JP2002053594A - New method for synthesizing oligopeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for synthesizing an oligopeptide having a longer chain length from an oligopeptide of an amino acid in a high-temperature water. SOLUTION: This method for producing the oligopeptide having the longer chain length is characterized in that the oligopeptide of the amino acid in a prescribed concentration is allowed to be present as a reactant in the high- temperature water at >=230 deg.C to synthesize the oligopeptide having the longer chain length in a short time of 0-10 s. In other aspect, the method for producing the oligopeptide is characterized in that the oligopeptide in the prescribed concentration and the amino acid formed from the oligopeptide are allowed to be present in the high-temperature water at >=230 deg.C to synthesize the objective oligopeptide having the longer chain length. As a result, the oligopeptide having the longer chain length is synthesized in a short time of <=10 s (e.g. about 0.7 s reaction time).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for synthesizing an oligopeptide, and more particularly, to a method for producing an oligopeptide at 230 ° C.
The present invention relates to a novel method for producing an oligopeptide, which makes it possible to synthesize an oligopeptide having a longer chain length from an amino acid oligopeptide in high-temperature hot water in an extremely short time. The present invention uses chemically stable, non-toxic, inexpensive high-temperature hot water of 230 ° C. or higher in high-temperature hot water, subcritical water or supercritical water to reduce the chain length from oligopeptides of amino acids and the like. This is useful as a method for synthesizing oligopeptides having a long length in a very short time.

[0002]

2. Description of the Related Art In recent years, the physiological activities of peptides and their important functions have been attracting attention. As a production technique, a method for producing various peptides having physiological activities by hydrolyzing proteins with enzymes or the like. Furthermore, methods for synthesizing various bioactive peptides by organic chemical means, genetic engineering techniques, and the like have been developed. These bioactive peptides are, for example, functional food materials, pharmaceuticals, and research reagents. Widely used as such. On the other hand, in recent years, various reaction systems using supercritical fluids and application examples thereof have attracted attention, and various research results have been reported so far. When a liquid exceeds a certain pressure and temperature, it becomes difficult to distinguish whether it is a gas or a liquid.For example, it exhibits a liquid-like behavior in dissolving various substances, and exhibits compression, In terms of expansion, it exhibits gaseous behavior such that its density can be arbitrarily changed by pressure. Fluids in such a state are different from ordinary properties of liquids and gases, and are called supercritical fluids, and various new reaction systems using the supercritical fluids and application examples thereof have been proposed.

[0003] Specifically, for example, a method of decaffeinating coffee beans with supercritical carbon dioxide,
Utilization of supercritical fluid for liquid chromatography, reduction of molecular weight of lignin samples in supercritical water, hydrolysis of chlorofluorocarbons and PCBs with supercritical water, ester synthesis reaction with lipase in supercritical carbon dioxide, etc. Have been reported in some research cases. On the other hand, it is well known that plastics and biomass can be decomposed by using supercritical water, and there are many research examples regarding them. Therefore, the present inventors have also attempted to obtain amino acids by decomposing plant-derived proteins in supercritical water. However, analysis of the decomposition products revealed that oligopeptides were formed in addition to the monomeric amino acids. The cause at that time was thought to be that oligopeptides were formed in the pathway in which proteins were hydrolyzed to produce amino acids.

A report from Science Magazine in 1999 (S
science, Vol. 283, p831-833, 1
999) under hydrothermal conditions (24 MPa, 200-25
(0 ° C) using a circulation type flow reactor, while circulating the monomeric amino acid (glycine) in the device and performing hydrothermal treatment and 0 ° C.
It has been reported that an oligopeptide (a peptide composed of about 10 or less amino acids) was successfully synthesized by repeating cooling for several tens of minutes. The paper states that high temperature alone induces dissociation reactions such as dehydration, deamination, and decarboxylation, which is rather disadvantageous for peptide synthesis. It is described as mandatory. As described above, various reaction systems using supercritical fluids and high-temperature hot water have been reported, and various methods for peptide synthesis have been reported. In general, there is no report that a peptide having a longer chain length was synthesized from an oligopeptide as a reaction substrate.

[0005]

Under such circumstances, in view of the above-mentioned prior art, the present inventors have made it possible to make only oligopeptides of amino acids exist in high-temperature hot water, and to prepare an oligopeptide having a longer chain length. As a result of examining the presence or absence of peptide synthesis, 2
Having obtained the knowledge that an oligopeptide having a longer chain length is produced in a very short time (for example, a reaction time of 0.72 seconds) in high-temperature hot water of 30 ° C. or higher, the present inventors have further studied and Was completed. That is, an object of the present invention is to provide a method for efficiently synthesizing an oligopeptide having a longer chain length from an amino acid oligopeptide by a short-time reaction in high-temperature hot water at 230 ° C. or higher. is there. Another object of the present invention is to provide a method for synthesizing an oligopeptide having a longer chain length by allowing an oligopeptide and an amino acid generated from the oligopeptide to exist in high-temperature hot water.

[0006]

The present invention for solving the above-mentioned problems comprises the following technical means. (1) A method for producing an oligopeptide, wherein an oligopeptide having a predetermined concentration of an amino acid is present as a reaction substrate in high-temperature hot water at 230 ° C. or higher to synthesize an oligopeptide having a longer chain length. (2) An oligopeptide having a predetermined concentration of an oligopeptide and an amino acid generated from the oligopeptide as a reaction substrate in high-temperature hot water of 230 ° C. or higher to synthesize an oligopeptide having a longer chain length. Manufacturing method. (3) The method for producing an oligopeptide according to the above (1) or (2), wherein the oligopeptide having a longer chain length is synthesized by adjusting the concentration of the oligopeptide as a reaction substrate. (4) The method for producing an oligopeptide according to the above (1) or (2), wherein the oligopeptide having a longer chain length is synthesized from the oligopeptide by adjusting the temperature of the reaction system. (5) The oligopeptide according to (1) or (2), wherein the oligopeptide having a longer chain length is synthesized from the oligopeptide by adjusting the reaction time between 0 and 10 seconds. Manufacturing method. (6) The method for producing an oligopeptide according to the above (1) or (2), wherein the synthesis reaction is performed in a high-temperature hot water state at a temperature of 230 to 450 ° C.

[0007]

Next, the present invention will be described in more detail. The present invention provides an oligopeptide having a predetermined concentration of an amino acid as a reaction substrate in high-temperature hot water, or an oligopeptide and a monomeric amino acid generated from the oligopeptide, and in a short time, an oligopeptide having a longer chain length. Is synthesized. The high-temperature hot water used is 230 ° C. or higher, preferably 230 ° C. to 45 ° C.
High-temperature hot water subcritical water or supercritical water in the temperature range of 0 ° C (critical temperature 375 ° C, water with a critical pressure of 22.05 MPa or more)
You can choose. In the present invention, the reaction often proceeds quite efficiently near the critical point, but the reaction proceeds even under hydrothermal conditions of 230 ° C. or higher. The pressure may be higher than the autogenous pressure at the operating temperature. The optimum temperature condition varies depending on the treatment time, but generally, high-temperature hot water of 230 ° C. or higher may be used, and more preferably 300 ° C. to 400 ° C.
Subcritical water or supercritical water (critical temperature 37
5 ° C. and a critical pressure of 22.05 MPa or more) can be selected. Further, appropriate temperature and pressure conditions can be adopted depending on the throughput and the reactor. As the reaction device, for example, a high-temperature and high-pressure reaction device is used. However, the type of the reaction device is not limited as long as it can set a reaction system of high-temperature hot water.

Next, in the present invention, an oligopeptide of an amino acid at a predetermined concentration or an oligopeptide and an amino acid (including the peptide) formed from the oligopeptide are present as a reaction substrate in high-temperature hot water at 230 ° C. or higher. Examples of these include, for example, various amino acids, di-, tri-, tetra-, penta-amino acids, α-amino acids, β-amino acids, γ-amino acids, δ-amino acids, monoaminomonocarboxylic acids, Examples thereof include those oligopeptides having aminodicarboxylic acid, diaminomonocarboxylic acid, and the like as constituent elements, and the constituent amino acids (including the peptides) generated from the oligopeptides. The amino acid is generated in the reaction system by the decomposition reaction of the oligopeptide, and is involved in the peptide binding reaction in the reaction system, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, Methionine, phenylalanine,
Examples include tryptophan, tyrosine, proline, cystine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, histidine and the like.
These amino acids are not added to the reaction system, but are present in the reaction system in a form generated by a decomposition reaction of the oligopeptide as a reaction substrate. The types of these oligopeptides and amino acids are not particularly limited.

In the reaction system of the present invention, it is sufficient that the above reaction substrate is present in high-temperature hot water of 230 ° C. or higher. For example, it is not necessary to add a catalyst such as an enzyme, a metal ion, an acid or a base. Absent. The present invention, the reaction substrate is present in high-temperature hot water of 230 ° C. or higher, and without a catalyst, an oligopeptide of an amino acid, or an oligopeptide having a longer chain length from an oligopeptide and an amino acid generated from the oligopeptide. The greatest feature is that synthesis is performed, but in this case, if necessary, even if a catalyst such as an enzyme, a metal ion, an acid, or a base is added and allowed to react, the reaction cannot be avoided. In the present invention, the above reaction system provides a very short reaction time of, for example, a reaction time of 0.01 to 10 seconds, preferably 0.01 to 5 seconds, and most preferably 0.01 to 2 seconds. Are generated. In addition, in the present invention, by increasing the oligopeptide concentration of the amino acid that is the reaction substrate, an oligopeptide having a longer chain length can be efficiently synthesized. Thus, various oligopeptides having longer chain lengths can be synthesized.
Preferably, the concentration of the reaction substrate is between 100 mM and 500 mM.
However, the present invention is not limited to this. Further, in the present invention, it is possible to synthesize various oligopeptides having a long chain length by adjusting the temperature and reaction time of the reaction system.

As shown in the examples described below, the present inventors have found that in high-temperature hot water, a very short time (for example, a reaction time of about 0.7 seconds) and the production of various oligopeptides having a longer chain length can be obtained. Was confirmed using a high performance liquid chromatography-mass spectrometer (LC-MS).
For example, it has been found that in high-temperature hot water, 0.113 mM tetraglycine can be synthesized at 344 ° C. and 0.86 seconds in the presence of diglycine as a reaction substrate. It was also found that by increasing the concentration of diglycine as a reaction substrate, oligopeptides having a longer chain length, such as tetraglycine and pentaglycine, can be synthesized.

When a diamino acid (diglycine) is present in high-temperature hot water, an enzyme such as ribosome or ribozyme, a metal ion such as Cu 2+, or a catalyst such as acid or base can be added without any addition. It was found that an oligopeptide having a longer chain length could be synthesized in a very short time.
Also, as an operation usually performed to generate a peptide bond, a so-called amino acid activation treatment for increasing an electron-withdrawing property by attaching an electron-withdrawing substituent such as a nitrophenyl group or an ester group to the carbonyl carbon of the amino acid is unnecessary. . The method of the present invention is, of course, applicable to the synthesis of various oligopeptides having longer chain lengths using oligopeptides of various amino acids.

[0012]

According to the present invention, for example, the presence of an oligopeptide of an amino acid at a predetermined concentration as a reaction substrate, or an oligopeptide and a monomer amino acid generated from the oligopeptide, as a reaction substrate in high-temperature hot water at 230 ° C. or higher, A tetraamino acid is synthesized from a diamino acid. in this case,
By increasing the concentration of the reaction substrate, various oligopeptides such as triamino acids, tetraamino acids, and pentaamino acids are efficiently synthesized. In this case, a decomposition reaction of a diamino acid also occurs to produce a monomeric amino acid, and is often involved in a peptide binding reaction. Further, in the above-mentioned reaction system, for example, a triamino acid is generated by a peptide bond between a diamino acid and a monomer amino acid generated from the diamino acid depending on the reaction temperature, the type and the concentration of the reaction substrate. Therefore, the present invention makes it possible to synthesize a desired oligopeptide in a very short time by adjusting the reaction temperature, the type and concentration of the reaction substrate in the above reaction system, and is useful as a method for synthesizing an oligopeptide. is there.

[0013]

EXAMPLES Next, the present invention will be specifically described based on examples, but the present invention is not limited to the following examples. Example 1 (1) Method and Apparatus The experiment was carried out by changing the temperature, reaction time, and diglycine concentration of an amino acid oligopeptide (diglycine) aqueous solution using a flow-type high-temperature and high-pressure reactor to obtain an oligo with a longer chain length. Peptide synthesis was performed. FIG. 1 shows the flow type high temperature / high pressure reactor used in this experiment. This reactor is provided with a salt bath AS140 of a potassium nitrate-sodium nitrate system in a salt bath.
The salt bath temperature can be controlled to 160 to 550 ° C. using (product of Parka Heat Treatment Co., Ltd.). The temperature measured at the salt bath outlet of the pipeline was used. The line is SUS3
Sixteen 1/16 inch (0.5 mm inner diameter) pipes were used. The reaction tube volume can be adjusted by changing the length of the portion to be put in the salt bath. Pump is 0.001-100m
Although it can be used at a flow rate of 1 / min, it is usually 1 to 1
Used at 00 ml / min. The reaction time is determined from the volume of the reaction tube, the pump flow rate, and the density of the liquid. The pressure can be adjusted by a back pressure valve. Amino acid oligopeptides are prepared as an aqueous solution in a raw material supply section, and nitrogen gas is bubbled to remove dissolved oxygen gas, and then supplied to the pipeline by a pump. The pipeline is circulated at a predetermined pressure and salt bath temperature, and after passing through the salt bath,
After cooling with water, the sample is collected in the collection section. The product of the obtained sample was confirmed using a high performance liquid chromatography mass spectrometer.

(2) Synthesis of Oligopeptide with Longer Chain Length from Reaction Substrate
1 mM aqueous diglycine solution
The reaction was carried out at 371 ° C. at a flow rate of 5 ml / min.
The length of the reaction tube was 170 cm and the volume of the reaction tube was 0.334.
ml. The density during the reaction is 0.5334 g / ml
The reaction time was 0.71 seconds. When the obtained sample was analyzed, along with unreacted diglycine, 0.118 m
M concentration of triglycine and 0.
063 mM concentration of tetraglycine (tetraglyc)
ine) was obtained. It is considered that the peptide bond between diglycines in tetraglycine was generated by a short-time reaction in hot water at 371 ° C.

Example 2 A 100 mM diglycine aqueous solution was reacted in the same manner as in Example 1. However, the reaction temperature was 344 ° C. The density during the reaction was 0.6441 g / ml, and the reaction time was 0.86 seconds. When the obtained sample was analyzed, the triglycine concentration was 0.074 mM and the tetraglycine concentration was 0.113.
mM and pentaglycine (pentaglycin)
e) The concentration was 0.002 mM. It is presumed that pentaglycine was formed by the peptide bond between diglycine and triglycine.

Example 3 A 100 mM aqueous diglycine solution was reacted in the same manner as in Example 1. However, the reaction temperature was 294 ° C. The density during the reaction was 0.7539 g / ml, and the reaction time was 1.01.
Seconds. When the obtained sample was analyzed, the triglycine concentration was 0.104 mM, and the tetradiglycine concentration was 0.0
75 mM, and pentaglycine concentration of 0.0006 mM
Met.

Example 4 A 100 mM aqueous diglycine solution was reacted in the same manner as in Example 1. However, the reaction temperature was 244 ° C. The density during the reaction was 0.8290 g / ml, and the reaction time was 1.11.
Seconds. When the obtained sample was analyzed, the concentration of triglycine was 0.084 mM, and the concentration of tetraglycine was 0.08 mM.
018 mM.

Comparative Example 1 In the same manner as in Example 1, 100 mM was used instead of diglycine.
A glycine aqueous solution was reacted. However, when the reaction temperature is 37
0 ° C. The density during the reaction is 0.54037 g / ml
The reaction time was 0.72 seconds. When the obtained sample was analyzed, only a triglycine concentration of 0.004 mM was detected. It has been found that the use of monomeric glycine causes less generation of peptide bonds having a longer chain length, which is not suitable for synthesis of oligopeptides having a longer chain length.

Example 5 A 100 mM diglycine aqueous solution was reacted in the same manner as in Example 1. However, the reaction temperature was 395 ° C. The density during the reaction was 0.18458 g / ml and the reaction time was 0.2
5 seconds. When the obtained sample was analyzed, the triglycine concentration was 0.147 mM and the tetraglycine concentration was 0.056 mM.

Example 6 An aqueous diglycine solution was reacted in the same manner as in Example 1.
However, the diglycine concentration was 500 mM, and the reaction temperature was 363 ° C. The density at the time of reaction is 0.57609 g
/ Ml, the reaction time was 0.77 seconds. When the obtained sample was analyzed, the triglycine concentration was 5.26 mM, the tetraglycine concentration was 3.11 mM, and the pentaglycine concentration was 0.627 mM.

[0021]

As described in detail above, the present invention provides a 230
The present invention relates to a method for producing an oligopeptide, characterized in that an oligopeptide of an amino acid having a predetermined concentration is present as a reaction substrate in high-temperature hot water at a temperature of at least ℃, and an oligopeptide having a longer chain length is synthesized. According to the invention, 1) 10
An oligopeptide having a longer chain length is produced in a very short time of 2 seconds or less. 2) An oligopeptide having a longer chain length can be easily synthesized by increasing the concentration of a reaction substrate. 3) A reaction system In the above, various oligopeptides having longer chain lengths are produced by adjusting the reaction temperature, the concentration of the reaction substrate, the reaction time, and the like. 4) The oligopeptide can be prepared without adding a catalyst or activating the oligopeptide. It is possible to convert a peptide into an oligopeptide having a longer chain length. 5) It is particularly effective as a new method for peptide synthesis.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of a flow-type high-temperature high-pressure reactor used in Examples.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Kazuo Torii 1-19-1 Nishinakada, Taihaku-ku, Sendai-shi, Miyagi F-term (reference) 4H045 AA20 BA12 BA13 FA32

Claims (6)

[Claims] 1. A method for producing an oligopeptide, characterized in that an oligopeptide of an amino acid having a predetermined concentration is present as a reaction substrate in high-temperature hot water at 230 ° C. or higher to synthesize an oligopeptide having a longer chain length. 2. A method comprising synthesizing an oligopeptide having a longer chain length by allowing a predetermined concentration of an oligopeptide and an amino acid generated from the oligopeptide to be present as a reaction substrate in high-temperature hot water at 230 ° C. or higher. A method for producing an oligopeptide. 3. The method for producing an oligopeptide according to claim 1, wherein the oligopeptide having a longer chain length is synthesized by adjusting the concentration of the oligopeptide as a reaction substrate. 4. By adjusting the temperature of the reaction system,
3. The method for producing an oligopeptide according to claim 1, wherein an oligopeptide having a longer chain length is synthesized from the oligopeptide serving as a reaction substrate. 5. The method according to claim 1, wherein an oligopeptide having a longer chain length is synthesized from the oligopeptide as a reaction substrate by adjusting the reaction time between 0 and 10 seconds. A method for producing an oligopeptide. 6. The method for producing an oligopeptide according to claim 1, wherein the synthesis reaction is performed in a high-temperature hot water state at a temperature of 230 to 450 ° C.