JPH051719B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to the production of oligopeptides by a combination of peptide synthesis reactions using specific proteolytic enzymes.
Tyrosyl-glycyl-glycyl-phenylalanyl-leucine [Tyr-Gly-Gly-Phe-Leu,
This invention relates to a new method for producing Leu-enkephalin.

Prior art The oligopeptide Leu-enkephalin is
Met-Enkephalin is a morphine-like analgesic peptide isolated from the brains of several mammalian species including pigs, and has attracted interest as a non-narcotic analgesic with fewer side effects due to its unique physiological activity. ing.

In recent years, attempts to synthesize useful peptides using the reverse reaction of proteolytic enzymes have become active.
It has been reported that the entire process is carried out by enzymatic synthesis [Kullman, W., Biochem.Biophys.
Res.Commun., 91 , 693 (1979); J.Biol.Chem.,
255(17), 8235 (1980) and 256(3), 1301 (1981)].

According to the above reports, a wide variety of synthetic routes using chymotrypsin and papain have been shown, but all of them are extremely complex and require multi-step processes, and they require protection from the raw materials. Selection and preparation of amino acids or protected peptides, deprotection reactions from the obtained peptides, etc. also require complicated operations. They have also reported that depending on the selection of the enzymes, protecting groups, etc., the desired synthesis reaction does not proceed, but rather a decomposition reaction occurs. Furthermore, the method reported above is never satisfied with the yield of the enzymatic reaction alone, and the total yield of the entire process, which also takes into account the yields of the above protection reactions, deprotection reactions, etc., is extremely low, making it impossible to implement it industrially. Not suitable for

However, compared to chemical synthesis methods, peptide synthesis methods that generally use enzymes (enzymatic synthesis methods) do not necessarily require protection of the side chain functional groups of raw material amino acids, etc., and the reaction is stereoselective. Although it has excellent characteristics such as the ability to use inexpensive racemic raw materials, no racemization during the reaction, and the reaction proceeding at room temperature and pressure, as is clear from the above report, the substrate specificity of the enzyme is Due to its nature, the enzymes that can be used are limited depending on the type of raw material amino acid, etc., and it is not the case that a single enzyme can be used for all peptide synthesis, but rather that enzymes that can catalyze the desired peptide synthesis reaction are used. The choice itself is extremely difficult. Furthermore, even with established enzymatic peptide synthesis methods, the reaction equilibrium is generally largely biased toward the substrate, which has the drawback of relatively low yields, reaction rates, etc. In particular, when enzymatically synthesizing oligopeptides in which three or more different amino acids are linked, such as Leu-Enkephalin,
After binding two amino acids in advance, it is necessary to perform a binding reaction with another amino acid, but the dipeptides, tripeptides, etc. used as raw material substrates for the reactions after this second step are It is conceivable that the protein may be hydrolyzed and cleaved by an enzyme, or that the amino acids generated by the cleavage may further participate in the synthesis reaction. In fact, in the above report, H-Leu-N ₂ H ₂ Ph + Boc- using papain
Gly-Phe-OH reaction, H-Phe-Leu-
In the reaction of N ₂ H ₂ Ph + Boc-Tyr (Bzl)-Gly-Gly-OH, the expected peptide is not synthesized, but a decomposition reaction of the raw peptide and a substitution reaction of peptide bonds occur. If such a side reaction occurs, the target product will naturally not be obtained, or a large amount of byproducts will be mixed in, making separation of the target product difficult and significantly reducing the purity of the target product.

Problems to be Solved by the Invention The purpose of the present invention is to
The object of the present invention is to provide a new method for producing Leu-enkephalin. In particular, it is an object of the present invention to provide a practical technique that allows the above-mentioned oligopeptide to be produced efficiently, with high yield, and with high purity through simple operations and steps.

Means for Solving the Problems The above purpose is to use α-chymotrypsin to
Substituted tyrosine (Tyr) and glycyl-glycine (Gly-Gly)-alkyl ester are subjected to a condensation reaction, and then the protected tripeptide (N
-Substituted Tyr-Gly-Gly-alkyl ester) was deesterified, and then phenylalanyl-leucine (Phe-Leu) was synthesized using Bacillus metalloprotease.
- Achieved by a method for producing oligopeptides characterized by carrying out a condensation reaction with an alkyl ester.

In this specification, descriptions of amino acids, peptides, protective groups, etc. shall follow the abbreviations proposed by the IUPAC committee or common symbols in the field. Furthermore, amino acids are in the L-form unless otherwise specified.

In the course of intensive research into the synthesis of peptides using proteases, the present inventors first discovered a method for enzymatic synthesis of dipeptide Phe-Phe and Asp-Phe-OMe precursors [see Japanese Patent Application Laid-Open No. 60-45596].
and des-, an inhibitor of enkephalinase.
Tyr ¹ −enkephalin (des−Tyr ¹ −
We have established an enzymatic synthesis method for enkephalin, Gly-Gly-Phe-Leu) using Bacillus metalloprotease (thermolysin) [see Japanese Patent Application Laid-open No. 96096/1983].

The present invention was completed as a result of research following these and others.

In the method of the present invention, first, N-substituted Tyr and Gly-Gly-alkyl ester are subjected to a condensation reaction using α-chymotrypsin (first step). The N-substituent in the N-substituted Tyr used as one substrate here is an amino group-protecting group commonly used in peptide synthesis reactions. A representative example thereof is benzyloxycarbonyl group (Z), and other examples include p-methoxybenzyloxycarbonyl group (pMZ), t
-butoxycarbonyl group (Boc), 2-chlorobenzyloxycarbonyl group, etc. are also included. The alkyl group in the Gly-Gly-alkyl ester used as the other substrate is also a commonly used carboxyl protecting group for amino acids. Specific examples include methyl (OMe), ethyl (OEt), propyl (OPr), butyl (OBu), and tert-butyl (OBu ^t ).
an alkyl group having 1 to 4 carbon atoms, preferably
Can give examples of ^OBut .

Used as one of the raw material substrates in the first step above
Gly-Gly is commercially available and can be easily produced by enzymatic synthesis using papain.
Since Gly does not have optical isomers, it can be easily synthesized using chemical synthesis methods. A specific example of the enzymatic synthesis method using papain will be described in detail in Examples below.

The enzymatic reaction of the above N-substituted Tyr and Gly-Gly-alkyl ester with α-chymotrypsin is as follows:
The reaction can be carried out using the former as an acid component and the latter as a base component, preferably in a water-ethyl acetate two-phase system. Here, the water-ethyl acetate two-phase system means using a water phase and an ethyl acetate phase that are prepared separately; in the actual reaction, both phases are uniformly mixed in an emulsion state by stirring, etc. Ru.

As the aqueous phase, an appropriate buffer is preferably used, such as an aqueous solution of (2-diaminomorpholino)ethanesulfonic acid (MES) or a Tris-HCl buffer. The aqueous phase also contains
For example, sodium hydroxide can be added to adjust the pH to about 4 to 5, and calcium chloride, etc., which is known as a stabilizing factor for the enzyme used, can also be dissolved.

More specifically, in the first step of the present invention, a base component is first dissolved in the aqueous phase and an acid component is dissolved in the ethyl acetate phase to prepare a solution of both substrates. The substrate concentration in each of the above substrate solutions is determined appropriately,
From the viewpoint of reaction rate, it is preferable to keep the concentration as high as possible, but it is usually in the range of about 10 to 200 mM for the acid component and about 10 to 1200 mM for the base component.
In particular, the concentration ratio of acid components to base components should be set to approximately 0.1.
-3, usually about 0.4-3, and within this range, the lower the acid component concentration, the higher the yield of the desired oligopeptide tends to be. In addition, the usage ratio (volume ratio) of each of the above substrate solutions should be such that the amount of ethyl acetate phase is at least equal to that of the aqueous phase, so that the desired synthesis reaction proceeds and the desired product is obtained in high yield. . Usually, the above volume ratio is preferably selected in a range where the amount of ethyl acetate phase is about 1 to 10 times that of the aqueous phase, and within this range, the yield of the target product tends to improve as the amount of ethyl acetate phase is increased. It is in.

In the above step, α-chymotrypsin is also added to the aqueous phase substrate solution. The amount used varies depending on the titer of the enzyme used, reaction conditions, etc., but it is usually about 0.2w/of the total volume of the aqueous phase used.
It is preferable that the amount is at least v%, preferably about 1 to 2 w/v%. Of course, it can be used at a high concentration above this range, but even if it is used at a high concentration, the yield of the target product etc. will not improve, and it is rather economically unfavorable.

The condensation reaction in the first step of the present invention can be carried out by adding and mixing the ethyl acetate phase containing the base component and the aqueous phase containing the acid component and enzyme, or by simultaneously adding the ethyl acetate phase and the enzyme containing the acid component. This is carried out by adding and mixing to an aqueous phase, or by mixing an ethyl acetate phase containing a base component and an acid component and an aqueous phase containing an enzyme, and stirring the mixture at a predetermined temperature. The temperature during the above reaction is usually about 20 to 50°C, and the higher the temperature, the shorter the reaction time, but it is usually appropriate to keep it around 40°C. The pH of the aqueous phase during the reaction is
It is usually in the range of about 5 to 8, preferably about 6.5 to 7.0. In order to maintain the PH of the aqueous phase within the above range, which may change as the reaction progresses, an acid such as hydrochloric acid or an alkali such as sodium hydroxide may be sequentially added to the reaction system. . Further, the above-mentioned stirring is usually carried out under relatively gentle conditions or can be carried out with shaking so that the reaction system maintains a homogeneous state. Furthermore, the above-mentioned stirring does not have to be carried out continuously during the reaction, but can also be carried out intermittently.

Through the above condensation reaction, the desired protected tripeptide is obtained as a solution in an organic solvent. this is,
It can be easily separated by separating the organic phase and performing operations such as concentration crystallization or extraction according to a conventional method, and can also be purified by conventional isolation and purification means.

The thus obtained protected tripeptide is useful as an intermediate for the synthesis of Leu-enkephalin of the present invention, either as it is or after removing the protecting group according to a conventional method, and is also useful as an intermediate for the synthesis of Leu-enkephalin of the present invention. It is useful as a synthetic intermediate for various opioid peptide hormones such as endorphin, dynorphin, etc. [see Science and Industry, Vol. 59, No. 11, 458-468 (1985)], and as a raw material for amino acid formulations for amino acid infusions. It is.

In the method of the present invention, after removing the carboxyl group protecting group of the protected tripeptide obtained above, this and Phe-Leu-alkyl ester are
Condensation reaction using metalloprotease (second
process).

The above-mentioned elimination reaction of the carboxyl group-protecting group can be easily carried out according to a conventional method, for example, a hydrolysis reaction using formic acid or the like.

In the second step, the deesterified N-substituted Tyr-Gly-Gly obtained in the first step is used as an acid component, the Phe-Leu-alkyl ester is used as a base component, and preferably an immobilized enzyme is used. The reaction can be carried out by subjecting both of the above components to a condensation reaction in an aqueous solution or in an organic solvent such as ethyl acetate.
The substrate concentrations in each of the above components can be determined as appropriate, but usually the acid component is about 5-20 mM and the base component is about 5-20 mM.
The concentration is preferably in the range of about 100 mM, and the temperature conditions, pH conditions of the aqueous phase, stirring conditions, etc. during the condensation reaction can be approximately the same as those in the first step.

According to the research conducted by the present inventors, the use of an immobilized enzyme (immobilized Bacillus metalloprotease) is most important in the second step. The Bacillus metalloprotease used here is typically commercially available, such as thermolysin (manufactured by Daiwa Kasei Co., Ltd.), but it does not have to be this commercially available product, and can be prepared separately from Bacillus bacteria. It may be a crude enzyme solution, a purified product thereof, or a Bacillus metalloprotease having similar enzyme properties. Moreover, the immobilization can be carried out according to various conventional methods using a suitable support (see Japanese Patent Publication No. 1719/1983). A particularly preferable support is Amberlite XAD7
(manufactured by Rohm and Haas) is an example.
The amount of the immobilized enzyme used can be selected as appropriate, but
Usually, the amount present in the reaction solution is preferably about 1 to 25 w/v%.

In addition, as a typical example of the Phe-Leu-alkyl ester used as the base component in the above second step, for example, the production of des-Tyr ¹ -enkephalin (Japanese Patent Application Laid-open No. 1983-1979), which was established by the present inventors,
96096 (see Publication No. 96096), that is, those obtained by enzymatic synthesis using thermolysicin, but are of course not limited to this, and various known enzymatic or chemical methods can be used. It may be obtained by a synthetic method. Further, as the carboxyl protecting group of Leu constituting the base component, for example, an OEt group can be preferably exemplified.

The condensation reaction in the second step yields the desired oligopeptide (Leu-enkephalin precursor), which can be separated by a conventional method. For example, when the above condensation reaction is carried out in an organic solvent, the target product can be easily separated by separating the organic phase according to a conventional method, concentrating and crystallizing it, or performing an operation such as extraction. . Also,
When the above condensation reaction is carried out in an aqueous solution, the target product is adsorbed on the immobilized enzyme carrier, and is extracted with a water-soluble or slightly water-soluble organic solvent such as acetonitrile, ethyl acetate, etc. , the target object can be separated. Furthermore, the target product thus separated can be purified by conventional isolation and purification means.

The oligopeptide obtained above can be converted into the desired Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) by removing its carboxyl group and amino group protecting group according to a conventional method. can.

EXAMPLES The present invention will be explained in more detail with reference to Examples below.

Example 1 (1) Production of Gly-Gly-OBu ^t Contains 0.1% 2-mercaptoethanol
0.05M-MES ((2-diaminomorpholino)ethanesulfonic acid monohydrate, manufactured by Dojindo Laboratories) solution and equal volume of ethyl acetate were equilibrated (40°C) using a separatory funnel. , MES−NaOH buffer saturated with ethyl acetate, and MES−NaOH buffer saturated with ethyl acetate.
An ethyl acetate solution saturated with NaOH solution was prepared.

An organic phase substrate solution was prepared by dissolving 84 mg (40 mM) of Z-Gly in 10 ml of an ethyl acetate solution saturated with the MES-NaOH solution obtained above.

On the other hand, the ethyl acetate-saturated MES− obtained above
201 mg of Gly-OBu ^t HCl salt in 5 ml of NaOH buffer
(final concentration 240mM) to prepare an aqueous phase substrate solution at pH 6.5.

Add 50 mg (1.0%) of papain (manufactured by Sigma) to the aqueous phase substrate solution, mix this with the organic phase substrate solution, and stir at 40°C.
The desired protected peptide was obtained by controlling the pH of the aqueous phase to 6.0 with 1N NaOH and carrying out the reaction.

Target product yield after 20 hours reaction (Z-Gly standard)
was 75%.

The protected peptide obtained above was dissolved in a mixed solution of methanol:water:acetic acid (5:3:2), and catalytic reduction was performed using hydrogen gas using palladium black as a catalyst to remove the Z group. −
Gly-OBu ^t was obtained.

(2) Production of Z-Tyr-Gly-Gly 0.25M Tris-HCl buffer containing 5mM CaCl ₂ and an equal volume of ethyl acetate were equilibrated (40°C) using a separating funnel, and saturated with ethyl acetate. A Tris-HCl buffer and an ethyl acetate solution saturated with the same Tris-HCl buffer were prepared.

An organic phase substrate solution was prepared by dissolving 378 mg (80 mM) of Z-Tyr in 15 ml of the ethyl acetate solution saturated with the Tris-HCl buffer obtained above.

On the other hand, in 3 ml of the ethyl acetate-saturated Tris-HCl buffer obtained above, the Gly-Gly-
After dissolving ^OBut to 600mM,
The pH was adjusted to 7.0 with 6N HCl. This is used as the aqueous phase substrate solution.

Add 36 mg of α-chymotrypsin (manufactured by Sigma) (final concentration 1.2%) to the above aqueous phase substrate solution, mix this with the above organic phase substrate solution, and add 1N NaOH to the aqueous phase while stirring at 40°C. The desired protected tripeptide was obtained by controlling the pH at 7.0.

Yield of target product after 20 hours reaction (Z-Tyr standard)
was 95%.

The protected tripeptide obtained above is dissolved in formic acid to remove the OBu ^t group, resulting in Z-Tyr-
I got Gly−Gly.

(3) Production of Phe-Leu-OEt A 0.25M Tris-HCl buffer containing 5mM CaCl ₂ and an equal volume of ethyl acetate were equilibrated (40°C) using a separating funnel, and the mixture was saturated with ethyl acetate. A Tris-HCl buffer and an ethyl acetate solution saturated with the Tris-HCl buffer were prepared.

313 mg (160 mM) of Leu-OEt HCl salt in 10 ml of ethyl acetate-saturated Tris-HCl buffer obtained above.
was dissolved to prepare an aqueous phase substrate solution with pH=7.5.

On the other hand, an organic phase substrate solution was prepared by dissolving 239.5 mg (80 mM) of Z-Phe in 10 ml of ethyl acetate saturated with the above Tris-HCl buffer.

Thermolysin (manufactured by Daiwa Kasei Co., Ltd., titer 9470 PU/mg) 20 mg (0.2
%), mixed with the substrate solution on the organic phase side, and reacted at 40°C with stirring to form Z-Phe.
−Leu−OEt was obtained.

The yield after 5 hours of reaction (based on Z-Phe) is
The yield after 24 hours reaction is 99.5%.
It was hot.

From the protected dipeptide obtained above, acetic acid and
Using a 25% HBr-acetic acid solution, the Z group was subjected to an elimination reaction to obtain Phe-Leu-OEt.

(4) Preparation of immobilized thermolysin 7.5 g of thermolysin was mixed with 5M-NaBr and
Dissolve in 120 ml of 0.25 M Tris-HCl buffer (PH7.5) containing 16.6 mM CaCl ₂ under ice cooling, add 30 g (wet weight) of Amberlite XAD7 (manufactured by Rohm and Haas), and incubate at 4°C. The enzyme was adsorbed onto the carrier by gentle shaking for 17 hours. As a result of quantifying the amount of enzyme protein remaining in the supernatant using the Biuret method, it was found that approximately 70% of the initial amount of enzyme was adsorbed.

After removing 75 ml of the above supernatant, 75 ml of 25% glutaraldehyde solution was added to the remaining immobilized enzyme suspension, and the mixture was shaken at 4°C for about 3 hours to perform a cross-linking reaction, and then cooled with 5 mM _CaCl2.
0.1M Tris-HCl buffer (PH7.5) approx.
Immobilized thermolysin was obtained by washing twice alternately with approximately 1 part of the same buffer containing 1M NaCl. The obtained immobilized enzyme was stored at 4°C.

(5) Synthesis of Z-Tyr-Gly-Gly-Leu-OEt 0.05M-MES solution containing 5mM _CaCl2 ,
Equilibrate (40 °C) with equal volumes of ethyl acetate in a separatory funnel and saturated with ethyl acetate.
A MES solution and an ethyl acetate solution saturated with the MES solution were prepared.

9 mg of Z-Tyr-Gly-Gly (final concentration
7.5mM) and Phe-Leu-OEt7mg (7.5mM)
A substrate solution was prepared by dissolving.

Meanwhile, the above ethyl acetate saturated MES solution (PH
5.0), 0.2 g of immobilized thermolysin was added, and after equilibration (40) for 30 minutes, the substrate solution was added thereto and the reaction was carried out by shaking at 40°C.

Sample small amounts of the reaction solution over time,
High performance liquid chromatography was performed under the conditions shown below to quantify the amount of product.

<High performance liquid chromatography> Equipment: High performance fluid chromatography (manufactured by Shimadzu Corporation)
LC-A type) Column: Inner diameter 4.6mm x length 150mm Cosmosil 5C ₁₈
-Ppacked colum manufactured by Hanui Chemical Co., Ltd.) Solvent: Acetonitrile - Water (60:40, PH with phosphoric acid)
(adjusted to 2.5) Detection: Ultraviolet absorption (254 nm) As a result, the desired product (Z
The yield of -Tyr-Gly-Gly-Phe-Leu-OEt, Leu-enkephalin precursor) based on the starting substrate amounted to approximately 28%.

The above object (Z-Tyr-Gly-Gly-Phe-Leu
-OEt) was extracted with ethyl acetate and dried in an evaporator, and a mixture of 5 ml of methanol, 3 ml of water, and 2 ml of acetic acid was added to each 1 mmol of the extracted product, a small amount of palladium black was added, and the mixture was extracted using hydrogen gas. The Z group was eliminated by catalytic reduction.

After the reaction, the palladium black was removed, the reaction solution was dried in an evaporator, and the resulting compound was
At 4°C, add an equal volume of 1M sodium hydroxide aqueous solution to cleave the ester bond, further neutralize with acetic acid,
Concentrate about 10 times using a rotary evaporator, wash the resulting precipitate with a small amount of distilled water, then with ether, and dry to obtain the desired Tyr-
Gly-Gly-Phe-Leu was obtained.

Example 2 In Example 1, the concentration of Z-Tyr-Gly-Gly was 5.5mM, the concentration of Phe-Leu-OEt was 55mM,
The pH of the MES-NaOH buffer used for equilibration of the immobilized enzyme was set to 4.5, and the reaction was performed by shaking in the same manner.

As a result, the desired product was produced with a yield of about 68%.

Example 3 Using the same immobilized enzyme as in Example 1, the synthesis reaction of Z-Tyr-Gly-Gly-Phe-Leu-OEt was carried out in an aqueous solution as follows.

i.e. 0.05M MES- containing 5mM _CaCl2
Add 26% of Z-Tyr-Gly-Gly to 6 ml of NaOH buffer.
mg (10mM) and 44mg of Phe-Leu-OEt・HBr
(20mM) to prepare a substrate solution at pH 5.0.

Weigh out 67 mg of immobilized thermolysin into 5 vials, and add 1 ml each of the above substrate solutions into each vial.
was added and shaken at 40°C to carry out the reaction.

For each reaction time, add 6N to one of the vials.
The reaction was stopped by adding 20μ of HCl, and the reactant in the immobilized enzyme was extracted with 2 ml of acetonitrile, which was used to quantify the amount of product under the same conditions as in Example 1.

As a result, it was found that the yield of the target product reached approximately 62% 3 hours after the start of the reaction.

Claims (1)

[Claims] 1 Condensation reaction of N-substituted tyrosine and glycyl-glycine alkyl ester using α-chymotrypsin, followed by de-esterification of the protected tripeptide obtained above, followed by phenylalanyl-leucine alkyl ester using Bacillus metalloprotease. A method for producing an oligopeptide, characterized by carrying out a condensation reaction.