The Synthesis and Structure Characterization of Deoxyalliin and Alliin

Advanced technologies 1(1) (2012), 38-46

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 (ORIGINAL SCIENTIFIC PAPER) UDC 615.322:582.573.16 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- Keywords: synthesis, alliin, deoxyalliin, struc- oxyalliin. It is purified by recrystallization from absolute ethanol. The obtained tural characterization. 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.

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 quan- and 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 chro- in 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 reso- 17] and hyperlipidemia [18, 19]. Also, garlic can reduce nance (NMR) and mass spectroscopy (MS) [38], high-per- blood 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 sec- enzymatic, 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.

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alliinase enzyme which, after destroying the cell structure for 24 h. The residual solvent was evaporated by using of the plant material, transforms allin to alliicin. Therefore, rotary evaporator and then dissolved in the mixture of there was a need to develop a procedure for allin synthe- acetone:water:glacial acetic acid (65:34:1, v/v/v). The sis. So, the aim of this study was to synthesize alliin from crystals were precipitated and washed using the cold mix- deoxyalliin by oxidation with hydrogen peroxide and to ob- ture of acetone:water:glacial acetic acid (65:34:1, v/v/v), tain deoxyallin itself, as well as their purification by using as well as the cold absolute ethanol. L(±)-aliin, the degree TLC method. purity of 95 %, was obtained after drying the crystals.

Experimental Characterization methods of synthesized compounds

Substances. L-cysteine standard (>99 %), allyl bromide UV spectrophotometric method. The UV spectra of (>98 %), sodium hydroxide, ninhydrin, cadmium(II) ace- aqueous solutions of L-cysteine, L-deoxyalliin and L(±)- tate were purchased from Merck Chemicals Ltd. (United alliin were recorded in the wavelength range of 190-350 nm Kingdom). Silica-gel G60 is a product of Wacker Chemie on the Cary 100 Conc. spectrophotometer. The spectrum AG (Germany). Absolute ethanol, glacial acetic acid, sul- of allyl bromide was recorded in the ethanol solution under furic acid (96 %), hydrogen peroxide (30 %), n-propanol, the the same conditions. n-butanol and acetone were bought from Zorka Pharma (Serbia). FTIR spectroscopic method. FTIR spectra of L-cysteine, L-deoxyalliin and L(±)-alliin were recorded by using a The preparation of ninhydrin-Cd-acetate reagent. Solu- potassium bromide pellet technique in the wavenumber tion I: ninhydrin standard (0.3 g) was dissolved in n-propa- range of 4000-600 cm-1 on a Bomem Hartmann & Braun nol (100 cm3); Solution II: cadmium(II) acetate (1 g) was MB-series FTIR spectrophotometer. The technique of a dissolved in glacial acetic acid (50 cm3) under reflux in a thin film between potassium bromide plates was applied boiling water bath. The solvents I and II were immediately for recording FTIR spectrum of allyl bromide on the same mixed in the ratio of 5:1 (v/v) before using. apparatus.

The preparation of thin layer. Silica gel G60 (30 g) was Mass spectrometry. Mass spectra of L-cysteine, L-deoxy- mixed with distilled water (65 cm3) for 1 min, and then ap- alliin and L(±)-aliin were obtained on the model 8230 mass plied to the glass plates (20×20 cm) in the thickness of spectrometer by using the electron ionization method. The 0.25 mm. applied electron energy was 70 eV, while the temperature of ionic source was 250 0C. The synthesis procedure of L-deoxyalliin (S-allyl-L- cysteine). L-cysteine and allyl bromide were used as a Thin layer chromatography. The aqueous solutions of L- precursor 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 de- the 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 sulfu- the 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 L- deoxyalliin (>95 %) were obtained after evaporating the Results and discussion absolute ethanol to half of the volume under reduced pressure. Synthesis and structural characterization of L-deoxyalliin. 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 Advanced technologies 1(1) (2012), 38-46

A crude L-deoxyalliin was purified by precipitation from at the wavelength of 200 nm which indicates n→π* and absolute ethanol. The obtained L-deoxyalliin (>95 %) was π→π* transition of the carboxylic chromophore group. The used for chemical synthesis of alliin by oxidation with hy- presence of auxochrome NH2 group with a free-electron drogen peroxide. L(±)-aliin was the product of that oxida- pair close to the chromophore group affects the maximus tion process. The structure characterization of deoxyalliin movement to higher wavelengths. The SH auxochrome was performed by the use of UV, FTIR and MS spectros- group has a weaker effect on the mentioned maximum copy. movement because it is more distant than a chromophore UV analysis. UV spectra of recrystallized deoxyalliin, group in the structure of L-cysteine. The secondary max- L- cysteine and allyl bromide are presented in Fig. 2. An imum absorbance at 225 nm due to π→π* transition at intensive and wide maximum can be noticed at 220 nm, C=C bond in the structure allyl bromide. This maximum which originates from n→π* and n→σ* transition in the is moved to higher values of the wavelength, because Br carbonyl group of deoxyalliin. In this range, π→π* transi- group has a batochromic effect on the absorption of C=C tion is appeared due to the presence of C=C bond in the bond. Precursors were transformed to deoxyalliin during structure of deoxyallliin. Moving these maximums to high- the synthesis process, which was confirmed by differenc- er values of wavelength is caused by the presence of NH2 es in the absorption of the UV spectra. The purity of the auxochrome, which has a significant impact on n→π* tran- obtained deoxyalliin was acceptable for a further synthesis sition and smaller effect on π→π* transition. Unlike deoxy- process of alliin. allin, UV spectrum of L-cysteine has a narrower maximum

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 -1 + bromide (Fig. 4) were recorded in the aim of the structure intensity band at 1423 cm is from δs(NH3 ). In the wave- -1 characterization of synthesized and purified deoxyalliin. number range of 3500-3200 cm , 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 -1 + 2600 cm 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 + -1 and deformation asymmetric vibration of NH3 should be presence of the band at 928 cm 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 precur- C=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

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+ -1 + of ν(NH3 ) in the range of 3100-2600 cm covers the low nates from δs(NH3 ), while a lower intensity band at 1417 -1 intensity bands of allyl group, νas(CH) and νs(CH). In the cm comes from vibrations of C=O group. -1 + range of 1600-1560 cm , the vibration of δas(NH3 ) is cov- -1 ered by the band of νas(C=O). A band at 1499 cm origi-

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 fragment. As the results of CH2=CH-CH=S fragment elimi- Fig. 6. The dominant peak at m/e 162 presents (M+1) peak nation from the molecule of deoxyalliin, the peak at m/e 90 in the case of deoxyalliin. The peak at m/e 145 appeared occurred in MS spectrum. The other peaks are not signifi- after removing hydroxyl or ammonia fragment, while the cant for consideration due to low intensities. peak at m/e 122 was obtained by removing CH2=C=CH2

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 follow- cess, 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

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Figure 8. UV spectrum of L(±)-alliin

FTIR analysis. FTIR spectrum of L(±)-alliin (Fig. 9) are in order to completely characterize the synthetic alliin, MS the similar in the intensity, shape and position of bands to spectrometry was applied. MS spectrum of L(±)-alliin is FTIR spectrum of deoxyalliin (Fig. 5). The existence of a presented in Fig. 10. As it can be seen in the spectrum, a strong intensity band at 1091 cm-1 in the spectrum of allin dominant peak at m/e 178 refers to (M+1) and indicates originates from the valence vibration of S=O group. This that the molar mass of synthetic L±)-alliin is 177. A peak band is the evidence that alliin was obtained by the oxida- at m/e 355 originates from alliin dimer that occurs during tion of deoxyalliin. the synthesis. MS analysis. In the addition to mentioned methods and

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 both cases of synthesis. The optical isomers of alliin can synthesis, the reaction mixture of deoxyalliin, pure recrys- be successfully separated by using this method. All com- tallized deoxyalliin, the reaction mixture of alliin and pure pounds were identified comparing the obtained Rf-values recrystallized alliin were analyzed by TLC method. The with the literature values [41] under identical experimental results of the investigation indicate that the reaction mix- conditions of TLC. The results of these investigations are tures did not have a significant amount of by-products in 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- Compounds Spot color 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

The control chromatogram which was sprayed with Conclussion sulfuric acid (50 %) was used as the confirmation of the obtained results. The number and position of spots at the An optimal procedure for deoxyalliin synthesis was control chromatogram correspond to the chromatogram developed as a main precursor for the synthesis of alliin. caused by the nynhidrin reagent. Also, the synthesis procedure of L(±)-alliin from deoxyalliin The biosynthetic procedures of obtaining alliin [25,26] using hydrogen peroxide was successfully defined. The require an incubation of the callus and the extraction of the synthetic alliin presents the precursor for the synthesis of obtained alliin. Unlike these routes of alliin biosynthesis, the biologically active compound of allicin. The purified de- the proposed synthetic procedure is faster and simpler. oxyalliin 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 ap- of anticoagulation factors in rats, Food and Chemical plying TLC method. The identification of alliin isomers and Toxicology, 45(3) (2007) 502-507. deoxyalliin was performed on the basis of literature data [16] F. Borrelli, R. Capasso, A. A. Izzo, Garlic (Allium sativum L.): adverse effects and drug interactions in humans, Molecular of Rf-values. Nutrition and Food Research, 51(11) (2007) 1386-1397. [17] M. Steiner, R. S. Lin, Changes in platelet function and Acknowledgements susceptibility of lipoproteins to oxidation associated with administration of aged garlic extract, Journal of This work was supported by the Ministry of Education and Cardiovascular Pharmacology, 31(6) (1998) 904–908. Science of the Republic of Serbia under the project TR- [18] J. Kojuri, A. R. Vosoughi, M. Akrami, Effects of Anethum 34012. graveolens and garlic on lipid profile in hyperlipidemic patients, Lipids in Health and Disease, 1(6) (2007) 5-10. 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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 (ORIGINALAN NAUČNI RAD) UDK 615.322:582.573.16 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 Ključne reči: sinteza, aliin, deoksialiin, struk- polazni prekursori za sintezu deoksialiina koristišćeni su L-cistein i alilbromid, turna karakterizacija. 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.

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