alliin. On the basis of a comparison of NMR
measurements, this method provided diastereomerically pure alliin.
RESULTS AND DISCUSSION
Synthesis of Diastereomerically Pure Alliin Deriva-
tives. To determine the absolute configuration of the sulfur
■
Figure 6. Syntheses of 1 and 2: (a) sodium methoxide, MeOH, 28.2%;
(b) sodium methoxide, MeOH, 16.4%.
sulfoxide derivatives 10 and 11 that were obtained from a
diastereoselective oxidation with H2O2, which pre-empts the
need for further HPLC purification. An L-cysteine methyl ester
hydrochloride was chosen as starting material for the syntheses
of compounds 10 and 11. Treatment of this starting material
with triethylamine and allyl bromide in EtOH/H2O yielded S-
allyl compound 9. The oxidation of the sulfur atom in 9 using a
30% H2O2 solution in MeCN furnished a diastereomeric
mixture of sulfoxides 10 and 11. After separation by column
chromatography on silica gel (MeOH/2-propanol, 1:2), 10 and
11 were obtained in pure form (Figure 3A). The easy
separation of these diastereomers by conventional column
chromatography on silica gel is probably facilitated by the
formation of an intramolecular hydrogen bond between the
hydrogen atoms of the amino groups and the oxygen atoms of
the sulfoxide moieties, which may induce a significant polarity
difference between conformers 10 and 11 (Figure 3B). Thus,
simple access to 10 and 11 is provided, which is especially
important for biological assays.
Figure 4. Synthesis of N-Boc derivatives 12 and 13: (a) Boc2O, DMF,
31.9%; (b) Boc2O, DMF, 70.0%.
atom in alliin, 1, and allo-alliin, 2, the availability of
diastereomerically pure samples of 1 and 2, suitable for
single-crystal X-ray diffraction analysis, is essential. As protected
amino acid derivatives generally crystallize easily, we focused on
the synthesis of diastereomerically pure protected derivatives of
1 and 2. Herein, we report a strategy for the preparation of
Single-Crystal X-ray Structure of Protected allo-Alliin,
11. To determine the absolute configurations of the sulfur
atoms in 10 and 11, we decided to prepare the corresponding
protected derivatives to obtain crystals suitable for X-ray
diffraction studies. Accordingly, the amino groups of 10 and 11
were protected using Boc2O to afford their N-Boc derivatives,
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1
Figure 7. Comparison of the amino acid region in the H NMR spectra of synthetic allo-alliin (2), synthetic alliin (1a), and natural alliin (1b).
that is, (Rc,Ss)-N-(tert-butoxycarbonyl)-S-allyl-L-cysteine sulf-
oxide methyl ester, 12, and (Rc,Rs)-N-(tert-butoxycarbonyl)-S-
allyl-L-cysteine sulfoxide methyl ester, 13, respectively (Figure
4). After recrystallization from hexane/EtOAc, colorless needles
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of 13 were obtained. On the basis of a single-crystal X-ray
diffraction analysis, the absolute configuration of the sulfur
atom and the α-carbon atom of 13 were determined as R and R,
respectively; the molecular structure of 13, which has the
empirical formula C12H21NO5S, is shown in Figure 5. It can
thus be concluded that the absolute configurations of the sulfur
and α-carbon atoms of 11 are also R and R, respectively.
Consequently, the absolute configuration of the sulfur and α-
carbon atoms in 10 and 12 should be S and R, respectively.
Absolute Configuration of Natural Alliin. Having
determined the absolute configurations of 10 and 11, we
subsequently focused on the elucidation of the absolute
configuration of natural alliin obtained from garlic. Diaster-
eomerically pure alliin and allo-alliin were synthesized from 10
and 11, respectively, and deprotection of 10 and 11 was
achieved with sodium methoxide in methanol (Figure 6).
Subsequently, garlic-derived alliin was extracted from the garlic
lyophilization powder and purified by column chromatography
on silica gel. An NMR analysis of synthetic alliin from 10,
synthetic allo-alliin from 11, and garlic-derived alliin, was
carried out to determine the absolute configuration of natural
carrying out the elemental analyses, as well as Bruker AXS
Japan Co., Ltd., for carrying out the X-ray crystallographic
studies.
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1
alliin by comparison. As shown in Figure 7, the H NMR
spectrum of garlic-derived alliin is identical to that of synthetic
alliin. From this result, it can be deduced that the absolute
configurations of the sulfur and α-carbon atom of garlic-derived
alliin are S and R, respectively. This is consistent with the
configuration of alliin reported by Barnsley in 1968,28 which
was based on enzyme reactivity, as well as ORD and CD curves.
Although the absolute configuration of garlic-derived alliin
has been the subject of research for many years, the focus of
these studies was mainly on its reactivity toward alliinase and its
ORD spectra. However, to better understand its biological
activity, a thorough investigation of the absolute configuration
was required. Therefore, we carefully determined the absolute
configuration of alliin as (Rc,Ss)-S-allyl-L-cysteine sulfoxide and
that of allo-alliin as (Rc,Rs)-S-allyl-L-cysteine sulfoxide in this
study.
Moreover, a simple method for the preparation of
diastereomerically pure alliin and allo-alliin on a laboratory
scale is provided herein; we believe that this may be useful for
the evaluation of the biological activity of alliin and allo-alliin. In
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.5b05501.
■
S
(17) Choi, M. K.; Chae, K. Y.; Lee, J. Y.; Kyung, K. H. Antimicrobial
activity of chemical substances derived from S-alk(en)yl-L-cysteine
sulfoxide (alliin) in garlic, Allium sativum L. Food Sci. Biotechnol. 2007,
16, 1−7.
Crystallographic data of compound 13 (CIF)
AUTHOR INFORMATION
Corresponding Author
*(W.H.) Phone: +81-466-84-3960. Fax: +81-466-84-3960. E-
mail: hakamata.wataru@nihon-u.ac.jp.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We express our sincere gratitude to the General Research
Institute of the College of Bioresource Sciences of Nihon
University for NMR and MS measurements. We also gratefully
acknowledge A. Sato of A-Rabbit-Science Japan Co., Ltd., for
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DOI: 10.1021/acs.jafc.5b05501
J. Agric. Food Chem. 2015, 63, 10778−10784

Journal of Agricultural and Food Chemistry
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DOI: 10.1021/acs.jafc.5b05501
J. Agric. Food Chem. 2015, 63, 10778−10784