Method for selectively forming substituted pyrazine

Abstract

To provide methods of selectively forming substituted pyrazines.SOLUTION: The methods can include receiving a reaction solution including at least one carbon source and at least one nitrogen source, and heating the reaction solution to a reaction temperature and holding the reaction solution at the reaction temperature for a time sufficient to produce a reaction product comprising at least one substituted pyrazine. The carbon source can be selected from the group consisting of a hydroxy ketone, a sugar treated with at least one buffer, and a combination thereof. Tobacco products incorporating substituted pyrazines are also provided.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for forming substituted pyrazines. Of particular interest is a method for selectively forming a target substituted pyrazine.

Background Art

[0002] Pyrazine is produced by the reaction (e.g., the Maillard reaction) of a carbon source with a nitrogen compound, such as an amino acid and a base (e.g., diammonium phosphate (DAP), NaOH). In many conventional reaction pathways aimed at the production of pyrazine, sugars (e.g., fructose, glucose, fructose / glucose mixture, rhamnose) are used as the carbon source. So far, most of the model reactions and fortified natural products that result in formulations rich in pyrazine upon heating have used sugars such as fructose, glucose, fructose / glucose mixture, and rhamnose as the carbon source component of the formulation. These sugars have been shown to act as a carbon source for forming the pyrazine aromatic ring structure.For example, U.S. Patent Application Publication No. 2004 / 0173228 by Coleman, U.S. Patent Application Publication No. 2010 / 0037903 by Coleman et al., U.S. Patent Application Publication No. 2012 / 0152265 by Dube et al., U.S. Patent Application Publication No. 2012 / 0260929 by Coleman et al., U.S. Patent Application Publication No. 2013 / 0125907 by Dube et al., U.S. Patent Application Publication No. 2013 / 0337418 by Anuradha et al., U.S. Patent Application Publication No. 2015 / 0040922 by Dube et al., and U.S. Patent Application Publication No. 2015 / 0122271 by Chen et al., each of which is hereby incorporated by reference in its entirety; U.S. Patent No. 5,258,194 by Anderson et al., U.S. Patent No. 6,298,858 by Coleman; U.S. Patent No. 6,325,860 by Coleman, U.S. Patent No. 6,440,223 by Dube et al., U.S. Patent No. 6,499,489 by Coleman; U.S. Patent No. 6,591,841 by White et al., U.S. Patent No. 6,695,924 by Dube et al., U.S. Patent No. 8,434,496 by Chen et al., U.S. Patent No. 8,944,072 by Brinkley et al., U.S. Patent No. 8,955,523 by Coleman et al., U.S. Patent No. 8,991,403 by Chen et al., U.S. Patent No. 9,010,339 by Dube et al., U.S. Patent No. 9,254,001 by Byrd et al., U.S. Patent No. 9,265,284 by Junker et al., and U.S. Patent No. 9,402,415 by Coleman 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, pp135 - 182, CRC Press, Boca Raton, 2012.

[0003] In most cases, these reactions of sugars and nitrogen sources have used ammonium hydroxide and / or free amino acids as the nitrogen source that provides the nitrogen bond within 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. This reaction produces a complex mixture of many substituted pyrazines. When sugar acts as the sole carbon source in the pyrazine formation reaction, pyrazine molecules and methylpyrazine molecules are the major pyrazines produced and often greatly exceed 60% of the total pyrazine yield. This tendency is evident even when free amino acids are used as a co-reagent in attempts to reduce the amount of pyrazine molecules and methylpyrazine molecules produced.

[0004] From a sensory perspective, pyrazine molecules and methylpyrazine molecules do not have the desirable sensory aroma notes for use in tobacco products, nor do they have acceptable volatility characteristics. Thus, their presence in a mixture of pyrazines is inferior to the desired characteristics for certain applications.

[0005] Therefore, it is desirable to provide a method for more selectively producing a desired substituted pyrazine in a greater amount.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Document

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0008] The present invention provides a method for selectively forming a certain substituted pyrazine. The method for selectively producing pyrazine includes obtaining a reaction solution containing at least one tobacco-derived carbon source (e.g., hydroxyketone and / or at least one sugar treated with a buffer) and at least one tobacco-derived nitrogen source (e.g., protein and / or amino acid), heating the reaction solution to a reaction temperature, and maintaining the reaction solution at the reaction temperature for a time sufficient to produce a reaction product containing at least one substituted pyrazine. The nitrogen source can be selected from the group consisting of amino acids, ammonium ions, and combinations thereof.

[0009] In various embodiments, at least one substituted pyrazine can 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(dimethylpryazine); trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; 2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrrazine; and 2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

[0010] In some embodiments, at least one substituted pyrazine is 2-substituted, 3-substituted or 4-substituted. In certain embodiments, at least one substituted pyrazine contains at least one substituent having two or more carbon atoms. In various embodiments, at least one substituted pyrazine contains at least one substituent having three or more carbon atoms.

[0011] In various embodiments, a method of selectively forming a substituted pyrazine can further include isolating at least one substituted pyrazine from the reaction product. The step of isolating at least one tobacco-derived pyrazine from the reaction product can include, for example, at least one of liquid-liquid extraction of the reaction product, liquid-solid extraction of the reaction product, and simple distillation of the reaction product.

[0012] The method of the present invention can further include incorporating at least one substituted pyrazine into a tobacco product. In certain embodiments, the tobacco product can be a smoking product. In some embodiments, the tobacco product can be a smokeless tobacco product.

[0013] In various embodiments of the present invention, a method of forming pyrazine is provided that includes obtaining a reactant solution comprising at least one α-hydroxy ketone and at least one nitrogen source, heating the reactant solution to a reactant temperature, and maintaining the reactant solution at the reactant temperature for a time sufficient to produce a reactant product comprising at least one substituted pyrazine. Various embodiments of the method can further include isolating at least one substituted pyrazine from the reactant product. In certain embodiments, the at least one hydroxy ketone can include acetol, and the at least one substituted pyrazine can 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 (dimethylypyrazine), 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, the at least one hydroxy ketone can include acetoin, and the at least one substituted pyrazine can be tetramethylpyrazine. In various embodiments, the at least one hydroxy ketone can include 1-hydroxy-2-butanone, and the at least one substituted pyrazine can 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-methylpyrazine, 2,3-diethyl-5,6-dimethylpyrazine (dimethylpryazine), trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine, 2,5-dimethyl-3-ethylpyrazine, and combinations thereof.

[0014] In various embodiments of the present invention, the method can further include adding a free amino acid to a reaction solution containing at least one α-hydroxy ketone and at least one nitrogen source. In some embodiments, the method can further include adding at least one aldehyde to a reaction solution containing at least one α-hydroxy ketone and at least one nitrogen source.

[0015] In various embodiments of the present invention, provided is a method for forming pyrazine, comprising obtaining a carbon source solution containing at least one sugar and at least one buffer such that an optimal amount of at least one hydroxy ketone is supplied from at least one sugar, mixing the carbon source solution with at least a nitrogen source to form a reaction solution, heating the reaction solution to a reaction temperature, and holding the reaction solution at the reaction temperature for a time sufficient to produce a reaction product containing at least one substituted pyrazine. Various embodiments of the methods herein can further include isolating at least one substituted pyrazine from the reaction product. The at least one sugar can be selected from the group consisting of glucose, fructose, rhamnose, and combinations thereof. In certain embodiments, the buffer can be selected from the group consisting of sodium hydroxide, phosphate buffer, and combinations thereof. In certain embodiments, the buffer can buffer in a pH range of about 6.5 to about 7.5. The method of the present invention can further include adding ammonium ions to a reaction solution containing a carbon source containing at least one sugar and at least one buffer.

[0016] The present invention includes, but is not limited to, the following embodiments.

[0017] Embodiment 1: A method for forming pyrazine, comprising obtaining a reaction solution containing at least one α-hydroxy ketone and at least one nitrogen source, heating the reaction solution to a reaction temperature, and holding the reaction solution at the reaction temperature for a time sufficient to produce a reaction product containing at least one substituted pyrazine.

[0018] Embodiment 2: The method according to Embodiment 1, wherein at least one hydroxy ketone contains acetol.

[0019] Embodiment 3: The method according to Embodiment 1 or 2, wherein at least one 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-dimethylpyrazine (dimethylypyrazine); 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.

[0020] Embodiment 4: The method according to any one of Embodiments 1 to 3, wherein at least one hydroxy ketone contains acetoin.

[0021] Embodiment 5: The method according to any one of Embodiments 1 to 4, wherein at least one substituted pyrazine is tetramethylpyrazine.

[0022] Embodiment 6: The method according to any one of Embodiments 1 to 5, wherein at least one hydroxy ketone contains 1-hydroxy-2-butanone.

[0023] Embodiment 7: The method according to any one of Embodiments 1 to 6, wherein 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 (dimethylpryazine); trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; 2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

[0024] Embodiment 8: The method according to any one of Embodiments 1 to 7, wherein the nitrogen source is selected from the group consisting of an amino acid, an ammonium ion, and a combination thereof.

[0025] Embodiment 9: The method according to any one of Embodiments 1 to 8, further comprising adding a free amino acid to the reaction solution.

[0026] Embodiment 10: The method according to any one of Embodiments 1 to 9, further comprising adding at least one aldehyde to the reaction solution.

[0027] Embodiment 11: The method according to any one of Embodiments 1 to 10, further comprising isolating at least one substituted pyrazine from the reaction product.

[0028] Embodiment 12: The method according to any one of Embodiments 1 to 11, wherein the step of isolating at least one substituted pyrazine from the reaction product includes at least one of liquid-liquid extraction of the reaction product, liquid-solid extraction of the reaction product, and simple distillation of the reaction product.

[0029] Embodiment 13: The method according to any one of Embodiments 1 to 12, wherein the reaction temperature is from about 90 °C to about 150 °C.

[0030] Embodiment 14: The method according to any one of Embodiments 1 to 13, wherein 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-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; 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 (dimethylpryazine); trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; 2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrrazine; and 2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

[0031] Embodiment 15: The method according to any one of Embodiments 1 to 14, wherein at least one substituted pyrazine is disubstituted.

[0032] Embodiment 16: The method according to any one of Embodiments 1 to 15, wherein at least one substituted pyrazine is trisubstituted.

[0033] Embodiment 17: The method according to any one of Embodiments 1 to 16, wherein at least one substituted pyrazine is tetrasubstituted.

[0034] Embodiment 18: The method according to any one of Embodiments 1 to 17, wherein at least one substituted pyrazine contains at least one substituent having 2 or more carbon atoms.

[0035] Embodiment 19: The method according to any one of Embodiments 1 to 18, wherein at least one substituted pyrazine contains at least one substituent having 3 or more carbon atoms.

[0036] Embodiment 20: The method according to any one of Embodiments 1 to 19, wherein the reaction product substantially does not contain pyrazine molecules and methylpyrazine molecules.

[0037] Embodiment 21: The method according to any one of Embodiments 1 to 20, further comprising incorporating at least one substituted pyrazine into a tobacco product.

[0038] Embodiment 22: The method according to any one of Embodiments 1 to 21, further comprising incorporating at least one substituted pyrazine into a tobacco product, wherein the tobacco product is a smoking product or a smokeless tobacco product.

[0039] Embodiment 23: A method for forming pyrazine, comprising obtaining 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, heating the reaction solution to a reaction temperature, and holding the reaction solution at the reaction temperature for a time sufficient to produce a reaction product containing at least one substituted pyrazine.

[0040] Embodiment 24: The method according to any one of Embodiments 1 to 23, wherein at least one sugar is selected from the group consisting of glucose, fructose, rhamnose, and combinations thereof.

[0041] Embodiment 25: The method according to any one of Embodiments 1 to 24, wherein the nitrogen source is selected from the group consisting of amino acids, ammonium ions, and combinations thereof.

[0042] Embodiment 26: The method according to any one of Embodiments 1 to 25, wherein the buffer is selected from the group consisting of sodium hydroxide, phosphate buffer, and combinations thereof.

[0043] Embodiment 27: The method according to any one of Embodiments 1 to 26, wherein the buffer buffers in a pH range of about 6.5 to about 7.5.

[0044] Embodiment 28: The method according to any one of Embodiments 1 to 27, further comprising adding ammonium ions to the reaction solution.

[0045] Embodiment 29: The method according to any one of Embodiments 1 to 28, further comprising isolating at least one substituted pyrazine from the reaction product.

[0046] Embodiment 30: The method according to any one of Embodiments 1 to 29, wherein the step of isolating at least one substituted pyrazine from the reaction product includes at least one of liquid-liquid extraction of the reaction product, liquid-solid extraction of the reaction product, and simple distillation of the reaction product.

[0047] Embodiment 31: The method according to any one of Embodiments 1 to 30, wherein the reaction temperature is about 90°C to about 150°C.

[0048] Embodiment 32: The method according to any one of Embodiments 1 to 31, wherein 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-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; 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 (dimethylpryazine); trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; 2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrrazine; and 2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

[0049] Embodiment 33: The method according to any one of Embodiments 1 to 32, wherein at least one substituted pyrazine is disubstituted.

[0050] Embodiment 34: The method according to any one of Embodiments 1 to 33, wherein at least one substituted pyrazine is trisubstituted.

[0051] Embodiment 35: The method according to any one of Embodiments 1 to 34, wherein at least one substituted pyrazine is tetrasubstituted.

[0052] Embodiment 36: The method according to any one of Embodiments 1 to 35, wherein at least one substituted pyrazine contains at least one substituent having 2 or more carbon atoms.

[0053] Embodiment 37: The method according to any one of Embodiments 1 to 36, wherein at least one substituted pyrazine contains at least one substituent having 3 or more carbon atoms.

[0054] Embodiment 38: The method according to any one of Embodiments 1 to 37, wherein the reaction product substantially does not contain pyrazine molecules and methylpyrazine molecules.

[0055] Embodiment 39: The method according to any one of Embodiments 1 to 38, further comprising incorporating at least one substituted pyrazine into a tobacco product.

[0056] Embodiment 40: The method according to any one of Embodiments 1 to 39, further comprising incorporating at least one substituted pyrazine into a tobacco product, wherein the tobacco product is a smoking product or a smokeless tobacco product.

[0057] These and other features, aspects, and advantages of the present disclosure will become apparent from the following detailed description, read in conjunction with the accompanying drawings described briefly below. The present invention includes any combination of two, three, four, or more of the above-described embodiments, as well as any combination of two, three, four, or more features or elements described in the present disclosure, whether or not such features or elements are explicitly combined in the description of a particular embodiment herein. The present disclosure is to be read as a whole such that, in any of its various aspects and embodiments, separable features or elements of the disclosed invention are to be construed as combinable unless the context clearly dictates otherwise.

[0058] To provide knowledge of embodiments of the present invention, reference is made to the accompanying drawings, in which reference numerals indicate components of exemplary embodiments of the present invention and which are not necessarily drawn to scale. The drawings are merely illustrative and are not to be construed as limiting the present invention.

Brief Description of the Drawings

[0059]

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DETAILED DESCRIPTION OF THE INVENTION

[0060] In the following, the present invention will be described more fully. However, the present invention can be implemented in many different forms and should not be construed as limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and the scope of the present invention will be fully conveyed to those skilled in the art. As used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. When referring to "percent dry weight" or "dry weight basis", it indicates the weight based on the dry components (i.e., all components excluding water).

[0061] The present invention provides a method for forming selected pyrazines. Pyrazines exhibit many different flavor profiles including roasted, toasted, and nutty flavors, but are not limited thereto. For example, pyrazines containing cyclopentyl derivatives are known for their positive sensory properties at very low levels in ppb. Pyrazines are formed by heating a mixture of a carbon source and a nitrogen source. The method of the present invention not only minimizes the formation of pyrazine molecules and methylpyrazine molecules, but also adjusts the reaction so that other desired substituted pyrazines can be produced in a controlled manner.

[0062] Selective formation of pyrazine using carbon sources other than sugars Conventionally, sugars have been used as carbon sources in reactions to form pyrazines. Several reaction pathways for generating pyrazine-rich formulations using sugars are known in the art and include: 1) hydrolysis of proteins to free amino acids and subsequent reaction of these free amino acids with sugars such as glucose and / or high fructose syrup; and 2) biotechnological synthesis of free amino acids using glucose and nitrogen (e.g., ammonium ions) and subsequent reaction of these free amino acids with sugars such as glucose and / or high fructose syrup. When sugars are used as intact molecules and reacted with a nitrogen source (e.g., ammonium hydroxide, amino acids), a series of pyrazines are produced that include pyrazine and methylpyrazine molecules as the major pyrazines, far smaller amounts of dimethylpyrazine molecules, and significantly smaller amounts of high molecular weight pyrazine molecules.

[0063] As used herein, the term "pyrazine molecule" refers to a heterocyclic organic compound of the chemical formula C4H4N2. This is different from the general term "pyrazine" used herein, which refers to a group of compounds produced from the reaction of a carbon source and a nitrogen source.

[0064] As used herein, the term "nitrogen source" refers to a nitrogen-containing compound that reacts with a carbon source to form at least one pyrazine. In various embodiments, the nitrogen source can include ammonium ions (NH4 + ), amino acids, proteins, or combinations thereof. In some embodiments, ammonium ions (NH4 +) can be provided. Amino acids can be derived, for example, from the hydrolysis of proteins. In some embodiments, as discussed in U.S. Patent Application Publication No. 15 / 009,199 to Dube et al., filed on January 28, 2016, which is incorporated herein by reference in its entirety, amino acids can be derived from the hydrolysis of tobacco-derived proteins. Other nitrogen-containing compounds that are known in the art and reactive with a carbon source and form at least one pyrazine can also be used as a nitrogen source in the embodiments of the invention disclosed herein.

[0065] In various embodiments of the present invention, the carbon source can include a hydroxyketone. A hydroxyketone is a functional group of a ketone where a hydroxyl group is adjacent. In two main classes of hydroxyketones, the hydroxyl group can be substituted at the α-position (i.e., α-hydroxyketone having the formula RCR’(OH)(CO)R) or the β-position (i.e., β-hydroxyketone having the formula RCR’(OH)CR2(CO)R). The structures of α- and β-hydroxyketones are shown below.

[0066]

Chemical formula

[0067] In various embodiments of the present invention, the carbon source can include at least one α-hydroxyketone. In various embodiments of the present invention, the R and R’ functional groups of at least one hydroxyketone can be independently selected from the group consisting of H or halo (e.g., Cl, F, or Br), OH, optionally substituted C1-10 alkyl, optionally substituted C1-10 alkoxy, optionally substituted C2-4 alkenyl, optionally substituted C2-4 alkynyl, NR6R7, NR6COR7, NR6CO2R7, CR6R7OR8, CONR6R7, CO2R6, CN, CF3, NO2, N3, C1-3 alkylthio, R9SO, R9SO2, CF3S, and CF3SO2. In certain embodiments of the present invention, the R and / or R’ functional group is C1-6 alkyl.

[0068] As used herein, the term "alkyl" means a saturated straight-chain, branched or cyclic hydrocarbon group (i.e., cycloalkyl group) and an unsaturated version of the saturated example (e.g., propenyl). In certain embodiments, alkyl represents a group containing 1 to 10 carbon atoms ("C1-10 alkyl"). In further embodiments, alkyl represents a group containing 1 to 8 carbon atoms ("C1-8 alkyl"), 1 to 6 carbon atoms ("C1-6 alkyl") or 1 to 4 carbon atoms ("C1-4 alkyl"). In other embodiments, alkyl represents a group containing 3 to 10 carbon atoms ("C3-10 alkyl"), 3 to 8 carbon atoms ("C3-8 alkyl") or 3 to 6 carbon atoms ("C3-6 alkyl"). In specific embodiments, alkyl represents 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.

[0069] With respect to a substituent, "optionally substituted" means, for example, halo (e.g., Cl, F, Br and I); alkyl (e.g., C1-10 alkyl), halogenated alkyl (e.g., CF3, 2-Br-ethyl, CH2F, CH2Cl, CH2CF3 or CF2CF3); C2-4 alkenyl, C2-4 alkynyl; hydroxyl; amino; amide; carboxylate; carboxamide; carbamate; carbonate; urea; acetate; alkylamino; arylamino; C1-10 alkoxy; aryl; aralkyl, aryloxy; nitro; azide; cyano; thio; alkylthio; sulfonate; sulfide; sulfinyl; sulfo; sulfate; sulfoxide; sulfamide; sulfonamide; phosphonic acid; phosphate; and / or a substituent optionally substituted by one or more moieties selected from the group consisting of phosphonate.

[0070] As used herein, the term "alkenyl" means an alkyl moiety in which at least one saturated C-C bond is replaced by a double bond. In certain embodiments, alkenyl refers to a group containing 2 to 10 carbon atoms ("C2-10 alkenyl"). In further embodiments, alkenyl refers to a group 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, alkenyl can 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.

[0071] As used herein, the term "alkynyl" means an alkyl moiety in which at least one saturated C-C bond is replaced by a triple bond. In certain embodiments, alkynyl refers to a group containing 2 to 10 carbon atoms ("C2-10 alkynyl"). In further embodiments, alkynyl refers to a group containing 2 to 8 carbon atoms ("C2-8 alkynyl"), 2 to 6 carbon atoms ("C2-6 alkynyl") or 2 to 4 carbon atoms ("C2-4 alkynyl"). In certain embodiments, alkynyl can 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.

[0072] As used herein, the term "alkoxy" means a straight or branched alkyl group bonded by an oxygen atom (i.e., -O-alkyl), and alkyl is as defined above. In certain embodiments, alkoxy refers to an oxygen-bonded group containing 1 to 10 carbon atoms ("C1-10 alkoxy"). In further embodiments, alkoxy refers to an oxygen-bonded group 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 1 to 3 carbon atoms ("C1-3 alkoxy").

[0073] As used herein, the term "halo" or "halogen" means fluorine, chlorine, bromine, or iodine.

[0074] As used herein, the term "amino" means a moiety represented by the structure NR2 and includes primary amines as well as secondary and tertiary amines substituted with alkyl or aryl (i.e., alkylamino or arylamino, respectively). Thus, R2 can 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.

[0075] Alkyl(amino) is a moiety represented by the structure -RNR2 and includes an alkyl group as defined above bonded to an amino group as defined above, and this moiety is bonded to another part of the molecule via the alkyl group.

[0076] As used herein, the term "cycloalkyl" means a non-aromatic, monocyclic or polycyclic ring containing carbon atoms and hydrogen atoms.

[0077] As used herein, the term "derivative" means a compound formed from a similar starting compound by bonding another molecule or atom to the starting compound. Further, derivatives according to the present invention include one or more compounds formed from a precursor compound by the addition of one or more atoms or molecules or by the combination of two or more precursor compounds.

[0078] In one embodiment of the present invention, acetoin has been successfully used as a precursor for producing tetramethylpyrazine (TMP). Specifically, the reaction of acetoin with ammonium hydroxide and phosphoric acid (or diammonium phosphate) has been successful, and tetramethylpyrazine (TMP) has been produced in an essentially quantitative yield (a yield exceeding 80%) with a considerable purity. The reaction of acetoin with ammonium hydroxide produces almost exclusively TMP, and the amounts of pyrazine molecules and methylpyrazine molecules are hardly or not at all detectable. As used herein, the terms "hardly or not at all detectable in amount", "substantially absent", and "substantially zero" indicate that the identified compound is present in an amount less than 1.0 wt%, less than 0.5 wt%, or less than 0.1 wt% based on the total weight of the reaction product.

[0079] Without being bound by theory, it has surprisingly been shown that the distribution of pyrazines in the reaction of a carbon source with a nitrogen source (e.g., ammonium ions and / or amino acids) is determined to a considerable extent by the carbon source, by various pyrazines synthesized from the reaction of a sugar as a carbon source with acetoin. In particular, it has surprisingly been found that when different hydroxyketones are used as the carbon source in the reaction to form pyrazines, a series of specific substituted pyrazines can be produced in a controlled manner.

[0080] In certain embodiments, at least one substituted pyrazine produced according to the methods described herein is disubstituted. In some embodiments, at least one substituted pyrazine produced according to the methods described herein is trisubstituted. In various embodiments, at least one substituted pyrazine produced according to the methods described herein is tetrasubstituted. In various embodiments, at least one substituted pyrazine produced according to the methods of the present invention comprises at least one substituent having two or more carbon atoms. In certain embodiments, at least one substituted pyrazine produced according to the methods of the present invention comprises at least one substituent having three or more carbon atoms.

[0081] In some embodiments, at least one substituted pyrazine produced according to the methods described herein is a branched pyrazine. As used herein, the term "branched pyrazine" refers to a pyrazine ring that is not essentially linear and includes an alkyl group thereon. For example, instead of an n-butyl group, isobutyl, sec-butyl, and tert-butyl groups (which is similarly applicable to propyl and pentyl groups). As will be described in more detail below, it has surprisingly been found that by varying the α-hydroxyketone, the distribution of the produced pyrazines can be determined. For example, using acetoin in the reaction results in only tetramethylpyrazine (purity of about 99.5% or more). Using acetol in the reaction results mainly in dimethyl-substituted pyrazines (pyrazine yield of about 95% or more).

[0082] As shown in FIG. 1, for example, a heating formulation containing a nitrogen source and at least one hydroxyketone can produce pyrazine. A solution rich in pyrazine can be prepared in various ways. For example, one method can include microwave heat treatment of a solution containing at least one amino acid and at least one hydroxyketone. As shown, for example, in operation 100 of FIG. 1, an aqueous reaction solution containing at least one amino acid and at least one hydroxyketone can be formed. As shown, for example, in operation 104 of FIG. 1, the reaction solution can be heated to the reaction temperature and held at the reaction temperature for a reaction time sufficient for the reactants to undergo a reaction to form pyrazine. As shown, for example, in operation 106 of FIG. 1, pyrazine can then be optionally isolated from the reaction product using simple distillation or other separation techniques known in the art.

[0083] In one embodiment, first, pyrazine is isolated from the reaction product by using simple distillation to give a distillate containing substantially water and pyrazine. Next, this distillate can be subjected to liquid-liquid extraction with cyclohexane. The amount of cyclohexane used in the liquid-liquid extraction can be equal to about half the amount of the distillate used. For example, when 10 L of the distillate is used, 5 L of cyclohexane can be used. The liquid-liquid extraction of the distillate with cyclohexane can be repeated a plurality of times. In some embodiments, the liquid-liquid extraction of the distillate with cyclohexane can be repeated at least 5 times. After the liquid-liquid extraction, the cyclohexane containing the extracted pyrazine can be dehydrated with any dehydrating agent known in the art. For example, sodium sulfate, magnesium hydroxide, and / or molecular sieves can be used to dehydrate the cyclohexane. After dehydration, pyrazine can be isolated by simple distillation and / or rotary evaporation (i.e., the cyclohexane can be removed).

[0084] As discussed above, using different hydroxyketones as the carbon source for the reaction to form pyrazine can generate a series of specific substituted pyrazines. In certain embodiments, the carbon source includes acetoin. As will be described in detail in Example 1 below, for example, when acetoin acts as the sole carbon source in a reaction with a nitrogen source (such as ammonium hydroxide (NH4OH) and phosphoric acid (H3PO4), diammonium phosphate, etc.) to produce pyrazine, the only pyrazine produced is tetramethylpyrazine. Furthermore, branched pyrazines such as isopropylpyrazine are not synthesized by adding an amino acid (such as leucine or free amino acids from hydrolyzed F1 protein) to a reaction containing acetoin, NH4OH, and H3PO4. Heating the reactants at a high temperature or for a long time does not change the result (i.e., tetramethylpyrazine (TMP) is the only pyrazine synthesized from acetoin). Therefore, if pyrazines other than TMP are required from this synthetic method, carbon sources other than acetoin can be synthesized.

[0085] In some embodiments, the carbon source can include 1-hydroxyacetone (also called 1-OH-acetone, acetol, or 1-hydroxy-2-propanone). Acetol, which is an α-hydroxyketone, is the simplest hydroxyketone. As will be described in detail below, acetol can be produced by the decomposition of various sugars. For example, acetol can be formed as an intermediate in the Maillard reaction (i.e., the reaction of sugar and amino acid to form pyrazine), and then acetol can further react to form other compounds. In some embodiments of the present invention, the carbon source can include 1-hydroxy-2-butanone. The structures of 1-OH-acetone and 1-OH-2-butanone are shown below. Note that in the case of 1-OH-acetone, a methyl group is bonded to one side of the carbonyl, and in the case of 1-OH-2-butanone, an ethyl group is bonded to one side of the carbonyl. Without being limited by theory, these structural features of the two α,β-hydroxyketones can determine the structure of the pyrazine produced.

[0086] [Chemical formula]

[0087] As shown in Example 2 below, for example, a series of pyrazines can be produced by the reaction of 1-hydroxyacetone as a carbon source with NH4OH as a source of base and nitrogen. When 1-hydroxyacetone (acetol) acts as the carbon source, a series of specific alkyl-substituted pyrazines can be produced. Exemplary pyrazines provided from the reaction of 1-hydroxyacetone and NH4OH include 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; 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.

[0088] When 1-hydroxy-2-butanone is used as the sole carbon source, pyrazines with an ethyl group attached can be produced. For example, as shown in Example 4 below, pyrazines synthesized from the reaction using 1-OH-2-butanone and NH4OH 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 (dimethylpryazine); trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; and 2,5-dimethyl-3-ethylpyrazine.

[0089] In the reaction with a nitrogen source, when 1-hydroxyacetone (acetol), 2-hydroxy-3-butanone (acetoin), and 1-hydroxy-2-butanone are used as the carbon source, pyrazine and methylpyrazine molecules do not appear in the series of structures and pyrazines produced. Note that other hydroxyketones can be used to produce an alternative series of pyrazines. Using different hydroxyketones as the carbon source for the reaction to produce pyrazines can not only minimize the formation of pyrazine and methylpyrazine, but also adjust the reaction so that other desirable substituted pyrazines are produced in a controlled manner.

[0090] For example, as shown in operation 102 of FIG. 1, an amino acid and / or an aldehyde can be optionally added to a reaction solution containing at least one hydroxyketone and a nitrogen source. As shown in Example 3 below, for example, the addition of an amino acid or a selected aldehyde can not only increase the number of synthetic pyrazines but also increase the yield of pyrazine. For example, in certain embodiments, the reaction solution can contain an amino acid selected from the group consisting of serine, alanine, leucine, isoleucine, valine, threonine, phenylalanine, and combinations thereof. Similarly, any alkyl aldehyde can be used to increase the number of synthetic pyrazines and the yield of pyrazine. For example, in certain embodiments, the reaction solution can contain an alkyl aldehyde selected from the group consisting of acetaldehyde, propanal, isopropanol, butanal, isobutanal, sec-butanol, and combinations thereof.

[0091] By extending the reaction time and increasing the temperature, the yield of pyrazine improves until the point at which a black tarry substance is produced. The reaction temperature can be, for example, about 30°C or higher, about 90°C or higher, about 100°C or higher, about 120°C or higher, or about 140°C or higher. In some embodiments, the reaction temperature can be about 90°C to about 150°C or about 120°C to about 140°C. The reaction time can be, for example, about 4 hours or longer, 8 hours or longer, about 12 hours or longer, 16 hours or longer, or about 24 hours or longer. In various embodiments, the reaction time can be about 4 to about 24 hours or about 12 to about 20 hours. In one embodiment, the reaction time can be about 16 hours.

[0092] In various embodiments, the molar ratio of hydroxyketone to nitrogen source can affect the yield of pyrazine. The molar ratio of hydroxyketone to nitrogen source (e.g., NH4OH) can be, for example, about 1:0.5, about 1:1, about 1:2, or about 1:2.5. In one embodiment, the ratio of hydroxyketone to nitrogen source can be about 1:0.5 to about 1:2.5 or about 1:1 to about 1:2. In one embodiment, the ratio of hydroxyketone to nitrogen source can be about 1:2.

[0093] An increase in the pH of the reaction solution may also cause an increase in the amount of pyrazine. The preferred pH range can 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 can be about 8.0 or higher, about 8.5 or higher, about 9.0 or higher, or about 10.0 or higher. For example, a small addition of NaOH or KOH can be used to increase the pH of the reaction solution.

[0094] Selective formation of pyrazine using sugars as carbon sources In various embodiments of the present invention, the selective formation of substituted pyrazines is optimized using at least one sugar (e.g., glucose, high fructose tobacco syrup (HFTS)) as a carbon source and ammonium ions, proteins, and / or amino acids as nitrogen sources. As discussed above, various reaction pathways to pyrazine-rich formulations have been previously used to produce pyrazine using sugar as a carbon source. For example, see U.S. Patent Application Publication No. 15 / 009,199 to Dube et al., filed January 28, 2016, which is hereby incorporated by reference in its entirety. However, when using sugar as the sole source in a reaction with a nitrogen source (e.g., ammonium hydroxide) to produce pyrazine, pyrazine molecules and methylpyrazine molecules may be the major pyrazines produced. This tendency is evident even when free amino acids are used as co-reagents. It has surprisingly been found that substituted pyrazines can be selectively produced by adjusting the pH of the carbon source before introducing the nitrogen source.

[0095] For example, as shown in FIG. 2, a heat - blended composition containing an amino acid and a sugar can produce pyrazine. For example, see U.S. Patent Application Publication No. 2010 / 0037903 to 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, pp135 - 182, CRC Press, Boca Raton, 2012, which are hereby incorporated by reference in their entirety. Solutions rich in pyrazine can be prepared in various ways. For example, one method can include microwave - heating treatment of a solution containing at least one amino acid and at least one sugar. As shown in operation 120 of FIG. 2, an aqueous reaction solution containing at least one amino acid and at least one sugar can be formed. As shown in operation 124 of FIG. 1, the reaction solution can be heated to the reaction temperature and held at the reaction temperature for a reaction time sufficient for the reactants to undergo a reaction to form pyrazine.

[0096] As discussed above, acetol, which is a hydroxyketone, can be produced by the decomposition of various sugars. Further, when used as a carbon source in a reaction with a nitrogen source, a series of substituted pyrazines that do not contain pyrazine molecules and methylpyrazine molecules can be produced. Therefore, acetol serves as an important intermediate in the reaction of sugars with free amino acids and / or ammonium ions and can produce pyrazines containing branched - alkyl side chains.

[0097] It has been shown by the literature that 1-hydroxy-2-propanone (acetol) can be produced from C6 sugars such as glucose and sorbitol, a derivative of glucose. 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 sugars 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, each of which is incorporated herein by reference in its entirety. These references describe the conversion of sugars to 1-hydroxy-2-propanone using phosphate buffer at high temperature, strong bases such as NaOH at pH 11.5 under reflux, Ni and palladium catalysts under hydrogen pressure, and copper chromite catalyst in the gas phase of heterogeneous reactions. Copper chromite has been considered the best catalyst. In most reactions, the conversion rate of the substrate exceeded 91%.When glycerol was used as the carbon source, the yield of acetol was 32.2% at 220 °C. When sorbitol was used as the carbon source, the highest yield was 11.8% at 280 °C, and when glucose was used as the carbon source, the highest yield of acetol was 8.99% at 280 °C.

[0098] Cellulose is converted to acetol in a yield of 30% using an 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 Groups 8 to 11, U.S. Patent Application Publication No. US2013 / 028174A1, 2013, which is incorporated herein by reference in its entirety.

[0099] In recent years, Novotny et al. (Czech J. Food Sci., 25, 119 - 130, 2007, incorporated herein by reference) synthesized α-hydroxycarbonyl compounds and α-dicarbonyl compounds via the decomposition of monosaccharides. They used three different models, each composed of an aqueous solution of potassium peroxodisulfate, an alkaline solution of potassium peroxodisulfate, and a solution of sodium hydroxide. Using GC / MS, a total of six α-hydroxycarbonyl compounds and six α-dicarbonyl compounds were identified. The maximum yields of α-dihydroxycarbonyls (glycolaldehyde, acetol, lactaldehyde, glyceraldehyde, 1,3-dihydroxyacetone, and acetoin) when glucose or fructose reacted with sodium hydroxide were approximately 4%. The yields were much lower in the aqueous solution of potassium peroxodisulfate (0.32%) and the alkaline solution of potassium peroxodisulfate (1.1%). Acetol and 1,3-dihydroxyacetone showed the highest yields (2.52% and 1.02%, respectively) when sodium hydroxide was used.

[0100] As shown in Example 6 below, α-hydroxyketones (e.g., acetol) can be produced from sugars and ultimately used as a carbon source for the sugar-ammonia reaction. Acetol can be separated by both distillation and column chromatography and then used as a carbon source in the reactions described herein. However, both methods are time-consuming (distillation) and expensive (chromatography). Therefore, it may be preferable to avoid the need to isolate hydroxyketones derived from sugar sources before using these hydroxyketones in reactions with nitrogen sources to produce pyrazines.

[0101] Surprisingly, it has been discovered that substituted pyrazines can be selectively formed from a sugar carbon source without first isolating the hydroxyketones (such as acetol, acetoin, etc.) formed from the decomposition of the sugar. For example, as shown in operation 110 of FIG. 2, by treating the sugar with a buffer before combining it with a nitrogen source, a series of pyrazines different from the series of pyrazines produced from the reaction of the sugar not pretreated with the buffer and the nitrogen source can be produced. The reaction can be optimized such that the maximum amount of acetol and acetol-like compounds is produced from the sugar carbon source. Without being limited by theory, the role of the buffer is to control the pH so that the maximum amount of hydroxyketones (such as acetol, acetoin, etc.) is produced from the decomposition of the sugar.

[0102] In some embodiments, the buffer can be a sodium hydrogen phosphate / sodium hydroxide buffer having a pH of about 12. In various embodiments, the buffer can include a potassium phosphate buffer having a pH of about 6.5 to about 7.5. In one embodiment, the buffer can include sodium carbonate, sodium sulfite, peroxodisulfate, sodium phosphate, or a combination thereof. The choice of buffer type, buffering capacity, pH, and reaction temperature can affect the synthesis of hydroxyketones from the sugar carbon source and thus can affect the series of pyrazines produced and the amount of pyrazines produced from the subsequent reaction with the nitrogen source.

[0103] In various embodiments, the buffer can buffer at a pH in the near-neutral or alkaline range, such as at a pH higher than about 6, higher than about 8, or higher than about 10 (such as about 6 to about 12). For example, in one embodiment, the pH of the sugar carbon source can be buffered to about 11 to about 12. In some embodiments, the pH of the sugar carbon source can be buffered to about 6.5 to about 7.5.

[0104] For example, glucose is a known carbon source for pyrazine production. When used as an intact molecule and reacted with ammonium hydroxide, a series of pyrazines are produced, including pyrazine molecules and methylpyrazine molecules as the main pyrazines, much smaller amounts of dimethylpyrazine molecules, and significantly smaller amounts of high molecular weight pyrazine molecules. When glucose is pre-reacted with NaOH at pH 12 and then reacted with ammonium hydroxide, a very similar series of pyrazines is produced. However, it was surprisingly discovered that when glucose is treated with a potassium phosphate buffer at pH 6.5 and subsequently reacted with ammonium hydroxide, only dimethylpyrazine and high molecular weight pyrazine are produced, thereby excluding the production of less desirable pyrazine molecules and methylpyrazine molecules.

[0105] By extending the reaction time and increasing the temperature of the reaction between the buffered sugar carbon source and the nitrogen source, the yield of pyrazine can be increased until a black tarry substance is produced. The reaction temperature can be, for example, about 30 °C or higher, about 90 °C or higher, about 100 °C or higher, about 120 °C or higher, or about 140 °C or higher. In some embodiments, the reaction temperature can be about 90 °C to about 150 °C or about 120 °C to about 140 °C. The reaction time can be, for example, about 30 minutes or longer, about 60 minutes or longer, about 90 minutes or longer, or about 120 minutes or longer. In various embodiments, the reaction time can be about 30 minutes to about 150 minutes or about 60 minutes to about 120 minutes.

[0106] An increase in the pH of the reaction solution may also result in an increase in the amount of pyrazine. The preferred pH range can 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 can be about 8.0 or higher, about 8.5 or higher, about 9.0 or higher, or about 10.0 or higher. For example, a small addition of NaOH or KOH can be used to increase the pH of the reaction solution.

[0107] In various embodiments of the present invention, for example, as shown in operation 122 of FIG. 2, by adding NH4OH to the amino acid / sugar reaction solution, the yield of pyrazine can be improved. The molar ratio of NH4OH / sugar can have a dramatic impact on the yield of pyrazine. For example, a molar ratio of sugar to NH4OH of 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), along with subsequent heat treatment, can produce a formulation rich in pyrazine. In some embodiments, an aqueous NH4OH solution can be slowly added to the amino acid / sugar solution during the reaction.

[0108] Different sugars and amino acids affect the types of pyrazines formed. For example, see 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, which is hereby incorporated by reference in its entirety. For example, leucine and valine produce more branched pyrazines with highly substituted side chains and lower odor thresholds. Highly substituted pyrazines may be desirable in some applications because they are relatively more potent than less branched pyrazines. The substitution of pyrazine can be the result of the amino acids used in the reaction. Therefore, it may be advantageous to select amino acids with branched and highly substituted side chains. Regarding sugars, rhamnose can be an ideal sugar for pyrazine formation, followed by fructose and glucose.

[0109] For example, as shown in operation 126 of FIG. 2, after the reaction, optionally, simple distillation, rotary evaporation or other separation techniques known in the art can be used to isolate pyrazine from the reaction product. In certain embodiments, rotary evaporation can be a preferred isolation technique in a scale - up process for inducing tobacco - derived pyrazine.

[0110] Use of substituted pyrazines in tobacco products As described above, the pyrazine produced according to the present invention may be useful, for example, as a component (such as a flavorant) contained in a tobacco product. The tobacco products to which the materials of the present invention are added are diverse and can include any product configured or adapted to deliver tobacco or certain components thereof to a product user. Exemplary tobacco products include smoking articles (such as cigarettes), smokeless tobacco products, and aerosol-generating devices containing tobacco materials or other plant materials that do not burn during use.

[0111] In various embodiments of the present invention, the pyrazine can be incorporated into a smoking article in the form of a flavorant in a tobacco composition and / or a filter element of a smoking article. For example, the pyrazine can be incorporated into a top dressing or casing of a tobacco product. Referring to FIG. 6, a smoking article 10 is shown having a representative configuration of a smoking article in the form of a cigarette and capable of containing a product derived from the cellulose-based sugar material of the present invention. The cigarette 10 includes a substantially cylindrical rod 12 of a charge or roll of a smokable filler (such as a smokable filler of about 0.3 to about 1.0 g of tobacco material, etc.) contained in a surrounding packaging material 16. The rod 12 is conventionally referred to as a "tobacco rod". The ends of the tobacco rod 12 are open and the smokable filler is exposed. The cigarette 10 has an optional band 22 (such as a printed coating containing a film-forming agent such as starch, ethyl cellulose, or sodium alginate) applied to the packaging material 16, and this band surrounds the cigarette rod in a direction transverse to the longitudinal axis of the cigarette. The band 22 can be printed on the inner surface of the packaging material (i.e., facing the smokable filler) or, although less preferably, on the outer surface of the packaging material.

[0112] One end of the tobacco rod 12 has a lighting end 18, and a filter element 26 is arranged at the mouth end 20. The filter element 26 is arranged adjacent to one end of the tobacco rod 12 such that the filter element and the ends of the tobacco rod are axially aligned, preferably in contact with each other. The filter element 26 may generally have a cylindrical shape, and its diameter may be essentially equivalent to the diameter of the tobacco rod. The ends of the filter element 26 can allow air and smoke to pass through the filter element. The plug wrap 28 wraps the filter element, and the tip material (not shown) wraps the plug wrap and a part of the outer wrapper 16 of the rod 12, thereby fixing the rod to the filter element 26.

[0113] The filter element of the present invention typically comprises a plurality of longitudinally extending segments. Each segment can have various properties and can include various materials capable of filtering or adsorbing particulate matter and / or gas-phase compounds. Typically, the filter element of the present invention includes 2 to 6 segments, often 2 to 4 segments. In a preferred embodiment, the filter element includes a mouth-end segment, a tobacco-end segment, and a compartment therebetween. This filter arrangement may be referred to as a "compartment filter" or a "plug / space / plug" filter. As will be described in more detail below, the compartment can be divided into two or more compartments.

[0114] In various embodiments, the filter element can comprise an adsorbent in the form of an activated carbon material, and the activated carbon can remove at least one gas-phase component of the mainstream smoke taken into the filter element. In one embodiment, the filter element 26 can include ventilation holes 30 extending through the tip paper (not shown) and the plug wrap 28, so that the mainstream smoke is diluted with air. The ventilation holes 30 can be configured as a row of perforations extending circumferentially around the filter element 26, or can comprise multiple rows of perforation rows. As will be understood, the exact number and size of the ventilation holes 30 will vary depending on the desired level of air dilution.

[0115] In various embodiments of the present invention, the pyrazines obtained by the methods disclosed herein can be incorporated into smokeless tobacco products in the form of flavorants in smokeless tobacco formulations. The form of the smokeless tobacco products of the present invention can be diverse. In a particular embodiment, the product is in the form of a snus-type product containing particulate tobacco material and a flavorant containing pyrazines obtained by the method of the present invention. The manner and method of formulating the snus-type tobacco formulation will be apparent to those skilled in the manufacture of snus tobacco products. For example, as shown in FIG. 7, an exemplary pouch product 300 can include an outer permeable container 320 in the form of a pouch containing a particulate mixture 315 adapted for oral use. The orientation, size, and type of the outer permeable pouch shown herein, as well as the type and nature of the composition adapted for oral use, are not to be construed as limiting.

[0116] In various embodiments, the moisture-permeable packet or pouch can function as a container for use of the composition internally. The composition / structure of the packet or pouch, such as the container pouch 320 in the embodiment shown in FIG. 7, can be varied as described herein. For example, suitable packets, pouches, or containers of the type used in the manufacture of smokeless tobacco products that can be modified in accordance with the present invention are available under the trade names CatchDry, Ettan, General, Granit, Goteborgs Rape, Grovsnus White, Metropol Kaktus, Mocca Anis, Mocca Mint, Mocca Wintergreen, Kicks, Probe, Prince, Skruf, and TreAnkrare. Pouch-type products having various embodiments, shapes, and forms similar to those of the pouch products described herein are commercially available as ZONNIC (sold by Niconovum AB). Further, pouch-type products having generally similar shapes and forms to various embodiments of the pouch products are described as snap bag compositions E-J in Example 1 of PCT WO2007 / 104573 of Axelsson et al., which is incorporated herein by reference and which are manufactured using excipient components and processing conditions that can be used in the manufacture of the pouch products described herein.

[0117] The amount of material contained in each pouch can vary. In more small-scale embodiments, the dry weight of the material in each pouch is at least about 50 mg to about 150 mg. In more large-scale embodiments, the dry weight of the material in each pouch preferably does not exceed about 300 mg to about 500 mg.

[0118] In some embodiments, each pouch / container can have a flavorant member disposed therein, as described in more detail by U.S. Patent No. 7,861,728 to Holton, Jr. et al., which is incorporated herein by reference. The flavorant member can include a flavoring agent that includes pyrazines derived by the method of the present invention, as described above. Optionally, other components can be included in each pouch. For example, at least one flavored strip, piece, or sheet of a flavored water-dispersible or water-soluble material (e.g., an oral malodor reducing edible film-type material) can be disposed within each pouch, with or without at least one capsule. Such strips or sheets can be folded or crumpled for easy incorporation into the pouch. See, for example, the types of materials and techniques described in U.S. Patent No. 6,887,307 to Scott et al., U.S. Patent No. 6,923,981 to Leung et al., and The EFSA Journal (2004) 85,1-32, which are incorporated herein by reference.

[0119] In various embodiments, the outer permeable pouch can comprise PLA or other pouch materials known in the art. Descriptions of various components of the snus-type product and its components are also described in U.S. Patent Application No. 2004 / 0118422 to Lundin et al., which is incorporated herein by reference. See, for example, U.S. Patent No. 4,607,479 to Linden; U.S. Patent No. 4,631,899 to Nielsen; U.S. Patent No. 5,346,734 to Wydick et al.; and U.S. Patent No. 6,162,516 to Derr and U.S. Patent Application Publication No. 2005 / 0061339 to Hansson et al., each of which is incorporated herein by reference. See also the types of pouches described in U.S. Patent No. 5,167,244 to Kjerstad et al., which is incorporated herein by reference. The snus-type product can be manufactured using equipment available from Merz Verpackungmaschinen GmBH such as the SB 51-1 / T, SBL 50 and SB 53-2 / T. The snus pouch can be provided as an individual pouch or a plurality of pouches (e.g., 2, 4, 5, 10, 12, 15, 20, 25 or 30 pouches) can be joined or linked together (e.g., end to end) such that a single pouch or individual portions can be easily removed from the one-piece strand or matrix of the pouch for use.

[0120] The present invention is not limited to smooth-type smokeless tobacco products. For example, a mixture of tobacco materials and a flavorant containing at least one pyrazine obtained by the methods described herein can be incorporated into various smokeless tobacco forms, such as loose wet snuff, loose dry snuff, chewing tobacco, pelletized tobacco pieces, extruded tobacco strips or pieces, powdered pieces or micronized or comminuted aggregates of components, flake-like pieces (which can be formed, for example, by agglomerating tobacco formulation components in a fluidized bed), formed tobacco pieces (formed in common shapes such as coins, cylinders, beans, cubes, etc.), products containing a mixture of edible materials combined with tobacco pieces and / or tobacco extracts, products containing tobacco (in the form of, for example, a tobacco extract) supported on a solid non-edible substrate, etc. For example, the smokeless tobacco product can be in the form of compressed tobacco pellets, multi-layer extruded pieces, extruded or formed rods or sticks, compositions having one type of tobacco formulation surrounded by different types of tobacco formulations, rolls of tape-like films, readily water-soluble or water-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 (which can be, for example, transparent, colorless, translucent or essentially darkly colored, and can be a flexible or rigid outer shell) and an internal region having tobacco or the flavor of tobacco (such as a Newtonian fluid or a thixotropic fluid containing a certain form of tobacco).

[0121] In some embodiments, the smokeless tobacco product of the present invention can be in the form of lozenges, tablets, microtabs or other tableted products. For example, see the types of lozenge formulations and techniques for formulating or manufacturing lozenges described in Shaw U.S. Patent No. 4,967,773; Acharya U.S. Patent No. 5,110,605; Dam U.S. Patent No. 5,733,574; Santus U.S. Patent No. 6,280,761; Andersson et al. U.S. Patent No. 6,676,959; Wilhelmsen et al. U.S. Patent No. 6,248,760; and U.S. Patent No. 7,374,779; Wilhelmsen U.S. Patent Application Publication No. 2001 / 0016593; Liu et al. U.S. Patent Application Publication No. 2004 / 0101543; Mcneight et al. U.S. Patent Application Publication No. 2006 / 0120974; Chau et al. U.S. Patent Application Publication No. 2008 / 0020050; Gin et al. U.S. Patent Application Publication No. 2009 / 0081291; and Axelsson et al. U.S. Patent Application Publication No. 2010 / 0004294, which are incorporated herein by reference.

[0122] Depending on the type of smokeless tobacco product being processed, the tobacco product can include one or more additional components in addition to the tobacco material and a flavorant comprising at least one pyrazine derived from the method of the present invention. For example, the tobacco material and tobacco-derived flavorants can be processed, blended, formulated, combined and / or mixed with other materials or ingredients, such as other tobacco materials or flavorants, fillers, binders, pH adjusters, buffers, salts, sweeteners, colorants, disintegrants, humectants and preservatives (any of which may be encapsulated components). For example, see their representative components, combinations of components, the relative amounts of these components and ingredients with respect to tobacco, and methods of using these components described in Mua et al. U.S. Patent Application Publication No. 2011 / 0315154; Holton, Jr. et al. U.S. Patent Application Publication No. 2007 / 0062549; and Holton, Jr. et al. U.S. Patent No. 7,861,728, each of which is incorporated herein by reference.

[0123] In various embodiments, at least one pyrazine derived from the methods described herein can be incorporated into a smokeless tobacco product in the form of an e-cigarette flavorant. A number of smoking products, flavor generators, and medical inhalers have been proposed that utilize electrical energy to volatilize or heat a volatile substance, or to provide the sensation of smoking a cigarette, cigar, or pipe without burning the tobacco to a significant extent. See, for example, the background art described in U.S. Patent No. 7,726,320 to Robinson et al., U.S. Patent Application Publication No. 2013 / 0255702 to Griffith Jr. et al., U.S. Patent Application Publication No. 2014 / 0000638 to Sebastian et al., U.S. Patent Application Publication No. 2014 / 0060554 to Collett et al., U.S. Patent Application Publication No. 2014 / 0096781 to Sears et al., U.S. Patent Application Publication No. 2014 / 0096782 to Ampolini et al., and U.S. Patent Application Publication No. 2015 / 0059780 to Davis et al., each of which is incorporated herein by reference in its entirety. Also refer to various alternative smoking products, aerosol delivery devices, and heat sources described in the background art therein.

[0124] An exemplary embodiment of the electronic smoking article 200 is shown in FIG. 8. As shown in FIG. 8, the control body 202 can be formed of a control body shell 201 that can include a control component 206, a flow sensor 208, a battery 210, and an LED 212. The cartridge 204 can be formed of a cartridge shell 203 that surrounds a reservoir housing 244, and the reservoir housing 244 is in fluid communication with a liquid transport element 236 suitable for sucking or otherwise transferring the aerosol precursor composition stored in the reservoir housing 244 to the heater 234. An opening 228 may be present in the cartridge shell 203 for discharging the aerosol formed from the cartridge 204. Such components are representative of the components that may be present within the cartridge and are not intended to limit the scope of the cartridge components included by the present disclosure. The cartridge 204 can be adapted to engage with the control body 202 by a press-fit engagement between a control body projection 224 and a cartridge receptacle 240. Such engagement can facilitate a stable connection between the control body 202 and the cartridge 204 and can establish an electrical connection between the battery 210 and the control component 206 within the control body and the heater 234 within the cartridge. The cartridge 204 can also include one or more electronic components 250 that can include an IC, a memory component, a sensor, and the like. The electronic component 250 can be adapted for communication with the control component 206. The various components of the electronic smoking device according to the present disclosure can be selected from components described and commercially available in the art.

[0125] In various embodiments, the aerosol precursor composition can include a flavorant comprising at least one pyrazine derived according to the method of the present invention. Exemplary formulations of aerosol precursor materials that can be used according to the present disclosure are described in U.S. Patent No. 7,217,320 to Robinson et al.; U.S. Patent Application Publication No. 2013 / 0008457 to Zheng et al.; U.S. Patent Application Publication No. 2013 / 0213417 to Chong et al.; U.S. Patent Application Publication No. 2014 / 0060554 to Collett et al.; and U.S. Patent Application Publication No. 2014 / 0000638 to Sebastian et al., the disclosures of which are hereby incorporated by reference in their entireties. Other aerosol precursors that can include the tobacco-derived pyrazines described herein include the aerosol precursors incorporated in VUSE(R) by R.J. Reynolds Vapor Company, BLU(TM) products by Imperial Tobacco, MISTIC MENTHOL products by Mistic Ecigs, and VYPE products by CN Creative Ltd. Also desirable are so-called "smoke juices" for electronic cigarettes available from Johnson Creek Enterprises LLC.

[0126] Experiment Aspects of the present invention are more fully illustrated by the following examples, which are set forth to illustrate certain aspects of the present invention and should not be construed as limiting thereof.

Examples

[0127] [Example 1] Pyrazine is produced in the reaction with ammonia using acetoin (3-hydroxy-2-butanone) as a carbon source instead of sugar.

[0128] Acetoin, ammonium hydroxide (28 - 30%), leucine, dichloromethane, and phosphoric acid (H3PO4) are obtained from Sigma - Aldrich (St. Louis, Missouri). The F1 protein is obtained from R.J. Reynolds Tobacco Co. (Winston - Salem, North Carolina) and hydrolyzed. The weight percentage of hydrolyzed amino acids in all hydrolyzed solutions ranges from 50 - 55%. All pyrazine synthesis reactions are carried out in a 40 mL Parr vessel. In each reaction, 0.8 grams of acetoin is mixed with 1.8 mL of NH4OH and 0.6 mL of H3PO4, and then sufficient hydrolyzed F1 protein (20 mL) is added to make the mass of amino acids 0.4 grams. For example, when 40 grams of F1 protein is hydrolyzed in 1 liter of solution, the weight percentage of amino acids in the solution is equal to 50%, which corresponds to 20 grams of amino acids in 1 liter of solution. To use 0.4 grams of amino acids in the reaction, 20 mL of the above solution is added to the reaction vessel. In some reactions, instead of hydrolyzed F1 protein, leucine is used as the amino acid source and only 20 mL of water is added to adjust the volume. After adjusting the pH of all solutions to 8.0, the reaction is started. After completion of each reaction, the mixture is extracted with 30 mL of dichloromethane (DCM). Next, 200 μL of the DCM extract is diluted to 1 mL using DCM and analyzed by GC / MS.

[0129] All GC / MS analyses are performed using a 6890 GC equipped with an Agilent (Wilmington, Delaware) 5973 mass - selective detector (MSD). The separations are obtained using a J&W (Wilmington, Delaware) DB - WAXTER capillary column (length 30 m × inner diameter 250 μm, film thickness 0.25 μm). The following operating parameters are used for each analysis.

[0130] Injector port temperature 260 °C Purge valve 3 mL / min Purge time 1 min Total flow 24 mL / min Constant flow 1 mL / min Injection volume: 2 μL, split 1:20 Initial column oven temperature: 50 °C Initial time of column oven: 3 minutes Column oven ramp rate: 15 °C / min Final column oven temperature: 250 °C Final time of column oven: 1 minute MSD transfer line temperature: 260 °C Each pyrazine is identified using the MS Wiley library.

[0131] In the first reaction, acetoin (0.8 grams) is reacted with NH4OH (1.8 mL) and H3PO4 (0.6 mL) at pH = 8 for 12 - 15 hours at 90 °C. More than 90% of the acetoin is converted to tetramethylpyrazine (TMP). Next, the reaction is repeated using the same conditions, but instead of heating at 90 °C for 12 - 15 hours, it is heated at 120 °C for only 4 hours. The results are the same as those obtained at 90 °C and 12 - 15 hours.

[0132] Next, to determine the possibility of synthesizing branched pyrazine, an amino acid (leucine) is added to the reaction reagent mixture. For this purpose, 0.8 grams of acetoin + 1.8 mL of NH4OH + 0.6 mL of H3PO4 and 0.25 grams of leucine are mixed with 20 mL of H2O, and the pH is adjusted to 8. Then the reaction mixture is heated at 120 °C for 18 hours, and then the reaction mixture is extracted with 30 mL of DCM and analyzed by GC / MS. Only TMP (t R = 8.2 minutes) and some acetoin (t R = 6.1 minutes) are detected. Branched pyrazine is not detected.

[0133] Next, a similar reaction is carried out, but instead of leucine and H2O, 20 mL of hydrolyzed F1 protein is used in the reaction. The reaction is carried out at 120 °C for 18 hours in a Parr container. After cooling the reaction mixture, 30 mL of DCM is used to extract pyrazine. Again, no branched pyrazine is observed, and only TMP is observed.

[0134] To determine whether ammonia consumes all of the acetoin and thus prevents the amino acids from reacting with acetoin, another base source (NaOH) is used instead of NH4OH to maintain basic reaction conditions with pH > 8. For this purpose, two reactions are carried out. In the first reaction, 0.8 of acetoin is mixed with 0.25 grams of leucine and 20 mL of 0.1N NaOH (pH = 12), and in the second reaction, H3PO4 is used to adjust the pH to 8.2. Both reactants are heated at 120 °C for 8 hours using a Parr vessel. After cooling the reactants, the reactants are extracted with DCM and analyzed by GC / MS. Even for TMP, no pyrazine is detected.

[0135] The above reaction shows that the addition of amino acids to the reaction of acetoin and NH4OH does not produce branched pyrazines. Furthermore, it can be seen that acetoin is not an important intermediate in the reaction of sugars and free amino acids to produce pyrazines containing branched alkyl side chains.

[0136] [Example 2] To selectively produce pyrazines other than TMP, a hydroxyketone carbon source other than acetoin is utilized.

[0137] 1-OH-acetone, 1-OH-2-butanone, ammonium hydroxide (28 - 30%), phosphoric acid (H3PO4), isoleucine, threonine and isovaleraldehyde are obtained from Sigma-Aldrich (St. Louis, Missouri). The F1 protein derived from Nicotiana plants is obtained from R.J. Reynolds Tobacco Co. (Winston-Salem, North Carolina) and hydrolyzed to form amino acids. The weight percentage of hydrolyzed amino acids in all hydrolyzed solutions ranges from 50 - 55%.

[0138] All pyrazine synthesis reactions are carried out in a 40 mL Parr vessel. In each reaction, 1 gram of 1-OH-acetone or 1-OH-2-butanone is mixed with 0.25, 0.5, 1, and 1.25 mL of NH4OH and 10 mL of H2O. Each reaction is mixed and heated at different temperatures (100 - 140 °C) for 4 - 24 hours. For most of the reactions, the pH level is about 11 (no adjustment is made). However, for the reaction where the pH level is adjusted to 8, concentrated H3PO4 is used to lower the pH.

[0139] After completion of each reaction, the mixture is extracted with 20 - 25 mL of dichloromethane. In each extraction, 250 μg of deuterated 2-methylpyrazine is used as the internal standard for all quantifications. For all reactions, a magnetic stirrer is used to stir the mixture during the reaction process.

[0140] All GC / MS analyses are performed using the same equipment and operating parameters as those used in Example 1 above. The MS Wiley library is used to identify each pyrazine. For quantitative analysis, the pyrazine is quantified using the single ion monitoring mode. Each pyrazine is quantified relative to the mass of the internal standard (250 μg) added to the extraction solvent.

[0141] First, 1-hydroxyacetone is reacted with NH4OH at different ratios, temperatures, pH levels, and reaction times to maximize the percent yield of pyrazine. Pyrazines detected in the reaction of 1-OH-acetone with NH4OH include 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.

[0142] In the first part of this study, reactions are carried out at two different pH levels (8.0 and 11.0) to determine which pH level gives the maximum yield and maximum number of pyrazines. In the first reaction, 1 mL of 1-OH-acetone is reacted with 0.5 mL of NH4OH and 10 mL of H2O. The pH level of this reaction is measured to be approximately 11.0. In the second reaction, the same amounts of reactants are mixed, but concentrated H3PO4 is used to adjust the pH level to 8.0. Both reactants are heated at 120 °C for 12 hours. The percent yield of pyrazine is higher when the pH level is approximately 11.0. Therefore, the pH level of subsequent experiments is not adjusted and the reaction is carried out at pH 11 or higher.

[0143] The effect of temperature (100, 110, 120, 130, 140 °C) on the synthesis of pyrazine using 1-OH-acetone (1 g) and NH4OH (1 g) at a C:N molar ratio of 1:2 in 10 mL of H2O (reaction time 12 h) is described below. As the temperature increases, the yield of pyrazine increases, and even when the temperature reaches 140 °C, an increase in pyrazine yield is still observed.

[0144] Tests are conducted on the effect of various reaction times (4, 8, 12, 16, and 24 h) on the synthesis of pyrazine using 1-OH-acetone (1 g) and NH4OH (1 g) at a C:N molar ratio of 1:2 in 10 mL of H2O. When the reaction time is 16 h, the maximum yield of pyrazine is obtained.

[0145] The effect of varying the 1-OH-acetone:NH4OH molar ratio (1:0.5, 1:1, 1:2, 1:2.5,... 1-OH-acetone and NH4OH) reaction in 10 mL of H2O on the yield of pyrazine after 12 h at 120 °C is tested. The optimal ratio is 1:2, corresponding to 1 gram of 1-OH acetone and 1 mL of NH4OH. Using a larger amount of NH4OH in the reaction causes the yield of the reaction to decrease by more than 10%.

[0146] In summary, when the reaction conditions (temperature, time, C:N ratio, pH) are optimized, the amount of pyrazine is maximized. The results show that at least 19 - 20 types of pyrazines are synthesized using hydroxyacetone as the sole carbon source under the optimized conditions (C:N = 1:2, temperature = 120 °C, reaction time = 16 h, pH = 11 - 12). The absence of detectable amounts of pyrazine molecules and / or methylpyrazine molecules from the synthesized pyrazine supports the discovery that the carbon source (i.e., α,β-hydroxyketone) affects the structure of the pyrazine produced from the reaction of the carbon source and the nitrogen source.

[0147] [Example 3] Measure the effect of the addition of amino acids and aldehydes to the individual reactions of 1-OH-acetone and NH4OH using the parameters of Example 2 above, with a C:N ratio of 1:2, mixed with 10 mL of H2O at 120°C for 12 hours.

[0148] Test two different amino acids as additional nitrogen sources. In each reaction, 0.2 grams of amino acid is added individually to each reaction, so the effect on pyrazine synthesis and its yield of the additional amino acid can be examined. In another reaction, isovaleraldehyde is added to the optimized reaction to examine the effect on pyrazine synthesis and yield. When using hydrolyzed F1 protein as an additional nitrogen source, 10 mL of hydrolyzed F1 protein (containing about 0.2 grams of another amino acid) is used. In this reaction, since the hydrolyzed F1 protein is contained in 10 mL of H2O, no H2O is added.

[0149] When using isoleucine as a possible additional nitrogen source, it has been observed that the concentrations of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl)pyrazine increase. In another reaction, when using threonine as an additional nitrogen source, the concentrations of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl)pyrazine increase, and the yield of 2,6-dimethyl-3-(2-methylbutyl)pyrazine increases. It is interesting to note that when threonine or isoleucine is added to the reaction, the total yield of pyrazine is similar. Both compounds increase the total yield of pyrazine by more than 7%.

[0150] When isovaleraldehyde is added to another reaction of 1-OH-acetone and NH4OH, the percent yields of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl)pyrazine increase from 0 mg to 14 mg and 13 mg respectively. The total yield of pyrazine increases by more than 20%.

[0151] Instead of pure amino acids, a mixture of amino acids prepared from the hydrolysis of F1 protein is used in the reaction. Since the hydrolyzed F1 protein is already in an aqueous solution, no water is added to the mixture. For this purpose, 10 mL of hydrolyzed F1 protein containing about 0.2 g of amino acids and 10 mL of H2O is reacted with -1-OH-acetone and NH4OH with a C:N ratio of 1:2 at 120 °C for 16 hours. The yield of 2,5-dimethylpyrazine rises above 80%, and the yields of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl)pyrazine rise from 0 mg to above 1 mg. Without being limited by theory, the alkyl part of the amino acid is converted to a Strecker aldehyde, which reacts with ammonium hydroxide to form an imine, which is then incorporated into the pyrazine structure.

[0152] Tests are conducted on the effects of different temperatures and C:N ratios (1:1 and 1:2) on the synthesis of pyrazine using 1-OH-acetone and NH4OH in the presence of additional amino acid / aldehyde. In these studies, increasing the temperature from 100 to 120 °C and increasing the C:N ratio result in an increase in the yield of pyrazine.

[0153] In summary, adding amino acids, selected aldehydes, or hydrolyzed F1 protein not only increases the percent yield of certain pyrazines but also increases the number of synthetic pyrazines.

[0154] [Example 4] Pyrazine is synthesized according to Examples 2 and 3 above, except that 1-OH-2-butanone is used as the carbon source instead of 1-OH-acetone.

[0155] For this purpose, 1 gram of 1-OH-2-butanone is reacted with 1 mL of NH4OH and 10 mL of H2O at 120 °C for 16 hours. Methylpyrazine is not formed. All of the pyrazines formed contain ethyl or more branched alkanes. However, the yield of pyrazine is not as high as when 1-OH-acetone is used. Pyrazines synthesized from the reaction using 1-OH-2-butanone and NH4OH with a C:N ratio of 1:2 over 16 hours at 120 °C 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 (dimethylpryazine); trans-3-M-2,n-propyl-6(1-butenyl)pyrazine; 2,5-dimethyl-3-ethylpyrazine.

[0156] As described above, when 1-OH-acetone is used as the carbon source, pyrazine molecules and methylpyrazine molecules are not generated. When 1-OH-2-butanone is used, pyrazine molecules, methylpyrazine, and dimethylpyrazine are not generated, confirming that the carbon source overwhelmingly dominates the pyrazine structure. The results further show that by changing the carbon source from 1-OH-acetone to 1-OH-2-butanone, the types of pyrazines synthesized can be controlled.

[0157] [Example 5] The reaction of 1-OH-acetone and NH4OH according to Example 2 above is carried out on a larger scale using a larger Parr reactor with a larger reaction volume.

[0158] In a 1.5-liter Parr high-pressure vessel, 100 grams of 1-OH-acetone is reacted with 100 mL of NH4OH and 1000 mL of H2O at 120 °C for 16 hours. After completion of the reaction, the mixture is cooled and transferred to a glass bottle.

[0159] It should be noted that there is a considerable amount of tarry substance that dissolves only in MeOH at the bottom of the container. By adding H2O on top of this material, the material hardens. It is found that the concentration plays an important role in the presence or absence of the tar-like substance. In all optimization studies, the amount of tar at the bottom of the reaction vessel is small. Therefore, a small amount of methanol (1 mL) is sufficient to dissolve everything and include the remaining reactants. In the case of large-scale reactions, the mass of the tar is larger and at least 100 - 200 mL of methanol is required to dissolve it.

[0160] After completion of the reaction, the aqueous solution is distilled at 130 - 140 °C (3 × 375 mL). At each distillation (375 mL), approximately 175 mL of an aqueous solution containing various pyrazines (light yellow - total amount approximately 500 mL) is collected. Next, to remove pyrazine from water, the distilled materials (3 × 175 mL) are combined and passed through a C 18 column (15 × 2.5 cm packed with SPE material). C 18 After removing water from the column, the trapped pyrazine is eluted using ethanol. Next, ethanol is removed using rotary evaporation and vacuum. Since there is some water in the final product, pyrazine is extracted into MTBE and dried over sodium sulfate. Next, MTBE is removed using a rotary evaporator and vacuum. The vial labeled Solution 1 contains most of the pyrazine after MTBE removal.

[0161] It is important to note here that three pyrazines, due to their high boiling points, remain in the reaction product without being distilled. This results in a decrease in yield. These pyrazines are identified as 2-(2-methylpropyl)3,5-dimethylpyrazine (12.57 minutes), 2,6-dimethyl-3-isobutylpyrazine (12.74 minutes), and 2(2-methylpropyl)3,5,6-trimethylpyrazine (12.95 minutes).

[0162] Use DCM (200 - 250 mL) to extract the remaining three pyrazines from the reaction solution after distillation. Next, use a rotary evaporator and vacuum to remove the DCM from the solution, while transferring the concentrated dark - colored solution to a second vial later and labeling it as solution 2. Figure 3 shows the chromatogram of this sample.

[0163] The yield of total pyrazines by distillation of 1200 mL of reaction solution (100 grams of 1 - OH - acetone+100 mL of NH4OH) is about 60% compared to 12 mL of reactants (1 gram of 1 - OH - acetone + 1 mL of NH4OH). This yield does not include the three pyrazines remaining in the reaction mixture after distillation.

[0164] The above solution 1 is used for gas chromatography-olfactometry (GCO) evaluation. The pyrazine samples are analyzed using an ODP3 equipped with an Agilent 7890A series GC with a 5975C MSD and a Gerstel multipurpose sampler with SPME function. An instrumental method for the samples is developed to obtain better separation of pyrazine and an analysis time suitable for olfactory analysts. The transfer line of the ODP3 is heated to 260 °C. Two drops of the sample are pipetted into a 20 mL SPME screw-cap vial to prepare the sample. An empty vial is analyzed before and after the sample. The pyrazine is separated by the gas chromatography column, and when these come out of the column, a subjective olfactory test is used for humans to detect / evaluate the pyrazine.As the identified pyrazines, there are 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-dimethylpyrazine isomer 1; 5H-cyclopentapyrazine, 6,7-dihydro-2,5-dimethylpyrazine 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-propenylpyrazine (Z)-isomer 6; trimethyl-1-propenylpyrazine (E)-isomer 1; trimethyl-2-propenylpyrazine; trimethyl-1-propenylpyrazine (E)-isomer 2. It should be noted that methylpyrazine compounds exist, but in an amount less than that which is typical in conventional sugar carbon source reactions.

[0165] Four flavor analysts evaluate the individual olfactory characteristics from the ODP portal in four separate sessions. The individual pyrazines based on the four independent evaluations are extremely positive. Descriptors predicted for the aroma of the substituted pyrazines are found. Nutty, roasted, toasty, chocolate, peanut, musty, brown, complex are descriptors that are frequently used. These are all positive aroma characteristics.

[0166] [Example 6] α-Hydroxy ketone (acetol) is produced from sugar and is ultimately used as a carbon source for the sugar ammonia reaction. Sodium hydroxide is used as a base to optimize various parameters such as sugar type, temperature, reaction time, pH, and base concentration to maximize the yield of acetol.

[0167] Glucose, fructose, 1-OH-acetone (acetol), 1-OH-2-butanone (acetoin), sodium hydroxide, sodium chloride, sodium sulfate anhydrous, hydrochloric acid, methanol, and dichloromethane are obtained from Sigma-Aldrich (St. Louis, Missouri). The synthetic reaction is carried out in a 40 mL Parr container or an open round-bottom flask. In all reactions where the pH is controlled, the reaction is carried out under reflux at 100 °C in a round-bottom flask. In each reaction, different sugars (glucose, fructose, or a mixture of both), 0.25 grams, 0.5 grams, or 1.0 grams are mixed with 25 mL of 0.025 M, 0.05 M, 0.1 M, 0.2 M NaOH. Each reactant is stirred and heated at different temperatures (90 - 140 °C) for 1 - 12 hours. The initial pH level of most reactions is about 12 and no adjustment is made during the reaction. However, for reactions where the pH level is adjusted to 9, hydrochloric acid is used. For reactions where the pH is kept constant at 12 during the process, 10 - 40 μL of 10 M NaOH is used. After completion of each reaction, the mixture is cooled and the pH is adjusted to 6.0 - 6.5 using 1 M HCl. Next, 1 mg of acetoin is added to the reaction mixture as an internal standard. The mixture is extracted 4 times with 8 - 10 mL of dichloromethane. All four extraction solvents are combined and dehydrated with sodium sulfate.

[0168] All GC / MS analyses are performed using the same equipment and operating parameters as those used in Example 1 above. The peaks are identified using the MS Wiley library. For quantitative analysis, a calibration curve is created using the concentration of acetol and the response coefficient ratio of acetol / acetoin. It is important to note that no acetoin is detected in any of the reactions carried out here at 90 °C, 100 °C and 120 °C. However, acetoin residues are found in the reaction where the temperature is set at 140 °C. For this purpose, the acetoin concentration in the reaction is determined and this value is taken into account in all calculations.

[0169] A calibration curve for the quantification of acetol in all reactions is created using GC / FID. Next, the following parameters are varied to determine the optimal conditions for the synthesis of acetol.

[0170] First, two different types of sugars (glucose, fructose and a mixture of both) are used to determine the sugar that gives a higher yield of acetol. In each reaction, 0.5 grams of sugar is mixed with 25 mL of 0.05 M NaOH. Each reactant is heated at 100 °C for 60 minutes. Table 1 below shows the reaction conditions and the corresponding number of mg of acetol obtained from each reaction. When glucose is used in the reaction, it is observed that a large amount of acetol is produced by glucose.

[0171]

Table 1

[0172] In this part of the study, since it is necessary to adjust the pH during the reaction process, all reactions are carried out under reflux in an open round-bottom flask rather than in a closed Parr reactor. Three different experiments are conducted. In the first experiment, the pH is measured every 10 minutes and no pH adjustment is made. In the second experiment, the pH is measured every 10 minutes and, if necessary, 10M NaOH is used to adjust the pH to the initial value (12.0). In the third experiment, the initial pH is adjusted to 9.0, then the reaction is started, and pH measurements are taken every 10 minutes (without adjustment).

[0173] The results of the pH measurements show that the pH drops from 12.0 to approximately 9 within the first 10 minutes of the reaction at 100°C. At the end of 30 minutes, the pH of the reaction is approximately 7 - 8 and remains constant for the remaining reaction time. In the experiment where the pH is maintained at approximately 12.0, it is necessary to add approximately 15 - 20 μL of 10M NaOH to the reaction every 10 minutes. After 40 minutes, the pH remains approximately 11 - 12 and constant.

[0174] Table 2 below shows the reaction conditions and the number of milligrams of acetol obtained from each reaction using various pH conditions. The maximum amount of acetol is obtained when the pH is maintained approximately constant at 12.

[0175]

Table 2

[0176] To determine the effect of sugar concentration on acetol production, three tests with different sugar concentrations are conducted. Thus, the reactions are carried out using 0.25 grams, 0.5 grams, and 1 gram of glucose in 25 mL of 0.05M NaOH solution. Using a Parr reactor, each reaction is heated at 100°C for 60 minutes. Table 3 below shows the results of this study. It is observed that the maximum amount of acetol is obtained when 0.25 grams of glucose is used in the reaction.

[0177] Without being limited to theory, it is considered that when the sugar concentration is high, the formation of acetol is minimized by acid formation during the reaction process. When the sugar concentration is low, it takes time for acid formation while the pH is high enough to cause the formation of acetol.

[0178]

Table 3

[0179] Four different concentrations of NaOH (0.2, 0.1, 0.05, and 0.025 M) are used for the preparation of acetol. Table 4 below shows the results of this study. As can be seen, the minimum amount of acetol is obtained when the NaOH concentration is 0.025 M. However, when the concentration increases from 0.025 M to 0.1 M, the mass of synthetic acetol also increases. When the base concentration increases to 0.2 M, the amount of acetol decreases. It should be noted that the reaction solution has the smell of burnt sugar compared to other reactions with lower base concentrations.

[0180]

Table 4

[0181] Investigate the effect of temperature on the synthesis of acetol using NaOH and glucose. For this purpose, various temperatures from 90 to 140 °C are being examined. The results at reaction temperatures of 90 °C, 100 °C, and 120 °C are similar, but in the reaction at 140 °C, about 25% more acetol is produced than in other reactions at lower temperatures. Table 5 below shows the results of this study.

[0182]

Table 5

[0183] The effect of reaction time on the synthesis of acetol is also examined. While varying the reaction time from 60 to 720 minutes, the reaction is carried out using the same conditions. Table 6 below shows the results of this examination. The results indicate that there is no significant change in the yield of acetol with the extension of the reaction time.

[0184]

Table 6

[0185] In the last part of this examination, based on the results obtained from previous reactions, various reactions are carried out using optimized conditions. In Reaction A, while maintaining the pH at approximately 11 - 12 constant, 1 gram of glucose is reacted with 0.05 M NaOH under reflux at 100 °C. This reaction continues for 120 minutes until the pH no longer changes while remaining constant. The yield of acetol is 6.7 mg. In Reaction B, a Parr vessel is used at 100 °C, and 0.5 gram of HFTS is reacted with 25 mL of 0.05 M NaOH for 60 minutes. The yield of acetol is 3.42 mg. When the same reaction is carried out under optimized conditions (0.1 M NaOH and 140 °C, 60 minutes), the yield of acetol increases by 100% to 6.69 mg (Rxn D). When the optimized conditions are applied to 0.5 gram of glucose (Rxn C), similar results are obtained. The acetol yield increases to 8.2 mg. When using the same reaction conditions but reducing the amount of glucose used by 50% (0.25 gram), the yield of acetol decreases to 5.32 mg.

[0186]

Table 7

[0187] In summary, various parameters such as sugar type, reaction temperature, reaction time, pH, base concentration, and sugar concentration are optimized for synthesizing acetol from reactive sugar and sodium hydroxide. It has been shown that the highest yield of acetol can be obtained by reacting 0.1 M sodium hydroxide and 0.5 grams of glucose at 140 °C for 60 minutes. Note that when the base concentration is low or the sugar concentration is high, the pH of the reaction can change rapidly (within 10 minutes) from 12 to 6.5. Also, note that at high temperature (140 °C), the reaction product contains a small amount of acetoin (along with acetol). Importantly, note that hydroxyketone (acetoin) is prepared from glucose using biotechnology techniques with a yield of over 90%. However, the published chemical conversion from glucose to hydroxyketone is much lower, at about 9%.

[0188] [Example 7] Pyrazine is produced using glucose as a carbon source.

[0189] As shown in Example 6 above, acetol can be synthesized by reacting 0.1 N NaOH and glucose using optimal conditions (reacting 0.5 grams of glucose with 25 mL of 0.1 N NaOH at 140 °C for 60 minutes). The yield of acetol was about 2% based on the weight of glucose.

[0190] Publications by 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 reference) showed that acetol could be synthesized in a yield of 4% (based on the weight of glucose) from the reaction of a 40% phosphate buffer solution at pH 6.5 - 6.8 with glucose at a temperature of 100 - 120 °C. Higher yields of acetol were obtained at pH 7.0 - 7.1, and it was shown that the yield of acetol decreased with a decrease in pH.

[0191] This example first describes the preparation of acetol by the reaction of 0.1 N NaOH with glucose under optimized conditions. Next, a method for isolating acetol from the reaction mixture is shown. Finally, it is shown that different branched pyrazines can be synthesized by reacting the above mixture with NH4OH without isolating acetolacetone.

[0192] Glucose, 1-OH-acetone (acetol), 1-OH-2-butanone (acetolacetone), sodium hydroxide, disodium hydrogen phosphate / sodium hydroxide buffer solution pH = 12, dibasic potassium phosphate, monobasic potassium phosphate, sodium chloride, anhydrous sodium sulfate, hydrochloric acid, methanol, and dichloromethane are obtained from Sigma-Aldrich (St. Louis, Missouri).

[0193] The synthesis of acetol is carried out in a 40 mL or 1.5 L Parr vessel. To synthesize acetol, 0.5 grams of glucose is mixed per 25 mL of 0.1 N NaOH or buffer solution. Each reactant is stirred and heated at 140 °C for a period of 60 minutes. The initial pH level of all reactions is approximately 12 and no adjustment is made during the reaction. After completion of each reaction, the mixture is cooled and the amounts of both acetol and sugar are measured using GC and HPLC. For the quantification of acetol, 1 mg of acetoin is added as an internal standard to 25 mL of the solution and extracted with 30 - 35 mL of DCM. Next, the extracted DCM solution is dehydrated with sodium sulfate and analyzed and quantified by GC / FID.

[0194] The synthesis of pyrazine is carried out in the same Parr vessel. The reaction is carried out by reacting acetol synthesized from the reaction of sugar and 0.1 N NaOH. In this reaction, it is reacted with either 0.25, 0.5 or 1 mL of NH4OH per 25 mL of the solution. The reactants are obtained at 120 °C or 140 °C for a period of 17 hours. Then, each reaction mixture is cooled and pyrazine is extracted and quantified. In each extraction, 0.25 mg of d6 - 2 - methylpyrazine is added as an internal standard and the solution is extracted with 30 - 35 mL of DCM as a solvent. Next, the DCM solution is dehydrated with sodium sulfate and analyzed and quantified by GC / MS. The extracted ions are used for the quantification of each pyrazine.

[0195] All GC / MS analyses are performed using the same equipment and operating parameters as those used in Example 1 above. Each pyrazine is identified using the MS Wiley library. For quantitative analysis, a calibration curve is created using the concentration of acetol and the response coefficient ratio of acetol / acetoin. It should be noted that no acetoin is detected in any of the reactions carried out at 90, 100 and 120 °C. However, acetoin residues are found in the reaction where the temperature is set at 140 °C. Therefore, the acetoin concentration is determined in the reaction and this value is taken into account in all calculations.

[0196] All HPLC / RI separations are performed using a Sugar-Pak (300×6.5 mm) column from Waters (Milford, MA). An Agilent 1100 series HPLC equipped with a quaternary pump, refractive index (RI) detector, autosampler, and oven heater set at 80 °C is used. The isocratic mobile phase for sugar analysis is 0.005% disodium EDTA dihydrate. The flow rate is set at 0.5 mL / min for these analyses.

[0197] First, acetol is synthesized using a buffer solution. Due to the rapid change in the pH of the solution in the reaction of glucose and 0.1 N NaOH solution for acetol synthesis, a buffer solution with a pH of 12 (purchased from Sigma Aldrich) is used instead of the 0.1 N NaOH solution in the synthesis of acetol. In this study, 25 mL of sodium hydrogen phosphate / sodium hydroxide buffer solution pH = 12 is reacted with 0.25 or 0.5 grams of glucose at 140 °C for 60 minutes. The results show that when a buffer solution is used for synthesis, the concentration of acetol in the reaction product is much higher (2×). Table 8 below shows the results of this study.

[0198]

Table 8

[0199] When a similar buffer (7.1 grams of Na2HPO4 and 1 gram of NaOH are mixed in 100 mL of H2O, pH about 12 and buffering capacity 0.05 N) is prepared and reacted with glucose at 140 °C for 60 minutes, the reaction mixture did not show the presence of acetol. When this reaction was repeated three times, acetol was not found in any of the reaction products. However, when a similar buffer with a lower buffering capacity (0.025 N) at the same pH was used, acetol was found in the reaction product, and the concentration was much higher than when 0.1 N NaOH was used. Therefore, it is found that the type, volume, pH, and reaction temperature of the buffer have an effect on the synthesis of acetol using glucose.

[0200] Next, determine the acetol concentration in the reaction of glucose with a 40% phosphate buffer at pH 6.5 - 7.0. React 25 mL of a 40% potassium phosphate buffer pH = 6.5 - 6.8 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 from the reaction of a 40% phosphate buffer (25 mL) at pH 6.5 - 6.8 with glucose at various concentrations. Note that other types of hydroxyketones such as 3 - OH - 2 - butanone and 1 - OH - 2 - butanone are also formed in this reaction. The amounts of these hydroxyketones are not measured. Figure 3 shows the GC / MS analysis of the reaction of glucose with a phosphate buffer at 140 °C for 60 minutes and extraction with DCM.

[0201]

Table 9

[0202] Next, measure the 300 mL of the reactant and the glucose concentration and acetol concentration in 1 L. React 6 grams of glucose with 300 mL of 0.1 N NaOH at 140 °C for 60 minutes. After the reactant is cooled, obtain both the acetol concentration and the glucose concentration in the solution. A small amount (0.964 mg / mL) of glucose is detected in the reactant. This corresponds to less than about 5% of the unreacted glucose remaining. Table 10 below shows the acetol concentration in this reaction. The acetol concentration is found to be about 12 mg per 25 mL. Using a high - pressure reaction vessel, perform two additional reactions using 1 liter of 0.1 N NaOH and 20 grams of glucose. Both reactions are carried out at 130 - 140 °C for 60 minutes. Measure both the glucose concentration and the acetol concentration. In both 1 - liter reactions, the glucose concentration is less than 1 mg / mL (0.945 and 0.958 mg / mL). Table 10 below also shows the acetol concentration in each 1 - liter reaction. Here too, the acetol concentration is about 12 mg per 25 mL.

[0203]

Table 10

[0204] Next, acetol is isolated from the reaction of glucose and 0.1N NaOH. Various methods are used to isolate acetol from the reaction of 0.1N NaOH and glucose. Previous results regarding the isolation of pyrazine by distillation showed that pyrazine with a boiling point of 140 °C was isolated from the reaction mixture. Therefore, an acetol is isolated from the reaction mixture using a distillation apparatus set at 120 - 140 °C. For this purpose, 100 mL of the reaction mixture of 0.1N NaOH and glucose is distilled at 140 °C. After collecting 40 mL of the distillate, both the distilled substance and the remaining substance are extracted with DCM. GC / FID analysis shows that only 15 - 20% of acetol is distilled, while 80 - 85% of acetol remains in the reaction mixture.

[0205] When the reaction mixture of glucose and 40% phosphate buffer is distilled at 140 °C for 60 minutes, similar results are obtained. In this distillation, water is continuously added to the flask during distillation to keep the volume of the distillate constant. After collecting approximately 10 mL of the solution by distillation, approximately 10 mL of H2O is added to the distillation flask to compensate for the lost volume. A total of 4 × 10 mL of fractions are collected. Acetol is observed in each fraction. Analysis of the remaining reaction mixture using DCM shows that there is further acetol in the solution. When this distillation is continued for several hours, acetol can be isolated.

[0206] In the second method, column chromatography is used to isolate acetol from the reaction mixture. For this purpose, C 18Isolate acetol using a 30×2.0 cm glass (or metal) column with (particle size 40 - 60 μm and pore size 90 Å), filled only up to a maximum of 12 - 15 cm. Wash the column with methanol and then with 0.1% FA solution, and then pass 25 mL of the reaction mixture containing acetol through the column. During this isolation, collect all of the solution. After pushing the solution through the column (fraction 1, 25 mL), wash the material from the column with 25 mL of deionized H₂O (fraction 2). Next, wash the column with an additional 25 mL of H₂O until clean H₂O elutes from the column (fraction 3). Then, dry the column with N₂ and elute the remaining analyte trapped in the column with 100% MeOH (fraction 4). Extract all fractions with DCM and analyze by GC / FID. The results show that most of the acetol is eluted by fractions 1 and 2, and fractions 3 and 4 do not contain acetol. Here, the reaction mixture is C 18 Note that it is very difficult to use the packing material for washing another reaction mixture after passing the reaction mixture through the packing material. It is very difficult to wash the packing material. In summary, isolating hydroxyketone from the reaction product can be timely and costly.

[0207] Next, synthesize pyrazine from the reaction product of glucose and sodium hydroxide with NH₄OH without first isolating hydroxyketone. After reacting 0.5 grams of glucose and 25 mL of 0.1 N NaOH at 140 °C for 60 minutes to prepare acetol, it is found that the reaction mixture contained an acetol solution of approximately 10 - 12 mg / 25 mL. For this purpose, react 25 mL of the solution with 0.5 or 1 mL of NH₄OH at 120 - 140 °C for 17 - 18 hours to determine the type and percent yield of pyrazine. Repeat each reaction twice. Table 11 below shows a list of the detected pyrazines, including the elution times from the GC column. Changing the amount of NH₄OH from 0.5 mL to 1 mL gives the same results.

[0208]

Table 11

[0209] The same reaction is carried out on a 1-liter scale. Prepare 2 batches of 1 liter of solution. In each batch, react 20 grams of glucose with 1000 mL of 0.1N NaOH solution at 140 °C for 60 minutes. Cool each solution and measure the acetol concentration. Next, add 20 mL of NH4OH to one reaction mixture and 40 mL of NH4OH to the second reaction mixture. Heat at 120 - 130 °C for 17 hours while continuously mixing each reactant. After cooling the reactants, add an internal standard (d6-2-methylpyrazine) to 25 mL of each reactant, extract with DCM, and determine the concentration and distribution of each pyrazine. Distill each remaining reaction solution and collect them individually. Collect approximately 125 mL of distillate solution per 500 mL of reaction mixture. Next, extract the distillate solution (10 mL) with DCM and quantify by GC / MS. The total mass of pyrazine is greater than when the solution was not extracted with DCM because the concentration of pyrazine in the distillate solution is high. The distribution of pyrazine is almost the same in both extracts. Note that when NH4OH is reacted with the reaction mixture of glucose and 0.1N NaOH, both 2-methylpyrazine and pyrazine are detected in large amounts in all reactions. Pyrazine is not detected in the extract of the remaining solution after distillation.

[0210] Next, pyrazine is synthesized from the reaction product of glucose, phosphate buffer, and NH4OH without first isolating the hydroxyketone. 25 mL of a reaction mixture of phosphate buffer (pH = 6.5 - 6.8) and glucose (0.5 or 1 gram each, solutions A and B) is reacted with 1 mL of NH4OH at 140 °C for 17 hours. After cooling each reaction product, 0.25 mg of deuterated 2-methylpyrazine (internal standard) is added to each reaction mixture, and the solution is extracted with DCM. Each extract is analyzed using GC / MS to calculate the mass of pyrazine and its percent distribution. Note that both pyrazine and 2-methylpyrazine are not synthesized in these reactions. Also, when 1 gram of glucose is reacted with the phosphate buffer, the total mass of pyrazine is 20% higher in solution B. Although not limited by theory, this is thought to be due to the higher concentration of acetol in solution B compared to solution A. Figure 4 shows the GC / MS analysis of pyrazine extracted using DCM from a reaction mixture of solution B (1 gram of glucose reacted with 25 mL of 40% phosphate buffer at 140 °C for 60 minutes) and 1 mL of NH4OH at 140 °C for 17 hours. For comparison, Figure 5 shows the GC / MS analysis of pyrazine extracted using DCM from 25 mL of a reaction mixture of 0.5 gram of glucose and 0.1 N NaOH reacted at 140 °C for 60 minutes, and then reacted with 1 mL of NH4OH at 140 °C. Both pyrazine and 2-methylpyrazine are detected in this reaction.

[0211] In summary, acetol (and other hydroxyketones) can be isolated from the reaction mixture of glucose and 0.1N NaOH using both distillation and column chromatography. Distillation consumes a lot of time and energy, and at the same time chromatography can be very costly. However, it is possible to synthesize pyrazine without isolating acetol. When acetol prepared from the reaction of 0.1N NaOH and glucose was reacted with NH4OH at 140 °C for 17 hours, an array of a series of pyrazines was produced. The pyrazine was isolated by distillation. It should be noted that pyrazine and 2-methylpyrazine were synthesized in this step. However, when acetol prepared from a mixture of glucose and phosphate buffer was reacted with NH4OH, the same pyrazines were produced in the same distribution, but no pyrazine or 2-methylpyrazine was detected in the reaction mixture.

[0212] Those skilled in the art of the field related to the present invention having the advantages of the teachings shown in the above description will envision many modifications and other embodiments of the present invention. Accordingly, it is understood that the present invention is not limited to the specific embodiments disclosed, and that modifications and other embodiments are included within the scope of the appended claims. Specific terms are used in this specification, but the terms are used only in a general and descriptive sense and not for purposes of limitation.

Claims

**Claim 1** A method for forming pyrazine, comprising: obtaining a carbon source solution comprising at least one sugar and at least one buffer selected from the group consisting of sodium hydroxide, phosphate buffer, and combinations thereof; mixing the carbon source solution with at least one nitrogen source selected from the group consisting of amino acids, ammonium ions, and combinations thereof to form a reaction solution; and heating the reaction solution to a reaction temperature and maintaining the reaction solution at the reaction temperature for a time sufficient to produce a reaction product comprising at least one substituted pyrazine. **Claim 2** The method according to claim 1, wherein the at least one sugar is selected from the group consisting of glucose, fructose, rhamnose, and combinations thereof. **Claim 3** The method according to claim 1 or 2, wherein the buffer buffers in a pH range of 6.5 to 7.

5. **Claim 4** The method according to any one of claims 1 to 3, wherein the at least one substituted pyrazine is disubstituted. **Claim 5** The method according to any one of claims 1 to 3, wherein the at least one substituted pyrazine is trisubstituted. **Claim 6** The method according to any one of claims 1 to 3, wherein the at least one substituted pyrazine is tetrasubstituted. **Claim 7** The method according to any one of claims 1 to 3, wherein the at least one substituted pyrazine comprises at least one substituent having two or more carbon atoms. **Claim 8** The method according to any one of claims 1 to 3, wherein the at least one substituted pyrazine comprises at least one substituent having three or more carbon atoms. **Claim 9** The at least one substituted pyrazine is 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-propyl)pyrazine; 3,6-Dimethyl-2-(n-propyl)pyrazine; 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 The method according to any one of claims 1 to 3, selected from the group consisting of combinations thereof.

10. The method according to any one of claims 1 to 9, wherein the at least one sugar is derived from tobacco.

11. The method according to any one of claims 1 to 10, wherein the at least one nitrogen source is derived from tobacco.

12. The method according to any one of claims 1 to 11, further comprising isolating the at least one substituted pyrazine from the reaction product.

13. The method according to claim 12, wherein the step of isolating the at least one substituted pyrazine from the reaction product comprises at least one of liquid-liquid extraction of the reaction product, liquid-solid extraction of the reaction product, and simple distillation of the reaction product.

14. The method according to any one of claims 1 to 13, wherein the reaction temperature is 90 °C to 150 °C.

15. The method according to any one of claims 1 to 14, wherein the pyrazine molecules and methylpyrazine molecules in the reaction product are less than 1.0% of the reaction product.

16. The method according to any one of claims 1 to 15, further comprising incorporating said at least one substituted pyrazine into a tobacco product.

17. The method according to claim 16, wherein said tobacco product is a smoking product or a smokeless tobacco product.

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