ES2554563B2 - Bacterial strains and their uses in acylation and / or deacilation reactions - Google Patents

Abstract

Bacterial strains and their uses in acylation and / or deactivation reactions. # The present invention describes microbial strains capable of carrying out regioselective alcoholysis and esterification reactions and the use thereof in the production of lipophilic carbohydrates and polyphenols of interest in The pharmaceutical and food industry. Also, the invention discloses the method of obtaining microbial strains and the method of obtaining lipophilic carbohydrates and polyphenols.

Description

The present invention relates to the microbial strains of the genera Terribacillus called Terribacillus sp. AE28122, Bacillus, called Bacillus sp. HR21-6, Enterobacler, called Enterobacter sp. AE1889 and Pseudomonas, called Pseudomonas sp. AE28120 with the ability to carry out regioselective alcoholysis and esterification reactions and the use thereof in acylation and / or deacilation reactions, in general and more specifically, in acylation and / or deacilation reactions for the production of lipophilic carbohydrates and polyphenols of interest in the pharmaceutical and food industry. Also, the invention also relates to a method of obtaining these microbial strains capable of carrying out these reactions. As well as obtaining regioisomers of partially acylated sugars and polyphenols. Therefore, the invention is useful in the fields of Industrial Microbiology and Chemical Engineering, in particular, in the sector of the production of compounds of interest in the pharmaceutical and food industry.

STATE OF THE TECHNIQUE

The chemo-, regal-and stereoselective protection and deprotection constitutes one of the most used strategies in organic synthesis, especially valuable in the synthesis of complex and polyfunctional molecules, among which are carbohydrates and polyphenols. Although there are many advances that have been achieved in this field in recent decades, the natural reactions are still far from being achieved, so this aspect remains today one of the great challenges in organic synthesis.

Biocatalysis, and particularly the use of hydrolytic enzymes such as lipases, is an alternative to conventional methods in organic synthesis and may be useful for stereo-and regioselective protections and protections in carbohydrates (Filice, M. et al. Nat. Protoc. .7, 1783-1796; 2012) And polyphenols (Torres, P. et al. J. Agric. Food ehem. 58, 807-813; 2010). Certain carbohydrates and polyphenols are of interest in the pharmaceutical and food industry as explained below.

Various epidemiological studies have shown the beneficial health effects that result from the consumption of antioxidant-rich foods, since they prevent the development of chronic degenerative diseases, including cardiovascular and cane diseases (Grez el aL, CurrOpin Pharmaco / 104, 362 -368; 2010). These compounds prevent damage caused by oxidative stress in certain biomolecules (AON, lipids, proteins), which are associated with an increased risk of cardiovascular problems, cancer and other chronic diseases (Cilla el al., Ann Nutr

Metab 54, 35-42; 2009).

Some studies show that natural polyphenolic compounds are, in vitro, more effective as antioxidants than vitamin A oe (Miller et aL, J Nutr Enviran Med 12, 39-51; 2002), so there is growing interest of this kind of compounds, especially those that can be obtained from by-products generated in the food industry. In this context, hydroxytyrosol stands out, which predominates in the olive tree (European Olea), both in the leaves and in its fruit, either free or as a derivative, and which is a phenolic compound of recognized efficacy as an antioxidant

(Fernández-Bolaños el al., CurrOrg Chem 12, 442-463; 2008).

In recent years, it has been proven that this natural antioxidant also has

important biological properties: platelet anliagreganle (González Correa el al. Br J Nutr, 101, 1157-1164, 2009), anllinflamalorio (Gong el al., Phytother Res 23, 646-650, 2009), cardioprolechlor (Poudyal, HJ Nutrition 140 , 946-953: 2010), anlimicrobial (Medina el al. J Agric Food Chem 55, 9817-9823; 2007), and anli-HIV (Lee-Huang el al., Biochem Biophys Res Commun 354, 872-878; 2007 ). Currently, there are numerous

research that attempts to demonstrate the benefit of this compound in the treatment of certain neurodegenerative diseases (Schaffer et aL, J Agric Food Chem 55,

5043-5049, 2007), and of the skin (Guo el al., Phytother Res 24, 352-359; 2010), in the

prevention of ischemic stroke and cancer prevention and treatment

(Sirianni el al., Mol Nutr Food Res 54, 833-640, 2010).

The olive contains, in addition to hydroxytyrosol, a wide variety of compounds

phenolics, such as 3,4-dihydroxyphenylglycol (Brenes, M.J. Agric Food Chem 47, 35353540; 1999) and prolocalcoic alcohol (Saina, M. el al. Food Chem 112, 525-532, 2009).

OH HO OH

HO ~ OH

HO ~ OH

HOA / HO HOA /

Hydroxytyrosol 3,4-Dihydroxyphenylglycol Protocol alcohol

Various studies on the phenolic compounds of the olive tree show the importance of the adl derivatives in the side chain of these natural polyphenolic compounds, since the lipophilic character allows their dispersion in oil, crossing cell membranes and cell uptake, and protection of membrane constituents and LDL (Gupta el al., Adv. Food Nu / r. Res. 69, 183-217; 2013). That is, the physical-chemical properties are improved and the antioxidant properties are preserved. Therefore, it is of great interest for the food industry to obtain new lipophilic antioxidants 10 by modifying natural polyphenols. These lipophilic structural analogues have a better bioavailability, are more effective than the natural polyphenols used as a starting material, and are useful in the formulation of "food

functional "that have beneficial health effects.

15 Bouallagui et al. (Bouallagui al., App / Biochem Bio / echno / 163, 592-599, 2011) have described the enzymatic production of hydroxytyrosol acetate and hydroxytyrosol oleate using C. antarctica lipase (Novozym 435), obtaining compounds with optimized biological activities in terms of bioavailability and antioxidant activity. The acylation catalyzed with G. antarctica B lipase takes place chemoselectively on the

20 hydroxyl of the aliphatic chain against aromatic hydroxyls (Torres de Pineda et al., Te / rahedron 61, 7654-7660; 2005).

On the other hand, sugars partially esterified or with different functions in the different carbons constitute important "building blocks" for the synthesis of a wide variety of more complex derivatives. Thus, for example, monosaccharides and oligosaccharides with a single free hydroxyl are intermediate compounds for the synthesis of heterooligosaccharides of biomedical interest and pharmacological activity as antitumor agents,

anti-inflammatory drugs, elc. (Plou el al., J Bio / echno / 96, 55-66; 2002).

Unfortunately, the high number of hydroxyl groups in sugars and their similar reactivity means that in many cases it is not possible to introduce regioselectively delenninated functions, or to resort to a long and expensive sequence of synthesis, to carry out transformations in only some of the hydroxyl groups.

Despite the interest of biocatalyzed carbohydrate reactions with lipases, the history of cases of regioselective protection or protection of monosaccharides in positions other than C-1 and e-6 are very scarce. Likewise, there are also very few examples in which regioselective protection / deprotection reactions in one of the two primary carbons (C-6 and C-6 ') of hexose disaccharides are described.

Therefore, there is a need in the state of the art to find new enzymes capable of carrying out regioselective acylation and / or deacylation reactions in order to obtain new compounds of interest in the pharmaceutical and food industry.

DESCRIPTION OF THE INVENTION

To overcome the problems in the prior art mentioned above, a first aspect of the present invention describes microbial strains selected from the list comprising Baci / lus strain deposited in the Spanish Type Culture Collection (CECT) with the number of Access CECT 8484, Enterobacter strain deposited in the Spanish Type Culture Collection (CECT) with the access number CECT 8462, Pseudomonas strain deposited in the Spanish Type Culture Collection (CECT) with the access number CECT 8463 and strain of Terribacilfus deposited in the Spanish Type Culture Collection (CECT) with the accession number CECT 8231 that have the ability to carry out regioselective reactions of acylation (esterification) and / or deacilation (alcoholysis), preferably on polyphenols and / or carbohydrates, preferably natural, which allows obtaining a collection of acylated and / or etherified compounds of interest in different po Sitions Thus, based on the ability of these bacteria to carry out these esterification and alcoholysis reactions, a series of inventive aspects have been developed that will be explained in detail below.

Another aspect described in the present invention relates to the enzyme extract obtainable from the culture of any of the bacterial strains described in the present invention.

5 Another aspect described in the present invention relates to the use of "bacterial strains

or to the use of the enzyme extract of the invention as catalysts for the enzymatic reactions of acylation and / or deacilation of compounds, with polyphenol compounds and / or carbohydrates or their acylated derivatives being preferred.

Another aspect described in the present invention relates to a method for obtaining acylated derivatives of polyphenols and / or carbohydrates by enzymatic acylation or deacylation reactions on polyphenols and / or carbohydrates, which comprises the following steps: a) placing the enzyme extract according to described in the present invention, in

Contact with polyphenols, carbohydrates or any of its acylated derivatives, in the presence of an acylating agent or an aliphatic alcohol to carry out enzymatic reactions of acylation or deacylation, respectively; and b) purifying the acylated derivatives of polyphenols and / or carbohydrates obtained in step (a).

Another aspect described in the present invention relates to the compound of the general formula

(one)

25 (1)

where: R1 is an alkyl group (Cl-C20), an alkenyl group (C2-Cm), an alkynyl group (C2-Cm), or an arylalkyl group; and R2 is an acyl group (-COR3), where R3 is a C1-C20 alkyl group, or a hydrogen.

Another aspect described in the present invention relates to the use of the compound of general formula (1) previously described for the preparation of a medicament.

Another aspect described in the present invention relates to a pharmaceutical composition comprising at least one compound of general formula (1) as previously described together with a pharmaceutically acceptable carrier.

Another aspect described in the present invention refers to the use of at least one compound of general formula (1) as previously described for the preparation of a food composition.

Another aspect described in the present invention refers to the non-therapeutic use of at least one compound of general formula (1) as previously described, as an antioxidant, preferably food, or as a component of cosmetic compositions.

Another aspect described in the present invention relates to a food or cosmetic composition comprising at least one compound of general formula (1) as previously described.

Another aspect described in the present invention relates to a process for obtaining the compounds of formula (1) as previously described throughout the present invention, which comprises:

to.

the enzymatic reaction of the peracylated 3,4-dihydroxyphenyl glycol compound with R, OH; or

b.

the enzymatic reaction of the compound 3,4-dihydroxyphenylglycol perazylated with R10H and subsequently the reaction with methanol of the compound obtained in step (a) under basic catalysis;

wherein R1 is a (C1-C20) alkyl group, an alkenyl (Cr C2o) group, an alkynyl (Cr C2o) group, or an arylalkyl group; as defined for substituent R1 of the general compound of formula (1).

Another aspect described in the present invention relates to a compound of formula 2,3,4,6,6'-penta-O-acetyl-a, ao-trehalose or 2,3,4,6-tetra-O-acetyl -a, aD-trehalose, as well as the use of said compounds as reaction intermediates for the synthesis of trehalose derivatives, as well as in the preparation of glycolipid analogs present in Mycobacterium tuberculosis.

Another aspect described in the present invention relates to a process for obtaining the compounds 2,3,4,6,6'-penta-O-acetyl-a, ao-trehalose or 2,3,4,6-tetra- O-acetyl-a, aD-trehalose comprising:

to. the enzymatic reaction of per-O-acetyl-Ct, a-D-trehalose with methanol or deuterated methanol at a temperature between 16-28 ° C.

DESCRIPTION OF THE FIGURES

Figure 1. Phylogenetic relationship of Terribaci / lus sp. AE2B 122, Bacillus sp. HR21-6 with other Gram positive bacteria (A), and Enterobactersp strains. AE1889 and Pseudomonas sp. AE28120 with other Gram negative bacteria (B) phylogenetically related. For its implementation, the Neighbor-Joining method has been used. Next to the branches is the percentage of replicas of the trees that present these taxa together in the same grouping in the bootstrap statistical test (500 replicas). Evolutionary distances have been calculated using the Maximum Composite Likelihood method, and are expressed in units of the number of base substitutions per site. The sequences comprised between 640 and 650 positions. For the phylogenetic analyzes, the MEGA4 program (http: //www.megasoftware.neUmega4/mega.html) has been used.

Figure 2. The anti-radical activity (free radical uptake by donation of hydrogen atoms) against the free radical DPPH (2,2-diphenyl-1-pyrrilehydracil) expressed in EC50 (~ M) values of hydroxytyrosyl acetate 2, of phenolic ethers 4a-4d, 5a-5d, and 3,4-dihydroxyphenylglyc14.

Figure 3. Nuclear Magnetic Resonance Spectrum CH-NMR) (A) and spectrum of

Extended nuclear magnetic resonance (1 H-NMR) (B) of the deacylation process of compound 27 (methyl 2,3,4,6-tetra-O-acetyl-Ct-D-glucopyranoside) by the enzyme extract of the invention.

Figure 4. Nuclear Magnetic Resonance Spectrum CH-NMR (A) and extended nuclear magnetic resonance spectrum (1 H-NMR) (B) of the deacylation process of compound 30 (per-O-acetyl-Ct, Ct-D- trehalose) by the enzymatic extract of the present invention.

Figure 5. Nuclear Magnetic Resonance Spectrum CH-NMR (A) and extended nuclear magnetic resonance spectrum (1 H-NMR) (B) of the deacylation procedure of compound 34 (per-O-acetylsacarose) by means of the enzyme extract of the present invention

Detailed description of the invention

The inventors of the present invention have identified a series of microbial strains, in particular, the bacterial strains Terribacil / us sp. AE2B 122, Bacil / us sp. HR21-6, Enterobacter sp. AE1889 and Pseudomonas sp. AE28120 deposited in the Spanish Type Crops Collection (CECT) with registration numbers 8231 (with deposit date 11/20/2012), 8464, 8462, 8463 (with deposit date of October 22, 2013), respectively, which have the ability to carry out regioselective reactions of acylation (for example stelification) and / or deacilation (for example alcoholysis). Said acylation and / or deacylation reactions are preferably carried out on polyphenols and / or natural carbohydrates, and etherification in benzyl positions of polyphenols during the alcoholysis of phenolic hydroxyl esters, which allows obtaining a collection of compounds of interest. acylated and / or etherified in different positions. Thus, based on the ability of these bacteria to carry out these esterification and alcoholysis reactions, a series of inventive aspects have been developed that will be explained in detail below.

Microbial strains of the invention and their uses

Through screening methods on samples obtained from different places of origin, such as oil mills, fish canning industries, etc., a series of microbial strains with lipolytic activity and ability to carry out regioselective esterification reactions (acylation) have been selected and / or alcoholysis (deacylation), in particular on carbohydrates and / or polyphenols, preferably natural.

These microbial strains were subjected to a phylogenetic analysis by analyzing the sequence of the 168 rRNA gene. From this analysis, the strains with the highest identity value were the microbial strains of the invention, being Terribacillus goriensis CL-GR16 (GeneBank accession number: NR_043895), Bacl1lus stratosphericus JCM 13349 (GeneBank accession number NR_042336) , Enterobaeter ludwigii EN-119 (GeneBank accession number AJ853891) and Pseudomonas stutzeri ATCC 17588 (Gene8ank accession number NR_103934).

Therefore, one aspect of the invention relates to the microbial strains of the invention, which belong to the genera Terribacillus, Bacillus, Enterobacter and Pseudomonas which, in another even more particular embodiment, are the microbial strains called Terribaei / lus sp. strain AE28122 (CECT 8231), Baei / lus sp. HR21-6 (CECT 6464), En / erobae / er sp. AE1889 (CECT 6462) and Pseudomonas sp. AE28120 (CECT 6463).

The microbial strains of the invention were deposited under the Budapest Treaty in the Spanish Type Culture Collection (CECT) [Parc Científic Universitat de Valencia, Agustín Escardino Professor Street, 9, 46980 Paterna (Valencia)] on November 20, 2012 ( CECT 8231), and October 22, 2013 (CECT 6464, CECT 6462 and CECT 8463).

Therefore, one aspect of the invention relates to the four microbial strains, capable of carrying out acylation I deacylation reactions that are deposited in the CECT with the registration numbers CECT 8231, CECT 8464, CECT 8462 and CECT 8463 In a particular embodiment, the preferred strains are: CECT 8464, CECT 6462 and CECT 6463.

As the person skilled in the art understands, the microbial strains of the invention can be grown in a liquid culture medium to obtain a supernatant which, after being subjected to concentration processes and optionally, lyophilization and / or dialysis, an extract is obtained enzymatic which, like any of the strains of the invention, can be used to carry out acylation and / or deacylation reactions, being useful in the production of the compounds of interest.

In addition, the microbial strains of the invention can be cultured in the presence or absence of a lipid substrate in order to obtain supernatants containing

the enzymes and compounds necessary to carry out the acylation reaction and deacylation.

For the purposes of the present invention, the term "lipid substrate 8" refers to a compound of a lipid nature, on which the lipases of the microbial strains will act. Examples of lipid substrates include, without limitation, surfactants, triglycerides, essential oils, more particularly, tributyrin, Tween 80, Tween 20 and vegetable oils, such as olive oil and sunflower oil. In a particular embodiment, the lipid substrate is tributyrin. The concentration of the lipid substrate in the culture medium can be variable, and concentrations of between 0.1 4% and even higher can be used. Preferably, the concentration of the lipid substrate in the culture medium is about 2% by weight with respect to the final culture.

Thus, in another aspect, the invention relates to an enzymatic extract obtainable from the culture of the microbial strains of the invention, capable of carrying out acylation and / or deacylation reactions. For the purposes of the present invention the terms supernatant and enzyme extract are used interchangeably.

For the purposes of the present invention, the term "enzyme extract" or "supernatant" refers to the supernatant obtainable from the culture of the strains of the invention, which may optionally be subjected to concentration, lyophilization and / or dialysis procedures and which comprises the Enzymes that the microbial strains described in the present invention excrete into the culture medium and that have lipolytic activity and that are capable of carrying out regioselective reactions of esterification (acylation) and / or alcoholysis (deacilation), that is, they are used as biological catalysts The enzyme extract or supernatant of the invention is, for the purposes of the present invention, an enzyme cocktail obtained from the cultures of the strains of the invention.

Without wishing to be bound by any theory, the inventors consider that, in the supernatant obtained from the cultures of the strains of the invention, in liquid medium, the enzymes necessary to carry out said acylation and / or deacilation reactions are included. Among the enzymes that can be part of said supernatant are lipases that are enzymes that catalyze a wide range of reactions (hydrolysis, intereslerifications, alcoholysis, acidolysis, slerification and

aminolysis, among others), including the reactions that are detected and shown in the tests and examples described throughout the present invention.

The supernatant obtained by culturing the strains of the invention may contain not only the enzymes necessary to carry out acylation and / or deacilation reactions, but may also contain other types of compounds that can interact with said enzymes, decreasing or enhancing the efficiency of conversion of the substrate to the products resulting from the reactions. Therefore, it may be convenient to subject the supernatant to a concentration and dialysis process to remove those undesirable particles or compounds.

In the present invention, "concentration and / or dialysis" means the process of separating the molecules in a solution by the difference in their diffusion rates through a semi-permeable membrane. Typically, a solution of several types of molecules is placed in a semipermeable dialysis bag, such as in a cellulose membrane with pores, and the bag is sealed.The sealed dialysis bag is placed in a container with a different solution, or pure water.The molecules small enough as to pass through the pores (often water, salts and other small molecules) tend to move in or out of the dialysis bag in the direction of the lowest concentration.The dialysis technique is widely known in the state of the technique and its implementation is routine for the person skilled in the art.In the practice of the present invention, the supernatant of the invention was poured in a 12 KDa dialysis bag and after being concentrated with 8 KDa polyethylene glycol, it was dialyzed with potassium phosphate buffer.

Therefore, in a particular embodiment, the enzyme extract described in the present invention, obtained from the cultures of the strains of the invention, is concentrated and / or dialyzed.

Additionally, whether the enzyme extract of the invention is concentrated and / or dialyzed or not, it can be subjected to a lyophilization process. Therefore, in another particular embodiment, the supernatant of the invention is lyophilized.

Freeze-drying is a process in which the product is frozen and subsequently introduced into a vacuum chamber to perform water separation by sublimation. In this way the water is removed from the solid state to the gaseous environment without going through the liquid state. The lyophilization technique is widely known in the state of the art and its application is routine practice for the person skilled in the art.

Once both the microbial strains of the invention are obtained, as well as the enzymatic extracts produced from their cultivation, and proven that they are capable of carrying out acylation reactions and deacilation, the person skilled in the art understands that both the microbial strains , like the enzymatic extracts of the invention, can be used to produce the compounds of interest indicated below.

Thus, in another aspect, the invention relates to the use of microbial strains or enzymatic extracts obtained from cultures of said strains, according to the invention, for the production of adhered derivatives, preferably the starting substrates, polyphenols and / or carbohydrates, natural or not.

In a particular embodiment, the production of acylated derivatives of polyphenols and / or carbohydrates is carried out by regioselective reactions of acylation or deacylation of polyphenols and / or carbohydrates, preferably natural.

For the purposes of the present invention the terms "regioselective" or "regioselectivity" refer to the preference of an esterification or hydrolysis reaction in an aliphatic hydroxyl of several possible, or an aromatic hydroxyl of several possible.

For the purposes of the present invention the terms "chemoselective" or "chemoselectrivity" refer to the preference of an esterification or alcoholysis reaction in an aliphatic hydroxyl (alcohol) over an aromatic hydroxyl (phenal) or vice versa.

More details on the production of acylated derivatives of polyphenols and / or carbohydrates through the use of microbial strains or enzymatic extracts thereof, described in the present invention, can be found in Examples 3 and 5 where the method of production of them.

Biotransformac; ones regiase / ectivas for obtaining acylated derivatives II polophilic polyphene / es and / or carbohydrates.

As explained above, the inventors have discovered new microbial strains capable of carrying out acylation and / or deacylation reactions on polyphenols and / or carbohydrates, preferably natural or not, whereby both microbial strains, such as enzymatic extracts obtained from the cultivation thereof, can be used in obtaining the compounds indicated below,

Thus, in another aspect, the invention relates to a method for obtaining acirated derivatives of polyphenols and / or carbohydrates, preferably from polyphenols and / or natural carbohydrates, by acylation or deacylation reactions on polyphenols and / or carbohydrates, which comprises the following stages:

a) placing the enzyme extract as defined throughout the present invention, in

contact with pOlifenoles, carbohydrates or any of its acylated derivatives, in

presence of an acylating agent or an aliphatic alcohol to carry out reactions

enzymatic acylation or deacilation, respectively and, optionally

b) purify the acylated derivatives of polyphenols and / or carbohydrates obtained in the

stage (a).

For the purposes of the present invention, the term "purify" or "purification" refers, as used in the description, to the separation of the clarified derivatives of polyphenols and / or carbohydrates obtained by the method described in the invention and its concentration. The separation of said compounds from the rest of the products that can be flared and that are not of interest can be carried out by means of differential solubility techniques, chromatography, electrophoresis or isoelectric focusing. Chromatography techniques can be based on molecular weight, loading or affinity of the protein and can be performed on a column, on paper or on a plate. The separation of the compounds can be carried out, for example, by fast liquid chromatography (FPLC), in an automated system that significantly reduces the purification time and increases the purification performance.

In a stage prior to step a) of the method of the invention, the microbial strains of the invention are grown in the presence or absence of a lipid substrate in order to obtain enzymatic extracts containing the enzymes and compounds necessary to carry carry out the acylation and / or deacylation reaction on the

5 polyphenols and / or carbohydrates.

The bacterial strains of the invention are grown in a liquid culture medium to extract the supernatant and subsequently obtain the enzyme extracts. For Terribacillus sp. AE2B 122 (CECT 8231), Bacillus sp. HR21-6 (CECT 8234) has been

10 used as a culture medium, PYA (agar, peptone and yeast extract). In the case of Enterobactersp. AE1B89 (CECT 8232) and Pseudomonas sp. AE2B120 (CECT 8233) has been used as a culture medium, LB (Luria-Bertani), although it is important to mention that any other culture medium could be used in which the strains of the invention can grow.

Optionally, the liquid culture can be supplemented with a lipid substrate to increase the secretion of the enzymes present in the enzyme extract of the invention, secreted by the strains of the invention and which are responsible for carrying out the acylation reactions. deacilation on polyphenols and / or carbohydrates.

As previously mentioned, said enzymes are considered to be, preferably, lipases. Thus, in a particular embodiment, the culture medium may be supplemented with a lipid substrate, according to as previously described in the present description of the invention.

25 Cultures are carried out at a temperature between 22 ° C and 37 ° C, with stirring for 2-3 days. After these days, the supernatant which, as mentioned above, will contain the enzymes, preferably lipases, responsible for carrying out the reactions described in the present invention.

30 The supernatant resulting from the cultures can be isolated by routine techniques for the person skilled in the art. For example, after the incubation period has elapsed, the culture can be allowed to stand so that the cells decant or it can be subjected to centrifugation, thus obtaining both ways to separate the cells from the liquid culture medium. Subsequently, the supernatant can be obtained, for example by aspiration with a pipette.

Once the enzyme extract is obtained, it is contacted with the compounds

5 who want to undergo acylation / deacilation reactions [step (a)]. Suitable conditions for carrying out these reactions are explained in detail in each of the examples contained in the present invention.

In a preferred embodiment, the starting compounds are preferably polyphenols or carbohydrates.

In another preferred embodiment, the phenolic type substrates of step a) of the method of the invention are preferably selected from any of the following: hydroxytyrosol, 3,4-dihydroxyphenylglycol, protocatecholic alcohol and acetylated derivatives thereof.

15, preferably: triacetylated hydroxytyrosol, 3,4-dihydroxyphenylglocol tetraacylated and peracylated protocatecholic alcohol.

In another preferred embodiment, the carbohydrate type substrates of step a) of the method of the invention are selected from mono-, di-, oligo-, polysaccharides or any of

20 its acylated derivatives, preferably: per-O-acetylated methyl a-D-glucopyranoside, octaacetylated trehalose and octaacetylated sucrose.

By "acetylated derivatives" refers to phenolic compounds or carbohydrates with at least one -OH group substituted by a -OCOCH3 group.

As previously explained, herein, supernatants obtained at a stage prior to step a) of the method of the invention may be concentrated and / or dialyzed, as previously explained, or may be used as is.

Therefore, in a particular embodiment, the method of obtaining acylated derivatives of polyphenols and / or carbohydrates of the invention comprises between steps (a) and (b), an optional step directed to the concentration and / or dialysis of the supernatants as well as

to its subsequent lyophilization. Concentration, dialysis and lyophilization techniques are described in detail in Example 2.

The different acylation reactions of phenolic compounds and / or carbohydrates are detailed below by means of the enzymatic reactions catalyzed by the enzymes present in the supernatants or enzymatic extracts collected from the cultures of the strains described in the present invention, to obtain phenolic compounds CQ5 and / or acylated lipafilic carbohydrates.

In a preferred embodiment, the enzymatic reaction is deacetylation, the polyphenol of step (a) is an acetylated derivative of hydroxytyrosol, 3,4-dihydroxyphenyl glycol or protocatecholic alcohol, preferably triacetylated hydroxytyrosol, 3,4-dihydroxyphenylglocol tetraacylated alcohol perazylated, and the reaction is carried out in the presence of an aliphatic alcohol.

By "aliphatic alcohol" is meant in the present invention a linear (branched-C7) alkyl group, with at least one -OH radical, for example, but not limited to methanol, ethanol, propanol, butanol, among others Preferably the aliphatic alcohol is methanol (MeOH) The aliphatic alcohol may be optionally deuterated at room temperature (approx. 18-30'C).

In a more preferred embodiment, the enzymatic reaction is deacylation, the polyphenol of step (a) is a triacetylated hydroxytyrosol and the reaction is carried out in the presence of methane!

In another preferred embodiment, the enzymatic reaction is of deacylation of the aromatic hydroxyls, the polyphenol of step (a) is 3,4-dihydroxyphenyl glycol tetraacylated or peracylated protocatechogric alcohol and the reaction is carried out in the presence of an aliphatic alcohol to produce the etherification in the benzyl position.

In another preferred embodiment, the enzymatic reaction is acylation, the step polyphenol

(a) it is hydroxytyrosol, 3,4-dihydroxyphenyl glycol or protocatechic alcohol and the reaction is carried out in the presence of an acylating group and optionally a ca-solvent.

Accepting group means a reagent capable of reacting with a compound by adding an acyl group to its structure, for example, the acylating group can be without being limited to isopropenyl acetate or vinyl acetate, which in turn acts as a solvent. .

The acylation reaction can be done in the absence or presence of an adsorbent of the culture supernatant of the strains of the invention, such as celite. Better results are obtained with the enzyme extract supported on celite. If the phenolic compound used is poorly soluble in the acylating agent, a CQ can be used

10 solvent, for example DMF (N, N-dimethyl formamide).

In another preferred embodiment, the enzymatic reaction is deacetylation, the carbohydrate of step (a) is methyl per-O-acetylated aD-glucopyranoside, per-O-acetyl-a, aD-trehalose, per-O-acetyl sucrose, octaacetylated trehalose or octaacetylated sucrose and the reaction is

15 carried out in the presence of aliphatic alcohol, preferably methanol or deuterated methanol at room temperature (16-28 ° C).

In a more preferred embodiment, the enzymatic reaction is from per-O-acetyl-a, a-Dtrehalosa with methanol or deuterated methanol at a temperature between 16-28 ° C,

The compounds 2,3,4,6,6'-penta-O-acetyl-a, ao-trehalose or 2,3,4,6-tetra-0acetyl-a, aD-trehalose or the enzymatic reaction of per -O-acetyl-sucrose with methanol or deuterated methanol at a temperature between 16-28 ° C, obtaining the compounds 2,3,4,6, l ', 6'-hexa-O-acetyl-sucrose or 2,3 , 4,6,6'-penta-G-acetyl-sucrose.

In another preferred embodiment, the enzymatic reaction is acylation, the carbohydrate of step (a) is methyl a-D-glucopyranoside and the reaction is carried out in the presence of isopropenyl acetate and OMF at a temperature between 50-700C.

Another aspect of the present invention describes a compound of general formula (1)

30 OR,

HO ~ OR2

HOJvJ

(one)

where: R1 is an alkyl group (Cr C2o), an alkenyl group (C2-C20), an alkynyl group (C, -C ,,), or an arylalkyl group; and R2 is an acyl group (-COR3, where R3 is a C, · C20 alkyl group) or a hydrogen.

By "alkyl" refers in the present invention to an aliphatic chain, linear or branched, having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms and more preferably 1 to 5 carbon atoms, for example , methyl, ethyl, n-propyl, propyl, n-butite, t-butyl, n-pentyl, among others.

By "alkenyl" in the present invention refers to a carbon chain, linear or branched, having at least one double bond and containing between 2 and 20 carbon atoms, preferably between 2 and 10 carbon atoms and more preferably from 2 to 5 carbon atoms

By "alkynyl" refers in the present invention to a carbon chain, linear or branched, which has at least one triple bond and contains between 2 and 20 carbon atoms, preferably between 2 and 10 carbon atoms and more preferably from 2 to 5

carbon atoms

By "arylalkyl" is meant in the present invention an aryl group attached to the rest of the molecule by an alkyl group (C, -C20), as defined above. A non-limiting example of arylalkyl is a benzyl group.

In a preferred embodiment, the compound of general formula (1) of the present invention is characterized in that R2 is an acetyl group (-COCH3).

In a preferred embodiment, the compound of general formula (1) of the present invention is characterized in that R2 is a hydrogen.

In another more preferred embodiment, the compounds of the general formula (1) are selected from the list comprising: 2- (3,4-dihydroxyphenyl) -2-methoxyethyl acetate, 2 (3,4- <1-hydroxyphenyl) acetate - 2-ethoxyethyl, 2- (3,4-dihydroxyphenyl) -2-propoxyethyl acetate, and acetate

2- (3,4-dihydroxyphenyl) -2-butoxyethyl.

In another preferred embodiment, the compounds of the general formula (1) are selected from the list comprising: 2- (3,4-dihydroxyphenyl) -2-methoxyethanol, 2- (3,4-dihydroxyphenyl) -2ethoxyethanol, 2- ( 3,4-dihydroxyphenyl) -2-propoxyethanol and 2- (3,4-dihydroxyphenyl) -2-butoxyethanol.

The present invention also describes the process of obtaining the compounds of formula (1) described above, comprising:

to. the enzymatic reaction of the peracylated 3,4-dihydroxyphenyl glycol compound with

R, OH; or

b. the enzymatic reaction of the compound 3,4-dihydroxyphenyl glycol perazylated with, or R 10 H and subsequently the reaction of the compound obtained in step (a)

by basic catalysis, preferably with C ~ C031 in methanol; where R, is an alkyl group (C, -C ,,), an alkenyl group (C, -C ,,), an alkynyl group (C, C20). or an arylalkyl group.

OR1

OAc OR, C t • 'd

HO ~ OAc a. aCI to HO ~ OH

AcO ~ OAC Enzymatic Extract,

M

HOV MeOH HO

M

15 AcO

In a preferred embodiment, the process described above is characterized in that the enzymatic reaction is carried out in the presence of the enzymatic extract described in the present invention.

The present invention also relates to the use of at least one general formula (1), as previously described, for the preparation of a medicament or a cosmetic composition or a food composition, where the composition

25 food is selected from a food, food supplement, functional food or nutraceutical.

For the purposes of the present invention the term "medicament" refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals.

For the purposes of the present invention the term "pharmaceutical composition" as defined in the present invention refers to a set of components that are formed by at least one product of general formula (1) as described in the present invention, in any concentration, which has at least one application in improving the physical or physiological or psychological well-being of a subject, which implies an improvement in the general state of his health. Said compositions further comprise at least one pharmaceutically acceptable carrier.

The pharmaceutical composition may comprise a "vehicle" or carrier, which is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the pharmaceutical composition.

The compounds and compositions of the present invention can be used together with other medicaments in combination therapies. The other drugs may be part of the same composition or of a different composition, for administration at the same time or at different times.

For the purposes of the present invention the term "cosmetic or cosmetic composition" refers to those preparations consisting of natural or synthetic substances or mixtures thereof, for external use in the various parts of the human body.

For the purposes of the present invention the term "food composition" refers to that food that, regardless of providing nutrients to the subject who takes it, beneficially affects one or more functions of the organism, so that it provides a better state of health and wellness. As a consequence, said composition may be intended for the prevention and / or treatment of a disease or the causative factor of a disease. Therefore, the term "food composition" of the present invention can be used as a synonym for functional food or food for specific nutritional purposes or medicinal food. For the purposes of the present invention, the term "food composition" is, or is part of a food, functional, nutraceutical or food supplement.

For the purposes of the present invention the term "nutraceutical" as used in the present invention refers to substances isolated from a food and used in a dosage form that have a beneficial effect on health.

For the purposes of the present invention the term "supplement", synonymous with any of the terms "dietary supplement", "nutritional supplement" or "food supplement" is a "food ingredient" intended to supplement the diet.

The present invention also relates to the non-therapeutic use of at least one compound of general formula (1), as previously described, as an antioxidant, preferably as a food antioxidant.

The term "antioxidant" means a composition capable of retarding or preventing the oxidation of other molecules,

Another object described in the present invention relates to a pharmaceutical composition comprising at least one compound of general formula (1), as previously described, together with a pharmaceutically acceptable carrier.

Another object described in the present invention relates to a food or cosmetic composition comprising at least one compound of general formula (1), as previously described,

Another aspect described in the present invention relates to acylated carbohydrate derivatives, preferably compounds of the formula 2,3.4.6,6 '· penta · O-acetyl · a, a · O · trehalose or 2,3,4 , f3..tetra · O-acetyl ... a, ao - trehalose.

Another aspect described in the present invention relates to the use of said acylated carbohydrate derivatives previously described as reaction intermediates for the synthesis of trehalose derivatives or for the elaboration of glycolipid analogs present in Mycobacterium tuberculosis.

Another aspect described in the present invention relates to the method of obtaining the glycolipid analogs present in Mycobacten'um tuberculosis comprising:

to. the enzymatic reaction of per-O-acetyl-a., a-o-trehalose with methanol or deuterated methanol at a temperature between 16-28 ° C.

In a preferred embodiment, said process is carried out in the presence of an enzyme extract as defined in the present invention.

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 are

10 will come 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.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.

Example 1: Phylogenetic analysis of Terribaci / lus sp. AE28 122 (CECT

20 8231), Bacillus sp. HR21-6 (CECT 8464), Enlerobacler sp. AE1B89 (CECT 8462) and Pseudomonas sp. AE2B120 (CECT 8463).

The phylogenetic study was carried out by sequence analysis of the ribosomal ribonucleic acid (rRNA) 16S gene. For this, the 16S rRNA gene of each of the strains of the invention was amplified by polymerase chain reaction (PCR) using primers 16F27, whose sequence is 5'-AGAGTTTGATCMTGGCTCAG-3 '(SEO ID NO: 1 ). and 16R1488, with the sequence 5 · -CGGTTACCTTGTTAGGACTTCACC-3 '(SEO ID NO: 2) (Moreno M.L., et al. FEMS Microbio / Eco /, 2009; 68, 59-71.). The PCR reaction conditions were as follows: a cycle of 95 oC for 5 minutes, 30 25 cycles of 94 oC for 1 minutes, 50 oC for 1 minutes and 72 oC for 2 minutes, and then an extension step of 10 minutes at 72 oC. The amplification products were analyzed by agarose gel electrophoresis. The amplified AON is sequenced by the Sanger method. Between 640 and 650 bp are used to search for similar sequences between sequences deposited in GenBank, using the algorithm

BlastN, proving to be Terribacillus goriensis CL · GR16 (GeneBank accession number NR_043895), Bacillus stratosphericus JCM 13349 (GeneBank accession number

NR_042336), Enterobae / er ludwigii EN-119 (GeneBank accession number AJ853891) and

Pseudomonas stulzeri ATCC 17588 (GeneBank accession number NR_103934) which show the highest identity value with the sequences of the strains of the invention. Therefore, the strains of the invention were assigned to the Terribaciffus genera as

Terribaeillus sp. AE2B 122 (CECT 8231), Baeillus, as Baeillus sp. HR21-6 (CECT 8484), Enterobaeter, as Enterobaetersp. AE1B89 (CECT 8462) and Pseudomonas as Pseudomonas sp. AE2B120 (CECT 8463).

To carry out the phylogenetic tree, the MEGA4 program package was used. First, a multiple alignment was performed, using Clustal W, of the 650 bp sequences (corresponding to the fragment between positions 53 to 703 (Figure 1A) and between positions 80 to 730 (Figure 1B) of Escherichia coll rRNA 168) of the strains and the sequences of greater identity obtained with BlastN. Finally the trees were built using several methods, obtaining similar results with all of them. Trees constructed by the Neighbor-Joining method are shown in the present invention (Figure 1).

Example 2: Preparation of the supernatants of the Terribacillus sp. AE2B

122 (CECT 8231), Baeitlus sp. HR21-6 (CECT8464), Enterobactersp. AE1B89 (CECT 8462) and Pseudomonas sp. AE2B120 (CECT 8463).

For the preparation of the enzyme extracts or supernatants, a pre-circle of each of the four strains described in the present invention was grown. Terribaciffus sp. AE2B 122 (CECT 8231) was grown in PYA medium overnight. The next day, said culture was used to inoculate PYA medium. After growing the culture in PYA medium, the supernatant was collected.

Baeillus sp. HR21-6 (CECT8464) was grown in LB medium overnight. To the day

next, said culture was used to inoculate PYA medium After growing the culture in PYA medium, the supernatant was collected.

In / erobae / er sp. AE1B89 (CECT 8462) and Pseudomonas sp. AE2B120 (CECT 8463) were grown in LB medium overnight. The next day, said culture was used to inoculate LB + tributyrin (TRI) medium 2%. After growing the culture in LB + 2% TRI medium, the supernatant was collected.

Each of the supernatants was poured into a 12 KDa dialysis bag (SIGMA, 09527). The dialysis bags were left overnight at 4 oC covered in 8 KDa polyethylene glycol and when the dialysis bags were completely dry, the exterior was cleaned with distilled water and dialyzed with potassium phosphate buffer O, 05M pH 7.8 a 4 oC and magnetic stirring. These dialysates were frozen at -80 oC.

Such supernatants can be lyophilized in order to prevent hydrolysis reactions from occurring. In addition, lyophilizing the supernatant helps to concentrate and preserve the enzyme of the microbial strain, and also to be stored.

Example 3: Preparation of lipophilic polyphenols. Acylation de-acylation reactions catalyzed by the supernatants / enzymatic extracts of Terribaeillus strains .p. AE2B 122 (CECT 8231), Baeillus .p. HR21-6 (CECT 8464), Enterobae / er .p. AE1B89 (CECT 8462) and Pseudomonas .p. AE2B120 (CECT 8463).

The following are illustrative and non-limiting examples of synthetic routes that lead to obtaining phenolic derivatives by regioselective deacylation catalyzed by enzymatic extracts / supernatants in an alcoholic medium.

Example 3.1 Reaction of deacilation of phenolic compounds by enzymatic catalysis to obtain lipophilic polyphenols.

Example 3.1.1.

Hydroxytyrosyl acetate (2) The deacylation of compound 1 (peracetylated hydroxytyrosol) by alcoholysis (deacylation) catalyzed by enzymatic extracts, obtained from the cultures of the strains of the invention, using as an solvent an aliphatic alcohol, such as MeOH, gives

place the monoacy derivative (compound 2) by a fully regioselective reaction, as described in Scheme 1.

ACO ~ OAC enzymatic extract HO ~ OAC

I • I

AcO .ó MeOH HO .ó

1 2

5 Scheme 1

To a solution of compound 1 (triacetylated hydroxytyrosol) (50 mg, 0.18 mmol) in MeOH is added the enzyme extract obtained from the culture of the bacterial strain Terribaciffus sp. strain AE2B122 (CEC T8231) (50 mg) and silica gel (63-200 ~ m, 100

10 mg) Stir at 40 'C for 18 hours. Filter on eelite and concentrate to dryness. The residue is purified by column chromatography (AcOEt-hexane 1: 2 _ 1: 1) to obtain hydroxytyrosyl acetate 2 (31 mg, 90%). (See Scheme 1). The yield for obtaining compound 2 purified by chromatographic techniques was 75-91% depending on the enzyme extract used.

15 Example 3.1 .2. The deacylation of compound 3 (3,4-dihydroxyphenyl glycol peracetylated) by alcoholysis catalyzed by the enzymatic extracts described in the present invention and using as an solvent an aliphatic alcohol of the R'OH type, takes place in a manner

20 fully regioselective in phenotic esters (Scheme 2). In the benzylic position the acyloxy group is substituted by the alkoxy group R, O from the alcohol resulting in the formation of compound 4 which can be isolated with yields of 40-90%. The radical R 'of the aliphatic alcohol of type R10H is preferably selected from any of the following: -CH, (methyl), -C, H, (ethyl), -C, H, (propyl) or -C, H, (butyl).

Depending on the aliphatic alcohol used as solvent, compounds 4a, 4b, 4c or 4d are obtained (Scheme 2). Reaction times range from 24 h for obtaining compound 4a to 6 days for obtaining compound 4d. The acetyl group of compound 4 can be removed by an alcoholysis (deacylation) with basic catalysis, using as catalyst any basic catalyst, preferably CS2C03

30 in methane!

Thus, compounds 4a-4d by a deacetylation reaction give rise to compounds 5a-5d with yields of 31-62% (Scheme 2).

OAc ACO ~ OAC ACOV ROH enzyme extract

OR HO ~ OAC Cs, C03 HOV MeOH OR HO ~ OH HOV

3

4 5

4. R = Me

53 R = Me

4b R = Et

5b R = Et

4c R = Pr

5c R = Pr

4d R = Bu

5d R = Bu

5

Scheme 2

2- {3,4-Dihydroxyphenyl) -2-methoxyethyl acetate (4a)

OMe

OAc

10 HO

Compound 3 (tetraacetylated) (28 mg, 0.08 mmol) is dissolved in MeOH (3 mL) and

add 28 mg of enzyme extract obtained from the culture of the bacterial strain

Terribacillus sp. strain AE2B122 (CEC T8231) and 56 mg of silica gel (63-200 ~ m). Be

15 stir at room temperature for two days. Another 28 mg of enzyme extract obtained from the culture of the bacterial strain Terribacillus sp. strain AE28122 (CEe T8231) and stirring is continued at room temperature for two days. It is concentrated to dryness and the product is purified by column chromatography (CH2CI2

MeOH 60: 1). Yield: 17 mg, 90%, R, 0.6 (CH, CI, .. MeOH 10: 1).

'H-NMR (300 MHz, CD, OD): ~ 6.80 (d 1 H, J,. • = 8.0 Hz, Ar-5), 6.79 (d, 1 H, J,. • = 2.0 Hz, Ar- 2), 6.68 (dd, 1H, J •., = 8.0 Hz, J •., = 2.0 Hz, Ar-6), 4.32 (dd, 1H, J = 8.0 Hz, J = 3.9 Hz, CH), 4.18 (dd, 1H, J = 11.4 Hz, J = 8.0 Hz, CH,), 4.07 (dd, 1H, J = 11, 4 Hz, J = 3.9 Hz, CH,), 3.25 (s 3H, OMe) , 2.06 (s 3H, OAc). "C-NMR (75.5 MHz, CD, OD): ~ 173.6 (CO),

145.7 (Ar-3 and 4), 131.4 (Ar-1), 120.7 (Ar-6), 117.2 (Ar-5), 115.8 (Ar-2), 83.4 (CH), 69.5 (CH,), 57.7 ( OMe), 21.6 (OAc).

5 2- (3,4-Dihydroxyphenyl) -2-ethoxyethyl acetate (4b) OE! HO OAe

HO

Compound 3 (60 mg, 0.18 mmol) is dissolved in EtOH (6 mL) and 60 mg of enzyme extract obtained from the bacterial strain Bacillus sp. HR21-6 (CECT

10 8464) And 120 mg of silica gel (63-200 ~ m). It is heated at 60 oC for 24 hours and

after that time it is concentrated to dryness and the product is purified by silica gel column chromatography (CH2Cb-EtOH 60: 1). Yield: 24 mg, 55%, RF 0.5

(CH, CI, -MeOH 10: 1).

15 'H-NMR (300 MHz, CDCI,): 5 6.89 (d, 1H, J,., = 2.0 Hz, Ar-2), 6.83 (d, 1H, J5 = 8.1 Hz, Ar5), 6.72 (dd, 1H, J, .5 = 8.1 Hz, J ,,, = 2.0 Hz, Ar-6), 4.42 (dd, 1H, J = 6.9 Hz, J = 5.0 Hz, CH), 4.20-4.12 (m, 2H, CH,), 3.43 (2dd, 1H each, 'J = 16.4 Hz,' J = 7.0 Hz, CH, CH,), 2.06 (s, 3H, OAc), 1.17 (t, 3H, J = 7.0 Hz, CH, CH,). 13C-NMR (75.5 MHz, CDCI,): 5 172.0 (CO),

144.5 (Ar-4), 144.4 (Ar-3), 131.0 (Ar-1), 119.9 (Ar-6), 115.5 (Ar-5), 113.9 (Ar-2), 79.6 20 (CH), 68.4 ( CH,), 64.8 (CH, CH,), 21.3 (OAe), 15.4 (CH, CH,).

2- (3,4-Dihydroxyphenyl) -2-propoxyethyl acetate (4c) OPr HO OAe

HO

Compound 3 (190 mg, 0.56 mmol) is dissolved in PrOH (4 mL) and 190 mg of enzyme extract obtained from the bacterial strain Bacillus sp. HR21-6 (CECT

8464) and 380 mg of silica gel (63-200 ~ m). Stir at 60 oC for two days. Be

add another 190 mg of the same extract and stirring is continued for two days.

It is concentrated to dryness and the product is purified by column chromatography (AcOEt

hexane 1: 3 ~ 1: 1). Yield: 81.4 mg, 57%, RF 0.6 (AeOEt-hexane 1: 1).

'H-NMR (300 MHz, CDCI,): 6 6.90 (d, lH, J,. • = 2.0 Hz, Ar-2), 6.84 (d, lH, J,. • = 8.1 Hz, Ar

5 5), 6.73 (dd, 1H, J •., = 8.1 Hz. J,., = 2.0 Hz, Ar-6). 4.41 (dd, lH, J = 7.6 Hz, J = 4.5 Hz, CH), 4.21-4.10 (m, 2H, CH,), 3.40-3.24 (m, 2H, OCH, CH,), 2.06 (s 3H, OAe), 1.59 (sextet, 2H, J = 7.4 Hz, OCH, CH,), 0.88 (t, 3H, J = 7.4 Hz, CH,). "C-NMR (75.5 MHz, CDCI,): 6

171.9 (CO), 144.4 (Ar-4), 144.3 (Ar-3), 131.2 (AH), 120.0 (Ar-6), 115.5 (Ar-5), 113.9 (Ar2), 79.7 (CH), 71.1 ( CH,), 68.4 (OCH, CH,), 23.1 (CH, CH,), 21.3 (OAe), 10.8 (CH,).

2 ~ 3,4-dihydroxyphenyl) -2-butoxyether (4d) acetate OBu HO OAe

HO

Compound 3 (190ng, 0.56 mmol) is dissolved in BuOH (4 mL) and 190 mg of enzyme extract obtained from the bacterial strain Bacillus sp. HR21-6

(CECT 8464) and 380 mg of siliee gel (6 ~ 200 ~ m), heating at 60 oC for 2

days. It is concentrated to dryness and purified by column chromatography with AcOEt

Hex 1: 2 Yield: 91 mg, 61%, RF 0.6 (CH, CI, -MeOH 10: 1).

20 'H-NMR (300 MHz, CDCI,): 6 6.90 (d, 1H, J,., = 1.9 Hz, Ar-2), 6.84 (d, lH, J,., = 8.0 Hz, Ar5), 6.73 (dd, lH, J,., = 8.0 Hz, J •., = 1.9 Hz, Ar-6), 6.50 (s, lH, OH), 5.95 (s, lH, OH), 4.44 (dd, lH , J = 7.7 Hz, J = 4.4 Hz, CH), 4.22-4.10 (m, 2H, CH,), 3.45-3.28 (m, 2H, OCH, CH,),

2.07 (s, 3H. OAe), 1.55 (m, 2H, J = 6.6 Hz. OCH, CH,), 1.39-1 .25 (m, 2H, CH, CH,). 0.86 (t, 3H, J = 7.3 Hz, CH, CH,). "C-NMR (75.5 MHz, CDCI,): 6 172.1 (CO), 144.4 (Ar-4),

25 144.3 (Ar-3), 131.1 (Ar-l), 120.0 (Ar-6), 115.5 (Ar-5), 113.9 (Ar-2), 79.7 (CH), 69.2 (OCH, CH,), 68.4 (CH,), 32.0 (OCH, CH,), 21.3 (OAe), 19.6 (CH, CH,), 14.2 (CH, CH,).

2- (3,4-Dihydroxyphenyl) -2-methoxyethanol (5a)

OMe

HO OH

HO

To a solution of compound 4a (54 mg, 0.24 mmol) in dry MeOH (1 ml) is added

Cs, CO, (231 mg, 0.72 mmol) and sodium ascorbate (48 mg, 0.24 mmol) in the presence of Ar

and in the dark Stir at room temperature for 4 hours. When the reaction is terminated, the compound is purified, without neutralizing or evaporating the solvent, by column chromatography using CH2Clr EtOH as eluent (1: 0 _ 1:10).

10 Yield: 23 mg, 53%, R, 0.2 (CH, CI, -MeOH 10: 1).

'H-NMR (300 MHz, 0.0): 66.94 (d, 1H, J ", = 8.1 Hz, Ar-5), 6.89 (d, 1H, J,., = 2.0 Hz , Ar2), 6.80 (dd, 1 H, J ", = 8.1 Hz, J", = 2.0 Hz, Ar-6), 4.29 (dd, 1 H, J = 7.5 Hz, J = 4.6 Hz, CH),

3.71 (dd, 1H, J = 11.9 Hz, J = 7.5 Hz, CH,), 3.64 (dd, 1H, J = 11.9 Hz, J = 4.6 Hz,

15 CH,), 3.27 (s, 3H, OMe), "C-NMR (75.5 MHz, 0.0): 6 144.1 (Ar-1), 144.0 (Ar-2), 130, 5 (Ar-4), 119.8 (Ar-5), 116.1 (Ar-6), 114.7 (Ar-3), 83.6 (CH), 65.0 (CH,), 56.0 (OMe),

2- (3,4-0ihydroxyphenyl) -2-ethoxyetanot (5b) OE!

HO OH

HO

To a solution of compound 4b (25 mg, 0.10 mmol) in dry MeOH (1 ml) is added

Cs, CO, (66 mg, 0.20 mmol) and sodium ascorbate (20 mg, 0.10 mmol) in the presence of Ar and

in the dark Stir at room temperature for 3 hours. When the reaction is terminated, the compound is purified, without neutralizing or evaporating the solvent, by column chromatography using CH2CI2-EtOH as eluent (1: 0 _ 1:10).

Yield: 12.4 mg, 62%, R, 0.3 (CH, CI, -MeOH 10: 1).

'H-NMR (300 MHz, CO, OO): ~ 6.91 (d, 1H, J •. • = 8.1 Hz, Ar-5), 6.88 (d, 1H, J,., = 1.8 Hz, Ar-2 ), 6.80 (dd, 1H, J,. • = 8.1 Hz, J,., = 2.0 Hz, Ar-6), 4.38 (dd, 1H, J = 7.7 Hz, J = 4.4 Hz, CH), 3.70 ( dd, 1H, J = 11 .7 Hz, J = 8.0 Hz, CH,), 3.61 (dd, 1H, J = 11 .7 Hz, J = 4.4 Hz, CH,) 3.48 (e, 2H, J = 6.9 Hz, CH, CH,), 1.19 (t, 3H, J = 7.1 Hz, CH, CH,). 13C-NMR (75.5

5 MHz, CO, OO): ~ 146.1 (Ar-4), 145.9 (Ar-3), 133.1 (Ar-1), 121.3 (Ar-6), 117.7 (Ar-5), 116.3 (Ar-2) , 83.9 (CH), 67.3 (CH,), 66.3 (CH, CH,). 16.1 (CH, CH,).

2- (3,4-0 i hid roxife nil) -2-pro poxielanol (Se) OPr

HO ~ OH

HO) lJ

To a solution of compound 4c (55 mg, 0.22 mmol) in dry MeOH (1 mL) is added

Cs, CO, (66 mg, 0.20 mmol) and sodium ascobate (44 mg, 0.22 mmol) in the presence of Ar and

in the dark Stir at room temperature for 2 hours. When the reaction is terminated the compound is purified, without neutralizing or evaporating the solvent,

15 by column chromatography using as eluent (AcOEt-hexane 1: 1). Yield: 14.5 mg, 31%, R, 0.1 (AcOEt-Hex 1: 1).

'H-NMR (300 MHz, CO, OO): ~ 6.78 (d, 1H, J,., = 1.9 Hz, Ar-2), 6.76 (d, 1H, J •., = 8.0 Hz, Ar-5 ), 6.65 (dd, 1H, J,. • = 8.0 Hz, J,., = 2.0 Hz, Ar-6), 4.22 (dd, 1H, J = 8.0 Hz, J = 4.1 Hz, 20 CH), 3.64 (dd, 1 H, J = 11 .6 Hz, J = 8.0 Hz, CH,), 3.51 (dd, 1H, J = 11.6 Hz, J = 4.1 Hz, CH,), 3.37-3.29 (m, 2H, OCH, CH,), 1.61 (s, 2H, J = 7.2 Hz, OCH, CH,), 0.93 (t, 3H, J =

7.4 Hz, CH,). 13C-NMR (75.5 MHz, CO, OD): ~ 147.3 (Ar-4), 146.9 (Ar-3), 133.3 (Ar-1),

120.6 (Ar-6), 117.0 (Ar-5), 115.8 (Ar-2), 85.2 (CH), 72.5 (CH,), 68.9 (OCH, CH,), 24.9 (OCH, CH,), 11.8 ( CH,).

2- (3,4-di h i droxifen iI) -2 -butoxiela n 01 (5d)

OBu

HO ~ OH

JlJ

HO

To a solution of compound 4d (78 mg, 0.29 mmol) in dry MeOH (2 ml), are added

sodium ascorbate (58 mg, 0.29 mmol) and Cs, COJ (280 mg, 0.88 mmol) and stirred at

room temperature, in darkness and argon atmosphere for 3 hours. When the reaction is terminated, the compound is purified, without neutralizing or evaporating the solvent, by column chromatography using AcOEt-Hex (1: 1) as eluent.

Yield: 28.1 mg, 43%, R, 0.3 (CH, CI, -MeOH 10: 1). 'H-NMR (300 MHz, CDCIJ): 6

6.84 (d, 1H, J,., = 1.8 Hz, Ar-2), 6.83 (d, 1H, J,., = 8.0 Hz, Ar-5), 6.72 (dd, 1H, J,., = 8.0 Hz, J ,,, = 1.8 Hz, Ar-6), 6.04 (sa, 3H, OH), 4.29 (dd, 1H, J = 7.8 Hz, J = 4.4 Hz, CH), 3.69-3.57

10 (m, 2H, CH,), 3.47-3.28 (m, 2H, OCH, CH,), 1.6G-1.51 (m, 2H, OCH, CH,), 1.43-1 .26 (m, 2H, CH , CHJ), 0.88 (t, 3H, J = 7.3 Hz, CH, CH3). 13C-NMR (75.5 MHz, CDCIJ): 6 144.3 (Ar4), 144.2 (Ar-3), 131.8 (Ar-1), 119.9 (Ar-6), 115.7 (Ar-5), 114.1 (Ar-2) , 82.5 (CH), 69.2 (OCH, CH,), 67.6 (CH,), 32.2 (OCH, CH, j, 19.7 (CH, CHJ), 14.2 (CH, CHJ).

15 Example 3.1.3.

The deacilation (alcoholysis) of compound 6 (peracylated prolocatecholic alcohol) with an aceloxyl group in a benzyl position calalized by enzymatic extracts using an aliphatic alcohol of the ROH type as solvent, also takes place totally regioselectively in the phenolic esters (Scheme 3) . Similar to the behavior observed in compound 3, in the benzyl position the acyloxy group is substituted by the alkoxy group from the alcohol used as solvent. Thus, for example, using MeOH as solvent, compound 7 can be obtained quantitatively in 24 h. Compound 7 has been previously isolated from the broth of

fermentation of marine fungus Y26-02 (1N, H.-H. et al. J Asian Nat Prod Res 11, 74725 750; 2009).

ACOrrOAC HOrrOMe Enzyme Extract

• AcO ~ MeOH HO ~

6 7

Scheme 3

4- (Methoxymethyl) benzene-1,2-diol (7)

H0X)

I "" OMe HO ~

5 To a solution of compound 6 (30 mg, 0.19 mmol) in MeOH (1 mL) 30 mg is added

of enzyme extract obtained from the culture of the bacterial strain Bacillus sp. HR21-6

(CECT 8464) and 60 mg of silica gel (63-200 ~ m) and heated at 40 oC for 24 h. Be

concentrated to dryness and purified by column chromatography (AcOEt-hexane 1: 1) to obtain the product as a reddish syrup. Yield: 29 mg, quantitative RF

10 0.6 (AcOEt-hexane 1: 1).

'H-NMR (300 MHz, COCI,): or 6.83-6.78 (m, 2H, Ar-3, Ar-5), 6.70 (d, 1 H, J,., = 7.7 Hz, Ar6), 4.30 ( s, 2H, CH,), 3.33 (s, 3H, OMe).

15 Example 3.2. Aeylation reaction of phenolic compounds by enzymatic catalysis to obtain lipophilic polyphenols.

Regioselective acylation (esterification) reactions of various polyphenols, preferably of polyphenols present in olive oil (Scheme 4) were carried out.

HO ~ OH

I

HO ¿

HO ~ I '~~ OH

HOU

Abstract

enzymatic HO ~ OH Ae0Y¡Y '- "oH! l. J ... • AeO I ¿+ HO ~

Or 9

HO ~ OAe

I¿ +

HO 2

HO ~ OAe Ac0Y¡Y '- "oAe AeO ~ + HO ~

12 13

Extract OH ~ ./.> .. ~~

Enzyme, HOifOH + AeOD _OH

! l.oJ ...

AeO ¿HO ¿

DMF 15 16

OH HO OAe AeoifoH

1 "" + I

HO ~ ¿

¿AcO

17 18

HO_I ~~~ OAC

AeoU

Scheme 4

Abstract

HO ~ OH HO "" 20

enzymatic, ~ O ~ HO ~ OH AcO "" 21 +

HO ~ OAC HO "" 23

+

HO ~ OAC + ACO ~ 25

ACO ~ OAC ACO ~ 6 Scheme 4 (continued)

5

Sayings polyphenols They are he compound 8 (hydroxytyrosol),

ACO ~ OH

HO ""

ACO ~ OH

AcO ""

ACO ~ OAC HO ~

compound 14 (3.4

dihydroxyphenyl glycol) and compound 20 (protocatechic alcohol). As an acylating agent, isopropenyl acetate was used, which in turn acts as a solvent for polythene and its acylated derivatives. The acylation can be done in the absence or presence of an adsorbenle of the culture supernatant of the strains of the invention, such as celite.

10 Better results are obtained with the enzyme extract supported on celite. If the phenolic compound used is poorly soluble in the achating agent DMF (N, N-dimethyl form measure) can be used as ca-solvent. The result of the acylation is quantified by 'H-NMR after elimination of the supernatant or enzyme extract containing the enzymes responsible for carrying out the acylation reaction.

The major product obtained from the acylation (esterification) reaction of the polyphenol hydroxytyrosol (compound 8) after 24 h (Table 1) is the compound 9 which is monoacylated derivative in position 4 of the aromatic ring. Compound 9 and its regioisomer in position 3 (compound 10), are formed in the ratio of 2: 1. In addition, of the monoacylated derivative compounds 2, 9, and 10, three other compounds that are the diacylated derivatives 11, 12, and 13 are detected in lesser amounts. The degree of conversion ranges from 88 to 96%. The results contrast with the regioselectivity that is obtained with the Hpase from C. antarctica, which leads exclusively to the regioisomer compound 2, acylated in the side chain.

The acylation (esterification) reaction of compound 14: 3,4-dihydroxyphenyl glycol after 24 h (Table 1) leads to a mixture of compounds 15 and 16 which are the monoacylated derivatives in the aromatic ring, and of compound 17 which is the monoacylated derivative at the end of the side chain, in a 1: 1: 1 ratio, with the extracts of Tembacillus sp. AE28 122 (CECT 8231) and En / erobac / er sp. AE1889 (CECT 8462), and in the 1: 1: 2 ratio with Pseudomonas sp. AE28120 (CECT 8462), Bacillus sp. HR21-6 (CECT 8464) and the pure lipase of C. antarctica. The degree of conversion ranges from 53 to 70%. The lowest degree of conversion is achieved with C. antarctica. The adladon of the benzyl hydroxyl of compound 14 is not detected with any of the enzymes or enzymatic extracts.

The adladon reaction (esterification) of compound 20: protocatechonic alcohol after 24 h (Table 1) leads to a mixture of mono, di and triacylated derivatives $, The reladon of monoadlated derivatives in position 4, 3 and chain lateral is sensitive to the enzyme extract used; thus, the proportions are 1: 1: 1 for the culture supernatant of Terribacillus sp. AE28 122 (CECT 8231), 1: 2: 2 for the culture supernatant of En / erobac / er sp. AE 1 889 (CECT 8462), 1: 3.5: 1 for the culture supernatant of Pseudomonas sp. AE28120 (CECT 8462), 1: 3: 2 for the culture supernatant of Bacillus sp. HR21-6 (CECT 8484) and 1: 0: 16 for C. anlarctica lipase. The degree of conversion ranges from 77 to 81% for the 4 supernatants tested, while with C. antarctic lipase, the conversion is quantitative. The formation of compound 6: triacetylated derivative is only detected with C. antarctic lipase.

Table 1. Percentage determined by 1 H NMR in the reaction mixture formed by acylation of compounds 8, 14 and 20 with extracts of the supernatants obtained from the culture of the bacterial strains of the invention or of a commercial lipase.

Bacterial Extracts and Lipase

Acylation of hydroxytyrosol (compound 8). Hydroxytyrosol (50ng) is dissolved in isopropenyl acetate (3.0 ml) and ketone (50 mg) and the corresponding enzyme extract obtained from the cultures of the strains of the invention (50 mg) are added. Stir to

10 40 oC and in darkness 24 hours. The solution is filtered and concentrated to dryness. The result of the aeylation is analyzed by 'H-NMR (See Table 1).

Acylation of 3,4-dihydroxyphenyl glycol (compound 14). 3,4-dihydroxyphenyl glycol (50 mg) is dissolved in a mixture of DMF / isopropenyl acetate 1: 1 (6.Q mi) and the corresponding enzyme extract obtained from the cultures of the strains of the invention (50 mg) is added. Stir at 40 oC and in darkness 24 hours. The solvent is removed and

5 The resulting residue is redissolved in dry acetone and filtered. Finally, it is concentrated to dryness. The acylation result is analyzed by 1 H-NMR (See Table 1)

Acylation of proto-caloric alcohol (compound 20). The alcohol (50 mg) is dissolved in isopropenyl acetate (3.0 ml) and the corresponding enzyme extract is added

10 obtained from the cultures of the strains of the invention (50 mg). Stir at 40 oC and in darkness 24 hours. The solution is filtered and concentrated to dryness. The acylation result is analyzed by lH_ NMR (See Table 1)

Example 4. Evaluation of the antioxidant capacity of lipophilic polyphenols described in the present invention.

The results obtained in the anti-radical activity test against DPPH for compounds 4a-4d, 5a-5d, 2 and 14. are represented below. The anti-radical activity expressed as EC5Q against DPPH (radical 2,2-diphenyl-1- picrilhydrazyl)

20 represents the amount of antioxidant needed to decrease the DPPH concentration to 50%. The Sánchez-Moreno method (Sánchez-Moreno, C. et al. J. Sci. Food Agric. 1998, 76, 270-276) was used with the modifications of Jiménez et al. J. Agric. And food ehem. 2005, 53, 5212-5217).

Of the new synthesized compounds, the most active was compound 4a with an activity similar to hydroxytyrosyl acetate (Compound 2), although less than 3,4-dihydroxyphenyl glycol (compound 14) (Table 2 and Figure 2)

Table 2. Anti-radical activity against OPPH expressed in EC values. (11M)

4th 14.5 ± 0.500

4c 21.7 ± 0.480

Table 2. Anti-radical activity against DPPH expressed in ECso values (J.lM)

5b 20.3 ± 1.73

14 12.0 ± 0.550

Example 5: Preparation of partially acylated carbohydrates. Acylation / deactivation reactions of carbohydrates catalyzed by enzymatic extracts 5 obtained from the cultures of Terribacil / us sp. AE2B 122 (CECT 8231), Bacil / us sp. HR21-6 (CECT 8464), Enterobactersp. AE1B89 (CECT 8462).

Example 5.1

The deacetylation reaction of compound 27 (methyl (l-O) has been carried out

10 per-O-acetylated glucopyranoside) by alcoholysis in methanolic medium catalyzed by the supernatants obtained from the cultures of the bacterial strains Terribacillus sp. AE28 '122 (C ECT 823 1), In / erobae / ersp. AE1B89 (CECT 8462) and Baeillus sp. HR21-6 (CECT 8464). The results obtained with the strains of the invention have been compared with the

results obtained with commercial lipases from Pseudomonas cepacea 15 30 U / mg (Sigma-Aldrich, EC 232-619-9) and C. antarctica supported on acrylic resin 5,000 U / g (Sigma-Aldrich, EC 3.1.1.3, Novozyme 435) (Scheme 5).

The reaction medium consists of a suspension of compound 27 and extract

Enzymatic or corresponding lipase, in methanol or in deuterated methanol (CD30 0).

Once the reaction is terminated, the proportions between the different compounds detected in the reaction crude (compounds 27-29) are obtained from the corresponding spectrum of 'H-NMR at C 0 30 0, after elimination of the Enzymatic extract or corresponding enzymes.

OH

HO Enzymatic Extract •

C0300, t.a.

28 29

Scheme 5

The enzymatic extrachlole comes from Terribacillus sp. AE28 122 (CECT 8231) only

5 allowed a 28% conversion (Table 3) after 12 h of reaction. Said conversion turned out to be totally regioselective, since only the presence of compound 28 (methyl derivative 6-0-acetyl-ex-o-glucopyranoside) was observed, together with compound 27 used as

raw material (72%).

10 The enzymatic extracts from Enterobacter sp. AE1B89 (CECT 8462) and B.cil / us sp. HR21-6 (CECT 8464), which showed a 64 and a

86% conversion, respectively in a significantly lower reaction time (6 h). Said enzymatic extracts show a high tendency to complete deacetylation, to give compound 29 (methyl a-o-glucopyranoside), which is in a proportion in the mixture of 64% or 65%, respectively. Compound 28 which is the acetylated derivative in e-6 is in a proportion of 20 and 21%,

respectively.

Commercial lipases from P. cepacea and C. antarctica, the latter being

20 supported on acrylic resin, did not lead to any reaction, despite carrying out the reaction at a significantly higher temperature (up to 60 oC) and using a higher proportion of enzyme (up to 1.5 times the weight of the monosaccharide).

Table 3. Proportions found for deacetylation of compound 27

27

catalyzed by the culture supernatants of the strains of the invention or by commercial lipases

Lipasa P. cepacea 100% 0% 0%

22 (Sigma-Aldrich, EC 232-619-9)

~! · 'TJ.;, I,' '-.> ~>' 'Y ",. [~ J4 ~ -f" "' :; - ~: c: ''" "~~ '-" " "-,", - ~ ,., 'il' "" ""), ..., •.;;

. • '. ~. ~. , 'J ", j ~, • ~' _ r., ,,, '1"' - ", ~

'; 1 ~ _'-.: .. ~ _ ~; "-,;." "" _ "" ",,, ~ .. ::) _ ~~ _. ~ -, -__ • ~~ J ..: .. l: l ...: '~

Oesacylation of compound 27 (methyl 2,3,4,6-tetra-O-acetyl-a · o-glucopyranoside).

A suspension of compound 27 (6.0 mg, 17 ... mol) and of the culture supernatant of the corresponding inventive strains (3.0 mg) in COJOD (1.0 mL) is stirred at room temperature for the time indicated in each case. In the case of lipases from P. cepacea and C. anfarctica supported on acrylic resin, a 1: 1.5 monosaccharide / lipase ratio, a temperature of 60 oC and a reaction time of

10 22 h. Figure 1 shows the 1 H-NMR spectrum of the reaction mixture [1 H NMR (300 MHz, CD30D)); The results are shown in Table 3.

Example 5.2. The culture supernatants of the strains described in the present invention also allow partial deacetylation of compound 30 (octaacetylated trehalose), using the same conditions as in the case of monosaccharide 27 (Scheme 6).

OAC

AcO O

~

AcO HO OAe OACO __---- '' "I

Aeo ~

AeO

Abstract

OAc AcO 31

enzymatic

OR

, + AcO

C030D, t.a. ~ OR 'R'O O R'O HO

AcO

OR '__..-- "' \

OR

32 R '= Ac 33 R' = H HO HO

Scheme 6

5 Enzymatic extracts of Tenibacillus sp. AE2B 122 (CECT 8231), Enterobacter sp. AE1B89 (CECT8462) and Bacillus sp. HR21 -6 (CECT 8484), allowed a conversion

between 89-91% in a reaction time between 7-12 h (Table 4). The preference of the enzymes present in the supernatants of the cultures of the above-mentioned strains was observed for carrying out the decellilation on one of 10 $ 10 glucopyranose rings (Table 4), allowing the detection of two new compounds,

compound 31 (2,3,4,6,6'-penta-O-acetyl-u, u-D-trehalose) (26-35%) and compound 32

(2,3,4,6-tetra-O-acetyl-u, u-D-trehalose) (38-40%), of which there is no history in

The state of the art.

15 It should also be noted that compound 32 is the result of a deimetrization in

so only two stages (acetylation and selective deacetylation) of trehalose. The

Deimetrization is the process by which the trehalose per-O is achieved

Acetylated 30 loses its original symmetry and becomes 31 and 32, where a unit of

Sugar is acetylated and the other totally or partially deacetylated. This process has been achieved in two stages (commercial trehalose acetylation and deacetylation

regioselective with the enzyme extract of the invention and represents a number of

Very inferior stages with respect to any purely chemical route known and used in the state of the art for the same purpose.

Table 4. Proportions found for deacetylation of compound 30 (octaacetylated trehalose) catalyzed by culture supernatants of bacterial strains of the invention or by commercial lipases

Percentage calculated

(Sigma-Aldrich, EC 232-619-9)

This same test was also carried out for quantitative purposes using the enzymatic extract of Bacillus sp. HR21-6 (CECT 8484), which allowed the isolation of the compounds 31 and 32 (29% and 49%, respectively, after chromatographic purification) and their characterization.

It is noteworthy that compounds 31 and 32 can be used as raw materials in the preparation of trehalose derived glycolipids, which are of interest as vaccines against tuberculosis, caused by the pathogen Mycobaclerium tuberculosis

(M. Gitteron et al. J. Exp. Med. 2004, 199, 849-659).

Again, when the reaction was carried out using commercial lipases from P. cepacea and C. anlarctica supported on acrylic resin, no reaction was observed.

Oesaclation of compound 30 (per-O-acetyl-a, a.-o-trehalose)

Method A. A suspension of per-O-acetyl-a, a-D-trehalose (compound 30) (6.0 mg, 8.8 µmol) and of the culture supernatant of the strains of the corresponding invention (3.0

5 mg) on CD, OD (1.0 mL) is stirred at room temperature for the time indicated in

each case. The 1 H-NMR spectrum of the reaction mixture ('H-NMR (500 MHz, CD, OD)] is shown in Figure 4; the results are shown in Table 3.

Method B. A suspension of per-O-acetyl-a, a-D-trehalose 30 (60.0 mg, 88.0 mol) and the

10 culture supernatant of the bacterial strain Bacillus sp. HR21-6 CECT 8464 (30.0 mg) in MeOH (20 mL) is stirred at room temperature for 3.5 h. It is then purified by column chromatography (CH2CI2 - + CH2Clr MeOH 1: 2) to give 31 and 32 as syrups.

15 2,3,4,6,6'-Penta-O-acetyl- <t, a-D-trehalose (compound 31)

AcO '------..--: "-"

AcO ~ - ~.:;

HO

OH

AcO OH

Yield: 14 mg (29%). 'H-NMR (500 MHz, CD, OD) B5.57 (dd, 1H, J,., = 10.2 Hz, J,. • = 9.5 Hz, H-3), 5.30 (d, 1H, J,., = 3.7 Hz, H-1), 5.07 (dd, 1H, J •., = 10.3 Hz, H-4) , 5.04 20 (d.1H, J, .. • = 3.7 Hz, H-1 '), 4.94 (dd, 1H, H-2), 4.43 (ddd, 1H, J, ... = 4.1 Hz, J ,. ", = 2.4 Hz, H-5), 4.28 (dd, 1H, J, ..... = 2.0 Hz, J .... 6b. = 11.7 Hz, H-6a '), 4.24 (dd, 1H, J ..., , = 12.4 Hz, H6a), 4.15 (dd, 1H, J, .. ". = 7.3 Hz, H-6b '), 4.12 (dd, 1H, H-6b), 3.82 (ddd, 1H, J •. .,. = 9.7 Hz, H-5 '), 3.74 (dd, 1H, J, ..,. = 9.7 Hz, J, .. •. = 9.1 Hz, H-3'), 3.51 (dd, 1H, H-2 '), 3.26 (dd, 1H, H4 '), 2.08, 2.05 (x2), 2.01.2.00 (5s, 3H each, 50Ac); 13C-NMR (125.7 MHz, CD, OD) 1) 25 172.7,172.4,171.7, 171.5, 171.3 (5 CO), 96.3 (C-1 '), 92.8 (C-1), 74.6 (C-3'), 72.8 (C-2 '),

72.2 (C-5 '), 72.0 (C-4'), 71 .9 (C-2), 71.5 (C-3), 69.9 (C-4), 69.1 (C-5), 65.0 (C- 6 '), 62.9 (C

6).

2,3,4,6-Tetra-O-acetyl-a, a-o-trehalose (compound 32).

AcO "'---' ~" - AcO ~ _ ~ ":: '

HO

Yield: 22 mg (49%). 'H-NMR (500 MHz, COJOO) 85.55 (dd, 1 H, J'.J = 10.2 Hz,

5 JJ. ' = 9.5 Hz, H-3), 5.34 (d, 1H, J ,, = 3.7 Hz, H-1), 5.07 (dd, 1H, J •. 5 = 10.3 Hz, H-4), 5.05 (d, 1H, J, · .z = 3.3 Hz, H-1 '), 4.96 (dd, 1H, H-2), 4.46 (ddd, 1H, J5 ... = 3.8 Hz, J5 ... = 2.4 Hz, H-5), 4.23 (dd, 1H, J ..... = 12.4 Hz, H-6a), 4.12 (dd, 1H, H-6b), 3.75 (dd, 1H, J5 ·. ,, · = 2.1 Hz, J .... 6b. = 11.3 Hz, H-6a '), 3.75 (t, 1H, JJ .. •. = 9.7 Hz, H-3'), 3.70 (ddd, 1H, Jo '= 9.8 Hz, J5 • ... · =

5.7 Hz, H-5 '), 3.65 (dd, 1H, H-6b'), 3.50 (dd, 1H, H-2 '), 3.31 (m, 1H, H-4'), 2.06, 2.05,

10 2.01.1.99 (45, 3H each, 40Ac); 13C-NMR (125.7 MHz, COJOO) 8172.4.171.7, 171.6,

171.3 (4CO), 96-8 (C-1 '), 93.3 (C-1), 74.7 (C-3', C-5 '), 72.9 (C-2'). 71.7 (x2), 71.6 (C-2, C3, C-4 '), 69.9 (C-4), 69.0 (C-S), 62.9 (C-6), 62.6 (C-6').

Example 5.3

In the present invention, the partial deacetylation process of compound 34 (octaacetylated sucrose) (Scheme 7) was also carried out, using the same conditions as in the case of rnonosaccharide 27.

OAe

OAe Aeo ~ AeO AeO

OAe OAe A "Rc ~ Ae O Enzymatic Extract 35 + CD30D, t.a. OAe OAe OH H Ae

3. 4

Ae OH OH

OAe

OH36

Scheme 7

Supernatants obtained from the crops of Terribacillus sp. AE28122 (CECT 8231),

In / erobae / er sp. AE1 B89 (CECT 8462) and Baeil / us sp. HR21-6 (C ECT 8484) allowed

a conversion between 82-90% (Table 5). The preference of the enzymes present in said supernatants was observed for carrying out the decellilation of the ring

10 of fruetofuranosa (Table 5), allowing the detection of compounds 35 (2,3,4,6,1 ', 6'

hexa-O-acetylsacarose) and 36 (2,3,4,6,6'-penta-O-acetylsacarose).

Additionally, a higher conversion (90%) was observed in a reaction time

lower (4h) in the case of the supernatant obtained from the cultivation of the strain of Terribaeillus 15 sp. AE2B122 (CECT 8231).

Table 5. Proportions found for deacetylation of compound 34 (octaacetylated sucrose) catalyzed by culture supernatants of bacterial strains of the invention or by commercial lipases

Percentage calculated (NMR)

Microorganism / Lipase

t (h)

34 35 36

I I

CECT 8231 10% 38% 52% 4

CECT 8462 18% 43% 39% 7.5

CECT8464 14% 44% 42% 8.5

Lipasa P. cepacea 100% 0% 0% 10 (Sigma-Aldrich, EC 232-619-9) Lipase C. Antarctic 100% 0% 0% 10 (Novozyme 435)

For the isolation of the aforementioned compounds, this same test was also performed with the supernatant obtained from the cultures of BBcillus sp_ HR21-6 5 (CECT 8464) and a chromatographic purification was carried out, to give rise to the compounds 35 (26% ) And 36 (27%).

With the commercial lipases from P. cepacea and C. antaretica supported on acrylic resin, no reaction was observed for this substrate.

Per-O-acetylsacarose deacylation (compound 34)

Method A. A suspension of per-O-acetylsacarose (compound 34) (6.0 mg, 8.8 Jmol) and the culture supernatant of the corresponding invention strains (3.0 mg) in

15 C0 30D (1.0 mL) is stirred at room temperature for the time indicated in each case. Figure 1 shows the 1 H-NMR spectrum of the CHRMN reaction mixture (500 MHz. C0300)}; The results are shown in Table 4.

Method B. A suspension of per-o-acetylsacarose (compound 34) (50.0 mg, 74 Jmol) and

20 the supernatant of the cuttive of the strain Bacillus sp. HR21-6 CECT 8484 (25.0 mg) in MeOH (16.0 mL) is stirred at room temperature for 4h. It is then purified by column chromatography (CH2CI2 CH: zCh-MeOH 1: 2) to give rise to

-

the

compound 35 (11.2 mg, 26%) and compound 36 (10.9 mg, 27%) as syrups.

Example 5.4. Regioselective carbohydrate acylation The enzymatic extracts of the bacterial strains described in the present invention also allow for the regioselective carbohydrate acylation reactions. So by

example acylation, preferably acetylation of compound 29 (methyl a-D

5 glucopyranoside) using isopropenyl acetate as an acylating agent, the enzymatic extract of Bacillus stratosphericus as an enzyme catalyst and DMF as a co-solvent (Scheme 8), has given Play to obtain compounds 37 and 28, which have aneyl groups in positions 4 and 6, respectively.

OH JO ~

HO HO .....,. ~~ -----'''-------- ... HO HO .....,. ~~ + AcO HO ....., . ~~

~ Enzymatic extract ~ ~ OH OM Baci / lus stratosphericus and DMF

29 28 37

60 oC

Scheme 8

Despite prolonged heating (60 oC for 105 h), conversion 15 is 49%, although with high regioselectivity, since the presence of the monoacetylated product in C-6 (compound 28) is observed (13% ), And compound 37 (methy4,6-di-O

acetyl-a-D-glucopyranoside) as a major acetylated derivative (36%), not detected in the corresponding deacetylation tests.

20 Acylation of methyl 2,3,4,6-tetra-O-acetyl .. (Jo-glucopyranoside (compound 29) A solution of compound 29 (6.0 mg, 0.017 mmol), the culture supernatant of the strain Bacillus sp HR21-6 CECT 8464 (3.0 mg) and isopropenyl acetal (0.5 mL) in DMF (0.3 mL) is heated with stirring at 60 ° C for 105 h, then concentrated to dryness and the residue is analyzed by 'H- NMR, showing a mixture

25 of compounds 28, 29 and 37 in a proportion of 13%, 51% and 36%. respectively.

Claims (17)

one.

Microbial strain of Pseudomonas deposited in the Spanish Type Culture Collection (CECT) with the accession number CECT 8463.

2.

Enzymatic extract obtainable from the culture of the bacterial strain described according to claim 1.

3.

Enzymatic extract according to claim 2, characterized in that it is concentrated and / or dialyzed.

Four.

Enzymatic extract according to any of claims 2 or 3 characterized in that the enzyme extract is lyophilized.

5.

Enzymatic extract according to any of claims 2 to 4, characterized in that the culture further comprises a lipid substrate.

6.

Enzymatic extract according to claim 5 wherein the lipid substrate is selected from surfactants, triglycerides or essential oils.

7.

Use of the bacterial strain according to claim 1, or of the enzyme extract according to any one of claims 2 to 6 as a catalyst for enzymatic reactions of acylation and / or deacilation of compounds.

8.

Use according to the preceding claim, wherein the compounds are polyphenols

9.

Use according to any of claims 7 to 8, wherein the reactions are regioselective.

10.

Method for obtaining acylated derivatives of polyphenols by enzymatic reactions of acylation or deacylation on polyphenols, comprising the following steps:

a) bringing the enzymatic extract described according to any of claims 2 to 6 into contact with polyphenols, or any of its acid derivatives, in the presence of an acylating agent or an aliphatic alcohol to carry out enzymatic acylation or deacylation reactions, respectively; Y b) purify the acylated derivatives of polyphenols obtained in step a). eleven . Method according to claim 10, wherein the polyphenols of step a) are hydroxytyrosol, 3,4-dihydroxyphenyl glycol, protocateuric alcohol or any of its acylated derivatives.

12.

Method according to claim 10 or 11, wherein the enzymatic reaction is deacylating, the polyethenol of step a) is an acetylated derivative of hydroxytyrosol, 3,4-dihydroxyphenyl glycol or protocateuic alcohol and the reaction is carried out in the presence of an aliphatic alcohol .

13.

Method according to claim 12, wherein the enzymatic reaction is deacylation, the polyenol of step a) is triacetylated hydroxytyrosol and the reaction is carried out in the presence of methanol.

14.

Method according to claim 12, wherein the enzymatic reaction is of deacylation of the aromatic hydroxyls, the polyphenol of step a) is 3,4-dihydroxyphenyl glycol tetraacylated and the reaction is carried out in the presence of an aliphatic alcohol to produce etherification in the benzyl position. .

fifteen.

Method according to claim 12, wherein the enzymatic reaction is deacylation, the polyphenol of step a) is peracylated protocatecholic alcohol and the reaction is carried out in the presence of an aliphatic alcohol to produce etherification of the benzyl position.

16.

Method according to claim 10 or 11, wherein the enzymatic reaction is acylation, the polyphenol of step a) is hydroxytyrosol, 3,4-dihydroxyphenylgicol or protocateuric alcohol and the reaction is carried out in the presence of an acylating agent and optionally a co-solvent

17.

Method according to claim 16 wherein the acylating agent is isopropyl acetate or vinyl acetate and / or the co-solvent is DMF.