JP2001178489A - Method of producing cyclic phosphatidic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzymatic production process for cyclic phosphatidic acid. SOLUTION: In this production process for cyclic phosphatidic acid(cPA), a phospholipase D is allowed to react with the lysophospholipid. According to this invention, cyclic phosphatidic acid that is useful as a medicine or a phy.siologically active substance can be obtained simply in high yield.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing cyclic phosphatidic acid. More specifically, the present invention relates to a method for enzymatic production of cyclic phosphatidic acid.

[0002]

2. Description of the Related Art Cyclic phosphatidic acid (hereinafter, also referred to as cPA) contains glycerophospholipid expected to be used as an antitumor agent as an active ingredient and has a tumor metastasis inhibitory effect (Int. J. Cancer: 81 , 918-922, 1999). Also,
Cyclic phosphatidic acid is expected as a new bioactive substance including functional foods in the future. Such a cyclic phosphatidic acid has been conventionally prepared by a chemical synthesis method (JP-A-5-230088, JP-A-7-1497).
No. 72, JP-A-7-258278, JP-A-9-25235, etc.).

[0003]

As described above, cyclic phosphatidic acid has been produced by a chemical synthesis method, but cyclic phosphatidic acid is unstable. Is difficult to obtain. In view of the problems of the related art, the present inventors have conducted intensive studies on a method for producing cyclic phosphatidic acid, and as a result, have found that phospholipase D efficiently produces cyclic phosphatidic acid. That is, phospholipase D
(Phosphatidylcholine phosphatide hydrolase, EC3.
1.4.4) acts on phospholipids, hydrolyzes the ester bond between phosphoric acid and the base moiety, and catalyzes the phosphatidyl group transfer reaction (phosphatidyl group transfer reaction) in addition to the simple hydrolysis. It is known to However, a method for producing cyclic phosphatidic acid using phospholipase D has not been conventionally known. The present invention has been made based on such findings, and an object of the present invention is to provide a method capable of producing cyclic phosphatidic acid easily and with high yield.

[0004]

The gist of the present invention, which has been made to solve the above-mentioned problems, is a method for producing cyclic phosphatidic acid, which comprises causing phospholipase D to act on lyso-type phospholipids.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, the present invention is a method for producing cyclic phosphatidic acid, which comprises reacting phospholipase D with a lyso-type phospholipid. The lyso-type phospholipid used in the present invention is not particularly limited as long as it is a lyso-type phospholipid on which phospholipase D can act. A wide variety of lyso-type phospholipids are already known, and those having different fatty acid species, molecular species having an ether or vinyl ether bond, and the like are known, and these are also available as commercial products.

[0006] Phospholipase D acts on lyso-type phospholipids as reaction substrates to produce two types of products, lysophosphatidic acid and cyclic phosphatidic acid. FIG. 1 shows an example of lysophosphatidylcholine. As shown in the figure, when phospholipase D acts on lysophosphatidylcholine (LPC), the ester bond between phosphoric acid and choline is hydrolyzed, and lysophosphatidic acid (LP) is hydrolyzed.
A reaction that generates A) and a reaction that generates cyclic phosphatidic acid (cPA) by a phosphatidyl group transfer reaction occur. By the latter reaction, cyclic phosphatidic acid is obtained from the lyso-type phospholipid.

The phospholipase D used in the above reaction
As such, those derived from higher plants such as cabbage and peanuts and those derived from microorganisms such as Streptomyces chromofuscus can be used, and these can be obtained as commercial reagents.
However, the relative strengths of the hydrolysis reaction and the phosphatidyl transfer reaction are different for each enzyme. In the present invention,
In particular, phospholipase D derived from Actinomadura sp. Strain No. 362 is preferably used, and it has been found that various cyclic phosphatidic acid analogs can be efficiently prepared by acting on lyso-type phospholipid.

[0008] The reaction between the lyso-type phospholipid and phospholipase D is not particularly limited as long as the enzyme can exhibit its activity. For example, the reaction is performed in an acetate buffer containing calcium chloride (about pH 5-6) at room temperature. ~ Under heating (preferably 37 ° C
About 1-5 hours). The generated cyclic phosphatidic acid is prepared according to a conventional method.
It can be purified by extraction, column chromatography, thin layer chromatography (TLC) and the like.

[0009] The cyclic phosphatidic acid thus prepared can be used for pharmaceuticals or various industries. In particular, since it has a tumor metastasis inhibitory activity, it is expected as a new antitumor agent. In addition, cyclic phosphatidic acid is expected as a new physiologically active substance including functional foods in the future.

[0010]

According to the present invention, cyclic phosphatidic acid can be easily and efficiently obtained by reacting phospholipase D with lyso-type phospholipid. In addition, there are various types of phospholipids, and it is possible to prepare a cyclic phosphatidic acid having a corresponding structure by acting phospholipase D on any lyso-type phospholipid. Cyclic phosphatidic acid is unstable in organic synthesis and is difficult to prepare. In particular, plasmalogen type cyclic phosphatidic acid having a vinyl ether bond is difficult to prepare in organic synthesis, but has an advantage that it can be easily prepared by an enzymatic method.

[0011]

EXAMPLES Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples. Example 1 Lysophosphatidylcholine (LPC) 100 mM acetate buffer (pH: 10 mM) containing about 50 μmole and 10 mM calcium chloride as a reaction solvent
5.6) Add 50 ml and suspend the LPC in the ultrasonic cleaner.
After adding 0.5 ml of a phospholipase D enzyme solution to the obtained suspension and mixing well, the mixture is reacted at 37 ° C. with shaking for 2 hours. The phospholipase D enzyme solution used was prepared by dissolving 100 units of phospholipase D (derived from Actinomadura sp. Strain No. 362, manufactured by Meito Sangyo Co., Ltd., and released by Seikagaku Corporation) in 1 ml of distilled water.

Next, after the progress of the reaction is confirmed by thin layer chromatography (TLC), 1.7 ml of a 1 M aqueous citric acid solution is added to the reaction solution to terminate the reaction. Note that T
LC was developed using TLC (Silica Gel 60) with a developing solvent of chloroform: methanol: acetic acid: 5% aqueous sodium disulfite solution (100: 40: 12: 5, volume ratio). The Rf value of LPC is about 0.
1, The Rf value of cyclic phosphatidic acid is about 0.5. The Rf value of LPA, which is formed as a small by-product, is about 0.3.

After the completion of the reaction, the reaction solution is ice-cooled and 26 ml of a mixed solution of chloroform: methanol (2: 1) is added. After stirring well, centrifuge at 1,400 xg for 5 minutes to separate the aqueous methanol layer (upper layer) and chloroform (lower layer). Transfer only the lower chloroform layer to another container, and re-add the remaining methanol-water layer with chloroform: methanol (2: 1)
Add 260 ml of the mixture. After centrifugation as before,
The lower chloroform layer is separated and combined with the previous chloroform layer. This is concentrated on a rotary evaporator,
A small amount of benzene was added to the remaining liquid and completely dried under a nitrogen stream to obtain cyclic phosphatidic acid. The cyclic phosphatidic acid thus obtained was dissolved in a few ml of a chloroform: methanol (1: 1) solution and stored at -20 ° C or lower.

The cyclic phosphatidic acid thus obtained was 41 μmole
And the yield is about 82%. The purity is about 85-90%, and LPA as a by-product and LPC as a starting material can be removed using TLC for preparation or silica column chromatography as necessary.

[Brief description of the drawings]

FIG. 1 shows the reaction between lysophosphatidylcholine (LPC) and phospholipase D.

Claims (1)

[Claims] 1. A method for producing cyclic phosphatidic acid, which comprises reacting phospholipase D with a lyso-type phospholipid.