RU2781198C2 - Methods for selective production of substituted pyrazines - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method for the production of pyrazines substituted with two, three, or four C₁-C₆ alkyl groups, including: obtaining a reaction solution containing one alpha-hydroxyketone including 1-hydroxy-2-butanone, and one nitrogen source containing NH₄OH, addition of free amino acids to the reaction solution; heating of the specified reaction solution to a reaction temperature of 100-140°C, and exposure of the reaction solution at the specified reaction temperature for 4-24 hours to obtain a reaction product containing substituted pyrazine; and isolation of the specified pyrazine substituted with two, three, or four C₁-C₆ alkyl groups from the reaction product, including liquid-liquid extraction of the reaction product, solid-liquid extraction of the reaction product, or simple distillation of the reaction product. The invention also relates to a method for the production of pyrazines substituted with two, three, or four C₁-C₆ alkyl groups, including: obtaining a solution of a carbon source, containing one C₆ sugar containing glucose or fructose and one buffer containing sodium hydroxide or phosphate buffer; mixing of the specified solution of the carbon source with one nitrogen source containing NH₄OH to obtain a reaction solution; heating of the reaction solution to a reaction temperature of 120-140°C, and exposure of the reaction solution at the specified reaction temperature for 17-18 hours to obtain a reaction product containing pyrazine substituted with two, three, or four C₁-C₆ alkyl groups; and isolation of the specified pyrazine substituted with two, three, or four C₁-C₆ alkyl groups from the reaction product, including liquid-liquid extraction of the reaction product, solid-liquid extraction of the reaction product, or simple distillation of the reaction product.

EFFECT: obtaining substituted pyrazines.

27 cl, 8 dwg, 11 tbl, 7 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to methods for preparing substituted pyrazines. Of particular interest are methods for the selective preparation of certain substituted pyrazines.

BACKGROUND OF THE INVENTION

Pyrazines are prepared by reactions (eg Maillard) of a carbon source with nitrogen containing compounds such as amino acids and bases (eg diammonium phosphate (DAP), NaOH). Many known synthesis schemes for preparing pyrazines use sugars (eg fructose, glucose, fructose/glucose mixtures, rhamnose) as the carbon source. Currently, in the absolute majority of model reactions and enriched products of natural origin, from which pyrazine-rich compositions can be obtained by heating, the carbon source components are sugars, such as fructose, glucose, fructose/glucose mixtures, and rhamnose. These sugars have been shown to serve as a carbon source for the formation of pyrazine aromatic ring structures. See, for example, U.S. Patent Application No. 2004/0173228 Coleman 2010/0037903 Coleman et al. 2012/0152265 Dube et al. 2012/0260929 Coleman et al. et al., 2013/0337418 by Anuradha, 2015/0040922 by Dube et al., and 2015/0122271 by Chen et al., US Pat. Nos. 5258194 by Anderson et al., 6298858 by Coleman, 6325860 by Coleman, 6440223 by Dube et al., 6499489 by Coleman, 6591841 by White et al., 6695924 by Dube et al., 8434496 by Chen et al., 8944072 by Brinkley et al., 8955523 Coleman et al., 8991403 Chen et al., 9010339 Dube et al., 9254001 Byrd et al., 9265284 Junker et al. the synthesis and characteristics of aqueous formulations rich in pyrazines, in Flavor Fragrance and Odor Analysis, Second Edition, Ray Marsili, ed., Chapter 7, pp 135-182, CRC Press, Boca Raton, 2012, full contents of each of these publications are included to this application by reference.

Most often in these reactions with sugar(s) and a nitrogen source, ammonium hydroxide and/or free amino acids are used as nitrogen source, which provide a nitrogen bond in the pyrazine structure. See, for example, Effect of Time, Temperature, and reactant ratio on pyrazine formation in model system, T. Shibamoto, R.A. Bernhard, J. Agric. Food Chem., 24, (1976) p. 847. In this reaction, a complex mixture of many substituted pyrazines is formed. If sugar acts as the only carbon source in the reaction of obtaining pyrazines, the molecules of pyrazine and methylpyrazine prevail among the obtained pyrazines, often making up much more than 60% of the total yield of pyrazines. This trend is evident even if free amino acids are used as co-reagents to reduce the amount of pyrazine and methylpyrazine molecules obtained.

From a sensory standpoint, the pyrazine and methylpyrazine molecules do not have the desired sensory notes or acceptable volatility characteristics for use in tobacco products. Thus, in certain applications, their presence in a mixture of pyrazines is an undesirable characteristic.

In this regard, there is a need to develop methods for the selective production of certain desirable substituted pyrazines on a large scale.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides methods for the selective preparation of certain substituted pyrazines. The process for selectively producing pyrazines may include providing a reaction solution containing at least one tobacco-derived carbon source (e.g., hydroxyketone and/or at least one buffered sugar) and at least one tobacco-derived nitrogen source (e.g. , protein and/or amino acid), and heating the reaction solution to the reaction temperature and maintaining the reaction solution at the reaction temperature for a time sufficient to obtain a reaction product containing at least one substituted pyrazine. The nitrogen source may be selected from the group consisting of amino acids, ammonium ions, and combinations thereof.

In various embodiments, said at least one substituted pyrazine may be selected from the group consisting of 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2-ethyl-6-methylpyrazine, 2,3 ,5-trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-2,5-dimethylpyrazine, 2,3,5,6-trimethylpyrazine, 2,3,5-trimethyl-6-ethylpyrazine, 2,6 -dimethyl-3-propylpyrazine, 2,5-diethyl-3,6-dimethylpyrazine, 2,6-dimethyl-3-(2-methylbutyl)pyrazine, 2,5-dimethyl-3-(2-methylbutyl)pyrazine, 2 ,5-dimethyl-3-(3-methylbutyl)pyrazine, 2,5-dimethyl-3-propylpyrazine, 2,5-dimethyl-3-cis-propenylpyrazine, 2-isopropenyl-3,6-dimethylpyrazine, 2-(2 -methylpropyl)-3,5-dimethylpyrazine, 2,6-dimethyl-3-isobutylpyrazine, 2-(2-methylpropyl)-3,5,6-trimethylpyrazine, 2,3-dimethylpyrazine, trimethylpyrazine, furaneol, 2,5- dimethyl-3-ethylpyrazine, tetramethylpyrazine, 2,3-diethyl-5-methylpyrazine, 2,5-dimethyl-3-propenylpyrazine, 2,3,5-trimethyl-6-isopropylpyrazine, 2-acetyl-4,5-dimethylpyrazine, 3,5-dimethyl-2-methylpropylpyrazine, 2,6-diethylpyrazine, 2,5-diethylpyrazine, 2-ethyl-3,5,6-trimethylpyrazine, 3,5(3,6-dimethyl)-2,N-propylpyrazine, 2,5-diethyl-3-methylpyrazine , 2,3-diethyl-5,6-dimethylpyrazine, trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine, 2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrazine , and 2,5-dimethyl-3-ethylpyrazine, and combinations thereof.

In some embodiments, said at least one substituted pyrazine is disubstituted, trisubstituted, or tetrasubstituted. In some embodiments, the implementation of the specified at least one substituted pyrazine contains at least one substituent group containing 2 or more carbon atoms. In various embodiments, said at least one substituted pyrazine contains at least one substituent group containing 3 or more carbon atoms.

In various embodiments, the method for selectively producing certain substituted pyrazines may further comprise isolating said at least one substituted pyrazine from the reaction product. The step of isolating said at least one tobacco-derived pyrazine from the reaction product may, for example, include at least one of reaction product liquid-liquid extraction, reaction product solid-liquid extraction, and reaction product simple distillation.

The methods of the present invention may further include incorporating at least one substituted pyrazine into the tobacco product. In some embodiments, said tobacco product may be a smoking article. In some embodiments, said tobacco product may be a smokeless tobacco product.

In various embodiments, the present invention provides a method for producing pyrazines, comprising obtaining a reaction solution containing at least one alpha-hydroxy ketone and at least one source of nitrogen, and heating the reaction solution to a reaction temperature and keeping the reaction solution at the reaction temperature for a time, sufficient to obtain a reaction product containing at least one substituted pyrazine. Various embodiments of the method may further include isolating said at least one substituted pyrazine from the reaction product. In some embodiments, said at least one hydroxy ketone may contain acetol, and said at least one substituted pyrazine may be selected from the group consisting of 2,3-dimethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-5-methylpyrazine, trimethylpyrazine, furaneol, 2,5-dimethyl-3-ethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, tetramethylpyrazine, 2,5-dimethyl-3-propenylpyrazine, 2,3,5- trimethyl-6-isopropylpyrazine, 2-acetyl-4,5-dimethylpyrazine, 3,5-dimethyl-2-methylpropylpyrazine, and combinations thereof. In some embodiments, said at least one hydroxy ketone may contain acetoin, and said at least one substituted pyrazine may be tetramethylpyrazine. In various embodiments, said at least one hydroxy ketone may contain 1-hydroxy-2-butanone, and said at least one substituted pyrazine may be selected from the group consisting of: 2,6-diethylpyrazine, 2,5-diethylpyrazine, 2 -ethyl-3,5,6-trimethylpyrazine, 3,5(3,6-dimethyl)-2,N-propylpyrazine, 2,5-diethyl-3-methylpyrazine, 2,3-diethyl-5-methyl pyrazine, 2 ,3-diethyl-5,6-dimethylpyrazine, trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine, 2,5-dimethyl-3-ethylpyrazine, and combinations thereof.

In various embodiments of the present invention, the method may further include adding the free amino acid to the reaction solution containing at least one alpha-hydroxy ketone and at least one nitrogen source. In some embodiments, the method may further include adding at least one aldehyde to the reaction solution containing at least one alpha-hydroxy ketone and at least one nitrogen source.

In various embodiments of the present invention, a process is provided for the preparation of pyrazines, comprising obtaining a carbon source solution containing at least one sugar and at least one buffer, while an optimized amount of at least one hydroxyketone can be obtained from said at least one sugar. ; mixing the carbon source solution with at least a nitrogen source to form a reaction solution; and heating the reaction solution to the reaction temperature and keeping the reaction solution at the reaction temperature for a time sufficient to obtain a reaction product containing at least one substituted pyrazine. Various embodiments of the methods herein may further include isolating said at least one substituted pyrazine from the reaction product. Said at least one sugar may be selected from the group consisting of glucose, fructose, rhamnose, and combinations thereof. In some embodiments, the buffer may be selected from the group consisting of: sodium hydroxide, phosphate buffer, and combinations thereof. In some embodiments, the buffer may create a pH in the range of about 6.5 to about 7.5. The methods of the present invention may further include adding ammonium ions to a reaction solution comprising a carbon source that contains at least one sugar and at least one buffer.

The present invention includes, but is not limited to, the following embodiments:

Embodiment 1: A method for producing pyrazines, comprising: obtaining a reaction solution containing at least one alpha-hydroxy ketone and at least one source of nitrogen, and heating the reaction solution to a reaction temperature, and keeping the reaction solution at the reaction temperature for a time sufficient to obtaining a reaction product containing at least one substituted pyrazine.

Embodiment 2: The method of any of the previous embodiments wherein at least one said hydroxy ketone comprises acetol.

Embodiment 3: The method of any of the previous embodiments wherein at least one said substituted pyrazine is selected from the group consisting of: 2,3-dimethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2- ethyl-5-methylpyrazine, trimethylpyrazine, furaneol, 2,5-dimethyl-3-ethylpyrazine, 2-ethyl-3,5-dimethylupyrazine, tetramethylpyrazine, 2,5-dimethyl-3-propenylpyrazine, 2,3,5-trimethyl- 6-isopropylpyrazine, 2-acetyl-4,5-dimethylpyrazine, 3,5-dimethyl-2-methylpropylpyrazine, and combinations thereof.

Embodiment 4: The method of any of the previous embodiments wherein at least one said hydroxy ketone comprises acetoin.

Embodiment 5: The method of any of the previous embodiments wherein at least one of said substituted pyrazine is tetramethylpyrazine.

Embodiment 6: The method of any of the previous embodiments wherein at least one said hydroxy ketone comprises 1-hydroxy-2-butanone.

Embodiment 7: The method of any of the previous embodiments, wherein said at least one substituted pyrazine is selected from the group consisting of: 2,6-diethylpyrazine, 2,5-diethylpyrazine, 2-ethyl-3,5,6- trimethylpyrazine, 3,5(3,6-dimethyl)-2,N-propylpyrazine, 2,5-diethyl-3-methylpyrazine, 2,3-diethyl-5-methylpyrazine, 2,3-diethyl-5,6-dimethylpyrazine , trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine, 2,5-dimethyl-3-ethylpyrazine, and combinations thereof.

Embodiment 8: The method of any of the previous embodiments wherein said nitrogen source is selected from the group consisting of amino acid, ammonium ions, and combinations thereof.

Embodiment 9: The method of any of the previous embodiments, further comprising adding the free amino acid to the reaction solution.

Embodiment 10: The method of any of the previous embodiments, further comprising adding at least one aldehyde to the reaction solution.

Embodiment 11: The method of any of the previous embodiments, further comprising isolating said at least one substituted pyrazine from the reaction product.

Embodiment 12: The method of any of the previous embodiments, wherein said step of isolating said at least one substituted pyrazine from the reaction product comprises at least one of reaction product liquid-liquid extraction, reaction product solid-liquid extraction, and simple distillation of the product reactions.

Embodiment 13: The method of any of the previous embodiments wherein said reaction temperature is from about 90°C to about 150°C.

Embodiment 14: The method of any of the previous embodiments wherein at least one said substituted pyrazine is selected from the group consisting of: 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2- ethyl-6-methylpyrazine, 2,3,5-trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-2,5-dimethylpyrazine, 2,3,5,6-trimethylpyrazine, 2,3,5- trimethyl-6-ethylpyrazine, 2,6-dimethyl-3-propylpyrazine, 2,5-diethyl-3,6-dimethylpyrazine, 2,6-dimethyl-3-(2-methylbutyl)pyrazine, 2,5-dimethyl-3 -(2-methylbutyl)pyrazine, 2,5-dimethyl-3-(3-methylbutyl)pyrazine, 2,5-dimethyl-3-propylpyrazine, 2,5-dimethyl-3-cis-propenylpyrazine, 2-isopropenyl-3 ,6-dimethylpyrazine, 2-(2-methylpropyl)-3,5-dimethylpyrazine, 2,6-dimethyl-3-isobutylpyrazine, 2-(2-methylpropyl)-3,5,6-trimethylpyrazine, 2,3-dimethylpyrazine , trimethylpyrazine, furaneol, 2,5-dimethyl-3-ethylpyrazine, tetramethylpyrazine, 2,3-diethyl-5-methylpyrazine, 2,5-dimethyl-3-propenylpyrazine, 2,3,5-trimethyl-6-isopropylpyrazine, 2 -acetyl-4,5-dimethylpi razine, 3,5-dimethyl-2-methylpropylpyrazine, 2,6-diethylpyrazine, 2,5-diethylpyrazine, 2-ethyl-3,5,6-trimethylpyrazine, 3,5(3,6-dimethyl)-2,N -propylpyrazine, 2,5-diethyl-3-methylpyrazine, 2,3-diethyl-5,6-dimethylpyrazine, trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine, 2,5,7 -trimethyl-6,7-dihydro-5H-cyclopentapyrazine, 2,5-dimethyl-3-ethylpyrazine and combinations thereof.

Embodiment 15: The method of any of the previous embodiments wherein at least one of said substituted pyrazine is disubstituted.

Embodiment 16: The method of any of the previous embodiments wherein at least one of said substituted pyrazine is trisubstituted.

Embodiment 17: The method of any of the previous embodiments wherein at least one substituted pyrazine is tetrasubstituted.

Embodiment 18: The method of any of the previous embodiments, wherein said at least one substituted pyrazine contains at least one substituent group containing 2 or more carbon atoms.

Embodiment 19: The method of any of the previous embodiments, wherein said at least one substituted pyrazine contains at least one substituent group containing 3 or more carbon atoms.

Embodiment 20: The method of any of the previous embodiments wherein said reaction product is substantially free of pyrazine molecules and methyl pyrazine molecules.

Embodiment 21: The method of any of the previous embodiments, further comprising incorporating said at least one substituted pyrazine into the tobacco product.

Embodiment 22: The method of any of the previous embodiments, further comprising incorporating said at least one substituted pyrazine into a tobacco product, said tobacco product being a smokeable product or a smokeless tobacco product.

Embodiment 23: A process for producing pyrazines, comprising: providing a carbon source solution containing at least one sugar and at least one buffer; mixing the carbon source solution with at least a nitrogen source to form a reaction solution; and heating the reaction solution to the reaction temperature and keeping the reaction solution at the reaction temperature for a time sufficient to obtain a reaction product containing at least one substituted pyrazine.

Embodiment 24: The method of any of the previous embodiments, wherein said at least one sugar is selected from the group consisting of glucose, fructose, rhamnose, and combinations thereof.

Embodiment 25: The method of any of the previous embodiments, wherein said nitrogen source is selected from the group consisting of amino acid, ammonium ions, and combinations thereof.

Embodiment 26: The method of any of the previous embodiments, wherein said buffer is selected from the group consisting of: sodium hydroxide, phosphate buffer, and combinations thereof.

Embodiment 27: The method of any of the previous embodiments wherein said buffer produces a pH in the range of about 6.5 to about 7.5.

Embodiment 28: The method of any of the previous embodiments, further comprising adding ammonium ions to the reaction solution.

Embodiment 29: The method of any of the previous embodiments, further comprising isolating said at least one substituted pyrazine from the reaction product.

Embodiment 30: The method of any of the previous embodiments, wherein said step of separating said at least one substituted pyrazine from the reaction product comprises at least one of reaction product liquid-liquid extraction, reaction product solid-liquid extraction, and simple distillation of the product reactions.

Embodiment 31: The method of any of the previous embodiments wherein said reaction temperature is from about 90°C to about 150°C.

Embodiment 32: The method of any of the previous embodiments, wherein said at least one substituted pyrazine is selected from the group consisting of: 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2- ethyl-6-methylpyrazine, 2,3,5-trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-2,5-dimethylpyrazine, 2,3,5,6-trimethylpyrazine, 2,3,5- trimethyl-6-ethylpyrazine, 2,6-dimethyl-3-propylpyrazine, 2,5-diethyl-3,6-dimethylpyrazine, 2,6-dimethyl-3-(2-methylbutyl)pyrazine, 2,5-dimethyl-3 -(2-methylbutyl)pyrazine, 2,5-dimethyl-3-(3-methylbutyl)pyrazine, 2,5-dimethyl-3-propylpyrazine, 2,5-dimethyl-3-cis-propenylpyrazine, 2-isopropenyl-3 ,6-dimethylpyrazine, 2-(2-methylpropyl)-3,5-dimethylpyrazine, 2,6-dimethyl-3-isobutylpyrazine, 2-(2-methylpropyl)-3,5,6-trimethylpyrazine, 2,3-dimethylpyrazine , trimethylpyrazine, furaneol, 2,5-dimethyl-3-ethylpyrazine, tetramethylpyrazine, 2,3-diethyl-5-methylpyrazine, 2,5-dimethyl-3-propenylpyrazine, 2,3,5-trimethyl-6-isopropylpyrazine, 2 -acetyl-4,5-dimethylpi razine, 3,5-dimethyl-2-methylpropylpyrazine, 2,6-diethylpyrazine, 2,5-diethylpyrazine, 2-ethyl-3,5,6-trimethylpyrazine, 3,5(3,6-dimethyl)-2,N -propylpyrazine, 2,5-diethyl-3-methylpyrazine, 2,3-diethyl-5,6-dimethylpyrazine, trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine, 2,5,7 -trimethyl-6,7-dihydro-5H-cyclopentapyrazine, 2,5-dimethyl-3-ethylpyrazine and combinations thereof.

Embodiment 33: The method of any of the previous embodiments wherein at least one of said substituted pyrazine is disubstituted.

Embodiment 34: The method of any of the previous embodiments wherein at least one of said substituted pyrazine is trisubstituted.

Embodiment 35: The method of any of the previous embodiments wherein at least one substituted pyrazine is tetrasubstituted.

Embodiment 36: The method of any of the previous embodiments, wherein said at least one substituted pyrazine contains at least one substituent group containing 2 or more carbon atoms.

Embodiment 37: The method of any of the previous embodiments, wherein said at least one substituted pyrazine contains at least one substituent group containing 3 or more carbon atoms.

Embodiment 38: The method of any of the previous embodiments, wherein said reaction product is substantially free of pyrazine and methylpyrazine molecules.

Embodiment 39: The method of any of the previous embodiments, further comprising incorporating said at least one substituted pyrazine into the tobacco product.

Embodiment 40: The method of any of the previous embodiments, further comprising incorporating said at least one substituted pyrazine into a tobacco product, said tobacco product being a smokeable product or a smokeless tobacco product.

These and other features, aspects and advantages of the present invention will be apparent upon reading the following detailed description in conjunction with the accompanying drawings, which are briefly described below. The present invention includes any combination of two, three, four or more of the above embodiments, as well as combinations of any two, three, four or more features or elements set forth in the present description, regardless of whether such features or elements are explicitly combined in description of a specific implementation option in the present description. This description is intended to be read as a whole, so any separable features or elements of the disclosed invention in any of its various aspects and embodiments should be considered as intended to be combined unless the context clearly indicates otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to provide an understanding of the embodiments of the present invention, reference is made to the accompanying drawings, which are not necessarily to scale, and in which the item numbers refer to the components of the exemplary embodiments of the present invention. The drawings are for illustrative purposes only and should not be construed as limiting the present invention.

FIG. 1 is a diagram describing methods for the selective preparation of substituted pyrazines;

in FIG. 2 is a diagram describing methods for the selective preparation of substituted pyrazines;

in FIG. 3 shows GC/MS analysis of glucose in reaction with phosphate buffer at 140° C. for 60 min and extraction with dichloromethane (DCM);

in FIG. 4 shows GC/MS analysis of pyrazines extracted with DCM from the reaction mixture (1 gram of glucose reacted with 25 ml of 40% phosphate buffer at 140°C for 60 minutes) with 1 ml of NH ₄ OH at 140°C for 17 hours ;

in FIG. 5 shows the GC/MS analysis of pyrazines extracted with DCM from 25 ml of the reaction mixture (0.1 N NaOH reacted with 0.5 g of glucose at 140°C for 60 min) after reaction with 1 ml of NH ₄ OH at 140 °C;

in FIG. 6 is an expanded perspective view of a cigarette-shaped smoking article showing the smoking material, wrapping material components, and filter elements of the cigarette;

in FIG. 7 is a plan view of an embodiment of a smokeless tobacco product taken across the width of the product showing an outer pouch filled with tobacco material; and

in FIG. 8 is a sectional view of an electronic smoking article comprising a cartridge and a control including a reservoir, according to an exemplary embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, a more detailed description of the present invention will be given. However, the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein; rather, these embodiments are provided so that the present disclosure is detailed and complete and fully conveys the scope of the present invention to those skilled in the art. Used in the present description and the claims, indefinite and definite forms of the singular include reference to the plural, unless the context clearly indicates otherwise. The reference to "percent dry matter" or "on a dry weight basis" refers to the weight in terms of dry ingredients (ie, all ingredients except water).

The present invention provides methods for obtaining certain pyrazines. Pyrazines have a variety of flavor profiles including, but not limited to, fried, toasted, and nutty notes. For example, pyrazines with cyclopentyl derivatives are known for their positive organoleptic properties, present at very low levels, ppb (parts per billion). Pyrazines are produced by heating a mixture of carbon sources and nitrogen sources. The methods of the present invention not only minimize the production of pyrazine and methylpyrazine molecules, but direct the reaction so that other desired substituted pyrazines can be produced in a controlled manner.

Selective production of pyrazines using a carbon source other than sugar(s)

Sugar(a) is used as a standard carbon source in pyrazine reactions. Several reaction routes are known in the art using sugar(s) to produce pyrazine-rich formulations, including: 1) hydrolysis of proteins to free amino acids followed by reaction of these free amino acids with sugar(s) such as glucose and/or high fructose syrup ; and 2) biotechnological synthesis of free amino acids using glucose and nitrogen (eg, ammonium ions) amino acids, followed by reaction of these free amino acids with sugar(s) such as glucose and/or high fructose syrup. When sugars are used as a whole molecule and react with a nitrogen source (e.g. ammonium hydroxide, amino acids), a range of pyrazines are obtained, including pyrazine and methylpyrazine molecules as the predominant pyrazines, with much less dimethylpyrazines and much less high molecular weight pyrazines.

In the context of the present description, the term "molecular pyrazine" refers to a heterocyclic organic compound with the chemical formula C ₄ H ₄ N ₂ . In the context of the present description, it differs from the general term "pyrazine(s)", which refers to a group of compounds obtained in the reaction of a carbon source with a nitrogen source.

In the context of the present description, the term "nitrogen source" refers to a nitrogen-containing compound that is capable of reacting with a carbon source to obtain at least one pyrazine. In various embodiments, the nitrogen source may comprise ammonium ions (NH ₄ ⁺ ), amino acids, proteins, or a combination thereof. In some embodiments, ammonium ions (NH ₄ ⁺ ) may be present through compounds such as ammonium hydroxide, diammonium phosphate (DAP), etc. Amino acids can be obtained by hydrolysis of a protein, for example, in some embodiments, amino acids can be isolated by hydrolysis of proteins derived from tobacco, as described in U.S. Patent Application No. 15/009199 to Dube et al., filed January 28, 2016 , the full content of which is incorporated into the present description by reference. Other nitrogen-containing compounds that are known in the art and are capable of reacting with a carbon source to produce at least one pyrazine can also be used as a nitrogen source in the embodiments of the present invention, which are disclosed in the present description.

In various embodiments of the present invention, the carbon source may include hydroxyketone. Hydroxyketone is a ketone functional group with a hydroxyl group adjacent to it (flanked by). In the two main classes of hydroxy ketones, the hydroxyl group can be located in the alpha position (i.e., the alpha hydroxy ketone, having the formula RCR'(OH)(CO)R), or in the beta position (i.e., the beta hydroxy ketone, having the formula formula RCR'(OH)CR ₂ (CO)R). The structure of alpha and beta hydroxy ketones is shown below.

In various embodiments of the present invention, the carbon source may include at least one alpha-hydroxy ketone. In various embodiments of the present invention, the functional groups R and R' of the specified at least one hydroxyketone may be H or a substituent independently selected from the group consisting of halogen (for example, Cl, F or Br), OH, optionally substituted with C1-10 alkyl, optionally substituted C1-10 alkoxy, optionally substituted C2-4 alkenyl, optionally substituted C2-4 alkynyl, NR ₆ R ₇ , NR ₆ COR ₇ , NR ₆ CO ₂ R ₇ , CR ₆ R ₇ OR ₈ , CONR ₆ R ₇ , CO ₂ R ₆ , CN, CF ₃ , NO ₂ , N ₃ , C1-3 alkylthio, R ₉ SO, R ₉ SO ₂ , CF ₃ S and CF ₃ SO ₂ . In certain embodiments of the present invention, the functional groups R and/or R' are C1-6 alkyl.

The term "alkyl" in the context of the present description means a saturated straight, branched or cyclic hydrocarbon group (ie cycloalkyl groups), as well as unsaturated variants of examples of saturated compounds (eg propenyl). In specific embodiments, alkyl refers to groups containing from 1 to 10 carbon atoms ("C1-10 alkyl"). In further embodiments, alkyl refers to groups containing 1 to 8 carbon atoms (“C 1-8 alkyl”), 1 to 6 carbon atoms (“C 1-6 alkyl”), or 1 to 4 carbon atoms (“C 1 -4 alkyl"). In other embodiments, alkyl refers to groups containing 3-10 carbon atoms ("C3-10 alkyl"), 3-8 carbon atoms ("C3-8 alkyl"), or 3-6 carbon atoms ("C3-6 alkyl" ). In certain embodiments, alkyl refers to methyl, trifluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2 -dimethylbutyl and 2,3-dimethylbutyl.

"Optionally substituted" with respect to a substituent group refers to substituent groups optionally substituted with one or more moieties selected from the group consisting of, for example, halogen {eg, Cl, F, Br, and I); alkyl {eg C1-10 alkyl), halogenated alkyl {eg CF ₃ , 2-Br-ethyl, CH ₂ F, CH ₂ Cl, CH ₂ CF ₃ or CF ₂ CF ₃₎ ; C2-4 alkenyl, C2-4 alkynyl, hydroxyl, amino, amido, carboxylate, carboxamido, carbamate, carbonate, urea, acetate, alkylamino, arylamino, C1-10 alkoxy, aryl, aralkyl, aryloxy, nitro, azido, cyano, thio , alkylthio, sulfonate, sulfide, sulfinyl, sulfo, sulfate, sulfoxide, sulfamide, sulfonamide, phosphonic acid, phosphate, and/or phosphonate.

The term "alkenyl" in the context of the present description means alkyl fragments, and at least one saturated C-C bond is replaced by a double bond. In specific embodiments, alkenyl refers to groups containing from 2 to 10 carbon atoms (“C2-10 alkenyl”). In further embodiments, alkenyl refers to groups containing 2 to 8 carbon atoms ("C2-8 alkenyl"), 2 to 6 carbon atoms ("C2-6 alkenyl"), or 2 to 4 carbon atoms ("C2- 4 alkenyl"). In certain embodiments, the alkenyl may be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1- hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl or 5-hexenyl.

The term "alkynyl" in the context of the present description means alkyl fragments, and at least one saturated C-C bond is replaced by a triple bond. In specific embodiments, alkynyl refers to groups containing from 2 to 10 carbon atoms (“C2-10 alkynyl”). In additional embodiments, alkynyl refers to groups containing 2 to 8 carbon atoms (“C2-8 alkynyl”), 2 to 6 carbon atoms (“C2-6 alkynyl”), or 2 to 4 carbon atoms (“C2-8 alkynyl”). 4 alkynyl"). In certain embodiments, the alkynyl may be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl or 5-hexynyl.

The term "alkoxy" in the context of the present description means straight or branched alkyl groups that are linked through an oxygen atom (ie -O-alkyl), with alkyl as described above. In particular embodiments, alkoxy refers to oxygen-bonded groups containing from 1 to 10 carbon atoms ("C1-10 alkoxy"). In additional embodiments, alkoxy refers to oxygen-bonded groups containing 1 to 8 carbon atoms ("C1-8 alkoxy"), 1 to 6 carbon atoms ("C1-6 alkoxy"), 1 to 4 carbon atoms ( "C1-4 alkoxy") or from 1 to 3 carbon atoms ("C1-3 alkoxy").

The term "halo" or "halogen" in the context of the present description means fluorine, chlorine, bromine or iodine.

The term "amino" in the context of the present description means a fragment represented by the structure NR ₂ and includes primary amines, and secondary and tertiary amines substituted with alkyl or aryl (ie, alkylamino or arylamino, respectively). Thus, R ₂ may represent two hydrogen atoms, two alkyl moieties, two aryl moieties, one aryl moiety and one alkyl moiety, one hydrogen atom and one alkyl moiety, or one hydrogen atom and one aryl moiety.

Alkyl (amino) is a moiety represented by the structure -RNR ₂ and includes an alkyl group as defined above attached to an amino group as defined above, said moiety being attached to another moiety via an alkyl group.

The term "cycloalkyl" means a non-aromatic, monocyclic or polycyclic ring containing carbon and hydrogen atoms.

The term "derivative" in the context of the present description means a compound that is obtained from a similar parent compound by adding a different molecule or atom to the parent compound. Additionally, derivatives according to the present invention include one or more compounds derived from a precursor compound by adding one or more atoms or molecules, or by combining two or more precursor compounds.

In one embodiment of the present invention, acetoin has been successfully used as a precursor for the preparation of tetramethylpyrazine (TMP). In particular, acetoin was easily (successfully) reacted with ammonium hydroxide and phosphoric acid (or diammonium phosphate) to give tetramethylpyrazine (TMP) in substantial quantitative yield (>80% yield) and a significant degree of purity. The reaction between acetoin and ammonium hydroxide produces almost exclusively TMP with few or no detectable pyrazine and methylpyrazine molecules. As used herein, the terms "little to undetectable", "substantially absent", and "substantially zero" are used to indicate that an identified compound is present in an amount of less than 1.0% by weight, less than 0.5% by volume or less than 0.1% by weight, based on the total weight of the reaction product.

Without wishing to be bound by any particular theory, it is believed that the various resulting pyrazines synthesized from reactions with sugar(s) versus acetoin as the carbon source unexpectedly indicate that the carbon source determines to a large extent the distribution of pyrazines in the reactions. between carbon sources and nitrogen sources such as ammonium ions and/or amino acids. In particular, it has been unexpectedly found that the use of various hydroxyketones as a carbon source in reactions to prepare pyrazines allows a number of specific substituted pyrazines to be prepared in a given manner.

In some embodiments, at least one substituted pyrazine obtained according to the methods described herein is disubstituted. In some embodiments, at least one substituted pyrazine obtained according to the methods described herein is trisubstituted. In various embodiments, the implementation of at least one substituted pyrazine, obtained in accordance with the methods described in the present description, is tetrasubstituted. In various embodiments, the implementation of at least one substituted pyrazine, obtained in accordance with the methods described in the present description, contains at least one substituent group containing 2 or more carbon atoms. In some embodiments, the implementation of at least one substituted pyrazine, obtained in accordance with the methods described in the present description, contains at least one substituent group containing 3 or more carbon atoms.

In some embodiments, the implementation of at least one substituted pyrazine, obtained in accordance with the methods described in the present description, is a branched pyrazine. In the context of the present description, the term "branched pyrazine" refers to the inclusion of an alkyl group to the pyrazine ring, which is non-linear in nature, for example, isobutyl, sec-butyl and tert-butyl groups instead of n-butyl group (this also applies in a similar way to propyl and pentyl groups). As described in more detail below, it was unexpectedly found that by changing the alpha-hydroxy ketone, the distribution of the resulting pyrazines can be set. For example, using acetoin in the reaction results in only tetramethylpyrazine (about 99.5% or higher purity). The use of acetol in the reaction results in a predominantly dimethyl-substituted pyrazine (about 95% or higher yield of pyrazines).

As shown in FIG. 1, for example, heated formulations containing nitrogen sources and at least one hydroxyketone allow pyrazines to be prepared. Pyrazine-rich solutions can be prepared in a variety of ways. For example, one method may include heating with microwave radiation a solution containing at least one amino acid and at least one hydroxyketone. As shown at operation 100 in FIG. 1, for example, an aqueous reaction solution containing at least one amino acid and at least one hydroxy ketone can be obtained. As shown in operation 104 in FIG. 1, the reaction solution, for example, may be heated to the reaction temperature and maintained at the reaction temperature for a reaction time that is sufficient for the reactants to react to form pyrazines. As shown in operation 106 in FIG. 1, for example, pyrazines can then optionally be isolated from the reaction product using simple distillation or other separation technology known in the art.

In one embodiment, the pyrazines are isolated from the reaction product by first using simple distillation to obtain a distillate containing predominantly water and pyrazines. This distillate can then be subjected to liquid phase extraction with cyclohexane. The amount of cyclohexane used in the liquid phase extraction may be about half the amount of distillate used. For example, when using 10 liters of distillate, 5 liters of cyclohexane can be used. The liquid-phase extraction of the distillate with cyclohexane can be repeated many times. In some embodiments, the liquid phase extraction of the distillate with cyclohexane may be repeated at least 5 times. After the liquid phase extraction, the cyclohexane containing the extracted pyrazines can be dried with any drying agent known in the art. For example, sodium sulfate, magnesium hydroxide and/or molecular sieves can be used to dry cyclohexane. After drying, the pyrazines can be isolated (ie the cyclohexane can be removed) by simple distillation and/or rotary evaporation.

As described above, the use of various hydroxy ketones as carbon sources in the reaction to produce pyrazines allows the production of a number of specific substituted pyrazines. In some embodiments, the carbon source includes acetoin. As described in more detail below in Example 1, for example, if acetoin is used as the sole carbon source for the preparation of pyrazines in reactions with a nitrogen source (for example, ammonium hydroxide (NH ₄ OH) and phosphoric acid (H ₃ PO ₄ ), phosphate diammonium, etc.), then the only pyrazine obtained is tetramethylpyrazine. In addition, branched pyrazines such as isopropylpyrazine are not synthesized by adding amino acids (such as, for example, leucine or free amino acids from hydrolyzed F1 protein) to a reaction mixture that contains acetoin, NH ₄ OH and H ₃ PO ₄ . Heating the reaction mixture at higher temperatures or for a longer period of time does not affect the result (ie, tetramethylpyrazine (TMP) will be the only pyrazine synthesized from acetoin). Therefore, carbon sources other than acetoin can be synthesized if pyrazines other than TMP are to be prepared by this synthetic method.

In some embodiments, the carbon source may contain 1-hydroxyacetone (also referred to as 1-OH-acetone, acetol, or 1-hydroxy-2-propanone). Acetol, an alpha hydroxy ketone, is the simplest hydroxy ketone. As described in more detail below, acetol can be obtained by decomposition of various sugars. For example, it can be prepared as an intermediate in the Maillard reaction (i.e., the reaction of sugar(s) and amino acid(s) to produce pyrazine), and the acetol can then be reacted further to produce other compounds. In some embodiments of the present invention, the carbon source may contain 1-hydroxy-2-butanone. The structure of 1-OH-acetone and 1-OH-2-butanone is presented below. It should be noted that in 1-OH-acetone the carboxyl group is linked on one side to the methyl group, while in 1-OH-2-butanone the carboxyl group is linked to the ethyl group on one side. Without being limited by theory, these structural features of both α,β-hydroxy ketones can determine the structure of the resulting pyrazines.

As shown in Example 2 below, for example, the reaction of 1-hydroxyacetone as a source of carbon and NH ₄ OH as a source of base and nitrogen produces a series of pyrazines. If 1-hydroxyacetone (acetol) serves as a carbon source, a number of specific alkyl-substituted pyrazines can be obtained. For example, examples of pyrazines produced by the reaction of 1-hydroxyacetone with NH ₄ OH may include: 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2-ethyl-6-methylpyrazine, ,5-trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-2,5-dimethylpyrazine, 2,3,5,6-trimethylpyrazine, 2,3,5-trimethyl-6-ethylpyrazine, 2,6 -dimethyl-3-propylpyrazine, 2,5-diethyl-3,6-dimethylpyrazine, 2,6-dimethyl-3-(2-methylbutyl)pyrazine, 2,5-dimethyl-3-(2-methylbutyl)pyrazine, 2 ,5-dimethyl-3-(3-methylbutyl)pyrazine, 2,5-dimethyl-3-propylpyrazine, 2,5-dimethyl-3-cis-propenylpyrazine, 2-isopropenyl-3,6-dimethylpyrazine, 2-(2 -methylpropyl)-3,5-dimethylpyrazine, 2,6-dimethyl-3-isobutylpyrazine, 2-(2-methylpropyl)-3,5,6-trimethylpyrazine, 2,3-dimethylpyrazine, trimethylpyrazine, furaneol, 2,5- dimethyl-3-ethylpyrazine, tetramethylpyrazine, 2,3-diethyl-5-methylpyrazine, 2,5-dimethyl-3-propenylpyrazine, 2,3,5-trimethyl-6-isopropylpyrazine, 2-acetyl-4,5-dimethylpyrazine and 3,5-dimethyl-2-methylpropylpyrazine.

When 1-hydroxy-2-butanone is used as the sole carbon source, pyrazines having attached ethyl groups can be obtained. For example, as shown in Example 4 below, pyrazines synthesized in the reaction using 1-OH-2-butanone and NH ₄ OH may include 2,6-diethylpyrazine, 2,5-diethylpyrazine, 2-ethyl-3,5, 6-trimethylpyrazine, 3,5(3,6-dimethyl)-2,N-propylpyrazine, 2,5-diethyl-3-methylpyrazine, 2,3-diethyl-5,6-dimethylpyrazine, trans-3-methyl-2 ,n-propyl-6(1-butenyl)pyrazine; and 2,5-dimethyl-3-ethylpyrazine.

If 1-hydroxyacetone (acetol), 2-hydroxy-3-butanone (acetoin) and 1-hydroxy-2-butanone are used as carbon sources in reactions with nitrogen sources, then among the structures and pyrazines obtained, the molecules of pyrazine and methylpyrazine are not formed. It should be noted that other hydroxyketones can be used to obtain alternative series of pyrazines. The use of various hydroxy ketones as a carbon source in pyrazine reactions can not only minimize the production of pyrazines and methyl pyrazines, but also allow the reaction to be directed such that the desired substituted pyrazine is produced in the desired manner.

As shown in FIG. 1 in step 102, amino acids and/or aldehydes, for example, may optionally be added to the reaction mixture solution containing at least one hydroxyketone and a source of nitrogen. As shown in Example 3 below, for example, the addition of an amino acid or a given aldehyde(s) can increase not only the number of pyrazines synthesized, but can also increase the yield of pyrazines. For example, in certain embodiments, the reaction solution may contain an amino acid selected from the group consisting of serine, alanine, leucine, isoleucine, valine, threonine, phenylalanine, and combinations thereof. Similarly, any alkylaldehyde can be used to increase the number of pyrazines synthesized as well as the yield of pyrazines. For example, in certain embodiments, the reaction solution may contain an alkylaldehyde selected from the group consisting of acetaldehyde, propanal, isopropanol, butanal, isobutanal, sec-butanal, and combinations thereof.

The increased reaction time and temperature allow for an increased yield of pyrazines until a black tarry mass is obtained. The reaction temperature may be, for example, about 30°C or more, about 90°C or more, about 100°C or more, about 120°C or more, or about 140°C or more. In some embodiments, the reaction temperature may be, for example, from about 90°C to about 150°C, or from about 120°C to about 140°C. The reaction time may be about 4 hours or more, 8 hours or more, about 12 hours or more, 16 hours or more, or about 24 hours or more. In various embodiments, the reaction time may be from about 4 to about 24, or from about 12 to about 20 hours. In some embodiments, the reaction time may be about 16 hours.

In various embodiments, the molar ratio of hydroxyketone to nitrogen source can affect the yield of pyrazines. The molar ratio of hydroxyketone: nitrogen source (eg, NH ₄ OH) may be, for example, about 1:0.5, about 1:1, about 1:2, or about 1:2.5. In some embodiments, the ratio of hydroxy ketone to nitrogen source may be from about 1:0.5 to about 1:2.5, or from about 1:1 to about 1:2. In some embodiments, the hydroxy ketone to nitrogen source may be about 1:2.

An increase in the pH of the reaction solution can also lead to an increase in the amount of pyrazines. A desirable pH range may be about 7.5 to about 10.5, or about 8.5 to about 9.5. In some embodiments, the pH of the reaction solution may be about 8.0 or greater, about 8.5 or greater, about 9.0 or greater, or about 10.0 or greater. A small addition of eg NaOH or KOH can be used to raise the pH of the reaction solution.

Selective Preparation of Pyrazines Using Sugar(s) as a Carbon Source

In various embodiments of the present invention, the selective production of substituted pyrazines is optimized using at least one sugar (eg, glucose, high fructose tobacco syrup (HFTS)) as a carbon source and ammonium ions, proteins and/or amino acids as a nitrogen source. As described above, various reaction routes of pyrazine-rich compounds have been used in the past to prepare pyrazines using sugar as a carbon source. See, for example, US Patent Application No. 15/009199, by Dube et al., filed January 28, 2016, the entire contents of which are incorporated herein by reference. However, if sugars are used as the only source in reactions with a nitrogen source (for example, ammonium hydroxide) to obtain pyrazines, pyrazine and methylpyrazine molecules can prevail among the pyrazines obtained. This trend is evident even if free amino acids are used as co-reagents. It has been unexpectedly found that substituted pyrazines can be selectively obtained by adjusting the pH of the carbon source prior to introducing it to the nitrogen source.

As shown in FIG. 2 heated compositions containing amino acids and sugars, for example, make it possible to obtain pyrazine. See, for example, U.S. Patent Publication No. 2010/0037903 by Coleman III et al. and Coleman III, On the synthesis and characteristics of aqueous formulations rich in pyrazines, in Flavor Fragrance and Odor Analysis, Second Edition, Ray Marsili, ed. ., Chapter 7, pp 135-182, CRC Press, Boca Raton, 2012, the entire contents of which are incorporated herein by reference. Solutions rich in pyrazines can be prepared in a variety of ways. For example, one method may include heating with microwave radiation a solution containing at least one amino acid and at least one sugar. As shown at operation 120 in FIG. 2, an aqueous reaction solution containing at least one amino acid and at least one sugar can be obtained. As shown at operation 124 in FIG. 1, the reaction solution, for example, may be heated to the reaction temperature and maintained at the reaction temperature for a reaction time sufficient for the reactants to react to form pyrazines.

As described above, acetol, a hydroxyketone, can be obtained by decomposing various sugars. In addition, when used as a carbon source in reactions with a nitrogen source, a number of substituted pyrazines can be obtained that do not include pyrazine and methylpyrazine molecules. Accordingly, acetol can be the main intermediate in reactions between sugar(s) and free amino acids and/or ammonium ions to produce pyrazines containing branched alkyl side chains.

The literature indicates that 1-hydroxy-2-propanone (acetol) can be obtained from a C6 sugar such as glucose and a glucose derivative, sorbitol. See, for example, M. H. Mohamad, et. al., A review of acetol: application and production)), Amer. J. Appl. Sci., 8, 1135-1139 (2011); M. A. Dasari, "Catalytic conversion of glycerol and sugar alcohols to value added products)", Univ. Missouri-Columbia, ISBN-10, 0549727582, pp, 264; W. Yan, Gas phase conversion of sugar to C3 chemicals, PhD Thesis, University of Missouri-Columbia, 2008; J. Hayami, Mechanism of acetol formation)), Bull. Chem. soc. Japan, 34, 927-932 (1961); P. F. Shaw, et. al., Base catalyzed Fructose degradation)), J. Agric. Food Chem., 16, 979-982 (1968); H. Weenen and W. Apeldoon, "Carbohydrate Cleavage in the Maillard Reaction)", Flavor Science, Recent Developments, A. Taylor and D. Mottran, eds., Royal Society of Chemistry, Special Publication # 197, Cambridge, 1996; the full content of each of which is incorporated into the present description by reference. These references report the conversion of sugars to 1-hydroxy-2-propanone using phosphate buffers at elevated temperatures, a strong base such as NaOH at pH 11.5 under reflux, pressurized Ni and palladium catalysts in hydrogen, and copper chromite catalyst. in the gaseous state in heterogeneous reactions. Copper chromite is considered the best catalyst. For most reactions, the conversion of substrates was more than 91%. The yield of acetol, in the case of using glycerol as a carbon source, was 32.2% at 220°C, in the case of using sorbitol as a carbon source, the highest yield was 11.8% at 280°C, and in the case of using glucose in as a carbon source, the highest yield of acetol was 8.99% at 280°C.

Cellulose was converted to acetol in 30% yield using a Sn-based catalyst system. See, for example, F. Chambon et al., Process for transformation of lignocellulose biomass or cellulose by catalysts based on Sn oxide and/or Sb oxide and a metal, that is selected from the Groups 8 to 11, US patent application, US 2013/028174 A1, 2013, the entire content of which is incorporated herein by reference.

Recently, Novotny et al., (Czech J. Food Sci., 25, 119-130, 2007, incorporated herein by reference) synthesized α-hydroxycarbonyl and α-dicarbonyl compounds by degradation of monosaccharides. They used three different models, including an aqueous solution of potassium persulfate, an alkaline solution of potassium persulfate, and a solution of sodium hydroxide, respectively. In total, six α-hydroxycarbonyl and six α-dicarbonyl compounds were detected by GC/MS. The maximum yield of α-hydroxycarbonyl (glycolaldehyde, acetol, lactaldehyde, glyceraldehyde, 1,3-dihydroxyacetone and acetoin), in the case of the reaction of glucose or fructose with sodium hydroxide, was approximately 4%. The yield was much lower in aqueous solutions of potassium persulfate (0.32%) and alkaline solution of potassium persulfate (1.1%). Acetol and 1,3-dihydroxyacetone had the highest yield (2.52% and 1.02%, respectively) when sodium hydroxide was used.

As shown in Example 6 below, alpha-hydroxy ketones (eg, acetol) can be prepared from sugar to ultimately be used as a carbon source in sugar and ammonium reactions. Acetol can be isolated by both distillation and column chromatography for its subsequent use as a carbon source in the reactions described in the present description. However, both technologies are time consuming (distillation) and expensive (chromatography). In this regard, for the preparation of pyrazines, it may be more preferable to eliminate the need to isolate hydroxy ketones obtained from a sugar source before using these hydroxy ketones in reactions with a nitrogen source.

As shown in Example 7 below, for example, it has been unexpectedly found that substituted pyrazines can be selectively produced from a sugar carbon source without first isolating the hydroxy ketone (eg, acetol, acetoin, etc.) formed from the decomposition of the sugar(s). As shown at operation 110 in FIG. 2, for example, by treating the sugar(s) with a buffer prior to combining the sugar(s) with a nitrogen source, a different range of pyrazines can be obtained from the range of pyrazines resulting from the reaction of sugar(s) that have not been pre-treated with a buffer and a nitrogen source. These reactions can be optimized so that the maximum amount of acetol and acetol-like compounds is obtained from the sugar carbon source. Without being limited by theory, the role of the buffer is to maintain the pH level so that the maximum amount of hydroxyketone(s) (eg, acetol, acetoin, etc.) is obtained during the decomposition of sugar.

In some embodiments, the buffer may be a sodium hydrogen phosphate/sodium hydroxide buffer with a pH of about 12. In various embodiments, the buffer may include a potassium phosphate buffer with a pH of about 6.5 to about 7.5. In some embodiments, the buffer may include sodium carbonate, sodium sulfite, peroxodisulfate, sodium phosphate, or combinations thereof. The choice of buffer type, buffer capacity, pH and reaction temperature can influence the synthesis of hydroxyketones from the sugar carbon source and can thus determine the range of pyrazines produced as well as the amount of pyrazines produced from the subsequent reaction with the nitrogen source.

In various embodiments, the buffer may create a pH in the approximately neutral or alkaline range, in particular at a pH greater than about 6, greater than about 8, or greater than about 10 (eg, about 6 to about 12). For example, in some embodiments, a sugar carbon source pH of about 11 to about 12 can be created. In some embodiments, a sugar carbon source pH of about 6.5 to about 7.5 can be created.

For example, glucose is a known carbon source for the production of pyrazines. When used as a molecule unchanged and reacted with ammonium hydroxide, a series of pyrazines are obtained, including pyrazine and methylpyrazine molecules as the predominant pyrazines with much smaller amounts of dimethylpyrazines and much smaller amounts of high molecular weight pyrazines. If glucose is first reacted with NaOH at pH 12, followed by reaction with ammonium hydroxide, a similar series of pyrazines is obtained. However, it has been surprisingly found that if glucose is treated with potassium phosphate buffer at pH 6.5 followed by reaction with ammonium hydroxide, then only dimethylpyrazines and high molecular weight pyrazines are formed, and, as a result, the production of less desirable pyrazine and methylpyrazine molecules is excluded.

The increased reaction time between the buffered carbon source from the buffered sugar(s) and the nitrogen source, as well as the increased temperature, allows an increased yield of pyrazines to be achieved up to the point of obtaining a black tarry mass. The reaction temperature may be, for example, about 30°C or more, about 90°C or more, about 100°C or more, about 120°C or more, or about 140°C or more. In some embodiments, the reaction temperature may be from about 90°C to about 150°C, or from about 120°C to about 140°C. The reaction time may be, for example, about 30 minutes or more, about 60 minutes or more, about 90 minutes or more, or about 120 minutes or more. In various embodiments, said reaction time may be from about 30 minutes to about 150 minutes, or from about 60 minutes to about 120 minutes.

An increased pH of the reaction solution can also lead to an increase in the amount of pyrazines. A desirable pH range may be from about 7.5 to about 10.5, or from about 8.5 to about 9.5. In some embodiments, the pH of the reaction solution may be about 8.0 or greater, about 8.5 or greater, about 9.0 or greater, or about 10.0 or greater. A small addition of NaOH or KOH, for example, can be used to raise the pH of the reaction solution.

In various embodiments of the present invention, such as represented at operation 122 in FIG. 2, addition of NH ₄ OH to the amino acid/sugar reaction solution can increase the yield of pyrazines. The molar ratio of NH ₄ OH/caxap can have a significant effect on the yield of pyrazines. For example, the molar ratio of sugar to NH ₄ OH is about 6:1 to about 1:1, or about 5:1 to about 2:1 (e.g., about 5:1, about 2.5:1, about 2:1, or about 1.5:1 ) followed by heating makes it possible to obtain compositions rich in pyrazines. In some embodiments, aqueous NH ₄ OH may be added slowly during the course of the reaction to the amino acid/sugar solution.

Various sugars and amino acids influence the type of pyrazines formed. See, for example, Coleman and Steichen, 2006, Sugar and selected amino acid influences on the structure of pyrazines in microwave heat treated formulations, J. Sci. Food Agric., 86, 380-391, the entire content of which is incorporated herein by reference. For example, leucine and valine make it possible to obtain more branched pyrazines with polysubstituted side chains (subchains) and a low odor threshold. Polysubstituted pyrazines are relatively more active than pyrazines that are not branched and therefore may be desirable in some applications. Substitutions in the pyrazine may result from the amino acid used in the reaction. Thus, an advantage may lie with the choice of branched amino acids with highly substituted side chains. In terms of sugars, rhamnose may be the ideal sugar for making pyrazines, followed by fructose and then glucose.

As shown at step 126 in FIG. 2, for example, after completion of the reaction, pyrazines can in some cases be isolated from the reaction product using simple distillation, rotary evaporation, or other separation technology known in the art. In some embodiments, rotary evaporation may be the preferred isolation method when scaling up the process for producing tobacco-derived pyrazines.

Use of substituted pyrazines in tobacco products

As described above, the pyrazines obtained in accordance with the present invention can be used as components (eg, flavoring substances), included in, for example, tobacco products. The tobacco product to which the materials of the present invention are added can vary and include any product configured or adapted to deliver tobacco or certain components thereof to the user of the product. Examples of tobacco products include smoking products (eg, cigarettes), smokeless tobacco products, and aerosol generating devices that contain tobacco material or other plant material that does not burn during use.

In various embodiments of the present invention, the pyrazines may be incorporated into smoking articles as a flavoring agent in the tobacco composition and/or in the filter element of the smoking article. For example, pyrazines can be added to flavorings (top dressing) or sauces in a tobacco product. FIG. 6 is an illustration of a smoking article 10 in the form of a cigarette having certain exemplary components of a smoking article that may contain products derived from the cellulosic sugar materials of the present invention. Cigarette 10 includes a generally cylindrical rod 12 with a charged (charge) or rolled (roll) smokeable filling material (e.g., about 0.3 to about 1.0 g of smokeable filling material, such as tobacco material) contained in a wrapper around it. around the wrapping material 16. The rod 12 is traditionally called a "tobacco rod". The ends of the tobacco rod 12 are open to exposure to the filling material being smoked. The cigarette 10 is shown with one optional band 22 (for example, a coating including a film former such as starch, ethyl cellulose, or sodium alginate) applied to the wrapper 16, and this band encircles the cigarette rod in a direction transverse to the longitudinal axis of the cigarette. The strip 22 may be applied to the inner surface of the wrapping material (ie, opposite the filling material being smoked), or, less preferably, to the outer surface of the wrapping material.

At one end of the tobacco rod 12 is an incendiary end 18 and on the side of the mouthpiece 20 is a filter element 26. The filter element 26 is located adjacent to one end of the tobacco rod 12, so that the filter element and the tobacco rod are located coaxially butt, preferably end to end. The filter element 26 may have a generally cylindrical shape, and its diameter may be substantially equal to the diameter of the tobacco rod. The ends of the filter element 26 allow air and smoke to pass through. Fitella 28 wraps the filter element, and a rim material (not shown) wraps the ficell and part of the outer wrap material 16 of the stem 12, thus securing the stem to the filter element 26.

The filter element according to the present invention usually contains multiple longitudinally extending segments. Each segment may have different properties and may include different materials to filter or adsorb particulate matter and/or gas phase compounds. Usually the filter element according to the present invention includes from 2 to 6 segments, often from 2 to 4 segments. In one preferred embodiment, the filter element includes an oral end segment, a tobacco end segment, and a cavity therebetween. This filter arrangement is sometimes referred to as a "compartment filter" or "plug/space/plug" filter. This cavity may be divided into two or more cavities, as described in more detail below.

In various embodiments, the filter element may comprise an adsorbent in the form of an activated carbon material, wherein said activated carbon is capable of removing at least one mainstream smoke gaseous phase component and is incorporated into the filter element. In some embodiments, the filter element 26 may include vents 30 that extend through the rim paper (not shown) and the ficell 28 and thus provide air dilution of the inhaled smoke. The vent holes 30 may be formed as a single perforation line around the circumference of the filter element 26, or may be composed of several perforation lines. Obviously, the exact number and size of the vents 30 may vary depending on the level of air dilution desired.

In various embodiments of the present invention, the pyrazines obtained using the methods disclosed herein may be included in smokeless tobacco products in the form of a flavor in a smokeless tobacco formulation. The form of the smokeless tobacco product of the present invention may vary. In one particular embodiment, the product is in the form of a snus product containing certain tobacco material and a pyrazine-containing flavor produced by the methods of the present invention. Forms and methods of making snus tobacco formulations will be apparent to those skilled in the art of making snus tobacco products. For example, as shown in FIG. 7, an exemplary pouch product 300 may include an outer permeable pouch-shaped container 320 that contains a specific mixture 315 adapted for oral administration. It is not to be construed as limiting the orientation, size, and type of the outer permeable pouch described herein, or the type and nature of the composition adapted for oral administration.

In various embodiments, the liquid-pervious sachet or pouch may function as a container for the application of the composition contained therein. As noted herein, the composition/design of such sachets or pouches may vary, such as the container pouch 320 in the embodiment shown in FIG. 7. For example, appropriate pouches, pouches or containers of the type indicated for use in the manufacture of smokeless tobacco products which may be modified in accordance with the present invention are available under the trademarks CatchDry, Ettan, General, Granit, Goteborgs Rape, Grovsnus White, Metropol Kaktus , Mocca Anis, Mocca Mint, Mocca Wintergreen, Kicks, Probe, Prince, Skruf and TreAnkrare. A type of product pouch similar in shape and appearance to various embodiments of the pouched product described herein is available on the market under the name ZONNIC (marketed by Niconovum AB). Additionally, pouch-type products generally similar in shape and appearance to the various pouched product embodiments are presented as E-J pouch snuff compositions in Example 1 of PCT Application Publication 2007/104573 to Axelsson et al., incorporated in the present description by reference, which are made using excipients and processing conditions suitable for use in the manufacture of packaged products as shown in the present description.

The amount of material contained in each bag may vary. In smaller embodiments, the dry weight of each pouch is at least about 50 mg to about 150 mg. In larger embodiments, the dry weight of each pouch preferably does not exceed about 300 mg to about 500 mg.

In some embodiments, each pouch/container may contain a flavoring agent, as described in more detail in US patent No. 7861728, author Holton, Jr. and others, which is included in the present description by reference. The flavoring may contain a pyrazine-containing flavor obtained by the methods of the present invention as described above. Other components may be contained within each pouch as needed. For example, at least one flavored strip, piece, or sheet of a flavored water-dispersible or water-soluble material (eg, a breath freshening edible film-like material) may be placed within each pouch, with or without at least one capsule. Such strips or sheets may be folded or crumpled for direct placement within the pouch. See, for example, the types of materials and technologies given in US Pat. which are included in the present description by reference.

In various embodiments, the outer permeable pouch may comprise PLA or other pouch materials known in the art. Descriptions of the various components of snus type products and their components are also provided in US Patent Application Publication No. 2004/0118422 to Lundin et al., which is incorporated herein by reference. See also, for example, US Pat. of which is included in the present description by reference. See also the types of packages shown in US patent No. 5167244, the author of Kjerstad, which is incorporated into this description by reference. Snus type products can be produced using equipment such as those available as SB 51-1/T, SBL 50 and SB 53-2/T from Merz Verpackungmaschinen GmBH. Snus pouches may be presented as individual pouches or multiple pouches (e.g., 2, 4, 5, 10, 12, 15, 20, 25, or 30 pouches) and may be joined or tied together (e.g., end-to-end) so that a single sachet or individual portion can be easily separated for use from a single strip or pouches matrix.

The present invention is not limited to snus-type smokeless tobacco products. For example, a mixture of tobacco material and flavoring substances containing at least one pyrazine, obtained by the methods described herein, can be introduced into various smokeless tobacco forms, such as loose moist snuff, loose dry snuff, chewing tobacco, granular tobacco particles, extruded tobacco strips or particles, finely divided or milled agglomerates of powdered particles and components, flake-like pieces (for example, which can be formed by agglomeration of tobacco formulation components in a fluidized bed plant), molded tobacco pieces (for example, formed in the shape of a coin, cylinder, ball, cube or the like), pieces of chewing gum containing tobacco, products with the introduction of a mixture of edible material combined with pieces of tobacco and / or tobacco extract, products with the introduction of tobacco (for example, in the form of tobacco extract) applied to the body. I blow an inedible base and the like. For example, a smokeless tobacco product may be in the form of compressed tobacco pellets, multilayer extruded particles, extruded or molded rods or sticks, compositions having one type of tobacco formulation surrounded by another type of tobacco formulation, rolled ribbon-like films, readily water-soluble or dispersible films, or strips. (See, for example, U.S. Patent Application Publication No. 2006/0198873 to Chan et al.), or capsule-like materials having an outer shell (e.g., a soft or hard outer shell that can be clear, colorless, transparent or intensely colored in nature) and an internal portion containing tobacco or tobacco flavoring (eg, Newtonian fluid or thixotropic fluid incorporating some kind of tobacco).

In some embodiments, the smokeless tobacco products of the present invention may be in the form of a lozenge, tablet, microtablet, or other tablet product. See, for example, the types of lozenge formulations and techniques for making molds or producing lozenges in U.S. Patent Nos. 4,967,773 to Shaw, 5,110,605; 6248760 by Wilhelmsen and 7374779 by U.S. Patent Publication Nos. 2001/0016593 by Wilhelmsen 2004/0101543 by Liu et al. 2006/0120974 by Mcneight 2008/0020050 by Chau et al. 2009 /0081291 by Gin et al. and 2010/0004294 by Axelsson et al., which are incorporated herein by reference.

Depending on the type of smokeless tobacco product being produced, the tobacco product may contain one or more additional components in addition to the tobacco material and flavoring agents containing at least one pyrazine obtained by the process of the present invention. For example, the tobacco material and tobacco-derived flavors may be processed, blended, formulated, combined, and/or blended with other materials or ingredients such as other tobacco materials or flavors, fillers, binders, regulators. pH, buffering agents, salts, sweeteners, colors, disintegrants, wetting agents, and preservatives (any of which may be an encapsulated ingredient). See, for example, said typical ingredients, combinations of ingredients, relative amounts of said ingredients and tobacco-related ingredients, and uses and uses of said ingredients in U.S. Patent Publication No. 2011/0315154 by Mua et al., and 2007/0062549 by Holton, Jr. et al. and US Pat. No. 7,861,728 to Holton, Jr. and others, each of which is included in the present description by reference.

In various embodiments, at least one pyrazine produced by the methods described herein may be incorporated into smokeless tobacco products as a flavoring agent in an electronic smoking article. A variety of smoking products, odor generators, and medical inhalers have now been proposed that use electrical energy to vaporize or heat highly volatile materials, or aim to provide the sensation of smoking a cigarette, cigar, or pipe without burning tobacco to a great extent. See, for example, various alternative smoking articles, aerosol delivery devices, and heating sources in the background section of U.S. Patent No. 7,726,320 to Robinson et al. et al., 2014/0000638 by Sebastian et al., 2014/0060554, by Collett et al., 2014/0096781, by Sears et al. Davis et al., the entire contents of which are incorporated herein by reference.

FIG. 8 depicts an exemplary embodiment of an electronic smoking article 200. As shown herein, the control 202 may be formed by a control body 201 that includes a control 206, a flow sensor 208, a battery 210, and an LED 212. The cartridge 204 may be formed a cartridge body 203 comprising a reservoir 244 that is in fluid communication with a fluid transport member 236 attached to the wick, or otherwise conveys to the heater 234 the aerosol precursor composition located in the reservoir. An opening 228 may be present in the cartridge shell 203 to allow the formed aerosol to escape from the cartridge 204. Such components are typical components that may be present in a cartridge and are intended to limit the scope of the components in the cartridge that are contained in the present disclosure. The cartridge 204 may be adapted to attach to the control 202 by a tight fit between the projection of the control 224 and the cartridge socket 240. This attachment allows for a stable connection between the control 202 and the cartridge 204, and also allows electrical contact between the battery 210 and the control element 206 in the control and heater 234 in the cartridge. The cartridge 204 may also include one or more electronic components 250, which may include an IC, a memory device, a sensor, or the like. The electronic component 250 may be adapted to communicate with the control element 206. Various components of the electronic smoking device in accordance with the present disclosure may be selected from those described in the art and are commercially available.

In various embodiments, the aerosol precursor composition may contain a flavoring agent containing at least one pyrazine obtained by the methods of the present invention. Examples of formulations for aerosol precursor materials that can be used in accordance with the present disclosure are described in US Patent No. 7,217,320 to Robinson et al., US Patent Publication Nos. 2013/0008457 by Zheng et al., 2013/0213417 by Chong et al., 2014/0060554 by Collett et al., and 2014/0000638 by Sebastian et al. . Other aerosol precursors that may contain the tobacco-derived pyrazines described herein include the aerosol precursors found in R. J. Reynolds Vapor Company's VUSE® product, Imperial Tobacco's BLU™ product, Mistic Ecigs' MISTIC MENTHOL product, and VYPE by CN Creative Ltd. Also suitable are the so-called "smoking juices" for electronic cigarettes, which are available from Johnson Creek Enterprises LLC.

EXPERIMENTAL PART

Aspects of the present invention are explained in more detail in the following examples, which are provided for the purpose of describing certain illustrative aspects of the present invention and should not be construed as limiting it.

Example 1

Pyrazines were prepared using acetoin (3-hydroxy-2-butanone) instead of sugar(s) as the carbon source in reactions with ammonia.

Acetoin, ammonium hydroxide (28-30%), leucine, dichloromethane and phosphoric acid (H ₃ PO ₄ ) were purchased from Sigma-Aldrich (St. Louis, MO). F1 protein was purchased from RJ Reynolds Tobacco Co. (Winston-Salem, NC) and subjected to hydrolysis. The mass fraction of hydrolyzed amino acids in the total hydrolyzed solution was in the range of 50-55%. All pyrazine synthesis reactions were carried out in a 40 ml Parr flask. In each reaction, 0.8 grams of acetoin was mixed with 1.8 ml of NH ₄ OH and 0.6 ml of H ₃ PO ₄ and then enough hydrolysed F1 protein (20 ml) was added to bring the mass of amino acids to 0.4 grams. For example, if 40 grams of F1 protein was hydrolyzed in 1 liter of solution, then the mass fraction of amino acids in the solution was up to 50%, which is equal to 20 grams of amino acids in 1 liter of solution. In order to use 0.4 g of amino acids in the reaction, only 20 ml of the above solution was added to the chemical reactor. In some reactions, leucine was used as the amino acid source instead of hydrolyzed F1 protein and only 20 ml of water was added to adjust the volume. For all solutions, the pH was adjusted to 8.0 and then the reaction was started. After completion of each reaction, the mixture was extracted with 30 ml of dichloromethane (DCM). Next, 200 μl of the DCM extract was diluted to 1 ml with DCM and analyzed by GC/MS.

All GC/MS analyzes were performed on a 6890 GC equipped with a 5973 Mass Selective Detector (MSD) from Agilent (Wilmington, DE). Separation was performed using a DB-WAXTER capillary column (30 m long x 250 µm I.D. with a film thickness of 0.25 µm) from J&W (Wilmington, DE). Each of the assays was performed under the following operating parameters:

Injector temperature 260°С flush valve 3 ml/min Flushing time 1 minute General flow 24 ml/min Constant flow 1 ml/min Injection volume 2 µl, flow split 1:20 Initial thermostat temperature 50°С Thermostat start time 3 min Thermostat heating rate 15°C/min End thermostat temperature 250°С Thermostat end time 1 minute Temp, transition line MSD 260°С

The MS Wiley library was used to identify each of the pyrazines.

In the first reaction, acetoin (0.8 grams) was reacted with NH ₄ OH (1.8 ml) and H ₃ PO ₄ (0.6 ml) at pH=8 at 90°C for 12-15 hours. More than 90% of the acetoin was converted to tetramethylpyrazine (TMP). Next, the reaction was repeated using identical conditions but instead of heating the reaction mixture at 90°C for 12-15 hours, it was heated only for 4 hours at 120°C. The results were similar to those obtained at 90°C and 12-15 hours.

Further, in order to determine whether branched pyrazines were formed, amino acids (leucine) were added to the reaction mixture. For this purpose, 0.8 grams of acetoin + 1.8 ml of NH ₄ OH + 0.6 ml of H ₃ PO ₄ and 0.25 grams of leucine were mixed with 20 ml of H ₂ O and the pH was adjusted to 8. The reaction mixture was then heated for 18 hours at 120°C and then the reaction mixture was extracted with 30 ml of DCM and analyzed using the GC/MS method. Only TMP was detected (t _R = 8.2 min) and a small amount of acetoin (t _R = 6.1 min). The presence of branched pyrazines was not detected.

Next, a similar reaction was carried out, but instead of leucine and H ₂ O, 20 ml of hydrolyzed F1 protein was used in the reaction. This reaction was carried out in a Parr vessel at 120° C. for 18 hours. After cooling the reaction mixture, 30 ml of DCM was used to extract the pyrazines. This time, branched pyrazines were also not detected, only TMP was present.

To determine if the ammonium had reacted with all of the acetoin, in order to prevent the amino acid(s) from reacting with acetoin, another source of base (NaOH) was used instead of NH ₄ OH to maintain the basic reaction conditions, pH > 8. For this purpose, two reactions. In the first reaction, 0.8 grams of acetoin was mixed with 0.25 grams of leucine and 20 ml of 0.1 N. NaOH (pH= 12), in the second reaction, the pH was adjusted to a value of 8.2 with H ₃ PO ₄ . Both reaction mixtures were heated at 120° C. for 8 hours using a Parr vessel. After cooling the reaction mixture, the contents of the reaction mixture were subjected to extraction with DCM and analyzed by GC/MS. The presence of pyrazines, and even TMP, was not detected.

The above reactions show that the addition of amino acids to the reaction mixture of acetoin and NH ₄ OH does not lead to branched pyrazines. In addition, it has been found that acetoin is not a key intermediate in reactions between sugar(s) and free amino acids to produce pyrazines containing branched alkyl side chains.

Example 2

Hydroxyketone carbon sources other than acetoin have been used to selectively prepare pyrazines other than TMP.

1-OH-acetone, 1-OH-2-butanone, ammonium hydroxide (28-30%), phosphoric acid (H ₃ PO ₄ ), isoleucine, threonine and isovaleric aldehyde were obtained from Sigma-Aldrich (St. Louis, MO ). F1 proteins isolated from a plant of the genus Nicotiana were obtained from RJ Reynolds Tobacco Co. (Winston-Salem, NC) and hydrolyzed to obtain amino acids. The mass fraction of hydrolyzed amino acids in the total hydrolyzed solution was in the range of 50-55%.

All pyrazine synthesis reactions were carried out in a 40 ml Parr flask. In each reaction, 1 gram of 1-OH-acetone or 1-OH-2-butanone was mixed with 0.25, 0.5, 1 and 1.25 ml of NH ₄ OH and 10 ml of H ₂ O. Each reaction mixture was stirred and heated at various temperatures (100-140°C) for a period of 4-24 hours. The pH level for most of the reactions was about 11 (no adjustment was made). However, in reactions in which the pH was adjusted to 8, H ₃ PO ₄ was used to lower the pH.

After completion of each reaction, the mixture was extracted with 20-25 ml of dichloromethane. For each extraction, 250 µm deuterated 2-methylpyrazine was used as an internal standard for all assays. In all reactions, the mixture was stirred using a magnetic stirrer during the reaction process.

All GC/MS analyzes were performed using the same equipment and operating parameters as those used in Example 1 above. The Wiley MS library was used to determine each pyrazine. In quantitation, pyrazines were quantified using the selected ion monitoring mode. Each pyrazine was quantified in comparison to the weight of the internal standard (250 μg) added to the extractant.

First, 1-hydroxyacetone was reacted with NH ₄ OH at various ratios, temperatures, pH levels, and reaction times to maximize the percentage yield of pyrazines. The pyrazines found after the reaction of 1-OH-acetone with NH ₄ OH included: 2,6-dimethylpyrazine; 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2-ethyl-6-methylpyrazine, 2,3,5-trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-2,5-dimethylpyrazine, 2,3,5,6-trimethylpyrazine, 2,3,5-trimethyl-6-ethylpyrazine, 2,6-dimethyl-3-propylpyrazine, 2,5-diethyl-3,6-dimethylpyrazine, 2,6-dimethyl- 3-(2-methylbutyl)pyrazine, 2,5-dimethyl-3-(2-methylbutyl)pyrazine, 2,5-dimethyl-3-(3-methylbutyl)pyrazine, 2,5-dimethyl-3-propylpyrazine, 2 ,5-dimethyl-3-cis-propenylpyrazine, 2-isopropenyl-3,6-dimethylpyrazine, 2-(2-methylpropyl)-3,5-dimethylpyrazine, 2,6-dimethyl-3-isobutylpyrazine, 2-(2- methylpropyl)-3,5,6-trimethylpyrazine.

In the first part of this study, reactions were run at two different pH levels (8.0 and 11.0) to determine which pH level resulted in the highest yield and the highest number of pyrazines. In the first reaction, 1 ml of 1-OH-acetone was reacted with 0.5 ml of NH ₄ OH and 10 ml of H ₂ O. The pH for this reaction was measured at approximately 11.0. In the second reaction, the same volume of reactant was stirred, but the pH was adjusted to 8.0 with concentrated H ₃ PO ₄ . Both reactions were heated at 120° C. for 12 hours. The percentage yield of pyrazines was higher at about pH 11.0. For this purpose, the pH level in subsequent experiments was not adjusted and the reactions were carried out at pH 11 or higher.

Further in the text, the effect of temperature (100, 110, 120, 130 and 140°C) on the synthesis of pyrazines using 1-OH-acetone (1 g) and NH ₄ OH (1 g) with a C:N molar ratio of 1 is given: 2 in 10 ml of H ₂ O (reaction time - 12 hours). The yield of pyrazines increases with increasing temperature, still showing an increase in the yield of pyrazines upon reaching a temperature of 140°C.

The effect of different reaction times (4, 8, 12, 16, and 24 hours) on the synthesis of pyrazines was studied using 1-OH-acetone (1 g) and NH ₄ OH (1 g) with a C:N molar ratio of 1:2 in 10 ml H ₂ O. The maximum yields of pyrazines were obtained when the reaction time was 16 hours.

The influence of different molar ratios of 1-OH-acetone:NH ₄ OH (1:0.5; 1:1; 1:2 and 1:2.5; 1-OH-acetone and NH ₄ OH) in the reaction in 10 ml H ₂ O on the output of pyrazines at 120°C after 12 hours. The optimal ratio is 1:2, which corresponds to 1 gram of 1-OH-acetone and 1 ml of NH ₄ OH.

In the case of using a larger volume of NH ₄ OH in the reaction, the yield in the reaction fell by more than 10%.

In summary, it should be noted that the reaction conditions (temperature, time, C:N ratio and pH) are optimized to maximize the quantitative content of pyrazines. The results showed that under optimal conditions (C:N = 1:2, temperature = 120°C, reaction time = 16 hours and pH = 11-12), at least 19-20 different pyrazines are synthesized using hydroxyacetone as the only source of carbon. The absence of any detectable amounts of pyrazine and/or methylpyrazine molecules among the synthesized pyrazines confirms the finding that the carbon source (ie, α,β-hydroxy ketone) influences the structure of the pyrazines produced by the reaction of the carbon source with a nitrogen source.

Example 3

The effect of adding amino acids and aldehyde in a separate reaction of 1-OH-acetone and NH ₄ OH was studied according to the above parameters in Example 2 at a C:N ratio of 1:2, when mixed with 10 ml of H ₂ O at 120°C for 12 hours.

Two different amino acids have been investigated as additional sources of nitrogen. In each reaction, 0.2 grams of an amino acid was added separately to each reaction mixture so that it was possible to investigate how additional amino acids affect pyrazine synthesis and yield. In a separate reaction, isovaleraldehyde was added to an optimized reaction mixture to investigate the effect on pyrazine synthesis and yield. In the case where hydrolyzed F1 protein was used as an additional source of nitrogen, 10 ml of hydrolyzed F1 proteins (which contain approximately 0.2 grams of various amino acids) were used. In this reaction, H ₂ O was not added, since the hydrolyzed F1 proteins were in 10 ml of H ₂ O.

It has been found that if isoleucine is used as a possible additional nitrogen source, the concentration of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl)pyrazine increases. In a separate reaction, if threonine is used as an additional source of nitrogen, then the concentration of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl)pyrazine increases, and the yield of 2 ,6-dimethyl-3-(2-methylbutyl)pyrazine. It is interesting to note that the total yields of pyrazines are similar when threonine or isoleucine is added to the reaction mixture. Both compounds increase the total yield of pyrazines by more than 7%.

The addition of isovaleraldehyde to a separate reaction mixture of 1-OH-acetone and NH ₄ OH leads to the fact that the percentage yield of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl )pyrazine increases from 0 to 14 and 13 mg, respectively. The total yield of pyrazines increases by more than 20%.

Instead of a pure amino acid, a mixture of amino acids obtained by hydrolysis of F1 proteins can be used in the reaction. Since the hydrolysed F1 proteins are already in aqueous solution, no water was added to the mixture. For this purpose, 10 ml of hydrolysed F1 proteins, which contained approximately 0.2 g of the amino acid and 10 ml of H ₂ O, were reacted with 1-OH-acetone and NH ₄ OH at a C:N = 1:2 ratio at 120°C in within 16 hours. The yield of 2,5-dimethylpyrazine increased by more than 80%, and the yield of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl)pyrazine increased from 0 to more than 1 mg. Without being limited by theory, the alkyl portion of the amino acid is converted to Strecker's aldehyde, which reacts with ammonium hydroxide to form an imine, which in turn adds to the pyrazine structure.

The effect of various temperatures and C:N ratios (1:1 and 1:2) on the synthesis of pyrazines using 1-OH-acetone and NH ₄ OH in the presence of additional amino acids/aldehyde was evaluated. In these studies, increasing the temperature from 100 to 120°C and the ratio C:N leads to an increase in the yield of pyrazines.

In summary, the addition of amino acids, individual aldehydes, or hydrolysed F1 protein not only increases the percentage yield of certain pyrazines, but also increases the number of pyrazines synthesized.

Example 4

Pyrazines were synthesized in accordance with the above examples 2 and 3, except that 1-OH-2-butanone was used as a carbon source instead of 1-OH-acetone.

For this purpose, 1 gram of 1-OH-2-butanone was reacted with 1 ml of NH ₄ OH and 10 ml of H ₂ O at 120° C. for 16 hours. Methylpyrazines were not formed. All the resulting pyrazines contained ethyl or higher branched alkanes. The yield of pyrazines, however, was not as high as with 1-OH-acetone. Pyrazines synthesized in the reaction using 1-OH-2-butanone and NH ₄ OH with C:N = 1:2 at 120°C for 16 hours included 2,6-diethylpyrazine, 2,5-diethylpyrazine, 2- ethyl-3,5,6-trimethylpyrazine, 3,5(3,6-dimethyl)-2,N-propylpyrazine, 2,5-diethyl-3-methylpyrazine, 2,3-diethyl-5,6-dimethylpyrazine, trans -3-methyl-2,n-propyl-6(1-butenyl)pyrazine, 2,5-dimethyl-3-ethylpyrazine.

As mentioned earlier, with 1-OH-acetone as a carbon source, pyrazine and methylpyrazine molecules are not formed. With 1-OH-2-butanone, pyrazine, methylpyrazine, and dimethylpyrazine molecules are not formed, confirming that the carbon source completely determines the structure of pyrazines. The results further show that by changing the carbon source from 1-OH-acetone to 1-OH-2-butanone, the type of pyrazines synthesized can be controlled.

Example 5

The reaction of 1-OH-acetone and NH ₄ OH according to Example 2 above was carried out on a larger scale using a Parr reactor with a large reaction volume.

100 grams of 1-OH-acetone was reacted with 100 ml of NH ₄ OH and 1000 ml of H ₂ O at 120° C. for 16 hours in a 1.5 liter Parr pressure reactor. After completion of the reaction, the mixture was cooled and transferred to a glass bottle.

It should be noted that at the bottom of the reactor there was a significant amount of resinous material, which is soluble only in MeOH. The addition of H ₂ O on top of this material caused it to solidify. It has been found that concentration can play an important role in the presence or absence of resinous material. In all optimization studies, the amount of resin at the bottom of the reaction vessel was low. For this reason, a small volume of methanol (1 ml) was sufficient to dissolve all of the material and incorporate it into the remaining reaction material. In larger scale reactions, the mass of resin was greater and at least 100-200 ml of methanol was needed to dissolve it.

After completion of the reaction, the aqueous solution was distilled (3 × 375 ml) at 130-140°C. At each distillation (375 ml), about 175 ml of an aqueous solution containing various pyrazines (light yellow in total volume of about 500 ml) was collected. Next, the distilled materials (3 x 175 ml) were combined and passed through a C ₁₈ column (15 x 2.5 cm with SPE material as a filler) in order to extract pyrazines from water. After removal of water from the C ₁₈ column, retained pyrazines were eluted with ethanol. Next, ethanol was removed by rotary evaporation and vacuum. Due to the presence of some water in the final product, the pyrazines were extracted into MTBE and dried over sodium sulfate. The MTBE was then removed by rotary evaporation and vacuum. The labeled vial solution 1 contained most of the pyrazines after MTBE had been removed.

It is important to note here that the three types of pyrazines cannot be isolated by distillation and remain in the reaction product due to their high boiling points. This contributes to their low percentage yield. These pyrazines were identified as 2-(2-methylpropyl) 3,5-dimethylpyrazine (12.57 min), 2,6-dimethyl-3-isobutylpyrazine (12.74 min) and 2(2-methylpropyl) 3.5, 6-trimethylpyrazine (12.95 min).

DCM (200-250 ml) was used to extract the remaining three pyrazines from the reaction solution after distillation. The DCM was then removed from the solution by rotary evaporation and evacuation, while the thick dark solution was then transferred to a second vial and labeled as Solution 2. Figure 3 shows the chromatogram of this sample.

The yield of all pyrazines upon distillation of 1200 ml of the reaction solution (100 grams of 1-OH-acetone + 100 ml of NH ₄ OH) was approximately 60% compared to 12 ml of the reaction mixture (1 gram of 1-OH-acetone + 1 ml of NH ₄ OH) . This yield did not include the three pyrazines that remain in the reaction mixture after distillation.

The above solution 1 was used for research by gas chromatographic olfactometry (GCO). This pyrazine sample was analyzed using an Agilent 7890A Series GC with 5975C MSD and ODP3 equipped with a Gerstel multipurpose sampler with SPME capability. For this sample, an instrumental method was developed to achieve a better separation of pyrazines and a suitable duration of analysis for olfactory research analysts. The transition line in ODP3 was heated to 260°C. The sample was prepared by pipetting two drops of the sample into a 20 ml SPME screw cap vial. The empty vial was analyzed before and after sample placement. Pyrazines were separated on a gas chromatographic column, and during the process of leaving the column, they were detected/determined by people using subjective odor recognition. Pyrazines identified included: methylpyrazine, 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2-ethyl-6-methylpyrazine, trimethylpyrazine, 2,5-dimethyl-3-propylpyrazine, 3-ethyl- 2,5-dimethylpyrazine, 2,5-dimethyl-3-isopropylpyrazine, 2-ethyl-3,5-dimethylpyrazine, tetramethylpyrazine, 2-methyl-5-propylpyrazine, 2,3,5-trimethyl-6-propylpyrazine isomer 1, 2,3-diethyl-5-methylpyrazine, 2,3,5-trimethyl-6-ethylpyrazine, 3,5-dimethyl-2-propylpyrazine, 2,3,5-trimethyl-6-propylpyrazine isomer 2, 2,3, 5-trimethyl-6-propylpyrazine isomer 3, trimethyl-1-propenylpyrazine (Z)-isomer 1, 5H-cyclopentapyrazine, 6,7-dihydro-2,5-dimethyl pyrazine isomer 1, 5H-cyclopentapyrazine, 6,7-dihydro -2,5-dimethyl pyrazine isomer 2, trimethyl-1-propenylpyrazine (Z)-isomer 2, 2,3-dimethyl-3-(1-propenyl)pyrazine (Z) isomer 1, trimethyl-1-propenylpyrazine (Z) -isomer 3, 2,3-dimethyl-3-(1-propenyl)pyrazine (Z), 2-isopropenyl-3,6-dimethylpyrazine, trimethyl-1-propenylpyrazine (Z) -isomer 4, trimethyl-1-propenylpyrazine ( Z)-isomer 5, trimethyl-1-propenylpyr zine (Z)-isomer 6, trimethyl-1-propenylpyrazine (E)-isomer 1, trimethyl-2-propenylpyrazine, trimethyl-1-propenylpyrazine (E)-isomer 2. It should be noted that the methylpyrazine compound, although available, is present in a smaller amount than is typical for standard reactions with sugar as a carbon source.

Four odor research analysts assessed individual olfactory characteristics via the ODP port in four separate sessions. Based on four independent evaluations, individual pyrazines were determined to be highly positive. The expected characteristics in the description of the aroma of substituted pyrazines were determined. Nutty, toasted, toasted, chocolatey, peanutty, aged, smoky, and complex were common descriptions. All these flavor characteristics are positive.

Example 6

From sugar, alpha-hydroxy ketones (acetol) were obtained, ultimately to be used as a carbon source in the reactions of sugar and ammonium. Sodium hydroxide was used as a base to optimize various parameters such as sugar type, temperature, reaction time, pH, and base concentration in order to maximize acetol yield.

Glucose, fructose, 1-OH-acetone (acetol), 1-OH-2-butanone (acetoin), sodium hydroxide, sodium chloride, anhydrous sodium sulfate, hydrochloric acid, methanol and dichloromethane were obtained from Sigma-Aldrich (St. Louis , MO). Synthesis reactions were carried out in a 40 ml Parr reactor or an open round bottom flask. For all reactions in which the pH was controlled, the reactions were carried out in a round bottom flask under reflux at 100°C. In each reaction, 0.25, 0.5, or 1.0 grams of various sugars (glucose, fructose, or a mixture of both) were mixed with 25 ml of 0.025, 0.05, 0.1, and 0.2 M NaOH. Each reaction mixture was stirred and heated at various temperatures (90-140° C.) for a period of 1-12 hours. The initial pH for most of the reaction mixtures was about 12, and the pH was not adjusted during the course of the reaction. However, for reaction mixtures in which the pH was adjusted to 9, hydrochloric acid was used. For reaction mixtures in which the pH was kept constant at 12 during the process, 10-40 µl of 10M NaOH was used. After completion of each reaction, the mixture was cooled and adjusted to pH 6.0-6.5 with 1 M HCl. Next, 1 mg of acetoin was added to the reaction mixture as an internal standard. The mixture was subjected to extraction 4 times with dichloromethane with a volume of 8-10 ml. All four extraction solutions were combined and dried with sodium sulfate.

All GC/MS analyzes were performed using the same equipment and operating parameters as those used in Example 1 above. The Wiley MS library was used for peak identification. For quantitative analysis, a calibration curve was built using the concentration of acetol and the ratio of response factors acetol/acetoin. It is important to note that acetoin was not determined in any of the reactions carried out at 90, 100 and 120°C. However, a residual amount of acetoin was found in the reaction when the temperature reached 140°C. For this purpose, the concentration of acetoin in the reaction mixture was determined and this value was used in the calculation for all calculations.

The GC/FID method was used to construct a calibration curve for the quantitative determination of acetol in all reactions. Next, to determine the optimal conditions for the synthesis of acetol, the following parameters were changed.

First, two different types of sugars (glucose, fructose, and a mixture of both) were used to determine which sugar provided the highest yield of acetol. In each reaction, 0.5 grams of sugar was mixed with 25 ml of 0.05 M NaOH. Each reaction mixture was heated for 60 minutes at 100°C. Table 1 below shows the reaction conditions and the corresponding mass of acetol in mg, which was obtained in each of the reactions. It can be seen that when glucose was used in the reaction, a large mass of acetol was formed.

In this part of the study, since pH adjustment was necessary during the reaction, instead of a closed Parr vessel, all reactions were carried out under reflux in an open round flask. Three different experiments were performed. In the first experiment, pH was measured every 10 minutes and no pH adjustment was made. In the second experiment, the pH was measured every 10 minutes and, if necessary, the pH was adjusted to the initial value (12.0) with 10 M NaOH. In the third experiment, the initial pH value was adjusted to 9.0 and then the reaction was started with pH measurement every 10 minutes (without adjustment).

The pH measurement results show that the pH drops from 12.0 to about 9 in the first 10 minutes of the reaction at 100°C. At the end of 30 minutes, the pH of the reaction mixture was about 7-8 and remained constant for the remainder of the reaction time. For experiments where the pH was maintained at about 12.0, it was necessary to add about 15-20 µl of 10 M NaOH to the reaction mixture every 10 minutes. After 40 minutes, the pH remained constant at a value of approximately 11-12.

Table 2 below shows the reaction conditions and the mass of acetol in mg obtained in each of the reactions using different pH conditions. If the pH was maintained at approximately 12, then the largest mass of acetol was obtained.

A test was carried out with three different concentrations of sugar in order to determine the effect of sugar concentration on the formation of acetol. Thus, the reactions were carried out using 0.25, 0.5 and 1 gram of glucose in 25 ml of 0.05 M NaOH solution. Each reaction mixture was heated at 100° C. for 60 minutes using a Parr reactor. Table 3 below presents the results of this study. It was demonstrated that when 0.25 grams of glucose was used in the reaction, the largest mass of acetol was obtained.

Without being limited by theory, there is reason to believe that when the sugar concentration is high, the formation of acid during the reaction minimizes the formation of acetol. In the case where the sugar concentration is lower, the formation of acid takes longer, while the pH value is high enough to allow the formation of acetol.

Four different concentrations of NaOH (0.2, 0.1, 0.05 and 0.025 M) were used to prepare acetol. Table 4 below presents the results of this study. As can be seen, when the NaOH concentration is 0.025 M, the smallest amount of acetol was formed. However, as the concentration increased from 0.025 to 0.1 M, the mass of synthesized acetol also increased. When the base concentration was increased to 0.2 M, the amount of acetol decreased. It should be noted that the reaction solution had a burnt sugar odor compared to other reactions where the base concentration was lower.

The effect of temperature on acetol synthesis was investigated using NaOH and glucose. For this purpose, different temperatures were studied in the range from 90 to 140°C. The results for the reaction temperature at 90, 100 and 120°C are similar, while the reaction at 140°C produces about 25% more acetol than other lower temperature reactions. Table 5 below presents the results of this study.

The effect of reaction time on the synthesis of acetol was studied. The reactions were carried out using identical conditions, while the reaction time was changed from 60 to 720 minutes. Table 6 below presents the results of this study. The results show that there is no significant change in the acetol yield with increasing reaction time.

In the last part of this study, based on the results obtained in the previous reactions, various reactions were carried out using optimal conditions. In Reaction A, 1 gram of glucose was reacted with 0.05 M NaOH under reflux at 100° C. while maintaining the pH at approximately 11-12. This reaction lasted for 120 minutes until the pH remained constant and did not change. The yield of acetol was 6.7 mg. Reaction B used a Parr vessel at 100° C. to react 0.5 grams of HFTS with 25 ml of 0.05 M NaOH for 60 minutes. The yield of acetol was 3.42 mg. When the same reaction was carried out under optimal conditions (0.1 M NaOH and 140°C for 60 min), the yield of acetol increased to 100% to 6.69 mg (Reaction D). Similar results were obtained when optimal conditions were applied with 0.5 grams of glucose (Reaction C). The yield of acetol reached 8.2 mg. Using identical reaction conditions, but using 50% less glucose (0.25 grams), the yield of acetol was reduced to 5.32 mg.

In summary, various parameters such as sugar type, reaction temperature, reaction time, pH, base and sugar concentration have been optimized for the synthesis of acetol in the reaction of sugars and sodium hydroxide. It has been demonstrated that the highest yield of acetol can be obtained when the reaction is carried out at 140° C. using 0.1 M sodium hydroxide and 0.5 grams of glucose for 60 minutes. It should be noted that the pH value of the reaction can change dramatically (within 10 minutes) from 12 to 6.5 if the base concentration is low or if the sugar concentration is high. It should also be noted that at high temperatures (140°C) the reaction product contains small amounts of acetoin (together with acetol). It is important to note that hydroxyketones (acetoin) are produced from glucose using biotechnological approaches with yields above 90%. However, published chemical conversions of glucose to hydroxyketones are much less than ~9%.

Example 7

Pyrazines were obtained using glucose as a carbon source.

As shown above in example 6, acetol can be synthesized in the reaction of 0.1 N. NaOH and glucose using optimal conditions (0.5 gram of glucose is reacted with 25 ml of 0.1 N NaOH at 140°C for 60 minutes). The yield of acetol was about 2% based on the mass of glucose.

Nodzu (R. Nodzu, On the action of phosphate upon hexoses, The formation of acetol from glucose in acidic solution of potassium phosphate. Bull Chem. Soc. Japan, 10, 122-130, 1935, which is incorporated herein by references) it is shown that acetol with a yield of 4% (based on the weight of glucose) can be synthesized in the reaction of a 40% solution of phosphate buffer with pH 6.5-6.8 with glucose at a temperature of 100-120°C. It has been shown that a higher yield of acetol can be obtained at pH 7.0-7.1, and as the pH decreases, the yield of acetol decreases.

This example primarily demonstrates the preparation of acetol in the reaction of 0.1 N. NaOH and glucose under optimal conditions. The following is a method for separating acetol from the reaction mixture. In conclusion, it was shown that the above mixture, without isolation of acetoin acetol, can react with NH ₄ OH for the synthesis of various branched pyrazines.

Glucose, 1-OH-acetone (acetol), 1-OH-2-butanone (acetoin), sodium hydroxide, sodium hydrogen phosphate/sodium hydroxide buffer pH 12, dipotassium phosphate, dipotassium phosphate, sodium chloride, sulfate anhydrous sodium, hydrochloric acid, methanol and dichloromethane were obtained from Sigma-Aldrich (St. Louis, MO).

The synthesis of acetol was carried out in a 40 ml or 1.5 L Parr vessel. For the synthesis of acetol, 0.5 grams of glucose was mixed with every 25 ml of 0.1 N. NaOH or buffer solution. Each reaction mixture was stirred and heated at 140° C. for a period of 60 minutes. The initial pH in all reactions was about 12 and no pH adjustments were made during the reaction. After completion of each of the reactions, the mixture was cooled and measured the amount of both acetol and sugar using GC and HPLC. For the quantitative determination of acetol, 1 mg of acetoin was added to 25 ml of the solution as an internal standard and extracted with 30–35 ml of DCM. The DCM-extracted solution was then dried over sodium sulfate and analyzed by GC/FID for quantitation.

Synthesis of pyrazines was carried out in the same vessel Parr. The reactions were carried out by reacting acetol, synthesized during the reaction with sugar, with 0.1 N. NaOH. In this reaction, every 25 ml of solution was reacted with either 0.25, 0.5 or 1 ml of NH ₄ OH. Reactions were carried out at 120 or 140°C for 17 hours. Next, each reaction mixture was cooled and the pyrazines were subjected to extraction and quantitative determination of the content. At each extraction, 0.25 mg d ₆ -2 methylpyrazine was added as an internal standard and the solution was extracted with 30-35 ml DCM as solvent. Next, the DCM solution was dried over sodium sulfate and analyzed by GC/MS to quantify the content. The extracted ions were used to quantify each pyrazine.

All GC/MS analyzes were performed using the same equipment and operating parameters as those used in Example 1 above. The Wiley MS library was used to identify each pyrazine. For quantitative analysis, a calibration curve was built using the concentration of acetol and the ratio of response factors acetol/acetoin. It should be noted here that acetoin was not detected in any of the reactions carried out at 90, 100 and 120°C. However, a residual amount of acetoin was found in the reaction, where the temperature reached 140°C. Thus, the concentration of acetoin was determined in the reaction mixture and this value was used in the calculation for all calculations.

All HPLC/RI separations were performed using Sugar-Pak (300 x 6.5 mm) columns from Waters (Milford, MA). The analysis was performed on an Agilent 1100 series HPLC equipped with a four channel pump, refractive index detectors (RT), autosampler and heater set at 80°C. The mobile phase for isocratic sugar analysis was EDTA, disodium salt, dihydrate 0.005%. The flow rate was set to 0.5 ml/min for these assays.

First, acetol was synthesized using a buffer solution. Due to a sharp change in the pH of the solution in the reaction of glucose from 0.1 N. NaOH solution for the synthesis of acetol instead of 0.1 N. NaOH solution in the synthesis of acetol used a buffer solution with pH 12 (purchased from Sigma Aldrich). In this study, 25 ml of sodium hydrogen phosphate/sodium hydroxide buffer pH = 12 was reacted with 0.25 or 0.5 grams of glucose at 140°C for 60 minutes. The results show that the concentration of acetol in the reaction product is much higher (by a factor of 2) in the case when a buffer solution was used for the synthesis. Table 8 below presents the results of this study.

In the case where a similar buffer was prepared (by mixing 7.1 grams of Na ₂ HPO ₄ and 1 gram of NaOH in 100 ml of H ₂ O at a pH of about 12 and a capacity of 0.05 N.) and reacted with glucose at 140 ° C within 60 minutes, acetol was absent from the reaction mixture. This reaction was repeated three times, and acetol was not detected in any of the reaction products. However, when a similar buffer with the same pH but lower capacity (0.025 N) was used, acetol was found in the reaction product and its concentration was much higher than when 0.1 N was used. NaOH. In this regard, it was found that when glucose is used, the synthesis of acetol is affected by the type of buffer, capacity, pH, and reaction temperature.

Next, the concentration of acetol was determined in the reaction with glucose with 40% phosphate buffer at pH 6.5-7.0. 25 ml of 40% potassium phosphate buffer pH=6.5-6.8 was reacted with 0.5 or 1 gram of glucose at 140° C. for 60 minutes using a Parr reactor. Table 9 below shows the mass of acetol obtained in the reaction of 40% phosphate buffer (25 ml) at pH 6.5-6.8 at various concentrations of glucose. It should be noted here that other types of hydroxyketones are also formed in the reaction, in particular 3-OH-2-butanone and 1-OH-2-butanone. The amount of these hydroxyketones was measured. Figure 3 shows GC/MS analysis of glucose reacted with phosphate buffer at 140° C. for 60 minutes and extracted with DCM.

Further, the concentrations of glucose and acetol were determined in 300 ml and 1 l of the reaction mixture. 6 grams of glucose were reacted with 300 ml of 0.1 N. NaOH for 60 min at 140°C. Next, the reaction mixture was cooled, and the concentrations of acetol and glucose were measured in the solution. A small amount of glucose (0.964 mg/ml) was found in the resulting product. Which corresponds to approximately less than 5% of the glucose that remains unreacted. Table 10 below shows the concentrations of acetol in this reaction. It can be determined that the concentration of acetol was approximately 12 mg for every 25 ml. Two additional reactions were carried out using 1 liter of 0.1 N. NaOH and 20 grams of glucose in a pressure washer. Both reactions were carried out at 130-140°C for 60 minutes. The concentration of glucose and acetol was determined. In both reactions at 1 liter, the glucose concentration was less than 1 mg/ml (0.945 and 0.958 mg/ml). Table 10 below also shows the concentration of acetol in each 1 liter of the reaction mixture. Again the concentration of acetol is approximately 12 mg for every 25 ml.

Further, acetol was isolated after the reaction of glucose with 0.1 N. NaOH. To isolate acetol from the reaction mixture, 0.1 N. NaOH and glucose were used in various ways. Previous results on the isolation of pyrazines by distillation show that pyrazines with a boiling point of 140°C were isolated from the reaction mixture. Thus, a distillation apparatus set at 120-140°C was used to isolate acetol from the reaction mixture. For this purpose, distillation of 100 ml of the reaction mixture of 0.1 N. NaOH and glucose at 140°C. After collecting 40 ml of the distillation solution, both the distilled and the remaining material were extracted with DCM. GC/FID analysis revealed that only 15-20% acetol was obtained by distillation, with more than 80-85% acetol remaining in the reaction mixture.

Similar results were obtained when the reaction mixture of glucose was distilled with 40% phosphate buffer at 140° C. for 60 minutes. In this distillation, water was constantly added to the flask during distillation to maintain a constant volume of distilled solution. After collecting about 10 ml of the solution by distillation, about 10 ml of H ₂ O was added to the distillation flask to compensate for the lost volume. A total of 4 fractions of 10 ml were collected. Acetol was found in every fraction. Analysis of the remaining reaction mixture with DCM showed the presence of more acetol in solution. Isolation of the acetol can be carried out if this distillation lasts for several hours.

) использовали для выделения ацетола. После промывания колонки метанолом и далее 0,1% раствором FA 25 мл реакционной смеси, содержащей ацетол, пропускали через колонку. В ходе данного выделения собирали весь раствор. После пропускания раствора через колонку (фракция 1, 25 мл), 25 мл деионизированной H₂O использовали для промывки материала из колонки (фракция 2). Затем колонку промывали дополнительно 25 мл H₂O до тех пор, пока из колонки не была элюирована чистая Н₂О (фракция 3). Далее колонку сушили N₂ и оставшиеся аналиты, удерживаемые на колонке, элюировали с помощью 100% МеОН (Фракция 4). Все фракции экстрагировали ДХМ и анализировали ГХ/ПИД. Результаты показали, что большую часть ацетола элюируют в виде фракций 1 и 2, в то время как фракции 3 и 4 не содержат ацетол. Здесь следует отметить, что после прохождения реакционной смеси через наполнитель C₁₈ очень сложно использовать сорбент для очистки следующей реакционной смеси. Очистить сорбент крайне сложно. Подводя итог, следует отметить, что выделение гидроксикетонов из продукта реакции может быть времязатратно и дорого.In the second method, column chromatography was used to isolate acetol from the reaction mixture. For this purpose, a 30 x 2.0 cm glass (or metal) column (filled only to a level of 12-15 cm) with C ₁₈ (particle size 40-60 µm and pore size 90

) was used to isolate acetol. After washing the column with methanol and then with 0.1% FA solution, 25 ml of the reaction mixture containing acetol was passed through the column. During this isolation, the entire solution was collected. After passing the solution through the column (fraction 1, 25 ml), 25 ml of deionized H ₂ O was used to wash the material from the column (fraction 2). The column was then washed with an additional 25 ml H ₂ O until pure H ₂ O was eluted from the column (fraction 3). The column was then dried over N ₂ and the remaining analytes retained on the column were eluted with 100% MeOH (Fraction 4). All fractions were extracted with DCM and analyzed by GC/FID. The results showed that most of the acetol was eluted as fractions 1 and 2, while fractions 3 and 4 contained no acetol. It should be noted here that after passing the reaction mixture through the C ₁₈ filler, it is very difficult to use the sorbent to purify the next reaction mixture. It is extremely difficult to clean the sorbent. In summary, it should be noted that the separation of hydroxyketones from the reaction product can be time consuming and expensive.

Next, pyrazines were synthesized from the product of the reaction of glucose and sodium hydroxide with NH ₄ OH without the primary isolation of hydroxyketones. After obtaining acetol during the reaction of 0.5 grams of glucose with 25 ml of 0.1 N. NaOH at 140°C for 60 minutes, it was found that the reaction mixture contained about 10-12 mg of acetol/25 ml of solution. For this purpose, 25 ml of the solution was reacted with 0.5 or 1 ml of NH ₄ OH for 17-18 hours at 120-140°C to establish the type and percentage yield of pyrazines. Each reaction was repeated twice. Table 11 below lists the pyrazines that were found along with their elution times from the GC column. The results are similar when the volume of NH ₄ OH is changed from 0.5 to 1 ml.

Similar reactions were carried out at a volume of 1 liter. Two batches of 1 liter solution were prepared. In each series, 20 grams of glucose was reacted with 1000 ml of 0.1 N. NaOH solution at 140°C for 60 min. Each solution was cooled and the concentration of acetol was measured. Next, 20 ml of NH ₄ OH was added to one of the reaction mixtures, and 40 ml of NH ₄ OH was added to the second reaction mixture. Each reaction mixture was heated at 120-130°C for 17 hours with continuous stirring. Then the reaction mixture was cooled, an internal standard (d ₆ -2-methylpyrazine) was added to 25 ml of each reaction mixture and extracted with DCM to determine the concentration and distribution of each pyrazine. Each remaining reaction solution was subjected to distillation and collected separately. Approximately 125 ml of the distilled solution was collected for every 500 ml of the reaction mixture. The distilled solution (10 ml) was then extracted with DCM and quantitated by GC/MS. The total mass of pyrazines was higher than when the solution was not extracted with DCM, which causes a higher concentration of pyrazines in the distilled solution. The distribution of pyrazines is almost identical in both extracts. It should be noted that both 2-methylpyrazine and pyrazine are found in large amounts in all reactions when NH ₄ OH is reacted with the reaction mixture of glucose and 0.1 N sodium hydroxide. NaOH. Pyrazines were not found in the extract of the solution remaining after distillation.

Next, pyrazines were synthesized from the product of the reaction of glucose and phosphate buffer with NH ₄ OH, without the primary isolation of hydroxyketones. 25 ml of the reaction mixture of phosphate buffer pH=6.5-6.8 and glucose (0.5 or 1 gram of solutions A and B, respectively) was subjected to interaction with 1 ml of NH ₄ OH at 140°C for 17 hours. Next, each reaction mixture was cooled, 0.25 mg of deuterated 2-methylpyrazine (internal standard) was added to each reaction mixture, and the solutions were extracted with DCM. Each extract was analyzed by GC/MS and the mass of pyrazines and their percentage distribution were calculated. It should be noted that neither pyrazine nor 2-methylpyrazine was synthesized in these reactions. Also, the total mass of pyrazines in solution B is 20% higher when 1 gram of glucose is reacted with phosphate buffer. Without being limited by theory, this is believed to be the reason for the higher concentration of acetol in solution B compared to solution A. 40% phosphate buffer at 140°C for 60 min) with 1 ml of NH ₄ OH at 140°C for 17 hours. For comparison, Figure 5 shows a GC/MS analysis of pyrazines extracted with DCM from a 25 ml reaction mixture of 0.1N. NaOH reacted with 0.5 grams of glucose at 140°C for 60 min and then reacted with 1 ml NH4OH at 140°C. Both pyrazine and 2-methylpyrazine were found in this reaction mixture.

In summary, acetol (and other hydroxyketones) can be isolated from a reaction mixture of glucose and 0.1 N sodium hydroxide. NaOH by distillation and column chromatography. Distillation is very time consuming and energy intensive, while chromatography can be extremely expensive. However, it is possible to synthesize pyrazines without isolation of acetol. A number of pyrazines were obtained when acetol was obtained from the reaction of 0.1 N. NaOH and glucose, the reaction was carried out with NH ₄ OH at 140°C for 17 hours. The pyrazines were isolated by distillation. It should be noted that pyrazine and 2-methylpyrazine were synthesized during this process. However, when acetol was prepared from a mixture of glucose and phosphate buffer reacted with NH ₄ OH, similar pyrazines were obtained with a similar distribution, but no pyrazine or 2-methylpyrazine was found in the reaction mixture.

Specialists in the art to which the present invention relates, can offer many modifications and other embodiments of the present invention based on what is set forth in the present description. Therefore, it should be understood that the present invention is not to be limited to the disclosed individual embodiments, and modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are used in the present description, they are used only in a general and descriptive sense, and not by way of limitation.

Claims (35)

1. A method for producing pyrazines substituted with two, three or four C ₁ -C ₆ alkyl groups, including: obtaining a reaction solution containing one alpha-hydroxy ketone, including 1-hydroxy-2-butanone, and one source of nitrogen containing NH ₄ OH, adding free amino acids to the reaction solution; heating said reaction solution to a reaction temperature of 100-140° C. and keeping the reaction solution at said reaction temperature for 4-24 hours to obtain a reaction product containing a substituted pyrazine; and isolation of said pyrazine substituted with two, three or four C ₁ -C ₆ alkyl groups from the reaction product, including liquid-liquid extraction of the reaction product, solid-liquid extraction of the reaction product, or simple distillation of the reaction product. 2. The method of claim 1 wherein said substituted pyrazine is tetramethylpyrazine. 3. The method according to claim 1 or 2, wherein said substituted pyrazine is selected from the group consisting of 2,6-diethylpyrazine, 2,5-diethylpyrazine, 2-ethyl-3,5,6-trimethylpyrazine, 3,5-dimethyl -2,N-propylpyrazine, 3,6-dimethyl-2,N-propylpyrazine, 2,5-diethyl-3-methylpyrazine, 2,3-diethyl-5-methylpyrazine, 2,3-diethyl-5,6-dimethylpyrazine , trans-3-methyl-2,N-propyl-6(1-butenyl)pyrazine, 2,5-dimethyl-3-ethylpyrazine, and combinations thereof. 4. The method of claim 1, further comprising adding at least one aldehyde to the reaction solution. 5. The method according to claim 1, wherein said reaction temperature is between 90°C and 150°C. 6. The method of claim 1 wherein said substituted pyrazine is selected from the group consisting of 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2-ethyl-6-methylpyrazine, 2,3 ,5-trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-2,5-dimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2,3,5-trimethyl-6-ethylpyrazine, 2,6 -dimethyl-3-propylpyrazine, 2,5-diethyl-3,6-dimethylpyrazine, 2,6-dimethyl-3-(2-methylbutyl)pyrazine, 2,5-dimethyl-3-(2-methylbutyl)pyrazine, 2 ,5-dimethyl-3-(3-methylbutyl)pyrazine, 2,5-dimethyl-3-propylpyrazine, 2,5-dimethyl-3-cis-propenylpyrazine, 2-isopropenyl-3,6-dimethylpyrazine, 2-(2 -methylpropyl)-3,5-dimethylpyrazine, 2,6-dimethyl-3-isobutylpyrazine, 2-(2-methylpropyl)-3,5,6-trimethylpyrazine, 2,3-dimethylpyrazine, trimethylpyrazine, 2,5-dimethyl- 3-ethylpyrazine, tetramethylpyrazine, 2,3-diethyl-5-methylpyrazine, 2,5-dimethyl-3-propenylpyrazine, 2,3,5-trimethyl-6-isopropylpyrazine, 2-acetyl-4,5-dimethylpyrazine, 3, 5-dimethyl-2-methylpropylpyrazine, 2,6-diethylpyrazine, 2,5-diethylpyrazine, 2-et yl-3,5,6-trimethylpyrazine, 3,5-dimethyl-2,N-propylpyrazine, 3,6-dimethyl-2,N-propylpyrazine, 2,5-diethyl-3-methylpyrazine, 2,3-diethyl- 5,6-dimethylpyrazine, trans-3-methyl-2,N-propyl-6(butenyl)pyrazine, 2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrazine and 2,5-dimethyl-3- ethylpyrazine; and combinations thereof. 7. The method of claim 1 wherein said substituted pyrazine is disubstituted. 8. The method of claim 1 wherein said substituted pyrazine is trisubstituted. 9. The method of claim 1 wherein said substituted pyrazine is tetrasubstituted. 10. The method of claim 1, wherein said substituted pyrazine contains at least one substituent group containing 2 or more carbon atoms. 11. The method of claim. 1, in which the specified substituted pyrazine contains at least one substituent group containing 3 or more carbon atoms. 12. The method of claim 1 wherein said reaction product is substantially free of a pyrazine molecule and a methyl pyrazine molecule. 13. The method according to any one of paragraphs. 1-12, further comprising introducing said at least one substituted pyrazine into the tobacco product. 14. The method of claim 13, wherein said tobacco product is a smoking product or a smokeless tobacco product. 15. A method for producing pyrazines substituted with two, three or four C ₁ -C ₆ alkyl groups, including: obtaining a carbon source solution containing one C ₆ sugar containing glucose or fructose, and one buffer containing sodium hydroxide or phosphate buffer; mixing said carbon source solution with one nitrogen source containing NH ₄ OH to form a reaction solution; heating the reaction solution to a reaction temperature of 120-140°C and holding the reaction solution at the indicated reaction temperature for 17-18 hours to obtain a reaction product containing pyrazine substituted with two, three or four C ₁ -C ₆ alkyl groups; and isolation of said pyrazine substituted with two, three or four C ₁ -C ₆ alkyl groups from the reaction product, including liquid-liquid extraction of the reaction product, solid-liquid extraction of the reaction product, or simple distillation of the reaction product. 16. The method of claim 15 wherein said buffer provides a pH in the range of 6.5 to 7.5. 17. The method of claim 15, further comprising adding ammonium ions to the reaction solution. 18. The method of claim 15, wherein said reaction temperature is between 90°C and 150°C. 19. The method of claim 15 wherein said substituted pyrazine is selected from the group consisting of 2,6-dimethylpyrazine, 2,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2-ethyl-6-methylpyrazine, 2,3 ,5-trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-2,5-dimethylpyrazine, 2,3,5,6-tetramethylpyrazine, 2,3,5-trimethyl-6-ethylpyrazine, 2,6 -dimethyl-3-propylpyrazine, 2,5-diethyl-3,6-dimethylpyrazine, 2,6-dimethyl-3-(2-methylbutyl)pyrazine, 2,5-dimethyl-3-(2-methylbutyl)pyrazine, 2 ,5-dimethyl-3-(3-methylbutyl)pyrazine, 2,5-dimethyl-3-propylpyrazine, 2,5-dimethyl-3-cis-propenylpyrazine, 2-isopropenyl-3,6-dimethylpyrazine, 2-(2 -methylpropyl)-3,5-dimethylpyrazine, 2,6-dimethyl-3-isobutylpyrazine, 2-(2-methylpropyl)-3,5,6-trimethylpyrazine, 2,3-dimethylpyrazine, trimethylpyrazine, 2,5-dimethyl- 3-ethylpyrazine, tetramethylpyrazine, 2,3-diethyl-5-methylpyrazine, 2,5-dimethyl-3-propenylpyrazine, 2,3,5-trimethyl-6-isopropylpyrazine, 2-acetyl-4,5-dimethylpyrazine, 3, 5-dimethyl-2-methylpropylpyrazine, 2,6-diethylpyrazine, 2,5-diethylpyrazine, 2- ethyl-3,5,6-trimethylpyrazine, 3,5-dimethyl-2,N-propylpyrazine, 3,6-dimethyl-2,N-propylpyrazine, 2,5-diethyl-3-methylpyrazine, 2,3-diethyl- 5,6-dimethylpyrazine, trans-3-methyl-2,N-propyl-6(butenyl)pyrazine, 2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrazine and 2,5-dimethyl-3- ethylpyrazine; and combinations thereof. 20. The method of claim 15 wherein said substituted pyrazine is disubstituted. 21. The method of claim 15 wherein said substituted pyrazine is trisubstituted. 22. The method of claim 15 wherein said substituted pyrazine is tetrasubstituted. 23. The method of claim 15, wherein said substituted pyrazine contains at least one substituent group containing 2 or more carbon atoms. 24. The method of claim 15, wherein said substituted pyrazine contains at least one substituent group containing 3 or more carbon atoms. 25. The method of claim 15, wherein said reaction product is substantially free of pyrazine and methylpyrazine molecules. 26. The method according to any one of paragraphs. 15-25, further comprising introducing said substituted pyrazine into the tobacco product. 27. The method of claim 26, wherein said tobacco product is a smoking product or a smokeless tobacco product.

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