JPS59187786A - Production of phospholipid secondary alcohol derivative using enzymic method - Google Patents

Abstract

PURPOSE:To produce a phospholipid secondary alcohol derivative, by reacting phosphoric acid structural part of a phospholipid with an ester bond of an alcohol structural part of a secondary alcohol in the presence of phospholipase DM. CONSTITUTION:A phospholipid expressed by formula I is reacted with a secondary alcohol having a 3-10C straight chain or branched alkyl group (R) which may be substituted by a substituent group selected from a halogen, amino, acetyl hydroxyl group, <=5C monomer or di-alkylamino or phenyl or 4-8C alicyclic hydrocarbon group (R) which may be substituted by the above-mentioned substituent group in the presence of phospholipase DM to produce the aimed phospholipid secondary alcohol derivative expressed by formula II. In the formulas I and II, A is formula III or IV, (R1 and R2 represent -O-COR11 or -O-R12; R11 and R12 represent 7-21C saturated or unsaturated aliphatic hydrocarbon group; B represents -(CH2)2N(CH3)3, -(CH2)2NH2 or -CH2(CH(NH2)COOH, etc.).

Description

[Detailed description of the invention]

The present invention relates to a method for producing phospholipid secondary alcohol derivatives by an enzymatic method, and provides a wide range of methods for producing phospholipid secondary alcohol derivatives, including phospholipid secondary alcohol derivatives that have conventionally been thought to be impossible to produce by enzymatic methods. This invention relates to a method for producing a phospholipid secondary alcohol conductor using the squid method. More specifically, the cabbage-derived phosphoryl 9-ase D used in the conventional enzymatic method (optimum temperature 40°C, below, optimum p.
The optimum temperature is 60-70, which is higher than H5, 4-56).
°C, optimum 7) It relates to a method for producing a phospholipid secondary alcohol derivative in which a phospholipid and a secondary alcohol are reacted in the presence of phospholipase DM near H7. In the present invention, a phospholipid secondary alcohol derivative refers to an ester bond between a phosphoric acid structure moiety and an alcohol structure moiety of a phospholipid as a starting material.
It refers to a new phospholipid that is different from the starting material and is derived by being hydrolyzed by the action of and simultaneously transferred to the secondary alcohol used in the second reaction. In particular, the present invention provides the following method:
(1) ■ R However, in the formula, A represents the following (1) or (11), where both R5 and R2 are 10-COR1+, or both are 104□, or the formula In (1), R1 and R2 represent the numbers -11-19], and in the above, R11 and R□ may be the same or different,
Each represents a C2 to C21 saturated or unsaturated aliphatic hydrocarbon group, and Bij, -(CHt)y# (CHsrs, -(C
Hz) t”2, -CM, CH(NH,)COOH
, -CH,CH2NH(CH,), -CH,CH,N
(CH,), -CH2CHOHCH20H or -
(Cut) rrLH (where m is a number from 1 to 5), a phospholipid represented by 5-, a halokene, amino, acetinope hydroxyl group, C31, mono- or di-alkylamino and C optionally substituted with a substituent selected from the group consisting of phenyl
A secondary alcohol having a C4-C6 alicyclic hydrocarbon group R which may be substituted with a 3-C7° straight or branched alkyl group or a -H surface substituent, l) The following formula (1) is characterized in that the reaction is carried out in the presence of H. Concerning the manufacturing method of conductors. It has been known that phospholipase D hydrolyzes choline base-phosphorus and esters of phospholipids, such as 6-phosphatidylcholine, and catalyzes the reaction to generate free bases and phosphatidic acid. Kates Cau J, Biochem, phys.
iol 32.571 (1954)'l]. Furthermore, when phospholipids such as lecithin and ethyl alcohol are reacted in the presence of phospholipase D, the ester bond between the phosphoric acid structure of the phospholipid and the YA group or alcohol structure of the phospholipid is simultaneously hydrolyzed. C Daus, which has been reported to produce phosphatidylethanol by transphosphatidyl group action.
son;Biochem. T., 102.205 (1965)]: [Y
Ana:,! , Biol, C'hetn, , ,
242.477 (1967)). Since the above-mentioned phosphatidyl group transfer effect of phosphorinse D was known, research in this field has been advanced, and the proposal of British patent AI, 581,810 (corresponding West German publication A 271754.7) has been made known. According to this proposal, L7f' and the phospholipid indicated by the -# hole, and a straight chain up to C6 which may be substituted with a hydroxyl group, a heloquinone, an amino or other substituted Jq+ group. Alternatively, the enzymatic action of the cabbage-derived phospholipase D with a primary alcohol having a +d-branched argyl group may be
A primary alcohol transfer reaction using No. 1 is disclosed. The reaction then contains more than 5 elemental atoms (7
In the case of alcohols containing more than 5 carbon atoms, the reaction is described as occurring only with primary alcohols containing more than 5 carbon atoms, and in the case of those alcohols containing more than 5 carbon atoms, the most important product of the reaction is the corresponding phosphatidic acid. Furthermore, the proposal 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. Furthermore, R, M, and Dawson used lecithin as the lipid (phospholipid) and 2-propanol as the secondary alcohol, and as a result of experiments with the known phosphorine (-ze D) derived from cabbage, they found that the phosphatidyl group of the lecithin substrate was reported that no transfer to secondary alcohols occurred [EioChem, J, vol, 102.
205 (1967)). As mentioned above, conventionally, the transfer reaction between phospholipids and alcohols by phospholipase D occurs only with primary alcohols, especially primary alcohols with a relatively small number of carbon atoms, and does not occur with secondary alcohols. Technical common sense was that Others other than the present inventor discovered the existence of a microorganism that has the ability to produce phospholipase D, which differs from the conventionally known phospholipase D in terms of its optimum humidity, optimum pH, etc., and has already filed a patent application in 1983.
-161076 and Japanese Patent Application No. 56-163475. As a result of further research, it was found that the enzyme produced by phospholipase-producing organisms is capable of forming phospholipid secondary alcohol derivatives, although it was previously thought that phospholipid secondary alcohol derivatives could only be formed. We have discovered the remarkable fact that it exhibits an enzymatic catalytic action that enables the transfer reaction between alcohols. According to research by the inventors, an enzyme newly named phospholipase DM is used in the present invention to catalyze the formation of a phospholipid secondary alcohol derivative between a phospholipid and a C4 secondary alcohol, such as 2-butanol. By reacting the phospholipid represented by the formula (1) with the secondary alcohol in the presence of this phospholipase DM, we can produce a phospholipid secondary alcohol component, which was previously said to be impossible to produce. It has been discovered that new derivatives can be prepared including: In this way, chemical synthesis methods that are both unreliable and disadvantageous are used. -1 under mild and easy conditions and means without
0- It has been found that new phospholipid secondary alcohol derivatives can be produced in good yields by an enzymatic method without the risk of side reactions. Therefore, an object of the present invention is to provide a new enzymatic method for producing phospholipid secondary alcohol derivatives. The above object and many more objects and advantages of the present invention will become clearer from the following journal entries. The raw material phospholipid used in the straw method of the present invention has the following formula ( 1
)% formula% (1) However, in the formula, A is the following (+) or (11) C'E, -C
H, -R. , where both R1 and Rt are 10-CO, R,
, or both are 10-R,2, or in the formula (1), RI and R3 represent the numbers -11-19], and in 2, R71 and R82 may be the same or different and each represents a 67 to C21 saturated or unsaturated aliphatic hydrocarbon group, and 10B is -(CH2), N (CD, ), -(CD, ),
NE,,-CD2CH(NE2)COOH,-CD,C
B2NH (CE, ), -CE20H2N (C'H3)
,, -CH2CBOHCH,,OH or -(CH2
)H [Here, Kabuto represents the number 11'L from 1 to 5]. The raw material phospholipid of the formula (1) is a known compound and is available on the market, and can be extracted from natural products or synthesized by methods known per se. for example,
Lecithin, cephalin, phosphatulserin, and phosphatidyl obtained by extraction from animal and plant tissues by known means.
N-methylethanolamine, phosphatidylglycerol, phosphatidyl-N, N-dimethylethanolamine, phosphatic acid alkyl ester, etc. can be used alone or as a mixture or as a mixture, and β-type phospholipids and alkyl ether-type phospholipids can be used. Also, part or all of its structure can be chemically synthesized and utilized by methods known per se. In the method of the present invention, the secondary alcohol to be reacted with the raw material phospholipid of formula (I) in the presence of phosphoIJ zR-zeDM has a linear or branched alkyl group R of 03 to C1o.
or a secondary alcohol having an alicyclic hydrocarbon group R of 04 to C8, which further comprises mono- or di-alkylamino of Cs or less and phenyl, amine, acetyl, hydroxyl group, 13 - A substituent selected from the group consisting of -r-P may be substituted. As a specific example of such a secondary alcohol, a secondary alcohol λ such as a manure can be exemplified. That is, the aliphatic alcohols include 2-propatol, 2-butanol, 2-penta/-/1=3-pentanol, 4-methyl-2-pentanol, 2-hegizanol, 3-hexanol, l- hexen-3-ol, 2-heptatool, 3-heptatool, 2-octatool, 2-nonanol, 3-nonanol, 2-decanol, 3-decanol, 2.3-butanediol, 2-methyl-2, 4-bentanediol, ■-chloro 2-pronogunono 1-promo 2-butanol, ■-7
Examples include mi/-2-pronognol, diimprono, nolamine, 1-amino-2-butanol, 3-human-14-logycin-2-butanone, yanityl, β-hydroxybutyric acid, dipropylene dalicol, etc. I can do it again.
Aromatic alcohols include l-phenylethanol, 1
-Phenyl-2-7'lonognol, p-chlorophenylmethylcarbinol α-(1-aminoethyl)-p
-Hydroxybenzyl alcohol, diphenylmethanol, etc. can be exemplified, and further examples of alicyclic alcohols include cyclobutano-/L=, cyclooctatool, cyclohexanol, cyclooctatool, 2-chlorocyclohexanol, and 1,4-dihydroxycyclohexane. An example is Nato. The secondary alcohols exemplified above can be either natural products or synthetic products, but they must be purified in advance using appropriate known means so that they do not contain any alcohol other than the desired secondary alcohol. Examples of such purification means include I':j, : , A W
＋、? ) Column chromatography using crystals, alumina, silica glue, activated carbon, etc., thin layer chromatography, and appropriate combinations of cloth-making methods such as Q. According to the method of the present invention, the formula (1) as illustrated above! A lipid and a secondary alcohol as exemplified above are mixed in the presence of a phospholipase (1). The phospholipase I) M used in this case is the conventionally known phospholipase D extracted from cabbage with an optimum temperature of 40°C or less, an optimum pH of 5.4 to 5.6, and an optimum temperature of 60 to 70°C. , yR-ase DAf, which can be distinguished from known phospholipase D by having an optimum pH of around 7.
An example of this is the phosphorylose IBM produced by Namae fungi. The phospholipase DM is a C′4 secondary alcohol 2-
Phospholipid secondary alcohol W' between ethanol and the formula (1) phospholipid such as lecithin! It can also be distinguished from known phospholipase D in that it catalyzes the formation of a 31-body. For example, the phosphorinse DAF bacterium produced in cattle is disclosed in Japanese Patent Application No. 56-161076 filed by the same applicant.
Nocardiopsis (Nocardiopsis) disclosed in No.
For example, phospholipase 9M-producing bacteria belonging to the genus Norcadeiopsis NO7'19 [1#RM-
Pbian 6'l 33), fi related to the application of the same applicant
- Phospholipase 9M-producing bacteria belonging to the genus Actinomadura (ylCt 1norn, adura J) disclosed in Application No. 56-163475, such as the genus Actinomadura NO362 strain [FERM-PA 6132]
etc. can be mentioned. Table 1 below shows the differences in enzymatic properties between the phospholipase DM used in the method of the present invention and the known phospholipase D, as well as the differences in optimal temperature and pH, as well as some other differences. -17- A phospholipid secondary alcohol derivative that could not be obtained using the known phospholipase D can be formed by the method of the present invention. It is presumed that this is due to the difference in enzymatic properties as described above between the known ospholipase D, which is involved in the mediating reaction, and the phospholipase J) M used in this induction method. Of course, the method of the present invention is not subject to any restrictions based on the assumption of such effects. -191 The phospholipase DM-producing bacterium Nocardiopsis genus N that produces phospholipase DM that can be used in the method of the present invention
0779 strain IFERM-P No. 6133] and Acti7 Madura sp. NO362 strain [FERM-P No.
The mycological properties of 61321, the method for measuring the titer of phospholipase DM produced by them, and the physicochemical properties are described below. /Caldeiopsis strain N0779 [FERM-PNo.
, 6133] form (a) grows well on glucose asparagine agar, glycerin asparagine agar, yeast malt agar, etc., and grows moderately on starch inorganic salt medium, forming colonies of aerial mycelia. . The color of the bacterial flora on which the spores have settled varies slightly depending on the type of culture medium and the time of observation, but it is generally white or grayish-white to light gray. On sucrose nitrate agar, nutrient agar, and oatmeal agar media, aerial mycelium does not attach or only attaches poorly to −20=. When this strain grown on an agar medium was observed under a microscope,
Aerial hyphae are 0.5 to 1.8 microns long and extend in straight lines with loosely waved or curved branches, and the entire aerial hyphae are spores in the range of 1 to 1. It is formed by a chain consisting of. The size of the spore is (), 5 ~ 0.8 x 0.7 It elongates with branches at .5 to 0, sa, and although it does not necessarily divide on agar medium, it is finely divided in most cases by liquid culture. However, zoospores, sporangia, sclerotia, etc. are not formed. (1)) Properties on various media The experimental methods described below are mainly based on E.B. Shearing (Tnl, , J, SysL, Ba
cleriol, vol. 16, 313-340.1966
It was carried out according to the method of 2010). 21- Color tone is [Color Standard-I (Japan Color Research Institute, 19
1964), and the hue symbol was written in parentheses along with the hue name in the order of hue name, saturation number, and brightness number. Cultivation was performed at 25°C, and Table 2 shows the observation results on each medium during the 2nd to 3rd week when growth was most vigorous. However, the second
In the table, the color of the surface of the substrate hyphae described in the growth item shows the observation result during the first week of culture before spore settlement. No description is given of a medium with fixed llI. 22- (c) Physiological properties 1 Raw W? g degree 25°C ~ 3 (growth rate around 1'C 2
Grows best at 0 to 30"C. 2 Liquefaction of gelatin: No liquefaction (glucose peptone gelatin medium - 1 - cultured at 125°C for 13 weeks). 3 Hydrolysis and bilysis of starch (also turch agar medium 1 - 1. Coagulation and peptonization of 7L of defatted beef: Neither coagulation nor peptonization was performed (3o'c, : (~4 weeks culture). 5 Live I# of melanin-like pigment, : Peptone Yase Generate with HX agar and tyrosine agar (at 25°C for 40 minutes)
. (d) Carbon in assimilability (:40'C1H'l-16F
ltR nutrition) L-Arapiace - Sucrose -
D-xylose - I7citol -D-glucose + 1, -rhamnose -D-7lactose
- Raffi7-su - (e) Chemical analysis of cells The diaminopimelic acid of this strain is of the meso type and does not contain 24-hyroxydiaminopimelic acid. The sugar composition of the cell wall is arapias, xylose, madulose,
It does not contain rhamnose, etc., but contains galaxe, mannose, etc. Moreover, this strain does not have nocal V mifulic acid. Based on the results of the second analysis, Bergey's Manu
al of mouth] e Dpterminative B
ecLeriology 8th edition, pp. 657-65
8 pages (1974), Lechevalier (Tinter,
J, System, Bacteriol,
20, pp. 435-443, 1970), Mayer (Inl:, J. SysL, Bacteriol, vol. 26, 487)
According to the classification method of p.
ype) type III, sugar composition type (cell u+all
sugar pat, tern) becomes type C. The cell wall type of this bacterium is III, the type is I#, and the type is C.According to Chevalier's taxonomy, this bacterium is of the Gusonvillei type, genus Madura, genus Thermoactiamyces, and genus Actinosa. It belongs to either the genus Biifigus or the genus Geodermatophyllus. ~25- However, in its morphology, this fungus either cuts off all of the aerial hyphae, or cuts the spores into long needles, or divides the aerial hyphae into small pieces.
Endospores, zoospores, and spores are not found.
Genus Ac1. inomadura dass-
onvillei 1. It is appropriate for classification to identify it as ype). In addition, in recent years, the genus Dasongbireikipu/Madeula has been integrated into the new genus Nocardiopsis proposed by Mayer, and is generally treated under the name of the genus Nocardiopsis. Therefore, this bacterium is Nocardiopsis genus NO? 79 (G
enus Nocardiopsis sp N 07
79). This bacterium has been deposited in the National Institute of Microbiology, Agency of Industrial Science and Technology, and its accession number is [-Feikoken Bacterial Submission No. 6133]. Bacteria used to produce the phospholipase DM used in the present invention include not only the genus Nocardiopsis N0V79 and a mutant strain obtained by mutating this strain, but also the genus Nocardiopsis (genus name Aku26-Chi7 Madeura). Any bacterium that belongs to the genus Gusonbirei and produces phospholipase DM can be used. Actinomadura genus NO362 strain [FERM-PNo
, 6132] Form (a) Form Grows well on starch inorganic salt agar, tyrosine agar, yeast/malt agar, oatmeal agar, etc., and grows moderately on glycerin-asparagine agar, forming a colony of aerial mycelia. Epiphytes. The color of the bacterial flora that has attached spores varies slightly depending on the type of culture medium and the time of observation, but it is generally grayish-white to gray with a slight purplish color. On sucrose nitrate agar, nutrient agar, and glucose asparagine agar, aerial mycelia do not attach or attach only poorly. When this strain grown on agar medium-1 was observed under a microscope, the aerial hyphae were branched at a diameter of 0.8 to 1.2μ, with some loops or spirals, and some curves. - It mainly extends in two long straight lines, and the tip is often loosely coiled into a loop. The spores settle in a chain of several tens to 1 +-, + t-, ) K month-, forming the entire hyphae. The size of the spores is ('1.8~i, 2 x 1.2~1.7μ
It is short cylindrical or oval, and is somewhat irregular in size and shape. The hyphae are 6 to 1.0 microns in width, have irregular branches, are not bent, and are elongated with zoospores and spores! ), sclerotia, etc. are not formed. In addition, septa and hyphal division are not usually observed, but hyphal division may be observed in liquid culture. (1)) Properties of various media The experimental methods described below are mainly based on E.B., Shearing (1nt, , , J, Sys↑, Bac
teriol. 16, 313-340 (1966). The color tone is F color standard-1 (Japan Color Research Institute to Foundation Law, 1
964), and the hue symbol was written in parentheses along with the hue name in the order of hue name, saturation number, and brightness number. Cultivation was carried out at 25° C., and Table 3 shows the observation results on the pot medium for 2 to 3 weeks, when the growth was most vigorous. However, the third
In the table, the color of the surface of the Jukuyo mycelia described in the growth items shows the observation results during the culture before spore settlement, and does not include media where spores are attached quickly and it is difficult to determine the color of the Jukuyo mycelium surface. do not have. Ha 8 Pe 1 He
Be; to 1 to capital 1
1- The chest
←(c) Physiological properties 1 Grows at a temperature of 10℃ to 37℃, 20 to 3℃.
Grows best at 0°C. 2 Liquefaction of gelatin: Not liquefied (cultivated on glucose peptone gelatin medium at 25°C for 13 weeks). 3. Hydrolyze and bi-decompose starch (culture on starch agar medium at 25°C for 3 weeks). 4 Coagulation and peptonization of skimmed milk: No coagulation, Pep)> conversion. (Culture at 30°C for 13-4 weeks). 5 Production of melanin-like pigment: peptone yeast iron agar,
It is produced by tyrosine agarization (25°C for 12-4 days). (d) Assimilation of carbon source (30°C 11O-16 days iC>
L-arapias + sucrose -D-xylose + i7citol ±D-glufus
+ L-lam 7-su - D-7 lactose - Lahui 7-su - (e) Chemical analysis of cells The diaminopimelic acid of this strain is men type 1), 31
- The sugar composition of the cell wall does not contain arabinose, xylose, rhamnose, etc., but contains madulose, lactose, and mammose. Based on the above analysis results, 7ey'sManua
l ofLl+e Deter+oinajive B
ecl, eriology fl's B edition, pages 657~
658 pages (197/1), Regieva're-worker (T n
ter, J, S y≦〕m, Baclerio
l, vol. 20, p. 135-/I p. 43, 197 (1
According to classification methods such as
le-rn) [becomes type 3. 1? Ouemotobacterium has a cell wall type of lll and a sugar composition type of lll.
3, it belongs to the genus Microbispora, Streptosborangium, Spirillosvora, Planomonospora, Dermadophilus, and Actinomadura. In its morphology, this fungus forms a spore chain consisting of a large number of spores, and since no sclerotia, sporangia, or zoospores were found, it is classified as a species of the genus Acti7 Madura (G.
Taxonomically, it is most appropriate to identify it as e-hada IS Acti+do32-omadura). Therefore, this bacterium was identified as Actinomadera NO362 (Ac
tinomadura sp N O362). This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its accession number is FERM P-6132 J. The bacteria to be used for this purpose are not only the Actinomadura genus NO362 mentioned above and mutant strains obtained by mutating this strain, but also any bacteria that belong to the Actinomadura genus and produce phospholipase D. Go out of your way. To produce phospholipase DM used in the method of the present invention using phospholipase DM-producing bacteria as exemplified in ``-'', phospholipase DM-producing bacteria as exemplified in ``-'' are cultured in a medium, and phospholipase DM is extracted from the culture. All you have to do is collect it. As for the culture form, both liquid culture and solid culture can be used, but from an industrial perspective, deep aeration agitation culture is advantageous. In addition, the culture sources used include - carbon sources, nitrogen sources, inorganic salts, and other nutrients used in microbial culture in the shell, as well as phospholipases belonging to the genus Chaldiopsis and Acti7 Madura 1) Any nutrient source available to the M-producing microorganism can be used. Examples of carbon sources for the medium include butyl sugar, fructose, sucrose, lactose, starch, glycerin, dextrin, molasses, sorbitol, etc., as well as fatty acids, fats and oils, crude lecithin, alcohol, and organic acids. can be used alone or in combination. As a nitrogen source, both inorganic nitrogen sources and organic nitrogen sources can be used. Examples of inorganic nitrogen sources include ammonium nitrate, ammonium sulfate, urea, sodium nitrate, and phosphoric acid.
Examples include ammonium, diammonium phosphate, ammonium chloride, etc., and organic nitrogen sources include flour, bran, defatted lees of soybean, rice, corn, cottonseed, rapeseed, wheat, corn steep liquor, Examples include pea')n, yeast extract, meat extract, casein, and amino acids. Examples of inorganic salts and macronutrients include salts such as phosphoric acid, magnesium, potassium, iron, aluminum, calcium, manganese, and zinc, as well as vitamins, nonionic surfactants, antifoaming agents, bacteriostatic growth, and phospholipase DM. Suitable substances that promote the production of can be used as necessary, other than those shown in the examples. Cultivation is carried out under aerobic conditions. The culture temperature can be changed as appropriate within the temperature range at which the bacteria can grow and produce phospholipase DM, but is particularly preferably about 20 to about 35°C. Although the culture time varies depending on the conditions, it is sufficient to culture until the maximum production amount of phospholipase DM is reached. In the case of liquid culture, the period is, for example, about 1 to 3 days. Since the phospholipase D produced in the culture is mainly dissolved in the culture solution in submerged culture, 1) Phospholipase DM can be collected from the culture solution obtained by separating the solid matter from the culture solution. When the culture is in liquid, I) Phospholipase I) M can be collected by any method commonly used for enzyme purification. For example, salting out with ammonium sulfate, common salt, etc., acetone,
Methods such as precipitation with organic solvents such as ethanol and methanol, dialysis, ion exchange chromatography, adsorption chromatography, del filtration, adsorbent, and isoelectric precipitation can be used. Furthermore, if the purification effect of phospholipase DM becomes negative by appropriately combining these methods, they can be carried out in combination. Phospholipase DM, which can be used by the method, can be used for example by concentration under reduced pressure, drying under pressure, with or without addition of various salts, carbohydrates, proteins, lipids, surfactants, etc. for stabilization.
It can be made into a liquid or solid form of phospholipase DM by a method such as freeze-drying. The enzymatic activity of phospholipase I) M used in the method of the present invention is determined by acting on the substrate glycerophospholipid to decompose the ester bond between phosphoric acid and a nitrogen-containing base and measuring the amount of base produced. The activity of phospholipase DM was measured by the choline oxidase method described below unless otherwise specified 1). Titer determination method: 1% egg Tlj Ii lecithin emulsion (0,1
g lecithin, 1+nl ethyl ether, 10ml distilled water ultrasonic emulsion) to 0.1ml, 0.2M pH 7.2)
Lis-hydrochloric acid buffer ('), 1 ml, I), IMC
Mix aCl, aqueous solution 0° (15 ml, distilled water 0.15 ml), add enzyme solution 0.1 ml, and incubate at 37°C for 2 hours.
After 0 minute reaction, 1M containing 50mM EDTA-2Na)
Add 0.2 ml of Lis-HCl buffer (pH 8,0) and immediately boil for 5 minutes to completely stop the reaction. Next, 4 ml of a solution of the choline coloring agent included in the cholinesterase measurement reagent kit (manufactured by Nippon Shoji Co., Ltd.) dissolved in the coloring solution was added, and after reacting at 37°C for 20 minutes, the absorbance of 337-5OOn was added. Measure. Month! As for 11t, the absorbance of a reaction was carried out in the same manner using an enzyme solution that had been preheated and inactivated. The enzyme activity that releases 1 μmol of choline per minute is defined as 1 unit. It can be used in the method of the present invention using enzyme preparations purified by the method described in 9. Purification method below using each of the aforementioned 7 Caldeiopsis sp. N ('1779 strain and Acti 7 Madura sp. The physical and chemical properties of phospholipase DM are described below. 1 Action: Decomposes the ester bond between the phosphoric acid of glycerophospholipid and the nitrogen-containing base to liberate phos-7atynoic acid and base. CH,0CORCH20COR 1 38- 2 Substrate specificity emulsion 0, containing 0.5 μmol of any one of lecithin, lysolecithin, and sphingomyelin as a sexual substrate.
using 1 ml and 1% Triton X- instead of distilled water.
The amount of choline liberated by the reaction was measured in the same manner as the 2-note titer measurement method, except that an aqueous solution containing 10 (,) was used,
Phospholipase DM activity for each substrate was measured. As a result, when the activity against lecithin was set as 100, the relative activity was as follows: Mecardiopsis phospholipase DM nee lysolecithin 4.9; sphingomyelin 0.3; Actinomadula phospholipase DM ne lysolecithin 3.6: sphingomyelin 0. ,3. 3 Optimal DH Instead of the buffer used in the titer measurement method, use pH 3.0~
For 4.0, formic acid/sodium formate buffer, pH 4.0-5
．． 5, acetic acid/sodium acetate buffer, pH 5.5 to 8.5
So Tris-maleic acid-caustic sorb buffer, +1 t
I7, f', 1 to 9.0, Tris-HCl buffer, l
])l9,f',)~N1j), glycine/
The activity of phospholipase DM was measured using a caustic soda buffer, and the optimal 1]I] was determined.
When using 0.1.5 ml of X-100 (Wako Pure Chemical Industries) aqueous solution, the optimum I- was also determined.
(around 7 (6,5-7, (1), Actinomadura phospholipase I') M knee optimum 1) around l-17, and 1% Tri1. on
(near 5, Makuti 7 Madura phosphorapase r) M knee optimum 1
) I-15. 4 In the optimal temperature titer measurement method, the reaction temperature conditions were set to 10.20.25.
．． 37.40, 50.55.60.70.80 and 9
Enzyme activity was measured at 0°C. As a result, the optimum temperature for Nocardiopsis phospholipase I'') M knee is 60°C to 80°C, especially 60°C to 7(')'C.
. Actino → Dura phospholipase DM knee optimum temperature was recognized to be 55°C to 80°C, particularly 60°C to 70°C. 5 To 0.1 ml of pH-stable enzyme solution, add 0.2 ml of Acti for Nocardiopsis phospholipase DM, 0.9 ml of 011M various buffers for Madura phospholipase DM, i.e. pH 3,0-3. For pH 5.5, use glycine/hydrochloric acid buffer, for pH 3.5 to 7.0, use acetic acid/sodium acetate buffer, for pH 5.0 to 8.041, use Tris/maleic acid/caustic soda buffer, pH 7.
, (l to 9, f') Tris-HCl buffer, l)
I-19. 0~! In J and 5, glycine and 7M soda buffer were respectively added and kept at 25°C for 2 hours. Then, add +1.5 M )')su・hydrochloric acid buffer (+) to these enzyme buffer solutions.
1-17.2), 1.2 ml in the case of Z Cardiopsis phospholipase DM, 9.0 ml in the case of Macchi 7 Madura phospholipase DM, and the pH was adjusted to 7.
．． It was set as 0 to 7.3. Using 0.1 ml of this solution, the titer was measured according to the titration method and the stable I-1 range was investigated. 1 to 7.0, especially stable pH 4, (
1 to 8.0. Also, instead of 0.15 ml of distilled water used in the titer measurement method, 0.1% Triton X-1f') f) aqueous solution was added. ]S
Except for using ml, the same procedure as described in ``2'' was performed to examine the stability range -42=, but the results were almost the same as those described in ``1.'' G Thermally stable oxygen solution 0 0.1 ml to 0.1 ml) IJ Soot-Acid Buffer (
+1117, 2) in the case of Nocardiopsis phospholipase DM, 9.9 ml in the case of Actinomadura phospholipase I) M, and 20
．． 30.37.40.50.60 and 30 to 65°C
After standing for a minute, the remaining enzyme activity was measured. As a result, Nocardiopsis phospholipase DMS-30°C
There was almost no deactivation with heat treatment for 30 minutes at 50'C.
80% of the activity remains after heat treatment for 30 minutes at 50°C, 80% activity remains after heat treatment for 30 minutes with Actinomadura phospholipase DM knee 3+1'C, and 60% remains after heat treatment at 50°C for 30 minutes.
The result was that % of the activity remained. 7 Substitute for CaCl2 aqueous solution in influence potency measurement method using various substances

[f') 05 +nl of aqueous solutions of the various substances were added to the enzyme reaction system to reach a concentration of i+nM, and the activity was measured. The result is that the activity when water is added is 100, and the activity is +1
Those that had an activating effect on Nocardiopsis phospholipase I) M nee, for example, A I C,
3, CuSO4,7,nSO4, CoCl2, CaCl
7, FeCl7,l;'e S starvation, MgC17,5n
C13, sodium deoxycholate, ethanol, isopropyl, 1-butyl, Triton X-10
0° Acti 7 Madura phospholipase DM knee, for example, A, lC1:1. CaCl3, FeCl2, FeC
1,, M g C+ 2.5nC17, sodium deoxycholate, ethanol, inpropar 7-ol, dobuta/-
On the other hand, those that had a choking effect were Nocardiopsis phospholipase T')M, such as dodecyl sodium sulfate, cetylpyridinium chloride, Acti7 Madura phospholipase DM, such as cetylpyridinium chloride, Met. 8. Method for measuring titer As described above. 9 Purification method Soybean flour 3.0g, cornstarch liquor 1°0%,
Peptone 0.5g, powdered yeast extract (), 1%, glutenose 1.0g-N) (4N O30, 25%, K
2 HPO40.4%, MgSO4・714200.
Approximately 151 of a medium (146,0) consisting of 01%, Tu+een-850, and 1% was placed in a 301 jar fermenter, and after sterilization at 120°C for 15 minutes, seed culture solution 1°51 was inoculated. and cultured at 27°C for 40 hours. The above culture solution contains 1% starch, (NI(,)H
2PO, 0.25%, peptone 0.25%, K 2HP
○Put 100ml of an aqueous medium (pl-(6,8) containing 30.2% and 40.01% MgSO into a 500ml Sakalo flask, and after steam sterilization,
9 strains rFERM-P No. 61331 or Actinomadura sp. No. 362 strains [FERM-P No. 613
A loopful of 45-21 spores was inoculated, and the culture temperature was 30°C.
It was prepared by culturing with shaking for 2 days at 0 rotations/min. After culturing, the solid bacterial cells are removed by centrifugation (1).
Sei 131 (/Caldeiopsis FERM-P No.
61, using the '43 strain? , z Basuke is (1,54,u/
ml; Acti 7 Madura FERM-P No. 6
When strain 131 was used, the concentration was 1.7 u/ml. ) was obtained. After cooling this centrifugation-1 supernatant to 5°C,
C. acetone was added, and the precipitate containing phospholipase I) M corresponding to a fraction with an acetone concentration of 30 to 70% was evaporated by centrifugation. This precipitate was converted to pi(6,('l, Acti/Madura genus Y7ERM
-P No. 6 ] 3 ] When using the strain (formerly 16
DEA dissolved in Tris-maleic acid at 5°C, dialyzed against 0.02M of the same buffer, and equilibrated with the same buffer.
E-cellulose was passed through the column and the flow-through fraction was collected. Next, the method of Kawauchi et al. CJ, Biochem, -81-214
6-639 (1,977): Fill a column with valmitoyl gauze prepared in [1,977], wash thoroughly with water, and then apply
A E-cellulose permeate was injected to adsorb the activity. This was added to 0.05M Tris-HCl buffer (pJ-17°2
), the same buffer containing 1,2% Trit:on
eG-], OT), poured into Toyopart (W~55F [manufactured by Toyo Soda Co., Ltd.]) as a gel filtration phase, and passed through the column using distilled water to separate the active fraction. The dried powder was mixed with Q, 025M imidazole-hydrochloric acid (pH
7,4), then in the case of Actinomadura phospholipase DM ('1,025 M trisacetic acid (pH
After dissolving in 8,3), polybuffer exchanger PRE 94 (20ml) manufactured by Pharmacia Fine Chemicals.
After adsorbing the activity through a packed column, p! -1 gradient. The active fraction of eluted phospholipase l) M was collected and concentrated using an ultrafiltration membrane, and then filtered with Sephadex G-
The phospholipase 1-)M active fraction was collected and lyophilized. Thus, in the case of Nocardiopsis phospholipase ')M, the specific activity was 1786 with an activity recovery rate of about 10%.
．． 4 for M; Recovered. ＼ O Isoelectric Point Nocardiopsis Phosphorinoguichize DMney 4.8
5 ± 0.1 (measured by ampholine electrophoresis) Actinomadura phospholinograse DM knee 6.4
±01 (measured by ampholine electrophoresis) O-transfer action The conventionally known phospholipase D produces phosphatidic acid from lecithin, as described in R, and converts it into prime number 1.
It is known that esters are transferred to Hakunaka's primary alcohols at 5 cm to form esters, but not with secondary alcohols. We similarly investigated the transfer effect of phospholipase DM, and found that this enzyme transfers to a wide range of surrounding alcohols, including primary alcohols for which known phospholipase D does not cause transfer. It has been found that in addition to the formation of C49-esters, esters are also formed by transfer to the secondary alcohols. Great wisdom y1? :'“S dehe] Kata-Nakaru Boss Tsuki Sugu-
ZDMI, f, Actual method of action on the rear ρ transformation I [7' I,
The -C reaction was carried out according to the method for the production of the rearrangement product by C, and the conversion of C4 secondary anocol 2-butanol and phosphorus)! ,i? Example t The secondary alcohol derivative of the phospholipid is formed by catalyzing a phospholipid secondary alcohol conductor formation reaction with lecithin. Known phospholipase D is described in "1.1. It forms a conductor. According to the method of the present invention, l:1', the formula (T
) By reacting an IJ lipid and a secondary alcohol as exemplified in ms in the presence of the phospholipase 7)-M described in detail above, the lower phospholipid (I+) I A-0-p-0- A phospholipid secondary alcohol derivative represented by R (seal) R -50- in which A and R have the same meanings as above can be produced. At this time, phospholipase DM does not need to be used as a purified product, and may be a crude product. Furthermore, suitable immobilizing phases such as polypropylene membranes, celite particles,
It can also be used by being supported and fixed on various polymer resins such as glass peas, or on granular or film-like materials of inorganic materials. The reaction can be carried out by bringing the IJ lipid of formula (1) into total contact with the secondary alcohol in the presence of phospholipase DM, preferably in the presence of a solvent. Examples of the solvent to be used include aqueous solvents and mixed solvents of aqueous solvents and organic solvents. The secondary alcohol itself can also serve as a solvent. In addition, a solution containing any other additives which do not inhibit the enzymatic catalytic action of P. coude DM can also be used, e.g. suitable additions that promote said action or serve to stabilize the yeast. It can be a solvent containing an agent. for example,
The solvent may be an aqueous solvent containing a buffer such as acetic acid, citric acid, or phosphoric acid, or a neutral salt such as calcium chloride. Examples of organic solvents include the secondary alcohol itself, such as n
-Aliphatic hydrocarbons such as hebutane, n-hexane, etc.Alicyclic hydrocarbons such as cyclopenkune, cyclohexane, cyclobutane, etc.;Aromatic hydrocarbons such as benzene, toluene, xylene, etc.;Acetone, methyl isocarbons, etc. Ketones such as zorobyl ketone; ethers such as dimethyl ether, diethyl ether, diisopropyl ether, etc.; esters such as methyl acetate, ethyl acetate, etc.; halodanized hydrocarbons such as carbon tetrachloride, chloroform, methylene chloride, etc.; Examples include amide bath media such as somethylformamide; sulfoxide solvents such as dimethyl sulfoxide. When using a mixed solvent of an aqueous solvent and an organic solvent, the mixing ratio of both can be selected appropriately, but for example, an aqueous solvent:
An example of such a mixing ratio is 50:1 to 1:10 in terms of the organic ratio (CV/V ratio). Anti-7 molar ratio, phospho-IJ no? -The amount of ZeDM to be used, the amount of medium to be used, etc. can be adjusted as appropriate, but for example,
A reaction molar ratio of about 1:10 to about 1:100 mole of secondary alcohol of formula (I+) to 1 mole of phospholipid of formula (1) can be exemplified. In addition, as the usage sound of phosphoringase DM, for example, about 10
An example of the usage amount is about 0 to about 1000 units. Furthermore, as the amount of the solvent used, for example, formula (I)
An example of the amount used is about 10 to about 500 times the amount of IJ lipid. -53- Since the reaction proceeds at room temperature, there is no particular need for cooling or heating, but cooling or vortexing conditions can be adopted as desired. For example, about 20 to about 60
A reaction temperature such as °C can be exemplified. Further, the reaction time can be selected as appropriate, and may be, for example, about 1 hour to about 24 hours. As desired,
For example, the reaction time can be changed as appropriate by tracking the reaction progress using a technique such as TLC (thin layer chromatography) and confirming the formation of the desired target product. The manner in which the IJ lipid of formula (T) is brought into contact with the secondary alcohol in the presence of phospholipase DM can be selected as appropriate, but it is usually carried out under stirring or shaking conditions. In addition, as mentioned above, phospholipase D can be used in the form of an identified enzyme supported and immobilized on a suitable granular or film-like carrier.
When M is used, for example, the reaction composition can be passed through a fixed enzyme membrane or an immobilized enzyme particle layer using a circulation pump. After carrying out the reaction in two series, the formed formula (I
[) The phospholipid negative secondary alcohol derivative can be used as it is or in the form of a salt after precipitation separation. Furthermore, separation and purification can be carried out using appropriate known methods such as silicic acid column chromatography, alumina column chromatography, high performance liquid chromatography, countercurrent distribution, ripple filtration, and adsorption chromatography. According to the method of the present invention, as described above, the phospholipid of formula (1) and the secondary alcohol are combined with phospholipase DM.
A 1,1 phospholipid secondary alcohol visiting conductor of formula (11) can be produced by reacting in the presence of . The resulting lipid secondary alcohol derivative of formula (II) has an excellent surfactant effect and has a large effect on the permeability of cell membranes. In this sense, the formula (I[) derivatives can be used as liposome-forming base materials, and as emulsifiers useful for skin physiology when incorporated into cosmetics such as creams and emulsions, and as emulsifiers for fatty drugs, insecticides, herbicides, etc. It is useful for a wide range of emulsifier applications such as All. Furthermore, in many cases, it is known that each phospholipid has a unique physiological activity, but since most of the formula (1) 3 conductors obtained by the method of the present invention have a structure similar to that, various types of phospholipids can be used. Physiological activity can be expected. By transferring a pharmacologically active compound having a secondary alcohol hydroxyl group or into which all secondary alcohol hydroxyl groups have been introduced to a phospholipid, the pharmacological side effects of the compound can be weakened or the therapeutic effects can be increased to reduce the dosage. It can also be expected to reduce the amount of damage. Furthermore, the above-mentioned pharmacologically active compound is transferred to phospholipids, and plays a useful role as a carrier for the pharmacologically active compound to accurately concentrate the compound in the affected area, and furthermore as a storage group for the pharmacologically active compound. You can also expect it. Furthermore, it is useful as an intermediate for chemical synthesis including various pharmaceuticals, and for example, derivatives obtained by transferring alcohols having highly reactive halogeno or amine substituents can be used. Furthermore, labeled phospholipid derivatives can be obtained by transferring a secondary alcohol labeled with triple water purple or I4C, and can be used to elucidate the metabolic pathway of phospholipids. Hereinafter, the embodiments of the method of the present invention will be explained according to the examples.
A more detailed example will be given. Reference Example 1 Preparation of phosphorino-ase I)H According to the section ③ Purification method above, Nocardiopsis sp. strain N0779 [
FERM-P status 6133) and Actinomadura sp.
Using each strain i10362 [FERM-P Muroxa2], phosphory/lyse DM was obtained with the same activity recovery rate and specific activity as described in the above section. -57- Space Example 1 (Run A 1 to A 21) Posted No. 4
The following formula (1) shown in the table IJ lipid substrate 1: L-α-
Lecithin, β, γ-nomiristoyl (Sigma) (1,2-ditetradenor-sn-glycerol-
3-phosphorylcholine) group t@3: L-α-lecithin,
β,γ-dihexadecyl (Calbiochemhe-IJ 7
(1,2-dihexadecyl-sn-gly70)
-L-3-phosphorylcholine) %m: L-α-lecithin, β, γ-hexadecylidine (same as above) (1,2-cyclohexadecylidene-Sn-ri IJ
Cellulose-3-phosphorylcholine) Substrate! 1': β-lecithin-α, γ-dinolumitoyl (same as above) -58- (], 3-dihexadecanoyl cerol-2-phosphorylcholine) and numerous types of 2 as shown in Table 4 below. grade alcohol,
According to the method for confirming the formation of a transfer product (phospholipid secondary alcohol) by TLC described later, the reaction was carried out in the presence of phospholipase DM, and the formation of a transfer product was confirmed. That R
The t values are shown in Table 4 below. Method for confirming the formation of transfer products by TLC: 1% phospholipid emulsion with the following composition: 0.1ml 10,4
M acetate buffer (pH 5,7) 0.1 mm10
8I calcium chloride aqueous solution 0.05 m6 Distilled water 0.1 10% secondary alcohol solution 0.1- to the reaction solution, phosphoryl/e-
ZeDM aqueous solution 0.1 m (0.2-1.2μ/0.1
d) was added and allowed to stand at 37°C for 1 to 5 bladders. The phospholipid emulsion for surface and upper placement is phospholipid lOQ M9
1 me of diethyl ether and 10 fn of distilled water were added to the solution, and the mixture was heated under ice-cooling conditions at 600 W, 2o, and 5 KHz.
Form by sonicating for minutes. At this time, if necessary, water or an organic solvent such as diethyl ether or acetone was added to form the reaction solution to make the 10 secondary alcohol solution FfQ>J. After the above-mentioned standing, 5o mMt7) E DT A (xfv
Add 0.2 mA f of an aqueous solution (cyaminetetraacetic acid) and add a chloroform-methanol mixture (2: IV/V) 5-
was added and stirred vigorously to extract the lipid (product). This suspension was centrifuged at 12,000Xs' for 10 minutes, the lower chloroform layer was separated, and a chloroform-methanol mixture (l: IV/V) was added. It was dissolved in sμt and used as a TLC sample. Of these, ioμt was spotted on a thin layer of silica dull (Funagle 6oA, 20X20α, Funakoshi Pharmaceutical), and diimbutylketone-acetic acid-water (4
0:25:5) as a developing solvent. The following reagents were used to detect spots. When a spot of phospholipid other than undecomposed substrate and its hydrolyzate (phosphatidic acid and its analogs) was detected, this was recognized as a transfer product. Color development of detection reagent phosphoric acid: Zinzade (7) reagent (Bei
ss, U, J. ch, τ0fn (LtO (1, 13104, 1964) Color development of primary amine: 0 of ninhydrin in ninhydrin reagent
．． 25 thiacetone solution) Coloration of secondary amines Secondary chlorous acid-benzidine reagent (Bi
sahel) if C. et al. Biochim. Biophys, Acta 7 0 5 9 81
．． 963) -61- Comparative Example 1 In Example 1, a known phospholipase D derived from cabbage (P-LBioch, e
The same procedure as in Example 1 was carried out except that mi-cars Inc.) was used. As a result, no transfer product was observed for any of the secondary alcohols shown in Table 4 below. -62- Example 2 (7?U Gatomu 1NJI67) L-α-Lecithin, β, γ-Similistoyl (manufactured by Sigma Chemical Company, purity 98%) 400 ■ Ditechyl ether 1 m
J, distilled water 10 F, /! was placed in an ultrasonic cell and subjected to ultrasonication at 600F and 2oKHz for 5 minutes while cooling on ice to obtain a milky white emulsion. This lecithin emulsion 2fnl (lecithin 80~), 0,
4M acetate buffer (pH 5,7) 2M! ,,0
．． Put 1M calcium chloride aqueous solution 1- and 10% l-amino-2-propatur hydrochloride aqueous solution 2 into a test tube with a stopper, and add Phospholy Guze DM aqueous solution (5z/go) 2-
After adding and mixing thoroughly, the mixture was allowed to stand at 37°C for 4 hours. Add 0.5N hydrochloric acid to the reaction solution! In addition, after stopping the reaction, chloroform-methanol (2:1) mixture 15
fnt was added and mixed vigorously to extract the phospholipids. This mixture was centrifuged at 2000×flO for 17 minutes, and the lower chloroform layer was separated. Add another 10 g of chloroform to the upper layer of water, perform the same extraction operation, combine the chloroform layers, and then add 1 g of chloroform to the water.
01 nl of 002N monic acid was added for washing. The chloroform layer was separated again from this mixture by centrifugation, dried under reduced pressure, and then dissolved in a mixture of l-n-hexane-2-propanol-water (60:80). When 20μt of this sample was spotted on a thin layer of silica gel (Funaguru, Funakoshi Pharmaceutical) and developed in a bath medium system of diingthyl ketone-acetic acid-water (40:25:5), phospholipids of three types were detected. Ta. Among them, the spot with the largest Rf value matched that of phosphatidic acid, while the spot with the smallest Rf value matched that of lecithin. In addition, only the center spot developed color with the ninhydrin reagent. -65- This sample was purified by high performance liquid chromatography. Column is Radial Pack Cartridge Silica 8II
1mX10oy+ (manufactured by Waters), eluent (f), n-hexane-2-prono and nor-water (60%
: 80 near 1, the flow rate was 2 mA 1 min, and peak detection was carried out using a 441 ultraviolet detector (Waters Corporation) at 4 wm absorption and an R401 differential refractometer (Waters).
Same) for Bab ζ. The sample was divided into 4 injections and 0.25 d each. Through this operation, two types of phospholipids, phosphatidine C11 sand and 1-amino-2-pronognole ester of phosphatidic acid, could be fractionated. Next, the solution solution was changed to η7-hexane-2-propanol-water (60:80:14) to bathe out the undecomposed lecithin adsorbed on the column.
It was confirmed by TLC and high performance liquid chromatography that the type 1 phosphorus substance was single. 3′! 100 phospholipids (about 30% of phosphatidic acid, about 30% of rearrangement products, about 40% of lecithin (molar ratio), about 20% of 1-amino-2- of phosphatidic acid)
Pronogol ester was obtained. JR of this compound
The spectra were measured using the thin film method using a JASCO model A202 infrared spectrophotometer. The results are shown in the 5th edition (Rtbn 4). The procedure was carried out in the same manner as above, using the same alcohol as shown in the gp, -5 table. The conclusion! , 1! are shown in Table 5 (Rqbn-ya 1-3 and 5-7). /ζ
However, when adding the secondary alcohol to the reaction solution, it was added as a solution in water, diethyl ether, or acetone, depending on the solubility of the alcohol. Example 3 (Run Haruka 7) L-α-lecithin, β, γ-nohexadecyl (manufactured by Carpioke Mubering) 400~, diethyl ether 1m7! and 10ml of distilled water. The mixture was emulsified in the same manner as in Example 2, and 2 ml of this emulsion was used to carry out a reaction in the same manner as in Example 2. however,
A 10% diethyl ether solution of cyclohexanol was used as the secondary alcohol. This reaction solution was treated in the same manner as in Example 2 to obtain transfer products 18-. The JR spectrum of this compound is shown in Run 6 (Run & 7
). The same procedure as above was carried out using other secondary alcohols shown in Table 6. The results are shown in Table 6 (Run
Al~6). Example 4 (Ru'n, IFh 1~&7)L
-α-lecithin, β, γ-hexadesylidine (manufactured by Calbiochem Behring) 40 (1,?'
1-phenyl-2-broknol was added as an acceptor for the rearrangement reaction, and the same method as in Example 2 was prepared as Y2. : On the other hand, extraction and purification were performed. As a result, the transfer product 22 is Xun. The IR spectrum of this compound is shown in Table 7 (Run A6). The same procedure as above was carried out using other secondary alcohols shown in Table 7. The results are shown in Table 7 (RwnA1~
5 and 7). Example 5 (RsM, A1 to A7) β-lecithin, α, γ-dihexadecanoyl (manufactured by Calbiochem Behring) 40 (ly was emulsified in the same manner as in Example 2,
Using an emulsion equivalent to 80μ, 1 as a receptor for the transfer reaction.
-Amino-2-propatol was added, and the reaction, extraction, and purification were carried out in the same manner as in Example 2. As a result, 10 μm of a transfer product was obtained. The JR spectrum of this compound is shown in Table 8 (Rrbn section 4). The same procedure as above was carried out using other secondary alcohols shown in Table 8. The results are shown in Table 8 (Rs n
A 1-3 and 5-7). Example 6 (7?u?7A1-&5) L-α-phosphatidylethanolamine, β. r-nomiristoyl (ri, L-α-phosphatidyl N-
Methylethanolamine, β, γ-cimyristoyl (+
1), L-α-phosphatidyl-DL-glycerol,
β, γ-nomiristoyl (I[l) (hereinafter referred to as J2, both manufactured by Calbiochem Behring), L-α-phosphatidylserine (■) (Sigma), and S. F. Yang et al. (J. Biol. Chttm.
L-α adjusted by the method of 242.477.1967)
-Phosphatitulethanol, β, γ-cimilistoyl (V) were emulsified in the same manner as in Example 2. Pour 1.5 ml of an emulsion containing ~50% of each phospholipid into separate stoppered test tubes, and add l-phenyl-2 as alcohol.
-pro/e-nol in 10% diethyl ether 1++
+A, 1ml of 0.4M acetate buffer, 1fnl of distilled water, 0
．． A 0.5-ml aqueous solution of O1m calcium chloride was added, and the reaction solution was further subjected to ultrasonication at 600F and 20KH2 for 1 minute. Next, 1 mA of a 0M phospholipase aqueous solution (8 u/ml') was added to each reaction solution, and the mixture was allowed to stand at 37°C for 6 hours. Hereinafter, extraction and purification of phospholipids will be described in Example 2.
A common rearrangement product, phosphatidic acid-1-phenyl-2-pronophenol ester, was obtained. The yield was 6 ■ for 1, 8 ■ for ■, 7 ■ for m, 8 ~ for ■, and 10 IQ for V. The JR spectrum is shown in Table 9. Table 9 Patent Applicant Meito Sangyo Co., Ltd. -77- Procedural Amendment July 11, 1980 Patent Chief I Shiji Gaku 1, Case Description Sui 58-6 +'l 305 Consideration 2, Title of the invention (Cover 3 of the alcohol defense offering, the person making the amendment, the relationship with the case, the address of the patent applicant: 0. the name of the applicant, the city of 1) 2-414 Sasanoya-cho, Agent 〒107 ((-1 person) (Attachment) (If On page 6, line 6 of the specification, [branched alkyl group j is followed by ``Rj.'') (2) In line 4 of page 7 of the specification, correct [Kates CawJ to F Ka, tes Can.'' (3) In lines 6 to 5 from the bottom of page 7 of specification, [Da1Lss
o″IL;Biochem, T,, J, F
R, M, C, Dawson; Biochem;
Jr,, correct it as j. (4) On page 25, line 7 of the specification, “Becteri
Correct "J Bacteriology" instead of "J Bacteriology". (5) On page 27, lines 2-3 of the Meishinbiki, ``Everything is profitable.''
Correct the statement to read, ``All can be used.'' (6) Akira 11? On the last line of page 27, the word ``spiral'' is corrected to ``rod-like j.'' (7) Seiwa - On page 28, lines 9-10, the word ``spore glue'' is written as ``spore glue''. 'Spore spores' - Correct. (8) In the table on page 30 of the 1st son of Akira, in the 4th line from the bottom, [Y
-1-194 is corrected as [i'r)'-1-19 j. (9) On page 32, line 5 of the specification, [Becteriolo
gy J is corrected to ``Bacteriology Jl''.
(11) The formula at the bottom of page 38 of the specification box is corrected as follows: 17 CH2QC(J)R CM, 0COR 0, -j 3-(12) On page 43, line 4 of the specification, the phrase "oxygen liquor solution" is corrected to "enzyme bath solution." (13) On page 44, line 7 of the specification, ``P'eC1,J'' is corrected to ``FeC1,j.'' (14) On page 44, line 11 of the specification 'i, ``Fec12,F
Correct the sentence ``eC1,'' by saying ``I Fe5O4XFeC1,''. (15) “MgSO4” in the 5th line from the bottom of page 45 of the specification
The statement has been corrected to read “MQ, SO4・7H20j.
Correct the E94J to 4-TN "PBE 94". (17) Meiji 111tI, page 53, line 10K, ``Two-bar alcohol'' VC1 1' About 1 = 1 to about 1: 1000, preferably,'' Toguchi said. (18) On page 53 of the specification list, lines 5-4 from the bottom, “Lipid IV
Shun says, "It's about 10 to about 100,000. Preferably." (19) In the 4th line of Teiho No. 54 in the specification, before it says [f] 20"CJ, add "about O"C to about 90'C, good trench." (20) Myeongshu+V, page 54, 6, before it says "about 1 hour", it says "about 1 minute to about 10 days, preferably about 01 hours to about 72 hours, more preferably about 1 hour to about 72 hours, especially 1" added. (21) In lines 3 and 4 of page 556 of the specification, the phrase ``herbicides, emulsifiers, etc.'' is corrected to If'If' j is an emulsifier of agricultural products such as herbicides. (22) In bright line 11, page 58, line 5, the phrase ``1,2-ditetradecanol'' is corrected to ``1,2-sotetradecanol.'' . (23) On page 60, lines 5-6 of the detailed statement, [In this case...
...in the formation of the reaction solution," should be corrected to read, "in the formation of the above reaction solution." (24) In the fourth line from the bottom of page 60 of the specification, after the phrase "preparation," add "after drying under reduced pressure at 30°C." (25) “60.
4.20X20cWLJ should be corrected to ``60/7.20cmX20cm Jl. (26) On page 66, line 7 of the specification, 12.4 fights'' should be corrected to ``T 214 fin.'' (27) On page 67, line 6 of the specification, it says "thin film method",
Corrected to ``liquid film method.'' 7-

Claims (1)

[Claims] 1. The following formula (I) 0 1 A -0-P -0-B ...... (1) CH In the formula, A is the following (1) or (11) C1i2. −
CH2-R2, where R1 and R2 are both 10-CUR,, or both are 10-R1, or in formula (i) R1
and R3 indicate -11-190 osci], and in the above, R1 and R12 may be the same or different,
Each represents a C, ~C2, saturated or unsaturated aliphatic hydrocarbon group, B is -(CH2)2NCCIfs)s, -(CH3),
NFI,,-Chi,CH(IVH,)COOfl,
-CH, C, H, NH (C1l, ), -cino, cH
,IV (C1f3),, -CH2CHOHCI
I2011 also L < U − ((1?/12)
II, where m is the number 771° of 1 to 5], and halogen, amine, acetyl, hydroxyl group, Cs, mono- or di-alkylamino and phenyl C3~, which may be optionally substituted with a substituent selected from the group consisting of
React with a secondary alcohol having a linear or branched alkyl group R of CIO or an alicyclic hydrocarbon group R of C°4 to C6 which may be substituted with the above substituent, and gold in the presence of phospholipase DiW. It is represented by the following formula (I+) 0 (I A -0-P -0-R-・-・-・(+r)CH, where A and R have the same meanings as above. Method for producing phospholipid secondary alcohol derivatives.