1(1) (2012), 38-46
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THE SYNTHESIS AND STRUCTURE CHARACTERIZATION OF DEOXYALLIIN AND ALLIIN
Vesna D. Nikolić*, Dusica P. Ilić, Ljubisa B. Nikolić, Mihajlo Z. Stanković,
Ljiljana P. Stanojević, Ivan M. Savić, Ivana M. Savić
Faculty of Technology, University of Nis, Leskovac, Serbia
Medical properties of garlic are mainly attributed to organosulfur compounds
which are formed by enzymatic, chemical and thermal transformations of
­S-allyl-L-cysteine during crushing, drying or processing the bulb. Garlic has
a bactericidal, bacteriostatic, antimicotic, antiviral, antisclerotic, antihypertensive, anti-aggregation and anticancer activity. The aim of this paper was to
synthesize alliin from a genuine compound of deoxyalliin. Deoxyalliin is a main
precursor for obtaining alliin which is contained in the garlic cloves. L-Cysteine
and allyl bromide were used as the initial precursors for the synthesis of deoxyalliin. It is purified by recrystallization from absolute ethanol. The obtained
deoxyalliin (>95 %) was used for the synthesis of alliin by oxidation with hydrogen peroxide. The structural characterization of synthesized deoxyalliin and
alliin was studied by using UV, FTIR and MS spectrometry. The separation of
optical alliin isomers was carried out by using a thin layer chromatography. The
identification of synthetic compounds was achieved on the basis of literature
data for Rf-values.
(ORIGINAL SCIENTIFIC PAPER)
UDC 615.322:582.573.16
Keywords: synthesis, alliin, deoxyalliin, structural characterization.
Introduction
Garlic (Allium sativum) is valued in many parts of the alkyl derivates of sulfur. The most significant amino acid in
world for its pungent aroma and flavor. However, most in- the mixture is a distereoisomer of alliin, S-allyl-L-cysteine
vestigations of health benefits of the garlic have consid- sulfoxide, an organosulfur compound that contributes to
ered its medicinal rather than culinary uses. Medicinal use its therapeutic value and pharmacological importance [23,
of garlic goes back to Greek and Egyptian antiquity. In in 24]. It is a derivative of the cysteine amino acid.
vitro studies, garlic has been found to have antibacterial
The ways proposed for the biosynthesis of alliin are de[1-3], antiviral, and antifungal (fungal infections of the skin scribed [25-29]. For the purpose of identification and quanand the ear) activity [4–9]. Garlic is widely used for its car- titative determination of alliin in garlic and garlic products
diovascular benefits [10]. It may also lower blood pressure different analytical methods were used, such as: liquid
since it helps to keep blood vessels to the heart flexible chromatography (LC) [30], high performance liquid chroin older people. One of the most intriguing possibilities of matography (HPLC) [31,32,33], liquid chromatography
garlic is that it helps in the prevention of cancer. It is used coupled with mass spectrometry detection (LC/MS) [34],
to prevent stomach and colon cancers [11-13]. Allium sa- gas chromatography (GC) [35], high-throughput method
tivum has been found to reduce platelet aggregation [14- [36], spectrophotometric method [37], nuclear mass reso17] and hyperlipidemia [18, 19]. Also, garlic can reduce nance (NMR) and mass spectroscopy (MS) [38], high-perblood sugar levels and may improve the insulin response formance thin layer chromatography (HPTLC) [39]. A rapid
[20]. Sulfur compounds of garlic (alkyl-cysteine derivates, and sensitive HPLC-electrospray/MS method has been
alkyl-sulfide, alkyl-disulfide and alkyl-polysulfide, thio- developed to determine alliin in rat plasma [40].
sulfonate, etc.) [21] are responsible for most medicinal
Even though it is a pharmacologically inactive, alliin
properties of this herb. These compounds are formed by represents the initial compound for a large number of secenzymatic, chemical and thermal transformation of alliin ondary reactions where therapeutic important products
after processing the bulb. Stoll and Seebeck [22] isolated containing sulfur (alliicin, vinyldithiine) are obtained. Alliin
the mixture of amino acids with the content of sulfur and can be isolated from garlic, but the main problem is an
*Author address: Vesna Nikolić, Faculty of Technology,
16000 Leskovac, Bulevar oslobođenja 124, Serbia
e-mail: [email protected]
The manucsript received: May, 16, 2012.
Paper accepted: Jun, 18, 2012.
38 1(1) (2012), 38-46
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alliinase enzyme which, after destroying the cell structure
of the plant material, transforms allin to alliicin. Therefore,
there was a need to develop a procedure for allin synthesis. So, the aim of this study was to synthesize alliin from
deoxyalliin by oxidation with hydrogen peroxide and to obtain deoxyallin itself, as well as their purification by using
TLC method.
Experimental
for 24 h. The residual solvent was evaporated by using
rotary evaporator and then dissolved in the mixture of
acetone:water:glacial acetic acid (65:34:1, v/v/v). The
crystals were precipitated and washed using the cold mixture of acetone:water:glacial acetic acid (65:34:1, v/v/v),
as well as the cold absolute ethanol. L(±)-aliin, the degree
purity of 95 %, was obtained after drying the crystals.
Characterization methods of synthesized compounds
Substances. L-cysteine standard (>99 %), allyl bromide
(>98 %), sodium hydroxide, ninhydrin, cadmium(II) acetate were purchased from Merck Chemicals Ltd. (United
Kingdom). Silica-gel G60 is a product of Wacker Chemie
AG (Germany). Absolute ethanol, glacial acetic acid, sulfuric acid (96 %), hydrogen peroxide (30 %), n-propanol,
n-butanol and acetone were bought from Zorka Pharma
(Serbia).
The preparation of ninhydrin-Cd-acetate reagent. Solution I: ninhydrin standard (0.3 g) was dissolved in n-propanol (100 cm3); Solution II: cadmium(II) acetate (1 g) was
dissolved in glacial acetic acid (50 cm3) under reflux in a
boiling water bath. The solvents I and II were immediately
mixed in the ratio of 5:1 (v/v) before using.
The preparation of thin layer. Silica gel G60 (30 g) was
mixed with distilled water (65 cm3) for 1 min, and then applied to the glass plates (20×20 cm) in the thickness of
0.25 mm.
UV spectrophotometric method. The UV spectra of
aqueous solutions of L-cysteine, L-deoxyalliin and L(±)alliin were recorded in the wavelength range of 190-350 nm
on the Cary 100 Conc. spectrophotometer. The spectrum
of allyl bromide was recorded in the ethanol solution under
the the same conditions.
FTIR spectroscopic method. FTIR spectra of L-cysteine,
L-deoxyalliin and L(±)-alliin were recorded by using a
potassium bromide pellet technique in the wavenumber
range of 4000-600 cm-1 on a Bomem Hartmann & Braun
MB-series FTIR spectrophotometer. The technique of a
thin film between potassium bromide plates was applied
for recording FTIR spectrum of allyl bromide on the same
apparatus.
Mass spectrometry. Mass spectra of L-cysteine, L-deoxyalliin and L(±)-aliin were obtained on the model 8230 mass
spectrometer by using the electron ionization method. The
applied electron energy was 70 eV, while the temperature
of ionic source was 250 0C.
The synthesis procedure of L-deoxyalliin (S-allyl-Lcysteine). L-cysteine and allyl bromide were used as a Thin layer chromatography. The aqueous solutions of Lprecursor for the synthesis of L-deoxyalliin. Firstly, L- cysteine, L-deoxyalliin and L(±)-aliin (20 µl) were added
cysteine was suspended in absolute ethanol, and then so- on a thin layer of Silica-gel G (a sheet of glass 20×20 cm,
dium hydroxide (20 mol dm-3) was added to achieve a ba- the layer thickness of 0.25 mm). The chromatograms were
sic medium. After that, allyl bromide was slightly added in developed with n-butanol:glacial acetic acid:water (2:1:1,
the suspension. The reaction was performed in the cold in v/v/v). The spots on the chromatographic plate were dethe first 1 h, and then at ambient temperature for 2 h. The tected by ninhydrin reagent spraying. In the case of the
reaction mixture was neutralized to pH 5.5 and placed in control chromatogram the spots were detected with sulfuthe cold place to the appearance of L-deoxyalliin crystals. ric acid (50 %). After drying at the temperature of 105 0C,
it came to the appearance of spots that were identified by
Recrystallization of L-deoxyalliin. After dissolving crude using literature data for the Rf values of the investigated
L-deoxyalliin in acetic acid (1 %), it was transferred in 15 compounds.
fold higher volume of absolute ethanol. The crystals of LResults and discussion
deoxyalliin (>95 %) were obtained after evaporating the
absolute ethanol to half of the volume under reduced presSynthesis and structural characterization of L-deoxysure.
alliin. L-deoxyalliin, as a precursor for obtaining L(±)-alliin,
Synthesis of alliin (S-allyl-L-cysteine sulfoxide). Al- was synthesized from L-cysteine and allyl bromide in the
liin was synthesized by oxidation of synthesized and following chemical reaction (Fig. 1):
pre-crystallized L-deoxyalliin at ambient temperature
Figure 1. The chemical reaction of L-deoxyalliin synthesis
39
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A crude L-deoxyalliin was purified by precipitation from
absolute ethanol. The obtained L-deoxyalliin (>95 %) was
used for chemical synthesis of alliin by oxidation with hydrogen peroxide. L(±)-aliin was the product of that oxidation process. The structure characterization of deoxyalliin
was performed by the use of UV, FTIR and MS spectroscopy.
UV analysis. UV spectra of recrystallized deoxyalliin,
L- cysteine and allyl bromide are presented in Fig. 2. An
intensive and wide maximum can be noticed at 220 nm,
which originates from n→π* and n→σ* transition in the
carbonyl group of deoxyalliin. In this range, π→π* transition is appeared due to the presence of C=C bond in the
structure of deoxyallliin. Moving these maximums to higher values of wavelength is caused by the presence of NH2
auxochrome, which has a significant impact on n→π* transition and smaller effect on π→π* transition. Unlike deoxyallin, UV spectrum of L-cysteine has a narrower maximum
at the wavelength of 200 nm which indicates n→π* and
π→π* transition of the carboxylic chromophore group. The
presence of auxochrome NH2 group with a free-electron
pair close to the chromophore group affects the maximus
movement to higher wavelengths. The SH auxochrome
group has a weaker effect on the mentioned maximum
movement because it is more distant than a chromophore
group in the structure of L-cysteine. The secondary maximum absorbance at 225 nm due to π→π* transition at
C=C bond in the structure allyl bromide. This maximum
is moved to higher values of the wavelength, because Br
group has a batochromic effect on the absorption of C=C
bond. Precursors were transformed to deoxyalliin during
the synthesis process, which was confirmed by differences in the absorption of the UV spectra. The purity of the
obtained deoxyalliin was acceptable for a further synthesis
process of alliin.
Figure 2. UV spectra of deoxyalliin, L-cysteine and allyl bromide
FTIR analysis. FTIR spectra of L-cysteine (Fig. 3) and allyl clearly observed due to the overlap with νas(C=O). A low
bromide (Fig. 4) were recorded in the aim of the structure intensity band at 1423 cm-1 is from δs(NH3+). In the wavecharacterization of synthesized and purified deoxyalliin. number range of 3500-3200 cm-1, the bands of νas(CH)
The characteristic band in the wavenumber range of 3100- and νs(CH) vibration of terminal allyl group appeared in the
2600 cm-1 is due to ν(NH3+) vibration at L-cysteine. A wide IR spectrum of allyl bromide (Fig. 4). The valence vibration
and medium intensity band was expanded as the results of C=C group at 1637 cm-1, and the deformation vibration
of combining bands and over tones which are placed from γ(CH) out-of-plane in the form of two bands at 928 and 986 cm-1
2000 cm-1. A valence asymmetric vibration of C=O group are also noticed. Over tones at 1800 cm-1, as well as the
and deformation asymmetric vibration of NH3+ should be presence of the band at 928 cm-1 are the confirmation of
expected in the range of 1600-1560 cm-1. A strong band at allyl group in the molecular structure. The FTIR spectrum
1590 cm-1 originates from valence asymmetric vibration of of deoxyalliin (Fig. 5) is different than spectra of precurC=O bond from L-cysteine. In this range of wavenumber, sors (Fig. 3 and 4), indicating their transformation during
there is a low intensity band of δas(NH3+) and cannot be the chemical reaction. A wide and medium intensity band
40 1(1) (2012), 38-46
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of ν(NH3+) in the range of 3100-2600 cm-1 covers the low
intensity bands of allyl group, νas(CH) and νs(CH). In the
range of 1600-1560 cm-1, the vibration of δas(NH3+) is covered by the band of νas(C=O). A band at 1499 cm-1 origi-
nates from δs(NH3+), while a lower intensity band at 1417
cm-1 comes from vibrations of C=O group.
Figure 3. FTIR spectrum of L- cysteine
Figure 4. FTIR spectrum of allyl bromide
Figure 5. FTIR spectrum of L-deoxyalliin
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MS analysis. Mass spectrum of L-deoxyalliin is shown in
Fig. 6. The dominant peak at m/e 162 presents (M+1) peak
in the case of deoxyalliin. The peak at m/e 145 appeared
after removing hydroxyl or ammonia fragment, while the
peak at m/e 122 was obtained by removing CH2=C=CH2
fragment. As the results of CH2=CH-CH=S fragment elimination from the molecule of deoxyalliin, the peak at m/e 90
occurred in MS spectrum. The other peaks are not significant for consideration due to low intensities.
Figure 6. MS spectrum of L-deoxyalliin
Synthesis and structural characterization of alliin
was confirmed by applying UV, FTIR and MS methods.
The synthesis of alliin was performed by the oxidation pro- The reaction of L(±)-alliin synthesis is shown in the followcess, where deoxyalliin and hydrogen peroxide were used ing chemical equation (Fig. 7):
as the initial reactants. The structure of obtained L(±)-alliin
Figure 7. The chemical reaction of L(±)-aliin synthesis
UV analysis. UV spectrum of the water solution of L(±)- originates from π→π* transition of terminal C=C bond and
alliin is presented in Fig. 8. A significant difference between n→σ* transition of S=O group. A low intensity saddle at
UV spectra of the obtained product and its precursors, 254 nm is due to n→π* transition in the allyl group.
­L-cysteine and allyl bromide (Fig. 2) can be noticed. This
was expected, considering that there is a difference in the
structure of the observed compounds. Namely, the maximum at 198 nm and slightly defined saddle at 254 nm exist
in the UV spectrum of alliin (Fig. 8). The maximum at 198 nm
42 1(1) (2012), 38-46
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Figure 8. UV spectrum of L(±)-alliin
FTIR analysis. FTIR spectrum of L(±)-alliin (Fig. 9) are
the similar in the intensity, shape and position of bands to
FTIR spectrum of deoxyalliin (Fig. 5). The existence of a
strong intensity band at 1091 cm-1 in the spectrum of allin
originates from the valence vibration of S=O group. This
band is the evidence that alliin was obtained by the oxidation of deoxyalliin.
MS analysis. In the addition to mentioned methods and
in order to completely characterize the synthetic alliin, MS
spectrometry was applied. MS spectrum of L(±)-alliin is
presented in Fig. 10. As it can be seen in the spectrum, a
dominant peak at m/e 178 refers to (M+1) and indicates
that the molar mass of synthetic L±)-alliin is 177. A peak
at m/e 355 originates from alliin dimer that occurs during
the synthesis.
Figure 9. FTIR spectrum of L(±)-alliin
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Figure 10. MS spectrum of L(±)-alliin
TLC analysis. L-cysteine as a precursor in deoxyalliin
synthesis, the reaction mixture of deoxyalliin, pure recrystallized deoxyalliin, the reaction mixture of alliin and pure
recrystallized alliin were analyzed by TLC method. The
results of the investigation indicate that the reaction mixtures did not have a significant amount of by-products in
both cases of synthesis. The optical isomers of alliin can
be successfully separated by using this method. All compounds were identified comparing the obtained Rf-values
with the literature values [41] under identical experimental
conditions of TLC. The results of these investigations are
presented in Table 1.
Table 1. The literature and obtained data of Rf data, as well as the color of spots
for L- cysteine, deoxyalliin and optical isomers of alliin
Literature
Obtained Rf-
Rf-values
values
deoxyalliin
0.68
0.67
orange
L(+)-alliin
0.58
0.58
pink
L(-)-alliin
0.49
0.45
orange
L- cysteine
0.56
0.55
yellow
Compounds
The control chromatogram which was sprayed with
sulfuric acid (50 %) was used as the confirmation of the
obtained results. The number and position of spots at the
control chromatogram correspond to the chromatogram
caused by the nynhidrin reagent.
The biosynthetic procedures of obtaining alliin [25,26]
require an incubation of the callus and the extraction of the
obtained alliin. Unlike these routes of alliin biosynthesis,
the proposed synthetic procedure is faster and simpler.
44 Spot color
Conclussion
An optimal procedure for deoxyalliin synthesis was
developed as a main precursor for the synthesis of alliin.
Also, the synthesis procedure of L(±)-alliin from deoxyalliin
using hydrogen peroxide was successfully defined. The
synthetic alliin presents the precursor for the synthesis of
the biologically active compound of allicin. The purified deoxyalliin and alliin were structurally characterized by using
UV, FTIR and MS spectroscopic methods. The separation
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of alliin optical isomer was successfully achieved by applying TLC method. The identification of alliin isomers and
deoxyalliin was performed on the basis of literature data
of Rf-values.
Acknowledgements
This work was supported by the Ministry of Education and
Science of the Republic of Serbia under the project TR34012.
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Izvod
SINTEZA I STRUKTURNA KARAKTERIZACIJA DEOKSIALIINA I ALIINA
Vesna D. Nikolić*, Dusica P. Ilić, Ljubisa B. Nikolić, Mihajlo Z. Stanković,
Ljiljana P. Stanojević, Ivan M. Savić, Ivana M. Savić
Tehnološki fakultet, Univerzitet u Nišu, Leskovac, Srbija
Lekovita svojstva belog luka najvećim delom se pripisuju specifičnim sumpororganskim jedinjenjima, koja nastaju enzimskim, hemijskim i termičkim transformacijama S-alil-L-cisteina u toku lagerovanja, sušenja ili prerade lukovice.
Poznato je da beli luk pokazuje baktericidna, bakteriostatska, antimikotična,
antiviralna, antisklerotična, antihipertenzivna, antiagregaciona i antitumotna
dejstva. Cilj ovog rada je sinteza deoksialiina kao glavnog prekursora za dobijanje aliina, koji se nalazi u česnjevima belog luka kao genuino jedinjenje. Kao
polazni prekursori za sintezu deoksialiina koristišćeni su L-cistein i alilbromid,
a njegovo prečišćavanje vršeno je prekristalizacijom iz apsolutnog etanola.
Dobijeni deoksialiin korišćen je za hemijsku sintezu aliina postupkom oksidacije sa vodonik-peroksidom. Strukturna karakterizacija sintetisanog deoksialiina
i aliina izvršena je primenom UV, FTIC i MS metoda. Razdvajanje optičkih
izomera aliina izvršeno je primenom tankoslojne hromatografije a njihova identifikacija upoređivanjem dobijenih Rf-vrednosti sa literaturnim.
46 (ORIGINALAN NAUČNI RAD)
UDK 615.322:582.573.16
Ključne reči: sinteza, aliin, deoksialiin, strukturna karakterizacija.