EP1567655A2

EP1567655A2 - Enzymatic production of acyl flavonoid derivatives

Info

Publication number: EP1567655A2
Application number: EP03812154A
Authority: EP
Inventors: Mohamed Ghoul; Jean-Marc Engasser; Philippe Moussou; Gilles Pauly; Melika Ardhaoui; Aude Falcimaigne
Original assignee: Cognis France SAS
Current assignee: BASF Health and Care Products France SAS
Priority date: 2002-12-03
Filing date: 2003-11-22
Publication date: 2005-08-31
Also published as: KR20050085377A; EP1426445A1; JP2006508654A; WO2004050889A3; US20060115880A1; WO2004050889A2

Abstract

Disclosed is a method for the enzymatic synthesis of flavonoid esters and flavonoid derivatives, according to which a) a reaction medium containing an organic solvent, a glycosylated flavonoid or aglycone flavonoid, an acyl group donor, and an enzymatic catalyst is produced, b) additional quantities of flavonoid and/or acyl donor are optionally added during the reaction, and c) the obtained esters are purified by eliminating enzymatic particles and the solvent. The inventive method is characterized by the fact that the concentration of water and/or alcohol which are formed during the reaction is controlled so as to be maintained below 150 mM.

Description

Manufacture of flavonoid derivatives

Field of the Invention

The invention is in the field of phyto- and biochemistry and relates to a process for the enzymatic production of flavonoid derivatives which are used in the food sector, in cosmetics and in pharmaceutical preparations.

State of the art

The biological effects of flavonoids have been well known for many years. By trapping various oxidizing species, they prevent oxidative damage to biomolecules such as DNA, lipids and proteins. In antioxidant assays, some flavonoids are more effective than nitamines C and E. In addition to this main property, several other biological effects have been demonstrated, including inhibiting the action of enzymes and the proliferation of animal cells, viruses and bacteria. They also have an effect on the vascular system and a strong antioxidant capacity.

Because of their skin-protecting and cleansing properties and their effects against aging, against skin discoloration and on the appearance of the skin, flavonoids have also been used as components of cosmetic or dermopharmaceutical compositions. Furthermore, they affect the mechanical properties of the hair.

The anti-oxidant properties of the flavonoids depend on the molecular principle. Studies of the structure-activity relationship have shown that the antioxidant effect on ortho-hydroxylation on ring B of the molecule, the number of free hydroxyl groups, the presence of a double bond between carbon 2 and 3 in ring C and the presence of a hydroxyl group on carbon 3 (Figure I) is based.

(I)

In principle, the use of these molecules is limited on the one hand by a very low solubility in aqueous and organic media and on the other hand by a low stability. Flavonoids are broken down by light, oxygen or oxidizing agents and temperature increases. These restrictions prevent their effective use in food and cosmetic and pharmaceutical compositions.

There are several well-known strategies to address this stability problem: encapsulation or antioxidant formulation. However, none of these solutions is completely satisfactory and there is still a need for new glycosylated flavonoids and aglyconflavonoids with increased stability.

Enzymatic and chemical modifications have been proposed to improve the properties of these molecules. The patents JP55157580 and JP58131911 mention the acylation of quercetin with fatty acid chlorides in dioxane in the presence of pyridine. In these patents, however, acylation is carried out in the presence of toxic solvents. The substrate conversion rates are low. The acylation of flavone, flavonol and flavanone in the same way has been described in the Coletica patents FR 2778663 (US6235294). This reaction was carried out chemically in the presence of a fatty acid chloride or anhydride. These reactions require the use of activated fatty acids and toxic solvents (pyridine, chloroform and toluene). Furthermore, high temperatures (100 ° C) were used and the substrate conversion rates were low (about 10 to 60%). In addition, these reactions are not selective, which leads to polyacylated products. Patent WO 09966062 mentions the chemical acylation of flavonoids (quercetin, galangin, (+) - catechin) by fatty acids (lauric acid, butyric acid, acetic acid ...) and a subsequent enzymatic hydrolysis step by means of a Mucor miehei lipase. This invention has the same disadvantages as the patent FR2778663 (US6235294). After the first performed acylation reaction, the products formed are polyacylated. If monoesters are desired, an enzymatic hydrolysis is required in a second step, which is carried out after the removal of the solvents from the first reaction to avoid deactivation of the enzyme. Flavonoid modifications have also been described in patent EP0618203, which mentions the acylation of (+/-) catechin by ethyl acetate and ethyl propionate and the acylation of epigallocatechin by phenyl propionate and butyrate. An expensive enzyme (the carboxylase from Streptomyces rochei) is required to carry out this reaction, and the conversion rates of the two substrates are very low (less than 1% of the acyl donor). Finally, in the Henkel / Cognis patent WO 0179245, an enzymatic acylation of flavonoids (naringin, rutin, aspartin, orientin, quercetin, camphor oil, cis-orientin, isoquercitrin) is carried out using various acids (p-chlorophenylacetic acid, stearic acid, 12-hydroxystearic acid, palmitic acid, palmitic acid, Lauric acid, capric acid, 4-hydroxyphenylacetic acid, 5-phenylvaleric acid, cumaric acid, oleic acid, linoleic acid). This patent describes a process in which a high concentration of Candida antarctica (40 g / 1) and, based on the flavonoids, an excess of acyl donor is used. The conversion rate of the substrates is low (10 to 20%).

All of the work described above is characterized by low yields. In addition, toxic solvents are used in the chemical modifications, the reactions are non-specific, which leads to product mixtures, and the enzymatic reactions described are limited to glycosylated flavonoids. The low conversion yields mentioned in the prior art are based on the fact that the processes used are not adapted to reactions with poorly soluble substrates and are drastically impaired by the water formed during the reaction.

Description of the invention

The invention relates to a process for the enzymatic synthesis of flavonoid esters and derivatives, in which a) a reaction medium is prepared comprising an organic solvent, a glycosylated flavonoid or aglyconflavonoid, an acyl group donor and an enzymatic catalyst, b) optionally adding further amounts of flavonoid and / or acyl donor during the reaction and c) purifying the esters thus obtained by separating enzymatic particles and the solvent, characterized in that the concentration of water formed during the reaction and / or alcohol is controlled so that it is kept below 150 mM.

The present invention - a process for the selective acylation of glycosylated flavonoids and aglyconflavonoids - leads to the improvement of the flavonoid derivatives with regard to their stability and solubility in various preparations, while their antioxidative properties are retained or improved. Another particular advantage achieved by these modified flavonoids is that bifunctional molecules with higher biological effectiveness are formed.

Compared to the methods known from the prior art, this method can achieve a significant improvement in terms of the final concentrations of flavonoid esters, the conversion yields (both for the flavonoids and the acyl donor originally present) and in particular the productivity, while the complicated, elaborate purification operations after synthesis can be reduced.

This process uses enzyme technology under mild temperature and pressure conditions without the use of any dangerous solvents, the flavonoid esters being formed by direct esterification or transesterification according to the following reaction schemes:

Flavonoid + RCOOH → Flavonoid-OCOR + H ₂ O Flavonoid + RCOOR '→ Flavonoid-OCOR + ROH

where R 'represents a Cl-C4-alkyl group, preferably a Cl-C2-alkyl group.

This process is characterized in that the reaction medium is first dewatered is used to remove any water present before the start of the reaction, and the water or alcohol formed during the reaction is removed online. Water and / or alcohol are kept at concentrations which are suitable for the solvents and substrates used, preferably at concentrations below 150 mM, particularly preferably below 100 mM.

The process is suitable for a variety of aglycon flavonoids and glycosylated flavonoids, and the conversion yields achieved with this mild enzymatic process are above those obtained so far: in the range of 50 to 99%.

The enzymatic synthesis is carried out under milder conditions than the chemical syntheses, avoiding the use of toxic solvents such as pyridine, benzene and THF, high temperatures and the accumulation of by-products such as salts or flavonoid degradation products, which would require additional purification steps.

Compared to the known methods mentioned above, this method can achieve a significant improvement in terms of the final concentrations of flavonoid esters, the conversion yields (for both the flavonoids and the acyl donor originally present) and, in particular, the productivity, while the complicated, expensive purification operations after the Synthesis can be reduced.

A difference between the method according to the invention and the known methods of the prior art is the way in which the enzymatic reaction is carried out, which enables considerably higher yields, and the large number of different flavonoids that can be used (both glycosylated forms and aglycon forms) ,

The main aim of the invention is to reduce all of the above-mentioned disadvantages of existing acylation methods and to propose a method for the enzymatic synthesis of flavonoid esters which, compared to the known methods mentioned above, offers a significant improvement in terms of the final concentrations of flavonoid esters, the conversion yields (both for the flavonoids as well as the originally existing acyl donor) and in particular the productivity, while the complicated, expensive purification operations after the synthesis are reduced. For this purpose, the invention relates to a process for the enzymatic synthesis of flavonoid esters, which is characterized in that predetermined amounts of a flavonoid (glycosylated forms and aglycon forms) or flavonoid derivative, an acyl group donor, an organic solvent, in a reactor designed for the formation of a reaction medium it can be the acyl donor and introduces an enzymatic catalyst, under conditions in which the reaction medium can first be dried to a water concentration of less than 150 mM, preferably less than 100 mM and in which the concentration of water formed during the reaction and / or alcohol can be kept below a predetermined point of 150 mM, preferably 100 mM. This predetermined point is met by on-line removal of the water and / or alcohol formed by adsorption on molecular sieves, by distillation or by pervaporation. This reaction can be carried out in the batch process or also in the fed-batch process with one or more substrates. In the fed-batch process, the molar ratio of flavonoid to acyl donor can be kept constant during the reaction by means of a suitable substrate addition profile. It is thus possible to control how the composition of the reaction medium develops over time, and thus to direct the enzymatic reaction towards maximum production of mono- or multiacylated compounds and at the same time to limit interfering reactions. The flavonoid esters thus obtained are finally purified by separating at least enzymatic particles (for example by decanting, filtering or centrifuging) and the solvent (for example by evaporation, distillation or membrane filtration).

The reaction is carried out according to the invention in such a way that the inhibition or deactivation of the enzyme reaction which is observed in the presence of high concentrations of flavonoids, acyl donors or water accumulations is initially restricted. The substrates can be fed gradually in a controlled manner as the reaction proceeds, thereby avoiding reaching concentrations that would inhibit the enzyme reaction.

The reaction can be carried out so that the flavonoid: acyl donor molar ratio of 0.01 to 20, preferably from 0.02 to 10. By keeping the values for the molar ratio in the reaction medium in the ranges given above, it is possible to achieve either higher reaction rates or maximum proportions of monoacylated flavonoids. By controlling the type and quantity of the reagents added in the course of time, the molar ratio can be kept constant during the reaction or can be varied in a controlled manner, so that it runs through a defined variation profile over time, but is nevertheless in the above-mentioned range of values throughout the reaction , The synthesis reaction can be optimized by temporarily or continuously withdrawing at least one component of the reaction medium. The component (s) withdrawn may possibly be returned to the reactor after fractionation.

According to one embodiment of the invention, provision can be made, in the meantime or continuously, to withdraw the entire reaction medium and, after fractionation, to inject one or more constituents of the medium removed into the reactor.

The reaction vessel or the reactor used for carrying out the process according to the invention is preferably provided with devices for controlling the temperature, the water and / or alcohol content and the pressure, with devices for adding reagents and with devices for withdrawing products.

During the course of the synthesis reaction, the temperature is advantageously set to 20-100 ° C. and the partial pressure above the reaction medium is advantageously set to 10 mbar (10 ³ Pa) to 1000 mbar (10 ⁵ Pa), starting from one to a concentration of less than 150 mM , preferably below 100 mM set initial water content, the amount of water and / or alcohol is kept below 150 mM, preferably below 100 mM and the reaction medium is advantageously stirred gently.

To obtain preparations from high-purity flavonoid esters, additional final fractionation operations can be carried out, for example by separating the remaining flavonoids or fats by extraction with organic solvents or supercritical fluids, by distillation or molecular distillation, by precipitation or by crystallization. The aglycon flavonoid or glycosylated flavonoid or flavonoid derivative used in the invention can be any compound selected from the group consisting of chalcon, flavon, flavanol, anthocyanin and flavanone, flavonol, coumarin, isoflavones and xanthones.

The acyl donor compound is selected from known fatty acids or their methyl, ethyl, propyl or butyl esters. This fatty acid is preferably selected from that of a straight-chain or branched aliphatic acid, saturated, unsaturated or cyclic, having up to 22 carbon atoms, optionally substituted by one or more substituents, from that of hydroxyl, amino, mercapto, halogen and alkyl-SS-alkyl existing Grappe, for example palmitic acid, 16-hydroxyhexadecanoic acid, 12-hydroxystearic acid, 11-mercaptoundecanoic acid, thioctanoic acid or quinic acid, straight-chain or branched aliphatic diacids, saturated or unsaturated, with up to 22 carbon atoms, for example hexadecanedioic acid or azelaic acid, and an aryl aliphatic acid and an aryl aliphatic acid dimeric acid, a cinnamic acid, optionally substituted by one or more substituents selected from the group consisting of hydroxyl, nitro, alkyl, alkoxy and halogen atoms, for example caffeic acid (3,4-dihydroxycinnamic acid), ferulic acid (4-hydroxy-3-meth oxycinnamic acid) or cumaric acid (4-hydroxycinnamic acid), a benzoic acid, optionally substituted by one or more substituents selected from the group consisting of hydroxyl, nitro, alkyl, alkoxy and halogen atoms, for example gallic acid (3,4,5-trihydroxybenzoic acid), vanillic acid (4- Hydroxy-3-methoxybenzoic acid) or protocatechic acid (3,4-dihydroxybenzoic acid) formed group selected. The fatty acid esters are preferably selected from the methyl or ethyl esters of the above compounds.

The reaction can be carried out with the acyl donor as solvent or in a suitable solvent, which can be any organic compound or any mixture of organic compounds in which the selected flavonoids or flavonoid derivatives and acyl donors are wholly or partly solubilized , The solvent (s) is selected in particular from the following substances: propan-2-ol, butan-2-ol, isobutanol, acetone, propanone, butanone, pentan-2-one, 1,2-ethanediol, 2,3-butanediol , Dioxane, acetonitrile, 2-methylbutan-2-ol, tert-butanol, 2-methylpropanol and 4- Hydroxy-2-methylpentanone, aliphatic hydrocarbons such as heptane, hexane or a mixture of two or more of these solvents.

The enzymatic catalyst used must of course effect and promote the transfer of an acyl group from an acyl donor to a flavonoid or flavonoid derivative and is advantageously a protease or lipase, e.g. from Candida antarctica, Rhizomucor miehei, Candida cylindracea, Rhizopus arrhizus, preferably immobilized on a support.

In view of the various features mentioned above, several different embodiments of the invention can be imagined, particularly with regard to the type of reagents used and the preferred objectives to be achieved.

In a first embodiment, one can imagine a synthesis process in a batch reactor (both substrates are added to the reactor with solvent and enzyme). In this case the reactor first contains the solvent, the total amount of flavonoids (generally from 1 g / 1 to 200 g / 1) required to obtain the desired final amount of modified flavonoids and the amount of free acid as Acyl donor, which corresponds to the molar ratio (of dissolved flavonoid / acyl donor) originally required (generally from 0.01 to 20). In order to achieve a fixed point in the amount of water in the reactor of less than 100 mM, the medium is brought to a temperature of 20-100 ° C., preferably 40-80 ° C. under vacuum (10-500 mbar, preferably 50-250 mbar) , heated, and the steam mixture produced is dried in a column filled with molecular sieves and then condensed and returned to the reactor. If necessary, the condensate is returned via a second column filled with molecular sieves. Then the enzyme is added in soluble or immobilized form (from 1 g / 1 to 100 g 1, preferably from 5 g / 1 to 20 g / 1). Water formed during the reaction is removed via the column filled with molecular sieves by adjusting the vacuum and the temperature in the reactor accordingly.

In a second embodiment, one can thus imagine a synthetic process in which the acyl donor and solvent are added during the reaction. In this case, the reactor first contains the solvent, the total amount of flavonoids (in generally from 1 g / 1 to 200 g / 1), which is required to obtain the desired final amount of modified flavonoids, and the amount of free acid as acyl donor, which corresponds to the originally required molar ratio (of dissolved flavonoid / acyl donor) (im generally from 0.01 to 20). In order to achieve a fixed point in the amount of water in the reactor of less than 100 mM, the medium is brought under vacuum (10-500 mbar, preferably 50-250 mbar) to a temperature of 20-100 ° C., preferably 40-80 ° C. , heated, and the steam mixture produced is dried in a column filled with molecular sieves and then condensed and returned to the reactor. If necessary, the condensate is led back through a second column filled with molecular sieves. Then the enzyme is added in soluble or immobilized form (from 1 g / 1 to 100 g / 1, preferably from 5 g / 1 to 20 g / 1). During the course of the reaction, solvent is added so that part of the solvent is evaporated off through a column with molecular sieves. The water is removed by exchange in the vapor phase. The steam is condensed and collected in a collecting container.

In order to keep the amount of solvent relatively constant, acyl donor is added online in such an amount per unit of time during the reaction that the molar ratio (of dissolved flavonoid / acyl donor) is kept at the required value. Thus, if it is advantageous to keep this molar ratio constant during the reaction, the acyl donor is added at a rate which corresponds to the rate of its consumption in the reaction; this rate of consumption can be determined by a preparatory kinetic examination of the enzyme reaction used. The amount of acyl donor added per unit time during the reaction is generally from 0.01 to 10 grams of acyl donor per hour per gram of enzyme catalyst in the reactor.

In a third embodiment of the invention, the synthesis process can also be carried out with the addition of flavonoids and solvents. In this case the reactor first contains the solvent, the total amount of free acid as acyl donor (generally from 1 g / 1 to 500 g / 1) required to obtain the desired final amount of modified flavonoids and the amount of Flavonoid, which corresponds to the molar ratio (of dissolved flavonoid acyl donor) originally required (generally from 1 g / 1 to 200 g / 1). In order to achieve a fixed point in the amount of water in the reactor of less than 100 mM, the medium is vacuum (10-500 mbar, preferably 50- 250 mbar) to a temperature of 20-100 ° C, preferably 40-80 ° C, and the steam mixture generated is dried in a column filled with molecular sieves and then condensed and returned to the reactor. If necessary, the condensate is recirculated through a second column filled with molecular sieves. Then the enzyme is added in soluble or immobilized form (from 1 g / 1 to 100 g / 1, preferably from 5 g / 1 to 20 g / 1).

During the course of the reaction, solvent is added and a vacuum is applied, so that part of the solvent and the water formed are evaporated off. The amount of evaporation is adjusted by controlling the vacuum and the temperature accordingly. The vapors formed are passed over a column filled with molecular sieves. The water is removed by contact with the molecular sieves. After the water has been removed, the steam is condensed and collected in a collecting tank, in order to be subsequently returned to the reactor. If necessary, anhydrous solvent is added during the reaction to compensate for evaporation losses and to keep the amount of solvent relatively constant. Furthermore, flavonoid is added in an amount per unit of time such that the molar ratio (of dissolved flavonoid / acyl donor) is kept at the required value. Thus, if it is advantageous to keep this molar ratio constant during the reaction, the flavonoid is added at a rate which corresponds to the rate of its consumption in the reaction; this rate of consumption can be determined by a preparatory kinetic examination of the enzyme reaction used. The amount of flavonoid added per unit time during the reaction is generally from 0.01 to 10 grams of flavonoid per hour per gram of enzyme catalyst in the reactor.

In a fourth embodiment of the invention, the synthesis process can also be carried out with the addition of flavonoid, acyl donor and solvent. In this fourth case, the reactor first contains the solvent, a variable concentration of flavonoid (preferably higher than the solubility of the flavonoid in the solvent) and the amount of free acid as acyl donor, which corresponds to the molar ratio (of dissolved flavonoid / acyl donor) originally required. In order to achieve a fixed point in the amount of water in the reactor of less than 100 mM, the medium is brought under vacuum (10-500 mbar, preferably 50-250 mbar) to a temperature of 20-100 ° C., preferably 40- 80 ° C, heated, and the steam mixture generated is dried in a column filled with molecular sieves and then condensed and returned to the reactor. If necessary, the condensate is returned via a second column filled with molecular sieves. Then the enzyme is added in soluble or immobilized form (from 1 g / 1 to 100 g / 1, preferably from 5 g / 1 to 20 g / 1).

During the course of the reaction, solvents are added and a vacuum in the range of 10-500 mbar, preferably 100-250 mbar, is applied. To remove the water, the vapors formed are passed over a column with molecular sieves. The steam is condensed and collected in a collecting container. If necessary, anhydrous solvent is added during the reaction to compensate for evaporation losses and to keep the amount of solvent relatively constant. Furthermore, flavonoid is added in an amount per unit of time such that the molar ratio (of dissolved flavonoid / acyl donor) is kept at the required value.

If it is advantageous to keep this molar ratio constant during the reaction, flavonoid and acyl donor are added in amounts per unit of time, each corresponding to the rate at which they are consumed in the reaction; these consumption rates can be determined by a preparatory kinetic examination of the enzyme reaction used.

In a fifth embodiment of the invention, the continuous synthesis process can alternatively be carried out with the addition and removal of flavonoid, acyl donor and / or solvent and possibly enzyme catalyst. In this fifth case, the reactor first contains the solvent, a variable concentration of flavonoid (preferably higher than the solubility of the flavonoid in the solvent) and the amount of free acid as acyl donor, which corresponds to the molar ratio (of dissolved flavonoid / acyl donor) originally required. In order to achieve a fixed point in the amount of water in the reactor of less than 100 mM, the medium is brought under vacuum (10-500 mbar, preferably 50-250 mbar) to a temperature of 20-100 ° C., preferably 40-80 ° C. , heated, and the steam mixture produced is dried in a column filled with molecular sieves and then condensed and returned to the reactor. If necessary, the condensate is recirculated through a second column filled with molecular sieves. Then the enzyme is added in soluble or immobilized form. While the implementation is taking place, substances are continuously or temporarily removed from the Stripped reaction medium. If the enzyme is in immobilized form, it can be retained in the reactor. After separation, the solvent and possibly the flavonoid and / or the acyl donor can be returned to the reactor. Anhydrous solvent is added during the reaction to compensate for evaporation and stripping losses, and it is still possible to add flavonoid and acyl donor in amounts per unit time to maintain the molar ratio of these two components at the required level. The water is removed through molecular sieves as described above. After dewatering, the evaporated solvent is condensed and returned to the reactor --uriick. If it is advantageous to keep this molar ratio constant during the reaction, flavonoid and acyl donor are added in amounts per unit of time which correspond to their respective consumption rates in the reaction and their respective withdrawal rates.

In a sixth embodiment, the reaction is carried out as above, but the free acid as acyl donor is replaced by its methyl, ethyl, propyl or butyl ester, preferably its methyl or ethyl ester. The alcohol formed is removed in the same way as above.

In a seventh embodiment, the acyl donor is used as the solvent.

In an eighth embodiment, water and / or alcohol present or present in the medium and / or formed during the reaction is removed through a pervaporation membrane, in the vapor or liquid phase.

Examples

Example 1:

Rutin monopalmitate synthesis was carried out in a 250 ml batch reactor using Candida antarctica lipase (Novozym 435). This is a lipase immobilized on a macroporous acrylic resin. The lipase is supplied with an activity of 7000 PLE xg ^'1 (propyl laurate synthesis), a water content of 1-2% by weight and an enzymatic protein content in the range between 1 and 10% by weight. For this synthesis 0.75 g (1.2 mmol) of rutin, 0.315 g (1.2 mmol) of palmitic acid and 250 ml were tert. -Amyl alcohol used. The medium was heated to 60 ° C. under vacuum (150 mbar) and the steam formed was passed over a column heated to 60 ° C. and filled with 50 g molecular sieves. The water was removed in the gas phase, which is much more effective than in the liquid phase. The dewatered steam was condensed and returned to the reactor through a second column filled with the same amount of molecular sieves. This procedure made it possible to obtain an initial water content of less than 100 mM after 6 hours and to solubilize the substrates. Then the enzyme (2.5 g) was added. The reaction was carried out at 60 ° C under vacuum (150 mbar) and the water formed was removed in the same manner as for the original drying, keeping its concentration below 100 mM.

This concentration can be varied by adjusting the vacuum and cooling the cooler accordingly. The pressures examined fluctuate between 10 and 700 mbar, and the temperature of the cooler between -20 and 5 ° C. With this procedure it is possible to adjust the water concentration in the reactor to between 5 and 400 mM.

After 48 hours of reaction, product analysis by HPLC showed that the conversion yields for the two substrates were approximately 90%.

At the end of the reaction, the enzyme was recovered by filtration. The medium was then concentrated by evaporating the solvent. Two extraction systems were used to eliminate substrate residues. A mixture of acetonitrile / heptane (3/5, v / v) was used to remove the palmitic acid, while the rutin was separated by extraction with water / heptane (2/3, v / v).

The structure of the product was confirmed by ¹ H-NMR analysis.

1H-NMR: (400 MHz, DMSO-d ₆ ): δ 0.8 (t, 3H), 1 (d, 3H), 1.25 (m, 24H), 1.45 (m, 2H), 2 , 1 (m, 2H), 3.1-3.6 (broad, CH sugar), 3.7 (d, 1H), 4.45 (s, 1H), 4.65 (t, 1H), 5 , 3 (broad, OH sugar), 5.1 (broad, OH sugar), 5.45 (d, 1H), 6.2 (s, 1H), 6.4 (s, 1H), 6.8 ( d, 1H), 7.6 (m, 2H), 12.6 (s, 1H, C ₅ -OH) ppm

Example 2:

The acylation of hesperidin (0.75 g, 1.2 mmol) with palmitic acid (0.315 g, 1.2 mmol) was carried out as described above.

HPLC analysis showed that after 48 hours 95% of the acyl donor was used up. The hesperidin monopalmitate could be obtained by liquid-liquid extraction using the same purification instructions as above. The structure of this hesperidin ester was confirmed by ¹ H-NMR analysis.

1H-NMR: (400 MHz, DMSO-d ₆ ): δ 0.83 (t, 3H), 1.0 (d, 3H), 1.05 (broad, 24H), 1.20 (m, 2H) , 2.25 (m, 2H), 3.4-3.6 (broad, CH sugar), 3.8 (s, 3H), 4.15 (s, 1H), 4.58 (s, 1H) , 4.75 (m, 2H), 5.0 (m, 1H), 5.18 (dd, 1H), 5.4 (d, 1H), 5.48 (d, 1H), 6.14 ( m, 1H), 6.18 (s, 1H), 7.0 (m, 3H), 9.15 (s, 1H), 12.05 (s, 1H) ppm

Example 3:

The acylation of esculin (0.75 g, 2 mmol) with palmitic acid (0.523 g, 2 mmol) was carried out as described in Example 1.

Liquid chromatography analysis showed that 80% of the acyl donor was consumed after 48 hours. The same purification instructions as above enabled the esculin monopalmitate to be obtained using liquid-liquid extraction. The structure of this esculin ester was confirmed by 1H NMR analysis.

1H-NMR: (400 MHz, DMSO-d ₆ ): δ 0.8 (t, 3H), 1.15 (broad, 24H), 1.4 (m, 2H), 2.25 (m, 2H) , 3.2 (m, 1H), 3.65 (m, 1H), 4.1 (dd, 1H), 4.35 (d, 1H), 4.85 (d, 1H), 5.25 ( s, 1H), 5.35 (d, 1H), 6.2 (d, 1H), 6.8 (s, 1H), 7.3 (s, 1H), 7.85 (d, 1H) ppm

Example 4 An acylation of rutin with lauric acid was carried out in a 27 ml reactor. Rutin (100 mg, 0.16 mmol) and lauric acid (20 mg, 0.10 mol) were in 20 ml of dried tert-amyl alcohol at 60 ° C. solved. A controlled water content in the reaction medium was adjusted to below 100 mM by adding molecular sieves (4 g). The esterification reaction was started by adding 0.2 g of Candida antartica lipase (Novozym 435). The HPLC analysis showed that the conversion of rutin to the monoester was 76%.

Example 5

The acylation of esculin (100 mg, 0.27mmol) with lauric acid (54 mg, 0.27mmol) was carried out as described in Example 4.

The HPLC analysis showed a conversion rate to the esculin monoester of 82%.

Example 6

The acylation of esculin (100 mg, 0.27 mmol) with l l-aminoundecanoic acid (55 mg,

0.27 mmol) was carried out as described in Example 4.

The HPLC analysis showed a conversion rate to the monoester of 61%.

Example 7

The acylation of esculin (100 mg, 0.27mmol) with 11-mercaptoundecanoic acid (59mg,

0.27 mmol) was carried out as described in Example 4.

The conversion of esculin to the monoester was 68% (HPLC analysis).

Example 8

The acylation of esculin (100 mg, 0.27 mmol) with adipic acid (40 mg, 0.27 mmol) was carried out as described in Example 4.

The HPLC analysis showed a conversion rate of 70% to the esculin monoester. Example 9

The acylation of rutin (100 mg, 0.16mmol) with dodecanedioic acid (38 mg, O.lόmmol - equimolar) was carried out as described in Example 4.

The HPLC analysis showed a conversion rate to the rutin monoester of 75%.

Example 10

The acylation of rutin (100 mg, 0.16 mmol) with dodecanedioic acid (754 mg, 3.27 mmol - excess acid) was carried out as described in Example 4. The HPLC analysis showed 75% conversion of rutin, the ratio of diesters to monoesters being 4: 1.

Example 11

The reaction of rutin with dodecanedioic acid with an excess of rutin was carried out in a 250 ml reactor.

Rutin (8, 13 mmol) and dodecanedioic acid (0.3 g, 1.3 mmol) were dissolved in 200 ml of tert-amyl alcohol and heated to 60 ° C. under a vacuum of 150-200 mbars). The vapors formed are passed over a column filled with molecular sieves and recovered. In this way, a low water content of less than 100 mm was achieved in the reactor after a few hours. 2 g of Candida antarctica ipase (Novozym 435) were added. After 4 days, a conversion rate of dodecanedioic acid to dodecanedioyl di-rutin (2 molecules of rutin linked by a diacid molecule) and to dodecanedioyl ratin (monoester) of 100% was achieved (HPLC analysis).

Example 12

The acylation of esculin (8, 21.7 mmol) with dodecanedioic acid (5 g, 21.7 mmol - excess acid) - was carried out as described in Example 11. The HPLC analysis showed that after 5 days the conversion rate to esculin monoester was 92%. Example 13

The acylation of rutin (100 mg, 0.16mmol) with hexadecanedioic acid (47 mg, O.lόmmol) - was carried out as described in Example 4. 87% of the rutin was converted (HPLC analysis).

The cleaning method of the liquid-liquid extraction according to Example 1 enabled the recovery of hexadecanedioyl rutin (monoester). The structure of the product was confirmed by 1H-NMR analysis.

1H NMR: (400 MHz, DMSO d ₆ ): δ 0.76 (d, 3H), 1.2 (m, 60H), 1.5 (m, 12H), 2.2 (m, 12H), 3.1-3.6 (broad, 8H), 3.7 (d, 1H), 4.45 (s, 1H), 4.65 (t, 1H), 5.43 (d, 1H), 6.18 (d, 1H), 6.36 (d, 1H), 6.84 (d, 1H), 7.50 (m, 2H) ppm.

Example 14

The acylation of esculin (100 mg, 0.27mmol) with hexadecanedioic acid (78mg, 0.27mmol) was carried out as described in Example 4.

The HPLC analysis showed a conversion rate of 89% to the esculin monoester.

Example 15

Esculin was reacted with thioctanoic acid in a 250 ml reactor. Esculin (0.87g, 2.5mmol) and thioctanoic acid (1.23g, 6mmol) were dissolved in 250 ml of tert-amyl alcohol and heated to 60 ° C under vacuum (150-200 mbars). The vapors formed are passed over a column filled with molecular sieves and recovered. In this way, a low water content of less than 100 mm was achieved in the reactor after 21 hours. 2.5 g of Candida antarctica lipase (Novozym 435) was added. After 70 hours, 50% of the esculin had been converted (HPLC analysis). After the reaction, the enzyme was filtered and the reaction medium was concentrated by evaporating the solvent. To remove excess thioctanoic acid, a mixture of water / heptane / acetonitrile (2/3 / 0.4, v / v / v) was used for extraction, and the ester was then obtained by extraction with dichloromethane. The structure of the ester was verified by 1H NMR. 1H MR: (400 MHz, DMSO d ₆ ): 1.2-1.9 (broad, 8H), 2.1-2.4 (broad, 4H), 3.2 (m, 2H), 3.5 (m, 1H), 3.7 (m, 1H) , 4.12 (dd, 1H), 4.35 (d, 1H), 4.85 (d, 1H), 5.23 (d, 1H), 5.33 (d, 1H), 6.26 (d, 1H), 6.84 (s, 1H), 7.33 (s, 1H), 7.86 (d, 1H) ppm.

Example 16

The reaction of rutin with phenylpropionic acid was carried out in a 250 ml reactor. Rutin (8g, 13 mmol) and phenylpropionic acid (10 g, 67 mmol) were dissolved in 200 ml of tert-amyl alcohol and heated to 60 ° C. under vacuum (150-200 mbars). The vapors formed are passed over a column filled with molecular sieves and recovered. In this way, a low water content of less than 100 mM was achieved in the reactor after 17 hours. 13 g of Candida antarctica-L pase (Novozym 435) were added. The HPLC analysis of the product showed a reaction to the rutin monoester of 55% after a reaction time of 105 hours.

Claims

claims

1. A process for the enzymatic synthesis of flavonoid esters and derivatives, in which a) a reaction medium is prepared comprising an organic solvent, a glycosylated flavonoid or aglycon flavonoid, an acyl group donor and an enzymatic catalyst, b) optionally further amounts of flavonoid and / or adding acyl donor and c) purifying the esters thus obtained by separating enzymatic particles and the solvent, characterized in that the concentration of water and / or alcohol formed during the reaction is controlled so that it is kept below 150 mM.

2. The method according to spoke 1, characterized in that the concentration of water and / or alcohol formed during the reaction is controlled so that it is kept below 100 mM.

3. The method according spoke 1, characterized in that the molar ratio of flavonoid to acyl donor in the reaction medium is adjusted so that it is in the range of 0.01 to 20.00 during the process.

4. The method according spoke 1, characterized in that the molar ratio of flavonoid to acyl donor in the reaction medium is adjusted so that it is in the range of 0.02 to 10.00 during the process.

5. The method according to claim 1, in which further additions of the reaction medium or in the meantime further amounts of at least one flavonoid in solid form or in the form of a liquid solution, solvent, enzymatic catalyst in soluble or immobilized form and azyl donor compound are added alone or solubilized in the solvent.

6. The method of spoke 1, in which, in the meantime or continuously, at least one component of the reaction medium is withdrawn and this at least one withdrawn component is returned to the reactor after fractionation.

7. The method of spoke 6, in which one continues to withdraw the entire reaction medium in the meantime or continuously and after fractionation one or more constituents of the withdrawn medium are injected back into the reactor.

8. The method according to claim 1, wherein the temperature is set to 20-100 ° C during the reaction, the partial pressure over the reaction medium is set to 10 mbar to 1000 mbar and the reaction medium is stirred.

9. The method of approached 1, in which remaining flavonoids or remaining acyl donor are eliminated by extraction with organic solvents or supercritical fluids, by distillation, by crystallization, by adsorption or by precipitation.

10. The method of spoke 1, in which one continues to fractionate the flavonoid esters shown by precipitation or chromatographic separation.

11. The method according spoke 1, in which the flavonoid is selected from the group formed by chalcon, flavon, flavonol, flavanone, anthocyanin, flavanol, coumarin, isoflavone and xanthone.

12. The method of approached 1, wherein the acyl donor compound is selected from the grapple, which is formed by straight-chain or branched aliphatic acids, saturated, unsaturated or cyclic, with up to 22 carbon atoms, optionally substituted by one or more substituents selected from the group consisting of hydroxyl , Amino, mercapto, halogen and alkyl-SS-alkyl group, for example palmitic acid, 16-hydroxyhexadecanoic acid, 12-hydroxystearic acid, 11-mercaptoundecanoic acid, thioctanoic acid or quinic acid, straight-chain or branched aliphatic diacids, saturated or unsaturated, with up to 22 Carbon atoms, for example hexadecanedioic acid or A-zelaic acid, an arylaliphatic acid and a dimeric acid derived therefrom, a cinnamic acid, optionally substituted by one or more substituents selected from the group consisting of hydroxyl, nitro, alkyl, alkoxy and halogen atoms, for example caffeic acid (3, 4-dihydroxycinnamic acid), ferulic acid (4-hydroxy-3-methoxycinnamic acid) or cumaric acid (4-hydroxycinnamic acid), a benzoic acid, optionally substituted by one or more substituents selected from the group consisting of hydroxyl, nitro, alkyl, alkoxy and halogen atoms, e.g. gallic acid ( 3,4,5-trihydroxybenzoic acid), vanillic acid (4-hydroxy-3-methoxybenzoic acid) or protocatechic acid (3,4-dihydroxybenzoic acid), or their methyl, ethyl, propyl or butyl esters.

13. The method according to claim 1, wherein the organic solvent is selected from the grapple, which is formed from propan-2-ol, butan-2-ol, isobutanol, acetone, propanone, butanone, pentan-2-one, 1,2 -Ethanediol, 2,3-butanediol, dioxane, acetonitrile, 2-methylbutan-2-ol, tert-butanol, 2-methylpropanol and 4-hydroxy-2-methylpentanone, aliphatic hydrocarbons such as heptane and hexane and mixtures of at least two of these components.

14. The method of claim 1, wherein the organic solvent is the acyl donor.

15. The method of claim 1, wherein the enzymatic catalyst comprises a protease and / or lipase.

16. The method according to claim 15, wherein the protease and / or lipase is immobilized on a carrier.

17. The method according to claim 1, wherein the water and / or the alcohol in the gas or liquid phase is removed from the medium by molecular sieves.

18. The method according to claim 1, wherein the water and / or the alcohol in the gas or liquid phase is removed from the medium by pervaporation.