Synthesis of flavor modulators

Abstract

The present invention relates to a process for the preparation of a compound of formula (III) or an edible salt thereof, in particular to an enzymatic process. Such compounds are preferably flavor modulating compounds.

Description

Technical Field

[0001] The present invention relates to a process for preparing a compound of formula (III) or an edible salt thereof, in particular an enzyme-catalyzed process. Such compounds are preferably flavor-adjusting compounds. Background Art

[0002] The synthesis of compounds of formula (III) generally involves harsh reaction conditions, low material and energy efficiency, and low yields.

[0003] Therefore, there is a continuing need in the art for efficient synthesis of compounds of formula (III) involving mild reaction conditions, improved material and energy efficiency, and improved yields. Summary of the invention

[0004] In one aspect, the present invention provides a method for preparing a compound of formula (III) or an edible salt thereof, comprising reacting a carboxylic acid derivative (I) with an amine (II) in a solvent, wherein the reaction is catalyzed by an enzyme:

[0005]

[0006] Among them, Ar 1 and Ar 2 Each comprises a heteroaromatic moiety, wherein LG is a leaving group or OH, and wherein R is H or COOH. Such a method may be referred to herein as a method of the present invention or a method according to the present invention. Unless otherwise expressly stated, any method or feature thereof referred to herein refers to a method according to the present invention.

[0007] Compounds and Reactants

[0008] The heteroaromatic moiety is an aromatic ring, which may be substituted, comprising at least one heteroatom within the ring (i.e., an atom different from carbon or hydrogen). Preferably, the heteroatom is nitrogen, oxygen or sulfur, more preferably nitrogen. Examples of heteroaromatic moieties are pyrrole, furan, thiophene, indole, benzofuran, benzothiophene, imidazole, oxazole, thiazole, pyrazole, isoxazole, isothiazole, pyridine, pyrilium, thiapyrilium, quinoline, isoquinoline, carbazole and purine, or any suitable derivative thereof.

[0009] Edible salts are readily understood by those skilled in the art to include those commonly used in the food and beverage industry, including chlorides, sulfates, phosphates, gluconates, sodium salts, citrates, carbonates, acetates, and lactates.

[0010] Preferably, Ar 1 and Ar 2 At least one heteroaromatic moiety contained in contains a nitrogen atom. Preferably, Ar 1 and Ar2 The total number of nitrogen atoms contained in is 1 to a maximum of 8, 1 to a maximum of 7, 1 to a maximum of 6, 1 to a maximum of 5, 1 to a maximum of 4, 2 to a maximum of 8, 2 to a maximum of 7, 2 to a maximum of 6, 2 to a maximum of 5, 2 to a maximum of 4, 3 to a maximum of 8, 3 to a maximum of 7, 3 to a maximum of 6, 3 to a maximum of 5, 3 to a maximum of 4.

[0011] Preferably, Ar 1 and Ar 2 At least one of them includes imidazolyl, indolyl, pyrimidinyl or thiazolyl, preferably 1H-4-imidazolyl, 1H-5-indolyl, pyrimidin-2-yl or 1,3-thiazol-2-yl.

[0012] Preferably, Ar 1 Contains a heteroaromatic moiety containing a nitrogen atom. Preferably, Ar 1 The number of nitrogen atoms contained in is 1 to a maximum of 4, 1 to a maximum of 3, 1 to a maximum of 2, 2 to a maximum of 4, 2 to a maximum of 3. Preferably, Ar 1 The number of nitrogen atoms contained in is 1, 2, 3, 4, 5 or 6.

[0013] Ar 1 The group includes imidazolyl, indolyl, pyrimidinyl or thiazolyl, preferably 1H-4-imidazolyl, 1H-5-indolyl, pyrimidin-2-yl or 1,3-thiazol-2-yl.

[0014] Preferably, the carboxylic acid derivative (I) can be represented by the formula (I-link), wherein Ar 1R is a heteroaromatic moiety and L1 is a linker.

[0015]

[0016] Preferably, Ar 1R Contains a heteroaromatic moiety containing a nitrogen atom. Preferably, Ar 1R The number of nitrogen atoms contained in is 1 to a maximum of 4, 1 to a maximum of 3, 1 to a maximum of 2, 2 to a maximum of 4, 2 to a maximum of 3. Preferably, Ar 1R The number of nitrogen atoms contained in is 1, 2, 3, 4, 5 or 6.

[0017] Preferably, Ar 1R It is imidazolyl, indolyl, pyrimidinyl or thiazolyl, preferably 1H-4-imidazolyl, 1H-5-indolyl, pyrimidin-2-yl or 1,3-thiazol-2-yl.

[0018] Preferably, L 1 It is C 1-3 Alkylene or C 1-3 Alkenylene. C 1-3Alkylene is a saturated carbohydrate diradical containing 1, 2 or 3 carbon atoms. 1-3 Alkenylene is a monounsaturated carbohydrate diradical containing 1, 2 or 3 carbon atoms. 1 is methylene (—CH2—), ethylene (—CH2—CH2—), trimethylene (—CH2—CH2—CH2—), —CH(CH3)—CH2—, vinylene (—CH═CH—), propenylene (—C(CH3)═CH—), wherein each H may be independently substituted by —NH2, —OH or halogen, preferably by —NH2. Preferably, L 1 It is vinylene or 1-amino-ethylene.

[0019] Preferably, Ar 1 It is 2-(1H-4-imidazolyl)-vinyl, 1H-5-indolyl, 2-(1H-5-imidazolyl)-vinyl, 1-amino-2-(1H-4-imidazolyl)-ethyl, (1,3-thiazol-2-yl)-vinyl, 2,3-dihydro-1H-indol-2-yl or 2-(pyrimidin-2-yl)vinyl.

[0020] Preferably, the carboxylic acid derivative (I) may be represented by formula (IV), which may be referred to as urocanic acid (LG=OH) or urocanyl ester (LG=OR'):

[0021]

[0022] Preferably, Ar 2 Contains a heteroaromatic moiety containing a nitrogen atom. Preferably, Ar 2 The number of nitrogen atoms contained in is 1 to a maximum of 4, 1 to a maximum of 3, 1 to a maximum of 2, 2 to a maximum of 4, 2 to a maximum of 3. Preferably, Ar 2 The number of nitrogen atoms contained in is 1, 2, 3, 4, 5 or 6.

[0023] Preferably, Ar 2 The group includes imidazolyl, indolyl, pyrimidinyl or thiazolyl, preferably 1H-4-imidazolyl, 1H-5-indolyl, pyrimidin-2-yl or 1,3-thiazol-2-yl.

[0024] Preferably, the amine (II) can be represented by the formula (II-link), wherein Ar 2R is a heteroaromatic moiety, and L2 is a linker:

[0025]

[0026] More preferably, the amine (II) can be represented by the formula (II-H-link), wherein Ar 2Ris a heteroaromatic moiety, and L2 is a linker:

[0027]

[0028] Preferably, Ar 2R Contains a heteroaromatic moiety containing a nitrogen atom. Preferably, Ar 2R The number of nitrogen atoms contained in is 1 to a maximum of 4, 1 to a maximum of 3, 1 to a maximum of 2, 2 to a maximum of 4, 2 to a maximum of 3. Preferably, Ar 2R The number of nitrogen atoms contained in is 1, 2, 3, 4, 5 or 6.

[0029] Preferably, Ar 2R It is imidazolyl, indolyl, pyrimidinyl or thiazolyl, preferably 1H-4-imidazolyl, 1H-5-indolyl, pyrimidin-2-yl or 1,3-thiazol-2-yl.

[0030] Preferably, L 2 It is C 1-3 Alkylene or C 1-3 Preferably, L 2 is methylene (—CH2—), ethylene (—CH2—CH2—), trimethylene (—CH2—CH2—CH2—), —CH(CH3)—CH2—, vinylene (—CH═CH—), propenylene (—C(CH3)═CH—), or —C(CH3)═CH—, wherein each H may be independently substituted by —NH2, —OH or halogen, preferably by —NH2. Preferably, L 2 It is a methylene group.

[0031] Preferably, Ar 2 It is (1H-imidazol-4-yl)-methyl, (1H-3-indol-3-yl)-methyl, (pyridin-4-yl)methyl, (pyridin-2-yl)methyl or 2-(pyrimidin-2-yl)methyl.

[0032] Preferably, the amine (II) may be represented by formula (V), which may be referred to as histamine:

[0033]

[0034] Preferably, Ar 1 and Ar 2 Each independently comprises a heteroaromatic moiety containing a nitrogen atom. Preferably, each Ar 1 and Ar 2 The number of nitrogen atoms contained in is independently 1 to a maximum of 4, 1 to a maximum of 3, 1 to a maximum of 2, 2 to a maximum of 4, 2 to a maximum of 3. Preferably, Ar 1 and Ar 2The number of nitrogen atoms contained in each is independently 1, 2, 3, 4, 5 or 6.

[0035] Preferably, Ar 1 and Ar 2 Each independently comprises an imidazolyl group, an indolyl group, a pyrimidinyl group or a thiazolyl group, preferably a 1H-4-imidazolyl group, a 1H-5-indolyl group, a pyrimidin-2-yl group or a 1,3-thiazol-2-yl group.

[0036] Preferably, the carboxylic acid derivative (I) can be represented by the formula (I-link), and the amine (II) can be represented by the formula (II-link), wherein Ar 1R and Ar 2R is independently a heteroaromatic moiety, wherein L 1 and L 2 Independently is a joint.

[0037] It will be appreciated that in the preferred case where the carboxylic acid derivative (I) may be represented by formula (I-link) and the amine (II) may be represented by formula (II-link), the compound may be represented by formula (III-link):

[0038]

[0039] Preferably, the carboxylic acid derivative (I) can be represented by the formula (I-linked), and the amine (II) can be represented by the formula (II-H-linked), wherein Ar 1R and Ar 2R are independently heteroaromatic moieties, wherein L 1 and L 2 Independently is a joint.

[0040] It will be appreciated that in the preferred case where the carboxylic acid derivative (I) can be represented by formula (I-link) and the amine (II) can be represented by formula (II-H-link), the compound can be represented by formula (III-H-link):

[0041]

[0042] Preferably, Ar 1R and Ar 2R and 1H-4-imidazolyl, 1H-5-indolyl, pyrimidin-2-yl or 1,3-thiazol-2-yl.

[0043] Preferably, L 1 and L 2 Independently C 1-3 Alkylene or C 1-3 Preferably, L 1 and L 2is independently methylene (—CH2—), ethylene (—CH2-CH2—), trimethylene (—CH2-CH2-CH2—), —CH(CH3)-CH2—, vinylene (—CH═CH—), propenylene (—C(CH3)═CH—), or —C(CH3)═CH—, wherein each H may be independently substituted by —NH2, —OH or halogen, preferably by —NH2. Preferably, L 1 is vinylidene or 1-amino-ethylene, L 2 More preferably, L 1 is vinylidene, L 2 It is a methylene group.

[0044] Preferably, the carboxylic acid derivative (I) can be represented by formula (IV), that is, Ar 1 is 2-(1H-4-imidazolyl)-vinyl, and the amine (II) can be represented by formula (V), that is, Ar 2 is (1H-imidazolyl-4-yl)-methyl, and the compound can therefore be represented by formula (VI), which can be referred to as N-urocanylhistamine:

[0045]

[0046] Preferably, the compound of formula (III) can be represented by formula (VI-E). Alternatively, the compound of formula (III) can be represented by formula (VI-Z):

[0047]

[0048] Unless otherwise expressly stated, the above preferences for certain substituents apply to all Markush formulas in which these substituents appear. 1 All parameter settings of LG can be applied to formula (I) and (III). All parameter settings of LG can be applied to formula (I), (I-link) and (IV). 2 All parameter settings of R mentioned herein are applicable to formula (II), (II-link) and (III). 1R and L 1 All parameter settings of can be applied to formula (I-link), (III-link) and (III-H-link). 2R and L 2 All parameter settings can be applied to formula (II-link), (II-H-link), (III-link) and (III-H-link).

[0049] It should also be understood that the molecular formulas and chemical names given herein mean all compounds or moieties that are generally considered by the technician to be represented by those molecular formulas or chemical names. Specifically, unless otherwise explicitly stated, and without limitation, molecular formulas or chemical formulas refer to all relevant stereoisomers (diastereomers, E / Z isomers, cis / trans isomers, conformers, rotational isomers, enantiomers, etc.), isotopologues, isotope configurations, tautomers, protonated species, (edible) salts, etc. known to the technician. As an important example, if a specific E / Z isomer is not specified in the chemical name, or if the molecular formula does not contain the letters E or Z next to the double bond, the double bond that is not part of the ring system can be E or Z. As another example, for Ar 1 and Ar 2 The structures drawn with heteroaromatic moieties contained therein refer to all tautomers of these moieties.

[0050] Reactions catalyzed by hydrolases

[0051] Preferably, the enzyme used in the method according to the present invention is a hydrolase. More preferably, the enzyme is a hydrolase and LG is a leaving group. Most preferably, the enzyme is a hydrolase, LG is a leaving group, and the solvent is a non-aqueous solvent, preferably a tertiary alcohol.

[0052] Hydrolases are polypeptides which have hydrolase activity, ie are capable of catalyzing hydrolysis and (trans)esterification, preferably in non-aqueous media.

[0053] Leaving groups have their usual meanings here. In the present specification, leaving groups can be easily displaced under the conditions of carrying out the reaction (I) + (II) → (III). The skilled person can select a suitable leaving group. Preferably, the leaving group leaves as an anion by heterolytic cleavage and can stabilize the additional electron density generated by the heterolytic cleavage of the bond. Preferred leaving groups are alkoxy (LG = OR', wherein R is an alkyl) and halogen (preferably LG = Cl, Br or I).

[0054] Preferably, LG is OR', wherein R' is alkyl. More preferably, R' is C 1-8 Alkyl, C 1-7 Alkyl, C 1-6 Alkyl, C 1-5 Alkyl, C 1-4 Alkyl, C 1-3 Alkyl, C 2-6 Alkyl, C 2-5 Alkyl, C 2-4 Alkyl, C 2-3 Alkyl. C x-yAlkyl is a saturated carbohydrate monoradical containing from x to y carbon atoms, which may be linear, branched or cyclic. Most preferably, R' is methyl.

[0055] When the enzyme is a hydrolase such as lipase or an amide synthase, the solvent is preferably a non-aqueous solvent. It should be understood that this does not exclude the presence of water or trace amounts of water during the reaction between the carboxylic acid derivative (I) and the amine (II), but simply specifies that the solvent is not water.

[0056] Preferably, the carboxylic acid derivative (I) and the amine (II) are soluble in the non-aqueous solvent, more preferably expressed as one of the following solubility values, while allowing the enzyme to catalyze the reaction according to the present invention.

[0057] Preferably, the solubility of the carboxylic acid derivative (I) in the non-aqueous solvent is at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g / L at a temperature of 20°C and a pressure of 1 atm.

[0058] Preferably, the solubility of amine (II) in the non-aqueous solvent is at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g / L at a temperature of 20° C. and a pressure of 1 atm.

[0059] Preferably, the solubility of the carboxylic acid derivative (I) and the amine (II) in the non-aqueous solvent is at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g / L at a temperature of 20° C. and a pressure of 1 atm.

[0060] Preferably, the non-aqueous solvent is a tertiary alcohol. Preferably, the tertiary alcohol is 2-methyl-2-butanol, tert-amyl alcohol, tert-butanol or a mixture thereof. More preferably, the tertiary alcohol is 2-methyl-2-butanol.

[0061] Preferably, the enzyme used in the method according to the invention is a lipase (a type of hydrolase). More preferably, the enzyme is a lipase and LG is a leaving group. Most preferably, the enzyme is a lipase, LG is a leaving group and the solvent is a non-aqueous solvent, preferably a tertiary alcohol.

[0062] Lipase is a polypeptide, preferably a serine hydrolase, which preferably has lipase activity in a non-aqueous medium. Their physiological function is to hydrolyze triglycerides into diglycerides or monoglycerides, fatty acids and glycerol. Lipase can be any type of lipase from EC 3.1 class. For example, lipase can be sn-1 specific diacylglycerol lipase (EC3.1.1.116), acylglycerol lipase (EC 3.1.1.23), galactose lipase (EC 3.1.1.26), triacylglycerol lipase (EC 3.1.1.3), phospholipase A1 (EC 3.1.1.32), lipoprotein lipase (EC 3.1.1.34), phospholipase A2 (EC3.1.1.4), lysophospholipase (EC 3.1.1.5), hormone sensitive lipase (EC 3.1.1.79). EC numbers refer to Enzyme Nomenclature 1992 published by NC-IUBMB, Academic Press, San Diego, Calif., including Supplements 1-5 published in Eur. J. Biochem., 1994, 223: 1-5; Eur. J. Biochem., 1995, 232: 1-6; Eur. J. Biochem., 1996, 237: 1-5; Eur. J. Biochem., 1997, 250: 1-6; and Eur. J. Biochem., 1999, 264: 610-650, respectively. The nomenclature is regularly supplemented and updated; see, for example, the World Wide Web at chem.qmw.ac.uk / iubmb / enzyme / index.html.

[0063] Lipase can be obtained by culturing a suitable microorganism and then separating the lipase expressed by the microorganism. Suitable microorganisms for this purpose belong to Mucor, Aspergillus, Rhizopus, Rhizomucor, Pseudomonas, Candida, Humicola, Thermomyces, Burkholderiaubonensis and Penicillium. Preferred species are Candida antarctica, Thermomyces lanuginosus and Rhizomucor miehei. Lipase obtained by culturing a suitable microorganism and then separating the lipase expressed by the microorganism may also be referred to herein as lipase from microbial sources.

[0064] Preferably, the lipase is Candida antarctica lipase-B (Cal-B).

[0065] Preferably, the enzyme used in the method according to the invention is a protease (a type of hydrolase). More preferably, the enzyme is a protease and LG is a leaving group. Most preferably, the enzyme is a protease, LG is a leaving group and the solvent is a non-aqueous solvent, preferably a tertiary alcohol.

[0066] Proteases are polypeptides that preferably have protease activity in non-aqueous media. Their physiological function is to hydrolyze proteins or peptides into smaller peptides or single amino acids. Proteases can be any type of peptidase from the EC 3.4 class. For example, a protease can be a subtilisin (EC 3.4.21.62).

[0067] Protease can be obtained by culturing a suitable microorganism and then separating the protease expressed by the microorganism. Suitable microorganisms for this purpose belong to Mucor, Aspergillus, Rhizopus, Rhizomucor, Pseudomonas, Candida, Humicola, Thermoeudomonas, Penicillium, Burkholderia and Bacillus. Preferred species are Antarctic Candida, Thermomyces lanuginosus, Rhizomucor miehei and Burkholderia uboni, more preferably Antarctic Candida, Thermomyces lanuginosus, Rhizomucor miehei and Bacillus licheniformes. Protease that can be obtained by culturing a suitable microorganism and then separating the protease expressed by the microorganism may also be referred to herein as a protease obtained from a microbial source.

[0068] Preferably, the lipase or protease is immobilized ("immobilized lipase" or "immobilized protease"). The immobilized lipase or protease is a lipase or protease adsorbed on a carrier. The carrier is an inert solid material. The immobilized protease or lipase can be kept suspended (for example by stirring) in the phase in which the reaction (I) + (II) → (III) is carried out. In the case of an immobilized enzyme, the expression "reaction (...) in a solvent refers to the fact that species (I), (II) and (III) react mainly in the solvent phase and / or are dissolved in the solvent phase, rather than to the fact that the enzyme should be dissolved in the solvent. Without being bound by this theory, the carrier forms a separate phase with the immobilized enzyme, catalyzing the reaction (I) + (II) → (III) at its boundary with the solvent phase. Preferably, the immobilized lipase or protease is used in combination with a non-aqueous solvent.

[0069] The lipase or protease may be immobilized by any common immobilization technique known to the skilled person. Examples of immobilization techniques can be found in J.Mol.Cat.B:Enzymatic 6(1999)29-39; Chibata et al. Biocatalysis:Immobilized cells and enzymes, J.Mol.Cat.37(1986)1-24; Sharma et al., Immobilized Biomaterials Techniques and Applications, Angew.Chem.Int.Ed.Engl.21(1982)837-854; Laskin (ed.), Enzymes and Immobilized Cells in Biotechnology, which are expressly incorporated herein by reference in their entirety. It is generally known that immobilized enzymes generally show increased resistance to organic solvents compared to enzymes in an unbound state. Therefore, it can be assumed that once the enzyme is immobilized or stabilized by other methods such as cross-linked enzyme crystals (Altus Biologics, Inc.), higher concentrations of enzyme can be used. When fixed on a solid support (e.g., on a column), the concentration of the enzyme in the reaction mixture can approach 100%. Therefore, various changes, modifications or substitutions may be made to the enzyme or reaction conditions without departing from the spirit or scope of the invention.

[0070] Preferably, the support is a particulate material having a particle size of from 100 to 2000 microns. Alternatively, the support material may be a membrane.

[0071] Preferably, the support is a porous material. The porous material preferably has an average pore size of at least 20 nanometers.

[0072] Preferably, the support is a hydrophobic material or an ion exchange resin.

[0073] The hydrophobic material can be selected from polypropylene, polyolefin, polystyrene, polyacrylate, inorganic materials such as silicate, silica, glass, zeolite, diatomaceous earth, bentonite, vermiculite, hydrotalcite, agarose, cellulose, etc. or a combination thereof. Materials such as silica, which are usually hydrophilic, must be treated with a suitable compound such as silane to make them hydrophobic.

[0074] The ion exchange resin may be selected from ion exchange resins based on polystyrene, polyacrylates, phenolic resins and silica. Ideally, the ion exchange resin is an anion exchanger, especially a macroporous weak anion exchange resin. Suitable examples include phenolic resins, polystyrene resins and styrene-DVB resins, such as the resins available under the trademarks DUOLITE ES568 and AMBERLYST A21.

[0075] Preferably, the lipase is immobilized and obtained from a microbial source. More preferably, the lipase is immobilized and obtained from Candida. Most preferably, the lipase is immobilized and obtained from Candida antarctica.

[0076] Preferably, the immobilized lipase is Novozym 435, Lipozyme 435, Lipozyme RM IM, AlcalaseCLEA, CalB immo 8806, CalB immo plus.

[0077] Novozym 435 (N435) is a commercially available immobilized lipase produced by Novozymes. It is based on the immobilization by interfacial activation of lipase B from Candida antarctica on the resin Lewatit VP OC 1600. The resin is a macroporous support formed by crosslinking poly(methyl methacrylate) with divinylbenzene.

[0078] Lipozyme 435 is an immobilized lipase commercially available from Novozymes, which is a food grade (activity > 9000 PLU) version of Novozyme 435, whereas Novozyme 435 is an industrial grade (activity > 10000 PLU).

[0079] Lipozyme RM IM is a commercially available immobilized lipase from Novozyme (derived from the Rhizomucor miehei fungus) immobilized in phenolic resin.

[0080] Alkaline protease CLEA[4] is an immobilized protease obtained from CLEA Technologies.

[0081] Reactions catalyzed by amide synthases / synthetases

[0082] Preferably, the enzyme used in the method according to the invention is an amide synthase. More preferably, the enzyme is an amide synthase and LG is OH. Most preferably, the enzyme is an amide synthase and LG is OH and the solvent is an aqueous solvent.

[0083] The aqueous solvent may be water, any aqueous buffer, a mixture of water and a water-miscible solvent, or any mixture thereof.

[0084] An amide synthase or amide synthetase is a polypeptide having amide synthase activity, preferably in aqueous medium, i.e., being capable of catalyzing the formation of an amide bond starting from a carboxylic acid or carboxylate / ester and an amine. The amide synthase may be any type of amide synthase from class EC 6.3. For example, the amide synthase may be a ribosomal peptide synthetase or a non-ribosomal amide synthetase. Alternatively, the amide synthetase may be from another EC class. For example, the amide synthetase may use an amide precursor biosynthesized by an acid-thiol ligase [5].

[0085] Amide synthase can be obtained by culturing a suitable microorganism and then isolating the amide synthase expressed by the microorganism. Suitable microorganisms for this purpose belong to the genera Mucor, Aspergillus, Rhizopus, Rhizomucor, Pseudomonas, Candida, Humicola, Thermomyces, Penicillium, Marinactinospora and Burkholderia. Preferred species are Candida antarctica, Thermomyces lanuginosa, Rhizomucor miehei, Marinactinospora and Burkholderia ebonyii.

[0086] Preferably, the amide synthase is Marinactinospora thermotolerans ATP-dependent amide bond synthase (McbA).

[0087] Other process parameters

[0088] Preferably, the process according to the invention is carried out in the presence of a molecular sieve. Molecular sieves can be zeolite materials with pores of precise and uniform structure and size. This enables them to preferentially adsorb gases and liquids according to molecular size and polarity. Zeolites are naturally occurring, highly porous crystalline solids belonging to the class of chemicals known as aluminosilicates. Preferably, the molecular sieve is a zeolite of one of the following four types: 3A, 4A, 5A and 13X. Their chemical formula is 2 / 3K2O13·Na 22O·Al2O3·2SiO2.·4.5H2O(3A), Na2O·Al2O3·2SiO2·4.5H2O(4A), 3 / 4CaO1 / 4Na2OAl2O3·2SiO2·4.5H2O(5A), Na2O·Al2O3·2.45SiO2·6.0H2O(13X).

[0089] Without being bound by this theory, the presence of a molecular sieve such as 4A helps remove water by adsorption during the reaction (I) + (II) → (III). Since the presence of water interferes with the reaction (e.g., by hydrolyzing (III) or competing with (II) for binding to (I), this adsorption can increase the yield and rate of the reaction.

[0090] Preferably, the process according to the invention is carried out at a temperature of 20°C to maximum 100°C, 20°C to maximum 90°C, 20°C to maximum 80°C, 20°C to maximum 70°C, 20°C to maximum 60°C, 20°C to maximum 50°C, 20°C to maximum 40°C, 30°C to maximum 100°C, 30°C to maximum 90°C, 30°C to maximum 80°C, 30°C to maximum 70°C, 30°C to maximum 60°C, 30°C to maximum 50°C, 30°C to maximum 40°C, 40°C to maximum 100°C, 40°C to maximum 90°C, 40°C to maximum 80°C, 40°C to maximum 70°C, 40°C to maximum 60°C, 40°C to maximum 50°C, 50°C to maximum 100°C, 50°C to maximum 90°C, 50°C to maximum 80°C, 50°C to maximum 70°C, 50°C to maximum 60°C. More preferably, the process according to the invention is carried out at a temperature of 40°C to a maximum of 80°C. Most preferably, the process according to the invention is carried out at a temperature of 50°C to a maximum of 70°C.

[0091] Preferably, the molar concentration ratio of (I) to (II) in the solvent is 1 / 2.00 to a maximum of 2.00, 1 / 1.95 to a maximum of 1.95, 1 / 1.90 to a maximum of 1.90, 1 / 1.85 to a maximum of 1.85, 1 / 1.80 to a maximum of 1.80, 1 / 1.75 to a maximum of 1.75, 1 / 1.70 to a maximum of 1.70, 1 / 1.65 to a maximum of 1.65, 1 / 1.60 to a maximum of 1.60, 1 / 1.75 to a maximum of 1.75, 1 / 1.80 to a maximum of 1.80 ... 1 / 1.55 ​​to maximum 1.55, 1 / 1.50 to maximum 1.50, 1 / 1.45 to maximum 1.45, 1 / 1.40 to maximum 1.40, 1 / 1.35 to maximum 1.35, 1 / 1.30 to maximum 1.30, 1 / 1.25 to maximum 1.25, 1 / 1.20 to maximum 1.20, 1 / 1.15 to maximum 1.15, 1 / 1.10 to maximum 1.10 or 1 / 1.05 to maximum 1.05.

[0092] Preferably, the ratio of the molar concentrations of (I) to (II) in the solvent is 0.50 to a maximum of 2.0, 0.55 to a maximum of 1.9, 0.60 to a maximum of 1.8, 0.65 to a maximum of 1.7, 0.70 to a maximum of 1.6, 0.75 to a maximum of 1.5, 0.80 to a maximum of 1.4, 0.85 to a maximum of 1.3 or 0.90 to a maximum of 1.2.

[0093] Preferably, the molar concentration of (I) in the solvent is 1 mM to a maximum of 100 mM, 1 mM to a maximum of 90 mM, 1 mM to a maximum of 80 mM, 1 mM to a maximum of 70 mM, 1 mM to a maximum of 60 mM, 1 mM to a maximum of 50 mM, 1 mM to a maximum of 40 mM, 1 mM to a maximum of 30 mM, 5 mM to a maximum of 100 mM, 5 mM to a maximum of 90 mM, 5 mM to a maximum of 80 mM, 5 mM to a maximum of 70 mM, 5 mM to a maximum of 60 mM, 5 mM to a maximum of 50 mM, 5 mM to a maximum of 40 mM, 5 mM to a maximum of 30 mM, 10mM to up to 100mM, 10mM to up to 90mM, 10mM to up to 80mM, 10mM to up to 70mM, 10mM to up to 60mM, 10mM to up to 50mM, 10mM to up to 40mM, 10mM to up to 30mM, 10mM to up to 100mM, 10mM to up to 90mM, 10mM to up to 80mM, 10mM to up to 70mM, 10mM to up to 60mM, 10mM to up to 50mM, 10mM to up to 40mM, 10mM to up to 30mM.

[0094] Preferably, the molar concentration of (II) in the solvent is 1 mM to a maximum of 100 mM, 1 mM to a maximum of 90 mM, 1 mM to a maximum of 80 mM, 1 mM to a maximum of 70 mM, 1 mM to a maximum of 60 mM, 1 mM to a maximum of 50 mM, 1 mM to a maximum of 40 mM, 1 mM to a maximum of 30 mM, 5 mM to a maximum of 100 mM, 5 mM to a maximum of 90 mM, 5 mM to a maximum of 80 mM, 5 mM to a maximum of 70 mM, 5 mM to a maximum of 60 mM, 5 mM to a maximum of 50 mM, 5 mM to a maximum of 40 mM, 5 mM to a maximum of 30 mM. ,10mM to up to 100mM, 10mM to up to 90mM, 10mM to up to 80mM, 10mM to up to 70mM, 10mM to up to 60mM, 10mM to up to 50mM, 10mM to up to 40mM, 10mM to up to 30mM, 10mM to up to 100mM, 10mM to up to 90mM, 10mM to up to 80mM, 10mM to up to 70mM, 10mM to up to 60mM, 10mM to up to 50mM, 10mM to up to 40mM, 10mM to up to 30mM.

[0095] Preferably, the ratio of the molar concentrations of (I) to (II) in the solvent is from 0.5 to a maximum of 2.0, preferably, wherein the molar concentration of (I) in the solvent is from 1 mM to a maximum of 100 mM; or the ratio of the molar concentrations of (I) to (II) in the solvent is from 0.8 to a maximum of 1.4, preferably wherein the molar concentration of (I) in the solvent is from 10 mM to a maximum of 50 mM; or the ratio of the molar concentrations of (I) to (II) in the solvent is from 0.9 to a maximum of 1.2, preferably wherein the molar concentration of (I) in the solvent is from 20 mM to a maximum of 30 mM.

[0096] Preferably, the concentration of the enzyme in the solvent is 1 g / L up to 100 g / L, 1 g / L up to 90 g / L, 1 g / L up to 80 g / L, 1 g / L up to 70 g / L, 1 g / L up to 60 g / L, 1 g / L up to 50 g / L, 10 g / L up to 100 g / L, 10 g / L up to 90 g / L, 10 g / L up to 80 g / L, 10 g / L up to 70 g / L, 10 g / L up to 60 g / L, 10 g / L up to 50 g / L. / L,20g / L to maximum 100g / L,20g / L to maximum 90g / L,20g / L to maximum 80g / L,20g / L to maximum 70g / L,20g / L to maximum 60g / L,20g / L to maximum 50g / L,30g / L to maximum 100g / L,30g / L to maximum 90g / L,30g / L to maximum 80g / L,30g / L to maximum 70g / L,30g / L to maximum 60g / L,30g / L to maximum 50g / L.

[0097] The concentration of the immobilized enzyme in the solvent is defined herein as the ratio of the mass of the immobilized enzyme (including the mass of the support) to the volume of the solution (or suspension) containing the solvent. In other words, the concentration does not refer to the concentration of the dissolved enzyme.

[0098] The concentration of the non-immobilized enzyme in the solvent is simply defined herein as the ratio of the mass of the non-immobilized enzyme to the volume of the solution (or suspension) comprising the solvent.

[0099] A method according to any one of the preceding embodiments, wherein the compound has the formula (VI), wherein the enzyme is an immobilized lipase, wherein LG is OR', wherein R' is C 1-4 An alkyl group, wherein the solvent is a tertiary alcohol, wherein the reaction is carried out at a temperature of 20°C to a maximum of 100°C, and wherein the molar concentration of (I) and (II) in the solvent is 0.5 to a maximum of 2.0, preferably wherein the molar concentration of (I) in the solvent is 1 mM to a maximum of 100 mM.

[0100] A method according to any one of the preceding embodiments, wherein the compound has formula (VI), wherein the enzyme is an immobilized lipase, wherein LG is OMe, wherein the solvent is a tertiary alcohol, wherein the reaction is carried out at a temperature of 20°C to a maximum of 100°C, and wherein the molar concentration of (I) and (II) in the solvent is 0.5 to a maximum of 2.0, preferably wherein the molar concentration of (I) in the solvent is 1 mM to a maximum of 100 mM.

[0101] A method according to any one of the preceding embodiments, wherein the compound has formula (VI), wherein the enzyme is an immobilized lipase, wherein LG is OMe, wherein the solvent is a tertiary alcohol, wherein the reaction is carried out at a temperature of 40°C to a maximum of 80°C, and wherein the molar concentration of (I) and (II) in the solvent is 0.5 to a maximum of 2.0, preferably wherein the molar concentration of (I) in the solvent is 20 mM to a maximum of 30 mM.

[0102] A method according to any one of the preceding embodiments, wherein the compound has formula (VI), wherein the enzyme is an immobilized lipase, wherein LG is OMe, wherein the solvent is 2-methyl-2-butanol, wherein the reaction is carried out at a temperature of 40°C to a maximum of 80°C, and wherein the molar concentration of (I) and (II) in the solvent is 0.5 to a maximum of 2.0, preferably wherein the molar concentration of (I) in the solvent is 20 mM to a maximum of 30 mM.

[0103] A method according to any one of the preceding embodiments, wherein the compound has formula (VI), wherein the enzyme is an immobilized lipase, wherein LG is OMe, wherein the solvent is a tertiary alcohol, wherein the reaction is carried out at a temperature of 40°C to a maximum of 80°C, and wherein the molar concentration of (I) and (II) in the solvent is 0.9 to a maximum of 1.2, preferably wherein the molar concentration of (I) in the solvent is 20 mM to a maximum of 30 mM.

[0104] Flavour adjustment and other aspects

[0105] In an embodiment, the method according to the invention comprises the separation of compounds. Any conventional method known to the skilled person can be used for separation. For example, the crude reaction mixture produced by the enzymatic reaction between (I) and (II) can be filtered or centrifuged to remove solids (e.g., molecular sieves and / or immobilized enzymes), and (III) can be separated and purified from the liquid phase of the enzymatic reaction mixture using silica gel flash column chromatography or LC-MS equipped with a preparative amide column. Finally, the reaction mixture can be separated and purified by vacuum distillation (e.g., using Rotovap TM) to remove any residual solvent (e.g., water miscible solvent), and water can be removed by freeze drying. The final product is further analyzed using LC-MS or HPLC and compared with authentic standards. More details of this exemplary separation can be found in Example 1.

[0106] In one aspect, the present invention provides a compound of any formula (III) as described herein (preferably obtainable by any process according to the invention as described herein).

[0107] In one aspect, the present invention provides a method for preparing a consumable or flavor composition comprising a compound of formula (III), comprising the step of preparing the compound of formula (III) by a method according to the present invention.

[0108] Examples of consumer goods include tasty foods, unpalatable foods such as dairy products, beverages and candies. Examples of flavor compositions include savoury flavourings and sour / acidic flavourings. Other examples of consumer goods are soups, sauces, broths, cheese products, seasonings, condiments, margarines, noodles and beverages.

[0109] Preferably, the consumable product is a food or beverage, said consumable product comprising at least 1 ppm, preferably at least 20 ppm, more preferably at least 50 ppm or 70 ppm ppb of one or more compounds of formula (III) and / or edible salts thereof.

[0110] Preferably, the consumables as food or beverage contain at least 0.0001 wt %, more preferably at least 0.0003 wt %, even more preferably at least 0.001 wt %, most preferably at least 0.003 wt % of one or more compounds of formula (III). Typically, the above-mentioned products will contain a concentration of no more than 1 wt %, preferably no more than 0.5 wt % of the compound of formula (III).

[0111] Preferably, the consumable product as food or beverage is a low sodium food. Without being bound by this theory, the compound of formula (III) contained in the low sodium food can mask the bitter taste of KCl therein.

[0112] Preferably, the compound of formula (III) is a flavor-modulating compound, ie has flavor-modulating properties. For example, some compounds may enhance the salty taste perception, and / or reduce bitterness and / or enhance umami taste.

[0113] In one aspect, the present invention provides a method for imparting, enhancing, improving or changing the flavor properties of a flavor composition or a consumable, comprising the steps of preparing a compound of formula (III) by a method according to the present invention and adding the compound of formula (III) to the flavor composition or the consumable.

[0114] Throughout this document, the terms "taste" and "flavor" are used interchangeably to describe the sensory effects perceived through the olfactory epithelium in the mouth (especially the tongue) and nasal cavity.

[0115] The term "flavor modulating compound" refers to a compound that has no flavor properties of its own. However, the substance is able to change or supplement or modulate the taste impact of other flavoring substances contained in a flavor composition or consumable, including salty impact, sour impact, bitter taste and / or umami impact.

[0116] For example, the flavor modulating compound has no salty taste at all up to levels above 1000 ppm. In combination with salty compounds (eg NaCl), it can enhance the salty taste perception.

[0117] For example, the flavor modulating compounds at levels above 1000 ppm do not have any discernible taste to counteract or mask the bitter taste. In combination with compounds having a bitter off taste, such as KCl, the bitter taste is mitigated.

[0118] For example, the flavor adjusting compound has no discernible taste at levels above 1000 ppm to counteract or mask the sour taste. In combination with compounds that may have a sour taste, such as NH4Cl, the sour taste is mitigated.

[0119] For example, the flavor modulating compounds do not impart any umami taste at levels above 1000 ppm. In combination with compounds capable of imparting umami taste, such as MSG, the umami taste is enhanced.

[0120] Flavour modulating compounds can be advantageously used to impart desired flavour attributes to the above-mentioned consumer products. In addition, taste improving substances can modulate the flavour impact of other flavour ingredients contained in these same products, thereby improving the overall flavour quality of these products.

[0121] The term "flavoring substance" refers to any substance that can impart (especially at a concentration of less than 0.1% by weight, more preferably less than 0.01% by weight) a perceptible flavor impact. For example, such flavoring substances can be selected from natural flavorings, artificial flavorings, spices, seasonings, etc., synthetic flavor oils and aromatic seasonings and / or oils, oleoresins, essences, distillates, and extracts from plants, leaves, flowers, fruits, etc. In general, any flavoring agent or food additive can be used, such as those described in the U.S. National Academy of Sciences in Chemicals Used in Food Processing publication 1274, pages 63-258. This publication is incorporated herein by reference.

[0122] In addition to flavor substances, the flavor adjustment compound can be used in the flavor composition in combination with one or more ingredients or excipients conventionally used in flavor compositions (e.g., carrier materials and other adjuvants commonly used in the art). Suitable excipients for flavor compositions are well known in the art and include, for example, but not limited to, solvents (including water, alcohol, ethanol, oil, fat, vegetable oil and miglyol), binders, diluents, disintegrants, lubricants, flavoring agents, colorants, preservatives, antioxidants, emulsifiers, stabilizers, flavor enhancers, anticaking agents, etc.

[0123] Examples of such carriers or diluents for flavor compositions can be found, for example, in "Perfume and Flavour Materials of Natural Origin", S. Arctander, ed., Elizabeth, NJ, 1960; in "Perfume and Flavour Chemicals", S. Arctander, ed., Vol. I and II, Allured Publishing Corporation, Carol Stream, USA, 1994; in "Flavourings", E. Ziegler and H. Ziegler (eds.), Wiley-VCH Weinheim, 1998 and in "CTFA Cosmetic Ingredient Handbook", JM Nikitakis (ed.), 1st edition, The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1988.

[0124] Other suitable and desirable ingredients of flavor compositions are described in standard textbooks such as "Handbook of Industrial Chemical Additives," eds. M. and I. Ash, 2nd edition, (Synapse 2000).

[0125] General Definition

[0126] In this document and its claims, the verb "comprise" and its conjugations are used in its non-limiting sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. Furthermore, the verb "comprise" can be replaced with "consisting essentially of", meaning that the product, assay device, respective method or use defined herein may include additional one or more components or respective additional one or more steps in addition to those specifically identified, which additional one or more components or respective one or more steps do not change the unique characteristics of the invention.

[0127] Furthermore, referring to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the element. Thus, the indefinite article "a" or "an" usually means "at least one".

[0128] All patent and literature references cited in this specification are hereby incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 - Enzymatic synthesis kinetics of compound (VI) in 2-methyl-2-butanol with (a) or without (w / o) molecular sieve 4A (b)

[0130] Figure 2 —Molar yield of compound (VI) synthesized enzymatically with or without molecular sieve 4A

[0131] References

[0132] 1.Brault,G.,et al.,Short-chain flavor ester synthesis in organicmedia by an E.coli whole-cell biocatalyst expressing a newly characterizedheterologous lipase.PLoS One,2014.9(3):p.e91872.

[0133] 2. Longo, MA and MA Sanromán, Production of food aroma compounds: microbial and enzymatic methodologies. Food Technology and Biotechnology, 2006.44(3):p.335-353.

[0134] 3. Vandamme, EJand W. Soetaert, Bioflavours and fragrances viafermentation and biocatalysis. Journal of Chemical Technology&Biotechnology: International Research in Process, Environmental&Clean Technology, 2002.77(12):p.1323-1332.

[0135] 4. Nuijens, T., et al., Enzymatic C-terminal amidation of aminoacids and peptides. Tetrahedron Letters, 2012.53(29):p.3777-3779.

[0136] 5.Petchey, MR and G. Grogan, Enzyme-catalysed synthesis of secondary and tertiary amides. Advanced Synthesis&Catalysis, 2019.361(17):p.3895-3914.

[0137] 6. Kokusho, Y., H. Machida, and S. Iwasaki, Studies on alkalinelipase: isolation and identification of lipase producing microorganisms. Agricultural and Biological Chemistry, 1982.46(5): p.1159-1164. Example

[0138] The present invention is explained in more detail below with some embodiments, which should not be interpreted as limiting the scope of the present invention. The present invention is not limited to the implementation described in the case given as an example. The present invention also extends to each combination of measures independent of each other as described above.

[0139] Example 1

[0140] The enzymatic synthesis of compound (VI) was carried out in an organic solvent of 2-methyl-2-butanol at 60°C using 25 mM methyl urocanate and 25 mM compound (V) (as raw materials) and 40 g / L Lipozyme 435 (Novozymes) for 73 h. The enzymatic kinetics are shown in Figures 1(a) and 1(b). There was no significant difference in the synthesis of compound (VI) between (a) and (b). Figure 2 The enzymatic molar conversion of compound (VI) is summarized. There are two treatments, one without adding molecular sieve 4A and the other with additional 100 g / L molecular sieve 4A. Molecular sieve 4A absorbs excess water and byproduct methanol that are usually undesirable during enzymatic synthesis. According to Figure 1, the enzymatic reaction using 100 g / L molecular sieve 4A not only has a slightly higher product molar yield (0.58 mol / mol) than the reaction without additional molecular sieve 4A (0.55 mol / mol), but also has less byproduct formation.

[0141] Compound (VI) in the crude reaction mixture produced by the enzymatic reaction between urocanic acid methyl ester and Compound (V) is separated and purified using silica flash column chromatography or preparative amide column with LC-MS.

[0142] Biotage SNAP Ultra 10g silica gel column (Biotage HP-Sphere 25um) was used for flash column chromatography. The elution rate was 20mL / min, and the mobile phase was a mixture of dichloromethane and methanol (containing 0.1% ammonia). The gradient elution of the mixed solvent was from 10% dichloromethane to 20% dichloromethane (90% methanol to 80% methanol), eluting 170mL, from 20% dichloromethane to 30% dichloromethane (80% methanol to 70% methanol), eluting 170mL, from 30% dichloromethane to 50% dichloromethane (70% methanol to 50% methanol), eluting 170mL, from 50% dichloromethane to 70% dichloromethane (50% methanol to 30% methanol), eluting 170mL. The glass tubes containing the product N-urocanyl histamine were collected and subjected to rotary evaporation to remove the solvent and freeze drying to remove the water.

[0143] A Waters preparative amide column (XBridge BEHPrep OBD Amide 5 μm, 19x250 mm) equipped with a single quadrupole LC-MS was used in the separation process. An isocratic elution with a solvent mixture of 80% acetonitrile (containing 0.1% formic acid) and 20% water (containing 0.1% formic acid) was used at a flow rate of 16 mL / min. The glass tube containing the product N-urocanyl histamine was collected and subjected to rotary evaporation to remove the solvent and freeze drying to remove water before NMR analysis.

[0144] Example 2

[0145] The enzymatic synthesis of compound (VI) was carried out in an organic solvent of 2-methyl-2-butanol at 60° C. using 25 mM methyl urocanate and 25 mM compound (V) (as raw materials) and 40 g / L lipase TLIM (Novozymes) and 100 g / L molecular sieve 4A for 72 h. The molar yield of compound (VI) was about 0.018 mol / mol.

[0146] Example 3

[0147] The enzymatic synthesis of compound (VI) was carried out in an organic solvent of 2-methyl-2-butanol at 50° C. using 25 mM methyl urocanate and 25 mM compound (V) (as starting materials) and 40 g / L lipase TL (Meito sanyo) and 100 g / L molecular sieve 4A for 72 h. The molar yield of compound (VI) was about 0.14 mol / mol.

[0148] Example 4

[0149] The enzymatic synthesis of compound (VI) was carried out in an organic solvent of 2-methyl-2-butanol using 25 mM methyl urocanate and 25 mM compound (V) (as raw materials) and 40 g / L lipase QLM (Meito sanyo) and 100 g / L molecular sieve 4A at 60° C. for 72 h. The molar yield of compound (VI) was about 0.017 mol / mol.

[0150] Example 5

[0151] The enzymatic synthesis of compound (VI) was carried out in an organic solvent of 2-methyl-2-butanol using 25 mM methyl urocanate and 25 mM compound (V) (as raw materials) and 40 g / L CalB immo plus (Purolite) and 100 g / L molecular sieve 4A at 60° C. for 71 h. The molar yield of compound (VI) was about 0.21 mol / mol.

[0152] Example 6

[0153] The enzymatic synthesis of compound (VI) was carried out in an organic solvent of 2-methyl-2-butanol using 25 mM methyl urocanate and 25 mM compound (V) (as raw materials) and 40 g / L CalB immo 8806 (Purolite) and 100 g / L molecular sieve 4A at 60° C. for 71 h. The molar yield of compound (VI) was about 0.18 mol / mol.

[0154] Example 7

[0155] The enzymatic synthesis of compound (VI) was carried out in an organic solvent of 2-methyl-2-butanol at 60° C. using 25 mM methyl urocanate and 25 mM compound (V) (as raw materials) and 40 g / L Lipozyme CalB and 100 g / L molecular sieves 4A for 72 h. The molar yield of compound (VI) was about 0.12 mol / mol.

[0156] Example 8

[0157] The enzymatic synthesis of compound (VI) was carried out in an organic solvent of 2-methyl-2-butanol at 60° C. using 25 mM methyl urocanate and 25 mM compound (V) (as raw materials) and 40 g / L subtilisin A and 100 g / L molecular sieves 4A for 72 h. The molar yield of compound (VI) was about 0.21 mol / mol.

[0158] Example 9

[0159] Tables I and II below list suitable enzymes that can be used in the methods of the present invention, but are not limited thereto.

[0160] Table I

[0161]

[0162] Table II

[0163]

[0164]

[0165] (a): PLU = propyl laurate unit. 1 PLU is the amount of enzyme activity that produces 1 μmol of propyl laurate per minute under specified standard conditions.

[0166] (b): IUN = transesterification unit. 1 PLU is equal to 1 IUN.

[0167] (c): Determination by the method of Kokusho et al., using olive oil emulsified with polyvinyl alcohol as substrate [6]. 1 U corresponds to the amount of enzyme that releases 1 μmol fatty acid per minute at 37°C

[0168] (d) PLU: One unit corresponds to the synthesis of 1 μmol of propyl laurate from lauric acid and 1-propanol per minute at 60°C.

[0169] (e) LU: lipase unit, 1LU is the amount of enzyme activity that releases 1 μmol of titratable butyrate from the substrate tributyrin per minute under specified standard conditions.

[0170] (f): Activity expressed in Anson units.

[0171] Embodiment 11:

[0172] The enzymatic synthesis of compound (VI) was carried out at 60°C in an organic solvent of 4-methyl-2-pentanol (secondary alcohol) using 25 mM methyl urocanate and 25 mM compound (V) (as raw materials) and 40 g / L Lipozyme 435 (Novozymes) for 72 h. There were two treatments, one without the addition of molecular sieve 4A and the other with the addition of 100 g / L molecular sieve 4A. Molecular sieve 4A absorbs excess water and byproduct methanol, which are usually undesirable during enzymatic synthesis.

[0173] Embodiment 12:

[0174] The amidation reaction was carried out in an aqueous system (pH 7-7.5 buffer) containing amide synthetase at 37°C using 25 mM urocanic acid and 25 mM compound (V) for 24-72 h.

Claims

1. A method for preparing a compound of formula (III) or an edible salt thereof, the method comprising reacting a carboxylic acid derivative (I) with an amine (II) in a solvent, wherein the reaction is catalyzed by an enzyme: Among them, Ar 1 and Ar 2 each comprising a heteroaromatic moiety, Where LG is a leaving group or OH, Wherein R is H or COOH.

2. The method according to claim 1, wherein Ar 1 and Ar 2 At least one heteroaromatic moiety contained in comprises a nitrogen atom.

3. A method according to any one of the preceding claims, wherein Ar 1 and Ar 2 Each independently comprises an imidazolyl group, an indolyl group, a pyrimidinyl group or a thiazolyl group, preferably a 1H-4-imidazolyl group, a 1H-5-indolyl group, a pyrimidin-2-yl group or a 1,3-thiazol-2-yl group.

4. A method according to any one of the preceding claims, wherein Ar 1 It is 2-(1H-4-imidazolyl)-vinyl, 1H-5-indolyl, 2-(1H-5-imidazolyl)-vinyl, 1-amino-2-(1H-4-imidazolyl)-ethyl, (1,3-thiazol-2-yl)-vinyl, 2,3-dihydro-1H-indol-2-yl or 2-(pyrimidin-2-yl)vinyl.

5. A method according to any one of the preceding claims, wherein Ar 2 It is (1H-imidazol-4-yl)-methyl, (1H-3-indol-3-yl)-methyl, (pyridin-4-yl)methyl, (pyridin-2-yl)methyl or 2-(pyrimidin-2-yl)methyl.

6. A method according to any one of the preceding claims, wherein Ar 1 is 2-(1H-4-imidazolyl)-vinyl, wherein Ar 2 is (1H-imidazolyl-4-yl)-methyl, wherein the compound has the formula (IV):

7. A method according to any one of the preceding claims, wherein the enzyme is a hydrolase and wherein LG is a leaving group.

8. The method of claim 7, wherein LG is OR', wherein R' is C 1-4 Alkyl, preferably wherein R' is methyl.

9. The method according to claim 7 or 8, wherein the solvent is a non-aqueous solvent and the hydrolase is a lipase, preferably an immobilized lipase, more preferably an immobilized lipase obtained from a microbial source, even more preferably an immobilized lipase obtained from Candida, and most preferably an immobilized lipase obtained from Candida antarctica.

10. The method according to any one of claims 7 to 9, wherein the non-aqueous solvent is a tertiary alcohol, preferably 2-methyl-2-butanol.

11. The method according to any one of claims 1 to 6, wherein the enzyme is an amide synthase, LG is OH, and the solvent is an aqueous solvent.

12. A process according to any one of the preceding claims, wherein the reaction is carried out in the presence of a molecular sieve, preferably wherein the molecular sieve is 4A.

13. The process according to any one of the preceding claims, wherein the reaction is carried out at a temperature of 40°C to a maximum of 80°C, preferably 50°C to a maximum of 70°C.

14. A method according to any one of the preceding claims, wherein the ratio of the molar concentrations of (I) to (II) in the solvent is from 0.9 to 1.2, preferably wherein the molar concentration of (I) in the solvent is from 20 mM to a maximum of 30 mM.

15. A method according to any one of the preceding claims, wherein the concentration of the enzyme in the solvent is from 30 g / L up to 50 g / L.

16. A method according to any one of the preceding claims, wherein the method comprises isolating the compound.

17. A method according to any preceding claim, wherein the compound is a flavour modulating compound.

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