JPH09221524A - Functional sugar-chain polymer and method for using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sugar-chain polymer which can be immobilized on a substrate more firmly without fail than the conventional ones. SOLUTION: This polymer is prepared by grafting a sugar molecule and a photoreactive functional group onto the main chain of a vinyl polymer. The photoreactive functional group is desirably an azido group of, e.g. azidobenzene. The polymer is desirably synthesized by copolymerizing a vinyl monomer having a sugar molecule with a vinyl monomer containing a photoreactive functional group.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a functionalized sugar chain polymer. In particular, the present invention relates to a sugar chain polymer having a photoreactive functional group and capable of being reliably immobilized on a substrate or the like by simply crosslinking by light irradiation.

[0002]

2. Description of the Related Art A sugar molecule having a unique three-dimensional structure, in which abundant polar sites and hydrophobic sites are balanced, is deeply involved in the biorecognition mechanism and is It is used in materials such as cell walls and crustacean shells that make up the biological skeleton. The present inventors have focused their attention on the functions of such sugar molecules, have synthesized sugar chain polymers in which sugar molecules have been introduced into polyvinyl polymers, and have conducted vigorous research (eg, Hirofumi Yura, Akaike). Toshihiro, "Cell Culture", Volume 19, 3
17-322, 1993).

The inventors of the present invention have proposed that the surface modification with these sugar chain polymers makes use of the organ-, tissue- or cell-specific properties of sugar. However, in the conventional methods for immobilizing sugar chain polymers, they have been immobilized on a base material by utilizing physical adsorption of a main chain composed mainly of vinyl polymers.

[0004]

In the immobilization by physical adsorption, the material that can be immobilized may be limited depending on the affinity between the vinyl polymer forming the main chain and the surface of the base material. In some cases, the chain polymer is easily desorbed due to environmental changes such as temperature and humidity.

[0005]

In order to solve the above problems, the present invention provides a functional sugar chain polymer in which a sugar molecule and a photoreactive functional group are grafted onto a vinyl polymer main chain. This photoreactive functional group is preferably an azido group such as azidobenzene.

This functional sugar chain polymer can be suitably synthesized by copolymerizing a vinyl monomer containing a sugar molecule and a vinyl monomer containing a photoreactive functional group.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION As an example of the functionalized sugar chain polymer of the present invention, poly (3-azidostyrene-co- {Np-vinylbenzyl- [O-β is represented by the following formula (1). -D-galactopyranosyl- (1 → 4) -D-gluconamide]})
(Hereinafter, abbreviated as Azo-PVLA).

[0008]

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This Azo-PVLA is a poly (N-p-vinylbenzyl- [O-β-D- having a β-galactose residue.
It has a structure in which galactopyranosyl- (1 → 4) -D-gluconamide]) (abbreviated as PVLA) and 3-azidostyrene are copolymerized.

Another example of the functional sugar chain polymer of the present invention is poly (3-azidostyrene-co- {Np-vinylbenzyl- [O-α] represented by the following formula (2). -D-glucopyranosyl- (1 → 4) -D-gluconamide]}) (hereinafter abbreviated as Azo-PVMA).
This PVMA has a glucose residue.

[0011]

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Further, the functional sugar chain polymer of the present invention is, for example, poly (Np-vinylbenzyl- [O-β-D-mannopyranosyl- (1 → 4) -D-mannanade having a mannose residue. ]) (Abbreviated as PVMan); poly (Np-vinylbenzyl- [O-α-D-galactopyranosyl- (1 → 6) -D-gluconamide]] having a galactose residue)
(Abbreviated as PVMeA); poly (Np-vinylbenzyl- [O-6- having a carboxymethylated galactose residue
Carboxymethyl-β-D-galactopyranosyl- (1 →
4) -OD-6-carboxymethyl-gluconamide])
(Abbreviated as PVLACOOH); poly (3-O-4′-vinylbenzyl-D-glucose) having a glucose residue
(Abbreviated as PVG); poly (Np-vinylbenzyl- [O-2-acetamido-2-deoxy-β-D-glucopyranosyl- (1 → 4)-) having N-acetylglucosamine residue
OD-2-acetamido-2-deoxy-β-D-glucopyranosyl- (1 → 4) -OD-2-acetamido-2-deoxy-β-D-gluconamide]), and poly (N- p-Vinylbenzyl- [O-2-acetamido-2-deoxy-β-D-
Glucopyranosyl- (1 → 4) -OD-2-acetamido-
2-deoxy-β-D-gluconamide]), or a mixture thereof (both abbreviated as PVGlcNAc); Np-
Vinylbenzyl- [O-β-D-glucopyranosyl- (1 →
3) -D-Gluconamide] (abbreviated as PVLam); or poly (N- having a structure in which D-glucose is opened.
It may have a structure in which a vinyl-based polymer containing a sugar chain such as p-vinylbenzyl-D-gluconamide) (abbreviated as PVGA) and the polyazidostyrene are copolymerized.

Here, Azo- represented by the above formula (1)
A method for synthesizing PVLA will be described. First, the following formula (3)
As shown in Figure 3, 3-nitrostyrene was azinated to give 3-
Use azidostyrene.

[0014]

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Separately, from p-aminomethylstyrene and lactose, Np-vinylbenzyl- [O-β-D-galactopyranosyl- (1 →
4) -D-Gluconamide]) (abbreviated as VLA) and by copolymerizing this VLA with the 3-azidostyrene, Azo-PVLA can be obtained.

[0016]

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PVMA other than Azo-PVLA,
PVMan, PVMeA, PVLACOOH, PVG,
A functional sugar chain polymer having a PNGlcNAc or PVGA structure can also be obtained by copolymerizing each monomer with 3-azidostyrene.

The functional polymer of the present invention preferably has a photoreactive functional group and is capable of photocrosslinking. This photoreactive functional group does not leave an amino group that may have non-specific interactions with cells etc. after the crosslinking reaction, and is reactive enough to cause the crosslinking reaction easily even without using a catalyst. Preferred are those having a cross-linking reaction which is completed at a temperature of about 120 ° C. from room temperature at a high temperature, those which can be easily introduced into sugar chain polymers, and those which have a versatile and non-limiting reaction. As the functional group that meets such conditions, a functional group that causes photoazo coupling such as an azide group is preferable. Further, instead of the photoreactive functional group, 6
A functional group that crosslinks by heating at 0 to 120 ° C. may be used.

The photoreactive functional group such as the azide group is preferably bound to the vinyl group-based monomer through a covalent bond. This vinyl-based monomer is not particularly limited, but it is preferable to use a styrene monomer when copolymerizing with a monomer containing a sugar chain in which a sugar molecule is bound to aminomethylstyrene as described above. Further, the photoreactive functional group can be easily derived to natural polysaccharides, oligosaccharides, preferably those having an amino group or those derived from an amino group.

The functional sugar chain polymer thus synthesized has a polyvinyl polymer as a main chain, and a sugar molecule and a photoreactive functional group are grafted to the main chain. Therefore,
This functional sugar chain polymer can be dissolved or dispersed in a suitable solvent, and the solution or dispersion can be applied to a substrate, dried if necessary, and then crosslinked by irradiation with light.

The crosslinked functional sugar chain polymer is more firmly and surely immobilized on the substrate as compared with the conventional immobilization by physical adsorption. Therefore, the functional sugar chain polymer of the present invention can be suitably used for firmly and surely immobilizing sugar molecules on the surface of a base material and modifying the surface of the base material.

Examples of the substrate surface-modified with the functional sugar chain polymer of the present invention include various medical materials, clinical diagnostic substrates, solid materials such as carriers for affinity chromatography, and the like. To be Further, by immobilizing the functional sugar chain polymer of the present invention on the surface of a substance that is poorly soluble in water, sugar molecules can be introduced on the surface of the substance and solubilized. Furthermore, by including a non-specifically acting drug with the functional sugar chain polymer of the present invention, local administration of the drug based on the specificity of the sugar molecule, so-called drug
It is also possible to build a delivery system (DDS).

[0023]

Industrial Applicability The functional sugar chain polymer of the present invention contains a sugar molecule having a specific affinity with a living organ, tissue, cell or the like. Surface functionalization of various base materials coated with this functional sugar chain polymer can be achieved.

Since the sugar chain polymer of the present invention has a photoreactive functional group introduced into the molecule, it is more firmly and surely immobilized on the substrate by photocrosslinking via the functional group. can do.

[0025]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1: Synthesis of Azo-PVLA (1) Synthesis of 3-azidostyrene 3-Nitrostyrene (5 g) was dispersed in 10 mL of ethanol, and 20 mL of concentrated hydrochloric acid was added, followed by tin chloride (6). Hydrate) (22.5 g / 25 mL) was added, and the mixture was stirred for 12 hours. Sodium hydroxide solution was added to this to neutralize it, and then ether was added for extraction. The ether layer was dried over magnesium sulfate and chlorine gas was added to obtain a crude compound.

Then, 20 mL of 2.4 g of this crude compound was added.
2 ml of concentrated sulfuric acid was added to the solution of
A solution of L 1N sodium nitrite was added. Three hours later,
200 g of an aqueous solution of 1.6 g of sodium azide was slowly added and reacted for 5 hours. After completion of the reaction, extraction with ether was carried out, washing with a 0.1N sodium hydrogen carbonate solution four times was performed, and then the ether layer was dried and the solvent was distilled off. The residue is silica gel column (chloroform: hexane = 20:
80) to obtain 0.8 g of 3-azidostyrene (Rf = 0.85).

(2) Synthesis of Azo-PVLA The above 3-azidostyrene (10, 5 mol%) and V
Azo-PVLA, which is an example of the functional sugar chain polymer of the present invention, was synthesized by copolymerizing with LA (90, 95 mol%). The IR spectrum of this Azo-PVLA is shown in FIG. An absorption peak peculiar to an azide group is seen near 2010 cm ^-1 .

Example 2: Synthesis of Azo-PVMA Np-vinylbenzyl- [O-α-D-glucopyranosyl- (1 → 4) -D-gluconamide] (V
Azo-PVM, which is another example of the functional sugar chain polymer of the present invention, is carried out in the same manner as in Example 1 except that MA is abbreviated).
A was synthesized. The IR spectrum of this Azo-PVMA is shown in FIG. An absorption peak peculiar to an azide group is seen near 2010 cm ^-1 .

Example 3 Azo-PVLA was dissolved in a small amount of water and then added with 0.0 each.
It diluted with the mixed solvent of isopropanol: water (7: 3) so that it might become the density | concentration of 5, 0.025, and 0.005 weight%. A vinyl chloride (vinyl chloride) sheet was molded into a circular shape having a diameter of 20 mm, washed with ethanol, and then fixed on the bottom surface of a 12-well cell culture dish using a silicone resin. 250 μL of each Azo-PVLA solution of each concentration was added to this petri dish and cast at 60 ° C. for 1 hour. Then, these cast films were irradiated with UV irradiation for 3 hours at 15 cm under a sterilizing UV lamp. After the cast membrane was washed several times with distilled water, each petri dish was immersed in distilled water in a beaker and sonicated with a sonicator for cleaning equipment for 20 minutes.

A 100 μg / mL BSA solution was added to these petri dishes, left for 15 minutes, and then thoroughly washed with PBS. After that, β- contained in Azo-PVLA
All, a lectin that specifically binds to galactose
P containing 1 μg of peroxidase-modified oA
BS was added to each dish and incubated at 37 ° C for 30 minutes. 3,3 ', 5,5'-Tetramethylbenzidine (TMB) substrate solution was added and incubated for 20 minutes. By measuring the absorption of this supernatant at a wavelength of 450 nm, the fixed amount of Allo-A, that is, the amount of sugar introduced to the surface of the vinyl chloride sheet was calculated. The results are shown in Table 1.

[0032]

[Table 1]

Example 4 The immobilization of Azo-PVLA on the surface of an epoxy resin sheet was evaluated according to the method of Example 3. As a result, the amount of immobilization before ultrasonic treatment was 161 ± 59 ng, and the amount of immobilization after ultrasonic treatment was 176 ± 50 ng. It was found that the coating of the functional sugar chain polymer of the present invention is not detached even if a severe treatment such as ultrasonic treatment is performed even if a measurement error is included.

Example 5 Crosslinked polystyrene (divinylbenzene) beads (DVB
The immobilization of Azo-PVLA on the surface was also evaluated in the same manner. The particle size of this bead is 0.3 mm
And the surface area per 0.1 g of beads is 1000 mm
^{Was 2} . As a result, only 2.1 ng / cm ² was immobilized on the surface of the DVB beads not coated with anything, whereas 5.2 ng / cm ² was immobilized on the DVB beads coated with Azo-PVLA. . In general, microbeads that are greatly affected by nonspecific adsorption
-The adsorption of AlloA more than 3 times was observed based on the coating effect of PVLA.

From the results of Examples 3 to 5, by using the functional sugar chain polymer of the present invention, it is possible to obtain a planar shape or a spherical shape irrespective of the base material such as vinyl chloride, epoxy resin or polystyrene. It was found that the sugar molecule can be firmly immobilized regardless of the shape of the substrate such as.

Example 6 Azo-PVLA and Azo-P as functional sugar chain polymers
VMA was selected. Each sugar chain polymer solution was prepared in the same manner as in Example 1. 1000 IU / m for those solutions
L of heparin was added. Each solution was added to a petri dish made of polystyrene and dried. After UV irradiation, ultrasonic treatment was carried out for 20 minutes with ultrapure water, and the solution was further washed 3 times and then dried.

Normal human blood was centrifuged at room temperature (100 rp).
m, 10 minutes), and the supernatant was added to PRP (Plate Rich Pla
sma). P containing 0.1% BSA in PRP
It was diluted with BS and prepared so as to have a cell number of 1 to 5 × 10 ⁶ per cm ³ , and this was added to each petri dish.
After the addition, centrifuge at 1500 rpm for 10 minutes and at 37 ° C for 1
Plates were allowed to adhere by standing for a time. The amount of adhesion was calculated by collecting the supernatant after adhesion by pipetting and counting the number of platelets remaining in the supernatant with a hemocytometer (manufactured by Sysmex). The results are shown in FIG. However, the data described is an average value of three measurement results.

As a control, FIG. 3 also shows the result of a petri dish not coated with anything. As is clear from the figure, even when the heparin concentration is not fixed, A
It is possible to reduce platelet adhesion by coating with zo-PVLA or Azo-PVMA,
Immobilization with high concentrations of heparin (1000 IU / mL) makes this effect even more pronounced.

[Brief description of drawings]

FIG. 1 IR of Azo-PVLA synthesized in Example 1.
It is a figure showing a spectrum.

FIG. 2 IR of Azo-PVMA synthesized in Example 2
It is a figure showing a spectrum.

FIG. 3 is a graph showing the results of heparin adsorption experiments of Examples.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yasuko Sakurai 3-6-2-907 Hisamoto, Takatsu-ku, Kawasaki-shi, Kanagawa

Claims (6)

[Claims] 1. A functional sugar chain polymer in which a sugar molecule and a photoreactive functional group are grafted onto a vinyl-based polymer main chain. 2. The functional sugar chain polymer according to claim 1, wherein the photoreactive functional group is an azido group. 3. A vinyl-based monomer containing a sugar molecule,
The functional sugar chain polymer according to claim 1 or 2, which is obtained by copolymerizing with a vinyl-based monomer having a photoreactive functional group. 4. The vinyl monomer having a sugar chain is Np-vinylbenzyl- [O-β-D-galactopyranosyl
-(1 → 4) -D-Gluconamide]; Np-vinylbenzyl- [O-α-D-glucopyranosyl- (1 → 4) -D-Gluconamide]; Np-vinylbenzyl- [O -β-D-mannopyranosyl- (1 → 4) -D-mannamide]; Np
-Vinylbenzyl- [O-α-D-galactopyranosyl- (1
→ 6) -D-Gluconamide]; N-p-vinylbenzyl-
[O-6-carboxymethyl-β-D-galactopyranosyl-
(1 → 4) -OD-6-carboxymethyl-gluconamide]; 3-O-4′-vinylbenzyl-D-glucose; N-
p-Vinylbenzyl- [O-2-acetamido-2-deoxy
-β-D-Glucopyranosyl- (1 → 4) -O-D-2-acetamido-2-deoxy-β-D-glucopyranosyl- (1 →
4) -OD-2-acetamido-2-deoxy-β-D-gluconamide], and Np-vinylbenzyl- [O-2-acetamido-2-deoxy-β-D-glucopyranosyl- (1
→ 4) -O-D-2-acetamido-2-deoxy-β-D-gluconamide], or a mixture thereof; Np-vinylbenzyl- [O-β-D-glucopyranosyl- (1 → 3) -D
-Gluconamide]; and Np-vinylbenzyl-D-gluconamide, at least one monomer selected from the group consisting of the functional sugar chain polymer according to claim 3. 5. The functional sugar chain polymer according to claim 3, wherein the monomer having a photoreactive functional group is 3-azidostyrene. 6. A method for using a functional sugar chain polymer, which comprises applying the solution of the functional polymer according to any one of claims 1 to 5 to a substrate and irradiating with light or heating.