CN113135988A

CN113135988A - Preparation method of thymosin beta 4

Info

Publication number: CN113135988A
Application number: CN202110377089.3A
Authority: CN
Inventors: 姬胜利; 殷金岗; 蒋康健; 邢昊
Original assignee: Reali Tide Biological Technology Weihai Co ltd
Current assignee: Reali Tide Biological Technology Weihai Co ltd
Priority date: 2021-04-08
Filing date: 2021-04-08
Publication date: 2021-07-20
Anticipated expiration: 2041-04-08
Also published as: CN113135988B

Abstract

The invention provides a preparation method of thymosin beta 4, which comprises the following steps: the method is applied to the Fomc/tBu method, and adopts a mode of combining a solid phase and a liquid phase to synthesize the thymosin beta 4 on resin by linear condensation, wherein Lys in an amino acid sequence of the thymosin beta 4³⁰、Asp³¹And Lys³³The condensation is carried out in the form of dipeptide or tripeptide fragments to introduce the sequence, the resin being an HMP-amino resin. The invention sequentially condenses corresponding protective amino acids or peptide fragments according to the amino acid sequence of the thymosin beta 4 to obtain the fully-protected thymosin beta 4 peptide resin, and the thymosin beta 4 pure product is obtained through cracking, purification and freeze-drying. The inventionSolves the problems of more synthesis byproducts, high purification difficulty and low yield in the prior art, and can be widely applied to the technical field of polypeptide drug preparation.

Description

Preparation method of thymosin beta 4

Technical Field

The invention relates to the technical field of medicine preparation, in particular to a preparation method of thymosin beta 4.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Thymosin beta 4 is originally an actin-sequestering protein isolated and extracted from bovine thymus and has high sequence conservation. Thymosin β 4 has important physiological functions in the body, such as: can promote angiogenesis and human vascular endothelial cell migration, and has effects of promoting wound healing, repairing cornea, inhibiting inflammatory reaction, preventing alopecia, regulating tumor and protecting heart.

The thymosin beta 4 is polypeptide with 43 amino acid residues constituting N-terminal Ser and acetylated amino group and molecular formula of C₂₁₂H₃₅₀N₅₆O₇₈S, molecular weight is 4963.5, and the amino acid sequence is: Ac-Ser⁴³-Asp⁴²-Lys⁴¹-Pro⁴⁰-Asp³⁹-Met³⁸-Ala³⁷-Glu³⁶-Ile³⁵-Glu³⁴-Lys³³-Phe³²-Asp³¹-Lys³⁰-Ser²⁹-Lys²⁸-Leu²⁷-Lys²⁶-Lys²⁵-Thr²⁴-Glu²³-Thr²²-Gln²¹-Glu²⁰-Lys¹⁹-Asn¹⁸-Pro¹⁷-Leu¹⁶-Pro¹⁵-Ser¹⁴-Lys¹³-Glu¹²-Thr¹¹-Ile¹⁰-Glu⁹-Gln⁸-Glu⁷-Lys⁶-Gln⁵-Ala⁴-Gly³-Glu²-Ser¹-OH。

The method for extracting the thymosin beta 4 from the tissues in the animal has the defects of low content of the thymosin beta 4 in the organism and complex extraction process; the prokaryotic gene recombination expression method has low yield and complex strain construction, the expression product needs to be further subjected to N-terminal acetylation modification, and thymosin beta 4 contains more Lys, which easily causes Lys side chain epsilon-NH₂The non-specific modification defect is not suitable for large-scale industrial production. Patent CN101119741 discloses a methionine-containing beta thymosin modified by oxidation or peroxidation using patent US5512656. The method disclosed in the patent US5512656 adopts a Boc synthesis strategy to synthesize peptide resin, DIC is directly used as a condensing agent without HOBT, so that amino acid racemization is easily caused to generate epimeric impurities, the epimeric impurities are similar to the physicochemical properties of main components, the separation and purification difficulty is increased, and the purity and yield of the product are reduced; and after condensation of each amino acid, 50% TFA is used for removing Boc protecting groups, 10% triethylamine is used for neutralization, a large amount of TFA and triethylamine are consumed, and finally, the peptide resin is cracked by using dangerous HF. In the patent CN101484467A, 2-chlorotrityl resin is used as a solid phase synthesis carrier to synthesize thymic beta 4, however, the 2-chlorotrityl resin has poor stability, and polypeptide chains are easy to break and fall off from the resin in the polypeptide synthesis process, so that the yield is reduced; the Lys-Ser and Glu-Thr present in the sequence are treated with a pseudoproline dipeptide: Fmoc-Lys (Boc) -Ser (Ψ Me, Me Pro) -OH, Fmoc-Glu (Otbu) -Thr (Ψ Me, Me Pro) -OH are raw materials which are not conventional amino acids in polypeptide synthesis, need special customization and are expensive; and wherein Asn and one Ile require a secondary condensation reaction; in addition, 3 Gln and 1 Asn used in the patent do not protect the side chains, so that side reactions are easy to occur in the synthesis process, and the purification difficulty of the product is reduced. Patent CN101412755 discloses a solid phase synthesis method of thymosin beta 4, which comprises dividing peptide chain into 4 fragments, firstly synthesizing 3 fragments with CTC resin, then synthesizing to obtain full-length peptide chain with Wang resin as carrier, cracking, and purifying to obtain product with total yield of 49%, but does not mention HPLC purity of the product and HPLC content of minimum single impurity. According to the synthesis method, HBTU and TBTU are used as condensing agents, compared with a conventional condensing agent HOBT for solid-phase polypeptide synthesis, the prices of HBTU and TBTU are higher, the molar molecular weights of HBTU and TBTU are respectively 2.8 times and 2.4 times of that of HOBT, and the feeding amounts of the condensing agent and protective amino acid are 5 times of molar equivalent, so that the use amounts of HBTU and TBTU are larger, and the production cost is increased; in addition, the charging amount of the segment D, the segment C and the segment B is 5 times of the molar equivalent, which is equivalent to that of each amino acid and the condensing agent in the segment D, the segment C and the segment B is 25 times of the molar equivalent, and the back of the current people paying attention to environmental protection and advocating green chemical synthesisUnder the scene, the preparation method has low atom economy, consumes a large amount of materials and generates a large amount of waste.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides the preparation method of the thymosin beta 4, which solves the technical problems of more synthesis byproducts, high purification difficulty and low yield in the prior art, greatly reduces reaction steps, is simple to operate, economical and efficient, greatly improves the synthesis efficiency, effectively reduces the generation of impurities, has low product purification difficulty and high product yield and purity, and is suitable for industrial large-scale production and preparation.

Specifically, the technical scheme of the invention is as follows:

the invention provides a preparation method of thymosin beta 4, which comprises the following steps: the method is applied to the Fomc/tBu method, and adopts a mode of combining a solid phase and a liquid phase to synthesize the thymosin beta 4 on resin by linear condensation, wherein Lys in an amino acid sequence of the thymosin beta 4³⁰、Asp³¹And Lys³³The condensation is carried out in the form of dipeptide and/or tripeptide fragments to introduce the sequence, the resin being an HMP-amino resin.

In an embodiment of the present invention, Lys is substituted³⁰、Asp³¹Condensing tripeptide fragment Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH³³The condensation was carried out as the dipeptide fragment Fmoc-Lys (Boc) -Phe-OH.

In an embodiment of the invention, the repeating dipeptide unit Ile in the thymosin beta 4 amino acid sequence¹⁰-Glu⁹And Ile³⁵-Glu³⁴Respectively carrying out condensation introduction sequences in the form of dipeptide fragments Fmoc-Ile-Glu (OtBu) -OH;

repetitive dipeptide unit Glu in thymosin beta 4 amino acid sequence¹²-Thr¹¹And Glu²³-Thr²²The condensed introduction sequences are respectively carried out in the form of dipeptide fragment Fmoc-Glu (OtBu) -Thr (tBu) -OH;

repeating dipeptide unit Gln in thymosin beta 4 amino acid sequence⁸-Glu⁷And Gln²¹-Glu²⁰Respectively condensing the dipeptide fragments in the forms of Fmoc-Gln (Trt) -Glu (OtBu) -OHIntroducing the sequence.

In an embodiment of the present invention, a method for preparing thymosin beta 4 comprises: synthesizing dipeptide fragment Fmoc-Gln (Trt) -Glu (OtBu) -OH, Fmoc-Ile-Glu (OtBu) -OH, Fmoc-Glu (OtBu) -Thr (tBu) -OH, Fmoc-Lys (Boc) -Phe-OH and tripeptide fragment Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH by liquid phase synthesis;

carrying out condensation reaction I on Fmoc-Ser (tBu) -OH and HMP-amino resin to obtain Fmoc-Ser (tBu) -HMP-amino resin, and sealing;

removing the Fmoc protecting group of the Fmoc-Ser (tBu) -HMP-amino resin, and adding the following Fmoc protected amino acids and peptide fragments into the Fmoc protected resin in sequence according to the amino acid sequence of thymosin beta 4 for condensation reaction II:

Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Gln(Trt)-Glu(OtBu)-OH、Fmoc-Ile-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-Thr(tBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Leu-OH、Fmoc-Pro-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Gln(Trt)-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-Thr(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asp(OtBu)-Lys(Boc)-Ser(tBu)-OH、Fmoc-Lys(Boc)-Phe-OH、Fmoc-Ile-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala-OH、Fmoc-Met-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Pro-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH；

sequentially adding a first Fmoc-protected amino acid Fmoc-Glu (OtBu) -OH into the Fmoc-protected-removed resin for condensation reaction II, then continuously performing Fmoc-removal treatment on the resin, sequentially adding a second Fmoc-protected Fmoc-Gly-OH into the Fmoc-protected-removed resin for condensation reaction II, sequentially repeating the Fmoc-removal treatment on the resin, and then sequentially adding subsequent Fmoc-protected amino acids or peptide fragments until the last Fmoc-Ser (tBu) -OH is condensed and introduced;

wherein the Fmoc-protected amino acids or peptide fragments involved in the condensation are all subjected to an activation treatment prior to addition to the Fmoc-protected resin.

In the embodiment of the invention, amino acids or peptide fragments protected by Fmoc are condensed and introduced in sequence according to the amino acid sequence of the thymosin beta 4, then N-terminal Ser amino group is acetylated, and then peptide resin is shrunk to obtain the thymosin beta 4 full-protection peptide resin, which has the structure that:

Ac-Ser (tBu) -Asp (OtBu) -Lys (Boc) -Pro-Asp (OtBu) -Met-Ala-Glu (OtBu) -Ile-Glu (OtBu) -Lys (Boc) -Phe-Asp (OtBu) -Lys (Boc) -Ser (tBu) -Lys (Boc) -Leu-Lys (Boc) -Thr (tBu) -Glu (OtBu) -Thr (Thr) -Gln (Trt) -Glu (OtBu) -Lys (Boc) -Asn Trt) -Pro-Leu-Pro-Ser (tBu) -Lys (Boc) -Glu (OtBu) -Thr (tBu) -Ile-Glu (OtBu) -Gln (Trt) -Glu (OtBu) -Lys (Boc), (Trt) -Gly-Glu (Tr-Ala-Gly-Ser (Boc) -Glu) (Boc) (OtBu) -Glu) (Met-Lys (Boc) -Glu) (Mett-Lys (Boc) -Glu-Lys (Boc) -Glu.

In some embodiments of the invention, the method of synthesizing Fmoc-gln (trt) -glu (otbu) -OH comprises: Fmoc-Gln (trt) -OH and HOSu in the presence of DCC to produce Fmoc-Gln (trt) -OSu; Fmoc-Gln (Trt) -OSu and H-Glu (OtBu) -OH are subjected to base catalysis to generate Fmoc-Gln (Trt) -Glu (OtBu) -OH;

the synthesis method of Fmoc-Ile-Glu (OtBu) -OH comprises the following steps: Fmoc-Ile-OH and HOSu are subjected to DCC to generate Fmoc-Ile-OSu; Fmoc-Ile-OSu and H-Glu (OtBu) -OH are catalyzed by alkali to generate Fmoc-Ile-Glu (OtBu) -OH;

the synthesis method of Fmoc-Glu (OtBu) -Thr (tBu) -OH comprises the following steps: Fmoc-Glu (OtBu) -OH and HOSu in the presence of DCC to form Fmoc-Glu (OtBu) -OSu; Fmoc-Glu (OtBu) -OSu and H-Thr (tBu) -OH are catalyzed by alkali to generate Fmoc-Glu (OtBu) -Thr (tBu) -OH;

the synthesis method of Fmoc-Lys (Boc) -Phe-OH comprises the following steps: Fmoc-Lys (Boc) -OH and HOSu in the presence of DCC to yield Fmoc-Lys (Boc) -OSu; Fmoc-Lys (Boc) -OSu and H-Phe-OH are subjected to base catalysis to generate Fmoc-Lys (Boc) -Phe-OH;

the synthesis method of Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH comprises the following steps: Fmoc-Asp (OtBu) -OH and HOSu in the presence of DCC to form Fmoc-Asp (OtBu) -OSu; Fmoc-Asp (OtBu) -OSu and H-Lys (Boc) -OH are subjected to base catalysis to generate Fmoc-Asp (OtBu) -Lys (Boc) -OH; Fmoc-Asp (OtBu) -Lys (Boc) -OH and HOSu in the presence of DCC to yield Fmoc-Asp (OtBu) -Lys (Boc) -OSu; Fmoc-Asp (OtBu) -Lys (Boc) -OSu and H-Ser (tBu) -OH are subjected to base catalysis to generate Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH;

wherein, in the above embodiment, the alkali is selected from Na₂CO₃、NaHCO₃、K₂CO₃、KHCO₃One or more of diethylamine and triethylamine.

In some embodiments of the invention, the HMP-amino resin is selected from any one of an HMP-AM resin, an HMP-MBHA resin, and an HMP-BHA resin, and the degree of resin substitution is from 0.2 to 0.8 mmol/g.

In some embodiments of the invention, the condensation reaction I comprises: dissolving Fmoc-Ser (tBu) -OH and HOBT in DMF, adding DIC to activate to obtain activated amino acid, adding the activated amino acid and DMAP into resin for condensation reaction, and then sealing the resin by using a mixed solution system of DIEA, acetic anhydride and DMF;

wherein, the molar equivalent ratio of Fmoc-Ser (tBu) -OH, HOBT, DIC, DMAP and resin is 2-5:2-5:2.2-5.5:0.1-0.3: 1.

In the condensation reaction I, the reaction condition during condensation is 15-35 ℃ for 6-24 h;

in the condensation reaction I, the sealing reaction condition is that the reaction is carried out for 6 to 24 hours at the temperature of between 15 and 35 ℃.

In the embodiment of the invention, a PIP/DMF solution system is adopted as a deprotection solution when the Fmoc protecting group of Fmoc-Ser (tBu) -HMP-amino resin is removed;

for example, in some embodiments, the PIP/DMF solution system ratio is 20% PIP/DMF (v/v).

In an embodiment of the present invention, the method for removing the Fmoc-protecting group of Fmoc-ser (tbu) -HMP-amino resin comprises: adding PIP/DMF solution into the swelled Fmoc-Ser (tBu) -HMP-MBHA resin, performing Fmoc deprotection at 15-35 ℃ for at least two times, and washing the resin by using DMF until the pH is neutral after the Fmoc deprotection.

In an embodiment of the invention, the Asp reaching the Asp for condensation synthesis of thymosin ss 4 is obtained in the course of sequential condensation³¹To Ser⁴³(i.e., introduction of Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH, Fmoc-Lys (Boc) -Phe-OH, Fmoc-Ile-Glu (OtBu) -OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Asp (OtBu) -OHOtBu) -OH, Fmoc-Pro-OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (OtBu) -OH and Fmoc-Ser (tBu) -OH, wherein 0.1-0.5mol/L HOBT is added into a PIP/DMF solution system when the Fmoc protecting group is removed each time;

in an embodiment of the invention, the activation of the Fmoc-protected amino acid or peptide fragment involved in the condensation comprises: mixing and activating the Fmoc protected amino acid or peptide fragment with a condensation system at 15-35 deg.C for 5-10 min;

in the embodiment of the invention, the condensation system is an A/D system or an A/B/C system, wherein A is HOBT or HOAT, B is one of HATU, HBTU, TBTU or PyBOP, C is DIEA or TMP, and D is DIC;

in an embodiment of the present invention, the activated Fmoc-protected amino acid or peptide fragment to be condensed is added to the Fmoc-protected resin, and condensation reaction II is performed at 15-35 ℃ for 1-5 hours.

In the embodiment of the invention, amino acids or peptide fragments protected by Fmoc are condensed and introduced according to the amino acid sequence of thymosin beta 4 in sequence, N-terminal Ser amino group is acetylated, and then peptide resin is shrunk;

in some embodiments, the act of acetylating the N-terminal Ser amino group comprises: adding a mixed solution of DIEA, acetic anhydride and DMF into a system of the condensation reaction II, and reacting for 1-5h at 15-35 ℃; the operation of peptide resin shrinkage includes washing the resin and then vacuum drying by pumping out the solvent.

In an embodiment of the present invention, the method for preparing thymosin beta 4 further comprises: cracking and condensing the obtained thymosin beta 4 full-protection peptide resin to obtain crude thymosin beta 4 peptide, and then purifying the crude thymosin beta 4 peptide;

in some embodiments of the invention, the lysing comprises: adding a cracking reagent into the thymosin beta 4 full-protection peptide resin, reacting for 2-5 hours at 15-35 ℃, performing suction filtration, adding the filtrate into precooled MTBE, precipitating, centrifuging, washing the precipitate, and performing vacuum drying to obtain crude thymosin beta 4 peptide;

preferably, the cleavage reagent is aqueous solution of TFA, TIS and EDT, and the volume ratio of TFA to TIS to EDT to H₂O＝85-95:1.5-5:2-5:1.5-5；

Preferably, the purification of the crude thymosin beta 4 peptide comprises: the crude peptide is purified by two steps by adopting a reversed-phase high-performance liquid-phase color boiling process, and gradient elution is adopted, wherein the mobile phase adopted in the first step of purification is as follows: mobile phase A phase was 0.1% TFA/H by volume fraction₂O (v/v) solution, and the mobile phase B is a volume fraction 0.1% TFA/ACN (v/v) solution; the second step of purification is salt conversion operation, and the adopted mobile phase is as follows: mobile phase A phase is 0.1% acetic acid/H by volume fraction₂O (v/v) solution, and the mobile phase B is 0.1% acetic acid/ACN (v/v) solution in volume fraction.

Compared with the prior art, the invention has the advantages that:

the invention has the beneficial effects that:

(1) the present invention is directed to amino acids Lys difficult to condense³⁰、Asp³¹And Lys³³To convert Lys into³⁰、Asp³¹Condensing tripeptide fragment Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH³³The dipeptide fragment Fmoc-Lys (Boc) -Phe-OH is condensed, so that the deletion of peptide impurities [ -Lys]-thymosin beta 4, [ -Asp ]]-thymosin beta 4 and [ -Lys]+[-Asp]]The generation of thymosin beta 4 reduces the purification difficulty and improves the product purity and yield.

(2) Aiming at the characteristics that thymosin beta 4 is a long peptide consisting of 43 amino acids, the production period is long, an intermediate product cannot be separated and purified in the solid-phase synthesis process, and impurities generated in each step can be accumulated in a final product, the invention comprehensively considers from the aspects of green chemical atom economy and production efficiency, and adopts a method combining solid-phase synthesis and liquid-phase synthesis to combine the repetitive dipeptide unit Ile in a peptide sequence¹⁰-Glu⁹And Ile³⁵-Glu³⁴Condensing the dipeptide fragments in the forms of Fmoc-Ile-Glu (OtBu) -OH and Glu^12-Thr¹¹And Glu²³-Thr²²Gln, respectively in the form of a dipeptide fragment Fmoc-Glu (OtBu) -Thr (tBu) -OH⁸-Glu⁷And Gln²¹-Glu²⁰The condensation is carried out in the form of dipeptide fragments Fmoc-Gln (Trt) -Glu (OtBu) -OH respectively, so that 9 steps of solid phase are reducedThe condensation reaction is simple to operate, economical and efficient, greatly improves the synthesis efficiency, effectively reduces the generation of impurities, has low product purification difficulty and high product yield and purity, and is suitable for industrial mass production and preparation.

(3) Asp for the present invention³¹Start to Ser⁴³0.1-0.5mol/L HOBT is added into the deprotection solution for removing the Fmoc protecting group each time, so that impurities (asparagine-linked imide) with similar polarity to thymosin beta 4 in the deprotection process are obviously inhibited]The generation of thymosin beta 4 reduces the difficulty of separation and purification and improves the product yield.

(4) The HMP-amino resin carrier adopted by the invention effectively avoids the problem of cross-linking polymerization of the long peptide in the synthesis process, and compared with CTC resin and Wang resin, the product purity and the product yield are obviously improved.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

The specific meanings of the abbreviations used in the present invention are listed in the following table:

example 1

Liquid phase synthesis of dipeptide, tripeptide and tetrapeptide fragments

(1) Preparation of dipeptide fragment Fmoc-Ile-Glu (OtBu) -OH: (a) synthesis of Fmoc-Ile-OSu: dissolving 0.2mol of Fmoc-Ile-OH and 0.24mol of HOSu in 0.4L of THF, and placing in an ice-water bath; dissolving 0.24mol of DCC in 0.2L of THF, dropwise adding into the mixed solution in the last step, continuing to react for 1h after dropwise adding is finished, and then raising the temperature to 25 ℃ to continue to react for 3 h; filtering the reaction solution, evaporating to dryness, adding DCM for dissolving, filtering and evaporating to dryness; and adding ethyl acetate to dissolve the solid, and recrystallizing to obtain Fmoc-Ile-OSu. (b) Synthesis of Fmoc-Ile-Glu (OtBu) -OH: 0.15mol of H-Glu (OtBu) -OH and 0.15mol of Na₂CO₃Dissolved in 0.2L of 50% THF/H₂O (v/v) solution to obtain mixed solution; dissolving 0.1mol of Fmoc-Ile-OSu prepared in the step (a) in THF, dropwise adding into the mixed solution in the previous step, reacting at 25 ℃ overnight, rotary evaporating the reaction liquid, adding 15% citric acid to adjust the pH to 3, extracting with ethyl acetate for 3 times, combining organic phases, washing with saturated NaCl solution for 3 times, adding anhydrous Na₂SO₄Drying overnight, evaporating the solvent to dryness, adding ethyl acetate to dissolve the solid, and recrystallizing to obtain Fmoc-Ile-Glu (OtBu) -OH.

(2) Preparation of the dipeptide fragment Fmoc-Lys (Boc) -Phe-OH: (a) synthesis of Fmoc-Lys (Boc) -OSu: 0.2mol of Fmoc-Lys (Boc) -OH and 0.24mol of HOSu were dissolved in 0.4L of THF and placed in an ice-water bath; dissolving 0.24mol of DCC in 0.2L of THF, dropwise adding into the mixed solution in the last step, continuing to react for 1h after dropwise adding is finished, and then raising the temperature to 25 ℃ to continue to react for 3 h; filtering the reaction solution, evaporating to dryness, adding DCM for dissolving, filtering and evaporating to dryness; the solid was dissolved with ethyl acetate and recrystallized to yield Fmoc-Lys (Boc) -OSu. (b) Synthesis of Fmoc-Lys (Boc) -Phe-OH: 0.15mol of H-Phe-OH and 0.15mol of Na are added₂CO₃Dissolved in 0.2L of 50% THF/H₂O (v/v) solution to obtain mixed solution; dissolving 0.1mol of Fmoc-Lys (Boc) -OSu prepared in the step (a) in THF, adding dropwise to the mixed solution in the previous step, reacting at 25 ℃ overnight, evaporating the reaction solution by rotary evaporation, addingAdjusting pH to 3 with 15% citric acid, extracting with ethyl acetate for 3 times, mixing organic phases, washing with saturated NaCl solution for 3 times, and adding anhydrous Na₂SO₄Drying overnight, evaporating the solvent to dryness, adding ethyl acetate to dissolve the solid, and recrystallizing to obtain Fmoc-Lys (Boc) -Phe-OH.

(3) Preparation of the tripeptide fragment Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH: (a) synthesis of Fmoc-Asp (OtBu) -OSu: 0.4mol of Fmoc-Asp (OtBu) -OH and 0.48mol of HOSu were dissolved in 0.8L of THF and placed in an ice-water bath; dissolving 0.48mol of DCC in 0.4L of THF, dropwise adding into the mixed solution in the last step, continuing to react for 1h after dropwise adding is finished, and then raising the temperature to 25 ℃ to continue to react for 3 h; filtering the reaction solution, evaporating to dryness, adding DCM for dissolving, filtering and evaporating to dryness; the solid was dissolved with ethyl acetate and recrystallized to yield Fmoc-Asp (OtBu) -OSu. (b) Synthesis of Fmoc-Asp (OtBu) -Lys (Boc) -OH: 0.45mol of H-Lys (Boc) -OH and 0.45mol of Na₂CO₃Dissolving in 0.6L 50% THF-H2O solution to obtain mixed solution; dissolving 0.3mol of Fmoc-Asp (OtBu) -OSu prepared in the step (a) in THF, dropwise adding into the mixed solution in the previous step, reacting at 25 ℃ overnight, rotary evaporating the reaction liquid, adding 15% citric acid to adjust the pH to 3, extracting with ethyl acetate for 3 times, combining organic phases, washing with saturated NaCl solution for 3 times, adding anhydrous Na₂SO₄Drying overnight, evaporating the solvent to dryness, adding ethyl acetate to dissolve the solid, and recrystallizing to obtain Fmoc-Asp (OtBu) -Lys (Boc) -OH. (c) Synthesis of Fmoc-Asp (OtBu) -Lys (Boc) -OSu: dissolving 0.2mol of Fmoc-Asp (OtBu) -Lys (Boc) -OH and 0.24mol of HOSu prepared in step (b) in 0.4L of THF, placing in an ice-water bath; dissolving 0.24mol of DCC in 0.2L of THF, dropwise adding into the mixed solution in the last step, continuing to react for 1h after dropwise adding is finished, and then raising the temperature to 25 ℃ to continue to react for 3 h; filtering the reaction solution, evaporating to dryness, adding DCM for dissolving, filtering and evaporating to dryness; the solid was dissolved with ethyl acetate and recrystallized to yield Fmoc-Asp (OtBu) -Lys (Boc) -OSu. (d) Synthesis of Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH: 0.15mol of H-Ser (tBu) -OH and 0.15mol of Na₂CO₃Dissolved in 0.2L of 50% THF/H₂O (v/v) solution to obtain mixed solution; dissolving 0.1mol of Fmoc-Asp (OtBu) -Lys (Boc) -OSu prepared in the step (c) in THF, and adding dropwise the solution to the mixed solution in the previous stepReacting at 25 deg.C overnight, rotary evaporating the reaction solution, adding 15% citric acid to adjust pH to 3, extracting with ethyl acetate for 3 times, mixing organic phases, washing with saturated NaCl solution for 3 times, adding anhydrous Na₂SO₄Drying overnight, evaporating the solvent to dryness, adding ethyl acetate to dissolve the solid, and recrystallizing to obtain Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH.

(4) Preparation of dipeptide fragment Fmoc-Glu (OtBu) -Thr (tBu) -OH: (a) synthesis of Fmoc-Ile-OSu: dissolving 0.2mol of Fmoc-Ile-OH and 0.24mol of HOSu in 0.4L of THF, and placing in an ice-water bath; dissolving 0.24mol of DCC in 0.2L of THF, dropwise adding into the mixed solution in the last step, continuing to react for 1h after dropwise adding is finished, and then raising the temperature to 25 ℃ to continue to react for 3 h; filtering the reaction solution, evaporating to dryness, adding DCM for dissolving, filtering and evaporating to dryness; and adding ethyl acetate to dissolve the solid, and recrystallizing to obtain Fmoc-Ile-OSu. (b) Synthesis of Fmoc-Ile-Glu (OtBu) -OH: 0.15mol of H-Glu (OtBu) -OH and 0.15mol of Na₂CO₃Dissolved in 0.2L of 50% THF/H₂O (v/v) solution to obtain mixed solution; dissolving 0.1mol of Fmoc-Ile-OSu prepared in the step (a) in THF, dropwise adding into the mixed solution in the previous step, reacting at 25 ℃ overnight, rotary evaporating the reaction liquid, adding 15% citric acid to adjust the pH to 3, extracting with ethyl acetate for 3 times, combining organic phases, washing with saturated NaCl solution for 3 times, adding anhydrous Na₂SO₄Drying overnight, evaporating the solvent to dryness, adding ethyl acetate to dissolve the solid, and recrystallizing to obtain Fmoc-Ile-Glu (OtBu) -OH.

(5) Preparation of dipeptide fragment Fmoc-Gln (Trt) -Glu (OtBu) -OH: (a) synthesis of Fmoc-Gln (Trt) -OSu: 0.2mol of Fmoc-Gln (Trt) -OH and 0.24mol of HOSu were dissolved in 0.4L of THF and placed in an ice-water bath; dissolving 0.24mol of DCC in 0.2L of THF, dropwise adding into the mixed solution in the last step, continuing to react for 1h after dropwise adding is finished, and then raising the temperature to 25 ℃ to continue to react for 3 h; filtering the reaction solution, evaporating to dryness, adding DCM for dissolving, filtering and evaporating to dryness; the solid was dissolved with ethyl acetate and recrystallized to yield Fmoc-Gln (Trt) -OSu. (b) Synthesis of Fmoc-Gln (Trt) -Glu (OtBu) -OH: 0.15mol of H-Glu (OtBu) -OH and 0.15mol of Na₂CO₃Dissolved in 0.2L of 50% THF/H₂O (v/v) solution to obtain mixed solution; subjecting 0.1mol of Fmoc prepared in step (a)-Gln (Trt) -OSu is dissolved in THF, and added dropwise to the mixed solution of the previous step, reacted at 25 ℃ overnight, the reaction solution is rotary evaporated, 15% citric acid is added to adjust pH to 3, extracted 3 times with ethyl acetate, the organic phases are combined, washed 3 times with saturated NaCl solution, and anhydrous Na is added₂SO₄The mixture is dried overnight, the solvent is evaporated, and the solid is dissolved by adding ethyl acetate and recrystallized to obtain Fmoc-Gln (Trt) -Glu (OtBu) -OH.

Example 2

Preparation of Fmoc-Ser (tBu) -HMP-MBHA resin

(1) Swelling of amino resin: taking 41.7g of HMP-MBHA resin with the substitution degree of 0.48mmol/g, adding 200ml of DCM, stirring for 0.5h under the protection of nitrogen to swell the resin, draining the solvent, washing the resin twice with DMF, and draining the solvent.

(2) Activation and condensation of Fmoc-Ser (tBu) -OH

Amino acid activation: dissolving 45mmol of Fmoc-Ser (tBu) -OH and 45mmol of HOBT in DMF, cooling to 0-10 ℃, adding 50mmol of DIC, and reacting for 5min to obtain activated amino acid.

Condensation reaction: adding activated amino acid into the resin in the step 1, adding 3mmol of DMAP, reacting overnight at 30 ℃ under the protection of nitrogen for 12h, emptying reaction liquid, washing the resin twice by using DMF, and draining the solvent.

And (3) blocking reaction: and (3) adding 90mmol DIEA/90mmol acetic anhydride/DMF solution into the resin in the step (2), reacting for 6 hours at 30 ℃ under the protection of nitrogen, emptying the reaction liquid, washing the resin three times by using DMF, draining the solvent, washing the resin three times by using MeOH, draining the solvent, and drying overnight at 25 ℃ in a vacuum drying oven to obtain the dried Fmoc-Ser (tBu) -HMP-MBHA resin.

(3) Fmoc-Ser (tBu) -HMP-MBHA resin substitution assay: the Fmoc protecting group of the Fmoc-Ser (tBu) -HMP-MBHA resin is removed by using 20% PIP/DMF, and the degree of substitution of the Fmoc-Ser (tBu) -HMP-MBHA resin is calculated to be 0.32mmol/g by measuring the absorbance at 595nm by using an ultraviolet spectrophotometry method.

Example 3

Preparation of thymosin beta 4 peptide resin

(1) Fmoc-Ser (tBu) -HMP-MBHA resin swelling: taking 31.3g of Fmoc-Ser (tBu) -HMP-MBHA resin, adding DCM, stirring for 0.5h under the protection of nitrogen, swelling the resin, draining the solvent, washing the resin twice with DMF, and draining the solvent.

(2) Removing Fmoc protecting group: to the resin swollen in step (1) was added a 20% PIP/DMF (v/v) solution (from Asp) 2 times the height of the resin³¹Start to Ser⁴³The Fmoc protection removal in 20% PIP/DMF (v/v) containing 0.25mol/L HOBT was performed twice at 30 ℃ for each Fmoc protection removal: removing Fmoc protection for the first time and Fmoc protection for the second time, washing the resin with DMF until the pH value is 7, and draining the solvent to obtain the resin with the Fmoc protection group removed; the first Fmoc removal protection time is 5min, and the second Fmoc removal protection time is 10 min.

(3) Ninhydrin detection: placing a small amount of resin in a test tube, and washing twice with DMF; adding two drops of 85% phenol-ethanol solution, two drops of pyridine and 5% ninhydrin-ethanol solution, respectively, heating at 110 deg.C for 3min, washing with DMF twice, and observing resin color.

(4) Activation of Fmoc-Glu (OtBu) -OH: weighing 30mmol of HOBT, dissolving the HOBT in DMF, cooling to 0-10 ℃, adding 33mmol of DIC, and reacting at 30 ℃ for 5min to obtain activated amino acid.

(5) Condensation reaction: adding activated amino acid into the resin in the step (2), reacting overnight for 1-2 h at 30 ℃ under the protection of nitrogen, detecting ninhydrin, emptying reaction liquid after the resin is colorless, washing the resin with DMF for three times, and draining the solvent.

(6) Repeating the steps (2), (3), (4) and (5) in order and sequentially aligning Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ile-OH, Fmoc-Thr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ser tBu) -OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Glu), (Fmoc-Glu), (OtBu) -OH, Fmoc-Leu-Lys (Boc) -OH, Fmoc-Glu (Boc) and Fmoc-Glu (Boc) in sequence according to the amino acid sequence of thymosin beta 4, Fmoc-Gln (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH, Fmoc-Lys (Boc) -Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH.

(7) Acetylation of the N-terminal Ser amino: removing the Fmoc protecting group at the N end, washing with DMF to neutrality, adding 60mmol DIEA/60mmol acetic anhydride/DMF solution, reacting at 30 deg.C under nitrogen protection for 2h, detecting ninhydrin to complete reaction, evacuating the reaction solution, washing the resin with DMF for three times, and draining off the solvent.

(8) Peptide resin shrinkage: washing the resin three times with MeOH for 5 min/time, draining the solvent, and drying overnight at 25 ℃ in a vacuum oven to obtain Ac-Ser (tBu) -Asp (OtBu) -Lys (Boc) -Pro-Asp (OtBu) -Met-Ala-Glu (OtBu) -Ile-Glu (OtBu) -Lys (Boc) -Phe-Asp (OtBu) -Lys (Boc) -Ser (tBu) -Lys (Boc) -Leu-Lys (Boc) -Thr-tBu (Glu) (OtBu) -Thr (Gln) (t) -Glu (OtBu) -Lys (Trt) -Pro-Asn-Leu-Pro-Ser (Lys) (Boc) -Boc-Glu (Boc) -Glu (OtBu) -Thr (Thr) (Gln) (Glt) -Glu (Glu) (GltBu) -Lys (Trt) -Lys (Leu) -Lys (Boc) -Lys (Thr) -Thr) (t-Glu) (t) -Ile-Glu) (t-Glu) (Boc) -Ala) The weight gain rate of the (E) -Gly-Glu (OtBu) -Ser (tBu) -HMP-MBHA resin, namely the thymosin beta 4 fully-protected peptide resin is 85 percent.

Example 4

Preparation of crude thymosin beta 4 peptide

(1) Placing the thymosin beta 4 fully-protected peptide resin prepared in the example 1 into a round-bottom flask, adding a 10-time volume of a cracking reagent, reacting for 3 hours at 25 ℃, and performing suction filtration to obtain a filtrate; the ratio of the volume parts of the cracking reagent is TFA to EDT to H₂O:TIS＝94:2:2:2。

(2) And (2) adding the filtrate prepared in the step (1) into 10 times volume of precooled MTBE for precipitation and centrifugation, washing the precipitate for four times by using the MTBE, centrifuging, and drying in vacuum at the temperature of 25 ℃ to obtain the crude linear thymosin beta 4 peptide. The MS detection shows that the missing peptide impurities of [ -Lys ] -thymosin beta 4, [ -Asp ] -thymosin beta 4 and [ -Lys ] + [ -Asp ] ] -thymosin beta 4 are not found.

Example 5

Purification of crude thymosin beta 4 peptide

The thymosin beta 4 crude peptide prepared in example 4 was purified by reversed-phase high performance liquid chromatography in two steps: a first purification step and a second purification step; first-step purification: mobile phase A phase was 0.1% TFA/H by volume fraction₂O (v/v) solution, mobile phase B as a volume fraction of 0.1% TFA/ACN (v/v) solution, second purification for pure product trans-salt: mobile phase A phase is 0.1% acetic acid/H by volume fraction₂O (v/v) solution, and the mobile phase B is 0.1% acetic acid/ACN (v/v) solution in volume fraction.

The thymosin beta 4 product is obtained by purification and freeze-drying, the HPLC purity of the product is 99.1 percent, the impurity [ asparaginyl imide ] -thymosin beta 4 is less than 0.1 percent, and the total yield is 50.6 percent.

Comparative example 1

The Wang resin (with the substitution degree of 0.35mmol/g) is used as a solid phase synthesis carrier to synthesize the thymosin beta 4, a dipeptide or tripeptide fragment is not adopted, all 43 amino acids are condensed in the form of single Fmoc protected amino acid, and a ninhydrin detection experiment shows that Lys is³⁰、Asp³¹And Lys³³Are all difficult amino acids to condense, Lys³⁰By carrying out a secondary condensation reaction and a blocking reaction, Asp³¹And Lys³³The secondary condensation reaction is required. The weight gain rate of the finally obtained thymosin beta 4 peptide resin is 58%, the HPLC purity of the peptide resin after cracking and purification is 97.8%, and impurities [ asparagine imide]Thymosin beta 4 was 0.9%, the overall yield was 10.3%.

Comparative example 2

The thymosin beta 4 is synthesized by using CTC resin (with the substitution degree of 0.36mmol/g) as a solid-phase synthesis carrier, dipeptide or tripeptide fragments are not adopted, all 43 amino acids are condensed in a single Fmoc protected amino acid form, the weight gain rate of the peptide resin is 35%, the HPLC purity of the peptide resin after cracking and purification is 98.0%, the impurity [ asparagine ] -thymosin beta 4 is 0.8%, and the total yield is 5.5%.

In the above-described embodiment of the present invention, the amino resin is changed to HMP-AM-resin or HMP-BHA-resin; the charging proportion of the raw materials is changed as follows: the molar mass ratio of the fully-protected amino acid to the condensation system to the resin is 2-5:2-5: 1; changing the reaction solvent, the reaction temperature to 15-35 ℃ and the reaction time; the system of the changed condensation system is as follows: A/D or A/B/C, wherein A is HOBT or HOAT, B is any one of HATU, HBTU, TBTU or PyBOP, C is DIEA or TMP, D is DIC and the like, and the direct synthesis of the fully-protected linear thymosin beta 4 peptide resin by the solid-phase synthesis method can be realized.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing thymosin beta 4, comprising: the method is applied to the Fomc/tBu method, and adopts a mode of combining a solid phase and a liquid phase to synthesize the thymosin beta 4 on resin by linear condensation, wherein Lys in an amino acid sequence of the thymosin beta 4³⁰、Asp³¹And Lys³³The tripeptide and dipeptide fragments are condensed to introduce the sequence, and HMP-amino resin is adopted as the resin.

2. The process according to claim 1, wherein Lys is substituted³⁰、Asp³¹Condensing tripeptide fragment Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH³³The condensation was carried out as the dipeptide fragment Fmoc-Lys (Boc) -Phe-OH.

3. The method according to claim 1, wherein the repeating dipeptide unit Ile in the thymosin beta 4 amino acid sequence¹⁰-Glu⁹And Ile³⁵-Glu³⁴Respectively carrying out condensation introduction sequences in the form of dipeptide fragments Fmoc-Ile-Glu (OtBu) -OH;

repeating dipeptide unit Gl in thymosin beta 4 amino acid sequenceu^12-Thr¹¹And Glu²³-Thr²²The condensed introduction sequences are respectively carried out in the form of dipeptide fragment Fmoc-Glu (OtBu) -Thr (tBu) -OH;

repeating dipeptide unit Gln in thymosin beta 4 amino acid sequence⁸-Glu⁷And Gln²¹-Glu²⁰The sequence was introduced by condensation in the form of the dipeptide fragment Fmoc-Gln (Trt) -Glu (OtBu) -OH, respectively.

4. The method for preparing according to any one of claims 1 to 3, characterized in that it comprises:

synthesizing dipeptide fragment Fmoc-Gln (Trt) -Glu (OtBu) -OH, Fmoc-Ile-Glu (OtBu) -OH, Fmoc-Glu (OtBu) -Thr (tBu) -OH, Fmoc-Lys (Boc) -Phe-OH and tripeptide fragment Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH by liquid phase synthesis;

5. The preparation method according to claim 4, wherein the thymosin beta 4 fully-protected peptide resin is obtained by introducing Fmoc-protected amino acids or peptide fragments into the thymosin beta 4 amino acid sequence in sequence, acetylating the Ser amino group at the N terminal, and then shrinking the peptide resin, and has the structure:

Ac-Ser (tBu) -Asp (OtBu) -Lys (Boc) -Pro-Asp (OtBu) -Met-Ala-Glu (OtBu) -Ile-Glu (OtBu) -Lys (Boc) -Phe-Asp (OtBu) -Lys (Boc) -Ser (tBu) -Lys (Boc) -Leu-Lys (Boc) -Thr (tBu) -Glu (OtBu) -Thr (Thr) -Gln (Trt) -Glu (OtBu) -Lys (Boc) -Asn Trt) -Pro-Leu-Pro-Ser (tBu) -Lys (Boc) -Glu (OtBu) -Thr (tBu) -Ile-Glu (OtBu) -Gln (Trt) -Glu (OtBu) -Lys (Boc), (Trt) -Gly-Glu (Tr-Ala-Gly-Ser (Boc) -Glu) (Boc) (OtBu) -Glu) (Met-Lys (Boc) -Glu) (Mett-Lys (Boc) -Glu) (OtBu) -Glu (Mett-Glu) (HMtBu-Glu).

6. The method of claim 4, wherein the Fmoc-Gln (Trt) -Glu (OtBu) -OH is synthesized by a method comprising: Fmoc-Gln (trt) -OH and HOSu in the presence of DCC to produce Fmoc-Gln (trt) -OSu; Fmoc-Gln (Trt) -OSu and H-Glu (OtBu) -OH are subjected to base catalysis to generate Fmoc-Gln (Trt) -Glu (OtBu) -OH;

the synthesis method of Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH comprises the following steps: Fmoc-Asp (OtBu) -OH and HOSu in the presence of DCC to form Fmoc-Asp (OtBu) -OSu; Fmoc-Asp (OtBu) -OSu and H-Lys (Boc) -OH are subjected to base catalysis to generate Fmoc-Asp (OtBu) -Lys (Boc) -OH; Fmoc-Asp (OtBu) -Lys (Boc) -OH and HOSu in the presence of DCC to yield Fmoc-Asp (OtBu) -Lys (Boc) -OSu; Fmoc-Asp (OtBu) -Lys (Boc) -OSu and H-Ser (tBu) -OH are subjected to base catalysis to generate Fmoc-Asp (OtBu) -Lys (Boc) -Ser (tBu) -OH.

7. The method according to claim 4, wherein the HMP-amino resin is selected from any one of HMP-AM-resin, HMP-MBHA-resin and HMP-BHA-resin, and the degree of resin substitution is 0.2 to 0.8 mmol/g;

preferably, the condensation reaction I comprises: dissolving Fmoc-Ser (tBu) -OH and HOBT in DMF, adding DIC to activate to obtain activated amino acid, adding the activated amino acid and DMAP into resin for condensation reaction, and then sealing the resin by using a mixed solution system of DIEA, acetic anhydride and DMF;

preferably, the molar equivalent ratio of Fmoc-Ser (tBu) -OH, HOBT, DIC, DMAP to resin is 2-5:2-5:2.2-5.5:0.1-0.3: 1;

8. The method according to claim 4, wherein a PIP/DMF solution system is used as a deprotection solution when removing the Fmoc protecting group of the Fmoc-Ser (tBu) -HMP-amino resin;

preferably, the method for removing the Fmoc-protecting group of the Fmoc-ser (tbu) -HMP-amino resin comprises: adding PIP/DMF solution into the swelled Fmoc-Ser (tBu) -HMP-MBHA resin, performing Fmoc protection removal at 15-35 ℃ for at least two times, and washing the resin by using DMF until the pH is neutral after the Fmoc protection removal;

preferably, the Asp reaching the condensation synthesis of thymosin ss 4 is obtained in the course of sequential condensation³¹To Ser⁴³When the Fmoc protecting group is removed each time, 0.1-0.5mol/L HOBT is added into a PIP/DMF solution system;

preferably, the activation of the Fmoc-protected amino acid or peptide fragment involved in the condensation comprises: mixing and activating the Fmoc protected amino acid or peptide fragment with a condensation system at 15-35 deg.C for 5-10 min;

preferably, the condensation system is an A/D system or an A/B/C system, wherein A is HOBT or HOAT, B is one of HATU, HBTU, TBTU or PyBOP, C is DIEA or TMP, and D is DIC;

preferably, the activated Fmoc-protected amino acid or peptide fragment to be condensed is added to the Fmoc-protected resin and subjected to condensation reaction II at 15-35 deg.C for 1-5 h.

9. The method according to claim 5, wherein the peptide resin is shrunk after acetylation of the Ser amino group at the N-terminus after introduction of Fmoc-protected amino acids or peptide fragments by condensation in the order of the amino acid sequence of thymosin β 4;

preferably, the acetylation of the N-terminal Ser amino group comprises: adding a mixed solution of DIEA, acetic anhydride and DMF into a system of the condensation reaction II, and reacting for 1-5h at 15-35 ℃;

preferably, the shrinking of the peptide resin comprises washing the resin and then vacuum drying the resin by drawing off the solvent.

10. The method of manufacturing according to claim 5, further comprising: cracking the thymosin beta 4 full-protection peptide resin to obtain crude thymosin beta 4 peptide, and then purifying the crude thymosin beta 4 peptide;

preferably, the lysing comprises: adding a cracking reagent into the thymosin beta 4 full-protection peptide resin, reacting for 2-5 hours at 15-35 ℃, performing suction filtration, adding the filtrate into precooled MTBE, precipitating, centrifuging, washing the precipitate, and performing vacuum drying to obtain crude thymosin beta 4 peptide;