WO2010052356A1

WO2010052356A1 - Enzymatic process for acylation of resveratrol at position 3

Info

Publication number: WO2010052356A1
Application number: PCT/ES2009/070476
Authority: WO
Inventors: Pamela Torres Salas; Francisco José PLOU GASCA; Antonio Ballesteros Olmo
Original assignee: Consejo Superior De Investigaciones Cientificas (Csic)
Priority date: 2008-11-07
Filing date: 2009-10-29
Publication date: 2010-05-14
Also published as: ES2343773B1; ES2343773A1

Abstract

Enzymatic procedure for the regioselective acylation at position 3 of resveratrol utilising a vinyl ester and specific fungal and bacterial lipases, immobilised, as biocatalyst. The lipases utilised in said procedure come from bacteria or fungi selected from among Alcaligenes, Pseudomonas or Thermomyces.

Description

ENZYMATIC PROCEDURE FOR ACILATION IN POSITION 3-

OF RESVERATROL

The present invention relates to an enzymatic process of regioselective acylation in position 3 of the resveratrol and in a single step, using a vinyl ester and an immobilized lipase as a biocatalyst. Given the varied applications of antioxidants, the invention can be included within the field of the biotechnology industry and, in particular, in the pharmaceutical sector, functional food, nutraceutical or cosmetic industries.

STATE OF THE TECHNIQUE

In recent years, different studies have shown the beneficial effect on health that derives from the intake of plant-based foods (fruits, vegetables, virgin olive oil, red wine, tea, etc.). The healthy properties that these foods exert go beyond what one would expect for their nutrients, vitamins and mineral salts, so it has been postulated that they are due to the secondary metabolites they contain, occupying a prominent place among these polyphenols. Of the different biological activities of the polyphenols, the antioxidant is the one that has generated the greatest interest since antioxidants are used to counteract the effects of oxidative processes in vivo, which have been related among others with some inflammatory, cardiovascular, cancer or even of aging (YZ Fang et al., "Free radicáis, antioxidants, and nutrition", Nutrition 2002, vol. 18, pp. 872-879).

Among the polyphenolic compounds, the case of resveratrol (trans-3,5,4'-trihydroxystilbene), a natural product present in the grape, is very remarkable. It has been described that resveratrol can critically interfere in a multitude of events associated with the development of degenerative diseases, including cardiovascular and cancer (AR Martin et al., "Resveratrol, a polyphenol found in grapes, suppresses oxidative damage and stimulates apoptosis during early colonic inflammation in rats ", Biochem. Pharmacol. 2004, vol. 67, pp. 1399-1410; JM Wu et al.," Mechanism of cardioprotection by resveratrol, a phenolic antioxidant present in red wine (Review) ", Int. J. Mol. Med. 2001, vol. 8, pp. 3-17).

In general, the desirable properties in the antioxidant compounds to be used as health promoting additives are: free radical scavenging capacity, stability and bioavailability. The main problem of the use of phenolic compounds is their low stability and / or the modification they undergo in vivo in detoxification processes, where the most antioxidant clusters, such as ortho-dihydroxylic acid, are blocked. Therefore, it is necessary to find compounds that are sufficiently stable both at room temperature and at body temperature, and that are functional long enough before being degraded and / or metabolized. One of the approaches that has been used to increase the stability of resveratrol, without decreasing its biological activity, is the preparation of modified derivatives with a glycosyl moiety or with a lipophilic chain (V. Cardile et al., "Chemoenzymatic synthesis and cell- growth inhibition activity of resveratrol analogues ", Bioorg. Chem. 2005, vol. 33, pp. 22-33; F. Orsini et al.," Isolation, synthesis, and antiplatelet aggregation activity of resveratrol 3-O-β -D- glucopyranoside and related compounds ", J. Nat. Prod. 1997, vol. 60, pp. 1082-1087; and G. Regev-Shoshani et al.," Glycosylation of resveratrol protects it from enzymic oxidation ", Biochem. J. 2003 , vol. 374, pp. 157-163).

On the other hand, it is very interesting to try to modulate the bioavailability of antioxidants by the addition of a lipophilic chain, a strategy that allows a simple way to modify their hydrophilic-lipophilic balance. These acylated derivatives maintain the stability of the antioxidant, are soluble in fatty media and also tend to be very permeable in human skin cell models (A. Tai et al., "Permeation and metabolism of a series of novel lipophilic ascorbic acid derivatives, 6 -O-acyl-2-O-α-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain, in a human living skin equivalent model "Bioorg. Med. Chem. Lett. 2004, vol. 14, pp. 623-27). These modifications can play a critical role in terms of residence time in the body, degree of metabolism, efficiency in absorption, and ultimately, effectiveness as new possible antioxidants.

In addition, minimal chemical modifications in the stilbene nucleus of resveratrol can cause major changes in its biological activity and, more specifically, in its antitumor properties (R. Chillemi et al., "Antitumor properties of stilbene-based resveratrol analogues: Recent results", Nat. Prod. Commun. 2007, vol. 2, pp. 499-513). Thus, some resveratrol derivatives with acyl chains have shown greater inhibition on the cell growth of DU-145 prostate cancer cells than resveratrol itself (V. Cardile et al., "Chemo-enzymatic synthesis and cell-growth inhibition activity of resveratrol analogues ", Bioorg. Chem. 2005, vol. 33, pp. 22-33).

It is, therefore, desirable to have simple procedures for lipophilization of natural antioxidants to give them greater stability and / or bioavailability. The lipophilization by enzymatic methods (generally using the enzymatic catalyst in immobilized form on supports that increase its mechanical and thermal resistance) offers very remarkable yields and selectivities, implicit environmental improvements, and offers the possibility of working in mild operating conditions (low temperatures and atmospheric pressure) What decreases energy consumption, leading to a significant reduction in costs. Due to the enormous interest of derivatives of natural antioxidants such as therapeutic substances, functional ingredients, nutraceuticals or cosmetic agents, enzymatic methods have been postulated as a "sustainable" alternative for their production.

When a polyphenolic compound is reacted with an acyl donor in the presence of an enzyme, the acylation position or positions can vary substantially depending on the biocatalyst tested, and may, in principle, take place on any of the phenolic OH groups. Thus, in the case of resveratrol, which has three phenolic groups (in positions 3-, 4'- and 5- of the stilbene core) with a similar chemical reactivity, it has been described that the reaction with vinyl acetate in the presence of Ia Candida lipase B selectively gives rise to 4'-O-acetyl-resveratrol with a yield of 50% (RW Teng et al., "Regioselective acylation of several polyhydroxylated natural compounds by Candida antárctica lipase", Biocatal. Biotransform. 2005, vol. 23, pp. 109-116). However, as the chain length of the fatty acid increases, the reaction rate decreases markedly. However, it is difficult to obtain a selective enzymatic esterification in the phenolic group in position 3- (equivalent to position 5- since it is a symmetric molecule) probably due to the difficulties of selectively acylating the most sterically hindered phenolic group in the molecule. Obtaining this acetylated product in position 3- is described by chemoenzymatic esterification. In that case, a chemical peracetylation is initially performed, followed by a regioselective alcohololysis catalyzed by lipase B of Candida Antarctica. With this same procedure, resveratrol diacetate is also obtained in the phenolic groups of positions 3,5- at shorter reaction times (G. Nicolosi et al., "Chemo-enzymatic preparation of resveratrol derivatives", J. Mol. Catal. B Enzym. 2002, vol. 16, pp. 223-229).

EXPLANATION OF THE INVENTION

The present invention relates to the enzymatic modification of the antioxidant, natural or synthetic, resveratrol by regioselective acylation in position 3-, using a vinyl ester, preferably of fatty acids of different chain length. Specifically, the enzymes used, as biocatalysts, are immobilized lipases. Acylation with fatty acids can minimize the oxidation and photodestruction of resveratrol, increase its life time and improve its bioavailability., In addition to increasing its biological properties (for example, as an antitumor).

Therefore, a first aspect of the present invention relates to a procedure for acylation of resveratrol, characterized in that it comprises the incubation of resveratrol with a vinyl ester (C ₂ -C ₂₆ ) in the presence of an immobilized lipase, where said lipase comes from bacteria or fungi that are selected from the list comprising bacteria or fungi of the genus Alcaligenes, Pseudomonas or Thermomyces.

As the acylating agent, in the process of the invention, a vinyl ester is used, where the number of carbon atoms in the ester can be between 2 and 26. Preferably the vinyl ester is a vinyl acetate or a vinyl ester of an acid fatty of different chain length, where the number of carbons can be between 4 to 26, more preferably the number of carbon atoms of the fatty acid ester is between 16 and 22, even more preferably it is vinyl stearate.

In another preferred embodiment of the process of the invention, the immobilized lipase comes from the species Alcaligenes sp., Pseudomonas cepacia or Thermomyces lanuginosus.

The lipase is immobilized in any convenient means and known to a person skilled in the art, such as but not limited to silica, alumina, controlled pore glass or diatomaceous earth, preferably the lipase is immobilized in diatomaceous earth.

Normally, the resveratrol solution (from, for example but not limited to the Polygonum cuspidatum plant) is prepared in the absence of the solvent, using the vinyl ester that acts as a solvent (or reaction medium) and which is an acyl donor at the same time . However, in a particular embodiment of the invention, a solution of resveratrol in an intermediate polarity solvent, which constitutes the reaction medium, can be prepared prior to incubation of the resveratrol.

By "intermediate polarity solvent" means in the present invention a solvent with an average polarity of 2 <log P '<4, where P' is the index developed by L. R. Synder. As examples, but not limited, these solvents can be selected from the list comprising 2-methyl-2-butanol, tert-butanol, isopropyl ether or 2-pentanone.

To the reaction medium containing the intermediate polarity solvent, water can be added in a proportion less than 0.2% (w / v) of the solvent.

Any of the above mixtures is heated to a temperature in the range between 25 and 65 ⁰ C, more preferably between 3O ⁰ C and 5O ⁰ C, and is added as catalyst (preferably lipases Alcaligenes sp bacterial lipase. Or Pseudomonas cepacia) or fungal (preferably Thermomyces lanuginosus lipase) immobilized (preferably in diatomaceous earth). Preferably, the addition of the enzyme is carried out in a proportion of 50-150 mg per ml of solution, which corresponds to 30-600 units (U) of tripropionin hydrolysis activity per ml of solution, defining a unit of activity as Ia that catalyzes the conversion of 1 μmol of substrate per minute. The system is maintained between 8 and 72 hours, depending on the chain length of the fatty acid, or the ester in general, and the degree of substitution desired, preferably the incubation is carried out with orbital agitation and more preferably at a stirring of between 100rpm and 250 rpm. Under these conditions, the majority product is the resveratrol ester in position 3-.

When other compounds such as triglycerides, ethyl esters or free fatty acids are used as acyl donors, maintaining all the conditions described above, the acylation reaction does not take place. Likewise, when other enzymes such as lipase B of Candida Antarctica and lipase of Rhizomucor miehei are used, maintaining all the conditions described above, the acetylated product is obtained mainly in the phenolic group of position 4'-, in a molar proportion with respect to to 3-0-acetyl-resveratrol 2.5: 1 and 2: 1, respectively.

The monitoring of the reaction of the present invention can be carried out by thin layer chromatography or by reverse phase high performance liquid chromatography (HPLC) (using a photodiode detector and an evaporative light scattering detector).

Another aspect of the invention relates to the products of the enzymatic reaction. Thus, once the reaction is complete, the organic phase is removed by evaporation under reduced pressure, and the residue obtained is purified. For example, by semi-preparative HPLC or by preparative silica gel chromatography eluting with heptane: ethyl acetate (in a proportion dependent on the length of the fatty acid). The inventors have characterized the products structurally synthesized by nuclear magnetic resonance and mass spectrometry.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Shows an HPLC chromatogram of the acetylation reaction of resveratrol in 2-methyl-2-butanol catalyzed by lipase of: (I) Alcaligenes sp. immobilized in diatomaceous earth, (II) Pseudomonas cepacia immobilized in diatomaceous earth, and (III) Thermomyces lanuginosus immobilized by granulation with silica. (1) Resveratrol; (2) 3-0-acetyl-resveratrol; (3) 4'-0-acetyl-resveratrol; (4) 3,5-di-O-acetyl-resveratrol; (5) 3,4'-di-O-acetyl-resveratrol.

Fig. 2. Shows a semi-preparative HPLC chromatogram of the acetylation reaction of resveratrol in anhydrous 2-methyl-2-butanol catalyzed by the lipase of Alcaligenes sp. immobilized in diatomaceous earth. A photodiode detector and quantification at 308 nm was used. (1) Resveratrol; (2) 3-O-acetyl-resveratrol; (3) 4'-O-acetyl-resveratrol; (4) 3,5-di-O-acetyl-resveratrol (5) 3,4'-di-O-acetyl-resveratrol; (6) 3,4 ', 5-tri-O-acetyl-resveratrol.

Fig. 3. Shows the kinetics of the acetylation reaction of resveratrol in 2- methyl-2-butanol catalyzed by the lipase of Alcaligenes sp. immobilized in diatomaceous earth. The different concentrations (mM) of 3-O-acetyl-resveratrol (•) are represented; 3,4'-di-O-acetyl-resveratrol (O); and 3,4,5-tri-O-acetyl-resveratrol

(OR).

Fig. 4. Shows an HPLC chromatogram of the acylation reaction of resveratrol with vinyl stearate in 2-methyl-2-butanol, catalyzed by the lipase of: (I) Alcaligenes sp. immobilized in diatomaceous earth, (II) Pseudomonas cepacia immobilized in diatomaceous earth, and (III) the Thermomyces lanuginosus lipase immobilized by granulation. A photodiode detector and quantification at 308 nm was used. The maximums corresponding to 3-0-acetyl-resveratrol (7) and 4'-O-acetyl-resveratrol (8) are indicated.

Fig. 5. Shows the kinetics of the acylation reaction of resveratrol with vinyl stearate in 2-methyl-2-butanol catalyzed by the lipase of Alcaligenes sp. immobilized in diatomaceous earth. The concentration (mM) of 3-O-stearyl-resveratrol produced at different intervals over a 167 hour reaction is represented. EXAMPLES OF REALIZATION

The materials and methods that were used for the development of the present invention, as well as examples of realization thereof, are detailed below. Said examples do not limit the invention, but its purpose is to illustrate it, showing the ability to synthesize in a single step of 3-0-acyl-resveratrol following the procedure described herein.

EXAMPLE 1: Synthesis of 3-O-acetyl-resveratrol with different lipases.

5.8 mg of resveratrol was weighed and dissolved in 0.5 ml of anhydrous 2-methyl-2-butanol (water content less than 0.2%). The solution was heated to 40 ⁰ C, and 35 .mu.l of vinyl acetate (molar ratio vinyl acetate: Resveratrol 15: 1) were added. 100 mg of lipase from different organisms were added to the mixture: 1) Alcaligenes sp. fixed assets (QLG, Meito Sangyo Co., Ltd.); 2) Pseudomonas cepacia immobilized (PS "Amano" IM, Amano Enzyme Europe Ltd.); 3) Thermomyces lanuginosus immobilized (Lipozyme TL IM, Novozymes A / S). The mixture was incubated at 40 ⁰ C with orbital shaking at 150 rpm for about 9 days, after which it was cooled Io. The reaction was followed by reverse phase high performance liquid chromatography (HPLC), using a photodiode detector and quantification at 308 nm. Under these conditions the following reaction compositions were obtained, (calculated by HPLC), according to the lipase used (Figure 1): I) 24.4% of 3-O-acetyl-resveratrol, 1.3% of 4'-O-acetyl-resveratrol , 70.3% of 3,4'-di-O-acetyl-resveratrol, 3.7% of 3,5-di-O-acetyl-resveratrol (the 3-O-acetyl-resveratrol molar ratio: 4'-O-acetyl- Resveratrol is 18: 1); II) 24.9% of 3-O-acetyl-resveratrol, 10.2% of 4'-O-acetyl-resveratrol, 54.8% of 3,4'-di-O-acetyl-resveratrol and 7.5% of 3,5-di- O-acetyl-resveratrol (the molar ratio 3-O-acetyl-resveratrol: 4'-O-acetyl-resveratrol is 2.5: 1); and III) Thermomyces lanuginosus: 35.5% of 3-O-acetyl-resveratrol, 21.3% of 4'-O-acetyl-resveratrol, 28.7% of 3,4'-di-O-acetyl-resveratrol and 0.3% of 3, 5-di-O-acetyl-resveratrol (the molar ratio of synthesis 3- O-acetyl-resveratrol: 4'-O-acetyl-resveratrol is 1.5: 1). In this figure 1 the maximums corresponding to each of the compounds are indicated according to the numbering used in Scheme 1.

Scheme 1.- Resveratrol acetylation reaction. The different compounds that can be obtained therein from (1) resveratrol are indicated. (2) 3-0-acetyl-resveratrol; (3) 4'-O-acetyl-resveratrol; (4) 3,5-di-O-acetyl-resveratrol (5) 3,4'-di-O-acetyl-resveratrol.

EXAMPLE 2: Production of 3-O-acetyl-resveratrol.

570 mg of resveratrol were weighed and dissolved in 6.5 ml of anhydrous 2-methyl-2-butanol (water content less than 0.2%). The solution was heated to 40 ⁰ C, and 3.5 ml of vinyl acetate (molar ratio vinyl acetate: resveratrol 15: 1) was added. To the mixture was added 1.5 g of lipase from Alcaligenes sp. immobilized in diatomaceous earth. The mixture was incubated at 40 ⁰ C with orbital shaking at 150 rpm for about 30 hours, after which it was cooled Io. The reaction was followed by thin layer chromatography, using heptane: ethyl acetate 1: 1 (v / v) as eluent, the detection being by UV light. Likewise, the mixture was subsequently analyzed by high performance liquid chromatography (HPLC) in reverse phase, with a column (125 x 4.6 mm) filled with Lichospher 100 RP8 (5 μm). Methanol: water 30:70 (v / v) was used as the initial mobile phase (the aqueous phase contains 0.1% v / v glacial acetic acid) at a flow of 1 ml / min; then a gradient of 5 minutes was made until a methanol composition: water 50:50 (v / v), keeping it for 15 minutes and finally it was returned to the initial composition methanol: water 30:70 (v / v) in 2 min. A photodiode detector and quantification at 308 nm was used. Under these conditions, a 3-O-acetyl-resveratrol yield of 39% (calculated by HPLC) is obtained. The synthesis of this product took place preferably until the 55 hours of reaction, although the maximum production is at 12 hours (approximate yield of 75%). The reaction mixture was filtered, and the liquid phase was subjected to evaporation under reduced pressure, obtaining a white solid residue.

The residue obtained in the previous step was subjected to silica gel column chromatography, using heptane: ethyl acetate 3: 1 (v / v) as eluent. Under these conditions, 3-O-acetylresveratrol is obtained with a high degree of purity. The determination of the chemical structure of the compound obtained was carried out by electrospray mass spectrometry (HPLC-ESI) and 2D- ¹ H- ¹³ C multiple correlation nuclear magnetic resonance experiments.

With this same procedure, the compound 3,4'-di-O-acetyl-resveratrol is also obtained in a yield of 40% (calculated by HPLC) at 30 hours of reaction. However, the synthesis of this product occurs preferably after 55 hours of reaction. To obtain a superior yield of this compound (approximately 60%) it is necessary to maintain the reaction until 160 hours. The determination of the chemical structure of the compound obtained was carried out by electrospray mass spectrometry (HPLC-ESI) and 2D- ¹ H- ¹³ C multiple correlation nuclear magnetic resonance experiments.

The results of this exemplary embodiment are represented in Figures 2 and 3. Where the reaction conditions in fig. 2 were described above: 50 mM resveratrol, 750 mM vinyl acetate, 150 mg / ml lipase, incubation at 40 ⁰ C with orbital shaking. A photodiode detector and quantification at 308 nm was used. The results of Figure 3 were produced at different intervals throughout a reaction of 167 hours under the same conditions described for Figure 2.

EXAMPLE 3: Production of 3-O-stearyl-resveratrol

342 mg of resveratrol were weighed and dissolved in 10 ml of anhydrous 2-methyl-2-butanol (water content less than 0.2%). The solution was heated to 40 ⁰ C, and 6.98 g of vinyl stearate (molar ratio vinyl stearate: Resveratrol 15: 1) were added. To the mixture was added 750 mg of lipase from Alcaligenes sp. immobilized in diatomaceous earth. The mixture was incubated at 40 ⁰ C with orbital shaking at 150 rpm for about 72 hours, after which it was cooled Io. The reaction was followed by thin layer chromatography, using heptane: ethyl acetate 4: 1 (v / v) as eluent, the detection being by UV light. The mixture was subsequently analyzed by reverse phase high performance liquid chromatography (HPLC), with a Mediterranea-C18 column (5 μm) (150 x 4.6 mm). Methanol: water 90:10 (v / v) was used as the mobile phase (the aqueous phase contains 0.1% v / v acetic acid) at a flow of 1.5 ml / min. A photodiode detector and quantification at 308 nm was used. Under these conditions a yield of 84% (calculated by HPLC) was obtained. The reaction mixture was filtered, and the liquid phase was subjected to evaporation under reduced pressure, obtaining a white solid residue. The residue obtained in the previous step was subjected to silica gel column chromatography, using heptane: ethyl acetate 2: 1 (v / v) as eluent. Under these conditions, 3-0-stearyl-resveratrol was obtained with a high degree of purity. The determination of the chemical structure of the compound obtained was carried out by mass spectrometry (HPLC-ESI) and 2D- ¹ H- ¹³ C multiple correlation nuclear magnetic resonance experiments.

The results of example 3 are represented in Figures 4 and 5.

Claims

1. Resveratrol acylation procedure, characterized in that it comprises the incubation of resveratrol with a vinyl ester in the presence of an immobilized lipase, where said lipase comes from bacteria or fungi that are selected from the list comprising Alkalgenes, Pseudomonas or Thermomyces.

2. Method according to claim 1, wherein the vinyl ester is vinyl acetate.

3. Method according to claim 1, wherein the vinyl ester is a vinyl ester of a fatty acid (C ₄ -C ₂₆ ).

4. Method according to claim 3, wherein the vinyl ester is a vinyl ester of a fatty acid (Ci6-C ₂₂ ).

5. Method according to any of claims 1 to 4, wherein the lipase comes from a bacterium of the species Alcaligenes sp.

6. Method according to any of claims 1 to 4, wherein the lipase comes from a bacterium of the species Pseudomonas cepacia.

7. Method according to any of claims 1 to 4, wherein the lipase comes from a fungus of the species Thermomyces lanuginosus.

8. Method according to any of claims 1 to 7, wherein the lipase is immobilized in diatomaceous earth.

9. Method according to any of claims 1 to 8, wherein the resveratrol is dissolved prior to incubation in a solvent of medium polarity.

10. Method according to claim 9, wherein the medium polar solvent is selected from the list comprising 2-methyl-2-butanol, tert-butanol, isopropyl ether or 2-pentanone.

11. Method according to any of claims 1 to 10, wherein the incubation is carried out at a temperature between 25 ⁰ C and 65 ⁰ C.

12. Method according to claim 11, wherein the incubation is carried out at a temperature between 3O ⁰ C and 5O ⁰ C.

13. Method according to any of claims 1 to 12, wherein the incubation is carried out with stirring.

14. Method according to claim 13, wherein the agitation is orbital and between 100 rpm and 250 rpm.