WO2019243656A1 - Method for enzymatic synthesis of monoesters of polyhydroxylated compounds - Google Patents

Method for enzymatic synthesis of monoesters of polyhydroxylated compounds Download PDF

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WO2019243656A1
WO2019243656A1 PCT/ES2019/070493 ES2019070493W WO2019243656A1 WO 2019243656 A1 WO2019243656 A1 WO 2019243656A1 ES 2019070493 W ES2019070493 W ES 2019070493W WO 2019243656 A1 WO2019243656 A1 WO 2019243656A1
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acid
synthesis
carried out
formula
synthesis process
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PCT/ES2019/070493
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Spanish (es)
French (fr)
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Pedro LOZANO RODRÍGUEZ
Elena ÁLVAREZ GONZÁLEZ
Juana María BERNAL PALAZÓN
Susana NIETO CERÓN
Celia GÓMEZ GARCÍA
Antonio DONAIRE GONZÁLEZ
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Universidad De Murcia
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C235/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C31/00—Saturated compounds having hydroxy or O-metal groups bound to acyclic carbon atoms
    • C07C31/18—Polyhydroxylic acyclic alcohols

Definitions

  • the present invention relates to a method of enzymatic synthesis of monoesters of polyhydroxy compounds, the monoster product being able to be isolated and obtained with high yield, without mixtures of other possible asters such as, for example, bi-esters or tri-esters.
  • the process is carried out in means free of organic solvents.
  • esters of polyhydroxylated compounds are bioactive products widely used in the cosmetic and pharmaceutical industries, with panthenyl triacetate being the most commonly used product.
  • the esters are currently obtained by chemical or enzymatic catalysis from derivatives of the carboxylic acids (eg acyl chlorides, vinyl esters, acetic anhydride, etc.), in reaction media based on organic solvents.
  • panthenyl triacetate is synthesized chemically, by means of, for example, a mixture of dimethyl aminopyridine and anhydrous acetic acid as a catalyst, and using temperatures of up to 110 ° C, and successive stages of purification, which implies a cost in its manufacture as described in the patent with publication number US6982346.
  • the acylation reaction of panthenol by acetic anhydride is catalyzed by strong acids, such as p-toluenesulfonyl as described in the patent with application number CN201611254844.
  • these classic organic synthesis processes are not very selective, since they do not allow obtaining mono- or diester derivatives, because the extent of the acylation reaction occurs without control.
  • soluble acids or bases as catalysts, it can generate the appearance of unwanted products, given the exothermic nature of the reactions, and the poor selectivity of the catalysts. For this reason, a purification stage that increases production costs is necessary, in addition to generating residual products that negatively affect the sustainability and / or “green” character of production processes, as contemplated by the principles of Green Chemistry
  • Enzymatic catalysis is the most selective synthesis strategy for the synthesis of chemical products, being one of the pillars in the development of Green Chemistry and / or Sustainable Chemistry.
  • panthenol monoesters by transesterification reactions has been carried out using various acyl esters as acyl donor substrate, e.g. ex. methyl acrylate, methyl methylacrylate, etc., and panthenol, as described in patent application W02008053051.
  • acyl donor substrate e.g. ex. methyl acrylate, methyl methylacrylate, etc.
  • panthenol as described in patent application W02008053051.
  • the presence of two primary hydroxyl groups in panthenol determines the uncontrolled obtaining of panthenol diesters as a result of two consecutive catalytic processes on the same panthenol molecule.
  • carboxylic acid derivatives as acyl donors in transesterification reactions (eg vinyl esters, isopropenyl esters, alkyl esters, anhydrides, etc.), does not only increase the cost of processes against the use of fatty acids. free, but also a considerable loss of sustainability of the processes, since said compounds are necessary to obtain them by chemical synthesis from the free carboxylic acids, which results in a consumption of reagents and solvents.
  • reaction media containing organic solvents generally aprotic (eg acetonitrile, tetrahydrofuran, acetone, etc.) in order to allow the dissolution of both substrates, panthenol and acyl donor esters, and thus be able to obtain a single-phase liquid system, where chemical reactions can be verified.
  • organic solvents generally aprotic (eg acetonitrile, tetrahydrofuran, acetone, etc.) in order to allow the dissolution of both substrates, panthenol and acyl donor esters, and thus be able to obtain a single-phase liquid system, where chemical reactions can be verified.
  • the present invention describes the process of selective enzymatic synthesis of monoesters of polyhydroxy compounds, said synthesis being verified by direct esterification between free carboxylic acids and the polyhydroxy compound, and without the use of any type of derivatization and / or chemical functionalization in both molecules / substrates, and without using any volatile organic solvent in the process.
  • the present invention relates to a method of enzymatic synthesis of monoesters of polyhydroxy compounds, comprising the steps of:
  • a) Form a monophasic eutectic mixture by mixing and melting by heating, in the presence of water of a carboxylic acid of formula Ri-COOH where R1 is an aliphatic radical of 10 to 25 carbon atoms, which can be saturated or unsaturated with a polyhydroxy compound of the formula R 2 -OH or R 3 CONHR 4 -OH where R 2 is a three to 20 carbon aliphatic radical containing at least two free hydroxyl groups and may be substituted in any of the C of the aliphatic radical with a linear or branched (Ci-C3) alkyl;
  • Ri-COOH where R1 is an aliphatic radical of 10 to 25 carbon atoms, which can be saturated or unsaturated with a polyhydroxy compound of the formula R 2 -OH or R 3 CONHR 4 -OH where R 2 is a three to 20 carbon aliphatic radical containing at least two free hydroxyl groups and may be substituted in any of the C of the aliphatic radical with a linear or
  • R 3 CONHR4-OH contain at least two free hydroxyl groups and where R 3 and R 4 can be the same or different and are an aliphatic radical of three to 10 carbons that can be substituted in any of the C of the aliphatic radical with a linear or branched (C-C 3 ) alkyl;
  • step b) add a hydrolytic enzyme (EC3.1) to the mixture of step a) at a temperature between 30 ° C and 100 ° C for the formation of the corresponding monoster and remove the H2O as formed in the reaction.
  • a hydrolytic enzyme EC3.1
  • both substrates are mixed, with heating in order to produce the fusion of all solid substrates.
  • the mixing is carried out with continuous stirring, more preferably mechanical, or magnetic stirring so that a liquid and single phase eutectic mixture is generated.
  • Said liquid mixture generated after melting is free of all types of volatile organic solvent, and is presented as a single homogeneous, colorless and transparent liquid phase, presenting a certain gel character, and which is stable at temperatures between 20 ° C and 90 ° C for periods of time longer than one week.
  • R1 has between 2 and 18 carbon atoms, more preferably between 10 and 18 carbon atoms. Even more preferably, it is selected from: decanoic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid or linolenic acid, among others, used without any modification and / or chemical derivatization on its carboxylic functional group.
  • the polyhydroxy compound of formula R2-OH or R 3 CONHR4OH is understood to cover substances that have at least two esterifiable hydroxyl groups independently of any carboxylic group that may be in the same molecule.
  • the polyhydroxy compound of formula R2-OH or R 3 CONHR4 has three, four, five or six free hydroxyl groups.
  • R2 is an aliphatic radical of four to 10 carbons containing four, five or six free hydroxyl groups.
  • the polyhydroxy compound of formula R 2 -OH or R 3 CONHR 4 -OH is selected from panthenol, xylitol and sorbitol.
  • R 3 CONHR 4 -OH is panthenol.
  • R 2 -OH is selected from xylitol and sorbitol.
  • hydrolytic enzymes are selected from esterases (EC3.1.1.), Lipases (EC3.1.1.3) and proteases (EC3.4.) They may be either free or immobilized on solid supports (eg Lewatit, polystyrene, etc).
  • esterases EC3.1.1.
  • Lipases EC3.1.1.3
  • proteases EC3.4.
  • Some particularly representative examples are: Antarctic Candida lipase (CALA), Antarctic Candida lipase (CALB), Alkalygenes sp. Lipases, Aspergillus sp.
  • Mucor sp. Geotricum sp., Ricopus sp., Burkholderia sp., Candida sp., Candida cylinderracea
  • Thermomices lanuginous, Mucor mihei, Rizomucor mihei, Pseudomonas fluorescens, Mucor javanicus, or lipasas of pig panceras, both in their free form , as fixed on solid supports (eg Lewatit, polystyrene, etc.), some sold under the brand Novozyme® 868, Novozyme® 525L, Novozyme® 435, Novozyme® 388, Lypozyme® RM, Novozyme® 871 and Lypozyme® TL .
  • lipase is Candida Antarctica.
  • the polyhydroxy compound and the carboxylic acid are in any molar ratio, it being preferable to use a molar ratio between the carboxylic acid and the polyhydroxy compound of 1: 1 (mol: mol), it being especially favorable to employ a molar ratio between the carboxylic acid and the 1: 2 polyhydroxy compound (mol: mol).
  • the amount of water in step a) is comprised between 0.1 and 20% (weight / weight) with respect to the content by weight of the polyhydroxy compound, with an amount of water between 0.1% and 10% (weight / weight) of the hydroxylated compound, and especially recommended an amount of water equivalent to 0.5% (weight / weight).
  • step a) is carried out at a temperature between 60 ° C and 200 ° C.
  • step a) is carried out within a reactor with agitation and capacity to perform vacuum.
  • the corresponding amounts of both substrates, carboxylic acid and hydroxylated compound in the desired molar ratio, as well as the amount of water required, will be mixed in the reactor.
  • the system will close tightly and a vacuum pressure of -1 bar will be applied, and it will be kept under constant agitation (orbital, mechanically or magnetically) at a temperature between 60 ° C and 200 ° C, a temperature between 70 being preferable ° C and 150 ° C, a temperature of 100 ° C being especially favorable.
  • Step a) is preferably performed for a time between 30 min and 24 h, a time between 1 and 2 hours being preferable.
  • step a) may be heated with microwave irradiation at a power between 300-900 watts.
  • microwave heating at a power of 600 watts for 1-2 min is preferable.
  • Step b) is preferably carried out at a temperature between 40 ° C and 80 ° C, and more favorably at 75 ° C for at least one hour.
  • step b) since in the esterification reaction catalyzed by the enzyme water is released as a byproduct thereof, preferably in step b) the removal of water is carried out by hygroscopic molecular sieves, which act as desiccants, in order to ensure that the process is carried out under anhydrous conditions, and thus favor the synthesis reaction by improving the yields of the product obtained.
  • an amount of agent will be added desiccant and / or molecular sieve between 5 and 50% (weight / weight) with respect to the weight of the reaction eutectic mixture, with an amount of desiccant between 10 and 30% being preferable, and particularly favorable the addition of 15% (weight / weight) of drying agent, with respect to the total mass of the substrates.
  • step b) where the amount of eutectic substrate mix is present, together with the desiccant agent and the immobilized enzyme, must be maintained under mechanical or magnetic stirring (100-500 rpm), for a time between 1 h and 72 h, a time between 6 h and 24 h being preferred.
  • Example 1 Example of enzymatic synthesis of panthenyl monodecanoate in the absence of solvent
  • Example 2 Example of attic synthesis of panthenyl monolaurate in the absence of solvent
  • Example 3 Example of enzymatic synthesis of panthenyl monooleate in the absence of solvent
  • Example 4 Example of enzymatic synthesis of panthenyl monopalmitate in the absence of solvent
  • Example 5 Example of enzymatic synthesis of panthenyl monodecanoate in the absence of solvent
  • panthenyl monodecanoate is determined by obtaining the following results:
  • Example 6 Example of enzymatic synthesis of panthenyl monomiristate in the absence of solvent
  • Example 7 Example of enzymatic synthesis of panthenyl monooleate in the absence of solvent
  • Example 8 Example of enzymatic synthesis of xylitol monolaurate in the absence of solvent
  • the mixture is filtered through a 0.45 micron membrane to separate the solid fraction (enzyme and molecular sieves), and the resulting liquid fraction is analyzed by HPLC-DAD.
  • the retention times of the substrates and products are: Xylitol: 2.1 min; lauric acid: 10.6 min; xylitol monolaurate: 6.5 min; Xylitol dilaurate: 19.6 min
  • the yield and selectivity of the xylitol monolaurate synthesis process is determined by obtaining the following results:
  • Sorbitol 2.4 min; lauric acid: 10.6 min; sorbitol monolaurate: 7.0: sorbitol dilaurate: 20.2 min

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Abstract

The present invention relates to a method for the enzymatic synthesis of monoesters of polyhydroxylated compounds, it being possible to isolate and obtain a product with high yield, without mixtures of other possible esters such as biesters or triesters. The method is carried out in mediums free of organic solvents.

Description

PROCEDIMIENTO DE SÍNTESIS ENZIMÁTICA DE MONOÉSTERES DE COMPUESTOS POLIHIDROXILADOS  PROCESS OF ENZYMATIC SYNTHESIS OF MONOESTERS OF POLYHYDROXYLED COMPOUNDS
DESCRIPCIÓN DESCRIPTION
La presente invención se refiere a un procedimiento de síntesis enzimática de monoésteres de compuestos polihidroxilados, pudiéndose aislar y obtener con alto rendimiento el producto monoester, sin mezclas de otros posibles ásteres como por ejemplo biésteres o triésteres. El procedimiento se lleva a cabo en medios libres de disolventes orgánicos. The present invention relates to a method of enzymatic synthesis of monoesters of polyhydroxy compounds, the monoster product being able to be isolated and obtained with high yield, without mixtures of other possible asters such as, for example, bi-esters or tri-esters. The process is carried out in means free of organic solvents.
Antecedentes de la invención Background of the invention
Los ásteres de compuestos polihidroxilados (i.e. pantenol,) son productos bioactivos de aplicación muy extendida en las industrias cosmética y farmacéutica, siendo el triacetato de pantenilo el producto más comúnmente utilizado. Los ásteres se obtienen actualmente por catálisis química o enzimática a partir de derivados de los ácidos carboxílicos (p. ej. cloruros de acilo, ásteres de vinilo, anhídrido acético, etc.), en medios de reacción basados en disolventes orgánicos. The esters of polyhydroxylated compounds (i.e. panthenol,) are bioactive products widely used in the cosmetic and pharmaceutical industries, with panthenyl triacetate being the most commonly used product. The esters are currently obtained by chemical or enzymatic catalysis from derivatives of the carboxylic acids (eg acyl chlorides, vinyl esters, acetic anhydride, etc.), in reaction media based on organic solvents.
Por ejemplo el triacetato de pantenilo se sintetiza por vía química, mediante, por ejemplo, una mezcla de dimetil aminopiridina y ácido acético anhidro como catalizador, y empleando temperaturas de hasta 110°C, y sucesivas etapas de purificación, lo que supone un coste en su manufactura como se describe en la patente con número de publicación US6982346. For example, panthenyl triacetate is synthesized chemically, by means of, for example, a mixture of dimethyl aminopyridine and anhydrous acetic acid as a catalyst, and using temperatures of up to 110 ° C, and successive stages of purification, which implies a cost in its manufacture as described in the patent with publication number US6982346.
En otros casos, la reacción de acilación del pantenol por el anhídrido acético está catalizada por ácidos fuertes, como el p-toluenosulfonilo como se describe en la patente con número de solicitud CN201611254844. En cualquier caso, estos procesos clásicos de síntesis orgánica son poco selectivos, ya que no permiten la obtención de los derivados mono- o diésteres, debido a que la extensión de la reacción de acilación ocurre sin control. Además, como consecuencia del empleo de ácidos o bases solubles como catalizadores, puede generar la aparición de productos indeseados, habida cuenta del carácter exotérmico de las reacciones, y la poca selectividad de los catalizadores. Es por ello, por lo que es necesaria una etapa de purificación que incrementa los costes de producción, además de generar productos residuales que afectan negativamente a la sostenibilidad y/o carácter “verde” de los procesos de producción, tal como contemplan los principios de la Química Verde. In other cases, the acylation reaction of panthenol by acetic anhydride is catalyzed by strong acids, such as p-toluenesulfonyl as described in the patent with application number CN201611254844. In any case, these classic organic synthesis processes are not very selective, since they do not allow obtaining mono- or diester derivatives, because the extent of the acylation reaction occurs without control. In addition, as a consequence of the use of soluble acids or bases as catalysts, it can generate the appearance of unwanted products, given the exothermic nature of the reactions, and the poor selectivity of the catalysts. For this reason, a purification stage that increases production costs is necessary, in addition to generating residual products that negatively affect the sustainability and / or “green” character of production processes, as contemplated by the principles of Green Chemistry
La catálisis enzimática es la estrategia de síntesis más selectiva para la síntesis de productos químicos, siendo uno de los pilares en el desarrollo de la Química Verde y/o Química Sostenible. Enzymatic catalysis is the most selective synthesis strategy for the synthesis of chemical products, being one of the pillars in the development of Green Chemistry and / or Sustainable Chemistry.
La síntesis enzimática de monoésteres de pantenol mediante reacciones de transesterificación ha sido realizada utilizando como sustrato donador de acilo a diversos ásteres metílicos, p. ej. acrilato de metilo, metilacrilato de metilo, etc., y el pantenol, como se describe en la solicitud de patente W02008053051. Además, la presencia de dos grupos hidroxilo primarios en el pantenol determina la obtención incontrolada de diésteres de pantenol como consecuencia de dos procesos catalíticos consecutivos sobre la misma molécula de pantenol. El empleo de derivados de ácidos carboxílicos como donadores de acilo en las reacciones de transesterificación (p. ej. ásteres vinílicos, ásteres isopropenílicos, ásteres alquílicos, anhídridos, etc.), no solo supone un encarecimiento de los procesos frente al empleo de ácidos grasos libres, sino que también una pérdida considerable de sostenibilidad de los procesos, dado que dichos compuestos es necesario obtenerlos por síntesis química a partir de los ácidos carboxílicos libres, lo que redunda en un consumo de reactivos y disolventes. The enzymatic synthesis of panthenol monoesters by transesterification reactions has been carried out using various acyl esters as acyl donor substrate, e.g. ex. methyl acrylate, methyl methylacrylate, etc., and panthenol, as described in patent application W02008053051. In addition, the presence of two primary hydroxyl groups in panthenol determines the uncontrolled obtaining of panthenol diesters as a result of two consecutive catalytic processes on the same panthenol molecule. The use of carboxylic acid derivatives as acyl donors in transesterification reactions (eg vinyl esters, isopropenyl esters, alkyl esters, anhydrides, etc.), does not only increase the cost of processes against the use of fatty acids. free, but also a considerable loss of sustainability of the processes, since said compounds are necessary to obtain them by chemical synthesis from the free carboxylic acids, which results in a consumption of reagents and solvents.
En todos los casos descritos, la síntesis enzimática de monoésteres de compuestos hidroxilados mediante reacciones de transesterificación, se ha realizado en medios de reacción que contenían disolventes orgánicos, generalmente apróticos (p.ej. acetonitrilo, tetrahidrofurano, acetona, etc.) con el fin de permitir la disolución de ambos sustratos, pantenol y los ásteres donadores de acilo, y así poder obtener un sistema líquido monofásico, donde poder verificarse las reacciones químicas. In all the cases described, the enzymatic synthesis of monoesters of hydroxylated compounds by transesterification reactions, has been carried out in reaction media containing organic solvents, generally aprotic (eg acetonitrile, tetrahydrofuran, acetone, etc.) in order to allow the dissolution of both substrates, panthenol and acyl donor esters, and thus be able to obtain a single-phase liquid system, where chemical reactions can be verified.
Ambos sustratos, pantenol y ásteres de ácidos carboxílicos, presentan propiedades solventes claramente contrapuestas, ya que el carácter hidrofílico e higroscópico del pantenol contrasta con el carácter hidrofóbico e insoluble en agua de los ásteres de los donadores de acilo utilizados. Estas características determinan que sea necesario emplear disolventes orgánicos de polaridad intermedia para conseguir la solubilización de los reactantes. Both substrates, panthenol and esters of carboxylic acids, have clearly contrasted solvent properties, since the hydrophilic and hygroscopic character Panthenol contrasts with the hydrophobic and water-insoluble character of the esters of the acyl donors used. These characteristics determine that it is necessary to use organic solvents of intermediate polarity to achieve solubilization of the reactants.
Además, tras la reacción catalítica, es necesario eliminar toda traza de disolvente orgánico empleado, así como de los productos secundarios de la reacción (p.ej. metanol, acetaldehído, etc.), para la recuperación y utilización del correspondiente monoéster de pantenilo para la preparación de cualquier producto de consumo. In addition, after the catalytic reaction, it is necessary to remove any trace of the organic solvent used, as well as the secondary products of the reaction (eg methanol, acetaldehyde, etc.), for the recovery and use of the corresponding panthenyl monoester for the preparation of any consumer product.
Por todo lo dicho sería muy interesante poder desarrollar un procedimiento de síntesis de monoésteres de compuestos hidroxilados mediante la esterificación directa entre un ácido carboxílico libre y un alcohol sin disolventes orgánicos y en presencia de agua y sin ser necesario el empleo de sustratos derivatizados/funcionalizados, con objeto de permitir la preparación de medios de reacción monofásicos/homogéneos, así como la mejora en los rendimientos de los monoésteres obtenidos. For all the above, it would be very interesting to be able to develop a process of synthesis of monoesters of hydroxylated compounds by direct esterification between a free carboxylic acid and an alcohol without organic solvents and in the presence of water and without the use of derivatized / functionalized substrates, in order to allow the preparation of monophasic / homogeneous reaction media, as well as the improvement in the yields of the obtained monoesters.
Descripción de la invención Description of the invention
La presente invención describe el procedimiento de síntesis enzimática selectiva de monoésteres de compuestos polihidroxilados, verificándose dicha síntesis mediante la esterificación directa entre los ácidos carboxílicos libres y el compuesto polihidroxilado, y sin el empleo de ningún tipo de derivatización y/o funcionalización química en ambas moléculas / sustratos, y sin emplear ningún tipo de disolvente orgánico volátil en el proceso. The present invention describes the process of selective enzymatic synthesis of monoesters of polyhydroxy compounds, said synthesis being verified by direct esterification between free carboxylic acids and the polyhydroxy compound, and without the use of any type of derivatization and / or chemical functionalization in both molecules / substrates, and without using any volatile organic solvent in the process.
La presente invención se refiere a un procedimiento de síntesis enzimática de monoésteres de compuestos polihidroxilados, que comprende las etapas de: The present invention relates to a method of enzymatic synthesis of monoesters of polyhydroxy compounds, comprising the steps of:
a) Formar una mezcla eutéctica monofásica mediante la mezcla y fusión por calentamiento, en presencia de agua de un ácido carboxílico de fórmula Ri-COOH donde R1 es un radical alifático de 10 a 25 átomos de carbono, el cual puede ser saturado o insaturado con un compuesto polihidroxilado de fórmula R2-OH o R3CONHR4-OH donde R2 es un radical alifático de tres a 20 carbonos que contiene al menos dos grupos hidroxilos libres y puede estar sustituido en alguno de los C del radical alifático con un alquil(Ci-C3) lineal o ramificado; a) Form a monophasic eutectic mixture by mixing and melting by heating, in the presence of water of a carboxylic acid of formula Ri-COOH where R1 is an aliphatic radical of 10 to 25 carbon atoms, which can be saturated or unsaturated with a polyhydroxy compound of the formula R 2 -OH or R 3 CONHR 4 -OH where R 2 is a three to 20 carbon aliphatic radical containing at least two free hydroxyl groups and may be substituted in any of the C of the aliphatic radical with a linear or branched (Ci-C3) alkyl;
y donde R3CONHR4-OH contienen al menos dos grupos hidroxilos libres y donde R3 y R4 pueden ser iguales o diferentes y son un radical alifático de tres a 10 carbonos que puede estar sustituido en alguno de los C del radical alifático con un alquil(Ci- C3) lineal o ramificado; and where R 3 CONHR4-OH contain at least two free hydroxyl groups and where R 3 and R 4 can be the same or different and are an aliphatic radical of three to 10 carbons that can be substituted in any of the C of the aliphatic radical with a linear or branched (C-C 3 ) alkyl;
b) adicionar una enzima hidrolítica (EC3.1) a la mezcla de la etapa a) a una temperatura comprendida entre 30°C y 100°C para la formación del monoester correspondiente y retirar el H2O según se forma en la reacción. b) add a hydrolytic enzyme (EC3.1) to the mixture of step a) at a temperature between 30 ° C and 100 ° C for the formation of the corresponding monoster and remove the H2O as formed in the reaction.
En la presente invención en la etapa a) se mezclan ambos sustratos, con calentamiento con el fin de producir la fusión de todos los sustratos sólidos. Preferentemente la mezcla se realiza con agitación continua, más preferentemente agitación mecánica, o magnética de manera que se genera una mezcla eutéctica líquida y monofásica. Dicha mezcla líquida generada tras la fusión está libre de todo tipo de disolvente orgánico volátil, y se presenta como una única fase líquida homogénea, incolora y transparente, presentando un cierto carácter de gel, y que resulta estable a temperaturas comprendidas entre 20°C y 90 °C durante periodos de tiempo superiores a una semana. In the present invention in step a) both substrates are mixed, with heating in order to produce the fusion of all solid substrates. Preferably the mixing is carried out with continuous stirring, more preferably mechanical, or magnetic stirring so that a liquid and single phase eutectic mixture is generated. Said liquid mixture generated after melting is free of all types of volatile organic solvent, and is presented as a single homogeneous, colorless and transparent liquid phase, presenting a certain gel character, and which is stable at temperatures between 20 ° C and 90 ° C for periods of time longer than one week.
En la presente invención, como sustratos donadores de acilo se emplean ácido grasos libres, de fórmula R1-COOH. Preferentemente R1 tiene entre 2 y 18 átomos de carbono, más preferentemente entre 10 y 18 átomos de carbono. Aun más preferentemente se selecciona entre: el ácido decanoico, ácido láurico, ácido mirístico, ácido palmítico, ácido palmitoleico, ácido esteárico, ácido oleico, ácido linoleico o ácido linolénico, entre otros, utilizados sin ningún tipo de modificación y/o derivatización química sobre su grupo funcional carboxílico. In the present invention, free fatty acids of the formula R1-COOH are used as acyl donor substrates. Preferably R1 has between 2 and 18 carbon atoms, more preferably between 10 and 18 carbon atoms. Even more preferably, it is selected from: decanoic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid or linolenic acid, among others, used without any modification and / or chemical derivatization on its carboxylic functional group.
En la presente invención de manera preferente el compuesto polihidroxilado de fórmula R2-OH o R3CONHR4OH se entiende que cubre substancias que tienen al menos dos grupos hidroxilo esterificables independientemente de cualquier grupo carboxílico que pueda estar en la misma molécula. Preferentemente el compuesto polihidroxilado de fórmula R2-OH o R3CONHR4 tiene tres, cuatro, cinco o seis grupos hidroxilos libres. Preferentemente R2 es un radical alifático de cuatro a 10 carbonos que contiene cuatro, cinco o seis grupos hidroxilos libres. In the present invention, preferably the polyhydroxy compound of formula R2-OH or R 3 CONHR4OH is understood to cover substances that have at least two esterifiable hydroxyl groups independently of any carboxylic group that may be in the same molecule. Preferably the polyhydroxy compound of formula R2-OH or R 3 CONHR4 has three, four, five or six free hydroxyl groups. Preferably R2 is an aliphatic radical of four to 10 carbons containing four, five or six free hydroxyl groups.
Más preferentemente el compuesto polihidroxilado de fórmula R2-OH o R3CONHR4- OH se selecciona entre pantenol, xilitol y sorbitol. De manera particular R3CONHR4-OH es pantenol. El pantenol, tanto en su enantiomero D, como la mezcla racémica D,L, siendo utilizado sin ningún tipo de modificación química sobre sus grupos funcionales hidroxilo. De manera particular R2-OH se selecciona entre xilitol y sorbitol. More preferably the polyhydroxy compound of formula R 2 -OH or R 3 CONHR 4 -OH is selected from panthenol, xylitol and sorbitol. In particular, R 3 CONHR 4 -OH is panthenol. Panthenol, both in its enantiomer D, and the racemic mixture D, L, being used without any chemical modification on its hydroxyl functional groups. In particular, R 2 -OH is selected from xylitol and sorbitol.
En la presente invención, de manera preferente las enzimas hidrolíticas se seleccionan entre las esterasas (EC3.1.1.), las lipasas (EC3.1.1.3) y las proteasas (EC3.4.) Pueden estar bien en forma libre o inmovilizadas sobre soportes sólidos (p.ej. Lewatit, poliestireno, etc). Para la presente invención, algunos ejemplos particularmente representativos son: lipasa A de Candida antárctica (CALA), lipasa B de Candida antárctica (CALB), lipasas de Alkalygenes sp., Aspergillus sp. Mucor sp., Geotricum sp., Ricopus sp., Burkholderia sp., Candida sp., Candida cilindracea, Thermomices lanuginous, Mucor mihei, Rizomucor mihei, Pseudomonas fluorescens, Mucor javanicus, o lipasas de panceras de porcino, tanto en su forma libre, como inmovilizadas sobre soportes sólidos (p.ej. Lewatit, poliestireno, etc), algunas comercializadas bajo la marca Novozyme® 868, Novozyme® 525L, Novozyme® 435, Novozyme® 388, Lypozyme® RM, Novozyme® 871 y Lypozyme® TL. De manera particular la lipasa es Candida antárctica. In the present invention, preferably hydrolytic enzymes are selected from esterases (EC3.1.1.), Lipases (EC3.1.1.3) and proteases (EC3.4.) They may be either free or immobilized on solid supports (eg Lewatit, polystyrene, etc). For the present invention, some particularly representative examples are: Antarctic Candida lipase (CALA), Antarctic Candida lipase (CALB), Alkalygenes sp. Lipases, Aspergillus sp. Mucor sp., Geotricum sp., Ricopus sp., Burkholderia sp., Candida sp., Candida cylinderracea, Thermomices lanuginous, Mucor mihei, Rizomucor mihei, Pseudomonas fluorescens, Mucor javanicus, or lipasas of pig panceras, both in their free form , as fixed on solid supports (eg Lewatit, polystyrene, etc.), some sold under the brand Novozyme® 868, Novozyme® 525L, Novozyme® 435, Novozyme® 388, Lypozyme® RM, Novozyme® 871 and Lypozyme® TL . In particular, lipase is Candida Antarctica.
En la presente invención es especialmente deseable para utilizar la lipasa B de Candida antárctica (CALB) libre (Novozym® 525 L), o la lipasa B de Candida antárctica inmovilizada por adsorción sobre Lewatit WO n° 89/02916 (Novozym® 435). In the present invention it is especially desirable to use the free Candida Antarctic Lipase (CALB) (Novozym® 525 L), or the Candida B Antarctic Lipase immobilized by adsorption on Lewatit WO No. 89/02916 (Novozym® 435).
El compuesto polihidroxilado y el ácido carboxílico están en cualquier relación molar, siendo preferible emplear una relación molar entre el ácido carboxílico y el compuesto polihidroxilado de 1 :1 (mol:mol), siendo especialmente favorable emplear una relación molar entre el ácido carboxílico y el compuesto polihidroxilado de 1 :2 (mol:mol). Preferentemente la cantidad de agua en la etapa a) está comprendida entre el 0.1 y el 20% (peso/peso) respecto al contenido en peso del compuesto polihidroxilado siendo preferible una cantidad de agua comprendida entre el 0.1 % y el 10% (peso/peso) del compuesto hidroxilado, y especialmente recomendable una cantidad de agua equivalente al 0.5% (peso/peso). The polyhydroxy compound and the carboxylic acid are in any molar ratio, it being preferable to use a molar ratio between the carboxylic acid and the polyhydroxy compound of 1: 1 (mol: mol), it being especially favorable to employ a molar ratio between the carboxylic acid and the 1: 2 polyhydroxy compound (mol: mol). Preferably the amount of water in step a) is comprised between 0.1 and 20% (weight / weight) with respect to the content by weight of the polyhydroxy compound, with an amount of water between 0.1% and 10% (weight / weight) of the hydroxylated compound, and especially recommended an amount of water equivalent to 0.5% (weight / weight).
Preferentemente la etapa a) se lleva a cabo a una temperatura comprendida entre 60°C y 200°C. Preferentemente la etapa a) se realiza dentro de un reactor con agitación y capacidad para realizar vacío. En el reactor se mezclarán las cantidades correspondientes de ambos sustratos, ácido carboxílico y compuesto hidroxilado en la relación molar deseada, así como la cantidad de agua necesaria. El sistema se cerrará herméticamente y se le aplicará una presión de vacío de -1 bar, y se mantendrá en agitación (orbital, mecánica o magnéticamente) constante a una temperatura comprendida entre 60 °C y 200°C, siendo preferible una temperatura entre 70 °C y 150 °C, siendo especialmente favorable una temperatura de 100 °C. La etapa a) se realiza preferentemente durante un tiempo comprendido entre 30 min y 24 h, siendo preferible un tiempo comprendido entre 1 y 2 horas. Preferably step a) is carried out at a temperature between 60 ° C and 200 ° C. Preferably step a) is carried out within a reactor with agitation and capacity to perform vacuum. The corresponding amounts of both substrates, carboxylic acid and hydroxylated compound in the desired molar ratio, as well as the amount of water required, will be mixed in the reactor. The system will close tightly and a vacuum pressure of -1 bar will be applied, and it will be kept under constant agitation (orbital, mechanically or magnetically) at a temperature between 60 ° C and 200 ° C, a temperature between 70 being preferable ° C and 150 ° C, a temperature of 100 ° C being especially favorable. Step a) is preferably performed for a time between 30 min and 24 h, a time between 1 and 2 hours being preferable.
Preferentemente, la etapa a) podrá calentarse con irradiación microondas a una potencia comprendida entre 300-900 wat. Preferentemente durante un tiempo comprendido entre 1-30 min, siendo preferible un calentamiento con microondas a una potencia de 600 wat durante 1-2 min. Preferably, step a) may be heated with microwave irradiation at a power between 300-900 watts. Preferably for a time between 1-30 min, microwave heating at a power of 600 watts for 1-2 min is preferable.
La etapa b) se realiza de manera preferible una temperatura comprendida entre 40°C y 80 °C, y más favorablemente a 75 °C durante al menos una hora. Step b) is preferably carried out at a temperature between 40 ° C and 80 ° C, and more favorably at 75 ° C for at least one hour.
En la presente invención, y dado que en la reacción de esterificación catalizada por la enzima se libera agua como subproducto de la misma, preferentemente en la etapa b) la retirada del agua se realiza mediante tamices moleculares higroscópicos, que actúan a modo de desecantes, con el fin de asegurar que el proceso se desarrolle en condiciones anhidras, y así favorecer la reacción de síntesis mejorando los rendimientos de producto obtenido. In the present invention, and since in the esterification reaction catalyzed by the enzyme water is released as a byproduct thereof, preferably in step b) the removal of water is carried out by hygroscopic molecular sieves, which act as desiccants, in order to ensure that the process is carried out under anhydrous conditions, and thus favor the synthesis reaction by improving the yields of the product obtained.
De manera preferente en la etapa b) se adicionará una cantidad de agente desecante y/o tamiz molecular comprendida entre el 5 y el 50% (peso/peso) respecto al peso de la mezcla eutéctica de reacción, siendo preferible una cantidad de agente desecante comprendida entre el 10 y el 30%, y particularmente favorable la adición de un 15% (peso/peso) de agente desecante, respecto a la masa total de los sustratos. Preferably in step b) an amount of agent will be added desiccant and / or molecular sieve between 5 and 50% (weight / weight) with respect to the weight of the reaction eutectic mixture, with an amount of desiccant between 10 and 30% being preferable, and particularly favorable the addition of 15% (weight / weight) of drying agent, with respect to the total mass of the substrates.
Preferentemente la etapa b) donde está presente la cantidad de mezcla eutéctica de sustratos, junto al agente desecante y la enzima inmovilizada, debe ser mantenida en agitación mecánica o magnética (100-500 rpm), durante un tiempo comprendido entre 1 h y 72 h, siendo preferido un tiempo comprendido entre 6 h y 24 h. Preferably step b) where the amount of eutectic substrate mix is present, together with the desiccant agent and the immobilized enzyme, must be maintained under mechanical or magnetic stirring (100-500 rpm), for a time between 1 h and 72 h, a time between 6 h and 24 h being preferred.
Ejemplos Examples
EJEMPLO DE REALIZACIÓN EXAMPLE OF REALIZATION
Los siguientes ejemplos ilustran, pero no limitan, la presente invención. The following examples illustrate, but do not limit, the present invention.
Ejemplo 1. Ejemplo de síntesis enzimática de monodecanoato de pantenilo en ausencia de solvente Example 1. Example of enzymatic synthesis of panthenyl monodecanoate in the absence of solvent
En tubo eppendorf® de 2 ml_ de capacidad, se adicionan 0.2 gramos de ácido decanoico, 0.5 g de pantenol y 0.05 gramos de agua, la mezcla resultante se calienta a 70°C hasta total fundición de los mismos. Posteriormente, se adicionan 0.08 gramos de agente desecante, molecular sieves MS 13-X (Sigma) y a mezcla se acondiciona a una temperatura de 45 °C durante 30 minutos. In an eppendorf® tube of 2 ml_ capacity, 0.2 grams of decanoic acid, 0.5 g of panthenol and 0.05 grams of water are added, the resulting mixture is heated at 70 ° C until they are completely melted. Subsequently, 0.08 grams of desiccant, molecular sieves MS 13-X (Sigma) are added and the mixture is conditioned at a temperature of 45 ° C for 30 minutes.
Finalmente se adicionan 0.08 gramos de biocatalizador, Novozyme® 435 dando inicio a la reacción de síntesis, manteniendo el vial de reacción a 45 °C aplicando pulsos de ultrasonidos de 45 segundos, con una amplitud de 70. Transcurrida una hora de reacción, la mezcla se filtra a través de una membrana de 0.45 mieras para separar la fracción sólida, y la fracción líquida resultante se analiza por CG-FID. El rendimiento y la Selectividad del proceso de síntesis de Monodecanoato de pantenilo se determina, obteniéndose los siguientes resultados: Rendimiento (%): 69.90 Finally, 0.08 grams of biocatalyst, Novozyme® 435 are added, starting the synthesis reaction, keeping the reaction vial at 45 ° C applying ultrasound pulses of 45 seconds, with an amplitude of 70. After one hour of reaction, the mixture it is filtered through a 0.45 micron membrane to separate the solid fraction, and the resulting liquid fraction is analyzed by CG-FID. The yield and selectivity of the synthesis process of panthenyl monodecanoate is determined, obtaining the following results: Yield (%): 69.90
Selectividad (%): 92.40 Selectivity (%): 92.40
Ejemplo 2. Ejemplo de síntesis enzi ática de monolaurato de pantenilo en ausencia de solvente Example 2. Example of attic synthesis of panthenyl monolaurate in the absence of solvent
En un matraz de 100 ml_ se adicionan 34 gramos de Pantenol, 17 gramos de ácido láurico y 0.2 gramos de agua, la mezcla resultante se calienta a 75°C hasta total fundición de los mismos. Posteriormente, se adicionan 3.5 g de agente desecante molecular sieves MS 13-X (Sigma) y a mezcla se acondiciona a una temperatura de 60°C durante 3 h. Finalmente se adicionan 3.5 g de biocatalizador, Novozyme® 435 dando inicio a la reacción de síntesis, manteniendo el matraz en agitación constante de 250 rpm y a una temperatura de 60°C. Transcurridas seis horas de reacción, la mezcla se filtra a través de una membrana de 0.45 mieras para separar la fracción sólida, y la fracción líquida resultante se analiza por CG-FID. El rendimiento y la selectividad del proceso de síntesis de monolaurato de pantenilo se determina, obteniéndose los resultados siguientes: In a 100 ml flask, 34 grams of Panthenol, 17 grams of lauric acid and 0.2 grams of water are added, the resulting mixture is heated at 75 ° C until they are completely melted. Subsequently, 3.5 g of molecular drying agent MS 13-X (Sigma) are added and the mixture is conditioned at a temperature of 60 ° C for 3 h. Finally, 3.5 g of biocatalyst, Novozyme® 435 are added, starting the synthesis reaction, keeping the flask under constant agitation of 250 rpm and at a temperature of 60 ° C. After six hours of reaction, the mixture is filtered through a 0.45 micron membrane to separate the solid fraction, and the resulting liquid fraction is analyzed by CG-FID. The yield and selectivity of the synthesis process of panthenyl monolaurate is determined, obtaining the following results:
Rendimiento (%): 80.93 Yield (%): 80.93
Selectividad (%): 96.99 Selectivity (%): 96.99
Ejemplo 3. Ejemplo de síntesis enzimática de monooleato de pantenilo en ausencia de solvente Example 3. Example of enzymatic synthesis of panthenyl monooleate in the absence of solvent
En un vial de 4 ml_ se adicionan 0.4 gramos de Pantenol, 117 mI_ de ácido oleico y 0.02 gramos de agua, la mezcla resultante se mantiene bajo irradiación microondas a 600 wat durante 2 minutos. In a 4 ml vial, 0.4 grams of Panthenol, 117 ml of oleic acid and 0.02 grams of water are added, the resulting mixture is kept under microwave irradiation at 600 watts for 2 minutes.
Posteriormente, se adicionan 0.6 g de agente desecante molecular sieves MS 13-X (Sigma) y a mezcla se acondiciona a una temperatura de 60°C durante 15 minutos. Finalmente se adicionan 0.60 g de biocatalizador, Novozyme® 435 dando inicio a la reacción de síntesis, manteniendo el vial a temperatura de 60°C pulsos de ultrasonidos de 45 segundos de duración. Transcurridas 2 horas de reacción, la mezcla se filtra a través de una membrana de 0.45 mieras para separar la fracción sólida, y la fracción líquida resultante se analiza por CG-FID. El rendimiento y la selectividad del proceso de síntesis de monooleato de pantenilo se determina obteniéndose los resultados siguientes: Subsequently, 0.6 g of molecular drying agent MS 13-X (Sigma) is added and the mixture is conditioned at a temperature of 60 ° C for 15 minutes. Finally, 0.60 g of biocatalyst, Novozyme® 435 are added, starting the synthesis reaction, keeping the vial at a temperature of 60 ° C with ultrasound pulses of 45 seconds duration. After 2 hours of reaction, the mixture is filtered through a 0.45 micron membrane to separate the fraction solid, and the resulting liquid fraction is analyzed by CG-FID. The yield and selectivity of the synthesis process of panthenyl monooleate is determined by obtaining the following results:
Rendimiento (%): 87.41 Yield (%): 87.41
Selectividad (%): 99.49 Selectivity (%): 99.49
Ejemplo 4. Ejemplo de síntesis enzimática de monopalmitato de pantenilo en ausencia de solvente Example 4. Example of enzymatic synthesis of panthenyl monopalmitate in the absence of solvent
En un vial de 4 ml_ se adicionan 0.4 gramos de Pantenol, 0.170 gramos de ácido palmítico y 0.02 gramos de agua. La mezcla resultante se mantiene bajo irradiación microondas a 600 wat durante 2 minutos. Posteriormente, se adicionan 0.6 gramos de agente desencante MS-13X (Sigma) y se acondiciona a una temperatura de 60 °C durante 30 minutos. Finalmente se adicionan 0.02 gramos de biocatalizador, Novozyme® 435 dando inicio a la reacción de síntesis, manteniendo el matraz en agitación constante de 250 rpm y a una temperatura de 60 °C. Transcurridas seis horas de reacción, la mezcla se filtra a través de una membrana de 0.45 mieras para separar la fracción sólida, y la fracción líquida resultante se analiza por CG- FID. El rendimiento y la Selectividad del proceso de síntesis de monopalmitato de pantenilo se determina obteniéndose los resultados siguientes: In a 4 ml vial, 0.4 grams of Panthenol, 0.170 grams of palmitic acid and 0.02 grams of water are added. The resulting mixture is kept under microwave irradiation at 600 watts for 2 minutes. Subsequently, 0.6 grams of MS-13X (Sigma) desiccant agent are added and conditioned at a temperature of 60 ° C for 30 minutes. Finally, 0.02 grams of biocatalyst, Novozyme® 435 are added, starting the synthesis reaction, keeping the flask under constant agitation of 250 rpm and at a temperature of 60 ° C. After six hours of reaction, the mixture is filtered through a 0.45 micron membrane to separate the solid fraction, and the resulting liquid fraction is analyzed by CG-FID. The yield and selectivity of the synthesis process of panthenyl monopalmitate is determined by obtaining the following results:
Rendimiento (%): 85.93 Yield (%): 85.93
Selectividad (%): 99.00 Selectivity (%): 99.00
Ejemplo 5. Ejemplo de síntesis enzimática de monodecanoato de pantenilo en ausencia de solvente Example 5. Example of enzymatic synthesis of panthenyl monodecanoate in the absence of solvent
En un matraz de 50 mL se adicionan 11.30 gramos de ácido decanoico, 13.50 gramos de Pantenol y 0.1 gramos de agua, la mezcla resultante se calienta a 70°C hasta total fundición de los mismos. Posteriormente se adicionan 2.30 gramos de agente desecante molecular sieves MS 13-X (Sigma) y a mezcla se acondiciona a una temperatura de 60 °C durante 2 h. Finalmente se adicionan 2.6 gramos de biocatalizador, Novozyme® 435 dando inicio a la reacción de síntesis. La reacción tiene lugar en un reactor cerrado herméticamente, con una presión de - 1 bar y se mantiene en agitación constante de 250 rpm y 60 °C. Transcurridas seis horas de reacción, la mezcla se filtra a través de una membrana de 0.45 mieras para separar la fracción sólida, y la fracción líquida resultante se analiza por CG-FID. El rendimiento y la selectividad del proceso de síntesis de monodecanoato de pantenilo se determina obteniéndose los siguientes resultados: In a 50 mL flask 1130 grams of decanoic acid, 13.50 grams of Panthenol and 0.1 grams of water are added, the resulting mixture is heated at 70 ° C until they are completely melted. Subsequently, 2.30 grams of molecular desiccant are added, MS 13-X (Sigma) and the mixture is conditioned at a temperature of 60 ° C for 2 h. Finally 2.6 grams of biocatalyst, Novozyme® 435 are added, starting the synthesis reaction. The reaction It takes place in a tightly sealed reactor, with a pressure of - 1 bar and is kept under constant agitation of 250 rpm and 60 ° C. After six hours of reaction, the mixture is filtered through a 0.45 micron membrane to separate the solid fraction, and the resulting liquid fraction is analyzed by CG-FID. The yield and selectivity of the synthesis process of panthenyl monodecanoate is determined by obtaining the following results:
Rendimiento (%): 71.82 Yield (%): 71.82
Selectividad (%): 96.10 Selectivity (%): 96.10
Ejemplo 6. Ejemplo de síntesis enzimática de monomiristato de pantenilo en ausencia de solvente Example 6. Example of enzymatic synthesis of panthenyl monomiristate in the absence of solvent
En un vial de 4 ml_ se adicionan 0.42 gramos de Pantenol, 0.23 gramos de ácido mirístico y 0.01 gramos de agua. La mezcla resultante se calienta a 70°C hasta total fundición de los mismos. Posteriormente se adicionan 0.50 gramos de agente desecante (molecular sieves MS 13-X, Sigma) y a mezcla se acondiciona a una temperatura de 60 °C durante 30 minutos. Finalmente se adicionan 0.50 gramos de biocatalizador, Novozyme® 435 dando inicio a la reacción de síntesis. La reacción tiene lugar en un reactor cerrado herméticamente, con una presión de - 1 bar y se mantiene en agitación constante de 250 rpm y 60 °C. Transcurridas seis horas de reacción, la mezcla se filtra a través de una membrana de 0.45 mieras para separar la fracción sólida, y la fracción líquida resultante se analiza por CG- FID. El rendimiento y la selectividad del proceso de síntesis de monomiristato de pantenilo se determina obteniéndose los siguientes resultados: In a 4 ml vial, 0.42 grams of Panthenol, 0.23 grams of myristic acid and 0.01 grams of water are added. The resulting mixture is heated at 70 ° C until they are completely melted. Subsequently, 0.50 grams of drying agent (molecular sieves MS 13-X, Sigma) are added and the mixture is conditioned at a temperature of 60 ° C for 30 minutes. Finally, 0.50 grams of biocatalyst, Novozyme® 435 are added, starting the synthesis reaction. The reaction takes place in a tightly sealed reactor, with a pressure of - 1 bar and is kept under constant stirring of 250 rpm and 60 ° C. After six hours of reaction, the mixture is filtered through a 0.45 micron membrane to separate the solid fraction, and the resulting liquid fraction is analyzed by CG-FID. The yield and selectivity of the synthesis process of panthenyl monomiristate is determined by obtaining the following results:
Rendimiento (%): 82.27 Yield (%): 82.27
Selectividad (%): 93.90 Selectivity (%): 93.90
Ejemplo 7. Ejemplo de síntesis enzimática de monooleato de pantenilo en ausencia de solvente Example 7. Example of enzymatic synthesis of panthenyl monooleate in the absence of solvent
En un vial de 4 mL se adicionan 0.42 gramos de Pantenol, 0.71 mL de ácido oleico y 0.02 gramos de agua. La mezcla resultante se funde irradiando microondas a 400 wat, hasta total fundición de los mismos. A continuación se adicionan 0.80 gramos de agente desecante molecular sieves MS 13-X (Sigma) y la mezcla se acondiciona a 60 °C y 250 rpm durante 30 minutos. Finalmente, se adicionan 0.80 gramos de biocatalizador, Novozyme® 435 dando inicio a la reacción de síntesis. La reacción tiene lugar, en un reactor a temperatura constante de 60 °C y 250 rpm. Transcurridas seis horas de reacción, la mezcla se filtra a través de una membrana de 0.45 mieras para separar la fracción sólida, y la fracción líquida resultante se analiza por HPLC-DAD. El rendimiento y la selectividad del proceso de síntesis de monooleato de pantenilo se determina, obteniéndose los siguientes resultados: In a 4 mL vial, 0.42 grams of Panthenol, 0.71 mL of oleic acid and 0.02 grams of water are added. The resulting mixture is melted by irradiating microwave at 400 wat, until they are completely cast. Next, 0.80 grams of molecular desiccant are added to MS 13-X (Sigma) and the mixture is conditioned at 60 ° C and 250 rpm for 30 minutes. Finally, 0.80 grams of biocatalyst, Novozyme® 435 are added, starting the synthesis reaction. The reaction takes place in a reactor at a constant temperature of 60 ° C and 250 rpm. After six hours of reaction, the mixture is filtered through a 0.45 micron membrane to separate the solid fraction, and the resulting liquid fraction is analyzed by HPLC-DAD. The yield and selectivity of the synthesis process of panthenyl monooleate is determined, obtaining the following results:
Rendimiento (%): 60.43 Yield (%): 60.43
Selectividad (%): 99.22 Selectivity (%): 99.22
Ejemplo 8. Ejemplo de síntesis enzimática de monolaurato de xilitol en ausencia de solvente Example 8. Example of enzymatic synthesis of xylitol monolaurate in the absence of solvent
En un matraz de 25 ml_ se adicionan 3 gramos de xilitol, 8 gramos de ácido láurico y 0.06 gramos de agua. La mezcla sólida resultante se calienta a 85 °C hasta total fundición de los sustratos. Posteriormente, se adicionan 0.6 g de agente desecante molecular sieves MS 13-X (Sigma) y a mezcla se acondiciona a una temperatura de 60 °C durante 12 h. Finalmente se adicionan 0.6 g de biocatalizador, Novozyme® 435 dando inicio a la reacción de síntesis, manteniendo el matraz en agitación constante de 250 rpm y a una temperatura de 60 °C. Transcurridas doce horas de reacción, la mezcla se filtra a través de una membrana de 0.45 mieras para separar la fracción sólida (enzima y molecular sieves), y la fracción líquida resultante se analiza por HPLC-DAD. Los tiempos de retención de los sustratos y productos son: Xilitol:2.1 min; ácido láurico: 10.6 min; monolaurato de xilitol: 6.5 min; dilaurato de xilitol: 19.6 min In a 25 ml flask, 3 grams of xylitol, 8 grams of lauric acid and 0.06 grams of water are added. The resulting solid mixture is heated at 85 ° C until the substrates are completely melted. Subsequently, 0.6 g of molecular drying agent MS 13-X (Sigma) is added and the mixture is conditioned at a temperature of 60 ° C for 12 h. Finally, 0.6 g of biocatalyst, Novozyme® 435 are added, starting the synthesis reaction, keeping the flask under constant agitation of 250 rpm and at a temperature of 60 ° C. After twelve hours of reaction, the mixture is filtered through a 0.45 micron membrane to separate the solid fraction (enzyme and molecular sieves), and the resulting liquid fraction is analyzed by HPLC-DAD. The retention times of the substrates and products are: Xylitol: 2.1 min; lauric acid: 10.6 min; xylitol monolaurate: 6.5 min; Xylitol dilaurate: 19.6 min
El rendimiento y la selectividad del proceso de síntesis de monolaurato de xilitol se determina obteniéndose los resultados siguientes:  The yield and selectivity of the xylitol monolaurate synthesis process is determined by obtaining the following results:
Rendimiento (%): 90 % Yield (%): 90%
Selectividad (%): 60 % Ejemplo 9. Ejemplo de síntesis enzimática de monolauarato de sorbitol en ausencia de solvente Selectivity (%): 60% Example 9. Example of enzymatic synthesis of sorbitol monolauarate in the absence of solvent
En un matraz de 25 ml_ se adicionan 3.7 gramos de sorbitol, 8 gramos de ácido láurico y 0.11 gramos de agua. La mezcla sólida resultante se calienta a 95 °C hasta total fundición de los sustratos. Posteriormente, se adicionan 0.74 g de agente desecante molecular sieves MS 13-X (Sigma) y a mezcla se acondiciona a una temperatura de 60 °C durante 12 h. Finalmente se adicionan 0.74 g de biocatalizador, Novozyme® 435 dando inicio a la reacción de síntesis, manteniendo el matraz en agitación constante de 250 rpm y a una temperatura de 60 °C.In a 25 ml flask, 3.7 grams of sorbitol, 8 grams of lauric acid and 0.11 grams of water are added. The resulting solid mixture is heated at 95 ° C until the substrates are completely melted. Subsequently, 0.74 g of molecular drying agent MS 13-X (Sigma) are added and the mixture is conditioned at a temperature of 60 ° C for 12 h. Finally, 0.74 g of biocatalyst, Novozyme® 435 are added, starting the synthesis reaction, keeping the flask under constant agitation of 250 rpm and at a temperature of 60 ° C.
Transcurridas doce horas de reacción, la mezcla se filtra a través de una membrana de 0.45 mieras para separar la fracción sólida (enzima y molecular sieves), y la fracción líquida resultante se analiza por HPLC-DAD. Los tiempos de retención de los sustratos y productos son: After twelve hours of reaction, the mixture is filtered through a 0.45 micron membrane to separate the solid fraction (enzyme and molecular sieves), and the resulting liquid fraction is analyzed by HPLC-DAD. The retention times of substrates and products are:
Sorbitol: 2.4 min ; ácido láurico: 10.6 min; monolaurato de sorbitol: 7.0: dilaurato de sorbitol: 20.2 min Sorbitol: 2.4 min; lauric acid: 10.6 min; sorbitol monolaurate: 7.0: sorbitol dilaurate: 20.2 min
El rendimiento y la selectividad del proceso de síntesis de monolaurato de sorbitol se determina obteniéndose los resultados siguientes: Rendimiento (%): 60.4  The yield and selectivity of the sorbitol monolaurate synthesis process is determined by obtaining the following results: Yield (%): 60.4
Selectividad (%): 61.0  Selectivity (%): 61.0

Claims

REIVINDICACIONES
1. Procedimiento de síntesis enzimática de monoésteres de compuestos polihidroxilados, caracterizado porque las etapas de: 1. Enzymatic synthesis process of monoesters of polyhydroxy compounds, characterized in that the steps of:
a) Formar una mezcla eutéctica monofásica mediante la mezcla y fusión por calentamiento, en presencia de agua de un ácido carboxílico de fórmula Ri-COOH donde R1 es un radical alifático de 10 a 25 átomos de carbono, el cual puede ser saturado o insaturado con un compuesto polihidroxilado de fórmula R2-OH o R3CONHR4-OH a) Form a monophasic eutectic mixture by mixing and melting by heating, in the presence of water of a carboxylic acid of the formula Ri-COOH where R 1 is an aliphatic radical of 10 to 25 carbon atoms, which can be saturated or unsaturated with a polyhydroxy compound of formula R 2 -OH or R 3 CONHR 4 -OH
donde R2 es un radical alifático de tres a 20 carbonos que contiene al menos dos grupos hidroxilos libres y puede estar sustituido en alguno de los C del radical alifático con un alquil(Ci-C3) lineal o ramificado; where R 2 is a three to 20 carbon aliphatic radical containing at least two free hydroxyl groups and may be substituted in any of the C of the aliphatic radical with a linear or branched (Ci-C 3 ) alkyl;
y donde R3CONHR4-OH contienen al menos dos grupos hidroxilos libres y donde R3 y R4 pueden ser iguales o diferentes y son un radical alifático de tres a 10 carbonos que puede estar sustituido en alguno de los C del radical alifático con un alquil(Ci- C3) lineal o ramificado; and where R 3 CONHR 4 -OH contain at least two free hydroxyl groups and where R 3 and R 4 can be the same or different and are an aliphatic radical of three to 10 carbons that can be substituted at any of the C of the aliphatic radical with a linear or branched (C-C 3 ) alkyl;
b) adicionar una enzima hidrolítica (EC3.1) a la mezcla de la etapa a) a una temperatura comprendida entre 30°C y 100°C para la formación del monoester correspondiente y retirar el H2O según se forma en la reacción. b) add a hydrolytic enzyme (EC3.1) to the mixture of step a) at a temperature between 30 ° C and 100 ° C for the formation of the corresponding monoster and remove the H 2 O as formed in the reaction.
2. Procedimiento de síntesis según la reivindicación 1 caracterizado porque la etapa a) se lleva a cabo a una temperatura comprendida entre 60°C y 200 °C. 2. Synthesis process according to claim 1 characterized in that step a) is carried out at a temperature between 60 ° C and 200 ° C.
3. Procedimiento de síntesis según una cualquiera de las reivindicaciones 1 a 2 caracterizada porque la enzima hidrolítica (EC3.1.) se selecciona entre: esterasas (EC3.1.1.), las lipasas (EC3.1.1.3.) y las proteasas (EC3.4.). 3. Synthesis method according to any one of claims 1 to 2 characterized in that the hydrolytic enzyme (EC3.1.) Is selected from: esterases (EC3.1.1.), Lipases (EC3.1.1.3.) And proteases (EC3.4.).
4. Procedimiento de síntesis según la reivindicación 3 caracterizado porque la lipasa es Candida antárctica. 4. Synthesis method according to claim 3 characterized in that the lipase is Candida Antarctica.
5. Procedimiento de síntesis según una cualquiera de las reivindicaciones 1-4 caracterizado porque el ácido carboxílico de fórmula R1-COOH se selecciona entre: ácido decanoico, ácido láurico, ácido mirístico, ácido palmítico, ácido palmitoleico, ácido esteárico, ácido oleico, ácido linoleico y ácido linolénico. 5. Synthesis process according to any one of claims 1-4 characterized in that the carboxylic acid of formula R 1 -COOH is selected from: decanoic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid and linolenic acid.
6. Procedimiento de síntesis según una cualquiera de las reivindicaciones 1-5 caracterizado porque el compuesto polihidroxilado de fórmula R2-OH se selecciona entre xilitol y sorbitol. 6. Synthesis process according to any one of claims 1-5 characterized in that the polyhydroxy compound of formula R2-OH is selected from xylitol and sorbitol.
7. Procedimiento de síntesis según una cualquiera de las reivindicaciones 1-5 caracterizado porque el compuesto polihidroxilado de fórmula R3CONHR4-OH es el pantenol. 7. Synthesis process according to any one of claims 1-5 characterized in that the polyhydroxy compound of formula R 3 CONHR 4 -OH is panthenol.
8. Procedimiento de síntesis según una cualquiera de las reivindicaciones 1-7 caracterizado porque la retirada del H2O según se forma en la etapa b) se realiza mediante tamices moleculares higroscópicos. 8. Synthesis method according to any one of claims 1-7 characterized in that the removal of H2O as formed in step b) is carried out by hygroscopic molecular sieves.
9. Procedimiento de síntesis según una cualquiera de las reivindicaciones 1-8 caracterizado porque la etapa a) se realiza en un reactor con agitación y capacidad para realizar vacío. 9. Synthesis process according to any one of claims 1-8 characterized in that step a) is carried out in a reactor with agitation and capacity to perform vacuum.
10. Procedimiento de síntesis según una cualquiera de las reivindicaciones 1-9 caracterizado porque la etapa a) se realiza durante un tiempo comprendido entre 1 hora y 2 horas. 10. Synthesis method according to any one of claims 1-9 characterized in that step a) is carried out for a time between 1 hour and 2 hours.
11. Procedimiento de síntesis según una cualquiera de las reivindicaciones 1-10 caracterizado porque en la etapa a) la fusión se realiza mediante irradiación microondas a una potencia comprendida entre 300-900 wat, durante un tiempo comprendido entre 1-30 min. 11. Synthesis method according to any one of claims 1-10 characterized in that in step a) the fusion is carried out by microwave irradiation at a power comprised between 300-900 watts, for a time between 1-30 min.
PCT/ES2019/070493 2018-06-19 2019-07-15 Method for enzymatic synthesis of monoesters of polyhydroxylated compounds WO2019243656A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008053051A2 (en) * 2006-12-15 2008-05-08 Haering Dietmar Panthenol esters of unsaturated carboxylic acids
CN103114108A (en) * 2012-09-03 2013-05-22 常州大学 Method for preparing D-panthenol 16 ether monopalmitate by using lipase as catalyst

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008053051A2 (en) * 2006-12-15 2008-05-08 Haering Dietmar Panthenol esters of unsaturated carboxylic acids
CN103114108A (en) * 2012-09-03 2013-05-22 常州大学 Method for preparing D-panthenol 16 ether monopalmitate by using lipase as catalyst

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
T DE DIEGO ET AL.: "Selective synthesis of panthenyl esters", BIOCATALYSIS AND BIOTRANSFORMATIONS, vol. 31, 2013, pages 175 - 180, XP055620462, DOI: 10.3109/10242422.2013.814644 *

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