47

E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

519

Mechanistic Studies on the Formation of Thiazolidine and Structurely Related Volatiles in Cysteamine /Carbonyls Model System Tzou-Chi Huang', Y-M. Su', L.Z. Huang' and Chi-Tang Ho'* 'Department of Food Science and Technology, National Pingtung Polytechnic Institute, 912, Pingtung, Taiwan 'T^epartment of Food Science, Rutgers University, New Brunswick, NJ 08901-8520, USA

Abstract Phosphate was found to dramatically enhance the formation of thiazolidine in a cysteamine/carbonyl model system. Phosphate tends to stabilize the primary carbocation formation which may lead to the completion of the cyclization by attacking the amino nitrogen on the activated carbon. Protic solvent further enhances thiazolidine formation by removing the water molecule. Thiazolidine formation is completed by combining the phosphate buffer with the protic solvent. The redox reaction catalyzed by phosphate ions results in the conversion of thiazolidine to the corresponding thiazoline through hydride transfer. The conversion of 2-acetyl-2,3,5,6-tetrahydro-l,4-thiazine to 5-acetyl-2,3-dihydro-l,4-thiazine via a proton transfer reaction catalyzed by azodicarbonamide was evidenced as well. A formation mechanism for thiazolidine and structurely related tetrahydro-l,4-thiazine and 2,3-dihydro-l,4thiazine is proposed.

1.

EVTRODUCTION

Schiff base formation between the amino group and the aldehyde group has been the subject of numerous studies [1]. The amino group on a cysteamine may react with an aldehyde group to form a Schiff base as well. In addition to the Schiff base formation, a subsequent ring closing reaction leading to the formation of a thiazolidine deserves special interest. Thiazolidines generally possess a characteristic popcorn flavor [2]. Model systems composed of D-glucose and L-cysteine have long been used to study the thermal generation of thiazolines and thiazines [3-4]. The reaction between cysteamine, the decarboxylated cysteine and 2,3-butanedione, a glucose degradation product, may lead to the formation of 2-acetyl-2methylthiazolidine [5]. Recently, a thiazolidine derivative method for the determination of trace aldehydes in foods and beverages has been developed [6-7]. These methods are based on the reaction of volatile carbonyl compounds with cysteamine (2-aminoethanethiol) to form stable thiazolidine derivatives under mild conditions (room temperature and neutral pH). The thiazolidine

520

derivatives formed were subsequently determined by gas chromatography. However, the formation pathways of thiazolidines are not yet well documented. On the other hand, intense roasted, popcorn-like odorant 5-acetyl-2,3-dihydro-l,4thiazine was identified in the D-ribose/L-cysteine model system [8-9]. It was proposed that a SchifF base is formed from the condensation between the amino group in cysteamine and the carbonyl group in 2,3-butanedione. Tautomerization and subsequent cyclization by a Michaeltype nucleophilic attack of the thiol group at the activated methyl carbon atom yield 5-(2hydroxyethenyl)-2,3,6-trihydro-l,4-thiazine. Oxidation of this enaminol results in 5-acetyl-2,3dihydro-l,4-thiazine which, due to the electronegativity of the sulfiir atom, tautomerizes into the more stable 5-acetyl-2,3-dihydro-l,4-thiazine, which is structurely related to 2-acetyl-2methylthiazolidine [8]. This paper focuses on the reactivity of cysteamine to a carbonyl compound involving 2,3butanedione and aliphatic short-chain aldehydes. A discussion on the formation mechanism of thiazolidine and structurally related volatile compounds will be provided.

2.

THE EFFECT OF A PHOSPHATE BUFFER SYSTEM ON THIAZOLmiNE FORMATION

Quantitative data obtained revealed that phosphate is a very effective buffer system for the promotion of the thiazolidine formation. The addition of a phosphate ion resulted in a 16fold, 12-fold and 21-fold increase for 2-acetyl-2-methylthiazolidine, 5-acetyl-2,3-dihydro-l,4thiazine and 2-acetyl-2,3,5,6-tetrahydro-l,4-thiazine formation respectively as compared to a water at pH 7.2 in cysteamine/2,3-butanedione system (Figure 1). The phosphate may act as both a hydrogen acceptor and donor, which catalyzes the Schiflf base formation during the generation of a thiazolidine and thiazines. Formation of thiazolidines was affected dramatically by the concentration of the phosphate buffer. Figure 2 shows the effect of different buffers on the formation of alkylthiazolidines in a cysteamine/aldehydes model system. A limited amount of unsubstituted thiazolidine was detected in the model system of cysteamine/aldehydes (pH 7.2) without phosphate. Concentrations of individual alkylthiazolidines increased with the increasing chain length of the alkyl group. The molar recovery of the five thiazolidines formed from the corresponding aldehyde and cysteamine were found to be quite low. They were 13%, 5.4%, 18.8%, 27.2% and 37.2% for unsubstituted thiazolidine, 2-methylthiazolidine, 2ethylthiazolidine, 2-propylthiazolidine and 2-butylthiazolidine, respectively. The reactivity of the aldehydes increased with the increasing alkyl chain length as shown in Figures 2A. Quantitative data obtained in this experiment revealed that phosphate was an effective buffer system for the formation of a thiazolidine. The addition of the phosphate buffer results in a 32-fold, 11-fold, 3.8-fold, 3.2-fold and 3.2-fold increases for unsubstituted thiazolidine, 2methylthiazolidine, 2-ethylthiazolidine, 2-propylthiazolidine and 2-butylthiazolidine respectively as compared with that in an aqueous system at pH 7.2 in Figure 2D. And the molar recovery for all of the five thiazolidines increased with increasing phosphate concentration linearly from 0.025 M to 0.2 M as shown in Table 1. This observation correlates better with the higher reactivity of the aldehydes larger than C3 (C3-C5) than those from formaldehyde and acetaldehyde in the preparation of thiazolidine from various aldehydes and cysteamine [10].

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Figure 2. Gas chromatogram of derivatives from aldehydes: Peaks: 1 = 2-methylthiazoline from acetaldehyde; 2 = thiazolidine from formaldehyde; 3 = 2-methylthiazolidine from acetaldehyde; 4 = 2-ethylthiazoline from propionaldehyde; 5 = 2-ethylthiazolidine from propionaldehyde; 6 = 2-propylthiazodine from butyraldehyde; 7 = 2-propylthiazolidine from butyraldehyde; 8 = 2-butylthiazoline from valeraldehyde; and 8 = 2-butylthiazolidine from valeraldehyde.

Table 1. Effect of phosphate buflFer concentration on thiazolidine formation

Phosphate buffer (M)

0.025

0.05

Concentration (mM) 0.1 0.2

0.20 (3.0y 0.66(9.9) Thiazolidine 0.42(9.2) 1.01(22.2) 2-Methylthiazolidine 0.71(20.6) 1.16(33.6) 2-Ethylthiazolidine 0.88(31.7) 1.21(43.5) 2-Propylthiazolidine 0.94(40.3) 1.27(54.5) 2-Butylthiazolidine "* value in parenthesis are molar recovery (%)

1.68(25.2) 1.79(39.3) 1.69(49.0) 1.64(59.0) 1.67(71.7)

2.78(41.7) 2.64(58.0) 2.53(73.3) 2.43 (87.4) 2.67(114.6)

nil 0.09(1.3) 0,25(5.5) 0.65(18.8) 0.76 (27.2) 0.87(37.3)

523 3. OXIDO-REDOX SYSTEM

REACTION

IN A CYSTEAMINE/CABONYLS

MODEL

Considerable amounts of 2-methylthiazoIine, 2-ethylthiazoline, 2-propylthiazoline and 2butylthiazoline were characterized in the heated cysteamine/aldehydes model system with a carbonate buffer (pH 10.3, 0.2 M) as shown in Figure 2C. These compounds were eiuted before the corresponding thiazolidine. No detectable amounts of thiazolines were observed in the experimental condition without buffer sah. The effect of azodicarbonamide, a well known hydrogen acceptor, was added to the reacted model system to study the influence of the oxido-redox reaction on the formation of 5acetyl-2,3-dihydro-l,4-thiazine. The formation of 5-acetyl-2,3-dihydro-l,4-thiazine was found to increase lineariy with increasing concentrations of azodicarbonamide in the range of 1 to 3 mM. The amount of 2-acetyl-2,3,5,6-tetrahydro-l,4-thiazine decreased with increasing concentration of azodicarbonamide. The formation of 2-acetyl-2-methylthiazolidine was found to be independent of azodicarbonamide as shown in Figure 3. The redox reaction seems to lead to the conversion of 5-acetyl-2,3,5,6-tetrahydro-l,4-thiazine to 5-acetyl-2,3-dihydro-l,4thiazine via a proton transfer reaction similar to that in the conversion of tetramethyldihydropyrazine to tetramethylpyrazine [11].

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524 The redox reaction was proposed by Huyghues-Despointes and Yaylayan [12] in a Maillard model system composed of D-glucose and proline. They reported that an adiketone/enediol redox couple participated in the generation of oxidation-reduction products in the Maillard system. Oxidation of thiazolidines in the presence of atmospheric oxygen may lead to the formation of the corresponding thiazoline [13]. Similarly it was also observed in a cysteamine/methylglyoxal model system [8]. They attributed methylglyoxal as a proton acceptor in the formation of 2-acetyl-2-thiazoline from 2-acetylthiazolidine. Two volatile compounds, 2-acetyl-2-thiazoline and 2-acetylthiazole were characterized in a heated aqueous L-cysteine solution [13]. They postulated that these two acetyl derivatives were the dehydrogenation products of 2-acetylthiazolidine. A significant amount of 2-acetylthiazolidine has also been observed in the reaction mixture of aldehydes and cysteamine by Hayashi et al. [6]. A redox reaction catalyzed either by phosphate or azodicarbonamide may facilitate the proton transfer from 2-acetyl-2,3,5,6-tetrahydro-l,4-thiazine to form a 5-acetyl-2,3-dihydro1,4-thiazine and from thiazolidines to the corresponding thiazolines. The phosphate or azodicarbonamide may act as both hydrogen acceptor and donor, which catalyzes the Schifif base formation during the generation of a thiazolidine and thiazines. 4. THE COMBBVED EFFECTS OF A BUFFER AND SOLVENT ON THIAZOLIDINE FORMATION IN A CYSTEAMBVE/CARBONYLS MODEL SYSTEM An interesting effect of combining the phosphate buffer and protic solvent on thiazolidine formation was found. As shown in Figure 4, a 1.13-fold increase of the unsubstituted thiazolidine was observed when 60 % ethanol in a phosphate buffer was utilized as the reaction medium as compared with that without ethanol. More ethanol did not enhance thiazolidine formation. A similar effect on the formation of 2-methylthiazolidine, 2-ethylthiazolidine, 2propylthiazolidine and 2-butylthiazolidine was observed as well when the solvent and buffer were combined as shown in Figure 4. These findings are in complete accordance with those published by Lin et al. [14]. An aqueous solution of 50% methanol containing 100 mM phosphate at pH 7.0 was found to be very effective for converting an orange pigment into a red amino acid pigment. A Schiff base was formed between the a-amino nitrogen of the amino acid and the carbonyl carbon of the orange Monascus pigment. A high yield of 60-70% of l-deoxy-l-p-toluino-D-fiuctose was obtained by Rosen et al. [15] although no detailed elucidation of the mechanism was provided in their paper. The phosphate buffer system served as either a proton donor or acceptor, which catalyzed the Schiff base formation [16]. Extra phosphate may also have catalyzed the dehydrogenation reaction. The protic solvent attracted the water molecule, which led to completion of the Schiff base formation.

525

2-butyithia2clidlne 2-propyithia2oiidine 2-ethyithiazoiidine methyithiazolidine thiazolidine

Ethanoi concantraticn (%)

Figure 4. Effect of ethanoi on the formation of thiazolidines in aldehydes/cysteamine model system.

5.

PROPOSED MECHANISM FOR THIAZOLIDINE FORMATION

The proposed mechanism for the formation of thiazolidine in a cysteamine/carbonyl model system is shown in Figure 5. In a reaction medium with a buffer, the nucleophilic amino group on a cysteamine molecule tends to attack the positively induced carbonyl carbon. A general acid catalyzed the elimination of a water molecule which gives a secondary carbocation ion for 2,3-butanedione and aliphatic aldehydes with a chain length longer than C2. Conversely, formaldehyde gives an extremely unstable primary carbocation. Phosphate tends to stabilize this primary carbocation. Another nucleophilic attack of the thiolate on carbocationic carbon leads to the formation of a thiazolidine.

6.

REFERENCES P.M.T. de Kok and E.A.E. Rosing, In Thermally Generated Flavors, Maillard, Microwave and Extrusion Process, American Chemical Society, Washington, DC, Series, (1994) 158-179. C.H. Yeo and T. Shibamoto, J. Agric. Food Chem., 39 (1991) 370-373.

526

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aldehydes R-fsH, R2=0H, Ci-C2 2,3-butanedione Ri=CH3, R2= C—CH3 !l O

H

c Figure 5. Proposed formation mechanism for thiazolidines from cysteamine and carbonyl compounds.

527 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

R.A. Scanlan, S.C. Kayer, L.M. Libbey and M.E. Morgan, J. Agric. Food Chem, 21 (1973) 673-675. EJ. Mulders, Z. Lebensm. Unters. Forsch. 152 (1973) 193-201. K. Umano, Y. Hagi, K. Nakahara, A. Shyoji and T. Shibamoto, J. Agric. Food Chem. 43 (1995)2212-2218. T. Hayashi, C.A. Reece and T. Shibamoto, J. Assoc. Off. Anal. Chem. 69 (1986) 101105. K. Miyashifa, K Kanda and T. Takagi, J. Assoc. Off. Anal. Chem. 68 (1991) 748-751. T. Hofmann and P. Schieberle, J. Agric. Food Chem. 43 (1995) 2187-2194. T. Hofmann, R. Hassner and P. Schieberle, J. Agric. Food Chem. 43 (1995) 2195-2198. A. Yasuhara and T. Shibamoto, J. Chromatogr. 547 (1991) 291-298. T.C. Huang, H.Y. Fu and C.-T. Ho, J. Agric. Food Chem. 44 (1996) 240-246. A. Huyghues-Despointes and V.A. Yaylayan, J. Agric. Food Chem. 44 (1996) 672-681. S.A. Sheldon and T. Shibamoto, Agric Biol. Chem. 51 (1987) 2473-2477. T.F. Lin, K. Yakushijin, G.H. Buchi and AL. Demain, J. Indust. Micro. (1992) 173-179. L. Rosen, J.W. Woods and W. Pigman, J. Amer. Chem. Soc. 80 (1958) 4697-4702. T.C. Huang, Biosci. Biotech. Biochem. 61 (1997) 1013-1015.