ES2386918B1 - A PROCEDURE FOR OBTAINING 1,2-DIACETINE - Google Patents

Abstract

The invention relates to a method for producing 1,2-diacetin, characterized in that it comprises hydrolysis of triacetin catalysed by an enzyme, preferably a lipase, at a pH of between 2 and 6, both inclusive, and at variable temperatures, preferably at a temperature of between 4 and 30°C.

Description

A procedure for obtaining 1,2-diacetin

Technical sector: Chemist-pharmacist. STATE OF THE TECHNIQUE

Lipases are the enzymes most used in biocatalysis, because they recognize many different substrates but, in many cases, with a high regio-or enantio-selectivity or specificity. In this way, lipases have been used in very different areas of chemistry, for example energy

(production of biodiesel), food (production of structured lipids)) or fine chemistry (for example resolution of racemic mixtures) In addition, lipases are quite robust biocatalysts, which allows their use in unconventional media, such as organic solvents, ionic liquids or supercritical fluids, further increasing the range of possible applications. However, there is no need

to lose

from view that a from the advantages from the enzymes

compared

with others catalysts is that they can be

used

in media totally aqueous.

The

substrates natural from the lipases They are the

oils

Y fat From done, a from the main

Applications

industrial from the lipases is the hydrolysis from

these natural substrates, usually to obtain free fatty acids or modified oils. Due to the nature of their substrates, most lipases have a peculiar reaction mechanism, with the active site isolated from the reaction medium by a peptide chain called quot; lidquot ;. In the presence of a hydrophobic surface (for example, a drop of oil) this lid moves to expose the active center and the lipase is adsorbed to the hydrophobic surface of the substrate drop.

Triacetin is apparently a very simple molecule, which can be easily produced by the complete acetylation of glycerol. This molecule is commonly used in lipase activity determinations, but the detailed study of this reaction has not been published, although its regioselective hydrolysis could produce compounds

multifunctional and even chiral. For example, regioselective hydrolysis of a single acetyl group of triacetin allows diacetin to be produced, which has a single free hydroxyl group at position 2 or 3. This compound may be useful for some uses, such as the synthesis of 0- ( 1,2-di-0-acetyl-glycerol-3-phosphoryl) ethanolamine [1]. Reference lb uses regiospecific lipases 1.3 to achieve 2-monoacetin, which is subsequently chemically acylated to achieve stereospecific diglycerides. But if this hydrolysis occurs only in position 1 or position 3, the carbon 2 of the diacetin will be a chiral center, further increasing the interest of the process.

However, the migration of acyl groups and racemization can produce a mixture of regal and enantioisomers, decreasing the interest of the process, so that experimental conditions must be selected in which this does not occur.

Although triglycerides usually have a low water solubility as they are formed by long acyl chains, the short triacetin chain significantly increases its water solubility (70 g / L at 25 ° C), allowing hydrolysis in aqueous systems at moderately high concentrations. However, it has been described that its low hydrophobicity causes this to be a bad substrate for lipases when it is completely soluble.

[2-3] and is even quite inefficient in inducing interfacial activation of lipases [4]. Even in the absence of migration, initially, as shown in the lb reference, the use of lipases in triacetin hydrolysis would be expected to lead to the formation of 2-monoacetin, passing through a 1,2-diacetin mixing phase 1 2-monoacetin. However, if triacetin is no longer very hydrophobic and is not a very good enzyme substrate, it is very possible that 1, 2 (2, 3) diacetin is even worse substrate and under certain conditions its almost total accumulation can be achieved.

References

[1] a) Puricelli, L. 0- (1,2di-O-acetyl-glycero-3-phosphoryl) ethanolamine, a process for its preparation and its therapeutic use. European patent O 335 331. 1989. b). Mazur et al. quot; REGIO ANO STEREOSELECTIVE SYNTHESIS OF TRIGLYCERIDESquot; W09116442, 1991

[2] Sarda, L. Desnuelle P. Action of the lipase pancreatique sur les esters in Biochim emulsion. Biophys Acta, 1958; 30: 513-521

[3] Ferrato, F., Carriere, F., Sarda, L., Verger R., in :, Methods in Enzymology, B. Rubin, E.A. Dennis (Eds.) Academic Press, New York. 1997; 286: 327-34

[4] Pernas, M.A., Pastrana, L., Fuciños, P., Rúa, M. L. Regulation of the interfacial activation within the Candida rugosa lipase family. J. Phys. Org. Chem., 2009; 22: 508-514.

Brief Description of the Invention

This patent application describes a regioselective hydrolysis process, preferably in aqueous media, of soluble triacetin to produce 1,2-diacetin in high yields catalyzed by different enzymes, preferably lipases. The reaction is designed so that the migration of acyl groups is minimized and thanks to the different hydrophobicity of diacetin and triacetin, the use of specific enzymes 1, 3, 1,2-diacetin is coupled.

Thus, this application refers to a process for obtaining 1,2-diacetin characterized in that it comprises performing a hydrolysis of triacetin catalyzed by a

enzyme,

preferably a lipase, to a pH understood between

2

Y 6, both of them values included

The

hydrolysis be makes to temperatures variables Y

preferably at a temperature between 4 and 30 ° C. According to this procedure the pH is preferably between 4 and 5.5. The enzyme, such as a lipase, for use in this procedure may be immobilized on a support. Saying

support can be by way of example an acrylic support,

porous glass, agarose, silica, cellulose, polystyrene,

polystyrene divinylbenzene or polyurethane.

In addition, lipases may be immobilized by

5

physical adsorption, for example, by physical adsorption on

hydrophobic supports.

According to other embodiments, the lipase is immobilized as

crosslinked covalent enzymatic aggregate or crystal

cross-linked enzyme.

1 o

Useful lipases can be lipases from microorganisms of

the genera Can di da, Thermomyces, Bacillus, Aspergillus,

Mucor, Rhizomucor.

According to particular embodiments, they can also be added

water soluble solvents between 10-90% v / v. Those

fifteen

solvents can be for example acetonitrile, dioxane,

tetrahydrofuran, dimethyl formamide, methanol, ethanol or

propanol

Hydrolysis can also be performed in a solvent

immiscible with water, saturated in water.

twenty

According to other particular embodiments it is used as

diethyl ether solvent.

According to other additional particular embodiments the

process comprises the addition of solvents soluble in

Water between 10-90%. v / v, which may be

25

selected from, for example, acetonitrile, dioxane,

tetrahydrofuran, dimethyl formamide, methanol, ethanol, propanol

and mixtures of them.

Brief description of the figures

30

Figure 1 shows the reaction as well as the possible

unwanted reactions

Figure 2 shows the reaction course of the hydrolysis of

triacetin at pH 5.5 and 22 ° C catalyzed by immobilized CALE

in Glutaraldehyde Sepabeads. (Circles: triacetin,

35

   Triangles: diacetin; Squares: monoacetin.)

Figure 3 shows the reaction course of the hydrolysis of triacetin at pH 5.5 and 22 ° C catalyzed by immobilized CALE in Sepabeads C18. Circles: triacetin, Triangles: diacetin; Squares: monoacetin. Figure 4 shows the hydrolysis reaction course of triacetin at pH 5.5 and 22 ° C catalyzed by RML immobilized in C18 sepabeads. (Circles: triacetin, Triangles: diacetin; squares: monoacetin). Figure 5 shows the hydrolysis reaction course of triacetin at pH 5, 5 and 22 ° C in 20% aceonitrile catalyzed by RML immobilized in Sepabeads C18. Circles: triacetin, Triangles: diacetin; Squares: monoacetin. Figure 6 shows the hydrolysis reaction course of triacetin at pH 5.5 and 22 ° C in 20% aceonitrile catalyzed by CALE immobilized in Glutaraldehyde Sepabeads (Circles: triacetin, Triangles: diacetin; squares: monoacetin).

Detailed description of the invention

It is intended to stop the hydrolysis of triacetin catalyzed by different specific lipases 1, 3

(eg, those of the genera Candida, Thermomyces, Bacillus, Aspergillus, Mucor, Rhizomucor) freely or immobilized on different supports (eg acrylic supports, porous glass, agarose, silica, cellulose, polystyrene, divinylbenzene polystyrene, polyurethane), either covalently or by physical absorption, in 1, 2-diacetin (figure 1), for later use in the design of different regal and enantiospecific products.

At pH 7 and 4 ° C or 25 ° C, acyl migration occurs, obtaining at the moment that all triacetin is consumed a mixture of 1,2- and 1,3-diacetin, in addition to a certain amount of 1 -and 2-monoacetin and even glycerol.

However, in the range of pH 6-3 and in the temperature range of 4 to 37 ° C it is possible to avoid the formation of 1,3-diacetin by migration and in addition the accumulation of 1,2-diacetin occurs, with very high yield (above 80% fully soluble) at 5 M triacetin (in this case a biphasic system is formed with part of the soluble triacetin and the rest in an insoluble triacetin phase, which is consumed until producing between 80% > 98% 1,2-diacetin).

and

even near to the 100%), for all the preparations from

the

different lipases used The results no be

they affected

barely by he use from different concentrations from

triacetin,

since 100 mM (in he that the triacetin is

PH control is critical. It is recommended to use very concentrated buffers (eg, 1 molar phosphate or acetate) and only raise the pH to 5.5 when the pH drops below pH 3 using a saturated buffer at pH 7 and very good agitation to avoid pH gradients where the migration of the acyl groups will occur

The reaction slowed down but increased yields of 1,2-diacetin if miscible co-solvents (5-75% v / v) were used with water (for example acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol, propanol, etc. ), possibly by inhibiting the hydrolysis of 1,2 diacetin more strongly than that of triacetin.

The hydrolysis reaction was also tested in diethyl ether saturated with water, a solvent described as an inhibitor of acyl migration. In this case only immobilized or aggregated enzymes were used, the solvent is previously saturated in acetate buffer at pH 5, this buffer being added progressively throughout the reaction to compensate for the water consumed by hydrolysis. The reaction rate was lower with most of the lipase preparations, but the yields of 1.2 diacetin were similar (80-gt; 98%).

For all the cases tested, using the different lipases described above free or immobilized on different supports, under conditions where the acyl migration was reduced or eliminated yields greater than 80% of 1,2-diacetin were achieved.

Examples of the invention

Example l. Hydrolysis of triacetin at pH 5.5 by lipase B

of antarctic Candida immobilized on Sepabeads C 18.

5

Glutaraldehyde-Sepabeads was prepared by suspending 10 g

EC-HA-Sepabeads wet in 20 mL of 15%

glutaraldehyde (v / v) in 200 mM phosphate at pH 7.0. The

suspension was kept under gentle agitation at 25 oc for 15

h. After that time, the activated support was filtered and

1 o

washed thoroughly with distilled water. This treatment is

performed to activate all primary support terms

with two glutaraldehyde molecules. The activated support is

used immediately after preparation.

10 g of glutaraldehyde-Sepabeads were added to 400 mL of

fifteen

a solution of lipase (0.5 mg / mL) in 10 mM sodium phosphate

at pH 7 in the presence of 0.1% (v / v) Triton X -100. After 7 hours

at 22 oc under stirring at 250 rpm, the suspension was filtered and the

immobilized lipase was washed 5 times with 10 volumes of water

distilled, the wet biocatalyst was stored at 4 ° C.

twenty

On the other hand, a solution of 100 mM of

triacetin in 500 mM sodium phosphate and its pH was adjusted to pH

5.5. The substrate was completely soluble in these

terms.

A 1.5 g sample of wet biocatalyst is added to

25

40 mL of the triacetin solution and suspensions of

reaction was gently stirred on a stirrer at 250 rpm and

22 ° C Samples were periodically taken from this suspension,

the biocatalyst was discarded by centrifugation and the

concentration of reaction products was analyzed by

30

HPLC Diacetin and triacetin were analyzed using 10%

acetonitrile / 90% water (v / v) as mobile phase at a flow of 1

ml / min and an RP-HPLC column (with a Spectra Physic SP pump

100 and a Spectra Physic SP 8450 UV detector) using a

Kromasil C18 column (15 cm x 0.46 cm), the volumes of

35

retention were 32. O ml for triacetin, 5.8 ml for 1, 2-

   diacetin, 4. 8 ml for 1,3-diacetin. The concentrations of

Triacetins and both diacetins were calculated using curved reference samples using commercial samples. Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.

After 2 hours of reaction, all triacetin had been consumed, with 95% of 1,2-diacetin and 5% of 2-monoacetin. The catalyst could be reused in 5 cycles without any variation in speed or performance. Figure 2 shows the reaction course of CALE catalyzed triacetin hydrolysis immobilized in Sepabeads glutaraldehyde at pH 5.5 and 22 ° C. (Circles: triacetin, Triangles: diacetin; squares: monoacetin.)

Example 2. Hydrolysis of triacetin at pH 5.5 by Lipase B from Candida antarctica immobilized on Sepabeads-C 18.

10 grams of octadecyl-Sepabeads were added to 400 mL of CALE in a lipase solution (O. 5 mg / mL) in 1 M mM phosphate buffer at pH 7 and 22 oc. After 7 h under stirring at 250 rpm, the suspension was filtered and the lipase was washed 5 times with 10 volumes of distilled water. The wet preparation was stored at 4 ° C.

On the other hand, a solution of 100 mM triacetin in 500 mM sodium phosphate was prepared and its pH adjusted to pH

5. 5. The substrate was completely soluble under these conditions.

A sample of 1.5 g of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred in a Shaker at 250 rpm and 22 ° C.

Periodically

be they took out samples from is suspension, be

discarded

he biocatalyst by centrifugation Y the

concentration

from the products from reaction be analyzed by

HPLC

Diacetin Y triacetin be analyzed using 10%

acetonitrile / 90% water (v / v) as a mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP pump

100 and a Spectra Physic SP 8450 UV detector) using a Kromasil C18 column (15 cm x 0.46 cm), retention volumes were 32.0 ml for triacetin, 5.8 ml for 1,2-diacetin, 4.8 ml for 1, 3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved reference samples using commercial samples. Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.

After 60 minutes of reaction, triacetin is depleted and a yield of 87% of 1.2 diacetin and 13% of 2-monoacetin is achieved. Figure 3 shows the reaction course of CALE catalyzed triacetin hydrolysis immobilized in Sepabeads C18 at pH 5.5 and 22 ° C. Circles: triacetin, Triangles: diacetin; Squares: monoacetin.

Example 3. Hydrolysis of triacetin at pH 5.5 by Rhizomucor miehei lipase (RML) immobilized on Sepabeads-C 18.

10 grams of octadecyl-Sepabeads were added to 55 mL (RML) of a lipase solution (0.5 mg / mL) in 10 mM phosphate buffer at pH 7 and 22 ° C. After 7 h under stirring at 250 rpm, the suspension was filtered and the lipase was washed 5 times with 1 O volumes of distilled water. The wet preparation was stored at 4 ° C.

On the other hand, a solution of 100 mM triacetin in 500 mM sodium phosphate was prepared and its pH adjusted to pH

5.5. The substrate was completely soluble under these conditions.

A 1.5 g sample of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred in a Shaker at 250 rpm and 22 ° C. Samples of this suspension were taken periodically

be

discard he biocatalyst by centrifugation Y the

concentration

from the products from reaction be analyzed by

HPLC

Diacetin Y triacetin be analyzed using 10%

acetonitrile / 90% water (v / v) as mobile phase at a flow of 1

ml / min and an RP-HPLC column (with a Spectra Physic SP 100 pump and a Spectra Physic SP 8450 UV detector) using a Kromasil C18 column (15 cm x 0.46 cm), retention volumes were 32. O ml for triacetin, 5.8 ml for 1,2-diacetin, 4. 8 ml for 1,3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved reference samples using commercial samples. Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.

After 1 hour, 88% of 1.2 diacetin and 12% of 2-monoacetin are achieved. Figure 4 shows the reaction course of the RML catalyzed hydrolysis of immobilized triacetin in Sepabeads C18 at pH 5.5 and 22 ° C. (Circles: triacetin, Triangles: diacetin; squares: monoacetin).

Example 4. Hydrolysis of triacetin at pH 5.5 in the presence of 20% acetonitrile by Rhizomucor miehei lipase (RML) immobilized on Sepabeads-C 18.

10 grams of octadecyl-Sepabeads were added to 55 mL (RML) of a lipase solution (0.5 mg / mL) in mO 1 O phosphate buffer at pH 7 and 22 oc. After 7 h under stirring at 250 rpm, the suspension was filtered and the lipase was washed 5 times.

with

1 o volumes from Water distilled The preparation wet be

stored

to 4 ° C

By

other side be prepared a dissolution from 100 mM from

triacetin in 500 mM sodium phosphate / 20% acetonitrile (v / v) and its pH was adjusted to pH 5. 5. The substrate was completely soluble under these conditions.

A 1.5 g sample of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred in a Shaker at 250 rpm and 22 ° C. Samples were periodically taken from this suspension, the biocatalyst was discarded by centrifugation and the concentration of the reaction products was analyzed by HPLC. Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as a mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP 100 pump and a Spectra Physic SP UV detector 8450) using a Kromasil C18 column (15 cm x 0.46 cm), retention volumes were 32.0 ml for triacetin, 5.8 ml for 1.2 diacetin, 4.8 ml for 1,3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved reference samples using commercial samples. Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.

After 4 h of reaction, the yield exceeds 95%.

Figure 5 shows the reaction course of the hydrolysis of triacetin catalyzed by RML immobilized in Sepabeads C18 at pH 5.5 and 22 ° C in 20% aceonitrile. Circles: triacetin, Triangles: diacetin; Squares: monoacetin.

Example 5. Hydrolysis of triacetin at pH 5.5 in 20% acetonitrile catalyzed by lipase B from Candida antarctica immobilized on Sepabeads-glutaraldehyde.

10 grams of octadecyl-Sepabeads were added to 400 mL of CALE in a lipase solution (O. 5 mg / mL) in 10 mM phosphate buffer at pH 7 and 22 ° C. After 7 h under stirring at 250 rpm, the suspension was filtered and the lipase was washed 5 times with 1O volumes of distilled water. The wet preparation was stored at 4 ° C.

On the other hand, a solution of 100 mM triacetin in 500 mM sodium phosphate was prepared and its pH adjusted to pH

5.5. The substrate was completely soluble under these conditions.

A 1.5 g sample of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred in a Shaker at 250 rpm and 22 ° C. Samples were periodically taken from this suspension, the biocatalyst was discarded by centrifugation and the

Concentration of reaction products was analyzed by HPLC. Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as a mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP 5 100 pump and a Spectra Physic UV detector SP 8450) using a Kromasil C18 column (15 cm x 0.46 cm), retention volumes were 32.0 ml for triacetin, 5.8 ml for 1,2-diacetin, 4.8 ml for 1,3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved samples of

10 reference using commercial samples. Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.

After 2 97% of 1.2 diacetin could be observed with a

15 1% of triacetin still present in the reaction medium, at 3 h 98% of diacetin could be observed and at 4 o'clock it still maintains 87%. Figure 6 shows the reaction course of CALE catalyzed triacetin hydrolysis immobilized in Sepabeads glutaraldehyde at pH 5.5 and 22 ° C at 20%

20 aceonitrile. (Circles: triacetin, Triangles: diacetin; squares: monoacetin).

Claims (14)

 Claims

one.

A process for obtaining 1,2-diacetin characterized in that it comprises performing a hydrolysis of triacetin catalyzed by an enzyme at a pH between 2 and 6, both included.

2.

A method according to claim 1, characterized in that the enzyme is a lipase.

3.

A process according to claim 1, characterized in that it is carried out at a temperature between 4 and 30 ° C.

Four.

A process according to claim 1, characterized in that it comprises hydrolyzing triacetin at a pH between 4 and 5.5.

5.

A method according to claim 1 or 2, characterized in that the lipase is immobilized on a support.

6.

A process according to claim 5, characterized in that the support is selected from acrylic supports, porous glass, agarose, silica, cellulose, polystyrene, polystyrene divinylbenzene and polyurethane.

7.

A process according to claim 5, characterized in that the hydrolysis is carried out using lipases immobilized by physical adsorption.

8.

A method according to claim 7, characterized in that the hydrolysis is carried out using lipases immobilized by physical adsorption on hydrophobic supports.

9.

A method according to claim 5, characterized in that the enzyme is a lipase and is immobilized as a cross-linked covalent enzyme aggregate or cross-linked enzymatic crystal.

1O. A method according to claim 2, characterized in that the lipase is selected from lipases of microorganisms of the genera Candida, Thermomyces, Bacillus, Aspergillus, Mucor and Rhizomucor. A method according to claim 1, characterized in that it further comprises adding water soluble solvents in an amount between 10-90% v / v. A method according to claim 11, characterized in that said solvents are selected from acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol and propanol. 13. A method according to claim 1, 1 O characterized in that it is carried out in a solvent immiscible with water, saturated in water. 14. A process according to claim 13, characterized in that diethyl ether is used as solvent. 15. A method according to claim 13, 15 characterized in that it also includes the addition of water-soluble solvents in an amount between 10-90%. v / v. 16. A process according to claim 13, characterized in that said solvents are selected 20 between acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol, propanol and mixtures thereof.