ES2554565B2 - 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 their use 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 "ibacillus termed Terribacillus sp. AE2B122, Bacillus, termed Bacillus sp. HR21-6, Entembae / er, termed En / erobae / er sp. AE1B89 and Pseudomonas, termed Pseudomonas sp. AE2B120 with the ability to carry out regioselective alcoholysis and esterification reactions and their use in acylation and / or deacilation reactions, in general and more specifically, in acylation and / or deacilation reactions for the production of carbohydrates and polyphenols lipophiles of interest in the pharmaceutical and food industry, the invention also relates to a method for 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 s ector 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, it is still far from reaching the perfection of natural reactions, so this aspect remains today one of the great challenges in organic synthesis.

Biocatalysis, and particularly the use of hydrolytic enzymes such as lipases, constitutes an alternative to conventional methods in organic synthesis and may be useful for carrying out protections and stereo-and regioselective protections in carbohydrates (Filice, M. et al. Na !. Protoc. 7, 1783-1796; 2012) And polyphenols (Torres, P. et al. J. Agric. Food ehem. 58, 807-813; 2010). Detained 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 diseases and cancer (Grez el al., Curr Opin Pharmaco / 1 04, 362-368; 2010). These compounds avoid the damage caused by oxidative stress in certain biomolecules (DNA, lipids, proteins), which are associated with an increased risk of cardiovascular problems, cancer and 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 or C (Miller et al., J Nutr Enviran Med 12, 39-51; 2002), so there is a growing interest in these types 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 et al., Curr Org Chem 12, 442-463; 2008).

In recent years, it has been proven that this natural antioxidant also has important biological properties: platelet antiaggregant (González Correa et al. Br

J Nutr, 101, 1157-1164, 2009), anti-inflammatory (Gong et al., Phytother Res 23, 646-650, 2009), cardioprolechlor (Poudyal, HJ Nutrition 140, 946-953; 2010), anlimicrobial (Medina el al J Agric Food Ghem 55, 9817-9823; 2007), and anli-HIV (Lee-Huang al., Biochem Biophys Res Gommun 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 el al., J Agric Food Chem 55,

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

prevention of ischemic stroke and cancer prevention and treatment

(Sirianni et al., Mol Nutr Food Res 54, 833-840, 2010).

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

phenolics, such as 3,4-dihydrox ~ enylglycol (Brenes, M.J. Agric Food Chem 47, 35353540; 1999) and protocatechic alcohol (Saitla, M. et al. Food Ghem 112, 525-532, 2009).

HO OH

HO ~ OH

OH H0X)

I "" "OH

HO ~ HO HO J

Hydroxytyrosol 3,4-Dihydroxyphenylglycol Alcohol protoeateeuieo

Several studies on the phenolic compounds of the olive tree show the importance of the acyl 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 et al., Adv. Food Nutr. 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 modification of 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 "functional foods" that have beneficial health effects.

15 Bouallagui et al. (Bouallagui et al., Appl Biochem Biotechnol 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. Acylation catalyzed with C. antarctica B lipase takes place chemoselectively on the

20 hydroxyl of the aliphatic chain against aromatic hydroxyls (Torres de Pinedo et al., Tetrahedron 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

25 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 such as antitumor, anti-inflammatory, etc. (Plou et al., J Biotechno / 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 certain functions regioselectively, or to resort to a long and expensive sequence of synthesis, to carry out transformations in only one of the hydroxyl groups.

Despite the interest of biocatalyzed carbohydrate reactions with lipases, the history of cases of regioselective protection or deprotection 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 Baciflus strain deposited in the Spanish Type Culture Collection (CECT) with the CECT access number 8484, Enterobacfer strain deposited in the Espa Collection "Wave of Type Crops (CECn with accession number CECT 8462, Pseudomonas strain deposited in the Spanish Type Culture Collection (CECT) with accession number CECT 8463 and Terribacillus strain deposited in the Spanish Type Culture Collection (CECT) with the accession number CECT 8231 that have the ability to carry out acrylection (esterification) and / or deacrylation (alcoholysis), preferably on polyphenols and / or carbohydrates, preferably natural, which allows obtaining a collection of acylated and / or etherified compounds of interest in different pos In this way, based on the ability of these bacteria to carry out these esterification and alcoholysis reactions, a series of inventive aspects have been developed and 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 pollfenoles and / or carbohydrate compounds or their acylated derivatives being preferred.

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

Contact with pOliphenols, carbohydrates or any of their acylated derivatives, in the presence of a watering agent or an aliphatic alcohol to carry out enzymatic reactions of acylation or deacylation, respectively; Y

b) purify 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

(one)

30

where: R, is an alkyl group (C, -C2lI), an alkenyl group (C2-C2Q), an alkynyl group (Cr ClO), or an arylalkyl group; and R2 is an acyl group (-COR3), where R3 is a C, -ClO 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 peracylated 3,4-dihydroxyphenyl glycol compound with

R, OH and subsequently the reaction with methanol of the compound obtained in

step (a) under basic catalysis;

where R, is a (C, -C20) alkyl group, an alkenyl (Cr C2o) group, an alkynyl (C2-C20) group, or an arylalkyl group; as defined for substituent R, 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, aD-trehalose or 2,3,4,6-tet ra-O- acetyl-o:, 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, S'-penta-O-acetyl-a, aD-trehalose or 2,3,4,6-tetra- O-acetyl-a, a

D-trehalose comprising:

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

DESCRIPTION OF THE FIGURES

Figure 1. Phylogenetic relationship of Terribacillus sp. AE28 122, Bacillus sp. HR21-6 with other Gram positive bacteria (A), and Enterobaeter sp. AE1889 and Pseudomonas sp. AE2B120 with other phylogenetically related Gram negative bacteria (B). 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 Boolslrap statistical test (500 replicas). Evolutionary distances have been calculated using the Maximum Composile 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 ECso values (f.lM) of hydroxytyrosyl acetate 2, is represented. of the phenolic ethers 4a-4d, Sa-Sd. and 3,4-dihydroxyphenylglyc14.

Figure 3. Nuclear Magnetic Resonance Spectrum CH-NMR (A) and extended nuclear magnetic resonance spectrum (1 H-NMR) (8) of the deacylation procedure of compound 27 (methyl 2,3,4,6-tetra-O -acetyl-ao-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 procedure of compound 30 (per-O-acetyl-a, ao-trehalose ) by the enzyme extract of the present invention.

Figure 5. Nuclear Magnetic Resonance Spectrum CH-NMR) (A) and spectrum of

Extended nuclear magnetic resonance (1 H-NMR) (B) of the deacylation process of compound 34 (per-O-acetylsacarose) by 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 Terribaeillus sp. AE2B 122, Baeillus sp. HR21-6, Enlerobaeler sp. AE1B89 and Pseudomonas sp. AE28120 deposited in the Spanish Type Crops Collection (CECT) with registration numbers 8231 (with deposit date 20.11.2012), 8484, 8462, 8463 (with deposit date of October 22, 2013), respectively, which have the ability to carry out regioselective reactions of acylation (for example esterification) 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 pOliphenols during 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 (aGilation) 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 16S rRNA gene. From this analysis, the strains with the highest identity value of the microbial strains of the invention were identified, being

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 stul 175 number (175 of access to GeneBank NR_'03934).

Therefore, one aspect of the invention relates to the microbial strains of the invention, which belong to the genera Terribacillus, BaciJlus, Enterobaeter and Pseudomonas which, in another still more particular embodiment, are the microbial strains termed Terríbaeil / us sp. strain AE2B122 (CECT 8231), Baeillus sp. HR21 -6 (CECT 8484), En / erobae / er sp. AE1B89 (CECT 8462) and Pseudomonas sp. AE2B120 (CECT 8463).

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 8484, CECT 8462 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 8462 and CECT 8463.

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 and / or deacylation reaction.

For the purposes of the present invention, the term "lipid substrate" 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, surfactanles, triglycerides, essential oils, more particularly, tributyrin, Tween 80, Tween 20 and vegetable oils, such as olive oil and / or sunflower oil. In a particular embodiment, the lipid substrate is tribulirin. The concentration of the lipid substrate in the culture medium can be variable, and concentrations of between 0.1% 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 comprising 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 catalysts Biological 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, interesterifications, alcoholysis, acidolysis, esterification 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 can

5 contain not only the enzymes necessary to carry out acylation reactions and deacylation, but also may 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

10 remove those undesirable particles or compounds.

In the present invention, "concentration and / or dialysis is understood as 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 various types of 15 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 bag

20 dialysis in the direction of the lowest concentration. The dialysis technique is widely known in the state of the art 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 into 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 expert in the maleria.

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 and / or deacilation reactions, the person skilled in the art understands that both Microbial strains, such as 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 acylated 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 pOliphenols and / or carbohydrates, preferably natural.

For the purposes of the present invention, the terms regioselective / an 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 "chemoselectivity" 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 by using microbial strains or enzymatic extracts thereof, described in the present invention, can be found in Examples 3 and 5 where the method of describing production of them.

Regioselective siotransformations for obtaining lipophilic acylated derivatives of polyphene / s 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, so that both microbial strains and 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 aethylated derivatives of polyphenols and carbohydrates, preferably from polyphenols and / or natural carbohydrates, by acylation or deacylation reactions on polyphenols and / or carbohydrates, comprising the following stages:

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

contact with polyphenols, 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 purpose of the present invention, the term "purify" or "purification" refers to, as used in the description, the separation of acylated 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 may accompany it 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 rapid 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

The invention is cultivated in the presence or absence of a lipid substrate with the aim of obtaining enzymatic extracts containing the enzymes and compounds necessary to carry out the acylation and / or deacylation reaction on the 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 6231), Bacil / us sp. HR21-6 (CECT 6234) has been used as a culture medium, PYA (agar, peptone and yeast extract). In the case of Enterobactersp. AE1B69 (CECT 6232) and Pseudomonas sp. AE2B120 (CECT 6233) has been used as a culture medium, LB (Luria-Bertani), although it is important to mention that any other culture medium in which the strains of the invention can grow can be used.

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.

The 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.

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 can be subjected to centrifugation, thus achieving both ways to separate the cells from the culture medium.

liquid. 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 that are to be subjected to the acylation / deacylation 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 carbohydrate them.

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: hydroxyrosol, 3,4-dihydroxyphenyl glycol, protocatecholic alcohol and acetylated derivatives thereof, preferably: triacellylated hydroxytyrosol, 3,4-dihydroxyphenylglocol telraacylated 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 their acylated derivatives, preferably: methyl aD-glucopyranoside per-O -acetylated, trehalose octaacetylated and sucrose octaaceized.

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 such.

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 / deacilation reactions of phenolic compounds and / or carbohydrates by 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 are detailed below, in order to obtain Phenolic compounds and / or lipophilic acylated carbohydrates.

In a preferred embodiment, the enzymatic reaction is deacylating, the polyenol of step (a) is an acetylated derivative of hydroxytyrosol, 3,4-dihydroxyphenylglycol or protocatecholic alcohol, preferably triacetylated hydroxytyrosol, 3,4-dihydroxyphenylglocol tetraacetyl or peracylated protocatecholic alcohol, and the reaction is carried out in the presence of an aliphatic alcohol.

By "aliphatic alcohol" in the present invention is meant a linear (or 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 pOliphenol of step (a) is a triacetylated hydroxytyrosol and the reaction is carried out in the presence of methanol.

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) 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 co-solvent.

By "acylating group" is meant a reagent capable of reacting with a compound by adding an acyl group to its structure, for example, the acylating group may be without being limited to isopropenyl acetate or vinyl acetate, which in turn act as 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 eyebrow. Better results are obtained with the enzyme extract supported on celite. If the phenolic compound used is poorly soluble in the acylating agent, a cosolvent can be used, for example DMF (N, N-dimethyl formamide).

In another preferred embodiment, the enzymatic reaction is deacylating, the carbohydrate of step (a) is per-O-acetylated methyl ao-glucopyranoside, per-O-acetyl-a., A-D-trehalose, per-O-acetyl-sucrose , octaacetylated trehalose or octaacetylated sucrose and the reaction is 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 16-28OC, obtaining compounds 2,3,4,6,6'- penta-O-acetyl- <x, <xo-trehalose or 2,3,4,6-tetra-0acetyl-a, ao-trehalose or the enzymatic reaction of per-O-acetyl-sucrose with methanol or deuterated methanol to a temperature between 16-28 ° C, obtaining the compounds 2,3,4,6,1 ', 6' -hexa-O-acetyl-sucrose or 2,3,4,6,6'-penta-O-acetyl -saccharose.

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

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

OR,

HO ~ OR2

HO) l)

(one)

where: R1 is a (C1-C20) alkyl group, an alkenyl (Cr e20) group, an alkynyl (C, -C ,,) group, an arylalkyl group; and R2 is an acid group (-COR3 • where R3 is a 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-butyl, t-butyl, n-pentyl, among others.

By "alkenyl" in the present invention refers to a carbon chain, linear or branched, which has at least one double 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 "alkynylon" in the present invention refers to a carbon chain, linear or branched, having at least one triple 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 "arylalkyl" is meant in the present invention an aryl group attached to the rest of the molecule by an alkyl group (Cr C2o), as defined above. A non-limiting example of arylalkyl is a benzyl group.

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

In a preferred embodiment, the general formula compound (1) of the present invention is characterized in that the 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-dihydroxyphenyl) -2 acetate -ethoxyethyl, 2- (3,4-dihydroxyphenyl) -2-propoxyethyl acetate, and 2- (3, 4-dihydroxyphenyl 1) -2-butoxyethyl acetate.

In another preferred embodiment, the compounds of the general formula (1) are selected from the lysia 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 peracylated with 10 R10H and subsequently the reaction of the compound obtained in step (a)

by basic catalysis, preferably with CS2C03, in methanol; where R1 is an alkyl group (Cr C2o), an alkenyl group (C2-C20), an alkynyl group (C2C20), or an arylalkyl group.

OR,

OAe OR, CtO Od

HO ~ OAe a oaCI .. HO ~ OH

AeO ~ OAe Enzymatic Extract

•

V HOJl) MeOH HOV

15 AeO

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 compound of 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 or nutraceutical food.

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 the improvement of the physical or physiological or psychological well-being of a subject, which implies an improvement of 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 can 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 composition" or "cosmetic" 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 of the factor causing 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 "nullification" as used in the present invention refers to substances isolated from a food and used in a dosage form having a beneficial effect on health.

For the purposes of the present invention the term "supplement", synonymous with any of the terms "dietary supplement", "nulricional 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.

By the term "antioxidant" is meant 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 formula 2,3,4,6,6'-penta-O-acetylCl, aD-trehalose or 2,3,4,6 -tetra-O-acetyl-a, treolosa.

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 Mycobacterium tuberculosis comprising:

8. The enzymatic reaction of per-O-acetyl-a, a-D-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 shows the effectiveness of the product of the invention.

Example 1: Phylogenetic analysis of Terribacillus sp. AE2B 122 (CECT

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

The phylogenetic study was carried out by analysis of the acid gene sequence

ribosomal ribonucleic (rRNA) 16S. For this, the 16S rRNA gene of each

One of the strains of the invention 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 ML, 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 a 10 minute extension step at 72 OC. The amplification products were analyzed by agarose gel electrophoresis. The amplified DNA 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 access number NR_043895), Bacillus stratosphericus JCM 13349 (GeneBank access number NR_042336), En / erobac / er ludwigii EN-119 (GeneBank access number AJ85om91) and Pseududom stutzeri ATCC 17588 (GeneBank accession number NR_103934)

5 that show greater identity value with the sequences of the strains of the invention. Therefore, the strains of the invention were assigned to the genera Terribacillus as Terribacil / us sp. AE2B 122 (CECT 8231), Bacil / us, as Bacil / us sp. HR21-6 (CECT 8464), En / erobac / er, as En / erobac / er sp. AE1 B89 (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 1 B) of Escherichia coll rRNA 165 ) of the strains and

15 of 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).

20 Example 2: Preparation of the supernatants of the Terribacillus sp. AE2B 122 (CECT 8231), Bacillus .p. HR21-6 (CECT8464), Enterobacter sp. AE1B89 (CECT 8462) and Pseudomonas sp. AE2B120 (CECT 8463).

For the preparation of enzyme extracts or supernatants, a pre

Inocula of each of the four strains described in the present invention. Terribacillus 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.

30 Bacil / us sp. HR21-6 (CECT8484) was grown in LB medium overnight. The next day, said culture was used to inoculate PYA medium. After growing the culture in PYA medium, the supernatant was collected.

Enterobaeter sp. AE1889 (CECT 8462) and Pseudomonas sp. AE28120 (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 pollfenoles. Acylation / deactivation reactions catalyzed by the supernatants / enzymatic extracts of the strains of Terribacillus sp. AE28 122 (CECT 8231), Bacillus sp. HR21-6 (CECT 8464), Enterobacter sp. AE1889 (CECT 8462) and Pseudomonas sp. AE2B120 (CECT 8463).

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

Example 3.1 Reaction of deacilation of phenotic 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 allophic alcohol, such as MeOH, gives

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

ACO ~ OAC enzymatic extract HO ~ OAC

I • I AcO h MeOH HO h

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 oC for 18 hours. It is filtered on kettle and concentrated to dryness. The residue is purified by column chromatography (AcOEt-hexane 1: 2 _ 1: 1) to obtain hydroxytyrosyl acetate 2 (31ng, 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 (peracetylated 3,4-dihydroxyphenyl glycol) by catalyzed alcoholysis 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 phenolic 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 R' OH is preferably selected from any of the following: -CH, (methyl), -C, H, (elyl), -C, H, (propyl) or -C, H, (bulilo).

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 methanol.

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

OAc OR ACO ~ OAC Enzymatic Extract HO ~ OAC Cs2C03

AcO) lJ ROH HO) lJ MeOH

4th R = Me 4b R = Et 4c R = Pr 4d R = Bu

Scheme 2

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

OMe HO

OAe

10 HO

OR

HO ~ OH

Ho) lJ

5. R = Me 5b R = Et Se R = Pr 5d R = Bu

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 TeffibaciJIus sp. strain AE2B122 (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 (CH2Cb

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

20 'H-NMR (300 MHz, CD30D): 06.80 (d 1H, J,. • = 8.0 Hz, Ar-5), 6.79 (d, 1H, 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 = 6.0 Hz, J = 3.9 Hz, CH), 4.16 (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 (5 3H, OMe), 2.06 (5 3H, OAc). "C-NMR (75.5 MHz, CD30D): O 173.6 (CO),

145.7 (Ar-3 and 4), 131.4 (Ar-l), 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) OEt 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 culture of the bacteMana Baci "us sp. HR21-6 strain (CECT 10 8464) and 120 mg of silica gel (6 ~ 200 ~ m.) Heated at 60 oC for 24 hours and

after that time it is concentrated to dryness and the product is purified by chromatography

in silica gel column (CH, CI, -EtOH 60: 1). Yield: 24 mg, 55%, R, 0.5 (CH, CI, -MeOH 10: 1).

15 'H-NMR (300 MHz, CDCI,): OR 6.89 (d, lH, J,. • = 2.0 Hz, Ar-2), 6.83 (d, lH, J,. • = 8.1 Hz, Ar5), 6.72 (dd, lH, J •., = 8.1 Hz, J •., = 2.0 Hz, Ar-6), 4.42 (dd, lH, J = 6.9 Hz, J = 5.0 Hz, CH), 4.20-4.12 ( m, 2H, CH,), 3.43 (2dd, lH 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,). "C-NMR (75.5 MHz, CDCI,): or 172.0 (CO),

144.5 (Ar-4), 144.4 (Ar-3), 131.0 (Ar-l), 119.9 (Ar-6), 115.5 (Ar-5), 113.9 (Ar-2), 79.6 20 (CH), 68.4 ( CH,), 84.8 (CH, CH,), 21.3 (OAc), 15.4 (CH, CH,).

2- {3,4-dihydroxyphenyl) -2-propoxyethyl acetate (4c)

OPr HO OAe

HO

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

8484) and 380 mg of silica gel (6 ~ 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%, R, 0.6 (AeOEt-hexane 1: 1).

'H-NMR (300 MHz, COCI,): ~ 6.90 (d, 1 H, J,., = 2.0 Hz, Ar-2), 6.84 (d, 1H, J,., = 8.1 Hz, Ar

5 5), 6.73 (dd, 1H, J,., = 8.1 Hz, J,., = 2.0 Hz, Ar-6), 4.41 (dd, 1H, 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 (sextete, 2H, J = 7.4 Hz, OCH, CH,) , 0.88 (t, 3H, J = 7.4 Hz, CH,). "C-NMR (75.5 MHz, COCI,): ~

171.9 (CO), 144.4 (Ar-4), 144.3 (Ar-3), 131.2 (Ar-1), 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-dihydroxylfenyl) -2-butoxylethyl (4d) acetate OBu HO OAe

HO

Compound 3 (190 mg, 0.56 mmol) is dissolved in BuOH (4 mL) and 190 mg is added

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

(CECT 8464) and 380 mg of silica gel (63--200 ~ m), heating at 60 'C for 2

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

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

20 'H-NMR (300 MHz, COCI,): ~ 6.90 (d, 1H, J,., = 1.9 Hz, Ar-2), 6.84 (d, 1H, J,., = 8.0 Hz, Ar5), 6.73 (dd, 1H, J ", = 8.0 Hz, J,., = 1.9 Hz, Ar-6), 6.50 (s, 1H, OH), 5.95 (s, 1H, OH), 4.44 (dd, 1 H , 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, OAc), 1.55 (m, 2H, J = 6.6 Hz, OCH, CH,), 1.39-1.25 (m, 2H, CH, CH,), 0.86 (1, 3H, J = 7.3 Hz , CH, CH,). "C-NMR (75.5 MHz, COCI,): ~ 172.1 (CO), 144.4 (Ar-4),

25 144.3 (Ar-3), 131.1 (Ar-1), 120.0 (Ar-6), 115.5 (Ar-S), 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-meloxethanol (5a)

OMe

HO OH

HO

To a solution of compound 4a (54 mg, 0.24 mmol) in dry MeOH (1 mL) is added Cs, C03 (231 mg, 0.72 mmol) and sodium ascorbal (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 as eluent CH, CI, -EtOH (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, 1 H, J,. • = 8.1 Hz, Ar-5), 6.89 (d, 1 H, J,. • = 2.0 Hz, Ar2) , 6.80 (dd, 1H, J •., = 8.1 Hz, J6., = 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-eloxyelanol (5b) OE!

HO OH

HO

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

Cs, C03 (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 CH2Clr 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, lH, J,. • = 8.1 Hz, Ar-5), 6.88 (d, lH, J ". = 1.8 Hz, Ar-2) , 6.80 (dd, lH, J. ,, = 8.1 Hz, J. ". 2.0 Hz, Ar-6), 4.38 (dd, lH, 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_RMN (75.5

5 MHz, CO, QO): 15 146.1 (Ar-4), 145.9 (Ar-3), 133.1 (Ar-l), 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- (3,4-0ihydroxyphenyl) -2-propoxyetanot (5c) OPr

HO ~ OH HO) l)

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

Cs, CO, (66 mg, 0.20 mmol) and sodium ascobalo (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 chromography using eluyenle (AcOEI-hexane 1: 1). Yield: 14.5 mg, 31%, RF 0.1 (AcOEl-Hex 1: 1).

'H-NMR (300 MHz, CO, OO): ~ 6.78 (d, lH, J ". = 1.9 Hz, Ar-2), 6.76 (d, lH, J,. • = 8.0 Hz, Ar-5) , 6.65 (dd, lH, J •., = 8.0 Hz, J. ,, = 2.0 Hz, Ar-6), 4.22 (dd, lH, J = 8.0 Hz, J = 4.1 Hz, 20 CH), 3.64 ( dd, 1H, J = 11 .6 Hz, J = 8.0 Hz, CH,), 3.51 (dd, lH, 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 (1, 3H, J =

7.4 Hz, CH,). 13C-NMR (75.5 MHz, CO, OO): ~ 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-dih idroxyphenyl) -2-butoxyethanol (5d)

OBu

HO ~ OH

HO) l)

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, CO, (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. t mg, 43%, R, 0.3 (CH, CI, -MeOH 10: 1). 'H-NMR (300 MHz, CDCI,): ~

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,), 1.60-1 .51 (m, 2H, OCH, CH,), 1.43-1.26 (m, 2H, CH, CH,), 0.88 (t, 3H, J = 7.3 Hz, CH, CH3). 13C-NMR (75.5 MHz, CDCI,): ~ 144.3 (Ar4), 144.2 (Ar-3), 131.8 (Ar-1), 119.9 (Ar-6), 115.7 (Ar-S), 114.1 (Ar-2 ), 82.5 (CH), 69.2 (OCH, CH,), 67.6 (CH,), 32.2 (OCH, CH,), 19.7 (CH, CH,), 14.2 (CH, CH,).

15 Example 3.1.3.

The deacilation (alcoholysis) of compound 6 (peracylated protocatechonic alcohol) with an acetyl group in benzyl position catalyzed by enzymatic extracts using an aliphatic alcohol of the ROH type as solvent, also takes place completely regioselectively in 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

sea fungus lenmentation Y26-02 iY'Iu, H.-H. et al. J Asian Nat Prod Res ", 74725 750; 2009).

ACO ~ OAC HO ~ OMe Enzymatic Extract

•

ACO ~ MeOH HO ~

6 7

Esq

uema 3

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

H0X) Io me HO - &

To a solution of compound 6 (30 mg, 0.19 mmol) in MeOH (1 ml) 30 mg are 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 (AcOEI-hexane 1: 1).

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

15 Example 3.2. Acylation reaction of phenolic compounds by enzymatic catalysis to obtain polyphenols II.

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

Extract HO ~ OH Enzyme HO ~ OH ACO ~ OH

I • I

HO h! L .. J- .. AcO ¿HO ~ +

8 or 9 10

HO ~ OAC ACO ~ OH

I¿ +

HO AcO ~

HO ~ OAC AcO ~ OAC

AcO ~ + HO ~

12 13

OH

Abstract if

OH

HO ~ OH LEAF

Enzymatic HO.! l..oJ- .. AcO ~~ l ' OH ACO ~ OH + I HO A

14

DMF fifteen 16

OH OH

HO ~ OACACO ~ OH

I + I

HO ¿AcO A

17 18

OH

HO ~ OAC

AcOÁJ

19 Scheme 4

Abstract

HO ~ OH HO h 20

Enzymatic •) CoA HO ~ OH AcO h 21 +

HO ~ OAC HO h 23

+

HO ~ OAC + ACO ~ 25

ACO ~ OAC AcO ~ 6 Scheme 4 (continued)

5

Sayings polyphenols They are he compound 8 (hydroxytyrosol),

ACO ~ OH

HO h

ACO ~ OH

AcO .d

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 aelated derivatives. Acylation can be done in the absence or presence of an adsorbent 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, OMF (N, N-dimethyl formamide) can be used as ca-solvent. The acylation result is quantified by 1 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 polyenol 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 lipase 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 10 monoacylated derivatives in the aromatic ring, and of the compound 17 which is the monoacylated derivative at the end of the side chain, in a 1: 1: 1 ratio, with the extracts of Terribacil / us sp. AE2B 122 (CECT8231) and Enterabacter sp. AE1 B89 (CECT 8462), and in the 1: 1: 2 ratio with Pseudomonas sp. AE2B120 (CECT 8462), Bacil / us 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 acylation of the benzyl hydroxyl of compound 14 with any of the enzymes or enzymatic extracts is not detected.

The acylation (esterification) reaction of compound 20: protocatechic alcohol after 24 h (Table 1) leads to a mixture of mono, di and triacylated derivatives. The ratio of monoacylated derivatives in position 4, 3 and side chain is sensitive to the enzyme extract used; thus, the ratio is 1: 1: 1 for the culture supernatant of Terribacil / us sp. AE2B 122 (CECT 8231), 1: 2: 2 for the culture supernatant of Enterabacter sp. AE1B89 (CECT 8462), 1: 3.5: 1 for the culture supernatant of Pseudomonas sp. AE2B120 (CECT 8462), 1: 3: 2 for the culture supernatant of Bacil / us sp. HR21-6 (CECT 8484) and 1: 0: 16 for the lipase of C. antareliea. 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 'H-NMR in the reaction mixture formed by acylation of 105 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.

Acylation of hydroxytyrosol (compound 8). Hydroxytyrosol (50 mg) is dissolved in isopropenyl acetate (3.0 ml) and celite (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 acylation result 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 DMF / isopropenyl acetate 1: 1 mixture (6.0 ml) 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 protocatechic 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 1H_ NMR (See Table 1)

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

The results obtained in the test of anti-radical activity against OPPH for compounds 4a-4d, Sa-Sd, 2 and 14. are represented below. The anti-radical activity expressed as EC50 against OPPH (radical 2,2-diphenyl-1- picrilhydrazyl)

20 represents the amount of antioxidant needed to decrease the concentration of OPPH 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 Chem. 2005, 53, 5212-5217).

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

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

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

5d 16.7 ± 1.81

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), Enterobacter sp. AE1 B89 (CECT 8462).

Example 5.1 The deacetylation reaction of compound 27 (methyl (t-D10 per-O-acetylated glucopyran) has been carried out by alcoholysis in methanolic medium catalyzed by

supernatants obtained from the cultures of the bacterial strains Terribacillus sp. AE2B 122 (CECT 8231), Enterobaeter sp. 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 the corresponding enzyme extract or lipase, in methanol or in deuterated methanol (COJOO).

Once the reaction is terminated, the proportions between the different compounds detected in the reaction crude (compounds 27-29) are obtained from the corresponding 1H_RMN spectrum in CDJOD, after the removal of the enzyme extract or the corresponding enzymes.

OH

HO enzyme extract

• HO ~ ....., .....

CD30D, t.a.

27 28 29

Scheme 5

The enzyme extract from TelTibacil / us sp. AE2B 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-O-acetyl-a.-D-glucopyranoside) was observed, together with compound 27 used as

raw material (72%).

10 The enzymatic extracts from the crops of Enlerobacter sp. AE1B89 (CECT 8462) and Bacil / us sp. HR21-6 (CECT 8484), which showed an 84 and 86 '% conversion, respectively at a significantly lower reaction time (6 h). Such enzymatic extracts show a high tendency to complete deacetylation, to give compound 29 (methyl a-D-glucopyranoside), which is found in

15 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 the deacetylation of compound 27 catalyzed by the culture supernatants of the strains of the invention or by commercial lipases.

Percentage calculated (NMR)

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

..., -. • • V ": '!' - rw- '~ ~ f" -, ""' '"' '., - ~ ~ ~

..... ,, ",, '~' .- '". ~ ~' ~

-

"í: -." '1' \ <, A..,!

"" ".. ~,.,

,. '

~~ _ ~ _. _. _______ ... ~ - <-. ~. ~. ~ ____...... ~~ -¡h., _ ': ..: .. __, -,' "___

Oesacylation of compound 27 (methy2,3,4,6-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 COlOD (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. antaretica 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 3 shows the lH-NMR spectrum of the CHRMN reaction mixture (300 MHz, CD, OD)]; The results are shown in Table 3.

Example 5.2. The culture supernatants of the strains described in the present invention,

15 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

OAc ~ ....- 'I

A ~~ O \ O

AcO 'CO ~~: .-- Extract 31

enzymatic

OR

• + CD30D, t.a. ~ OR1 AcO R'0 O R0

30 'HO

AcO

OR '~ ..-- "\

OR

32 = Ac

R '33 R = H HO HO

'

Scheme 6

5 Enzymatic extracts of Terribacíllus sp. AE28 122 (CECT 8231), Enterobacter sp. AE1889 (CECT8462) and Bacillus sp. HR21-6 (CECT 8464), 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 105 cultures of the previously mendonated strains was observed for carrying out deacetylation on one of the

10 glucopyranose rings (Table 4), allowing the detection of two new compounds,

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

(2,3,4,6-tetra-O-acetyl-a, a-D-trehalose) (38-40%), of which there is no background in the prior art.

15 It should also be noted that compound 32 is the result of a demetrization in only two stages (acetylation and selective deacetylation) of trehalose. Demetrization is the process by which it is achieved that the per-oacetylated trehalose 30 loses its original symmetry and becomes 31 and 32, where one unit of sugar is acetylated and the other totally or partially deacetylated. This process is

20 has achieved in two stages (commercial trehalose acetylation and regioselective deacetylation 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 the culture supernatants of the bacterial strains of the invention or by commercial steps.

Llipasa microorganism t (h)

Lipasa P. cepacea

100%

0%

0%

0%

(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 8464), which allowed the isolation of 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 glycolipids derived from trehalose, which have an interest as vaccines against tuberculosis, caused by the pathogen Mycobacterium tuberculosis

(M. Gilleron al. J. Exp. Med. 2004, 199, 649-659).

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

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

Method A. A suspension of per-O-acetyl-a, aD-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) in C030D (1 .0 rnL) is stirred at room temperature for the time indicated in each case. The lH-NMR spectrum of the reaction mixture is shown in Figure 4

['H-NMR (500 MHz, CO, OO)]; The results are shown in Table 3.

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

10 culture supernatant of the bacterial strain Bacillus sp. HR21-6 CECT 8464 (30.0 mg)

in MeOH (20 mL) it is stirred at room temperature for 3.5 h. Then it

Purify by column chromatography (CH2CI2_CH2Cb-MeOH 1: 2) to give 31 and 32 as syrups.

15 2,3,4,6,6 'Penta · O · acetyl-a, a · D · trehalose (compound 31)

AcO ...., ¡--...: ~ AcO ~ .....,. ~

HO

AcO OH

Yield: 14 mg (29%). 'H-NMR (500 MHz, CO, OO) ¡; 5.57 (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, J1 ', 2' = 3.7 Hz, H-1 '), 4.94 (dd, 1H, H-2), 4.43 (ddd, 1H, J5.e;, = 4.1 Hz, J ~. 6b = 2.4 Hz, H-5). 4.28 (dd, 1H. J,. ,,,. = 2.0 Hz. J ", ", = 11.7 Hz. H-6a ·), 4.24 (dd, 1H, J" ,,, = 12.4 Hz, H · 6a). 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 (55, 3H each, 50Ac); 13C-NMR (125.7 MHz. CO, OO) 8

25 172.7, 172.4, 171.7. 171.5.171.3 (5 COl. 96.3 (C-1 '). 92.8 (C-1). 74.6 (C-3'). 72.8 (C-Z '),

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-o-trehalose (compound 32).

AcO .--- "lN: ...--

AcO ~ _ ~ "" '"

HO

HO HO

Yield: 22 mg (49%). 'H-NMR (500 MHz, CD, OD) S 5.55 (dd, lH, J,., = 10.2 Hz,

5 J, .. = 9.5 Hz, H-3), 5.34 (d, lH, J ", = 3.7 Hz, Hl), 5.07 (dd, lH, J., '= 10.3 Hz, H- 4), 5.05 (d, lH, J ", z = 3.3 Hz, H-l '), 4.96 (dd, lH, H-2), 4.46 (ddd, lH, J" ,, = 3 , 8 Hz, J ,, 5O = 2.4 Hz, H-5), 4.23 (dd, lH, J ", 5O = 12.4 Hz, H-6a), 4.12 (dd, lH, H-6b), 3 , 75 (dd, lH, J,. ,,,, = 2.1 Hz, J,. ,, 5O '= 11.3 HZ, H-6a'), 3.75 (t, lH, J ",., = 9.7 Hz, H-3 '), 3.70 (ddd, lH, J. ",' = 9.8 Hz, J", 5O '=

5.7 Hz, H-5 '), 3.65 (dd, lH, H-6b'), 3.50 (dd, lH, H-2 '), 3.31 (m, lH, H-4'), 2.06 , 2.05, 10 2.01, 1.99 (45, 3H each, 40Ac); "C-NMR (125.7 MHz, CD, OD) S 172.4, 171.7, 171 .6,

171.3 (4CO), 96-8 (C-l '), 93.3 (Cl), 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-5), 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 monosaccharide 27.

AC, Rc ~ OAc

co

OH

AcO O

OAc

Ac ~ Extract

ACACO

enzymatic 35 OH

OAc + AcO OR CD30D, t.a. OAc

OAc OH

OAc ACRc ~

OH

AcO or OAc

OH

Scheme 7

Supernatants obtained from the cultures of Teffibacillus sp. AE28122 (CECT 8231),

In / erobac / er sp. AE1B89 (CECT 8462) and Bacillus sp. HR21 -6 (CECT 8464) allowed

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

10 of truetoturane (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 lower reaction time (4h) in the case of the supernatant obtained from the culture of the Terribaci / lus strain

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

Calculated percentage (NMR) Microorganism / lipase

t (h)

34 35 36

II CECT 8231 10% 38% 52% 4 CECT 8462 18% 43% 39% 7.5 CECT 8464 14% 44% 42% 8.5 Upasa P. cepacea 100% 0% 0% 10 (Sigma-Aldrich, EC 232 - <> 19 -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 supernadanle obtained from the cultures of Bacillus sp. HR21-6 5 (CECT 8464) and chromatographic purification was performed to give rise to the

compounds 35 (26%) Y36 (27%).

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

Per-O-acetylsacarose deactivation (compound 34)

Method A. A suspension of per-O-acetylsacarose (compound 34) (6.0 mg, 8.8IJmol) and

the culture supernatant of the strains of the corresponding invention (3.0 mg) in

15 C0300 (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-D-acetylsacarose (compound 34) (50.0 mg, 74 IJmol) and

20 the supernatant of the cuttive of the strain Baciffus sp. HR21-6 CECT 6464 (25.0 mg) in MeOH (16.0 mL) is stirred at room temperature for 4h. Then, it is purified by column chromatography (CH2Ch - + CH2Cb-MeOH 1: 2) to give 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 regioselectric acylation reactions of carbohydrates. Thus, for example acylation, preferably acetylation of compound 29 (melil a-O

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

OH JO ~

HO HO "", .. ~.:,. --- = ----> HO HO + AcO

~ Enzymatic extract "'.... ~ ....:,. HO"' .... ~ ....:,. OH Bacilfus stratosphericus OMe DMF

29 28 37 60'C

Scheme 8

Despite prolonged heating (60 oC for 105 h), the conversion

15 is 49%, although with high regioselectivity, since the presence of the monoacetylated product in C-6 (compound 28) (13%), Yel compound 37 (methyl4,6-di-Oacetyl-Cl-D-) is observed glucopyranoside) as a major acetylated derivative (36%), not detected in the corresponding deacetylation tests.

Acylation of methy2,3,4,6-tetra-O-acetyl-a-o-glucopyranoside (compound 29) A solution of compound 29 (6.0 mg, 0.017 mmol), the culture supernatant of the strain Bacil / us sp. HR21-6 CECT 8464 (3.0 mg) and isopropenyl acetate (0.5 mL) in DMF (0.3 mL) is heated with stirring at 60 oC for 105 h. It is then concentrated to dryness and the residue is analyzed by 1 H-NMR, showing a mixture

25 of the compounds 28.29 and 37 in a proportion of 13%, 51% and 36%. respectively.

Claims (19)

one.

Microbial strain of Terribacilus deposited in the Spanish Type Culture Collection (CECT) with the accession number CECT 8231.

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 and / or carbohydrates.

9.

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

10.

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

a) bringing the enzymatic extract described according to any of claims 2 to 6 into 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 acylation or deacylation reactions, respectively ; Y b) purify the acylated derivatives of polyphenols and / or carbohydrates obtained in step a).

eleven.

Method according to claim 10. wherein the polyphenols of step a) are hydroxytyrosol, 3,4-dihydroxyphenyl glycol, protocateuchic alcohol or any of its adlated derivatives.

12.

A method according to claim 10 wherein the carbohydrates of step a) are selected from mono-, di-, oligo-, polysaccharides or any of their acylated derivatives, preferably tetraacetylated methyl-a-D-glucopyranoside, octaacetylated tetrahelosa or octaacetylated sucrose.

13.

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

14.

Method according to claim 13, wherein the enzymatic reaction is deacylation, the polyenol of step a) is triacetylated hydroxytyrosol and the reaction is carried out in the presence of methane!

fifteen.

Method according to claim 13, wherein the enzymatic reaction is of deacylation of the aromatic hydroxyls, the polyenol of step a) is 3,4-dihydroxytenyl glycol tetraacylated and the reaction is carried out in the presence of an aliphatic alcohol to produce etherification in the benzyl position. .

16.

Method according to claim 13, wherein the enzymatic reaction is deacylating, the polyenol of step a) is peracylated protocatecholic alcohol and the reaction is carried out in the presence of an alitatic alcohol to produce the etherification of the benzyl position.

17.

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

18.

Method according to claim 17 wherein the acylating agent is isopropyl acetate or vinyl acetate and / or the co-solvent is DMF.

19.

Method according to any of claims 10 or 12, wherein the enzymatic reaction is deacylation, the carbohydrate of step (a) is methyl to tetracetylated D-glucopyranoside, oclaacetylated trehalose or oclaacetylated sucrose and

The reaction is carried out in the presence of optionally aliphatic alcohol deuterated methanol at room temperature.