WO2020124975A1

WO2020124975A1 - Method for preparing thymalfasin

Info

Publication number: WO2020124975A1
Application number: PCT/CN2019/092741
Authority: WO
Inventors: 姚志军; 尹传龙; 宓鹏程; 陶安进; 袁建成
Original assignee: 深圳翰宇药业股份有限公司
Priority date: 2018-12-20
Filing date: 2019-06-25
Publication date: 2020-06-25
Also published as: CN111349152A; CN111349152B

Abstract

Disclosed is a method for preparing thymalfasin, wherein acetyl-salicylaldehyde and acetyl-salicylaldehyde analogs are used for preparing thymalfasin by performing selective N-terminal acetylation of the thymalfasin 28 peptide. The method does not require the other aminos of the thymalfasin 28 peptide to be protected, thereby avoiding expensive raw materials and tedious removal operations for the special protection of lysine side chain aminos required by other acetylation methods.

Description

A new method for preparing thymus

This application requires the priority of the Chinese patent application filed on December 20, 2018 in the Chinese Patent Office with the application number 201811564487.0 and the invention titled "A New Method for the Preparation of the Thymus Method", the entire contents of which are incorporated by reference in this application in.

Technical field

The invention belongs to the technical field of medicinal chemistry, and in particular relates to a new method for preparing thymus, in particular to a new method for preparing thymus by selective acetylation.

Background technique

Thymus Fascin (Tαl) is an acidic polypeptide containing 28 amino acids, with a pI of 4.2, a relative molecular weight of 3108, and N-terminal acetylation. The structural formula is:

Thymus Fasin is a biological response regulator, mainly an immune enhancer of the T lymphatic system. Tαl can restore the function of T lymphocytes and promote the proliferation of mature T cells, promote the accumulation of lymphocytes around pathogenic tissues, and promote the production of lymphokines and lymphokine receptors. It also has the effects of stimulating the migration of vascular endothelial cells, promoting angiogenesis and wound healing. In clinical practice, Tαl is often used as an immunopotentiator or immunomodulator for the treatment of various immunodeficiency diseases and immunosuppressed diseases, as well as the treatment and research of hepatitis B, hepatitis C, and malignant tumors.

At present, the new synthesis method of thymus method mainly uses Fmoc solid phase synthesis. Since thymus is a difficult peptide, β-sheets are formed during the synthesis process, which leads to difficult synthesis and low purity and yield. Although the method of using a skeleton protecting group can greatly improve the production of β-sheets, it cannot be acetylated on the solid phase, and only the 28 peptide can be synthesized by the method of the skeleton protecting group, and then the N-terminal acetylation is performed in the liquid phase. Since the thymus method contains 4 lysines, the lysine side chain amino group will also be acetylated during acetylation, which in turn involves orthogonal protection and deprotection steps of the lysine side chain amino group. Greatly increase material cost and operation difficulty.

Due to the high cost of chemical synthesis and low yield, and the environment is not friendly. There are a lot of literatures that try to use gene engineering recombination to prepare thymus method. Among them, E. coli, as a common strain in genetic engineering, has the characteristics of clear genetic background, complete carrier receptor system, rapid growth, simple culture, and stable recombinants. Therefore, many researchers regard E. coli as the preferred strain. However, although Tαl expressed by E. coli has the same amino acid composition as Tαl, the N-terminal lacks acetylation modification, which severely limits the new application of genetic engineering recombinant preparation of thymus. So far, there is no new application of drugs prepared by genetic engineering thymus method.

Summary of the invention

In view of this, the object of the present invention is to provide a new method for preparing the thymus method for the problems in the prior art, which can selectively select the 28-peptide thymus method synthesized by chemical methods and the gene recombinant preparation 28 peptide thymus N-terminal acetylation.

To achieve the purpose of the present invention, the present invention adopts the following technical solutions:

A new method for preparing thymus includes:

Step 1: Coupling reaction of acetylsalicylic aldehyde or acetylsalicylic aldehyde analogue with thymus fascin 28 peptide in pyridine/acetic acid buffer, and then removing the reaction solvent;

Step 2: Mix the product obtained in Step 1 with the TFA solution to obtain a TFA solution of crude thymus;

Step 3: Add the TFA solution of the crude thymus method to the poor solvent to settle, centrifuge to separate the precipitated solid, wash with the poor solvent, purge and dry with nitrogen to prepare the crude thymus method.

Preferably, the acetyl salicylaldehyde analog has the structure shown in formula I,

Wherein, R1 is independently selected from -H, -C _n H _2n+1 , -OC _n H _2n+1 , n= ₁ , 2, 3 or 4;

R2 is independently selected from -H, -NO ₂ , -S(=O)CH ₃ ;

R3 is independently selected from -H, -C _n H _2n+1 , -OC _n H _2n+1 , n= ₁ , 2, 3 or 4;

R4 is independently selected from -H, -NO ₂ , -S(=O)CH ₃ .

Preferably, the pyridine/acetic acid buffer in step 1 is a mixed solution of pyridine and acetic acid, and the molar ratio of pyridine to acetic acid is 1: (1-10).

Preferably, the reaction concentration of thymus fascin 28 peptide in step 1 is 5-50 mM.

Preferably, the molar ratio of the thymus fascin 28 peptide to acetylsalicylic aldehyde or acetylsalicylic aldehyde analog in step 1 is 1: (1-10); the coupling reaction time in step 1 is 2-5h.

Preferably, the TFA solution in step 2 is an aqueous solution containing 50-100% TFA.

Preferably, the amount of the TFA solution is 5-15 ml of TFA solution per gram of thymus fasin 28 peptide.

Preferably, in the novel method for preparing thymus of the present invention, the poor solvent in step 3 is one or two of ether, isopropyl ether, methyl tert-butyl ether, n-heptane, n-hexane, and petroleum ether More than one mixed solvent.

Preferably, the amount of the poor solvent used in step 3 is 5 to 15 times that of the TFA solution used in step 2.

Further, as a preference, the method for preparing a thymus method according to the present invention further includes the step of preparing thymus method sperm peptide after purification by reverse-phase high-performance liquid chromatography and drying.

It can be seen from the above technical solution that the present invention provides a novel method for preparing thymus method. The thymus method 28 peptide is subjected to selective N-terminal acetylation to prepare thymus method method using acetylsalicylic aldehyde or acetylsalicylic acid analogue. The present invention does not need to protect other amino groups of the thymosin 28 peptide, avoiding the expensive raw materials and cumbersome removal operations required for the special protection of the lysine side chain amino groups required by other acetylation methods, which greatly simplifies The difficulty of acetylation of the N-terminal of 28 peptides of thymus method improves the yield of thymus method and reduces the cost. It can prepare the thymus method for fermentation method and solve the last obstacle. It is widely suitable for 28 peptide thymus synthesized by chemical method. Fascin and 28-peptide thymus Fascin prepared by gene recombination prepared acetylated thymus Fasin.

BRIEF DESCRIPTION

In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required in the embodiments or the description of the prior art.

Figure 1 shows the mass spectrogram of the new crude thymus prepared in Example 2;

2 shows a mass spectrogram of the new crude thymus prepared in Example 4;

FIG. 3 shows a mass spectrogram of the new crude thymus prepared in Example 6;

4 shows a mass spectrogram of the new crude thymus prepared in Example 7;

FIG. 5 shows a mass spectrogram of the crude thymus prepared in Example 8.

detailed description

The invention discloses a new method for preparing thymus. Those skilled in the art can learn from this article and appropriately improve the process parameters to achieve. In particular, it should be noted that all similar substitutions and modifications will be obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and products of the present invention have been described through preferred embodiments, and it is obvious that relevant personnel can modify or appropriately modify and combine the methods described herein without departing from the content, spirit, and scope of the present invention to implement and apply this method. Invention technology.

A new method for preparing thymus includes:

Step 3: Add the TFA solution of crude thymus method to the poor solvent to settle, centrifuge to separate the precipitated solid, wash with the poor solvent, purge and dry with nitrogen to prepare the crude thymus method.

The method of the present invention adopts acetylsalicylic aldehyde or acetylsalicylic aldehyde analogue to prepare thymus fascin by selective N-terminal acetylation of thymus fascin 28 peptide.

R2 is independently selected from -H, -NO ₂ , -S(=O)CH ₃ ;

R4 is independently selected from -H, -NO ₂ , -S(=O)CH ₃ .

In some embodiments, the acetylsalicylic aldehyde can be prepared by acetylation of salicylaldehyde and acetic anhydride. The specific method can refer to the relevant manual or literature.

In some embodiments, the acetyl salicylaldehyde analog may be prepared by acetylation of a benzene ring substituted derivative of salicylaldehyde and acetic anhydride. The benzene ring substituted derivative of salicylaldehyde is a phenol having the structure shown in Formula II:

R2 is independently selected from -H, -NO ₂ , -S(=O)CH ₃ ;

R4 is independently selected from -H, -NO ₂ , -S(=O)CH ₃ .

In some embodiments, the acetyl salicylaldehyde analog is 4-methoxyacetyl salicylaldehyde, 5-nitroacetyl salicylaldehyde, 4-methoxy 5-nitroacetyl salicylaldehyde, or 6- Methoxyacetyl salicylaldehyde.

Preferably, the pyridine/acetic acid buffer solution in the novel method for preparing a thymus in Step 1 of the present invention is a mixed solution of pyridine and acetic acid, and the molar ratio of pyridine to acetic acid is 1: (1-10). In some specific embodiments, the molar ratio of pyridine to acetic acid is 1:1. In some specific embodiments, the molar ratio of pyridine to acetic acid is 1:5. In some specific embodiments, the molar ratio of pyridine to acetic acid is 1:2. In some specific embodiments, the molar ratio of pyridine to acetic acid is 1:3. In some specific embodiments, the molar ratio of pyridine to acetic acid is 1:4.

Preferably, in the method for preparing a thymus method according to the present invention, the reaction concentration of the thymus method 28 peptide in step 1 is 5-50 mM. In some specific embodiments, the reaction concentration of the thymus fasin 28 peptide is 20 mM; in some specific embodiments, the reaction concentration of the thymus fasin 28 peptide is 30 mM; in some specific embodiments, the thymus The reaction concentration of the fascin 28 peptide is 29 mM; in some embodiments, the reaction concentration of the thymus fascin 28 peptide is 26 mM.

Preferably, in the method for preparing a thymus method according to the present invention, the molar ratio of the thymus method 28 peptide to acetylsalicylic aldehyde or acetylsalicylic acid analogue in step 1 is 1: (1-10).

Preferably, in the novel method for preparing thymus of the present invention, the coupling reaction time in step 1 is 2-5 hours.

Preferably, in the method for preparing a thymus in the present invention, the method for removing the solvent in step 1 is distillation under reduced pressure or lyophilization.

Preferably, in the novel method for preparing thymus of the present invention, the TFA solution in step 2 is an aqueous solution containing 50-100% TFA. In some specific embodiments, the TFA solution is an aqueous solution containing TFA and TIS, wherein the volume ratio of TFA:TIS:H ₂ O is 90:5:5.

Preferably, in the method for preparing a thymus method according to the present invention, the amount of the TFA solution is 5-15 ml of TFA solution per gram of thymus method 28 peptide. In some specific embodiments, the amount of the TFA solution is 10 ml of TFA solution per gram of thymus fasin 28 peptide. In some specific embodiments, the amount of the TFA solution is 8 ml of TFA solution added per 1 g of thymus fasin 28 peptide. In some specific embodiments, the amount of the TFA solution is 8.7 ml of TFA solution per 1 g of thymus fasin 28 peptide. In some specific embodiments, the amount of the TFA solution is 6.7 ml of TFA solution per 1 g of thymus fasin 28 peptide.

Step 3 of the new method for preparing the thymus method according to the present invention uses a poor solvent to settle the TFA solution of the new crude thymus method.

Preferably, in the novel method for preparing thymus of the present invention, the poor solvent in step 3 is one or two of diethyl ether, isopropyl ether, methyl tert-butyl ether, n-heptane, n-hexane, and petroleum ether More than one mixed solvent. In some specific embodiments, the poor solvent is diethyl ether; in some specific embodiments, the poor solvent is a mixed solvent of methyl tert-butyl ether and n-hexane in a volume ratio of 1:1; in some specific In the examples, the poor solvent is isopropyl ether.

Preferably, in the novel method for preparing a thymus of the present invention, the amount of the poor solvent used in step 3 is 5 to 15 times that of the TFA solution used in step 2. In some specific embodiments, the amount of poor solvent is 6.7 times the TFA solution described in Step 2; in some specific embodiments, the amount of poor solvent is 10 times the TFA solution described in Step 2. In some embodiments, the amount of poor solvent is 12 times the TFA solution described in step 2. In some specific embodiments, the amount of poor solvent is 8 times the TFA solution described in step 2.

In some embodiments, the reversed-phase high-performance liquid chromatography purification specifically involves twice purification and salt conversion through a C18 column. In some specific embodiments, the first purification condition is that the mobile phase A phase is 0.1% TFA, the B phase is acetonitrile, the detection wavelength is 220 nm, and the flow rate is 80 ml/min; the second purification condition is the mobile phase A phase It is 0.3% acetic acid, phase B is acetonitrile, the detection wavelength is 220 nm, and the flow rate is 80 ml/min; the salt conversion condition is that the mobile phase A phase is 20 mM ammonium acetate/water solution, and phase B is acetonitrile, the detection wavelength is 220 nm, and the flow rate is 80 ml/min.

It can be known from the above technical solution that the present invention provides a novel method for preparing thymus gland by using acetylsalicylic aldehyde or acetyl salicylaldehyde analogue to selectively Th-fascin 28 peptide by N-terminal acetylation. The present invention does not need to protect other amino groups of the thymosin 28 peptide, avoiding the expensive raw materials and cumbersome removal operations required for the special protection of the lysine side chain amino groups required by other acetylation methods, which greatly simplifies The difficulty of acetylation of the N-terminal of 28 peptides of thymus method improves the yield of thymus method and reduces the cost. It can prepare the thymus method for fermentation method and solve the last obstacle. It is widely suitable for 28 peptide thymus synthesized by chemical method. Fascin and 28-peptide thymus Fascin prepared by gene recombination prepared acetylated thymus Fasin.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be described clearly and completely in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all the embodiments . Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels. The TFA involved in the examples is trifluoroacetic acid, and TIS is triisopropylsilane.

Example 1. Preparation of acetylsalicylic aldehyde

To a 100ml round bottom flask was added 4.884g/40mmol of salicylaldehyde, 30ml of dichloromethane was added, and dissolved with magnetic stirring. Weigh 6.126g/60mmol acetic anhydride and 4.746g/60mmol pyridine, and after stirring at room temperature for 3 hours, the raw material disappeared after TLC detection. The reaction system was added to 70 ml of 1N hydrochloric acid, and extracted and separated. The organic phase was extracted twice with 1N hydrochloric acid, washed once with water, washed twice with saturated sodium bicarbonate solution and then washed three times with water. After the obtained organic phase was dried over anhydrous sodium sulfate, the solvent was removed in vacuo and dried in vacuo to obtain 6.354 g of white waxy solid with a yield of 96.8%.

Example 2. N-terminal acetylation of acetylsalicylic aldehyde to prepare new crude product of thymus method

Weigh 1.5g/0.49mmol of thymus fasin 28 peptide (prepared by solid-phase coupling), and add 24.5ml of pyridine/acetic acid solution (mole: mole=1:1) (the reaction concentration of thymus fasin 28 peptide is 0.49mmol/ 0.0245L=20mmol/L), add 0.33g/2mmol acetylsalicylic aldehyde after dissolving with magnetic stirring. After stirring and reacting for 2h, the reaction solution was spinned off under reduced pressure, and the remaining viscous solid was added with 15ml TFA solution (TFA: TIS: H ₂ O=90:5:5), after magnetic stirring for 5 minutes, about half of TFA was removed under reduced pressure. Pour the residual liquid into 100 ml of ether, centrifuge to separate out the solid, and then wash the obtained solid with an appropriate amount of ether. After purging the resulting solid with nitrogen to dryness, it was vacuum dried to obtain 1.46 g of crude thymus, with a purity of 90.23%, and mass detection [M+H ^]+ : 3108.598.

Example 3. Preparation of 4-methoxyacetylsalicylic aldehyde

Add 6.1g/40mmol 4-methoxysalicylic aldehyde to a 100ml round bottom flask, add 30ml of dichloromethane, and dissolve with magnetic stirring. Weigh 6.1g/60mmol of acetic anhydride and 4.7g/60mmol of pyridine, and after stirring at room temperature for 3 hours, TLC detected that the raw material disappeared. The reaction system was added to 70 ml of 1N hydrochloric acid, and extracted and separated. The organic phase was extracted twice with 1N hydrochloric acid, washed once with water, washed twice with saturated sodium bicarbonate solution and then washed three times with water. After the obtained organic phase was dried over anhydrous sodium sulfate, the solvent was removed in vacuo and dried in vacuo to obtain 7.54 g of white solid with a yield of 97.1%.

Example 4, N-terminal acetylation to prepare crude thymus

Weigh 1.5g/0.49mmol of thymus fasin 28 peptide (prepared by fermentation method), add 16.5ml pyridine/acetic acid solution (mole:mole=1:5) (the reaction concentration of thymus fasin 28 peptide is 0.49mmol/0.0165L = 30mmol/L), add 0.78g/4mmol 4-methoxyacetylsalicylic aldehyde after dissolving with magnetic stirring, and after stirring and reacting for 5h, remove the solvent by lyophilization, and add 12ml of TFA solution to the resulting solid (TFA: TIS: H ₂ O=90:5:5), after magnetic stirring for 5 minutes, the reaction solution was poured into 120 ml of diethyl ether, the precipitated solid was centrifuged, and the solid was washed with an appropriate amount of diethyl ether. After purging the resulting solid with nitrogen to dryness, it was vacuum dried to obtain 1.42 g of crude thymus, with a purity of 91.01%, and mass spectrometry detection [M+H] ⁺ :3108.574.

Example 5. Preparation of 5-nitroacetylsalicylic aldehyde

Add 6.7g/40mmol 5-nitrosalicylic aldehyde to a 100ml round bottom flask, add 30ml of dichloromethane, and dissolve with magnetic stirring. Weigh 6.1g/60mmol of acetic anhydride and 4.7g/60mmol of pyridine, and stir at room temperature overnight after TLC detected the raw material disappeared. The reaction system was added to 70 ml of 1N hydrochloric acid, extracted and separated. The organic phase was extracted twice with 1N hydrochloric acid, washed once with water, washed twice with saturated sodium bicarbonate solution and then washed three times with water. After the obtained organic phase was dried over anhydrous sodium sulfate, the solvent was removed in vacuo and dried in vacuo to obtain 7.24 g of a yellow solid with a yield of 86.6%.

Example 6, N-terminal acetylation to prepare new crude thymus

Weigh 1.5g/0.49mmol of thymus fasin 28 peptide (prepared by fermentation method), add 16.8ml of pyridine/acetic acid solution (mole:mole=1:2) (the reaction concentration of thymus fasin 28 peptide is 0.49mmol/0.0168L =29mmol/L), add 0.15g/0.72mmol 5-nitroacetylsalicylic aldehyde after dissolving under magnetic stirring, and after stirring and reacting for 2h, remove the solvent by lyophilization, and add 13ml of TFA solution to the resulting solid (TFA: TIS: H ₂ O=90:5:5), after magnetic stirring for 5 minutes, the reaction solution was poured into 120 ml of methyl tert-butyl ether: n-hexane = 1:1 (V:V), the precipitated solid was separated by centrifugation, and then appropriate amount The solid was washed with methyl tert-butyl ether. After purging the resulting solid with nitrogen to dryness, it was vacuum dried to obtain 1.38 g crude thymus, with a purity of 89.93%, and mass spectrometry detection [M+H] ⁺ :3108.283.

Example 7. N-terminal acetylation of 4-methoxy 5-nitroacetylsalicylic aldehyde to prepare new crude thymus

Weigh 1.5g/0.49mmol of thymus-fasin 28 peptide (prepared by fermentation method), add 16.8ml of pyridine/acetic acid solution (mole:mole=1:3) (the reaction concentration of thymus-fasin 28 peptide is 0.49mmol/0.0168L = 29mmol/L), add 0.21g/0.88mmol 4-methoxy 5-nitroacetylsalicylic aldehyde after dissolving under magnetic stirring, and after stirring and reacting for 2h, remove the solvent by lyophilization, and add 10ml TFA solution to the resulting solid ( TFA: TIS: H ₂ O=90:5:5), after stirring magnetically for 5 minutes, the reaction solution was poured into 120 ml of isopropyl ether, the solid precipitated out was centrifuged, and the solid was washed with an appropriate amount of isopropyl ether. After purging the resulting solid with nitrogen to dryness, it was vacuum dried to obtain 1.35 g crude thymus, with a purity of 91.21%, and mass spectrometry detection [M+H] ⁺ :3108.390.

Example 8. N-terminal acetylation of 6-methoxyacetylsalicylic aldehyde to prepare new crude thymus

Weigh 1.5g/0.49mmol of thymus fasin 28 peptide (prepared by fermentation method), add 18.8ml pyridine/acetic acid solution (mole:mole=1:4) (the reaction concentration of thymus fasin 28 peptide is 0.49mmol/0.0188L = 26mmol/L), add 0.33g/1.7mmol 6-methoxyacetylsalicylic aldehyde after dissolving with magnetic stirring, and after stirring and react for 2h, remove the solvent by lyophilization, and add 10ml TFA solution to the resulting solid (TFA: TIS: H ₂ O=90:5:5), after magnetic stirring for 5 minutes, the reaction solution was poured into 80 ml of isopropyl ether, the solid precipitated out was centrifuged, and the solid was washed with an appropriate amount of isopropyl ether. After purging the resulting solid with nitrogen to dryness, it was vacuum dried to obtain 1.40 g crude thymus, with a purity of 91.32%, and mass spectrometry detection [M+H] ⁺ :3108.259.

Example 9. Purification and preparation of thymus

The crude thymus method 2.4g was purified twice by the C18 column, converted to salt, and freeze-dried to obtain the target product. The first step purification conditions: mobile phase A phase is 0.1% TFA, phase B is acetonitrile, detection wavelength is 220nm, flow rate is 80ml/min. The second step purification conditions: mobile phase A is 0.3% acetic acid, phase B is acetonitrile, the detection wavelength is 220nm, the flow rate is 80ml/min. Salt conversion conditions: mobile phase A phase is 20mM ammonium acetate/water solution, phase B is acetonitrile, detection wavelength is 220nm, flow rate is 80ml/min. After freeze-drying, 2.03 g of thymosin was obtained, and the purity of HPLC was 99.79%.

Claims

A new method for preparing thymus includes:

Step 1: Coupling reaction of acetylsalicylic aldehyde or acetylsalicylic aldehyde analogue with thymus fascin 28 peptide in pyridine/acetic acid buffer, and then removing the reaction solvent;

Step 2: Mix the product obtained in Step 1 with the TFA solution to obtain a TFA solution of crude thymus;

Step 3: Add the TFA solution of crude thymus method to the poor solvent to settle, centrifuge to separate the precipitated solid, wash with the poor solvent, purge and dry with nitrogen to prepare the crude thymus method.
The method according to claim 1, wherein the acetylsalicylic aldehyde analog has the structure shown in formula I,

Wherein, R1 is independently selected from -H, -C n H 2n+1 , -OC n H 2n+1 , n= 1 , 2, 3 or 4;

R2 is independently selected from -H, -NO 2 , -S(=O)CH 3 ;

R3 is independently selected from -H, -C n H 2n+1 , -OC n H 2n+1 , n= 1 , 2, 3 or 4;

R4 is independently selected from -H, -NO 2 , -S(=O)CH 3 .
The method according to claim 1, wherein the pyridine/acetic acid buffer in step 1 is a mixed solution of pyridine and acetic acid, and the molar ratio is 1: (1-10).
The method according to claim 1, wherein the reaction concentration of the thymus fascin 28 peptide in step 1 is 5-50 mM.
The method according to claim 1, the molar ratio of the thymus fascin 28 peptide to acetylsalicylic aldehyde or acetylsalicylic aldehyde analogue in step 1 is 1: (1-10); the reaction time is 2-5h .
According to the method of claim 1, the TFA solution in step 2 is an aqueous solution containing 50-100% TFA.
The method according to claim 1, wherein the amount of the TFA solution is 5-15 ml of TFA solution per 1 g of thymus fasin 28 peptide.
The method according to claim 1, wherein the poor solvent in step 3 is one or a mixture of two or more of ether, isopropyl ether, methyl tert-butyl ether, n-heptane, n-hexane, and petroleum ether .
According to the method of claim 1, the amount of the poor solvent in step 3 is 5 to 15 times that of the TFA solution in step 2.
The method according to any one of claims 1-9, further comprising the step of preparing thymus fasin peptide after purification by reverse-phase high-performance liquid chromatography and drying.