JPS63254994A - Production of n-substituted leucine enkephalinamide - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the titled compound useful as an anesthetic agent, etc., by successively reacting an N-substituted Tyr ester with a Gly ester in the presence of a specific enzyme in an aqueous medium, then reacting the resultant reaction product with a Gly ester, Phe ester and then Leu amide. CONSTITUTION:An N-substituted tyrosine (Tyr) lower alkyl ester is reacted with glycine (Gly) lower alkyl ester in the presence of serine protease (e.g. alpha-chymotrypsin) in an aqueous medium containing a water-soluble organic solvent (e.g. dimethylformamide) and the resultant product is then reacted a glycine lower alkyl ester and phenylalanine (Phe) lower ester in the presence of thiol protease (e.g. papain). The obtained reaction product is subsequently reacted with leucinamide (Leu amide) in the presence of serine protease to extend the peptide and afford the aimed compound.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to N-substituted leucine enkephalinamide, that is, N-substituted tyrosyl-glycyl-glycyl-phenylalanyl-leucinamide (hereinafter referred to as X-Tyr-Gly-G
The present invention relates to a method for producing ly-Phe-Leu-Nl (abbreviated as 2). More specifically, in an aqueous medium containing a water-soluble organic solvent, N-substituted tyrosine lower alkyl ester (hereinafter referred to as X-Tyr-OY), glycine lower alkyl ester (hereinafter referred to as
he-OY3), and leucine amide (hereinafter referred to as Le
The present invention relates to a method for producing N-substituted leucine enkephalinamide, which is characterized by carrying out a peptide elongation reaction by sequentially reacting u-NH2) with a protease.

N-substituted leucine enkephalin amide is converted to leucine enkephalin by removing its N substituent and C-terminal amino group by an appropriate method. Leucine enkephalin exhibits a morphine-like anesthetic effect, and its pharmacological activity is said to be equal to or greater than that of morphine.

[Prior Art] Various methods have been proposed for peptide synthesis, most of which are based on so-called chemical methods. In the method of chemically forming peptide bonds, in the reaction with the condensation reagent used and in the deprotection step,
Amino acids may racemize, and careful reaction design is generally required. Furthermore, it is necessary to introduce an appropriate protecting group into the hydroxyl group, carboxyl group or amino group that is not to be condensed in the side chain of the amino acid prior to condensation. Furthermore, a type of chemical method called a solid phase method is known.

Although this method has the advantage of simplifying the purification of intermediates, it is limited to the synthesis of relatively small amounts of peptides and is not suitable as a total method on an industrial scale.

In contrast, enzymatic peptide synthesis methods have attracted attention in recent years. It has been known for a long time that proteolytic enzymes (hereinafter referred to as proteases) such as papain and chymotrypsin can be involved in the reverse reaction, which is the formation of peptide bonds. Fruton revealed that a monoaminocarboxylic acid such as leucine whose amino group is protected with a benzoyl group or the like and leucine or glycine whose carboxyl group is protected as an amide or anilide can be peptide-bonded using papain or chymotrypsin. “Advances in Prota”
in Che+nistry″″, Volume 5, page 33 (1949), Academic Press in
c, New York), and in the case of chymotrypsin, ^c-Ty under optimal conditions for its esterase activity.
It has been revealed by Morihara et al. that it is possible to form a peptide bond between an N-substituted amino acid alkyl ester such as r-OEt and leucine amide (Biochem. J. Vol. 163, p. 531, (1977). )).

A method is known for enzymatically synthesizing enkephalin by combining and improving these known facts. Ullmann et al. used papain and chymotrypsin to detect t-butoxycarbonyl
Tyrosyl-glycylphenyl hydrazide (Boc-T
yr-Gly-NtlNHPh) and t-butoxycarbonyl-glycyl-phenylalanyl-L-leucine phenylhydrazide (Boc-Gly-Phe-Le
u-NHNHPh) is first synthesized, and the former is removed from N2N.
After Boc removal of HPh and the latter, both were condensed using papain to obtain leucine enkephalin (B
iochem.

Biophys. Res. Com. , Article 91(2)
roll. 693 pages, (1979)).

In addition, Johansen et al. reported a method to obtain methionine enkephalin by sequentially elongating one amino acid at a time from the N-terminus using carboxypeptidase-Y, a type of exoprotease (Pe
ptides 1980.

Scriptor. Kopenhagen. 46 pages, (1981)).

[Problems to be solved by the invention Generally, even in peptide synthesis using protease,
Protecting groups are required for amino and carboxyl groups that are not fused. In the method conducted by Ullmann et al., the reverse reaction of hydrolysis with papain is used, so if the once-protected amino and carboxyl groups participate in condensation in the next step, the protecting groups must be removed. No. Furthermore, in order to reduce the solubility of the product and prevent hydrolysis by papain, a protecting group must be introduced into the side chain of tyrosine in advance, but ultimately this protecting group must also be removed. In this way, the deprotection step is unavoidable, which not only complicates the synthesis process, but also causes racemization due to the radical reagents used for deprotection, making selective deprotection difficult. There was a point.

Furthermore, in the method carried out by Johansen et al., carboxypeptidase-Y is used as the protease, but due to its broad substrate specificity, side reactions are likely to occur and reaction control is difficult. Furthermore, carboxypeptidase-Y is not easy to handle and is expensive, so it has not always been satisfactory from an industrial standpoint.

[Means and effects for solving the problems] In view of the current situation, the present inventors have conducted intensive research to establish a simpler synthetic method using a general-purpose protease that does not have the above problems. As a result of repeated efforts, we have arrived at the present invention. That is, the present invention provides the following steps: A: A step of reacting an N-substituted tyrosine lower alkyl ester with a glycine lower alkyl ester in the presence of a serine protease in an aqueous medium containing a water-soluble organic solvent; B: In the presence of a thiol protease. , a step of reacting the product of step A with glycine lower alkyl ester and then reacting with phenylalanine lower alkyl ester, C: a step of reacting the product of step B with leucine amide in the presence of serine protease. This method is characterized by producing N-substituted leucine enkephalinamide by elongating the peptide through steps C to C.

The proteases used in the present invention are selected by considering their substrate specificity, with serine proteases forming peptide bonds between tyrosine and glycine, and phenylalanine and leucine; Thiol proteases are used for the formation of peptide bonds between and glycine, and between glycine and phenylalanine. Among these, it is preferable to use α-chymotrypsin as the serine protease and papain as the thiol protease.

In the present invention, since the esterase activities of α-chymotrypsin and papain are used to form peptide bonds,
Peptide synthesis can be performed without performing a reaction to remove an ester group, which is a protecting group at the C-terminus to be bonded to the peptide.

The concentration of these enzymes used is not limited, but if the concentration used is high, the reaction will be completed in a short time. However, extremely high enzyme concentrations tend to lead to enzyme autolysis. Generally, about 0.2 to 0.3mM is sufficient, preferably 0.24mM for both papain and α-chymotrypsin.

The concentration of the starting material Nfi-substituted tyrosine lower alkyl ester (X-Tyr-OY) for the process of the present invention is within its solubility in the solvent, and each intermediate, i.e., N-substituted tyrosylglycine Lower alkyl ester (X-Tyr-Gly-OY+), N-substituted tyrosylglycylglycine lower alkyl ester (X-Tyr
-Gly-Gly-OY2) and the solubility and yield of N-substituted tyrosylglycylglycyl phenylalanine lower alkyl ester (X-Tyr-Gly-Gly-Phe-OY3). Generally, 0.05M to 0.1M is convenient.

The concentration of each substrate used varies depending on the protease; when papain is used, glycine lower alkyl ester and phenylalanine lower alkyl ester are used.
Once, the generated N-substituted tyrosylglycine lower alkyl ester (X-Tyr-Gly-OY+) and N-substituted tyrosylglycylglycine lower alkyl ester (X-T
1 of each concentration of yr-Gly-Gly-OY2)
It is preferable to maintain the amount at 0 times. This is important when using papain's esterase activity for peptide synthesis. On the other hand, when α-chymotrypsin is used, the concentration of glycine lower alkyl ester and leucine amide used is not particularly limited other than setting it within the solubility of the solvent used; ester (X-Tyr-OY) and the generated N-substituted tyrosylglycylglycyl phenylalanine lower alkyl ester (X-Tyr-Gly-Gly
-Phe-OYs).

The pH range in which the protease used in the present invention exhibits esterase activity varies depending on the individual protease, but
For α-chymotrypsin, pH 9 to pH 11,
p) 110 is preferred. In addition, papain has a pH of 5 to p.
H7, p) IS is most desirable. Figure 1 shows N
-benzoyltyrosylglycine methyl ester (Bz-
Tyr-G, y-OMe) and glycine methyl ester (
The composition of the reaction solution of each pl (in the reaction with papain with GIy-OMe) was investigated by high performance liquid chromatography. At pH 6, the target product benzoyltyrosylglycylglycine methyl ester (Bz-T
It can be seen that yr-Gly-Gly-OMe) is produced in high yield.

As the water-soluble organic solvent of the present invention, dimethylformamide, acetonitrile, methanol, ethanol, dimethyl sulfoxide, glycerin, ethylene glycol, etc. can be used, but dimethylformamide is most preferred. The aqueous medium used in the present invention is an aqueous solution containing these organic solvents in an appropriate mixing ratio and whose pl+ is adjusted.

The mixing ratio of the organic solvent and water in the aqueous medium is limited from the viewpoint of maintaining the solubility and enzyme activity of the substrates and products used, but the organic solvent is preferably 40% to 80% papain and α-chymotrypsin. 50 if you use
% is most desirable. As described above, the mixing ratio of organic solvent and water has a great influence on the utilization of papain's esterase activity.

Figure 2 shows (Bz-Tyr-Gly-OMe) and (Gl
In the reaction of papain with y-OMe), the target product N- is obtained when the latter is reacted in a DMF 40% aqueous solution at twice the concentration of the former and when it is reacted in a DMF 50% aqueous solution at 10 times the concentration of the former. Benzoyltyrosylglycylglycine methyl ester (Bx-Tyr-Gly-Gly-
This figure illustrates the production rate of OMe). 10 times amount D
When reacting in a 50% MF aqueous solution, 70% in 3 hours
The target product (Br-Tyr-Gly-Gly-O
Me) is generated.

The reaction temperature is preferably 20°C from the viewpoint of maintaining enzyme activity.
It is carried out at a temperature of 40°C to 40°C.

The reaction time varies depending on the reaction temperature and the amount of enzyme used, so it cannot be uniquely limited, but it can be
X-Tyr-Gly-Gly-Phe-OYs) +
In the reaction of (Leu-NH2), papain was used for (X-Tyr-Gly-OY+)+ (Gly
-OY2), and (X-Tyr-Gly-Gly-OY
2) ” (Phe-OY3) reaction from 3 hours to 5 hours
Time is preferable.

First, X-Tyr-Gly-OY is synthesized using α-chymotrypsin under the above conditions. Subsequently, Gty-oy2 is reacted with papain, but residual α-chymotrypsin is removed in advance by ultrafiltration using IJFII. This is because when α-chymotrypsin and papain coexist, α-chymotrypsin is degraded by papain's action as a hydrolase, causing impurities. The first reaction, that is, X-
It is also possible to add Gly-0'h in advance to the reaction of Tyr-OY and Guy-OY with α-chymotrypsin, and omit the addition of Gty-oy2 during the second stage reaction. In this way, X-Tyr-G
After synthesizing ly-Gly-OY, Phe-OY is added to synthesize X-Tyr-Gly-Gly-Phe-OYs.
get. Leu-N) then by α-chymotrypsin
I2 is reacted, but as before, it is an ultraviolet reaction using an OF membrane in advance? Papain is removed by F A or the like. Final product X-Tyr-Gl/-G1y-Phe-L
eu-N)I2 can be obtained from the reaction solution by conventional treatments such as extraction and purification. Also, the intermediate X-Tyr-Gly-Gl
When y-Phe-OY3 is produced, it becomes insolubilized by adding more than an equivalent amount of water to the reaction solution and precipitates, so it can be roughly purified at that stage.

Since the enzyme used in the present invention has a high degree of substrate specificity, the next step reaction can be carried out even in a reaction solution where the reaction substrate from the previous step remains, which greatly simplifies the purification of intermediates. can be converted into

[Examples] The present invention will be described below based on Examples, but the present invention is not limited thereto.

Bz-Tyr-OEt 626mg and Gly-OMe
496 mg of hydrochloride was dissolved in 20+n4 50% DMF/water mixed solvent and adjusted to pH 10. Add α-chymotrypsin (manufactured by Wako Pure Chemical Industries, titer 1000115 derived from bovine pancreas) to this reaction solution.
ts/mg) was added, the p)I was readjusted to 10, and the mixture was stirred at room temperature. The composition of the reaction solution after 1 hour was analyzed by high performance liquid chromatography, and it was found that the concentration of Bz-Tyr-Gly-OMe had reached 0.8M. After shifting the p)I of this reaction solution to 6, in order to remove insolubilized proteins, etc.
The mixture was passed through a lex-5R (trade name) 5.5 μm filter unit (manufactured by Millibore). Then, ultraviolet filtration was performed using an IIF membrane (Toyo Roshi U-1101 molecular weight cutoff 10,000). A, (Gly-OMe to D f solution) hydrochloride 1.70 g, 2-mercaptoethanol 0. IJ,
and papain (manufactured by Sigma, Papaya Lat
ex origin, titer 24 units/mg) 100+ng
was added, the pH was readjusted to 6, and the mixture was stirred. The composition of the reaction solution after 3 hours was analyzed in the same manner as above, and the concentration of Bz-Tyr-G 1y-G ly-OMe reached 0.058M.

Immediately add 2.55 g of Phe-OEt hydrochloride to this reaction solution.
was added and continued stirring. When 50 mR of water was added to this reaction solution after 5 hours, Bz-Tyr-Gly-Gl
y-Phe-OEt was precipitated. This product was collected by centrifugation. The procedure of suspending this in water and recovering it by centrifugation was repeated several times to perform rough purification. Bz
The yield from -Tyr-OEt was 39%. This recovered material and 257 mg of hydrochloride of Leu-N) 12 were added to 20+
Redissolve J in 50% DMF/water mixed solvent, and reduce the p of the reaction solution.
H was adjusted to lO. After adding 120 mg of α-chymotrypsin to this reaction solution and readjusting the pH to 10,
Stir at room temperature. After 1 hour, 2N-hydrochloric acid was added and p
The reaction was stopped by raising the temperature to H4. From this reaction solution, Bz-Tyr-
Gly-Gly-Phe-Leu-NO3 was extracted and concentrated under reduced pressure. Bz-Tyr-Gly-Gly-Phe-L was dissolved in methanol, separated by high-performance liquid chromatography, and freeze-dried.
A powder of eu-NO3 was obtained.

The final yield from Bz-Tyr-OEt was 35%.

The obtained Bz-Tyr-Gly-Gly-Ph
The e-Leu-NH2 physical property values matched those of the standard product synthesized by the liquid phase method within the error range.

The measurement device and measurement conditions for high performance liquid chromatography analysis are as follows.

tlV-8modelll (Detector) GE-4 (Product name) (Gradient device) (Product name) (Preparative separation: φ21.
5mm, 300+nm) Elution mode: Linear gradient mode CH3CN 10%i, IC statement 0.19
6-〇)Is(:N' 60*:I(C or 0.1*25
Divided flow rate: 1 m/min Detection wavelength: 210 nm [Effects of the invention] According to the method of the present invention, water-soluble WA? In an aqueous medium containing @ medium, under pH conditions in which the protease used exhibits esterase activity, one amino acid ester is sequentially elongated from the N-terminus without deesterifying the intermediate. is significantly shortened, and
It is possible to avoid ceramization and decrease in yield that occur in the process of deprotecting intermediates.

Furthermore, according to the method of the present invention, the peptide bond is formed by an enzyme and its high substrate selectivity is utilized, so that the next reaction step can be carried out even in a reaction solution containing unreacted substrate. This not only simplifies the purification of complicated intermediates, but also allows the use of a mixture of 0- and 5-amino acid esters as raw material amino acid esters, which satisfies the economic efficiency required for the manufacturing process. Can be done.

[Brief explanation of drawings]

Figure 1 shows Bz-Tyr-Gly-OMe and Gly-O
1 is a high performance liquid chromatograph of the composition of a reaction solution after 3 hours at each pH in a reaction of papain with Me. Figure 2 shows Bz-Tyr-Gly-OMe and Gly-O
In the reaction of papain with Me, the target product Bz-T when the latter was set at twice and 10 times the concentration of the former.
2 is a diagram illustrating the production rate of yr-Gly-Gly-OMe. Figure 1 Retention time fmtn,)

Claims (6)

[Claims] (1) A step of reacting an N-substituted tyrosine lower alkyl ester and a glycine lower alkyl ester in an aqueous medium containing a water-soluble organic solvent, A: in the presence of a serine protease, B: in the presence of a thiol protease, A A step of reacting the product of step B with glycine lower alkyl ester and then reacting with phenylalanine lower alkyl ester; C: reacting the product of step B with leucine amide in the presence of serine protease; A method for producing N-substituted leucine enkephalinamide, which comprises elongating the peptide in a step. (2) The production method according to claim (1), wherein the N-substituted tyrosine lower alkyl ester, phenylalanine lower alkyl ester, and leucine amide are in the L-form. (3) The N substituent of the N-substituted tyrosine lower alkyl ester is a benzoyl group, a benzyloxycarbonyl group,
Claim No. 1 which is a t-butoxycarbonyl group
) or (2). (4) The production method according to any one of claims (1) to (3), wherein the serine protease is α-chymotrypsin. (5) The production method according to any one of claims (1) to (4), wherein the thiol protease is papain. (6) The manufacturing method according to any one of claims (1) to (5), wherein the water-soluble organic solvent is dimethylformamide.