JP3053403B1 - Polymer containing tyrosine residue and method for producing the same - Google Patents

Abstract

Abstract: PROBLEM TO BE SOLVED: To provide a highly water-soluble polymer containing a tyrosine compound as a constituent. Further, the present invention provides a method for producing a highly water-soluble polymer containing the tyrosine compound as a constituent. SOLUTION: A highly water-soluble tyrosine residue having a polymerization degree of 3 to 500 obtained by oxidatively polymerizing an oxidatively polymerizable compound containing a compound represented by the following general formula (1) and then hydrolyzing it. Containing polymer. General formula (1) (In the formula, R ₁ represents an amino group, a substituted amino group, an amide group, or an amine salt, and R ₂ represents a hydrocarbon group having 1 to 25 carbon atoms.)

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a water-soluble polymer containing a residue of a tyrosine compound and a method for producing the same, and relates to a reactive polymer, an antioxidant, a starting material for a functional polymer, an antibacterial material, Polymers and tyrosine residue-containing polymers useful as biocompatible materials are provided.

[0002]

2. Description of the Related Art Tyrosine is a constituent amino acid of a protein and has a phenol group. On the other hand, it is known that phenols are polymerized with various oxidation catalysts. For example, Materials Science and E
Nginering C4, 189 (1996) shows an example in which dodecyl ester of tyrosine is polymerized using an enzyme catalyst (peroxidase) and an oxidizing agent (hydrogen peroxide). However, the polymer was insoluble in water. On the other hand, proteins consisting of amino acids are a kind of biological macromolecules, and if a water-soluble polymer containing residues of a tyrosine compound, which is a kind of amino acids, can be obtained, raw materials for materials with good biodegradability and molding It is very preferable as a raw material for producing a material having good biocompatibility, which has excellent properties and processability. However, no highly water-soluble polymer containing such a tyrosine compound residue has been proposed so far.

[0003]

Accordingly, the present invention provides
An object is to provide a polymer having high water solubility, which contains a tyrosine compound as a constituent. Furthermore, the present invention
It is an object of the present invention to provide a method for producing a highly water-soluble polymer containing a tyrosine compound as a component.

[0004]

The object of the present invention has been attained by the following means. That is, the present invention is characterized in that (1) an oxidatively polymerizable compound containing a compound represented by the following general formula (1) is oxidatively polymerized and then hydrolyzed to have a degree of polymerization of 3 to 500. Highly water-soluble polymer containing tyrosine residue, general formula (1)

[0005]

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(Wherein R ₁ represents an amino group, a substituted amino group, an amide group, or an amine salt, and R ₂ represents a hydrocarbon group having 1 to 25 carbon atoms.) (2) a peptide containing a tyrosine residue; (3) a highly water-soluble tyrosine residue-containing polymer having a degree of polymerization of 3 to 500, which is obtained by oxidatively polymerizing an oxidatively polymerizable compound using an oxidizing agent and an oxidation catalyst, and then hydrolyzing the compound; An oxidatively polymerizable compound containing a compound represented by the following general formula (1) is oxidatively polymerized using an oxidizing agent and an oxidizing catalyst, and is then hydrolyzed.
A method for producing a tyrosine residue-containing polymer represented by formula (1):

[0007]

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(Wherein R ₁ represents an amino group, a substituted amino group, an amide group, or an amine salt, and R ₂ represents a hydrocarbon group having 1 to 25 carbon atoms.) (4) a peptide containing a tyrosine residue; A method for producing a tyrosine residue-containing polymer having a degree of polymerization of 3 to 500, wherein the oxidatively polymerizable compound is oxidatively polymerized using an oxidizing agent and an oxidizing catalyst, and then hydrolyzed; (3) or (4), wherein the enzyme catalyst is a peroxidase; (6) the tyrosine residue-containing polymer according to (5), wherein the enzyme catalyst is peroxidase; ) An object of the present invention is to provide a method for producing a tyrosine residue-containing polymer according to (3) or (4), wherein the oxidizing agent is hydrogen peroxide. The high water solubility of the polymer of the present invention means that the polymer has a water solubility of at least 5% by weight (the solubility in water is 5 or more).

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS First, a method for producing a polymer comprising a tyrosine compound according to the present invention as a constituent will be described. In the production method of the present invention, as the oxidatively polymerizable compound, a tyrosine compound represented by the following general formula (1) can be used. General formula (1)

[0010]

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(In the formula, R ₁ represents an amino group, a substituted amino group, an amide group, or an amine salt, and R ₂ represents a hydrocarbon group having 1 to 25 carbon atoms.) R ₁ in the general formula (1) is represented by R ₁ . The substituted amino group to be formed is not particularly limited. For example, an alkyl-substituted amino group such as a methylamino group or a dimethylamino group, an anilino group, an o-toluidino group, an m-toluidino group, a p-toluidino group, And an aromatic substituted amino group such as a group. The amide group represented by R ₁ in the general formula (1) is not particularly limited. For example, an amide group in which an amino group such as N-acetamide is substituted with an acyl group, or an amino group such as N-chloroacetylamine An amide group in which a group is substituted with a substituted acyl group, an amide group in which an amino group is substituted with a substituted sulfonyl group such as Np-toluenesulfonylamine, and the like can be given.

The amine salt represented by R ₁ in the above general formula (1) can be obtained from an amine compound and various acids as described in “Chemical Encyclopedia”, Tokyo Kagaku Dojin, 1st edition. Is the salt that is used. The amine salt used in the present invention is not particularly limited, but salts formed with hydrochloric acid, sulfuric acid, nitric acid, etc. are generally well soluble in water, and are obtained from these acids in a system using water as a reaction solvent in the present invention. Salts such as hydrochloride, sulfate and nitrate can be preferably used. More preferably, it is a hydrochloride. -O of the above general formula (1)
The bonding position of the phenolic hydroxyl group represented by H is not particularly limited, but is a meta position relative to a substituent having R ₁ and R ₂ ,
The para-position is preferred, and more preferably those bonded to the para-position can be used. R _{2 of the} above general formula (1)
Examples of the hydrocarbon group having 1 to 25 carbon atoms represented by
There is no particular limitation. Specific examples include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group,
Alkyl groups such as heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, pentadecyl group, octadecyl group, methylbutyl group, ethylbutyl group, isopropyl group, isobutyl group, sec-butyl group and tert-butyl group. Although it can be mentioned, preferably, it is an alkyl group having 1 to 6 carbon atoms, methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, methylbutyl group, ethylbutyl group, isopropyl group, isobutyl group, It is preferable to use a lower substituent such as a sec-butyl group and a tert-butyl group, and more preferably a methyl group and an ethyl group.

The oxidatively polymerizable compound represented by the general formula (1) is an optically active oxidatively polymerizable compound because it has at least one asymmetric carbon atom. A compound may be used, or a racemate may be used. In the oxidatively polymerizable compound represented by the general formula (1), the benzene ring may have a substituent other than a phenolic hydroxyl group that is inactive to the oxidative polymerization reaction. Examples of such a substituent include an alkyl group such as a methyl group and an ethyl group, an alkoxy group such as a methoxy group, a halogen atom such as iodine and bromine, and a phenolic hydroxyl group and a group other than these. It is preferred that at least one other than a substituent is a hydrogen atom.

Specific examples of the oxidatively polymerizable compound represented by the above general formula (1) include L-tyrosine methyl ester hydrochloride, L-tyrosine ethyl ester hydrochloride, D-methyl tyrosine hydrochloride, D, L- Tyrosine methyl ester hydrochloride, L-tyrosine copper salt, N-acetyl-L-tyrosine,
In addition to N-chloroacetyl-L-tyrosine, Np-toluenesulfonyl-L-tyrosine, DL-m-tyrosine, dipeptides formed from various amino acid residues including tyrosine residues, and the like, Preferably,
L-tyrosine methyl ester hydrochloride, L-tyrosine ethyl ester hydrochloride, D-methyl tyrosine hydrochloride, D, L
-Tyrosine methyl ester hydrochloride can be used.

In the present invention, a peptide containing a tyrosine residue may be used as the starting oxidatively polymerizable compound instead of the compound represented by the general formula (1). In the present invention, a peptide is a generic term for compounds formed by peptide bonding of two or more amino acids and includes a tyrosine residue, as described in the “Chemical Encyclopedia”, Tokyo Kagaku Dojin, 1st edition. A compound. In a narrow sense, those having an amino acid residue number (n) of 100 or less are called oligopeptides having n of 2 to about 10 are called polypeptides, those having about 10 to 50 are called polypeptides, and those having about 50 or more are called proteins. The ratio of tyrosine residues in these amino acid residues is preferably 30
Mol% or more, more preferably 50 to 100 mol%. In the present invention, it is preferable to use an oligopeptide containing a tyrosine residue.
More preferably, 2) is used.

Among the oxidatively polymerizable compounds used in the present invention, various oxidatively polymerizable compounds such as phenols, in addition to the above-mentioned general formula (1) and the peptide containing a tyrosine residue,
A copolymerization reaction can be performed using naphthols and aromatic amines. The phenols, naphthols, aromatic amines used in the copolymerization reaction and the amount used can be appropriately selected based on various physical properties required according to the intended use of the obtained tyrosine residue-containing polymer. Singly or in combination of two or more. The proportion of the constituent components to be copolymerized is not particularly limited, but is 99 mol% or less, preferably 50 mol% or less based on the total amount of the oxidatively polymerizable compound. More preferably, it is at most 20 mol%.

Specific examples of such phenols include o-cresol, m-cresol, p-cresol, p-octylphenol, p-dodecylphenol, 2,3-dimethylphenol, 2,4-dimethylphenol, , 5-dimethylphenol, 2,6-dimethylphenol, 3,4-dimethylphenol, 3,5
-Dimethylphenol, o-ethylphenol, m-ethylphenol, 2,4,6-dimethylphenol, p
-T-butylphenol, 2,4-di-t-butylphenol, 2,6-di-t-butylphenol, pt-
Alkylphenols such as amylphenol, hydroquinone, catechol, resorcinol, pyrogallol, urushiol, thiol, polyhydric phenols such as laccol, o-chlorophenol, m-chlorophenol,
p-chlorophenol, 2,4,6-trichlorophenol, o-bromophenol, m-bromophenol,
halogenated phenols such as p-bromophenol, o-fluorinated phenol, m-fluorinated phenol, p-fluorinated phenol, aminophenols such as o-aminophenol, m-aminophenol and p-aminophenol, bisphenol A, p- (α-cumyl) phenol, p-
Examples thereof include phenylphenol, guaiacol, guetor, phenol, tyramine, and tyramine hydrochloride.

Specific examples of naphthols include α-naphthol, β-naphthol, 1,4-dihydroxynaphthalene and the like.

Specific examples of the aromatic amines include aniline, o-anisidine, p-anisidine, 2,4-xylidine, 3,4-xylidine, p-cresidine, and 4,4 ′.
-Diamino-3,3'-diethyldiphenylmethane,
Examples thereof include 4,4'-diaminobenzanilide, diaminodiphenyl ether, 3,3'-dimethyl-4,4'-diaminodiphenylmethane, and sulfanilic acid.

In the present invention, any oxidizing agent can be used, but preferably oxygen or peroxide can be used. Oxygen may be a mixture with an inert gas or air. Examples of peroxides include:
Hydrogen peroxide, t-butyl hydroperoxide, di-
Examples thereof include t-butyl peroxide, cumene hydroperoxide, dicumyl peroxide, peracetic acid, perbenzoic acid, and the like. More preferably, peroxide can be used, and hydrogen peroxide is particularly preferable. In the present invention, the amount of the oxidizing agent is not particularly limited. When oxygen is used, it is generally used in an excess of an equivalent or more with respect to the oxidatively polymerizable compound. When a peroxide is used, it is usually used in an amount of at least 3 equivalents but preferably at least 2 equivalents to the oxidatively polymerizable compound.

The oxidation catalyst used in the present invention is not particularly limited as long as it has the ability to oxidize and polymerize the above-mentioned oxidation-polymerizable compound, but it is preferable to use an enzyme catalyst. As an enzyme catalyst, peroxidase, laccase, tyrosinase and the like are known as catalysts for oxidative polymerization of phenols. In this study, it is more preferable to use peroxidase. Peroxidases of various origins (for example, of plant origin, of bacterial origin, of carrier fungi) can be used, but those of horseradish, soybean, and Coprinus cinerius origin are particularly preferred.

This oxidative polymerization is based on a coupling reaction of a phenoxy radical generated by a dehydrogenation reaction of a phenolic hydroxyl group in an oxidatively polymerizable compound. And a polymer having phenylene units and oxyphenylene units at random.

Although the reaction of the present invention can be carried out in the absence of a reaction solvent, it is generally preferable to use a solvent. The solvent is not particularly limited as long as it is inert to the oxidatively polymerizable compound and is a liquid at the reaction temperature, but is preferably inactive in the oxidation catalyst and dissolves the oxidatively polymerizable compound and the oxidizing agent oxidation catalyst. Solvents are preferred. Preferred examples of the solvent include aromatic hydrocarbons such as benzene, toluene and xylene; linear and cyclic aliphatic hydrocarbons such as heptane and cyclohexane; halogenated hydrocarbons such as chlorobenzene, dichlorobenzene and dichloromethane; acetonitrile , Nitriles such as benzonitrile; alcohols such as methanol, ethanol, n-propyl alcohol and isopropyl alcohol;
Ethers such as dioxane, tetrahydrofuran and ethylene glycol dimethyl ether; amides such as N, N-dimethylformamide and N-methylpyrrolidone; nitro compounds such as nitromethane and nitrobenzene; water; and the like. it can. These can be used alone or as a mixture.

When using an enzyme as an oxidation catalyst, it is preferable to use a solvent containing water.
More preferably, water is used, and particularly preferably, a buffer can be used.

The pH of the buffer used at this time is desirably near the optimum pH of the oxidation catalyst to be used. However, the type of the oxidatively polymerizable compound, the type of the oxidation catalyst to be used, the type of the solvent to be used, and the buffer to be used. It depends on the type of salt in the liquid, etc.
It may be appropriately selected according to conditions, and usually, pH 3 to 12
Is preferable. When horseradish peroxidase is used as the oxidation catalyst, preferably pH 5 to 9, and more preferably pH 6 to 8 can be used. As the type of the buffer, an acetate buffer, a phosphate buffer, a carbonate buffer, and a Tris buffer are preferable, and a Tris buffer is more preferable.

The salt concentration of the buffer varies depending on the type of the oxidatively polymerizable compound used, the type of the oxidation catalyst used, the type of the solvent used, the type of the salt of the buffer used, and the like. Suffices, usually 0.001-1
A range of 0 molarity is preferred. If horseradish peroxidase is used as the oxidation catalyst,
-5 mol, more preferably 0.1-3 mol, can be used.

In the present invention, many different methods can be used for the reaction between the oxidation-polymerizable compound and the oxidation catalyst. For example, a solution of an oxidation-polymerizable compound and an oxidation catalyst may be individually prepared and then poured into the same container, or an oxidation catalyst may be added to a solution of the oxidation-polymerizable compound. Various other combinations are possible, but a method in which the oxidation catalyst is inactivated is not preferable.

In the present invention, the temperature in the reaction between the oxidation-polymerizable compound and the oxidation catalyst is preferably a temperature at which the activity of the oxidation catalyst is not inactivated. The reaction temperature is -10
To 180 ° C, more preferably 10 to 10 ° C.
0 ° C. is preferred. In particular, when using an enzyme catalyst for the oxidation catalyst, care must be taken that the enzyme catalyst loses its activity when the reaction temperature is too high. However, the allowable range of the reaction temperature varies depending on the solvent used, and the enzyme catalyst is stabilized depending on the solvent, so that a high reaction temperature can be adopted.

In the present invention, the addition amount of the oxidation catalyst in the reaction between the oxidation-polymerizable compound and the oxidation catalyst may be appropriately adjusted depending on the oxidation polymerization activity of the oxidation catalyst to be used, and is preferably represented by the formula (1). 1 to
It may be about 1000% by weight, more preferably 1 to 100% by weight. The tyrosine residue-containing polymer of the present invention is obtained by subjecting an oxidatively polymerizable compound to oxidative polymerization using an oxidizing agent and an oxidizing catalyst, followed by hydrolysis. The hydrolysis reaction may be performed using only water, but is preferably performed in the presence of an acid such as hydrochloric acid or sulfuric acid or an alkali such as sodium hydroxide in order to promote the reaction. More preferred. Further, the hydrolysis reaction is accelerated by a hydrolase, but there is no problem with using this. More specifically, this hydrolysis is performed by subjecting the reaction solution after the oxidative polymerization to usually 20 to 100 in the presence of water.
It can be performed by heating at 10 ° C. for 10 minutes to 100 hours. When the reaction is performed in the presence of an acid as described above, 0.1N or more, preferably 1N or more of hydrochloric acid, sulfuric acid,
When using nitric acid in the presence of alkali, 0.1
It is possible to use N or more, preferably 1 N or more of sodium hydroxide and potassium hydroxide. It is considered that the —COOR ₂ group in the residue corresponding to the compound represented by the general formula (1) in the polymer was hydrolyzed to a carboxyl group by this hydrolysis treatment. The number average molecular weight of the tyrosine residue-containing polymer obtained in the present invention is usually in the range of 300 to 100,000, preferably 500 to 30,000, more preferably 500 to 10,000.
000.

In the tyrosine residue-containing polymer obtained in the present invention, for example, a tyrosine residue-containing polymer containing only a tyrosine compound as a constituent is soluble in water.
The tyrosine monomer has an extremely low solubility in water of 0.5 g or less at 25 ° C., but the polymer containing a tyrosine residue obtained in the present invention is soluble in water at 30 ° C. in an amount of about 5 to 10% by weight. is there. Further, the polymer of the present invention has a high solubility in a buffer solution, and may exhibit a solubility higher than that of distilled water. The solubility in this case is about 5 to 20% by weight in water at 30 ° C. The pH of the buffer used is preferably pH 3 to 10, and examples thereof include a phosphate buffer, a carbonate buffer, and an acetate buffer. By increasing the solubility in water, the polymer becomes a reactive polymer, an antioxidant, a starting material for a functional polymer, an antibacterial material, a biodegradable polymer,
When used as a biocompatible material, various effects that cannot be obtained by using a monomer alone can be expected. For example, when used as a biodegradable polymer, acceleration of the biodegradation rate can be expected.

[0031]

The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples. (Example-1) In a 50 ml eggplant flask, 61
Take 4 milligrams of L-tyrosine ethyl ester hydrochloride and add 25 milliliters of Tris buffer (1 molar, p
H7.6) was added and the mixture was stirred at 25 ° C. Further, 10 milligrams of horseradish peroxidase was added, and the mixture was stirred for a while, and then 5% aqueous hydrogen peroxide was added dropwise. 1.7 ml of a 5% hydrogen peroxide solution was added dropwise over 2 hours. The oxidative polymerization reaction was performed at 25 ° C. After completion of the dropwise addition, the reaction was further continued at 25 ° C. for 1 hour while stirring. As the reaction progresses,
A powdery polymer precipitated in the reaction system. After completion of the reaction, the polymer was separated and collected by centrifugation and dried under reduced pressure (6).
09 milligrams). The resulting polymer is pale yellow,
Soluble in acidic water, alkaline water, methanol, DMF and DMSO, and insoluble in distilled water, THF and chloroform. The number average molecular weight of the polymer determined by GPC was 890 (in terms of polystyrene). From NMR and IR analysis, it was estimated that the produced polymer was a random copolymer of the phenylene unit derived from the tyrosine compound and the oxyphenylene unit of the tyrosine compound. 61 mg of the obtained polymer was added to a 1 N aqueous solution of sodium hydroxide
Heat in milliliter at 60 ° C. for 24 hours. After cooling to room temperature, the mixture was neutralized with 1 N hydrochloric acid. The reaction solution was subjected to molecular weight fraction 5
Dialysis using a dialysis membrane of No. 00 to isolate the polymer
Purified. The yield was 54 milligrams from drying under reduced pressure.
Proton NMR confirmed that the produced polymer had its ester group in the original polymer hydrolyzed quantitatively and was converted to polytyrosine. The hydrolyzate was 7% by weight soluble in distilled water at 30 ° C.

Example 2 The same operation as in Example 1 was carried out except that 290 mg of L-tyrosine methyl ester hydrochloride was used instead of L-tyrosine ethyl ester hydrochloride, to obtain a polymer of 170 mg of tyrosine ester. I got This was 5% by weight soluble in distilled water.

(Example 3) The same operation as in Example 1 was carried out except that 290 mg of D-methyltyrosine hydrochloride was used instead of L-tyrosine ethyl ester hydrochloride, to obtain 200 mg of a tyrosine ester polymer. Obtained. The number average molecular weight of the polymer was 5,500.
This was 5% by weight soluble in distilled water.

Example 4 A tyrosine ester polymer was prepared in the same manner as in Example 1 except that 290 mg of D, L-tyrosine methyl ester hydrochloride was used instead of L-tyrosine ethyl ester hydrochloride. 170 milligrams were obtained. The number average molecular weight of the polymer is 4500
Met. This was 5% by weight soluble in distilled water.

Example 5 The same operation as in Example 1 was carried out except that 50 mg of phenol was added to obtain 630 mg of a copolymer of tyrosine ester and phenol. This was 5% by weight soluble in distilled water.

[0036]

According to the present invention, a polymer containing a tyrosine residue having high water solubility can be easily obtained in a high yield. The high water-soluble tyrosine residue-containing polymer thus obtained is suitable for applications such as biodegradable polymers, biocompatible materials, reactive polymers, antioxidants, starting materials for functional polymers, and antibacterial materials. It is.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Ryohei Ikeda 32-18-503 Tsukidaidai, Hodogaya-ku, Yokohama, Kanagawa Prefecture Examiner ▲ Eiichi Yoshi ▼ (56) References Biotechnology and Bioengineering (1999), 63 (4) , P. 449-458 Analytical Letters (1992), 25 (3), p. 443-452 (58) Fields investigated (Int. Cl. ⁷ , DB name) C08G 65/00-67/02 C12P 13/00 CA (STN)

Claims (7)

(57) [Claims] 1. A water-soluble polymer having a degree of polymerization of 3 to 500, which is obtained by oxidatively polymerizing an oxidatively polymerizable compound containing a compound represented by the following general formula (1) and then hydrolyzing it. High tyrosine residue-containing polymer. General formula (1) (In the formula, R ₁ represents an amino group, a substituted amino group, an amide group, or an amine salt, and R ₂ represents a hydrocarbon group having 1 to 25 carbon atoms.) 2. A method according to claim 1, wherein the peptide containing a tyrosine residue and the oxidatively polymerizable compound are oxidatively polymerized using an oxidizing agent and an oxidizing catalyst, and then hydrolyzed.
A water-soluble, tyrosine residue-containing polymer. 3. An oxidatively polymerizable compound containing a compound represented by the following general formula (1) is oxidatively polymerized using an oxidizing agent and an oxidizing catalyst, and is then hydrolyzed. A method for producing a tyrosine residue-containing polymer having a molecular weight of from 500 to 500. General formula (1) (In the formula, R ₁ represents an amino group, a substituted amino group, an amide group, or an amine salt, and R ₂ represents a hydrocarbon group having 1 to 25 carbon atoms.) 4. A polymerization degree of from 3 to 500, wherein an oxidatively polymerizable compound and a peptide containing a tyrosine residue are oxidatively polymerized using an oxidizing agent and an oxidation catalyst, and then hydrolyzed.
A method for producing a tyrosine residue-containing polymer as described above. 5. The method for producing a tyrosine residue-containing polymer according to claim 3, wherein the oxidation catalyst is an enzyme catalyst. 6. The method for producing a tyrosine residue-containing polymer according to claim 5, wherein the enzyme catalyst is peroxidase. 7. The method for producing a tyrosine residue-containing polymer according to claim 3, wherein the oxidizing agent is hydrogen peroxide.