2-Methoxy-3-isopropyl-pyrazines

Musty or potato-like flavor and aroma have been observed as a defect in milk (Hammer and Babel 1957) and Gruyere de Comte cheese (Dumont et al. 1975). This off-flavor results from the production of nitrogenous cyclic compounds by Pseudomonas taetrolens and P. perolens (Morgan 1976). Musty-flavored compounds produced by these organisms include 2,5-dimethylpyrazine and 2-methoxy-3-isopropyl-pyrazine. The Gruyere de Comte with potato off-flavor contained 3-methoxy-2-propyl pyridine, as well as alkyl pyrazine compounds (Dumont et al. 1975). Murray and Whitfield (1975) postulated that alkyl pyrazines are formed in vegetables by condensation of amino acids such as valine, isoleucine, and leucine with a 2-carbon compound. Details of the synthetic mechanism in pseudomonads are unknown. 

There are many reports of Pseudomonas cultures producing musty, earthy, and potato-like odors ( l-6). The work of Morgan et al. (T) established 2-methoxy-3-isopropyl pyrazine to be partially responsible for these odors. Subsequently, 2-methyoxy-3-isopropyl pyrazine was found in bell peppers (8), a similar compound 2-methoxy-3-secbutyl pyrazine was identified in galbanum oil (9), and several 2-methoxy-3-alkyl pyrazines were identified in various raw botanicals (10). The odor threshold exhibited by 2-methoxy-3-isobutyl pyrazine (1 part in 10 indicates flavor significance for these compounds even at the exceptionally low concentrations in which they occur in foods and other natural products Producing these compounds from microbial fermentations could be an economical source of flavor for the food industry. 

Because the potential for producing "natural" 2-methoxy-3-alkyl pyrazines via fermentation might be improved by optimizing media composition, we studied the influence of media components on the synthesis 2-methoxy-3-isopropyl pyrazine by cultures of Pseudomonas perolens and selected mutant strains. 

Effect of Carbon Source. The growth of Pseudomonas perolens as well as culture levels of 2-methoxy-3-isopropyl pyrazine in minimal salts plus 1% pyruvate are shown in Figure 1. High cell density was obtained but the presence of 2-methoxy-3-isopropyl pyrazine was not detected until after cell numbers were no longer increasing. At this point, the amount of pyrazine increased rapidly reaching a maximum on the fifth day. The growth and pyrazine synthesis with other carbon sources showed similar behavior. The results are presented in Table I, and plotted for selected carbon sources at 1%... 

Carbon Source % (w/v) 2-Methoxy 3-isoPropyl Pyrazine (ng/mL) % vs. 1% Lactate... 

As can be seen from Table I, pyruvate and lactate at 1% resulted in the highest yield of 2-methoxy-3-isopropyl pyrazine. At the 2% level these substrates resulted in a decrease of approximately 30% in the amount of pyrazine with a much greater drop at 0.2% and 0.5% levels. This is in contrast to glucose and fructose as carbon source where the amount of pyrazine synthesized was inversely related to the initial concentration of sugar in the media. Sodium acetate yielded about 20 ng/mL which is impressive considering that the cell numbers were 3-4 orders of magnitude lower than that found... 

Table II. The influence of nitrogen source and concentration on the production of 2-methoxy-3-isopropyl pyrazine by P. petal ens.

Although clear differences were observed in the amounts of 2-methoxy-3-isopropyl pyrazine produced, the data were more striking when population density was considered. When compared on the basis of pyrazine synthesized per 10 0 cfu/mL, the culture grown in media containing 0.2 mM phosphate produced almost 50 fold more pyrazine than the one grown in 50 mM phosphate. 

Actinomycetes, primarily streptomycetes, are capable of producing highly odorous volatile metabolites in low yields in submerged culture. This subject has been reviewed in detail(37). Among the more important volatiles identified ares geosmin, the earthy odor, methyl isoborneol, having a camphor or menthol odor 2-methoxy-3-isopropyl pyrazine, with a musty vegetable odor and miscellaneous compounds such as sesquiterpenoids and lactones. 

Figure 5 Extraction curves for A, IPMP, and B, TCA, isolated from grain and water (white and black dots on the graph, respectively). Extraction was performed at 50°C four times ranging from 1 min to 120 min. IPMP, 2-methoxy-3-isopropyl-pyrazine TCA, 2,4,6-trichloroanisole.

Maltol Isobutyrate 2-Methoxy 3-(or 5- or 6-) Isopropyl Pyrazine 5H-5-Methyl-6,7 -dihydrocyclopenta[b]pyrazine 5-Methyl Furfural Methyl Furoate Methyl Hexanoate Methyl Isovalerate 5-Methyl 2-Phenyl 2-Hexenal Methyl Thiobutyrate Methyl Valerate P-Naphthyl Ethyl Ether Phenyl Ethyl Cinnamate Phenyl Ethyl Propionate Propyl Formate Propyl Mercaptan Salicylaldehyde 8-T etradecalactone 2-Tridecanone... 

Geosmin, 2-Methyl isobomeol (MIB) and 2-isopropyl methoxy pyrazine are known to be produced by various types of actinomycete cultures (10-15). Geosmin and MIB are saturated tertiary alcohols and resist oxidation. The steric configuration of the hydroxyl and methyl groups in both compounds are believed to interact with receptors in the nose, imparting their characteristic earthy odour (16). The four compounds itemised as the key osmogenes in this odorous emission have extremely low odour threshold concentrations. Their occasional occurence in drinking water can lead to widespread complaints and are routinely monitored for within this Authority. 

Alkyl-2-methoxypyrazines exhibit a base peak at miz 124 in the mass spectrum. The peak corresponds to a molecular ion in 2-methoxy-3-methylpyrazine (24a) and to a fragment ion, resulting from a McLafferty rearrangement of an alkyl group, in 3-isopropyl-2-methoxypyrazine (24b) and 3-5cc-butyl-2-methoxy pyrazine (24d) (97). 

Methoxypyrazines 3-isobutyl-2-methoxy-pyrazine, 3-isopropyl-2-methoxypyrazine Strong X No ... 

Pyrazine, 3-isobutyl-2-methoxy-occurrence, 3, 192-193 Pyrazine, 3-isopropyl-2-methoxy-occurrence, 3, 193... 

Isopropyl-2-methoxy pyrazine (2- Isopropy 1 - 3- methoxy pyrazine) och3 i i-C3H7... 

Isopropyl-5-methoxy pyrazine (5-Isopropyl-2-methoxypyrazine) i-C3H7 OCH3... 

Methoxy-3-isopropyl-5-methyl-pyrazine OCH3 i-C3H7 ch ... 

Methoxy -3-isopropyl-6-methyl-pyrazine OCH3 i-C3H7 ch3... 

Geosmin, 2-isopropyl, 3-methyox pyrazine, 2-isobutyl-3-methoxy-pyrazine, 2-methyl isobomeol, 2,3, 6 trichloroanisole ... 

Isopropyl-3-methoxy-5-methylpyrazine gave 2-isopropyl-3-methoxy-5-[3-(pyran-2-yloxy)propyl]pyrazine (289) [2-(3-bromopropoxy)pyran, PT2NK (made in situ), THF, A, -78°C, 3 h 83%] 298 also analogues likewise.295,298... 

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