JP2525253B2 - Enzymatic method for the synthesis of amino acid oligomers with a single molecular weight - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for enzymatically synthesizing an amino acid oligomer having a single molecular weight using a protease. Amino acid oligomers are biocompatible materials, functional carriers, membrane materials,
Besides being used as an electronic material, it is important as a base material for drug delivery systems.

[Prior Art] As a method for producing an amino acid oligomer, a chemical method, a microbial fermentation method, or an enzymatic synthesis method is known. Among them, a typical chemical synthesis method is the NCA method in which N-carboxy-α-amino acid anhydride (abbreviated as NCA) is synthesized from α-amino acid and polymerized in the presence of a suitable polymerization initiator. .

Chemical synthesis methods other than the NCA method include a method using an active ester and a method using a condensing agent such as mixed acid anhydride and N, N'-dicyclohexylcarbodiimide. As an example of microbial fermentation, polyglutamic acid, polylysine synthesis,
It has been reported. In addition, as an enzymatic synthesis method, a synthetic method utilizing a reverse reaction of a protein hydrolysis reaction by a proteolytic enzyme (protease) or a transesterification reaction has been reported.

[Problems to be Solved by the Invention] Among the chemical synthesis methods, the NCA method is the most widely used method for the synthesis of amino acid oligomers or polymers, but there are some limitations in its use. That is, when an amino group phosgenation step using extremely toxic phosgene is included, or when an amino acid derivative having a functional group such as an amino group, a carboxyl group, a hydroxyl group, or a thiol group in a side chain is used, before synthesis. These functional groups are protected by an appropriate protecting group, and deprotection is required after the completion of the synthesis, which complicates the synthesis process.

Further, it is necessary to select an appropriate organic solvent that dissolves phosgene or a polymerization initiator and an oligomer to be formed. In addition, generally, in the chemical synthesis method, it is difficult to obtain an optically active polymer because the operation involves many steps and is accompanied by racemization of the product.

On the other hand, in the synthesis of amino acid oligomers and polymers by microbial fermentation, enormous efforts are required for screening and modification of microorganisms that efficiently produce the target product, and the product depends on the components of the medium and the resulting oligomer is In general, it has a disadvantage that it has an extremely broad molecular weight distribution.

On the other hand, in peptide synthesis by an enzyme, the reaction can be carried out under mild conditions in an aqueous solution system or an aqueous solution system containing an organic solvent, and a product having high optical purity without racemization is obtained. Is said to be possible.

However, the peptide synthesis reaction by the proteases disclosed so far utilizes a reverse reaction of hydrolysis reaction between the same or different amino acids or their derivatives to form a peptide bond. Since it is difficult to dissolve, there are few examples of isolation and structural analysis without analytical means.

As described above, in the chemical synthesis of the amino acid oligomer or polymer, the synthetic steps such as the use of a highly toxic gas, the introduction of a protective group on the amino acid side chain, the deprotection and the racemization of the product are complicated.

Therefore, the present inventor has studied a reaction system capable of synthesizing an amino acid oligomer having a uniform degree of polymerization by a single operation. As a result of various examinations of various conditions (pH, type of buffer solution, salt concentration, reaction temperature, reaction time, substrate / enzyme ratio, etc.) of an enzymatic reaction using a protease with an amino acid derivative as a substrate, a single operation was performed. A synthetic method for obtaining an amino acid oligomer having a single molecular weight was established. As shown in the examples, the present invention makes it possible to obtain an optically active amino acid oligomer using an optically impure amino acid ester such as a racemate as a starting material. See also (Helvetica Chimic
a Acta, 62,488 (1979) and J. Chem. Tech. Biotechnol., 3
5B, 282 (1985)), it is said that leucine or phenylalanine methyl ester alone does not form an oligomer. However, the present invention enables the production of homooligopeptides using methyl esters of these amino acids as substrates.

[Means for Solving Problems] The present invention has the following configuration (1).

“(1) A method for producing an α-amino acid oligomer having a single molecular weight as an amino acid derivative, wherein a hydrophobic natural or synthetic amino acid having a carboxyl terminus esterified is used as a substrate, and cysteine protease or thiol is used in a buffer solution. A method for enzymatically synthesizing an optically active amino acid oligomer, which comprises reacting in the presence of a protease and synthesizing it in one pot. ”The constitution and effects of the present invention are described in detail below.

The present invention is summarized as follows. A simple enzymatic method for producing an amino acid oligomer having a single molecular weight by an enzyme exemplified by serine protease or cysteine protease, which comprises various amino acids or their carboxyl terminal esterified as a substrate. The method of synthesizing a homopolyamino acid having a uniform molecular weight by reacting the natural amino acid of 1) in a buffer solution in the presence of a protease and discharging the product as a precipitate out of the reaction system.

Examples of the enzyme used in the present invention include chymotrypsin,
Serine proteases such as trypsin, subtilisin and carboxypeptidase, cysteine proteases such as papain, promain and ficin, and metalloproteases such as thermolysin. These enzymes as they are,
Alternatively, it can be used after being modified with a modifier such as polyethylene glycol.

As the substrate, 20 kinds of natural amino acids and other synthetic amino acids or esters thereof can be used.
However, it is necessary to select the substrate depending on the substrate specificity of the enzyme. As the ester, alkyl, aryl or phenyl ester can be used.

The pH of the buffer solution can be used in the range of 2-10,
Each individual enzyme has an optimum pH. For example, it is 6-8 for papain, around 7 for chymotrypsin, and around 4 for pepsin. As the salt for the buffer solution, acetate, phosphate, succinate, and carbonate can be used in the range of 0.1M-5M, but preferably 0.5-2M.

In the method of the present invention, the ratio of substrate to enzyme used is 100.
It is from 1 to 10,000 times, preferably from 1,000 to 2,500 times.

The reaction temperature is 0 to 80 degrees Celsius, and preferably 20 degrees.
From 40 to 40 degrees Celsius, the end of the reaction is reached when the product is completely precipitated.

According to the present invention, an optically active amino acid oligomer can be obtained using a racemic amino acid ester as a starting material.

 Hereinafter, the present invention will be described with reference to examples.

Example 1 (Synthesis of oligomer of L-leucine) 182 mg of L-leucine methyl ester hydrochloride was dissolved in 10 ml of 0.5M sodium carbonate buffer (pH 8.5), and previously dissolved with reducing agent dithiothreitol and ethylenediaminetetraacetic acid. Activated papain (Sigma, Type III)
To 10 μM, and gently shaken at 25 ° C. for 2 days.

The precipitate deposited from the reaction solution is collected by filtration and washed several times with pure water. The obtained crystals were naturally dried to obtain 93 mg of an L-leucine oligomer (yield 51%). The product was analyzed by FT-NMR, F
When analyzed by AB-MS or the like, it was a hexameric oligopeptide as apparent from the spectrum. ¹ HNMR (CF ₃ COOD-TMS) δ: 4.46 (1H, t, -CH-) N-terminal methine, 4.85-4.76
Methine outside the (1H, m, -CH-) N-terminal position, the ratio of the former to the latter is 1: 5.

FAB-MS m / e: 711 (M + H), 582,568,427,371,354,337,284,256,
All of 243,200,145,86 are hexameric fragments.

Example 2 (Synthesis of L-phenylalanine oligomer) Enzyme, reaction temperature, reaction time, substrate / substrate except that 216 mg of L-phenylalanine methyl ester hydrochloride was used.
The reaction was performed under the same conditions as in Example 1, such as the enzyme ratio.

88 mg of L-phenylalanine methyl ester oligomer was obtained (yield 40.7%). What was obtained as a result of the analysis by FT-NMR and FAB-MS was a pentamer oligomer. ¹ HNMR (CF ₃ COOD-TMS) δ: 4.63 (1H, t, -CH-) N-terminal methine, 4.83-5.00
Methine outside the (1H, m, -CH-) N-terminal position, the ratio of the former to the latter is 1: 4.

FAB-MS m / e: 754 ((M + CH 3) + 2H) 708,621,295,267,149,121,
Both 119 and 57 are pentameric fragments.

Example 3 (D, L-leucine methyl ester (racemic form)
Synthesis of L-leucine methyl ester oligomer) The reaction conditions were the same as in Example 1 except that 364 mg of D, L-leucine methyl ester hydrochloride was used. 76 mg of L-leucine methyl ester oligomer was obtained. It is clear from the ¹ H NMR of the oligomer that it is a hexamer. Yield 20.8%.

FIG. 1 shows ¹ H NMR of the oligomer obtained in Example 1 and the oligomer obtained in Example 4.

[Brief description of drawings]

FIG. 1 (A) is the ¹ H NMR spectrum of the L-leucine methyl ester hexamer obtained in Example 1, and FIG. 1 (B) is the L-leucine methyl ester hexamer obtained in Example 3. 2 shows the ¹ H NMR spectrum of

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Claims (1)

(57) [Claims] 1. A method for producing an α-amino acid oligomer having a single molecular weight as an amino acid derivative, wherein a hydrophobic natural or synthetic amino acid having a carboxyl terminal esterified as a substrate is used, and cysteine protease or A method for enzymatically synthesizing an optically active amino acid oligomer, which comprises synthesizing in one pot by reacting in the presence of a thiol protease.