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WO2010052356A1 - Procédé enzymatique pour l'acylation de resvératrol en position 3- - Google Patents

Procédé enzymatique pour l'acylation de resvératrol en position 3- Download PDF

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Publication number
WO2010052356A1
WO2010052356A1 PCT/ES2009/070476 ES2009070476W WO2010052356A1 WO 2010052356 A1 WO2010052356 A1 WO 2010052356A1 ES 2009070476 W ES2009070476 W ES 2009070476W WO 2010052356 A1 WO2010052356 A1 WO 2010052356A1
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WO
WIPO (PCT)
Prior art keywords
resveratrol
lipase
acetyl
vinyl ester
immobilized
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/ES2009/070476
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English (en)
Spanish (es)
Inventor
Pamela Torres Salas
Francisco José PLOU GASCA
Antonio Ballesteros Olmo
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Consejo Superior de Investigaciones Cientificas CSIC
Original Assignee
Consejo Superior de Investigaciones Cientificas CSIC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Consejo Superior de Investigaciones Cientificas CSIC filed Critical Consejo Superior de Investigaciones Cientificas CSIC
Publication of WO2010052356A1 publication Critical patent/WO2010052356A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12P7/62—Carboxylic acid esters

Definitions

  • 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.
  • 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.
  • resveratrol trans-3,5,4'-trihydroxystilbene
  • resveratrol trans-3,5,4'-trihydroxystilbene
  • 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).
  • 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.
  • 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.
  • 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.
  • the enzymes used 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).
  • 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.
  • a vinyl ester is used, where the number of carbon atoms in the ester can be between 2 and 26.
  • 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.
  • 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.
  • 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 .
  • a solution of resveratrol in an intermediate polarity solvent, which constitutes the reaction medium can be prepared prior to incubation of the resveratrol.
  • 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.
  • these solvents can be selected from the list comprising 2-methyl-2-butanol, tert-butanol, isopropyl ether or 2-pentanone.
  • 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).
  • 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 ⁇ mol 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-.
  • acylation reaction does not take place.
  • 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).
  • HPLC reverse phase high performance liquid chromatography
  • Another aspect of the invention relates to the products of the enzymatic reaction.
  • the organic phase is removed by evaporation under reduced pressure, and the residue obtained is purified.
  • the residue obtained is purified.
  • the inventors have characterized the products structurally synthesized by nuclear magnetic resonance and mass spectrometry.
  • 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.
  • 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.
  • 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.
  • concentrations (mM) of 3-O-acetyl-resveratrol (•) are represented; 3,4'-di-O-acetyl-resveratrol (O); and 3,4,5-tri-O-acetyl-resveratrol
  • 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.
  • EXAMPLE 1 Synthesis of 3-O-acetyl-resveratrol with different lipases.
  • 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.
  • resveratrol 570 mg 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 compound 3,4'-di-O-acetyl-resveratrol is also obtained in a yield of 40% (calculated by HPLC) at 30 hours of reaction.
  • 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.
  • resveratrol 342 mg 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 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.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé enzymatique permettant l'acylation régiosélective de resvératrol en position 3- à l'aide d'un ester vinylique et de lipases fongiques et bactériennes spécifiques immobilisées en tant que biocatalyseur. Les lipases utilisées au cours de ce procédé proviennent de bactéries ou de champignons choisis parmi Alcaligenes, Pseudomonas ou Thermomyces.
PCT/ES2009/070476 2008-11-07 2009-10-29 Procédé enzymatique pour l'acylation de resvératrol en position 3- Ceased WO2010052356A1 (fr)

Applications Claiming Priority (2)

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ES200803184A ES2343773B1 (es) 2008-11-07 2008-11-07 Procedimiento enzimatico para la acilacion en posicion 3- del resveratrol.
ESP200803184 2008-11-07

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106834369A (zh) * 2016-11-29 2017-06-13 大连工业大学 一种白藜芦醇‑二十碳五烯酸酯的制备方法
WO2019236765A1 (fr) * 2018-06-05 2019-12-12 Flagship Pioneering Innovations V, Inc. Polyphénols de catéchine acylés et leurs procédés d'utilisation pour le traitement du cancer
US10953027B2 (en) 2018-06-05 2021-03-23 Flagship Pioneering Innovations V, Inc. Active agents and methods of their use for the treatment of metabolic disorders and nonalcoholic fatty liver disease

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2554563B2 (es) * 2014-05-05 2016-08-16 Universidad De Sevilla Cepas bacterianas y sus usos en reacciones de acilación y/o desacilación

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5387514A (en) * 1990-12-24 1995-02-07 Hoechst Aktiengesellschaft Acylation of alcohols with Pseudomonas lipase immobilized on a polystyrene resin

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US5387514A (en) * 1990-12-24 1995-02-07 Hoechst Aktiengesellschaft Acylation of alcohols with Pseudomonas lipase immobilized on a polystyrene resin

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CARDILE, V. ET AL.: "Chemo-enzymatic synthesis and cell-growth inhibition activity of resveratrol analogues.", BIOORGANIC CHEMISTRY, vol. 33, no. 1, 2005, pages 22 - 33 *
NICOLOSI, G. ET AL.: "Chemo-enzymatic preparation of resveratrol derivatives.", JOURNAL OF MOLECULAR CATALYSIS B ENZYMATIC, vol. 16, no. 5-6, 2002, pages 223 - 229 *
SANTANIELLO, E. ET AL.: "Lipase-catalyzed deacylation by alcoholysis: A selective, useful transesterification reaction", CURRENT ORGANIC CHEMISTRY, vol. 10, no. 10, 2006, pages 1095 - 1123 *
SANTANIELLO, E. ET AL.: "Selective lipase- catalyzed preparation of diol monobenzoates by transesterification and alcoholysis reactions in organic solvents.", JOURNAL OF MOLECULAR CATALYSIS B ENZYMATIC, vol. 40, no. 3-4, 2006, pages 81 - 85 *
TENG, R.W. ET AL.: "Regioselective acylation of several polyhydroxylated natural compounds by Candida antarctica lipase B", BIOCATALYSIS AND BIOTRANSFORMATION, vol. 23, no. 2, 2005, pages 109 - 116 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106834369A (zh) * 2016-11-29 2017-06-13 大连工业大学 一种白藜芦醇‑二十碳五烯酸酯的制备方法
WO2019236765A1 (fr) * 2018-06-05 2019-12-12 Flagship Pioneering Innovations V, Inc. Polyphénols de catéchine acylés et leurs procédés d'utilisation pour le traitement du cancer
US10953027B2 (en) 2018-06-05 2021-03-23 Flagship Pioneering Innovations V, Inc. Active agents and methods of their use for the treatment of metabolic disorders and nonalcoholic fatty liver disease
US11813272B2 (en) 2018-06-05 2023-11-14 Flagship Pioneering Innovations V, Inc. Active agents and methods of their use for the treatment of metabolic disorders and nonalcoholic fatty liver disease

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ES2343773A1 (es) 2010-08-09

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