JPS5837840B2 - 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 carboxyl-terminal amino acid residues, temperature control,
There are various industrial problems that must be taken into consideration, including the selection of a solvent, the properties of the amino protecting group and carboxyl protecting group, and the effects on the functional groups of the amino acid side chains.

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, the only amino acid that is unlikely to cause racemization, is at the carboxyl terminal, but racemization is unavoidable with other amino acids. This has a serious drawback.

Avoiding racemization is the most important issue in the production of peptides, and if racemization occurs during the reaction, it impures the product and requires removal of unnecessary isomers, which is disadvantageous industrially. becomes.

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 the yield cannot necessarily be said to be excellent.

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

(For example, J.S., Fruton//Advance
es in protein Chemistry
vol 5, Academic Press Jn
c, New York N. Y. (1949) This method is shown in the following reaction formula.

The common point in the conventional methods (1)-{3) above is that the phenylamino group bonded to the end of the so-called amine precipitate in (n) is a group that cannot be easily removed, and once formed, Detachment from the peptide requires hydrolysis under severe conditions, resulting in cleavage of the peptide chain, which is a major drawback; therefore, it is considered impossible to use these methods for peptide synthesis. It was getting worse.

Furthermore, it has been reported that method (4) involves side reactions of transamidation and transpeptidation and is not practical.

[For example, R.B. Johnston et al.: J. Bi
ol, Chem, 185, 629 (1950) and J. ol. S. Fruton et al.: J, Biol. C
Hem, 204, 891 (1953)), that is, in the method (4), the primary amino group of the acid amide bonded to the terminal of the amine promotes the amidase action of papain.

Therefore, the above-mentioned method has academic significance in showing that papain or chymotrypsin has a catalytic action for forming a peptide bond 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 show the possibility of synthesizing oligopeptides and 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.

The present invention is based on the knowledge that enzymes belonging to metalloproteinases have a catalytic effect on dehydration condensation reactions that produce peptide bonds. An amino acid or peptide having a protecting group at the N-terminus represented by OH (in the formula, X is a protecting group for the terminal amino group, and A is an amino acid residue or a peptide residue) and the general formula Y is a protecting group for the terminal carboxyl group, and B is the same or different amino acid residue or peptide residue as A). In a reaction apparatus equipped with a means for detecting the reaction mixture and a means for adding an acid or base to the reaction mixture to adjust the pH of the reaction mixture, the reaction is carried out within the pH range in which the metalloproteinase exhibits enzymatic activity in an aqueous solution. This is a method for producing a peptide represented by the general formula X-A-B-Y (wherein X, A, B, and Y are the same as defined above), characterized by the following.

To outline the present invention, the metalloproteinase used in the present invention (Metallo proteinas
Examples of enzymes belonging to this group include neutral proteinase originating from Streptomyces, and Bacillus subtilis varietus amyliquifaciens.
is var. amyloliquefaciens
origin), Bacillus thermoproteoliticus (Ba
cillus thermoproteolitic
US origin) thermolysin ('I' hermoly
s in ), collagenase ( Collagenas
e) and Crotalus atrox proteinase (
Crotulu atrox proteinase
) is exemplified.

It has been reported that these enzymes have regularity in hydrolyzing amino acid residues, i.e., hydrophobic residues such as leucine, isoleucine, phenylalanine, and valine, and that there is no regularity in those on the carboxyl side. .

C H, Matsubara et al; B
iochem. Biophys, Res. Comm
, 2 1, 242 (1965)) General 2-X-t-OH (in the formula
is a protecting group for the terminal amino group, and A is an amino4 residue or a peptide residue) The amino acid or peptide represented by the above corresponds to (I) shown in the known method of (1)-{4). In this specification, this compound is hereinafter referred to as an acid component.

In the acid component, A means an amino acid or a residue of its peptide, and examples of the amino acid include monoaminocanolebonic acids such as glycine, alanine, palin, norvaline, leucine, isoleucine, and norleucine, which are used in the field of peptide synthesis. , sulfur-containing amino acids such as seramine, threonine, homoserine, methionine, cystine or cysteine with side chain functional groups protected, aspartic acid or glutamic acid with side chain carboxyl groups protected. monoaminodicarboxylic acid,
Aliphatic amino acids such as diaminomonocarboxylic acids such as ornithine, lysine or arginine (with protected chain amino groups) as well as aromatic nuclei such as phenylalanine tyrosine or heterocycles such as histidine and tryptophan. Amino acids 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 an acid, a tertiary
alkoxy carbonyl, such as tertiary butyloxycarbonyl (Boc-), tertiary amyloxycarbonyl (t-Aoc-), substituted or unsubstituted pendyloxycarbonyl, such as penzyloxycarbonyl (Z
-), p-methoxybenzyloxycarbonyl (PMZ
-), 3.5 dimethoxybenzyloxycarbonyl C Z (OMe)2'''J, 2-4.6-}limethylhenzyloxycarbonyl (TMZ-), p-
Phenyl asohenzyloxycarponyl (PZ-), p
Representative examples include toluenesulfonyl (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 (If) shown by the known method of 1)-{4), hereinafter referred to as an amine component, and B represents the same or different amino acid or peptide residue as A. .

Protecting groups for carboxyl groups in the amine component include alkoxy residues of esters, such as methoxy (OM
e), ethoxy (-0Et), tertiary alkoxy groups such as tertiary putoxy (-0But), substituted or unsubstituted penzyloxy groups such as penzyloxy (-0B
zl), a substituted or unsubstituted penzhydryloxy group, such as benzhydryloxy (-0Bh), an amide group, a substituted or unsubstituted pendylamino group, such as 2.4
-dimethoxybenzylamino (-NHDMB), a substituted or unsubstituted penzhydrylamino group such as penzhydrylamino (-NHBh), and the like.

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, such as Nω-penzyloxycarbonyl (Nω-Z) to protect the ω-amino group (N(I)).
, t-ptoxycarbonyl (Nω-Boc) and tosyl (Eω-TOS), etc., and to protect the N-guanidino group (NG) of arginine, in addition to the nitro group, NG-benzyloxycarbonyl (NG-Z) and N0・NG-dibenzyloxycarbonyl (N'-Z-Z), etc.
For protection of the imidazole nucleus (Nlm), Nlm −
Benzyl (Nlm-BZl) and tosyl (Nlm-To
s) etc., ω-benzyloxy (-0Bzl) etc. to protect the ω-carboxyl group, and 0-ether when the hydroxyl group of the oxyamino acid needs to be protected, regardless of whether it is an aliphatic or aromatic amino acid. For example, an O-benzyl group (OBzl) can be used, and an S-benzyl group (SBzl) can be used as the S-protecting group for the mercapto group of cysteine.

The term "protecting group" as used herein means that at least the introduced group satisfies the conditions that it is stable during the reaction, that it can be easily removed from the product, and that no side reactions occur during the removal. do.

In addition to the acid component and amine component of the starting material having the above-mentioned protecting groups, the Nα-amino group of some of the amines can of course be free, as well as hydrochloric acid, hydrobromide, oxalate, p-toluenesulfone. Any inorganic or organic acid salt form may be used, such as acid salts and acetates.

In the present invention, the dehydration condensation reaction that produces 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 6 to 8.

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 a base. Depending on the pH, 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 88 degrees, acid is supplied.

The starting materials are generally 0.00% acid component per mole amine component.
It is used in a proportion of 8 to 2 moles, preferably 1 to 1.5 moles.

If these starting materials are difficult to dissolve in an aqueous solvent, their solubility can be improved by adding a solvent such as an alcohol such as methanol or ethanol, dimethylformamide, dioxane, tetrahydrofuran, or dimethyl sulfoxide.

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 400rn9, preferably 50 to 300rn9.

The reaction temperature is generally 20~20°C from the viewpoint of maintaining enzyme activity.
The temperature is 45°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.

Next, the present invention will be described with reference to Examples, but the present invention is not limited thereto in any way.

Example I Z -Gln -OH 2 8 0. 2m9 (1
mmol) and H-Phe-Phe-OBut
268.5 μm (lmmol) was suspended in the 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 6.5.
100 m9 of thermoase was added adjusting the temperature to ~7.0 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 separate the precipitated crystals. Water, dilute hydrochloric acid (1 normal),
It was washed with water, aqueous ammonia (7%) and water, and then dried.

This was recrystallized from ethyl acetate to give m, p, 1 9 7
Z-G1nPhe-Phe with ~200°C
-OBut330m9 (52.3%) was obtained.

Example 2 Z -Phe -Val -OH 3 9 8.5m
9 (1 mmol) and H-Phe-Val-
OBut3 2 0. 4 m9 (1 mmol
) was suspended in 10rrll of water in a flask.

Next, a glass electrode of a pH meter was inserted into this, and sodium hydroxide aqueous solution (1/1 0 normal) was added dropwise to the pH meter.
Thermoase 100 while adjusting H to 6.5-7.5
Irl9 was added 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 after the reaction was measured using a glass electrode pH meter, and hydrochloric acid (1/10 normal) was added to maintain the pH of the reaction solution at 6.5 to 7.5.

The precipitated crystals were crushed, 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, m and p.
, Z-Ph having 1 3 0 to 1 4 5'C
e-Val -Phe -Val -OBu t2 4
Obtained 0m9.

Yield 34.2%.

Claims (1)

[Claims] 1. In the presence of an enzyme belonging to metalloproteinases, a compound of the general formula X-A-OH (wherein X is a protecting group for the terminal amino group,
A is an amino acid residue or a peptide residue) and an amino acid or peptide having a protecting group at the N-terminus;
Amino acids having a protecting group at the C-terminus represented by the general formula H-B-Y (wherein Y is a protecting group for the terminal carboxyl group, and B is the same or different amino acid residue or peptide residue as A) or peptide, 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, so that the enzymatic activity of the metalloproteinase in an aqueous solution is detected. General formula X-A-B, characterized in that the reaction is carried out within a pH range showing
A method for producing a peptide represented by -Y (wherein X, A, B, and Y are the same as defined above). 2. The manufacturing method according to claim 1, wherein the reaction is carried out at pH 6 to 8. 3. A patent claim in which the metalloproteinase is a neutral proteinase of Streptomyces origin, Bacillus subtilis varietus amyliquifaciens, Bacillus thermoproteoliticus, thermolysin, collagenase, Kurtulus atrox proteinase, or a mixture thereof. The manufacturing method according to scope 1 or 2. 4. Aliphatic α-monoaminomonocarboxylic acids, oxyamino acids, sulfur-containing amino acids, in which A and B representing amino acid residues or peptide residues are the same or different, respectively;
A patent claim that is a residue of a monoamino dicarboxylic acid or a diamino monocarboxylic acid, a residue of an amino acid having an aromatic ring, a residue of an amino acid having a heterocycle, or an oligo or polypeptide residue consisting of these amino acid residues The manufacturing method according to any one of items 1 to 3. 5 The aliphatic monoamino monocarboxylic acid is glycine, alanine, palin, norvaline, leucine, inleucine, or norleucine, the aliphatic oxyamino acid is serine, threonine, or homoserine, and the aliphatic sulfur-containing amino acid is methionine. , cystine or cysteine with side chain functional group protected, aliphatic monoamino dicarboxylic acid is aspartic acid or glutamic acid with side chain functional group protected, and aliphatic diaminocarboxylic acid is cystine or cysteine with side chain functional group protected. 5. The method according to claim 4, wherein the amino acid having an aromatic ring is ornithine, lysine or arginine, the amino acid having an aromatic ring is 7-enylalanine or tyrosine, and the amino acid having a heterocycle is histidine or tryptophan.