JP3198239B2 - Surface modification method for polymer substrate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for modifying a polymer surface. More specifically, aspartic acid β-allyl ester or aspartic acid β
The present invention relates to a method for immobilizing vinyl ester or glutamic acid γ-allyl ester or glutamic acid γ-vinyl ester and introducing a functional group onto the polymer surface, thereby modifying the polymer surface to facilitate introduction of a functional peptide.

[0002]

2. Description of the Related Art In recent years, peptides having various functions have been developed with the progress of synthetic techniques. However, most of them focus on the development of pharmaceuticals and reagents that make use of physiological activities, and few attempts have been made to immobilize peptides on polymer substrates and convert them into materials. As an attempt to convert a peptide into a material, it is particularly important to modify the surface of an artificial skin material or an artificial blood vessel with a biocompatible peptide, or modify the surface of an electrode with a conductive peptide. However, the direct chemical bonding between proteins, including peptides, and the polymer substrate is due to the difference in reactivity and stability between the former, which is rich in reactivity and poor in stability, and the latter, which lacks reactivity and is more stable. Extremely difficult. For this reason, usually, a pretreatment on the substrate side is first performed to impart reactivity to the substrate surface, and then the peptide is bound and immobilized (Polym. Prepr. Japn., 40, 661 (1992)
1), Biomaterials 8 , 194 (1990)), and immobilization using affinity (Polym. Prepr. Japn., 40, 672).
(1991)). In this process, the pretreatment operation is complicated, the active site of the enzyme or the peptide is damaged, or the activity expression is sterically inhibited, and the objective is not sufficiently achieved at present. In recent years, Matsuda and colleagues have synthesized a functional polypeptide with a vinyl group introduced at the N-terminus, copolymerized with styrene by radical polymerization, and succeeded in immobilizing it on polystyrene without impairing its function (Polym . Prep
r. Japn., 39, 594 (1990)) However, there are problems such as the inability to accumulate polypeptides on the polystyrene surface at a high density, and a more general method of introducing a functional polypeptide onto a polymer substrate is still required. Development is desired.

[0003]

Accordingly, the first aspect of the present invention is as follows.
It is an object of the present invention to provide a method for modifying the surface of a polymer substrate so that a functional peptide can be introduced more easily. A second object of the present invention is to provide such a surface modified polymer. A third object of the present invention is to provide a method for introducing a functional peptide into such a surface-modified polymer substrate.

[0004]

Means for Solving the Problems In view of the above circumstances, the present inventors have been intensively researching to develop a method for modifying the surface of a polymer substrate, and have found that aspartic acid and glutamic acid in gelatin have been developed. It was recognized that the radical reaction between the allylated gelatin obtained by esterifying the residue with allyl alcohol and polylactic acid causes a chemical bond of gelatin to the surface of polylactic acid. The present inventors have paid attention to this phenomenon, and as a result of further research, as a result of radical reaction, aspartic acid β-allyl ester or aspartic acid β-vinyl ester on the polymer surface, or glutamic acid γ-allyl ester or glutamic acid γ-vinyl ester And efficiently reacting the functional peptide with the α-position amino or carboxyl group of aspartic acid or glutamic acid immobilized on the surface of the polymer substrate to introduce the functional peptide onto the polymer surface. I found that I can do it. The present invention has been completed based on such facts by further research.

That is, the gist of the present invention is to provide (1) aspartic acid β-allyl ester or aspartic acid β-vinyl ester, or glutamic acid γ-allyl ester or glutamic acid γ-vinyl ester via an alcohol residue of the ester, (2) aspartic acid β-allyl ester or aspartic acid β-vinyl ester, or glutamic acid γ-allyl ester or γ-vinyl glutamic acid; The ester is linked via the alcohol residue of the ester
A polymer substrate obtained by immobilizing aspartic acid or glutamic acid obtained by immobilization on a polymer substrate by a radical reaction, (3) a vinyl monomer and β-allyl aspartate or β-vinyl aspartate, or γ-allyl glutamate (4) a method for producing a vinyl polymer substrate having a modified polymer surface, comprising copolymerizing an ester or glutamic acid γ-vinyl ester by a radical reaction to fix aspartic acid or glutamic acid on the vinyl polymer surface; The functional peptide is introduced onto the surface, wherein the functional peptide is introduced via the α-position amino group or carboxyl group of aspartic acid or glutamic acid immobilized on the polymer substrate according to any one of the above (1) to (3). Has a functional peptide A method of manufacturing a polymer substrate,
(5) aspartic acid β-allyl ester or aspartic acid β-vinyl ester, or glutamic acid γ-allyl ester or glutamic acid γ-vinyl ester and a functional peptide previously bound via an α-position amino group or carboxyl group; Next, a method for producing a polymer substrate having a functional peptide on the surface thereof by copolymerizing this with a vinyl monomer, and (6) the functional peptide comprises an RGD-containing cell adhesive peptide, collagen, trypsin, amylase and protease The method according to (4) or (5), wherein the method is selected from the group.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. The aspartic acid β-allyl ester, aspartic acid β-vinyl ester, glutamic acid γ-allyl ester and glutamic acid γ-allyl ester used in the present invention are all manufactured by the method of Brook and Chan (Synthesis,
1983, 201) or the method of Belshaw et al. (Synth. Commu
n., 20 , 3157 (1990)).
That is, aspartic acid or glutamic acid is suspended in dry allyl alcohol or vinyl alcohol, and chlorotrimethylsilane is gradually dropped under a nitrogen gas atmosphere,
After stirring at room temperature for 18 hours, ethyl ether is added to obtain precipitated crystals, whereby the desired allyl ester or vinyl ester of aspartic acid or glutamic acid can be produced.

[0007] The polymer which can be used in the present invention is not particularly limited as long as it is a polymer capable of binding to a β-ester of aspartic acid or a γ-ester of glutamic acid by a radical reaction. For example, those selected from the group consisting of polystyrene, polyvinyl chloride, polylactic acid, poly (1-vinylethylene), polyethylene, polypropylene, and silicone resin can be used. These polymers can be used commercially, or can be synthesized and used by a usual method.

Radical reaction of a polymer with aspartic acid β-allyl ester or aspartic acid β-vinyl ester or glutamic acid γ-allyl ester or glutamic acid γ-vinyl ester (hereinafter sometimes abbreviated as amino acid ester) used in the present invention. Is
Performed in a suitable solvent. Such solvents include water, D
MSO, MeOH, CHCl _{3 and the} like. The concentration of these amino acid esters in the solvent is usually 1 to 1
0% by weight, preferably 1-4% by weight.

The radical reaction used in the present invention is a reaction between a solid or liquid polymer and an amino acid ester solubilized in a solvent. Therefore, the former is a heterogeneous reaction, while the latter is a heterogeneous reaction. Becomes a homogeneous reaction.

The radical reaction usually occurs by the action of heat, light, radiation or a polymerization initiator. As such a polymerization initiator, a peroxide, an azo compound or a redox initiator is generally and widely used. In the present invention, when an aqueous solvent is used, hydrogen peroxide, DMSO, MeOH
When an organic solvent such as CHCl ₃ is used, benzoyl peroxide is particularly preferred, but not limited thereto.

The temperature of the radical reaction used in the present invention is usually 50 to 60 ° C. when hydrogen peroxide is used, and usually 60 to 80 ° C. when benzoyl peroxide is used.
C is preferred. The reaction time depends on the reaction temperature.
~ 18 hours. When the polymer is reacted in a liquid state after completion of the reaction, methanol and the like are added to the reaction solution to precipitate and separate the polymer, and the unreacted amino acid ester is removed by hot water extraction using a Soxhlet extractor or the like. Also,
When the polymer is reacted in a solid state, the polymer is washed with the used solvent, and further washed and dried with MeOH or the like. Thus, the desired aspartic acid β-ester or glutamic acid γ
A polymer with an immobilized ester can be obtained.

The copolymerization reaction of the vinyl monomer and the aspartic acid β-allyl ester or aspartic acid β-vinyl ester or glutamic acid γ-allyl ester or glutamic acid γ-vinyl ester to be used in the present invention is also basically carried out by the radical reaction. Aspartic acid β-allyl ester or the like or glutamic acid γ-
The reaction can be carried out by employing the conditions used in a radical reaction between an allyl ester or the like and a polymer. That is, water, DMSO, MeOH, CHCl _{3 and the} like are usually used as the solvent. The concentration of the vinyl monomer and the aspartic acid β-allyl ester or glutamic acid γ-allyl ester in the solvent is usually 2
10 to 10% by weight and 1 to 10% by weight, preferably 5 to 6%
% And 1-4% by weight. The radical reaction usually occurs by the action of heat, light, radiation or a polymerization initiator. As such a polymerization initiator, a peroxide, an azo compound or a redox initiator is generally and widely used.
In this reaction, benzoyl peroxide and hydrogen peroxide can be conveniently used. The temperature of the radical reaction is 50 to 6 when benzoyl peroxide or hydrogen peroxide is used, respectively.
0 ° C or 60-80 ° C is preferred. The reaction time depends on the reaction temperature, but is usually 1 to 16 hours.

For the reaction between the functional peptide and the aspartic acid β-ester or glutamic acid γ-ester or the aspartic acid or glutamic acid residue immobilized on the polymer substrate, a method used in a usual peptide synthesis reaction can be adopted. For example, aspartic acid β-
When reacting an α-carboxyl group of an ester or glutamic acid γ-ester or an α-carboxyl group of an aspartic acid or glutamic acid residue immobilized on a polymer substrate with a free amino group of a functional peptide, an aspartic acid β- Α-amino group of ester or glutamic acid γ-ester or α of aspartic acid or glutamic acid residue immobilized on a polymer substrate
After protecting the amino group and the reactive group of the functional peptide, a peptide bond can be formed between them by reacting with a cyclohexylcarbodiimide derivative or the like as a catalyst.

The functional peptides used in the present invention include RGD-containing cell adhesive peptides (Pierschbacher,
MD and E. Ruoslahti: Nature, 309, 30-33 (198
4)), collagen, trypsin, amylase, protease and the like, but are not limited thereto.

[0015]

The present invention will be described in more detail with reference to the following Examples and Reference Examples, but the present invention is not limited by these Examples and the like.

Reference Example 1 (Synthesis of β-allyl ester of aspartic acid) Method of Belshaw et al. (Synth. Commun., 20 , 3157 (1990))
L-aspartic acid (4.0 g, 0.03 mol) was suspended in dry allyl alcohol (150 ml, 2.20 mol), and chlorotrimethylsilane (9.5 ml, 0.057 mol) was added under a nitrogen gas atmosphere. ) Gradually,
The mixture was stirred at room temperature for 18 hours. Thereafter, 1 liter of ethyl ether was added to obtain 5.8 of needle-like crystalline aspartic acid β-allyl ester hydrochloride as a white precipitate.
0 g was obtained.

Example 1 (Immobilization by Reaction with Polymer Solution) Polystyrene (manufactured by Wako Pure Chemical Industries, Ltd.) 0.5 g and Reference Example 1
0.27 aspartic acid β-allyl ester obtained in
g was dissolved in 20 ml of chloroform, and 10 mmol /
Benzoyl peroxide was added to a concentration of 1 liter and reacted at 60 ° C. for 16 hours. The reaction was stopped by adding 500 ml of methanol. Unreacted aspartic acid β-allyl ester not involved in the immobilization was removed by hot water extraction using a Soxhlet extractor for about 14 hours, and a part of the obtained white powder polymer was dissolved in chloroform. It was confirmed by thin layer chromatography that no monomer or homopolymer was present. The amount of aspartic acid β-ester bonded to polystyrene can be semi-quantitatively determined by the micro Kjeldahl method and the measurement of the absorption (1737 cm ⁻¹ ) area of the ester bond in the infrared absorption spectrum, and is about 6.3% by weight. Met.

Example 2 0.5 g of polyvinyl chloride (manufactured by Kanegafuchi Chemical Industry Co., Ltd.) and 0.2 parts of β-allyl aspartate obtained in Reference Example 1
Was dissolved in 20 ml of DMSO, and benzoyl peroxide was added to a concentration of 10 mmol / l,
The reaction was performed at 60 ° C. for 6 hours. The reaction was stopped by adding 500 ml of methanol. Unreacted aspartic acid β-allyl ester which did not participate in the immobilization was removed by hot water extraction for about 14 hours using a Soxhlet extractor,
One part of the obtained white powder polymer was dissolved in DMSO, and it was confirmed by thin layer chromatography that no monomer or homopolymer was present.

The amount of aspartic acid β-ester bonded to polyvinyl chloride can be determined by measuring the absorption of ester bond (1737 cm
^-1 ) It could be known semi-quantitatively by measuring the area, and was about 2.0% by weight.

Example 3 (Immobilization by reaction with a polymer film) Polystyrene film (0.2 mm thick) 0.15 g
Was immersed in 10 ml of methanol in which 0.40 g of aspartic acid β-allyl ester obtained in Reference Example 1 was dissolved, benzoyl peroxide was added to a concentration of 10 mmol / L, and the reaction was carried out at 60 ° C. for 9 hours. I let it. The polystyrene film was taken out of the reaction solution, and washed with methanol to remove unreacted β-allyl aspartate that did not participate in the immobilization, thereby obtaining an immobilized polystyrene film of aspartic acid β-ester.

The obtained polystyrene film was used for ESC
A (Electron Spectroscopy for Chemical Analysis), and the polystyrene film was subjected to aspartic acid β-
It could be confirmed that the ester was bonded. That is, the polystyrene film obtained as described above was immersed in an aqueous hydrochloric acid solution to convert the α-amino group of aspartic acid into a hydrochloride salt, and then subjected to ESCA to remove nitrogen atoms not found in the raw material polystyrene film. Generation of a photoelectron spectrum derived from a chlorine atom was confirmed.

Example 4 Poly (1-vinylethylene) (manufactured by Nippon Synthetic Rubber Industry Co., Ltd.)
0.12 g of a film (thickness: 0.2 mm) of Reference Example 1
0.27 aspartic acid β-allyl ester obtained in
g was dissolved in 20 ml of methanol, and benzoyl peroxide was added so as to have a concentration of 10 mmol / l, followed by reaction at 60 ° C. for 16 hours. The polystyrene film was taken out of the reaction solution, and washed with methanol to remove unreacted β-allyl aspartate that did not participate in the immobilization, thereby obtaining an immobilized polystyrene film of aspartic acid β-ester.

Example 5 0.11 g of a polystyrene film (0.2 mm thick)
Was immersed in 20 ml of distilled water in which 0.27 g of aspartic acid β-allyl ester obtained in Reference Example 1 was dissolved.
Hydrogen peroxide was added to a concentration of 0 mmol / liter and reacted at 60 ° C. for 16 hours. The polystyrene film was taken out of the reaction solution, and washed with methanol to remove unreacted β-allyl aspartate that did not participate in the immobilization, thereby obtaining an immobilized polystyrene film of aspartic acid β-ester.

[0024]

According to the present invention, the polymer surface is modified, and it becomes easy to immobilize useful functional peptides on the polymer surface. In addition, it is possible to provide an easy method for modifying the surface of the polymer and to provide a polymer whose surface is modified.

──────────────────────────────────────────────────続 き Continuing from the front page (56) References Mitsuo Yasui, et al., “Immobilization of aspartic acid β-monoallyl ester to synthetic polymer” Proceedings of the 69th Annual Meeting of the Chemical Society of Japan ▲ I ▼ , The Chemical Society of Japan, March 29, 1995, 3C315, p. 204 (58) Fields investigated (Int. Cl. ⁷ , DB name) C08J 7/16 C07K 17/08 C08F 8/30-8/32 C08F 218/02-218/12 CA (STN)

Claims (6)

(57) [Claims] 1. An aspartic acid β-allyl ester or aspartic acid β-vinyl ester, or a glutamic acid γ-allyl ester or a glutamic acid γ-vinyl ester, via an alcohol residue of the ester,
A method for modifying a polymer surface, wherein the method is immobilized on a polymer substrate by a radical reaction. 2. An aspartic acid β-allyl ester or aspartic acid β-vinyl ester, or a glutamic acid γ-allyl ester or a glutamic acid γ-vinyl ester, via an alcohol residue of the ester,
A polymer substrate on which aspartic acid or glutamic acid obtained by immobilizing on a polymer substrate by a radical reaction is immobilized. 3. A vinyl polymer obtained by copolymerizing a vinyl monomer with a β-allyl aspartic acid ester, a β-vinyl aspartic acid ester, a γ-allyl glutamic acid ester or a γ-vinyl glutamic acid by a radical reaction, and converting the aspartic acid or glutamic acid into a vinyl polymer. A method for producing a vinyl polymer substrate having a modified polymer surface, wherein the substrate is immobilized on the surface. 4. Introducing a functional peptide via an α-position amino group or carboxyl group of aspartic acid or glutamic acid immobilized on the polymer substrate according to any one of claims 1 to 3. A method for producing a polymer substrate having a functional peptide on the surface thereof. 5. A functional peptide previously bound to aspartic acid β-allyl ester, aspartic acid β-vinyl ester, or glutamic acid γ-allyl ester or glutamic acid γ-vinyl ester via an α-position amino group or carboxyl group. A method for producing a polymer substrate having a functional peptide on the surface by copolymerizing the polymer with a vinyl monomer. 6. The method according to claim 4, wherein the functional peptide is selected from the group consisting of an RGD-containing cell adhesion peptide, collagen, trypsin, amylase and protease.