THE SYNTHESIS AND STRUCTURE …Thin layer chromatography. The aqueous solutions of L-cysteine, L-deoxyalliin and L(±)-aliin (20 µl) were added on a thin layer of Silica-gel G (a

38

Advanced technologies

Introduction

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, antihyperten-sive, 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 de-oxyalliin. It is purified by recrystallization from absolute ethanol. The obtained deoxyalliin (>95 %) was used for the synthesis of alliin by oxidation with hydro-gen 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.

Keywords: synthesis, alliin, deoxyalliin, struc-tural characterization.

Garlic (Allium sativum) is valued in many parts of the world for its pungent aroma and flavor. However, most in-vestigations of health benefits of the garlic have consid-ered its medicinal rather than culinary uses. Medicinal use of garlic goes back to Greek and Egyptian antiquity. In in vitro studies, garlic has been found to have antibacterial [1-3], antiviral, and antifungal (fungal infections of the skin and the ear) activity [4–9]. Garlic is widely used for its car-diovascular benefits [10]. It may also lower blood pressure since it helps to keep blood vessels to the heart flexible in older people. One of the most intriguing possibilities of garlic is that it helps in the prevention of cancer. It is used to prevent stomach and colon cancers [11-13]. Allium sa-tivum has been found to reduce platelet aggregation [14-17] and hyperlipidemia [18, 19]. Also, garlic can reduce blood sugar levels and may improve the insulin response [20]. Sulfur compounds of garlic (alkyl-cysteine derivates, alkyl-sulfide, alkyl-disulfide and alkyl-polysulfide, thio-sulfonate, etc.) [21] are responsible for most medicinal properties of this herb. These compounds are formed by enzymatic, chemical and thermal transformation of alliin after processing the bulb. Stoll and Seebeck [22] isolated the mixture of amino acids with the content of sulfur and

alkyl derivates of sulfur. The most significant amino acid in the mixture is a distereoisomer of alliin, S-allyl-L-cysteine sulfoxide, an organosulfur compound that contributes to its therapeutic value and pharmacological importance [23, 24]. It is a derivative of the cysteine amino acid.

The ways proposed for the biosynthesis of alliin are de-scribed [25-29]. For the purpose of identification and quan-titative determination of alliin in garlic and garlic products different analytical methods were used, such as: liquid chromatography (LC) [30], high performance liquid chro-matography (HPLC) [31,32,33], liquid chromatography coupled with mass spectrometry detection (LC/MS) [34], gas chromatography (GC) [35], high-throughput method [36], spectrophotometric method [37], nuclear mass reso-nance (NMR) and mass spectroscopy (MS) [38], high-per-formance thin layer chromatography (HPTLC) [39]. A rapid and sensitive HPLC-electrospray/MS method has been developed to determine alliin in rat plasma [40].

Even though it is a pharmacologically inactive, alliin represents the initial compound for a large number of sec-ondary reactions where therapeutic important products containing sulfur (alliicin, vinyldithiine) are obtained. Alliin can be isolated from garlic, but the main problem is an

(ORIGINAL SCIENTIFIC PAPER)UDC 615.322:582.573.16

*Author address: Vesna Nikolić, Faculty of Technology, 16000 Leskovac, Bulevar oslobođenja 124, Serbiae-mail: [email protected] The manucsript received: May, 16, 2012.Paper accepted: Jun, 18, 2012.

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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 synthe-sis. So, the aim of this study was to synthesize alliin from deoxyalliin by oxidation with hydrogen peroxide and to ob-tain deoxyallin itself, as well as their purification by using TLC method.

Experimental

Substances. L-cysteine standard (>99 %), allyl bromide (>98 %), sodium hydroxide, ninhydrin, cadmium(II) ace-tate were purchased from Merck Chemicals Ltd. (United Kingdom). Silica-gel G60 is a product of Wacker Chemie AG (Germany). Absolute ethanol, glacial acetic acid, sul-furic acid (96 %), hydrogen peroxide (30 %), n-propanol, n-butanol and acetone were bought from Zorka Pharma (Serbia).

The preparation of ninhydrin-Cd-acetate reagent. Solu-tion I: ninhydrin standard (0.3 g) was dissolved in n-propa-nol (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 ap-plied to the glass plates (20×20 cm) in the thickness of 0.25 mm.

The synthesis procedure of L-deoxyalliin (S-allyl-L-cysteine). L-cysteine and allyl bromide were used as a precursor for the synthesis of L-deoxyalliin. Firstly, L-cysteine was suspended in absolute ethanol, and then so-dium hydroxide (20 mol dm-3) was added to achieve a ba-sic medium. After that, allyl bromide was slightly added in the suspension. The reaction was performed in the cold in the first 1 h, and then at ambient temperature for 2 h. The reaction mixture was neutralized to pH 5.5 and placed in the cold place to the appearance of L-deoxyalliin crystals.

Recrystallization of L-deoxyalliin. After dissolving crude L-deoxyalliin in acetic acid (1 %), it was transferred in 15 fold higher volume of absolute ethanol. The crystals of L-deoxyalliin (>95 %) were obtained after evaporating the absolute ethanol to half of the volume under reduced pres-sure.

Synthesis of alliin (S-allyl-L-cysteine sulfoxide). Al-liin was synthesized by oxidation of synthesized and pre-crystallized L-deoxyalliin at ambient temperature

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 mix-ture 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

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-deoxy-alliin 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.

Thin layer chromatography. The aqueous solutions of L-cysteine, L-deoxyalliin and L(±)-aliin (20 µl) were added on a thin layer of Silica-gel G (a sheet of glass 20×20 cm, the layer thickness of 0.25 mm). The chromatograms were developed with n-butanol:glacial acetic acid:water (2:1:1, v/v/v). The spots on the chromatographic plate were de-tected by ninhydrin reagent spraying. In the case of the control chromatogram the spots were detected with sulfu-ric acid (50 %). After drying at the temperature of 105 0C, it came to the appearance of spots that were identified by using literature data for the Rf values of the investigated compounds.

Results and discussion

Synthesis and structural characterization of L-deoxy-alliin. L-deoxyalliin, as a precursor for obtaining L(±)-alliin, was synthesized from L-cysteine and allyl bromide in the following chemical reaction (Fig. 1):

Figure 1. The chemical reaction of L-deoxyalliin synthesis

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Figure 2. UV spectra of deoxyalliin, L-cysteine and allyl bromide

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 hy-drogen peroxide. L(±)-aliin was the product of that oxida-tion process. The structure characterization of deoxyalliin was performed by the use of UV, FTIR and MS spectros-copy.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* transi-tion is appeared due to the presence of C=C bond in the structure of deoxyallliin. Moving these maximums to high-er values of wavelength is caused by the presence of NH2

auxochrome, which has a significant impact on n→π* tran-sition and smaller effect on π→π* transition. Unlike deoxy-allin, 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 max-imum 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 differenc-es in the absorption of the UV spectra. The purity of the obtained deoxyalliin was acceptable for a further synthesis process of alliin.

FTIR analysis. FTIR spectra of L-cysteine (Fig. 3) and allyl bromide (Fig. 4) were recorded in the aim of the structure characterization of synthesized and purified deoxyalliin. The characteristic band in the wavenumber range of 3100-2600 cm-1 is due to ν(NH3

+) vibration at L-cysteine. A wide and medium intensity band was expanded as the results of combining bands and over tones which are placed from 2000 cm-1. A valence asymmetric vibration of C=O group and deformation asymmetric vibration of NH3

+ should be expected in the range of 1600-1560 cm-1. A strong band at 1590 cm-1 originates from valence asymmetric vibration of C=O bond from L-cysteine. In this range of wavenumber, there is a low intensity band of δas(NH3

+) and cannot be

clearly observed due to the overlap with νas(C=O). A low intensity band at 1423 cm-1 is from δs(NH3

+). In the wave-number range of 3500-3200 cm-1, the bands of νas(CH) and νs(CH) vibration of terminal allyl group appeared in the IR spectrum of allyl bromide (Fig. 4). The valence vibration of C=C group at 1637 cm-1, and the deformation vibration γ(CH) out-of-plane in the form of two bands at 928 and 986 cm-1 are also noticed. Over tones at 1800 cm-1, as well as the presence of the band at 928 cm-1 are the confirmation of allyl group in the molecular structure. The FTIR spectrum of deoxyalliin (Fig. 5) is different than spectra of precur-sors (Fig. 3 and 4), indicating their transformation during the chemical reaction. A wide and medium intensity band

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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 cov-ered 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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Figure 6. MS spectrum of L-deoxyalliin

Figure 7. The chemical reaction of L(±)-aliin synthesis

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 elimi-nation from the molecule of deoxyalliin, the peak at m/e 90 occurred in MS spectrum. The other peaks are not signifi-cant for consideration due to low intensities.

Synthesis and structural characterization of alliinThe synthesis of alliin was performed by the oxidation pro-cess, where deoxyalliin and hydrogen peroxide were used as the initial reactants. The structure of obtained L(±)-alliin

was confirmed by applying UV, FTIR and MS methods. The reaction of L(±)-alliin synthesis is shown in the follow-ing chemical equation (Fig. 7):

UV analysis. UV spectrum of the water solution of L(±)-alliin is presented in Fig. 8. A significant difference between UV spectra of the obtained product and its precursors, 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 maxi-mum at 198 nm and slightly defined saddle at 254 nm exist in the UV spectrum of alliin (Fig. 8). The maximum at 198 nm

originates from π→π* transition of terminal C=C bond and n→σ* transition of S=O group. A low intensity saddle at 254 nm is due to n→π* transition in the allyl group.

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Figure 8. UV spectrum of L(±)-alliin

Figure 9. FTIR 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 oxida-tion 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.

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Figure 10. MS spectrum of L(±)-alliin

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

TLC analysis. L-cysteine as a precursor in deoxyalliin synthesis, the reaction mixture of deoxyalliin, pure recrys-tallized deoxyalliin, the reaction mixture of alliin and pure recrystallized alliin were analyzed by TLC method. The results of the investigation indicate that the reaction mix-tures 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 com-pounds 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.

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.

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 de-oxyalliin and alliin were structurally characterized by using UV, FTIR and MS spectroscopic methods. The separation

Compounds

deoxyalliin

L(+)-alliin

L(-)-alliin

L- cysteine

Literature Rf-values

0.68

0.58

0.49 0.56

Obtained Rf-values

0.67

0.58

0.45 0.55

Spot color

orange

pink

orange

yellow

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of alliin optical isomer was successfully achieved by ap-plying 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 TR-34012.

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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 sumporor-ganskim jedinjenjima, koja nastaju enzimskim, hemijskim i termičkim trans-formacijama 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 dobi-janje 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 oksidaci-je 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 iden-tifikacija upoređivanjem dobijenih Rf-vrednosti sa literaturnim.

Ključne reči: sinteza, aliin, deoksialiin, struk-turna karakterizacija.

(ORIGINALAN NAUČNI RAD)UDK 615.322:582.573.16

Izvod

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Analytical Biochemistry, 307(1) (2002) 76-83.[38] A. Sendl, H. Wagner, Isolation and Identification of

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Documents

THE SYNTHESIS AND STRUCTURE …Thin layer chromatography. The aqueous solutions of L-cysteine, L-deoxyalliin and L(±)-aliin (20 µl) were added on a thin layer of Silica-gel G (a