JPS5834120B2 - Method for producing peptides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a peptide, and particularly to a method for synthesizing a peptide using an enzyme as a catalyst.

Typical conventional methods for synthesizing peptides include the azide method based on the carboxyl activity method, the mixed acid anhydride method, the carbodiimide method, the active ester method, and the acid chloride method.

However, these conventional methods require racemization and side reactions at the carboxyl-terminal amino acid residue, temperature control,
Industrially, various problems arise, including the selection of a solvent, the properties of the □-protecting group and the carboxyl protecting group, and the influence on the functional group of the amino acid residue.

The fragment condensation method not only allows the peptide synthesis reaction to be divided into fragments and minimizes losses due to unexpected failures, but also has many industrial advantages.

However, in the fragment condensation method, it is good if glycine, which is the only amino acid that does not have the possibility of causing racemization, is at the carboxyl terminal, but racemization can be avoided if other amino acids are used. It has the serious drawback that it is not.

Avoiding racemization is the most important issue in the production of peptides, and if racemization occurs during the reaction, it will impure the bioacid and require removal of unnecessary isomers, making it difficult for industrial use. It will be disadvantageous.

Among the conventional methods for forming peptide bonds mentioned above, the azide method has high utility value in that it is the only method that is unlikely to cause racemization, but it is more complicated to operate than other methods, and side reactions occur during the hydrazide production process. occurs, and therefore it cannot necessarily be said that the yield is superior*.

Apart from the purely organic chemical methods mentioned above, the production of peptide bonds by papain and chymotrypsin has been reported.

(For example, J, S, Fruton"Advance
es in Protein Chemistry”
Vol 5, Academic Press Jnc
, New York N, Y, 1949) The reaction formula for this method is as follows.

What is common in the conventional methods (1) to (3) above is that the phenylamine group bonded to the end of the so-called ane component in (II) is a group that cannot be easily removed; Separation from the formed peptides requires hydrolysis under harsh conditions, resulting in cleavage of the peptide chain, a major drawback, and therefore, these methods cannot be used for peptide synthesis. It was thought that

Furthermore, method (4) is reported to be impractical because it involves side reactions of transamidation and transpeptidation [for example, RoB].

Johnston et al.: J. Biol. Chem.
, 185.629 (1950) and J.S.
Fruton et al.: J. Biol.

Chem, 204.891 (1953)).

That is, in method (4), the primary amino group of the acid amide compound bonded to the terminal of the amine component promotes the amidase action of papain.

Therefore, the above method is based on scientific evidence showing that papain or chymotrypsin has a catalytic action for peptide bond formation even when the protecting group of the terminal carboxyl group of the amine component forms an anilide or primary amide bond. However, it did not indicate the possibility of synthesizing oligopeptides or polypeptides.

The present invention was made in view of the current situation, and
The purpose is to provide a method for producing a desired oligopeptide or polypeptide with simple operations and high purity and high yield.

According to the present invention, it has been recognized that peptide synthesis using an enzyme is possible by introducing a specific protecting group as a protecting group for the terminal carboxyl group of the amine component.

That is, the present invention provides an enzyme represented by the general formula Amino acids or peptides having a protecting group at the N-terminus and the general formula H-B-Y (where Y is a tertiary alkoxy group, a substituted or unsubstituted benzyloxy group, a substituted or unsubstituted benzylamino group, a substituted or unsubstituted A protecting group for a terminal carboxyl group selected from the group consisting of a benzhydryloxy group and a substituted or unsubstituted benzhydryloxy group, where B is the same or different amino acid residue or peptide residue as A). An amino acid or a peptide having a protecting group at the C-terminus is added to the reactor in a reaction apparatus equipped with a means for detecting the pH of the reaction solution and a means for adding an acid or base to the reaction solution to adjust the pH of the reaction solution. , general 2 X-A-B-Y (wherein X, A, B, and Y are the same as defined above), characterized in that the reaction is carried out within the pH range in which thiol proteinase exhibits enzymatic activity in an aqueous solution. This is a method for producing the peptide shown in

To outline the present invention, thiol proteinase used in the present invention
), papain is an enzyme that belongs to
), Stem bromelei
n), Ficin, Cathepsin B (C
athepsin B), chymopapain (Chymo
papain) and streptococcal proteinase (5treptococcal proteinase
e) is exemplified.

These enzymes have a fairly wide specificity when hydrolyzing proteins, and can cleave a large number of peptide bonds, amide bonds, and ester bonds, and also cleave benzoyl-argininamide, benzoylglycyleucylglycine, benzoyltyrosine glycinamide, etc. It is efficiently degraded and has both exo and endopeptidase functions (J,
R, Kimmel and E, L.

Sm1th,Advance Enzymology,
Vol 19.267 (1957)).

The general 2X-A-OH used as a starting material (in the formula
is a protecting group for the terminal amino group, and A is an amino acid residue or a peptide residue) The amino acid or peptide represented by (I) is shown in the known methods (1) to (4) above.
In this specification, this compound is hereinafter referred to as an acid component.

In the acid component, A means an amino acid and its peptide residue, and examples of the amino acid include monoamino monocarboxylic acids such as glycine, alanine, valine, norvaline, leucine, inleucine, and norleucine, which are used in the field of peptide synthesis; Oxyamino acids such as serine, threonine, homoserine, methionine, sulfur-containing amino acids such as cystine or cysteine with side chain functional groups, monoamino dicarboxylic acids such as aspartic acid or glutamic acid with side chain carboxyl groups protected. acid,(
Aliphatic amino acids such as diaminomonocarboxylic acids such as ornithine, lysine or arginine with protected side chain a□) groups, and amino acids with aromatic nuclei such as phenylalanine and tyrosine or heterocycles such as histidine and tryptophan. are exemplified and indicated herein by abbreviations commonly used in this field, and the same applies to peptides.

As a protecting group for the free terminal amino group of the acid component, a tertiary
substituted or unsubstituted benzyloxycarbonyl, such as benzyloxycarbonyl (Z-), p-methoxy Benzyloxycarbonyl (P
MZ-), 3,5-dimethoxybenzyloxycarbonyl (Z (OMe)2), 2-4,6-)limethylbenzyloxycarbonyl (TMZ-), p-phenylazobenzyloxycarbonyl (PZ-), Representative examples include p-)luenesulfonyl (Tos-) and o-nitrophenylsulfenyl (Nps-).

The acid component is added to the reaction system as a free acid, but may also be added as a salt that can be ionized under the pH conditions of the reaction system.

The amino acid or peptide represented by the general formula H-BY used as one of the starting materials is the above-mentioned (
It is a compound corresponding to the compound (n) shown by the known methods of 1) to (4), hereinafter referred to as an amine component, and B is the same or different amino acid or peptide residue from A. .

As a protecting group for the carboxyl group in the amine component,
Tertiary alkoxy groups, such as tertiary butoxy (-0B
u-t), a substituted or unsubstituted benzyloxy group such as benzyloxy (-()Bzl), a substituted or unsubstituted benzhydryloxy group such as benzhydryloxy (
-0Bh), a substituted or unsubstituted benzylamino group such as 2,4-dimethoxybenzylamino (-NHDMB)
, a substituted or unsubstituted benzhydrylamino group such as benzhydrylamino (-NHBh).

The protecting group at the carboxyl terminal of these amine components must be free from esterase and amidase effects, since the enzymes used in the present invention, ie, enzymes belonging to thiol proteinases, such as papain, are associated with esterase and amidase effects.

As is clear from the above, the acid component and amine component used in the present invention include cases where the side chain of the amino acid or peptide residue has a functional group.

In this case, it is often desirable to protect these functional groups, and the protecting groups include N(″)-benzyloxycarbonyl (No) to protect the ω-amino group (N″J).
-Z), t-butoxycarbonyl (N(1')-BOC
) and tosyl (N”-TO8), etc., to the N of arginine.
-To protect the guanidino group (N'), in addition to the nitro group, NG
-benzyloxycarbonyl (N'-Z) and No.
No-dibenzyloxycarbonyl (No-Z-Z), etc., and N1m − to protect the imidazole nucleus (N1m).
Benzyl (Nim-BZl) and tosyl (Nim-TO
8), etc., to protect the ω-carboxyl group, use ω-benzyloxy (-OBzl), and when the hydroxyl group of an oxyamino acid needs to be protected, regardless of whether it is an aliphatic or aromatic amino acid, use 0. -Ether types such as 0-benzyl group (OBzl) can be used.

As the S-protecting group for the mercapto group of cysteine, an S-benzyl group (SBzl) or the like can be used.

The protecting group referred to in this specification must at least satisfy the conditions that the introduced group is stable during the reaction, can be easily removed from the product, and does not cause any side reactions during the removal. It is.

In addition to the acid component and amine component of the starting material having the above-mentioned protecting groups, the N''-amino group of the amine component may be free, as well as hydrochloride, hydrobromide, oxalate, p- Any inorganic or organic acid salt form may be used, such as 1-luenesulfonate and acetate.

In the present invention, the dehydration condensation reaction to generate peptide bonds is performed in an aqueous solution at a pH at which the enzyme used in the present invention exhibits enzymatic activity.
It is necessary to implement this within the scope of.

This range is approximately pH 4 to 7.5.

When carrying out the present invention industrially, in order to maintain the pH of the reaction solution within this range, the reaction apparatus is equipped with a means for detecting the pH of the reaction solution and a means for supplying an acid or base, and the detected pH of the reaction solution is Depending on the conditions, an acid or a base is supplied to this to maintain the pH of the reaction solution within a desired range.

Specifically, a controlled amount of base is supplied depending on the detected pH of the reaction solution, and when the pH exceeds about 7.5, acid is supplied.

The starting materials are generally 0.00% acid component per mole amine component.
When these starting materials used in a ratio of 8 to 2 moles, preferably 1 to 1.5 moles, are difficult to dissolve in aqueous solvents, solvents such as alcohols such as methanol and ethanol, dimethylformamide, dioxane, tetrahydrofuran, and dimethyl sulfoxide are used. can be added to improve its solubility.

In this case, it is necessary to limit the amount added to an extent that does not inhibit the enzyme reaction of the present invention, and usually 1 part by weight of water to 1 part by weight of solvent.
Use parts by weight or less.

The reaction of the present invention proceeds in an aqueous solvent, and the reaction product must have a relatively low solubility therein, preferably a system in which the reaction product is poorly soluble or insoluble.

The amount of enzyme required for the reaction is 1 for 1 mmol of amine component.
0 to 400 mm, preferably 50 to 300 mm.

Further, cysteine or a salt thereof, 2-mercaptoethanol or a salt thereof, etc. may be added to the solution as an enzyme activator.

The reaction temperature is generally 20~20°C from the viewpoint of maintaining enzyme activity.
The temperature is 55°C, preferably 30-40°C.

By carrying out the reaction under the above conditions, the reaction proceeds smoothly and is usually completed in 1 to 24 hours.

Since the product precipitates outside the reaction system, it can be easily isolated.

To explain the present invention more specifically, the smallest peptide, ie, dipeptide represented by the general formula XABY can be easily produced using an enzyme belonging to thiol proteinase, such as papain.

For example, when producing a dipeptide such as lysyl lysine, the 2-derivative obtained from lysine with the 6-position amino group protected with a carbobenzoxyl group (2-) is used as the acid component, and the t-butyl ester is used as the amine component. By applying the above method, lysyl lysine with a side chain amino group protected can be obtained.

In the case of arginylleucine as a dipeptide containing arginine, IJ-Z-arginine (protected with Z, which is the guanyl group and amino group of arginine) is used as the acid component, and as the amine component, benzhydryl ester of leucine is used to prepare papain. A dipeptide derivative can be easily obtained by reacting in the presence of the dipeptide.

In the case of an anoic acid having a functional group in its side chain, the reaction can be carried out with it protected as in the case of the above two sides, or the reaction can be carried out without protection.

Taking histidine as an example, (a) is an example with side chain protection, and (b) is an unprotected example, but good results were obtained in both cases when the reaction was carried out using papain.

As described above regarding dipeptides, when the acid component contains one peptide bond, that is, the reaction between the acyl dipeptide and the amino acid amide derivative also proceeds due to papain without side reactions.

- For example, the reaction proceeded smoothly in this case as well, and carbobenzoxy-prolyl-glycyl-leucine-penthydrylamide could be obtained as a product.

When both the acid component and the amine component are dipeptides.

- For example, it can be applied to the synthesis of the 3-6 tetrapeptide of Val 5-angiotensin-■, which is known as a peptide hormone.

That is, when a paryl-histidine ester is used as the amine component and the reaction is carried out in the presence of papain, a product is obtained in which the benzyl ester group of the tetrapeptide is eliminated.

These are thought to be the result of esterase action, which is one of the properties of papain.

Such a reaction is extremely advantageous when a peptide synthesis plan envisages this tetrapeptide as the next acid component.

As is clear from the above, although the present invention uses enzymes belonging to thiol proteinases with esterase and amidase actions, oligopeptides and It has a unique effect in that it enables the synthesis of polypeptides.

Next, the present invention will be described in more detail with reference to embodiments, but the present invention is not limited thereto.

Example I Z -Glm -OH280,2 (1 mmol) and H-Phe -Phe-OBut368.5771 (
1 mmol) was suspended in 10 TrLl of water in a flask.

Insert the glass electrode of a pH meter into this and drop a sodium hydroxide aqueous solution (1/10 normal) to adjust the pH to approximately 5.0.
o. im ml of papain 1507Q and 2-mercaptoethanol were added while adjusting the temperature, and the flask was shaken to dissolve the solids.

This was shaken and reacted at 38° C. for 20 hours.

During this time, the pH of the reaction solution was measured using a glass electrode pH meter, and an aqueous sodium hydroxide solution (1/10 normal) was added to maintain the pH of the reaction solution at 5 to 6.

The precipitated crystals were separated daily, washed successively with water, dilute hydrochloric acid (1N), water, aqueous ammonia (7%) and water, and then dried.

This was recrystallized from ethyl acetate, m, p, 197-20
Z-Glm-Phe-Phe-O with 0 °C
But510 ■ was obtained.

Yield 80.8%. Example 2 Z-Phe-Val-OH398,5m9 and H
320.4 m9 of Phe-Val-OBut was suspended in 10 ml of water in a flask.

Next, a glass electrode of a pH meter was inserted into this, and while adjusting the pH of the system to approximately 4.55.0 with dilute hydrochloric acid, 0.1 ml of papain 150 and 2 mercaptoethanol was added, and the flask was shaken to reduce the solid content. Dissolve this and add 3
The mixture was shaken and reacted at 8°C for 20 hours.

During this time, the pH of the reaction solution was measured using a glass electrode pH meter, and hydrochloric acid was added to adjust the pH of the reaction solution to 4.5 to 5.0.
was held at

The precipitated crystals were separated from each other, washed successively with water, dilute hydrochloric acid (1N), water, aqueous ammonia (7%), and water, and then dried.

This was recrystallized from ethyl acetate/petroleum ether to give m, p,
Z-PheVal-Ph with 130-145°C
e-Val-OBut3 Qm9 was obtained.

Yield 4.3%.

Claims (1)

[Claims] 1 In the presence of an enzyme belonging to thiol proteinase, the N-terminus is protected by a general 2X-A-OH (wherein X is a terminal amino group protecting group and A is an amino acid residue or a peptide residue) an anoic acid or a peptide having the general formula H-B-Y (wherein Y is a tertiary alkoxy group, a substituted or unsubstituted benzyloxy group, a substituted or unsubstituted benzyloxy group), a substituted or unsubstituted benzyloxy group, A protecting group for a terminal carboxyl group selected from the group consisting of a substituted benzhydryloxy group and a substituted or unsubstituted benzhydryl amino group, and B is the same or different amino acid residue or peptide residue as A) A reaction device equipped with means for detecting the pH of a reaction solution, and means for adding an acid or base to the reaction solution in order to adjust the pH of the reaction solution. Among them, the general 2-X-
A method for producing a peptide represented by A-BY (wherein X, A, B, and Y are the same as defined above).