JPS62232393A - Enzymatic production of peptide derivative - Google Patents

Abstract

PURPOSE:To produce a peptide derivative in high efficiency at a low cost, by reacting an N-terminal-protected amino acid with a C-terminal-protected amino acid ester, amino acid amide, etc., in the presence of serine carboxypeptidase. CONSTITUTION:The objective peptide of formula III can be produced by the condensation reaction of (A) an N-terminal-protected amino acid of formula II with (B) a C-terminal-protected amino acid ester, amino acid amide, peptide ester or peptide amide of formula III in an aqueous solution or a mixture of water and a hydrophilic organic solvent at <=6.5pH, preferably 4-6pH at 5-50 deg.C, preferably 30-40 deg.C in the presence of serine carboxypeptidase. The enzyme used in the above reaction is carboxypeptidase Y originated from yeast and the hydrophilic organic solvent is e.g. methanol, propanol, isopropanol, dioxane, ethylene glycol, etc.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for the enzymatic production of peptides.

(Prior art) In recent years, the physiological activity and sweetening effects of peptides have been recognized as pharmaceutical and food products.
Interested in? In addition to Abiruni, cheaper and safer methods for producing infarctide are now being considered as research subjects. In particular, enzymatic synthesis of peptides using protease reverse reactions: ■ does not involve racemates; ■ does not require protection of side chain functional groups; ■ has few side reactions and is easy to purify; What reagent? Many advantages over chemical synthesis methods such as no need to use In recent years, research has progressed rapidly, and a review article (Eng., M., Chaiken et al., Ap.
pli'el Biochemistry and B
iotechnology, 7+385-399(1
982); Aiyuki Morihara, Proteins/Nucleic Acids/Enzymes, 29.4
9-56 (1984) etc.) have also come into widespread use.

(1) #Calsberub (Ca
The method (Japanese Patent Publication No. 56-500519) uses serine carboxypeptidase and a reverse reaction of the esterase or amidase activity that the enzyme exhibits at pH 8 or higher, and can be expressed by the following reaction formula.

X'-A'-Y' + H-B'-Z' -+
X'-A'-B'-Z'10M' (acid component)
(Amine component) (In the formula, X' is a protecting group for the terminal amine group, and Y/ and Z' are protecting groups for the terminal carboxyl group.

A' and B' are amino acid residues or peptide residues, and B' is the same or different amino acid residue or peptide residue from A'. It is something that can be done,
Explaining with the above formula, Y' of X/ A/ Y/ and B/
z/ is substituted to form the compound X/ A/ B/
Y/ is generated.

This reaction mechanism differs from the present invention in that it is not a synthesis method that utilizes a so-called condensation reaction in which an acid component and an amine component are directly dehydrated and condensed. In fact, no condensation reaction occurs under these conditions.

The following problems are known in the Rusberg method. (2) It is said that the amine component can be used in the form of a free amino acid, but in reality, unless it is protected with Z', the synthesis rate and yield will be extremely low. ■ Substitution reaction or iptide transfer reaction is carried out on the alkaline side (pH 8 to 1).
05), racemization of amino acids and cleavage of substrate components and product esters are performed non-enzymatically. ■
Furthermore, such ester cleavage also occurs enzymatically, and it is thought that the addition of amine components to the product occurs one after another, resulting in a large number of side reaction products, making it difficult to secure a stable target product. It is the cause of this.

(2) On the other hand, callus (apart from the LUG method, Isowa et al. are also attempting to synthesize callus using protease.
a, Bull, Chem, Soc, Japan,
50+2766 (1977). The method of Isowa et al. can be expressed by the following formula, and the protease used is Bacillus thermoproteolyticus.
Endopeptidases such as metalloprotease derived from P. prote-olyticus and thermolysin are used.

X// A"-OH+H-B"-Z"→X"-Al1-
'f3''-Z'' (Ami / Sei゛fishing (in the formula, " is a protecting group for the terminal carboxyl group.) Compared to the Kalsberb method, the method of Isokazu et al. only needs to protect the amino group of the acid component, and the synthetic material is cheaper. However, thermolysin is an endo-type protease. Therefore, there is a problem that the reaction product becomes a substrate for hydrolysis.For example, Z-A-B-
The * part of OMe is cleaved and decomposed, which not only lowers the yield of the desired product, but also theoretically may cause side reactions such as the decomposed product becoming a synthetic substrate. Therefore, the reaction pH 1
￥Is this side reaction product around 50 instead of the optimal 7.0-8.0? How to make the precipitate insoluble? I'm taking it. Therefore, there is a problem that the efficiency of the synthesis reaction is significantly lower than the optimal conditions. Furthermore, the specificity of thermolysin is high for amino acids with aliphatic side chains; what about other side chains? There are also problems, such as difficulties in the synthesis reaction of amino acids.

(3) In addition to these enzyme synthesis methods, Hahain (Kul
lmann.

W, Biochem, Biophys, Res
, Commun,, 91.

693 (1979); Takai et al., Pep
tide Chemistryl 980 (Okaw
a, K, Ea,) Protein Res, F
'ound,.

0saka P-213) Encarin and caerulein are synthesized by thiol proteases↓, but the site of hydrolytic action is Phe (Vaρ, Leu)-
A- (A means no specificity), and there is a problem that it cannot be applied to a wide range of peptide synthesis. In addition, limited enzyme synthesis methods have been developed using tolysine/Kimo) IJpusin, Takupusin, etc., which have even more limited substrate specificity, but similar to papain, it is difficult to expand the use (Kazuyuki Morihara, Protein・Nucleic acids/enzymes 26゜1979-1998 (1981)
).

(Problems to be Solved by the Invention) The present invention aims to solve the problems of the above-mentioned prior art and to provide a basic and universal method for synthesizing peptides using an enzymatic method.

That is, the present invention provides a method for synthesizing takuputide using an enzymatic method,
The substrate specificity of the enzyme is wide, so it can be applied to a variety of amino acids; the substrate material is inexpensive because there is little need to protect the starting amino acids; the yield and purity of the product are high because no side reactions occur; Another object of the present invention is to provide a method for synthesizing R-ptedo in which there is no fear that the reaction product becomes a substrate for an enzyme and is degraded.

In order to achieve the above objective, the present inventors conducted various studies and found that serine carboxy (butidase (hereinafter referred to as S) has a wide substrate specificity.
(Sometimes abbreviated as CP) is selected, and the substrate raw material is cheaper than the substitution reaction of Kalsberb, and side reactions are less likely to occur. The present invention has been completed. This condensation reaction was previously thought not to occur in SCP (Kazuyuki Morihara, Proteins, Nucleic Acids, Enzymes, 49-56 (1984)).

(Structure of the Invention) The method of the present invention involves reacting a substrate component with an amine component in the presence of serine carboxypeptidase to produce a compound of general formula (I): X-A-B-Y (1) (formula X represents a protecting group for an amine group, A represents an amino acid residue, B represents an amino acid residue or a peptide residue, Y represents a protecting group for a carboxyl group or an amino group) ? In the manufacturing method, N having the general formula (■): X-A-OH(I[) (wherein X and A are as defined above)
- terminally protected amino acids (acid components) with a p of less than 6.5
H, preferably in an aqueous solution or a mixed solvent of water and a hydrophilic organic solvent having a pH of 4 to 6.0, in the presence of serine carboxypeptidase, 5° to 50°C, preferably 30 to
At a temperature of 40° C., a C-terminally protected amino acid ester, amino acid amide having the general formula (■): H-B-'I' ([1), where B and Y are as defined above. -11,3 ptedoester or peptide amide.

As mentioned above, the method of the present invention is different from the aforementioned Kalsburg method in that the amine group-protected acid component of formula (1) and the carboxyl group-protected amine component of formula (2) are directly dehydrated and condensed in a weakly acidic environment. , is a novel SCP-catalyzed synthesis reaction ◎ The enzyme (SCP) used in the present invention may be any serine carboxypeptidase that is widely distributed in eukaryotic cells, but yeast carboxypeptidase (SCP), which is particularly easily available, can be used. Those derived from yeast such as butidase Y, those derived from animals such as cathepsin A, and K-11ium lupoxypeptidase (Penici-11ium Janthinel).
l-derived), Ascirugillus carboxyl is butidase (derived from
Aspergillus saitoi.

As. These include those derived from fungi such as those derived from pergillus oryzae, carboxy R-butidase C1 from orange and tangerine leaves and peels, and serine carboxy R-butidases derived from plants such as germinating barley, tomato, and pineapple flour.

SCP exists ubiquitously in eukaryotic cells, has extremely broad substrate specificity, and is an exo-type R-butidase that liberates one diamino acid from the carbo-1 end. SCP
Butidase Y (hereinafter CP
Y) is (optimum pH for gotidase activity is slightly acidic (pH 5,
5-65), and esterase activity is present in Alka! It has been revealed that it exists in J (pH 8) (
Rikimaru Hayashi, Proteins/Nucleic Acids/Enzymes, 28.1421-143
1 (1983)).

To use these 5CPIs, it is not necessarily necessary to completely purify them; for example, yeast autolysis supernatant may be used, or protease A1 mixed in the supernatant may be used.
To inactivate amino (butidase), 0.1 mmol may be pretreated with pustatin or 1 mmol EDTA for 30 minutes and may be used.

The method of the present invention may be carried out by dissolving serine carboxypeptidase in a mixture of reaction components, or by dissolving the serine carboxypeptidase in a mixture of reaction components. It may be immobilized on a suitable support. The enzyme may be immobilized by any known immobilization method. The immobilization carrier is preferably one that does not dissolve in the aqueous medium used and exhibits little swelling.

Next, as the protecting group X for the terminal amino group in the acid component -), urethane-type protecting groups such as t-butyloxycarbonyl group (Boa-), acyl-type protecting groups such as benzoyl group (BZ-), tosyl group, acetyl group, p-toluenesulfonyl group (Toc-), o-
A nitrophenylsulfenyl group (Npθ-) or the like can be used. Particularly suitable protecting groups include carbobenzoxy and t-butyloxycarbonyl groups, since these derivatives are easy to make, economical to use, and can be easily cleaved off again.

The amino acids in the acid component
Aliphatic amino acids such as diaminomonocarboxylic acids as well as aromatic nuclei or heterocycles? Examples include amino acids with Amino acid residues are represented herein by abbreviations commonly used in this field.

In the amine component H-BY, B is the same or different amino acid or peptide residue from A above, and the amine group does not need to be protected. Is Y a protecting group for the terminal carboxyl group? It is best to protect it with an amide type or ester type (methyl, ethyl, benzyl, etc.).

In both the acid component and the amine component, the reaction generally proceeds in a substantially leap-like manner even if the functional groups of the side chains of amino acid residues and infarcted residues are not protected. When unprotected, the same advantages as when X is a hydrogen atom occur, but the solubility of the product must be taken into consideration. When protecting, ω
-To protect the amine group (No), No-benzyloxycarbonyl (No-Z), t-butoxycarbonyl (No-Z) is used to protect the amine group (No).
-Boc) and tosyl (No-Tos), but in addition to the nitro group, N-benzyloxycarbonyl (No-Z) and
G・No-dibenzyloxycarbonyl group (N -Z
-Z), but for the inspection of imidazole nucleus (Nim), Nl
m-benzyl group (N-BZI) and tosyl group (N-BZI)
-Tos), but for protection of ω-carboxyl group, ω
-benzyloxy group (-0BZI), O-ether type for the hydroxyl group of oxyamino acid, for example Q-benzyl group (0-BZI), S-protecting group for the mercapto group of cysteine, S-benzyl group ( S-Bzx) are respectively used as representative examples.

In the present invention, the condensation reaction for producing an iptide bond is carried out in an aqueous medium that dissolves the raw material system and insolubilizes the product system. After dissolving the raw material in water, the pH is adjusted using an appropriately diluted inorganic acid such as phosphoric acid, sulfuric acid, hydrochloric acid, caustic acid, inorganic alkali, or various buffer solutions. Almost all raw materials are easily dissolved at around pH 5, so it is difficult to dissolve them. It is not necessary to add a hydrophilic solvent to make it easier, but is it necessary to add a hydrophilic solvent to the raw material system and production system? Another advantage is that a hydrophilic organic solvent may be added for solubilization.

The hydrophilic organic solvents mentioned here include alcohol solvents such as methanol, ethanol, furopanol, and isopropanol, ether solvents such as tetrahydrofuran, dioxane, and dimethoxyethane, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, and ethylene glycol. Can be mentioned.

Next, the amounts of each component necessary for the reaction will be described. The amount of raw materials used is preferably 1 to 1.5 mmol of the amine component to 0.7 to 1.0 mmol of the acid component. Before the enzyme required for the reaction, it is preferable to use 0.1 .mu. or more, particularly 0.1 to 10 .mu.m of free enzyme per mmol of acid component. The concentration of the raw materials in the aqueous medium is not particularly limited in order to obtain a practical yield, but a high yield can be obtained if the concentration is several times or more higher than -Ka of the acid component and amine component.

The reaction temperature is generally 5-60℃ from the viewpoint of maintaining enzyme activity.
℃ is preferable, and the reaction pH is preferably weakly acidic 4.0 to 6.0.

By carrying out the reaction under the above conditions, the reaction proceeds smoothly. Normal reaction time is 1 to 24 hours, but immobilized enzyme? When used, the reaction completes more quickly.

After the reaction is complete and the immobilized enzyme is removed as necessary, to isolate the target product from the solution, separate the insolubilized precipitate and add an acid aqueous solution (e.g. citric acid, dilute hydrochloric acid) that does not cause removal of the protecting group. Aqueous solution, etc.), alkaline aqueous solution (for example, ammonia water, sodium bicarbonate aqueous solution, etc.), and water may be used to sequentially wash and dry. If necessary, it may be further recrystallized from, for example, hot methanol. Other purification means commonly used for purification of putide are also applicable.

Next, is the present invention based on examples? , will be explained more specifically, but it goes without saying that the present invention is not limited to the scope of these examples.

The abbreviations in the examples are as follows.

CbZ = benzyloxycarbonyl group Met: L-methionine residue phe: L-phenylalanine residue AQa: L-alanine residue Tyr: L-tyrosine residue Thr: L-threonine residue Le: L-isoleucine residue Vaρ :L-valine residue Example I Synthesis of CbZ-Met-PheNH2 CbZ-Me
t (acid component) 1 mmol and Ph e NH2 (amine component) 1.2 mmol fj! ::Dissolved in 10 ml of water
After adjusting the temperature to 5.3 with HYIN #4 acid or caustic soda, add carboxypeptidase Ylq and incubate at 35°C for 25 minutes.
Allowed time to react. The reaction solution was filtered through a flat 4-glass filter to collect the product, and then washed sequentially with a small amount of water, a small amount of 5% sodium bicarbonate solution, and 0.2N hydrochloric acid to remove unreacted materials, and finally with a sufficient amount of water. After washing and drying, the weight measurement and expected product condition were 98%.

The progress or purity of the reaction can be determined using silica gel 6O-F25.
4 (Merck & Co., Ltd.) by thin layer chromatography. The solvent system used was chloroform/n-tanol/acetic acid/water (11:5:2:1).

The synthesis reaction was further tracked using Shimaz 5A using an ODS column.
quantified by high performance chromatography. The solvent is 3
0% acetonitrile/buffer (10 mmol KH2PO4
, 10 mmol H3PO4, and 50 mmol Na2SO4) was used. The sulfur rate is 1.27 minutes.

Identification of the product was carried out in mass 7. Made by Hitachi, M-
Analysis was performed using a model 80 mass spectrometer using a secondary ionization method. -Next ion species is XE+, -nth ion acceleration voltage 8KV
, secondary ion acceleration voltage 3KW.

Glycerol was used as a matrix agent.

The expected mass quictor was obtained and the product was Cbz-M.
It was identified as et-PheNH2 (see figure).

Example 2゜Following the same method as in Example 1, the amine component was
Fixed with heNH2, acid component? A, [la.

C of Tyr, Thr, Met, Phe, Ipe
When a synthetic reaction was carried out as a 'bz derivative, Table 1
The following products were obtained, and the respective Km values and VmaX values were also obtained as described.

Table 1. CPY Catalyst Synthesis Reaction Example 3 Using Phenylalanine Amide as the Amine Component Following the same method as in Example 1, the acid component 4Me
and Vai and using the derivatives shown in Table 2 as the amine component, the products shown in the same table were obtained.
The respective Km values were also as described in the same table.

Table 2. Example 4 of CPY catalytic synthesis reaction using C'bz-methionine or Cbz-Va(l) as the ff component. Production of Cbz-Met-Phe-NH2 using crude enzyme.
Add chloroform, mix and liquefy, then stir at room temperature for 30 minutes. Next, add 20 ml of water and mix thoroughly.
H%! Near, make sure to set it to 0. About 2 hours later pay7.
After being left at 25°C for 18 hours, the supernatant was centrifuged. Obtained. this? The contents were pressurized into a cellophane tube and dialyzed under running water overnight, and the contents were used as a crude enzyme preparation of carboxybutidase Y.

Cbz-Met l + nmol and PheNHz L
2 mmol? Dissolved in 10 mt of water, pH fj! ::l
After adjusting to 53 with N hydrochloric acid or IN caustic soda, this crude enzyme preparation freeze-dried product 15■? Add and heat to 35℃ for 25 minutes.
Allowed time to react. The following treatment was carried out in the same manner as in Example 1 to obtain a similar synthetic product 420? Obtained. Yield 98%.

Example 5 Cbz-Met-Phe-NH using 1ml regulated enzyme
Morris (Morri
(Biochem, J, 147,593)
(1975)) 0-alkylated with triethyloquinonium tetrafluoroporate, followed by 1
．． 6- Diaminohexane treated with amine group? I put it in. This treated nylon net? After soaking in water and adjusting the pH to 60, dissolve 0.59 each of carbodiimide and carboquine (butidase Y) in a small amount of water, place in water containing a nylon net, and fix by leaving overnight at room temperature. The process has been completed. Wash thoroughly with water.
After that, I was careful not to let the surface of the net dry out.

Cut 1 g of this net, υ, and Cbz- as in Example 1.
1 mmol each of Met and Ph e NH2 are placed in a solubilized 20-proof aqueous solution (adjusted to pH 5.3), shaken gently, and netted and removed after 15 minutes. Below is this reaction completed solution? The same procedure as in Example 1 was carried out to obtain 380m91 of the same synthetic product.

Yield 90%.

(Effects of the Invention) (11) When the method of the present invention is compared with the Kalsberb method, which is a typical conventional method, it has the following advantages.

■ Synthesis of butidase by the Kalsberb method uses a reverse reaction of the esterase activity of SCP, but the present invention uses a reverse reaction of the butidase activity of SCP, which corresponds to the original use of CPY in the synthesis of R-butide, resulting in high efficiency. Also good.

(2) Since the method of the present invention is also a condensation reaction, it has the advantage that only amine groups need to be used as the substrate for the acid component, and the raw materials are inexpensive.

(2) Further, at a pH close to 5, there is an advantage that the reaction product tends to crystallize and the reaction equilibrium tends to proceed in the direction of synthesis.

■ The synthesis product is X-A-B-Y, which has a pH of 3.
At pH 10.5, it is predicted that it will be degraded by esterase activity or become a substrate for rearrangement reaction, whereas at pH 5, the carboxyl terminus is protected, so CPY's inherent property is that it is hydrated by butidase activity. A major feature is that there are almost no side reaction products because it does not become a decomposition substrate and no other reactions occur.

Features like that? The present invention utilizes the reverse reaction of butidase activity under weakly acidic conditions.
? It is more immersive than the method used.

(2) When the method of the present invention is compared with the method using thermolysin, it has the following advantages.

■ The thermolysin method uses the optimal pHB for butidase activity.
Since the synthesis reaction is carried out at a pH of 5, which is significantly far away from CPY, it cannot be said that the inherent activity of thermolysin is effectively utilized. Features? It can be utilized to the maximum and synthesized efficiently.

■ In terms of substrate specificity, CPY is by far the most versatile enzyme that can be used to synthesize a variety of 4-ptedos.

(2) Since thermolysin is an endo-type (butidase), the reaction product can serve as a substrate for hydrolysis, but in the present invention using CPY, the reaction product is not decomposed as a substrate.

(3) In the method of synthesizing Takuputide according to the present invention, the product is not decomposed as a substrate. Crystallize and separate (
It is not necessarily necessary to remove the solvent from the reaction system, and a hydrophilic organic solvent can also be used. If you use such a solvent, will the raw amine component and acid component be removed? It is also possible to carry out a continuous synthesis reaction by continuously supplying the immobilized SCP to an immobilized SCP column and continuously flowing out the produced peptide dissolved in a hydrophilic organic solvent from the column.

[Brief explanation of drawings]

The figure is a vector diagram for cell 7 of the product of Example 1. Patent applicant Sun) IJ-Co., Ltd. (5 other people)

Claims (5)

[Claims] (1) By reacting the substrate component with the amine component in the presence of serine carboxypeptidase, the general formula:
B-Y (in the formula, X represents a protecting group for an amino group, A represents an amino acid residue, B represents an amino acid residue or a peptide residue, and Y represents a protecting group for a carboxyl group or an amino group) A method for producing a peptide having the general formula: X-A-OH, in which X and A are as defined above.
- the terminally protected amino acid is prepared in an aqueous solution or a mixed solvent of water and a hydrophilic organic solvent with a pH of 4 to 6.5 in the presence of serine carboxypeptidase at a temperature of 5° to 50°C, with the general formula: H- C having B-Y (wherein B and Y are as defined above)
terminally protected amino acid esters, amino acid amides,
A method for producing a peptide, which comprises carrying out a condensation reaction with a peptide ester or peptide amide. (2) The method according to claim 1, which uses a serine carboxypeptidase enzyme that exhibits peptidase activity at a pH lower than 6.5. (3) The method according to claim 1, wherein carboxypeptidase Y from yeast is used as the serine carboxypeptidase. (4) The method according to claim 1, which uses immobilized serine carboxypeptidase. (5) Claims 1 and 2, wherein the hydrophilic organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, dioxane, tetrahydrofuran, dimethyl sulfoxide, and ethylene glycol;
The method described in item 3 or 4.