JPH0571236B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing organophosphoric acid ester derivatives by an enzymatic method, which was previously unknown to be able to be produced by an enzymatic method, and the derivatives are useful in the fields of, for example, liposome-forming substrates, emulsifiers, physiologically active substances, and carriers thereof. This invention relates to an enzymatic method for producing organophosphate ester derivatives. In particular, non-living systems such as alkylphosphorylcholine, alkyl polyethylene glycol phosphoryl choline, alkylphenyl polyethylene glycol phosphoryl choline, etc. included in the following formula () having a structure that does not belong to the category of biological organic phospholipids such as glycerophospholipids and sphingophospholipids An enzymatic organic phosphate ester, which was previously unknown to be able to be produced by an enzymatic method, by enzyme transfer using a synthetic organic phosphate ester as a substrate and a wide range of alcoholic OH-containing compounds as acceptors. The present invention relates to a method for producing a derivative. More specifically, the present invention provides the following formula ()

[Chemical formula] However, in the formula, A is R ₁ -X or R ₂ -O-
(CH ₂ CH ₂ O) _o , where X represents an oxygen atom or a sulfur atom; R ₁ represents a saturated or unsaturated C ₆ to C ₁₂₀ aliphatic hydrocarbon residue, or a group R ₃ -Y-R ₄ -; In the group R ₃ -Y-R ₄ -, Y is O, S, SO,
SO ₂ , O-CO-O, O-CO-NH or O-
CS-NH, R ₃ is halogen, C ₆ - C ₁₆ aryl group, C ₁ -
_C18 alkoxy group, C1 _-C18 alkylmercapto group, C1- _C18 alkanesulfinyl group, C1-C18 alkoxy group, C1 _{- C18} alkylmercapto group, C1 _-C18 alkanesulfinyl group
_C18 alkanesulfonyl group, _C2 - _C10 alkylcarbonylamide group, C2 _{- C6} alkylcarbamoyl group, _C2 - _C6 alkylthiocarbonylamide group, _C1 - _C6 alkanesulfonamide group,
a _saturated or unsaturated C1- _C24 fat, which may be substituted with at least one substituent selected from the group consisting _of a C2 _- C5 alkoxycarbonyl group and a _C3 - _C5 ureido group; represents a group hydrocarbon residue, where the above C ₁ to C ₁₈ alkoxy group is
It may be further substituted with a C ₁ -C ₄ alkoxy group, and a portion of the above C ₁ -C ₂₄ aliphatic hydrocarbon residue is composed of a 3- to 6-membered cycloalkane ring. and R ₄ is a halogen, hydroxyl group, nitrile group, C ₃ -
_C6 cycloalkane group, _C6 to _C8 aryl group,
_C1 - _C4 alkoxy group, _C1 - _C18 alkylmercapto group, _C1 - _C4 alkanesulfinyl group,
It has a _C1 to _C4 alkanesulfonyl group, a _C2 to _C5 alkoxycarbonyl group, an amino group which may have a _C2 to _C4 acyl group, and a _C1 to _C5 alkyl group. saturated or unsaturated C2 _{- C8} aliphatic carbonization optionally substituted with at least one substituent selected from the group consisting of an optional carbamoyl group and a _C1 - _C4 alkanesulfonamide group Indicates a hydrogen residue, where the above _C1 - _C4 alkoxy group is a _C1 - _C4 alkoxy group, a _C1 - _C4 alkylmercapto group, a _C1 - _C4 alkanesulfinyl group,
C1- _C4 alkanesulfonyl group, halogen, hydroxyl group, nitrile group, _C6 - _C8 aryl group, C1 _{-C4 alkanesulfonamide} group, _carbamoyl group, _C2
It may be further substituted with an amino group which may have ~ _C5 alkoxycarbonyl group and _C2 - _C4 acyl group, and the above _C2 - _C8 aliphatic hydrocarbon residue is A part of the inside may be composed of a 3- to 6-membered cycloalkane ring, or the aliphatic hydrocarbon residue may be an alicyclic hydrocarbon residue; R ₂ is A saturated or unsaturated C ₁ to C ₂₀ aliphatic hydrocarbon residue, a C ₆ to _{C 26} acyl group derived from a saturated or unsaturated aliphatic carboxylic acid, and a 2-N-acyl having the acyl group. - represents a group selected from the group consisting of an alkylphenyl group having an amino-ethyl group and a saturated or unsaturated C ₆ to C ₂₆ alkyl group; n represents an integer from 1 to 20, provided that R ₂ is above C ₆
When representing a ~ _C26 acyl group, n represents an integer of 2 to 20; furthermore, in the formula, A' represents an oxide anion or hydroxy, and B represents _{-CH2.CH2 +} N ( _CH3 ) ₃ , −CH ₂・CH ₂ NH ₂ , −CH ₂・CHOH・CH ₂ OH, −
A group selected from the group consisting of CH ₃ and -CH ₂ / CH ₃ , and an organic phosphoric acid ester represented by the following (1) to (3) (1) halogen, amino group, carboxyl group, and hydroxyl group. a saturated or unsaturated C ₁ -C ₂₇ aliphatic hydrocarbon residue optionally having a substituent selected from _the group consisting of; ₂₀ aromatic hydrocarbon residues, wherein the aromatic ring portion of the aromatic hydrocarbon residues may have a substituent selected from the group consisting of halogen, amino group, carboxyl group, and hydroxyl group. ; A C ₆ - _{C 20} alicyclic hydrocarbon residue having a side chain bonded to a primary alcoholic OH; Having a bond selected from an ether bond, an ester bond, or an amide bond in the molecule, and containing a halogen, A saturated or unsaturated C ₂ to _{C 27} aliphatic hydrocarbon residue that may have a substituent selected from the group consisting of an amino group, a carboxyl group, and a hydroxyl group; an ether bond, an ester bond, and a C ₇ to _{C 20} aromatic hydrocarbon residue having a bond selected from an amide bond and having a linear side chain bonded to a primary alcoholic OH,
The aromatic ring portion of the aromatic hydrocarbon residue may have a substituent selected from the group consisting of halogen, amino group, carboxyl group, and hydroxyl group; pregnane steroid compound residue; primary alcoholic A heterocyclic ring having a linear side chain bonded to OH and having the following: furan ring, tetrahydrofuran ring, dioxolane ring, thiophene ring, imidazole ring, imidazolidine ring, pyrrolidine ring, oxazole ring, thiazole ring, pyran ring, oxane ring, Thiane ring, pyridine ring, piperazine ring, piperidine ring, pyrimidine ring, triazine ring, morpholine ring, indole ring, purine ring, phthalimide ring, tetrahydrophthalimide ring, benzoxolane ring, hexamethyleneimine ring, pterin ring, homopiperidine ring and a homomorpholine ring, a _heterocyclic primary alcohol compound residue _having a heterocycle selected from _the group consisting _of , a hydroxyl group, a carboxyl group, a carbonyl group, an oxo group, a halogen, an amino group, a benzyl group, a phenyl group, and a C ₁ to _{C 3} alkylene sulfonic acid group; A primary alcohol compound having a residue selected from the group consisting of: (2) Saturated or unsaturated C ₁ to _{C 20} which may have a substituent selected from the group consisting of halogen, amino group, carboxyl group and phenyl group
aliphatic hydrocarbon residue; a saturated or unsaturated _C3 - _C20 aliphatic hydrocarbon residue having a bond selected from ether bonds, ester bonds, and amide bonds in the molecule; a _C4 to _C8 alicyclic hydrocarbon residue having a secondary alcoholic OH; having a secondary alcoholic OH directly bonded to the ring or a linear side chain bonded to the secondary alcoholic OH, and having a piperidine ring, pyrrolidine ring,
A heterocyclic secondary alcohol compound residue having a heterocyclic ring selected from the group consisting of a morpholine ring, a tetrahydrofuran ring, a phthalimide ring, a piperazine ring, a sorbitan ring, and an oxane ring, where the heterocyclic moiety of the residue is C ₁ - It may have a substituent selected from the group consisting of a C ₃ alkyl group, a carboxyl group, a C ₁₂ to _{C 18} acyl group, a C ₁ to _{C 3} alkylene sulfonic acid group, and a hydroxyl group; Secondary alcohol compounds with selected residues. (3) Amino group, acetylamino group, methylnitrosouride group, _C1 - _C3 alkyl group, phenyl group, phenol group, hydroxymethylphenyl group, nitrophenyl group, mercapto group, methylmercapto group, phenylmercapto group , nitrophenylmercapto group, halogen, carboxyl group, pyridyl group, pyrimidyl group, triazyl group, purine group, and hexose residue, which may have a substituent selected from 8-azapurine group; disaccharide residues, deoxypentose residues, deoxyhexose residues, heptose residues; however, among the above substituents, pyridyl groups, pyrimidyl groups, triazyl groups, purine groups and 8-azapurine groups are mercapto groups, halogens, hydroxyl groups, carboxyl group, oxo group, amino group, C1 _{-C3 alkyl} group, methoxy group,
A sugar compound having a sugar residue selected from the group consisting of an ethoxy group, a carboxymethyl group, an acetyl group, and anisoyl group. Phospholipase D (hereinafter referred to as phospholipase for convenience) catalyzes a transfer reaction between an organic phosphate ester and a primary or secondary alcoholic hydroxyl group-containing compound with an alcoholic hydroxyl group-containing compound selected from
The following formula () is characterized by being reacted in the presence of DM)

[C] However, A and A' in the formula have the same meanings as above,
C relates to a method for producing organic phosphate derivatives and salts thereof represented by the residues in (1) to (3) above. Conventionally, the production of known compounds in the above formula () organic phosphate ester derivatives has been carried out by chemical synthesis methods using synthetic reagents, regardless of the difficulty of the synthesis method. However, when attempting to manufacture chemical synthesis methods, depending on the target compound, it often requires harmful reagents, takes a long time and is expensive, and involves complex methods and multi-step synthetic routes. In many cases, it cannot be obtained without going through the following steps. In addition, in chemical synthesis methods, for example, reaction selectivity and stereospecificity are poor, and it is often difficult to synthesize something using a complicated synthesis method. Furthermore, it is often impossible to synthesize the reaction components because they are unstable to acids, alkalis, heat, etc., or are insoluble in the solvent used or undergo denaturation. In this regard, by using the enzyme transfer reaction according to the present invention, the above-mentioned drawbacks and difficulties can be advantageously overcome by a simple method. That is, according to the method of the present invention, after synthesizing a large amount of an organic phosphate ester of the formula (), such as an organic phosphoric acid choline ester, which can be easily synthesized using known synthesis means at a low cost and with high purity, Using this as a starting substrate, we selectively, stereospecifically, and uniformly carry out the transfer reaction to organophosphate derivatives, which could only be obtained through complicated synthetic methods or were difficult to synthesize. It is a step reaction and can be carried out under extremely mild conditions. Conventionally, only glycero-type phospholipids or sphingo-type phospholipids, both natural and non-natural types, have been known as substrates for the enzymatic action of phospholipase D. This time, the present inventors have developed phospholipase DM using a non-biologically synthesized organic phosphoric acid choline ester belonging to the above formula (), which has not been attempted before since it does not belong to the category of phospholipids, as a substrate. Surprisingly, the reaction not only cleaved the ester bond between phosphoric acid and choline, but also caused many types of alcoholic OH-containing substances to coexist in the reaction system, similar to when acting on glycerophospholipids. We discovered that a transfer reaction occurs using a compound as a receptor. Thus, according to the method of the present invention, an organic phosphoric acid is produced, which is characterized by reacting an easily obtained synthetic organic phosphoric acid ester with an alcoholic OH-containing compound serving as a receptor in the presence of phospholipase DM. It has been found that a method for producing ester derivatives can be provided. In the present invention, an organic phosphate ester derivative refers to an ester bond between the phosphoric acid moiety of the organic phosphate ester of the formula (), which is a synthetic substrate, and the moiety represented by B in the formula (), using phospholipase DM. refers to an organic phosphate ester derivative represented by the above formula (), which is a compound that is hydrolyzed by the action of and simultaneously induces the transfer of the phosphoric acid moiety to an alcoholic OH-containing compound that is a receptor coexisting in the reaction system. Conventionally known substrates on which phospholipase D acts are natural or non-natural glycerophospholipids and sphingophospholipids. For example, in the case of lecithin, the phospholipase D is known to act at the ester bond between the phosphate moiety of lecithin and choline, and hydrolyzes this bond to generate choline and phosphatidic acid. [M.Kates, Can.J.Biochem.physiol, 32, 571,
(1954)]. Also, regarding sphingomyelin,
It is known to act similarly to catalyze the reaction that produces choline and ceramide phosphate [Davidson, FM and Long, C.Biochem.J.69,
458-466 (1958)]. On the other hand, in the case of lecithin, known phospholipase C is known to catalyze the reaction that generates choline phosphate by cutting the ester bond between the glycerin structure of lecithin and phosphoric acid [T.Hori et al. ., J.Biochem., 64, 533 (1968)]. In addition, known sphingomyelin phosphodiesterase acts on sphingomyelin to reduce its N
- Known to catalyze the action of hydrolyzing the ester bond between acylsphingosine and phosphoric acid [IKeZaWa, H., Mori.M., et al.Biochim
Biophys acta, 528, 247-259 (1978)]. In this way, known phospholipase C and known swingomyelin phosphodiesterase have choline on the polar side of lecithin or sphingomyelin.
The position of action is clearly differentiated from phospholipase D, which cleaves phosphate bonds, across the phosphate.
Phospholipase D is also known to have the effect of cleaving the choline-phosphate ester bond and at the same time causing a transfer reaction with a coexisting alcohol compound. For example, it is known that when lecithin and ethanol are reacted in the presence of cabbage phospholipase D, phosphatidic acid is transferred to ethanol to produce phosphatidylethanol [RMCDawson; Biochem. J. 102, 205,
(1967)]; SF Yang; J.Biol.Chem., 242, 477
(1967)]. However, with regard to phospholipase C and sphingomyelin diesterase, it is not known that transesterification occurs with other alcohol compounds between the glycerin structure or the N-acylsphingosine structure. As mentioned above, since the alcohol transfer effect of phosphatidyl groups by phospholipase D was known, research in this field has progressed, and a British patent was granted.
The proposal No. 1581810 (corresponding West German publication No. 2717547) is known. According to this proposal, a glycero-type phospholipid represented by the general formula of this proposal and a straight or branched alkyl group up to C ₅ which may be substituted with a hydroxyl group, halogen, amino or other substituent are combined. A primary alcohol transfer reaction using the enzymatic action of a primary alcohol and cabbage-derived phospholipase D is disclosed. and the reaction takes place only with primary alcohols that do not contain more than 5 carbon atoms; in the case of alcohols that contain more than 5 carbon atoms, the main product of the reaction is the corresponding phosphatidic acid. It is stated that. Furthermore, the proposal also states that there are no particular restrictions on the selection of the alcohol component as long as it is a primary alcohol that satisfies the above requirements. In addition, the inventors of the above proposal, S. Kovatchev, H. Eibl, etc., Adv. Exp. Med. Biol., Vol. 101,
221 (1978), regarding the primary alcohol transfer reaction using the enzymatic action of phospholipase D.
They reported that no rearrangement reaction was observed with _C7 - _C10 alkanols, but a 20% rearrangement reaction occurred with _C6 hexanol. on the other hand,
Rokhimov, MM, Uzb.Biol.Zh., Vol.3, 6
-9 (1979), alcohols with _C5 or higher, e.g.
They reported that no rearrangement reaction occurred with _C6 hexanol. RMDawson used lecithin as the phospholipid and sucrose, glucose, and galactose as the saccharides, and as a result of experiments using known phospholipase D derived from cabbage, no transfer of the phosphatidyl group of the lecithin substrate to these saccharides occurred. reported [Biochem.J.
vol.102, 205-209 (1967)]. In addition, SFYang reported that as a result of a similar experiment to the above using glucose and inositol, no transfer to sugars occurred [J. Biol.
Chem., vol.242, 477-484 (1967)]. As mentioned above, conventionally, the transfer reaction between phospholipids and alcohols by phospholipase D has only occurred in the case of primary alcohols, especially chain primary alcohols that have a simple structure with a relatively small number of carbon atoms. It is conventional technical knowledge that neither pentoses nor hexoses occur with sugars. The present inventors have discovered the existence of a microorganism that has a phospholipase D production ability that differs from the conventionally known cabbage-derived phospholipase D in terms of its optimum temperature, optimum pH, etc., and has already published JP-A No. 58-63388. (Showa 58
(Published on April 15, 1983) and Japanese Patent Application Publication No. 1983-67183 (Published on April 21, 1983). As a result of further research, the phospholipase D obtained from the phospholipase D-producing bacteria was further disclosed in the above-mentioned JP-A-58-63388 and JP-A-58-67183. Unlike phospholipase D, it has a wide range of alcoholic properties, including primary alcohols below _C5 , and which has not been previously described to undergo rearrangement.
It has been described that OH-containing compounds also have the effect of transferring glycerophospholipids. The substrate phospholipids include lecithin, diacyl ester type, monoacyl ester type, plasmalogen type, cycloalkylidene type, dialkyl ether type, monoalkyl ether type α-glycerophospholipid and β
- It has been described that in addition to glycerophospholipids, sphingophospholipids can also be used as substrates. However, among the substrates indicated in the broad general concept above, those specifically disclosed are lecithin, lysolecithin, β,γ-dihexadecyl L-
Only α-lecithin and sphingomyelin. Furthermore, there is no mention or suggestion of a synthetic organic phosphoric acid ester having the structure of the formula (), which does not belong to the above-mentioned general concept used in the present invention. Furthermore, neither the hydrolysis reaction by phospholipase D using a non-biologically synthesized organic phosphate ester as a substrate, which is completely different from the phospholipids found in biological systems, nor the transfer action have been known. However, as mentioned above, the research conducted by the present inventors revealed that phospholipase DM acts on the non-biologically synthesized organic phosphate ester of the formula (), which does not belong to the category of phospholipids, resulting in the formation of the formula (). At the same time, the ester bond between the phosphoric acid part and the B part of the ester is hydrolyzed, and a wide range of alcoholic OH-containing compounds coexisting in this reaction system are used as acceptors.
It has been discovered that the phosphoric acid moiety can be transferred to these alcoholic OH-containing compounds. Therefore, according to the method of the present invention, the types of organic phosphate ester derivatives that were previously only obtainable by difficult synthetic methods can be expanded to include a wide variety of organic phosphate ester derivatives, and moreover, they can be obtained extremely easily. Benefits are obtained that allow preparation. In addition, by the method of the present invention, the target substrates for the transfer action by enzymes have expanded from conventionally known biological phospholipids to non-living synthetic organic phosphate esters, making this technical field more useful and abundant. This is significant in terms of making it a reality. This is because, compared to biological phospholipids, synthetic organic phosphate ester substrates can not only be produced in large quantities at low cost and with good purity, but also allow for the use of the resulting formula () derivatives, such as oxidation, This has the advantage of being a more stable derivative against reduction, ultraviolet light and temperature, and even enzymatic hydrolysis;
Cheap and stable organophosphate derivatives desired in the fields of medicine, cosmetics, food, agrochemicals, etc. can be produced under mild and easy conditions and by means without the need for complicated and disadvantageous chemical synthesis methods. This is because it can be produced in good yield by an enzymatic method with almost no risk of side reactions, and it also has the advantage of providing a wide range of desired organic phosphate derivatives. Therefore, an object of the present invention is to provide a process for producing many novel organophosphate derivatives by enzymatic methods, including organophosphate derivatives that could only be provided by conventionally disadvantageous synthetic methods. It is in. The above objects and many other objects and advantages of the present invention will become more apparent from the following description. The synthetic raw material organic phosphate ester used in the method of the present invention is represented by the following formula ().

[Chemical formula] However, in the formula, A is R ₁ -X or R ₂ -O-
(CH ₂ CH ₂ O) _o , where X represents an oxygen atom or a sulfur atom; R ₁ represents a saturated or unsaturated C ₆ to C ₁₂₀ aliphatic hydrocarbon residue, or a group R ₃ -Y-R ₄ -; In the group R ₃ -Y-R ₄ -, Y is O, S, SO,
SO ₂ , O-CO-O, O-CO-NH or O-
CS-NH, R ₃ is halogen, C ₆ - C ₁₆ aryl group, C ₁ -
_C18 alkoxy group, C1 _-C18 alkylmercapto group, C1- _C18 alkanesulfinyl group, C1-C18 alkoxy group, C1 _{- C18} alkylmercapto group, C1 _-C18 alkanesulfinyl group
_C18 alkanesulfonyl group, _C2 - _C10 alkylcarbonylamide group, C2 _{- C6} alkylcarbamoyl group, _C2 - _C6 alkylthiocarbonylamide group, _C1 - _C6 alkanesulfonamide group,
a _saturated or unsaturated C1- _C24 fat optionally substituted with at least one substituent selected from the group consisting _of a C2 _- C5 alkoxycarbonyl group and a _C3 - _C5 ureido group; represents a group hydrocarbon residue,
Here, the above _C1 to _C18 alkoxy group may be further substituted with a _C1 to _C4 alkoxy group, and the above C1 to C18 alkoxy group may be further substituted with a C1 to C4 alkoxy group.
A portion of the C ₁ to _{C 24} aliphatic hydrocarbon residue may be composed of a 3 to 6-membered cycloalkane ring, and R ₄ is a halogen, a hydroxyl group, a nitrile group, or a C 1 to C 24 aliphatic hydrocarbon residue. ₃ ~
_C6 cycloalkane group, _C6 to _C8 aryl group,
_C1 - _C4 alkoxy group, _C1 - _C18 alkylmercapto group, _C1 - _C4 alkanesulfinyl group,
It has a _C1 to _C4 alkanesulfonyl group, a _C2 to _C5 alkoxycarbonyl group, an amino group which may have a _C2 to _C4 acyl group, and a _C1 to _C5 alkyl group. saturated or unsaturated C2 _{- C8} aliphatic carbonization optionally substituted with at least one substituent selected from the group consisting of an optional carbamoyl group and a _C1 - _C4 alkanesulfonamide group Indicates a hydrogen residue, where the above _C1 - _C4 alkoxy group is a _C1 - _C4 alkoxy group, a _C1 - _C4 alkylmercapto group, a _C1 - _C4 alkanesulfinyl group,
C1- _C4 alkanesulfonyl group, halogen, hydroxyl group, nitrile group, _C6 - _C8 aryl group, C1 _{-C4 alkanesulfonamide} group, _carbamoyl group, _C2
It may be further substituted with an amino group which may have ~ _C5 alkoxycarbonyl group and _C2 - _C4 acyl group, and the above _C2 - _C8 aliphatic hydrocarbon residue is Indicates that a part of the inside may be composed of a 3- to 6-membered cycloalkane ring, or the aliphatic hydrocarbon residue may be an alicyclic hydrocarbon residue; R ₂ is A saturated or unsaturated C ₁ to C ₂₀ aliphatic hydrocarbon residue, a C ₆ to _{C 26} acyl group derived from a saturated or unsaturated aliphatic carboxylic acid, and a 2-N-acyl having the acyl group. - represents a group selected from the group consisting of an alkylphenyl group having an amino-ethyl group and a saturated or unsaturated C ₆ to C ₂₆ alkyl group; n represents an integer from 1 to 20, provided that R ₂ is above C ₆
When representing a ~ _C26 acyl group, n represents an integer of 2 to 20; Furthermore, in the formula, A' represents an oxide anion or hydroxy, and B represents _{-CH2.CH2 +} N ( _CH3 ) ₃ , −CH ₂・CH ₂ NH ₂ , −CH ₂・CHOH・CH ₂ OH, −
Indicates a group selected from the group consisting _{of CH3} and _-CH2.CH3 . The substrate of the above formula (), which is a starting material used in the present invention, such as organic phosphoric acid choline ester, can be easily obtained by a known synthesis method based on the following commercially available raw materials. Most of the commercially available raw materials are various alcohols and their polyoxyethylene derivatives produced from petroleum, natural animal and vegetable oils, whale oil, etc., as well as organic phosphates made by combining these with phosphoric acid, etc. in the emulsifier field. It is produced in large quantities in the field of detergents and is available commercially at a low price, and can be used to synthetically bind choline by known means. For example, various alcohols (R・OH) are easily available as reagents and industrial raw materials, and polyoxyethylene alkyl ether [RO
(CH ₂ CH ₂ O) _o H], polyoxyethylene alkyl phenol ether [

[Formula]], polyoxyethylene alkyl ester [RCOO (C ₂ H ₄ O) _o H], polyoxyethylene alkyl amide [RCONH (CH ₂ CH ₂ O) _o H], and various other alcohols are also surfactants. It is commercially available as an industrial raw material. Furthermore, monoorganic phosphoric acid esters synthesized from these are also produced in the field of detergents and surfactants and are commercially available in a wide variety, so that they can be appropriately selected from among these commercially available products for use. As means for synthesizing organic phosphoric acid choline ester of formula () using the above raw materials, for example, Okazaki et al.
The synthesis can be carried out using known synthesis methods such as those disclosed in JP-A-58-10592, and by appropriately selecting and combining them as necessary. Specific examples of the raw material formula () organic phosphate ester used in the present invention include the following formula () compounds. In R ₁ of the above formula (), the saturated or unsaturated C ₆ to _{C 120} aliphatic hydrocarbon residue may be linear or branched, and the linear aliphatic hydrocarbon residue As, hexyl group, octyl group, decyl group,
dodecyl group, tetradecyl group, hexadecyl group,
Examples include an octadecyl group, an eicosyl group, and a docosyl group, and preferred examples of the aliphatic hydrocarbon residue include a dodecyl group, a tetradecyl group, an octadecyl group, and a docosyl group. Examples of the branched aliphatic hydrocarbon residues include aliphatic hydrocarbon residues having an isoprene skeleton, such as a phytyl group,
Examples include a solanesyl group, a dekeprenyl group, and a dolykyl group. Among the above Y, preferred examples include O, S,
Examples include SO, SO ₂ and O-CO-O. The aliphatic hydrocarbon residue of R ₃ above may be linear or branched, and may include a methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group,
Octadecyl group, eicosyl group, 3-heptyl-
Examples include decyl group, 1-methyl-octadecyl group, 17-methyl-octadecyl group, oleyl group, linoleyl group, and 3,7,11,15-tetramethyl-hexadecyl group, which are included in the aliphatic hydrocarbon residue. An example of the cycloalkane ring is cyclohexane. Among the substituents that can be substituted on the aliphatic hydrocarbon residue of R ₃ above, fluorine and chlorine are preferable as the halogen, and phenyl group as the C ₆ to C ₁₆ aryl group,
Examples include octylphenyl group, and examples of _C1 to _C18 alkoxy groups include methoxy group, pentoxy group, heptoxy group, octyloxy group, nonyloxy group,
Examples include decyloxy group, undecyloxy group, dodecyloxy group, tridecyloxy group, and tetradecyloxy group, and examples of _C1 to _C18 alkylmercapto groups include methylmercapto group, ethylmercapto group, dodecylmercapto group, and pentadecylmercapto group. Examples of the C ₁ to _{C 18} alkanesulfinyl group include a pentadecane sulfinyl group and a hexadecane sulfinyl group; examples of the C ₁ to _{C 18} alkanesulfonyl group include a pentadecane sulfonyl group; , hexadecanesulfonyl group, examples of _C2 - _C10 alkylcarbonylamide group include caproylamide group, N-methyl-caproylamide group, N-butyl-acetamide group, and butylcarbamoyl group as _C2 - _C6 alkylcarbamoyl group. Examples include a butylcarbonylamide group as a C ₂ -C ₆ alkylthiocarbonylamide group, and a pentasulfonamide group and an N-methyl-sulfonamide group as an example of a C ₁ -C ₆ alkanesulfonamide group. Examples of the C ₂ to _{C 5} alkoxycarbonyl group include a methoxycarbonyl group and an ethoxycarbonyl group, and examples of the C ₃ to _{C 5} ureido group include a butyl urein group. Among these substituents, the above-mentioned alkoxy group, an alkylmercapto group, an alkanesulfinyl group, an alkanesulfonyl group, and an aryl group, and when substituted, the number of substituents is preferably one. Furthermore, it can be substituted with the above C ₁ to C ₁₈ alkoxy group.
Examples of the _C1 - _C4 alkoxy group include a methoxy group and a butoxy group. In R ₄ above, the aliphatic hydrocarbon residue may be linear or branched, and the linear aliphatic hydrocarbon residue may be an alkylene group such as an ethylene group, a propylene group, a butylene group, or a pentene group. However, as the branched aliphatic hydrocarbon residue, 1-methyl-propylene group, 2-methyl-propylene group, 2,2-dimethyl-propylene group, 2-methylene-propylene group, 2-ethyl-propylene group, 2 -Propyl-
Propylene group, 2-isopropyl-propylene group, 2-pentyl-propylene group, 1-pentyl-propylene group, 1-ethyl-ethylene group, 2-
Examples include alkylene groups such as (2-propynyl)-propylene group, 2-(1-propynyl)-propylene group, and 2-propanediene-propylene group,
When the aliphatic hydrocarbon residue is an alicyclic hydrocarbon residue, examples of the alicyclic hydrocarbon residue include a cyclopentene group, a cyclohexene group, and a 2-ethyl-cyclohexene group. Among the substituents for _R4 , examples of the halogen include fluorine, examples of the _C3 to _C6 cycloalkyl group include cyclopropyl, cyclopentyl, and cyclohexyl, and examples of the _C6 to _C8 aryl group include phenyl. group, 4-methyl-phenyl group, 2-methoxy-5-chloro-phenyl group,
Examples of _C1 to _C18 alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, and hexadecyloxy groups, and examples of _C1 to _C18 alkylmercapto groups include methylmercapto, ethylmercapto, and octadecyl. Examples include mercapto groups, methanesulfinyl groups as C ₁ -C ₄ alkanesulfinyl groups, and C ₁ -C ₄ alkanesulfinyl groups.
Examples of alkanesulfonyl groups include methanesulfonyl groups, examples of C ₁ to C ₄ alkoxycarbonyl groups include methoxy groups and ethoxy groups, and C ₂
Examples of the amino group which may have a _C5 acyl group include an amino group and an acetamido group, and a _C1 to _{C5 acyl} group.
Examples of the carbamoyl group which may have an alkyl group include a carbamoyl group and a dimethylcarbamoyl group, and examples of the C ₁ to _{C 4} alkanesulfonamide group include a methanesulfonamide group. Among these substituents, preferred substituents are Examples of the group include the above-mentioned alkoxy group, alkylmercapto group, alkoxycarbonyl group, and alkanesulfonamide group, and when substituted, the number of substituents is preferably 1 to 2. Among the substituents that may be further substituted on the C ₁ to C ₁₈ alkoxy group, examples of the _{C 1} to _{C 4} alkoxy group include a methoxy group and _an ethoxy group;
An example of an alkylmercapto group is a methylmercapto group, an example of a _C1 - _C4 alkanesulfinyl group is a methanesulfinyl group, and a _C1 - _C4
An example of the alkanesulfonyl group is a methanesulfonyl group, an example of the _C6 - _C8 aryl group is a phenyl group, an example of the _C1 - _C4 alkanesulfonamide group is a methanesulfonamide group,
Examples of the C ₂ to _{C 5} alkoxycarbonyl group include a methoxycarbonyl group and an ethoxycarbonyl group, and examples of the amino group which may have a C ₂ to _{C 4} acyl group include an amino group and an acetamido group, and these substituents Preferred substituents among these include the alkoxy group and aryl group described above. In the above R ₂ , saturated or unsaturated C ₁ ~
The _C26 aliphatic hydrocarbon residue may be linear or branched, and is preferably a methyl group, dodecyl group, tetradecyl group, hexadecyl group, octadecyl group, or oleyl group, and is a saturated or unsaturated aliphatic carboxylic acid. The C ₆ to _{C 26} acyl group derived from may be linear or branched, and preferably a lauroyl group, palmitoyl group, stearoyl group, or oleoyl group, and a saturated or unsaturated C ₆ to _{C 26} alkyl group The alkylphenyl group having the group may be linear or branched, and octylphenyl group and nonylphenyl group are preferable. In addition, n is preferably in the range of 1 to 15. As B in the above formula (), -CH ₂ ·CH ₂₊ N (CH ₃ ) ₃ , -CH ₂ ·CH ₂ NH ₂ , -CH ₂ ·CHOH · CH ₂ OH, -
Examples include CH ₃ and -CH ₂ .CH ₃ , and these compounds of formula () having B can be easily synthesized by known synthesis methods. In addition, a substrate obtained by chemical synthesis, such as organic phosphoric acid choline ester, is converted into choline by adding other alcohol compounds, such as ethanolamine, glycerol, ethanol, etc., and allowing the action of phospholipase DM to convert it into choline. Prepare ethanolamine ester, organic phosphoric acid glycerol ester, and organic phosphoric acid ethanol ester and use the formula ()
It can also be used as a compound. Also, even if B is a compound other than the above, phospholipase DM
Any substrate that can undergo a transfer reaction can be used. Specific examples of compounds of formula () in which R ₁ represents a saturated or unsaturated C ₆ to _{C 120} aliphatic hydrocarbon residue include, for example, octyl-phosphorylcholine, decyl-phosphorylcholine, dodecyl- Examples include 〃, tetradecyl-〃, hexadecyl-〃, octadecyl-〃, eicosyl-〃, docosyl-〃, tetradecylthioalcohol-1-phosphorylcholine, phytyl-phosphorylcholine, solanesyl-phosphorylcholine, decaprenyl-phosphorylcholine, dolikyl-phosphorylcholine, etc. . Moreover, as specific examples when the above R ₁ is represented by R ₃ -Y-R ₄ , the following compounds can be exemplified. 2-methoxymethyl-3-octadecyloxy-propanol-1-phosphorylcholine, 2-ethyl-3-octadecyloxy-propanol-1-phosphorylcholine, 2-methoxy-3-(2-methoxy-octadecyloxy)-propanol-1- Phosphorylcholine, 2-methoxy-3-[7-(4-octyl-phenyl)-heptyloxy]-propanol-1-phosphorylcholine, 2-methoxy-3-(5-dodecyloxy-pentoxy)-propanol-1-phosphorylcholine, 2-methoxy-3-(12-pentoxy-dodecyloxy)-propanol-1-phosphorylcholine, 2-methoxy-3-(5-dodecylmercapto-pentoxy)-propanol-1-phosphorylcholine, 2-methoxymethyl-2-methyl -3-octadecyloxy-propanol-1-phosphorylcholine, 2,2'-bis-methoxymethyl-3-hexadecyloxy-propanol-1-phosphorylcholine, 2-methoxy-3-octadecyloxy-thiopropanol-1-phosphorylcholine, 3-heptadecyloxy-thiopropanol-
1-phosphorylcholine, 2-methyl-3-hexadecyloxy-thiopropanol-1-phosphorylcholine, 2-benzyl-3-octadecyloxy-thiopropanol-1-phosphorylcholine, 3-(3-tetradecyloxy-propoxy)-
Thiopropanol-1-phosphorylcholine, 3-(2-pentadecylmercapto-ethoxy)
-thiopropanol-1-phosphorylcholine, 2-(2-methoxy-ethoxy)-3-octadecyloxy-thiopropanol-1-phosphorylcholine, 3-(12-pentyloxy-dodecyloxy)-
Propanol-1-phosphorylcholine, 3-(5-dodecyloxy-pentyloxy)-
Propanol-1-phosphorylcholine, 3-(2-pentadecylmercapto-ethoxy)
-propanol-1-phosphorylcholine, 3-(2-pentadecyloxy-ethoxy)-propanol-1-phosphorylcholine, 3-hexadecyloxy-carbonyloxy-
Propanol-1-phosphorylcholine, 2-octadecyloxy-ethanol-1-phosphorylcholine, 4-hexadecyloxy-butyl alcohol-
1-phosphorylcholine, 2-allyl-3-octadecyloxy-propanol-1-phosphorylcholine, 3-hexadecyloxy-2-(2-propynyl)-propanol-1-phosphorylcholine, 2-benzyloxymethyl-3-heptadecyloxy -propanol-1-phosphorylcholine, 2-methyl-3-(3-tetradecyloxy-
propoxy)-propanol-1-phosphorylcholine, 2-benzyl-3-octadecyloxy-propanol-1-phosphorylcholine, 3-(10-heptyloxy-decyloxy)-2
-(2-methoxy-ethoxy)-propanol-1
-phosphorylcholine, 4-(2-hexadecylmercapto-ethoxy)
-2-Butyl alcohol-2-phosphorylcholine, 3-heptadecyloxy-2-(methylmercapto-methyl)-propanol-1-phosphorylcholine, 2-n-butylmercapto-3-octadecyloxy-propanol-1-phosphorylcholine, 2 -hexadecylmercapto-3-methoxy-
Propanol-1-phosphorylcholine, 2-hydroxy-3-(3-tetradecyloxy-propoxy)-propanol-1-phosphorylcholine, 4-(2-hexadecanesulfinyl-ethoxy)-2-butylalcohol-2-phosphorylcholine, 3-octadecylmercapto-propanol-
1-phosphorylcholine, 3-octadecanesulfinyl-propanol-1-phosphorylcholine, 3-octadecanesulfonyl-propanol-
1-phosphorylcholine, 3-(octadec-9t-enylmercapto)-propanol-1-phosphorylcholine, 3-(octadec-9t-en-sulfonyl)-propanol-1-phosphorylcholine, 3-hexadecanesulfonyl-propanol-
1-phosphorylcholine, 3-hexadecylmercapto-propanol-
1-phosphorylcholine, 3-(3,7,11,15-tetramethyl-hexadecylmercapto)-propanol-1-phosphorylcholine, 3-(3,7,11,15-tetramethyl-hexadecanesulfonyl)-propanol-1 -Phosphorylcholine, 3-heptadecylmercapto-propanol-
1-phosphorylcholine, 3-heptadecanesulfonyl-propanol-
1-phosphorylcholine, 3-eicosylmercapto-propanol-1
-phosphorylcholine, 3-eicosansulfonyl-propanol-1
-phosphorylcholine, 3-dodecylmercapto-propanol-1-
Phosphorylcholine, 3-dodecanesulfonyl-propanol-1-
Phosphorylcholine, 3-(2-pentadecyloxy-ethylmercapto)-propanol-1-phosphorylcholine, 3-(2-pentadecyloxy-ethanesulfonyl)-propanol-1-phosphorylcholine, 2-methoxy-3-octadecylmercapto-
Propanol-1-phosphorylcholine, 2-methoxy-3-octadecanesulfinyl-propanol-1-phosphorylcholine, 2-methoxy-3-octadecanesulfonyl-
Propanol-1-phosphorylcholine, 3-(2-pentadecylmercapto-ethylmercapto)-propanol-1-phosphorylcholine 2-Methyl-3-octadecylmercapto-propanol-1-phosphorylcholine, 3-(2-pentadecanesulfonyl-ethylmercapto) -propanol-1-phosphorylcholine, 3-(2-methoxy-octadecylmercapto)
-Propanol-1-phosphorylcholine, 3-tetradecylmercapto-propanol-
1-phosphorylcholine, 2-methyl-3-octadecanesulfinyl-
Propanol-1-phosphorylcholine, 3-hexadecylmercapto-2-methyl-propanol-1-phosphorylcholine, 2,2-dimethyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 3-octadecyl-mercapto-cyclohexanol-1 -phosphorylcholine, 3-(14-methoxy-tetradecylmercapto)
-propanol-1-phosphorylcholine, 2-isopropyl-3-octadecylmercapto-propanol-1-phosphorylcholine, 4-heptadecylmercapto-3-methyl-n
-butanol-1-phosphorylcholine, 3-octadecylmercapto-butanol-1
-phosphorylcholine, 3-(9-octyloxy-)nonylmercapto)-propanol-1-phosphorylcholine, 3-(4-tridecyloxy-butylmercapto)-propanol-1-phosphorylcholine, 3-pentadecylmercapto-propanol-
1-phosphorylcholine, 3-(10-n-butoxy-decyloxy-ethylmercapto)-propanol-1-phosphorylcholine, 5-hexadecylmercapto-pentanol-
1-phosphorylcholine, 3-(11-hexyloxy-undecylmercapto)-propanol-1-phosphorylcholine, 3-(7-decyloxy-heptylmercapto)
-propanol-1-phosphorylcholine, 3-hexadecylmercapto-2-methoxy-
Propanol-1-phosphorylcholine, 2-ethoxy-3-heptadecylmercapto-
Propanol-1-phosphorylcholine, 2-methoxy-3-(trans-octadec-
9t-enylmercapto)-propanol-1-phosphorylcholine, 2-methoxy-3-(octadec-9,12-dienylmercapto)-propanol-1-phosphorylcholine, 2-methoxy-3-(1-methyl-octadecylmercapto) -propanol-1-phosphorylcholine, 3-(3-heptyl-decylmercapto)-2-
Methoxy-propanol-1-phosphorylcholine, 2-methoxy-3-(3-tetradecyloxy-propylmercapto)-propanol-1-phosphorylcholine, 3-(5-dodecyloxy-pentylmercapto)-2-methoxy-propanol-1 -phosphorylcholine, 2-methoxy-3-(8-nonyloxy-octylmercapto)-propanol-1-phosphorylcholine, 2-ethyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-propyl-3-hexadecylmercapto-
Propanol-1-phosphorylcholine, 2-n-butyl-3-octadecylmercapto-propanol-1-phosphorylcholine, 2-benzyloxy-3-octadecylmercapto-propanol-1-phosphorylcholine, 2-methoxymethyl-3-hexadecylmercapto- Propanol-1-phosphorylcholine, 2-ethoxymethyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2,2-bismethoxymethyl-3-octadecylmercapto-propanol-1-phosphorylcholine, 2-methoxy-3-heptadecyl Mercapto
Propanol-1-phosphorylcholine, 4-hexadecylmercapto-2-butanol-2-phosphorylcholine, 4-octadecylmercapto-2-pentanol-2-phosphorylcholine, 3-hexadenylmercapto-cyclopentanol-1-phosphorylcholine, 2 -methanesulfonamide-3-octadecylmercapto-propanol-1-phosphorylcholine, 2-methoxymethyl-3-hexadecylmercapto-thiopropanol-1-phosphorylcholine, 3-[2-(N-dodecyl-carboxamide)-
Ethylmercapto]-2-methoxy-propanol-1-phosphorylcholine, 2-methoxy-3-(9-phenyl-octadecylmercapto)-propanol-1-phosphorylcholine, 3-[11-(N-n-butyl-carboxamide)
-undecylmercapto]-2-methoxy-propanol-1-phosphorylcholine, 2-methoxy-3-octadecylmercapto-
Thiopropanol-1-phosphorylcholine, 4-octadecylmercapto-2-thiopentanol-2-phosphorylcholine, 2-methyl-2-methoxycarbonyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-methyl-3-hexa Decylmercapto-thiopropanol-1-phosphorylcholine, 3-hexadecylmercapto-2-(2-methoxy-ethoxy)-propanol-1-phosphorylcholine, 2-hexadecylmercapto-3-methylmercapto-propanol-1-phosphorylcholine, 2 -benzyl-3-hexadecylmercapto-
Propanol-1-phosphorylcholine, 2-phenyl-3-hexadecylmercapto-
Propanol-1-phosphorylcholine, 2-allyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-methylene-3-octadecylmercapto-
Propanol-1-phosphorylcholine, 3-(11-caproylamide-undecyloxy)-propanol-1-phosphorylcholine, 3-(11-N-methyl-caproylamide-undecyloxy)-propanol-1-phosphorylcholine, 3-(11 -pentanesulfonamide-undecyloxy)-propanol-1-phosphorylcholine, 3-(11-N-methyl-pentanesulfonamide-undecyloxy)-propanol-1-phosphorylcholine, 3-(11-N-n-butyl -acetamido-undecyloxy)-propanol-1-phosphorylcholine, 3-(11-N-n-butyl-carboxyamido-undecyloxy)-propanol-1-phosphorylcholine, 3-(11-N-n-butyl- Thiocarboxamide-undecyloxy)-propanol-1-
Phosphorylcholine, 3-(11-butoxycarbonyl-undecyloxy)-propanol-1-phosphorylcholine, 3-(3-tetradecyloxy-propoxy)-
Propanol-1-phosphorylcholine, 2-n-butyl-3-(11-N-n-butyl-
Carboxamide-undecyloxy)-propanol-1-phosphorylcholine, 2-n-butyl-3-(11-N-n-butyl-
N-Methyl-carboxamido-undecyloxy)-propanol-1-phosphorylcholine, 2-(2-methoxy-ethoxy)-methyl-3-
Octadecyloxy-propanol-1-phosphorylcholine, 3-heptadecyloxy-2-methanesulfinylmethyl-propanol-1-phosphorylcholine, 2-(2-amino-ethyl)-3-hexadecyloxy-propanol-1-phosphorylcholine , 2-(2-acetamido-ethyl)-3-hexadecyloxy-propanol-1-phosphorylcholine, 3-hexadecyloxy-2-(2-methanesulfonamido-ethyl)-propanol-1-phosphorylcholine, 2-cyan Methyl-3-octadecyloxy-
Propanol-1-phosphorylcholine, 2-ethoxycarbonylmethyl-3-octadecyloxy-propanol-1-phosphorylcholine, 2-carbamoylmethyl-3-octadecyloxy-propanol-1-phosphorylcholine, 2-cyclopentyl-3-(8-undecyl oxy-hexyloxy)-propanol-1-phosphorylcholine, 2-cyclohexylmethyl-1-(8-nonyloxy-octyloxy)-propanol-1-
Phosphorylcholine, 3-(7-decyloxy-heptyloxy)-2
-(4-methyl-benzyl)-propanol-1-
Phosphorylcholine, 2-(5-chloro-2-methoxy-benzyl)-
3-(4-tridecyloxy-butoxy)-propanol-1-phosphorylcholine, 3-octadecyloxy-2-phenyl-propanol-1-phosphorylcholine, 2-cyan-3-heptadecyloxy-propanol-1-phosphorylcholine, 2 -Carbamoyl-3-heptadecyloxy-
Propanol-1-phosphorylcholine, 2-hydroxy-3-[5-(3-octyloxy-propoxy)-pentyl]-propanol-1
-phosphorylcholine, 2-methoxy-3-[7-(4-octyl-cyclohexyl)-heptyloxy]-propanol-
1-phosphorylcholine, 2-isopropoxy-3-(8-nonyloxy-octyloxy)-propanol-1-phosphorylcholine, 2-(2-hydroxy-ethoxy)-3-octadecyloxy-propanol-1-phosphorylcholine, 3-hepta Decyloxy-2-(2-methylmercapto-ethoxy)-propanol-1-phosphrylcholine, 3-heptadecyloxy-2-(2-methanesulfinyl-ethoxy)-propanol-1-phosphorylcholine, 3-heptadecyloxy -2-(2-methanesulfonyl-ethoxy)-propanol-1-phosphorylcholine, 2-(2-amino-ethoxy)-3-hexadecyloxy-propanol-1-phosphorylcholine, 2-(2-acetamido-ethoxy)- 3-hexadecyloxy-propanol-1-phosphorylcholine, 3-hexadecyloxy-2-(2-methanesulfonamido-ethoxy)-propanol-1-
Phosphorylcholine, 2-ethoxycarbonylmethoxy-3-heptadecyloxy-propanol-1-phosphorylcholine, 2-cyanmethoxy-3-heptadecyloxy-propanol-1-phosphorylcholine, 2-N-dodecyl-carboxyamidomethoxy-3-hepta Decyloxy-propanol-1-
Phosphorylcholine, 3-heptadecyloxy-2-methylmercapto-propanol-1-phosphorylcholine, 3-hexadecyloxy-2-methanesulfinyl-propanol-1-phosphorylcholine, 3-hexadecyloxy-2-methanesulfonyl-propanol -1-phosphorylcholine, 2-amino-3-hexadecyloxy-propanol-1-phosphorylcholine, 2,2-dimethyl-3-(6-undecyloxy-hexyloxy)-propanol-1-phosphorylcholine, 2-benzyl- 2-methyl-3-[2-(10-n
-butoxy-decyloxy)-ethoxy]-propanol-1-phosphorylcholine, 2-ethoxycarbonyl-2-methyl-1-octadecyloxy-propanol-1-phosphorylcholine, 2-(2-pentadecanesulfinyl-ethoxy)-pentanol -1-phosphorylcholine, 2-(2-pentadecanesulfonyl-ethoxy)
-Pentanol-1-phosphorylcholine, 3-(4-tridecyloxy-butoxy)-cyclopentanol-1-phosphorylcholine, 3-hexadecylamino-thiocarbonyloxy-2-methyl-propanol-1-phosphorylcholine, 3- Hexadecyloxy-2-methoxymethyl-thiopropanol-1-phosphorylcholine, 3-heptadecyloxy-2-methylmercaptomethyl-thiopropanol-1-phosphorylcholine, 3-heptadecyloxy-2-isopropoxy-thiopropanol-1 -phosphorylcholine, 3-hexadecyloxy-2-methylmercapto-thiopropanol-1-phosphorylcholine, 2,2-dimethyl-3-hexadecyloxy-
Thiopropanol-1-phosphorylcholine, 2-ethoxycarbonyl-2-methyl-3-octadecyl-thiopropanol-1-phosphorylcholine, 2-hexadecyloxy-thiopentanol-
1-phosphorylcholine, 3-octadecyloxy-1-thiocyclopentanol-1-phosphorylcholine, 2,3-bis-octadecylmercapto-propanol-1-phosphorylcholine, 3-tetradecylmercapto-2-ethoxy-
Propanol-1-phosphorylcholine, 3-hexadecylmercapto-2-ethoxy-
Propanol-1-phosphorylcholine, 3-(14-methylmercapto-tetradecylmercapto)-propanol-1-phosphorylcholine, 3-(17-methyl-octadecylmercapto)-
Propanol-1-phosphorylcholine, 3-(8-fluoro-octadecylmercapto)
-Propanol-1-phosphorylcholine, 2-hydroxy-3-octadecylmercapto-propanol-1-phosphorylcholine, 2-hydroxy-3-octadecanesulfinyl-propanol-1-phosphorylcholine, 2-hydroxy-3-octadecanesulfonyl-propanol- 1-phosphorylcholine, 2-methoxy-3-[8-(4-octylcyclohexyl)octylmercapto]propanol-
1-phosphorylcholine, 3-hexadecylmercapto-2-methylmercapto-propanol-1-phosphorylcholine, 3-hexadecylmercapto-2-methanesulfinyl-propanol-1-phosphorylcholine, 2-carbamoylmethoxy-3-hexadecylmercapto -propanol-1-phosphorylcholine, 2-amino-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-acetylamino-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-methyl-3-(11-butylureylene) -undecylmercapto-propanol-1-phosphorylcholine, 2-N-dodecyl-carboxamide-methoxy-3-hexadecyl-mercapto-propanol-1-phosphorylcholine, 2-ethylidene-3-hexadecylmercapto-propanol-1-phosphorylcholine, cyclo Propane-1-hexadecylmercaptomethyl-1-methanol-1-phosphorylcholine, 3-hexadecylmercapto-2-methanesulfonyl-propanol-1-phosphorylcholine, 2-methoxy-3-(2-methoxy-octadecylmercapto)-propanol -1-phosphorylcholine, 2-methoxy-3-(2-tetodecyloxy-
octadecylmercapto)-propanol-1-
Phosphorylcholine, 2-methoxy-3-(14-methoxy-tetradecylmercapto)-propanol-1-phosphorylcholine, 2-methoxy-3-(14-methylmercapto-
Tetradecylmercapto)-propanol-1-
Phosphorylcholine, 2-methoxy-3-(17-methyl-octadecylmercapto)-propanol-1-phosphorylcholine, 3-(8-fluoro-octadecylmercapto)
-2-methoxy-propanol-1-phosphorylcholine, 2-fluoro-3-octadecylmercapto-
Propanol-1-phosphorylcholine, 2-fluoro-3-octadecanesulfinyl-propanol-1-phosphorylcholine, 2-fluoro-3-octadecanesulfonyl-
Propanol-1-phosphorylcholine, 2-ethylmercapto-3-octadecylmercapto-propanol-1-phosphorylcholine, 3-hexadecanesulfinyl-2-methyl-
Propanol-1-phosphorylcholine, 3-heptadecylmercapto-2-methyl-propanol-1-phosphorylcholine, cis-3-hexadecylmercapto-cyclohexanol-1-phosphorylcholine, 2,3-bis-octylmercapto-propanol-1- Phosphorylcholine, 2-methoxy-3-[8-(4-octylphenyl)octylmercapto]propanol-1-phosphorylcholine, 2,2-dimethyl-3-hexadecanesulfinyl-propanol-1-phosphorylcholine, 2,2- Dimethyl-3-hexadecanesulfonyl-propanol-1-phosphorylcholine, 2-methoxymethylene-3-octadecylmercapto-propanol-1-phosphorylcholine, 2,2-diethoxy-3-hexadecylmercapto-propanol-1-phosphorylcholine, 4-octadecyl Mercapto-n-butanol-1-phosphorylcholine, 4-octadecanesulfinyl-n-butanol-1-phosphorylcholine, 4-octadecanesulfonyl-n-butanol-1-phosphorylcholine, 3-hexadecanesulfinyl-cyclopentanol-1 -phosphorylcholine, 3-hexadecanesulfonyl-cyclopentanol-1-phosphorylcholine, 4-octadecylmercapto-n-butanol-1-phosphorylcholine, 3-octadecanesulfinyl-cyclohexanol-1-phosphorylcholine, 3-octadecanesulfonyl-cyclohexanol -1-phosphorylcholine, 2-ethyl-3-octadecylmercapto-cyclohexanol-1-phosphorylcholine, 2,2-(6-methyl-cyclohex-3-enylidene)-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2 -octadecylmercapto-ethanol-1
-phosphorylcholine, 2-(2-propynyl)-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-(1-propynyl)-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-propadienyl-3-hexa Decylmercapto-propanol-1-phosphorylcholine, 2-(1-propenyl)-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-(1-methyl-2-propynyl)-3-hexadecylmercapto-propanol-1 -phosphorylcholine, trans-3-hexadecylmercapto-cyclohexanol-1-phosphorylcholine, 2-isopropyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-methyl-3-heptadecymercapto-propanol-1-phosphorylcholine, 2-Methyl-3-hexadecanesulfonyl-propanol-1-phosphorylcholine, 2-methyl-2-methoxy-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2,2-bis-methoxymethyl-hexadecylmercapto-propanol- 1-phosphorylcholine, 2-cyclopropyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-methylmercaptomethyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-methanesulfinylmethyl-3 -hexadecylmercapto-propanol-1-phosphorylcholine, 2-methanesulfonylmethyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-methoxyethoxymethyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-cyanomethyl -3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-carbamoylmethyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-N,N-dimethyl-carbamoylmethyl-
3-hexadecylmercapto-propanol-1
-phosphorylcholine, 2-ethoxycarbonylmethyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-methyl-3-(1-methoxycarbonyl-
heptadecylmercapto)-propanol-1-
Phosphorylcholine, 2-(2,2,2-trifluoroethoxy)-3
-hexadecylmercapto-propanol-1-
Phosphorylcholine, 3-heptadecylmercapto-thiopropanol-1-phosphorylcholine, 2-methyl-3-hexadecanesulfinyl-
Thiopropanol-1-phosphorylcholine, 2-methyl-3-hexadecanesulfonyl-thiopropanol-1-phosphorylcholine, 2-benzyl-3-octadecylmercapto-
Thiopropanol-1-phosphorylcholine, 2,2-dimethyl-3-hexadecylmercapto-thiopropanol-1-phosphorylcholine, 2-aminoethyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-acetylaminoethyl-3 -hexadecylmercapto-propanol-1-phosphorylcholine, 2-methylaminoethyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-hydroxymethyl-3-heptadecylmercapto-propanol-1-phosphorylcholine, 2-cyclopentyl- 3-heptadecylmercapto-propanol-1-phosphorylcholine, 2-cyclohexylmethyl-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-p-methylbenzyl-3-octadecylmercapto-propanol-1-phosphorylcholine, 2-( 2-methoxy-5-chlorobenzyl)-3
-octadecylmercapto-propanol-1-
Phosphorylcholine, 2-cyano-3-heptadecylmercapto-propanol-1-phosphorylcholine, 2-carbamoyl-3-heptadecylmercapto-propanol-1-phosphorylcholine, 2-hydroxy-3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-isopropoxy-3-heptadecylmercapto-propanol-1-phosphorylcholine, 2-(2-phenethyloxy)-3-heptadecylmercapto-propanol-1-phosphorylcholine, 2-(2-hydroxyethoxy)-3-octadecylmercapto -Propanol-1-phosphorylcholine, 2-(2-methylmercaptoethoxy)-3-octadecylmercapto-propanol-1-phosphorylcholine, 2-(2-methanesulfinylethoxy)-3-
Octadecylmercapto-propanol-1-phosphorylcholine, 2-(2-methanesulfonylethoxy)-3-octadecylmercapto-propanol-1-phosphorylcholine, 2-(2-aminoethoxy)-3-heptadecylmercapto-propanol-1-phosphorylcholine , 2-(2-acetylaminoethoxy)-3-heptadecylmercapto-propanol-1-phosphorylcholine, 2-(2-methylaminoethoxy)-3-heptadecylmercapto-propanol-1-phosphorylcholine, 2-ethoxycarbonylmethoxy -3-hexadecylmercapto-propanol-1-phosphorylcholine, 2-cyanomethoxy-3-hexadecylmercapto-propanol-1-phosphorylcholine. Furthermore, the above R ₂ is saturated or unsaturated C ₁ to _{C 26}
Specific examples of aliphatic hydrocarbon residues include 2-decyloxy-ethyl alcohol-1-phosphorylcholine, 2-dodecyloxy-ethyl alcohol-1-
Phosphorylcholine, 2-tetradecyloxy-ethyl alcohol-
1-phosphorylcholine, 2-hexadecyloxy-ethyl alcohol-
1-phosphorylcholine, 2-octadecyloxy-ethyl alcohol-
1-phosphorylcholine, 2-eicosyloxy-ethyl alcohol-1
-phosphorylcholine, 2-docosyloxy-ethyl alcohol-1-
Phosphorylcholine, Methyl-tetradecylethylene glycol-phosphorylcholine, Decyl-pentaethylene glycol-phosphorylcholine, Dodecyl-pentaethylene glycol-phosphorylcholine, Tetradecyl-pentaethylene glycol-phosphorylcholine, Hexadecyl-pentaethylene glycol-phosphorylcholine, Eicosyl-pentaethylene glycol-phosphorylcholine , docosyl-pentaethylene glycol-phosphorylcholine, hexadecyl-diethylene glycol-phosphorylcholine, hexadecyl-tetraethylene glycol-phosphorylcholine, hexadecyl-octaethylene glycol-phosphorylcholine, hexadecyl-dodecaethylene glycol-phosphorylcholine, hexadecyl-tetradecaethylene glycol-phosphorylcholine, hexadecyl- Examples include octadecaethylene glycol-phosphorylcholine. Further, specific examples of the case where R ₂ is a C ₆ to _{C 26} acyl group derived from a saturated or unsaturated aliphatic carboxylic acid include lauroyl-decaethylene glycol-phosphorylcholine, stearoyl-diethylene glycol-phosphorylcholine , stearoyl-tetraethylene glycol-phosphorylcholine, stearoyl-decaethylene glycol-phosphorylcholine, oleoyl-pentadecaethylene glycol-
An example is phosphorylcholine. In addition, 2-N having the above acyl group
A specific example of the -acyl-amino-ethyl group is lauroylamidoethyl-diethylene glycol-phosphorylcholine. Furthermore, the above R ₂ is saturated or unsaturated C ₆ ~
Specific examples of alkylphenyl groups having a _C26 alkyl group include octylphenyl-ethylene glycol-phosphorylcholine, octylphenyl-diethylene glycol-phosphorylcholine, octylphenyl-tetraethylene glycol-phosphorylcholine, octylphenyl-tetraethylene glycol-phosphorylcholine, octylphenyl-ethylene glycol-phosphorylcholine, octylphenyl-tetraethylene glycol-phosphorylcholine, enyl-octaethylene glycol-phosphorylcholine, octylphenyl-dodecaethylene glycol-phosphorylcholine, octylphenyl-octadecaethylene glycol-phosphorylcholine, nonylphenyl-ethylene glycol-phosphorylcholine, nonylphenyl-diethylene glycol-phosphorylcholine, nonylphenyl-octaethylene glycol-
Examples include phosphorylcholine, nonylphenyl-tetradecaethylene glycol-phosphorylcholine, and nonylphenyl-eicosaethylene glycol-phosphorylcholine. Examples of alcohol compounds with which the raw organic phosphate ester of formula () above is reacted in the presence of phospholipase DM in the method of the present invention are as follows (1)
-(3) alcoholic hydroxyl group-containing compounds can be used. (1) The following compounds can be mentioned as primary alcohol compounds. A saturated or unsaturated C ₁ to C ₂₇ aliphatic hydrocarbon residue which may have a substituent selected from the group consisting of halogen, amino group, carboxyl group, and hydroxyl group: - the above aliphatic hydrocarbon residue Among them, when there is no substituent, a C ₁ to _{C 26} aliphatic hydrocarbon residue is preferable, and examples thereof include methanol, ethanol,
1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol,
1-octanol, 1-nonal, 1-decanol, 1-undecanol, 1-dodecanol, 1
-tetradecanol, 1-hexadecanol, 1
-octadecanol, 1-docosanol, 1-eicosanol, 1-hexacosanol, 2-ethyl-1-hexanol, isoamyl alcohol,
Examples include geraniol, citronellol, falnesol, phytol, and allyl alcohol. In the above, when having a halogen as a substituent, a C ₂ to C ₆ aliphatic hydrocarbon residue is preferable,
Examples include 2-chloroethanol, 4-chloro-1-butanol, 5-chloro-1-pentanol, 6-chloro-1-hexanol, 3-bromo-1-propanol, and the like. In the above, when having an amino group as a substituent, a C ₂ to C ₂₀ aliphatic hydrocarbon residue is preferable,
Examples include ethanolamine, 3-amino-1-propanol, 4-amino-1-butanol, 5-amino-1-heptanol, 6-amino-1-hexanol, diethanolamine, triethanolamine, tris(hydroxymethyl )
Aminomethane, N-(3-aminopropyl)diethanolamine, N-(β-aminoethyl)ethanolamine, dihydroxyethylglycine, sphingosine, N-methyldiethanolamine,
N-butyldiethanolamine, N-methylethanolamine, N-methyl-propanolamine, N-ethyl-ethanolamine, N-ethylhexanolamine, N-propylpropanolamine, N-propylhexanolamine, N-
Butylethanolamine, N-butylhexanolamine, N-pentylethanolamine, N-
Hexylethanolamine, N,N-dibutylethanolamine, N,N-diethylethalamine, N,N-diethylpentanolamine, N,
N-dipropylethanolamine, N,N-dibutylhexanolamine, N,N-dihexylethanolamine, N,N-dimethylethanolamine, N,N,N-trimethylpropanolamine, N,N,N-trimethylhexanolamine , N,N,N-triethylbutanolamine,
N,N,N-tripropylethanolamine,
N,N,N-tripropylpentanolamine,
N,N,N-tripethylhexanolamine,
Examples include N,N,N-tripentylethanolamine. In the above, when having a carboxyl group as a substituent, a _C2 to _C16 aliphatic hydrocarbon residue is preferable, and examples thereof include serine, homoserine,
Examples include β-hydroxypropionic acid, γ-hydroxybutanoic acid, 16-hydroxyhexadecanoic acid, gluconic acid, and galactonic acid. In the above, when having a hydroxyl group as a substituent, a C ₂ to C ₁₆ aliphatic hydrocarbon residue is preferable,
Examples include ethylene glycol, 1,2-
Propanediol, 1,3-propanediol,
glycerol, 1,4-butanediol, 1,6
-Hexanediol, 1,8-octanediol, 1,10-decanediol, 1,12-dodecanediol, 1,16-hexadecanediol, 1,
2,6-hexanetriol, propylene glycol, sorbitol, mannitol, galactitol, xylitol, arabitol, ribitol, xylulose, ribulose, erythritol, erythrulose, threose, erythrose, dihydroxyacetone, glyceraldehyde, trimethylolpropane, diglycerol,
Triglycerol, pentaglycerol, hexaglycerol, pentaerythritol, dipentaerythritol, 1,1,1-tris(hydroxymethyl)propane, 3,3-bis(hydroxymethyl)heptane, 2-n-butyl-1,3- Examples include propanediol and 2-ethyl-1,3-hexanediol. A _C7 to _C20 aromatic hydrocarbon residue having a linear side chain bonded to a primary alcoholic OH, where the aromatic ring portion of the aromatic hydrocarbon residue is a halogen, an amino group, a carboxyl group, and a hydroxyl group. It may have a substituent selected from the group consisting of: - Among the above aromatic hydrocarbon residues, if it does not have a substituent, it is preferably a C ₇ to C ₁₂ aromatic hydrocarbon residue, Examples include benzyl alcohol, β-
Examples include phenethyl alcohol, 3-phenyl-1-propanol, cinnamic alcohol, and 2-naphthalene ethanol. In the above, when having a halogen as a substituent, a C ₇ to C ₁₉ aromatic hydrocarbon residue is preferable,
Examples include chlorobenzyl alcohol,
Examples include O,O-bis(2-hydroxyethyl)tetrabromobisphenol. In the above, when having an amino group as a substituent, a C ₇ to C ₁₂ aromatic hydrocarbon residue is preferable,
Examples include p-aminophenethyl alcohol, N-ethyl-N-(2-hydroxyethyl)-
m-Toluidine, hydroxyethylaniline, N
Examples include -(2-cyanoethyl)-N-(2-hydroxyethyl)aniline and N-phenyldiethanolamine. In the above, when a carboxyl group is included as a substituent, a _C8 to _C12 aromatic hydrocarbon residue is preferable, and an example thereof is 2-hydroxyethylsalicylic acid. A saturated or unsaturated compound that has a bond selected from an ether bond, an ester bond, or an amide bond in its molecule, and may have a substituent selected from the group consisting of halogen, amino group, carboxyl group, and hydroxyl group. C ₂ ~
C ₂₇ aliphatic hydrocarbon residue: - Among the above aliphatic hydrocarbon residues, when it has an ester bond, a C ₄ to _{C 27} aliphatic hydrocarbon residue is preferred, examples of which include monolaurin, mono Olein, 1,2-dilaurin, methacrylic acid-2
-Hydroxyethyl, serine ethyl ester, ethylene glycol monolaurate, diethylene glycol monolaurate, ethylene glycol monostearate, diethylene glycol monooleate, ethylene glycol monopalmitate, diethylene glycol monopalmitate, and the like. Among the above aliphatic hydrocarbon residues, when they have an ether bond, _C4 to _C26 aliphatic hydrocarbon residues are preferred, examples of which include diethylene glycol, triethylene glycol, pentaethylene glycol, decaethylene glycol, Ethylene glycol monolauryl ether, diethylene glycol monolauryl ether, ethylene glycol monocetyl ether, diethylene glycol monocetyl ether, ethylene glycol monostearyl ether, diethylene glycol monostearyl ether, ethylene glycol monobutyl ether, ethylene glycol mono(2-diethylaminoethyl) ether, ethylene Examples include glycol monohexyl ether, ethylene glycol monotrile ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, and diethylene glycol monohexyl ether. In the above, if it has an amide bond, C ₅ ~
_C26 aliphatic hydrocarbon residues are preferred, examples include pantothenyl alcohol, pantetheine, tri-L-serine, L-seryl-L-leucine, L-seryl-L-methionine, L-seryl-
L-arginine, L-seryl-L-glutamine, lauryl diethanolamide, stearyl diethanolamide, trioxyethylene lauryl amide, palmitic acid diethanolamide, lauryl sulfate triethanolamine, tetraoxyethylene lauryl ether sulfate triethanolamine,
N-caproylethanolamine, N-lauroylethanolamine, N-palmitoylethanolamine, N-stearoylethanolamine,
Examples include N-oleoylethanolamine. C ₇ that has a bond selected from an ether bond, an ester bond, and an amide bond in the molecule, and a linear side chain bonded to a primary alcoholic OH.
~ _C20 aromatic aliphatic hydrocarbon residue, where the aromatic ring portion of the aromatic hydrocarbon residue may have a substituent selected from the group consisting of halogen, amino group, carboxyl group and hydroxyl group; Good: - When the aromatic hydrocarbon residue has an ether bond,
_C8 to _C20 aromatic hydrocarbon residues are preferred, examples of which include 1,4-di(2-hydroxyethoxy)benzene, anis alcohol, ethylene glycol monophenyl ether, ethylene glycol monooctyl phenol ether, diethylene glycol mono Examples include octyl phenol ether, ethylene glycol monononyl phenol ether, diethylene glycol monononyl phenol ether, ethylene glycol monochlorophenyl ether, ethylene glycol monobenzyl ether, mehuenecine, and the like. If the above has an ester bond,
_C8 to _C12 aromatic hydrocarbon residues are preferred, examples of which include serine benzyl ester. In the above case, if it has an amide bond, C ₈
~ _C12 aromatic hydrocarbon residues are preferred, examples of which include L-seryl-L-tyrosine, L-seryl-L-phenylalanine, and the like. C6 _~ with side chain attached to primary alcoholic OH
_C20 alicyclic hydrocarbon residue: -The above alicyclic hydrocarbon is preferably a cyclohexyl residue, examples of which include retinol, 1,4-dihydroxymethylcyclohexane, 3-cyclohexyl-1-
Examples include propanol and adamantane methanol. Pregnane steroid compound residues: - Examples of the above pregnane steroid compounds include aldosterone, corticosterone, cortisone, dehydrocorticosterone, deoxycorticosterone, idrocortisone, prednisolone, prednisone, tetrahydrocortisol, tetrahydrocotylzone, and triamcinolone. etc. can be mentioned. Heterocycles having a linear side chain bonded to a primary alcoholic OH and described below: furan ring, tetrahydrofuran ring, dioxolane ring, thiophene ring, pyrrolidine ring, imidazole ring, imidazolidine ring, oxazole ring, thiazole ring, pyran ring, Oxane ring, thiane ring, pyridine ring, piperazine ring, piperidine ring, pyrimidine ring, triazine ring, morpholine ring, indole ring, purine ring, phthalimide ring, tetrahydrophthalimide ring, benzoxolane ring, pterin ring, hexamethyleneimine ring, A heterocyclic primary alcohol compound residue having a heterocycle selected from the group consisting of a homopiperidine ring and a homomorpholine ring, where the heterocyclic portion of the residue is a C ₁ -C ₃ alkyl group, a C ₁ -C ₃ alkylene group. basis,
It may have a substituent selected from the group consisting of a hydroxyl group, a carboxyl group, a carbonyl group, a halogen, an amino group, an oxo group, a benzyl group, a phenyl group, and a C ₁ to C ₃ alkylene sulfonic acid group: When there is no substituent in the ring residue,
Furan ring, tetrahydrofuran ring, thiophene ring, pyrrolidine ring, imidazole ring, oxane ring, pyridine ring, piperazine ring, piperidine ring,
Morpholine ring, indole ring, phthalimido ring, tetrahydrophthalimide ring, benzoxolane ring, pteric acid, hexamethyleneimine ring,
Homopiperidine rings and homomorpholine rings are preferred, examples include N-hydroxyethylpyridine, 2-(2-hydroxyethyl)pyridine,
-Hydroxymethylpyridine, 3-hydroxymethylpyridine, 4-hydroxymethylpyridine,
2-(3-hydroxypropyl)pyridine, 3-
(3-hydroxypropyl)pyridine, 4-(3-
hydroxypropyl)) pyridine, N-(2-hydroxyethyl)piperazine, 2-(4-pyridyl)
Allyl alcohol, N-(2-hydroxyethyl)
piperidine, 2-(2-hydroxyethyl))piperidine, 2-hydroxymethylpiperidine, 3-
Hydroxymethylpiperidine, N-(2-hydroxyethyl)pyrrolidine, N-(2-hydroxyethyl)hexamethyleneimine, 2-thiophenemethanol, 2-thiopheneethanol, N-
(2-hydroxyethyl)morpholine, N-(2-
Hydroxyethyl)phthalimide, N-hydroxymethylphthalimide, N-hydroxymethyltetrahydrophthalimide, 2-(3-indole)ethanol, 4-hydroxymethylimdazole, tetrahydropyran-2-methanol, tetrahydrofurfuryl alcohol, furfuryl Alcohol, piperonyl alcohol, 6-hydroxymethylpterin, N-(2-hydroxyethyl)
Homomorpholine, N-(2-hydroxyethyl)
Examples include homopiperidine. In the above, when having a C ₁ to C ₃ alkyl group as a substituent, a dioxolane ring, a pyridine ring,
Preferred are a pyrrolidine ring, a furan ring, a thiazole ring, an oxazole ring, an imidazole ring, a pyrimidine ring, an imidazolidine ring, and a purine ring, examples of which include 5-methylfurfuryl alcohol, N-
Methyl-2-hydroxyethylpyrrolidine, 2,
2-dimethyl-1,3-dioxolane-4-methanol, 5-ethyl-2-pyridineethanol,
6-methyl-2-pyridineethanol, 1-(hydroxymethyl)-5,5-dimethylhydantoin, 2,4-dimethyl-4-hydroxyoxazoline, 5-(2-hydroxyethyl)-4-methylthiazole, thiamine, Examples include 7-(2-hydroxyethyl) theophylline, 7-(3-hydroxypropyl) theophylline, and the like. 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 a hydroxyl group is present as a substituent in the above, a morpholine ring, a furan ring, a pyridine ring, a piperazine ring, a piperidine ring, a purine ring, a tetrahydrofuran ring, an oxane ring, and a pyrimimidine ring are preferable, and examples thereof include 2-hydroxymethyl 2 -(4-pyridyl)-1,3-propanediol, 2,6-pyridinemethanol, 3-(1-piperazinyl)-1,2-propanediol, N-
(2-hydroxyethyl)-4-(hydroxypropyl)piperidine, 3-morpholino-1,2-propanediol, 2,5-furandimethanol,
5-thioglucose, 3,4-furandimethanol, 5-hydroxymethyluracil, 5-hydroxymethyl-6-methyluracil, kojic acid, ascorbic acid, isoascorbic acid, curcono-
δ-lactone, galactono-γ-lactone, α-
Glucoheptonic acid-γ-lactone, pyridoxine, pyridoxal, pyridoxamine,
etc. can be mentioned. In the above, when the substituent has an oxo group, a pyrrolidine ring is preferable, and examples thereof include N-(2-hydroxyethyl)-2-pyrrolidone, N-(3-hydroxypropyl)-2-pyrrolidone, etc. can give. In the above, when having an amino group as a substituent, a pyrimidine ring is preferable, and examples thereof include 5-hydroxymethylcytosine and toxopyrimidine. When a phenyl group is used as a substituent in the above, a piperidine ring and a piperazine ring are preferable, examples of which include scopolamine, atropine,
Examples include hydroxyzine. In the above, when having a C ₁ to C ₃ alkylene sulfonic acid as a substituent, a piperazine ring is preferable, and examples thereof include N-(2-hydroxyethyl)piperazine N'-2-ethanesulfonic acid,
N-(2-hydroxyethyl)piperazine-N'-
Examples include 3-propanesulfonic acid and N-(2-hydroxyethyl)piperazine-N'-2-hydroxypropane-3-sulfonic acid. In the above, when it has a carbonyl group as a substituent, a furan ring is preferable, and examples thereof include 5-hydroxymethyl-2-furfural. (2) The following compounds can be mentioned as secondary alcohol compounds. Saturated or unsaturated _C1 - _C20 aliphatic hydrocarbon residue which may have a substituent selected from the group consisting of halogen, amino group, carboxyl group and phenyl group: - the above aliphatic hydrocarbon Among them, _C3 to _C14 aliphatic hydrocarbon residues are preferred when they have no substituent, examples of which include 2-propanol, 2-butanol, 2-pentanol, 3-pentanol, 4-
Methyl-2-pentanol, 2-hexanol,
3-hexanol, 1-hexen-3-ol,
2-heptanol, 3-heptanol, 2-octanol, 2-nonanol, 3-nonanol, 2
-decanol, 3-decanol, 2,3-butanediol, 2-methyl-2,4-pentanediol, 2-undecanol, 3-undecanol,
Examples include 2-tridecanol and 2-tetradecanol. In the above, when having a halogen as a substituent, a _C3 to _C6 aliphatic hydrocarbon residue is preferable, examples of which include 1-chloro-2-propanol and 1-bromo-2-butanol. It will be done. In the above, when having an amino group as a substituent, a C ₃ to _{C 6} aliphatic hydrocarbon residue is preferable,
Examples include 1-amino-2-propanol, diisopropanolamine, 1-amino-2-propanol,
- Examples include butanol. In the above, when having a phenyl group as a substituent, a C ₁ to C ₆ aliphatic hydrocarbon residue is preferable, and examples thereof include 1-phenylethanol,
Examples include 1-phenyl-2-propanol, p-chlorophenylmethylcarbinol, α-(1-aminoethyl)-p-hydroxybenzyl alcohol, and diphenylmethanol. In the above, when having a carboxyl group as a substituent, a _C3 to _C18 aliphatic hydrocarbon residue is preferable, and examples thereof include β-hydroxybutyric acid. Saturated or unsaturated C ₃ to C ₂₀ aliphatic hydrocarbon residues having a bond selected from ether bonds, ester bonds and amide bonds in the molecule: - Examples of the above aliphatic hydrocarbon residues include Examples include propylene glycol, ethyl lactate, diethyl malate, ricinoleate methyl ester, ricinoleate ethyl ester, and N-lauroylisopropanolamine. _C4 - _C8 alicyclic hydrocarbon residue carbonyl having a secondary alcoholic OH directly attached to the ring: - Examples of the above alicyclic hydrocarbon residues include cyclobutanol, cyclopentanol, cyclohexanol , cyclooctanol, 2-chlorocyclohexanol, 1,4-dihydroxycyclohexane, and the like. A tetrahydrofuran ring, a piperidine ring, a morpholine ring, a piperazine ring, a pyrrolidine ring, an oxane ring, a sorbitan ring, and a phthalimide ring, which have a secondary alcoholic OH directly bonded to a ring or a chain side chain bonded to a secondary alcoholic OH. a heterocyclic secondary alcohol compound residue having a heterocyclic ring selected from the group consisting of
C1 _{- C3} alkyl group, hydroxyl group, carboxyl group
It may have a substituent selected from the group consisting of a C ₁₂ to _{C 18} acyl group and a C ₁ to C ₃ alkylene sulfonic acid group. As the heterocycle having an alcoholic OH, a piperidine ring and a pyrrolidine ring are preferable, and examples thereof include:
Examples include 3-hydroxypiperidine, 4-hydroxypiperidine, 3-hydroxypyrrolidine, and the like. In the above, the heterocycle having a linear side chain bonded to the secondary alcoholic OH is preferably a phthalimide ring or a morpholine ring, examples of which include:
N-(2-hydroxypropyl)phthalimide,
Examples include N-(2-hydroxypropyl)morpholine. In the above, the substituent is preferably a piperidine ring when it has a C ₁ to C ₃ alkyl group,
An example thereof is N-methyl-4-hydroxypiperidine. When a hydroxyl group is present as a substituent in the above, a tetrahydrofuran ring or an oxane ring is preferable, and examples thereof include lyxose and D-glucuronolactone. In the above, when the substituent has a C ₁₂ to C ₁₈ acyl group, a sorbitan ring is preferable, and examples thereof include sorbitan monolaurate and sorbitan monooleate. In the above, when having a C ₁ to C ₃ alkylene sulfonic acid group as a substituent, a piperazine ring is preferable, and examples thereof include piperazine-N, piperazine-N,
Examples include N'-bis(2-hydroxypropane-3-sulfonic acid). (3) The following compounds can be mentioned as sugar compounds. Amino group, acetylamino group, methylnitrosouride group, _C1 - _C3 alkyl group, phenyl group,
Phenol group, hydroxymethylphenyl group, nitrophenyl group, mercapto group, methylmercapto group, phenylmercapto group, nitrophenylmercapto group, halogen, carboxyl group, pyridyl group, pyrimidyl group, triazyl group, purine group,
and a pentose residue, a hexose residue, a disaccharide residue thereof, a deoxypentose residue, a deoxyhexose residue, a heptose residue, which may have a substituent selected from the 8-azapurine group, provided that the above-mentioned substituents Among them, pyridyl group, pyrimidyl group, triazyl group, purine group and 8-azapurine group are mercapto group,
halogen, hydroxyl group, carboxyl group, oxo group,
Amino group, _C1 - _C3 alkyl group, methoxy group,
Ethoxy group, carboxymethyl group, acetyl group,
It may have a substituent selected from the group consisting of and anisoyl group: - In the above, examples of cases without a substituent include lipose, xylose, arabinose, etc. as a pentose residue; and as a hexose residue. , glucose, galactose, mannose, sorbose, talose, fructose, etc.; these disaccharide residues include hexose disaccharides, such as sutucarose, maltose, cellbiose, gentiobiose, lactose, isomaltose, melibiose, etc.; deoxypentose residues As deoxyhexose residues, 2-deoxyhexose residues such as 2-deoxyglucose, 2-deoxygalactose, etc.; as heptose residues, glucoheptose, sedoheptulose, glucoheptose, etc. Examples include heptulose and mannoheptulose. When an amino group is present as a substituent in the above, a hexose residue substituted at the 2- or 3-position is preferable, and examples thereof include glucosamine, galactosamine, mannosamine, methyl-3-amino-3-deoxy-β- Glucoside, methyl-3
-amino-3-deoxy-β-mannoside, and the like. When having an acetylamino group as a substituent in the above, a hexose residue substituted at the 2-position is preferable, and examples thereof include N-acetyl-glucosamine, N-acetyl-galactosamine, N-acetyl-galactosamine, and
-acetyl-mannosamine, etc. When a methylnitrosouride group is present as a substituent in the above, a hexose residue substituted at the 2-position is preferable, and an example thereof is streptozotocin. In the case where a C ₁ to C ₃ alkyl group is used as a substituent in the above, a pentose residue substituted at the 1st or 3rd position and a hexose residue substituted at the 1st or 3rd position are preferable, examples of which include: , α-methylriboside, β-methylriboside,
β-Methylarabinoside, 2-o-methylxylose, α-methylxyloside, α-methylglucoside, β-methylglucoside, α-methylmannoside, β-methylmannoside, α-methylgalactoside, β-methylgalactoside, 3- Examples include o-methylglucose. In the case where a phenyl group or a nitrophenyl group is present as a substituent in the above, a pentose residue substituted at the 1-position and a hexylase residue substituted at the 1-position are preferable, such as phenyl-
α-glucoside, phenyl-β-glucoside, phenyl α-galactoside, phenyl-β-galactoside, o-nitrophenyl-α-galactoside, o-nitrophenyl-β-galactoside, m
-nitrophenyl-α-lactoside, m-nitrophenyl-β-galactoside, p-nitrophenyl-α-galactoside, p-nitrophenyl-β
-galactoside, o-nitrophenyl-β-glucoside, p-nitrophenyl-β-glucoside,
p-nitrophenyl-α-glucoside, o-nitrophenyl-β-xyloside, p-nitrophenyl-β-xyloside, p-nitrophenyl-α-
Examples include mannoside, p-nitrophenyl-β-mannoside, and the like. In the case where the substituent has a phenol group, hydroxymethylphenyl group, mercapto group, methylmercapto group, phenylmercapto group or nitrophenylmercapto group, a hexylase residue substituted at the 1-position is preferable; Examples include phenyl-β-thiogalactoside, phenyl-α-thiogalactoside, goldthioglucose, o-nitrophenyl-1-thio-β-
Galactoside, p-nitrophenyl-1-thio-
β-galactoside, p-nitrophenyl-1-thio-β-glucoside, β-thioglucose, β
-Thiogalactose, salicin, arbutin, etc. In the case where a halogen is included as a substituent in the above, a hexose residue is preferable, and an example thereof is chloralose. Furthermore, in the above, a pyridyl group as a substituent,
Pyrimidyl group, triazyl group, purine group or 8-
When it has an asaprine group, it is preferably a pentose residue or deoxypentose residue in which the 1st position is substituted with such a group. Furthermore, the mercapto group,
halogen, hydroxyl group, carboxyl group, oxo group,
Amino group, _C1 - _C3 alkyl group, methoxy group,
Preferably, it further has a substituent selected from the group consisting of ethoxy, carboxymethyl, acetyl and anisoyl groups. For example,
Cytidine, uridine, adenosine, arabinocytidine, guanosine, cyclocytidine, adenine deoxyriboside, cytosine deoxyriboside,
Guanine deoxyriboside, thymine deoxyriboside, uracil deoxyliposide, inosine, orotidine, 6-mercaptopurine riboside, 4-
Thiouridine, 5-bromouridine, 6-fluorodeoxyuridine, 6-methylaminopurine-
9-riboside, 8-azaadenosine, 5-azacytidine, 3-deazauridine, ^N4 -acetylcytidine, 5-carboxymethyluridine, ^N6-
Methyl-2'-deoxyadenosine, 5-bromo-
2'-deoxycytidine, 8-bromoadenosine,
5-hydroxyuridine, 5-methyluridine,
N ⁴ -anisoyl-2'-deoxycytidine, N ⁶ -
Ethanoadenosine, 5-methoxyuridine, 3-
Examples include methyluridine, 2'-chloroadenosine, 1-methyladenosine, ^N6 , ^N6 -dimethyladenosine, 2'-o-methyladenosine, and 3'-o-methylcytidine. Examples include alcoholic hydroxyl group-containing compounds selected from (1), (2), and (3) as exemplified above, but there are no restrictions on the selection of alcohols as long as they fall within the range from (1) to (3) above. do not have. The alcoholic hydroxyl group-containing compound (hereinafter sometimes abbreviated as acceptor alcohol) as exemplified above can be used as either a natural product or a synthetic product, but it must be prepared in advance so that it does not contain any alcohol other than the target acceptor alcohol. It is preferable to use it after purification using appropriate known 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 purification means. can. According to the method of the present invention, the formula () as exemplified above
An organic phosphate ester and an acceptor alcohol as exemplified above are reacted in the presence of phospholipase DM. Phospholipase D has been discovered in animals, plants, and many other microorganisms. Phospholipase D of plant origin can be easily obtained from, for example, cabbage, carrots, peanuts, etc. Phospholipase D of microbial origin can be obtained, for example, from phospholipase D produced by the genus Streptomyces
39918), (Japanese Patent Application Laid-Open No. 58-152481), as well as phospholipase D produced by Micromonospora
44094), etc. are known. As the phospholipase D that can be used in the present invention, any phospholipase D of any origin can be used as long as it acts on the organic phosphate ester of the above formula () as a starting material and causes a transfer to the acceptor alcohol. As an example of the phospholipase D having excellent translocation power used in the present invention, a phospholipase D-producing bacterium belonging to the genus Nocardiopsis disclosed in JP-A No. 58-63388 is exemplified by No. 779 strain of the genus Nocardiopsis. Phospholipase D-producing bacteria belonging to the genus Actinomadura disclosed in Kenjoyori No. 512 [FERM BP-512] and JP-A-58-67183, such as the genus Actinomadura strain No. 362, FERM BP-512. No. 511 [FERM
Examples include phospholipase D produced such as [BP-511]. Phospholipase D, which has the effect of causing a transfer reaction to various receptor alcohols using the synthetic organic phosphate ester of formula () as a starting material, is specifically referred to as phospholipase DM and is described herein. According to the method of the present invention, by reacting a synthetic organic phosphate ester of formula () as described above with a receptor alcohol included in the above (1) to (3) in the presence of the above phospholipase DM, the following can be achieved. formula()

[C] However, in the formula, A and A' have the same meanings as above,
C represents a residue in (1) to (3) above, and organic phosphate derivatives and salts thereof can be produced. Phospholipase DM used in the production includes, for example, phospholipase D produced by the Actinomadeura sp. No. 362 strain,
Alternatively, phospholipase D produced by the Nocardiopsis sp. No. 779 strain mentioned above can be exemplified, but for the aliphatic primary alcohol of up to _C6 , which is the alcohol mentioned in (1) above, phospholipase D derived from cabbage can also be used. 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 immobilized on a suitable immobilization carrier, such as a polypropylene membrane, celite grains, glass beads, etc., or on a granular or film-like material made of various polymer resins or inorganic materials. The reaction can be carried out by bringing the synthetic organic phosphate ester of formula () into contact with the acceptor alcohol in the presence of phospholipase DM, preferably in the presence of a solvent. Examples of the solvent to be used include water, an aqueous solvent, and a mixed solvent of an aqueous solvent and an organic solvent. The acceptor alcohol itself can also serve as a solvent. Also available are solvents containing any other additives that do not inhibit the enzymatic catalytic action of phospholipase DM, such as solvents containing suitable additives that promote this action or serve to stabilize the enzyme. be able to. For example, in addition to proteins such as albumin and casein, it contains buffering agents such as acetic acid, citric acid, and phosphoric acid, neutral salts such as calcium chloride, and bile salts such as sodium taurocholate. This can be done in a diluted aqueous solvent. Further examples of organic solvents include the acceptor alcohol itself; aliphatic hydrocarbons such as n-heptane, n-hexane, isooctane, etc.; alicyclics such as cyclopentane, cyclohexane, cyclobutane, etc. Group hydrocarbons;
Aromatic hydrocarbons such as benzene, toluene, xylene etc.; Ketones 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.; Examples include halogenated hydrocarbons such as carbon chloride, chloroform and methylene chloride; amide solvents such as dimethylformamide; and sulfoxide solvents such as dimethylsulfoxide. When using an aqueous solvent in the form of a mixed solvent with an organic solvent, the mixing ratio of both can be selected appropriately, but for example, the ratio of aqueous solvent: 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, about 1:1 to about 1:1000 mol of acceptor alcohol to 1 mol of organic phosphoric acid ester of formula (). The reaction molar ratio can be exemplified. In addition, the amount of phospholipase DM to be used is, for example, about 10 to about
An example usage amount is 100,000 units, preferably about 100 to about 1,000 units. Further, the amount of the solvent to be used may be, for example, about twice to about 100 times (by volume) the amount of the synthetic organic phosphate ester of formula (). 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 about 1 H to about 72 H. If desired, e.g.
By tracking the progress of the reaction using techniques such as TLC (thin layer chromatography) and confirming the formation of the desired target product, the reaction time can be changed as appropriate. The manner in which the organic phosphate ester of formula () is brought into contact with the acceptor alcohol in the presence of phospholipase DM can be selected as appropriate, but it is usually carried out under stirring or shaking conditions. Furthermore, when reacting with a substrate or acceptor alcohol that is susceptible to oxidative decomposition, 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 formed organic phosphate ester derivative of formula () can be separated and utilized as it is or in the form of a salt by precipitation. Here, examples of the salts of the organic phosphate derivatives of formula () include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, oxalic acid, maleic acid, lactic acid, tartaric acid, fumaric acid, and methanesulfonic acid. acids, salts with organic acids such as benzenesulfonic acid and toluenesulfonic acid, arginine, aspartic acid,
Salts with amino acids such as glutamic acid, sodium,
Examples include salts with alkali metals such as potassium, salts with alkaline earth metals such as magnesium and calcium, and ammonium salts. Furthermore, the derivatives and their salts can be separated and purified using appropriate known methods such as silicic acid column chromatography, alumina column chromatography, ion exchange chromatography, high performance liquid chromatography, countercurrent distribution, gel filtration, and adsorption chromatography. can. According to the method of the present invention, as described above, an organic phosphate ester derivative of formula () can be produced by reacting an organic phosphate ester of formula () with a receptor alcohol in the presence of phospholipase DM. . The resulting organic phosphate ester derivative of formula () has an excellent surfactant effect and has a large effect on the permeability of fat membranes. From this meaning, the expression ()
The derivatives can be used as liposome formation substrates or liposome surface modification substrates, and can also be expected to have the effect of inducing or promoting the production of useful substances by adding them to animal or plant fat culture media. It can also be used as an emulsifier for skin physiology when incorporated into cosmetics such as creams and emulsions, and as an emulsifier for fatty drugs, insecticides,
It is useful in pharmaceutical emulsifier applications such as herbicides. Furthermore, many of the formula () derivatives obtained by the method of the present invention are expected to have various physiological activities such as blood pressure lowering, anticancer action, and cholesterol dissolving action. or,
By transferring a pharmacologically active compound having an alcohol hydroxyl group or into which an alcohol hydroxyl group has been introduced into an organic phosphate ester, the pharmacological side effects of the compound can be weakened, or the pharmacological effects can be increased and the dosage can be reduced. You can also expect that. Furthermore, it can be used as a carrier for pharmacologically active compounds, such as vitamins, antibiotics, bactericides, etc., in order to transfer the above-mentioned pharmacologically active compounds to organophosphate esters and concentrate the compounds precisely in the affected area. It can also be expected to play a useful role as a protective group for pharmacologically active compounds. Furthermore, it is useful as an intermediate for chemical synthesis including various pharmaceutical products, and for example, derivatives obtained by transferring a receptor alcohol having a highly reactive halogen or amino substituent group can be used. Furthermore, labeled organophosphate derivatives can be obtained by transferring tritium- or ¹⁴ C-labeled acceptor alcohols. Furthermore, fluorescent derivatives can also be produced by inducing a fluorescent acceptor alcohol. 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} %,
_MgSO4・_7H2O0.01 %, Tween 85
Approximately 15 liters of a medium containing 0.1% (PH 6.0) was placed in a 30 liter jar fermentor, and after sterilization at 120°C for 15 minutes, 1.5 liters of the seed culture solution 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 an aqueous medium (PH6.8) containing 0.2% K ₂ HPO ₃ and 0.01% MgSO ₄ into a 500 ml Sakaguchi flask, and after steam sterilization, add Nocardiopsis sp. NO779 strain [FERM BP-512] or Actinomadeura sp.
Spores of NO362 strain [FERM BP-511] were inoculated with a platinum loop, and incubated at a culture temperature of 30℃ and 120 rpm for 2 hours.
It was prepared by shaking culture for 1 day. After culturing, the bacterial solids are removed by centrifugation,
Centrifugation supernatant 13l (Nocardiopsis sp. FERM BP−
0.54μ/ml when using the 512 strain; 1.7μ/ml when using the Actinomadeula FERM BP-511 strain.
It was hot in ml. ) was obtained. After cooling the centrifuged supernatant to 5°C, acetone at -20°C was added, and a precipitate containing phospholipase DM corresponding to an acetone concentration fraction of 30 to 70% was collected by centrifugation. When this precipitate was used with Nocardiopsis FERM BP-512 strain, it was PH6.0, and Actinomadeula genus FERM BP-512 strain was used.
When using the FERM BP-511 strain, it was dissolved in Tris-maleic acid at pH 6.5, dialyzed against 0.02M of the same buffer, and passed through DEAE-cellulose equilibrated with the same buffer. Collected transit classifications. Next, a column was filled with palmitoyl gauze prepared by the method of Horiuchi et al. [J.Biochem. 81 , 1639 (1977)], thoroughly washed with water, and the above DEAE-cellulose permeate was injected to adsorb the activity. Add this to 0.05M Tris
After washing with hydrochloric acid buffer (PH7.2), 0.2% Triton
The same buffer containing -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 HW-55F was used as a gel permeation carrier.
It was injected into a packed column [manufactured by Toyo Soda Co., Ltd.], passed through the column using distilled water, 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), poly buffer exchanger PBE ^TN 94 manufactured by Pharmacia Fine Chemicals was used.
(20ml) was passed through a packed column to desorb the activity, and then eluted with a pH gradient using a polybuffer for elution (PH5.0) manufactured by the same company. Eluted phospholipase DM
The active fraction was collected and concentrated using an ultrafiltration membrane, passed through a Sephadex G-75 packed column, and the phospholipase DM active fraction was collected and connected for drying. Thus, Nocardiopsis phospholipase
In the case of DM, the specific activity is approximately 40%.
Phospholipase DM was recovered as 178.3μ/mg protein, with an activity recovery rate of about 43% in the case of Actinomadeula phospholipase DM, with a specific activity of 218.3μ/mg protein. Reference Example 2 Preparation of Substrate As described above, the method of Okazaki and Hara [oil chemistry,
vol 30, p553-557 (1981)], JP-A-57-98291, JP-A-58-10592, and the like. Substrate (1) Tetradecyl-phosphorylcholine

[Chemical] Dissolve tetradecyl bromide (manufactured by Tokyo Kasei Co., Ltd.) in 150 ml of acetone, add 19.2 g of silver salt of dibenzyl phosphate, and reflux with vigorous stirring in the dark for 4 hours. Remove the silver bromide precipitate and remove the solvent under reduced pressure. After evaporation under water, 23 g of dibenzyltetradecyl phosphate crystals were obtained by dissolving in petroleum ether at below -20°C. 18.9 g of dibenzyltetradecyl phosphate and 9.3 g of sodium iodide were dissolved in 1500 ml of acetone, refluxed for 2.5 hours, left overnight, and the resulting crystals were collected, washed with cold acetone and dried to obtain 15.2 g of sodium salt of benzyl tetradecyl phosphate. I got it. Dissolve 14.2 g of sodium salt of benzyltetradecyl phosphate in 550 ml of 20% acetone aqueous solution,
280ml of 50% acetone solution containing 6.7g of silver nitrate
was added, stirred vigorously in the dark, and left overnight at 0°C.
The resulting crystals were collected and washed with cold water and cold acetone to obtain 13.4 g of silver salt of benzyltetradecyl phosphate. Dissolve the silver salt of benzyltetradecyl phosphate in 300 ml of anhydrous tetrahydrofuran solution and add 2-
Bromoethyltrimethylammonium picrate
After refluxing 15 g of the product in the dark for 7.5 hours, the silver bromide produced was removed by centrifugation and dried under reduced pressure, followed by extraction with benzene and evaporation to obtain crude benzyltetradecylphosphorylcholine pifrate. Dissolve this in 370 ml of acetone, add 9 g of sodium iodide, and reflux for 1 hour with stirring. The resulting precipitate is collected by filtration, washed several times with cold acetone, dried, and 11 g of O-sodium tetradecylphosphorylcholine iodide is obtained. Obtained. Dissolve O-sodium-tetradecylphosphorylcholine iodide in 90% ethanol aqueous solution.
Passing through a mixed column of Amberlite IRC50 and IR45 equilibrated with a 90% ethanol aqueous solution was deionized to obtain 7.3 g of crude tetradecylphosphorylcholine, which was then passed through a silicic acid column to remove chloroform-
Elute with methanol and recrystallize with chloroform-acetone to obtain purified tetradecyl-phosphorylcholine.
6.6g was obtained. Substrate (2) Dodecyl-phosphorylcholine

Dodecyl-phosphorylcholine was obtained in the same manner as in Substrate (1) using dodecyl bromide (manufactured by Tokyo Kasei Co., Ltd.) as a starting material. Substrate (3) Hexadecyl-phosphorylcholine

[Chemical formula] Hexadecyl-phosphorylcholine was obtained in the same manner as the substrate (1) using hexadecyl bromide (manufactured by Tokyo Kasei Co., Ltd.) as a starting material. Substrate (4) Octadecyl-phosphorylcholine

[Chemical formula] Octadecylphosphorylcholine was obtained in the same manner as for substrate (1) using octadecyl bromide (manufactured by Tokyo Kasei Co., Ltd.) as a starting material. Substrate (5) 2-dodecyloxy-ethyl alcohol-1-phosphorylcholine

[Chemical] 1 g of ethylene glycol monododecyl ether (manufactured by Tokyo Kasei Co., Ltd.), 20 ml of anhydrous toluene
To this was added a solution of 1.5 g of triethylamine and 1.5 g of 2-bromoethyl phosphate ester dichloride dissolved in 20 ml of anhydrous toluene.
The mixture was stirred at 0°C for 5 hours and further stirred at room temperature overnight. This mixture was then added with 0.1N potassium chloride solution at 0°C.
ml and stirred vigorously for 1 hour, further stirred at room temperature for 2 hours, and the solvent layer was dried over sodium sulfate and concentrated. This was dissolved in 60 ml of anhydrous methanol/60 ml of anhydrous chloroform, and further dissolved in anhydrous trimethylamine until saturated, and refluxed for 24 hours. This solution was concentrated using a rotary evaporator, and the residue was dissolved in 100 ml of anhydrous methanol, followed by adding 1.5 g of silver acetate and stirring at room temperature for 4 hours. The solution was filtered, concentrated, eluted with methylene chloride/methanol/water 65/25/4 using a silicic acid column, and recrystallized from chloroform-acetone to obtain 0.2 g of 2-dodecyloxy-ethyl alcohol-1-phosphorylcholine. Obtained. Substrate (6) 2-hexadecyloxy-ethyl alcohol-1-phosphorylcholine

embedded image 2-Octadecyloxy-ethanol was obtained from the sodium salt of ethylene glycol and octadecyl bromide, and 2-hexadecyloxy-ethyl alcohol-1-phosphorylcholine was obtained in the same manner as the substrate (5) using this as a starting material. Substrate (7) 4-hexadecyloxy-butyl alcohol-1-phosphorylcholine

[Chemical formula] 4-Hexadecyloxy-butyl alcohol was obtained from the sodium salt of 1,4-butanediol and hexadecyl bromide (manufactured by Tokyo Kasei Co., Ltd.), and 4-hexadecyloxy-butyl alcohol was obtained in the same manner as the substrate (5) using this as a starting material. -Hexadecyloxy-butyl alcohol-1-phosphorylcholine was obtained. Substrate (8) 3-(12-pentyloxy-dodecyloxy)-propanol-1-phosphorylcholine

[Chemical formula] The sodium salt of 1,12-dodecanediol was reacted with pentyl bromide, and then purified through a silicic acid column to obtain 12-pentyloxydotecanol. This was brominated with phosphorus tribromide in the presence of pyridine, separated and purified using a silicic acid column, and 12-pentyloxy-dodecyl bromide was obtained. 1 of this bromide,
It was reacted with sodium salt of 3-propanediol and separated and purified using a silicic acid column to obtain 3-(12-pentyloxy-dodecyloxy)-propanol.
Using this as a starting material, 3-
(12-pentyloxy-dodecyloxy)-propanol-1-phosphorylcholine was obtained. Substrate (9) 2-methoxymethyl-3-octadecyloxy-propanol-1-phosphorylcholine

[Chemical formula] The sodium salt of 2-methoxymethyl-1,3-propanediol was reacted with octadecyl bromide (manufactured by Tokyo Kasei Co., Ltd.) to produce 2-methoxymethyl-3.
-Octadecyloxy-1-propanol was obtained. Using this as a starting material, prepare 2 in the same manner as the substrate (5).
-Methoxymethyl-3-octadecyloxy-propanol-1-phosphorylcholine was obtained. Substrate (10) 3-hexadecyloxy-carbonyloxy-propanol-1-phosphorylcholine

[Chemical] Chloroformic acid-hexadecyl ester (obtained from cetyl alcohol and phosgene in the presence of dimethylaniline) is reacted with sodium salt of 1,3-propanediol, separated and purified using a silicic acid column, and hexadecyl-( 3-hydroxypropyl)
- Carbonic ester was obtained. 3-hexadecyloxy-carbonyloxy-
Propanol-1-phosphorylcholine was obtained. Substrate (11) 2-hydroxy-3-(3-tetradecyloxy-propoxy)-propanol-1-
Phosphorylcholine

[Chemical formula] Dissolve 1 g of 2-benzyloxy-3-(3-tetradecyloxy-propoxy)-1-propanol-phosphorylcholine in 200 ml of ethanol,
Hydrogenation was carried out at room temperature using palladium/charcoal as a catalyst. The solution was filtered, concentrated and dried over phosphorus pentoxide to yield 0.3 g of 2-hydroxy-3-(3-tetradecyloxy-propoxy)-propanol-1-phosphorylcholine. Starting material 2-benzyloxy-3-(3
-tetradecyloxy-propoxy)-propanol-1-phosphorylcholine was obtained as follows. Add 45 ml of anhydrous tetrahydrofuran to a solution of phosphorus oxytrichloride in 9 ml of anhydrous tetrahydrofuran.
2-benzyloxy-3-(3-tetradecyloxy-propoxy)-1-propanol 4.0 g per ml
A solution of was added dropwise at 0°C. This mixture was stirred at 0°C for 1 hour, further stirred at room temperature for 1 hour, cooled, 1N caustic soda solution was added thereto, stirred for 2 hours, alcoholic hydrochloric acid 45 ml, and chloroform 90 ml.
was added, and the solvent layer was separated and concentrated. The residue is dissolved in 90 ml of dioxane solution, heated under reflux for 4 hours and concentrated, and 45 ml of chloroform and 23 ml of 2N hydrochloric acid are added to the residue and separated. The solvent layer was separated, concentrated and dried, and phosphoric acid
3.8 g of mono-[2-benzyloxy-3-(3-tetradecyloxy-propoxy-1-propyl]-ester) was obtained. This was dissolved in 120 ml of anhydrous pyridine, and 0.9 g of 2-bromoethanol and 2,4, 6
-Triisopropylbenzole sulfochloride 51
To this was added 45 ml of 0.1N potassium chloride aqueous solution, stirred for 2 hours, acidified with concentrated hydrochloric acid, added methylene chloride and mixed. The solvent layer was dried and concentrated, and phosphoric acid (2- Bromoethyl)-[2
-benzyloxy-3-(3-tetradecyloxy-propoxy)-1-propyl]-ester 4.4
I got g. Add this to 60ml of chloroform/methanol.
The mixture was dissolved in 60 ml of water, anhydrous trimethylamine was added thereto, heated under reflux for 20 hours, and concentrated. The residue was dispersed in 85 ml of methanol, 1.7 g of silver acetate was added, and the mixture was stirred for 2 hours and overconcentrated. This was purified using a silicic acid column to obtain 1.6 g of the desired starting material. Substrate (12) 3-octadecylmercapto-propanol-1-phosphorylcholine

[Chemical] Dissolve 0.5 g of sodium in methanol, add 2.0 g of 3-mercaptopropionic acid methyl ester dropwise, and add 7.4 g of octadecyl bromide to 25 ml of methanol.
A solution containing g was added thereto, and the mixture was refluxed for 3 hours, cooled, and purified using a silicic acid column to obtain 5.9 g of 3-octadecylmercapto-propionic acid methyl ester. This was dissolved in ether and reduced with lithium aluminum hydride until no ester was detected by thin layer chromatography to obtain 4.0 g of 3-octadecylmercapto-propanol. Using the 3-octadecylmercapto-propanol obtained here as a starting material, 3-octadecyl-propanol-phosphorylcholine was obtained in the same manner as the substrate (5). Substrate (13) 2-methoxy-3-octadecylmercapto-propanol-1-phosphorylcholine

[Chemical] Dissolve 10 g of 2-O-methylglycerin in 100 ml of anhydrous pyridine and add benzol sulfochloride to this.
11.6 ml was added dropwise at 0° C. with stirring. The reaction solution was left at a low temperature of 0° to 4°C, 230 g of ice was added, and the aqueous layer was extracted with methylene chloride. 2N hydrochloric acid for the solvent layer,
The residue was washed with sodium bicarbonate solution and water, dried, concentrated, and purified using a silicic acid column to obtain 10.8 g of 2-O-methyl-glycerol-mono-benzenesulfonate. Solid potassium hydroxide in 90ml of absolute ethanol
A solution of 13.3 g of octadecyl mercaptan dissolved in 90 ml of absolute ethanol was added dropwise at room temperature, and after stirring for 1 hour, 10.4 g of 2-O-methyl-glycerin-mono-benzenesulfonate was dissolved in 45 ml of absolute methanol. The solution was added and stirred at room temperature for 1.5 hours. 1.5 liters of cold water at 0°C was added, acidified with 2N hydrochloric acid, extracted with ether, dried and concentrated, and purified with a silicic acid column to obtain 9.2 g of 2-methoxy-3-octadecylmercapto-propanol. Using this compound as a starting material, 2-methoxy-3-octadecylmercapto-propanol-1-phosphorylcholine was obtained in the same manner as for substrate (5). Substrate (14) 2-methyl-octadecylmercapto-
Propanol-1-phosphorylcholine

[Chemical formula] 0.1 g of sodium was dissolved in 60 ml of methanol, 11.4 g of octadecyl mercaptan was added thereto, and 4 g of methyl methacrylate was added in an ice bath. After stirring at room temperature for 1 hour, the mixture was filtered and dried to obtain α-methyl-β-octadecylmercaptopropionic acid methyl ester. This was reduced with lithium aluminum hydride in anhydrous ether to give 2-methyl-
3-octadecylmercapto-propanol was obtained. Using this compound as a starting material, 2-methyl-3-octadecylmercapto-
Propanol-1-phosphorylcholine was obtained. Substrate (15) 3-(2-pentadecyloxy-ethylmercapto)-propanol-1-phosphorylcholine

[Chemical formula] -Sodium salt of ethylene glycol and 1-bromopentadecane are reacted to obtain 2-pentadecyloxy-ethanol, which is reacted with phosphorus tribromide to form 2-bromoethoxypentadecane, and this compound is converted into 3-mercaptopropion. 3-(2-pentadecyloxy-ethylmercapto)-propanol-1-phosphorylcholine was obtained in the same manner as substrate (12) by reacting with acid methyl ester. Substrate (16) 3-[2-(N-dodecyl-carboxamide)-ethylmercapto]-2-methoxy-propanol-1-phosphorylcholine

[Chemical formula] 2-Methoxy-1,3-propanediol-mono-benzenesulfonate and the sodium salt of 2-(N-dodecyl-carboxamide)-ethyl mercaptan are reacted to form 3-[2-(N-dodecyl-
3-[2-(N-dodecyl-carboxamide)-ethylmercapto]-2-methoxy-propanol was obtained in the same manner as the substrate (5) using this as a starting material. -1-phosphorylcholine was obtained. Substrate (17) 3-heptadecyloxy-thiopropanol-1-phosphorylcholine

embedded image 3-Heptadecyloxy-thiopropanol-1-phosphorylcholine was obtained in the same manner as the substrate (5) using 3-heptadecyloxy-thiopropanol as a starting material. Substrate (18) 3-octadecanesulfinyl-propanol-1-phosphorylcholine

[Chemical formula] 1 g of 3-octadecylmercapto-propanol, the starting material for substrate (12), was mixed with 0.3 g of perhydrol.
ml of glacial acetic acid, and after stirring at room temperature for 1 hour, the mixture was made weakly alkaline with sodium hydroxide. 2
After standing for a period of time, the mixture was washed with filtered water, dried, and purified through a silicic acid column to obtain 0.5 g of 3-octadecylsulfinyl-propanol. Using this as a starting material, 3-octadecylsulfinyl-propanol-1-phosphorylcholine was obtained in the same manner as substrate (5). Substrate (19) 3-octadecanesulfonyl-1-phosphorylcholine

embedded image 1.0 g of 3-octadecylmercapto-propanol obtained in Substrate (12) was dissolved in 20 ml of glacial acetic acid, 0.26 ml of perhydrol was added, and the mixture was stirred at room temperature for 1 hour. Hydrogen peroxide was repeatedly added and the resulting crystals were purified with a silicic acid column to obtain 0.2 g of 3-octadecanesulfinyl-propanol. Using this as a starting material, 3-octadecanesulfonyl-propanol-1-phosphorylcholine was obtained in the same manner as substrate (5). Substrate = Tetradecylthioalcohol-1-phosphorylcholine

[Chemical formula] Tetradecylthioalcohol-1-phosphorylcholine was obtained in the same manner as for substrate (5) using myristyl mercaptan (manufactured by Tokyo Kasei Co., Ltd.) as a starting material. Substrate (21) Dodecyl-pentaethylene glycol-phosphorylcholine

Dodecyl-pentaethylene glycol-phosphorylcholine was obtained using pentaethylene glycol mono-dodecyl ether (manufactured by Tokyo Kasei Co., Ltd.) in the same manner as the substrate (5). Substrate (22) Octylphenyl-decaethylene glycol-phosphorylcholine

embedded image Octylphenyl-decaethylene glycol-phosphorylcholine was obtained using decaethylene glycol-mono-octylphenyl ether (manufactured by Tokyo Kasei Co., Ltd.) in the same manner as the substrate (5). Substrate (23) Nonylphenyl-pentaethylene glycol-phosphorylcholine

[Chemical formula] Nonylphenyl-pentaethylene glycol-phosphorylcholine was obtained using pentaethylene glycol-mono-nonylphenyl ether (manufactured by Tokyo Kasei Co., Ltd.) in the same manner as the substrate (5). Substrate (24) Stearoyl-tetraethylene glycol-phosphorylcholine

[Chemical formula] Stearoyl-tetraethylene glycol-phosphorylcholine was obtained using tetraethylene glycol mono-stearate (manufactured by Tokyo Kasei Co., Ltd.) in the same manner as the substrate (1). Substrate (25) 2-lauroylamidoethyl-diethylene glycol-phosphorylcholine

[Chemical] Triethylene glycol-lauroylamide [CH ₃ - (CH ₂ ) ₁₀ CONH (CH ₂・CH ₂ O) ₃ H, manufactured by Sanyo Chemical Industries, Ltd.] was prepared in the same manner as the substrate (1) to prepare 2-lauroylamide ethyl. -diethylene glycol-phosphorylcholine was obtained. Substrate (26) phytyl-phosphorylcholine

[Chemical formula] Phytylphosphorylcholine was obtained using phytol as the starting material in the same manner as the substrate (5). Example 1 The following chess card () prepared by the method of Reference Example 2
Organic phosphoric acid choline ester of () and substrate () Tetradecyl-phosphorylcholine substrate () 2-dodecyl-oxyethyl alcohol-1-phosphorylcholine Many types of alcohols shown in Table 1 below are listed below.
According to the method for confirming the formation of metastatic products by TLC, the reaction was performed in the presence of phospholipase DM to confirm the formation of metastatic products. The Rf values are shown in Table 1 below. Method for confirming the formation of metastases by TLC The following composition: 1% organic phosphoric acid choline ester aqueous solution 0.1ml 0.4M acetate buffer (5.7) PH 0.1ml 1M calcium chloride aqueous solution 0.01ml 20% alcohol solution 0.2ml phospholipase DM aqueous solution in the reaction solution 0.01ml (10
~100U/ml) was added and left to stand at 30°C for 2 days. When preparing the above 20% alcohol solution, water or an organic solvent such as diethyl ether or acetone was added as necessary, and the pH of the acidic alcohol was adjusted to 6.0 with 1N caustic soda or the amines with 1N hydrochloric acid. After the above-mentioned standing, 0.5 ml of 1/10N hydrochloric acid was added, and further 3 ml of a chloroform-methanol mixture (2:1 v/v) was added and the mixture was vigorously stirred to extract the product. The lower chloroform layer was separated and dried under reduced pressure at 30°C, then chloroform-methanol solution (2:1v/
v) 0.1 ml was added and dissolved to prepare a TLC sample. 10 μl of this was spotted on a thin layer of silica gel (Merck Silica Gel 60 TLC plate 20 x 2 cm) and diisobutyl ketone-acetic acid-water (40:25:5 v/v) was added.
Chloroform-methanol-water-(60:30:5v/
v) or chloroform-methanol-water-ammonia (50:20:1:2v/v) as a developing solvent. The following reagents were used to detect spots. If a spot other than the undecomposed substrate and its hydrolyzate was detected, this was recognized as a transfer product. Table 1 shows the Rf values of the transfer products when diisobutylketone-acetic acid-water (40:25:5v/v) was used as the developing solvent. Color development of detection reagent phosphoric acid: Zinzade reagent (Beiss UJChromatog. 13
104.1964) Color development of primary amines: Ninhydrin reagent (0.25% ninhydrin solution in acetone) Color development of secondary amines: Hypochlorous acid-benzidine reagent (Bischel MC)
Biochim, Biophys, Acta 70 598.1963) Coloration of sugars: Naphresorcinol-phosphate reagent (Hay G.
W. et al. J. Chromtog. 11 479.1963) Periodic acid-benzidine reagent (Wolform. ML et al. Anal. Chem. 28 1037.
(1956) Coloration of purines and pyrimidines: Fluoretcein-ammonia reagent (Wieland T. et al. Angew.Chem. 63 511.1951) Coloration of amino sugars: Acetylacetone-Ehrlich reagent (Ellson-Mongan reaction) (Partridge SM
Biochem.J. 42 238.1948)

【table】

[Table] Pyridine

【table】

【table】

【table】

[Table] All samples were colored with Zinzade's reagent to detect organic phosphate esters, and those with special functional groups were detected with the various reagents mentioned above. Example 2 Tetradecyl-phosphorylcholine 1% aqueous solution prepared by the method of Reference Example 2
1ml 0.4M acetate buffer (PH5.7) 1ml 1M calcium chloride aqueous solution 0.1ml Put 2ml of 20% sorbitol aqueous solution into a test tube with a stopper, and put 1ml of phospholipase DM aqueous solution (20u/
ml) was added, mixed well, and left to stand at 30°C for 2 days.
After adding 1 ml of 0.5N hydrochloric acid to the reaction solution to stop the reaction, a chloroform-methanol mixture (2:
1v/v) was added and mixed vigorously to extract the organic phosphate ester. 2000×g2 of this mixture
The mixture was centrifuged for a minute and the lower chloroform layer was separated. The separated extract was washed with 10 ml of water. The chloroform layer was separated again from this mixture by centrifugation, dried under reduced pressure, and then 0.5 ml of n-hexane-2
-Dissolved in a mixture of propanol and water (60:80:14v/v). 20 ul of this sample was spotted on a thin layer of silica gel (Merck Silica Gel 60TLC (plate)), diisobutylketone-acetic acid-water (40:25:5v/v).
When developed in the solvent system of
The values matched. This sample was purified by high performance liquid chromatography. The column was a radial pack cartridge made of silica 8 mm x 10 cm (manufactured by Waters), and the solvent was n-hexane-2-propanol-water (60:
80:14v/v), a flow rate of 2ml/min, and a 441 type ultraviolet absorption detector (manufactured by Waters) for peak detection.
Absorption at 214 nm and an R401 differential refractometer (manufactured by Waters) were used. The sample was divided into 5 injections of 0.1 ml each. Using this solvent, three components, tetradecyl phosphoric acid, tetradecyl-phosphorylcholine, and tetradecyl-phosphoryl sorbitol, were separated. The obtained teradecyl-phosphoryl sorbitol was purified once again by the same operation to obtain 1.5 mg of purified tetradecyl-phosphoryl sorbitol. 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 2. (Run No. 7) The same procedure as above was carried out using other alcohols shown in Table 2. The results are shown in Table 2.
(Run No. 1 to 6 and 8 to 51) However, when adding the alcohol to the reaction solution, use water, diethyl ether, or acetone solution as appropriate depending on the solubility of the alcohol, and use 1N caustic soda or amines as the acidic alcohol. was added after adjusting the pH to 6.0 with 1N hydrochloric acid.

【table】

[Table] Example 3 2-Dodecyloxy-ethyl alcohol-1- prepared by the method of Reference Example 2
A reaction was carried out in the same manner as in Example 2 using 1 ml of a 1% phosphorylcholine aqueous solution. However, 20% of 6-amino-1-hexanol as alcohol
2 ml of solution was used. The pH of the 20% solution of 6-amino-1-hexanol was adjusted to 6.0 with 1N hydrochloric acid. This reaction solution was treated in the same manner as in Example 2 to obtain 1.5 mg of 2-dodecyloxy-ethyl alcohol-1-phosphoryl-6-amino-1-hexanol.
The IR spectrum of this compound is shown in Table 3.
(Run No. 9) The same procedure as above was carried out using other alcohols shown in Table 3. The result is the third
Shown in the table. (Run No. 1-8 and 10-51)

【table】

[Table] Example 4 A reaction was carried out in the same manner as in Example 2 using 1 ml of a 1% aqueous solution of 4-hexadecyloxy-butyl alcohol-1-phosphorylcholine prepared by the method in Reference Example 2. However, 2 ml of a 20% solution of 1-octanol, fructose or pyridoxine was used as the alcohol. This reaction was treated in the same manner as in Example 2 to obtain a rearrangement product. The IR spectrum of this compound is shown in Table 4.
(Run No. 8) The same procedure as above was carried out using other organic phosphoric acid choline esters shown in Table 4.
The results are shown in Table 4. (Run No.1~7,
9-23)

【table】

【table】

[Table] Tetradecyl prepared by the method of Reference Example 2
Add 10ml of 5% aqueous solution of phosphorylcholine, 101ml of 0.4M acetate buffer, and 1ml of M calcium chloride aqueous solution to an Erlenmeyer flask with a stopper, add 5g of ethanol, and then add 10ml of phospholipase DM aqueous solution (20u/ml).
was added and allowed to stand at 30°C for 2 days. After allowing the above to stand still, add 1N hydrochloric acid to adjust the pH to 2.0, stop the reaction, and add 50ml of chloroform-methanol mixture (2:1v/v).
was added and stirred vigorously to extract the product. Separate the lower chloroform layer, dry it under reduced pressure at 30°C, and add 2 ml of n-hexane-2propanol.
It was dissolved in a mixture of water (60:80:14v/v). Thereafter, tetradecyl-phosphorylethanol was purified in the same manner as in Example 2 to obtain 150 mg of purified product. Tetradecyl-phosphorylethanolamine was obtained by a similar operation.
200mg of tetradecyl-phosphorylglucerol, 150mg of tetradecyl-phosphorylmethanol, and 150mg of tetradecyl-phosphorylmethanol were obtained. Put 1 ml of a 1% aqueous solution of tetradecyl-phosphorylethanolaminetetradecyl-phosphorylglyceroltetradecyl-phosphorylmethanoltetradecyl-phosphorylethanol prepared by the above method into separate test tubes with stoppers, and use 1,6-hexanediol as alcohol. Add 500mg
1 ml of 0.4M acetate buffer, 1M calcium chloride aqueous solution
Add 0.1ml and then add 1 ml of phospholipase DM aqueous solution.
ml (20u/ml) and left at 30°C for 2 days. This reaction solution was treated in the same manner as in Example 2 to obtain a rearrangement product. This IR spectrum is shown in Table 5. (Run
No.1~4)

[Table] Example 6 Tetradecyl prepared by the method of Reference Example 2
A reaction was carried out in the same manner as in Example 2 using 1 ml of a 1% phosphorylcholine solution. However, 100 mg (30 u) of cabbage phospholipase D (manufactured by Boehringer Mannheim Yamanouchi) was added as an enzyme.
Alcohols include methanol, ethanol, propanol, n-butanol, n-heptanol,
A 20% solution of n-hexanol or glycerol was used. These reaction solutions were treated in the same manner as in Example 2 to obtain transfer products. The IR spectra of these compounds are shown in Table 6. (Run No.1~7)

【table】

【table】

Claims (1)

[Claims] 1 The following formula () [Chemical formula] In the formula, A is R ₁ -X or R ₂ -O-(CH ₂ CH ₂ O) _o
, where X represents an oxygen atom or a sulfur atom; R ₁ represents a saturated or unsaturated C ₆ to C ₁₂₀ aliphatic hydrocarbon residue, or a group R ₃ −Y−R ₄ − In the group R ₃ -Y-R ₄ -, Y is O, S, SO,
SO ₂ , O-CO-O, O-CO-NH or O-
CS-NH, _R3 is halogen, _C6 to _C16 aryl group, _C1 to _C18 alkoxy group, _C1 to _C18 alkylmercapto group, _C1 to _C18 alkanesulfinyl group, _C1 - _C18 alkanesulfonyl group, _C2
~ _C10 alkylcarbonylamide group, C2 _- C6 alkylcarbamoyl group, C2 _{- C6} alkylthiocarbonylamide group, _{C1 -C6 alkanesulfonamide} group, _C2 - _C5 alkoxycarbonyl group and a saturated or unsaturated C ₁ to _{C 24} aliphatic hydrocarbon residue optionally substituted with at least one substituent selected from the group consisting of a C ₃ to C ₅ ureido group; The above C ₁ - C ₁₈ alkoxy group may be further substituted with a C ₁ - C ₄ alkoxy group, and the above C ₁ - _{C 24} aliphatic hydrocarbon residue may be partially substituted in the residue. may be composed of a 3- to 6-membered cycloalkane ring, and R ₄ is a halogen, hydroxyl group, nitrile group, C ₃ -
_C6 cycloalkyl group, _C6 to _C8 aryl group,
_C1 - _C4 alkoxy group, _C1 - _C18 alkylmercapto group, _C1 - _C4 alkanesulfinyl group,
It has a _C1 to _C4 alkanesulfonyl group, a _C2 to _C5 alkoxycarbonyl group, an amino group which may have a _C2 to _C4 acyl group, and a _C1 to _C5 alkyl group. saturated or unsaturated C2 _{- C8} aliphatic carbonization optionally substituted with at least one substituent selected from the group consisting of an optional carbamoyl group and a _C1 - _C4 alkanesulfonamide group Indicates a hydrogen residue, where the above _C1 - _C4 alkoxy group is a _C1 - _C4 alkoxy group, a _C1 - _C4 alkylmercapto group, a _C1 - _C4 alkanesulfinyl group,
C1- _C4 alkanesulfonyl group, halogen, hydroxyl group, nitrile group, _C6 - _C8 aryl group, C1 _{-C4 alkanesulfonamide} group, _carbamoyl group, _C2
It may be further substituted with an amino group which may have ~ _C5 alkoxycarbonyl group and _C2 - _C4 acyl group, and the above _C2 - _C8 aliphatic hydrocarbon residue is A portion thereof may be composed of a 3- to 6-membered cycloalkane ring, or the aliphatic hydrocarbon residue may be an alicyclic hydrocarbon residue; R ₂ is saturated or unsaturated. _C1 to _C20 aliphatic hydrocarbon residue, _C6 to _C26 acyl group derived from a saturated or unsaturated aliphatic carboxylic acid, 2-N-acyl-amino-ethyl having the acyl group and an alkylphenyl group having a saturated or unsaturated C ₆ to C ₂₆ alkyl group; n is an integer of 1 to 20; provided that R ₂ is the above C ₆ ~ When indicating a _C26 acyl group,
n represents an integer from 2 to 20, A' represents oxide anion or hydroxy, and B represents -CH ₂ .CH ₂₊ N (CH ₃ ) ₃ , -CH ₂ .
An organic phosphoric acid ester represented by, which represents a group selected from the group consisting of _CH2NH2 , _-CH2・CHOH・_{CH2OH , -CH3, and -CH2CH3} , and the following (1) to ( 3)
An alcoholic hydroxyl group-containing compound selected from
Namely, (1) a saturated or unsaturated _C1 - _C27 aliphatic hydrocarbon residue which may have a substituent selected from the group consisting of halogen, amino group, carboxyl group and hydroxyl group; primary alcoholic OH a _C7 to _C20 aromatic hydrocarbon residue having a chain-like side chain bonded to the aromatic hydrocarbon residue, where the aromatic ring portion of the aromatic hydrocarbon residue is composed of a halogen, an amino group, a carboxyl group, and a hydroxyl group. may have substituents selected from the group; _C6 to _C20 alicyclic hydrocarbon residues with side chains bonded to primary alcoholic OH; ether bonds, ester bonds and amides within the molecule A saturated or unsaturated _C2 - _C27 aliphatic carbon having a bond selected from a bond and optionally having a substituent selected from the group consisting of a halogen, an amino group, a carboxyl group, and a hydroxyl group. Hydrogen residue; a _C7 to _C20 aromatic hydrocarbon residue that has a bond selected from an ether bond, an ester bond, or an amide bond in the molecule, and a linear side chain bonded to a primary alcoholic OH. basis,
The aromatic ring portion of the aromatic hydrocarbon residue may have a substituent selected from the group consisting of halogen, amino group, carboxyl group, and hydroxyl group; pregnane steroid compound residue; primary alcoholic It has a linear side chain bonded to OH, and has a furan ring, a tetrahydrofuran ring, a dioxolane ring, a thiophene ring, an imidazole ring, an imidazolidine ring, a pyrrolidine ring, an oxazole ring, a thiazole ring, a pyran ring, an oxane ring, and a thian ring. , pyridine ring, piperazine ring, piperidine ring, pyrimidine ring, triazine ring, morpholine ring, indole ring, purine ring,
A heterocyclic primary alcohol compound residue having a heterocycle selected from the group consisting of a phthalimide ring, a tetrahydrophthalimide ring, a benzoxolane ring, a hexamethyleneimine ring, a pterin ring, a homopiperidine ring, and a homomorpholine ring, where the residue The heterocyclic portion of the group is a _C1 - _C3 alkyl group,
A substituent selected from the group consisting of a C ₁ to C ₃ alkylene group, a hydroxyl group, a carboxyl group, a carbonyl group, an oxo group, a halogen, an amino group, a benzyl group, a phenyl group, and a C ₁ to C ₃ alkylene sulfonic acid group. (2) A primary alcohol compound having a residue selected from the group consisting of; (2) May have a substituent selected from the group consisting of halogen, amino group, carboxyl group, and phenyl group; Saturated or unsaturated C ₁ to _{C 20}
aliphatic hydrocarbon residue; a saturated or unsaturated _C3 - _C20 aliphatic hydrocarbon residue having a bond selected from ether bonds, ester bonds, and amide bonds in the molecule; a _C4 to _C8 alicyclic hydrocarbon residue having a secondary alcoholic OH; having a secondary alcoholic OH directly bonded to the ring or a linear side chain bonded to the secondary alcoholic OH, and having a piperidine ring, pyrrolidine ring,
A heterocyclic secondary alcohol compound residue having a heterocyclic ring selected from the group consisting of a morpholine ring, a tetrahydrofuran ring, a phthalimide ring, a piperazine ring, a sorbitan ring, and an oxane ring, where the heterocyclic moiety of the residue is C ₁ - It may have a substituent selected from the group consisting of a C ₃ alkyl group, a carboxyl group, a C ₁₂ to _{C 18} acyl group, a C ₁ to _{C 3} alkylene sulfonic acid group, and a hydroxyl group; Secondary alcohol compound having selected residues, (3) amino group, acetylamino group, methylnitrosouride group, C1 _{- C3} alkyl group, phenyl group, phenol group, hydroxymethylphenyl group, nitrophenyl group , a mercapto group, a methylmercapto group, a phenylmercapto group, a nitrophenylmercapto group, a halogen, a carboxyl group, a pyridyl group, a pyrimidyl group, a triazyl group, a purine group, and an 8-azapurine group. optional pentose residue, hexose residue, these disaccharide residues, deoxypentose residue, deoxyhexose residue, heptose residue, provided that among the above substituents, pyridyl group, pyrimidyl group, triazyl group, purine group and -Azapurine group includes mercapto group, halogen, hydroxyl group, carboxyl group, oxo group, amino group, _C1 - _C3 alkyl group, methoxy group,
may have a substituent selected from the group consisting of an ethoxy group, a carboxymethyl group, an acetyl group, and anisoyl group; a sugar compound having a sugar residue selected from; an alcoholic hydroxyl group-containing compound selected from and is reacted in the presence of phospholipase D that catalyzes the transfer reaction between an organic phosphate ester and a primary or secondary alcoholic hydroxyl group-containing compound. and A′ have the same meanings as above,
C represents a residue in the alcoholic hydroxyl group-containing compounds (1) to (3). A method for producing an organic phosphoric acid ester derivative and its salts represented by