JP2002363131A - Method for x-y-x type structure lipid production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently produce oils and fats having a high content of an objective structure lipid, preferably about 80% at a low cost without a high purification operation. SOLUTION: This method is characterized in that in a method for X-Y-X type structure lipid production, firstly a fatty acid (X) is directly and selectively introduced into the 1- and 3-positions of glycerol to prepare a 1,3-diglyceride (X-O-X) and then a fatty acid (Y) is introduced into the 1,3-diglyceride (X-O-X) to prepare a triglyceride (X-Y-X). The direct and selective introduction of the fatty acid (X) into the 1- and 3-positions of glycerol is carried out by using a fatty acid chloride by a combination of a solvent, a base and a reaction temperature without using a protective group and a similar mechanism. In the intermediate process, neither a column purification nor recrystallization is performed. The fatty acid (Y) is docosahexaenoic acid or eicosapentaenoic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently producing an XYX type structural lipid.

[0002]

2. Description of the Related Art Conventionally, the nutritional evaluation of fats and oils is often discussed according to the type of fatty acids constituting triglycerides. For example, polyunsaturated fatty acids (PUFA) are good for the body, ω-3 and ω. The argument that the balance of -6 is important is also an argument that focuses only on constituent fatty acids.

[0003] However, the amount of fats and oils taken in the diet is not determined uniformly according to the proportion of fatty acids in the fats and oils.
It has been found that some fatty acids are easily absorbed and some are not, depending on the structure of triglycerides. In other words, it has been found that fatty acids that are absorbed and metabolized while being decomposed by enzymes in the digestive tract are absorbed differently depending on where they are bound to glycerin having three hydroxyl groups. . Based on these findings, studies focusing on the structure of fats and oils have been conducted from the viewpoint of actively absorbing fatty acids having high nutritional value.

[0004] Specifically, a fatty acid bonded to the outer one of the three hydroxyl groups of glycerin is susceptible to hydrolysis of lipase, leaving only one fatty acid at the second position. Focusing on the phenomenon in which monoglyceride is easily absorbed, the medium-chain fatty acids (8 and 10 carbon atoms) which are more easily decomposed by lipase into X of XYX type fats and oils
Are typical), and Y is nutritionally valuable P
UFA, in particular, CDC (caprylic acid-DHA-caprylic acid) provided with docosahexaenoic acid (DHA), which has attracted attention in recent years, is expected to increase the added value of DHA-containing fats and oils.

[0005] It is said that PUFA is advantageous for PUFA absorption.
The study of triglycerides with UFA has been relatively old. However, an efficient method for producing a structured fat has not been put into practical use due to technical problems and cost problems. In particular, with respect to DHA, which has been attracting attention in recent years, it is difficult to purify DHA itself, and it is difficult for enzymes derived from microorganisms (lipases) to act on very long chain fatty acids such as DHA. Had become more difficult to apply. Furthermore, the problem of cost and the problem of the industrial-level manufacturing method related to it remain as problems.

[0006]

As described above, the idea of the structural lipid itself is not so new, and many of these patent applications have actually existed for more than ten years. When chemically synthesizing such structural lipids, it is necessary to control which fatty acid is attached to which of the three hydroxyl groups of glycerin, so protection is usually carried out by a method commonly used in organic synthesis. By introducing a group, a method of restricting the introduction position of the fatty acid is employed.

Strictly speaking, the structure of glycerin is sterically different at the 1-position and the 3-position, so that a glycerin derivative having different substituents introduced therein is asymmetric, and is therefore distinguished from another compound. However, the structural lipid CDC, which is the objective here, is described symmetrically, with the premise that no asymmetry occurs because the structural lipid CDC is bilaterally symmetric. A synthetic method has been established to introduce a predetermined fatty acid at an arbitrary position by using a protecting group. Although it is not a protecting group,
Since dihydroxyacetone having a ketone structure at the hydroxyl group can be converted to glycerin by reducing the ketone, the same effect as when a protecting group is introduced is obtained, that is, a fatty acid can be introduced only at the 1,3-position. Later, the ketone can be converted to a hydroxyl group to introduce a fatty acid at the 2-position (JP-A-59-190948).

In these established prior art synthetic methods,
The structure of the finally formed triglyceride is clear, and the purity is as high as 90% or more. However, the number of steps is increased, and usually a purification operation is required for each step, which is expensive, and is not suitable for mass production because purification is difficult.

In view of the advantages and disadvantages of the prior art described above, the present invention provides a low-cost, high-efficiency, high-content, preferably about 80%, fat / oil without the need for sophisticated purification operations. It is intended to be manufactured.

[0010]

The present invention relates to a method for producing an XYX type structural lipid, which comprises firstly and directly introducing a fatty acid (X) into the glycerin at the 1,3-position. , 3
-Diglyceride (X-OX) is prepared, then 1,3-
A method comprising preparing a triglyceride (XYX) by introducing a fatty acid (Y) into diglyceride (XOX).

The direct and selective introduction of the fatty acid (X) at the 1,3-position is carried out using fatty acid chlorides, without using protecting groups or similar mechanisms, only by a combination of solvent, base and reaction temperature. In this case, the present invention relates to X-
In the method for producing a YX type structural lipid, a fatty acid chloride is used, without using a protecting group or a similar mechanism, and directly and selectively to glycerin only by a combination of a solvent, a base and a reaction temperature. A fatty acid (X) is introduced at the 1,3-position to prepare 1,3-diglyceride (XOX), and then a fatty acid (Y) is introduced into 1,3-diglyceride (XOX). And preparing triglyceride (XYX).

It is characterized in that no column purification or recrystallization is performed in the intermediate step, and in this case, the present invention provides an XYX
In the method for producing a type-structured lipid, first, a fatty acid (X) is directly and selectively introduced into glycerin at the 1,3-position to prepare 1,3-diglyceride (XOX). First, directly and selectively to glycerin, using only a combination of solvent, base and reaction temperature, using a fatty acid chloride, without using a protecting group or a similar mechanism.
A fatty acid (X) is introduced at the 1,3-position to prepare 1,3-diglyceride (XOX), and then a fatty acid (Y) is introduced into 1,3-diglyceride (XOX). This method is characterized in that triglyceride (XYX) is prepared, and column purification and recrystallization are not performed in an intermediate step.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a polyunsaturated fatty acid (PUFA) is preferably a fatty acid having 18 or more carbon atoms and 3 or more double bonds, more preferably 2 or more carbon atoms.
It is a fatty acid having 0 or more and 3 or more double bonds. Specifically, α-linolenic acid (18: 3, n-3), γ-linolenic acid (18: 3, n-6), arachidonic acid (20: 4, n-
6), eicosapentaenoic acid (20: 5, n-3), n
-6-docosapentaenoic acid (22: 5, n-6), docosahexaenoic acid (22: 6, n-3) and the like.

In order to efficiently and economically produce an oil or fat having a high content of the target structural lipid, preferably about 80%, (a) no protective group is used, (b) column purification in an intermediate step, This is a method in which recrystallization is not carried out. Specifically, (1) a fatty acid X is introduced directly into glycerin preferentially (selectively) at the 1,3-position to give 1,3-diglyceride (X-O -X), and (2) a fatty acid Y is introduced into 1,3-diglyceride (XOX) to prepare a triglyceride (X
-YX) is a production method employing a synthetic route of the procedure of preparing (YX).

The introduction (esterification,
Acylation) uses the most common acylation method, the acid chloride method. As a solvent, tetrahydrofuran (TH
F) or use acetonitrile. Solvents which are reactive with acid chlorides, for example alcoholic solvents, acetone, cannot be used. Not only consider acid chlorides and reactivity, but also highly polar compounds such as glycerin and fats and oils,
Alternatively, it must be capable of realizing the incorporation of a low-polarity compound such as a raw material fatty acid chloride. In the laboratory, dimethylformamide (DMF) and dimethylsulfoxide (DMS) are often used in such situations.
O) and the like, but these have a high boiling point and cannot be subjected to operations such as concentration under reduced pressure. Usually, by utilizing the property that these solvents are miscible with water, pour a large amount of water and extract fat-soluble components with water-immiscible solvents such as hexane, ethyl acetate, ether, toluene, methylene chloride and chloroform. Is performed. However, this method cannot recover the solvent, which is not preferable industrially because of cost and waste liquid treatment. From such a background, when the test was performed with several kinds of solvents, it was found that the object could be achieved by using tetrahydrofuran (THF) and acetonitrile.

The acid chloride method is carried out in the presence of a basic catalyst. Triethylamine (TE
A) is preferred. The selection of the basic catalyst required in the acid chloride method depends on whether or not the desired 1,3-diglyceride can be obtained. For example, when dimethylaminopyridine (DMAP), which is widely used in organic synthesis, is used, the selectivity to the 1,3-position is not obtained, and the content of the target product is extremely small. As a result of this study, the reaction temperature was maintained sufficiently low (0 ° C. or less),
It has been discovered that the use of TEA as a base catalyst has been successful.

That is, in the manufacturing method of the present invention, T
The present invention adopts a reaction condition in which glycerin is mixed with HF or acetonitrile as a solvent, TEA is added as a base, and fatty acid chloride (caprylic acid = octanoic acid) is diluted with the solvent and gradually added at a low temperature. Can achieve the purpose.

In this case, the unreacted acid chloride cannot completely react with monoglyceride or glycerin. Since the present invention does not need to be a pure CDC in the end, there is no problem as long as the content of 1,3-diglyceride is within the setting range of the product specification. The present invention aims at 80%
About to put.

It has also been found that diglyceride and monoglyceride can be easily concentrated (removal of acid chloride and triglyceride) by partitioning between hexane and acetonitrile. Alternatively, it is considered that the method can be carried out using hydrous ethanol and hexane. These operations can be introduced as needed, and the content of the target substance can be enhanced.

Further, the production of 1,3-diglyceride was
Divided into steps, the first time using a large excess of glycerin under the conditions described above to preferentially produce 1-diglyceride and after removing the excess glycerin (possible with partitioning of water and organic solvent), By changing the solvent to a general solvent having a lower polarity and performing the second acylation, the residual of monoglyceride and unreacted acid chloride could be reduced. In the examples, methylene chloride is used as the second solvent, but any solvent that can dissolve monoglyceride, triglyceride, and acid chloride (glycerin does not need to be dissolved) and does not react with acid chloride can be used. Therefore, many common organic solvents, such as hexane, ethyl acetate, chloroform, toluene, benzene, pyridine (in this case, also serving as a base) could be used. However, in practice, highly toxic solvents (benzene, chloroform, etc.) are not preferred, and expensive solvents are not selected.

In the method of producing 1,3-diglyceride by dividing it into two steps, it was also confirmed in Examples that the degree of purification of the intermediate affects the subsequent reaction. Experimentally, WSCD as used here,
Although a condensing agent such as DCC is simple and effective, it is not suitable for industrialization because the reagent is expensive and it is difficult to remove by-products. The acid chloride method similar to the first reaction in principle is the most realistic and can be realized by strengthening only the reaction conditions (use of DMAP, reaction temperature).

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail with reference to embodiments. The present invention is not limited by these examples.

EXAMPLES Experimental Example (1) Preparation of 1,3-Dicaprylglycerol 0.5 g (5.43 mmol) of glycerol, 10 ml of acetonitrile and 1.96 ml of triethylamine were mixed well with a stirrer and cooled on ice. 1.77 g (2.0 eq) of capryl chloride was diluted with 4 ml of acetonitrile and slowly added to the reaction solution. After completion of the dropwise addition, the mixture was returned to room temperature and stirred for 1 hour. After completion of the reaction, the solvent was distilled off under reduced pressure, and the mixture was partitioned between ethyl acetate and water. The ethyl acetate layer was washed successively with water, saturated NaHCO ₃ and saturated brine, dried over anhydrous MgSO 4, and concentrated to dryness under reduced pressure. Then, Sample 1 was obtained.

Experimental Example (2) Preparation of 1,3-dicapril-2-DHA-glycerol 2.83 g (30.7 mmol) of glycerol, 40 ml of acetonitrile and 1.02 ml of triethylamine were mixed well with a stirrer and cooled on ice. 1.0 g (0.2 eq) of capryl chloride was diluted with 4 ml of acetonitrile and slowly added to the reaction solution. After completion of the dropwise addition, the mixture was stirred on ice for 30 minutes, and the solvent was distilled off under reduced pressure. Methylene chloride and water were added to the residue, and the mixture was partitioned. The methylene chloride layer was washed successively with water and saturated brine, and dried over anhydrous MgSO ₄ . 1.02 ml of triethylamine was added to the filtrate, mixed well with a stirrer, and cooled on ice. 0.8 g of capryl chloride was diluted with 4 ml of methylene chloride and slowly added to the reaction solution. After completion of the dropwise addition, the mixture was stirred on ice for 30 minutes, and 0.811 g of dimethylaminopyridine (DMAP) and 1.91 g of docosahexaenoic acid (DHA) were added and dissolved. 0.944 g of water-soluble carbodiimide (WSCD) was added to 4 m of methylene chloride.
and slowly added dropwise to the reaction solution. Stirred on ice for 2 hours and at room temperature for 16 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the mixture was partitioned between ethyl acetate and water. The ethyl acetate layer was washed successively with water, 1N HCl and saturated saline, and dried over anhydrous Mg.
After drying over SO ₄ , the mixture was concentrated to dryness under reduced pressure to obtain Sample 2 (3.04 g). Sample 2 was applied to a column of about 20 ml of silica gel and eluted with hexane / ethyl acetate / triethylamine (100/10/1) to separate a triglyceride fraction (2.83 g).

<< Results and Discussion >> In order to selectively acylate the 1,3-positions of glycerin, a method of reacting an acid chloride at a low temperature using a relatively weak base, triethylamine (TEA), was used. The reaction solvent must be a solvent that has an affinity for both high-polarity glycerin and low-polarity fatty acid chloride, and must not react with the acid chloride. In this regard, alcohols,
Acetone was unsuitable, and DMF and DMSO were not used in the test because of their high boiling points and problems in solvent removal. THF was easily degraded, and unknown products were confirmed by TLC for a plurality of deteriorated solvents. However, it was found that good results were obtained by using the same solvent after passing through activated alumina. It was also shown that acetonitrile could be used for the same purpose. Table 1 shows the results of the lipid composition analysis of Sample 1 obtained in Experimental Example (1).

[0026]

[Table 1]

As is clear from Table 1, since the ratio of diglyceride is high and triglyceride and monoglyceride are low, it is understood that acylation occurs preferentially to the 1,3-position. That is, diglyceride is obviously 1,3-
It can be determined that diglyceride is mainly used. When this was partitioned between acetonitrile and hexane, it was found that less than diglyceride was extracted into the acetonitrile layer. The composition was as shown in Table 2.

[0028]

[Table 2]

It is considered that a structural lipid reflecting this glyceride composition can be obtained by acylating PUFA such as DHA to the partial glyceride obtained in Experimental Example (1). That is, an oil or fat containing about 88.9% of CDC is obtained.

In the reaction so far, even though monoglyceride and C8-Cl remained in the reaction system, the reaction could not be further advanced by extending the time or adding TEA. Naturally, reducing the residual amount of monoglyceride leads to increasing the purity of diglyceride,
It may be advantageous to complete the reaction as much as possible, as it may reduce the complexity of pre-processing for subsequent reactions, such as removal of unreacted raw materials. So, T
A test was conducted in which 0.1 equivalent of dimethylaminopyridine (DMAP), a stronger base than EA, was added before and after the addition of the acid chloride, but in any case, the selectivity to the 1,3-position was lacking. However, an increase in triglyceride and 1,2-diglyceride was shown. Since there is no general reason that the reaction of TEA alone is difficult to complete, it is possible that partial glyceride of the product associates and solvates so that the acid chloride does not easily come into contact with unreacted hydroxyl groups. Is done.

Taking these phenomena into consideration, Experimental Example (2) was a test in which the solvent was changed by two-step formation of diglyceride, and the subsequent acylation of DHA was tested under conditions without purification. The first-stage reaction mainly aimed at the production of 1-monoglyceride, which could be realized by using an excessive amount of glycerin. By removing the unreacted glycerin, the range of polarity of the compound used in the subsequent reaction system can be greatly reduced, and the choice of the solvent to be used is greatly expanded.

The following experiment was carried out as a modification of the experimental example (2). Methylene chloride was used to allow for partitioning with water for glycerin removal and for subsequent reactions to be performed on WSCD. After-treatment after the first-stage reaction, after washing with water, drying the methylene chloride layer and treating with anhydrous magnesium sulfate, adding the same amount of TEA as in the first stage, and reacting 80% of the acid chloride under cooling. Was. The progress so far was very good, and the production of triglyceride was small on TLC, and the residual of monoglyceride was also reduced. In addition, unreacted acid chloride was considerably reduced on TLC, and thus the reaction was proceeded to the third-stage reaction. DMAP was added to and dissolved in the second-stage reaction solution, DHA was added, the mixture was cooled on ice, and WS diluted with methylene chloride was used.
CD was added slowly. Addition of the third reaction reagent directly to the solution after the second reaction involves a large amount of the hydrochloride salt of TEA and concerns about how the remaining unreacted raw materials affect the final acylation. Was left,
The results of the reaction were good, and only traces of spots corresponding to unreacted diglyceride and monoglyceride were confirmed on TLC. However, since the molar ratio cannot be calculated precisely, the amount of DHA to be added had to be set to be slightly excessive, so that a large amount of unreacted DHA remained (in TLC-FID analysis, about 8. 8%). In this experiment, a sample obtained for the purpose of structural analysis of triglyceride was analyzed using a silica gel column (6 volumes) with hexane / ethyl acetate / TEA (100: 10: 1) to generate only a triglyceride fraction. Was. As a result, the CCC, CDC + CCD, and CDD in GC were 6.7%, 88.1%, and 5.2%, respectively. Further, when the ratio of CDC to CCD was determined by HPLC on a silver column, the ratio was 89.8: 9.2, indicating that the selective reaction in one or two steps was successful.

Here, the reason that all the compositions are not calculated only from the HPLC is that the concentration-light scattering detector used for the detection has a large contribution of the molecular weight in principle, and CCC and CDD which differ from each other are not quantitative. (Separation is good). As a result, the abundance ratio of CCC was higher than expected. However, by adding DMAP at the stage of shifting from the second reaction to the third reaction, unreacted acid chloride was not selected. It is considered the result of the reaction. In the subsequent stages of the reaction, the situation was confirmed only by TLC, but the detection sensitivity of saturated fatty acids was extremely low, so that it can be said that the estimation of the residual amount of unreacted raw materials was insufficient. That is, it is considered that the amount of CCC generated can be reduced by increasing the completion of the reaction at this stage or by removing or inactivating the remaining raw materials.

<Conclusion> It has been shown that selective acylation of glycerin at the 1,3-position can be performed at a practical level without using a protecting group or the like. The conditions of the reaction needed to be very mild, for which purpose the use of TEA and cooling of the reactor were effective. Further, THF and acetonitrile are preferable as the reaction solvent for the reaction. Diglyceride generation 2
It is possible to increase the diglyceride production efficiency by dividing the reaction into two stages. In this case, a solvent other than the above can be used in the second stage reaction, and ethyl acetate, methylene chloride, chloroform which can be distributed with water can be used. Are simple because there is no need to replace the solvent. By acylating PUFA such as DHA to the obtained 1,3-diglyceride, a CDC-type structured lipid could be prepared. This time, the acylation of WSCD, which can be easily carried out in a laboratory, was used. However, industrially, it is considered that the use of an acid chloride is simple and low in cost as in the first and second stage reactions. However, even if this method is used, advanced purification of DHA is necessary to increase the value of the final CDC product, and how to achieve this at low cost will be a major barrier to commercialization. .

[0035]

[Effect of the Invention] Low cost without advanced purification operation,
It is possible to efficiently produce a fat or oil having a high content of the target XYX type structural lipid (particularly Y: DHA), preferably about 80%.

Claims (4)

[Claims] In a method for producing an XYX type structural lipid, first, a fatty acid (X) is introduced directly and selectively into glycerin at the 1,3-position to prepare 1,3-diglyceride (X-
OX), and then 1,3-diglyceride (X-O)
-X) into which triglyceride (XY) is introduced.
-X). 2. The direct and selective introduction of the fatty acid (X) at the 1,3-position using a fatty acid chloride, without using protecting groups or similar mechanisms, and using only a combination of a solvent, a base and a reaction temperature. The method for producing an XYX type structural lipid according to claim 1, which is performed by: 3. The method according to claim 1, wherein column purification and recrystallization are not performed in the intermediate step. 4. The method for producing an XYX type structural lipid according to claim 1, wherein the fatty acid (Y) is docosahexaenoic acid or eicosapentaenoic acid.