JP2000128898A - Poly(l-aspartic acid) and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new polymer having a high ratio of α-peptide, high stereoregularity, high optical purity, excellent biodegradability, biocompatibility and functionality by treating an aspartic ester with an actinomyces-derived alkaline protease. SOLUTION: This polymer comprises structural units of formula I and formula II (R1 and R2 are each a 1-3C alkyl), contains >=80 mol % unit I and has >=2,000 molecular weight. The polymer is obtained by bringing an aspartic ester of formula III into contact with an actinomyces-derived alkaline protease (preferably derived from Streptomyces sp.). Preferably the reaction is carried out at 40-60 deg.C for 5 hours to 30 days by making an initial substrate concentration and enzyme concentration in the reaction solution 0.1-20% respectively. The amount of water added is preferably 5-10% based on the whole reaction solution. When the objective polymer is hydrolyzed, a poly(L-aspartic acid salt) having >=80 mol % βpeptide bond is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel aspartic acid polymer and a method for producing the same. Specifically, the present invention relates to a method for using poly-L-aspartic acid ester rich in α-peptide bonds and having high optical purity, poly-L-aspartic acid or a salt thereof rich in β-peptide bonds and having high optical purity, and an enzyme. Related to their manufacturing methods.

[0002]

2. Description of the Related Art Polycarboxylic acids represented by polyacrylic acid are widely used in various industrial fields as water-soluble functional polymers (for example, washing builders, dispersants, scale inhibitors, etc.). However, since these polymers are not biodegradable, there is a risk of contaminating them if they diffuse into rivers and soil from waste liquids and the like. Therefore, there is a strong demand for the development of an alternative polymer that is low in environmental load.

[0003] Among them, polyaspartic acid has biodegradability and advantageous characteristics such as biocompatibility and bioabsorbability, so that it can be used not only as a substitute for polyacrylic acid but also as a medical product. It is expected to be applied to various uses such as cosmetics and metal absorbents.

As a method for producing an aspartic acid polymer, chemical polymerization methods such as thermal polymerization and a method using a phosphoric acid catalyst have been reported. These methods all involve passing through a polyimide and then hydrolyzing to produce polyaspartic acid. However, in such a chemical polymerization, polyaspartic acid can be provided easily and in a large amount, but the resulting polymer is a D, L-mixture, and the α-peptide bond, β-peptide bond and unreacted Are not satisfactory in terms of biodegradability and biocompatibility. Further, since the polymer chain is a D, L-mixture, it has a random structure, and as a result, functions such as the ability to block Ca ions are reduced. Therefore, at present, sufficient functions have not been obtained in terms of functions.

On the other hand, synthesis of polyaspartic acid by enzymatic polymerization has been studied. According to the enzymatic method, the reaction can be performed under mild conditions, and it can be expected that a product having high optical purity without racemization is obtained. However, in the enzymatic synthesis of polyaspartic acid, only the production of oligomers (molecular weight of about 1030 or less) has been reported so far (Uemura et al., Agric. Biol. Che.
m., 54, 2277, 1990), no higher molecular weight polymer was obtained.

[0006]

Accordingly, an object of the present invention is to provide a high molecular weight polyaspartic acid having high optical purity and stereoregularity and a method for producing the same,
Accordingly, it is to provide a novel polymer excellent in biodegradability, biocompatibility and functionality.

[0007]

Means for Solving the Problems To achieve the above object, the present inventor has proposed an enzyme polymerization method in which the type and amount of an enzyme, the amount of water, the reaction temperature, the reaction time, and the type of aspartate ester used as a substrate are used. As a result of examining various conditions such as the above, by treating aspartic acid diester with an alkaline protease derived from actinomycetes, the α peptide bond was reduced to about 80%.
% Of poly L-aspartate having high stereoregularity. By further hydrolyzing this polymer, β-peptide bonds accounted for about 80% or more, and poly L-aspartate having high stereoregularity was successfully synthesized.
Furthermore, it was confirmed that these poly L-aspartic acids exhibited excellent biodegradability, and the present invention was completed.

That is, the present invention is as follows.
1. The following general formula (1)

[0009]

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Wherein R ¹ is an alkyl group having 1 to 3 carbon atoms, and a structural unit (I) represented by the following general formula (2):

[0011]

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(Wherein R ² is an alkyl group having 1 to 3 carbon atoms) comprising a structural unit (II) having a molecular weight of about 2000 or more and containing at least 80 mol% of the structural unit (I). Poly-L-aspartate. 2. The following general formula (3)

[0013]

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(Wherein R ¹ is hydrogen or an alkali metal) and a structural unit (III) represented by the following general formula (4)

[0015]

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(Wherein R ² is hydrogen or an alkali metal), poly-L-asparagine having a molecular weight of about 2,000 or more and containing at least 80 mol% of the structural unit (IV). Acid or salt thereof. 3. The following general formula (5)

[0017]

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Wherein R ¹ and R ² are the same or different and each is an alkyl group having 1 to 3 carbon atoms, and the aspartic acid ester represented by the formula (I) is an actinomycete, preferably belonging to the genus Streptomyces. 2. The method for producing a polyaspartic acid ester according to the above 1, which comprises a step of bringing into contact an actinomycete, particularly an alkaline protease derived from Streptomyces sp. 4. 2. The method for producing polyaspartic acid or a salt thereof according to 2 above, which comprises a step of hydrolyzing the polyaspartic acid ester according to 1 above, preferably the polyaspartic acid ester obtained by the above 3 method.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The poly-L-aspartate of the present invention has the following general formula (1)

[0020]

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The structural unit (I) represented by the following general formula (2)

[0022]

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It comprises the structural unit (II) represented by the formula: and contains the structural unit (I) in an amount of 80 mol% or more, preferably 90% or more. As R ¹ , commonly used protecting groups (eg, alkyl, aryl, and the like) as long as the final product polymer does not hinder the functionality, stereoregularity, and biocompatibility when applied to a living body. A lower alkyl group having 1 to 3 carbon atoms such as methyl, ethyl, propyl and isopropyl. In addition, R ² includes a substituent that does not reduce the polymerization reactivity by the hydrolase, and is preferably a lower alkyl group having 1 to 3 carbon atoms such as methyl, ethyl, propyl, and isopropyl. You.

Molecular weight of the polymer (number average molecular weight)
Is about 2000 or more, preferably about 3000 or more.

The poly-L-aspartate of the present invention is rich in α-peptide bonds and has high optical purity, so that it has excellent stereoregularity, is isotactic, and has a high helix-forming ability. Therefore, it is excellent in function as a polymer electrolyte and biodegradability. Also, since it does not cause racemization, it is also excellent in biocompatibility.

The poly-L-aspartic acid or a salt thereof of the present invention has the following general formula (3)

[0027]

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The structural unit (III) represented by the following general formula (4)

[0029]

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Wherein the structural unit (IV) is contained in an amount of at least 80 mol%, preferably at least 90%. R ¹ and R ² include hydrogen or an alkali metal such as sodium, potassium, lithium and the like.

Molecular weight of the polymer (number average molecular weight)
Is about 2000 or more, preferably about 3000 or more.

The poly-L-aspartic acid or a salt thereof of the present invention is rich in β-peptide bonds and has high optical purity, so that it has excellent stereoregularity and is isotactic.
High helix-forming ability. Therefore, it is excellent in function as a polymer electrolyte and biodegradability. Also, since it does not cause racemization, it is also excellent in biocompatibility.

The poly-L-aspartate of the present invention may be obtained by any method.
For example, the following general formula (5)

[0034]

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The aspartic acid ester represented by the formula can be used as a substrate and brought into contact with a hydrolase to produce the aspartic acid ester. As R ¹ , commonly used protecting groups (eg, alkyl, aryl, and the like) as long as the final product polymer does not hinder the functionality, stereoregularity, and biocompatibility when applied to a living body. A lower alkyl group having 1 to 3 carbon atoms such as methyl, ethyl, propyl and isopropyl. In addition, R ² includes a substituent that does not reduce the polymerization reactivity by the hydrolase, and is preferably a lower alkyl group having 1 to 3 carbon atoms such as methyl, ethyl, propyl, and isopropyl. You.

The enzyme used for the synthesis of the polymer is
The dehydration polymerization reaction between the carboxyl group and the amino group bonded to the α-carbon of the substrate is not particularly limited as long as it is a hydrolase that can more selectively catalyze the dehydration polymerization reaction. A preferred example is an alkaline protease derived therefrom. Particularly preferred are alkaline proteases derived from actinomycetes belonging to the genus Streptomyces, especially alkaline proteases derived from Streptomyces sp. Such enzymes are
It may be used as it is added to the reactor as it is, or may be used by immobilizing it on a conventionally known carrier by an adsorption method, an entrapment method, a crosslinking method, a covalent bonding method, or the like.

In the method of the present invention, the initial substrate concentration and the enzyme concentration in the reaction solution are both 0.01% to 50%.
%, Preferably 0.1% to 20%.

In the method of the present invention, the reaction temperature is appropriately selected within the range of 4 to 100 ° C., preferably 4 to 100 ° C.
0-60 ° C. The amount of water to be added is 0 to 20%, preferably 5 to 10% of the whole reaction solution. The reaction time is 5 hours to 30 days.

The poly-L-aspartic acid of the present invention or a salt thereof may be obtained by any method. For example, the poly-L-aspartic acid ester of the present invention, preferably, the poly-L-aspartic acid ester obtained by the method of the present invention. Poly L-
The aspartic acid ester can be produced by a hydrolysis treatment. The conditions of the hydrolysis treatment are not particularly limited, and for example, a method of reacting at room temperature for several hours under a strong alkali can be mentioned.

[0040]

The present invention will be described in more detail with reference to the following examples and test examples, which are merely examples and do not limit the scope of the present invention.

Example 1 Enzymatic synthesis of poly L-aspartate (1) L-aspartate diethyl ester (100 mg)
Streptomyces sp.-derived alkaline protease (1 mg) was added to distilled water (10 m).
g) was added, and the mixture was stirred at 40 ° C. for 3 days under an argon gas atmosphere. After completion of the reaction, the reaction solution was dissolved in chloroform,
The enzyme was filtered off. Chloroform was distilled off under reduced pressure to obtain a polymer (yield: 73%). IR (KBr): 3428 (NH), 2984 (CH ₂ ), 1738, 1188 (es
ter C = O), 1658,1560 ( CONH) cm -1 1 H-NMR (270 MHz, DMSO-d 6): δ = 1.18 (t, J = 1.7
Hz, 3H, CH ₃ of Et), 2.5-2.8 (m, 2H, CH ₂ ), 4.06 (q,
J = 1.7 Hz, 2H, CH ₂ of Et), 4.30 (m, 0.12H, β-linkage
CH), 4.56 (m, 0.88H, α-linkage CH), 8.0-8.3 (m, 1H,
NH) ¹³ C-NMR (75 MHz, DMSO-d ₆ ): δ = 14.1 (CH ₃ of
Et), 36.0 (CH ₂ ), 49.7 (CH), 60.3 (CH ₂ of Et), 169.9
(α-β, β-αC = O), 170.2 (α-αC = O) From the above analysis results, the obtained polymer had about 90% α-peptide bonds and about 10% β-peptide bonds. Poly L-
It was shown to be ethyl aspartate.

Example 2 Production of poly L-aspartate (1) The poly ethyl L-aspartate obtained in Example 1 was added to
1.3 times equivalent of a 0.5 N sodium hydroxide solution (water / methanol = 25/75 v / v) was added, and the mixture was stirred at 25 ° C. for 3 hours to perform saponification, thereby obtaining sodium poly-L-aspartate. Was. IR (KBr): 3450 (NH), 1639 (CONH), 1597, 1400 (C
^{OONa) cm -1 1 H-NMR} (300 MHz, D 2 O): δ = 2.6-3.1 (m, 2H, CH
₂ ), 4.53 (m, 0.81H, β-linkage CH), 4.70 (m, 0.19H,
α-linkage CH) ¹³ C-NMR (75 MHz, D ₂ O): δ = 39.7 (CH ₂ ), 52.3
(CH), 172.9 (β-β C = O), 173.5 (α-β, β-α C =
O), 174.0 (α-α C = O), 178.6 (COONa) Molecular weight (Mw): 2500 Molecular weight distribution (Mw / Mn): 1.4 From the above results, α of the obtained sodium poly-L-aspartate was obtained. The / β binding ratio was shown to be about 1/4.

Example 3 Enzymatic synthesis of poly L-aspartate (2) and production of poly L-aspartate (2) diethyl L-aspartate (100 mg)
Streptomyces sp.-derived alkaline protease (5 mg) was added to distilled water (10 m).
g) was added, and polymerization was carried out at 40 ° C. for 1 day in an argon gas atmosphere. Next, acetonitrile (10 m
g) to dissolve the polymer,
Polymerization was carried out at 2 ° C. for 2 days. After the reaction was completed, the polymer was dissolved in chloroform, and the enzyme was separated by filtration. Chloroform was distilled off under reduced pressure from the filtrate to obtain polyethyl L-aspartate. This was treated with alkali to obtain sodium poly-L-aspartate (yield: 71%). The polymer has a molecular weight (Mw) of 3,600 and a molecular weight distribution (Mw / Mn) of 1.
It was 3.

Test Example 1 Biodegradability Test of Poly L-Aspartate The biodegradability of the sodium poly L-aspartate obtained in Example 3 was measured according to the OECD guidelines (301C, MI
According to the TI method), the activated sludge of the Yokohama sewage treatment plant was used to measure the biological oxygen demand, and as a result, 60
% Or better biodegradability.

[0045]

The poly-L-aspartic acid and its ester of the present invention are rich in β- and α-peptide bonds and have high optical purity, respectively, and therefore have excellent stereoregularity and high helix-forming ability. Therefore, it is excellent in function and biodegradability as a polymer electrolyte, and is useful as a substitute for polyacrylic acid. Furthermore, since it does not cause racemization, it has excellent biocompatibility and is extremely useful as a polymer material in the fields of medical products and cosmetics. Further, the enzymatic polymerization method of the present invention can perform the reaction at normal temperature and normal pressure, which is useful in that the production cost can be reduced as compared with the chemical synthesis method.

Continued on front page F term (reference) 4B064 AG01 BA10 BA11 BH02 BH04 BH05 BJ12 CA04 CA21 CB02 CB30 CD13 CE01 DA01 DA16 DA20 4H045 AA10 AA20 BA05 EA15 EA34 EA61 FA16 FA70 HA03

Claims (5)

[Claims] [Claim 1] The following general formula (1) Wherein R ¹ is an alkyl group having 1 to 3 carbon atoms, and a structural unit (I) represented by the following general formula (2): (Wherein R ² is an alkyl group having 1 to 3 carbon atoms), and the structural unit (I) is represented by 8
Poly L- having a molecular weight of about 2000 or more containing 0 mol% or more
Aspartic acid esters. 2. The following general formula (3): (Wherein R ¹ is hydrogen or an alkali metal) and a structural unit (III) represented by the following general formula (4): (Wherein R ² is hydrogen or an alkali metal) comprising poly (L-aspartic acid) having a molecular weight of about 2,000 or more and containing the structural unit (IV) of 80 mol% or more, or poly (L-aspartic acid) thereof. salt. 3. The following general formula (5): (Wherein R ¹ and R ² are the same or different and have 1 to 1 carbon atoms)
The method for producing a poly-L-aspartate according to claim 1, comprising a step of contacting the aspartate represented by (3 alkyl groups) with an actinomycete-derived alkaline protease. 4. The method according to claim 3, wherein said actinomycete belongs to the genus Streptomyces. 5. The method for producing poly-L-aspartic acid or a salt thereof according to claim 2, comprising a step of hydrolyzing the poly-L-aspartic acid ester according to claim 1.