JP2023173908A - Method and device for producing peptide - Google Patents

Abstract

To provide a production method and a production device that can produce peptides without requiring large amounts of reaction reagents and with high yields.SOLUTION: The present invention relates to a production method of a peptide by solid phase synthesis using an automatic synthesizer, including repeating: a peptide elongation step of supplying an amino acid having a protecting group into a reaction system to react with a terminal amino group of a peptide; a reaction confirmation step of, after the peptide elongation step, supplying into the reaction system a test reagent capable of reversibly reacting with the terminal amino group of the peptide to cause coloring; and a deprotection step of supplying a de-protecting agent into the reaction system, after the reaction confirmation step, and relates to a peptide automatic synthesizer for carrying out the production method.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method and apparatus for producing peptides.

Peptides, which are polymers of amino acids, have high utility as pharmaceuticals, functional materials, etc., and are a group of compounds that have received the most attention in recent years.

Solid phase synthesis is known as a method for chemically synthesizing peptides. In the solid-phase synthesis method, the other end of the peptide chain to be elongated is bonded to a resin carrier, and then the carboxy group of the newly added amino acid is linked to the amino group of the peptide chain being elongated to form an amide bond. formation and elongation of the peptide chain. After the desired peptide is obtained, the peptide is separated from the carrier.

During the elongation reaction, reactive functional groups other than those used for the amide bond are protected with protective groups, thereby elongating the peptide chain while suppressing side reactions. More specifically, the amino group of the newly added amino acid is protected with a protecting group, and after the amino acid is linked to the peptide chain, the protecting group is removed (deprotection). The amino group thus exposed is linked to the carboxy group of a new amino acid that is subsequently added. By repeating this process, the peptide chain is elongated.

When synthesizing such peptides manually in a laboratory, etc., a method called the Kaiser test is used to check whether the condensation reaction between the amino group at the end of the peptide chain and the carboxy group of the added amino acid has been completed. It can be confirmed. The Kaiser test is an analytical method that performs a ninhydrin reaction on a solid phase and detects the residual terminal amino group. Ninhydrin reacts with the terminal amino group to produce a dye called Luhemann purple, but if the reaction has finished, the dye will not be produced, so if the reaction is colorless even after adding ninhydrin, the reaction has stopped. This is an indication of completion.

In this Kaiser test, the test reagent ninhydrin can only react with primary amino groups and specific secondary amino groups, and cannot react with secondary amino groups such as proline and N-methyl amino acids. . In addition, the Kaiser test requires heating to a high temperature for about 5 minutes to react with ninhydrin, and is a destructive test in which the used sample cannot be reused. The Kaiser test is a destructive test because it requires covalent bonding of ninhydrin to the terminal amino group of the peptide, and this bond does not react reversibly.

On the other hand, in recent years, a new test method has been proposed to replace the Kaiser test (Non-Patent Document 1). The test reagent used in this method is capable of reacting not only with all primary and secondary amino groups, but also with tertiary amino groups. Additionally, since this reagent can react reversibly with peptides, this test method is a non-destructive test in which the peptides used in the test can be reused, resulting in The yield of peptides can be increased. This testing method is very effective because peptides are very expensive.

Rio Suzuki et al. , Org. Lett. 2020, 22, 3309-3312

Peptide synthesis is performed manually at the laboratory level, and peptide synthesis is usually performed using a commercially available automatic peptide synthesizer. Automatic synthesis equipment can perform the above reactions in a short time, with high precision, and with simple operation.

Automatic peptide synthesizers do not extract the peptide from the inside and perform the Kaiser test each time the amino acid condensation reaction occurs. Therefore, in automatic synthesizers, the protecting groups generated during deprotection are detected by UV to indirectly monitor the condensation reaction. Therefore, with current automatic synthesis equipment, it is not possible to quantitatively monitor the condensation reaction between amino acids and peptides with high precision. Efforts are being made to improve yield.

An object of the present invention is to provide a production method and a production apparatus that can produce peptides in high yield without using a large amount of reaction reagents.

The present inventors have discovered that the above problem can be solved by the following aspects.
《Aspect 1》
A method for producing a peptide by solid phase synthesis using an automatic synthesizer, the method comprising:
a peptide elongation step in which an amino acid having a protecting group is supplied into the reaction system and reacted with the terminal amino group of the peptide;
After the peptide extension step, a reaction confirmation step of supplying a test reagent capable of reversibly reacting with the terminal amino group of the peptide to cause coloration into the reaction system, and after the reaction confirmation step, a deprotecting agent A method for producing a peptide, comprising repeating a deprotection step of supplying peptide into the reaction system.
《Aspect 2》
The method for producing a peptide according to aspect 1, wherein in the peptide elongation step, the amino acid is supplied in an amount that is 3.0 times or less the stoichiometric amount of the reaction.
《Aspect 3》
The method for producing a peptide according to aspect 1 or 2, wherein if the reaction solution is colored after the reaction confirmation step, the peptide extension step is further repeated.
《Aspect 4》
After the peptide extension step and before the reaction confirmation step, a first washing step of washing out the inside of the reaction system, and After the reaction confirmation step and before the deprotection step, the The method for producing a peptide according to any one of aspects 1 to 3, which does not include a third washing step for washing out the inside of the reaction system.
《Aspect 5》
After the deprotection step, a fourth washing step of washing out the inside of the reaction system is included, and a test reagent that can be colored by reversibly reacting with the terminal amino group of the peptide is supplied into the reaction system. The method for producing a peptide according to any one of aspects 1 to 4, comprising a step of confirming deprotection.
《Aspect 6》
A method for producing a peptide according to any one of aspects 1 to 5, which is the Fmoc method.
《Aspect 7》
The method for producing a peptide according to any one of aspects 1 to 6, wherein the test reagent is a compound having an N-hydroxy cyclic imide skeleton.
《Aspect 8》
An automatic peptide synthesizer for carrying out the production method according to any one of aspects 1 to 7, comprising:
a reaction vessel with an optically transparent window;
a plurality of reagent supply containers for supplying reagents to the reaction containers;
an optical device for observing the color inside the reaction container through the window, and a control device for controlling these;
An automatic peptide synthesis device comprising:
《Aspect 9》
9. The apparatus according to aspect 8, wherein the reaction container has the window at a position corresponding to a supply position of the test reagent.

According to the present invention, it is possible to provide a production method and production apparatus that do not require the use of large amounts of reaction reagents and can produce peptides in high yield.

FIG. 1 schematically shows a flowchart for one embodiment of the manufacturing method of the invention. FIG. 2 schematically shows one embodiment of the manufacturing apparatus of the present invention.

《Peptide production method》
The method of the present invention is a method for producing peptides by solid-phase synthesis using an automatic synthesizer, in which an amino acid having a protecting group is supplied into a reaction system and the peptide is subjected to a condensation reaction with the terminal amino group of the peptide. After the elongation step, the peptide elongation step, a reaction confirmation step of supplying a test reagent capable of reversibly reacting with the terminal amino group of the peptide to cause coloration into the reaction system, and after the coloring reagent supply step. , including repeating the deprotection step of supplying the deprotecting agent in a state where the reaction solution is not colored.

According to the method of the present invention, the reaction rate of the condensation reaction between an amino acid and a peptide can be quantitatively monitored in an automatic peptide synthesizer. When the above test reagent is supplied into the reaction system, if the condensation reaction has finished, the terminal amino group of the peptide is bound to a protecting group, so the peptide is colored because there is no terminal amino group. do not. On the other hand, if the condensation reaction is not completed, the peptide is colored by the test reagent because an amino group is present at the end of the peptide. By creating a calibration curve in advance between the reaction rate of the test reagent and the supplied amino acid (reaction rate by Kaiser test) and color (light reflectance or transmittance), it is possible to By examining the color tone, the reaction rate can be observed quantitatively.

Moreover, this test reagent can be easily washed out by supplying a solvent to the reaction system, and this step can be easily incorporated into commercially available automatic synthesis equipment.

Furthermore, in conventional automatic synthesis equipment, the condensation reaction was completed by adding reaction reagents such as amino acids 5 to 10 times the stoichiometric amount of the condensation reaction and heating them with microwaves for several minutes. However, according to the method of the present invention, large amounts of reaction reagents such as amino acids are not used, and the amount of reaction reagents is initially set to about 1.0 to 3.0 times the stoichiometric amount. It is now possible to monitor the reaction rate and, if the reaction is not completed, add more reaction reagents to carry out the peptide extension step.

As a result, the method of the present invention enables a significant cost reduction compared to the conventional method of discarding a large amount of unreacted reaction reagents. Specifically, in the case of a peptide with 10 amino acid residues, using the method of the present invention can significantly reduce the cost of synthesis by approximately 80% compared to conventional methods. I found out that it will happen. Conventionally, peptide synthesis has been extremely expensive, which has been a major problem, but the method of the present invention has been found to be an extremely important breakthrough.

As used herein, a peptide refers to a small protein consisting of three or more amino acids in which the carboxy group of one amino acid is bonded to the amino group of the other amino acid. Peptide synthesis involves the process of repeatedly adding amino acid molecules to a reaction system. For example, peptide drugs are usually manufactured by repeatedly reacting amino acid molecules 5 to 30 times, especially 10 to 20 times. It is. In this specification, an automatic synthesis device refers to a device that automatically controls the addition of amino acids, and an automatic synthesis device is capable of automatically adding solvents and the like and automatically cleaning the inside of the reaction system. is preferred.

As used herein, the solid phase synthesis method refers to a peptide synthesis method well known in the art, in which an amino acid is added to the terminal amino group of a peptide that is fixed to a solid support via a linker molecule. A synthetic method that involves a reaction.

FIG. 1 shows a schematic flowchart of the method of the invention.

<start>
At the beginning of peptide synthesis, the starting amino acids can be attached to a solid support, optionally via a linker molecule. This protects the so-called C-terminus of the peptide. This solid support-(linker molecule-) starting amino acid combination is commercially available and may be used. At the beginning of the process, the peptide extension step can react with the amino groups of the solid support (linker molecule) starting amino acid rather than the terminal amino groups of the peptide. Additionally, preparatory steps well known in the art, such as swelling the solid support with a solvent, can be included before proceeding to the peptide extension step.

As the solid carrier, well-known solid carriers used in solid-phase synthesis of peptides can be used, such as polystyrene resins, polyacrylamide resins, polyethylene glycol resins, glass, polysaccharides (e.g., cellulose, agarose), etc. can be mentioned. The solid support can be porous, for example in the form of beads, particles, fibers or any other suitable form.

As the linker molecule, well-known linker molecules used in solid phase synthesis of peptides can be used. For example, the linker molecule is designed such that the peptide eventually detaches from the solid support, thereby providing a free acid or free amide at the C-terminus. The linker molecule can be a peptide acid such as 4-hydroxymethylphenoxyacetyl-4'-methylbendihydrylamine, or a peptide amide such as a benzhydrylamine derivative, 2-chlorotolyl chloride, 4-(α-[2,4 -dimethoxyphenyl]Fmoc-aminomethyl)phenoxy, and the like.

As for the starting amino acid, the same amino acids as those used in the peptide extension step described below can be used.

<Peptide extension step>
The method of the present invention includes a peptide elongation step in which an amino acid having a protecting group is supplied into the reaction system and reacted with the terminal amino group of the peptide. A peptide bond can be formed by a condensation reaction between the carboxy group and amino group of an amino acid.

As the amino acid, well-known amino acids used in peptide synthesis can be used, and even if the amino terminal has a protecting group (main chain protecting group) and the side chain also has a side chain protecting group. good.

The term "protecting group" refers to an atomic group used for the purpose of temporarily protecting a highly reactive functional group such as an amino group, a carboxy group, or a hydroxy group. Examples of the protecting group include fluorenylmethoxycarbonyl (Fmoc) group, acetyl group, benzyl group, benzoyl group, t-butoxycarbonyl (Boc) group, t-butyl group, t-butyldimethyl group, silyl group, trimethylsilyl group. Examples include ethyl group, N-phthalimidyl group, trimethylsilylethyloxycarbonyl group, and carbamate group. Those skilled in the art can use various protecting groups depending on the reaction conditions and purpose.

Examples of amino acid residues to which the protecting group is attached include glycine, alanine, serine, threonine, valine, leucine, tyrosine, isoleucine, phenylalanine, histidine, cysteine, methionine, lysine, arginine, proline, tyrosine, asparagine, aspartic acid, Examples include glutamine, glutamic acid, tryptophan, etc., but the type thereof is not limited. Furthermore, a sugar amino acid in which an amino acid residue and a sugar residue are bonded through a glycosidic bond, an amide bond, or a carbon-carbon bond can also be used. In that case, the sugar residue may be N-acetylglucosamine, N-acetylglucosamine, - Acetylgalactosamine, xylose, mannose, galactose, glucose, fucose, sialic acid, iduronic acid, glucuronic acid, oligosaccharides containing constituent sugar units of these, and the like.

In this step, a coupling agent can further be used to react the amino acids. Such coupling agents are well known in the art and include, for example, phosphonium-based condensing agents, uronium-based condensing agents, in-situ activators (e.g., BOP, HATU, HOBt, HBTU, PyBOP, PyAOP, etc.), and the like. can be mentioned. It is known that these coupling agents are used in combination; for example, in the case of the Fmoc method, an organic base such as diisopropylethylamine can be further used in combination.

Furthermore, in order to react the amino acids, the inside of the reaction system can be heated, and in particular, the reaction time can be shortened by performing microwave heating.

In the method of the present invention, there is no need to supply a large excess of amino acids in the peptide elongation step as in the conventional method. For example, the amount of amino acid supplied is 5.0 times or less, 4.0 times or less, 3.0 times or less, 2.5 times or less, 2.0 times or less of the stoichiometric amount for reaction with the amino group of the peptide. , 1.5 times or less, or 1.2 times or less. Further, the coupling agent can also be used in a similar amount.

<First cleaning step>
After the peptide extension step, a first washing step for washing out the inside of the reaction system can be performed. For washing out, well-known solvents used in peptide synthesis can be used, such as N,N-dimethylformamide (DMF), methylene chloride, N-methylpyrrolidone, dimethylacetamide, and the like. Among these, DMF is particularly preferable as the solvent used in the method of the present invention, taking into account the coloring properties of the test reagent.

Note that the other cleaning steps described in this specification can also be performed in the same manner as the first cleaning step. This allows unreacted amino acids, coupling agents, etc. to be washed out. By performing the first washing step, it is possible to reliably confirm the color tone in the next reaction confirmation step.

<Reaction confirmation process>
The method of the present invention includes a reaction confirmation step of supplying a test reagent into the reaction system after the above steps. The test reagent used here is a reagent that can be colored by reversibly reacting with the terminal amino group of the peptide, and for example, a reagent as described in Non-Patent Document 1 can be used.

When this reagent is added, if the peptide has a terminal amino group, it will turn brown, for example. If not, do not color. In addition, this coloring becomes dark or light depending on the amount of the terminal amino group of the peptide remaining, so it is important to note that the reaction rate between the test reagent and the supplied amino acid (reaction rate by Kaiser test) and the color (reflection of light) By preparing a calibration curve in advance with respect to the reaction rate or transmittance, the reaction rate can be quantitatively observed by examining the color tone within the reaction system.

Therefore, if the reaction rate is low after performing the reaction confirmation step, the yield of the target peptide can be improved by repeating the peptide elongation step using the same amino acid. In addition, in an automatic synthesis apparatus, the color tone can be determined using, for example, a light source, a CCD camera, an image processing device, etc., and a control device can be made to determine whether to repeat the peptide extension step or proceed to the next step.

A test reagent that reversibly reacts with the terminal amino group of a peptide can be bound to the amino group not by a covalent bond but by, for example, an ionic bond, and may be colored by binding to the terminal amino group of the peptide. By washing out the peptide with a solvent, it can be removed from the terminal amino group of the peptide and decolorized. Examples of such test reagents include compounds having an N-hydroxy cyclic imide skeleton. Examples of such compounds include N-hydroxyphthalimide (NHPI) compounds and N-hydroxynaphthalimide compounds.

Examples of NHPI-based compounds include NHPI and compounds having substituents thereon, such as compounds having the following chemical formula and compounds containing the structure of this chemical formula as a part thereof.

Here, R1 to R4 are hydrogen; alkyl group; alicyclic group; aromatic group; heterocyclic group; hydroxy group; carboxy group; thiol group; sulfonic acid group; phosphonic acid group, etc.; nitro group; alkylamino Groups; each can be selected from halogen atoms such as fluorine, chlorine, bromine, and iodine, and these can jointly form a cyclic group containing a substituent or substituent element as described above, for example, R2 and R3 can jointly form an aromatic ring, an aliphatic ring, or a heterocycle, and may also be an N-hydroxysuccinimide group.

<Second cleaning process>
If the reaction confirmation step determines that the peptide extension step is not complete, a second washing step can be performed to wash out the test reagent. However, it has been found that this washing step does not have to be performed. In the synthesis of peptides, it is common to repeat a large number of washing steps, which generates a large amount of waste liquid and increases costs. In particular, ordinary solvents used in peptide synthesis are harmful substances that cause environmental pollution if discarded as is, and their recovery process also requires a large amount of cost. However, according to studies by the present inventors, the peptide elongation step could be performed depending on the type of amino acid even in the presence of a test reagent. The fact that there is no need to carry out a washing step is very advantageous considering that it is necessary to carry out the peptide extension step to deprotection step several dozen times in order to produce the desired peptide. .

It has been found that when the peptide extension step is performed without performing the second washing step, the colored reaction system loses its color as the reaction rate improves. Peptide synthesis usually requires thorough washing and careful reaction at each step, so it is extremely surprising that the peptide extension step was carried out without any problems even in the presence of test reagents. It was unexpected.

<Third cleaning process>
Once it is confirmed through the reaction confirmation step that the peptide extension step has been completed, a third washing step can be performed to wash out the test reagent. However, this cleaning step may not be performed. The fact that there is no need to carry out a cleaning step is, as mentioned above, very advantageous. According to the studies conducted by the present inventors, the deprotection step could be carried out without any problem without performing the third washing step, but this was also very unexpected. Note that if the third washing step is not performed, the deprotecting agent and test reagent introduced in the next step will react and the reaction system will be colored, but if the fourth washing step is performed, the reaction system will be colored. It was also found that it can also be made colorless.

<Deprotection process>
The method of the present invention includes a deprotection step of supplying a deprotecting agent into the reaction system after the above steps. Thereby, the protecting group of the peptide containing an amino acid-derived protecting group at the terminal is removed in the peptide elongation step, thereby preparing for the next peptide elongation step.

As the deprotecting agent, a well-known deprotecting agent used in peptide synthesis can be used, and the type of deprotecting agent is selected depending on the type of protecting group. For example, when the protecting group is an Fmoc group, a weak base such as piperidine can be selected as the deprotecting agent, and when the protecting group is a Boc group, trifluoroacetic acid (TFA) can be selected as the deprotecting agent. A strong acid such as can be used.

<Fourth cleaning step>
After the deprotection step, a fourth washing step can be performed to wash out the deprotecting agent. When performing the deprotection confirmation step, by performing this washing step, the color tone in the next deprotection confirmation step can be reliably confirmed.

<Deprotection confirmation process>
The method of the present invention can include, after the above steps, a deprotection confirmation step of supplying a test reagent into the reaction system. Generally, in peptide synthesis, it is believed that by selecting an appropriate deprotecting agent, complete deprotection of the peptide terminus can be achieved, so the next peptide extension can be carried out without a step to confirm deprotection. Although it is possible to proceed to other steps, it is preferable to confirm whether deprotection has been performed reliably. The test reagent used here can be the same as the reagent used in the reaction confirmation step described above.

When this reagent is added, if the terminal amino group is present in the peptide (due to removal of the protective group at the terminal end of the peptide), the peptide will be colored brown, for example, but the deprotection has not yet been completed. If a protecting group is present at the end of the peptide, it will not be colored. In this process, a calibration curve between the reaction rate of the test reagent and the supplied amino acid (reaction rate by Kaiser test) and color (light reflectance or transmittance) can be created in advance to ensure that the reaction system is By examining the color tone, the degree of deprotection can be quantitatively observed.

In the deprotection confirmation step, if it is confirmed that the deprotection is insufficient, the deprotection step can be performed again. There is no need to include a washing step when returning from the deprotection confirmation step to the deprotection step.

<Fifth cleaning step>
Once the deprotection confirmation step confirms that the deprotection step is complete, a fifth washing step can be performed to wash out the test reagent. However, depending on the type of amino acid used in the next peptide extension step, this washing step may not be performed. According to the studies conducted by the present inventors, the peptide elongation step could be performed even in the presence of a test reagent. The fact that there is no need to carry out a cleaning step is, as mentioned above, very advantageous.

<end>
At the end of peptide synthesis, not only the terminal end of the peptide is deprotected, but also the side chain protecting group is removed. In addition, in the same manner, it is also removed from the solid carrier using a strong acid or the like. This step can be carried out by methods well known in the art, depending on the type of protecting group and solid support used. Recovery of peptides can also be performed by methods well known in the art.

《Automatic peptide synthesizer》
The automatic peptide synthesis apparatus of the present invention is, for example, an apparatus for carrying out the above-described peptide production method. Therefore, for each configuration used in the automatic peptide synthesis apparatus of the present invention, reference can be made to the configurations described regarding the above-mentioned peptide manufacturing method. In addition, automatic peptide synthesizers are commercially available, and with respect to non-characteristic parts of the automatic peptide synthesizer of the present invention, unless there is any particular detailed mention of each device, mechanism, means, etc. in this specification, Those skilled in the art can adopt the configuration from a well-known automatic synthesis apparatus.

One embodiment of an automated peptide synthesizer is shown in FIG. 2.

This peptide automatic synthesis apparatus 100 includes a reaction container 10 having an optically transparent window 11, a plurality of reagent supply containers 20 for supplying reagents to the reaction container 10, and observation of the color inside the reaction container through the window 11. The optical device 30 is equipped with an optical device 30 for controlling the optical device 30, and a control device 40 for controlling the optical device 30.

The reaction vessel 10 is filled with a solid carrier to which amino acids are bound. An optically transparent window 11 is present in the reaction container 10, so that the colored state inside can be observed using an optical device 30.

The window 11 of the reaction vessel 10 is preferably located at a position corresponding to the position of the reaction vessel 10 to which the test reagent is supplied. According to studies conducted by the present inventors, it has been found that it takes some time for the colored state to spread throughout the reaction system. This is because when a test reagent is supplied into the reaction container 10, the reaction system becomes colored immediately after the test reagent comes into contact with the reaction system, but depending on the internal stirring state, it takes time for the coloring to spread. This is thought to be due to this. In contrast, in this embodiment, by installing the window 11 near the position where the test reagent is supplied or by supplying the test reagent near the window 11, the internal color tone can be determined immediately after the test reagent is supplied. This is preferable because it makes it possible.

The reaction vessel 10 can also be connected to a waste liquid container 12, and during each washing process, solvents and the like are washed out from inside the reaction vessel 10 into the waste liquid container 12.

The reaction container 10 can also be connected to a gas supply pipe 13 for supplying an inert gas to the inside, and thereby the inert gas can be supplied to the inside of the reaction container 10 and the inside can be stirred.

The plurality of reagent supply containers 20 can include, for example, an amino acid supply container 20a, a coupling agent supply container 20b, a solvent supply container 20d, and a test reagent supply container 20d, and further include a deprotection agent supply container. In addition to this, for example, amino acid supply containers can also include the number of amino acids depending on the type of amino acid. Reagents can be introduced from these reagent supply containers 20 into the reaction container 10 through reagent supply pipes 21a to 21d, and although not shown, there can be as many reagent supply pipes as there are reagent supply devices. .

The reagent supply container 20 is also connected to a control device 40, so that the chemical solution can be automatically supplied to the reaction container 10 at a timing and supply amount controlled by the control device 40.

As the optical device 30, for example, a combination of a light source and a CCD camera is used. The optical device 30 is connected to the control device 40, and can determine the internal color tone at necessary timing and cause the control device to determine whether to repeat the peptide extension step or proceed to the next step. .

The optical device 30 can further include an image processing device that processes images taken by the camera. The image processing device may be common to the control device 40 or may be an independent component.

The control device 40 controls each device of the automatic peptide synthesizer to execute the above-described peptide manufacturing method. The control device 40 may be an electronic control unit (ECU), which is configured as a microprocessor centered on a CPU, and in addition to the CPU, a ROM that stores processing programs and a ROM that temporarily stores data. It can be equipped with a RAM, an input/output port, and a communication port. Thereby, the control device 40 can control each device.

This automatic peptide synthesis apparatus 100 has a reaction promotion system 50 for adjusting the internal temperature of the reaction vessel 10. The reaction acceleration system 50 may merely have a temperature regulating function, but preferably can be a microwave heating device as is well known in the art. Further, this reaction promotion system 50 is preferably used in combination with a cooling water circulation device 51 for circulating cooling water through the jacket 52 around the reaction vessel 10, and a temperature measuring device 53 using an optical fiber or the like. The reaction can be controlled by these and the control device 40.

The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited thereto.

Experiment 1: Confirmation of yield by the method of the present invention A peptide having the following chemical formula was synthesized by solid phase synthesis using the Fmoc method.

Here, in the method of Example 1, at the end of the reaction of each amino acid, NHPI is added to the reaction system and after confirming that the reaction system is colorless, the deprotection step, the washing step, and the next peptide extension step are carried out. By repeating this process, the above peptide was synthesized. The obtained yield was 16 mg, and the yield was 25%. In this example, the synthesis was carried out manually, but it was suggested that similar yields and yields could be obtained in a similar manner on an automated synthesizer. Moreover, here, the synthesis was performed using 3.0 times the stoichiometric amount of reagents such as amino acids.

In the method of Comparative Example 1, the above peptide was synthesized in the same manner as in Example 1, except that at the end of the reaction of each amino acid, the completion of the reaction was confirmed by Kaiser test. The amount obtained was 13 mg, which was a yield of 20%.

In the method of Comparative Example 2, the above peptide was synthesized using a commercially available automatic synthesizer (EYELA GPS-1000CP, Tokyo Rikakikai Co., Ltd.). Here, although the amount of starting amino acids was the same as in Example 1, synthesis was performed using 10 times the stoichiometric amount of reagents for each amino acid, etc. The yield obtained is at most 16 mg, and the yield is at most 25%.

Experiment 2: Reference study of coloring by each test reagent Using NHPI, test reagent 1 shown in Table 1, and test reagents 2 to 8, which have a relatively similar structure to NHPI, each peptide having a Leu residue was We investigated whether coloring should occur after the deprotection step.

In test reagents 2 to 8, there was no significant difference in coloring between the state in which the amino acid was reacted and the peptide had a protector at its N-terminus, and the state after deprotection, but in test reagent 1 (NHPI), , colored bright orange only after deprotection.

Furthermore, using NHPI of test reagent 1 shown in Table 2 and test reagents 9 to 17 having a relatively similar structure to NHPI, we investigated whether each peptide having a Leu residue would be colored after the deprotection step. I did it.

For test reagents 9 to 17, a large difference in coloration was observed between the state in which a protector was present at the N-terminus of the peptide by reacting with amino acids and the state after deprotection. Test reagents 9 to 12 were colored pale orange after deprotection, and test reagents 13 to 17 were colored brown to black after deprotection.

These results suggested that electronic resonance between the aromatic ring and the carbonyl moiety of the imide is important for the imide to function as a test reagent.

10 Reaction container 11 Window 12 Waste liquid container 13 Gas supply pipe 20 Reagent supply container 21 Reagent supply pipe 30 Optical equipment 40 Control equipment 50 Reaction promotion system 51 Cooling water circulation device 52 Jacket 53 Temperature measurement device 100 Automatic synthesis device

Claims (9)

A method for producing a peptide by solid phase synthesis using an automatic synthesizer, the method comprising:
a peptide elongation step in which an amino acid having a protecting group is supplied into the reaction system and reacted with the terminal amino group of the peptide;
After the peptide extension step, a reaction confirmation step of supplying a test reagent capable of reversibly reacting with the terminal amino group of the peptide to cause coloration into the reaction system, and after the reaction confirmation step, a deprotecting agent A method for producing a peptide, comprising repeating a deprotection step of supplying peptide into the reaction system. The method for producing a peptide according to claim 1, wherein in the peptide elongation step, the amino acid is supplied in an amount that is 3.0 times or less the stoichiometric amount of the reaction. The method for producing a peptide according to claim 1, wherein if the reaction solution is colored after the reaction confirmation step, the peptide extension step is further repeated. After the peptide extension step and before the reaction confirmation step, a first washing step of washing out the inside of the reaction system, and After the reaction confirmation step and before the deprotection step, the The method for producing a peptide according to claim 1, which does not include a third washing step of washing out the inside of the reaction system. After the deprotection step, a fourth washing step of washing out the inside of the reaction system is included, and a test reagent that can be colored by reversibly reacting with the terminal amino group of the peptide is supplied into the reaction system. The method for producing a peptide according to claim 1, comprising a step of confirming deprotection. The method for producing the peptide according to claim 1, which is the Fmoc method. The method for producing a peptide according to claim 1, wherein the test reagent is a compound having an N-hydroxy cyclic imide skeleton. An automatic peptide synthesizer for carrying out the production method according to any one of claims 1 to 7, comprising:
a reaction vessel with an optically transparent window;
a plurality of reagent supply containers for supplying reagents to the reaction containers;
an optical device for observing the color inside the reaction container through the window, and a control device for controlling these;
An automatic peptide synthesis device comprising: 9. The apparatus according to claim 8, wherein the reaction container has the window at a position corresponding to a supply position of the test reagent.