JPH01294701A - Oligosaccharide ester of fatty acid - Google Patents

Abstract

PURPOSE:To attempt to utilize the title compd. in imparting a cytophilic property and preventing aggregation of phospholipid endoplasmic reticulum as a modifying agent for phospholipid aggregate by specifying the degree of polymn. of saccharides and the degree of substitution of acyl groups. CONSTITUTION:An oligosaccharide ester of a fatty acid is prepd. by esterifying an oligosaccharide having a degree of polymn. of 2-30, such as glucan, fructan, xylan, galactan, mannan, chitin, or chitosan with a 12-22C satd. fatty acid or a fatty acid having 1-4 unsatd. bonds in the range of degree of substitution of 1-4. This saccharide ester is useful as a modifying agent for phospholipid aggregate, an aggregation preventing agent for phospholipid endoplasmic reticulum, a fusion preventing agent for phospholipid endoplasmic reticulum, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a newly synthesized oligosaccharide fatty acid ester with a well-defined structure and a method for utilizing this ester in phospholipids.

Phospholipids self-assemble to form monolayers, bilayers, multilayers, or endoplasmic reticulum made of these. These are used for ultra-thin films, solid surface modification, hydrogels, carriers, etc., and by introducing the oligosaccharide fatty acid ester of the present invention, various functions can be imparted to them. For example, if this oligosaccharide fatty acid ester is introduced into a phospholipid monolayer with a fixed orientation, it will not only be possible to control the surface hydration state and hydrophilic gel region, but also to
It is also expected to control interactions with biomolecules, cells, etc., and can be applied to biosensors, cell culture bed modifiers, etc.

[Prior art and the problem to be solved by the invention] There are many methods for immobilizing the tang structure on the solid phase surface, but in order to operate effectively with a small amount, it is necessary to immobilize it on the solid phase surface with a covalent bond. There are known ways to fix it. However, this method requires
There are problems such as complicated processing and restrictions on surface chemical structure.

On the other hand, in connection with genetic engineering and cell engineering, the cultivation of many types of bacteria and cells, including Escherichia coli, has become important. In the past, various innovative culture bed designs have been developed, such as glass lining, polymer lining, and even ceramic lining. However, the culture effect has not yielded the desired results, mainly due in part to the lack of hydrophilicity between the culture bed or culture tank surface and the cell surface.

In addition, endoplasmic reticulum (liposomes), which are an example of phospholipid aggregates, are attracting attention as cell models or capsule materials, but in reality they are unstable, so the use of phospholipids alone is not suitable for use as microcapsules. do not have. Therefore, attempts have been made to improve the strength of the membrane by embedding cholesterol, proteins, etc. In these embedding examples, although the membrane strength is improved, it is necessary to introduce about 20% or more of cholesterol. However, if more than 20% cholesterol is introduced, the characteristics originally possessed by the phospholipid endoplasmic reticulum (for example,
gel-liquid crystal phase transition, changes in substance permeability in response to specific stimuli, etc.), and further restrictions on the preparation method (e.g., inability to prepare small-diameter phospholipid vesicles, inability to use organic solvents, etc.) It was only available for limited use. In addition, these phospholipid vesicles are susceptible to morphological changes such as fusion and aggregation after preparation, and there are problems with stability over time when commercialized and stability of dispersion in blood when administered by intravenous injection. As a solution to these problems, there is a method of adding a charged substance such as phosphatidic acid to the endoplasmic reticulum, but this method is not yet sufficient. On the other hand, water-soluble polymers are also used as additive stabilizers, but when phospholipid vesicles are used for in vivo administration, the addition efficiency for stabilization is low because many of these substances are dissolved with a high degree of freedom. Even with polymers with adjusted solubility, there were problems with biocompatibility and metabolism.

[Means to solve the problem]

The present invention aims to solve the problems seen in the prior art described above. That is, the present invention provides the following
This invention provides the following invention.

(2) An oligosaccharide fatty acid ester having a degree of sugar polymerization as an integer of 2 to 30 and a degree of acyl group substitution in the range of 1 to 5.

(2) The oligosaccharide fatty acid ester according to claim 1, comprising an acyl group having 12 to 22 carbon atoms and consisting of a saturated aliphatic chain or an unsaturated aliphatic chain containing 1 to 4 unsaturated bonds.

(2) A modifier for phospholipid aggregates comprising the oligosaccharide fatty acid ester according to claim 1 or 2.

(2) An anti-aggregation agent for phospholipid vesicles comprising the oligosaccharide fatty acid ester according to claim 1 or 2.

(2) A fusion inhibitor for phospholipid vesicles comprising the oligosaccharide fatty acid ester according to claim 1 or 2.

(2) A surface fixing agent for phospholipid membranes comprising the oligosaccharide fatty acid ester according to claim 1 or 2.

A more detailed explanation will be given below.

The method for producing the oligosaccharide fatty acid ester of the present invention is as follows.

Polymerization irregularities and type-specific fractionation of oligosaccharides, whose degree of sugar polymerization is an integer between 2 and 30, are performed using gel filtration with a heated column and a differential refractometer as a detector to determine the purity. High (can be done.

To confirm the quality and purity of the separated oligosaccharide, use a silica gel thin layer plate and develop it multiple times with a mixed solvent (1-butanol, pyridine, water) and develop the color with sulfuric acid, or mix using a Yamino column as the stationary phase. This can be carried out by subjecting it to liquid chromatography using a solvent (acetonitrile/water) as a mobile phase.

It should be noted that the oligosaccharide used in the present invention may be a commercially available pure product as long as the structure is clear.

The means for obtaining the oligosaccharide fatty acid ester includes (1) adding an organic base catalyst such as pyridine and a basic polymer catalyst such as vinylpyridine-styrene copolymer to the oligosaccharide obtained as described above; In the presence of
Method of adding fatty acid chloride or fatty acid anhydride, (2)
, a method in which oligosaccharide is dissolved in N,N-dimethylformamide, dimethyl sulfoxide, etc. and transesterified with fatty acid methyl, etc., and (3).

There is a synthesis method using lipase, but any method may be used.

After the reaction of (11, (2) or (3)) is completed, the solvent is distilled off under reduced pressure, impurities are dissolved in a nonpolar solvent such as diethyl ether or hexane, filtered, and the residue is dried. Crudely purified product.

The oligosaccharide fatty acid ester of the present invention has different polarity, solubility in various solvents, and affinity for various column carriers, mainly due to differences in the type and degree of substitution of fatty acids, and differences in degree of sugar polymerization. Because they have different sexes, it is necessary to separate and separate them so that they have a single character.

That is, the crude product of the oligosaccharide fatty acid ester described above is subjected to liquid chromatography using a reversed-phase column as a stationary phase and a polar solvent such as water, alcohol, or tetrahydrofuran as a mobile phase. By this method, the crude product is eluted in order of decreasing degree of substitution, so it is possible to separate fractions of each peak. Fractions can be confirmed using Fourier transform nuclear magnetic resonance measurements (hereinafter referred to as FT-
It is abbreviated as NMR. ).

The combination of polar solvent and reversed phase column types is as follows:
It differs depending on the type of oligosaccharide fatty acid ester to be separated and fractionated.

In this way, by using a reversed-phase preparative chromatograph, it is possible to obtain a large amount of oligosaccharide fatty acid ester with a clear structure and high purity.

The separation and fractionation of the oligosaccharide fatty acid ester of the present invention is not limited to the above-mentioned methods, but also includes, for example, normal phase liquid chromatography using silica gel as a stationary phase, supercritical chromatography, or solvent extraction. You can also choose your method.

The quality and purity of the oligosaccharide fatty acid ester obtained as described above is confirmed by subjecting it to reversed-phase analytical affinity chromatography or thin layer chromatography under the same conditions as liquid chromatography. be able to.

The method for producing phospholipid vesicles and phospholipid monolayers in the present invention is a commonly used one-ship method, and is not particularly special.

Oligosaccharides used in the present invention include glucan, fructan, xylan, galactan, mannan, chitin,
Among sugars of the chitosan series and sugars with different bonding modes, it is a polysaccharide whose degree of sugar polymerization is expressed as an integer between 2 and 30, and it also contains a sugar alcohol.

Examples of fatty acids used in the present invention include lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, and behenic acid as straight chain saturated fatty acids. In addition, there are linear unsaturated fatty acids, branched saturated fatty acids, and branched unsaturated fatty acids known as oleic acid, linoleic acid, lilunic acid, etc. Any fatty acid can be used as long as it has 22 fatty acids.

Table 1 shows the structures of typical oligosaccharide fatty acid esters produced as samples in the present invention. Correlation between sample number and structure of maltooligosaccharide fatty acid ester The structure of maltooligosaccharide is α-1,4-glucan.

Sample numbers 28-30 are maltopentaose 2,4-octadecadienate.

The structure of sophorooligosaccharide is β-1,2-glucan.

The structure of laminario oligosaccharide is β-1,3-glucan.

Table 4. Correlation between sample number and structure of cellooligosaccharide fatty acid ester The structure of cellooligosaccharide is β-1,4-glucan.

Table 5. Correlation between sample number and structure of isomaltooligosaccharide fatty acid ester The structure of isomaltooligosaccharide is α-1,6-glucan.

Table 6. Correlation between sample number and structure of inulooligosaccharide fatty acid ester The structure of inulooligosaccharide is β-2,1-fructan.

It is a genenate.

They are summarized in Tables 1 to 8 according to the tucharide structure.

[For production]

The oligosaccharide fatty acid ester of the present invention has very good affinity with phospholipids and is therefore suitable as a phospholipid modifier. Furthermore, the oligosaccharide fatty acid ester fixed to the phospholipid can keep the sugar structure exposed to the aqueous phase and can be uniformly dispersed on the membrane.

The oligosaccharide fatty acid ester of the present invention as a modifier for phospholipid aggregates is a compound with an adjusted hydrophilic and hydrophobic balance, so it can be used for various purposes (e.g., making cells parent, preventing phospholipid endoplasmic reticulum aggregation, preventing membrane fusion. , recognition labeling with specific antibodies, etc.). Both are sufficiently effective when added in small amounts, and do not change the basic physical properties of the phospholipid aggregate.

Moreover, the oligosaccharide fatty acid ester of the present invention is
This is ideal for use in combination with polymerizable phospholipids, for example, by coating and polymerizing them on the surface of a culture container to convert the container surface into a hydrophilic, cell-philic surface. This provides a surface on which dramatic culture effects not previously seen can be observed.

〔Example〕

Specific examples will be described below.

"Production of oligosaccharide fatty acid ester" polymerization degree 5
To 1 mole of maltopentaose, 7.5 times the mole of special grade and lysine and 100 times the mole of special grade N,N-dimethylformamide were added and dissolved. To this, 2.5 times the mole of special grade balmitic acid chloride (acyl chain length 16) was added dropwise to initiate the reaction. This reaction was carried out in an anhydrous system, and the reaction was carried out at 50° C. for 24 hours with stirring. This reaction solution was heated to 50°C.
, pyridine and N,N-dimethylformamide were distilled off under reduced pressure. Next, 1. In order to elute and remove impurities such as valmitic acid, the mixture was washed repeatedly with hexane and then filtered. The obtained residue was dried under reduced pressure at 50°C to obtain a crude product.

This crude product was fractionated using a column (Hibercolumn Ricrosolve RP-2, manufactured by Merck & Co., Ltd.). A 9:1 mixed solvent of methanol and water was used as the mobile phase. A differential refractometer was used for detection.

Each peak separated by the column was fractionated, and each peak was
The structure was confirmed by T-NMR.

As a result, as shown in the chromatogram shown in Figure 1, which shows a degree of substitution of maltopentaose palmitate of 4 or more, maltopentaose (degree of substitution 0(1)), maltopentaose monopalmitate (degree of substitution 1(2)) ), maltopentaose dipalmitate (degree of substitution 2 (3)), maltopentaose tripalmitate (degree of substitution 3 (4)), and then maltopentaose palmitate with a degree of substitution 4 or higher (5) were eluted in this order. It has become clear that it is also possible. By utilizing this knowledge, maltopentaose monopalmitate, maltopentaose dipalmitate, and maltopentaose tripalmitate can be separated from the reaction mixture with high precision.The separation and fractionation conditions are as described above. Not only Waters' Micro Bondasphere C8 and Shiseido's Capsule Back C1B can be used as columns,
Single or double mobile phase of tetrahydrofuran, 1-propanol, 2-propanol, methanol, water
Separation is also possible using a mixed solvent of more than one species.
A plurality of peaks are seen at the substitution degree of 2 (3) and the substitution degree of 3 (4), but these are all isomers mainly due to differences in the substitution positions.

Regarding the synthesis of other oligosaccharide fatty acid esters, 1 to 3 times the mole of special fatty acid chloride and 2 to 15 times the mole of special grade pyridine are added to 1 mole of oligosaccharide, and the mixture is kept at 70°C or lower for 12 hours or more. A crude product was obtained by the same method as above.These were fractionated in the same way, and each structure was confirmed to have the desired degree of substitution using FT-N.
Confirmed by MR.

However, for the synthesis of unsaturated fatty acid esters, this operation was performed under a nitrogen stream. In addition, the reaction temperature was 40°C
The reaction was allowed to proceed for 24 hours or more while being stirred, taking care not to exceed 24 hours, to ensure sufficient esterification. It was confirmed by FT-NMR that the deterioration of unsaturation in the thus obtained oligosasocalide unsaturated fatty acid esters was 3% or less in all cases.

Modification of phospholipids with oligosaccharide fatty acid esters” Example 1゜Maltopentaose monopalmitate (sample number 1) in which one molecule of palmitic acid is introduced into maltopentaose (pure product) with a degree of polymerization of 5 (sample number 1) 3■ and phospholipids Dipalmitoylphosphatidylcholine (hereinafter abbreviated as DPPC)
After dissolving 100 μm in methanol and mixing, a thin film was formed on the inner wall of a test tube using an evaporator. 5ml
of pure water and several glass beads were added, and the mixture was stirred at 50° C. using a portex to obtain a cloudy white liquid. This cloudy liquid was treated with an extruder. The pore size of the extruder filter is O.

4 μ lock, 0.2 μ steel, 0, 1 μ■,
A white transparent PPC endoplasmic reticulum dispersion was obtained by passing the solution 5 times each while changing the concentration to 0.05 .mu.-. The average particle diameter of the endoplasmic reticulum in this state was calculated from a negatively stained transmission electron microscope (hereinafter abbreviated as TEM) photograph, and was 55 n- in diameter.

After diluting the endoplasmic reticulum dispersion to 0.5% by weight, gel filtration (Pharmacia, Sepharose CL-4B, 20
After removing freely dissolved sugar fatty acid esters from the system, the fraction containing the endoplasmic reticulum was colored with sugar by the phenol-sulfuric acid method, and sample No. 1 was introduced into the endoplasmic reticulum. determined the rate. As a result, it was revealed that in sample number 1, about 50% of the additive was taken into the endoplasmic reticulum.

The morphological stability of the endoplasmic reticulum dispersion that had passed through a 0.05μ filter was evaluated by measuring the change in absorbance (turbidity) at 500 nm when the dispersion was allowed to stand at room temperature. In the endoplasmic reticulum without the addition of oligosaccharide fatty acid ester (control), the turbidity increased rapidly, and particle size distribution measurements showed that the particle size of the endoplasmic reticulum increased over time, indicating aggregation and fusion of the endoplasmic reticulum. It was confirmed. In the system in which 2 mol% of Sample No. 1 was added to the lipid, it was revealed that the dispersion state was kept stable and there was no change over time. When the diameters of these endoplasmic reticulum were measured using TEM photographs, it was found that in the control, 74n-
However, no change in particle size was observed in the system in which sample number 1 was introduced.

Sample No. 1/70 μl of a phospholipid vesicle dispersion having a composition of DPPC (1 morph: 50 mol) was sealed in an aluminum differential scanning calorimeter container, and differential scanning calorimeter measurement was performed. gel-
When the liquid crystal transition temperature was observed, an endothermic peak was observed at 41° C. similar to that of the DPPC vesicle, and no broadening of the peak was observed. From this, it was confirmed that even if the oligosaccharide fatty acid ester was introduced in an amount of about 2 mol %, it did not affect the transfer of the membrane at all. In addition, the introduction of oligosaccharide fatty acid ester into the DPPC endoplasmic reticulum was confirmed using 0.04% by weight of jack bean lectin (hereinafter referred to as
It is abbreviated as Con A. ) was also confirmed by the aggregation of endoplasmic reticulum.

Example 2 Samples Nos. 2 to 63 listed in Tables 1 to 8 were embedded in DPPC endoplasmic reticulum using the same method as in Example 1, and their ability to prevent aggregation and fusion was examined. Each of the above samples was introduced in an amount of 5 mol % relative to the lipid, and the results obtained by measurement are summarized in Table 9 together with the results of Example 1.

Sample number 2, 3.10~14.19~28°31.34
, 35.37-41, 43.46°49.52.54.
56 to 63 were added to the endoplasmic reticulum dispersion as powder, and 50
Embedding was possible by simply leaving it at ℃ for a few hours.

Sample number 2.3, 28.31, 40, 43°46.49
, 52, 54, and 56 have no effect on preventing endoplasmic reticulum aggregation when introduced at 1 mol % or less, but sample numbers 10 to 15.
24-27.60 Table 9. Explanation of the sample number and introduction effect code of oligosaccharide fatty acid ester introduced into the DPPC endoplasmic reticulum: ■ indicates extremely effective; O indicates effective; △ indicates somewhat effective; × indicates no effect show.

showed a preventive effect even at 0.2 mol%. For prevention of fusion, a smaller amount is sufficient.

Note that for oligosaccharides with a small degree of polymerization, for example, oligosaccharides with a degree of polymerization of 2 to 3, the degree of substitution is 3.
Above that, the rate of introduction into the endoplasmic reticulum decreased. Therefore,
For those with a small degree of polymerization, the degree of substitution is 1 or 2.
Oligosaccharide fatty acid esters with a reduced content are suitable for preventing endoplasmic reticulum fusion and aggregation.

In addition, the introduction of oligosaccharide fatty acid ester into the DPPC endoplasmic reticulum was confirmed using sample numbers 2-15 and 28-30.
Aggregation of endoplasmic reticulum by adding 0.04% by weight of Con A for samples No. 46 to 48, and 0.04% by weight of wheat germ lectin (hereinafter abbreviated as WGA) for sample number 56.57. It was also confirmed.

Example 3 Sample numbers 1 to 5 were prepared in the same manner as in Example 2 using egg yolk lecithin (hereinafter abbreviated as EYL) as the phospholipid.
7 was embedded in the endoplasmic reticulum and its effect was evaluated. Although the effects were almost the same, differences were observed in the following points.

In Example 2, fusion and aggregation measurements were performed at room temperature, but since EYL endoplasmic reticulum is difficult to aggregate at room temperature, measurements were performed at 4°C. If EYL is an ethanol solution product, it will develop color using the phenol-sulfuric acid method if ethanol is incompletely removed during endoplasmic reticulum preparation. Therefore, as a result of calculating the introduction rate for a system that was carefully adjusted, Table 1
Almost the same results were obtained. This indicates that the introduction rate and performance of the oligosaccharide fatty acid ester depend on the structure of the sugar fatty acid ester, regardless of the phospholipid structure. Introduction of oligosaccharide fatty acid ester into the endoplasmic reticulum was performed using sample numbers 1-15゜28-
30.46-48, 0.04% by weight Con
Add A, and in the case of sample number 56°57, 0.04
It was also confirmed from the aggregation of endoplasmic reticulum by adding % by weight of WGA.

Example 4゜ Sample numbers 28, 29.30 were polymerized phospholipid 1,
The mixture was introduced into a vesicle made of 2-bis(2,4-octadecagenoyl)glycerophosphocholine, and a radical initiator such as azobisisobutyronitrile was added thereto, followed by heating and polymerization. The polymerized vesicles obtained here were copolymers with oligosaccharide fatty acid esters, and stably supported the tank structure. These are the product name “Triton
-100J, sodium cholate, and other surfactants, and even when washed with organic solvents such as methanol, the shape was maintained extremely stably. Since this endoplasmic reticulum was aggregated by Con A, stable coverage of the wi chain was confirmed.

Example 5 Sample number 56.57 was introduced into the endoplasmic reticulum composed of 1,2-bis(2,4-octadecagenoyl)glycerophosphocholine according to Example 4, and radicals such as azobisisobutyronitrile were introduced into the endoplasmic reticulum. After adding the initiator, it was heated and polymerized. The polymerized vesicles obtained here stably support oligosaccharide fatty acid esters, and can be washed with surfactants such as the product name "Triton X-100J" and sodium cholate, and with organic solvents such as methanol. The endoplasmic reticulum maintained its shape extremely stably even when exposed to WGA.
The stable coating of sugar chains was confirmed by aggregation.

Example 6 Sample No. 28.29.30 was introduced into a phospholipid monolayer consisting of 1,2-bis(2,4-octadecagenoyl)glycerophosphocholine, and azobisisobutyronitrile, etc. After adding a radical initiator, a polymerized phospholipid thin film was obtained by heating and polymerizing. These membranes have oligosaccharide fatty acid esters fixed with covalent bonds, so they are prepared as membranes whose surfaces are stably modified with sugar chains. In such systems, sugars do not elute into water at all. Ta. Furthermore, it was revealed by fluorescence microscopy observation using Con A labeled with a fluorescent probe that this membrane surface was easily recognized by lectin.

Example 7 Sample No. 56.57 was introduced into a phospholipid monolayer consisting of 1,2-bis(2,4-octadecagenoyl)glycerophosphocholine. Furthermore, after adding a radical initiator such as azobisisobutyronitrile, the mixture was heated and polymerized to obtain polymerized phospholipid ffflll. These membranes have oligosaccharide fatty acid esters fixed by covalent bonds, so they are prepared as membranes whose surfaces are stably modified with sugar chains. Fluorescence microscopy observation using WGA labeled with a fluorescent probe revealed that in such a system, sugar does not elute into water at all, and that this membrane surface is easily recognized by lectin.

Example 8゜Sample numbers 28, 29.30 were treated with 1-valmitoyl 2-(
2,4-octadecagenoyl)glycerophosphocholine is introduced into the endoplasmic reticulum and copolymerized with ultraviolet irradiation or a radical initiator to prepare a phospholipid/oligosaccharide fatty acid ester copolymer with sugar chains introduced. did. After freeze-drying, a thin film can be easily obtained by dissolving this in an organic solvent such as chloroform, coating it on a glass plate, and drying it in a nitrogen stream, or redispersing it in water to remove sugar chains. Endoplasmic reticulum exposed to the aqueous phase was obtained. This endoplasmic reticulum structure was confirmed by TEM. The agglutination reaction due to the action of lectin was also confirmed in the same way.

Example 9 As examples of oligosaccharide fatty acid esters according to the present invention, sample numbers 1, 4, 18, 28 and 29 were used as polymerizable phospholipid 1゜2-bis(2,4-octadecagenoyl), respectively. ) It was mixed with glycerophosphocholine so that the sugar portion was about 2 mol% of the total. Using this, the inner surface of a typical container used for cell culture was coated with a monomolecular film. For examples of sample numbers 28 and 29, a radical initiator was further added and Table 10. The effect of coating with a phospholipid molecular film containing oligosaccharide fatty acid esters in E. coli culture increased the stability of the polymerized system.Next, the culture medium was placed in a culture vessel coated with a phospholipid monomolecular film containing these oligosaccharide fatty acid esters. The cells were filled with E. coli and cultured at 37° C. for 48 hours. The bacteria after culture were collected and the number of surfaces was compared by turbidimetry, and the results shown in Table 10 were obtained.

A higher culture efficiency was observed compared to the uncoated example. The culture efficiency was expressed as a multiplier of 10 for the number of E. coli cultured in each culture device, with the number of E. coli cultured in the non-coated culture device being 1.

Example 10゜Sample number 1, 4, 10.13.19.22゜61.62
．． Regarding No. 63, the effect of preventing endoplasmic reticulum fusion and aggregation was evaluated using the method of Example 1, using human plasma at 25° C. instead of pure water. As a result, the effect of preventing fusion and aggregation was observed in all samples compared to the additive-free system.

2I had no effect in pure water! It was found that maltose monopalmitate ester (sample number 62), a derivative, has the effect of preventing phospholipid endoplasmic reticulum fusion and aggregation in plasma.

[Test example]

The membrane stability of the oligosaccharide fatty acid ester-introduced phospholipid vesicles shown in Examples was tested by releasing carboxyfluorescein (hereinafter abbreviated as CF).

That is, the membrane stability of phospholipid vesicles into which oligosaccharide fatty acid esters were introduced was evaluated by measuring the release of CF encapsulated in endoplasmic reticulum using DPPC as the phospholipid. The performance was compared with that of

4ml to 1+wl of 0.5% phospholipid vesicle dispersion
100 mM CF (pI-I8.6) was added to encapsulate CF in the phospholipid endoplasmic reticulum. The connotation is previously reported (Ma
kromol Chem, Rapid Co5u+un
8+215 (1987)) Next, unencapsulated CF was removed by gel filtration, and then fluorescence measurement was performed. That is, 3111 Tris Buff Fur (50m
After adding 300 μl of the sample into M, pH 8,6),
The increase in fluorescence intensity accompanying the emission of CF was measured (excitation wavelength - 330 nm, fluorescence wavelength - 520n+++), at which time:
A surfactant (trade name: TRITON

As an example, sample number 10.13 was added to phospholipid endoplasmic reticulum (
Figure 2 shows the results of comparing the CF release behavior in the systems 7) and (8) with that of the phospholipid vesicles without oligosaccharide fatty acid ester (6).

As shown in this figure, the addition of oligosaccharide fatty acid ester suppresses the release of CF, no disturbance of the lipid packing structure is observed, and it is clear that more stable phospholipid vesicles can be obtained. Ta. This result was also observed in systems to which other samples were added.

As the amount of oligosaccharide fatty acid ester added increased, the CF release rate decreased and then increased again. Therefore, it was confirmed that there is an addition ratio of oligosaccharide fatty acid ester that is most effective in suppressing CF release. Generally this value is 50 molar ratios, as used in this study.
: It was around 1.

〔Effect of the invention〕

B, II long-chain fatty acid esters and polysaccharide long-chain fatty acid esters, which are amphipathic polymers, have low biotoxicity and are therefore widely used in cosmetics, food additives, and even protein solubilizers. There is. Rather than functional molecules, these can be thought of as materials in which a characteristic chemical environment can be set, and the amount used to derive the properties (the biochemical properties of such polysaccharide long-chain fatty acid esters) For example, Professor Shomoto and his colleagues at Nagasaki University are attempting to introduce long-chain fatty acids into polysaccharides with large molecular weight through ester bonds and use them as artificial cell walls and stabilizers for phospholipid endoplasmic reticulum. (Po17mers in Medicine)
”, Plesum Press (1983) p1
57) However, compared to such a polysancharide-coated vesicle system, the oligosaccharide fatty acid ester of the present invention is sufficiently effective just by adding a small amount to the phospholipid vesicles, so that the original small It is distinctive in that it maintains the basic physical properties of the cell body.

On the other hand, polysancalide fatty acid ester with a high degree of polymerization has its I! Since precise separation and purification according to chain length and degree of fatty acid ester substitution is difficult, its structure is unclear, and the degree of sugar polymerization and degree of fatty acid ester substitution are all average values. On the other hand, the oligosaccharide fatty acid ester of the present invention can be obtained as a pure product, and the degree of polymerization, degree of fatty acid ester substitution, etc. can be strictly specified, so the sugar chain length, type of fatty acid, and degree of substitution can be changed. By doing so, various physical properties such as the properties and solubility of the oligosaccharide fatty acid ester can be adjusted, and the oligosaccharide fatty acid ester can be used properly depending on the purpose.

Furthermore, the oligosaccharide fatty acid ester of the present invention is easy to separate and purify, so there is no contamination with impurities, and there are no factors that adversely affect the membrane properties of phospholipids.Many of them have relatively low solubility in water. Because of its low molecular weight, it is stably fixed to lipid assembly membranes. Some oligosaccharides have higher physiological activity than polysaccharides, so if they are immobilized on the surface of phospholipid endoplasmic reticulum, they can also be used as endoplasmic reticulum for recognition on the surface.

The oligosaccharide fatty acid ester of the present invention having an unsaturated bond in the acyl chain can be introduced into a polymerizable phospholipid aggregate and copolymerized by ultraviolet irradiation, addition of an initiator, etc. As a result, oligosaccharide fatty acid esters can be covalently immobilized on polymeric phospholipid aggregates. I like this!
Polymer phospholipid aggregates, in which chain parts are covalently fixed to the surface, are stable against physical and chemical stimuli such as heat, organic solvents, and surfactants, so they can effectively utilize the activity of sugars. For example, polymeric phospholipid monolayers or bilayers to which oligosaccharide fatty acid esters are covalently bonded bind extremely sensitively to lectins, which are agglutinins, and thus serve as sensors for these.

Therefore, oligosaccharide fatty acid esters with such many characteristics are unprecedented functional materials with high added value.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a chromatogram showing maltopentaose palmitate with a degree of substitution of 4 or more, and FIG. 2 is a diagram showing CF release behavior of phospholipid endoplasmic reticulum. The symbols in the figure have the following meanings. 1.1f conversion rate 0.2. Substitution degree 1.3. Substitution degree 2. 4.
Substitution degree 3.5. Substitution degree 4 or higher.

Claims (6)

[Claims] (1) An oligosaccharide fatty acid ester having a degree of sugar polymerization as an integer of 2 to 30 and a degree of acyl group substitution in the range of 1 to 5. (2) The oligosaccharide fatty acid ester according to claim 1, comprising an acyl group having 12 to 22 carbon atoms and consisting of a saturated aliphatic chain or an unsaturated aliphatic chain containing 1 to 4 unsaturated bonds. (3) A modifier for phospholipid aggregates comprising the oligosaccharide fatty acid ester according to claim 1 or 2. (4) An anti-aggregation agent for phospholipid vesicles comprising the oligosaccharide fatty acid ester according to claim 1 or 2. (5) A fusion inhibitor for phospholipid vesicles comprising the oligosaccharide fatty acid ester according to claim 1 or 2. (6) A surface fixing agent for phospholipid membranes comprising the oligosaccharide fatty acid ester according to claim 1 or 2.