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Mechanism of pyrazole formation in [13C-2] labeled glycine model systems: N–N bond formation during Maillard reaction
Food Research International 36 (2003) 571–577 www.elsevier.com/locate/foodres
Mechanism of pyrazole formation in [13C-2] labeled glycine model systems: N–N bond formation during Maillard reaction Varoujan A. Yaylayan*, Luke J.W. Haffenden McGill University, Department of Food Science and Agricultural Chemistry, 21,111 Lakeshore, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9 Received 27 May 2002; accepted 17 December 2002
Abstract Studies with 13C-2 labeled glycine in model systems containing 3-hydroxy-2-butanone or glyceraldehyde have indicated that the b-dicarbonyl compounds, the immediate precursors of pyrazoles, are produced in Maillard model systems through two pathways. One pathway involves dehydration of a,b-dihydroxy carbonyl compounds with elimination of the a-hydroxyl group and the other through aldol condensation of an a-hydroxy carbonyl compound with simple aldehydes to produce a,b-dihydroxy carbonyl moiety that can undergo the above-mentioned dehydration to produce b-dicarbonyl structures. The conversion of b-dicarbonyls into pyrazoles can be achieved through 1,3-diimine formation by reaction with either two ammonia molecules or with a primary amine and an ammonia. After imine–enamine isomerizations the resulting dienamine can be oxidized to form pyrazole rings similar to the oxidation of two thiol moieties into disulfide linkages. The a-dicarbonyl species can serve as hydrogen acceptors since in their absence no pyrazole formation was detected. Glycine/3-hydroxy-2-butanone system generated 3,4,5-trimethyl-pyrazole (associated with the aroma of tequila) and 1,3,4,5-tetramethyl-pyrazole whereas, glycine/glyceraldehyde generated 1,5-dimethyl and 1,3,5-trimethyl-pyrazoles. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Maillard reaction; 13C-labeled glycine; Mechanism of pyrazole formation; N–N bond formation; Py–GC/MS
1. Introduction Although pyrazoles are not considered among the common Maillard reaction products, however their presence in Maillard systems further indicates the diversity of molecular structures that could be formed during the reaction. In addition, their formation poses a challenge to researchers in this field, since available synthetic routes for pyrazoles require synthons with preformed N–N bonds such as hydrazines. Such precursors have not been identified in Maillard systems and are difficult to envisage their formation under the reaction conditions. Furthermore, the related heterocyclic compounds isoxazole and isothiazoles could also have similar mechanistic origins as pyrazoles. Elucidation of the mechanism of formation of different heterocyclic compounds in general can contribute to our understanding of reactive intermediates formed during the reaction and * Corresponding author. Tel.: +1-514-398-7918; fax: +1-514-3987977. E-mail address: [email protected] (V. A. Yaylayan).
enhance our ability to predict and control the formation of different flavor related compounds. The 3,4-dimethylpyrazole was first reported in tobacco smoke around 30 years ago (Schmeltz & Hoffmann, 1977). Recently, other methyl substituted pyrazoles have been also identified in trace amounts in liquid smoke flavorings (Guillen, Manzanos, & Ibargoita, 2001) in soy beans (Lee & Takayuki, 2000), in heated malt and beer (Narziss, Meidaner, & Koch, 1988), in roasted carob (Cantalego, 1997) and in heated licorice essential oil (Frattini, Bicchi, Barettini, & Nano, 1977). Lo´pez (1999) on the other hand, reported formation of Maillard generated 3,4,5trimethylpyrazole in significant amounts during cooking of Agave tequilana to manufacture tequila beverage. Mechanism of formation of pyrazoles during Maillard reaction is not reported, however, many pyrazole derivatives can be synthesized in the laboratory by the interaction of hydrazine or monosubstituted hydrazines with b-dicarbonyls or with a,b-unsaturated carbonyl compounds (Acheson, 1976). Mechanism of formation of heterocyclic compounds in Maillard model systems can be investigated through labeling studies using the
0963-9969/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0963-9969(03)00003-6
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convenient pyrolysis/gas chromatography/mass spectrometry (Py–GC/MS) as an integrated reaction, separation and identification system (Yaylayan, 1999). Although under pyrolytic conditions a higher number of products are formed compared with aqueous reactions, however, most of the products identified in aqueous systems are also formed under pyrolytic conditions albeit in different amounts. In addition, experimental evidence was provided that the position and label distribution in the common products observed in the same model systems, between aqueous and pyrolytic reactions, are identical (Yaylayan & Wnorowski, 2000). This indicates the similarity of mechanisms of formation of these common products under both conditions. Consequently, mechanistic conclusions derived from label incorporation in the products observed under pyrolytic conditions, that are common to both systems, have relevance to the aqueous reactions. Here we report the mechanism of formation of pyrazoles during Maillard reaction, using 13C-2 labeled glycine and 3-hydroxy-2-butanone/2,3-butanedione or glyceraldehyde as model systems
2. Materials and methods 2.1. Materials All reagents and chemicals were purchased from Aldrich Chemical Company (Milwaukee, WI). [2-13C]glycine (90% enriched) and [15N]glycine (98% enriched) were purchased from Cambridge Isotope Laboratories (Andover, MA). 2.2. Pyrolysis–GC/MS analysis A Hewlett-Packard GC/mass selective detector (5890 series II GC/5971B MSD) interfaced to a CDS Pyroprobe 2000 unit, through a valved interface (CDS 1500), was used for Py–GC/MS analysis. In all experiments, solid samples of a mixture of labeled or unlabeled glycine or ammonium carbonate/dicarbonyl compound (3:1 ratio, 2 mg) were introduced inside the quartz tube (0.3 mm thickness) and plugged with quartz wool and inserted inside the coil probe. The pyroprobe was set at 250 C at a heating rate of 50 C/ms with a total heating time of 20 s. The pyroprobe interface temperature was set at 250 C. The samples were introduced under splitless mode. The 3 ml/min constant flow was maintained by an Electronic Pressure Controller (Hewlett-Packard). Capillary direct MS interface temperature was 180 C; ion source temperature was 280 C. The ionization voltage was 70 eV and the electron multiplier was 2047 V. The mass range analyzed was 17 – 200 amu. The column was a CP-Sil 5CB (30 m 0.32 mm 0.25 mm; Varian, Mississagua, ON). The column initial temperature (30 C) was held for 2 min then increased to 100 C at a
rate of 30 C/min, then further increased to 250 C at a rate of 7 C/min, and held for 35 min. The reported percent label incorporation values (corrected for natural abundance and for % enrichment) are the average of doublicate analyses and are rounded off to the nearest multiple of 5%. The identity and purity of the peaks were determined using NIST AMDIS version 2.1 software.
3. Results and discussion Most of the heterocyclic compounds reported in Maillard model systems originate from a-dicarbonyl and a-hydroxycarbonyl intermediates formed from reducing sugars during the reaction (Weenen, 1998) (see Fig. 1). However, pyrazoles, isoxazoles and isothiazoles require b-dicarbonyl moieties (Acheson, 1976) in addition to the ability to form N–N, N–O or N–S bonds, respectively. These requirements for their formation perhaps explain the low concentrations detected in food products. Analysis of the data generated from the interaction of 13C-2 glycine and glycine with glyceraldehyde, 3-hydroxy-2-butanone or 2,3-butanedione have indicated that all three model systems can generate pyrazoles. Glycine/3-hydroxy-2-butanone or 2,3-butanedione system generated 3,4,5-trimethyl and 1,3,4,5tetramethylpyrazoles whereas, glycine/glyceraldehyde generated 1,5-dimethyl and 1,3,5-trimethylpyrazoles (see Fig. 2). Furthermore, the data from different model systems containing different precursors also indicated that both a-hydroxycarbonyl and a-dicarbonyl moieties are necessary for the formation of pyrazoles in addition to the presence of b-dicarbonyl moiety. Since a-hydroxycarbonyl and a-dicarbonyl moieties are mutually convertible through redox reactions in Maillard model systems, it is expected therefore that model systems containing either of the two precursors or both should generate pyrazoles. In theory, the a-hydroxycarbonyl compounds can be converted into b-dicarbonyl derivatives (precursors of pyrazoles) by two different pathways, depending on the substituents at the b-position (see Fig. 1). In glyceraldehyde for example, the presence of a b-hydroxyl group can facilitate dehydration reaction where the a-hydroxyl group is eliminated to produce a b-dicarbonyl derivative, whereas, the b-position in 3hydroxy-2-butanone is unsubstituted and thus incapable of dehydration to produce b-dicarbonyl moiety. Such intermediates require a different mechanism to be converted into b-dicarbonyl derivatives. One such mechanism is the aldol condensation with simple aldehydes to form a,b-dihydroxy carbonyl intermediates which upon dehydration in a similar fashion to that of glyceraldehyde can generate the required b-dicarbonyl compounds. Evidence for the above mentioned mechanisms comes from label incorporation studies and the detection
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Fig. 1. Origin of simple heterocyclic compounds. AR=Amadori rearrangement; SR=Strecker reaction, [O]=oxidation.
of specifically substituted pyrazoles identified in glyceraldehyde, 3-hydroxy-2-butanone or 2,3-butanedione/ glycine systems as elaborated below. 3.1. Preliminary experiments to identify precursors of pyrazoles in Maillard model systems Literature studies (Acheson, 1976) have indicated that for laboratory synthesis of pyrazoles, b-dicarbonyls are essential starting materials for the generation of the carbon backbone of pyrazoles in addition to hydrazine derivatives. Alternatively, if amines are used instead of hydrazines, oxidizing conditions are necessary to form the N–N single bond (Bates, Kohrt, Folk, & Xia, 1997) (see Fig. 3), similar to the oxidation of two thiol moieties into disulfide linkages. During Maillard reaction, amines and a-dicarbonyl intermediates are produced abundantly from amino acids and reducing sugars (Weenen, 1998). The a-dicarbonyl intermediates are known to undergo reductions (Huyghues-Despointes & Yaylayan, 1996) and therefore can serve as oxidizing species to form N–N bonds during the Maillard reaction. In order to test this hypothesis, a mixture consisting of
2,4-pentanedione, a commercially available b-dicarbonyl, and ammonium carbonate a source of ammonia was reacted with and without 2,3-butanedione. Both model systems produced 4-amino-3-pentene-2-one (see Fig. 4) as the major product, but only the model system containing an a-dicarbonyl species, produced 1,3,5-trimethylpyrazole and a trace amount of 1,3,4,5-tetramethylpyrazole. In addition, the mixture consisting of 2,4-pentanedione and glycine produced mainly 4-(methylamino)-3-penten-2one. Furthermore, when the b-dicarbonyl was also removed from the model system, again no pyrazole was detected. These studies have indicated the importance of both a- and b-dicarbonyl species in the generation of pyrazole, the former as an oxidizing species and the latter as the carbon backbone. 3.2. Mechanism of formation of 1,5-dimethyl and 1,3,5trimethylpyrazoles in [13C-2]glycine/glyceraldehyde model system To study the role of a-hydroxycarbonyl moiety in generation of pyrazoles, glyceraldehyde was reacted with [13C-2]-labeled and unlabeled glycine. This model
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Fig. 2. Summary of model systems studied and pyrazole derivatives identified in them.
Fig. 3. Proposed general mechanism of formation of pyrazole moiety from b-dicarbonyl precursors.
system produced 1,5-dimethyl and 1,3,5-trimethyl-pyrazoles as shown in Fig. 2. The specific b-dicarbonyl precursors required for their formation are 3-ketobutanal and 2,4-pentanedione respectively (see Fig. 4). These precursors can be formed from glyceraldehyde by an amino acid assisted chain elongation process identified earlier (Keyhani & Yaylayan, 1996) and shown in Fig. 4. Through this chain elongation process glyceraldehyde can be converted into 3,4-dihydroxy-2-butanone with the incorporation of one C-2 atom of glycine as a methyl group. Dehydration of this intermediate can generate 3-ketobutanal the b-dicarbonyl precursor of 1,5-dimethylpyrazole. A similar chain elongation reaction with the aldehyde end of 3-ketobutanal can convert it into 2,4-pentanedione with incorporation of two C-2 atoms of glycine. If these b-dicarbonyl precursors are eventually converted into pyrazoles, then 1,5-dimethyl-
pyrazole should incorporate at least one C-2 atom of glycine and 1,3,5-trimethylpyrazole should incorporate at least two C-2 atoms of glycine according to the proposed scheme (see Fig. 4). In order to verify the role of glycine in the generation of 3-ketobutanal and 2,4-pentanedione, a model system consisting only of glyceraldehyde and ammonium carbonate was studied. The data indicated that the model system did not produce the above pyrazoles (the main product was 2-hydroxymethyl-2-methylpyrazine). In model systems containing glyceraldehyde/glycine, the C-2 atoms of glycine will be incorporated as methyl groups into the pyrazole ring, this fact was also confirmed by examination of label (13C-2 atom of glycine) incorporation into the mass fragments originating from M-15 (loss of a methyl group). For example, in the case of 1,5-dimethylpyrazole, ion at m/z 81 represents a loss of a methyl group
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Fig. 4. Proposed mechanism of formation of 1,5-dimethyl and 1,3,5-trimethylpyrazoles in [13C-2]glycine/glyceraldehyde model system. (20 ) indicates C-2 atom of glycine, * indicates mixed origin.
from the molecular ion at m/z 96. The labeled spectrum generated two ions one at m/z 81 (45%) and the other at m/z 82 (55%) indicating the presence of a labeled carbon atom in the methyl group. The mechanism of conversion of b-dicarbonyls into pyrazoles is illustrated for 1,3,5-trimethylpyrazole in Fig. 4 and is based on our initial proposal shown in Fig. 3. Reaction of ammonia with one of the carbonyl groups of 2,4-pentanedione can produce 4-amino-3-penten-2-one. The formation of 4-amino-3-penten-2-one has already been verified in model systems containing 2,4-pentanedione and ammonia (see earlier). In order for this intermediate to form 1,3,5-trimethylpyrazole, it should react with methyl amine and form the corresponding dienamine. Methyl amine can be formed either through decarboxylation of glycine and hence with incorporation of labeled C-2 atom or without incorporation when formaldehyde (originating from glyceraldehyde) forms an imine with subsequent isomerization and hydrolysis (Davidek, Velisek, & Pokorny, 1990) (see Fig. 1). Formaldehyde originating from Strecker reaction will also be labeled with C-2 atom of glycine. The final step in the formation of pyrazole requires oxidation of dienamine (Fig. 4) through the intermediacy of a-dicarbonyl compounds such as pyruvaldehyde that is known to form from glyceraldehyde (Weenen, 1998).
We have already demonstrated above that the absence of such oxidizing compounds, prevents formation of pyrazoles. In addition, oxidative N–N bond formation have been recently reported in literature for pyrazole synthesis (Bates et al., 1997). The final number of C-2 atoms of glycine incorporated into both pyrazoles will therefore depend on the origin of methyl amine. Table 1 lists percent C-2 label incorporation in different pyrazoles identified in the Table 1 Percent 13C-label distribution in different pyrazolesa formed in glyceraldehyde and 2,3-butanedione model systems containing [13C-2]glycine Model system
M
M+1
M+2
M+3
0 0
45 0
55 30
70
100 15
85
100 15
85
13
[ C-2]glycine/glyceraldehyde 1,5-dimethyl-pyrazole 1,3,5-trimethyl-pyrazole 13
[ C-2]glycine/2,3-butanedione 3,4,5-trimethyl-pyrazole 1,3,4,5-tetramethyl-pyrazole 13
[ C-2]glycine/2-hydroxy-3-butanone 3,4,5-trimethyl-pyrazole 1,3,4,5-tetramethyl-pyrazole a
All listed pyrazoles also showed 100% incorporation of two nitrogen atoms, when model systems with [15N]glycine was used.
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Fig. 5. Proposed mechanism of formation of 3,4,5-trmethyl and 1,3,4,5-tetramethylpyrazoles in [13C-2]glycine/3-hydroxy-2-butanone or 2,3-butanedione model system.
model systems, consistent with the proposed mechanism shown in Fig. 4. 3.3. Mechanism of formation of 3,4,5-trimethyl and 1,3,4,5-tetramethylpyrazoles in [13C-2]glycine/3hydroxy-2-butanone or 2,3-butanedione model system To study the role of a-dicarbonyl moiety in generation of pyrazoles, 2,3-butanedione was reacted with [13C-2]-labeled and unlabeled glycine. This model system produced 3,4,5trimethyl and 1,3,4,5-tetramethylpyrazoles as shown in Figs. 2 and 5. Both pyrazoles have a common b-dicarbonyl precursor, 3-methyl-2,4-pentanedione, which can be formed by aldol condensation of acetaldehyde (generated from a-dicarbonyl cleavage of 2,3-butanedione) with 3-hydroxy-2-butanone as shown in Fig. 5. The formation of acetaldehyde and 3-hydroxy-2-butanone was confirmed when 2,3-butanedione was pyrolyzed alone. Furthermore, the addition of 3-hydroxy-2-butanone to the mixture of 2,3-butanedione/glycine before pyrolysis, increased the peak areas associated with pyrazoles. Further reaction of 3-methyl-2,4-pentanedione with two moles of ammonia can generate 3,4,5-trimethylpyrazole and with a mixture of ammonia and methylamine can generate 1,3,4,5-tetramethylpyrazole through the same mechanism depicted in Fig. 4. Since the b-dicarbonyl backbone of both pyrazoles is formed without the participation of glycine, 3,4,5-trimethylpyrazole is expected not to incorporate any C-2 atoms of glycine and 1,3,4,5-tetramethylpyrazole to incorporate only one C-2 atom originating from methylamine participation in the reaction. As was indicated earlier, methyl amine has a mixed origin in these model systems. Inspection of Table 1 indicates incorporation of C-2 atom in a manner consistent with the proposed mechanism. When excess ammonium carbonate was
added to 2,3-butanedione/glycine model system before pyrolysis, the peak representing the 1,3,4,5-tetramethyl pyrazole increased significantly (from 5 to 40% of total peak area of the pyrogram) confirming participation of ammonia in the formation of pyrazoles. It is interesting to note at this point that 3-hydroxy-2-butanone was one of the major Maillard intermediates reported during preparation of tequila beverage (Lo´pez, 1999; Mancilla-Margalli & Lo´pez, 2002) along with 3,4,5-trimethylpyrazole.
Acknowledgements V.Y. acknowledges funding for this research by the Natural Sciences and Engineering Research Council (NSERC) of Canada. References Acheson, R. M. (1976). An introduction to the chemistry of heterocyclic compounds (3rd ed.). New York: John Wiley. Bates, D. K., Kohrt, J. F., Folk, H., & Xia, M. (1997). Fused pyrazole synthesis by N–N bond formation: the pyrazolo[5,1]benzothiazole system. Heterocyclic Communications, 5, 201–206. Cantalego, M. J. (1997). Effects of roasting temperature on the aroma components of Carob (Ceratonia siliqua L.). Journal of Agricultural and Food Chemistry, 45, 1345–1350. Davidek, J., Velisek, J., Pokorny, J. (Eds.). (1990). Chemical changes during food processing. Amsterdam: Elsevier. Frattini, C., Bicchi, C., Barettini, C., & Nano, G. M. (1977). Volatile flavor components of Licorice. Journal of Agricultural and Food Chemistry, 25, 1238–1241. Guillen, M. D., Manzanos, M. J., & Ibargoita, M. L. (2001). Carbohydrate and nitrogenated compounds in Liquid smoke flavorings. Journal of Agricultural and Food Chemistry, 49, 2395–2403. Huyghues-Despointe, & Yaylayan, V. (1996). Retro-adol and redox reactions of Amadori compounds: Mechanistic studies with variously labeled D-[13C]glucose. Journal of Agricultural and Food Chemistry, 44, 672–681.
V.A. Yaylayan, L.J.W. Haffenden / Food Research International 36 (2003) 571–577 Keyhani, A., & Yaylayan, V. (1996). Elucidation of the mechanism of pyrazinone formation in glycine model systems using labeled sugars and amino acid. Journal of Agricultural and Food Chemistry, 44, 2511–2516. Lee, K.-G., & Takayuki, S. (2000). Antioxidant properties of aroma compounds isolated from soybeans and Mung beans. Journal of Agricultural and Food Chemistry, 48, 4290–4293. Lo´pez, M. G. (1999). Tequila aroma. In F. Shaidi, & C.-T. Ho (Eds.), Flavor chemistry of ethnic foods (pp. 211–217). New York: Plenum. Mancilla-Margalli, N. A., & Lo´pez, M. G. (2002). Generation of Maillard compounds from inulin during the thermal processing of Agave tequilana Weber Var. Azul. Journal of Agricultural and Food Chemistry, 50, 806–812. Narziss, L., Meidaner, H., & Koch, M. (1988). Volatile substances
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formed during malt and beer production, especially those formed by heating the product. Part 1. Analysis, identification and effect on aroma. Monatsschr. Brauwiss., 41, 344–352. Schmeltz, I., & Hoffmann, D. (1977). Nitrogen-containing compounds in tobacco and tobacco smoke. Chemical Reviews, 77, 295–311. Weenen, H. (1998). Reactive intermediates and carbohydrate fragmentation in Maillard chemistry. Food Chemistry, 62(4), 393–401. Yaylayan, V. A. (1999). Analysis of complex reaction mixtures: novel applications of Py–GC/MS and microwave assisted synthesis. American Laboratory, 31(9), 30–31. Yaylayan, V., & Wnorowski, A. (2000). The influence of pyrolytic and aqueous phase reactions on the mechanism of formation of Maillard products. Journal of Agricultural and Food Chemistry, 48, 3549– 3554.
Mechanism of pyrazole formation in [13C-2] labeled glycine model systems: N–N bond formation during Maillard reaction Varoujan A. Yaylayan*, Luke J.W. Haffenden McGill University, Department of Food Science and Agricultural Chemistry, 21,111 Lakeshore, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9 Received 27 May 2002; accepted 17 December 2002
Abstract Studies with 13C-2 labeled glycine in model systems containing 3-hydroxy-2-butanone or glyceraldehyde have indicated that the b-dicarbonyl compounds, the immediate precursors of pyrazoles, are produced in Maillard model systems through two pathways. One pathway involves dehydration of a,b-dihydroxy carbonyl compounds with elimination of the a-hydroxyl group and the other through aldol condensation of an a-hydroxy carbonyl compound with simple aldehydes to produce a,b-dihydroxy carbonyl moiety that can undergo the above-mentioned dehydration to produce b-dicarbonyl structures. The conversion of b-dicarbonyls into pyrazoles can be achieved through 1,3-diimine formation by reaction with either two ammonia molecules or with a primary amine and an ammonia. After imine–enamine isomerizations the resulting dienamine can be oxidized to form pyrazole rings similar to the oxidation of two thiol moieties into disulfide linkages. The a-dicarbonyl species can serve as hydrogen acceptors since in their absence no pyrazole formation was detected. Glycine/3-hydroxy-2-butanone system generated 3,4,5-trimethyl-pyrazole (associated with the aroma of tequila) and 1,3,4,5-tetramethyl-pyrazole whereas, glycine/glyceraldehyde generated 1,5-dimethyl and 1,3,5-trimethyl-pyrazoles. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Maillard reaction; 13C-labeled glycine; Mechanism of pyrazole formation; N–N bond formation; Py–GC/MS
1. Introduction Although pyrazoles are not considered among the common Maillard reaction products, however their presence in Maillard systems further indicates the diversity of molecular structures that could be formed during the reaction. In addition, their formation poses a challenge to researchers in this field, since available synthetic routes for pyrazoles require synthons with preformed N–N bonds such as hydrazines. Such precursors have not been identified in Maillard systems and are difficult to envisage their formation under the reaction conditions. Furthermore, the related heterocyclic compounds isoxazole and isothiazoles could also have similar mechanistic origins as pyrazoles. Elucidation of the mechanism of formation of different heterocyclic compounds in general can contribute to our understanding of reactive intermediates formed during the reaction and * Corresponding author. Tel.: +1-514-398-7918; fax: +1-514-3987977. E-mail address: [email protected] (V. A. Yaylayan).
enhance our ability to predict and control the formation of different flavor related compounds. The 3,4-dimethylpyrazole was first reported in tobacco smoke around 30 years ago (Schmeltz & Hoffmann, 1977). Recently, other methyl substituted pyrazoles have been also identified in trace amounts in liquid smoke flavorings (Guillen, Manzanos, & Ibargoita, 2001) in soy beans (Lee & Takayuki, 2000), in heated malt and beer (Narziss, Meidaner, & Koch, 1988), in roasted carob (Cantalego, 1997) and in heated licorice essential oil (Frattini, Bicchi, Barettini, & Nano, 1977). Lo´pez (1999) on the other hand, reported formation of Maillard generated 3,4,5trimethylpyrazole in significant amounts during cooking of Agave tequilana to manufacture tequila beverage. Mechanism of formation of pyrazoles during Maillard reaction is not reported, however, many pyrazole derivatives can be synthesized in the laboratory by the interaction of hydrazine or monosubstituted hydrazines with b-dicarbonyls or with a,b-unsaturated carbonyl compounds (Acheson, 1976). Mechanism of formation of heterocyclic compounds in Maillard model systems can be investigated through labeling studies using the
0963-9969/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0963-9969(03)00003-6
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convenient pyrolysis/gas chromatography/mass spectrometry (Py–GC/MS) as an integrated reaction, separation and identification system (Yaylayan, 1999). Although under pyrolytic conditions a higher number of products are formed compared with aqueous reactions, however, most of the products identified in aqueous systems are also formed under pyrolytic conditions albeit in different amounts. In addition, experimental evidence was provided that the position and label distribution in the common products observed in the same model systems, between aqueous and pyrolytic reactions, are identical (Yaylayan & Wnorowski, 2000). This indicates the similarity of mechanisms of formation of these common products under both conditions. Consequently, mechanistic conclusions derived from label incorporation in the products observed under pyrolytic conditions, that are common to both systems, have relevance to the aqueous reactions. Here we report the mechanism of formation of pyrazoles during Maillard reaction, using 13C-2 labeled glycine and 3-hydroxy-2-butanone/2,3-butanedione or glyceraldehyde as model systems
2. Materials and methods 2.1. Materials All reagents and chemicals were purchased from Aldrich Chemical Company (Milwaukee, WI). [2-13C]glycine (90% enriched) and [15N]glycine (98% enriched) were purchased from Cambridge Isotope Laboratories (Andover, MA). 2.2. Pyrolysis–GC/MS analysis A Hewlett-Packard GC/mass selective detector (5890 series II GC/5971B MSD) interfaced to a CDS Pyroprobe 2000 unit, through a valved interface (CDS 1500), was used for Py–GC/MS analysis. In all experiments, solid samples of a mixture of labeled or unlabeled glycine or ammonium carbonate/dicarbonyl compound (3:1 ratio, 2 mg) were introduced inside the quartz tube (0.3 mm thickness) and plugged with quartz wool and inserted inside the coil probe. The pyroprobe was set at 250 C at a heating rate of 50 C/ms with a total heating time of 20 s. The pyroprobe interface temperature was set at 250 C. The samples were introduced under splitless mode. The 3 ml/min constant flow was maintained by an Electronic Pressure Controller (Hewlett-Packard). Capillary direct MS interface temperature was 180 C; ion source temperature was 280 C. The ionization voltage was 70 eV and the electron multiplier was 2047 V. The mass range analyzed was 17 – 200 amu. The column was a CP-Sil 5CB (30 m 0.32 mm 0.25 mm; Varian, Mississagua, ON). The column initial temperature (30 C) was held for 2 min then increased to 100 C at a
rate of 30 C/min, then further increased to 250 C at a rate of 7 C/min, and held for 35 min. The reported percent label incorporation values (corrected for natural abundance and for % enrichment) are the average of doublicate analyses and are rounded off to the nearest multiple of 5%. The identity and purity of the peaks were determined using NIST AMDIS version 2.1 software.
3. Results and discussion Most of the heterocyclic compounds reported in Maillard model systems originate from a-dicarbonyl and a-hydroxycarbonyl intermediates formed from reducing sugars during the reaction (Weenen, 1998) (see Fig. 1). However, pyrazoles, isoxazoles and isothiazoles require b-dicarbonyl moieties (Acheson, 1976) in addition to the ability to form N–N, N–O or N–S bonds, respectively. These requirements for their formation perhaps explain the low concentrations detected in food products. Analysis of the data generated from the interaction of 13C-2 glycine and glycine with glyceraldehyde, 3-hydroxy-2-butanone or 2,3-butanedione have indicated that all three model systems can generate pyrazoles. Glycine/3-hydroxy-2-butanone or 2,3-butanedione system generated 3,4,5-trimethyl and 1,3,4,5tetramethylpyrazoles whereas, glycine/glyceraldehyde generated 1,5-dimethyl and 1,3,5-trimethylpyrazoles (see Fig. 2). Furthermore, the data from different model systems containing different precursors also indicated that both a-hydroxycarbonyl and a-dicarbonyl moieties are necessary for the formation of pyrazoles in addition to the presence of b-dicarbonyl moiety. Since a-hydroxycarbonyl and a-dicarbonyl moieties are mutually convertible through redox reactions in Maillard model systems, it is expected therefore that model systems containing either of the two precursors or both should generate pyrazoles. In theory, the a-hydroxycarbonyl compounds can be converted into b-dicarbonyl derivatives (precursors of pyrazoles) by two different pathways, depending on the substituents at the b-position (see Fig. 1). In glyceraldehyde for example, the presence of a b-hydroxyl group can facilitate dehydration reaction where the a-hydroxyl group is eliminated to produce a b-dicarbonyl derivative, whereas, the b-position in 3hydroxy-2-butanone is unsubstituted and thus incapable of dehydration to produce b-dicarbonyl moiety. Such intermediates require a different mechanism to be converted into b-dicarbonyl derivatives. One such mechanism is the aldol condensation with simple aldehydes to form a,b-dihydroxy carbonyl intermediates which upon dehydration in a similar fashion to that of glyceraldehyde can generate the required b-dicarbonyl compounds. Evidence for the above mentioned mechanisms comes from label incorporation studies and the detection
V.A. Yaylayan, L.J.W. Haffenden / Food Research International 36 (2003) 571–577
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Fig. 1. Origin of simple heterocyclic compounds. AR=Amadori rearrangement; SR=Strecker reaction, [O]=oxidation.
of specifically substituted pyrazoles identified in glyceraldehyde, 3-hydroxy-2-butanone or 2,3-butanedione/ glycine systems as elaborated below. 3.1. Preliminary experiments to identify precursors of pyrazoles in Maillard model systems Literature studies (Acheson, 1976) have indicated that for laboratory synthesis of pyrazoles, b-dicarbonyls are essential starting materials for the generation of the carbon backbone of pyrazoles in addition to hydrazine derivatives. Alternatively, if amines are used instead of hydrazines, oxidizing conditions are necessary to form the N–N single bond (Bates, Kohrt, Folk, & Xia, 1997) (see Fig. 3), similar to the oxidation of two thiol moieties into disulfide linkages. During Maillard reaction, amines and a-dicarbonyl intermediates are produced abundantly from amino acids and reducing sugars (Weenen, 1998). The a-dicarbonyl intermediates are known to undergo reductions (Huyghues-Despointes & Yaylayan, 1996) and therefore can serve as oxidizing species to form N–N bonds during the Maillard reaction. In order to test this hypothesis, a mixture consisting of
2,4-pentanedione, a commercially available b-dicarbonyl, and ammonium carbonate a source of ammonia was reacted with and without 2,3-butanedione. Both model systems produced 4-amino-3-pentene-2-one (see Fig. 4) as the major product, but only the model system containing an a-dicarbonyl species, produced 1,3,5-trimethylpyrazole and a trace amount of 1,3,4,5-tetramethylpyrazole. In addition, the mixture consisting of 2,4-pentanedione and glycine produced mainly 4-(methylamino)-3-penten-2one. Furthermore, when the b-dicarbonyl was also removed from the model system, again no pyrazole was detected. These studies have indicated the importance of both a- and b-dicarbonyl species in the generation of pyrazole, the former as an oxidizing species and the latter as the carbon backbone. 3.2. Mechanism of formation of 1,5-dimethyl and 1,3,5trimethylpyrazoles in [13C-2]glycine/glyceraldehyde model system To study the role of a-hydroxycarbonyl moiety in generation of pyrazoles, glyceraldehyde was reacted with [13C-2]-labeled and unlabeled glycine. This model
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Fig. 2. Summary of model systems studied and pyrazole derivatives identified in them.
Fig. 3. Proposed general mechanism of formation of pyrazole moiety from b-dicarbonyl precursors.
system produced 1,5-dimethyl and 1,3,5-trimethyl-pyrazoles as shown in Fig. 2. The specific b-dicarbonyl precursors required for their formation are 3-ketobutanal and 2,4-pentanedione respectively (see Fig. 4). These precursors can be formed from glyceraldehyde by an amino acid assisted chain elongation process identified earlier (Keyhani & Yaylayan, 1996) and shown in Fig. 4. Through this chain elongation process glyceraldehyde can be converted into 3,4-dihydroxy-2-butanone with the incorporation of one C-2 atom of glycine as a methyl group. Dehydration of this intermediate can generate 3-ketobutanal the b-dicarbonyl precursor of 1,5-dimethylpyrazole. A similar chain elongation reaction with the aldehyde end of 3-ketobutanal can convert it into 2,4-pentanedione with incorporation of two C-2 atoms of glycine. If these b-dicarbonyl precursors are eventually converted into pyrazoles, then 1,5-dimethyl-
pyrazole should incorporate at least one C-2 atom of glycine and 1,3,5-trimethylpyrazole should incorporate at least two C-2 atoms of glycine according to the proposed scheme (see Fig. 4). In order to verify the role of glycine in the generation of 3-ketobutanal and 2,4-pentanedione, a model system consisting only of glyceraldehyde and ammonium carbonate was studied. The data indicated that the model system did not produce the above pyrazoles (the main product was 2-hydroxymethyl-2-methylpyrazine). In model systems containing glyceraldehyde/glycine, the C-2 atoms of glycine will be incorporated as methyl groups into the pyrazole ring, this fact was also confirmed by examination of label (13C-2 atom of glycine) incorporation into the mass fragments originating from M-15 (loss of a methyl group). For example, in the case of 1,5-dimethylpyrazole, ion at m/z 81 represents a loss of a methyl group
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Fig. 4. Proposed mechanism of formation of 1,5-dimethyl and 1,3,5-trimethylpyrazoles in [13C-2]glycine/glyceraldehyde model system. (20 ) indicates C-2 atom of glycine, * indicates mixed origin.
from the molecular ion at m/z 96. The labeled spectrum generated two ions one at m/z 81 (45%) and the other at m/z 82 (55%) indicating the presence of a labeled carbon atom in the methyl group. The mechanism of conversion of b-dicarbonyls into pyrazoles is illustrated for 1,3,5-trimethylpyrazole in Fig. 4 and is based on our initial proposal shown in Fig. 3. Reaction of ammonia with one of the carbonyl groups of 2,4-pentanedione can produce 4-amino-3-penten-2-one. The formation of 4-amino-3-penten-2-one has already been verified in model systems containing 2,4-pentanedione and ammonia (see earlier). In order for this intermediate to form 1,3,5-trimethylpyrazole, it should react with methyl amine and form the corresponding dienamine. Methyl amine can be formed either through decarboxylation of glycine and hence with incorporation of labeled C-2 atom or without incorporation when formaldehyde (originating from glyceraldehyde) forms an imine with subsequent isomerization and hydrolysis (Davidek, Velisek, & Pokorny, 1990) (see Fig. 1). Formaldehyde originating from Strecker reaction will also be labeled with C-2 atom of glycine. The final step in the formation of pyrazole requires oxidation of dienamine (Fig. 4) through the intermediacy of a-dicarbonyl compounds such as pyruvaldehyde that is known to form from glyceraldehyde (Weenen, 1998).
We have already demonstrated above that the absence of such oxidizing compounds, prevents formation of pyrazoles. In addition, oxidative N–N bond formation have been recently reported in literature for pyrazole synthesis (Bates et al., 1997). The final number of C-2 atoms of glycine incorporated into both pyrazoles will therefore depend on the origin of methyl amine. Table 1 lists percent C-2 label incorporation in different pyrazoles identified in the Table 1 Percent 13C-label distribution in different pyrazolesa formed in glyceraldehyde and 2,3-butanedione model systems containing [13C-2]glycine Model system
M
M+1
M+2
M+3
0 0
45 0
55 30
70
100 15
85
100 15
85
13
[ C-2]glycine/glyceraldehyde 1,5-dimethyl-pyrazole 1,3,5-trimethyl-pyrazole 13
[ C-2]glycine/2,3-butanedione 3,4,5-trimethyl-pyrazole 1,3,4,5-tetramethyl-pyrazole 13
[ C-2]glycine/2-hydroxy-3-butanone 3,4,5-trimethyl-pyrazole 1,3,4,5-tetramethyl-pyrazole a
All listed pyrazoles also showed 100% incorporation of two nitrogen atoms, when model systems with [15N]glycine was used.
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Fig. 5. Proposed mechanism of formation of 3,4,5-trmethyl and 1,3,4,5-tetramethylpyrazoles in [13C-2]glycine/3-hydroxy-2-butanone or 2,3-butanedione model system.
model systems, consistent with the proposed mechanism shown in Fig. 4. 3.3. Mechanism of formation of 3,4,5-trimethyl and 1,3,4,5-tetramethylpyrazoles in [13C-2]glycine/3hydroxy-2-butanone or 2,3-butanedione model system To study the role of a-dicarbonyl moiety in generation of pyrazoles, 2,3-butanedione was reacted with [13C-2]-labeled and unlabeled glycine. This model system produced 3,4,5trimethyl and 1,3,4,5-tetramethylpyrazoles as shown in Figs. 2 and 5. Both pyrazoles have a common b-dicarbonyl precursor, 3-methyl-2,4-pentanedione, which can be formed by aldol condensation of acetaldehyde (generated from a-dicarbonyl cleavage of 2,3-butanedione) with 3-hydroxy-2-butanone as shown in Fig. 5. The formation of acetaldehyde and 3-hydroxy-2-butanone was confirmed when 2,3-butanedione was pyrolyzed alone. Furthermore, the addition of 3-hydroxy-2-butanone to the mixture of 2,3-butanedione/glycine before pyrolysis, increased the peak areas associated with pyrazoles. Further reaction of 3-methyl-2,4-pentanedione with two moles of ammonia can generate 3,4,5-trimethylpyrazole and with a mixture of ammonia and methylamine can generate 1,3,4,5-tetramethylpyrazole through the same mechanism depicted in Fig. 4. Since the b-dicarbonyl backbone of both pyrazoles is formed without the participation of glycine, 3,4,5-trimethylpyrazole is expected not to incorporate any C-2 atoms of glycine and 1,3,4,5-tetramethylpyrazole to incorporate only one C-2 atom originating from methylamine participation in the reaction. As was indicated earlier, methyl amine has a mixed origin in these model systems. Inspection of Table 1 indicates incorporation of C-2 atom in a manner consistent with the proposed mechanism. When excess ammonium carbonate was
added to 2,3-butanedione/glycine model system before pyrolysis, the peak representing the 1,3,4,5-tetramethyl pyrazole increased significantly (from 5 to 40% of total peak area of the pyrogram) confirming participation of ammonia in the formation of pyrazoles. It is interesting to note at this point that 3-hydroxy-2-butanone was one of the major Maillard intermediates reported during preparation of tequila beverage (Lo´pez, 1999; Mancilla-Margalli & Lo´pez, 2002) along with 3,4,5-trimethylpyrazole.
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