JP3530928B2 - New oligopeptide synthesis method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for synthesizing oligopeptides, more specifically, at 230 ° C.
The present invention relates to a novel method for producing an oligopeptide, which enables an oligopeptide having a longer chain length to be synthesized from an amino acid oligopeptide in high temperature hot water in an extremely short time. INDUSTRIAL APPLICABILITY The present invention uses chemically stable, non-toxic, and inexpensive water in high-temperature hot water conditions of 230 ° C. or higher, high-temperature hot water, subcritical water, or supercritical water to obtain a longer chain length from oligopeptides of amino acids and the like. It is useful as a method for synthesizing an oligopeptide having a long length in a very short time.

[0002]

2. Description of the Related Art In recent years, with attention paid to the physiological activity of peptides and their important functions, as a production technique thereof, a method of producing various peptides having physiological activity by hydrolyzing a protein with an enzyme or the like. In addition, methods for synthesizing various physiologically active peptides have been developed by organic chemical means, genetic engineering techniques, etc., and these physiologically active peptides are, for example, functional food materials, pharmaceuticals, research reagents. It is widely used as etc. On the other hand, in recent years, various reaction systems using supercritical fluids and their application examples have been receiving attention, and various research results have been reported so far. When a liquid exceeds a certain specific pressure and temperature, it becomes difficult to distinguish whether it is a gas or a liquid.For example, it shows a liquid-like behavior in that it dissolves various substances, compresses, At the point of expansion, it exhibits a gaseous behavior such that its density can be arbitrarily changed by pressure. A fluid in such a state is called a supercritical fluid because it is different from the normal properties of liquids and gases, and various new reaction systems using the supercritical fluid and various application examples thereof have been proposed.

Concretely, for example, a method of decaffeinating coffee beans with supercritical carbon dioxide,
Utilization of supercritical fluids for liquid chromatography, reduction of molecular weight of lignin-based samples in supercritical water, hydrolysis of CFCs with supercritical water, PCB hydrolysis, ester synthesis reaction with lipase in supercritical carbon dioxide, etc. The research example of is reported. On the other hand, it is well known that plastics and biomass can be decomposed by using supercritical water, and there are many studies on them. Therefore, the present inventors also attempted to decompose a plant-derived protein in supercritical water to obtain an amino acid. However, analysis of the decomposition products revealed that oligopeptides were formed in addition to the monomer amino acids. The knowledge at the time thought that the cause of this was that oligopeptides were produced in the pathway by which proteins were hydrolyzed to produce amino acids.

Report of Science magazine in 1999 (S
science, Vol. 283, p831-833,1
999), hydrothermal conditions (24 MPa, 200-25
Using a circulating flow reactor at 0 ° C), hydrothermal treatment and 0 ° C were performed while circulating the monomer amino acid (glycine) in the device.
It has been reported that the oligopeptide (oligopeptide: a peptide consisting of about 10 or less amino acids) was successfully synthesized by repeating cooling for several tens of minutes. In the same paper, it is stated that high temperature conditions alone induce dissociation reactions such as dehydration, deamination, and decarboxylation, and are rather unfavorable for peptide synthesis. It is described that it is 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. However, there is no report that a peptide having a longer chain length was synthesized from an oligopeptide as a reaction substrate.

[0005]

In view of the above-mentioned conventional technique, the present inventors have made only oligopeptides of amino acids present in high-temperature hot water to solve the problems described above. As a result of examining the presence or absence of peptide synthesis, 2
The present invention was obtained by further finding out that an oligopeptide having a longer chain length is produced in an extremely short time (for example, a reaction time of 0.72 seconds) in high-temperature hot water of 30 ° C. or higher, and the present invention is carried out. Has been completed. That is, the object of the present invention is to provide a method for efficiently synthesizing an oligopeptide having a longer chain length from an oligopeptide of an amino acid by a short-time reaction in hot water of 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 produced 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) Reaction in high temperature hot water for a short time of about 0.01 to 10 seconds
From oligopeptides to longer chain oligopeptides
A method for synthesizing a peptide, which comprises synthesizing an oligopeptide having a longer chain length by allowing an oligopeptide having a predetermined concentration of an amino acid to be present as a reaction substrate in hot water of 230 ° C. or higher. Manufacturing method. (2) Reaction in high temperature hot water for a short time of 0.01 to 10 seconds
In the form of oligopeptides and their degradation products
From the amino acids present in the reaction system, oligos with longer chain length
A method for synthesizing a peptide, which comprises allowing a predetermined concentration of an oligopeptide and an amino acid produced from the oligopeptide to be present as reaction substrates in hot water of 230 ° C. or higher,
A method for producing an oligopeptide, which comprises synthesizing an oligopeptide having a longer chain length. (3) The method for producing an oligopeptide according to (1) or (2) above, wherein an 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 (1) or (2) above, wherein an 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) above, wherein an 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 (1) or (2) above, wherein the synthetic reaction is performed in a high-temperature hot-water state at a temperature of 230 to 450 ° C.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be described in more detail. The present invention allows an oligopeptide having a predetermined concentration of an amino acid, or an oligopeptide and a monomer amino acid produced from the oligopeptide, to be present as a reaction substrate in high-temperature hot water, and the oligopeptide having a longer chain length can be obtained in a short time. Is characterized by synthesizing. The high temperature hot water to be used is high temperature hot water of 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 (water with a critical temperature of 375 ° C and a critical pressure of 22.05 MPa or more)
You can choose. In the present invention, the reaction often proceeds fairly efficiently near the critical point, but the reaction also proceeds under hydrothermal conditions of 230 ° C. or higher. The pressure may be at least the autogenous pressure at the operating temperature. The optimum temperature condition varies depending on the treatment time, but in general, 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 in the temperature range of ℃ (critical temperature 37
Water having a temperature of 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 reaction apparatus. As the reaction apparatus, for example, a high temperature / high pressure reaction apparatus is used, but the type is not limited as long as it is an apparatus capable of setting a reaction system of high temperature hot water.

Next, in the present invention, an oligopeptide having a predetermined concentration of an amino acid, or an oligopeptide and an amino acid (including the peptide) produced from the oligopeptide, are present as a reaction substrate in hot water of 230 ° C. or higher. Examples of these include various amino acids, di-, tri-, tetra-, penta-amino acids, α-amino acids, β-amino acids, γ-amino acids, δ-amino acids, monoaminomonocarboxylic acids, and monoamino acids. Examples thereof include those oligopeptides having aminodicarboxylic acid, diaminomonocarboxylic acid and the like as constituent elements, and the constituent amino acids (including the peptide) produced from the oligopeptides. The amino acid is produced in the reaction system by the decomposition reaction of the oligopeptide, and is involved in the peptide bond 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 produced by the decomposition reaction of the oligopeptide as the reaction substrate. The types of these oligopeptides and amino acids are not particularly limited.

In the reaction system of the present invention, the above reaction substrate may be present in 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 makes an oligopeptide of an amino acid, or an oligopeptide of an amino acid produced from the oligopeptide and the oligopeptide, with a longer chain length, in the absence of a catalyst, by allowing the above reaction substrate to exist in hot water of 230 ° C. or higher. The most important feature is to synthesize it, but in this case, if necessary, a catalyst such as an enzyme, a metal ion, an acid, or a base may be added and allowed to react with each other. In the present invention, the above reaction, for example, the reaction time from 0.01 to 10 seconds, preferably 0.01 to 5 seconds, most preferably between very short time of 0.01 seconds, more chain length Long oligopeptides are produced. Further, in the present invention, an oligopeptide having a longer chain length can be efficiently synthesized by increasing the concentration of the oligopeptide of the amino acid which is the reaction substrate. Therefore, by adjusting the concentration of the oligopeptide of the reaction substrate, It becomes possible to synthesize various oligopeptides having a longer chain length. Suitably, the concentration of the reaction substrate is 100 mM to 500 mM, but is not limited thereto. Further, in the present invention, it becomes possible to synthesize various oligopeptides having a long chain length by adjusting the temperature and reaction time of the reaction system.

As shown in Examples described later, the inventors of the present invention produced various oligopeptides having longer chain length in an extremely short time (for example, a reaction time of about 0.7 seconds) in high temperature hot water. It was confirmed using a high performance liquid chromatography-mass spectroscope (LC-MS) that it was possible.
For example, it was found that in high temperature hot water, 0.113 mM tetraglycine can be synthesized in the presence of diglycine as a reaction substrate at 344 ° C. for 0.86 seconds. 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 diamino acid (diglycine) is allowed to exist in high-temperature hot water, without adding any enzyme such as ribosome, ribozyme, metal ion such as Cu 2+, or catalyst such as acid or base, It was found that an oligopeptide having a longer chain length can be synthesized in an extremely short time.
Further, as a normal operation for forming a peptide bond, 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 a carbonyl carbon of an amino acid is not necessary. . The method of the present invention can, of course, be applied to the synthesis of various oligopeptides having longer chain lengths using oligopeptides of various amino acids.

[0012]

In the present invention, the presence of a predetermined concentration of an oligopeptide of an amino acid, or an oligopeptide and a monomer amino acid produced from the oligopeptide as a reaction substrate in hot water of 230 ° C. or higher, Tetraamino acids are synthesized from diamino acids. in this case,
By increasing the concentration of the reaction substrate, various oligopeptides such as triamino acid, tetraamino acid, pentaamino acid can be efficiently synthesized. In this case, a diamino acid decomposition reaction also occurs to produce a monomer amino acid, which is often involved in the peptide bond reaction. Further, in the above reaction system, depending on the reaction temperature, the type and concentration of the reaction substrate, for example, a triamino acid is produced by a peptide bond of a diamino acid and a monomer amino acid produced from the diamino acid. Therefore, the present invention makes it possible to synthesize a desired oligopeptide in an extremely 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 Experiments were carried out by using a flow type high temperature / high pressure reactor to change the temperature, reaction time and diglycine concentration of an amino acid oligopeptide (diglycine) aqueous solution to obtain oligos with longer chain length. The peptide was synthesized. Figure 1 shows the flow-type high-temperature and high-pressure reactor used in this experiment. This reactor is a potassium nitrate-sodium nitrate based salt bath agent AS140 in a salt bath.
(Product of Parka Heat Treatment Co., Ltd.) can be used to control the salt bath temperature to 160 to 550 ° C. As the measured temperature value, the temperature value at the salt bath outlet of the pipeline was used. Line is SUS3
Sixteen 1/16 inch (0.5 mm id) pipes were used. The reaction tube volume can be adjusted by changing the length of the portion put in the salt bath. Pump is 0.001-100m
It can be used at a flow rate of 1 / min, but usually 1 to 1
Used at 00 ml / min. The reaction time is calculated from the reaction tube volume, pump flow rate and liquid density. The pressure can be adjusted by the back pressure valve. An oligopeptide of an amino acid is prepared as an aqueous solution in a raw material supply part, nitrogen gas is bubbled to remove dissolved oxygen gas, and then supplied to a pipeline by a pump. The pipeline is circulated at a predetermined pressure and salt bath temperature, and after passing through the salt bath,
Cool with water and collect the sample in the recovery unit. 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 Using the above-mentioned high temperature / high pressure reactor, pressure 1 MPa at 25 MPa
1 mM of 00 mM diglycine aqueous solution
It was made to flow at 5 ml / min and reacted at 371 ° C.
The length of the reaction tube is 170 cm and the volume of the reaction tube is 0.334.
It was ml. Density during reaction is 0.5334 g / ml
The reaction time was 0.71 seconds. When the obtained sample was analyzed, it was found to be 0.118 m with unreacted diglycine.
M concentration of triglycine and 0.
Tetraglycine at a concentration of 063 mM
ine) was obtained. It is considered that in tetraglycine, peptide bonds between diglycines were generated by a short-time reaction in high temperature hot water of 371 ° C.

Example 2 In the same manner as in Example 1, 100 mM diglycine aqueous solution was reacted. 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. Pentaglycine is presumed to have been produced by the peptide bond between diglycine and triglycine.

Example 3 In the same manner as in Example 1, 100 mM diglycine aqueous solution was reacted. However, the reaction temperature was 294 ° C. The density during the reaction was 0.7539 g / ml, and the reaction time was 1.01.
It was 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 is 0.0006 mM
Met.

Example 4 A 100 mM diglycine aqueous 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.
It was seconds. When the obtained sample was analyzed, the triglycine concentration was 0.084 mM, and the tetraglycine concentration was 0.
It was 018 mM.

Comparative Example 1 In the same manner as in Example 1, 100 mM was used instead of diglycine.
An aqueous glycine solution was reacted. However, the reaction temperature should be 37
It was set to 0 ° C. Density during reaction is 0.54037g / ml
The reaction time was 0.72 seconds. When the obtained sample was analyzed, only triglycine concentration of 0.004 mM was detected. It was found that the use of monomeric glycine produced less long-chain peptide bonds and was not suitable for the synthesis of longer-chain oligopeptides.

Example 5 In the same manner as in Example 1, 100 mM diglycine aqueous solution was reacted. However, the reaction temperature was 395 ° C. The density during the reaction was 0.18458 g / ml, and the reaction time was 0.2.
It was 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 Diglycine aqueous solution was reacted in the same manner as in Example 1.
However, the concentration of diglycine was 500 mM, and the reaction temperature was 363 ° C. Density during reaction is 0.57609g
/ 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 above in detail, the present invention provides a 230
The present invention relates to a method for producing an oligopeptide, characterized in that an oligopeptide having a predetermined concentration of an amino acid is present as a reaction substrate in high-temperature hot water of ℃ or higher, 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 an extremely 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) Reaction system In, the reaction temperature, the concentration of the reaction substrate, the reaction time, etc. are adjusted to produce various oligopeptides having longer chain lengths. 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, and 5) it is useful as a new method for peptide synthesis, which is particularly effective.

[Brief description of drawings]

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

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazuo Torii 1-19-13 Nishinakada, Taichiro-ku, Sendai City, Miyagi Prefecture (56) References JP-A-2002-37799 (JP, A) Science, Vol. 283, No. 5403 (1999) p. 831-833 Proceedings of the 32nd Autumn Meeting of the Chemical Society of Japan (1999) p. 635 (58) Fields investigated (Int. Cl. ⁷ , DB name) BIOSIS / WPI (DIALOG) PubMed CA (STN) REGISTRY (STN) JSTPlus (JOIS)

Claims (6)

(57) [Claims] 1. A method for synthesizing an oligopeptide having a longer chain length from an oligopeptide by a short-time reaction of about 0.01 to 10 seconds in high-temperature hot water, which is performed in high-temperature hot water at 230 ° C. or higher. A method for producing an oligopeptide, which comprises allowing an oligopeptide having a predetermined concentration of an amino acid to be present as a reaction substrate to synthesize an oligopeptide having a longer chain length. 2. An oligopeptide having a longer chain length is synthesized from an oligopeptide and an amino acid present in the reaction system in a form of being decomposed from the oligopeptide by a short-time reaction of about 0.01 to 10 seconds in high-temperature hot water. A method for synthesizing an oligopeptide having a longer chain length by allowing a predetermined concentration of an oligopeptide and an amino acid produced from the oligopeptide to be present as reaction substrates in hot water of 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 the reaction substrate. 4. By adjusting the temperature of the reaction system,
The method for producing an oligopeptide according to claim 1 or 2, wherein an oligopeptide having a longer chain length is synthesized from the oligopeptide as a reaction substrate. 5. The 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 or 2, wherein the synthetic reaction is performed in a high temperature hot water state at a temperature of 230 to 450 ° C.