Thermally Generated Flavors - American Chemical Society

Chapter 12

Mechanism of Pyrazine Formation H. Weenen, S. B. Tjan, P. J. de Valois, N. Bouter, A. Pos, and H. Vonk

Downloaded by UCSF LIB CKM RSCS MGMT on November 26, 2014 | http://pubs.acs.org Publication Date: November 30, 1993 | doi: 10.1021/bk-1994-0543.ch012

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Pyrazine formation was studied as a model for carbohydrate fragmentation in the Maillard reaction. Thus 1- C-glucose, 2- C-glucose and 1- C-fructose were reacted with asparagine in 1,2-propanediol, and the volatile products isolated by steam distillation and extraction. The product mixture was analyzed by GC, NMR and GC-MS, and consisted mainly of dimethyl-, monomethyl, and to a lesser extent trimethylpyrazines. Although product yields and ratios for the reaction of asparagine with glucose differed to some extent from the same reaction with fructose, the C-distribution of the resulting pyrazines was not much different. The C-distribution in pyrazines originating from 2- C-glucose was quite different from the C-distribution in pyrazines which were formedfromglucose and fructose labeled at the C-1 position. The C-incorporation in the pyrazines obtained from all three labeled hexoses was in agreement with retro-aldolization of the intermediate deoxyglucosones as the main cleavage mechanism (scheme 5). Both 1- and 3-deoxyglucosone appear to play an approximately equally important role in the formation of the methylated pyrazines. 13

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The formation of many flavor substances in the Maillard reaction is initiated by carbohydrate cleavage. The resulting mono- and dicarbonyl products may react further to form pyrazines, thiazoles, carbocyclic compounds, etc., depending on conditions and other reactants (7-5). Asparagine appears to be a particularly good amino acid in the formation of pyrazines (5,6), and was used here to study pyrazine formation and the underlying carbohydrate cleavage mechanism.

0097-6156/94/0543-0142$06.00/0 © 1994 American Chemical Society

In Thermally Generated Flavors; Parliment, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

12. WEENENETAL.

Mechanism of Pyrazine Formation

143

In the literature three main carbohydrate cleavage mechanisms are described. Hayami (7) studied sugar decomposition which was not amine or amino acid catalyzed, but which took place in 40% aqueous phosphate buffer (pH 6.7). Acetol was reported as the main three-carbon fragmentation product. Its formation was explained by hydrolytic cleavage of a p-diketone intermediate, which was generated from 1-deoxy glucosone by isomerization. In the Maillard reaction the main cleavage mechanism is often considered to be retroaldolization (8). The structures of a number of Maillard reaction products permit one to assume that they have been formed by retroaldol cleavage. In a glucose plus p-alanine model reaction, production of C and C3 sugar fragments was negligible under acidic conditions, but increased with pH, also in agreement with the retro-aldol cleavage mechanism (9). In a glucose plus alkyl amine model system pyruvaldehyde and glyoxal diimines were identified as the main products (70,72). It was suggested that C fragments are formed directly from an aldose or the corresponding imine (70), and C fragments from deoxyosones (77) or Amadori rearrangement products (72). In addition to the retroaldolization mechanism, there seems to be some evidence for an alternative cleavage mechanism in the Maillard reaction: cleavage of a-dicarbonyl species (8). This mechanism is supposed to involve immonium ion formation, followed by hydrolytic cleavage, resulting in a carboxylic acid and an immoniumbetaine intermediate. The mechanism is based mainly on products formed from a-dicarbonyl species such as 3-deoxyglucosone. We recently reported on the reactivity of 3-deoxyglucosone and the formation of pyrazines from l- C-elucose (6). We now report on the formation of pyrazines from l- C-glucose, 2-* C-glucose and l- C-fructose, which were analysed by N M R and GC-MS.


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Experimental General. The reaction conditions for generating the pyrazines were as described before (6). Abbreviations in the text are as follows: Pyr: pyrazine; M M P : methylpyrazine; D M P : dimethylpyrazine; T M P : trimethylpyrazine; 1-done: 1-deoxy glucosone; 3-done: 3-deoxyglucosone; 4-done: 4-deoxyglucosone. NMR. N M R conditions have been described before (6). Because samples of the labeled pyrazines were relatively dilute, the chemical shifts of the pyrazine C-atoms varied considerably, making it impossible to assign each peak in the C - N M R to a specific pyrazine. For the same reason only some of the methyl signals for 2,5-DMP could be identified individually. 13

G C and GC-MS. The G C colums used were a 50 m CP-Sil 8CB (Chrompack) and a 60 m Stabilwax (Restek). The CP-Sil column was used in most of our studies, and did not separate 2,5-DMP and 2,6-DMP. The Stabilwax column gave almost complete resolution of 2,5-DMP and 2,6-DMP. The term dimethylpyrazine (DMP) was generally used to indicate a mixture of 2,5-DMP and 2,6-DMP. We used GC-MS data to identify the individual pyrazines, and to quantify the level of C-enrichment. The spectra of the labeled pyrazines were compared with the spectra of the unlabeled pyrazines. The [M]" , [M+l] - and [M-l] - patterns of the unlabeled pyrazines were used to determine the extent of ^ C-enrichment in the labeled pyrazines. MS-MS experiments of unlabeled D M P had indicated this to be an acceptable approach. Using this procedure the ratios of none, once and twice labeled pyrazines were obtained, and were used to calculate total C-content. When the same approach was used to determine the C-content of the 13

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THERMALLY GENERATED FLAVORS

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fragmentation patterns representing [M-HCN] - and [ C H C N H ] , values were obtained which were qualitatively comparable with the N M R results. However the C contents of the pyrazines calculated from the C contents of the fragments were significantly and consistently different from the C contents of the pyrazine molecules based on the molecular ion mass spectrum. We reasoned that the values for the fragments may be effected by isotope effects, and are therefore unreliable. 3

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Results and Discussion 13

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Pyrazines from l- C-glucose and l- C-fructose. Distribution of C in the pyrazine molecules was determined by *H-NMR, C - N M R and GC-MS (Tables 1—4). C-incorporation in pyrazines derived from l- C-fructose was found to be slightly higher than in pyrazines derived from l- C-glucose. N M R data clearly indicate that C - l of fructose and glucose is incorporated at the methyl and methine carbon atoms of the methylated pyrazines, but not at the quarternary carbon atom. C-incorporation at the methyl position of 2,5-DMP is equal for fructose and glucose, C-incorporation at the methine position is slightly higher for l- C-fructose derived pyrazines than for l- C-glucose derived pyrazines. A l l in all there is very little difference in C-incorporation into pyrazines from l- C-fructose and l- C-glucose, which is not necessarily what we expected. Fructose gives pyrazines in a significantly higher yield than glucose (Table 5), and because it is a ketose, it must follow a different route in the formation of the deoxyglucosones (Scheme 1 and 2). Comparison of the theoretically possible C containing retroaldol products from the l-^C-deoxyglucosones (Scheme 3) suggests that the position of the C-label in the methylated pyrazines is indicative for the relative importance of the intermediate deoxyglucosones (6). Since 2,3-dimethylpyrazine is formed only to a negligible extent, 4-deoxyglucosone is apparently not an important intermediate. Similarly intermediates 3 and 4 (Scheme 3) can be ruled out as contributing significantly to the product pyrazines. This allows one to assign the C H labeled pyrazines as coming from 3-deoxyglucosone, and the C H labeled pyrazines as originating from 1-deoxy glucosone. If we apply this reasoning to the pyrazines derived from l- C-glucose and 1- C-fructose, it can be concluded that 1- and 3-deoxyglucosone are participating approximately equally in the formation of pyrazines from fructose, and that 1-deoxyglucosone is slightly more important in the formation of pyrazines from glucose.


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Pyrazines from 2- C-glucose. Methylated pyrazines that were formed from 2- C-glucose, were labeled almost exclusively at the quarternary carbon atom, in agreement with the mechanism indicated in Scheme 4. C - N M R showed two small signals in the C H region (139.4 and 141.5 ppm) of the spectrum, indicating that there is an additional pathway leading to pyrazines. This can be explained by assuming that retroaldolization of glucose or glucose imine also takes place. This reaction which has been suggested by Hayashi et al. (10), results in glycolaldehyde or the corresponding imine, which in turn can lead to methylpyrazine or dimethylpyrazine with a C-label at the C H position. Comparison of the integrated signals in the C - N M R of 2- C-glucose derived 2,5-dimethylpyrazine with one of synthetic origin, allowed the quantitation of the methine C and the quarternary C (Table 4). 13

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Retroaldolization versus other cleavage mechanisms. A l l our observations are in agreement with retroaldolization of the intermediate 1- and 3-deoxyglucosones


12.

WEENENETAL.

Mechanism of Pyrazine Formation

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Table 1. C-Incorporation in Pyrazines Based on H-NMR

1

CH (%)

CH (%) hexose

2,5-DMP

13

l- C-glucose

13

l- C-fructose

13

2- C-glucose

1

all

2,5-DMP

all

20.4

22.4

25.8

29.8

(21.2-23.6)

(14.6-25.6)

(20.3-29.8)

(23.9-34.5)

26.8

27.0

27.1

30.0

(25.8-28.0)

(25.9-28.0)

(26.1-28.0)

(29.8-30.1)

Thermally Generated Flavors - American Chemical Society

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