WO2016171538A1 - Enzymatic-chemical method for the synthesis of vanillylamides of organic acids - Google Patents

Abstract

The invention relates to a method for synthesising vanillylamides of organic acids, based on vanillin oxime, inorganic salts of vanillylamine or based on organic salts of vanillylamine having organic acids. The method involves: the chemical synthesis of vanillylamine organic salts based on the hydrogenation of the vanillin oxime in the presence of an organic acid; the release of vanillylamine inorganic salts in the presence of a base or carbonate and an organic acid; and an enzymatic condensation of the two elements that form the organic salt, the vanillylamine and the organic acid, in order to bring about the synthesis of organic acid vanillylamides. The organic acid used for the reaction can be any organic acid: natural or synthetic, saturated, linear or branched, having substituents such as hydroxyl groups or others. The reactions can be carried out independently or in sequence. These capsaicinoid compounds, components similar to capsaicin or vanillylamides can be used as a pungent material, irritant, in pharmaceutical products, as additives in food, in cosmetics, among others.

Description

 CHILD TlCO-E OIATIFIC ASSESSMENT FOR THE VAWLSIS OF VAiNiLLt AMtiMS

OF ACIDOS OReÁM COS

CAMPO BE LA NVE CIÓN

The present invention relates to a method for the synthesis of amides of organic acids with vatnillinylamine {valniinamides} within which capsaidnoids γ are similar to capsaicin, responsible for the pungent power of c il s {ca sicum wnum). The present invention relates to the production of organic vaninylamine salts with organic acids and their enzymatic condensation to generate vainiiiioamides. The method can be started from the chemical synthesis of organic vanillinamine sai and continue in a sequence! with the approximate condensation of said organic salt to give place the corresponding vainiinamide, or it can start directly from the attic enzyme condensation of an organic vainiiinamine salt, to give rise to the corresponding vainiinamide, when the organic vainiiinamine sai has previously or independently synthesized as proposed in the present invention or when it becomes synthesized from other reactions that may arise. The synthesis step of the organic vaMIlinamine salt can be carried out by hydrogenating the vaMinoxiroa in the presence of an organic acid, or from the release of inorganic salts of vainiiinamine in the presence of an organic acid.

TO TECEDENTES

Capsaicinoids constitute a family of amides of particular interest to the food industry. These compounds are responsible for the pungent effect of chili peppers. Of this family, capsaioin {ím? Is- $ ~ metii-N ~ v¾iniliin-6-nonenamide} is the most pungent natural capsaicinoid. Three structural characteristics that confer the pungent activity to these compounds have been identified: the amide bond, the goodbye residue and the vatnilloid ring. Capsaicinoids are attributed various pharmacological activities within which they decrease the ability to stimulate e! respiratory, cardiovascular and digestive system, which is why they are applied as promoters of energy metabolism. Moreover, it has been shown that capsaicinoids have ani-Mamaíoria pharmacological activity and bioqueadora gives pain transmission; However, the latter is limited due to the irritation caused by the skin. It has also been observed in cultures in v $, that ia capsaicin is able to inhibit the growth of various cancer cell lines,

Typically, vainiinamrctas are usually prepared by chemical synthesis from vaininamine, commonly used as a vain feamine cioohydrate, and activated organic acids, usually acid chlorides. However, prior to the aeiation reaction, it is necessary to release vanillin nina from its dihydrochloride using different types of bases. There are several antecedents in the literature related to the production of vainiinamine salts and their condensation with organic acids, methodologies described below.

For the production of vanillin amine salts in US Pat. No. 4,388,250, a method for the preparation of p-droxybenzyl nitrides is protected from the corresponding p-ftydroxy benzathóehyde, where first the p »tsidroxybenaldhyde is reduced to p- Hydroxybenzylamine with hydrogen in an aqueous medium with ammonia. Preferably, this reaction is carried out at low pressure with small amounts of alcohol with Raney nickel as catalyst. In patents ÜS 8,329,948 B2 and US 8,329,948 a method is presented for the synthesis of rmamides and substituted amines by accelerating the leuokart reaction. This reaction is accelerated by reacting the formamide or / or affinity Formamtda and formic acid with an aldehyde or a ketone. In particular, for the production of vainiioamine hydrochloride, vainiinformamide is first produced, from vanillin. The vasniinfermamide is subsequently dissolved in dilute hydrochloric acid and methanol, and the reaction is rapidly heated to 12 CTC, immediately after reaching this temperature the reaction is cooled to 6 C to obtain quantitative yields of the vanillin hydrochloride. In the US 6,968,418 82, a method for producing vanillin amine, or salts thereof, is described, first carrying out a reaction with vanillin and hydroxyamine or one of its salts, in the presence of an organic ealinium oxime form, subsequently carried carried out a hydrogenation reaction in the presence of a suitable catalyst and an acid, organic or inorganic.

On the other hand, for the synthesis of capsaicin, capsaicinoids, capsaicin analogs and in general vainiinamides, there are several alternatives reported in the literature. In US 4, S32,139, a method for preparing N-phenylmethylquinamides with analgesic activity is described. The aquinaquides can be linear or branched, from 11 to 23 carbon atoms, preferably fV-vainiin-alquinamides. The method of synthesis of these amides consists, once synthesized ai saturated or unsaturated acid, in activating the residue only as acid chloride and dissolving the vanillin amine oohydrochloride in water with sodium hydroxide to release the vanillin amine from its hydrochloride and thus allowing the amidation reaction to be carried out. US Pat. No. 094,782 includes a method for producing nonanoyl-vainiiiinamide succlonate, an ester of the succeinic acid m position 4 of the slntene capsaicin {nonanoli yainiiiinamide). The esterification requires the succinct ann drkfo. In US patents 8,322,862 and US 5,488,910 a method is described for synthesizing capsaicin in a biphasic reaction medium, usually water-benzene, in which vanillin amine chloronidtate and activated organic acid are used in fo m chloride acslo. In order to release the vanillin amine from its toroorohydrate it is necessary to dissolve it in the aqueous phase that must contain potassium carbonate. In US patent application 2007/0293703 Ai a method of producing the isomer and of capsaicin is described. The novelty of this method is that the configuration of the organic acid is defined from the beginning of the synthesis process and thus remains the end. This patent application also refers to the use of acid chloride as an acylating agent. US 7,446,226 B2 refers to new compounds called "capsaiton derivatives", to their production method and to their use as amphimicrobial agents. According to the claims of this invention, it is intended to protect a method to produce new capsaicin derivatives called ai uino analogues or íenii-capsaicirta. The salting of vamsnaml m is carried out by means of the acid chloride, which is formed from thionyl chloride.

An important point in common of these methodologies that differentiates them from the present invention is the requirement to use vanillininamine chlorohydrate as the raw material for obtaining vanilliumamides. In fact, in order to facilitate the release, of the valnSinamine from m dorohydrate, commonly the chemical production of capsaieinoids is carried out in biphasic systems. Under these conditions, the vainiinamina is released in aqueous medium while the acylation takes place an Inmiscibl solvent in the aqueous medium. On the other hand, a common denominator of the previously described methodologies for the synthesis of vainitlinamides is the use of acid derivatives, usually acyl chlorides. To produce the acid derivatives it is normal to use thionium chloride as a precursor to acid chlorides. . This agent, besides being toxic. results from the risk given its reactivity. Another tool used as an alternative for the production of capsaieinoids, analogs and capsaicin and vainlinamides is enzymatic synthesis. Da! Hydrolsttese enzymes da! type amidasa in organic solvents to shift the equilibrium of the reaction towards synthesis. However, these enzymes have disadvantages, among which are their low stability in organic solvents. A very successful enzymatic tool due to its high stability in organic media and its efficiency in the synthesis of amides is the use of üpasas. For the synthesis of capsaieinoids by means of üpasas, several strategies have been proposed, such as the condensation of vaninitinamine and different organic acids using as a catalyst the lipase B of Candida Antarctica (CaL-8) in phosphate buffer as a reaction medium (a - Kenji Kobata ®t yes b- Kenji Kobata é ai). Another method for the production of these capsaicin analogs also using Cal «8 _« describes the transsacration of capsaicin with several naturally occurring oils in two different reaction media, β-hexane and CC¾ supefcrl ioo (c ~ Kenji Kobata el al, ú- Kenji Kobata af). In this sense, qm has been demonstrated in the enzymatic production of capsaicin analogues »yields are much higher in hydropic polar solvents than in hydrophobic appeals (Dolores Reyes-Duarte ef BÍ, Edmundo Castillo oí $ 3,

In the patent application US 2003/01 S787Q Al, enzymes present in enzymatic extracts of Sírapíomyüús g us are protected, which have the ability to hsdroiizar and / or synthesize capsaiana. The patent application covers the use of these enzymes for the synthesis of analogs in a water: n-hexane yarn. A case very similar to the previous one, is that of the patent application US 2004/0 06172, which protects the use of dopans of Si ptomyces strains for the synthesis of capsaicin analogs using various organic acids, also in reaction media. Biphasic water: n-hexane.

Although in the enamatic synthesis of vainiiinamides the activation of organic acids and consequently of acid chlorides is omitted, a great drawback that limits the reaction is the need to release valnillinamine from its hydrochloride. An altemaísva is to carry out acylation in biphasic systems. However, the presence of water should be avoided given the hydrocytic nature of the enzymes used. Given this restriction, the proposals describing the use of solvents other than water require the addition of tertiary amines to release valnillinamlna from its hydrochloride. In spite of this Vainiinamine release form from its feasible ciorohydrogen m, to reach a high yield in the reaction the need to use an excess of tertiary amin (molar ratios of tertiary hydrochloride and amine of up to 1 18) is reported. The foregoing hinders the commercial application and the economic viability of the method, not only because of the excess of used tertiary amtññ, but also because of the difficulty for its recovery and recovery,

B EVE DESCRIPTION OF THE FISURES

Figur 1 »Scheme of the sequential reactions involved m the aromatic chemical method proposed for the synthesis of organic acid vainiinamldas, the) General scheme of the sequence reaction! d production of organic acid vainiinamides preceded by the formation of organic vanillinarine salts obtained from the hydrogenation of the vainiinoxtma in the presence of an organic acid, 1b) General scheme of the reaction sequence! of production of organic acid vainiinamides preceded by the formation of organic vainamine salts obtained by the release of inorganic salts of vanin inarnine by the use of some base with the subsequent addition of an organic acid. Where, C t »Catalyst; Pis ^™ Solvent: Ez ~ Enzyme; A ~ acid; 8 ~ base; C ™ salt formed between A and B; D ™ water ø acid formed between A and 8.

Figure 2. Scheme of the reaction of the enzymatic chemical synthesis method of organic acid vainiinamides, from organic vanillinarine salts. Where, Dis * Solvent; Ez ~ Enzyme.

 Figure 3, Scheme of the reactions involved in the proposed synthesis routes for the chemical synthesis of vainsllinamine organic salts. 3a) Synthesis route for the production of organic vanillinarinine salts obtained by means of the vanillin oxime in the presence of an organic acid. 3b) Synthesis route for the production of organic vaninamine salts by means of the release of inorganic salts of vainiinamine with a base and the subsequent addition of an organic acid. Where, Dís- Solvent; Ez »Enzyme.

Figure 4. HPLC chromatogram in ai that two peaks corresponding to the vainiinamirsa can be observed in two different protonation states {TR 2.7 and 3.8 min) and the peak corresponding to the caprilvainiliinamlda {TR 10.3 min), the dark line belongs to a sample at the beginning of the reaction of example 12, while the gray line belongs to a sample taken 48 hours after the start of the reaction. Where, V »Vaírilinamfna; Or Caprilvalniinamide. F¾M 5. Ultraviolet light absorption profile typical of vainiinamides where absorption maximums at -230 and ~ 28G nm can be observed In this case the figure corresponds to the absorption of eapriivatninamide obtained with the synthesis method of Sa present invention .

Figure 8. HPLC chromomagram in which two peaks corresponding to vasnlinamine and two different prctonaction states (T 2.7 and 3.6 m-n) and the peak corresponding to iaurilvainylamide (TR 10.8 min) can be observed. The dark line belongs to a sample at the beginning of the reaction of! Example 15, while the clear line belongs to a sample taken 48 hours after the start of the reaction.

DETAILED DESCRIPTION

The present invention relates to a chemo-ensimitie method for the synthesis of vayniinamides from organic vanillin amine salts. These organic vanillin amine salts are currently not commercially available and no synthesis methods have been found in the prior art. To solve the problem of their lack of availability, the inventors propose two alternate routes of synthesis of such organic vainiinamine safes, but it is possible that in time other synthesis routes, whose products, can be used as inputs in the method of synthesis of vainiiiinaniides of the present invention. In this way _, the method of synthesizing vainylisnamides can be carried out: i) from an organic vanillinamine sai obtained by the hydrogenation of the vanillinimin in the presence of an organic acid, followed by the enzymatic condensation of said sai (Figure la); i) from an organic vainiinamine salt obtained by the release of inorganic salts of valniHinarnina through the use of some base and the subsequent addition of an organic acid, followed by the enzymatic condensation of said sai (Figure Ib) or 111 } by means of the condensation in direct elmatics of an organic saira of vairaííinamioa obtained by any other route of synthesis (Figure 2). In either case, the final product of the enzymatic condensation is a vainii amide. The production reactions of organic vanillin amine salts (Figure 3a and 3fc) and the condensation thereof can be carried out independently, or in sequence!

As described above, current methods of chemical synthesis of vanillinamides require the use of tphonium chloride and other activators of organic acids and solubslitation of vainiinamine salts in water for dissociation. On the other hand, the methods of synthetic synthesis require the use of tertiary amines for the release of vainiinamine from its salt or ie of biphasic reaction media, in which the presence of water prevents an efficient synthesis reaction.

 As will be clear, in the present invention the use of thionyl chloride and other activators of organic acids is avoided, the sdubi & aeióii of vainiiinamine salts in water for dissociation is dispensed with and the addition of tertiary amines is avoided. for the release of vainylrsarnine during enzymatic synthesis of vainiinamides.

 It is important to note that the present invention has substantial differences with the applications and patents described in the background and represents a fundamental innovation in the synthesis of vainiinamides. The differences relate in particular to the direct condensation of the inorganic sai of vainiinamine and the organic acid without the need for a tertiary amine for the release of salt, in one of its modalities, as well as the use of {linear or branched alcohols) As a reaction medium during the hydrogenation of vanillin oxime and the release of inorganic salts as a necessary condition for the formation of organic salts of vaini iinamine with water-insoluble organic acids in another of its modalities, for when starting from Inorganic Vainiinamine salts or vanininoxime, respectively.

In the present invention a chemical-anzyme method is described for the synthesis of vainiinamides from organic vainiinamine salts with organic acids, salts which at its z may be derived from the hydrogen peroxide or from the release of inorganic salts of vainiinamine in presence of an organic acid or other routes that come to be proposed. It should be emphasized that the use of thionyl chloride and other organic acid activators is avoided in this method, the soiubiiteaeión of the vanillin amine salts in water is discarded for their dissociation and the addition of tertiary amines for the release of the vanillin amine salts, elements that are essential in the synthesis methods described in the state of the art and that constitute the main difference of the method of the present invention with respect to the state of the art,

The present invention solves the problems described above thanks to the innovation that involves the ability to carry out the enzymatic condensation of a organic sai efe vainiinamina in the respective amide; previously said organic salt can be synthesized from vaninyloxyme, from inorganic salts of the vainiinamfrsa (both synthetic routes described in the present invention) or by other synthetic routes that (legume to arise and both steps; synthesis of the organic salts of Vainiinamines (by the routes of acp synthesis ^" described) and their ensmal condensation can be carried out in sequence or independent fornia. Thus, the method for the chemical-enzymatic synthesis of vatniinarnides from an organic sai of vainiinamine comprises the steps of:

 Contact an organic sai of vainiinamine with an enzyme belonging to the enzymatic classification EC 3.1.1 3 and or an organic solvent or unconventional medium,

 Incubate the memela at a temperature suitable for the functioning of the enzyme, allowing the enzymatic condensation of the organic salt in a vainiinamide,

 Gpaonaímente the purifíeacidn of the obtained vainiinamide.

With these steps the condensation of the organic salt is obtained him vainiinamina by effect of the enzyme, obtaining the corresponding vainiinamide. The reaction can be carried out in the presence of an organic solvent, in an unconventional medium or by directly using the organic vainiinamine salt as the reaction medium, provided that the temperature is that of its melting point or above this . Examples 2-8, 12, 13, 1S-1S and 21-26 illustrate the application of these steps for the aromatic synthesis of various vayniphylamides from the corresponding organic vainiinamine sai, previously synthesized by any of the synthetic routes described in the present invention.

As already mentioned, the organic salt of vainiinamine, as a previous step to its condensation in matic for the production of the corresponding vainiinamide, can be obtained by applying one of the synthetic routes A1 or A2, using various catalysts, temperatures, bases and Reaction solvents, which are described below.

The synthesis route A1) comprises the production of organic vanillinamine salts by hydrogenation of valnilinoxime in the presence of organic acids, examples 1, 0, 14 and 20 illustrate the application of this stage for the production of organic salts from vanillinimex and different acids organic These variations vary depending on the pressure of hydrogen, the reaction time and the reaction solvent. In all these cases, the process of obtaining organic vanillin amine salt is a previous step to enzymatic condensation (described above). This route consists of the steps of:

 Ala) Bring the vanillinimex and an organic acid into organic solvent or the mixture of two or more solvents in contact with molecular hydrogen in the presence of some transition metal as catalyst,

 Aib) inouba the medulla at a suitable temperature allowing the reaction to proceed until obtaining the organic salt of vaintinamine,

 A1e) the recovery of the transition metal used as a catalyst and,

Mu) Highly opted for purification of the organic vanillin amine obtained.

As already mentioned, alternatively to the synthesis route Al, the organic vaninylamine salt can be obtained by the synthetic route A2) which comprises the production of organic vanillin amine salts by the release of the inorganic salts of vanillin amine with some base and the subsequent addition of organic acids, examples 10, 11 and 28 illustrate the application of this first step for the production of organic salts from an inorganic vanillin amine salt, in this case vanillin amine hydrochloride, and an organic acid, in In this case, eeianoic acid, depending on the concentration of the base and the reaction solvent, as a previous step to the erszyme condensation, or showing that the presence of the inorganic salt or that is formed does not prevent said condensation ( example 25} This stage consists of the steps of:

 A2a Contact the vanillin amine inorganic salt with a base in an organic solvent or the mixture of two or more solvents,

 tbf incubating the mixture allowing the vanillin amine release reaction to proceed,

 Tie Add an organic acid,

 A2d incubate the mixture allowing the reaction of organic vanillin amine formation to proceed,

A2e Optionally the separation of the organic vanillin amine salt obtained, A2f Optional purify the organic vaniniine salt obtained.

The production reactions of the organic vanillin amine salt, either by the route Ai or A2, and the condensation in imatic of said salt can be carried out independently, or aunt sequentially! In this sense, in case the reactions are carried out sequentially!, On route A1 or A2, a solvent, or mixture of solvents, appropriate for the condensation of the organic vainiinamine sai should be used. Example 13

10 describes a sequence process! for the production of a vainiylinamide.

 The origin of vanillinoxime as well as that of eloroMinerate vanillin amine or some of its inorganic salts does not belong to the field of this invention and can be obtained by traditional chemistry methods in the public domain, from vanillin or commercially acquired in companies such as Advanced L. Syrrthesis (United States, CA).

 For the production of the organic vainiinamine salts by the route of synthesis A1), a reaction of aron hydrogene is required in an organic solvent that dissolves the vainiinoscim and the organic acid that will form the organic vanillin amine salt. AND! organic acid will be selected based on vanilla bean

It is desired to synthesize in step b) of the method of obtaining the vainiinamlda of the present invention.

For the synthetic routes Al) or A2) various organic solvents, preferably linear or branched alcohols or mixtures thereof (Examples 1 9, 14 _» 20 and 26), alcohols of 1 to 6 atoms can be used

25 carbon, whether linear or branched. For example, it is usual, but not essential, that methane mixtures be used for hydrogenated reactions! and tenvamii-aioonol with methane ratios! which can vary between 10 and 90% v / v. On the other hand, it is advisable, although not indispensable, that for the release reactions of the inorganic salt of vainiinamine of the stage A2,

30 use methane !, ethanol and piss of both in different proportions (Examples 10, 11 and 26}.

81 the reaction of production of organic vanillin amine and the enzymatic condensation of said salt will be carried out sequentially! Suitable solvents can be used for the correct functioning of the enzyme 35 (Example 13). In these cases it is not advisable to use primary alcohols or n

secondary as well as avoid primary amines.

 The organic acids suitable for the formation of the vanillin amine salts or glands are saturated, linear or branched organic acids of 6 to 22 carbon atoms. Examples of elves are oaproioo (or exanóteo), caprflico (or capric {& decanóico) octaneolic acid, léurico (or dodeeanósco), stearico {or octadecanóíco), pelargónico (on nanóic), S-methyl-nonanóico, araquidoníco and oleico, in re others.

Depending on the organic acid used for the formation of the organic salt of vaMiiinamine and of the solvent or solvent mixture used as an average of mc or, fe concentration efe vainíllirtoxima used in the synthetic route A1) _> as well as the concentration of the inorganic salt of vainiinamtna and fa base for the route of synthesis A2}, can be between 0.1 and 2M, and even exceed the limits of solubility (leaving an insoluble part, which will gradually dissolve as the reaction progresses). The selected organic acid can be used at any concentration with raspéete- valnillinoxime or inorganic salt of vainiinamine {between 0.5: 1 and 2 1), but preferably at the same molar concentration thus avoiding residual substrates at! reaction term.

 The reaction of vainiifeoxyma hydroregeneration for the synthesis of organic salts of vanillin in the synthesis route A1 is carried out in the presence of idol at pressures between 1 and 10 atmospheres and using some transition metal as catalyst. Suitable catalysts for reaction a) are Pd / C, Pt, ¾ Ru or Raney Ni. It is preferable to use Pd / C at a ratio between 2 and 20% by weight (based on the amount of vainiinoxlma),

The hydrogenation reaction requires a time of between 0.5 and 48 hours depending on the type of organic acid used to form the organic salt, the solvent or the mixture of solvents used as the reaction medium, as well as the stirring system and its efficiency . Normally, the vanillin is completely hydrogenated! After 3 hours of reaction (Examples 1, 9, 4 and 20), the hydrogenation of the vaninoxime for the pro-projection of the organic vainiinamine salts of the synthesis route Al) preferably occurs between 25 * C and 46 ^* C although know that the reaction can occur between 0 ^* C and 70 "C

The sacuenesaies reactions involved in the chemical chemical method proposed for the synthesis of organic acid vainiinarnides from valnillinoxime is schematized in Figure 3a. After carrying out the hydrogenation of the vaninyloxime by the synthetic route Ai, the transition metal used as catalyst is recovered, the reaction mixture is added. To avoid the loss or depletion of the organic salt of vaMlnamine, before separating the catalyst from the solution, it is advisable to raise the temperature to more than 50, preferably to βθ ^B C. Once this temperature is reached, the catalyst is separated from ia solution. The separation can be carried out by processes known in the state of the art, such as filtration, decantation or centrifugation, among others.

Once iterated out the production of the organic salt vainsinamina by the synthetic route A1), it is recommended the reaction mixture at room temperature ( ^"25 ° €). Once this temperature is reached, the catalyst is separated. The separation can be carried out by processes known in the state of the art, such as filtration, decantation or centrifugation, among others,

If necessary and optionally, it is possible to purify the organic vanillamine salt. by evaporating the solvent or the mixture of solvents used. Once this has been done, it is recommended to wash the organic salt produced in a solid state using 8 volumes (referred to the amount of solvent evaporated) of a solvent that does not dissolve the sai, for example ethyl acetate, separating the salt from the solvent by decantation, centrifugation or filtering or some other operation known in the state of the art. It is recommended that the washings be carried out at a temperature between 10 and 30 * C, which avoids the dissolution of the organic salt. Preferably around 20 * C. The organic sai can then be dried and stored indefinitely.

The release reaction of inorganic ia sai from vaninyl Synamine for the synthesis of organic vanillin salts from the synthesis route A2 is carried out in the presence of bases such as NaOH, OH, UOH, ammonia and alkali carbonates (from ü, a, K , hey). The preferable bases for synthesis of A2 are NaOH and KOH. It is preferable to start KOH at an equimolar relationship with respect to inorganic sai of vanillinarin (Examples 10, 11 and 28).

The release reaction of the inorganic salt of the synthesis mine A2 requires a 0.5 and 6 hour erodium depending on the type of inorganic sai of vainiinamine, before proceeding with the addition of the organic acid. Depending on the organic acid buffered to form the organic salt of vasnynaminamine, the solvent or the solvent mixture used as the reaction medium, so As with the agiation system and its efficiency, the reaction time requires between 1 and 8 hours. Normally vanillin amine organic sai is produced after 3 hours of reaction.

The temperature of the release reaction of the inorganic sai of vainiiinamine for the production of the organic salts of vainiinam na by the route of synthesis A2) depends on the base and the solvent used. Mormafmente, the zcl m Fuerte® bases in solvents are exothermic reactions so ue reactions ia synthesis route Á2 usually occur between 35 ° C ^and 80 C although it is known that the reaction can give between 20 ^* C and 80 X .

The synthesis route by means of the release of the inorganic salt of vanillin amine is schematized in Figure 3b.

Once the production of the organic salt of vantinamine has been carried out by the synthetic route A2), in order to avoid the loss or loss of the organic salt of vainiamine _; it is advisable to cool the reaction mixture to induce the precipitation of any inorganic salt, preferably at 15 ^* C. Once this temperature has been reached, the inorganic salts (the one formed between the base and the acid corresponding to the inorganic sai of vainiiinnantine, as well as the ia Inorganic salt of residual vanillinamine) are separated. The separation can be carried out by processes known in the state of the art, such as filtration, decantation or centrifugation, among others. This separation is optional since the inorganic salt does not prevent the subsequent reaction of erszimatic condensation {example 28),

 If necessary and optionally, it is possible to purify the organic vanillin amine salt, by evaporation of the solvent or the mixture of solvents used. Hec or the above it is recommended to wash the organic salt produced in solid state using up to 6 volumes (referring to the amount of solvent evaporated) of a solvent that does not dissolve the salt, for example ethyl acetate, separating the sai from the solvent by decantation, ø irado centrifugation or some other operation known in the state of the art. It is recommended that the washings be carried out in a caco at a temperature between 10 and 30 * C, which avoids the dissolution of the organic salt. Preferably at 20 ° C. The organic sai can then be dried and stored indefinitely.

As already mentioned, the vanilllinamide synthesis method of the present invention can be carried out sequentially! with any of the routes of synthesis A1 or A2. If it is desired to do in this way {sequence!}, To continue with f $ synthesis of vainiinamide by the method of the present invention, in case of having used a primary alcohol, a secondary alcohol or primary amine as solvent in the route of synthesis A1 or A2, it will be necessary to evaporate it to avoid the enzymatic aeration of alcohol. This requirement is eliminated if a primary or secondary alcohol or a primary amine is not used for the route of synthesis A1 or A2, and particularly if the same solvent used is used! and the method of the present invention for the synthesis of organic acid vanillin amides (Example 13).

 The enzymatic condensation of organic vanillin amine salts of the method of the present invention is preferably carried out at temperatures between 15 and 85 X. temperatures at fas that obtain the best performances of fas enzymes belonging to the EN 3.1 classification. 3.

For the enzymatic condensation of the valniinamine salts of the method of the present invention the selected valnünamlna salt can be used at any concentration or even above saturation m the solvent or unconventional medium, since the insoluole salt will dissolve as that the enzyme produces vanillylamide. Typically, the method of the present invention can be carried out at a concentration of vainiHinamine salts there between 0.02 M and 2, preferably at the highest possible concentration.

The vainiinamide synthesis method of the present invention can be carried out by solvent or medium or conventional crying, using as a reaction medium the same organic salts of vanillininamma in a liquid state at the temperatures of its melting point or above he, to the extent that said temperature is compatible with the stability of the enzyme used.

There is a wide range of organic solvents suitable for the vaninylamide synthesis method of the present invention, although tertiary alcohols are preferably used. Particularly it is recommended to use 2-methyl ~ 2-ouíanof (Example 5} or 2-methy-2-.propanoi, although the synthesis can also be carried out in other organic solvents such as pyrldine; dimethisulfoxide; dioxane; trlerilamine; di-isopropylamine ; benzene; cyclohexane; acetone (Example 8), aoetonlthlo; tetrahydrofuran (Example 2); hydrocarbons such as n-hexane and n-heptane, or in solvent mixtures. Likewise, some media can be used instead of organic solvents unconventional such as supercritical CO2 and ionic liquids. In order to carry out the method of vanillinamide synthesis of the present invention, the prior dehydration of the solvent is more essential, however it is preferred to remove the water either by evaporation, pervaporation, molecular sieve adsorption, in ion exchange resins or by the addition of you go out.

Enzymes suitable for use in the method of synthesis of vairtiyliriamides of the present invention belong to the enzymatic classification EC 3.1.1.3 and are simply known as lipases or iriaciigiicerol ispases or tnacylglycerol hydrofases. They can be used in any of these commercial presentations, in solution, or bindings, forming aggregates, catalytic crucibles or bie «immobilized in some support. Particularly, it is advisable to use the following enzymes: Antarctic Candida lipase, Antarctic Kikfa lipase 8, Candida üytktdracea lipase, Candida rugosa lipase, Burk oide a pacia lipase, Pssudo onas aeniginous lipase, Ps dormms sp lipase ., Pseudomonas fluo sc & ns lipase, Rhszomuwr mieftei iipase, Ustilage ma is lipase 621 and Yar wm! ipoiytica lipase. The best results have been obtained with lipase 8 from Candida Antarctica in its commercial form known as ovo2ym43¾. In the literature it is widely documented that these b ooatalteadores have the ability to synthesize amides (Sol im et ai, d uñ ú Castillo et al, Florían Le Joubíoux eí ai, Xiang ~ £ 3uo Li eí a /, T íerry Maugard ei ai , Abui Asim and the quotes found in those publications).

The amount of enzyme in the biocataNator or free era used for the vanillin synthesis method of the present invention will depend on the concentration of the vanillin amine salts, the enzymatic activity of the enzyme or the biocataler and the time required for the reaction. For the method of synthesizing vainiylinamides of the invention, it is advisable to use 2 to 50 mg ml of enzyme or biocaffactor, equivalent to an activity between 2 LU rrsl and 50 LU / mL (where LU are the Lipase activity Units, defined as co or the amount of enzyme necessary for the synthesis of 1 ^ mol / mln of propiMaurate from urauric acid and 1-propanol at 60 ^* C). It is preferable to use 10 to 20 lü / ml.

The organic vanillin amine salts to be used in the vanillin amine synthesis method of the present invention can be saturated, linear or branched organic acids of 6 to 22 carbon atoms. Examples of these are the organic salts of vanillin amine and caproic (or hexanoic) acids, caprieo (or octanoles): capric (or decanóico), iáurlco (or dodecanolco), stearic (and octadecanóico), pelargénico (or nonanóico), 8-meiif ~ nQnanóÍeo _; arachidonic © and heard, among others. The vainiylinamide synthesis method of the present invention from organic vanillin amine salts is found asquesriatode in Figure 2, while in Figure 1 (a and ø) the vainiinamide synthesis method of the present invention is shown sequentially coupled to the synthetic routes A1, where vainiinoxirna is started, and A2, where it is based on inorganic vainiinamine salts,

 Pyrficaciérs da vainiifinarnidas. Vaioiinamides are usually very soluble in alcohols and ethyl acetate, so the reaction product can be concentrated by evaporation of the reaction medium and purified by extraction with said solvents or by biphasic extraction with mixtures of immiscible solvents, although from this Ultimate scares yields are low. They can also be purified by preparative HPLC matography or by Flash chromatography.

MATERIALS AND «ÉTOOOS

Vainliiifioxime 95% was purchased from Advanced Synthesls (United States, CA), 99% vainsifinamine dofof idrate, eaproic {or rtexanoic), eapriolic (or octanoic), lauric (or dodecanoic), stearic (or octad candic) acids ) and tñftuoioacetic (TFA) as well as 5% and 10% Pd C were purchased in $ gma ~ A! drich (Edo. d © Mexico, Mexico), The following organic solvents were used: methane! 99%, ethanol%, tert-amii-alcohol 99% and terbutanol 99% of the JT Baker brand, 99% acetone and 99% tetrahydrofuran from Budríok & Jaeson and 99% acetoneitrile from Boney eií. The R 4M enzymes of Rh & omumr imete ^" movflkaáa m ansonic exchange resin, Duoilte% Ύ14Μ (Th @ rmomym $ imugmos lipase immobilized in granulated sica) and Novozym 435 (Candida B lipase immobilized in Lewatit) were also analyzed. Nevozymes®, the Coagertt column (Bidentate C18 2S0 mm x 4.0 mm) from MXC MieroSoiv Technology Corporation.

Cyantifleación by HPLC to estimate% «the conversion and entoto ento

For the quantification by HPLC, a Ultímate 3000 device equipped with a diode array detector (Dsonex) was used. In all cases, the conversion and performance data are based on the quantification of the disappearance of vainiíinamina, although it is also possible to observe the appearance of the reaction products. A Coagent Bidantate C18 column was used and a gradient in the mobile phase composed of {A) a phosphate solution {25 mM _s pH 3.8} and (B) acetonitriium was used at a rate of 1 mfJmin. The gradient is shown in the Table The identity of the vainslnamfdas obtained corresponds to what was reported (John. Janusz et al 1993, Edmundo Castillo eí to 2007).

Ta & la 1 Proportions of the gradient used for the analysis in HPLG. Phase A refers to the phosphate buffer solution (25 rn, pH 3.S) and phase 8 refers to acetonitri.

In order to better understand the invention below, some examples are presented without elves limiting the scope of the present invention. It is worth mentioning that "various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art. technique. However, in this document the inventors try to make it clear that said modifications are intended to fall within the scope of the appended claims. In the characterization of the reactions, the methodology described below is started.

EXAMPLES

 Ejeitspf 1 Synthesis of vanillilinamine capmat. (Synthesis path Ai)

In a 500 ml bottle of idrogenaclone, he dissolved 1 § (6 branches) of vanillinimex in 15 mL of a methanol-terbuyanol mixture {8: 2), achieving a clear and transparent solution; 0.7 g (11.96 mmol) of hexanoic acid and 100 mg of Pd C catalyst were added. The reaction was taken to complete hydrogen consumption, approximately in 1 hour and 15 minutes at 60 psi of hydrogen. At the end of the same, to recover the organic sai, the reaction piss was concentrated on a rotary evaporator under reduced pressure until it reached a flagpole consistency to which 10 mL of ethyl acetate was added. This operation {c concentration-solvent addition) was repeated once more, before cooling the piss to § * C to recover the precipitated salt by vacuum filtration. The solids were washed with 1S mL of ethyl acetate and dried at room temperature to constant weight. From this reaction 1 g of the vainiinarnin caproate was obtained.

Example 2 Enzymatic synthesis of caproylvainiliinamide. Variant 1

From 0.1 M of vainiinarnin caproate, as can be obtained in example 1, an enamel reaction was carried out in a 1.5 mt eppendorf tube, in a thermal agitator (Themiomixer compaet, eppendor1¾S> with ai In order to stir the system and keep the temperature controlled, the reaction contained 1 ml of tetra idrofuran (THF), 10 mg of Novo and m 435 (Lipase B of Candida Antarctica immobilized in Le aíii fíovGzymes®) and was carried out at 55 ^e C with stirring of 1400 rpm.Tmnscyrridas the 48 ñoras of reaction the tube was removed from the thermo ^» stirrer and the samples were analyzed at the initial and fine time! Of the reaction, by HP C, where the disappearance of vainiinarnin was cisanified and observed 92% conversion of vainiinamine caproate to caproylvalyiliinamide, Example 3 Enamatic synthesis of caproifvainitin mMa. Variant 2

 The procedure was similar to Example 2, except that the biocataiteator was replaced by Upozyme RU4M (R iiomuoor m lipase immobilized in Duoiite® ammonium exchange resin, Novoz mes®), obtaining a conversion of 70% of vainiinamine caproate to caprolivainiliinamide,

Example 4 Ermmáiica synthesis of caproiívainilllnamida. Variant 3

Example 2 was proceeded in a similar manner except that s iocatalyst was replaced by Upozyme ΤΙΛΜ (Ttwmomyces lanugmosm lipase immobilized in granulated silica, Novozymes ©), obtaining an 82% conversion of vainiflinamine caproate to caprolivainiliinamide.

Example S Enzymatic synthesis of caprolivainiliinamide. Variant 4

With 0.1 of vainiinamine caproate obtained in Example 1, an enzymatic reaction was carried out in an eppendorf tube of 1.S mL, in a thermal stirrer (Thermomixer compact, eppendorü) in order to stir the system and maintain the controlled temperature. The reaction was limited to 1 m of 2-metii-2-butanol (2M2B} _; 20 mg of Novoar m 435 and was carried out at 3S ^* C with stirring of 1400 rpm. After 48 hours of reaction the tube was removed from! thermo-agitator and samples taken at the initial and final time of the reaction were analyzed by HPLC, to quantify the disappearance of vairtiinamine.After 48 reaction a 95% conversion of valnit synamine to caproiívainiinamide was obtained.

Example 8 Enzymatic synthesis of caproiivainiliinamide Variant 5

The procedure was similar to example 5 unless! solvent 2 2B by acetone. After 48 hours of reaction, it obtained a 14% conversion of caproaium from vainiinamine to caproiívs inamide. Example 7 Without enzyme enzymes of caproylvainiinamide. Variant 8

Example 5 was similarly carried out except that the 2M2 & solvent was replaced by acetone and the FeiGeatafízaefor Novozym 435 with üpoz me R * M, obtaining a conversion of 25% in 48 hours of reaction,

Example 8 Enzymatic synthesis of caprofivaMiiínaíBída. Variant 7

The procedure was similar to Example 5 except that the Novozym 435 biocaiafeator was replaced by üpozyme Tt ~, obtaining a conversion of 10% after 48 hours of reaction.

EJenspí® § Synthesis of vainslylamine capriate. (Synthesis path Al)

S dissolved 4 g of valnillnoxíma (24 rrsmoles} in 20 ml of methane! And 3.8 ml of octanoic acid (1 eq.) In 30 ml of methane !. Both solutions were mixed and placed in the bottle of hydrogenator (Parr 3918EG) Afterwards, 200 mg of 5% Pd C was added Once the bottle was placed in the hydrogenator, the system was purged with hydrogen and then brought to a pressure of 60 psi or hydrogen at room temperature (25 * C). m stirring until complete hydrogen consumption Approximately in 3 hours the pressure of the hydrogenated was released and the solution was filtered under vacuum on paper Whatman 1 to recover the catalyst After separating the catalyst, the solution was concentrated by evaporating the methane At reduced pressure in a roaster evaporator until reaching a final volume of 16 ml, 30 mt of ethyl acetate was added to this solution and it was brought to Ú ^* C to crystallize the salt. hours at this temperature and the vaimliinam na capriiate crystals were filtered. When filtering this heterogeneous mixture, 4.9 g of the salt were obtained, which is equivalent to a yield of 89%. Example 10 Synthesis of Vainii amtna Capriiatus. (Synthesis path A2)

In a 1.2 L vessel, S8.1 g {1 mol) of potassium hydroxide was dissolved in 1 t of meíanol. achieving a clear and transparent solution. The temperature of the solution reached 55 ^e C. To this solution was added 189.8 g (1 mol) of dore-vainiinamlna hydrate and stirred vigorously for 30 min. Subsequently, 144.1 g (1 mol} of octanoic acid were added. In the reaction vigorous stirring was maintained for 2 hr, sufficient time for the reaction to reach an ambient temperature (~ 28 ° C). Subsequently, the solution was ro in a Büchner funnel with filter paper, the precipitated KCI and a homogeneous solution were obtained After the solids were separated, the methane was evaporated to dryness and 270 g of vainiinamine octanoate (0.9 mol) were obtained,

Example 11 Synthesis of vaisamine caproaium. (Synthesis path A2)

In a glass of 0.6 Lm dissolved 10 g (0.S mol) of sodium hydroxide in 0.5 L of ethanol, achieving a clear and transparent solution, the temperature of the solution reached 38 ^to C. To this solution were added 47.3 9 (0.5 mol) of valnidlnamlna hydrochloride and stirred vigorously for 30 min. Subsequently, 38 g (0.5 mol) of octanoic acid were added. In the reaction, fgomsa stirring was maintained for 2 hr, sufficient time for the reaction to reach an ambient temperature (* ^» 28 * €). Subsequently, the solution was fixed in a büehner funnel with filter paper passing the NaC! precipitate and a homogeneous solution. After separation of the solids, methane was evaporated! to dryness and 63.8 g of vanine Bnamine octanoate (0.43 mol) were obtained.

Example 12 Enzymatic synthesis of caprilvainíllínarnida. Variant 1

With the vanillin amine capriate obtained in example 9, 4 enzymatic reactions were carried out with a different concentration of sai (0.02 M, 0.1 M, 0.5 U and 1.0 M) in sealed 1S mL vials, m a Nbridator ( Thermo Scientific® S ake 'n Steck) in order to agitate the system and keep the temperature controlled. The reactions contained 10 ml of tert-ami-alcohol (2Μ2Β), 200 mg of Novozym 435 biocatalyst and were carried out at 55 ^to C with stirring at 280 rpm. The solutions at 0.02 and 0.1 they were homogeneous mixtures from the start of the reaction; on the contrary, the reactions at 0.5 and 1.0 M were heterogeneous at the time of the reaction, but they were roogene & aron after 12 hours of process. After 48 hours of reaction, the vials were removed from the ibridizator and the samples were analyzed by S HPLC at the start and end times of the 4 reactions, to cuanijfícar the disappearance of vanillin. The reactions at 0.02 and 0.1 M of the salt reached 99% conversion of capirex from vaireifinamine to capriívainiinamide, while the reactions that started with O.SM and 1.0M of salt reached 95 and 93% conversion to the amide respectively.

10 For the purification of the caprilvainiilinamide, the products of the 4 reactions were mixed, the biocataucer was filtered off and 2M2B m evaporated to a Büchi 461 mtavapor (at 80 ^e C, 100 rpm and -60 Pa), until obtaining a viscous and brown oil. Subsequently, with 1 g of said oil, purification was carried out by flash chromatography (eveleris® Preparative Flash

] 5 Chrornatograp and System) with a gradient from 0 to 0. S n-hexane / ethyl acetate from which 0.92 g of caprilva IMfoamide, Jamplo 13 Chemical synthesis of the calamine salt of vanillin amine (Rula of synthesis A1} and synthesis were obtained Enzymatic sequence of the eaprivainilinamide.

 20 1.57 g {9.4 mmdos) of valnilnoxime and 1.62 g of octanoic acid (1.2 eo) were dissolved in 15 ml of 2 28. The solution was placed in the fixtrogenator bottle {Parr 391 £ ø) and 78.6 mg were added of 5% Pd / C. Once the bottle was placed in the hydrogenator, the system was purged 3 times with nitrogen and 3 times with hydrogen after which the system was brought to 45 * C and 80 psi of hydrogen, Al

After 12 hours, the reaction of the iiidrogenator was removed and the solution was filtered under vacuum on atman paper 1 to recover the catalyst. From this sample, an aliquot of 200 μΐ was taken, 200 ≠ ~ e water was added and the amount of vamiylamine was quantified by HPLC, determining a yield of 81.8% in the reaction.

After hydrogenated use, the filtered solution was transferred to a 40 ml bottle which added 226 mg of 43S ovozym and was taken to an Incubator at 80 ^* 0 stirred at 300 rpm, after 72 hours of transduction of the reaction The bottle was removed from the incubator. A sample was taken from this solution and 92.1% conversion in the condensation enzyme reaction was determined by HPLC

35 for obtaining caprilvalniilinamide. The evolution of this reaction is <><) shown in Figure 4,

Example 14 Synthesis of vainiinamtna laurate, (Synthesis route A1)

2 g (12 mols) of vanillinoxyam m were placed in the 800 mL hydrogenation bottle and a mixture of mefanoi: ierbutane! (8: 2) was activated 30 mL. 2.4 g (1eq) of uric acid were added to this solution and 200 mg of Pd / C, Subsequently the system was purged 3 times with nitrogen and 3 times with hydrogen after which the system led to 60 psi of hydrogen and kept under stirring for approximately 1 hour until the complete consumption of hydrogen ,, After this time, the catalyst was filtered in vacuo on V¾haímar¾ 1 paper and the reaction medium was evaporated to dryness by distillation under reduced pressure on a rotary evaporator.Once the reaction medium acquired the consistency of a paste, they were added 10 mL of ethyl acetate and slipped again under reduced pressure to remove traces of methane !. After distillation, 10 mL of ethyl acetate was added and the mixture was cooled in an ice bath for 2 hours. , s and filtered in vacuo on paper and dried at room temperature to obtain a constant weight of which 2.8 g of valniinamine laurate were obtained. EJ roplo 1S Enzymatic synthesis of lauriivasnilriamida. Variant 1

 With 0.1 of the vamillinamine laurate obtained in example 14, an enzymatic reaction was carried out in a 1.5 mL eppendorf tube, in a thermal stirrer (Tbermomixer cornpaet, eppendorf®) in order to stir the system and keep the temperature controlled. The reaction contained 1 mL of tert-amylafcohol (2M2B), 10 mg of Movozyrn 435 and was carried out at SS * C with stirring at 1400 rpm After 48 hours of reaction the tube was removed from the thermo-stirrer and analyzed by HPLC samples at the initial and final time of the reaction, to quantify the disappearance of vainiylinamine (Figure 6). 9% was found at the conversion of vainiylinamine laurate to fauri! valni !! inarnida,

Example 1 § Enamatic synthesis of lauri! Vainiilinarrt¾a. Variant 2

 The procedure was similar to example 5 unless! solvent

2M2B per THF and a conversion of 97% was obtained. Example 1? Enzymatic synthesis of ia laurylvainiinamide. Variant 3

 Procedure 16 was carried out in a similar way to example 16, except that the bíoeaíaí & ador Movozym 43S was replaced by üpozyme JL-M, obtaining a conversion of

30%

Ijampfo 18 Enzymatic synthesis of laurylvainiinamide. Vary 4

The procedure was similar to Example 15 except that the solvent 21 ^ 28 was replaced by THF and the bioamptator by Upozyme -i, obtaining a conversion of 83%

Example 10 Synthesis in mathematics of ia lauñivainilinamkia. Variant 5

Example 16 was proceeded in a similar manner except that the 2M2B solvent was replaced by acetone, the Hovozym 435 iocatalyst by UpoEyme T14U and the reaction was carried out at 35 ^* C. After 48h of this reaction, a conversion of the 12%

Example 30 Synthesis of Vainiinamine Stearate. (Huta of synthesis A1)

3.4 § (11.96 mmoies) of stearic acid was dissolved in 50 ml of a methano-terbutanol mixture (50:50) in the 600 ml hydrogen bottle, achieving a clear and transparent solution, to which 2 g were added { 11.96 nimoies) of vainiinoxsma and 200 mg of FW. The system was purged 3 times with nitrogen and 3 times with hydrogen after which the system was brought to 60 psi of hydrogen and maintained with stirring for approximately 2 hours, until complete consumption of hydrogen, at which time the reaction was completed.

The reaction mixture was concentrated to a paste consistency in a rotary evaporator under reduced pressure. To this concentrate 10 ml of ethyl acetate were added and it was concentrated again, adding another 10 ml of ethyl acetate. The mixture was cooled to 5 ^¾ C, held at this temperature for 2 hours then vacuum filtering. The solids obtained were washed with 5 mL of ethyl acetate and dried at room temperature to constant weight. From this reaction, 3.8 g of valniiiinamine stearate were obtained.

SJernol © 21 Thematic synthesis of le steariivamilinamJda, Variant 1

With 0.1 M of vanillinamine esiarate obtained in Example 20, an enzymatic reaction was carried out in a 1.5 ml eppendorf tube, in a thermal stirrer (Thermomixer compact, eppendorf®} in order to stir the system ¥ keep the temperature controlled. The reaction contained 1 mt of tert-amyloleohoi (2WB) _f 10 mg of Novoxvm 43S and was carried out at S5 * C with stirring at 1400 rpm After 48 hours of reaction the tube was removed from the thermo-stirrer and the initial and final time of the reaction was analyzed by HPLC to quantify the disappearance of yainiinamlrsa, under these conditions a 74% conversion was obtained.

Example 22 Ensmátíoa synthesis of steariivalniliinamide. Variant 2

The procedure was similar to Example 21 except that the 2M2B solvent was replaced by acetone, obtaining a conversion of 62%,

Example 23 Aromatic synthesis of steanívaíniinamide. Variant 3

The procedure was similar to Example 21, except that the 2M28 solvent was replaced by tratrahydrofuran (THF), obtaining a conversion of 53%

Example 24 Ensimatic synthesis of astea rUvainlllinami fa. Variant 4

The procedure was similar to Example 21 except that solvent 21 2B was replaced with THF and the reaction was carried out with the Lipozyme R 4M biocataiizer. A conversion of 79% was obtained.

EJesw fe 2S Enzymatic synthesis of steariivvalniirsamide. S variant

The procedure was similar to example 21 except that the biocatalyst No sym 435 was replaced by Liposyme Ύ14, obtaining a conversion da! 31%

Example 28. Enzymatic synthesis of capnivalniliinamide from vainiliinarnine caprylate, without the removal of inorganic salt (KCI).

Synthesis of vainifcamsna caprilato. In a vial of 16 ml 0.2 bouquet was dissolved! Potassium hydroxy 10 ml of methane !, achieving a clear and transparent solution. The solution temperature reached §8 * C. To this solution was added 0.2 mmoi of vanillin amine hydrochloride and stirred vigorously for 30 m. Subsequently, 0.2 mmol of octanoioo acid was added. In the reaction vigorous stirring was maintained for 1 hr, sufficient time for the reaction to reach an ambient temperature {~ 28 * C). Subsequently, it was «vaporized methane! to dryness and a white solid (~ Ü.6 mg) corresponding to a mixture of KCI and vanillinnar capriinate was obtained.

Emimáfica condensation of caprilvainilinarnida. The white solid obtained in the previous step was resuspended in 10 ml of 2 ^» methyl ^« 2 ^» butanoi _! In addition, a heterogeneous mixture was obtained (the organic sai was dissolved leaving the KCI as undissolved solids), Λ said solution was added n 200 mg of Novozym 43S bio-analyzer and the mixture was taken to a hydro-kador (Thermo Sctentífic® Shake 'n Síack) with which the stirring was controlled and the temperature was maintained at 5Q. After 48 hours of reaction, the vial was removed from nibTidizadof and the samples corresponding to the initial and final time were analyzed by HPLC. HPLC analysis revealed the conversion of 85% caprilaio from vanillin mlna to caprüvainiilieamlda. This example demonstrates that the presence of inorganic sai (KCI) does not impede the activity of the blocatalteador,

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iohrs M. Janusz, Bñm L Buckwalíer, Patricia A. Young, Themas. laHann, Ral h W. Fa ser, Geraid B. Kasting, fterice E, Lo mans, Gary A. ercfcaert Che S. Madáin, Eíiza eth P, Sermam, Richard L Bohne, Tfto as L Cu s, Judlt R. Mslsteirv; Vaniiloid i, Anaíogs of Capsaícín with Antinocieeptive and AníBtofiammatory Aclivíty. Jmsm & í of Medici al C is y 38 (1SS3) 2595-2604.

Claims

1 A method for roduction tfuimieo ^ risi alkie de vainállinaroldas, characterized by the steps of:  s a) Contacting an inaccurate vanilla salt with an enzyme will belong to the EC 3.1.1.3 enzymatic classification and, optionally, an organic solvent or an unconventional medium,  ) incubate the mixture at a temperature suitable for the functioning of the enzyme, allowing the condensation of the organic salt m unaδ vainitnamide,  o) Qpcionalrnente the purification of the vaninyloarnide obtained, 2, The method of claim 1, characterized in that the organic vaniniine salt may be saturated, linear or branched organic acids of 6 15 to 22 carbon atoms. 3, The method of claim 1, characterized in that the concentration of the organic vanillin amine salt in step a) is between 0.01 M and 2 & t. The method of claim 1, characterized in that the organic solvent of step a} is selected from the group consisting of third-party alcohols, pyrldine, dimelsulfoxide, dioxane, triethiamine, di-di-opropiiamine, benzene, cyclohexane or, acetone, aceíonitriío, teíra íldrofurano, rvnexaho, ivheptano, and mixtures thereof.  25  5. The method of claim 1, characterized in that the unconventional means of step a) is € 0 ?. supercritical or an ionic liquid. 6. The method of claim 1, characterized in that the solvent of step a) is anhydrous at the start of the reaction. 7. The method of claim 1, characterized in that the enzyme from step a) is a triacylgiiceroi lipase selected from the group qm consists of: Candida antarcUce lipase A, Candida aníamiiea lipase 8, Candida oyHntíracea lipase. 35 rugged Candida lipase, coated Burktmidem lipase, Pseuá & monas ipase a & mginosa, Pseudomonas sp lipase sp „Pseutomonas ftuo $ c & r® lipase _> Rhizomucor mteh & i lipase, Ustilago maya lipase 521 and Yarroma lipofylioB lipase, regardless of whether it is soluble, iophilic or immobilized, 8. The method of claim 1, characterized in that the enzyme of! step a} is between 2 and 50 mg / ml. or gives 2 LU / role at 50 LU / mL 8. The method of claim 1, characterized in that the reaction of step t>) is carried out at a temperature between 15 ° C and 85 X. 10, The method of claim 1, characterized in that the purification of step c) is carried out by extraction with alcohols or ethyl acetate, by biphasic extraction with mixtures of immiscible solvents followed by solvent vaporization, or by c preparative HPIC omatography or bten by Flash chromatography. 11 The method of claim 1, characterized in that prior to step a), an organic salt synthesis step consisting of vanithynamine and an organic acid is optionally carried out, 12, The method of claim 11 _< characterized in that the step of synthesis of the organic salt formed by vainiinamine and an organic acid is achieved from the current vaininoxime current and an organic acid, by the following steps:  MaJ Contact the valniinneximma and an organic acid in some organic solvent or the mixture of two or more solvents, with a molecular hydrogen atmosphere in the presence of aigórs transition metal as catalyst,  A1b Incubate the piss at a suitable temperature allowing the reaction to proceed until obtaining the organic salt of Valnilifinamine,  Á1e) The recovery of the transition metal used as a catalyst and, A1dJ Optionally, the purification of the organic vainiiinamine salt obtained. 13. The method of claim 12, characterized in that the solvent of the step Ala) is an alcohol or mixture of alcohols, each selected from the group consisting of: linear alcohols of 1 to β carbon atoms and branched alcohols of 4 to β carbon atoms. 14. The method of claim 12, characterized in that the organic acid of step Al a) is selected from the group consisting of: organic acids of 8 to 22 carbon atoms, whether saturated or iosaturated, linear or branched, 15. The method of claim 12, characterized in that the concentration of the vayniinosdma and the organic acid in step Al a} ₍ is between 0.01 and 2. 18. The method of claim 12, characterized in that in step Ai a) the molar ratio vairtilfínoxíma: organic acid is between 1: 2 and 2: 1. one?. The method of claim 12, characterized in that the molecular hydrogen from step A a) is at a pressure between 1 and 10 atmospheres, 18. The method of claim 12, characterized in that e! catalyst employed in the Ala step) is a transition metal selected from the group consisting of; Pd / C, Pt Rh, or Raney Ni. 19. The method of claim 12, characterized in that the reaction of step Al) is carried at a temperature between 0 ^* C and 70 ^S C. 20. The method of claim 19, characterized by a macaon of weight A1b ) is preferably carried out at a temperature between 25 "C and 45 ^* C. 21, The method of claim 12, characterized in that the recovery of the transition metal is carried out by filtration, decantation or centrifugation. 22. The conformity method according to claim 12, characterized in that the optional purification step is carried out by evaporation of the organic solvent or solvent mixture to obtain the organic salt of vainiSinamine solid, followed by washing of the salt with some solvent in which it is not soluble, then recover the salt by decantation, centrifugation or filtration, after drying the organic sai of valniliinamine. 23. The method of claim 11, characterized in that the step of synthesis of the organic sai formed by valniiiinamine and an organic acid is achieved from the release of the corresponding inorganic salt of valnílisnamlna with a base and the subsequent addition of an acid organic, by the following steps:  § A2a) Contact the vanillin amine inorganic sai with a base in an organic solvent, the mixture gives two or more solvents,  A2b) incubate the mixture allowing the vanillin amine release reaction to proceed  A2c) Add im organic acid 0 Alé) Incubate the mixture allowing the formation reaction of the organic salt of vainiiStnamina to pass,  A2e G donalmenie the separation of the organic sai from vainiinamira obtained,  A2f) Optionally the purification of the organic vanillin amine salt5 obtained. 24. B method of claim 23, characterized in that the dissolvent of step A2a) is an alcolioi or mixture of alcohols, each selected from the group consisting of: linear alcohols of 1 to 6 carbon atoms and branched alcohols 0 of 4 to 6 volumes of carfconus, 25. The method of claim 23, characterized in that the concentration of the inorganic vanillin amine sai and at the base of step A2a), is between 0.01 and 2M, 5 26. The method of claim 23, characterized in that in the molar ratio Inorganic salt of vanifisnamine: base of step A2a), is between 1: 2 and 2: 1.  2?. The method of claim 23, characterized in that the base used in the Ata step) is selected from the group consisting of alkaline bases, alkaline earth bases and carbonates of any of the elements of group 1 of the periodic table0, 28. The method of claim 23, characterized in that the reaction of step A2b) is carried out at a temperature between 0 and? Q ^and C. 5 2S. The method of claim 28, characterized in that the reaction of the step A2b) is preferably carried out at a temperature between 2 ° C and 45 X, 30. The method of claim 23, characterized in that the organic acid from step A2c) is selected from the group consisting of: organic acids from 6 to 22 S carbon atoms, whether saturated or unsaturated, linear or branched. 31. The method of claim 23, characterized in that in step A2c) the organic acid is added to a molar ratio of the inorganic sai of inactive vanillin; organic acid, between 1: 2 and 2: 1, 0 32. The method of claim 23, characterized in that the concentration of the inorganic salt of vanillin amine and of the organic acid in step A2c1 is between 0.01 and 2M. The method of claim 23, characterized in that the reaction of step A2d) is carried out at a temperature between 20 ^S C and 80 X. 34. Method of claim 33, characterized in that the reaction of step A2d) is preferably carried out at a temperature between 35 and 80 X, 0  35. The method of claim 23, characterized in that the separation of the organic salt of vanillin amine is carried out by decantation, filtration or centrifugation, leaving "sai token in solution." The method according to claim 35, characterized in that the purification step is carried out by evaporation of the organic solvent or solvent mixture to obtain the organic solid vanillin amine salt, followed by washing the salt with some solvent. in which it is not soluble, then recover the salt by decantation, centrifugation or filtration, followed by drying of the organic vanillin amine salt.