Field

The invention relates to a method for preparing a compound of formula (III) or an edible salt thereof, specifically to an enzyme-catalysed method. Such compounds are preferably flavor modulation compounds.

Background

The synthesis of compounds of formula (III) often involves harsh reaction conditions, low materials and energy efficiencies, and a low yield [1 -3],

There is thus a continuing need in the art for efficient synthesis of compounds of formula (III) involving mild reaction conditions, increased materials and energy efficiencies, and increased yields.

Description of the invention

In an aspect, the invention provides a method for preparing a compound of formula (III) or an edible salt thereof, comprising the reaction between carboxylic acid derivative (I) and amine (II) in a solvent, wherein the reaction is catalysed by an enzyme:

(I) (II) (HI) wherein each of Ar ¹ and Ar ² comprise a heteroaromatic moiety, wherein LG is a leaving group or OH, and wherein R is H or COOH. Such a method may be called a method of or according to the invention herein. Unless explicitly mentioned otherwise, any method, or feature thereof, mentioned herein refers to a method according to the invention.

Compound and reactants

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

An edible salt is readily understood by the skilled person and includes those salts typically employed in the food and beverage industry including chlorides, sulphates, phosphates, gluconates, sodium, citrates, carbonates, acetates and lactates.

Preferably, at least one heteroaromatic moiety comprised in Ar ¹ and Ar ² comprises a nitrogen atom. Preferably, the combined number of nitrogen atoms comprised in Ar ¹ and Ar ² is from 1 up to 8, from 1 up to 7, from 1 up to 6, from 1 up to 5, from 1 up to 4, is from 2 up to 8, from 2 up to 7, from 2 up to 6, from 2 up to 5, from 2 up to 4, is from 3 up to 8, from 3 up to 7, from 3 up to 6, from 3 up to 5, from 3 up to 4.

Preferably, at least one of Ar ¹ and Ar ² comprises an imidazolyl, an indolyl, a pyrimidinyl or a thiazolyl, preferably 1 H-4-imidazolyl, 1 H-5-indolyl, pyrimidin-2-yl or 1 ,3-thiazol-2-yl.

Preferably, Ar ¹ comprises a heteroaromatic moiety comprising a nitrogen atom. Preferably, the number of nitrogen atoms comprised in Ar ¹ is from 1 up to 4, from 1 up to 3, from 1 up to 2, from 2 up to 4, from 2 up to 3. Preferably, the number of nitrogen atoms comprised in Ar ¹ is 1 , 2, 3, 4, 5 or 6.

Preferably, Ar ¹ comprises an imidazolyl, an indolyl, a pyrimidinyl or a thiazolyl, preferably 1 H-4- imidazolyl, 1 H-5-indolyl, pyrimidin-2-yl or 1 ,3-thiazol-2-yl.

Preferably, the carboxylic acid derivative (I) can be represented by formula (l-link), wherein Ar ^1R is a heteroaromatic moiety and Li is a linker.

(l-link)

Preferably, Ar ^1R comprises a hetero aromatic moiety comprising a nitrogen atom. Preferably, the number of nitrogen atoms comprised in Ar ^1R is from 1 up to 4, from 1 up to 3, from 1 up to 2, from 2 up to 4, from 2 up to 3. Preferably, the number of nitrogen atoms comprised in Ar ^1R is 1 , 2, 3, 4, 5 or 6.

Preferably, Ar ^1R is an imidazolyl, an indolyl, a pyrimidinyl or a thiazolyl, preferably 1 H-4-imidazolyl, 1 H-5-indolyl, pyrimidin-2-yl or 1 ,3-thiazol-2-yl.

Preferably, L ¹ is a C1-3 alkylene or C1-3 alkenylene. A C1-3 alkylene is an saturated carbohydrate diradical comprising 1 , 2 or 3 carbon atoms. A C1-3 alkenylene is an singly unsaturated carbohydrate diradical comprising 1 , 2 or 3 carbon atoms. Preferably, L ¹ is methylene (-CH2-), ethylene (-CH2-CH2-), trimethylene (-CH2-CH2-CH2-), -CH(CH3)-CH2-, ethenylene (-CH=CH-), propenylene (-C(CH3)=CH-), wherein each H may independently be substituted by -NH2, -OH or a halogen, preferably by -NH2. Preferably, L ¹ is ethenylene or 1 -amino-ethylene.

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

Preferably, the carboxylic acid derivative (I) can be represented by formula (IV), which may be called urocanic acid (LG=OH) or an urocanyl ester (LG=OR’):

Preferably, Ar ² comprises a heteroaromatic moiety comprising a nitrogen atom. Preferably, the number of nitrogen atoms comprised in Ar ² is from 1 up to 4, from 1 up to 3, from 1 up to 2, from 2 up to 4, from 2 up to 3. Preferably, the number of nitrogen atoms comprised in Ar ² is 1 , 2, 3, 4, 5 or 6.

Preferably, Ar ² comprises imidazolyl, indolyl, pyrimidinyl or thiazolyl, preferably 1 H-4-imidazolyl, 1 H- 5-indolyl, pyrimidin-2-yl or 1 ,3-thiazol-2-yl.

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

(ll-link)

More preferably, the amine (II) can be represented by formula (ll-H-link), wherein Ar ^2R is a heteroaromatic moiety and L2 is a linker:

(ll-H-link)

Preferably, Ar ^2R comprises a hetero aromatic moiety comprising a nitrogen atom. Preferably, the number of nitrogen atoms comprised in Ar ^2R is from 1 up to 4, from 1 up to 3, from 1 up to 2, from 2 up to 4, from 2 up to 3. Preferably, the number of nitrogen atoms comprised in Ar ^2R is 1 , 2, 3, 4, 5 or 6.

Preferably, Ar ^2R is an imidazolyl, an indolyl, a pyrimidinyl or a thiazolyl, preferably 1 H-4-imidazolyl, 1 H-5-indolyl, pyrimidin-2-yl or 1 ,3-thiazol-2-yl.

Preferably, L ² is a C1-3 alkylene or C1-3 alkenylene. Preferably, L ² is methylene (-CH2-), ethylene (- CH2-CH2-), trimethylene (-CH2-CH2-CH2-), -CH(CH3)-CH2-, ethenylene (-CH=CH-), propenylene (- C(CH3)=CH-), or -C(CH3)=CH-, wherein each H may independently be substituted by -NH2, -OH or a halogen, preferably by -NH2. Preferably, L ² is methylene.

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

Preferably, the amine (II) can be represented by formula (V), which may be called histamine:

Preferably, Ar ¹ and Ar ² each independently comprise a heteroaromatic moiety comprising a nitrogen atom. Preferably, the number of nitrogen atoms comprised in each of Ar ¹ and Ar ² is independently from 1 up to 4, from 1 up to 3, from 1 up to 2, from 2 up to 4, from 2 up to 3. Preferably, the number of nitrogen atoms comprised in each of Ar ¹ and Ar ² is independently 1 , 2, 3, 4, 5 or 6.

Preferably, each of Ar ¹ and Ar ² independently comprise an imidazolyl, an indolyl, a pyrimidinyl or a thiazolyl, preferably 1 H-4-imidazolyl, 1 H-5-indolyl, pyrimidin-2-yl or 1 ,3-thiazol-2-yl.

Preferably, the carboxylic acid derivative (I) can be represented by formula (l-link) and the amine (II) can be represented by formula (ll-link), wherein Ar ^1R and Ar ^2R are independently heteroaromatic moieties, wherein L ¹ and L ² are independently linkers.

It is understood that, in the preferable case that the carboxylic acid derivative (I) can be represented by formula (l-link) and the amine (II) can be represented by formula (ll-link), the compound can be represented by formula (Ill-link):

(Ill-link) Preferably, the carboxylic acid derivative (I) can be represented by formula (l-hnk) and the amine (II) can be represented by formula (ll-H-link), wherein Ar ^1R and Ar ^2R are independently heteroaromatic moieties, wherein L ¹ and L ² are independently linkers.

It is understood that, in the preferable case that the carboxylic acid derivative (I) can be represented by formula (l-link) and the amine (II) can be represented by formula (ll-H-link), the compound can be represented by formula (lll-H-link):

(lll-H-link)

Preferably, Ar ^1R and Ar ^2R are independently an imidazolyl, an indolyl, a pyrimidinyl or a thiazolyl, preferably 1 H-4-imidazolyl, 1 H-5-indolyl, pyrimidin-2-yl or 1 ,3-thiazol-2-yl.

Preferably, L ¹ and L ² are independently a C1-3 alkylene or C1-3 alkenylene. Preferably, L ¹ and L ² are independently methylene (-CH2-), ethylene (-CH2-CH2-), trimethylene (-CH2-CH2-CH2-), -CH(CH3)-CH2- , ethenylene (-CH=CH-), propenylene (-C(CH3)=CH-), or -C(CH3)=CH-, wherein each H may independently be substituted by -NH2, -OH or a halogen, preferably by -NH2. Preferably, L ¹ is ethenylene or 1 -amino-ethylene, and L ² is methylene. More preferably, L ¹ is ethenylene and L ² is methylene.

Preferably, the carboxylic acid derivative (I) can be represented by formula (IV), i.e. Ar ¹ is 2-(1 H-4- imidazolyl)-ethenyl, and the amine (II) can be represented by formula (V), i.e. Ar ² is (1 H-imidazol-4-yl)- methyl, and the compound can thus be represented by formula (VI), which may be called N- urocanylhistamine:

Preferably, the compound of formula (III) can be represented by formula (Vl-E). Alternatively, the compound of formula (III) can be represented by formula (Vl-Z):

Unless explicitly mentioned otherwise, the preferences for certain substituent groups mentioned above may be applied to all Markush formulas in which these substituent group appear. Hence, all preferences for Ar ¹ mentioned herein may be applied to formulas (I) and (III). All preferences for LG may be applied to formulas (I), (l-link) and (IV). All preferences for Ar ² mentioned herein may be applied to formulas (II) and (III). All preferences for R mentioned herein may be applied to formulas (II), (ll-link) and (III). All preferences for Ar ^1R and L ¹ mentioned herein may be applied to formulas (l-link), (Ill-link) and (lll-H-link). All preferences for Ar ^2R and L ² mentioned herein may be applied to formulas (ll-link), (ll- H-link), (Ill-link) and (lll-H-link). It is further understood that the formulae and chemical names presented herein are meant to refer to all compounds or moieties which are generally recognized by the skilled person to be represented by those formulae or chemical names. Specifically, unless explicitly mentioned otherwise, and without being limiting, a formula or chemical formula refers to all relevant stereoisomers (diastereoisomers, E/Z isomers, cis/trans isomers, conformers, rotamers, enantiomers, etc.), isotopologues, isotopomers, tautomers, protonated species, (edible) salts etc. as recognized by the skilled person. As an important example, a double bond not part of a ring system may be either E or Z if no specific E/Z-isomer is denoted in a chemical name or if a formula does not contain a letter E or Z next to the double bond. As another example, the structures drawn for the heteroaromatic moieties comprised in Ar ¹ and Ar ² refer to all tautomers of these moieties.

Hydrolase-catalysed reactions

Preferably, the enzyme used during a method according to the 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.

A hydrolase is a polypeptide having hydrolase activity, i.e. able to catalyse hydrolyses and (trans)esterifications, preferably in non-aqueous media.

A leaving group has its customary meaning here. In the context of the current invention, a leaving group is able to be displaced easily under the conditions at which the reaction (I) + (II) (III) is performed. A skilled person is capable of selecting appropriate leaving groups. Preferred leaving groups leave as anions via heterolysis, and can stabilize the additional electron density that results from bond heterolysis. Preferred leaving groups are alkoxy groups (LG=OR’, wherein R is an alkyl group) and halogens (preferably LG=CI, Br or I).

Preferably, LG is OR’, wherein R’ is an alkyl group. More preferably, R’ is a CI-B alkyl, C1-7 alkyl, C1- 6 alkyl, C1-5 alkyl, C1-4 alkyl, C1-3 alkyl, C2-6 alkyl, C2-5 alkyl, C2-4 alkyl, C2-3 alkyl. A C _{x y} alkyl is a saturated carbohydrate monoradical comprising from x up to y carbon atoms, which may be linear, branched or cyclic. Most preferably, R’ is methyl.

Wherever the enzyme is a hydrolase such as a 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 thereof during the reaction between the carboxylic acid derivative (I) and the amine (II), but simply specifies the solvent as not being water.

Preferably, the carboxylic acid derivative (I) and the amine (II) are soluble in the non-aqueous solvent, more preferably expressed by one of the solubility values below, while at the same time allowing the enzyme to catalyse the reaction according to the invention.

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 an a pressure of 1 atm.

Preferably, the solubility of 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, 1 1 , 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 g/L at a temperature of 20 °C and at a pressure of 1 atm. Preferably, the solubility of the carboxylic acid derivative (I) and the solubility of 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, 1 1 , 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 g/L at a temperature of 20 °C and an a pressure of 1 atm.

Preferably, the non-aqueous solvent is a tertiary alcohol. Preferably, a tertiary alcohol is 2-methyl-2- butanol, tert-amyl alcohol, tert-butyl alcohol or mixtures thereof. More preferably, a tertiary alcohol is 2- methyl-2-butanol.

Preferably, the enzyme used during a 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.

A lipase is a polypeptide, preferably a serine hydrolase, having lipase activity, preferably in nonaqueous media. Their physiological function is the hydrolysis of triglycerides into di- or monoglycerides, fatty acids and glycerol. The lipase may be any type of lipase from the EC 3.1 class. For example, a lipase may be a sn-1-specific diacylglycerol lipase (EC 3.1 .1 .116), an acylglycerol lipase (EC 3.1 .1 .23), a galactolipase (EC 3.1 .1 .26), a triacylglycerol lipase (EC 3.1 .1 .3), a phospholipase A1 (EC 3.1 .1 .32), a lipoprotein lipase (EC 3.1 .1 .34), a phospholipase A2 (EC 3.1 .1 .4), a lysophospholipase (EC 3.1 .1 .5), an hormone-sensitive lipase (EC 3.1.1.79). The EC number refers to Enzyme Nomenclature 1992 from 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, e.g., the World Wide Web at chem.qmw.ac.uk/iubmb/enzyme/index.html.

A lipase may be obtained by culturing a suitable microorganism and subsequently isolating a lipase expressed by the microorganism. Suitable microorganisms for this purpose belong to the genera Mucor, Aspergillus, Rhizopus, Rhizomucor, Pseudomonas, Candida, Humicola, Thermomyces, Burkholderia ubonensis and Penicillium. Preferred species are Candida antarctica, Thermomyces lanuginosus and Rhizomucor miehei. A lipase that may be obtained by culturing a suitable microorganism and subsequently isolating a lipase expressed by the microorganism, may also be called a lipase from a microbial source herein.

Preferably, a lipase is Candida antarctica lipase-B (Cal-B).

Preferably, the enzyme used during a 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.

A protease is a polypeptide having protease activity, preferably in non-aqueous media. Their physiological function is the hydrolysis of proteins or peptides into smaller peptides or single amino acids. The protease may be any type of peptidase from the EC 3.4 class. For example, a protease may be a subtilisin (EC 3.4.21 .62). A protease may be obtained by culturing a suitable microorganism and subsequently isolating a protease expressed by the microorganism. Suitable microorganisms for this purpose belong to the genera Mucor, Aspergillus, Rhizopus, Rhizomucor, Pseudomonas, Candida, Humicola, Thermomyces, Penicillium, Burkholderia and Bacillus. Preferred species are Candida antarctica, Thermomyces lanuginosus, Rhizomucor miehei and Burkholderia ubonensis, more preferably Candida antarctica, Thermomyces lanuginosus, Rhizomucor miehei and Bacillus licheniformes. A protease that may be obtained by culturing a suitable microorganism and subsequently isolating a protease expressed by the microorganism, may also be called a protease obtained from a microbial source herein.

Preferably a lipase or a protease is immobilized (“immobilized lipase” or “immobilized protease”). An immobilized lipase or protease is a lipase or protease which is adsorbed onto a carrier. A carrier is an inert, solid material. An immobilized protease or lipase may be held in suspension in the phase wherein the reaction (I) + (II) (III) is performed, e.g. via agitation. 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 and/or are dissolved in the solvent phase, and does not refer to the fact that the enzyme should be dissolved in the solvent. Without being bound to this theory, the carrier together with the immobilized enzyme forms a separate phase, on whose boundary with the solvent phase the reaction (I) + (II) (III) is catalysed. Preferably, an immobilized lipase or protease is used in combination with a non-aqueous solvent.

The lipase or protease may be immobilized via any common immobilization technique known to the skilled person. Examples of immobilization techniques are 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 by reference herein in its entirety. It is generally known that an immobilized enzyme often shows increased resistance to organic solvents as compared to the enzyme in the unbound state. Thus, it can be assumed that once the enzyme is immobilized or stabilized by other methods such as crosslinked enzyme crystals (Altus Biologies, Inc.), higher concentrations of the enzyme may be used. When immobilized on a solid support, for example on a column, the concentration of the enzyme in the reaction mixture could approach 100%. Therefore, various changes, modifications or alternations to the enzyme or the reaction conditions may be made without departing from the spirit or scope of the invention.

Preferably, a carrier is a particulate material having a particle size of 100 to 2000 micrometres. Alternatively, a carrier material may be a membrane.

Preferably, a carrier is a porous material. A porous material preferably has an average pore diameter of at least 20 nanometres.

Preferably, a carrier is a hydrophobic material or am ion-exchange resin.

A hydrophobic material may be selected from polypropylene, polyolefin, polystyrene, polyacrylate(ester), inorganic materials like silicate, silica, glass, zeolites, diatomaceous earth, bentonite, vermiculite, hydrotalcite, agarose, cellulose etc. or combinations thereof. Materials like silica etc. which are normally hydrophilic have to be treated with a suitable compound e.g. a silane to render them hydrophobic. An ion-exchange resin may be selected from ion-exchange resins based on polystyrene, polyacrylate, phenol-formaldehyde resins and silicas. Ideally, the ion-exchange resin is an anion exchanger, especially a macroporous weak anion exchange resin. Suitable examples include phenolformaldehyde, polystyrenic, and styrene-DVB resins such as are available under the Trade Marks DUOLITE ES568 and AMBERLYST A21 .

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.

Preferably, an immobilized lipase is Novozym 435, Lipozyme 435, Lipozyme RM IM, Alcalase CLEA, CalB immo 8806, CalB immo plus.

Novozym 435 (N435) is a commercially available immobilized lipase produced by Novozymes. It is based on immobilization via interfacial activation of lipase B from Candida antarctica on a resin, Lewatit VP OC 1600. This resin is a macroporous support formed by poly(methyl methacrylate) crosslinked with divinylbenzene.

Lipozyme 435 is a commercially available immobilized lipase from Novozymes, which is a food grade (activity > 9000 PLU) variant of Novozyme 435, which is a technical grade (activity > 10000 PLU)..

Lipozyme RM IM is a commercially available immobilized lipase from Novozymes derived from the Rhizomucor miehei fungus, immobilized in phenolic resin.

Alcalase CLEA [4] is an immobilized protease, which is from the company CLEA Technologies.

Amide synthase/synthetase-catalysed reactions

Preferably, the enzyme used during a 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, LG is OH and the solvent is an aqueous solvent.

An aqueous solvent may be water, any aqueous buffer, a mixture of water and a water-miscible solvent, or any mixtures thereof.

An amide synthase or amide synthetase is a polypeptide having amide synthase activity, i.e. able to catalyse the formation of an amide bond starting from a carboxylic acid or carboxylate and an amine, preferably in aqueous media. The amide synthase may be any type of amide synthase from the EC 6.3 class. For example, the amide synthase may be a ribosomal peptide synthetase or a nonribosomal amide synthetase. Alternatively, the amide synthetase may be from another EC class. For example, the amide synthetase may use an amide precursor which is biosynthesised by an acid-thiol ligases [5],

An amide synthase may be obtained by culturing a suitable microorganism and subsequently isolating an 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 lanuginosus, Rhizomucor miehei, Marinactinospora and Burkholderia ubonensis.

Preferably, an amide synthase is Marinactinospora thermotolerans ATP-dependent amide bond synthetase (McbA). Other process parameters

Preferably, a method according to the invention is performed in the presence of a molecular sieve. A molecular sieve may be a zeolite material with pores of precise and uniform structure and size. This allows them to preferentially adsorb gases and liquids based on molecular size and polarity. Zeolites are naturally existing, highly porous crystalline solids, belonging to the class of chemicals known as aluminosilicates. Preferably, a molecular sieve is a zeolite of one of four types: 3A, 4A, 5A, and 13X. Their chemical formulae are 2/3K ₂ Ol 3 Na22O Al2O3-2SiO2.-4.5H ₂ O (3A), Na ₂ O Al2O _:! -2SiO2-4.5H ₂ O (4A), 3/4CaO1/4Na ₂ OAl2O3-2SiO2-4.5H ₂ O (5A), Na2O AI2O3-2.45SiO2 6.OH2O (13X).

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

Preferably, a method according to the invention is performed at a temperature from 20°C up to 100°C, from 20°C up to 90°C, from 20°C up to 80°C, from 20°C up to 70°C, from 20°C up to 60°C, from 20°C up to 50°C, from 20°C up to 40°C, 30°C up to 100°C, from 30°C up to 90°C, from 30°C up to 80°C, from 30°C up to 70°C, from 30°C up to 60°C, from 30°C up to 50°C, from 30°C up to 40°C, 40°C up to 100°C, from 40°C up to 90°C, from 40°C up to 80°C, from 40°C up to 70°C, from 40°C up to 60°C, from 40°C up to 50°C, 50°C up to 100°C, from 50°C up to 90°C, from 50°C up to 80°C, from 50°C up to 70°C, from 50°C up to 60°C. More preferably, a method according to the invention is performed at a temperature from 40°C up to 80°C. Most preferably, a method according to the invention is performed at a temperature from 50°C up to 70°C.

Preferably, the ratio between the molar concentrations of (I) and (II) in the solvent is from 1/2.00 up to 2.00, from 1/1.95 up to 1 .95, from 1/1.90 up to 1 .90, from 1/1.85 up to 1.85, from 1/1 .80 up to 1.80, from 1/1.75 up to 1.75, from 1/1.70 up to 1.70, from 1/1.65 up to 1.65, from 1/1.60 up to 1.60, from 1/1 .55 up to 1 .55, from 1/1 .50 up to 1 .50, from 1/1 .45 up to 1 .45, from 1/1 .40 up to 1 .40, from 1/1 .35 up to 1.35, from 1/1.30 up to 1 .30, from 1/1.25 up to 1 .25, from 1/1.20 up to 1.20, from 1/1 .15 up to 1.15, from 1/1.10 up to 1.10, or from 1/1.05 up to 1.05.

Preferably, the ratio between the molar concentrations of (I) and (II) in the solvent is from 0.50 up to 2.0, from 0.55 up to 1 .9, from 0.60 up to 1 .8, from 0.65 up to 1 .7, from 0.70 up to 1 .6, from 0.75 up to 1 .5, from 0.80 up to 1 .4, from 0.85 up to 1 .3, or from 0.90 up to 1 .2.

Preferably, the molar concentration of (I) in the solvent is from 1 mM up to 100 mM, from 1 mM up to 90 mM, from 1 mM up to 80 mM, from 1 mM up to 70 mM, from 1 mM up to 60 mM, from 1 mM up to 50 mM, from 1 mM up to 40 mM, from 1 mM up to 30 mM, 5 mM up to 100 mM, from 5 mM up to 90 mM, from 5 mM up to 80 mM, from 5 mM up to 70 mM, from 5 mM up to 60 mM, from 5 mM up to 50 mM, from 5 mM up to 40 mM, from 5 mM up to 30 mM, 10 mM up to 100 mM, from 10 mM up to 90 mM, from 10 mM up to 80 mM, from 10 mM up to 70 mM, from 10 mM up to 60 mM, from 10 mM up to 50 mM, from 10 mM up to 40 mM, from 10 mM up to 30 mM, 10 mM up to 100 mM, from 10 mM up to 90 mM, from 10 mM up to 80 mM, from 10 mM up to 70 mM, from 10 mM up to 60 mM, from 10 mM up to 50 mM, from 10 mM up to 40 mM, from 10 mM up to 30 mM. Preferably, the molar concentration of (II) in the solvent is from 1 mM up to 100 mM, from 1 mM up to 90 mM, from 1 mM up to 80 mM, from 1 mM up to 70 mM, from 1 mM up to 60 mM, from 1 mM up to 50 mM, from 1 mM up to 40 mM, from 1 mM up to 30 mM, 5 mM up to 100 mM, from 5 mM up to 90 mM, from 5 mM up to 80 mM, from 5 mM up to 70 mM, from 5 mM up to 60 mM, from 5 mM up to 50 mM, from 5 mM up to 40 mM, from 5 mM up to 30 mM, 10 mM up to 100 mM, from 10 mM up to 90 mM, from 10 mM up to 80 mM, from 10 mM up to 70 mM, from 10 mM up to 60 mM, from 10 mM up to 50 mM, from 10 mM up to 40 mM, from 10 mM up to 30 mM, 10 mM up to 100 mM, from 10 mM up to 90 mM, from 10 mM up to 80 mM, from 10 mM up to 70 mM, from 10 mM up to 60 mM, from 10 mM up to

50 mM, from 10 mM up to 40 mM, from 10 mM up to 30 mM.

Preferably, the ratio between the molar concentrations of (I) and (II) in the solvent is from 0.5 up to 2.0, preferably wherein the molar concentration of (I) in the solvent is from 1 mM up to 100 mM; or the ratio between the molar concentrations of (I) and (II) in the solvent is from 0.8 up to 1.4, preferably wherein the molar concentration of (I) in the solvent is from 10 mM up to 50 mM; or the ratio between the molar concentrations of (I) and (II) in the solvent is from 0.9 up to 1 .2, preferably wherein the molar concentration of (I) in the solvent is from 20 mM up to 30 mM.

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

The concentration of an immobilized enzyme in the solvent is defined herein as the mass of the immobilized enzyme (including the mass of the carrier) to the volume of the solution (or suspension) comprising the solvent. In other words, the concentration does not referto the concentration of dissolved enzymes.

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

The method according to any one of the preceding claims, wherein the compound is of formula (VI), wherein the enzyme is an immobilized lipase, wherein LG is OR’, wherein R’ is a C1-4 alkyl, wherein the solvent is a tertiary alcohol, wherein the reaction is performed at a temperature from 20°C up to 100°C, and wherein the molar concentrations of (I) and (II) in the solvent is from 0.5 up to 2.0, preferably wherein the molar concentration of (I) in the solvent is from 1 mM up to 100 mM.

The method according to any one of the preceding claims, wherein the compound is of formula (VI), wherein the enzyme is an immobilized lipase, wherein LG is OMe, wherein the solvent is a tertiary alcohol, wherein the reaction is performed at a temperature from 20°C up to 100°C, and wherein the molar concentrations of (I) and (II) in the solvent is from 0.5 up to 2.0, preferably wherein the molar concentration of (I) in the solvent is from 1 mM up to 100 mM.

The method according to any one of the preceding claims, wherein the compound is of formula (VI), wherein the enzyme is an immobilized lipase, wherein LG is OMe, wherein the solvent is a tertiary alcohol, wherein the reaction is performed at a temperature from 40°C up to 80°C, and wherein the molar concentrations of (I) and (II) in the solvent is from 0.5 up to 2.0, preferably wherein the molar concentration of (I) in the solvent is from 20 mM up to 30 mM.

The method according to any one of the preceding claims, wherein the compound is of 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 performed at a temperature from 40°C up to 80°C, and wherein the molar concentrations of (I) and (II) in the solvent is from 0.5 up to 2.0, preferably wherein the molar concentration of (I) in the solvent is from 20 mM up to 30 mM.

The method according to any one of the preceding claims, wherein the compound is of formula (VI), wherein the enzyme is an immobilized lipase, wherein LG is OMe, wherein the solvent is a tertiary alcohol, wherein the reaction is performed at a temperature from 40°C up to 80°C, and wherein the molar concentrations of (I) and (II) in the solvent is from 0.9 up to 1.2, preferably wherein the molar concentration of (I) in the solvent is from 20 mM up to 30 mM.

Flavor modulation and other aspects

In embodiments, a method according to the invention comprises the isolation of the compound. The isolation may be performed using any common method known to the skilled person. For example, the crude reaction mixture resulting from the enzymatic reaction between (I) and (II) may be filtrated or centrifuged to remove the solids (e.g. the molecular sieve and/or immobilized enzyme), silica flash column chromatography or LC-MS equipped with prep amide column were applied to isolate and purify (III) from the liquid phase of the enzymatic reaction mixture.. Finally, any remaining solvent (e.g. water miscible solvent) may be removed by vacuum distillation (e.g. using a Rotovap™) and water may be removed by freeze drying. Further analytics are carried out on the final product using LC-MS or HPLC and compared with authentic standards. More details of this exemplary isolation can be found in Example 1 .

In an aspect, the invention provides any compound of formula (III) as described herein, preferably obtainable by any method according to the invention as described herein.

In an aspect, the invention provides a method for preparing a consumer product or flavor composition comprising a compound of formula (III), comprising the step of preparing the compound of formula (III) via a method according to the invention.

Examples of consumer products include a savoury food, a non-savoury food such as dairy, a beverage and confectionery. Examples of a flavor composition includes a savoury flavoring and a sour/acid flavoring. Further examples of consumer products are soups, sauces, stocks, bouillons, cheese products, dressings, seasonings, margarines, noodles and beverages.

Preferably, the consumer product is a food product or a beverage, said consumer 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.

Preferably, the consumer product being a food product or a beverage contains 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 the one or more compounds of formula (III). Typically, the aforementioned products will contain the compounds of formula (III) in a concentration of not more than 1 wt.%, preferably of not more than 0.5 wt.%.

Preferably, the consumer product being a food product or a beverage is a low-sodium food product. Without being bound to this theory, the compounds of formula (III) comprised in the low-sodium food product is able to mask the bitter taste of KCI therein.

Preferably, the compound of formula (III) is a flavor modulation compound, i.e. possesses flavor modulating properties. For example, some of the compounds can enhance the salty taste perception, and /or reduce bitter taste and/or enhance umami taste.

In an aspect, the invention provides a method to confer, enhance, improve or modify the flavor properties of a flavor composition or a consumer product, comprising the step of preparing a compound of formula (III) via a method according to the invention and the step of adding the compound of formula (III) to the flavor composition or the consumer product.

Throughout this document the terms "taste" and "flavor" are used interchangeably to describe the sensory impact that is perceived via the mouth, especially the tongue, and the olfactory epithelium in the nasal cavity.

The term “flavor modulating compound” refers to a compound that has no flavor properties as such. However, said substance is capable of altering or complementing or modulating the taste impact of other flavoring substances contained in a flavor composition or in a consumer product, including the salty taste impact, acidic taste impact, bitterness and/or umami taste impact.

For example, the flavor modulating compound does not have any salty taste at all up to levels above 1000 ppm. In combination with salty flavor compounds, for example with NaCI, it can enhance the salty taste perception.

For example, the flavor modulating compound does not have any recognizable taste at a level above 1000 ppm for counteracting or masking bitter taste. In combination with compounds which can have bitter off taste, for example KCI, the bitter taste is reduced.

For example, the flavor modulating compound does not have any recognizable taste at a level above 1000 ppm for counteracting or masking sour taste. In combination with compounds which can have sour off taste, for example NH4CI, the sour taste is reduced.

For example, the flavor modulating compound does not impart any umami taste at a level above 1000 ppm. In combination with compounds which can impart umami taste, for example MSG, the umami taste is enhanced.

The flavor modulating compounds can be applied advantageously to impart desirable taste attributes to the aforementioned consumer products. In addition, the taste improving substances are capable of modulating the taste impact of other flavor ingredients contained within these same products, thereby improving the overall flavor quality of these products.

The term “flavoring substance” refers to any substance that is capable of imparting a detectable flavor impact, especially at a concentration below 0.1 wt.%, more preferably below 0.01 wt.%. For example, such flavoring substance may be selected from natural flavors, artificial flavors, spices, seasonings, and the like, synthetic flavor oils and flavor aromatics and/or oils, oleoresins, essences, distillates, and extracts derived from plants, leaves, flowers, fruits, and so forth, Generally, any flavor or food additive such as those described in Chemicals Used in Food Processing, publication 1274, pages 63-258, by the National Academy of Sciences, can be used. This publication is incorporated herein by reference.

Flavor modulating compounds can be used in flavor compositions in conjunction with one or more ingredients or excipients conventionally used in flavor compositions beside flavoring substances, for example carrier materials and other auxiliary agents commonly used in the art. Suitable excipients for flavor compositions are well known in the art and include, for example, without limitation, solvents (including water, alcohol, ethanol, oils, fats, vegetable oil, and miglyol), binders, diluents, disintegrating agents, lubricants, flavor agents, coloring agents, preservatives, antioxidants, emulsifiers, stabilisers, flavor-enhancers, anti-caking agents, and the like.

Examples of such carriers or diluents for flavor compositions may be found in for example, ’’Perfume and Flavour Materials of Natural Origin”, S. Arctander, Ed., Elizabeth, N.J., 1960; in "Perfume and Flavour Chemicals", S. Arctander, Ed., Vol. I & II, Allured Publishing Corporation, Carol Stream, USA, 1994; in “Flavourings”, E. Ziegler and H. Ziegler (ed.), Wiley-VCH Weinheim, 1998, and “CTFA Cosmetic Ingredient Handbook”, J.M. Nikitakis (ed.), 1st ed., The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1988.

Other suitable and desirable ingredients of flavor compositions are described in standard texts, such as “Handbook of Industrial Chemical Additives”, ed. M. and I. Ash, 2nd Ed., (Synapse 2000).

General definitions

In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a product, an assay device respectively a method or a use as defined herein may comprise additional component(s) respectively additional step(s) than the ones specifically identified, said additional components), respectively step(s) not altering the unique characteristic of the invention.

In addition, reference to an element by 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 be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Legend to the figures

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

Figure 2 — Molar yield of compound (VI) by enzymatic synthesis with or w/o molecular sieve 4A

References

1. Brault, G., et al., Short-chain flavor ester synthesis in organic media by an E. coli whole-cell biocatalyst expressing a newly characterized heterologous lipase. PLoS One, 2014. 9(3): p. e91872. 2. Longo, M.A. and M.A. Sanroman, Production of food aroma compounds: microbial and enzymatic methodologies. Food Technology and Biotechnology, 2006. 44(3): p. 335-353.

3. Vandamme, E.J. and W. Soetaert, Bioflavours and fragrances via fermentation and biocatalysis. Journal of Chemical Technology & Biotechnology: International Research in Process, Environmental & Clean Technology, 2002. 77(12): p. 1323-1332.

4. Nuijens, T., et al., Enzymatic C-terminal amidation of amino acids and peptides. Tetrahedron Letters, 2012. 53(29): p. 3777-3779.

5. Petchey, M.R. and G. Grogan, Enzyme-catalysed synthesis of secondary and tertiary amides. Advanced Synthesis & Catalysis, 2019. 361 (17): p. 3895-3914. 6. Kokusho, Y., H. Machida, and S. Iwasaki, Studies on alkaline lipase: isolation and identification of lipase producing microorganisms. Agricultural and Biological Chemistry, 1982. 46(5): p. 1159-1164.

Examples

The invention is explained in more detail below with a number of examples, which are not to be construed as limiting the scope of the invention. The invention is not limited to the forms of implementation described in the cases given as examples. The invention also extends to each combination of measures as described above, independently from each other.

Example 1

The enzymatic synthesis of compound (VI) was carried out in organic solvent 2-methyl-2-butanol with 25mM methyl urocanate and 25mM compound (V) as the strafing material as well as 40g/L Lipozyme 435 (Novozymes) at 60°C for 73h, the enzymatic kinetics were shown in Figure 1 (a) and (b). There was no significant difference of compound (VI) synthesis between (a) and (b). The enzymatic molar conversion yield of compound (VI) was summarized in Figure 2. There were two treatments, one without the addition of molecular sieve 4A, while the other one had additional 100g/L molecular sieve 4A. Molecular sieve 4A absorbs excess water and by-product methanol, which are generally undesirable during enzymatic synthesis. According to Figure 1 , the enzymatic reaction with 100g/L molecular sieve 4A not only had slightly higher product molar yield (0.58mol/mol) than that without additional molecular sieve 4A (0.55mol/mol), but also lower by-product formation.

The compound (VI) in crude reaction mixture resulting from the enzymatic reaction between methyl urocanate and compound (V) was isolated and purified using either silica flash column chromatography or preparative amide column with LC-MS.

Biotage SNAP Ultra 10g silica column (Biotage HP-Sphere 25um) was used for flash column chromatography. The elution rate was 20mL/min and mobile phase was the mixture of dichloromethane and methanol (with 0.1 % ammonia). The gradient elution of mixed solvent was from 10 to 20% dichloromethane (90 to 80% methanol) for 170mL, 20 to 30% dichloromethane (80 to 70% methanol) for 170mL, 30 to 50% dichloromethane (70 to 50% methanol) for 170mL, and 50 to 70% dichloromethane (50 to 30% methanol) for 170mL. The glass tubes containing product N- urocanylhistamine were collected and went through roto-evaporation for solvent removal and freeze drying for water removal.

Waters preparative amide column (XBridge BEH Prep OBD Amide 5pm, 19x250mm) equipped with single quadrupole LC-MS was used during the separation. Isocratic elution of a solvent mixture of 80% acetonitrile with 0.1 % formic acid and 20% water with 0.1 % formic acid was used with a flow rate of 16mL/min. The glass tubes containing product N-urocanylhistamine were collected and went through roto-evaporation for solvent removal and freeze drying for water removal prior to NMR analysis.

Example 2

The enzymatic synthesis of compound (VI) was carried out in organic solvent 2-methyl-2-butanol with 25mM methyl urocanate and 25mM compound (V) as the starting material as well as 40g/L Lipase TLIM (Novozymes) and 100g/L molecular sieve 4A at 60°C for 72h. The molar yield of compound (VI) was around 0.018mol/mol. Example 3

The enzymatic synthesis of compound (VI) was carried out in organic solvent 2-methyl-2-butanol with 25mM methyl urocanate and 25mM compound (V) as the starting material as well as 40g/L Lipase TL (Meito sanyo) and 10Og/L molecular sieve 4A at 50°C for 72h. The molar yield of compound (VI) was around 0.14mol/mol.

Example 4

The enzymatic synthesis of compound (VI) was carried out in organic solvent 2-methyl-2-butanol with 25mM methyl urocanate and 25mM compound (V) as the starting material as well as 40g/L Lipase QLM (Meito sanyo) and 100g/L molecular sieve 4A at 60°C for 72h. The molar yield of compound (VI) was around 0.017mol/mol.

Example 5

The enzymatic synthesis of compound (VI) was carried out in organic solvent 2-methyl-2-butanol with 25mM methyl urocanate and 25mM compound (V) as the starting material as well as 40g/L CalB immo plus (Purolite) and 100g/L molecular sieve 4A at 60°C for 71 h. The molar yield of compound (VI) was around 0.21 mol/mol.

Example 6

The enzymatic synthesis of compound (VI) was carried out in organic solvent 2-methyl-2-butanol with 25mM methyl urocanate and 25mM compound (V) as the starting material as well as 40g/L CalB immo 8806 (Purolite) and 100g/L molecular sieve 4A at 60°C for 71 h. The molar yield of compound (VI) was around 0.18mol/mol.

Example 7

The enzymatic synthesis of compound (VI) was carried out in organic solvent 2-methyl-2-butanol with 25mM methyl urocanate and 25mM compound (V) as the starting material as well as 40g/L Lipozyme CalB and 10Og/L molecular sieve 4A at 60°C for 72h. The molar yield of compound (VI) was around 0.12mol/mol.

Example 8

The enzymatic synthesis of compound (VI) was carried out in organic solvent 2-methyl-2-butanol with 25mM methyl urocanate and 25mM compound (V) as the starting material as well as 40g/L Subtilisin A and 100g/L molecular sieve 4A at 60°C for 72h. The molar yield of compound (VI) was around 0.21 mol/mol.

Example 9

The Tables I and II below suitable enzymes that can be used in a method according to the invention, without being limiting. Table

Table (a): PLU = Propyl Laurate Unit, 1 PLU is the amount of enzyme activity which generates 1 pmol of propyl laurate per minute under defined standard conditions.

(b): IUN = Interesterification Unit. 1 PLU is equal to 1 IUN.

(c): assayed by the method of Kokusho et al using olive oil emulsified with polyvinylalcohol as a substrate [6], 1 U corresponds to the amount of enzyme which liberates 1 pmol fatty acid per min at 37°C.

(d): PLU: one unit corresponds to the synthesis of 1 pmol per minute of propyl laurate from lauric acid and 1 -propanol at 60°C.

(e): LU: Lipase Unit, 1 LU is the amount of enzyme activity which liberates 1 pmol oftritratable butyric acid from the substrate glycerol tributyrate per minute under defined standard conditions.

(f): Activity expressed in Anson units.

Example 1 1 :

The enzymatic synthesis of compound (VI) is carried out in organic solvent 4-methyl-2-pentanol (a secondary alcohol) with 25mM methyl urocanate and 25mM compound (V) as the strafing material as well as 40g/L Lipozyme 435 (Novozymes) at 60°C for 72h. There are two treatments, one without the addition of molecular sieve 4A, while the other one has additional 10Og/L molecular sieve 4A. Molecular sieve 4A absorbs excess water and by-product methanol, which are generally undesirable during enzymatic synthesis.

12:

The amidation reaction is performed with 25mM urocanic acid and 25mM compound (V) in aqueous system (pH 7-7.5 buffer solution) with amide synthetase at 37°C for 24- 72h.