REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (C149770060WO00-SEQ-WWZ.xml; Size: 14,268 bytes; and Date of Creation: July 13, 2022) is herein incorporated by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

Certain substituted pyrazines (e.g., alkylpyrazines (AlkPyrs), acylpyrazines (AcylPyrs), and alkyl/alkoxypyrazines (Alk/AlkO-Pyrs)) are useful compounds. Some of the substituted pyrazines are distributed in nature and have been studied, ^{1 4} isolated, and characterized from plants, ⁵ insects, ^6,7 fermentation broth, ⁸ coffee, roasted nuts, cereals, meat products, and wine. ^{9 13} Some of AlkPyrs and Alk/AlkO-Pyrs are used to flavor chocolate, roasted nuts, chocolate, and meat because of their aromas perceived as, for example, coffee, nutty, roasted, earthy, roasted cocoa, beef-like, and woody. ¹⁴ The growing demand for certain AlkPyrs and Alk/AlkO-Pyrs as additives or ingredients is limited by the low occurrence of substituted pyrazines in plants or animal sources, such as 0.003 - 0.2 pg/100 g in various cocoa bean varieties. ¹⁸ Generally, to solve this supply limitation, the industry looks to organic synthesis to address production bottlenecks. Some AlkPyrs can be formed during the thermal processing of foodstuff, ^{4 14 15} similar to the Maillard reaction, ^16,17 where a diketo compound and amino acid condense to form imine intermediates. There remains a need for improved methods of preparing substituted pyrazines.

SUMMARY OF THE DISCLOSURE

In one aspect, the present disclosure provides methods of producing a substituted pyrazine of Formula (Dl): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof, and optionally a substituted pyrazine of Formula (D2): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof; wherein: each of R ¹ and R ² is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted acyl, substituted or unsubstituted alkoxy, or substituted or unsubstituted carbocyclyl; each of R ³ and R ⁴ is independently hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl; provided that at least one of R ¹ and R ² is not hydrogen.

In certain embodiments, the methods comprise incubating a certain a-keto-acid, a certain aldehyde, and a pyruvate decarboxylase to form a certain a-hydroxyketone. In certain embodiments, the methods further comprise incubating the certain a-hydroxyketone with a certain amine to form a certain substituted dihydropyrazine. In certain embodiments, the methods further comprise incubating the certain substituted dihydropyrazine, a base, and an oxidant to form a certain substituted pyrazine.

In certain embodiments, the methods comprise the steps shown in Figure 1A.

In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence of SEQ ID NO: 1, 2, or 3, or a combination thereof. In certain embodiments, the methods of producing the substituted pyrazine comprise the steps shown in the scheme: The methods described herein may be advantageous over known methods because the former may produce regioselectively enriched (e.g., regio selectively (e.g., isomerically) enriched for the substituted pyrazine of Formula (Dl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, over the substituted pyrazine of Formula (D2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof) substituted pyrazines, whereas the latter may produce substituted pyrazines that are not regioselectively enriched. In certain embodiments, the molar ratio of the substituted pyrazine of Formula (Dl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the substituted pyrazine of Formula (D2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 55:45 and 95:5, inclusive. In certain embodiments, the molar ratio of the substituted pyrazine 10, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the substituted pyrazine 11, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is about 70:30.

The methods described herein may be advantageous over known methods also because the former may be simpler, faster, easier to purify, less expensive, less energy-demanding, higher-yielding, and/or more scalable, may involve fewer steps, lower reaction temperature, fewer types of side products, and/or less amount of side products, and/or may reduce side- product pollution.

In another aspect, the present disclosure provides substituted pyrazines produced by the methods described herein.

In another aspect, the present disclosure provides mixtures comprising: a first substituted pyrazine of Formula (Dl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof; and a second substituted pyrazine of Formula (D2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof; wherein: each of R ¹ , R ² , R ³ , and R ⁴ is as described herein; and the molar ratio of the first substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the second substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 51:49 and 99.9:0.1, inclusive; provided that: at least one of R ¹ and R ² is not hydrogen; and the first substituted pyrazine is different from the second substituted pyrazine.

In another aspect, the present disclosure provides compositions comprising: the substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, or the mixture; and optionally an excipient.

In another aspect, the present disclosure provides kits comprising: the substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, the mixture, or the composition; and instructions for using the substituted pyrazine, tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, co-crystal, mixture, or composition.

In another aspect, the present disclosure provides methods of altering the flavor of a food, drink, or cigarette comprising adding an effective amount of the substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, the mixture, or the composition, to the food, drink, or cigarette, or to a raw or intermediate material for producing the food, drink, or cigarette.

While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that the detailed description presented herein are not intended to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.

Other features and advantages of this disclosure will become apparent in the following detailed description of certain embodiments of the disclosure.

DEFINITIONS

Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 ^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5 ^th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3 ^rd Edition, Cambridge University Press, Cambridge, 1987. Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC), supercritical fluid chromatography (SFC), and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al, Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

Unless otherwise provided, a formula depicted herein includes compounds that do not include isotopically enriched atoms and also compounds that include isotopically enriched atoms. Compounds that include isotopically enriched atoms may be useful as, for example, analytical tools, and/or probes in biological assays.

When a range of values (“range”) is listed, it is intended to encompass each value and sub-range within the range. A range is inclusive of the values at the two ends of the range unless otherwise provided. For example, “an integer between 1 and 4” refers to 1, 2, 3, and 4. For example, “Ci-6 alkyl” is intended to encompass Ci, C2, C3, C4, C5, Ce, Ci-6, C1-5, Cm*, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.

“Alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“Ci-20 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“Ci-12 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“Ci-io alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“Cms alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“Ci- ₆ alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“Ci-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“Cm* alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Ci alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”). Examples of Ci- ₆ alkyl groups include methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (Cs), tertiary amyl (Cs), and n-hexyl (Co) . Additional examples of alkyl groups include n-heptyl (C7), n-octyl (Cs) and the like. Unless otherwise specified, each instance of an alkyl group is independently optionally substituted, e.g., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents. In certain embodiments, the alkyl group is unsubstituted Ci-12 alkyl (e.g., -CH3 (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted 77-propyl (77-Pr), unsubstituted isopropyl (/-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (77-Bu), unsubstituted / _{<? /7} -butyl (tert- Bu or /-Bu), unsubstituted .sec-butyl (.sec-Bu or s-Bu), unsubstituted isobutyl (/-Bu)). In certain embodiments, the alkyl group is substituted Ci-12 alkyl (such as substituted Ci-e alkyl, e.g., -CH ₂ F, -CHF ₂ , -CF ₃ , -CH ₂ CH ₂ F, -CH ₂ CHF ₂ , -CH ₂ CF ₃ , or benzyl (Bn)). The attachment point of alkyl may be a single bond (e.g., as in -CH3), double bond (e.g., as in =CH ₂ ), or triple bond (e.g., as in ºCH). The moieties =CH ₂ and ºCH are also alkyl.

In some embodiments, an alkyl group is substituted with one or more halogens. “Perhaloalkyl” is a substituted alkyl group as defined herein wherein all of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, the alkyl moiety has 1 to 8 carbon atoms (“Ci-s perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 6 carbon atoms (“Ci- ₆ perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 4 carbon atoms (“Ci^ perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 3 carbon atoms (“C1-3 perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 2 carbon atoms (“C1-2 perhaloalkyl”). In some embodiments, all of the hydrogen atoms are replaced with fluoro. In some embodiments, all of the hydrogen atoms are replaced with chloro. Examples of perhaloalkyl groups include -CF3, -CF ₂ CF ₃ , -CF ₂ CF ₂ CF ₃ , -CCI3, -CFCk, -CF2CI, and the like.

“Alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more (e.g., two, three, or four, as valency permits) carbon- carbon double bonds, and no triple bonds (“C _2-2 o alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C _2-i o alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C _2- 9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C _2-8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C _2- 7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C _2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C _2- s alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C _2- 4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C ₂ alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C _{2 4} alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C ₄ ), and the like. Examples of C _2-6 alkenyl groups include the aforementioned C _2-4 alkenyl groups as well as pentenyl (C ₅ ), pentadienyl (C ₅ ), hexenyl (Ce), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (Cs), octatrienyl (Cs), and the like. Unless otherwise specified, each instance of an alkenyl group is independently optionally substituted, e.g., unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is unsubstituted C _2-10 alkenyl. In certain embodiments, the alkenyl group is substituted C _2-10 alkenyl. In an alkenyl group, a C=C double bond for which the stereochemistry is not specified (e.g. , -CH=CHCH ₃ or ) may be in the (E)- or (Z)-configuration.

“Alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more (e.g., two, three, or four, as valency permits) carbon- carbon triple bonds, and optionally one or more double bonds (“C _2-20 alkynyl”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C _2-10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C _2-9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C _2-8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C _2-7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C _2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C _2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C ₂ alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1- butynyl). Examples of C _2-4 alkynyl groups include ethynyl (C ₂ ), 1-propynyl (C ₃ ), 2-propynyl (C ₃ ), 1-butynyl (C ₄ ), 2-butynyl (C ₄ ), and the like. Examples of C _2-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C ₇ ), octynyl (Cg), and the like. Unless otherwise specified, each instance of an alkynyl group is independently optionally substituted, e.g., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is unsubstituted C _2-10 alkynyl. In certain embodiments, the alkynyl group is substituted C _2-10 alkynyl.

“Carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 13 ring carbon atoms (“C _3-13 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C _3-8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C _3-7 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C _5-10 carbocyclyl”). Exemplary C _3-6 carbocyclyl groups include cyclopropyl (C ₃ ), cyclopropenyl (C ₃ ), cyclobutyl (C ₄ ), cyclobutenyl (C ₄ ), cyclopentyl (C ₅ ), cyclopentenyl (C ₅ ), cyclohexyl (Oό), cyclohexenyl (C6), cyclohexadienyl (Ce). and the like. Exemplary C3-8 carbocyclyl groups include the aforementioned C _3-6 carbocyclyl groups as well as cycloheptyl (C ₇ ), cycloheptenyl (C ₇ ), cycloheptadienyl (C ₇ ), cycloheptatrienyl (C ₇ ), cyclooctyl (Cs), cyclooctenyl (Cs), bicyclo[2.2.1]heptanyl (C ₇ ), bicyclo[2.2.2]octanyl (Cs), and the like. Exemplary C _3-10 carbocyclyl groups include the aforementioned C _3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C ₁₀ ), cyclodecenyl (C ₁₀ ), octahydro-li/- indenyl (C ₉ ), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged, or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”). Carbocyclyl can be saturated, and saturated carbocyclyl is referred to as “cycloalkyl.” In some embodiments, carbocyclyl is a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms (“C _3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C _3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C _3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C _5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C _5-10 cycloalkyl”). Examples of C _5-6 cycloalkyl groups include cyclopentyl (C ₅ ) and cyclohexyl (C ₅ ). Examples of C _3-6 cycloalkyl groups include the aforementioned C _5-6 cycloalkyl groups as well as cyclopropyl (C ₃ ) and cyclobutyl (C4). Examples of C3-8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C ₇ ) and cyclooctyl (Cs). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is unsubstituted C _3-10 cycloalkyl. In certain embodiments, the cycloalkyl group is substituted C _3-10 cycloalkyl. Carbocyclyl can be partially unsaturated. Carbocyclyl may include zero, one, or more (e.g., two, three, or four, as valency permits) C=C double bonds in all the rings of the carbocyclic ring system that are not aromatic or heteroaromatic. Carbocyclyl that includes one or more (e.g., two or three, as valency permits) C=C double bonds in the carbocyclic ring is referred to as “cycloalkenyl.” Carbocyclyl that includes one or more (e.g. , two or three, as valency permits) CºC triple bonds in the carbocyclic ring is referred to as “cycloalkynyl.” “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently optionally substituted, e.g., unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents. In certain embodiments, the carbocyclyl group is unsubstituted C3-10 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C3-10 carbocyclyl. In certain embodiments, the carbocyclyl is substituted or unsubstituted, 3- to 7-membered, and monocyclic. In certain embodiments, the carbocyclyl is substituted or unsubstituted, 5- to 13- membered, and bicyclic.

In some embodiments, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms (“C3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10 cycloalkyl”). Examples of C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-6 cycloalkyl groups include the aforementioned C5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (Cs). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is unsubstituted C3-10 cycloalkyl. In certain embodiments, the cycloalkyl group is substituted C3-10 cycloalkyl. In certain embodiments, the carbocyclyl includes oxo substituted thereon.

“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 13-membered non aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-13 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged, or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”). A heterocyclyl group can be saturated or can be partially unsaturated. Heterocyclyl may include zero, one, or more (e.g., two, three, or four, as valency permits) double bonds in all the rings of the heterocyclic ring system that are not aromatic or heteroaromatic. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently optionally substituted, e.g., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is unsubstituted 3- 10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl is substituted or unsubstituted, 3- to 7-membered, and monocyclic. In certain embodiments, the heterocyclyl is substituted or unsubstituted, 5- to 13-membered, and bicyclic. In certain embodiments, the heterocyclyl includes oxo substituted thereon.

In some embodiments, a heterocyclyl group is a 5 10 membered non aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5 8 membered non aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5 6 membered non aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing one heteroatom include aziridinyl, oxiranyl, or thiiranyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6- membered heterocyclyl groups containing one heteroatom include piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include azocanyl, oxecanyl, and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a Ce aryl ring (also referred to herein as a 5,6-bicyclic heterocyclic ring) include indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6- membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

“Aryl” refers to a radical of a monocyclic or polycyclic ( e.g ., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 p electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-i4 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C ₆ aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“Cio aryl”; e.g., naphthyl such as 1- naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“Ci4 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently optionally substituted, e.g., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is unsubstituted C6-14 aryl. In certain embodiments, the aryl group is substituted C6-14 aryl.

“Heteroaryl” refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 p electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continues to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, e.g., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1 4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1 4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently optionally substituted, e.g., unsubstituted (“unsubstituted heteroaryl”) or substituted (“substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is substituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is 5-6 membered, monocyclic. In certain embodiments, the heteroaryl group is 8-14 membered, bicyclic.

Exemplary 5-membered heteroaryl groups containing one heteroatom include pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5- membered heteroaryl groups containing three heteroatoms include triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

“Partially unsaturated” refers to a group that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation but is not intended to include aromatic groups ( e.g ., aryl or heteroaryl groups) as herein defined. Likewise, “saturated” refers to a group that does not contain a double or triple bond, i.e., contains all single bonds.

In some embodiments, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted,” whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound. The present disclosure contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.

Exemplary carbon atom substituents include halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -NR ^bb C(=NR ^bb )N(R ^bb ) ₂ , -C(=0)NR ^bb S0 ₂ R ^aa , -NR ^hb S0 ₂ R ^aa , -S0 ₂ N(R ^bb ) ₂ , -S0 ₂ R ^aa , -S0 ₂ 0R ^aa , -OSCkR^, -S(=0)R ^aa , -0S(=0)R ^aa , -Si(R ^aa ) ₃ , -OSi(R ^aa ) ₃ , -C(=S)N(R ^bb ) ₂ , -C(=0)SR ^aa , -C(=S)SR ^aa , -SC(=S)SR ^aa , -SC(=0)SR ^aa , -0C(=0)SR ^aa , -SC(=0)0R ^aa , -SC(=0)R ^aa , -P(=0)(R ^aa ) ₂ , -P(=0)(0R ^cc ) ₂ , -0P(=0)(R ^aa ) ₂ , -0P(=0)(0R ^cc ) ₂ , -P(=0)(N(R ^bb ) ₂ ) ₂ , -0P(=0)(N(R ^bb ) ₂ ) ₂ , -NR ^bb P(=0)(R ^aa ) ₂ , -NR ^bb P(=0)(0R ^cc ) ₂ , -NR ^bb P(=0)(N(R ^bb ) ₂ ) ₂ , -P(R ^CC ) ₂ , -P(OR ^cc ) ₂ , -P(R ^CC ) ₃ ⁺ X ^“ , -P(OR ^CC ) ₃ ⁺ X ^“ , -P(R ^CC ) ₄ , -P(OR ^CC ) ₄ ,-OP(R ^cc ) ₂ , -OP(R ^CC ) ₃ ⁺ X-, -OP(OR ^cc ) ₂ , -OR(OR ^¥ ) ₃ ⁺ c-, -OP(R ^cc ) ₄ , -OP(OR ^cc ) ₄ , -B(R ^aa ) ₂ , -B(OR ^cc ) ₂ , -BR^OR^), Ci-io alkyl, Ci-io perhaloalkyl, C _2-i o alkenyl, C _2-i o alkynyl, heteroCi-io alkyl, heteroC _2-i o alkenyl, heteroC _2-i o alkynyl, C _3-i o carbocyclyl, 3-14 membered heterocyclyl, Ce-u aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups; wherein X- is a counterion; or two geminal hydrogens on a carbon atom are replaced with the group =0, =S, =NN(R ^bb ) _¾ =NNR ^bb C(=0)R ^aa , =NNR ^bb C(=0)0R ^aa , =NNR ^bb S(=0) ₂ R ^aa , =NR ^hb , or =NOR ^cc ; each instance of R^ is, independently, selected from Ci-io alkyl, Ci-io perhaloalkyl, C _2-i o alkenyl, C _2-i o alkynyl, heteroCi-io alkyl, heteroC _2-i oalkenyl, heteroC _2-i oalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two R ^aa groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups; each instance of R ^bb is, independently, selected from hydrogen, -OH, -OR ^aa , -N(R ^CC ) ₂ , -CN, -C(=0)R ^aa , -C(=0)N(R ^cc ) ₂ , -C0 ₂ R ^aa , -S0 ₂ R ^aa , -C(=NR ^tc jOR ^aa , -0(=NR ^¥ )N(B ^e ¾, -S0 ₂ N(R ^cc ) ₂ , -SChR^, -S0 ₂ 0R ^cc , -SOR^, -C(=S)N(R ^cc ) ₂ , -C(=0)SR ^cc , -C^SjSR^, -R(=0)(R^) ₂ , -P(=0)(0R ^cc ) ₂ , -P(=0)(N(R ^cc ) ₂ ) ₂ , Ci-10 alkyl, Ci-10 perhaloalkyl, C _2-i o alkenyl, C _2- 10 alkynyl, heteroCi-ioalkyl, heteroC _2-i oalkenyl, heteroC _2-i oalkynyl, C _3-i o carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl, or two R ^bb groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups; wherein X- is a counterion; each instance of R ^cc is, independently, selected from hydrogen, Ci-10 alkyl, Ci-10 perhaloalkyl, C _2-i o alkenyl, C _2-i o alkynyl, heteroCi-io alkyl, heteroC _2-i o alkenyl, heteroC _2-i o alkynyl, C _3-i o carbocyclyl, 3-14 membered heterocyclyl, Ce-u aryl, and 5-14 membered heteroaryl, or two R ^cc groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl. carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups; each instance of R ^dd is, independently, selected from halogen, -CN, -NO2, -N3, -SO2H, -C(=0)SR ^ee , -C(=S)SR ^ee , -SC(=S)SR ^ee , -P(=0)(0R ^ee ) ₂ , -P(=0)(R ^ee ) ₂ , -0P(=0)(R ^ee ) ₂ , -0P(=0)(0R ^ee )2, Ci-6 alkyl, Ci-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, heteroCi-6alkyl, heteroC2-6alkenyl, heteroC2-6alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^8g groups, or two geminal R ^dd substituents can be joined to form =0 or =S; wherein X- is a counterion; each instance of R ^ee is, independently, selected from Ci- ₆ alkyl, Ci- ₆ perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, heteroCi-6 alkyl, heteroC2-6alkenyl, heteroC2-6 alkynyl, C3-10 carbocyclyl,

C ₆ 10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^8g groups; each instance of R ^ff is, independently, selected from hydrogen, Ci- ₆ alkyl, Ci- ₆ perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, heteroCi- ₆ alkyl, heteroC2-6alkenyl, heteroC2-6alkynyl, C3- 10 carbocyclyl, 3-10 membered heterocyclyl, Ce-io aryl and 5-10 membered heteroaryl, or two R ^ff groups are joined to form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^gg groups; and each instance of R ^gg is, independently, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OCi-6 alkyl, -ON(Ci-e alkyl) ₂ , -N(Ci-e alkyl) ₂ , -N(Ci-e alkyl) ₃ ⁺ X ^“ , -NH(Ci-e alkyl) ₂ ⁺ X ^“ , -NH ₂ (CI-6 alkyl) ⁺ X ^“ , -NH ₃ ⁺ X ^“ , -N(OCi- ₆ alkyl)(Ci- ₆ alkyl), -N(OH)(Ci- ₆ alkyl), -NH(OH), -SH, -SCi-6 alkyl, -SS(Ci-e alkyl), -C(=0)(Ci- ₆ alkyl), -CO2H, -C0 ₂ (Ci- ₆ alkyl), -OC(=0)(Ci- 6 alkyl), -0C0 ₂ (Ci- ₆ alkyl), -C(=0)NH ₂ , -C(=0)N(Ci- ₆ alkyl) ₂ , -OC(=0)NH(Ci-e alkyl), -NHC(=0)( Ci-6 alkyl), -N(Ci-e alkyl)C(=0)( Ci-e alkyl), -NHC0 ₂ (Ci- ₆ alkyl), -NHC(=0)N(Ci- 6 alkyl) ₂ , -NHC(=0)NH(CI-6 alkyl), -NHC(=0)NH ₂ , -C(=NH)0(Ci-e alkyl), -OC(=NH)(Ci-e alkyl), -OC(=NH)OCi-e alkyl, -C(=NH)N(Ci-e alkyl) ₂ , -C(=NH)NH(CI- ₆ alkyl), -C(=NH)NH ₂ , -OC(=NH)N(CI-6 alkyl) ₂ , -OC(NH)NH(CI- ₆ alkyl), -OC(NH)NH ₂ , -NHC(NH)N(CI- ₆ alkyl) ₂ , -NHC(=NH)NH ₂ , -NHS0 ₂ (CI-6 alkyl), -S0 ₂ N(Ci- ₆ alkyl) ₂ , -S0 ₂ NH(Ci- ₆ alkyl), -S0 ₂ NH ₂ , -S0 ₂ Ci-6 alkyl, -S0 ₂ 0Ci- ₆ alkyl, -0S0 ₂ Ci- ₆ alkyl, -SOCi-e alkyl, -Si(Ci- ₆ alkyl) ₃ , -OSi(Ci- ₆ alkyl) ₃ -C(=S)N(CI-6 alkyl) ₂ , C(=S)NH(Ci-e alkyl), C(=S)NH ₂ , -C(=0)S(Ci- ₆ alkyl), -C(=S)SCi- 6 alkyl, -SC(=S)SCi-6 alkyl, -P(=0)(0Ci- ₆ alkyl) ₂ , -P(=0)(Ci-e alkyl) ₂ , -0P(=0)(Ci- ₆ alkyl) ₂ , -0P(=0)(0Ci- ₆ alkyl) ₂ , Ci- ₆ alkyl, Ci- ₆ perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, heteroCi- ₆ alkyl, heteroC _2-6 alkenyl, heteroC _2-6 alkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal R ^gg substituents can be joined to form =0 or =S; wherein X- is a counterion.

In certain embodiments, the carbon atom substituents are independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, -OR ^aa , -SR ^aa , -N(R ^bb ) ₂ , -CN, -SCN, -N0 ₂ , -C(=0)R ^aa , -C0 ₂ R ^aa , -C(=0)N(R ^bb ) ₂ , -0C(=0)R ^aa , -0C0 ₂ R ^aa , -0C(=0)N(R ^bb ) ₂ , -NR ^bb C(=0)R ^aa , -NR ^bb C0 ₂ R ^aa , or -NR ^bb C(=0)N(R ^bb ) ₂ . In certain embodiments, the carbon atom substituents are independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, -OR ^aa , -SR ^aa . -N(R ^bb ) ₂ , -CN, SCN, N0 ₂ , -C(=0)R ^aa , -CChR ²² , -C(=0)N(R ^bb ) ₂ , -0C(=0)R ^aa , -0C0 ₂ R ^aa , -0C(=0)N(R ^bb ) ₂ , -NR ^bb C(=0)R ^aa , -NR ^bh C0 ₂ R ^aa . or -NR ^bb C(=0)N(R ^bb ) ₂ , wherein R ^aa is hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group (e.g., acetamidomethyl, t- Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl) when attached to a sulfur atom; and each R ^bb is independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, or a nitrogen protecting group. In certain embodiments, the carbon atom substituents are independently halogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, -OR ^aa , -SR ^aa , -N(R ^bb ) ₂ , -CN, -SCN, or -N0 ₂ . In certain embodiments, the carbon atom substituents are independently halogen, substituted (e.g., substituted with one or more halogen moieties) or unsubstituted Ci- ₆ alkyl, -OR ^aa , -SR ²²¹ , -N(R ^bb ) ₂ , -CN, -SCN, or-N0 ₂ , wherein R ^aa is hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group (e.g., acetamidomethyl, i-Bu, 3- nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl) when attached to a sulfur atom; and each R ^bb is independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, or a nitrogen protecting group.

A “counterion” or “anionic counterion” is a negatively charged group associated with a positively charged group in order to maintain electronic neutrality. An anionic counterion may be monovalent (i.e., including one formal negative charge). An anionic counterion may also be multivalent (i.e., including more than one formal negative charge), such as divalent or trivalent. Exemplary counterions include halide ions (e.g., F , Cl , Br , G), NCF , CIO _{4 ,} OH-, H ₂ PO _{4 ,} HCO ₃ - _, HSO ₄ , sulfonate ions (e.g., methanesulfonate, trifluoromethanesulfonate, p- toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene- 1 -sulfonic acid-5-sulfonate, ethan-1 -sulfonic acid-2-sulfonate, and the like), carboxylate ions (e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, and the like), BF4-, PF4 , PFY, , AsFY, , SbFV, , B[3,5-(CF ₃₎₂ C6H ₃ j4j . B(C6F5)4 ^_ , BPI14 , Al(OC(CF ₃ ) ₃ ) ₄ -, and carborane anions (e.g., CB 11 H 12 or (HCBi iPvlcsBn,) ). Exemplary counterions which may be multivalent include C0 ₃ ^2- , HPO ₄ ^2- , PO ₄ ^3- _, B ₄ O ₇ ^2- , SO ₄ ^2- , S ₂ 0 ₃ ^2- , carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates, aspartate, glutamate, and the like), and carboranes.

“Halo” or “halogen” refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include hydrogen, -OH, -OR ^aa , -N(R ^CC )2, -CN, -C(=0)R ^aa , -C(=0)N(R ^CC )2, -C0 ₂ R ^aa , -S0 ₂ R ^aa . -C(=NR ^bb )R ^aa , -C(=NR ^cc )OR ^aa , -C(=NR ^CC )N(R ^CC ) ₂ , -S0 ₂ N(R ^cc ) ₂ , -S0 ₂ R ^cc , -S0 ₂ 0R ^cc , -SOR ^aa , -C(=S)N(R ^cc ) ₂ , -C(=0)SR ^cc , -C(=S)SR ^cc , -P(=0)(0R ^cc ) ₂ , -P(=0)(R ^aa ) ₂ , -P(=0)(N(R ^cc ) ₂ ) ₂ , Ci-10 alkyl, Ci-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroCi-ioalkyl, heteroC ₂ -ioalkenyl, heteroC ₂ -ioalkynyl, C ₃ -io carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two R ^cc groups attached to an N atom are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups, and wherein R ^aa , R ^bb , R ^cc and R ^dd are as defined above.

In certain embodiments, the nitrogen atom substituents are independently substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, -C(=0)R ^aa , -C0 ₂ R ^aa , -C(=0)N(R ^bb ) ₂ , or a nitrogen protecting group. In certain embodiments, the nitrogen atom substituents are independently substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, -C(=0)R ^aa , -C0 ₂ R ^aa , -C(=0)N(R ^bb ) ₂ , or a nitrogen protecting group, wherein R ^aa is hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, or an oxygen protecting group when attached to an oxygen atom; and each R ^bb is independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, or a nitrogen protecting group. In certain embodiments, the nitrogen atom substituents are independently substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl or a nitrogen protecting group.

In certain embodiments, the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen protecting groups include -OH, -OR ^aa , -N(R ^CC ) ₂ , -C(=0)R ^aa , -C(=0)N(R ^cc ) ₂ , -C0 ₂ R ^aa , -S0 ₂ R ^aa , -C(=NR ^cc )R ^aa , - C(=NR ^cc )OR ^aa , -C(=NR ^CC )N(R ^cc ) ₂ , -S0 ₂ N(R ^cc ) ₂ , -S0 ₂ R ^cc , -S0 ₂ 0R ^cc , -SOR ^aa , -C(=S)N(R ^CC ) ₂ , - C(=0)SR ^cc , -C(=S)SR ^CC , Ci-io alkyl (e.g., aralkyl, heteroaralkyl), C _2-i o alkenyl, C _2-i o alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R ^dd groups, and wherein R ^aa , R ^bb , R ^cc , and R ^dd are as defined herein. Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 ^rd edition, John Wiley & Sons, 1999, incorporated herein by reference.

Amide nitrogen protecting groups (e.g., -C(=0)R ^aa ) include formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3- phenylpropanamide, picolinamide, 3-pyridylcarboxamide, A ben zo y 1 p h e n y 1 a 1 a n y 1 derivative, benzamide, p-phenylbenzamide, o-n i t ro phenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (A’-dithiobenzyloxyacylamino)acetamide, 3-(/?-hydroxyphenyl)propanamide,

3 (o nitrophenyljpropanamide, 2 methyl 2 (o nitrophenoxyjpropanamide, 2 methyl 2 (o phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o- nitrocinnamide, A-accty 1 meth ion i ne. o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

Carbamate nitrogen protecting groups (e.g., -C(=0)0R ^aa ) include methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2.7— cli— /— huty 1— [ 9— ( 10, 10-dioxo-l 0, 10, 10, 10- tetrahydrothioxanthyl)] methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2- phenylethyl carbamate, l-(l-adamantyl)-l-methylethyl carbamate (Adpoc), l,l-dimethyl-2- haloethyl carbamate, l,l-dimethyl-2,2-dibromoethyl carbamate (DB-/-BOC), 1,1-dimethyl- 2,2,2-trichloroethyl carbamate (TCBOC), 1 -methyl- l-(4-biphenylyl)ethyl carbamate (Bpoc),

1 — ( 3.5 -cl i— / — hu ty lpheny 1) — 1 — m e t h y 1 e t h y 1 carbamate (i-Bumeoc), 2-(2’- and 4’-pyridyl)ethyl carbamate (Pyoc), 2-(/V,A-dicyclohexylcarboxamido)ethyl carbamate, /-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, A-h y dro x y pi peri di n y 1 carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p- methoxybenzyl carbamate (Moz), p-nitrobcnzyl carbamate, p-bromobcnzyl carbamate, p- chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4- methylsulfmylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2- methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(l,3-dithianyl)] methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), l,l-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p- (dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6- chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o- nitrophenyl)methyl carbamate, t- amyl carbamate, .S' benzyl thiocarbamate, p cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, a (N,N dimethylcarboxamidojbenzyl carbamate, 1,1 dimethyl 3 (N.N dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobomyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p ’-methoxyphenylazo)benzyl carbamate, 1- methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-l-cyclopropylmethyl carbamate, l-methyl-l-(3,5-dimethoxyphenyl)ethyl carbamate, 1 -methyl- \-{p- phenylazophenyl)ethyl carbamate, 1-methyl-l-phenylethyl carbamate, l-methyl-l-(4- pyridyl)ethyl carbamate, phenyl carbamate, p-(p he n y 1 azo ) ben zy 1 carbamate, 2,4,6-tri-i- butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

Sulfonamide nitrogen protecting groups (e.g., -S(=0) ₂ R ^aa ) include p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6- trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), b- trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4’,8’- dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include phenothiazinyl-(10)-acyl derivative, N’-p- toluenesulfonylaminoacyl derivative, /V’-phenylaminothioacyl derivative, N- benzoylphenylalanyl derivative, /V-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2- one, /V-phthalimide, /V-dithiasuccinimide (Dts), /V-2,3-diphenylmaleimide, N-2,5- dimethylpyrrole, /V-l .1 ,4,4-tctramcthyldisilylazacyclopcntanc adduct (STABASE), 5- substituted 1 ,3-dimcthyl-l ,3,5-triazacyclohcxan-2-onc. 5-substituted 1 ,3-dibcnzyl-l ,3,5- triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, A-mcthyl amine, A-allylamine, A-[ 2-(tri methyl si lyl iethoxyj methy lamine (SEM), /V-3-acetoxypropylamine, A-( 1 -isopropyl-4- nitro-2-oxo-3-pyrrolin-3-yl)amine, quaternary ammonium salts, A-benzylamine, /V-di id- met h o x y p h e n y 1 ) m e t h y 1 a m i n e , A-5-dibenzosuberylamine, /V-triphenylmethylamine (Tr), A-[(4- methoxyphenyl)diphenylmethyl]amine (MMTr), A-9-p h c n y 1 P uo re n y 1 a m i n e (PhF), A-2,7- dichloro-9-fluorenylmethyleneamine, /V-fcrroccny 1 methy I am i no (Fcm), A-2-picolylamino A’- oxide, /V-l,l-dimethylthiomethyleneamine, A'-benzy 1 ideneam i ne. N-p- methoxybenzylideneamine, A d i p h e n y 1 m e t h y 1 e n e a m i n c , A [(2 pyridyl)mesityl] methy leneamine, A-(A’,A’-di methy laminomcthylcncjaminc, N, N - isopropylidenediamine, N-p-n i t ro b e n zy 1 i de n c a m i n e . /V-salicylideneamine, A- 5- chlorosalicylideneamine, N (5 chloro 2 hydroxyphenyljphenylmethyleneaminc, A cyclohexylideneamine, A-( 5.5 -d i m c t h y 1 -3 -o x o- 1 -c y c I o h e x c n y 1 ) a m i n c , A-boranc derivative, A-diphenylborinic acid derivative, A-[phenyl(pentaacylchromium- or tungsten)acyl]amine, A- copper chelate, A-zinc chelate, A-nitroamine, A-nitrosoamine, amine A-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o n i t ro b en ze n e s u 1 fe n a m i de (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nit ro^4- m c t h o x y be n zen c s u 1 fe n a m i de . triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, a nitrogen protecting group is Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts.

In certain embodiments, the oxygen atom substituents are independently substituted ( e.g ., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, -C(=0)R ^aa , -CC>2R ^aa , -C(=0)N(R ^bb ) ₂ , or an oxygen protecting group. In certain embodiments, the oxygen atom substituents are independently substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, -C(=0)R ^aa , -C0 ₂ R ^aa , -C(=0)N(R ^bb ) ₂ , or an oxygen protecting group, wherein R ^aa is hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, or an oxygen protecting group when attached to an oxygen atom; and each R ^bb is independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, or a nitrogen protecting group. In certain embodiments, the oxygen atom substituents are independently substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl or an oxygen protecting group. In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to herein as an “hydroxyl protecting group”). Oxygen protecting groups include -R ^aa , -N(R ^bb ) ₂ , -C(=0)SR ^aa , -C(=0)R ^aa , -C0 ₂ R ^aa . -C(=0)N(R ^bb ) ₂ , wherein X-, R ³ *, R ^bb , and R ^cc are as defined herein. Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 ^rd edition, John Wiley & Sons, 1999, incorporated herein by reference.

Exemplary oxygen protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), /-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-mcthoxybcnzyloxy methyl (PMBM), (4- methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), / butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4- methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, l-[(2-chloro-4- methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1 ,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzo furan-2- yl, 1-ethoxyethyl, l-(2-chloroethoxy)ethyl, 1-methyl-l-methoxyethyl, 1-methyl-l- benzyloxyethyl, l-methyl-l-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2- trimethylsilylethyl, 2-(phenylselenyl)ethyl, /-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxy benzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4- picolyl, 3-methyl-2-picolyl /V-oxido, diphenylmethyl, p,p ’-dinitrobenzhydryl, 5- dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl) methyl, 4-(4 bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4 ^r ,4"-tris(benzoyloxyphenyl)methyl, 3-(imidazol-l- yl)bis(4',4"-dimethoxyphenyl)methyl, l,l-bis(4-methoxyphenyl)-r-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1 ,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, /- butyldimethylsilyl (TBDMS), /-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), /-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-c h 1 o ro p hen o x y acetate, 3- phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p- phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2- (trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2- (triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophcnyl carbonate, alkyl benzyl carbonate, alkyl p- methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o nitrobenzyl carbonate, alkyl p-nitrobcnzyl carbonate, alkyl 5-benzyl thiocarbonate, 4-ethoxy-l-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o- (dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4- (methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4-(l,l,3,3-tetramethylbutyl)phenoxyacetate, 2,4— bis(l, 1— dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E) 2 methyl-2-butenoate, o-(methoxyacyl)benzoate, a-naphthoate, nitrate, alkyl N,N,N',N'- tetramethylphosphorodiamidate, alkyl /V-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

In certain embodiments, an oxygen protecting group is silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, ί-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl.

In certain embodiments, the sulfur atom substituents are independently substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, -C(=0)R ^aa , -C0 ₂ R ^aa , -C(=0)N(R ^bb ) ₂ , or a sulfur protecting group. In certain embodiments, the sulfur atom substituents are independently substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, -C(=0)R ^aa , -C0 ₂ R ^aa , -C(=0)N(R ^bb ) ₂ , or a sulfur protecting group, wherein R ^aa is hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, or an oxygen protecting group when attached to an oxygen atom; and each R ^bb is independently hydrogen, substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl, or a nitrogen protecting group. In certain embodiments, the sulfur atom substituents are independently substituted (e.g., substituted with one or more halogen) or unsubstituted Ci- ₆ alkyl or a sulfur protecting group.

In certain embodiments, the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a “thiol protecting group”). Sulfur protecting groups include -R ^aa , -N(R ^bb ) ₂ , -C(=0)SR“ -C(=0)R ^aa , -C0 ₂ R ^a \ -C(=0)N(R ^bb ) ₂ , -C(=NR ^bb )R ^aa , -C(=NR ^bb )OR ^aa , -C(=NR ^bb )N(R ^bb ) ₂ , -S(=0)R ^aa , -S0 ₂ R ^aa , -Si(R ^aa ) ₃ , -P(R ^CC ) _¾ -P(R ^CC ) ₃ ⁺ X ^“ , -P(OR ^cc ) ₂ , -P(OR ^CC ) ₃ ⁺ X ^“ , -P(=0)(R ^aa ) ₂ , -P(=0)(0R ^cc ) _¾ and -P(=0)(N(R ^bb ) ₂ ) ₂ , wherein R ^aa , R ^bb , and R ^cc are as defined herein. Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 ^rd edition, John Wiley & Sons, 1999, incorporated herein by reference. In certain embodiments, a sulfur protecting group is acetamidomethyl, i-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine- sulfenyl, or triphenylmethyl.

The “molecular weight” of -R, wherein -R is any monovalent moiety, is calculated by subtracting the atomic weight of a hydrogen atom from the molecular weight of the molecule R H. The “molecular weight” of -L-, wherein -L- is any divalent moiety, is calculated by subtracting the combined atomic weight of two hydrogen atoms from the molecular weight of the molecule H-L-H.

In certain embodiments, the molecular weight of a substituent is lower than 200, lower than 150, lower than 100, lower than 50, or lower than 25 g/mol. In certain embodiments, a substituent consists of carbon, hydrogen, fluorine, chlorine, bromine, iodine, oxygen, sulfur, nitrogen, and/or silicon atoms. In certain embodiments, a substituent consists of carbon, hydrogen, fluorine, chlorine, bromine, and/or iodine atoms. In certain embodiments, a substituent consists of carbon, hydrogen, and/or fluorine atoms. In certain embodiments, a substituent does not comprise one or more, two or more, or three or more hydrogen bond donors. In certain embodiments, a substituent does not comprise one or more, two or more, or three or more hydrogen bond acceptors.

The term “salt” refers to ionic compounds that result from the neutralization reaction of an acid and a base. A salt is composed of one or more cations (positively charged ions) and one or more anions (negative ions) so that the salt is electrically neutral (without a net charge). Salts of the compounds of this invention include those derived from inorganic and organic acids and bases. Examples of acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, hippurate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N ⁺ (Ci 4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further salts include ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

The term “solvate” refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The provided compounds may be prepared, e.g., in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of a crystalline solid. “Solvate” encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

The term “hydrate” refers to a compound that is associated with water. Typically, the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R x FhO, wherein R is the compound and wherein x is a number greater than 0. A given compound may form more than one type of hydrates, including, e.g., monohydrates (x is 1), lower hydrates (x is a number greater than 0 and smaller than 1, e.g., hemihydrates (R 0.5 FhO)), and polyhydrates (x is a number greater than 1, e.g., dihydrates (R-2 FhO) and hexahydrates (R-6 FhO)).

The term “tautomers” refers to compounds that are interchangeable forms of a particular compound stmcture, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of p electrons and an atom (usually H). For example, ends and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci- and nitro- forms of phenylnitromethane, that are likewi e formed by treatment with acid or base.

Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.

It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers.” Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.”

Stereoisomers that are not mirror images of one another are termed “diastereomers,” and those that are non- superimpo sable mirror images of each other are termed “enantiomers.” When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory ( i.e ., as (+) or (-)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture.”

The term “isotopically labeled compound” refers to a derivative of a compound that only structurally differs from the compound in that at least one atom of the derivative includes at least one isotope enriched above (e.g., enriched between 3- and 10-fold, between 10- and 30-fold, between 30- and 100-fold, between 100- and 300-fold, between 300- and 1,000-fold, between 1,000- and 3, 000- fold, or between 3,000- and 10,000-fold above) its natural abundance, whereas each atom of the compound includes isotopes at their natural abundances. In certain embodiments, the isotope enriched above its natural abundance is ¾. In certain embodiments, only one, two, three, four, or five hydrogen atoms of the isotopically labeled compound include ² H above its natural abundance. In certain embodiments, the isotope enriched above its natural abundance is ¹³ C, ¹⁵ N, or ¹⁸ 0. In certain embodiments, only one, two, or three carbon atoms of the isotopically labeled compound include ¹³ C above its natural abundance. In certain embodiments, only one, two, or three nitrogen atoms of the isotopically labeled compound include ^1S N above its natural abundance. In certain embodiments, only one, two, or three oxygen atoms of the isotopically labeled compound include ¹⁸ 0 above its natural abundance.

The term “polymorphs” refers to a crystalline form of a compound (or a salt, hydrate, or solvate thereof) in a particular crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Various polymorphs of a compound can be prepared by crystallization under different conditions.

The term “co-crystal” refers to a crystalline structure comprising at least two different components (e.g., a provided compound and an acid), wherein each of the components is independently an atom, ion, or molecule. In certain embodiments, none of the components is a solvent. In certain embodiments, at least one of the components is a solvent. A co-crystal of a provided compound and an acid is different from a salt formed from a provided compound and the acid. In the salt, a provided compound is complexed with the acid in a way that proton transfer (e.g., a complete proton transfer) from the acid to a provided compound easily occurs at room temperature. In the co-crystal, however, a provided compound is complexed with the acid in a way that proton transfer from the acid to a provided herein does not easily occur at room temperature. In certain embodiments, in the co-crystal, there is no proton transfer from the acid to a provided compound. In certain embodiments, in the co-crystal, there is partial proton transfer from the acid to a provided compound. Co-crystals may be useful to improve the properties (e.g., solubility, stability, and ease of formulation) of a provided compound.

The solid forms described herein (e.g., tautomers, stereoisomers, isotopically labeled compounds, salts, solvates, polymorphs, and co-crystals) include all combinations thereof. For example, a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal of a compound described herein includes, e.g., a polymorph of a solvate of a salt of an isotopically labeled compound of a tautomer of a stereoisomer of the compound described herein. In certain embodiments, the solid form is acceptable for use in foods, drinks, or cigarettes.

The terms "incubating" and "incubation" refer to a process of mixing two or more chemical or biological entities (such as a chemical compound and an enzyme) and allowing them to interact under conditions favorable for producing a desired product.

“Yeasts” refer to eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. Yeasts are unicellular organisms which are believed to have evolved from multicellular ancestors.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows an exemplary method described herein.

Figure IB shows the structure of certain alkylpyrazines (AlkPyrs), acylpyrazines (AcylPyrs), and alkyl/alkoxypyrazines (Alk/AlkO-Pyrs) useful in food chemistry. Each of the moieties recited in Figure IB (e.g., acetyl) is unsubstituted.

Figure 2 A shows HPFC chromatogram of 1:1 mixture of commercial 10 and 11 (mobile phase: 20:80 CH3CN/H2O on an Alltech Econosil C-18 column, IOm (250 mm x 22 mm) with a 5 mF/min flow rate with A278 monitoring of the effluent. Figure 2B shows GC/EI-MS profiles (selected-ion mode, m/z 136) of 1:1 mixture of commercial 10 and 11. Figure 2C shows a GC/EI- MS profile of purified 325-EDMP (11). Figure 2D shows a GC/EI-MS profile of purified 235- EDMP (10). Figures 3A and 3B show key structural ² 7CH and Vei l HMBC correlations within 235- EDMP (10) (Figure 3A) and 325-EDMP (11) (Figure 3B) between carbon (·) and hydrogen (shown explicitly as bold and underlined). The pyrazine ring numbering is shown.

Figure 4 shows a representative partial GC/EI-MS chromatogram with selected-ion monitoring (m/z 136) of 235- and 325-EDMP at 50:50 relative abundance in the product mixture after incubating 1 ,2-diaminopropane and pentan-2,3-dione (dropwise addition) at 0 °C for 1 h and then air oxidation.

Figure 5 A shows a GC/EI-MS selected-ion ( m/z 102) profile of the crude product extracted from the reaction mixture after incubating PDC, propanal, and pyruvate to make 3- hydroxypentan-2-one. Figure 5B shows a compound eluting at R _t = 5.29 min in Panel A had EI- MS fragment ions consistent with the a-cleavage sites of 3-hydroxypentan-2-one. The compound eluting at R _t = 5.43 min in Panel A had EI-MS fragment ions consistent with a-cleavage sites of 2-hydroxypentan-3-one (see Figure 6B). Figure 5C shows a GC/EI-MS total ion chromatogram of the crude product described for Panel A (top panel) and putative compounds (bottom panel). Putative compounds within the biocatalysis-derived sample whose peak thresholds were greater than or equal to those for the hydroxypentanones at R _t = 5.29 min and 5.43 min and had relative fragment ions abundances like those listed in a spectral database (see Figures 21 to 23 for fragment ion profiles. ^47,48 The coupled carbon-chain moieties originating from propanal are highlighted with bold C-C bonds.

Figure 6A shows a GC/EI-MS selected-ion ( m/z 102) profile of the crude product extracted from the reaction mixture after incubating PDC, acetaldehyde, and 2-oxobutanoate to make 2-hydroxypentan-3-one. Figure 6B shows a compound eluting at R _t = 5.43 min in Panel A had EI-MS fragment ions consistent with the a-cleavage sites of 2-hydroxypentan-3-one. The compound eluting at R _t = 5.29 min in Panel A had EI-MS fragment ions consistent with a- cleavage sites of 3-hydroxypentan-2-one (see Figure 5B). Figure 6C shows a GC/EI-MS total ion chromatogram of the crude product described for Panel A (top panel) and a putative compound (bottom panel). A compound (R _t = 4.85 min) within the biocatalysis-derived sample whose peak threshold was approximately equal to those for the hydroxypentanones at R _t = 5.29 min and 5.43 min and had relative fragment ions abundances like those listed in a spectral database ⁵⁰ (see Figures 24 A to 24C for fragment ion profile). The coupled carbon-chain moieties originating from acetaldehyde are highlighted with bold C-C bonds.

Figures 7A to 7D show partial GC/EI-MS chromatograms with selected-ion monitoring of the dihydropyrazines ( m/z 138) in the product mixture after incubating propane- 1,2-diamine (8) with 2-hydroxypentan-3-one (17) (Figure 7A) and 3-hydroxypentan-2-one (18) (Figure 7B). Partial GC/EI-MS profiles with selected-ion monitoring of the EDMPs ( m/z 136) after air- oxidation of the dihydropyrazines in the sample analyzed in Figure 7A (Figure 7C) and in Figure 7B (Figure 7D).

Figures 8A to 8F show GC/EI-MS total ion chromatogram (TIC) of products isolated from the crude reaction mixture after incubating 1,2-diaminopropane with 2-hydroxy-3- pentanone (Figure 8A) and 3-hydroxy-2-pentanone (Figure 8B). The EDMPs 10 (-5.40 min) and 11 (5.49 min) are highlighted. Figure 8C shows an EI-MS fragmentation profile of compound eluting from the GC at 4.60 min in the TIC profile of Figure 8A. Figure 8D shows a similar fragmentation ion profile was identified for 2,6-dimethylpiperazine in a mass spectral database. ⁵³ Figure 8E shows an EI-MS fragmentation profile of peak eluting from the GC at 4.79 min in the TIC profile of Figure 8A. Figure 8F shows a similar fragmentation ion profile was identified in a mass sp 5-EDMP. Correlation data is summarized in Table 3.

Figure 12 shows a ² /C,H, 7C.I I ( ^l3 C-' H HMBC) spectrum of 235-EDMP. Correlation data are summarized in Table 4. 25-EDMP. Correlation data are summarized in Table 5.

Figure 16 shows a ² /C,H, 7C.I I ( ^l3 C-' H HMBC) correlation spectrum of 325-EDMP. Correlation data are summarized in Table 6.

Figure 17 shows a ¹ H NMR analysis ( H NMR, 500 MHz, CDCb) of 2-hydroxy-3- pentanone in a crude sample extracted (Et ₂ 0) from a reaction mixture incubated with PDC, acetaldehyde, and 2-oxobutanoate sodium salt for 48 h. d: 4.86 (1H, quartet, J = 6.9 Hz), 3.01 (2H, quartet, / = 7.1 Hz), 1.43 (3H, doublet, 7 = 7.0 Hz), 1.18 (3H, triplet, / = 6.5 Hz).

Figure 18 shows reference homonuclear ¹ H- ¹ H COSY data for 3-hydroxy-2-hexanone ¹ used to predict the chemical shift correlations of 3-hydroxy-2-pentanone made via a carboligation reaction catalyzed by a pyruvate decarboxylase in the current study. Homonuclear correlations between H3 and HI, H4, H5, and H7 (bold circles) and annotated highlighted because these were the most diagnostic for identifying ¹ H- ¹ H correlations in 3-hydroxy-2-pentanone (Figure 19). Other correlations are circled and annotated.

Figure 19 shows a ¹ H- ¹ H COSY analysis (500 MHz, CDCI3) of the putative biocatalyzed 3-hydroxy-2-pentanone in a crude sample extracted (Et ₂ 0) from a reaction mixture in which PDC, propanal, and 2-oxopropanoate sodium salt were incubated for 48 h. Putative correlations between H5 and H4 are circled and annotated off-diagonal.

Figure 20A shows a GC/EI MS spectrum of the compound eluting at 5.74 min in the TIC of the crude 3-hydroxy-2-pentanone sample (Figure 5C) whose fragment ions correspond approximately with those for Figure 20B: 2-methyl-2-pentenal in the mass spectral database. ⁵⁸

Figure 21 A shows a GC/EI MS spectrum of the compound eluting at 7.93 min in the TIC of the crude 3-hydroxy-2- pentanone sample (Figure 5C) whose fragment ions correspond approximately with those for Figure 21B: 3-hydroxy-2-methylpentanal in the mass spectral database. ⁵⁹

Figure 22A shows a GC/EI MS spectrum of the compound eluting at 12.15 min in the TIC of the crude 3-hydroxy-2-pentanone sample (Figure 5C) whose fragment ions correspond approximately with those for Figure 22B: 2,6-diethyl-5-methyl-l,3-dioxane in the mass spectral database. ⁶⁰

Figure 23A shows a GC/EI MS spectrum of the compound eluting at 4.85 min in the TIC of the 2-hydroxy-3-pentanone sample (Figure 6C) whose fragment ions correspond approximately with those for Figure 23B: paraldehyde in the mass spectral database. ⁶¹

Figures 24A to 24C show GC/EI-MS analysis in total-ion count (TIC) mode (Figure 24A) and selected-ion monitoring (SIM) mode (Figure 24B) for the EDMPs ( mlz 136), and SIM mode {mlz 80) (Figure 24C) for the pyrazine core minus the alkyl substituents of the product mixture after incubating 1 ,2-diaminopropane and 2 -hydroxy- 3 -pentanone. The products (boxed) are characterized in Figures 8A to 8F.

Figures 25A to 25C show a GC/EI-MS analysis in TIC mode (Figure 25A) and SIM mode (Figure 25B) for the EDMPs ( mlz 136), and SIM mode {mlz 80) (Figure 25C) for the pyrazine core minus the alkyl substituents of the product mixture after incubating 1,2- diaminopropane and 3-hydroxy-2-pentanone. The products (boxed) are characterized in Figures 8A to 8F.

Figures 26A to 26F show an EI-MS fragmentation profile of peaks eluting from the GC (TIC profile in Figure 8A) at 3.87 min (Figure 26A), 4.12 min (Figure 26B), 4.52 min (Figure 26C), deduced from related azo-compounds, ⁶² and 7.52 min (Figure 26D), which corresponded to a putative alkyl ester with a molecular formula of C9H16O2. EI-MS fragmentation profile of peaks eluting from the GC (TIC profile in Figure 8B) at 3.73 min (Figure 26E) and 4.43 min (Figure 26F). The fragment ions in Figures 26A to 26C, 26E, and 26F are inferred from those in Figures 8A to 8F and a previous document. ⁶²

DET AIDED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE DISCLOSURE In one aspect, the present disclosure provides methods of producing a substituted pyrazine of Formula (Dl): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof, and optionally a substituted pyrazine of Formula (D2): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof; wherein: each of R ¹ and R ² is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted acyl, substituted or unsubstituted alkoxy, or substituted or unsubstituted carbocyclyl; each of R ³ and R ⁴ is independently hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl; provided that at least one of R ¹ and R ² is not hydrogen.

The methods described herein may be advantageous over known methods because the former may produce regioselectively enriched substituted pyrazines, whereas the latter may produce substituted pyrazines that are not regioselectively enriched.

Certain substituted pyrazines useful in food chemistry include the ones shown in Figure

IB.

The first synthesis of 2,3,5,6-tetraphenylpyrazine in 1897 involved self-condensation of 2-(benzylideneamino)-2-phenylacetonitrile. ¹⁹ Another early method of synthesizing pyrazine isomers was discovered fortuitously when nitrosated ketones (1) were reduced to an aminoketone (2) with hydrogen. This synthesis demonstrated the intermediate formation of dihydropyrazines by isolating them under anaerobic conditions and subsequent oxidation to the corresponding tetralkylpyrazines (3) (Scheme 1, panel A). ²⁰ One more approach to pyrazine (5) started with the deaminocyclisation of ethylenediamine (4), followed by dehydrogenation over copper chromite catalysts (Scheme 1, panel B). ²¹ Later, microwave irradiation was used to form substituted pyrazines by coupling 2-chloropyrazine (6) with nucleophiles of PhSNa, MeSNa, EtONa, and PhONa in A- met h y 1 p y rro 1 i do n c (NMP) that gave the desired monothioether/ether products (7) (Scheme 1, panel C). ²² Access to alkylpyrazines from the pyrazine core has been done with alkyllithium. However, one example demonstrated a downside to this approach using methylpyrazines where the alkyllithium either alkylated the pyrazine ring, the methyl group of methylpyrazine, or both, leading to undesired, low yielding product mixtures. ²³ Another alkylpyrazine, 2-alkyl-3,6- dimethylpyrazine, was made by thermal electrocyclization or zirconium-mediated alkenylation of 2,5-dimethylpyrazine with acetylenes. However, this method was of little practical value because the requisite zirconium catalyst is extremely air-sensitive, and the preparation of the cationic complex needs expensive inert atmosphere equipment. ²⁴

Today, pyrazines may be assembled synthetically by condensation between dicarbonyls and diamino compounds (Scheme 1, panel D) or biosynthetically between self-condensation of an aminocarbonyl (2) followed by oxidization on the proposed biosynthetic pathway to 325- EDMP (11) in Serratia marcescens (Scheme 1, panel E). ^25,26 Symmetry in one reagent in the condensation reaction gives the best isomeric selectivity in the product en route to pyrazines. ²⁵ Several variations of the condensation reactions have been developed by adding metal catalysts and making solvent alterations. However, the main drawbacks of the synthetic approaches are low yields, prolonged reaction time, toxic solvents and metals, and complicated separation techniques to isolate the products. ²⁵

8 9 10 o

15 16/14

Scheme 1. Exemplary synthesis of pyrazine and substituted pyrazines. Reagents and conditions:

Panel A) a) ¾, b) dehydration/oxidation. ²⁰

Panel B) a) deaminocyclisation and Cu ₂ Cr ₂ 0s dehydrogenation. ²¹

Panel C) a ) thioalk/phenoxide or alk/phenoxide nucleophiles, methylpyrrolidone, microwave radiation. ²²

Panel D) Example of coupling a diamine and diketone to make a 50:50 mixture of 235- EDMP/325-EDMP isomers, a) rt, 1 h, Et ₂ 0, KOH.

Panel E) Proposed 325-EDMP biosynthesis in Serratia marcescens from L-threonine (12), showing a self-condensation step of 13. a) oxidation/decarboxylation; b ) spontaneous iminization condensation; c ) tautomerization; d ) acetyltransferase/ Acetyl CoA, reductase (H7H ⁺ ; e) dehydratase, 0 ₂ .

Panel F) Proposed 235-EDMP biosynthesis from mixed aminocarbonyl compounds, showing intermediates and products from self- and cross condensation. Steps b - e) are the same as in Panel E.

While green chemistry is becoming important in the modern era, developing environment-friendly methods to synthesize pyrazines is a priority. Also, as the food and beverage industries continue to increase using pyrazines as ingredients for foodstuffs, a desire to develop "natural" pyrazines is growing. ²⁷ It is interesting to note that synthetically derived pyrazines are not considered "natural" despite being structurally identical to a natural product. ²⁸ Thus, a niche to address growing consumer demand for "natural" ingredients has emerged, and biocatalytic processes are sought as alternatives to produce specific substituted pyrazine isomers by enzymatic methods. ²⁷

Certain Alk/AlkO-Pyrs are used to improve the odor of cosmetics and toiletries in the perfume industry. ^27,29 Certain AlkPyrs have garnered interest recently and are supplied at -2200 kg/year, as of 2004. ^{27 0} The seven most extracted pyrazines from natural resources include 2,3,5- trimethylpyrazine (235-TMP); 2-ethyl-3-methylpyrazine (23-EMP); 2,5-dimethylpyrazine (25- DMP); 2,6-dimethylpyrazine (26-DMP); 2-ethyl-3,5-dimethylpyrazine (235-EDMP) and its constitutional isomer 3-ethyl-2, 5-dimethyl (325-EDMP); 2,3-dimethylpyrazine (23-DMP); and methylpyrazine (MP). These compounds are used to favorably augment taste notes in foods ( e.g ., sauces, breakfast cereals, chocolate, cocoa, meat, potatoes, peanut products, popcorn, and bread) and drinks (e.g., coffee, tea, and wine). ^27,31

Among the many alkylpyrazines, the ethyl-dimethylpyrazines are among the most potent odorants (perceived as roasty and sweet). For example, 235-EDMP and 325-EDMP are significant contributors to the desirable taste of Houjicha green tea. ³¹ These EDMPs are typically only available by extraction from natural sources as mixtures in foodstuff, such as coffee aroma and raw plants, vegetables, fish, poultry, and beef. ³

However, one account found 325-EDMP as a single isomer in the bacterium Serratia marcescens. The biosynthetic pathway to 325-EDMP was proposed to proceed through a C2- symmetrical 2,5-dimethyl-2,5-dihydropyrazine (25-DMDHP) intermediate derived after dehydration of a self-condensation product from l-aminopropan-2-one (13) (Scheme 1, panel E). ³² A subsequent Friedel Crafts reaction with acetyl CoA followed by a keto-reduction, dehydration, and tautomerization are proposed to add the ethyl substituent found in 325-EDMP. ³² Extrapolation of a similar biosynthetic route to 235-EDMP compared to that proposed for 325- EDMP is less probable. The biosynthetic route to 235-EDMP needs to proceed through an analogous 26-DMDHP, and the amino carbonyl coupling partners require l-aminopropan-2-one (13) and 2-aminopropanal (15) (Scheme 1, panel F), analogous to aminoacetone needed on the 325-EDMP pathway. The drawback, in this case, is that 13 and 15 will self- and cross-couple to generate 2,6-dimethyl-2,5-dihydropyrazine (16) and 3,6-dimethyl-2,5-dihydropyrazine (14), and the downstream enzymatic steps, keto-reduction, dehydration, and tautomerization, would, unfortunately, lead to a mixture of 235- and 325-EDMPs.

The broad market appeal of certain substituted pyrazines as additives is increasing; thus, additional resources for accessing substituted pyrazines (e.g., EDMPs) as single isomers are needed. 235- and 325-EDMPs are used as, e.g., flavoring agents, food additives, and fragrances, ³³ and are found as added ingredients in cigarettes, ³⁴ and related AlkPyrs are found in e-cigarettes. ³⁵ Given this broad chemosensory application of substituted pyrazines, several accounts highlight the synthesis and separation of these compounds. ^25,36,37 Commercially available mixtures of 235- and 325-EDMPs were chromatographic ally separated to show that each had a distinct effect on the behavioral response of fire ants in an electroantennogram study. ³⁸ Earlier synthetic attempts to make 235- and 325-EDMPs separately were successful but are challenged by multiple steps that increase the start-stop assembly sequences and affect the yield (Scheme 2). ²⁴ Likely, the most direct route to the pyrazine framework is coupling a carbonyl compound with an alkyl amine partner in a condensation reaction. However, the chemical synthesis of a single constitutional isomer of EDMP by condensing an amine with a carbonyl compound occurs rapidly without regioselectivity.

Scheme 2. An exemplary synthesis of Compounds 10 and 11. Reagents and conditions: a) isoamyl nitrite, cone HC1, Et ₂ 0. 10-20 °C, 2 h; b) allylamine, hexane, reflux; c) potassium tert- butoxide, DMSO, 50 °C, 1-2 h; d) CICOOCHg, Et ₃ N, CH ₂ C1 ₂ , 10-20 °C; e) short contact-time pyrolysis in toluene solution at -300 °C.

An earlier independent study evaluated the production of alkyl di- and tetrahydropyrazines, including the 5,6-dihydropyrazine precursors of 235- and 325-EDMPs from a-hydroxy ketone made in baker's yeast whole cells and diamines. ³⁹ This earlier study evaluated the products by GC-olfactometry (GC-O) and did not measure the selectivity of the condensation reaction.

We considered the benefits of synthetic strategies for assembling aromatic N- heterocycles, including metal-free and metal-catalyzed methods for multicomponent reactions. ⁴⁴ Specifically, many earlier successful efforts to synthesize 235- and 325-EDMP separately were less appealing because they progressed through multistep procedures, high temperature, and toxic solvents, resulting in moderate yield. ²⁴

In one aspect, the present disclosure provides a biocatalytic method for producing substituted pyrazines, such as 235-EDMP and 325-EDMP. In certain embodiments of the present disclosure, we evaluated the regioselectivity of the coupling reaction between a diamine and a- hydroxy ketone. In certain embodiments, we used the enzyme pyruvate decarboxylase (PDC) isolated from baker's yeast ( Saccharomyces cerevisiae ) to obtain hydroxypentanone reactants to couple with propane- 1 ,2-diamine, followed by KOH treatment to enrich each EDMP isomers regio selectively.

In certain embodiments, the present disclosure provides a method of producing an a- hydroxy ketone of Formula (Bl): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof, the method comprising incubating a first reaction mixture comprising:

(a) a compound of Formula (Al): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof;

(b) a compound of Formula (A2): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof; and

(c) a pyruvate decarboxylase; for a first time duration sufficient to produce the a-hydroxy ketone of Formula (B 1), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof; wherein:

R ¹ is substituted or unsubstituted alkyl, substituted or unsubstituted acyl, substituted or unsubstituted alkoxy, or substituted or unsubstituted carbocyclyl; and

R ² is substituted or unsubstituted alkyl, substituted or unsubstituted acyl, substituted or unsubstituted alkoxy, or substituted or unsubstituted carbocyclyl.

In certain embodiments, a chiral carbon atom of a formula described herein is of the R configuration. In certain embodiments, a chiral carbon atom of a formula described herein is of the S configuration.

In certain embodiments, the first time duration is sufficient to further produce an a- hydroxyketone of Formula (B2): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof.

In certain embodiments, the molar ratio of the a-hydroxy ketone of Formula (B 1), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the a-hydroxyketone of Formula (B2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 51:49 and 60:40, between 60:40 and 70:30, between 70:30 and 80:20, between 80:20 and 90:10, between 90:10 and 99: 1, inclusive. In certain embodiments, the molar ratio of the a-hydroxyketone of Formula (Bl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof, to the a-hydroxyketone of Formula (B2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 55:45 and 95:5, inclusive. In certain embodiments, the molar ratio of the a-hydroxyketone of Formula (Bl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the a-hydroxyketone of Formula (B2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 60:40 and 80:20, inclusive.

In certain embodiments, R ¹ is hydrogen. In certain embodiments, R ¹ is unsubstituted alkyl (e.g., unsubstituted Ci- ₆ alkyl). In certain embodiments, R ¹ is unsubstituted Ci-4 alkyl. In certain embodiments, R ¹ is unsubstituted methyl. In certain embodiments, R ¹ is unsubstituted ethyl. In certain embodiments, R ¹ is unsubstituted propyl (e.g., n- Pr or /-Pr). In certain embodiments, R ¹ is unsubstituted butyl. In certain embodiments, R ¹ is substituted alkyl (e.g., alkyl substituted with one or more instances of halogen (e.g., F)). In certain embodiments, R ¹ is substituted Ci- ₆ alkyl.

In certain embodiments, R ¹ is substituted methyl (e.g., fluorinated methyl or Bn). In certain embodiments, R ¹ is -CF3. In certain embodiments, R ¹ is substituted ethyl, substituted propyl, or substituted butyl. In certain embodiments, R ¹ is substituted or unsubstituted acyl. In certain embodiments, R ¹ is -C(=0)-( substituted or unsubstituted, Ci- ₆ alkyl). In certain embodiments, R ¹ is unsubstituted acetyl. In certain embodiments, R ¹ is substituted or unsubstituted alkoxy. In certain embodiments, R ¹ is -0-(substituted or unsubstituted, Ci- ₆ alkyl). In certain embodiments, R ¹ is -OMe. In certain embodiments, R ¹ is -OCF3. In certain embodiments, R ¹ is -OEt. In certain embodiments, R ¹ is -OPr or -OBu. In certain embodiments, R ¹ is substituted or unsubstituted carbocyclyl (e.g., substituted or unsubstituted, monocyclic, 3- to 7-membered carbocyclyl). In certain embodiments, R ¹ is substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, substituted or unsubstituted cyclopentyl, substituted or unsubstituted cyclohexyl, or substituted or unsubstituted cycloheptyl.

In certain embodiments, (a) of the first reaction mixture is 2-oxobutanoic acid, or a salt (e.g., an addition salt with a monovalent base, such as an alkaline metal salt) thereof. In certain embodiments, (a) of the first reaction mixture is sodium 2-oxobutanoate.

In certain embodiments, (a) of the first reaction mixture is pyruvic acid, or a salt ( e.g ., an addition salt with a monovalent base, such as an alkaline metal salt) thereof. In certain embodiments, (a) of the first reaction mixture is sodium pyruvate.

In certain embodiments, R ² is hydrogen. In certain embodiments, R ² is unsubstituted alkyl (e.g., unsubstituted Ci- ₆ alkyl). In certain embodiments, R ² is unsubstituted C1-4 alkyl. In certain embodiments, R ² is unsubstituted methyl. In certain embodiments, R ² is unsubstituted ethyl. In certain embodiments, R ² is unsubstituted propyl (e.g., n- Pr or z ^' -Pr). In certain embodiments, R ² is unsubstituted butyl. In certain embodiments, R ² is substituted alkyl (e.g., alkyl substituted with one or more instances of halogen (e.g., F)). In certain embodiments, R ² is substituted Ci- ₆ alkyl.

In certain embodiments, R ² is substituted methyl (e.g., fluorinated methyl or Bn). In certain embodiments, R ² is -CF3. In certain embodiments, R ² is substituted ethyl, substituted propyl, or substituted butyl. In certain embodiments, R ² is substituted or unsubstituted acyl. In certain embodiments, R ² is -C(=0)-( substituted or unsubstituted, Ci- ₆ alkyl). In certain embodiments, R ² is unsubstituted acetyl. In certain embodiments, R ² is substituted or unsubstituted alkoxy. In certain embodiments, R ² is -0(substituted or unsubstituted, Ci- ₆ alkyl). In certain embodiments, R ² is -OMe. In certain embodiments, R ² is -OCF3. In certain embodiments, R ² is -OEt. In certain embodiments, R ² is -OPr or -OBu. In certain embodiments, R ² is substituted or unsubstituted carbocyclyl (e.g., substituted or unsubstituted, monocyclic, 3- to 7-membered carbocyclyl). In certain embodiments, R ² is substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, substituted or unsubstituted cyclopentyl, substituted or unsubstituted cyclohexyl, or substituted or unsubstituted cycloheptyl.

In certain embodiments, R ¹ is hydrogen; and R ² is not hydrogen. In certain embodiments, R ¹ is not hydrogen; and R ² is hydrogen. In certain embodiments, each of R ¹ and R ² is not hydrogen.

In certain embodiments, (b) of the first reaction mixture is acetaldehyde.

In certain embodiments, (b) of the first reaction mixture is propionaldehyde.

In certain embodiments, R ¹ and R ² are different from each other. In certain embodiments, R ¹ and R ² are the same.

In certain embodiments, the molar ratio of (a) to (b) of the first reaction mixture is between 10:1 and 4:1, between 4:1 and 2:1, between 2:1 and 1:1, between 1:1 and 1:2, between 1:2 and 1:4, or between 1:4 and 1:10, inclusive. In certain embodiments, the molar ratio of (a) to (b) of the first reaction mixture is between 3:1 and 1:3, inclusive. In certain embodiments, the molar ratio of (a) to (b) of the first reaction mixture is between 1.5:1 and 1:1, inclusive.

In certain embodiments, the pyruvate decarboxylase is a pyruvate decarboxylase isolated from a species of the yeast Saccharomyces. In certain embodiments, the pyruvate decarboxylase is a pyruvate decarboxylase isolated from a strain of the yeast Saccharomyces cerevisiae ( e.g ., strain ATCC 204508 / S288c). In certain embodiments, the pyruvate decarboxylase is a pyruvate decarboxylase isolated from baker’s yeast. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence that is between 70% and 75%, between 75% and 80%, between 80% and 85%, between 85% and 90%, between 90% and 95%, or between 95% and 100%, inclusive, identical to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence of SEQ ID NO: 1.

In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence that is between 70% and 75%, between 75% and 80%, between 80% and 85%, between 85% and 90%, between 90% and 95%, or between 95% and 100%, inclusive, identical to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence of SEQ ID NO: 2.

In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence that is between 70% and 75%, between 75% and 80%, between 80% and 85%, between 85% and 90%, between 90% and 95%, or between 95% and 100%, inclusive, identical to the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence of SEQ ID NO: 3.

In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1, 2, or 3. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence of SEQ ID NO: 1, 2, or 3, or a combination thereof. In certain embodiments, the pyruvate decarboxylase comprises an amino acid sequence of SEQ ID NO: 1, 2, or 3.

In certain embodiments, the ratio of the pyruvate decarboxylase to (b) is between 10 mg:0.03 mmol and 10 mg:0.1 mmol, between 10 mg:0.1 mmol and 10 mg:0.3 mmol, between 10 mg:0.3 mmol and 10 mg:l mmol, between 10 mg:l mmol and 10 mg:3 mmol, between 10 mg:3 mmol and 10 mg: 10 mmol, between 10 mg: 10 mmol and 10 mg:30 mmol, between 10 mg:30 mmol and 10 mg: 100 mmol, or between 10 mg: 100 mmol and 10 mg:300 mmol, inclusive. In certain embodiments, the ratio of the pyruvate decarboxylase to (b) is between 10 mg:0.3 mmol and 10 mg:30 mmol, inclusive. In certain embodiments, the ratio of the pyruvate decarboxylase to (b) is between 10 mg:l mmol and 10 mg: 10 mmol, inclusive.

In certain embodiments, the first reaction mixture further comprises one or more cofactors. In certain embodiments, the first reaction mixture further comprises thiamine pyrophosphate, or a salt thereof. In certain embodiments, the first reaction mixture further comprises magnesium (II). In certain embodiments, the first reaction mixture further comprises an alkaline earth metal salt or alkali metal salt of magnesium (II) ( e.g ., MgSCE). In certain embodiments, thiamine pyrophosphate, or a salt thereof, and/or magnesium (II) are cofactors. In certain embodiments, the molar ratio of each of the cofactors to the compound of Formula (Al), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof, is independently between 0.0001:1 and 0.001:1, between 0.001:1 and 0.01:1, between 0.01:1 and 0.1:1, between 0.1:1 and 1 : 1 , or between 1:1 and 10:1, inclusive.

In certain embodiments, the first reaction mixture further comprises a buffer solution (e.g., an aqueous buffer solution). In certain embodiments, the first reaction mixture further comprises a buffer solution of pH between 4 and 5, between 5 and 6, between 6 and 7, or between 7 and 8, inclusive. In certain embodiments, the first reaction mixture further comprises a buffer solution of pH between 5 and 7, inclusive. In certain embodiments, the first reaction mixture further comprises a buffer solution of pH between 5.5 and 6.5, inclusive. In certain embodiments, the first reaction mixture further comprises a buffer solution of pH about 6. In certain embodiments, the buffer solution is a sodium citrate buffer solution.

In certain embodiments, the first reaction mixture is in vitro. In certain embodiments, the first reaction mixture is in vivo or ex vivo. In certain embodiments, the first reaction mixture is a cell-based reaction mixture. In certain embodiments, the cell-based reaction mixture comprises a cell selected from the group consisting of a yeast, a plant, an alga, a fungus, and a bacterium. In some embodiments, the cell-based reaction mixture comprises a cell of a species of the yeast Saccharomyces. In some embodiments, the cell-based reaction mixture comprises a cell of a strain of the yeast Saccharomyces cerevisiae. In some embodiments, the cell-based reaction mixture comprises a cell of Baker's yeast.

In certain embodiments, the cells used in the cell-based reaction mixture recombinantly express the pyruvate decarboxylase. For example, the cell used in the cell-based reaction mixture may be transformed with a nucleic acid molecule (e.g., a vector such as an expression vector) comprising a nucleotide sequence encoding the pyruvate decarboxylase. In certain embodiments, the nucleotide sequence encoding the pyruvate decarboxylase is operably linked to a promoter (e.g., an inducible promoter or a constitutive promoter). The transformed cells can be cultured under conditions that allow the expression of the pyruvate decarboxylase. The cells contain the expressed pyruvate decarboxylase can be collected and used in the cell-based reaction mixture. In certain embodiments, a nucleotide encoding SEQ ID NO: 1 comprises a nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, a nucleotide encoding SEQ ID NO: 2 comprises a nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, a nucleotide encoding SEQ ID NO: 3 comprises a nucleic acid sequence of SEQ ID NO: 6.

In certain embodiments, the cell used in the cell-based reaction mixture is transformed with a nucleotide comprising a nucleic acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the nucleic acid sequence of SEQ ID NO: 4, 5, or 6. In certain embodiments, the cell used in the cell-based reaction mixture is transformed with a nucleotide comprising a nucleic acid sequence that is between 70% and 75%, between 75% and 80%, between 80% and 85%, between 85% and 90%, between 90% and 95%, or between 95% and 100%, inclusive, identical to the nucleic acid sequence of SEQ ID NO: 4, 5, or 6. In certain embodiments, the cell used in the cell-based reaction mixture is transformed with a nucleotide comprising a nucleic acid sequence of SEQ ID NO: 4, 5, or 6.

In certain embodiments, the temperature of the first reaction mixture is between -20 and 0, between 0 and 20, between 20 and 25, between 25 and 40, or between 40 and 60 °C, inclusive. In certain embodiments, the temperature of the first reaction mixture is between 0 and 40 °C, inclusive. In certain embodiments, the temperature of the first reaction mixture is between 10 and 30 °C, inclusive. In certain embodiments, the temperature of the first reaction mixture is between 20 and 25 °C, inclusive. In certain embodiments, the temperature of the first reaction mixture is substantially constant over the first time duration. In certain embodiments, the first time duration is between 1 and 3 hours, between 3 and 6 hours, between 6 and 12 hours, between 12 and 24 hours, between 1 and 3 days, between 3 and 7 days, or between 7 and 14 days, inclusive. In certain embodiments, the first time duration is between 0.5 and 7 days, inclusive. In certain embodiments, the first time duration is between 1 and 3 days, inclusive.

In certain embodiments, the a-hydroxyketone of Formula (B 1) is of Formula (17): or a stereoisomer, isotopically labeled compound, solvate, polymorph, or co-crystal thereof. In certain embodiments, the a-hydroxyketone of Formula (B2) is of Formula (18): or a stereoisomer, isotopically labeled compound, solvate, polymorph, or co-crystal thereof.

In certain embodiments, the a-hydroxyketone of Formula (B 1) is of Formula (18), or a stereoisomer, isotopically labeled compound, solvate, polymorph, or co-crystal thereof. In certain embodiments, the a-hydroxyketone of Formula (B2) is of Formula (17), or a stereoisomer, isotopically labeled compound, solvate, polymorph, or co-crystal thereof.

In certain embodiments, the rate of conversion of (b) of the first reaction mixture to the a- hydroxyketone of Formula (Bl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, and to, if present, the a-hydroxyketone of Formula (B2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof, is between 10% and 95% ( e.g ., between 10% and 20%, between 20% and 40%, between 40% and 60%, between 60% and 80%, or between 80% and 95%), inclusive. In certain embodiments, the rate of conversion of (b) of the first reaction mixture to the a-hydroxyketone of Formula (Bl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, and to, if present, the a-hydroxyketone of Formula (B2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 30% and 70%, inclusive.

In certain embodiments, the method further comprises isolating the a-hydroxyketone of Formula (Bl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof; or a mixture of the a-hydroxyketone of Formula (B 1), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, and the a-hydroxyketone of Formula (B2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof. In certain embodiments, the step of isolating described in this paragraph comprises liquid-liquid phase separation, drying, filtration, concentration, chromatography, decolorization, or recrystallization, or a combination thereof. In certain embodiments, the step of isolating described in this paragraph comprises liquid-liquid phase separation, drying, filtration, concentration, decolorization, or recrystallization, or a combination thereof. In certain embodiments, the liquid-liquid phase separation is a separation of an organic phase and an aqueous phase. In certain embodiments, the drying is drying an organic phase over a solid drying agent ( e.g ., anhydrous NaiSCri, anhydrous MgSCU, anhydrous CaSCri, anhydrous CaCk, or activated molecular sieves). In certain embodiments, the filtration is a filtration of a mixture of an organic phase and a solid drying agent to remove the solid drying agent and hydrates thereof. In certain embodiments, the concentration is concentration of an organic phase to remove part or substantially all of the volatiles (e.g., organic solvents). In certain embodiments, the concentration is performed under a pressure lower than 1 atm (e.g., between 0.001 and 0.01, between 0.01 and 0.1, or between 0.1 and 1 atm, inclusive). In certain embodiments, the concentration is performed under a temperature of between 0 and 10, between 10 and 20, between 20 and 25, between 25 and 35, between 35 and 50, or between 50 and 80 °C, inclusive. In certain embodiments, the chromatography is flash chromatography (e.g., normal-phase flash chromatography (e.g., over silica gel)). In certain embodiments, the step of isolating described in this paragraph does not comprise chromatography. In certain embodiments, the decolorization comprises redissolving in an organic solvent, decolorization, and concentration. In certain embodiments, the decolorization comprises contacting with a solid decolorization agent (e.g., activated charcoal). In certain embodiments, the recrystallization is a single- solvent recrystallization. In certain embodiments, the recrystallization is a multi-solvent (e.g., bi-solvent or tri-solvent) recrystallization. In certain embodiments, the recrystallization is a hot filtration-recrystallization. In certain embodiments, the step of isolating described in this paragraph further comprises removing part or substantially all of the volatiles (e.g., organic solvents) by decreasing the pressure (e.g., to a pressure of lower than 1 atm (e.g., between 0.001 and 0.01, between 0.01 and 0.1, or between 0.1 and 1 atm, inclusive) and/or increasing the temperature (e.g., to a temperature between 25 and 35, between 35 and 50, or between 50 and 80 °C, inclusive).

In certain embodiments, the method does not further comprise isolating the a- hydroxyketone of Formula (Bl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof; or a mixture of the a-hydroxyketone of Formula (B 1), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co- crystal thereof, and, if present, the a-hydroxyketone of Formula (B2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof.

In certain embodiments, the methods further comprise incubating a second reaction mixture comprising:

(a) the compound of Formula (Bl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof; and

(b) an amine of Formula (B3): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof; for a second time duration sufficient to produce a substituted dihydropyrazine of Formula (Cl): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof; wherein:

R ³ is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl; and.

R ⁴ is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl.

In certain embodiments, the second reaction mixture further comprises (c) the a- hydroxyketone of Formula (B2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof; and the second time duration is sufficient to further produce a substituted dihydropyrazine of Formula (C2): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof.

In certain embodiments, the molar ratio of the substituted dihydropyrazine of Formula (Cl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof, to the substituted dihydropyrazine of Formula (C2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 51:49 and 60:40, between 60:40 and 70:30, between 70:30 and 80:20, between 80:20 and 90:10, between 90:10 and 99:1, inclusive. In certain embodiments, the molar ratio of the substituted dihydropyrazine of Formula (Cl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the substituted dihydropyrazine of Formula (C2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof, is between 55:45 and 95:5, inclusive. In certain embodiments, the molar ratio of the substituted dihydropyrazine of Formula (Cl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the substituted dihydropyrazine of Formula (C2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 60:40 and 80:20, inclusive.

In certain embodiments, R ³ is hydrogen. In certain embodiments, R ³ is unsubstituted alkyl (e.g., unsubstituted Ci- ₆ alkyl). In certain embodiments, R ³ is unsubstituted Ci-4 alkyl. In certain embodiments, R ³ is unsubstituted methyl. In certain embodiments, R ³ is unsubstituted ethyl. In certain embodiments, R ³ is unsubstituted propyl (e.g., n- Pr or z ^' -Pr). In certain embodiments, R ³ is unsubstituted butyl. In certain embodiments, R ³ is substituted alkyl (e.g., alkyl substituted with one or more instances of halogen (e.g., F)). In certain embodiments, R ³ is substituted Ci- ₆ alkyl.

In certain embodiments, R ³ is substituted methyl (e.g., fluorinated methyl or Bn). In certain embodiments, R ³ is -CF3. In certain embodiments, R ³ is substituted ethyl, substituted propyl, or substituted butyl. In certain embodiments, R ³ is substituted or unsubstituted carbocyclyl (e.g., substituted or unsubstituted, monocyclic, 3- to 7-membered carbocyclyl). In certain embodiments, R ³ is substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, substituted or unsubstituted cyclopentyl, substituted or unsubstituted cyclohexyl, or substituted or unsubstituted cycloheptyl.

In certain embodiments, R ⁴ is hydrogen. In certain embodiments, R ⁴ is unsubstituted alkyl (e.g., unsubstituted Ci- ₆ alkyl). In certain embodiments, R ⁴ is unsubstituted C1-4 alkyl. In certain embodiments, R ⁴ is unsubstituted methyl. In certain embodiments, R ⁴ is unsubstituted ethyl. In certain embodiments, R ⁴ is unsubstituted propyl (e.g., zz-Pr or z-Pr). In certain embodiments, R ⁴ is unsubstituted butyl. In certain embodiments, R ⁴ is substituted alkyl (e.g., alkyl substituted with one or more instances of halogen (e.g., F)). In certain embodiments, R ⁴ is substituted Ci- ₆ alkyl.

In certain embodiments, R ⁴ is substituted methyl (e.g., fluorinated methyl or Bn). In certain embodiments, R ⁴ is -CF3. In certain embodiments, R ⁴ is substituted ethyl, substituted propyl, or substituted butyl. In certain embodiments, R ⁴ is substituted or unsubstituted carbocyclyl (e.g., substituted or unsubstituted, monocyclic, 3- to 7-membered carbocyclyl). In certain embodiments, R ⁴ is substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, substituted or unsubstituted cyclopentyl, substituted or unsubstituted cyclohexyl, or substituted or unsubstituted cycloheptyl.

In certain embodiments, the substituted dihydropyrazine of Formula (Cl) is of the formula: or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof.

In certain embodiments, the substituted dihydropyrazine of Formula (C2) is of the formula: or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof.

In certain embodiments, the substituted dihydropyrazine of Formula (Cl) is of the formula: or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof.

In certain embodiments, the substituted dihydropyrazine of Formula (C2) is of the formula: or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof.

In certain embodiments, (b) of the second reaction mixture is propane- 1 ,2-diamine, or a salt thereof (e.g., an addition salt with an acid). In certain embodiments, (b) of the second reaction mixture is propane- 1,2-diamine.

In certain embodiments, the molar ratio of (a) to (b) of the second reaction mixture is between 10:1 and 4:1, between 4:1 and 2:1, between 2:1 and 1:1, between 1:1 and 1:2, between 1:2 and 1:4, or between 1:4 and 1:10, inclusive. In certain embodiments, the molar ratio of (a) to (b) of the second reaction mixture is between 3:1 and 1:3, inclusive. In certain embodiments, the molar ratio of (a) to (b) of the second reaction mixture is between 1:1 and 1:1.5, inclusive.

In certain embodiments, the second reaction mixture further comprises an organic solvent. In certain embodiments, the organic solvent of the second reaction mixture is one single organic solvent of the second reaction mixture. In certain embodiments, the organic solvent of the second reaction mixture is a mixture of two or more (e.g., three) organic solvents of the second reaction mixtures (e.g. , organic solvents of the second reaction mixtures described in this paragraph). In certain embodiments, the organic solvent of the second reaction mixture is an aprotic solvent. In certain embodiments, the organic solvent of the second reaction mixture is an ether solvent. In certain embodiments, the organic solvent of the second reaction mixture is a di(unsubstituted Ci- ₆ alkyl) ether. In certain embodiments, the organic solvent of the second reaction mixture is diethyl ether. In certain embodiments, the organic solvent of the second reaction mixture is methyl ierf-butyl ether, tetrahydrofuran, or 2-methyltetrahydrofuran. In certain embodiments, the organic solvent of the second reaction mixture is an alcohol solvent. In certain embodiments, the organic solvent of the second reaction mixture is ethanol or isopropanol, or a mixture thereof. In certain embodiments, the organic solvent of the second reaction mixture is a ketone solvent. In certain embodiments, the organic solvent of the second reaction mixture is acetone, acetonitrile, dichloromethane, or ethyl acetate. In certain embodiments, the organic solvent of the second reaction mixture is an alkane solvent. In certain embodiments, the organic solvent of the second reaction mixture is pentane, hexane, or heptane, or a mixture thereof. In certain embodiments, the boiling point of the organic solvent of the second reaction mixture at about 1 atm is between 30 and 50, between 50 and 70, between 70 and 100, between 100 and 130, between 130 and 160, or between 160 and 200 °C, inclusive.

In certain embodiments, the temperature of the second reaction mixture is between -20 and 0, between 0 and 20, between 20 and 25, between 25 and 40, or between 40 and 60 °C, inclusive. In certain embodiments, the temperature of the second reaction mixture is between 0 and 40 °C, inclusive. In certain embodiments, the temperature of the second reaction mixture is between 10 and 30 °C, inclusive. In certain embodiments, the temperature of the second reaction mixture is between 20 and 25 °C, inclusive. In certain embodiments, the temperature of the second reaction mixture is substantially constant over the second time duration.

In certain embodiments, the second time duration is between 1 and 10 minutes, between 10 and 60 minutes, between 1 and 3 hours, between 3 and 6 hours, between 6 and 12 hours, between 12 and 24 hours, or between 1 and 3 days, inclusive. In certain embodiments, the second time duration is between 10 minutes and 1 day, inclusive. In certain embodiments, the second time duration is between 10 minutes and 6 hours, inclusive. In certain embodiments, the rate of conversion of (a) of the second reaction mixture to the substituted dihydropyrazine of Formula (Cl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, and to, if present, the substituted dihydropyrazine of Formula (C2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 10% and 99% ( e.g ., between 10% and 20%, between 20% and 40%, between 40% and 60%, between 60% and 80%, or between 80% and 99%), inclusive. In certain embodiments, the rate of conversion of (a) of the second reaction mixture to the substituted dihydropyrazine of Formula (Cl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, and to, if present, the substituted dihydropyrazine of Formula (C2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 90% and 99%, inclusive.

In certain embodiments, the method further comprises isolating the substituted dihydropyrazine of Formula (Cl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof; or a mixture of the substituted dihydropyrazine of Formula (Cl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, and the substituted dihydropyrazine of Formula (C2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof. In certain embodiments, the step of isolating described in this paragraph comprises liquid-liquid phase separation, drying, filtration, concentration, chromatography, decolorization, or recrystallization, or a combination thereof. In certain embodiments, the step of isolating described in this paragraph comprises liquid-liquid phase separation, drying, filtration, concentration, decolorization, or recrystallization, or a combination thereof. In certain embodiments, the liquid-liquid phase separation is a separation of an organic phase and an aqueous phase. In certain embodiments, the drying is drying an organic phase over a solid drying agent (e.g., anhydrous Na2SC>4, anhydrous MgSCri, anhydrous CaSCri, anhydrous CaCF, or activated molecular sieves). In certain embodiments, the filtration is a filtration of a mixture of an organic phase and a solid drying agent to remove the solid drying agent and hydrates thereof. In certain embodiments, the concentration is concentration of an organic phase to remove part or substantially all of the volatiles (e.g., organic solvents). In certain embodiments, the concentration is performed under a pressure lower than 1 atm (e.g., between 0.001 and 0.01, between 0.01 and 0.1, or between 0.1 and 1 atm, inclusive). In certain embodiments, the concentration is performed under a temperature of between 0 and 10, between 10 and 20, between 20 and 25, between 25 and 35, between 35 and 50, or between 50 and 80 °C, inclusive. In certain embodiments, the chromatography is flash chromatography (e.g., normal-phase flash chromatography (e.g., over silica gel)). In certain embodiments, the step of isolating described in this paragraph does not comprise chromatography. In certain embodiments, the decolorization comprises redissolving in an organic solvent, decolorization, and concentration. In certain embodiments, the decolorization comprises contacting with a solid decolorization agent (e.g., activated charcoal). In certain embodiments, the recrystallization is a single-solvent recrystallization. In certain embodiments, the recrystallization is a multi-solvent (e.g., bi-solvent or tri-solvent) recrystallization. In certain embodiments, the recrystallization is a hot filtration- recrystallization. In certain embodiments, the step of isolating described in this paragraph further comprises removing part or substantially all of the volatiles (e.g., organic solvents) by decreasing the pressure (e.g., to a pressure of lower than 1 atm (e.g., between 0.001 and 0.01, between 0.01 and 0.1, or between 0.1 and 1 atm, inclusive) and/or increasing the temperature (e.g., to a temperature between 25 and 35, between 35 and 50, or between 50 and 80 °C, inclusive).

In certain embodiments, the method does not further comprise isolating the substituted dihydropyrazine of Formula (Cl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof; or a mixture of the substituted dihydropyrazine of Formula (Cl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, and the substituted dihydropyrazine of Formula (C2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof.

In certain embodiments, the methods further comprise incubating a third reaction mixture comprising:

(a) the substituted dihydropyrazine of Formula (Cl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof;

(b) a base; and

(c) an oxidant; for a third time duration sufficient to produce a substituted pyrazine of Formula (Dl): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof.

In certain embodiments, the third reaction mixture further comprises (c) the substituted dihydropyrazine of Formula (C2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof; and the third time duration is sufficient to further produce a substituted pyrazine of Formula

(D2): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof.

In certain embodiments, the molar ratio of the substituted pyrazine of Formula (Dl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the substituted pyrazine of Formula (D2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 51:49 and 60:40, between 60:40 and 70:30, between 70:30 and 80:20, between 80:20 and 90:10, between 90:10 and 99: 1, inclusive. In certain embodiments, the molar ratio of the substituted pyrazine of Formula (Dl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the substituted pyrazine of Formula (D2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 55:45 and 95:5, inclusive. In certain embodiments, the molar ratio of the substituted pyrazine of Formula (Dl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the substituted pyrazine of Formula (D2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 60:40 and 80:20, inclusive.

In certain embodiments, the substituted pyrazine of Formula (Dl) is of Formula (10): or a tautomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof. In certain embodiments, the substituted pyrazine of Formula (D2) is of Formula (11): or a tautomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof.

In certain embodiments, the substituted pyrazine of Formula (Dl) is of Formula (11), or a tautomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof. In certain embodiments, the substituted pyrazine of Formula (D2) is of Formula (10), or a tautomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof.

In certain embodiments, the substituted pyrazine of Formula (Dl) is of the formula: or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof.

In certain embodiments, the base is an inorganic base. In certain embodiments, the base is an alkali metal hydroxide or an alkaline earth metal hydroxide. In certain embodiments, the base is LiOH, NaOH, or KOH. In certain embodiments, the base is KOH. In certain embodiments, the base is L12CO3, NaiCCte, or K2CO3. In certain embodiments, the base is LiHCCh, NaHCCte, or KHCO3. In certain embodiments, the base is ammonia, ammonium carbonate, or ammonium hydroxide. In certain embodiments, the base is an organic base ( e.g ., mono-, di-, or tri- (unsubstituted Ci- ₆ alkyl) amine, cyclic non-aromatic amine, or aromatic amine (e.g., pyridine)).

In certain embodiments, the molar ratio of (a) to (b) of the third reaction mixture is between 10:1 and 4:1, between 4:1 and 2:1, between 2:1 and 1:1, between 1:1 and 1:2, between 1:2 and 1:4, or between 1:4 and 1:10, inclusive. In certain embodiments, the molar ratio of (a) to (b) of the third reaction mixture is between 3:1 and 1:3, inclusive. In certain embodiments, the molar ratio of (a) to (b) of the third reaction mixture is between 1.5:1 and 1:1.5, inclusive.

In certain embodiments, the oxidant is dioxygen. In certain embodiments, the oxidant is atmospheric air (e.g., atmospheric air at an altitude of between 0 and 100, between 100 and 1,000, or between 1,000 and 10,000 m, inclusive). In certain embodiments, the oxidant is silica- supported perchloric acid (see, e.g., Das et al., Tetrahedron Letters, 2007, 48, 5371-5374). In certain embodiments, the oxidant is a manganese complex or a manganese salt. In certain embodiments, the oxidant is a manganese complex comprising 4,5-bis(diphenylphosphino)- acridine or 4.5-bis(diphcnylphosphino)-9//-acridinc-10-idc (see, e.g., Daw et al., ACS Catalysis, 2018, 8, 7734-7741). In certain embodiments, the oxidant is manganese dioxide (see, e.g., Raw et al., Chemical Communications, 2002, 18, 2286-2287).

In certain embodiments, the molar ratio of (a) to (c) of the third reaction mixture is between 1:1 and 1:1.1, between 1:1.1 and 1:2, between 1:2 and 1:4, between 1:4 and 1:10, between 1:10 and 1:100, or between 1:100 and 1:1,000, inclusive. In certain embodiments, the molar ratio of (a) to (c) of the third reaction mixture is between 1:1 and 1:1,000, inclusive.

In certain embodiments, the third reaction mixture further comprises an organic solvent. In certain embodiments, the organic solvent of the third reaction mixture is one single organic solvent of the third reaction mixture. In certain embodiments, the organic solvent of the third reaction mixture is a mixture of two or more (e.g., three) organic solvent of the third reaction mixtures (e.g., organic solvent of the third reaction mixtures described in this paragraph). In certain embodiments, the organic solvent of the third reaction mixture is an aprotic solvent. In certain embodiments, the organic solvent of the third reaction mixture is an ether solvent. In certain embodiments, the organic solvent of the third reaction mixture is a di(unsubstituted Ci- ₆ alkyl) ether. In certain embodiments, the organic solvent of the third reaction mixture is diethyl ether. In certain embodiments, the organic solvent of the third reaction mixture is methyl tert- butyl ether, tetrahydrofuran, or 2-methyltetrahydrofuran. In certain embodiments, the organic solvent of the third reaction mixture is an alcohol solvent. In certain embodiments, the organic solvent of the third reaction mixture is ethanol or isopropanol, or a mixture thereof. In certain embodiments, the organic solvent of the third reaction mixture is a ketone solvent. In certain embodiments, the organic solvent of the third reaction mixture is acetone, acetonitrile, dichloromethane, or ethyl acetate. In certain embodiments, the organic solvent of the third reaction mixture is an alkane solvent. In certain embodiments, the organic solvent of the third reaction mixture is pentane, hexane, or heptane, or a mixture thereof. In certain embodiments, the organic solvent of the third reaction mixture is the same as the organic solvent of the second reaction mixture. In certain embodiments, the boiling point of the organic solvent of the third reaction mixture at about 1 atm is between 30 and 50, between 50 and 70, between 70 and 100, between 100 and 130, between 130 and 160, or between 160 and 200 °C, inclusive.

In certain embodiments, the temperature of the third reaction mixture is between -20 and 0, between 0 and 20, between 20 and 25, between 25 and 40, or between 40 and 60 °C, inclusive. In certain embodiments, the temperature of the third reaction mixture is between 0 and 40 °C, inclusive. In certain embodiments, the temperature of the third reaction mixture is between 10 and 30 °C, inclusive. In certain embodiments, the temperature of the third reaction mixture is between 20 and 25 °C, inclusive. In certain embodiments, the temperature of the third reaction mixture is substantially constant over the third time duration.

In certain embodiments, the third time duration is between 1 and 10 minutes, between 10 and 60 minutes, between 1 and 3 hours, between 3 and 6 hours, between 6 and 12 hours, between 12 and 24 hours, or between 1 and 3 days, inclusive. In certain embodiments, the third time duration is between 20 minutes and 1 day, inclusive. In certain embodiments, the third time duration is between 30 minutes and 8 hours, inclusive.

In certain embodiments, the pressure of the first, second, and third reaction mixtures is about 1 atm. In certain embodiments, the step of incubating the third reaction mixture is immediately after the step of incubating the second reaction mixture. In certain embodiments, (a) of the third reaction mixture is in the form of the second reaction mixture after the step of incubating the second reaction mixture.

In certain embodiments, the method further comprises isolating the substituted pyrazine of Formula (Dl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof; or a mixture of the substituted pyrazine of Formula (Dl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, and the substituted pyrazine of Formula (D2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof. In certain embodiments, the step of isolating described in this paragraph comprises liquid-liquid phase separation, drying, filtration, concentration, chromatography, decolorization, or recrystallization, or a combination thereof. In certain embodiments, the step of isolating described in this paragraph comprises liquid-liquid phase separation, drying, filtration, concentration, decolorization, or recrystallization, or a combination thereof. In certain embodiments, the liquid-liquid phase separation is a separation of an organic phase and an aqueous phase. In certain embodiments, the drying is drying an organic phase over a solid drying agent (e.g., anhydrous Na2S04, anhydrous MgSCU, anhydrous CaSCri, anhydrous CaCh, or activated molecular sieves). In certain embodiments, the filtration is a filtration of a mixture of an organic phase and a solid drying agent to remove the solid drying agent and hydrates thereof. In certain embodiments, the concentration is concentration of an organic phase to remove part or substantially all of the volatiles (e.g., organic solvents). In certain embodiments, the concentration is performed under a pressure lower than 1 atm (e.g., between 0.001 and 0.01, between 0.01 and 0.1, or between 0.1 and 1 atm, inclusive). In certain embodiments, the concentration is performed under a temperature of between 0 and 10, between 10 and 20, between 20 and 25, between 25 and 35, between 35 and 50, or between 50 and 80 °C, inclusive. In certain embodiments, the chromatography is flash chromatography (e.g., normal-phase flash chromatography (e.g., over silica gel)). In certain embodiments, the chromatography is high-performance liquid chromatography (HPLC) (e.g., reverse-phase HPLC or normal-phase HPLC). In certain embodiments, the step of isolating described in this paragraph does not comprise chromatography. In certain embodiments, the decolorization comprises redissolving in an organic solvent, decolorization, and concentration. In certain embodiments, the decolorization comprises contacting with a solid decolorization agent (e.g., activated charcoal). In certain embodiments, the recrystallization is a single-solvent recrystallization. In certain embodiments, the recrystallization is a multi-solvent (e.g., bi-solvent or tri-solvent) recrystallization. In certain embodiments, the recrystallization is a hot filtration-recrystallization. In certain embodiments, the step of isolating described in this paragraph further comprises removing part or substantially all of the volatiles (e.g., organic solvents) by decreasing the pressure (e.g., to a pressure of lower than 1 atm (e.g., between 0.001 and 0.01, between 0.01 and 0.1, or between 0.1 and 1 atm, inclusive) and/or increasing the temperature (e.g., to a temperature between 25 and 35, between 35 and 50, or between 50 and 80 °C, inclusive).

In another aspect, the present disclosure provides substituted pyrazines produced by the methods described herein. In certain embodiments, the substituted pyrazine produced by the methods is a substituted pyrazine of Formula (Dl), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof. In certain embodiments, the substituted pyrazine produced by the methods is a substituted pyrazine of Formula (D2), or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof.

In another aspect, the present disclosure provides compositions comprising: the substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, or the mixture; and optionally an excipient.

In certain embodiments, the excipient is acceptable for use in foods, drinks, or cigarettes. In another aspect, the present disclosure provides kits comprising: the substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, the mixture, or the composition; and instructions for using the substituted pyrazine, tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, co-crystal, mixture, or composition.

In certain embodiments, the kit comprises a first container, wherein the first container comprises the substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, the mixture, or the composition. In some embodiments, the kit further comprises a second container. In certain embodiments, the second container comprises an excipient. In certain embodiments, the second container comprises the instructions. In certain embodiments, each of the first and second containers is independently a vial, ampule, bottle, syringe, dispenser package, tube, or box.

In another aspect, the present disclosure provides mixtures comprising: a first substituted pyrazine of Formula (Dl): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof; and a second substituted pyrazine of Formula (D2): or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co crystal thereof; wherein: each R ¹ is the same and is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted acyl, substituted or unsubstituted alkoxy, or substituted or unsubstituted carbocyclyl; each R ² is the same and is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted acyl, substituted or unsubstituted alkoxy, or substituted or unsubstituted carbocyclyl; each R ³ is the same and is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl; each R ⁴ is the same and is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl; and the molar ratio of the first substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the second substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 51:49 and 99.9:0.1, inclusive; provided that: at least one of R ¹ and R ² is not hydrogen; and the first substituted pyrazine is different from the second substituted pyrazine.

In certain embodiments, R ¹ , R ² , R ³ , and R ⁴ are as described herein.

In certain embodiments, the molar ratio of the first substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the second substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 51:49 and 60:40, between 60:40 and 70:30, between 70:30 and 80:20, between 80:20 and 90:10, between 90:10 and 99: 1, inclusive. In certain embodiments, the molar ratio of the first substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the second substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 55:45 and 95:5, inclusive. In certain embodiments, the molar ratio of the first substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, to the second substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, is between 60:40 and 80:20, inclusive.

In certain embodiments, the mixture further comprises one or more additional compounds (e.g., additional substituted pyrazines). In certain embodiments, the mixture is substantially free of additional compounds (e.g., the combined molar concentration of the additional compounds in the mixture is between 0.01% and 0.1%, between 0.1% and 1%, between 1% and 3%, between 3% and 10%, inclusive).

In another aspect, the present disclosure provides methods of altering the flavor of a food, drink, or cigarette comprising adding an effective amount of the substituted pyrazine, or a tautomer, stereoisomer, isotopically labeled compound, salt, solvate, polymorph, or co-crystal thereof, the mixture, or the composition to the food, drink, or cigarette, or to a raw or intermediate material for producing the food, drink, or cigarette. In certain embodiments, the food is a sauce, cereal, chocolate, cocoa, meat product, fish product, potato, nut product, popcorn, or bread. In certain embodiments, the drink is a coffee, tea, liquor, wine, or beer. In certain embodiments, the cigarette is an electronic cigarette.

EXAMPLES

In order that the invention described herein may be more fully understood, the following examples are set forth. The examples are offered to illustrate the methods and uses described herein and are not to be construed in any way as limiting their scope.

Our first attempts explored how steric could direct the coupling orientations between diamines to diketones to access 235-EDMP. Also, various physical parameters of the reaction conditions were changed, such as reduced temperature, the order-of-addition of reactants, and supplementation with chiral zeolites (Montmorillonite phyllosilicates) to template the orientation of the coupling partners to direct the regiochemistry of the reaction. Each reaction trial resulted in 50:50 mixtures of the ethyl dimethylpyrazine regioisomers.

An alternative approach was explored to direct the regioselectivity of the reactions; acyloins (a-hydroxy ketone) replaced the diketone as the electrophilic coupling reactant used in the previous trial experiments. The hydroxy ketone reactants were made biocatalytically with pyruvate decarboxylase (E.C. 4.1.1.1). The coupling reaction between 2-hydroxypentan-3-one and propane- 1 ,2-diamine resulted in the desired 235-EDMP at >70% (~77 mg total) relative to 30% 325-EDMP in the product mixture. The 3-hydroxypentan-2-one acyloin congener bio catalyzed and reacted with propane- 1,2 -diamine to make 325-EDMP (-60% relative abundance, -73 mg) over the 235-EDMP. These results suggested a mechanism directed by the hydroxy ketone electrophilicity and the sterics at each nucleophilic center of the diamine.

Exemplary Synthesis of Substituted Pyrazines Chemicals, Reagents, and Instrumental Analysis

All chemicals and reagents were obtained from Sigma- Aldrich (St. Louis, MO) and used without further purification unless noted otherwise.

Gas chromatography /electron-impact mass spectrometry (GC/EI-MS) analysis was performed on an Agilent 6890N gas chromatograph equipped with a capillary GC column (30 m x 0.25 mm x 0.25 mM; HP-5MS; J & W Scientific) with He as the carrier gas (flow rate, 1 mL/min). The injector port (at 250 °C) was set to splitless injection mode. A 1-pL aliquot of each sample was injected using an Agilent 7683 auto-sampler (Agilent, Atlanta, GA). The column temperature was increased from 40 to 150 °C at 20 °C/min, then increased by 20 °C/min to 250 °C with a 5 min hold, and returned to 40 °C. The gas chromatograph was coupled to a mass selective detector (Agilent 5973 inert ) operated in electron impact mode (70 eV ionization voltage).

NMR experiments were recorded on an Agilent DDR2 500 MHz NMR spectrometer (500 MHz ( ' H)) at 25 °C. ¹ H NMR data was acquired using a recycle delay of 20 s and 32 scans. For Heteronuclear Single Quantum Correlation (HSQC) 2D-NMR experiments, the number of transients (nt) = 16 and the number of increments (ni) = 128. For acquiring Heteronuclear Multiple Bond Correlation (HMBC) 2D-NMR spectra nt = 16 and ni = 400. The ¹ H NMR chemical shifts were referenced to that of residual protonated solvent in CDCh (7.24 ppm).

An Agilent HP 1100 instrument (Agilent Technologies, San Diego, CA, USA) coupled to an analytical column (Alltech, 250 mm x 2.1 mm i.d., 5 pm particle size) or a semipreparative Alltech Econosil C-18 column (250 mm x 22 mm i.d., 10 pm particle size) attached to a UV- diode array detector were used to monitor (A278) and separate the EDMP isomers. Guided by earlier HPLC solvent systems to separate pyrazines by C-18 RP-HPLC, ^38,40,41 various isocratic mixtures of CH3CN (20% to 50%) in water were tested as the eluent to optimize the resolution of elution (i¾)· A volume of 1 pL of a 37 pM EDMP solution (dissolved in CH3CN) was loaded onto the analytical column to determine a suitable solvent system of ClUCN/water suitable for the semipreparative column. A 100 pL of a 37 pM EDMP solution (dissolved in CH3CN) was loaded onto the semipreparative column and eluted with an isocratic ClUCN/water mixture (20:80) at 5 mL/min. The elution volumes of the chromatographically separated EDMP isomers from multiple injections were collected, acidified to pH 1 (6M HC1) to convert the compounds to the pyrazinium chloride salts, and the solvent was removed under a stream of nitrogen.

Example 1. HPLC separation to isolate 235-EDMP from a commercially available EDMP mixture

The EDMP isomeric mixture (Sigma- Aldrich, St. Louis, MO) contained a 50:50 mixture of each isomer (235-EDMP and 325-EDMP), as determined by GC/MS analysis. A 1 pL-aliquot (from a 5 mg EDMP/mL sample dissolved in CH3CN) was loaded onto the analytical column (Alltech, Cl 8, 250 mm x 2.1 mm i.d., 5 pm particle size) to determine that a solvent mixture of CHsCN/water (20:80, isocratic) was suitable as the mobile phase for the larger-scale separation.

Example 2. Synthesis of 2-ethyl-3 ,5-dimethylpyrazine (10) through diketone and diamine coupling

Propane- 1 ,2-diamine (1 mM, 74 pL) and pentane-2, 3-dione (1 mM, 100 pL) were incubated in diethyl ether (2 mL) at 25 °C, 1 h, and the reaction was monitored by GC-MS analysis.

The coupling reaction between diketone and diamine was followed by different temperatures (25 °C, 0 °C, 30 °C) and changing the addition order of the reactants (pentane-2, 3- dione and propane- 1 ,2-diamine) to the reaction mixture. Different Zeolites (ZBH: Zeolite beta, hydrogen (SiOVAhCh -360:1); ZYH: Zeolite Y, hydrogen (S1O2/AI2O3 - 80:1); ZMA: Zeolite mordenite, ammonium (S1O2/AI2O3- 20:1); ZSM: Zeolite ZSM-5, ammonium (S1O2/AI2O3-2OO to 400:1)) were used as a blocking agent and followed the coupling reaction.

Example 3. Synthesis of 2-ethyl-3 ,5-dimethylpyrazine (10) by a method described herein

Synthesis of 17 and 18

A pyruvate decarboxylase (PDC) (E.C. 4.1.1.1) (10 mg, 5 U) from baker’s yeast (S. cerevisiae) was added to 0.1 M sodium citrate buffer (pH 6.0) (4 mL) containing 2 mM thiamine diphosphate (3.68 mg) and 20 mM MgSCC (9.6 mg). Acetaldehyde (3.1 mmol, 176 pL) and 2- oxobutanoate sodium salt (3.0 mmol, 370 mg) were added to the reaction mixture and incubated at 25 °C for 48 h. The reaction mixture was extracted with diethyl ether (3 x 2 mL). The organic fractions were combined, and the solvent was removed under a stream of nitrogen. The resulting residue (~82 mg) without further purification was dissolved in CDCI ₃ and analyzed by ¹ H NMR to judge the 2-hydroxypentan-3-one (17) at -54% converted yield (Ligure 17). An aliquot of the crude sample was dissolved in ether and analyzed by GC/EI-MS in selected-ion and total ion modes.

The synthesis and workup of 3-hydroxypentan-2-one (18) were similar to those described for 17 except that propionaldehyde (2.5 mmol, 145 pL) and 2-oxopropanoate (i.e., pymvate) sodium salt (2 mmol, 220 mg) were used in place of acetaldehyde and 2-oxobutanoate, respectively, during the synthesis. These cosubstrates were incubated with the PDC enzyme (10 mg, 10 U). After concentrating the sample, the resulting residue (~90 mg) without further purification was dissolved in CDCb and analyzed by ¹ H NMR (Figure 17). An aliquot of the crude sample was dissolved in ether and analyzed by GC/EI-MS in selected-ion and total ion modes.

Synthesis of EDMP isomers

Propane- 1 ,2-diamine (1 mmol, 74 pL) and 17 or 18 (-0.9 mmol, -90 mg) were mixed in ether (2 mL) and incubated at rt for 1 h.

KOH pellets (1 mmol, 56 mg) were stirred with the reaction mixture at rt in an open-air container for 2 h to convert the dihydropyrazine intermediates to the aromatic EDMP isomers. The resulting mixture was filtered, and the ether layer was separated from the residual water made during the dehydration steps of the reaction. An aliquot (1 pL) from the ether layer was analyzed by GC/EI-MS. The EDMP isomers were quantified using a standard curve with 2,5- DMP as the internal standard. The converted yields were calculated based on the amount of substrate added to the reaction mixture.

To estimate an approximate purity of the synthesized EDMP isomers in the crude sample, we assumed the average ionization potential for all analytes present in the crude EDMP sample mixtures were the same when analyzed by GC/EI-MS.

For the preparative-scale separation of the EDMP isomers, a 100 pL-aliquot (from a 5 mg EDMP/mL sample dissolved in CH3CN) was loaded onto a preparative column (Alltech Econosil, C18, 250 mm x 22 mm i.d., 10 pm particle size) and eluted with CEECN/water (20:80, isocratic) at 5 mL/min. The elution volume of each of the two peaks was separately collected.

The fractions from multiple 100 pL-injections for each peak were combined, acidified to pH 1 (6M HC1) to convert the pyrazines to their HC1 salts, and concentrated under a stream of nitrogen. The separated EDMP isomers were analyzed by NMR and GC-MS to characterize the 235-EDMP and 325-EDMP isomers.

Example 4. Separation of the EDMP isomers This example aimed to develop a method to make 2-ethyl-3,5-dimethylpyrazine (235- EDMP, 10) selectively over its 3-ethyl-2,5-dimethylpyrazine (325-EDMP, 11) isomer. Each individual isomer is not available commercially; thus, we needed to separate them and characterize their identities unequivocally to help differentiate the products from our synthetic trials. A commercial-grade 50:50 mixture of EDMP isomers was used to separate 10 from 11. Separation by fractional distillation was not practical because the boiling points of 10 and 11 are similar (181-182 °C). ⁴³ Therefore, we separated the isomers by preparative, reverse-phase HPLC with UV monitoring of the effluent (Figure 2A), based on a previous technique. ³⁸ We evaluated the relative purity of each isomer by GC/EI-MS before (Figure 2B) and after purification, judged to be -95% pure (Figure 2C, D). The shorter retention-time pyrazine on the GC/EI-MS was designated as 11 (Figure 2C), and the longer retention-time compound was assigned as 10 (Figure 2D) based on separate validation of each compound by NMR experiments.

Example 5. NMR characterization of the EDMP isomers

The separate eluent fractions isolated from the reverse-phase HPFC were acidified to convert the pyrazines to their nonvolatile HC1 salts. Each fraction was concentrated and analyzed by ID- ¹ !!- and ¹³ C-NMR, 2D-HSQC (Heteronuclear Single Quantum Coherence Spectroscopy), and 2D-HMBC (Heteronuclear Multiple Bond Correlation) to unambiguously characterize each EDMP. The HSQC NMR spectmm showed one-bond C-H (QCH) correlations for the methyl, ethyl, and aryl ring carbons and attached hydrogens and confirmed distinct chemical shift differences for each EDMP isomer (Figures 11 and 15). The HSQC data alone provided important information about the alkyl substitution on the aromatic ring but was not conclusive to distinguish one isomer from the other.

A combination of long-range, diagnostic correlations was made from the HMBC data that showed two and three through-bond C-H correlations C ^~ Jc _{\ i} and ³ /CH) that helped distinguish the purified EDMP isomers 10 and 11. The spectrum for one EDMP isomer had correlations between the aryl hydrogen (H-6), C-2 (ethyl attachment), and CIO (methyl), and between the ethyl substituent hydrogens (H-7 and H-8) and C-2 (Figure 3A, Table 4). This combination of Jew and ei _l HMBC correlations helped validate the alkyl substitution patterns on the pyrazine ring that were consistent with the 235-EDMP structure (10). The distinct long-range HMBC correlations in 10 (Figure 12) contrasted those in 11 (Figure 16), which were between the aryl hydrogen (H- 6), C-2 (methyl-7 attachment), and C-5 (methyl-10 attachment) (Figure 3B, Table 6). This spin- coupled system revealed the " ortho"/" meta" -locations of the methyl substituents relative to H-6. Reciprocally, H-10 correlated with C-6, the unsubstituted aryl carbon, and this spin system reinforced the C-10 (methyl) position. There was no crosstalk between H-6 and either of the ethyl carbons, suggesting a >3 -bond distance between these sets of atoms and supporting the 325- EDMP structure (11).

Example 6. Attempted synthesis of 235-EDMP

A direct route to the pyrazine framework is to couple a carbonyl compound with an alkyl amine partner in a condensation reaction, but this coupling occurs rapidly without regio selectivity. We explored different reaction conditions to couple pentan-2,3-dione and propane- 1,2-diamine to direct the regioselectivity. First, the coupling reaction was carried out at different temperatures (-25 °C, 0 °C, and room temperature (~23 °C) to assess if the temperature could, in part, control the steric approach of the reagents. We envisioned that at sub-ambient temperatures, the terminal amine of the propane- 1,2-diamine reactant could preferentially select for the less encumbered methyl-substituted ketone of the pentan-2,3-dione co-reactant. For each of the temperature control reactions, the order and rate (dropwise or single bolus) in which the reactants were added to the reaction mixture were changed. We also hypothesized that various microporous zeolites could provide cavitation to coordinate the least hindered, terminal ammonium group of the diamine and the least sterically blocked methyl keto group of the 2,3- diketone through protonation. These interactions were imagined to direct regioselective imine formation between the internal amino group and the ethyl keto group of the reactants toward the desired 235- EDMP. Undesirably, all these approaches resulted in 50:50 isomeric mixtures of 235- EDMP and 325-EDMP (Figure 4). It appeared that the sterics in each of the reactants provided little regioselective control in the transition state to yield the 235-EDMP preferentially under the tested conditions.

Example 7. Biocatalytic synthesis of isomerically enriched EDMP isomers

Biocatalysis of alkyl acyloins

Our approach looked at changing the functional group reactivity of the electrophilic reactant (pentan-2,3-dione, 9). The diketone electrophile substrate was replaced with a hydroxy ketone reactant to couple with the diamine (8) nucleophile. With the application of a-hydroxy ketones as the electrophile, we hoped the terminal amino group of diamine 8 to react selectively with the keto functional group of 2-hydroxypentan-3-one (17). We also hoped that this initial coupling preference to influence the overall reaction selectivity toward the 235-EDMP isomer (Scheme 3). We tested this hypothesis by preparing 17 biocatalytically (Scheme 3) with commercial pyruvate decarboxylase (PDC) isolated from S. cerevisiae. In nature, PDC catalyzes the nonoxidative decarboxylation of pyruvate to acetaldehyde using thiamine diphosphate and Mg (II) (e.g., Mg ²⁺ ions) as cofactors in ethanol production in plants and fungi. ³⁹ PDC can also catalyze off-pathway carboligation reactions to form acyloins by condensing an acetaldehyde and 2-oxoalkanoic acids to make dialkyl acyloins ^39,45 that are desired for this study. We incubated PDC with acetaldehyde (21, C2-moiety) and propanal (22, C3-moiety) and oxoalkanoate sodium salts 19 and 20 to make 17 and 18 hydroxypentanones (Scheme 4), respectively. In this study, the carbon building blocks (acetaldehyde for the C2-moiety and 2-oxobutanoic acid as a precursor for the C3-moiety) differed from those used as feedstocks (pyruvate as a precursor for the C2-moiety and propanal for the C3-moiety) in the earlier report on the whole-cell assembly of a mixture of hydroxy pentanones 17 (3.9% yield) and 18 (0.5% yield). ³⁹

Scheme 3. A proposed mechanism for the formation of 5,6-dihydropyrazine intermediates toward pyrazines from condensation between acyloins and 1,2-propanediamine: a) condensation between the less hindered amine with the keto group of the hydroxyketone substrate, b) proton transfer and dehydration, c) formation of the keto group, and d) condensation between the internal amine and the keto group.

Scheme 4. Synthesis of hydroxypentanones (17 and 18) from the appropriate aldehyde and oxoalkanoate incubated with a) pyruvate decarboxylase (PDC), MgSC>4, and thiamine diphosphate in sodium citrate buffer.

Here, we extracted the biocatalyzed 2-hydroxypentan-3-one (17) from the aqueous enzyme incubation buffer with ether and removed the organic solvent to obtain a crude product mixture. ¹ H NMR analysis of 17 without purification had diagnostic chemical shifts at d 1.18 (3H, triplet, J = 1.2 Hz) and d 3.01 (2H, quartet, J = 1.5 Hz) for the ethyl attached to the keto group and at d 1.43 (3H, doublet, J = 6.7 Hz) and d 4.86 (1H, quartet, 7= 6.1 Hz) for the hydroxy ethyl attached to the other side of the keto group (Figure 17 for product numbering). In the sample containing 17, 18 was not easily identified by signature ¹ H NMR signals.

The crude sample containing the biocatalyzed 3-hydroxypentan-2-one (18) was extracted and analyzed identically to the methods described for 17. ¹ H NMR data for the cmde sample of 18 was more challenging to interpret, because diagnostic chemical shifts were not readily discernible. The homonuclear COSY spectra of a reference compound 3-hydroxy-2-hexanone ⁴⁶ with similar structural features to 3-hydroxy-2-ketone 18, made in our study, were used to assign the expected chemical shift values for 18. The COSY spectrum for the putative 18 made biocatalytically had putative ¹ H- ¹ H correlations between H4 and H5 (the terminal ethyl group) but lack diagnostic correlations between H3 (attached to the C-OH) and neighboring protons (Figure 19) as seen for 3-hydroxy-2-hexanone (Figure 18). Therefore, the sample was analyzed by GC/EI-MS to determine if 18 had been made.

A selected ion chromatogram of the crude sample putatively containing 18 showed a chromatographic peak corresponding to a compound that fragmented into ions consistent with 3- hydroxypentan-2-one (Figures 5A and 5B). Diagnostic fragment ions for the 2-hydroxypentan-3- one isomer 17 (at 40% abundance relative to 18) were also detected for a peak eluting at 5.43 min (see Figure 6B for ion fragments). Evaluation of the GC/EI-MS total ion chromatogram for the crude sample, where 18 was the intended biocatalysis product, showed several other peaks with ion abundances above those for 17 and 18. The compound identities of the most abundant peaks were identified by comparing their fragment ion profiles against those in spectral databases (Figure 5C). Each off-pathway product can arise from the excess propanal substrate used to stimulate the reaction biocatalyzed by the PDC enzyme. These other compounds included 2- methylpent-2-enal (5.74 min) resulting from dehydration of 3-hydroxy-2-methylpentanal, 3- hydroxy-2-methylpentanal (7.93 min) derived from aldol condensation between two propanal molecules, and 2,6-diethyl-5-methyl-l,3-dioxan-4-ol (12.15 min) resulting from condensation between 3-hydroxy-2-methylpentanal and propanal (Figure 5C).

We evaluated the selected-ion chromatogram of the crude sample containing 17 to gain insight into why the H NMR for 17 biocatalyzed by PDC from 2-oxobutanoate and acetaldehyde was more interpretable than the ¹ H NMR for 18. The selected-ion chromatogram showed a chromatographic peak (at 5.42 min) corresponding to a compound that fragmented into ions consistent with 2-hydroxypentan-3-one (Figures 6A and 6B). Diagnostic fragment ions for the 3- hydroxypentan-2-one isomer 18 (at -30% abundance relative to 17) were also detected for a peak eluting at 5.29 min (see Figure 5B for ion fragments). GC/EI-MS total-ion analysis of the crude sample, where 17 was the intended biocatalysis product, showed other peaks with significantly lower ion abundances than those for 17 and 18 (Figure 6C). One relatively abundant peak eluting at 4.85 min was identified as paraldehyde resulting from self-condensation of the acetaldehyde cosubstrate.

We concluded that the PDC carboligation efficiency for substrates 2-oxobutanoate (19, non-natural substrate) and acetaldehyde (21, natural substrate) to make 17 was more significant (-43% converted yield) than for pyruvate (20, natural substrate) and propanal (22, non-natural substrate) substrates to make 18 (-15% converted yield) (Table 1). PDC may be more permissive for non-natural 2-oxo-alkanoate substrates than for non-native alkanal substrates longer than acetaldehyde. The greater relative abundance of 17 made biocatalytically within its crude sample matrix enabled diagnostic NMR analysis.

Table 1. Estimated yield of a-hydroxyketone (HK) syntheses

"Arb. Units: Arbitrary Units; *SM: starting material; ^c based on 1.5 mmol of 19 and 21 each; ^rf based on 2 mmol of 20 and 22 each.

Synthesis of isomerically enriched 235-EDMP

Diamine (8) and the crude sample of 17 (Scheme 3) were mixed in ether for 2 h. GC/EI- MS analysis of the product mixture showed that dihydropyrazine intermediates of the EDMP isomers were made at a 70:30 ratio, based on the peak area (Figure 7A); the identity of each putative trialkyl dihydropyrazines was not characterized by NMR or comparison against authentic standards. The sample containing the putative dihydropyrazines were oxidized in open- air conditions in a basic (KOH pellets) ether emulsion to obtain the EDMP isomers at a 70:30 ratio; the 235-EDMP isomer (10) predominated (Figure 7C). The products corresponding to the GC/EI-MS peaks were identified based on an earlier preparative HPLC separation and characterization of the EDMP isomers by NMR analysis. The converted yield of 235-EDMP (~77 mg) and 325-EDMP (~33 mg) was estimated from the relative ion abundance of each isomer in the sample and the 110 mg total calculated from a standard curve for EDMP. The approximate purity of the synthesized EDMP isomers in the crude sample was estimated at 29 % (Table 2).

Synthesis of isomerically enriched 325-EDMP

We biocatalyzed an isomerically enriched sample of 325-EDMP (11) from the hydroxyketone isomer (18, Scheme 3) incubated with diamine 8. GC/EI-MS analysis of the product mixture showed that dihydropyrazine intermediates of the EDMP isomers were obtained at a 40:60 ratio (Figure 7C). The dihydropyrazines were oxidized in open-air conditions in a basic (KOH pellets) ether emulsion and converted to a 40:60 mixture of the EDMP isomers with 325-EDMP (11) predominating over its isomer 10 (Figure 7D). The products corresponding to the GC/EI-MS peaks were identified based on an earlier preparative HPLC separation and characterization of the EDMP isomers by NMR analysis. The yield of 325-EDMP (~57 mg) and 235-EDMP (~38 mg) was estimated from the relative ion abundance of each isomer in the sample and the 95 mg total calculated from a standard curve for EDMP. The approximate purity of the synthesized EDMP isomers in the crude sample was estimated at 9 % (Table 2).

'HMBC correlations between carbon (·) and hydrogen (shown explicitly as bold and underlined) are shown in the structures.

We referenced the COSY spectrum for 3-hydroxy-2-hexanone (Figure 18) to help predict the chemical shifts for the biocatalyzed 3-hydroxy-2-pentanone (18) (Figure 19). H5 (CFE) of 18 was expect to resonate at -0.9 ppm, similar to H6 (CFb) of the hexanone homolog. H5 and H4 of to evaluate the potential correlations between H5 and H4 of. Putative correlation between H5 and H4 of 18 was provisionally assigned as a terminal ethyl group; the same correlation between H5 and H4 was also observed in the COSY spectrum for 3-hydroxy-2-hexanone (Figure 18).

Putative side-products in EDMP synthesis

The GC/EI-MS total ion chromatogram for the crude sample, where 10 was predominant at 70:30 over its EDMP isomer 11, showed several other peaks with ion abundances similar to those for 10 and 11 (Figure 8A). The side products putatively identified were either formed chemically during the coupling reaction between the diamine (8) and off-pathway products in the crude sample of 17 or by unexpected accelerated reactions of unreacted starting reagents in the hot injector port (235 °C) of the GC instrument. ⁵¹ The compound identities of some most abundant peaks were identified by comparing their fragment ion profiles against those in spectral databases. For example, the side products eluting from the GC (Figure 8 A) whose EI-MS fragmentation matched best to those in mass spectral databases included cis/trans- 2,6- dimethylpiperazine (Figures 8C and 8D) and (7 ^' .s//ra».s-2, 5-dimethyl piperazine (Figures 8E and 8F) resulting from parallel and antiparallel dimerization, respectively, of diamine (8). Other compounds eluting at 3.87, 4.12, 4.51, 4.60, and 4.79 min did not have fragment ion profiles matching those in the mass spectral databases but contained fragment ions that could be inferred from related azo-compounds ⁵² (Figures 26A to 26C). The compound eluting at 7.52 min (Figure 8A) fragmented into ions that were consistent with those for a putative alkyl ester with a molecular formula of C9H16O2 (Figure 26D).

The GC/EI-MS total ion chromatogram for the crude sample, where 11 was predominant at 60:40 over its EDMP isomer 10, showed other peaks with ion abundances greater than those for 10 and 11 (Figure 8B). Curiously, several of the side products coeluted from the GC for this crude sample compared to the more resolved compounds in the sample where 10 was predominant over its EDMP isomer 11 (Figure 8A). Many of the same fragment ions ( m/z 114, 99, 85, 70, 56, 58) consistent with azo-compounds were identified as before (Figures 26E and 26F). Conclusion

Pyrazines are used as synthetic precursors in medicinal chemistry and are of interest within the pharmaceutical sector. ⁵⁵ A SciFinder ¹¹ database query for all types of substituted pyrazines reports -950 compounds. Of these, -185 are a combination of tri-n-alkyl- / cycloalkyl- substituted pyrazines. This dearth of citations reflects limited methods for alkylpyrazine preparation. ⁵⁶ The application of a green, all-natural route to 325-EDMP alkylpyrazine via a viable, proposed biosynthetic route is promising toward potentially increasing titers. However, a parallel biosynthetic route to the 235-EDMP isomer would be difficult without invoking carbonyl/amine reagents that would self- and cross-couple to produce an uncontrolled mixture of intermediate products. Here, we reported a semibiocatalytic method that employed a decarboxylase to access a sample enriched in the prerequisite 2-hydroxypentan-3-one (17) reactant. The electrophile 17 was reacted with propane- 1 ,2-diamine (8) to make a 70:30 isomerically enriched mixture of 235-EDMP (10) over 325-EDMP (11). We intuit the 70:30 isomeric ratio of 10: 11 is established by the 70:30 ratio of hydroxypentanones (17:18) (Figure 6A) made in the carboligation reaction catalyzed by the decarboxylase from cosubstrates 2- oxobutanoate and acetaldehyde. We view this reaction as being controlled by the greater reactivity of the carbonyl group over the hydroxyl of the acyloin and the terminal amine over the internal amine of the diamine 8. In another example, 3-hydroxypentan-2-one (18) was used in place of 17 to make the 325-EDMP isomer when reacted with diamine 8 in modest 60:40 enrichment over the 235-isomer. Again, the 60:40 enrichment was considered to result mainly from the 60:40 ratio of hydroxypentanones 18:17 made in the carboligation reaction catalyzed by the decarboxylase from cosubstrates pyruvate and propanal (Figure 5A).

This synthetic enrichment step is a significant advance in the synthesis of substituted pyrazines from typical reagents, e.g., a diamine and bifunctional alkadiones, that produce statistical mixtures of all condensation products or self-condensation of an alkoxy amine that produces a limited number of symmetrical pyrazines.

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EQUIVALENTS AND SCOPE

In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the present disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.