JPS6258712B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing oligopeptides by condensation reaction of N-substituted glycyl-glycine and phenylalanyl-leucyl alkyl ester using a specific proteolytic enzyme. .

Prior Art Oligopeptide, glycyl-glycyl-phenylalanyl-leucine, i.e. Gly-Gly-Phe-
Leu is Des-Tyr ¹ -Enkephalin (des-
It is known as an inhibitor of ^enkephalin -degrading enzyme (enkephalinase).

However, enkephalin (L-
Tyr-Gly-Gly-L-Phe-L-Leu) is a morphine-like analgesic peptide isolated from the brains of several mammalian species, including pigs, and is of interest as a biological component analgesic. ing. On the other hand, enkephalin administered into a living body is extremely quickly degraded by enzymes in brain cells and loses its physiological activity.
Therefore, if the activity of enkephalinase, which is an enzyme that degrades enkephalin, is inhibited, the relative concentration of enkephalin in the body will increase, and it is expected that the analgesic effect will be enhanced and its duration will be extended. Des-Tyr ¹ -enkephalin is known as a substance having this enkephalinase inhibitory activity, but this compound is currently produced exclusively by chemical synthesis methods.

In recent years, attempts to synthesize useful peptides using the reverse reaction of proteolytic enzymes have become active, and methods for synthesizing peptides using such proteolytic enzymes (enzymatic synthesis method) are Compared to conventional synthesis methods, it is not necessary to protect the side chain functional group of the amino acid, the reaction proceeds stereoselectively, allowing the use of inexpensive racemic raw materials, and racemization does not occur during the reaction. It has very excellent characteristics, such as the fact that it does not react at all, and that the reaction proceeds at room temperature and pressure.

On the other hand, in the enzymatic synthesis method, the enzyme that can be used is determined depending on the type of amino acid used as a raw material due to the substrate specificity of the enzyme, and it is not the case that one enzyme can be used for any peptide synthesis.
It is extremely difficult to select an enzyme that can catalyze the desired peptide synthesis reaction, and even with established enzymatic peptide synthesis methods, the reaction equilibrium is generally heavily biased toward the substrate. However, there are problems in that the yield, reaction rate, etc. are quite low. In particular, when enzymatically synthesizing oligopeptides in which three or more different amino acids are bonded, the so-called stepwise method is usually used in which two amino acids are bonded in advance, and then additional amino acids are bonded in sequence. However, this method requires the use of dipeptides, tripeptides, etc. as raw material substrates for reactions from the second stage onwards, and these raw material substrates are hydrolyzed and cleaved by enzymes used in the synthesis reaction system. In many cases, the amino acids generated by the cleavage are further involved in the synthesis reaction. When such side reactions occur, the target product may not be obtained, a large amount of by-products are produced, making it difficult to separate the target product, or the purity of the target product is significantly reduced.

Problems to be Solved by the Invention An object of the present invention is to provide a new method for producing des-Tyr ¹ -enkephalin by making full use of enzymatic synthesis methods. 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 According to the present invention, a condensation reaction of N-substituted glycyl-glycine and phenylalanyl-leucyl alkyl ester is carried out using a Bacillus metalloprotease in a water-ethyl acetate two-phase system. A method for producing characterized oligopeptides is provided.

In this specification, descriptions of amino acids, peptides, protective groups, etc. shall follow symbols commonly used in the field.

The present inventors have previously established an enzymatic synthesis method for the dipeptides Phe-Phe and Asp-Phe in the course of extensive research into the synthesis of peptides using proteases (see Japanese Patent Laid-Open No. 45596/1983).
In subsequent research, the present inventors aimed to synthesize enkephalin, and as a first step, des-Tyr ¹ , an inhibitor of enkephalinase, was synthesized.
-Conducted extensive research into the enzymatic synthesis (condensation) reaction of enkephalin. As a result, new findings were obtained that the above condensation reaction is catalyzed by thermolysin, a Bacillus metalloprotease, and that this reaction is efficiently carried out in a water-ethyl acetate two-phase system. The present invention was completed based on this knowledge.

N-substitution as one substrate in the method of the present invention
Gly--N in Gly (hereinafter referred to as "acid component")
- The substituent 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, t
-butoxycarbonyl group (Boc), 2-chlorobenzyloxycarbonyl group, etc. are also included. The alkyl group in the other substrate Phe-Leu-alkyl ester (hereinafter referred to as "base component") is also a commonly used carboxyl protecting group for amino acids.
Specific examples thereof include alkyl groups having 1 to 4 carbon atoms;
For example, methyl, ethyl, propyl, and butyl groups can be exemplified, and other groups include benzyl, p-nitrobenzyl, and p-nitrobenzyl.
-Chlorobenzyl group etc. are also well known. Among these raw material substrates, Gly--Gly, which is the acid component, can be easily synthesized by chemical synthesis because Gly does not have optical isomers, and is also commercially available. Each amino acid of Phe-Leu, which is a base component, is usually used in the L-form, and the base component may be synthesized by chemical synthesis, but the enzymatic synthesis according to the present invention Preferably, it is synthesized using thermolysin in the same manner as in the method.

In the present invention, it is important to carry out the enzyme reaction 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.

A suitable buffer solution is preferably used as the aqueous phase, and for example, an aqueous solution of (2-diaminomorpholino)ethanesulfonic acid (MES) is preferably used. In addition, sodium hydroxide, etc., can be added to the aqueous phase in order 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. You can also do that.

In the method of the present invention, a solution of both substrates is prepared by first dissolving an acid component in the aqueous phase and dissolving a base component in the ethyl acetate phase. The substrate concentration in each of the above substrate solutions is determined appropriately, and 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 5 to 60 mM, especially for base components. Adjust the concentration ratio of acid components to approximately 0.2~
A range of 3, usually about 0.4 to 3, is suitable; in this range, the lower the concentration of the acid component, the higher the yield of the desired oligopeptide tends to be; Although the product yield decreases slightly, the production of side reaction products tends to be suppressed. Also, the usage ratio (volume) of each of the above substrate solutions
By using at least an equal amount of the aqueous phase to the ethyl acetate phase, the desired synthesis reaction proceeds and the desired product is obtained in high yield. Normally, 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 greater the amount of ethyl acetate phase used, the better the purity and yield of the target product will be. There is a tendency to

In the method of the present invention, Bacillus metalloprotease is used by adding it to the aqueous phase substrate solution. For example, thermolysin (manufactured by Daiwa Kasei Co., Ltd.) is commercially available as the enzyme agent, but in the present invention, it is not necessary to use this commercially available product, and a crude enzyme solution separately prepared from Bacillus bacteria is used. Bacillus metalloproteases having similar enzymatic properties can also be used. Its usage is
Although it varies depending on the titer of the enzyme used, reaction conditions, etc., in the case of thermolysin, the total volume of the aqueous phase used in the present invention is usually about 100 ml. It is good to set it to 2 W/V% or more, 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.

In the condensation reaction of the present invention, the ethyl acetate phase containing the base component and the aqueous phase containing the acid component and the enzyme are added and mixed, or the ethyl acetate phase and the enzyme are simultaneously added to the aqueous phase containing the acid component. A mixture (emulsion) is prepared by adding and mixing or by mixing an ethyl acetate phase containing a base component and an acid component and an aqueous phase containing an enzyme.
This is carried out by stirring 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 usually preferably in the range of about 4.5 to 6.5, and in order to maintain the PH of the aqueous phase within the above range, which may change as the reaction progresses, the reaction system An acid such as hydrochloric acid can also be added sequentially. 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, but can also be carried out intermittently.

Through the above condensation reaction, the desired oligopeptide is obtained as a solution in an organic solvent. This 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 further purified by conventional isolation and purification means.

The thus obtained oligopeptide is converted into des-Tyr ¹ -enkephalin (GIy-Gly-L-Per-L-Leu) by removing its carboxyl group and amino group protecting group according to a conventional method. Can be done. It is useful as an enkephalinase inhibitor and also as an intermediate in the synthesis of enkephalin.

Examples Examples will be given below to explain the present invention in more detail.

Example 1 (1) Preparation of L-Phe-LeuOEt 0.25M Tris-HCl buffer (containing _5mMCaCl2 ) and an equal volume of ethyl acetate were equilibrated (40°C) using a separating funnel, and ethyl acetate A Tris-HCl buffer saturated with Tris-HCl buffer and an ethyl acetate solution saturated with the same Tris-HCl buffer were prepared.

313 mg of L-LeuOEt HCl salt in 10 ml of ethyl acetate-saturated Tris-HCl buffer obtained above.
(160mM) 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 20mg in the above aqueous phase substrate solution
(0.2%), mixed with the organic phase substrate solution, reacted at 40℃ with stirring, and Z-L
-Phe-L-LeuOEt obtained.

Yield (Z-Phe standard) After 5 hours of reaction: 93.2% After 24 hours of reaction: 99.5% From the protected dipeptide obtained above, acetic acid and
The Z group was eliminated using a 25% HBr-acetic acid solution to obtain L-Phe-L-LeuOEt.

(2) Production of Z-Gly-Gly-L-Phe-L-LeuOEt First, 5mM which is a stabilizing factor of thermolysin.
Equilibrate a 0.05M MES ((2-diaminomorpholino)ethanesulfonic acid monohydrate, manufactured by Dojindo Laboratories) solution containing CaCl ₂ and an equal volume of ethyl acetate using a separatory funnel ( A MES solution saturated with ethyl acetate and an ethyl acetate solution saturated with the same MES solution were prepared.

MES solution obtained above saturated ethyl acetate solution 10
ml, 61.4 mg of L-Phe-L-LeuOEt was dissolved (final concentration 20 mM) to prepare an organic phase substrate solution.

Meanwhile, the ethyl acetate saturated MES solution obtained above
53.3 mg of the other substrate, Z-Gly-Gly (manufactured by Sigma), was dissolved in 10 ml (final concentration 20 mM), and the pH was adjusted to 4.5 with a 4N aqueous sodium hydroxide solution to prepare an aqueous phase substrate solution.

Thermolysin (manufactured by Daiwa Kasei Co., Ltd., Bacillus metalloprotease,
40 mg (potency 9470 PU/mg) was dissolved, and the organic phase substrate solution was added thereto and stirred at 40°C to carry out a reaction in an emulsion state. In addition, the reaction is in progress
1N hydrochloric acid was added to the solution to maintain the aqueous phase at 4.5. Sample small amounts of the organic phase over time;
High performance liquid chromatography was performed under the conditions shown below to quantify the amount of product. The amount of product in the aqueous phase was negligible compared to that in the organic phase.

<High performance liquid chromatography> Equipment: High performance fluid chromatography (manufactured by Shima Ritsu Seisakusho)
LC-3A type) Column: Inner diameter 10 mm x length 300 mm Packing material: TSK-GEL LS-410K (ODS-Silica manufactured by Toyo Soda Co., Ltd.) Solvent: Acetonitrile-Water (55:45, PH with phosphoric acid)
(adjusted to 2.5) Detection: Ultraviolet absorption (25 nm) The results are shown in Figure 1. In FIG. 1, the horizontal axis shows the reaction time (hours), the vertical axis shows the product yield (%), and curve 1 shows the target product Z-Gly-
Gly-L-Phe-L-LeuOEt, curve 2 is Z-Gly-Gly-L-Phe-L-Phe-
L-LeuOEt is shown respectively.

From FIG. 1, the yield of the desired product (precursor of des-Tyr ¹ -leucine enkephalin, shown in curve 1) is about 60%, based on the starting substrate, while the by-product is as shown in curve 2 above. It can be seen that only about 6% of the pentapeptide shown by is produced.

The above object (Z-Gly-Gly-L-Phe-L-
LeuOEt) was extracted with ethyl acetate, dried in an evaporator, and 5 mmol of acetic acid was extracted per 4 mmol of the
ml and 10 ml of 25% HBr-acetic acid solution was added, and the mixture was reacted at room temperature for 1 hour to eliminate and remove the Z group.

After the reaction, add diethyl ether to the system.
HBr-Gly-Gly-L-Phe-L-LeuOEt was precipitated. This was dissolved in distilled water with an equal amount of sodium carbonate, and shaken with chloroform in a separatory funnel to extract Gly-Gly-L-Phe-L-LeuOEt. Allowed to dry.

The obtained compound was added with an equal volume of 1N aqueous sodium hydroxide solution at 0 to 4°C to cleave the ester bond, further neutralized with acetic acid, concentrated about 10 times using a rotary evaporator, and the resulting precipitate was Wash each with a small amount of distilled water and then with ether and dry.
Obtained Gly-Gly-L-Phe-L-Leu.

Comparative Example 1 In order to clarify the effects observed in the method of the present invention in the water-ethyl acetate two-phase system shown in Example 1 above, the following comparative method in an aqueous phase system was carried out.

That is, 1/20M MES-NaOH saturated with ethyl acetate.
buffer (PH) 5.4), each with a final concentration of 20mM.
Each substrate (Z-Gly-Gly and L-Leu
-L-PheOEt) was added and dissolved, and the same enzyme (0.1%) used in Example 1 was added to this aqueous solution, and an enzyme reaction was carried out in the aqueous solution under the same conditions.

The reaction solution was sampled over time and subjected to high performance liquid chromatography under the same conditions as in Example 1, and the amount of product was quantified. The results are shown in FIG.

As is clear from Figure 2, in the reaction in aqueous solution, the yield of the target product is only 7.5% (see curve 1).
On the other hand, as a by-product, the pentapeptide Z-Gly-Gly-L-Phe-L-Phe-L-
LeuOEt is approximately 15% (shown as curve 2 in the figure) and Z-Gly-Gly-L, which is a higher molecular weight peptide
-Phe-L-Phe-L-Phe-L-LeuOEt is about 10
% (shown as curve (3) in the figure) was also confirmed to be generated.

Example 2 In Example 1, the final concentration of Z-Gly-Gly was
15mM, final concentration of L-Phe-L-LeuOEt
The reaction was carried out in the same manner in an emulsion state at 5 mM, organic phase volume/aqueous phase volume ratio of 10/1, aqueous phase enzyme concentration of 2%, and aqueous phase PH of 4.5.

As a result, the desired product was produced with a high yield of about 82%.

As described above, it can be seen that the enzyme reaction in the water-ethyl acetate two-phase system of the present invention can improve the yield of the target product. Depending on the selection of reaction conditions, approximately
The desired product can be synthesized with a high yield of around 90%.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between reaction time and yield in the method shown in Example 1, and FIG. 2 is the same graph in the method shown in Comparative Example 1.

Claims (1)

[Claims] 1. A method for producing an oligopeptide, which comprises carrying out a condensation reaction of N-substituted glycyl-glycine and phenylalanyl-leucyl alkyl ester in a water-ethyl acetate two-phase system using a Bacillus metalloprotease.