Methods of selectively forming substituted pyrazines

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 hydroxy ketones, sugars treated with at least one buffer, and combinations thereof. Tobacco products incorporating substituted pyrazines are also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to methods for forming substituted pyrazines. Of particular interest are methods for selectively forming targeted substituted pyrazines. [Background technology]

[0002] Pyrazines are produced by the reaction (e.g., Maillard reaction) of a carbon source with a nitrogen compound, such as an amino acid, and a base (e.g., diammonium phosphate (DAP), NaOH). Many conventional reaction pathways aimed at producing pyrazines use sugars (e.g., fructose, glucose, fructose / glucose mixtures, rhamnose) as the carbon source. To date, most model reactions and fortified natural products that result in pyrazine-rich formulations upon heating have used sugars such as fructose, glucose, fructose / glucose mixtures, and rhamnose as the carbon source components of the formulation. These sugars have been shown to act as carbon sources for the formation of pyrazine aromatic ring structures.See, for example, U.S. Patent Application Publication No. 2004 / 0173228 to Coleman, U.S. Patent Application Publication No. 2010 / 0037903 to Coleman et al., U.S. Patent Application Publication No. 2012 / 0152265 to Dube et al., U.S. Patent Application Publication No. 2012 / 0260929 to Coleman et al., U.S. Patent Application Publication No. 2013 / 0125907 to Dube et al., U.S. Patent Application Publication No. 2013 / 0337418 to Anuradha et al., U.S. Patent Application Publication No. 2015 / 0040922 to Dube et al., and U.S. Patent Application Publication No. 2015 / 0122271 to Chen et al., each of which is incorporated by reference in its entirety. No. 98,858 to Coleman, U.S. Pat. No. 6,325,860 to Dube et al., U.S. Pat. No. 6,440,223 to Dube et al., U.S. Pat. No. 6,499,489 to Coleman, U.S. Pat. No. 6,591,841 to White et al., U.S. Pat. No. 6,695,924 to Dube et al., U.S. Pat. No. 8,434,496 to Chen et al., U.S. Pat. No. 8,944,072 to Brinkley et al., U.S. Pat. No. 8,955,523 to Coleman et al., U.S. Pat. No. 8,991,403 to Chen et al., U.S. Pat. No. 9,010,339 to Dube et al., U.S. Pat. No. 9,254,001 to Byrd et al., U.S. Pat. No. 9,265,284 to Junker et al., and U.S. Pat. No. 9,402,415 to 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 to provide the nitrogen bond within the pyrazine structure. See, for example, "Effect of time, temperature, and reactant ratio on pyrazine formation in a model system," T. Shibamoto, RA Bernhard, J. Agric. Food Chem., 24, (1976) p. 847. This reaction produces a complex mixture of many substituted pyrazines. When sugars serve as the sole carbon source in pyrazine-forming reactions, pyrazine and methylpyrazine molecules are the predominant pyrazines produced, often accounting for well over 60% of the total pyrazine yield. This trend is evident even when free amino acids are used as co-reagents in an attempt to reduce the amount of pyrazine and methylpyrazine molecules produced.

[0004] From a sensory perspective, pyrazine and methylpyrazine molecules do not have the desired sensory notes or acceptable volatility characteristics for use in tobacco products, and therefore their presence in mixtures of pyrazines provides less than desirable properties for certain applications.

[0005] It would therefore be desirable to provide a method for selectively producing larger quantities of certain desired substituted pyrazines. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US Patent Application Publication No. 2004 / 0173228 [Patent Document 2] US Patent Application Publication No. 2010 / 0037903 [Patent Document 3] US Patent Application Publication No. 2012 / 0152265 [Patent Document 4] US Patent Application Publication No. 2012 / 0260929 [Patent Document 5] US Patent Application Publication No. 2013 / 0125907 [Patent Document 6] US Patent Application Publication No. 2013 / 0337418 [Patent Document 7] US Patent Application Publication No. 2015 / 0040922 [Patent Document 8] US Patent Application Publication No. 2015 / 0122271 [Patent Document 9] U.S. Patent No. 5,258,194 [Patent Document 10] U.S. Patent No. 6,298,858 [Patent Document 11] U.S. Patent No. 6,325,860 [Patent Document 12] U.S. Patent No. 6,440,223 [Patent Document 13] U.S. Patent No. 6,499,489 [Patent Document 14] U.S. Patent No. 6,591,841 [Patent Document 15] U.S. Patent No. 6,695,924 [Patent Document 16] U.S. Patent No. 8,434,496 [Patent Document 17] U.S. Patent No. 8,944,072 [Patent Document 18] U.S. Patent No. 8,955,523 [Patent Document 19] U.S. Patent No. 8,991,403 [Patent Document 20] U.S. Patent No. 9,010,339 [Patent Document 21] U.S. Patent No. 9,254,001 [Patent Document 22] U.S. Patent No. 9,265,284 [Patent Document 23] U.S. Patent No. 9,402,415 [Non-patent literature]

[0007] [Non-Patent Document 1] Coleman III, On the synthesis and characteristics of aqueous formulations rich in pyrazines, in Flavor Fragrance and Odor Analysis, Second Edition, Ray Marsili, ed., Chapter 7, pp 135-182, CRC Press, Boca Raton, 2012 [Non-patent document 2] Effect of time, temperature, and reactant ratio on pyrazine formation in model system, T. Shibamoto, RABernhard, J. Agric. Food Chem., 24, (1976) p.847 Summary of the Invention [Means for solving the problem]

[0008] The present invention provides a method for selectively forming certain substituted pyrazines. The method for selectively producing pyrazines includes obtaining a reaction solution containing at least one tobacco-derived carbon source (e.g., a hydroxyketone and / or at least one buffered sugar) and at least one tobacco-derived nitrogen source (e.g., a protein and / or an 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 comprising 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, 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-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, The pyrazine may be selected from the group consisting of 5,6-trimethylpyrazine; 3,5(3,6-dimethyl)-2,N-propylpyrazine; 2,5-diethyl-3-methylpyrazine; 2,3-diethyl-5,6-dimethylpyrazine; trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; 2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrazine; and 2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

[0010] In some embodiments, at least one substituted pyrazine is di-, tri-, or tetra-substituted. In certain embodiments, at least one substituted pyrazine includes at least one substituent having two or more carbon atoms. In various embodiments, at least one substituted pyrazine includes at least one substituent having three or more carbon atoms.

[0011] In various embodiments, the method for selectively forming certain substituted pyrazines 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 some embodiments, the tobacco product can be a smoking article. In some embodiments, the tobacco product can be a smokeless tobacco product.

[0013] In various embodiments of the present invention, a method of forming a pyrazine is provided, the method comprising obtaining a reactant solution comprising at least one α-hydroxyketone 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 comprise isolating the at least one substituted pyrazine from the reactant product. In some embodiments, the at least one hydroxyketone 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, 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 hydroxyketone can include acetoin, and the at least one substituted pyrazine can be tetramethylpyrazine. In various embodiments, the at least one hydroxyketone 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; 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 the reaction solution comprising at least one α-hydroxyketone and at least one nitrogen source. In some embodiments, the method can further include adding at least one aldehyde to the reaction solution comprising at least one α-hydroxyketone and at least one nitrogen source.

[0015] Various embodiments of the present invention provide a method for forming pyrazines, 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 hydroxyketone is provided from the 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 maintaining the reaction solution at the reaction temperature for a sufficient time to produce a reaction product comprising at least one substituted pyrazine. Various embodiments of the methods herein can further comprise isolating the 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 some embodiments, the buffer can be selected from the group consisting of sodium hydroxide, phosphate buffer, and combinations thereof. In some embodiments, the buffer can buffer at a pH range of about 6.5 to about 7.5. The methods of the present invention can further comprise adding ammonium ions to a reaction solution containing a carbon source including 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 of forming a pyrazine, the method comprising obtaining a reaction solution comprising at least one α-hydroxyketone and at least one nitrogen source, heating the reaction solution to a reaction temperature, and maintaining the reaction solution at the reaction temperature for a sufficient time to produce a reaction product comprising at least one substituted pyrazine.

[0018] Embodiment 2: The method of embodiment 1, wherein the at least one hydroxyketone comprises acetol.

[0019] Embodiment 3: The method of embodiment 1 or 2, wherein the 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; 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 of any one of embodiments 1-3, wherein the at least one hydroxyketone comprises acetoin.

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

[0022] Embodiment 6: The method of any one of embodiments 1-5, wherein the at least one hydroxyketone comprises 1-hydroxy-2-butanone.

[0023] Embodiment 7: The method of any one of embodiments 1-6, wherein the at least one substituted pyrazine is selected from the group consisting of 2,6-diethylpyrazine; 2,5-diethylpyrazine; 2-ethyl-3,5,6-trimethylpyrazine; 3,5(3,6-dimethyl)-2,N-propylpyrazine; 2,5-diethyl-3-methylpyrazine; 2,3-diethyl-5-methylpyrazine; 2,3-diethyl-5,6-dimethylpyrazine; trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; 2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

[0024] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the nitrogen source is selected from the group consisting of amino acids, ammonium ions, and combinations thereof.

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

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

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

[0028] Embodiment 12: The method of any one of embodiments 1 to 11, wherein 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.

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

[0030] Embodiment 14: 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-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 Pyrazine, 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 2,5-diethyl-3-methylpyrazine; 2,3-diethyl-5,6-dimethylpyrazine; trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; 2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrazine; and 2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

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

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

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

[0034] Embodiment 18: The method of any one of embodiments 1-17, wherein at least one substituted pyrazine comprises at least one substituent having two or more carbon atoms.

[0035] Embodiment 19: The method of any one of embodiments 1-18, wherein at least one substituted pyrazine comprises at least one substituent having 3 or more carbon atoms.

[0036] Embodiment 20: The method of any one of embodiments 1-19, wherein the reaction product is substantially free of pyrazine and methylpyrazine molecules.

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

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

[0039] Embodiment 23: A method of forming a pyrazine, the method comprising: obtaining a carbon source solution comprising at least one sugar and at least one buffer; combining the carbon source solution with at least a nitrogen source to form a reaction solution; heating the reaction solution to a reaction temperature; and maintaining the reaction solution at the reaction temperature for a sufficient time to produce a reaction product comprising at least one substituted pyrazine.

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

[0041] Embodiment 25: The method of 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 of any one of embodiments 1 to 25, wherein the buffering agent is selected from the group consisting of sodium hydroxide, phosphate buffer, and combinations thereof.

[0043] Embodiment 27: The method of any one of embodiments 1 to 26, wherein the buffering agent buffers at a pH range of about 6.5 to about 7.5.

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

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

[0046] Embodiment 30: The method of embodiments 1-29, wherein 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.

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

[0048] Embodiment 32: 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-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 Pyrazine, 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 2,5-Dimethyl-3-ethylpyrazine; 2,3-Diethyl-5,6-dimethylpyrazine; trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; 2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrazine; and 2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

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

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

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

[0052] Embodiment 36: The method of any one of embodiments 1 to 35, wherein at least one substituted pyrazine comprises at least one substituent having two or more carbon atoms.

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

[0054] Embodiment 38: The method of any one of embodiments 1 to 37, wherein the reaction product is substantially free of pyrazine and methylpyrazine molecules.

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

[0056] Embodiment 40: The method of any of embodiments 1-39, further comprising incorporating at least one substituted pyrazine into a tobacco product, wherein the tobacco product is a smoking article 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, which are briefly described below. The present invention includes any combination of two, three, four, or more of the above-described embodiments, as well as combinations of two, three, four, or more features or elements described in this disclosure, whether or not such features or elements are explicitly combined in the description of a particular embodiment herein. The present disclosure, in all its various aspects and embodiments, is to be read as a whole such that separable features or elements of the disclosed invention are construed as combinable unless the context clearly dictates otherwise.

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

[0059] [Figure 1] 1 is a flow chart illustrating a method for selectively forming a substituted pyrazine. [Figure 2] 1 is a flow chart illustrating a method for selectively forming a substituted pyrazine. [Figure 3] GC / MS analysis of glucose extracted with dichloromethane (DCM) after reacting with phosphate buffer at 140° C. for 60 minutes is shown. [Figure 4] GC / MS analysis of pyrazines extracted from a reaction mixture of (1 gram of glucose reacted with 25 mL of 40% phosphate buffer at 140° C. for 60 minutes) and 1 mL of NH 4 OH using DCM at 140° C. for 17 hours is shown. [Figure 5] GC / MS analysis of pyrazines extracted at 140° C. using DCM from 25 mL of a reaction mixture (0.1 N NaOH reacted with 0.5 grams of glucose at 140° C. for 60 minutes) reacted with 1 mL of NH 4 OH. [Figure 6]1 is an exploded perspective view of a smoking article in the form of a cigarette, showing the smoking material, wrapping material components and cigarette filter element; FIG. [Figure 7] FIG. 1 is a top view of an embodiment of a smokeless tobacco product viewed across the width of the product, showing an outer pouch filled with tobacco material. [Figure 8] 1 is a cross-sectional view of an electronic smoking article comprising a cartridge and a control body, including a reservoir housing, according to an exemplary embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention is described more fully below. However, this invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. References to "percent dry weight" or "dry weight basis" refer to weight based on dry ingredients (i.e., all ingredients excluding water).

[0061] The present invention provides a method for forming selected pyrazines. Pyrazines exhibit many different flavor profiles, including, but not limited to, roasted notes, toasted notes, and nutty notes. For example, pyrazines containing cyclopentyl derivatives are known for their positive sensory properties at very low ppb levels. 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 and methylpyrazine molecules, but also allows for tuning of the reaction to produce other desired substituted pyrazines in a controlled manner.

[0062] Selective formation of pyrazines using carbon sources other than sugars Traditionally, sugars have been used as carbon sources in reactions that form pyrazines. Several reaction pathways using sugars to produce pyrazine-rich compositions are known in the art, including: 1) hydrolysis of proteins to free amino acids, followed by 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), followed by 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 is produced, including pyrazine and methylpyrazine molecules as the major pyrazines, much less dimethylpyrazine molecules, and significantly less high-molecular-weight pyrazine molecules.

[0063] The term "pyrazine molecule" as used herein refers to a heterocyclic organic compound of the formula CHN. This is distinct from the general term "pyrazine," which is used herein to refer 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 is ammonium ion (NH + ), amino acids, proteins, or combinations thereof. In some embodiments, ammonium ions (NH4 +) can be provided. The amino acids can be derived, for example, from protein hydrolysis. In some embodiments, the amino acids can be derived from the hydrolysis of tobacco-derived proteins, as discussed in U.S. Patent Application Publication No. 15 / 009,199 to Dube et al., filed January 28, 2016, which is incorporated by reference in its entirety. Other nitrogen-containing compounds known in the art that are reactive with a carbon source to form at least one pyrazine can also be used as nitrogen sources in embodiments of the invention disclosed herein.

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

[0066] [ka]

[0067] In various embodiments of the present invention, the carbon source can comprise at least one α-hydroxyketone. In various embodiments of the present invention, the R and R′ functional groups of the at least one hydroxyketone can be substituents independently selected from the group consisting of H or halo (e.g., Cl, F, or Br), OH, optionally substituted C alkyl, optionally substituted C alkoxy, optionally substituted C alkenyl, optionally substituted C alkynyl, NR R, NRCOR, NRCOR, CR R OR, CONR R, CO R, CN, CF, NO, N, C alkylthio, R SO, R SO, CF S, and CF SO. In some embodiments of the present invention, the R and / or R′ functional groups are C alkyl.

[0068] The term "alkyl," as used herein, refers to saturated straight-chain, branched, or cyclic hydrocarbon groups (i.e., cycloalkyl groups) as well as unsaturated versions of saturated examples (e.g., propenyl). In certain embodiments, alkyl refers to groups containing 1 to 10 carbon atoms ("C1-10 alkyl"). In further embodiments, alkyl refers to groups 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 refers to groups 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 a specific embodiment, alkyl denotes 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] "Optionally substituted" with respect to substituents refers to substituents that are optionally substituted with one or more moieties selected from the group consisting of, for example, halo (e.g., Cl, F, Br, and I); alkyl (e.g., C alkyl), alkyl halide (e.g., CF, 2-Br-ethyl, CHF, CHCl, CHCF, or CFCF); C alkenyl, C alkynyl; hydroxyl; amino; amido; carboxylate; carboxamido; carbamate; carbonate; urea; acetate; alkylamino; arylamino; C alkoxy; aryl; aralkyl, aryloxy; nitro; azido; cyano; thio; alkylthio; sulfonate; sulfide; sulfinyl; sulfo; sulfate; sulfoxide; sulfamido; sulfonamide; phosphonic acid; phosphate; and / or phosphonate.

[0070] The term "alkenyl," as used herein, refers to 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 ("C-10 alkenyl"). In further embodiments, alkenyl refers to a group containing 2 to 8 carbon atoms ("C-8 alkenyl"), 2 to 6 carbon atoms ("C-6 alkenyl"), or 2 to 4 carbon atoms ("C-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] The term "alkynyl," as used herein, refers to an alkyl moiety in which at least one saturated C-C bond is replaced with 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] The term "alkoxy," as used herein, refers to a straight-chain or branched alkyl group linked through an oxygen atom (i.e., -O-alkyl), where alkyl is as defined above. In certain embodiments, alkoxy refers to an oxygen-linked group containing 1 to 10 carbon atoms ("C1-10 alkoxy"). In further embodiments, alkoxy refers to an oxygen-linked 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] The term "halo" or "halogen," as used herein, means fluorine, chlorine, bromine, or iodine.

[0074] The term "amino," as used herein, refers to a moiety represented by the structure NR2, and includes primary amines and alkyl- or aryl-substituted secondary and tertiary amines (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 that contains an alkyl group, as defined above, attached to an amino group, as defined above, which is attached to another part of the molecule through the alkyl group.

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

[0077] The term "derivative," as used herein, means a compound formed from a similar, starting compound by attaching another molecule or atom to the starting compound. Additionally, a derivative according to the present invention includes 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 to produce tetramethylpyrazine (TMP). Specifically, the reaction of acetoin with ammonium hydroxide and phosphoric acid (or diammonium phosphate) has been successful, producing tetramethylpyrazine (TMP) in essentially quantitative yield (greater than 80%) and with concomitant considerable purity. The reaction of acetoin with ammonium hydroxide produces almost exclusively TMP, with little or no detectable amounts of pyrazine and methylpyrazine molecules. As used herein, the terms "little or no detectable amounts," "substantially no," and "substantially zero" indicate that the identified compound is present in an amount of 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 limited by theory, the various pyrazines synthesized from the reaction of sugars with acetoin as a carbon source surprisingly demonstrate that the distribution of pyrazines in the reaction of a carbon source with a nitrogen source (e.g., ammonium ion and / or amino acid) is determined to a large extent by the carbon source. In particular, it has been surprisingly found that the use of different hydroxyketones as carbon sources in the pyrazine-forming reaction allows the production of a series of specifically substituted pyrazines 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 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 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 an alkyl group on the pyrazine ring that is not essentially linear. For example, isobutyl, sec-butyl, and tert-butyl groups are used in place of n-butyl groups (this also applies to propyl and pentyl groups). As described in more detail below, it has surprisingly been found that the distribution of pyrazines produced can be determined by varying the α-hydroxyketone. For example, using acetoin in the reaction yields only tetramethylpyrazine (purity of about 99.5% or greater). Using acetol in the reaction yields primarily dimethyl-substituted pyrazines (pyrazine yield of about 95% or greater).

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

[0083] In one embodiment, pyrazine is first isolated from the reaction product by simple distillation, which provides a distillate containing mostly water and pyrazine. This distillate can then 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 distillate used. For example, if 10 L of distillate is used, 5 L of cyclohexane can be used. The liquid-liquid extraction of the distillate with cyclohexane can be repeated multiple times. In some embodiments, the liquid-liquid extraction of the distillate with cyclohexane can be repeated at least five times. After liquid-liquid extraction, the extracted pyrazine-containing cyclohexane can be dehydrated using 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, the pyrazine can be isolated (i.e., cyclohexane can be removed) by simple distillation and / or rotary evaporation.

[0084] As discussed above, the use of different hydroxyketones as carbon sources in the pyrazine-forming reaction can produce a series of specifically substituted pyrazines. In some embodiments, the carbon source includes acetoin. As detailed in Example 1 below, for example, when acetoin serves as the sole carbon source in a reaction with a nitrogen source (e.g., ammonium hydroxide (NH4OH) and phosphoric acid (H3PO4), diammonium phosphate, etc.) to produce pyrazines, the only pyrazine produced is tetramethylpyrazine. Furthermore, branched pyrazines such as isopropylpyrazine are not synthesized by adding amino acids (e.g., leucine or free amino acids from hydrolyzed F1 protein) to a reaction containing acetoin, NH4OH, and H3PO4. Heating the reaction at high temperatures or for extended periods of time does not alter the results (i.e., tetramethylpyrazine (TMP) is the only pyrazine synthesized from acetoin). Therefore, if pyrazines other than TMP are desired from this synthetic approach, carbon sources other than acetoin can be synthesized.

[0085] In some embodiments, the carbon source can include 1-hydroxyacetone (also known as 1-OH-acetone, acetol, or 1-hydroxy-2-propanone). Acetol, an α-hydroxyketone, is the simplest hydroxyketone. As 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 a sugar with an amino acid to form a pyrazine), which can then be further reacted 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 attached to one side of the carbonyl, and in the case of 1-OH-2-butanone, an ethyl group is attached 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 that will be produced.

[0086] [ka]

[0087] For example, a series of pyrazines can be produced by the reaction of 1-hydroxyacetone as a carbon source with NHOH as a base and nitrogen source, as shown below in Example 2. When 1-hydroxyacetone (acetol) acts as the carbon source, a series of specific alkyl-substituted pyrazines can be produced. For example, exemplary pyrazines provided by the reaction of 1-hydroxyacetone 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- Examples include 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] Using 1-hydroxy-2-butanone as the sole carbon source can produce pyrazines with an ethyl group attached. For example, as shown in Example 4 below, pyrazines synthesized from the reaction of 1-OH-2-butanone with 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, trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine, and 2,5-dimethyl-3-ethylpyrazine.

[0089] When 1-hydroxyacetone (acetol), 2-hydroxy-3-butanone (acetoin), and 1-hydroxy-2-butanone are used as carbon sources in the reaction with a nitrogen 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 alternative series of pyrazines. Using a different hydroxyketone as the carbon source in the pyrazine-producing reaction not only minimizes the formation of pyrazine and methylpyrazine, but also allows the reaction to be tailored to produce other desirable substituted pyrazines in a controlled manner.

[0090] For example, as shown in operation 102 of Figure 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, the addition of, for example, an amino acid or a selected aldehyde can increase not only the number of pyrazines synthesized but also the yield of the pyrazines. For example, in one embodiment, the reaction solution can include 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 pyrazines synthesized and the yield of the pyrazines. For example, in one embodiment, the reaction solution can include an alkyl aldehyde selected from the group consisting of acetaldehyde, propanal, isopropanol, butanal, isobutanal, sec-butanal, and combinations thereof.

[0091] Increasing the reaction time and temperature improves the yield of pyrazine up to the point where black tar 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 higher, 8 hours or higher, about 12 hours or higher, 16 hours or higher, or about 24 hours or higher. In various embodiments, the reaction time can be about 4 to about 24 hours or about 12 to about 20 hours. In some embodiments, 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., NHOH) can be, for example, about 1:0.5, about 1:1, about 1:2, or about 1:2.5. In some embodiments, 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 some embodiments, the ratio of hydroxyketone to nitrogen source can be about 1:2.

[0093] Increasing the pH of the reaction solution can also result in an increase in the amount of pyrazine. A 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, the pH of the reaction solution can be increased using small amounts of NaOH or KOH.

[0094] Selective formation of pyrazines 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 a nitrogen source. As discussed above, various reaction routes to pyrazine-rich formulations have previously been used to produce pyrazines using sugars as carbon sources. See, for example, U.S. Patent Application Publication No. 15 / 009,199, filed January 28, 2016, to Dube et al., which is incorporated herein by reference in its entirety. However, when sugars are used as the sole source in a reaction with a nitrogen source (e.g., ammonium hydroxide) to produce pyrazines, pyrazine and methylpyrazine molecules can be the predominant pyrazines produced. This trend is evident even when free amino acids are used as co-reagents. It has surprisingly been discovered 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 heated formulation containing an amino acid and a sugar can produce pyrazines. See, for example, 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, pp. 135-182, CRC Press, Boca Raton, 2012, which are incorporated by reference in their entireties. Pyrazine-rich solutions can be prepared in a variety of ways. For example, one method can include microwave heating of a solution containing at least one amino acid and at least one sugar. For example, 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. For example, as shown in operation 124 of FIG. 1, the reaction solution can be heated to a reaction temperature and held at the reaction temperature for a reaction time sufficient for the reactants to undergo a reaction to form pyrazines.

[0096] As discussed above, the hydroxyketone acetol can be produced by the decomposition of various sugars. Furthermore, when used as a carbon source in a reaction with a nitrogen source, a series of substituted pyrazines, excluding pyrazine and methylpyrazine, can be produced. Therefore, acetol can be an important intermediate in the reaction of sugars with free amino acids and / or ammonium ions to produce pyrazines containing branched alkyl side chains.

[0097] Literature has shown that 1-hydroxy-2-propanone (acetol) can be produced from C6 sugars such as glucose and its derivative sorbitol. See, for example, M. H. Mohamad et al., "A review of acetol: application and production," Amer. J. Appl. Sci., 8, 1135-1139 (2011); M. A. Dasari, "Catalytic conversion of glycerol and sugar alcohols to value-added products," University of 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. These references describe the conversion of sugars to 1-hydroxy-2-propanone using phosphate buffer at high temperatures, strong bases such as NaOH at pH 11.5 under reflux, Ni and palladium catalysts under hydrogen pressure, and copper chromite catalysts in the gas phase of heterogeneous reactions. Copper chromite has been considered the best catalyst. In most reactions, the conversion of the substrate was greater than 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. When glucose was used as the carbon source, the highest yield of acetol was 8.99% at 280° C.

[0098] Cellulose has been converted to acetol in 30% yield using a Sn-based catalyst system. See, e.g., 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. US 2013 / 028174 A1, 2013, which is incorporated herein by reference in its entirety.

[0099] Recently, Novotny et al. (Czech J. Food Sci., 25, 119-130, 2007, incorporated herein by reference) synthesized α-hydroxycarbonyl and α-dicarbonyl compounds via the degradation of monosaccharides. They used three different models, consisting of an aqueous solution of potassium peroxodisulfate, an alkaline solution of potassium peroxodisulfate, and a solution of sodium hydroxide, respectively. Using GC / MS, a total of six α-hydroxycarbonyl and six α-dicarbonyl compounds were identified. The maximum yield of α-hydroxycarbonyls (glycolaldehyde, acetol, lactaldehyde, glyceraldehyde, 1,3-dihydroxyacetone, and acetoin) was approximately 4% when glucose or fructose was reacted with sodium hydroxide. The yields were much lower with aqueous potassium peroxodisulfate (0.32%) and alkaline potassium peroxodisulfate (1.1%) solutions. Acetol and 1,3-dihydroxyacetone gave 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 in 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 approaches 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 a nitrogen source to produce pyrazines.

[0101] It has surprisingly been discovered that substituted pyrazines can be selectively formed from a sugar carbon source without first isolating the hydroxyketones (e.g., acetol, acetoin, etc.) formed from the degradation of the sugar, as shown, for example, in Example 7 below. By treating the sugar with a buffer before combining it with a nitrogen source, as shown, for example, in operation 110 of FIG. 2, a series of pyrazines can be produced that differs from the series of pyrazines produced from the reaction of a sugar that has not been pretreated with a buffer and nitrogen source. The reaction can be optimized to maximize the production of acetol and acetol-like compounds 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 (e.g., acetol, acetoin, etc.) is produced from the degradation 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 certain embodiments, the buffer can include sodium carbonate, sodium sulfite, peroxodisulfate, sodium phosphate, or combinations thereof. The selection of the type of buffer, buffer capacity, pH, and reaction temperature can affect the synthesis of hydroxyketone from the sugar carbon source, and therefore can affect the amount of pyrazine produced in the series and from the subsequent reaction with the nitrogen source.

[0103] In various embodiments, the buffering agent can buffer at a pH in the approximately neutral or alkaline range, for example, at a pH greater than about 6, greater than about 8, or greater than about 10 (e.g., from about 6 to about 12). For example, in certain embodiments, 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 is produced, including pyrazine and methylpyrazine molecules as the predominant pyrazines, much less dimethylpyrazine molecules, and significantly less high molecular weight pyrazine molecules. Pre-reacting glucose with NaOH at pH 12 followed by reaction with ammonium hydroxide produces a very similar series of pyrazines. However, it has surprisingly been discovered that treating glucose with potassium phosphate buffer at pH 6.5 and subsequently reacting with ammonium hydroxide produces only dimethylpyrazines and high molecular weight pyrazines, thereby eliminating the production of the less desirable pyrazine and methylpyrazine molecules.

[0105] By extending the reaction time and increasing the temperature of the buffered sugar carbon source and nitrogen source, the yield of pyrazine can be increased up to the point where black tar 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 higher, about 60 minutes or higher, about 90 minutes or higher, or about 120 minutes or higher. In various embodiments, the reaction time can be about 30 minutes to about 150 minutes or about 60 minutes to about 120 minutes.

[0106] Increasing the pH of the reaction solution can also result in an increase in the amount of pyrazine. A 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, the pH of the reaction solution can be increased using small amounts of NaOH or KOH.

[0107] In various embodiments of the present invention, pyrazine yield can be improved by adding NHOH to the amino acid / sugar reaction solution, for example, as shown in operation 122 of Figure 2. The NHOH / sugar molar ratio can have a dramatic effect on pyrazine yield. For example, a sugar to NHOH molar ratio 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), followed by heat treatment, can produce a pyrazine-enriched formulation. In some embodiments, aqueous NHOH can be added slowly to the amino acid / sugar solution during the reaction.

[0108] Different sugars and amino acids affect the type of pyrazine formed. See, for example, Coleman and Steichen, 2006, "Sugar and selected amino acid influences on the structure of pyrazines in microwave heat-treated formulations," J. Sci. Food Agric., 86, 380-391, incorporated herein by reference in its entirety. For example, leucine and valine produce more branched pyrazines with highly substituted subchains 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 pyrazines may be a result of the amino acids used in the reaction. Therefore, it may be advantageous to select amino acids with branched and highly substituted subchains. With regard to sugars, rhamnose may be the ideal sugar for pyrazine formation, followed by fructose and glucose.

[0109] After the reaction, the pyrazines can optionally be isolated from the reaction products using simple distillation, rotary evaporation, or other separation techniques known in the art, for example, as shown in operation 126 in Figure 2. In some embodiments, rotary evaporation can be a preferred isolation technique in a scale-up process to derive tobacco-derived pyrazines.

[0110] Use of substituted pyrazines in tobacco products As mentioned above, pyrazines produced according to the present invention may be useful, for example, as components (e.g., flavorings) included in tobacco products. The tobacco products to which the materials of the present invention may be added vary and may include any product configured or adapted to deliver tobacco or certain components thereof to a user of the product. Exemplary tobacco products include smoking articles (e.g., cigarettes), smokeless tobacco products, and aerosol-generating devices that contain tobacco or other plant material that is not combusted during use.

[0111] In various embodiments of the present invention, pyrazines can be incorporated into a smoking article in the form of a flavoring agent in a tobacco composition and / or a filter element of the smoking article. For example, pyrazines can be incorporated into the top dressing or casing of a tobacco product. Referring to FIG. 6, a smoking article 10 is shown in the form of a cigarette and having certain representative components of a smoking article that can contain a product derived from a cellulosic sugar material of the present invention. The cigarette 10 includes a generally cylindrical rod 12 of a charge or roll of smokable filler material (e.g., about 0.3 to about 1.0 g of smokable filler material, such as tobacco material) contained in a surrounding wrapping material 16. The rod 12 is conventionally referred to as a "tobacco rod." The ends of the tobacco rod 12 are open, exposing the smokable filler material. The cigarette 10 includes an optional band 22 (e.g., a printed coating containing a film-forming agent, such as starch, ethyl cellulose, or sodium alginate) applied to the wrapping material 16, surrounding the cigarette rod transversely to the cigarette's longitudinal axis. The band 22 may be printed on the inside surface of the wrapper (ie, facing the smokable filler material) or, less preferably, on the outside surface of the wrapper.

[0112] The tobacco rod 12 has a lighting end 18 at one end and a filter element 26 disposed at the mouth end 20. The filter element 26 is disposed adjacent to one end of the tobacco rod 12, with the ends of the filter element and the tobacco rod axially aligned and preferably abutting each other. The filter element 26 may have a generally cylindrical shape, and its diameter may be essentially equal to the diameter of the tobacco rod. The ends of the filter element 26 allow air and smoke to pass through the filter element. A plug wrap 28 encases the filter element, and tipping material (not shown) encases the plug wrap and a portion of the outer wrapping material 16 of the rod 12, thereby securing the rod to the filter element 26.

[0113] Filter elements of the present invention typically comprise a plurality of longitudinally extending segments. Each segment can have a variety of properties and can comprise a variety of materials capable of filtering or adsorbing particulate matter and / or gas phase compounds. Typically, filter elements of the present invention comprise two to six segments, and more often two to four segments. In a preferred embodiment, the filter element comprises a mouth-end segment, a tobacco-end segment, and a compartment therebetween. This filter arrangement is sometimes referred to as a "compartment filter" or a "plug / space / plug" filter. As described in more detail below, the compartment can be divided into two or more compartments.

[0114] In various embodiments, the filter element can include a sorbent in the form of an activated carbon material, which can remove at least one gas-phase component of mainstream smoke entrained in the filter element. In certain embodiments, the filter element 26 can include vent holes 30 extending through the tipping paper (not shown) and plug wrap 28, thereby air diluting the mainstream smoke. The vent holes 30 can be configured as a single row of perforations extending circumferentially around the filter element 26, or can include multiple rows of perforations. As will be appreciated, the exact number and size of the vent holes 30 will vary depending on the level of air dilution desired.

[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 flavorings in smokeless tobacco formulations. The form of the smokeless tobacco products of the present invention can vary. In one particular embodiment, the product is in the form of a snus-type product containing particulate tobacco material and a flavoring containing pyrazines obtained by the methods of the present invention. The manner and method of formulating snus-type tobacco formulations will be apparent to those skilled in the art of snus tobacco product manufacturing. For example, as shown in FIG. 7 , an exemplary pouch product 300 can include an outer water-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 water-permeable pouch and the type and nature of the composition adapted for oral use shown herein are not intended to be limiting.

[0116] In various embodiments, a moisture-permeable packet or pouch can serve as a container for use of a composition therein. The composition / structure of a packet or pouch, such as container pouch 320 in the embodiment shown in FIG. 7, can be modified as described herein. For example, suitable packets, pouches, or containers of the type used to manufacture smokeless tobacco products that can be modified according to 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. A pouch-type product similar in shape and configuration to various embodiments of the pouch product described herein is commercially available as ZONNIC (sold by Niconovum AB). Additionally, pouch-type products generally similar in shape and form to various embodiments of the pouch products are described in Example 1 of PCT WO2007 / 104573 to Axelsson et al., which is incorporated herein by reference, as snuff bag compositions E-J, which are produced using excipient ingredients and processing conditions that can be used to produce the pouch products described herein.

[0117] The amount of material contained in each pouch may vary. In smaller embodiments, the dry weight of material in each pouch is at least about 50 mg to about 150 mg. In larger embodiments, the dry weight of 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 flavoring member disposed therein, as described in more detail in U.S. Patent No. 7,861,728 to Holton, Jr. et al., which is incorporated herein by reference. The flavoring member can include a flavoring agent containing a pyrazine derived by the method of the present invention, as described above. Other components can be included within each pouch, as desired. For example, at least one flavored strip, piece, or sheet of flavored water-dispersible or water-soluble material (e.g., a breath-freshening 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, U.S. Patent No. 6,887,307 to Scott et al., U.S. Patent No. 6,923,981 to Leung et al., and the types of materials and techniques described in The EFSA Journal (2004) 85, 1-32, which are incorporated herein by reference.

[0119] In various embodiments, the outer water-permeable pouch can comprise PLA or other pouch materials known in the art. A description of the various components of snus-type products and their components is also found in U.S. Patent Application Publication No. 2004 / 0118422 to Lundin et al., which is incorporated herein by reference. See also, 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, as well as U.S. Patent Application Publication No. 2005 / 0061339 to Hansson et al., each of which is incorporated herein by reference. See also pouches of the type described in U.S. Patent No. 5,167,244 to Kjerstad et al., which is incorporated herein by reference. Snus-type products can be manufactured using equipment such as that available from Merz Verpackungmaschinen GmBH as SB 51-1 / T, SBL 50, and SB 53-2 / T. Snus pouches can be provided as individual pouches, or multiple pouches (e.g., 2, 4, 5, 10, 12, 15, 20, 25, or 30 pouches) can be bonded or linked together (e.g., end to end) so that single pouches or individual portions can be easily removed from a one-piece strand or matrix of pouches for use.

[0120] The present invention is not limited to snus-type smokeless tobacco products. For example, the mixture of tobacco material and flavoring agent containing at least one pyrazine obtained by the method described herein can also be incorporated into various smokeless tobacco forms, such as loose moist snuff, loose dry snuff, chewing tobacco, pelletized tobacco pieces, extruded tobacco strips or pieces, pulverized pieces or finely ground or crushed agglomerates of components, flaked pieces (which can be formed, for example, by agglomerating tobacco formulation components in a fluidized bed), molded tobacco pieces (formed in common shapes, for example, coins, cylinders, beans, cubes, etc.), tobacco-containing gum pieces, products comprising a mixture of edible materials combined with tobacco pieces and / or tobacco extract, products comprising tobacco (for example, in the form of tobacco extract) supported on a solid inedible substrate, etc. For example, the smokeless tobacco product can have the form of compressed tobacco pellets, multi-layered extruded pieces, extruded or formed rods or sticks, compositions having one type of tobacco formulation surrounded by a different type of tobacco formulation, rolls of tape-like film, water-soluble or water-dispersible films or strips (see, e.g., Chan et al., U.S. Patent Application Publication No. 2006 / 0198873), or an encapsulant material having an outer shell (which can be, e.g., transparent, colorless, translucent, or essentially darkly colored, flexible, or rigid) and an inner region having tobacco or a tobacco flavor (e.g., a form of tobacco-containing Newtonian or thixotropic fluid).

[0121] In some embodiments, the smokeless tobacco products of the present invention can be in the form of lozenges, tablets, microtabs, or other tablet-type products (see, for example, U.S. Patent No. 4,967,773 to Shaw; U.S. Patent No. 5,110,605 to Acharya; U.S. Patent No. 5,733,574 to Dam; U.S. Patent No. 6,280,761 to Santus; U.S. Patent No. 6,676,959 to Andersson et al.; U.S. Patent No. 6,248,760 to Wilhelmsen et al.; and U.S. Patent Application Publication No. 2004-2009444, all of which are incorporated by reference). See U.S. Patent Application Publication No. 01 / 0016593 to Liu et al.; U.S. Patent Application Publication No. 2004 / 0101543 to Mcneight et al.; U.S. Patent Application Publication No. 2006 / 0120974 to Mcneight et al.; U.S. Patent Application Publication No. 2008 / 0020050 to Chau et al.; U.S. Patent Application Publication No. 2009 / 0081291 to Gin et al.; and U.S. Patent Application Publication No. 2010 / 0004294 to Axelsson et al., for types of lozenge formulations and techniques for formulating or manufacturing lozenges.

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

[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 a flavoring in an electronic smoking article. Numerous smoking products, flavor generators, and medicinal inhalers have been proposed that utilize electrical energy to vaporize or heat volatile substances or that attempt to provide the sensation of cigarette, cigar, or pipe smoking without burning tobacco to a significant extent. See, for example, the various alternative smoking articles, aerosol delivery devices, and heat sources described in the background art 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., which are incorporated by reference in their entireties.

[0124] An exemplary embodiment of an 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 encloses a reservoir housing 244 that is in fluid communication with a liquid transport element 236 suitable for wicking or otherwise transporting the aerosol precursor composition stored in the reservoir housing 244 to a heater 234. An opening 228 can be present in the cartridge shell 203 to allow for the evacuation of aerosol formed from the cartridge 204. Such components are representative of components that may be present in a cartridge and are not intended to limit the scope of cartridge components encompassed by the present disclosure. The cartridge 204 can be adapted to engage the control body 202 via a press-fit engagement between the control body protrusion 224 and the cartridge receptacle 240. Such engagement may facilitate a stable coupling between the control body 202 and the cartridge 204, as well as establish an electrical connection between the battery 210 and control component 206 in the control body and the heater 234 in the cartridge. The cartridge 204 may also include one or more electronic components 250, which may include ICs, memory components, sensors, etc. The electronic components 250 may be adapted to communicate with the control component 206. The various components of an electronic smoking apparatus according to the present disclosure may be selected from those described in the art and commercially available.

[0125] In various embodiments, the aerosol precursor composition can include a flavoring agent containing at least one pyrazine derived according to the methods 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 incorporated herein by reference in their entireties. Other aerosol precursors that can include the tobacco-derived pyrazines described herein include the aerosol precursors incorporated in VUSE® by RJ Reynolds Vapor Company, BLU™ 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 e-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 invention and are not to be construed as limitations thereof. [Example]

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

[0128] Acetoin, ammonium hydroxide (28-30%), leucine, dichloromethane, and phosphoric acid (H3PO4) were obtained from Sigma-Aldrich (St. Louis, MO). F1 protein was obtained from RJ Reynolds Tobacco Co. (Winston-Salem, NC) and hydrolyzed. The weight percentage of hydrolyzed amino acids in all hydrolyzed solutions ranged from 50-55%. All pyrazine synthesis reactions were performed in a 40 mL Parr vessel. For each reaction, 0.8 grams of acetoin was mixed with 1.8 mL of NH4OH and 0.6 mL of H3PO4, and then sufficient hydrolyzed F1 protein (20 mL) was added to achieve a mass of 0.4 grams of amino acid. For example, if 40 grams of F1 protein were hydrolyzed in 1 liter of solution, the weight percentage of amino acids in the solution was equal to 50%, which corresponds to 20 grams of amino acid per liter of solution. To use 0.4 grams of amino acid in the reaction, 20 mL of the above solution was added to the reaction vessel. In some reactions, leucine was used as the amino acid source instead of hydrolyzed F1 protein, and only 20 mL of water was added to adjust the volume. The pH of all solutions was adjusted to 8.0 before starting the reactions. After each reaction was completed, the mixture was extracted with 30 mL of dichloromethane (DCM). 200 μL of the DCM extract was then diluted to 1 mL with DCM and analyzed by GC / MS.

[0129] All GC / MS analyses are performed using an Agilent (Wilmington, DE) 6890 GC equipped with a 5973 Mass Selective Detector (MSD). Separations are obtained using a J&W (Wilmington, DE) DB-WAXTER capillary column (30 m length × 250 μm internal diameter, 0.25 μm film thickness). The following operating parameters are used for each analysis:

[0130] Injection port temperature 260℃ Purge valve 3mL / min Purge time: 1 minute Total flow rate 24mL / min Constant flow rate 1mL / min Injection volume 2 μL, split 1:20 Column oven initial temperature: 50°C Column oven initial time: 3 minutes Column oven ramp rate: 15°C / min Column oven final temperature: 250°C Column oven final time 1 min MSD transfer line temperature 260℃ The MS Wiley library is used to identify each pyrazine.

[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. Greater than 90% of the acetoin is converted to tetramethylpyrazine (TMP). The reaction is then 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 similar to those obtained at 90°C and 12-15 hours.

[0132] Next, an amino acid (leucine) is added to the reaction reagent mixture to determine whether branched pyrazines are synthesized. 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. The reaction is then heated at 120°C for 18 hours, after which the reaction mixture is extracted with 30 mL of DCM and analyzed by GC / MS. TMP(t R =8.2 min) and some acetoin (t R = 6.1 min) is detected. Branched pyrazines are not detected.

[0133] Next, a similar reaction was performed, but instead of leucine and HO, 20 mL of hydrolyzed F1 protein was used. The reaction was carried out in a Parr vessel at 120 °C for 18 hours. After the reaction was cooled, 30 mL of DCM was used to extract the pyrazines. Again, no branched pyrazines were observed, only TMP.

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

[0135] The above reactions demonstrate that the addition of amino acids to the reaction of acetoin with NH4OH does not produce branched pyrazines. Furthermore, acetoin is not a key intermediate in the reaction of sugars with 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 were obtained from Sigma-Aldrich (St. Louis, MO). Fl protein from Nicotiana spp. was obtained from RJ Reynolds Tobacco Co. (Winston-Salem, NC) and hydrolyzed to form amino acids. The weight percentage of hydrolyzed amino acids in all hydrolyzed solutions ranged from 50-55%.

[0138] All pyrazine synthesis reactions are carried out in 40 mL Parr vessels. For each reaction, 1 gram of 1-OH-acetone or 1-OH-2-butanone is mixed with 0.25, 0.5, 1, or 1.25 mL of NH4OH and 10 mL of HO. Each reaction is mixed and heated at different temperatures (100-140°C) for 4-24 hours. The pH level for the majority of reactions is approximately 11 (no adjustment is made). However, for reactions 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. For each extraction, 250 µg of deuterated 2-methylpyrazine is used as an internal standard for all quantifications. For all reactions, the mixture is stirred using a magnetic stirrer during the reaction process.

[0140] All GC / MS analyses were performed using the same instrumentation and operating parameters as those used in Example 1 above. The MS Wiley library was used to identify each pyrazine. For quantitative analysis, pyrazines were quantified using single ion monitoring mode. Each pyrazine was quantified relative to the mass of an 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, and 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; and 2-(2-methylpropyl)-3,5,6-trimethylpyrazine.

[0142] In the first part of this study, reactions are run at two different pH levels (8.0 and 11.0) to determine which pH level gives the greatest yield and 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 HO. The pH level of this reaction is measured to be approximately 11.0. In the second reaction, equal amounts of reactants are mixed, but the pH level is adjusted to 8.0 using concentrated H3PO4. Both reactions are heated at 120°C for 12 hours. The percent yield of pyrazines is higher when the pH level is approximately 11.0. For this reason, the pH level in subsequent experiments is not adjusted; the reactions are run at pH 11 or higher.

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

[0144] The effect of different reaction times (4, 8, 12, 16, and 24 hours) on the synthesis of pyrazine using 1-OH-acetone (1 g) and NHOH (1 g) in a C:N molar ratio of 1:2 in 10 mL of HO is studied. A reaction time of 16 hours gives the maximum yield of pyrazine.

[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 HO on the yield of pyrazine after 12 hours 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 reduces the yield of the reaction by more than 10%.

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

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

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

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

[0150] Addition of isovaleraldehyde to a separate reaction of 1-OH-acetone and NH4OH increases the percent yield of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl)pyrazine from 0 mg to 14 mg and 13 mg, respectively. The overall yield of pyrazines 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. Because the hydrolyzed F1 protein is already in aqueous solution, no water is added to the mixture. For this purpose, 10 mL of hydrolyzed F1 protein containing approximately 0.2 g of amino acids and 10 mL of HO is reacted with 1-OH-acetone and NH4OH in a C:N ratio of 1:2 at 120°C for 16 hours. The yield of 2,5-dimethylpyrazine increases to over 80%, and the yields of 2,5-dimethyl-3-(2-methylbutyl)pyrazine and 2,5-dimethyl-3-(3-methylbutyl)pyrazine increase from 0 mg to over 1 mg. Without being limited by theory, the alkyl moiety of the amino acid is converted to Strecker aldehyde, which reacts with ammonium hydroxide to form an imine, which is then incorporated into the pyrazine structure.

[0152] The effect 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 acids / aldehydes is examined. In these studies, increasing the temperature from 100 to 120°C and increasing the C:N ratio increases the yield of pyrazine.

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

[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 HO at 120°C for 16 hours. No methylpyrazines are formed. All pyrazines formed contain ethyl or higher branched alkanes. However, the yield of pyrazines is not as high as when 1-OH-acetone is used. Pyrazines synthesized from the reaction of 1-OH-2-butanone and NH4OH in a C:N ratio of 1:2 at 120°C for 16 hours include 2,6-diethylpyrazine; 2,5-diethylpyrazine; 2-ethyl-3,5,6-trimethylpyrazine; 3,5(3,6-dimethyl)-2,n-propylpyrazine; 2,5-diethyl-3-methylpyrazine; 2,3-diethyl-5,6-dimethylpyrazine; trans-3-M-2,n-propyl-6(1-butenyl)pyrazine; and 2,5-dimethyl-3-ethylpyrazine.

[0156] As previously mentioned, when 1-OH-acetone is used as the carbon source, pyrazine and methylpyrazine molecules are not produced. When 1-OH-2-butanone is used, pyrazine, methylpyrazine, and dimethylpyrazine molecules are not produced, confirming that the carbon source overwhelmingly controls the structure of pyrazines. The results further demonstrate that the type of pyrazine synthesized can be controlled by changing the carbon source from 1-OH-acetone to 1-OH-2-butanone.

[0157] [Example 5] The reaction of 1-OH-acetone with 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 HO for 16 hours at 120° C. After the reaction is complete, the mixture is cooled and transferred to a glass bottle.

[0159] Note that there is a significant amount of tar-like material at the bottom of the vessel that dissolves only in MeOH. Adding HO on top of this material makes it hard. Concentration is found to play a significant role in the presence or absence of tar-like material. 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 encompass the remaining reactants. For larger-scale reactions, the mass of tar is larger, and at least 100–200 mL of methanol is required to dissolve it.

[0160] After the reaction is complete, the aqueous solution is distilled at 130-140 °C (3 x 375 mL). During each distillation (375 mL), approximately 175 mL of aqueous solution containing various pyrazines (light yellow - total volume approximately 500 mL) is collected. Next, to remove the pyrazines from the water, the distilled material (3 x 175 mL) is combined and subjected to C 18 Pass through a column (15 x 2.5 cm packed with SPE material). 18 After removing the water from the column, the captured pyrazines are eluted using ethanol. The ethanol is then removed using rotary evaporation and vacuum. Because some water is present in the final product, the pyrazines are extracted into MTBE and dried with sodium sulfate. The MTBE is then removed using a rotary evaporator and vacuum. The vial labeled Solution 1 contains the majority of the pyrazines after MTBE removal.

[0161] It is important to note that three pyrazines were not distilled off and remained in the reaction mixture due to their high boiling points, resulting in a lower yield. These pyrazines are identified as 2-(2-methylpropyl)-3,5-dimethylpyrazine (12.57 min), 2,6-dimethyl-3-isobutylpyrazine (12.74 min), and 2(2-methylpropyl)-3,5,6-trimethylpyrazine (12.95 min).

[0162] DCM (200-250 mL) is used to extract the remaining three pyrazines from the reaction solution after distillation. Then, using a rotary evaporator and vacuum, the DCM is removed from the solution, while the resulting dark solution is later transferred to a second vial and labeled Solution 2. Figure 3 shows the chromatogram of this sample.

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

[0164] Solution 1 above was used for gas chromatography-olfactometry (GC / OLF). Pyrazine samples were analyzed using an ODP3 equipped with an Agilent 7890A Series GC with a 5975C MSD and a Gerstel multipurpose sampler with SPME capabilities. To obtain better separation of pyrazines and a suitable analysis time for olfactory analysts, an instrumental method for the samples was developed. The ODP3 transfer line was heated to 260°C. Two drops of sample were pipetted into a 20 mL SPME screw-cap vial to prepare the sample. Empty vials were analyzed before and after the sample. Pyrazines were separated by the gas chromatography column, and a subjective olfactory test was used to detect and evaluate them as they exited the column.The pyrazines identified include 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 These include 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, and trimethyl-1-propenylpyrazine (E)-isomer 2. Note that methylpyrazine compounds were present, but in lower amounts than is customary for conventional sugar carbon source reactions.

[0165] Four taste analysts evaluate each olfactory profile from the ODP portal in four separate sessions. Based on four independent evaluations, each pyrazine is highly positive. Expected descriptors for the flavor of substituted pyrazines are found. Nutty, roasted, toasty, chocolate, peanut, moldy, brown, and complex are commonly used descriptors. These are all positive flavor characteristics.

[0166] [Example 6] α-Hydroxyketone (acetol) is produced from sugars and ultimately used as a carbon source in the sugar-ammonia reaction. Sodium hydroxide is used as a base to optimize various parameters, such as the type of sugar, 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, anhydrous sodium sulfate, hydrochloric acid, methanol, and dichloromethane are obtained from Sigma-Aldrich (St. Louis, MO). Synthesis reactions are performed in 40 mL Parr vessels or open round-bottom flasks. For all pH-controlled reactions, the reactions are performed in round-bottom flasks under reflux at 100 °C. In each reaction, 0.25 grams, 0.5 grams, or 1.0 grams of a different sugar (glucose, fructose, or a mixture of both) are mixed with 25 mL of 0.025 M, 0.05 M, 0.1 M, or 0.2 M NaOH. Each reaction is stirred and heated at different temperatures (90–140 °C) for 1–12 hours. The initial pH level for most reactions is approximately 12, and no adjustments are 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 four times with 8-10 mL of dichloromethane. All four extraction solvents are combined and dehydrated over sodium sulfate.

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

[0169] Use GC / FID to create a calibration curve for quantification of acetol in all reactions. Then, vary the following parameters to determine the optimal conditions for acetol synthesis:

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

[0171] [Table 1]

[0172] In this part of the study, all reactions are carried out under reflux in open round-bottom flasks rather than in closed Parr reactors, as the pH needs to be adjusted during the reaction process. Three different experiments are performed. In the first experiment, the pH is measured every 10 minutes and no pH adjustments are made. In the second experiment, the pH is measured every 10 minutes and, if necessary, adjusted to an initial value (12.0) using 10 M NaOH. In the third experiment, the initial pH is adjusted to 9.0, then the reaction is started and pH measurements are made every 10 minutes (no adjustments).

[0173] pH measurements show that the pH drops from 12.0 to approximately 9 within the first 10 minutes of reaction at 100°C. At the end of 30 minutes, the pH of the reaction is approximately 7-8 and remains constant for the remainder of the reaction time. Experiments to maintain the pH at approximately 12.0 require the addition of approximately 15-20 μL of 10 M NaOH to the reaction every 10 minutes. After 40 minutes, the pH remains constant at approximately 11-12.

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

[0175] [Table 2]

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

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

[0178] [Table 3]

[0179] Four different concentrations of NaOH (0.2, 0.1, 0.05, and 0.025M) are used to prepare acetol. Table 4 below shows the results of this study. As can be seen, when the NaOH concentration is 0.025M, the least amount of acetol is obtained. However, as the concentration increases from 0.025M to 0.1M, the mass of acetol synthesized also increases. As the concentration of base increases to 0.2M, the amount of acetol decreases. Note that the reaction solution smells like burnt sugar compared to other reactions with lower base concentrations.

[0180] [Table 4]

[0181] The effect of temperature on the synthesis of acetol using NaOH and glucose is investigated. For this purpose, various temperatures between 90 and 140°C are investigated. Results are similar at reaction temperatures of 90°C, 100°C, and 120°C, but the reaction at 140°C produces approximately 25% more acetol than the 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 was also investigated. The reaction was carried out using the same conditions, with the reaction time varying from 60 to 720 minutes. Table 6 below shows the results of this study. The results show that there is no significant change in the yield of acetol with increasing reaction time.

[0184] [Table 6]

[0185] In the final part of this study, various reactions are performed using optimized conditions based on the results obtained from the previous reactions. In Reaction A, 1 gram of glucose is reacted with 0.05 M NaOH at 100 °C under reflux while maintaining a constant pH of approximately 11–12. The reaction is continued for 120 minutes until the pH remains constant and no longer changes. The yield of acetol is 6.7 mg. In Reaction B, 0.5 grams of HFTS is reacted with 25 mL of 0.05 M NaOH at 100 °C for 60 minutes in a Parr vessel. The yield of acetol is 3.42 mg. When the same reaction is performed under optimized conditions (0.1 M NaOH and 140 °C for 60 minutes), the yield of acetol increases 100% to 6.69 mg (Rxn D). Similar results are obtained when 0.5 grams of glucose is subjected to the optimized conditions (Rxn C). The yield of acetol increases to 8.2 mg. Using the same reaction conditions but using 50% less glucose (0.25 grams), 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, have been optimized to synthesize acetol from reacting sugars and sodium hydroxide. It has been shown that the highest yield of acetol is obtained when the reaction is carried out at 140 °C for 60 minutes using 0.1 M sodium hydroxide and 0.5 grams of glucose. It should be noted that the pH of the reaction can change rapidly (within 10 minutes) from 12 to 6.5 when the base concentration is low or the sugar concentration is high. It should also be noted that at high temperatures (140 °C), the reaction product contains a small amount of acetoin (along with acetol). It is important to note that hydroxyketone (acetoin) has been prepared from glucose using biotechnological methods in yields exceeding 90%. However, published chemical conversions of glucose to hydroxyketone are much lower, at approximately 9%.

[0188] [Example 7] Pyrazines are produced using glucose as a carbon source.

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

[0190] A publication by R. 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, 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 glucose with a 40% phosphate buffer solution at pH 6.5-6.8 at a temperature of 100-120°C. A higher yield of acetol was obtained at pH 7.0-7.1, and the yield of acetol decreased as the pH decreased.

[0191] This example first describes the preparation of acetol by reaction of 0.1 N NaOH with glucose under optimized conditions. It then demonstrates how acetol can be isolated from the reaction mixture. Finally, it demonstrates that the above mixture can be reacted with NH4OH without isolating acetol acetoin to synthesize different branched pyrazines.

[0192] Glucose, 1-OH-acetone (acetol), 1-OH-2-butanone (acetoin), 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, MO).

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

[0194] The synthesis of pyrazines was carried out in the same Parr vessel. The reaction was carried out by reacting acetol, synthesized from the reaction of sugar with 0.1N NaOH. This reaction involved adding either 0.25, 0.5, or 1 mL of NH4OH per 25 mL of solution. The reaction mixture was then heated at 120°C or 140°C for a period of 17 hours. Each reaction mixture was then cooled, and the pyrazines were extracted and quantified. For each extraction, 0.25 mg of d6-2-methylpyrazine was added as an internal standard, and the solution was extracted with 30–35 mL of DCM. The DCM solution was then dehydrated with sodium sulfate and analyzed by GC / MS for quantification. The extracted ions were used to quantify each pyrazine.

[0195] All GC / MS analyses were performed using the same instrumentation and operating parameters as those used in Example 1 above. The MS Wiley library was used to identify each pyrazine. For quantitative analysis, a calibration curve was constructed using the concentration of acetol and the acetol / acetoin response factor ratio. Note that no acetoin was detected in any of the reactions performed at 90, 100, and 120°C. However, acetoin residues were found in the reaction where the temperature was set to 140°C. Therefore, the acetoin concentration was determined in the reaction, and this value was considered in all calculations.

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

[0197] First, a buffer solution is used to synthesize acetol. Due to the sudden change in solution pH during the reaction of glucose with 0.1N NaOH solution for acetol synthesis, a pH 12 buffer solution (purchased from Sigma-Aldrich) is used in the synthesis of acetol instead of 0.1N NaOH solution. In this study, 25 mL of disodium 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 the concentration of acetol in the reaction product is much higher (2x) when a buffer solution is used to perform the synthesis. Table 8 below shows the results of this study.

[0198] [Table 8]

[0199] When a similar buffer (7.1 grams of NaHPO4 and 1 gram of NaOH mixed in 100 mL of HO, pH approximately 12, buffering capacity 0.05N) was prepared and reacted with glucose at 140°C for 60 minutes, the reaction mixture showed no acetol. When this reaction was repeated three times, acetol was not found in any of the reaction products. However, when a similar buffer with the same pH and lower buffering capacity (0.025N) was used, acetol was found in the reaction products, and the concentration was much higher than when 0.1N NaOH was used. Thus, it is clear that the type, volume, pH, and reaction temperature of the buffer have an effective influence on the synthesis of acetol using glucose.

[0200] Next, we determined the acetol concentration in the reaction of glucose with 40% phosphate buffer at pH 6.5-7.0. 25 mL of 40% potassium phosphate buffer (pH 6.5-6.8) was reacted with 0.5 or 1 gram of glucose for 60 minutes at 140°C using a Parr reactor. Table 9 below shows the mass of acetol obtained from the reaction of 25 mL of 40% phosphate buffer at pH 6.5-6.8 with various concentrations of glucose. Note that other hydroxyketones, such as 3-OH-2-butanone and 1-OH-2-butanone, are also formed in this reaction. The amounts of these hydroxyketones were not measured. Figure 3 shows the GC / MS analysis of glucose reacted with phosphate buffer at 140°C for 60 minutes, followed by extraction with DCM.

[0201] [Table 9]

[0202] Next, the glucose and acetol concentrations of the 300 mL and 1 L reactions are measured. Six grams of glucose are reacted with 300 mL of 0.1 N NaOH at 140°C for 60 minutes. After the reaction is cooled, both the acetol and glucose concentrations in the solution are obtained. A small amount of glucose (0.964 mg / mL) is detected in the reaction. This corresponds to less than approximately 5% of the glucose remaining unreacted. Table 10 below shows the acetol concentration in this reaction. The acetol concentration is determined to be approximately 12 mg per 25 mL. Two additional reactions are performed using a high-pressure reactor, using 1 liter of 0.1 N NaOH and 20 grams of glucose. Both reactions are performed at 130-140°C for 60 minutes. Both the glucose and acetol concentrations are measured. In both 1-liter reactions, the glucose concentrations are 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. Again, the acetol concentration is approximately 12 mg per 25 mL.

[0203] [Table 10]

[0204] Next, acetol is isolated from the reaction of glucose with 0.1N NaOH. Various methods are used to isolate acetol from the reaction of 0.1N NaOH with glucose. Previous results on the isolation of pyrazine by distillation showed that pyrazine, with a boiling point of 140 °C, is isolated from the reaction mixture. Therefore, acetol is isolated from the reaction mixture using a distillation apparatus set at 120–140 °C. To this end, 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 and remaining material are extracted with DCM. GC / FID analysis indicates that only 15–20% of the acetol is distilled, while 80–85% remains in the reaction mixture.

[0205] Similar results are obtained by distilling a reaction mixture of glucose and 40% phosphate buffer at 140 °C for 60 minutes. During this distillation, water is continuously added to the flask during the distillation to maintain a constant volume of the distilled solution. After collecting approximately 10 mL of solution through distillation, approximately 10 mL of HO is added to the distillation flask to replace the lost volume. A total of 4 × 10 mL fractions are collected. Acetol is present in each fraction. Analysis of the remaining reaction mixture with DCM indicates the presence of additional acetol in solution. The distillation can be continued for several hours, allowing the acetol to be isolated.

[0206] In the second method, column chromatography is used to isolate acetol from the reaction mixture. 18Acetol is isolated using a 30 x 2.0 cm glass (or metal) column (only packed up to 12-15 cm) equipped with a column (particle size 40-60 μm and pore size 90 Å). After washing the column with methanol followed by a 0.1% FA solution, 25 mL of the reaction mixture containing acetol is passed through the column. During this isolation, all of the solution is collected. After the solution is forced through the column (fraction 1, 25 mL), 25 mL of deionized HO is used to wash the material off the column (fraction 2). The column is then washed with an additional 25 mL of HO until pure HO is eluted from the column (fraction 3). The column is then dried with N2, and the remaining analytes trapped on the column are eluted with 100% MeOH (fraction 4). All fractions are extracted with DCM and analyzed by GC / FID. The results show that the majority of acetol is eluted by fractions 1 and 2, while fractions 3 and 4 contain no acetol. Here, the reaction mixture is C 18 It should be noted that after passing through the packing, it is very difficult to use the packing to wash another reaction mixture. Cleaning the packing is very difficult. In summary, isolating the hydroxyketone from the reaction product can be timely and expensive.

[0207] Next, pyrazines are synthesized from glucose and the reaction product of sodium hydroxide and NH4OH without first isolating the hydroxyketone. After preparing acetol by reacting 0.5 grams of glucose with 25 mL of 0.1 N NaOH at 140°C for 60 minutes, the reaction mixture is found to contain approximately 10–12 mg of acetol per 25 mL of solution. To this end, 25 mL of the solution is reacted with 0.5 or 1 mL of NH4OH at 120–140°C for 17–18 hours to determine the type and percent yield of pyrazines. Each reaction is repeated twice. Table 11 below lists the pyrazines detected, including their elution times from the GC column. Results are similar when the amount of NH4OH is changed from 0.5 mL to 1 mL.

[0208] [Table 11]

[0209] A similar reaction was performed on a 1-liter scale. Two 1-liter batches of solution were prepared. In each batch, 20 grams of glucose was reacted with 1,000 mL of 0.1 N NaOH solution at 140°C for 60 minutes. Each solution was cooled and the acetol concentration was measured. Next, 20 mL of NH4OH was added to one reaction mixture, and 40 mL of NH4OH was added to the second reaction mixture. Each reaction was heated at 120-130°C for 17 hours with continuous mixing. After the reactions cooled, an internal standard (d6-2-methylpyrazine) was added to 25 mL of each reaction mixture, and the mixture was extracted with DCM to determine the concentration and distribution of each pyrazine. The remaining reaction solutions were distilled and collected separately. Approximately 125 mL of the distilled solution was collected for each 500 mL of reaction mixture. The distilled solution (10 mL) was then extracted with DCM and quantified by GC / MS. The total mass of pyrazines was higher than when the solution was not extracted with DCM, due to the higher concentration of pyrazines in the distilled solution. The distribution of pyrazines is nearly identical in both extracts. Note that when NH4OH is reacted with the reaction mixture of glucose and 0.1 N NaOH, both 2-methylpyrazine and pyrazine are detected in large amounts in all reactions. No pyrazines are detected in the extract of the solution remaining after distillation.

[0210] Next, pyrazines were synthesized from the reaction products of glucose and phosphate buffer with NH4OH without first isolating the hydroxyketone. A 25 mL reaction mixture of phosphate buffer at pH 6.5–6.8 and glucose (0.5 or 1 gram, respectively, solutions A and B) was reacted with 1 mL of NH4OH at 140 °C for 17 hours. After cooling, 0.25 mg of deuterated 2-methylpyrazine (internal standard) was added to each reaction mixture, and the solution was extracted with DCM. GC / MS was used to analyze each extract, and the mass and percent distribution of pyrazines were calculated. Note that these reactions do not synthesize both pyrazine and 2-methylpyrazine. Furthermore, when 1 gram of glucose was reacted with phosphate buffer, the total mass of pyrazines was 20% higher in solution B. Without being 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 a GC / MS analysis of pyrazines extracted using DCM from a reaction mixture of Solution B (1 gram of glucose reacted with 25 mL of 40% phosphate buffer for 60 minutes at 140°C) and 1 mL of NH4OH for 17 hours at 140°C. For comparison, Figure 5 shows a GC / MS analysis of pyrazines extracted using DCM from a reaction mixture of 0.5 grams of glucose and 25 mL of 0.1 N NaOH reacted for 60 minutes at 140°C, 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 a reaction mixture of glucose and 0.1 N NaOH using both distillation and column chromatography. Distillation is time- and energy-consuming, while chromatography can be very expensive. However, it is possible to synthesize pyrazines without isolating acetol. Reacting acetol prepared from the reaction of glucose with 0.1 N NaOH with NH4OH at 140 °C for 17 hours produced an array of pyrazines. The pyrazines were isolated by distillation. Note that pyrazine and 2-methylpyrazine were synthesized in this process. However, when acetol prepared from a mixture of glucose and phosphate buffer was reacted with NH4OH, similar pyrazines were produced in a similar distribution, but no pyrazines or 2-methylpyrazines were detected in the reaction mixture.

[0212]

[0023] Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing description. It is understood, therefore, that the invention is not to be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. 1. A method for forming pyrazines, comprising: obtaining a reaction solution comprising at least one α-hydroxyketone and at least one nitrogen source; 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.

2. The method of claim 1 , wherein the at least one hydroxyketone comprises acetol.

3. The 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; tetramethylpyrazine; 2,5-dimethyl-3-propenylpyrazine; 3. The method of claim 2, wherein the pyrazine is selected from the group consisting of 2,3,5-trimethyl-6-isopropylpyrazine; 2-acetyl-4,5-dimethylpyrazine; 3,5-dimethyl-2-methylpropylpyrazine, and combinations thereof.

4. 10. The method of claim 1, wherein the at least one hydroxyketone comprises acetoin.

5. 5. The method of claim 4, wherein at least one substituted pyrazine is tetramethylpyrazine.

6. 10. The method of claim 1, wherein the at least one hydroxyketone comprises 1-hydroxy-2-butanone.

7. The 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; 7. The method of claim 6, wherein the pyrazine is selected from the group consisting of trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; 2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

8. 2. The method of claim 1, wherein the nitrogen source is selected from the group consisting of amino acids, ammonium ions, and combinations thereof.

9. 10. The method of claim 1, further comprising adding a free amino acid to the reaction solution.

10. 10. The method of claim 1, further comprising adding at least one aldehyde to the reaction solution.

11. 10. The method of claim 1, further comprising isolating the at least one substituted pyrazine from the reaction product.

12. 12. The method of claim 11, wherein the step of isolating 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.

13. 10. The method of claim 1, wherein the reaction temperature is from about 90°C to about 150°C.

14. 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-trimethylpyrazine; 2,3,5-trimethyl-6-ethylpyrazine; 2,6-dimethyl-3-propylpyrazine; 2,5-diethyl-3,6-dimethylpyrazine; 2,6-dimethyl-3-(2-methylbutyl)pyrazine; 2,5-dimethyl-3-(2-methylbutyl)pyrazine; 2,5-dimethyl-3-(3-methylbutyl)pyrazine; 2,5-dimethyl-3-propylpyrazine; 2,5-dimethyl-3-cis-propenylpyrazine; 2-isopropenyl-3,6-dimethylpyrazine; 2-(2-methylpropyl)-3,5-dimethylpyrazine; 2,6-dimethyl-3-isobutylpyrazine; 2-(2-methylpropyl)-3,5,6-trimethylpyrazine, 2,3-dimethylpyrazine; trimethylpyrazine; furaneol, 2,5-dimethyl-3-ethylpyrazine; tetramethylpyrazine; 2,3-diethyl-5-methylpyrazine; 2,5-dimethyl-3-propenylpyrazine; 2,3,5-trimethyl-6-isopropylpyrazine; 2-acetyl-4,5-dimethylpyrazine; 3,5-dimethyl-2-methylpropylpyrazine; 2,6-diethylpyrazine; 2,5-diethylpyrazine; 2-ethyl-3,5,6-trimethylpyrazine; 3,5(3,6-dimethyl)-2,N-propylpyrazine; 2,5-diethyl-3-methylpyrazine; 2,3-diethyl-5,6-dimethylpyrazine; trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; 2. The method of claim 1, wherein the compound is selected from the group consisting of 2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrazine; and 2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

15. 10. The method of claim 1, wherein at least one substituted pyrazine is disubstituted.

16. 10. The method of claim 1, wherein at least one substituted pyrazine is trisubstituted.

17. 10. The method of claim 1, wherein at least one substituted pyrazine is tetrasubstituted.

18. 10. The method of claim 1, wherein the at least one substituted pyrazine comprises at least one substituent having two or more carbon atoms.

19. 10. The method of claim 1, wherein the at least one substituted pyrazine comprises at least one substituent having three or more carbon atoms.

20. 10. The method of claim 1, wherein the reaction product is substantially free of pyrazine and methylpyrazine molecules.

21. 21. The method of any one of claims 1 to 20, further comprising incorporating at least one substituted pyrazine into a tobacco product.

22. 22. The method of claim 21, wherein the tobacco product is a smoking article or a smokeless tobacco product.

23. 1. A method for forming pyrazines, comprising: obtaining a carbon source solution comprising at least one sugar and at least one buffer; mixing the carbon source solution with at least a nitrogen source to form a reaction solution; and heating the reaction solution to 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.

24. 24. The method of claim 23, wherein the at least one sugar is selected from the group consisting of glucose, fructose, rhamnose, and combinations thereof.

25. 24. The method of claim 23, wherein the nitrogen source is selected from the group consisting of amino acids, ammonium ions, and combinations thereof.

26. 24. The method of claim 23, wherein the buffering agent is selected from the group consisting of sodium hydroxide, phosphate buffer, and combinations thereof.

27. 24. The method of claim 23, wherein the buffering agent buffers at a pH range of about 6.5 to about 7.

5.

28. 24. The method of claim 23, further comprising adding ammonium ions to the reaction solution.

29. 24. The method of claim 23, further comprising isolating the at least one substituted pyrazine from the reaction product.

30. 30. The method of claim 29, wherein the step of isolating 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.

31. 24. The method of claim 23, wherein the reaction temperature is from about 90°C to about 150°C.

32. 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-trimethylpyrazine; 2,3,5-trimethyl-6-ethylpyrazine; 2,6-dimethyl-3-propylpyrazine; 2,5-diethyl-3,6-dimethylpyrazine; 2,6-dimethyl-3-(2-methylbutyl)pyrazine; 2,5-dimethyl-3-(2-methylbutyl)pyrazine; 2,5-dimethyl-3-(3-methylbutyl)pyrazine; 2,5-dimethyl-3-propylpyrazine; 2,5-dimethyl-3-cis-propenylpyrazine; 2-isopropenyl-3,6-dimethylpyrazine; 2-(2-methylpropyl)-3,5-dimethylpyrazine; 2,6-dimethyl-3-isobutylpyrazine; 2-(2-methylpropyl)-3,5,6-trimethylpyrazine, 2,3-dimethylpyrazine; trimethylpyrazine; furaneol, 2,5-dimethyl-3-ethylpyrazine; tetramethylpyrazine; 2,3-diethyl-5-methylpyrazine; 2,5-dimethyl-3-propenylpyrazine; 2,3,5-trimethyl-6-isopropylpyrazine; 2-acetyl-4,5-dimethylpyrazine; 3,5-dimethyl-2-methylpropylpyrazine; 2,6-diethylpyrazine; 2,5-diethylpyrazine; 2-ethyl-3,5,6-trimethylpyrazine; 3,5(3,6-dimethyl)-2,N-propylpyrazine; 2,5-diethyl-3-methylpyrazine; 2,3-diethyl-5,6-dimethylpyrazine; trans-3-methyl-2,n-propyl-6(1-butenyl)pyrazine; 24. The method of claim 23, wherein the compound is selected from the group consisting of 2,5,7-trimethyl-6,7-dihydro-5H-cyclopentapyrazine; and 2,5-dimethyl-3-ethylpyrazine; and combinations thereof.

33. 24. The method of claim 23, wherein at least one substituted pyrazine is disubstituted.

34. 24. The method of claim 23, wherein at least one substituted pyrazine is trisubstituted.

35. 24. The method of claim 23, wherein at least one substituted pyrazine is tetrasubstituted.

36. 24. The method of claim 23, wherein the at least one substituted pyrazine comprises at least one substituent having two or more carbon atoms.

37. 24. The method of claim 23, wherein the at least one substituted pyrazine comprises at least one substituent having three or more carbon atoms.

38. 24. The method of claim 23, wherein the reaction product is substantially free of pyrazine and methylpyrazine molecules.

39. 39. The method of any one of claims 23 to 38, further comprising incorporating at least one substituted pyrazine into a tobacco product.

40. 40. The method of claim 39, wherein the tobacco product is a smoking article or a smokeless tobacco product.

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