JPH0588114B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a sphingophospholipid heterocyclic compound derivative by an oxygen method, which has not been previously known to be able to be produced by an oxygen method. This invention relates to a method for producing oxygen-method sphingophospholipid heterocyclic compound derivatives, which are useful in the field of active substances and their carriers. More specifically, unlike the cabbage-derived phospholipase D used in the conventional oxygen method for other phospholipids (optimal temperature below 40°C, optimum pH 5.4-5.6), the optimum temperature is 60-70°C. A method for producing a sphingophospholipid heterocyclic compound derivative by reacting a sphingophospholipid with a heterocyclic compound having an alcoholic hydroxyl group, which was conventionally thought to be unable to be produced by the oxygen method, in the presence of phospholipase DM at a temperature of approximately 7°C and an optimum pH of 7. Regarding. In the present invention, the sphingophospholipid heterocyclic compound derivative is defined as a sphingophospholipid heterocyclic compound derivative that is obtained by simultaneously hydrolyzing the ester bond between the phosphoric acid structure moiety of the sphingophospholipid as a starting material and the alcohol structure moiety of the sphingophospholipid by the action of phospholipase DM. It means a new sphingophospholipid different from the starting material, derived by transfer to a heterocyclic compound having an alcoholic hydroxyl group used in the above reaction. In particular, the present invention provides the following formula () [Formula] where A represents a group of the following formula (i) or (ii) [Chemical formula] or [Chemical formula], where R is a saturated or unsaturated
Represents a _C12 to _C24 aliphatic hydrocarbon group, A' represents an oxide anion or a hydroxyl group, and B represents -( _CH2 ) _2N +
( _CH3 ) ₃ , -( _CH2 ) _2NH2 or _{-CH2CH (} OH) _CH2
Indicates (OH). The sphingophospholipids represented by the following (1) to (3)
A heterocyclic compound having an alcoholic hydroxyl group shown in (1) having a chain side chain bonded to a primary alcoholic OH, and having a tetrahydrofuran ring, a dioxolane ring, a thiophene ring, a pyrrolidine ring, an imidazole ring, an imidazolidine ring , oxazole ring,
a heterocyclic primary alcohol compound having a heterocycle selected from the group consisting of a thiazole ring, a pyran ring, an oxane ring, a thian ring, a piperidine ring, a triazine ring, a tetrahydrophthalimide ring, a benzoxolane ring and a hexamethyleneimine ring;
Here, the heterocyclic moiety of the compound is a _C1 - _C3 alkyl group, a _C1 - _C3 alkylene group, a hydroxyl group, a carboxyl group, a carbol group, a halogen, an amino group, an oxo group, a benzyl group, a phenyl group, and
may have a substituent selected from the group consisting of C ₁ to _{C 3} alkylene sulfonic acid groups; (2) a secondary alcoholic OH directly bonded to the ring or a chain bonded to the secondary alcoholic OH; a heterocyclic secondary alcohol compound having a side chain and a heterocyclic ring selected from the group consisting of a piperidine ring, a morpholine ring, a piperazine ring, a pyrrolidine ring, an oxane ring, and a phthalimide ring, wherein the heterocyclic moiety of the compound is a _C1 - _C3 alkyl group,
It may have a substituent selected from the group consisting of a hydroxyl group and a C ₁ -C ₃ alkylene sulfone ring group. (3) Pyridine ring, pyrimidine ring, triazine ring,
A pentose derivative or deoxypentose derivative having a heterocycle selected from a purine ring and an 8-azapurine ring as a substituent, wherein the heterocyclic moiety of the derivative is a mercapto group, a halogen,
It may have a substituent selected from the group consisting of a hydroxyl group, a carboxyl group, an oxo group, an amino group, a _C1 - _C3 alkyl group, a methoxy group, an ethoxy group, a carboxymethyl group, an acetyl group, and an azoyl group. However, heterocyclic compounds having a sugar selected from the group consisting of adenosine, cytidine, uridine, guanosine and arabinocytidine are excluded, heterocyclic compounds having an alcoholic hydroxyl group selected from the group consisting of sphingophospholipid and heterocyclic Phospholipase D (hereinafter referred to as
The following formula () [Formula] wherein A and A' have the above meanings, and C is the alcohol shown in (1) to (3) above. The present invention relates to a method for producing a sphingophospholipid heterocyclic compound derivative represented by the following formula, which represents a residue obtained by removing the alcoholic hydroxyl group from a heterocyclic compound having an alcoholic hydroxyl group, and salts thereof. It has been known that phospholipase D catalyzes a reaction in which sphingophospholipid D hydrolyzes the choline base-phosphate ester of sphingophospholipids, such as sphingomyelin, to produce choline and N-acylsphingosine-1-phosphate. [FM Davidson, “Biochem, J.vol69, 458-466
(1958)” (cabbage phospholipase D); Y,
OKAWA et al. “J.Biochem., 78, 363-372 (1975)”
(Strepto-myces hachijoeusis phospholipase D)]. Furthermore, regarding glycerophospholipids, for example, lecithin and ethyl alcohol can be treated with phospholipase D.
It has been reported that when reacted in the presence of phospholipid, the ester bond between the phosphoric acid structure part of the phospholipid and the alcohol structure part of the phospholipid is hydrolyzed, and at the same time, phosphatidyl ethanol is produced by the phosphatidyl group transfer action. Publicly available [RM
Dawson; Biochem. J. 102, 205 (1967); Yaug;
J. Biol chem., 242, 477 (1967)]. Furthermore, British Patent No. 1581810 (corresponding to West German Publication No. 2717547), which uses the phospholipase D derived from cabbage to transfer the phospholipid group, describes a method for producing phospholipids.
has been proposed, but this only describes the generation of transfer derivatives by the action of phospholipase D between glycerophospholipids and chain primary alcohols with _C5 or less; There is no description or suggestion of a transfer effect with alcohol. That is, according to this proposal, a glycerophospholipid represented by the general formula of this proposal and a primary having a straight chain or branched alkyl group up to C ₅ which may be substituted with a hydroxyl group, halogen, amino or other substituent. It only discloses a primary alcohol transfer reaction with alcohol using the enzymatic action of the cabbage-derived phospholipase D, and there is no mention at all of sphingophospholipids whose nonpolar moiety structure is completely different from that of glycerinolipids. do not have. Only,
Robert J. CHALIFOUR et al. reported that they attempted to generate glycerol transfer by the action of phospholipase D using sphingomyelin as a substrate and glycerol as an acceptor alcohol, but it was not possible. (Can.J.Biochem., vol58, 1189 (1980)).
Conventionally, when sphingophospholipids are treated with phospholipase D in the presence of a compound having an alcoholic hydroxyl group, the ester bond between the phosphoric acid structure of the sphingophospholipid and the alcohol structure of the sphingophospholipid is hydrolyzed. At the same time, it was completely unknown that a new sphingophospholipid derivative different from the starting sphingophospholipid was produced by N-acylsphingosine-1-phosphoryl transfer. The present inventors have discovered the existence of a microorganism that has a phospholipase D production ability that differs from the conventionally known cabbage phospholipase D in terms of its optimum temperature, optimum pH, etc., and has already published JP-A No. 58-63388, Japanese Unexamined Patent Publication 1983-
Proposed in No. 67183. In this proposal, phospholipase D obtained from the phospholipase D-producing bacteria encapsulates primary alcohols with _C5 or less, and further transfers phospholipids to a wide range of alcohol compounds that have never been mentioned before. It has been described that it has an effect. Among these, glycerin lipids and sphingophospholipids are described as phospholipids that serve as substrates, and heterocyclic primary alcohols are described as heterocyclic alcohols among alcohols in which rearrangement occurs.
The heterocycle of the heterocyclic primary alcohol includes a furan ring, a phthalimide ring, a pyrrole ring, an indole ring, a pyridine ring, a morpholine ring, a pyrimidine ring,
A piperazine ring and an imidazopyrimidine ring (purine ring) are described. However, some of the above proposals include tetrahydrofuran ring, dioxolane ring, thiophene ring, pyrrolidine ring, imidazole ring, imidazolidine ring, thioxazole ring, thiazole ring, pyran ring, oxane ring, thian ring, piperidine ring, triazine ring, and tetrahydrophthalimide ring. Heterocyclic primary alcohols and heterocyclic secondary alcohols having a ring, benzoxolane ring, or hexamethyleneimine ring are neither described nor suggested at all. Regarding the heterocyclic alcohols mentioned in the above proposal, there is no particular mention of the types of substituents, but only three types of heterocyclic primary alcohols having direct substituent groups are described: pyridoxine, thiamine, and adenosine. has been done. As a result of further research, the present inventors found that the phospholipase DM hydrolyzes the sphingophospholipid represented by the above formula (), and at the same time, the above (1), (2) and ( 3) We discovered a new fact that this transfer occurs to heterocyclic compounds having an alcoholic hydroxyl group selected from the group consisting of: According to the research of the present inventors, examples of sphingophospholipids include sphingomyelin and the heterocyclic primary alcohol N-(2-hydroxyethyl).
In the present invention, there is an enzyme newly called phospholipase DM that catalyzes the formation of a sphingophospholipid heterocyclic compound derivative with pyrrolidine, and in the presence of this phospholipase DM, the sphingophospholipid represented by the formula ( and the above (1)
It has been discovered that a new sphingophospholipid heterocyclic compound derivative, which was previously unknown to be able to be produced, can be produced by reacting with the heterocyclic compound having an alcoholic hydroxyl group (3). In this way, a new sphingoline can be produced by an enzymatic method in a one-step reaction, without the need for complicated and disadvantageous chemical synthesis means, under mild and easy conditions, and without the fear of side reactions. It was found that lipid heterocyclic compound derivatives can be produced in good yield. Therefore, an object of the present invention is to provide a new enzymatic method for producing sphingophospholipid heterocyclic compound derivatives. The above objects and many other objects and advantages of the present invention will become more apparent from the following description. The sphingophospholipid used in the method of the present invention is represented by the following formula (). [C] However, in the formula, A represents the following (i) or (ii) [C] or [C], where R represents a saturated or unsaturated C ₁₂ to C ₂₄ aliphatic hydrocarbon group, and A ' represents an oxide anion or a hydroxyl group, B
represents -( _CH2 ) _{2N +} ( _CH3 ), -( _CH2 ) _2NH2 or _-CH2.CH (OH) _CH2 (OH). The atom sphingophospholipid of the formula () is a known compound and is available on the market, and can be extracted from natural products or synthesized by methods known per se. For example, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol, etc., which are extracted from animal, plant, and microbial tissues by known means, can be used alone or in mixtures, and can be used as they are or after being purified. Therefore, part or all of its structure can be chemically synthesized and utilized. Examples of the heterocyclic alcohols to be reacted with the raw material sphingophospholipid of formula () in the presence of phospholipase DM in the method of the present invention are listed below.
Heterocyclic compounds having an alcoholic hydroxyl group (1) to (3) can be used. (1) The following compounds can be mentioned as heterocyclic M-class alcohol compounds. A heterocyclic ring having a linear side chain bonded to a primary alcoholic OH and having the following heterocycles: tetrahydrofuran ring, dioxolane ring, thiophene ring, pyrrolidine ring, imidazole ring, imidazolidine ring, oxazole ring, thiazole ring, pyran ring, oxane ring, thiane ring. a heterocyclic primary alcohol compound residue having a heterocyclic ring selected from the group consisting of a ring, a piperidine ring, a triazine ring, a tetrahydrophthalimide ring, a benzoxolane ring, and a hexamethyleneimine ring, where the heterocyclic portion of the residue is Consisting of C ₁ - C ₃ alkyl group, C ₁ - C ₃ alkylene group, hydroxyl group, carboxyl group, carbonyl group, halogen, amino group, oxo group, benzyl group, phenyl group, and C ₁ - _{C 3} alkylene sulfonic acid group It may have a substituent selected from the group; In the above, when there is no substituent, a thiophene ring, a pyrrolidine ring, an imidazole ring, a piperidine ring, a tetrahydrophthalimide ring, a benzoxolane ring and a hexamethyleneimine ring are used. Preferred examples include N-(2-hydroxyethyl)
Pyrrolidine, N-(2-hydroxyethyl)piperidine, 2-(2-hydroxyethyl)piperidine, 2-hydroxymethylpiperidine, 3-hydroxymethylpiperidine, N-(2-hydroxyethyl)hexamethyleneimine, 2-thiophene Methanol, 2-thiopheneethanol, N-hydroxymethyltetrahydrophthalimide, 4-
Examples include hydroxymethylimidazole and piperonyl alcohol. In the above, when the substituent has a _C1 to _C3 alkyl group, dioxolane ring, pyrrolidine ring, thiazole ring, oxazole ring, imidazole ring and imidazolidine ring are preferable, examples of which include N-methyl-2-hydroxy Ethylpyrrolidine, 2,2-dimethyl-1,3-dioxolane-4-methanol, 1-(hydroxymethyl)-
Examples include 5,5-dimethylhydantoin, 2,4-dimethyl-4-hydroxyoxazoline, and 5-(2-hydroxyethyl)-4-methylthiazole. When a C ₁ to C ₃ alkylene group is used as a substituent in the above, the alkylene group means a bond connecting two heterocycles, and a piperidine ring is preferable, and an example thereof is 1,3-bis(N- 2-hydroxyethyl-4-piperidyl)propane, 1-
(N-2-hydroxyethyl-4-piperidyl)-
Examples include 3-(4'-piperidyl)propane. When having a hydroxyl group as a substituent in the above, a piperidine ring, a tetrahydrofuran ring and an oxane ring are preferable, and examples thereof include N-(2
-hydroxyethyl)-4-(hydroxypropyl)piperidine, 5-thioglucose, kojic acid, ascorbic acid, isoascorbic acid, glucono-δ-lactone, galacno-γ-lactone,
Examples include α-glucoheptonic acid-γ-lactone. In the above, when having an oxo group as a substituent, a pyrrolidine ring is preferable, and examples thereof include N-(2-hydroxyethyl)-2-pyrrolidone,
Examples include N-(2-hydroxypropyl)-2-pyrrolidone. In the above, when having an amino group as a substituent, a pyrimidine ring is preferable, as an example,
Examples include toxopyrimidine. In the above, when a phenyl group is included as a substituent, a piperidine ring is preferable, and examples thereof include scopolamine and atropine. (2) The following compounds can be mentioned as heterocyclic secondary alcohol compounds. It has a secondary alcoholic OH directly bonded to the ring or a linear side chain bonded to the secondary alcoholic OH, and is selected from the group consisting of a piperidine ring, a morpholine ring, a piperazine ring, a pyrrolidine ring, an oxane ring, and a phthalimide ring. a heterocyclic secondary alcohol compound residue having a heterocyclic ring, where the heterocyclic portion of the residue is C ₁ -
It may have substituents selected from the group consisting of C ₃ alkyl groups, hydroxyl groups and C ₁ to _{C 3} alkylene sulfonic acid groups: - a hetero with a secondary alcoholic OH directly bonded to the ring in the above The ring is preferably a piperidine ring or a pyrrolidine ring, examples of which include 3-hydroxypiperidine, 4-hydroxypiperidine, and 3-hydroxypyrrolidine. In the above, the heterocycle having a linear side chain bonded to the secondary alcoholic OH includes a phthaimide ring and a morpholine ring, and examples thereof include preferably N-(2-hydroxypropyl)phthalimide,
Examples include N-(2-hydroxypropyl)morpholine. In the above, when the substituent has a _C1 to _C3 alkyl group, a piperidine ring is preferable, and an example thereof is N-methyl-4-hydroxypiperidine. In the above, when a C ₁ to _{C 3} alkylene sulfonic acid group is used as a substituent, a piperazine ring and a morpholine ring are preferable, examples of which include piperazine-N,N'-bis(2-hydroxypropane-3-sulfone ring). , 3-(N-morpholine)-2
-Hydroxypropanesulfonic acid, etc. (3) Examples of heterocyclic compounds with sugar include the following: A pentose residue or deoxypentose residue having as a substituent a heterocycle selected from a pyridine ring, a pyrimidine ring, a triazine ring, a purine ring, and an 8-azapurine ring, where the heterocyclic portion of the residue is a mercapto group, a halogen ,
It may have a substituent selected from the group consisting of hydroxyl group, carboxyl group, oxo group, amino group, _C1 - _C3 alkyl group, methoxy group, ethoxy group, carboxymethyl group, acetyl group, and anisoyl group. :1 In the above, examples of the pentose residue and deoxypentose residue having a pyridine ring as a substituent include 3-deazauridine. In the above, examples of the pentose residue and deoxypentose residue having a triazine ring as a substituent include 5-azacytidine. In the above, the pentose residue and deoxypentose residue having a pyrimidine ring as a substituent include 4-thiouridine, 5-bromouridine, 5-hydroxyuridine, 6-fluorodeoxyuridine, 3-methyluridine, and 5-carboxymethyluridine. , 5-methyluridine, ^N4
-acetylcytidine, 55-methoxyuridine, 5
-Bromo-2'-deoxycytidine, ^N4 -anisoyl-2'-deoxycytidine, 3'-O-methylcytidine, orotidine, cyclocytidine, thymine deoxyriboside, laucyl deoxyriboside,
Examples include cytosine deoxyriboside. In the above, the pentose residue and deoxypentose residue having a purine ring as a substituent include 6-mercaptoguanosine, 1-methyl-guanosine, xanthosine, 6-mercaptopurine riboside, 6-methylaminopurine-9-riboside, N ⁶ -methyl-2'-deoxyadenosine, 8
- promoadenosine, N ⁶ - ethanoadenosine,
2'-chloroadenosine, 1-methyladenosine,
Examples include ^N6 , ^N6 -dimethyladenosine, 2'-O-methyladenosine, inosine, guanine deoxyriboside, and adenine deoxyriboside. In the above, examples of the pentose residue and deoxypentose residue having an 8-azapurine ring as a substituent include 8-azaadenosine. Heterocyclic compounds having an alcoholic hydroxyl group selected from the above examples (1) to (3) can be exemplified, but as long as the above ranges (1) to (3) are met, the selection of alcohols is There are no restrictions. Heterocyclic compounds having an alcoholic hydroxyl group as exemplified above (1) to (3) can be used as natural products or synthetic products, but do not include heterocyclic compounds having an alcoholic hydroxyl group other than the target compound. Therefore, it is preferable to use it after purifying it in advance using an appropriate means. Examples of such purification means include distillation, recrystallization, column chromatography using alumina, silica gel activated carbon, ion exchange resin, etc., thin layer chromatography, and appropriate combinations of these. . According to the method of the present invention, a sphingophospholipid of the formula () as exemplified above is reacted with a heterocyclic compound having an alcoholic hydroxyl group selected from the groups (1) to (3) as exemplified above in the presence of phospholipase DM. let The phospholipase DM used at this time is:
It is distinguished from known phospholipase D by having an optimum temperature of 60 to 70°C and an optimum pH of around 7, compared to the conventional phospholipase D extracted from cabbage which has an optimum temperature of 40°C or less and an optimum pH of 5.4 to 5.6. An example of this is phospholipase DM produced by a phospholipase DM-producing bacterium that can produce N-acylsphingosine lipids.
Phospholipase DM with 1-phosphoryl transfer action
All can be used regardless of their origin. The phospholipase DM is distinguishable from the known phospholipase D in that it catalyzes the formation of a sphingophospholipid derivative of the formula () between a sphingophospholipid and N-(2-hydroxyethyl)pyrrolidine. Examples of such phospholipase DM-producing bacteria include Japanese Patent Application Laid-Open No. 1983-1999 filed by the same applicant.
Phospholipase DM belonging to the genus Nocardiopsis disclosed in No. 63388
For example, the producing bacteria include the genus Norcadeiopsis strain No. 779 [FERM-BP-512], and the phospholipase belonging to the genus Actinomadu-ra disclosed in Japanese Patent Application Laid-open No. 58-67183 filed by the same applicant. DM-producing bacteria such as Actinomadeura spp.
No. 362 strain [FERM-BP-511], etc., which uses the phospholipid of the above formula () as a raw material to carry out the transfer reaction of the heterocyclic compound having an alcoholic hydroxyl group of the above (1) to (3). Phospholipase D of any origin can be used as phospholipase DM as long as it has an action. Table 1 below shows the differences in enzymatic properties between the phospholipase DM used in the method of the present invention and the known phospholipase D, as well as the differences in optimum temperature and optimum PH, as well as some other differences. The enzymatic properties were measured by the methods described in the above-mentioned JP-A-58-63388 and JP-A-58-67183. [Table] Product names of Sunlight Co., Ltd.
The reason why a sphingophospholipid heterocyclic compound derivative that cannot be obtained using the known phospholipase D can be formed in the method of the present invention is due to the combination of the known phospholipase D, which is involved in this enzymatic catalytic reaction, and the phospholipase DM used in the method of the present invention. It is presumed that the difference in enzymatic properties as described above is involved. Of course, the method of the present invention is not subject to any restrictions based on the assumption of such effects. The phospholipase DM used in the method of the present invention is prepared by reacting, for example, N-(2-hydroxyethyl)pyrrolidine, a heterocyclic primary alcohol, with sphingoline in accordance with the experimental method for rearrangement action described below [method for confirming the formation of rearrangement products by TLC]. The heterocyclic compound derivative of the sphingophospholipid is formed by catalyzing a sphingophospholipid derivative formation reaction with the lipid sphingomyelin. Known cabbage phospholipase D does not form the above derivatives. According to the method of the present invention, the formula () as exemplified above
By reacting a sphingophospholipid with a heterocyclic compound having an alcoholic hydroxyl group as exemplified above (1) to (3) in the presence of the phospholipase DM detailed above, the following formula () is obtained. A sphingophospholipid heterocyclic compound derivative represented by the following formula, where A, A' and C have the same meanings as described above, can be produced. At this time, phospholipase DM does not need to be used as a purified product, and may be a crude product. Furthermore, it can also be used by being supported and fixed on a suitable immobilization carrier, such as various polymer resins such as polypropylene membranes, celite particles, glass beads, etc., or granular or film-like materials of inorganic materials. The reaction can be carried out by bringing the sphingophospholipid of formula () into contact with the heterocyclic compound having an alcoholic hydroxyl group (1) to (3) in the presence of phospholipase DM, preferably in the presence of a solvent. . Examples of the solvent to be used include aqueous solvents and mixed solvents of aqueous solvents and organic solvents. Further, some heterocyclic compounds having an alcoholic hydroxyl group can themselves serve as a solvent. Also, phospholipase
Solvents containing any other additives that do not inhibit the enzymatic catalytic action of DM can also be used, such as solvents containing suitable additives that promote this action or help stabilize the enzyme. . For example, aqueous solutions containing proteins such as albumin and casein, buffers such as acetic acid, citric acid, and phosphoric acid, neutral salts such as calcium chloride, and bile salts such as sodium taurocholate. It can be a solvent. Furthermore, examples of organic solvents include aliphatic hydrocarbons such as n-heptane, n-hexane, and isooctane; alicyclic hydrocarbons such as cyclopentane, cyclohexane, and cyclobutane; benzene, toluene, and xylene. Aromatic hydrocarbons such as acetone, methyl isopropyl ketone, etc.; ethers such as dimethyl ether, diethyl ether, diisopropyl ether, etc.; esters such as methyl acetate, ethyl acetate, etc.; carbon tetrachloride, chloroform, Examples include halogenated hydrocarbons such as methylene chloride; amide solvents such as dimethylformamide; and sulfoxide solvents such as dimethylsulfoxide. When using a mixed solvent of an aqueous solvent and an organic solvent, the mixing ratio of both can be selected appropriately, but for example, the ratio of aqueous solvent to organic solvent (V/V ratio)
A mixing ratio such as 100:0 to 1:99 can be exemplified. The reaction molar ratio, the amount of phospholipase DM used, the amount of solvent used, etc. can be selected as appropriate, but for example, the above (1)-(3) heterocycle having an alcoholic hydroxyl group is A reaction molar ratio of about 1:1 to about 1:1000 moles of compound may be exemplified. Additionally, phospholipase DM
Examples of the usage amount include about 10 to about 100,000 units, preferably about 100 to about 1,000 units per gram of the sphingophospholipid of formula (). Furthermore, the amount of solvent to be used is, for example, about 2
An example of usage is approximately 100 times the capacity. Since the reaction proceeds at room temperature, there is no particular need for cooling or heating, but cooling or heating conditions can be adopted as appropriate, if desired. for example,
Examples of reaction temperatures include about 0°C to about 90°C, preferably about 20°C to about 60°C. The reaction time can also be selected as appropriate, for example from about 1 minute to about 10 minutes.
Examples of reaction times include days, preferably from about 1 hour to 72 hours. If desired, the reaction time can be changed as appropriate by tracking the reaction progress using a technique such as TLC (thin layer chromatography) and confirming the formation of the desired target product. The mode of contacting the sphingophospholipid of formula () with the heterocyclic compound having an alcoholic hydroxyl group (1) to (3) in the presence of phospholipase DM can be selected as appropriate, but it is usually carried out under stirring or shaking conditions. It is. Furthermore, when reacting using a substrate that is susceptible to oxidative decomposition or a heterocyclic compound having an alcoholic hydroxyl group, it is desirable to carry out the reaction in a nitrogen stream or the like. In addition, when phospholipase DM is used in the form of an immobilized enzyme supported and immobilized on a suitable granular or film-like carrier as described above, for example, it can be transferred via an immobilized enzyme membrane or an immobilized enzyme particle layer. The reaction can be carried out in such a manner that the reaction composition liquid is passed through using a circulation pump. After carrying out the reaction as described above, the sphingophospholipid complex derivative of the formula () formed is precipitated and separated as it is or in the form of a salt. and can be used. In addition, as the salt of the formula () sphingophospholipid heterocyclic compound derivative here,
For example, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, oxalic acid, maleic acid, lactic acid, tartaric acid,
Salts with organic acids such as fumaric acid, methanesulfonic acid, benvinsulfonic acid, and toluenesulfonic acid; salts with amino acids such as arginine, aspartic acid, and glutamic acid; salts with alkali metals such as sodium and potassium; magnesium and calcium. Examples include salts with alkali metals such as, ammonium salts, etc. Furthermore, the derivatives and their salts can be used in silicic acid column chromatography, alumina column chromatography, ion exchange chromatography, high performance liquid chromatography, countercurrent distribution,
Separation and purification can be carried out using appropriate known methods such as gel filtration and adsorption chromatography. According to the method of the present invention, as described above, the sphingophospholipid of the formula () and the heterocyclic compound having an alcoholic hydroxyl group of the above (1) to (3) are reacted in the presence of phospholipase DM, and the formula ( ) Sphingophospholipid complex derivatives can be produced. The resulting sphingophospholipid heterocyclic compound derivative of the formula () has excellent surfactant action and has a large effect on the permeability of cell membranes. In this sense, the derivatives of formula () can be used as liposome-forming base materials and liposome surface modification base materials, as well as as emulsifiers useful for skin physiology when incorporated into cosmetics such as creams and emulsions, and as emulsifiers for fatty drugs. It is useful for a wide range of emulsifier applications, including emulsifiers, insecticides, herbicides, etc. Furthermore, sphingophospholipids are important membrane components of plant cells and animal cells, and in animals, they are present in large amounts particularly in the brain and elongated tissues, as well as in organs and blood cells. The most abundant sphingophospholipid is sphingomyelin, and its derivatives include ceramide phosphorylethanolamine and ceramide 2-aminoethylphosphonate. Types of sphingophospholipids found in nature are still extremely rare. The physiological role of sphingophospholipids is thought to be involved in neurotransmission, but little has been elucidated in this field yet. Rather, its analogs, sphingoglycolipids, are known as various antigens or as receptors for bacterial toxins, interferons, hormones, etc. Recently, it has also been thought that it is involved in cell differentiation induction and proliferation. The sphingophospholipid heterocyclic compound derivatives of the formula () that can be derived in the present invention are novel sphingophospholipid derivatives having many different structures, and some of these sphingophospholipid analogs have effective effects as nervous system drugs. Derivatives can be expected. Furthermore, it is possible that some of these derivatives, especially sphingophospholipid analogues derived from sugar-bearing heterocycles, have the effect of promoting differentiation induction or promoting cell proliferation when used in cell culture. Possible gender. It is also conceivable that it may have the effect of inhibiting the progression of differentiation or canceration. Furthermore, by transferring a heterocyclic compound having a primary or secondary alcohol hydroxyl group or a heterocyclic pharmacologically active compound into which a primary or secondary alcohol hydroxyl group has been introduced to a sphingophospholipid, the pharmacological side effects of the compound may be weakened. Alternatively, it can be expected to enhance the pharmacological effect and reduce the dosage. Furthermore, the above-mentioned pharmacologically active compound may be transferred to sphingophospholipids, which may serve as a carrier for the pharmacologically active compound to accurately concentrate the compound in the affected area, and may also play a useful role as a protective group for the pharmacologically active compound. You can expect it. Furthermore, it is useful as an intermediate for chemical synthesis including various pharmaceuticals, and for example, derivatives obtained by transferring alcohols having highly reactive halogen or amino substituents can be used. Furthermore, tritium
By transferring a heterocyclic compound having an alcoholic hydroxyl group labeled with ¹⁴ C, a labeled sphingophospholipid heterocyclic compound derivative can be obtained, which can also be used to elucidate the metabolic pathway of sphingophospholipid. Hereinafter, several embodiments of carrying out the method of the present invention will be illustrated in more detail with reference to Examples. Reference Example 1 Preparation of phospholipase DM. Soy flour 3.0%, corn starch liquor 1.0
%, peptone 0.5%, powdered yeast extract 0.1%, glucose 1.0%, _{NH4NO3 0.25} %, _{K2HPO4 0.4} %,
MsSO ₄・7H ₂ O0.01%, Tween −85
A medium (PH6.0) consisting of approximately 0.1% was placed in a 30 jar fermentor, and after sterilization at 120°C for 15 minutes,
Seed culture solution 1.5 was inoculated and cultured at 27°C for 40 hours. In addition, the above seed culture solution contains 1% starch,
( _NH4 ) _{H2PO4 0.25} %, peptone 0.25%,
Pour 100 ml of aqueous medium (PH6.8) containing 0.2% K ₂ HPO ₃ and 0.01% MgSO ₄ 7H ₂ O into a 500 ml Sakaguchi flask, and after steam sterilization, Nocaldeiopsis sp. NO.779
Strain [FERM.BP512] or Actinomadeura sp.
A loopful of spores of the NO.362 strain [FERM・BP-511] was inoculated and cultured with shaking at a culture temperature of 30° C. and 120 rpm for 2 days. After culturing, the bacterial solids are removed by centrifugation,
Centrifugal supernatant 13 (Nocardiopsis FERM・
0.54u/ml when using BP-512; when using Actinomadeula sp. FERM/BP-511 strain
It was 1.7u/ml. ) was obtained. This centrifuged supernatant was
After cooling to °C, add acetone at -20 °C to collect phospholipase corresponding to the acetone concentration 30-70% fraction.
The precipitate containing DM was collected by centrifugation. This precipitate was collected from Nocardiopsis FERM・BP−.
When strain 512 was used, it was dissolved in Tris-maleic acid at pH 6.0, and when strain Actinomadeula FERM/BP-511 was used, it was dissolved in Tris-maleic acid at pH 6.5 and dialyzed against the same buffer at 0.02M. After that, it was equilibrated with the same buffer.
The DEAE-cellulose was passed through the column and the flow-through fraction was collected. Next, the method of Horiuchi et al. [J.Biochem. 81 , 1639
(1977)] was packed into a column, thoroughly washed with water, and the above DEAE-cellulose permeate was injected to adsorb the activity. this
After washing with 0.05M Tris-HCl buffer (PH7.2),
The same buffer containing 0.2% Triton X-100 was added to elute the activity. After collecting the active fraction and concentrating it using an ultrafiltration membrane (Type G-10T) manufactured by Bioengineering Co., Ltd., Toyopearl was used as a gel carrier.
Inject into HW-55F (manufactured by Toyo Soda Co., Ltd.) packed column,
Distilled water was used to pass through the column, and the active fraction was collected and freeze-dried. This dry powder was mixed with 0.025M imidazole in the case of Nocardiopsis phospholipase DM.
After dissolving in hydrochloric acid (PH7.4), add 0.025M Tris for Actinomadeula phospholipase DM.
After dissolving in acetic acid (PH8.3), use the polybuffer exchanger PBE ^TN 94 manufactured by Pharmacia Fine Chemicals.
(20 ml) was passed through a packed column to adsorb the activity, and then the pH
Eluted by gradient. Eluted phospholipase
The active fraction of DM is collected and concentrated using an ultrafiltration membrane.
The mixture was passed through a Sephadex G-75 packed column, and the phospholipase DM active fraction was collected and freeze-dried. Thus, Nocardiopsis phospholipase
In the case of DM, the specific activity is approximately 40%.
Phospholipase DM was recovered as 178.3 u/mg protein, with an activity recovery rate of about 43% in the case of Actimadeula phospholipase DM, and as specific activity 218.3 u/mg protein. Example 1 (Run No. 1 to No. 50) Sphingomyelin, derived from egg yolk (manufactured by Sigma)
and various types of heterocyclic alcohols shown in Table 2 below were reacted in the presence of phospholipase DM according to the method for confirming the formation of a transfer product by TLC described below, and the formation of a transfer product was confirmed. . That Rf
The values are shown in Table 2 below. Method for confirming the formation of transfer products by TLC: - The following composition: 1% sphingomyelin emulsion 0.1ml 0.2M acetate buffer (PH5.7), 0.1ml 1M calcium chloride aqueous solution 0.01ml 20% heterocyclic alcohol aqueous solution 0.2ml Add 0.01ml of phospholipase DM aqueous solution to the reaction solution.
(IU) was added and left at 30°C for 2 days. The above 1% sphingomyelin emulsion was prepared by adding 100 mg of sphingomyelin, 1 ml of diethyl ether-petroleum ether (1:1 V/V) and 10 ml of distilled water, and ultrasonicating at 600 W and 20 KHz for 5 minutes under ice-cooling conditions. It was formed by In addition, the above 20% heterocyclic alcohol aqueous solution may be diluted with 1N hydrochloric acid or 1N as necessary.
It was used after adjusting the pH to 6.0 with caustic soda. After the above-mentioned standing, 0.5 ml of 0.1N hydrochloric acid was added to stop the reaction, and the mixture was vigorously stirred to extract lipids (products).
This suspension was allowed to stand for several minutes, the lower chloroform layer was collected, dried under reduced pressure at 30°C, and then dissolved in 100% of a chloroform-methanol mixture (2:1 V/V) to be used as a TLC sample. did. Ten of these were spotted on a thin layer of silica gel (Merck, silica gel 60TLC plate, 20 x 20 cm), and chloroform-
Methanol-water (60:30:5V/V), chloroform-methanol-ammonia-water (50:20:1:
2V/V) or diisobutylketone-acetic acid-water (40:25:5V/V) as a developing solvent. The following reagents were used to detect spots. The substrate that was partially degraded at the detected spot and its hydrolyzate N-acylsphingosine-1-
If a spot of phospholipid other than phosphoric acid was detected, this was recognized as a transfer product. Table 2 shows the Rf values when chloroform-methanol-water (60:30:5V/V) was used as the developing solvent. (Run No. 1 to 50) Detection reagent: Phosphorus coloration: Zinzade reagent (Beiss UJ
Chromatog. 13 , 104, 1964) Color development of purines and pyrimidines: fluorescein-ammonia reagent (Wieland T. et al. Angew.
63, 511, 1951) Coloration of organic compounds: 50% sulfuric acid. [Table] [Table] Example 2 (Run No. 1 to No. 15) Sphingomyelin (derived from egg yolk, manufactured by Sigma)
500 mg, diethyl ether 1 ml, and distilled water 10 ml were placed in an ultrasonic cell, and sonicated at 600 W and 20 KHz for 5 minutes while cooling on ice to prepare an emulsion. 0.2ml of this sphingomyelin emulsion, 0.2ml of 0.2M acetate buffer (PH5.7), 0.1ml of 1M calcium chloride aqueous solution, 0.5ml of 20% N-(2-hydroxyethyl)pyrrolidine aqueous solution, and a test tube with a stopper. After adjusting the pH to 5.7, add 0.1 ml (20 U) of phospholipase DM aqueous solution, mix well, and heat at 30℃.
So I let it sit for 2 days. Next, add 2 0.1N hydrochloric acid to the reaction solution.
ml was added to stop the reaction, and then 10 ml of a chloroform-methanol mixture (2:1 V/V) was added and vigorously mixed to extract phospholipids. Add this mixture to 2000
After centrifugation at ×g for 10 minutes, the lower chloroform layer was separated, and the separated chloroform layer was further washed with 5 ml of water. From this mixture, separate the chloroform layer again by centrifugation, dry it under reduced pressure at 30°C, and then
of n-hexane-2-propanol-water (60:
80:14V/V) was dissolved in the mixed solution. 10ml of this sample was added to a thin layer of silica gel (Merck Silica Gel 60TLC).
Spot chloroform on a plate (20 x 20 cm).
When developed with a solvent system of methanol-water (60:30:5V/V), three types of phospholipids were detected, two of which were sphingomyelin, N-acylsphingosine-1-phosphate, and Rf values. matched. Therefore, this sample was separated and purified using high performance liquid chromatography. Column is radial pack cartridge silica 8
mm x 10cm, (manufactured by Waters), solvent: n-hexane-isopropanol-water (60:80:14V/
V), the peak was detected using a 441 type ultraviolet detector (manufactured by Wouters) at 214 nm, and
A model R401 differential spectrometer (manufactured by Waters) was used. The sample was divided into 5 injections of 0.1 ml each. With this solvent, N-acylsphingosine-1-phosphate, sphingomyelin, N-acylsphingosine-1-phosphate-N-(2-hydroxyethyl)
Three components of pyrrolidine ester were separated. The obtained N-acylsphingosine-1-phosphate-N-(2-hydroxyethyl)pyrrolidine ester was purified once again by the same operation to obtain 3 mg of purified product. This was confirmed to be single by TLC and high performance liquid chromatography. The IR spectrum of this compound was measured by the liquid film method using a JASCO Model A202 infrared spectrophotometer. The results are shown in Table 3 (Run No. 3). Furthermore, the rearrangement products of various heterocyclic alcohols shown in Table 3 were prepared in the same manner and their IR spectra were measured. The results are shown in Table 3. (Run 1~2, 4~
15) [Table] [Table] Example 3 (Run No. 1 to No. 2) Add 10 ml of 5% sphingomyelin emulsion prepared in the same manner as in Example 2 to 10 ml of 0.2 M acetate buffer (PH
5.7) and 1 ml of 1M calcium chloride aqueous solution were placed in an Erlenmeyer flask with a stopper, 5 g of ethanol hydrochloride was added, and 10 ml of phospholipase DM aqueous solution (20 U/ml) was added, and the flask was allowed to stand at 30°C for 2 days. After the above-mentioned standing, 1N hydrochloric acid was added to adjust the pH to 2.0 to stop the reaction, and 50 ml of a chloroform-methanol mixture (2:1 V/V) was added and vigorously stirred to extract the product. This mixture was centrifuged at 2000×g for 2 minutes, and the lower chloroform layer was separated. The fractionated extract was dried under reduced pressure at 30°C, and then 2 ml of n-hexane-2-propanol-water (60:80:
14V/V) was dissolved in a mixed solution. Hereinafter, N-acylsphingosine-1-phosphate ethanolamine ester was purified by the same operation as in Example 2, and the purified product was
Obtained 150 mg. 130 mg of purified N-acylsphingosine-1-phosphate glycerol ester was obtained by the same operation. Substrate obtained as above: N-acylsphingosine-1-phosphate ethanolamine ester, (ceramide phosphorylethanolamine) Substrate: N-acylsphingosine-1-phosphate glycerol ester (ceramide phosphorylglycerol) ) 0.2 ml of 5% emulsion of 20% N-(2-hydroxyethyl)-2 into separate stoppered test tubes.
−0.2 ml of pyrrolidone aqueous solution, 0.2 M acetate buffer (PH
5.7) Add 0.2ml of 1M calcium chloride aqueous solution to 0.1ml of phospholipase DM aqueous solution and adjust the pH to 5.7.
ml (20 U) was added and left to stand at 30°C for 2 days. This reaction solution was treated in the same manner as in Example 2, and the common rearrangement product, N-acylsphingosine-1, was
-Phosphate N-(2-hydroxyethyl)-2-pyrrolidone ester was obtained. Its IR spectrum is shown in Table 4. 【table】

Claims (1)

[Claims] 1 The following formula () [Formula] In the formula, A represents a group of the following formula (i) or (ii) [Chemical formula] or [Chemical formula], where R is a saturated or unsaturated group.
represents a _C12 to _C24 aliphatic hydrocarbon group, A' represents an oxide anion or a hydroxyl group, and B
represents −(CH ₂ ) ₂ N+(CH ₃ ) ₃ , −(CH ₂ ) ₂ NH ₂ or −CH ₂ CH(OH)CH ₂ (OH). (3)
A heterocyclic compound having an alcoholic hydroxyl group shown in (1) having a chain side chain bonded to a primary alcoholic OH, and having a tetrahydrofuran ring, a dioxolane ring, a thiophene ring, a pyrrolidine ring, an imidazole ring, an imidazolidine ring , oxazole ring,
a heterocyclic primary alcohol compound having a heterocycle selected from the group consisting of a thiazole ring, a pyran ring, an oxane ring, a thian ring, a piperidine ring, a triazine ring, a tetrahydrophthalimide ring, a benzoxolane ring and a hexamethyleneimine ring;
Here, the heterocyclic moiety of the compound is a _C1 - _C3 alkyl group, a _C1 - _C3 alkylene group, a hydroxyl group, a carboxyl group, a carbonyl group, a halogen, an amino group, an oxo group, a benzyl group, a phenyl group, and
may have a substituent selected from the group consisting of C ₁ to _{C 3} alkylene sulfonic acid groups; (2) a secondary alcoholic OH directly bonded to the ring or a chain bonded to the secondary alcoholic OH; A heterocyclic secondary alcohol compound having a side chain and a heterocycle selected from the group consisting of a piperidine ring, a morpholine ring, a piperazine ring, a pyrrolidine ring, an oxane ring, and a phthalimide ring, wherein the heterocyclic moiety of the compound is _C1 - _C3 alkyl group,
(3) a pyridine ring, a pyrimidine ring, a triazine ring, which may have a substituent selected from the group consisting of a hydroxyl group and a C ₁ to _{C 3} alkylene sulfonic acid group;
A pentose derivative or deoxypentose derivative having a heterocycle selected from a purine ring and an 8-azapurine ring as a substituent, wherein the heterocyclic moiety of the derivative is a mercapto group, a halogen,
It may have a substituent selected from the group consisting of hydroxyl group, carboxyl group, oxo group, amino group, _C1 - _C3 alkyl group, methoxy group, ethoxy group, carboxymethyl group, acetyl group and anisoyl group, However, heterocyclic compounds having a sugar selected from the group consisting of adenosine, cytidine, uridine, guanosine and arabinocytidine are excluded, heterocyclic compounds having an alcoholic hydroxyl group selected from the group consisting of sphingophospholipid and heterocyclic The following formula is characterized in that the reaction is carried out in the presence of phospholipase D activated by the nonionic surfactant polyoxyethylene (10) octyl phenyl ether, which can catalyze the transfer reaction between alcohol and alcohol. ) [Formula] In the formula, A and A' have the above meanings, and C
represents a residue obtained by removing the alcoholic hydroxyl group from the alcoholic hydroxyl group-containing heterocyclic compound shown in (1) to (3) above, and a method for producing a sphingophospholipid heterocyclic compound derivative and its base.