JPS6281394A - Mixed acid type polymerizable phospholipid derivative and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula [R<1> and R<2> are mutually different, one of them is 10-22C fatty acid residue and the other is acyl shown by the formula R<0>CH=CHCH=CHCO (R<0> is 5-17C alkyl]. EXAMPLE:1-Palmitoyl-2-(2E,4E-octadecadienoyl)-L-3-glycerophosphorylcho line. USE:Useful for simulation of biological reaction as a biomembrane model or a carrier sealable into a drug, enzyme, gene, etc. PREPARATION:A dienecarboxylic acid shown by the formula R<0>CH=CHCH= CHCOOH is reacted with N,N'-dicarbonyl diimidazole to give a 1-acyl imidazole, which is reacted with 1- or 2-monoacyl-L-3-glycerophosphorylcholine in the presence of sodium imidazole as a catalyst.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a mixed acid type polymerizable phospholipid derivative, more specifically a phosphorylcholine type polymerizable phospholipid derivative having a photopolymerizable diene group as a functional group; It relates to its manufacturing method.

[Conventional technology]

There is active research on molecular tissue engineering that utilizes the self-assembly ability of amphiphilic substances to form and apply monolayer films, bilayer films, laminated films, and vesicles. Among these amphipathic substances, phospholipids are the most representative and are the main constituents of biological membranes, and are widely used because they can be easily extracted from natural products.

When phospholipids are suspended in a water bath, they form liposomes, which are closed vesicles consisting of a lipid bilayer membrane with Mizushima gold inside. These liposomes are not only used as biological membrane models with excellent morphology for simulating biological reactions, but also can be used to completely encapsulate carriers such as drugs, enzymes, and genes.
In addition, liposomes are being actively used as carriers, with specific functions added to them, such as targeting target cells with liposomes loaded with specific labeled molecules, and application to sensors.

In addition, we are applying the bilayer membrane-forming ability of phospholipids to microcapsules by coating capsule membranes.
Applications are being made in a wide range of fields such as biochemistry, medicine, pharmacy, and engineering, including biosensors and medical materials in the form of monolayer films and laminated films, as well as molecular electronics.

However, organized molecular assemblies formed from amphiphilic substances have the difficult problem of instability because their structure is maintained solely by hydrophobic cohesive forces.

Therefore, in order to apply it more widely to the various fields mentioned above, it is essential to improve the instability of the organized molecular aggregates of phospholipids, and there is currently a strong demand for technology for this purpose. be.

In order to meet this demand, a method is proposed in which a photopolymerizable functional group is introduced into a phospholipid molecule, the molecules are organized, and then the structure is completely stabilized by polymerizing with covalent bonds by light irradiation. In particular, in applications such as medical liposomes, polymerizable phospholipid derivatives with a structure and composition similar to natural phospholipids and good biocompatibility are in high demand.

The commonly used phospholipids are cylinder-shaped molecules with a good balance of hydrophobic and hydrophilic groups.
It is a diacyl-3-glycerophosphorylcholine (hereinafter abbreviated as lecithin) type phospholipid that can break the bilayer membrane structure even when used alone. Examples of lecithin-type phospholipids in which photopolymerizable functional groups are introduced include those in which methacrylic groups, styrene groups, diacetylene groups, thuene groups, etc. are introduced, respectively.

Examples of introducing methacrylic funds: (For example, J
, Am, Chem, Soc,, 104.791 (19
82) ), examples of introducing styrene groups: (e.g. tp5
Publication No. 116689/1989, Japanese Patent Publication No. 1161/1982
No. 390), each polymerizable functional group can only be introduced at the end of a hydrophobic alkyl group, which has the disadvantage of greatly reducing the amount of heat required for the glue liquid crystal phase transition, which is an inherent property of bilayer membranes. There is. Furthermore, their molecular structure is far different from that of natural lipids,
In addition, toxicity becomes a problem when applied to living organisms.

Also, an example in which a diacetylene group is introduced; (for example, U.S. Patent 4
348329), but polymerizable liposomes using this diacetylene visiting conductor have a polymerizable functional group introduced into the central part of the alkyl group, and also have a rigid conformation because they form a conjugated unsaturated bond. , and no phase transition temperature (hereinafter abbreviated as Tc) is observed. Furthermore, Tc
It is a topochemical reaction that can be polymerized only in the following conditions, and has the disadvantage that many unsaturated bonds remain.

Examples of introducing tsene groups: (Angs+w, Cham,
Int, Ed.

Engl., 20.90 (1981)), a polymerizable liposome made of this diene derivative has a polymerizable functional group introduced at the root of a hydrophobic alkyl chain, that is, on the most hydrophilic group side, and the above-mentioned diacetylene derivative. Compared to gold, it has a more flexible structure with fewer restrictions on the movement of alkyl chains, can be polymerized in either the glue state below Tc or the enemy crystal state above Te, and has fewer unsaturated bonds remaining. Motsu. However, since the polymerizable functional group is introduced into both of the two alkyl chains of all lipids, the movement of the alkyl chains is considerably restricted after polymerization, and the heat of r-ru liquid crystal phase transition is also reduced (see Reference Example 2). . On the other hand, a mixed acid type 1,2-noacyl-L=3- consisting of long-chain fatty acid residues
A method for producing glycerophosphorylcholines is also known,
The fatty acid residues are residues of linoleic acid, lylunic acid, arachidonic acid, etc., and long-chain dienecargenic acid residues are not described at all (for example, Japanese Patent Publication No. 59-4083
Publication No. 9).

[Problem that the invention seeks to solve]

In view of the current situation, the present inventors conducted intensive research to obtain a polymerizable phospholipid derivative gold having a structure and composition similar to natural phospholipids as well as unique properties. The phospholipid derivative introduced as a diene-based photopolymerizable functional group is a compound that satisfies all of the above-mentioned requirements, and moreover, this compound is a monoacyl-L derivative that can be easily obtained from synthetic lecithin or natural lecithin. It has been discovered that it can be synthesized as a complete product with high purity and yield from -3-glycerophosphorylcholine, and the present invention has been completed. That is, an object of the present invention is to provide a photopolymerizable phospholipid derivative having a structure and composition closest to natural phospholipids, and to provide a method for producing the same economically and with good yield.

[Failure to solve the problem]

In the present invention, the general formula % formula % (2 [wherein R1 and R2 are different from each other, one of which represents all saturated or unsaturated C4-C22 aliphatic residues, and Ikekata represents R0CH=CHCH=CHCO (Here, FLO is C5
- C47 alkyl group) acyl group] is provided. As mentioned above, diene groups, which are photopolymerizable functional groups, are introduced into both of the two alkyl chains of the lecithin-type phospholipid. Phosphorylcholine (hereinafter abbreviated as didene) is well known. However, compounds in which a diene group is introduced into only one alkyl chain of lecithin (hereinafter abbreviated as monodiene)
There are no examples, and furthermore, it is completely unknown that monotene lecithin undergoes polymerization by light irradiation after being formed into molecular structures (for example, into somes/somes), resulting in polymerization and stabilization.

Specifically, the desired monodiene can be easily obtained by converting free monoacyl-L-3-glycerophosphorylcholine into total ashes in the presence of a sodium imidazole catalyst using long-chain tsenecarbic acid as an acylating agent. Lecithin can be obtained. This reaction takes place between 0°C and 30°C.
It is preferable to carry out the reaction at a temperature of, usually at room temperature, with stirring.

Moreover, the molar ratio of monoacyl-L-3-glycerophosphorylcholine and acylating agent is preferably 1:1.2 to 1:1.5.

The starting material monoacyl-L-3-glycerophosphorylcholine includes %1-monoacyl form and 2-monoacyl form, both of which are applicable to the present invention.

The 1-monoacyl-L-3-glycerophosphorylcholine is usually prepared by mixing synthetic lecithin or natural lecithin with a suitable solvent such as chloroform or ethyl ether, among others, in a suitable buffer (e.g. 0.2 M). Using phospholipase A2 or its related enzymes obtained from snake venom, such as Naja najlL, in the presence of Liss hydrochloride buffer (pH 7,4), etc., and an activator, such as calcium chloride solution. It can be obtained by carefully performing hydrolysis at room temperature so as not to cause acyl conversion.

In addition, 2-monoacyl-L-3-glycerophosphorylcholine can be used in various bacteria, such as E. schiericia coli (E, Co11), Mycobacterium freyi (M
It can also be obtained by hydrolyzing synthetic or natural lecithin using phospholipase A obtained from B. ycobactariutn fert, Bacillus subtilis, or related enzymes.

Furthermore, the obtained monoacyl-L-3-glycerophosphorylcholine was hydrogenated to eliminate unsaturated bonds in the alkyl chain, resulting in a saturated fully hydrogenated monoacyl-L-3.
- It can also be provided in the present invention in the form of glycerophosphorylcholine.

The long chain noene caldic acid used in the present invention is RCH=C)I
CH=CHCOOH (R is a C5-C17 alkyl group)
It is an α, β, γ, δ-unsaturated caldic acid represented by Examples of such dienecardanoic acids include 2,4-decadienoic acid, 2,4-undecadienoic acid, 2,4-dodecadienoic acid, 2,4-tridecatsuenoic acid, 2,4-tetradecatsuenoic acid, 2,4 -pentadecadienoic acid, 2.4
-hexadecadienoic acid, 2,4-heptadecadienoic acid,
All photopolymerizable geometric isomers such as 2.4-octadecadienoic acid, 2゜4-nonadecadienoic acid, 2,4-eicosadienoic acid, 2.4-heneicosatsuenoic acid and 2.4-tocosasienoic acid. I can lift my body. Such a long chain diencal? For example, JP-A-60-13
It can be obtained by a method such as that disclosed in Japanese Patent No. 737.

As the catalyst used in the present invention, sodium imidazole is preferred.

The manufacturing method of the present invention will be explained in detail by focusing on the gold side when using t-1-acylimidazole. First, N, N'-force? Nilji imida! - suspension in anhydrous chloroform,
To this was added long-chain phosphoric acid, and the mixture was reacted for about 1 hour at room temperature in a nitrogen gas flow and shielded from light.
Acylimidazole is obtained. After this compound is isolated,
Alternatively, the reaction solution can be used in the next reaction without isolation. Separately, synthetic lecithin or natural lecithin, such as egg yolk lecithin, soybean lecithin, etc.
In the presence of a buffer solution and an activator, 1-acyl-L-3-glycerophosphorylcholine is obtained by hydrolysis using phospholipase A2 at room temperature, taking care not to cause acyl transfer. 1-Acyl- thus obtained
lmN as L-3-glycerophosphorylcholine total catalyst
a - The above 1- with DMSO common solution and anhydrous pyridine
The reaction is completed by adding to the acylimidazole reaction solution and stirring at room temperature for several hours.

To isolate the target product from the reaction mixture, first, the reaction solution is neutralized with hydrochloric acid-methanol and concentrated under reduced pressure.

This concentrated solution is diluted with chloroform-methanol, water is added, and then dispersed, and the lower layer is further concentrated under reduced pressure. Chloroform-methanol-water and then ethanol gold were added to the obtained concentrate to add [Amberley] MB.
-3 type" resin column, washed with the same solvent, and the passed through solution and the washings were combined and concentrated under reduced pressure. The desired product can be obtained by processing and purifying this concentrate using a silica gel column chromatography method according to a conventional method.

〔Example〕

The present invention will be described in more detail below with reference to Examples, but the present invention is not limited thereto.

Example 1 Synthesis of 1-valmitoyl-2-(2E,4g-octadecajenoyl)-L-3-glycerophosphorylcholine (a) Preparation of 1-valmitoyl-L-3-glycerophosphorylcholine 1.2-') A Lumitoyl L-3-Glycerophosphozylco9F3 In addition, phosphoryl obtained from Nanoya Naja venom/#
-SeA230Tn9ftQ, 2M) Lis-HCl buffer iy. <s7, 4) Solution dissolved in 6- and IM
Calcium chloride @go. ti'6 plus about 20 at room temperature
Stir for hours.

Ethanol was added to this reaction solution and concentrated to dryness under reduced pressure.The dried product was dissolved in a small amount of chloroform, passed through a column of silica dal (40 g) activated with the same solvent, and 60 g of chloroform was added.
0flt, chloroform-meta/-water (6 5
: 2 5 : 4) 1.5 1te PF 1st order m was output. The target fraction was collected using gold thin layer chromatography (hereinafter abbreviated as TLC), concentrated under reduced pressure, and dried under reduced pressure over phosphorus pentoxide for about 20 hours to obtain 1-valmitoyl-L- 3-Glycerophosphorylcholine 1.8.9
(Yield: 87.8%) t-obtained.

(b) Synthesis of 1-valmitoyl-2-(2E.4E-octadecajenoyl)-L-3-glycerophosphorylcholine 2E,4g-octadecatsuenoic acid 3.5 #(1
2.5 mmol) and N, N'-force? Nildiimidazole 2, 4. ! 50- of dry chloroform was added to i+ (15 mmol) and reacted for about 1 hour at room temperature in a nitrogen stream and shielded from light. Next, Example 1- (1- obtained in ml) and Lumitoyl-L-3-glycerophosphorylcholine 5. IJ (10 mmol)'f. (Additionally hydrogenated as a catalyst under water cooling)
9 (50%) and imidazole (hereinafter abbreviated as ImH)
1. ! i' and dried dimethyl sulfoxide (DMS)
Sodium imidazole (hereinafter abbreviated as lmNa) prepared by reacting in 20 d of O (abbreviated as lmNa)-DMS
After adding 20 fIt of Og solution and 1-total amount of anhydrous pyridine, the mixture was stirred at room temperature for 2 hours.

After the completion of ashing, the reaction solution was neutralized with IN hydrochloric acid-methanol,
Concentrate under reduced pressure. The obtained concentrate was dissolved in 600 ml of total chloroform-methanol (2:1), and then 120 ml of water was added.
'lt and separate the layers using a separatory funnel, separate the lower layer and concentrate under reduced pressure.

Chloroform-methanol-water (65:
25:4) 200mg and 100mg of ethanol were added and dissolved, and then "Amberley) MB-31480mg'
After stirring for about 2 hours, the resin was taken out and washed with the above-mentioned medium system, and the resulting solution F and the washing were combined and concentrated under reduced pressure.

This concentrate was dissolved in 95% ethanol from Kintekisha, and the alumina (40%) was preactivated with 95% ethanol.
9) The mixture was applied to a column and extracted with 240-ml homologue, and the eluate was concentrated under reduced pressure.

This concentrate was dissolved in a small amount of chloroform, and a silica gel (1501i') column previously activated with the same solvent was added (VC or kj, l chloroform 11, chloroform-methanol-water (65:25:2))4. I made him come out with the office lady. The target fraction was collected from the obtained m fractions by TLC, concentrated and dried under reduced pressure, and 1-valmitoyl-2-(2E.
74.7M (yield: 60.6%) of 4E-octadecatzuenoic acid/l/)-L-3-glycerophosphorylco1J was obtained.

This substance was analyzed by TLC (Merck silica del plate).

Developing medium: chloroform-methanol-water (65:25
:4))? When performed, detection with ultraviolet light and phosphomolybdic acid gave a single spot whose value of 84 was dipalmitoyl-L-α-glycerophosphorylcholine (
Sigma). The optical rotation was as follows.

[α]D=+6.49 <cHct,, C=1> Also, elemental analysis values of this substance, nuclear magnetic resonance (NMR) spectrum, infrared (IR) absorption spectrum, and ultraviolet (tr)
v) The measurement results of absorption spectra are shown.

Elemental analysis value (C4□H8oO8NP-H20 molecule fi1
776, 1) Calculated value (ch): C: 65. OO. H
: 10.65, N: 1.805 Actual value (%): C: 64
．． 6, H:10.7. N: 2.215C-NM
R spectrum δ value (cDct,, ppm) 12
3 4 567BCH5CH2C
H2 (CH2) 1. CH2CH2CO2CH2'H
-NMR spectrum δ value (CDCA, , δ (ppm)
, TMS) FT-IR spectrum (crn-1)
:See Figure 1 (KBrf), □
, 6 (,.=.) 1645, 1624 (νCTC) 1246 (Si, =.) 1144 (Si p-o-c) 1090 (Si P-Oe) 1057 (δP-0-C) 997 (δC-H ) UVx vector (in ethanol): λ = 262.5
(nm) ax g = 24700 (A"mol-'z') Example 2 Synthesis of 1-acyl-2-(2B,4E-octadecagenoyl)-L,-3-glycerophosphorylcholine derived from egg yolk (! Ll Preparation of 1-acyl/L/-L-3-glycerophosphorylcholine derived from egg yolk. Phosphorinoyase A230Q'i obtained from the venom of Naja Naja in 0.2
M) Lis-HCl buffer 1 (pH 7,4) 81n1. The dissolved solution and IM calcium chloride [Q, 1d'i] were added and stirred at room temperature for about 16 hours.

After the reaction was completed, the ethyl ether layer was removed, ether was added to the residue, and the mixture was concentrated to dryness under reduced pressure. This dried product was dissolved in a small amount of chloroform and treated in the same manner as in Example 1-(a) to obtain 5.4# of 1-acyl-L-3-glycerophosphorylcholine derived from egg yolk (yield: 91%).

(b) Synthesis of 1-acyl-2-(2E,4E-octadecajenoyl)-L-3-glycerophosphorylcholine derived from egg yolk 2E,4E-octadecadienoic acid3. FM'(12,5
mmol) and N, N'-force? Nildiimidazole2.
4.9 (15 mmol) in dry chloroform 60 dt-
It was then turned into ashes under the same conditions as in Example 1-(b). Next, 1-acyl-L-glycerophosphorylcholine 5.11 derived from egg yolk obtained in Example 2-(a) was added to this reaction solution.
9 (10 mmol) and further under water cooling, Example 1
-(Catalyst lmNa prepared in the same manner as bl) -DMS
After adding OFj solution 20- and anhydrous pyridine 2-, the mixture was stirred at room temperature for 3 hours.

After the reaction was completed, the desired egg yolk-derived 1-acyl-2-(2E, 4g
4.5 g (yield: 58%) of L-3-octadecagenoyl-L-3-glycerophosphoryl coria was obtained.

TLC analysis of this substance was performed in the same manner as in Example 1 and showed a single spot, which was consistent with that of egg yolk lecithin. Moreover, the optical rotation was as follows.

[α]”=+5.69 (CHC2,,C=1) Furthermore, it was confirmed from the results of the same analysis method as in Example 1 that it was a pure substance.

13C-NMR spectrum δ value (CDCL, ppm
) 14.1.22.7, 25.0, 28.8, 29.
7, 32.033.2, 34.2, 54.4, 5
9.3, 63.1, 66.471.0, 118.
7.128.4.145.9.166.7.173.8
FT-IR spectrum (Fuchi-1): See Figure 2 (KBr
D3ri) 1718 (ν.-8) 1645
．． 1624 (νc=c) 1247 (1/P=o) 1145 (sheer 0O) 1092 (sheer 0O) 1068 (δp-o-c) 998 (δC-H) UV spectrum (in ethanol): λrllllLX =
262-1 (nm) g=23600(l-(2)1
-1・α-1) Example 3 Synthesis of soybean-derived 1-acyl-2-(2E,4E-octadecagenoyl)-L-3-glycerophosphorylcholine (&) Soybean-derived 1-acyl-L- Preparation of 3-glycerophosphorylcholine 7 g' of 1,2-diacyl-L-3-glycerophosphorylcholine derived from soybean (dissolved in 150-ethyl ether,
This is combined with phosphoryl 2- obtained from the poison of Nanoya Naja.
ZeA210m9 (0.2M) Lith-HCl buffer 1 (pH 7,
4) M decomposition solution and IM calcium chloride solution 01d'i- were added to 4trt, and the mixture was stirred at room temperature for about 16 hours.

After the reaction was completed, the ethyl ether layer was removed, ethanol was added to the residue, and the mixture was concentrated to dryness under reduced pressure.

This dried product was dissolved in a small amount of chloroform, and Example 1-
(Processed in the same manner as al, 1-acyl-derived from soybean
L-3-glycerophosphorylcholine 4, IJ (yield 86
．． 6%).

(b) Synthesis of 1-acyl-2-(21,4E-octadecajenoyl)-L-3-glycerophosphorylcholine from soybean 2E,4E-octadecatsuenoic acid 0.7.9 (2,5
Mi1) Mo#),! : Dry chloroform 10- was added to 0.48 J (3 mmol) of N,N'-mergonyl diimigusol and reacted under the same conditions as in Example 1-(b).

Next, the soybean-derived 1-acyl-L-3-glycerophosphorylcholine 1. obtained in Example 3-(a) was added to this reaction solution.
I mNa-DMSOD solution 3r prepared by adding 08 fIC, 2 mm IJ mol) and reacting 150 ~ (50%) sodium hydride as a catalyst with 300 m9 of ImH in dry DMSO6 for about 1 hour under water cooling ! L1.
After adding 0.5 tntt of anhydrous pyridine, the mixture was stirred at room temperature for about 2 hours.

After completion of the reaction, the desired soybean-derived 1-acyl-2-(2E,4
g-Octadecagenoyl)-L-3-glycerophosphorylcholine 0.71 (yield: 44%) was obtained ◎ TLC analysis results of this substance showed a single spot as in Examples 1 and 2, and soybean It was consistent with that of lecithin. Moreover, the optical rotation was as follows.

18゜[α)D=+6.99 (CHCL3.C=1) Furthermore, from the analysis results similar to Example 1.2 as shown below,
It was confirmed that this substance is a pure substance.

"C-NMR spectrum δ value (CDCL, ppm
)14. L, 22.8, 25.0.27.3, 29.
8, 32.0, 33.2° 34.2, 54.4.59.
2.63.1.66.4, 70.7, 118.7°12
8.5, 130.2, 146.0, 166.7
, 179.8 FT-IR spectrum (cIn-'): See Figure 3 (NaC1f'4X) 1718 (ν
c=o) 1641, 1618 (ν.=c) 1242 (ν, yo.) 1138 (v, -o-c) 1090 (SiP-0'') 1063 (δ, -8-C) 999 (δC- 11) trv spectrum (in ethanol) 2 -1L!-26
2,1(ntn)ε=21800(J・mol−1・c
m″) Example 4 Soybean-derived (hydrogenated) 1-acyl-2-(2F,,4E-
Synthesis of (octadecagenoyl)-L-3-glycerophosphorylcholine (a) Preparation of 1-acyl-L-3-glycerophosphorylcholine from soybean (hydrogenated) Example 3 - (1- from soybean obtained in ml Acyl-L
-3-Glycerophosphorylcholine (10.9 i) and emenol (200 g) were mixed with 200 μm of platinum dioxide and stirred at room temperature in a hydrogen gas atmosphere for about 20 hours.

After the reaction, the platinum dioxide was filtered off, and the resulting filtrate was concentrated to dryness under reduced pressure, and dried over phosphorus pentoxide under reduced pressure for about 20 hours to obtain 1-acyl-L-3-glycerol derived from soybeans (hydrogenated). Phosphorylcholine 9.8N (yield: 98%) was obtained.

(b) Soybean-derived (hydrogenated) 1-acyl-2-(2, 4
Synthesis of E-octadecadienoyl)-L-3-glycerophosphorylcholine 2E,4E-octadecadienoic acid 3.51! (12,5
mmol) and N,N'-carbonyldiimidazole2.
411 (15 mmol) to 60 tan dry chloroform
In addition, the reaction was carried out under the same conditions as in Example 1-(b). Next, 5.34.9 (10 mmol) of the soybean-derived (hydrogenated) 1-acyl & -L-3-glycerophosphorylcholine obtained in Example 4-(a) was added to this reaction solution.
The mixture was stirred at room temperature for 1 hour.

After completion of the reaction, treatment was performed in the same manner as in Example 1-(b) to obtain the desired soybean-derived (hydrogenated) 1-acyl-2-(2
Then, 3.9 g (yield: 49 g) of 4E-octadecajenoyl)-L-3-glycerophosphorylcholine was obtained.

The TLC analysis results of this substance showed a single sporatra as in Examples 1.2 and 3, which was consistent with that of soybean 7tin. Also, the optical rotation was as follows.

[α)29'= + 6.46 (CHCL3.C
= 1.08) Furthermore, it was confirmed from the analysis results similar to those of Examples 1, 2, and 3 as shown below that this substance was a pure substance.

13C-NMB spectrum δ value (cncz,, p
pm) 14.2, 22.8.25. O,=29.32
．． 0,33.1.34.24゜54.53.59.4.
63.1.63.6.66.5.70.8,118.6
゜128.3, 145.7, 146.1, 166
．． 5, 173.6FT-IR spectrum (crn):
See Figure 4 (NaCt disk) 1716 (
'c=o) 1641 , 1616 (Sh, = 0) 1246 (Sh P-0) 1140 (Shear 0-C) 1090 (Sill - 0θ) 1065 (δp-o-c) 1001 (δ, -IK) W Spectrum (ethanol): λ-, -262.6
(nm) ε-24300 (l-mol-cm)
Reference Example 1 A multilayer liposome water bath solution was prepared according to a conventional method so that the concentration of each of the phospholipid derivatives obtained in Examples 1, 2, and 3 was 10 mM. Furthermore, small unilamellar liposomes were prepared from this G solution by ultrasonication. Hereinafter, this will be referred to as a monomeric liposome.

In addition, when this liposome ρ solution was irradiated with ultraviolet rays in a nitrogen atmosphere using a high-pressure mercury run f (manufactured by Riko Kagaku Sangyo Co., Ltd.) and the change over time of the characteristic absorption spectrum in the ultraviolet region was measured, λrrllLx was 262 ( The absorption peak around 193 nm decreased and disappeared completely after about 1 hour, while the absorption around 193 nm increased. From this, it was confirmed that the polymerization was completed. Hereinafter, this will be referred to as a polymeric liposome.

Transmission electron microscopy using negative staining using uranyl acetate revealed that there were almost no morphological changes between monomeric liposomes and d-phosphoric liposomes. This was also confirmed by comparing the glue filtration patterns. Furthermore, the turbidity change at room temperature is
It was found that polymeric liposomes are much smaller than monomeric liposomes and have superior storage stability.

Reference Example 2 Monomeric and polymeric liposomes made of the egg yolk-derived monotene lecithin obtained in Example 2 and a similar glue made of dinoene lecithin? Measurements were made using a differential scanning calorimeter. As a result, in liposomes such as dinoene lecithin, the heat absorption peak (around 22°C) observed in monomeric liposomes almost completely disappeared (ΔH (kc
al/mol) decreased from 5.90 to 1.18). In contrast, liposomes made of monodiene lecithin
It was confirmed that even in the polymeric liposome, the heat absorption peak (near 29°C) that existed in the monomeric liposome did not completely disappear and remained.

Reference Example 3 Regarding liposomes prepared from the phospholipid derivatives obtained from Examples 1, 2.3 and 4, using glucose and 5(6)-calcexyfluorescein as encapsulation markers,
The retention ability and release behavior of the marker were investigated. As a result, for each lipid, polymeric liposomes had a lower release rate and improved retention capacity than monomeric liposomes. Additionally, heat, organic solvents (e.g. ethanol) and surfactants (e.g. Triton
100rSDS) was improved.

Furthermore, monomeric IJ and N-some marker retention ability at room temperature, which was hardly observed in dinoene lecithin, was observed in monodiene lecithin monomeric liposomes.

〔Effect of the invention〕

As is clear from the above description, according to the present invention, the desired polymerizable phospholipid derivative can be easily prepared from synthetic lecithin or natural lecithin, and can be obtained with high purity in a single reaction process from zolecithin and long-chain diene caldic acid. can be synthesized in high yields and economically produced on an industrial scale.

Furthermore, the monodiene lecithin of the present invention can be obtained as a whole, and its structure and composition are closest to natural lecithin compared to dinoene lecithin, making it safer and more biocompatible. In addition, it is a lecithin-type photopolymerizable phospholipid, which is a cylindrical molecule with a well-balanced hydrophobic group and hydrophilic group that can form a bilayer membrane structure even when used alone.

This phospholipid can be quickly polymerized by ultraviolet irradiation under mild conditions in a short period of time, whether below Tc or above Tc (see Reference Example 1). The polymerized bilayer membrane has improved stability and ability to retain inclusions, as well as improved resistance to surfactants, organic solvents, and temperature changes (see Reference Example 3). Further, even after polymerization, the liquid crystal phase transition of the liquid crystal does not disappear due to the inherent properties of lipid membranes (see Reference Example 2), making it suitable for forming flexible membranes.

Therefore, it is a photopolymerizable phospholipid derivative suitable for application fields such as medical liposomes that require consideration of biocompatibility and various compatibility with biomembranes, as well as other biosensors, microcapsules, and biomedical materials. It is expected that it will be used in a wide range of fields such as biochemistry, medicine, pharmacy, and engineering, including applications in cosmetics, not only in the form of bilayer membrane vesicles, but also in the form of monolayer membranes and stacked membranes. , its industrial value is great.

[Brief explanation of drawings]

Figures 1, 2, 3 and 4 show FT of the polymerizable phospholipid derivatives obtained in Examples 1.2.3 and 4, respectively.
- The measurement results of the IR spectrum are shown. L) 000 3200 2qoo
IGOOto. ↓ Circle number - (cy one figure 1 1 + OO0320024001cOO960 once L
<C party -1) Figure 2'+000 3200 2→00
1GOOto. 'JL number-(C package-1) Figure 3 Li4) Go '3200 2400
1GOO900 Agricultural Reform (Keri Figure 4)

Claims (6)

[Claims] (1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. A mixed acid type polymerizable phospholipid derivative represented by R^0CH=CHCH=CHCO (where R^0 represents an alkyl group of C_5 to C_1_7). (2) The mixed acid type polymerizable phospholipid derivative according to claim (1), wherein the fatty acid residue is selected from acyl groups derived from egg yolk phospholipids or soybean phospholipids. (3) The mixed acid type polymerizable phospholipid derivative is 1-acyl-
The mixed acid type polymerizable phospholipid derivative according to claim (1) or (2), which is 2-(2,4-alkadienoyl)-L-3-glycerophosphorylcholine. (4) The mixed acid type polymerizable phospholipid derivative is 1-(2,4
-alkadienoyl)-2-acyl-L-3-glycerophosphorylcholine. The mixed acid type polymerizable phospholipid derivative according to claim (1) or (2). (5) R^0CH=CHCH=CHCOOH (here R
^0 is an alkyl group of C_5 to C_1_7) 1-acylimidazole obtained by reacting a dienecarboxylic acid represented by C_5 to C_1_7 alkyl group with N,N'-dicarbonyldiimidazole is reacted with 1- or General formula characterized by reacting 2-monoacyl-L-3-glycerophosphorylcholine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, R^1 and R^2 are different from each other, and one of them is saturated. Alternatively, it represents an unsaturated C_1_0 to C_2_2 fatty acid residue, and the other represents an acyl group of R^0CH=CHCH=CHCO (here, R^0 is an alkyl group of C_5 to C_1_7). ] A method for producing a mixed acid type polymerizable phospholipid derivative represented by (6) Claim (5), wherein the monoacyl-L-3-glycerophosphorylcholine is obtained by hydrolyzing synthetic or natural diacyl-L-3-glycerophosphorylcholine with phospholipase A_1 or A_2. manufacturing method.