阅读说明：本技术 固液相结合制备胸腺法新的方法 (Method for preparing thymalfasin by solid-liquid combination ) 是由 王浩 冯福权 周建华 于 2019-10-18 设计创作，主要内容包括：本发明公开了固液相结合制备胸腺法新的方法,本发明所述方法包括如下步骤：以CTC Resin为固相载体合成胸腺法新1-16个氨基酸的全保护肽作为第一片段,并且在N末端乙酰化氨基；合成第16-28个氨基酸的全保护肽作为第二片段；在液相里将第一片段和第二片段偶联,得到胸腺法新全保护肽,经裂解、纯化、冻干得胸腺法新成品。该方法可以两片段同时固相合成,有效的缩短40％的合成周期、提高粗肽纯度,降低下游纯化制备难度,提高了总收率,适合于工业化生产。(The invention discloses a method for preparing thymalfasin by combining solid phase with liquid phase, which comprises the following steps: synthesizing a thymalfasin 1-16 amino acid full-protection peptide serving as a first fragment by taking CTC Resin as a solid phase carrier, and acetylating amino at the N terminal; synthesizing a full protection peptide with 16 th to 28 th amino acids as a second segment; coupling the first segment and the second segment in a liquid phase to obtain thymalfasin full-protection peptide, and cracking, purifying and freeze-drying to obtain a thymalfasin finished product. The method can realize simultaneous solid-phase synthesis of two segments, effectively shorten the synthesis period by 40 percent, improve the purity of crude peptide, reduce the difficulty of downstream purification preparation, improve the total yield and is suitable for industrial production.)

1. The method for preparing thymalfasin by combining solid and liquid is characterized by comprising the following steps:

(1) synthesizing 1-16 amino acids of thymalfasin in a solid phase, acetylating amino at the N end, and cracking full-protection peptide to obtain a first fragment;

(2) synthesizing 17-28 amino acids of thymalfasin by using a solid phase carrier, and cracking and fully protecting the thymalfasin to obtain a second fragment;

(3) coupling the first segment and the second segment in a liquid phase to synthesize thymalfasin full-protection peptide;

(4) cracking, purifying and freeze-drying to obtain the thymalfasin finished product.

2. The method for preparing thymalfasin according to claim 1, wherein: the Resin used in the step (1) and/or the step (2) is 2-Cl-CTC Resin, and the substitution degree is 0.6-1.2 mmol/g.

3. The method for preparing thymalfasin according to claim 1, wherein: when the peptide chain is synthesized in the step (1), AC is used₂O acetylates an amino group at the N-terminal end of a peptide chain under an alkaline condition.

4. The method for preparing thymalfasin according to claim 3, with solid-liquid combination, characterized in that: the base used for acetylating the N-terminal amino group of the first fragment is any one of Py, NMM, DIEA, TEA and TMP.

5. The method for preparing thymalfasin according to claim 1, wherein: the substitution degree of the amino acid resin prepared in the step (1) and/or the step (2) is 0.3-0.8 mmol/g.

6. The method for preparing thymalfasin according to claim 1, wherein: the cracking reagent of the total protection segment in the step (1) and/or the step (2) is 15 to 30 percent of V/V TFE/DCM or 0.5 to 3 percent of V/V TFA/DCM.

7. The method for preparing thymalfasin according to claim 1, wherein: in the step (3), the condensing agent combination is any one of DCC/HOSu and EDC & HCl/HOSU.

8. The method for preparing thymalfasin according to claim 1, wherein: the cracking reagent adopted in the step (4) is TFA and m-cresol which are 80-95: 5-20.

9. The method for preparing thymalfasin according to claim 8, wherein: the cleavage reagent used in step (4) was TFA to m-cresol 90: 10.

10. The method for preparing thymalfasin in solid-liquid phase combination as recited in claim 1, further comprising the steps of:

(1) preparation of Fmoc-Leu-CTC Resin

Adding 2-CTC resin into a solid phase reactor, and adding DCM swelling resin; draining the solvent, adding DMF solution of Fmoc-Gly-OH, adding DIEA, stirring for reaction, adding methanol, and sealing for 30 min; washing with methanol; after vacuum drying, obtaining Fmoc-Leu-CTC Resin;

(2) preparation of thymalfasin first fragment peptide:

a) adding Fmoc-Leu-CTC Resin into a solid phase reactor, adding DCM swelling Resin, draining the solvent, and adding 20% piperidine/DMF solution for reaction twice; washing with DMF; the result of indene test is positive; b) dissolving Fmoc-Asp (OtBu) -OH and HOBt in DMF, adding DIC, and activating in ice bath; adding the mixture into a reactor for reaction until the indene detection result is negative, and then draining the solvent; adding DMF for washing; c) repeating the above steps, and sequentially adding Fmoc-Lys (BOC) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu) -OH and Fmoc-Ser tBu-OH according to the amino acid sequence to perform coupling reaction; d) after the end of coupling, 20eq of AC was used₂O/NMM acetylates the N-terminal amino group; e) vacuum drying to obtain AC-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (BOC) -Asp (OtBu) -Leu-CTC Resin; f) adding peptide resin into 20% TFE/DCM for reaction, collecting filtrate, and performing vacuum spin-drying on the filtrate to obtain AC-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (BOC) -Asp (OtBu) -Leu-OH which is the first fragment of the thymalfasin;

(3) preparation of first fragment-succinimidyl ester:

AC-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (BOC) -Asp (OtBu) -Leu-OH is added into DCM for dissolution, DCC and HOSU are added for reaction under ice bath condition, TLC monitors that the raw material reaction is completely disappeared; alternately washing with 5% NaCO3 water solution and 5% citric acid water solution, washing with saturated sodium chloride solution at equal volume, filtering to remove insoluble substances, and drying the filtrate with anhydrous sodium sulfate; spin-drying the filtrate to obtain first segment-succinimidyl ester;

(4) preparation of Fmoc-Asn (trt) -CTC Resin

Adding 2-CTC resin into a solid phase reactor, and adding DCM swelling resin; draining the solvent, adding DMF solution of Fmoc-Asn (trt) -OH, adding DIEA, stirring for reaction, adding methanol, and sealing for 30 min; washing with methanol; after vacuum drying, obtaining Fmoc-Asn (trt) -CTC Resin;

(5) preparation of second fragment peptide of thymalfasin:

a) adding Fmoc-Asn (trt) -CTC Resin into a solid phase reactor, adding DCM swelling Resin, draining the solvent, and adding 20% piperidine/DMF solution for reaction twice; washing with DMF; the result of indene test is positive; b) dissolving Fmoc-Glu (OtBu) -OH and HOBt in DMF, adding DIC, and activating in ice bath; adding the mixture into a reactor for reaction until the indene detection result is negative, and then draining the solvent; adding DMF for washing; c) repeating the steps, and sequentially adding Fmoc-Ala-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Val-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (BOC) -OH, Fmoc-Glu (OtBu) -OH and Fmoc-Lys (BOC) -OH according to the amino acid sequence to perform coupling reaction; d) contracting the methanol and drying in vacuum to obtain NH₂-Lys (BOC) -Glu (OtBu) -Lys (BOC) -Glu (OtBu) -Val-Val-Glu (OtBu) -Ala-Glu (OtBu) -Asn (trt) -CTC Resin; f) adding peptide resin into 20% TFE/DCM for reaction, and spin-drying the filtrate to obtain NH₂-Lys (BOC) -Glu (OtBu) -Lys (BOC) -Glu (OtBu) -Val-Val-Glu (OtBu) -Ala-Glu (OtBu) -Asn (trt) -OH, which is the second fragment of thymalfasin;

(6) preparing thymalfasin full-protection peptide:

dissolving the first fragment-succinimidyl ester prepared in the step (3) in DCM solution, and slowly adding NH in ice-water bath₂DCM solution of-Lys (BOC) -Glu (OtBu) -Lys (BOC) -Glu (OtBu) -Val-Glu (OtBu) -Ala-Glu (OtBu) -Asn (trt) -OH is reacted, and solvent is rotated to obtain thymalfasin full-protection peptide after the reaction is finished;

(7) cracking

Preparing a cracking reagent of TFA and m-cresol of 95: 5, adding the reagent into the fully-protected peptide for reaction, and adding anhydrous ether for sedimentation after the reaction is finished; centrifuging and drying to obtain the thymalfasin finished product.

Technical Field

The invention relates to the field of solid-phase synthesis of polypeptide medicaments, in particular to a method for preparing thymalfasin by combining a solid phase with a liquid phase.

Background

Chinese name: thymalfasin (Thymalfasin)

English name: thymalfasin

The peptide sequence structure is as follows:

Ac-Ser1-Asp2-Ala3-Ala4-Val5-Asp6-Thr7-Ser8-Ser9-Glu10-Ile11-Thr12-Thr13-Lys14-Asp15-Leu16-Lys17-Glu18-Lys19-Lys20-Glu21-Val22-Val23-Glu24-Glu25-Ala26-Glu27-Asn28-OH

cas number: 69440-99-9

Molecular weight: 3108.3

The molecular formula is as follows: c₁₂₉H₂₁₅N₃₃O₅₅

Thymalfasin is developed and marketed by American Seisan company, is a freeze-dried powder injection and enters China before and after 1995. Thymalfasin has been approved in more than 30 countries for the current treatment of hepatitis b and c and as an immunopotentiator for use in oncology patients. The product has effects of promoting T cell differentiation and maturation, increasing NK cell activity, promoting T cell to secrete various lymphokines, increasing IL-2 receptor affinity to IL-2, increasing NK cell number and activity, and indirectly killing virus-infected liver cell and tumor cell by proliferation of helper T cell, cytotoxic lymphocyte and NK cell.

The prior preparation method of thymalfasin is mainly a solid-phase synthesis method, but a large number of beta-folded structures are formed in the molecular structure of thymalfasin, and the phenomenon of difficult coupling can occur if peptide grafting is carried out from C end to N end one by one, so that the coupling efficiency of amino acid is reduced, and the difficulty of separation and purification is increased.

CN 104987382A introduces a method of synthesizing dipeptide fragments of continuous amino acids by liquid phase method, and synthesizing thymalfasin by feeding dipeptide fragments into solid phase, which shortens the solid phase synthesis period to a certain extent, but a large amount of dipeptide fragments need to be synthesized, thus actually greatly increasing the whole synthesis period and having fussy working procedures; CN101484467 reports a process for synthesizing thymalfasin by using a pseudoproline dipeptide solid phase, which reduces the difficulty of difficult sequence coupling to a certain extent, but can not effectively avoid the generation of impurities such as missing peptide and the like; CN101104638A reports a solid-phase synthesis process of thymalfasin, namely, a method for sequentially carrying out solid-phase synthesis on residual amino acids in a sequence by using Fmoc-Rink Amide resin or Fmoc-Rink Amide MBHA resin as a carrier, connecting side chain carboxyl of Fmoc-Asp-X with resin amino and adopting an Fmoc strategy. The method is also a one-by-one coupling method essentially, and the difficulty of the difficult sequence part cannot be effectively reduced.

Disclosure of Invention

In order to overcome the defects in synthesis of thymalfasin, the invention provides a novel method for preparing thymalfasin by solid-liquid combination of a fragment method.

According to one aspect of the technology, the invention provides a method for preparing thymalfasin by combining a solid phase and a liquid phase, which comprises the following steps:

(1) synthesizing 1-16 amino acids of thymalfasin in a solid phase, acetylating amino at the N end, and cracking full-protection peptide to obtain a first fragment;

(2) synthesizing 17-28 amino acids of thymalfasin by using a solid phase carrier, and cracking and fully protecting the thymalfasin to obtain a second fragment;

(3) coupling the first segment and the second segment in a liquid phase to synthesize thymalfasin full-protection peptide;

(4) cracking, purifying and freeze-drying to obtain the thymalfasin finished product.

According to one aspect of the technology, the invention provides a method for preparing thymalfasin through solid-liquid phase combination, wherein the Resin used in the step (1) and/or the step (2) is 2-C1-CTC Resin, and the substitution degree is 0.6-1.2 mmol/g.

According to an aspect of the present invention, there is provided a method for preparing thymalfasin using solid-liquid phase combination, wherein AC is used when the peptide chain is synthesized in step (1)₂O acetylates an amino group at the N-terminal end of a peptide chain under an alkaline condition.

According to an aspect of the present invention, there is provided a method for preparing thymalfasin by solid-liquid phase combination, wherein the base used for acetylating the N-terminal amino group of the first fragment in step (1) is any one of Py, NMM, DIEA, TEA and TMP.

According to one aspect of the technology, the invention provides a method for preparing thymalfasin by solid-liquid phase combination, wherein the substitution degree of the amino acid resin prepared in the step (1) and/or the step (2) is 0.3-0.8 mmol/g.

According to one aspect of the technology, the invention provides a method for preparing thymalfasin by solid-liquid phase combination, wherein the cracking reagent of the total protection fragment in the step (1) and/or the step (2) is 15-30% V/V TFE/DCM or 0.5-3% V/V TFA/DCM.

According to one aspect of the technology, the invention provides a method for preparing thymalfasin by solid-liquid phase combination, wherein the condensing agent used in step (1) and/or step (2) is any one of HoBt/DIC, HoAt/DIC, Cl-HOBt/DIC and HOOBt/DIC, wherein DIC can be replaced by any one of DCC and EDC.HCl; or any one of HATU/HoBt/DIEA, HBTU/HoBt/DIEA, HCTU/HoBt/DIEA, TATU/HoBt/DIEA, TBTU/HoBt/DIEA, PyBOP/HoBt/DIEA or PyAOP/HoBt/DIEA, wherein DIEA can be replaced by any one of NMM, TMP or TEA; the reaction solvent is one or a combination of DMF, DCM, DMSO, DMA and NMP; the Fmoc removing reagent is 15-30% V/V piperidine/DMF solution or 1-5% V/V DBU/DMF solution, and the removing is carried out twice, each time for 5-20 min.

According to an aspect of the present invention, there is provided a method for preparing thymalfasin by solid-liquid phase combination, wherein a condensing agent combination is used in step (3) to prepare any one of DCC/HOSu and EDC. HCl/HOSu.

According to one aspect of the technology, the invention provides a method for preparing thymalfasin by combining a solid phase and a liquid phase, wherein a cracking reagent adopted in the step (4) of the method is TFA and m-cresol 80-95: 5-20

According to one aspect of the present invention, there is provided a method for preparing thymalfasin using solid-liquid phase combination, wherein the cleavage reagent used in step (4) is preferably TFA/m-cresol (90: 10)

According to one aspect of the technology, the invention provides a method for preparing thymalfasin by combining solid phase with liquid phase, and in the synthesis method of thymalfasin, the preferable technical scheme is that the method comprises the following steps:

(1) preparation of Fmoc-Leu-CTC Resin

Adding 2-CTC resin into a solid phase reactor, and adding DCM swelling resin; draining the solvent, adding DMF solution of Fmoc-Gly-OH, adding DIEA, stirring for reaction, adding methanol, and sealing for 30 min; washing with methanol; after vacuum drying, obtaining Fmoc-Leu-CTC Resin;

(2) preparation of thymalfasin first fragment peptide:

a) adding Fmoc-Leu-CTC Resin into solid phase reactionAdding DCM swelling resin into a reactor, draining the solvent, adding 20% piperidine/DMF solution, and reacting twice; washing with DMF; the result of indene test is positive; b) dissolving Fmoc-Asp (OtBu) -OH and HOBt in DMF, adding DIC, and activating in ice bath; adding the mixture into a reactor for reaction until the indene detection result is negative, and then draining the solvent; adding DMF for washing; c) repeating the above steps, and sequentially adding Fmoc-Lys (BOC) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu) -OH and Fmoc-Ser tBu-OH according to the amino acid sequence to perform coupling reaction; d) after the end of coupling, 20eq of AC was used₂O/NMM acetylates the N-terminal amino group; e) vacuum drying to obtain AC-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (BOC) -Asp (OtBu) -Leu-CTC Resin; f) adding peptide resin into 20% TFE/DCM for reaction, collecting filtrate, and performing vacuum spin-drying on the filtrate to obtain AC-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (BOC) -Asp (OtBu) -Leu-OH which is the first fragment of the thymalfasin;

(3) preparation of first fragment-succinimidyl ester:

AC-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (BOC) -Asp (OtBu) -Leu-OH is added into DCM for dissolution, DCC and HOSU are added for reaction under ice bath condition, TLC monitors that the raw material reaction is completely disappeared; alternately washing with 5% NaCO3 water solution and 5% citric acid water solution, washing with saturated sodium chloride solution at equal volume, filtering to remove insoluble substances, and drying the filtrate with anhydrous sodium sulfate; spin-drying the filtrate to obtain first segment-succinimidyl ester;

(4) preparation of Fmoc-Asn (trt) -CTC Resin

Adding 2-CTC resin into a solid phase reactor, and adding DCM swelling resin; draining the solvent, adding DMF solution of Fmoc-Asn (trt) -OH, adding DIEA, stirring for reaction, adding methanol, and sealing for 30 min; washing with methanol; after vacuum drying, obtaining Fmoc-Asn (trt) -CTC Resin;

(5) preparation of second fragment peptide of thymalfasin:

a) adding Fmoc-Asn (trt) -CTC Resin into a solid phase reactor, adding DCM swelling Resin, draining the solvent, and adding 20% piperidine/DMF solution for reaction twice; washing with DMF; the result of indene test is positive; b) dissolving Fmoc-Glu (OtBu) -OH and HOBt in DMF, adding DIC, and activating in ice bath; adding the mixture into a reactor for reaction until the indene detection result is negative, and then draining the solvent; adding DMF for washing; c) repeating the steps, and sequentially adding Fmoc-Ala-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Val-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (BOC) -OH, Fmoc-Glu (OtBu) -OH and Fmoc-Lys (BOC) -OH according to the amino acid sequence to perform coupling reaction; d) contracting the methanol and drying in vacuum to obtain NH₂-Lys (BOC) -Glu (OtBu) -Lys (BOC) -Glu (OtBu) -Val-Val-Glu (OtBu) -Ala-Glu (OtBu) -Asn (trt) -CTC Resin; f) adding peptide resin into 20% TFE/DCM for reaction, and spin-drying the filtrate to obtain NH₂-Lys (BOC) -Glu (OtBu) -Lys (BOC) -Glu (OtBu) -Val-Val-Glu (OtBu) -Ala-Glu (OtBu) -Asn (trt) -OH, which is the second fragment of thymalfasin;

(6) preparing thymalfasin full-protection peptide:

dissolving the first fragment-succinimidyl ester prepared in the step (3) in DCM solution, and slowly adding NH in ice-water bath₂DCM solution of-Lys (BOC) -Glu (OtBu) -Lys (BOC) -Glu (OtBu) -Val-Glu (OtBu) -Ala-Glu (OtBu) -Asn (trt) -OH is reacted, and solvent is rotated to obtain thymalfasin full-protection peptide after the reaction is finished;

(7) cracking

Preparing a cracking reagent of TFA and m-cresol of 95: 5, adding the reagent into the fully-protected peptide for reaction, and adding anhydrous ether for sedimentation after the reaction is finished; centrifuging and drying to obtain thymalfasin product

Drawings

FIG. 1: scheme 1 is a synthetic route diagram of the present invention;

FIG. 2: the method of the invention adopts HPLC chromatogram of thymalfasin finished products.

Detailed Description

The following examples are intended to further illustrate some, but not all, preferred embodiments of the present invention. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention. The invention will be further described with reference to the accompanying drawings.

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

example 1

Preparing a first fragment of thymalfasin full-protection peptide:

1) swelling resin: 40g of 2-Cl-CTC Resin with a substitution degree of 1.0mmol/g is weighed, 400ml of DCM swelling Resin is added for 0.5h, the solvent is drained, 200ml of DMF is added for washing the Resin twice, and the solvent is drained.

2) Preparation of Fmoc-Leu-CTC Resin: weighing 28.3g of Fmoc-Leu-OH, dissolving in 200ml of DMF, adding 42ml of DIEA, uniformly mixing, adding the solution into a reaction column for reaction for 2 hours, and draining the solvent; adding 200ml DMF to wash the resin for three times, and pumping out the washing solvent; preparing 400ml of sealing liquid according to the volume ratio of DCM, methanol and DIEA of 17: 2: 1, sealing the resin twice for 10min each time; adding 200ml DMF to wash the resin for three times, and pumping out the washing solvent; the solution was washed three times with methanol for 10min each time, and after drying the solvent by suction and vacuum drying, 52.8g of Fmoc-Leu-CTC Resin was obtained, and the degree of substitution was measured to be 0.68 mmol/g.

3) Preparation of thymalfasin first fragment: a) swelling amino acid Resin, weighing 44.1g of Fmoc-Leu-CTC Resin, adding 500ml of DCM swelling Resin for 0.5h, draining the solvent, adding 300ml of DMF, washing the Resin twice, and draining the solvent; b) removing Fmoc protecting group, adding 300ml of 20% piperidine/DMF solution into the resin, deprotecting twice, the first deprotection time is 10min, and the second deprotectionThe time is 15min, after the deprotection is finished, 300ml of DMF is added to wash the resin for 5 times, the solvent is drained, and the operation is repeated, and the indene detection is positive; c) weighing 37.0g of Fmoc-Asp (OtBu) -OH and 14.6g of HOB, measuring 17ml of DIC to dissolve in 300ml of DMF, activating in an ice-water bath for 5min, adding the solution into a reaction column for reaction at 30 ℃ for 2h, detecting negative in indene, draining the solvent, washing the resin with 300ml of DMF for 3 times, and draining the solvent; d) extending a peptide chain, adding Fmoc-Lys (BOC) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu) -OH and Fmoc-Ser (tBu) -OH in sequence according to the molar mass ratio of resin, amino acid, HOBt and DIC of 1: 3.6, repeating the above steps, sequentially according to the amino acid sequence, and carrying out coupling reaction by measuring 57ml₂Dissolving O and 66ml NMM in 300ml DMF, adding into a reactor, reacting for 0.5h, and washing the resin five times by 300ml DMF after the reaction is finished to obtain 106.9g of thymalfasin first fragment peptide resin; e) preparing 800ml of 20% TFE/DCM solution, adding the thymalfasin first fragment peptide resin, reacting for 2 hours, and spin-drying the filtrate to obtain 69.1g of thymalfasin first fragment.

Example 2

Preparation of first fragment of thymalfasin-succinimidyl ester:

adding the first fragment of thymalfasin into 200ml of DCM for dissolving, adding 6.8g of DCC and 3.4g of HOSU, reacting under an ice bath condition, and monitoring by TLC that the reaction of the raw materials completely disappears; concentrating the liquid to about 100ml, using an equal volume of 5% NaCO₃Alternately washing the aqueous solution and 5% citric acid aqueous solution, washing with saturated sodium chloride solution at equal volume, filtering to remove insoluble substances, and drying the filtrate with anhydrous sodium sulfate; the filtrate was spin-dried to obtain 70.8g of the first fragment-succinimidyl ester.

Example 3

Preparation of thymalfasin second fragment full-protection peptide:

1) swelling resin: 40g of CTC Resin with a substitution degree of 1.0mmol/g is weighed, 400ml of DCM swelling Resin is added for 0.5h, the solvent is drained, 200ml of DMF is added for washing the Resin twice, and the solvent is drained.

2) Preparation of Fmoc-Asn (trt) -CTC Resin: weighing 47.7g of Fmoc-Asn (trt) -OH, dissolving in 200ml of DMF, adding 42ml of DIEA, uniformly mixing, adding the solution into a reaction column for reaction for 2 hours, and draining the solvent; adding 200ml DMF to wash the resin for three times, and pumping out the washing solvent; preparing 400ml of sealing liquid according to the volume ratio of DCM, methanol and DIEA of 17: 2: 1, sealing the resin twice for 10min each time; adding 200ml DMF to wash the resin for three times, and pumping out the washing solvent; washing with methanol for three times, each time for 10min, draining the solvent, and drying in vacuum to obtain Fmoc-Asn (trt) -CTC Resin56.2g, and measuring the degree of substitution to be 0.59 mmol/g.

3) Preparation of a second fragment of thymalfasin: a) swelling amino acid Resin, weighing 50.8g of Fmoc-Asn (trt) -CTC Resin, adding 500ml of DCM swelling Resin for 0.5h, draining the solvent, adding 300ml of DMF, washing the Resin twice, and draining the solvent; b) removing Fmoc protecting groups, adding 300ml of 20% piperidine/DMF solution into the resin, deprotecting twice, wherein the first deprotection time is 10min, the second deprotection time is 15min, after the deprotection is finished, adding 300ml of DMF to wash the resin for 5 times, draining the solvent, repeating the steps, and detecting positive indene; c) weighing 38.3g of Fmoc-Glu (OtBu) -OH and 14.6g of HOB, measuring 17ml of DIC, dissolving in 300ml of DMF, activating in an ice-water bath for 5min, adding the solution into a reaction column, reacting for 2h at 30 ℃, performing negative indene detection, draining the solvent, washing the resin for 3 times by 300ml of DMF, and draining the solvent; d) extending a peptide chain, and sequentially adding Fmoc-Ala-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Val-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (BOC) -OH, Fmoc-Glu (OtBu) -OH and Fmoc-Lys (BOC) -OH according to the molar mass ratio of resin, amino acid, HOBt and DIC of 1: 3.6, performing coupling reaction by using one coupling, wherein the coupling cannot be completely indenylated and colorless, the feeding can be repeated by 2eq for one time, and after the completion, washing the resin by 300ml of DMF for five times to obtain 108.0g of the thymalfasin second; e) preparing 800ml of 20% TFE/DCM solution, adding the thymalfasin second fragment peptide resin, reacting for 2 hours, and spin-drying the filtrate to obtain 62.9g of thymalfasin second fragment.

Example 4

Preparation of thymalfasin deprotected peptides:

and (2) dissolving the second fragment and the first fragment of the thymalfasin in 200ml of DCM respectively, after the second fragment and the first fragment of the succinimide ester are dissolved completely, slowly adding the first fragment of the succinimide ester solution into the second fragment solution under the ice bath condition, reacting for 4 hours, and after the reaction is finished, spin-drying the reaction solution to obtain 118.8g of thymalfasin full-protection peptide.

Example 5

Cracking and purifying:

preparing 500ml of lysate according to lysate TFA/m-cresol 95/5, stirring uniformly, slowly adding thymalfasin full-protection peptide, dissolving, reacting at 30 ℃ for 3 hours, adding the reaction solution into 3600ml of isopropyl ether for settling, centrifuging and drying to obtain a thymalfasin crude product, purifying by using a C18 preparation column, using a mobile phase 0.3% acetic acid aqueous solution, enabling the flow rate to be 60-100 ml/min, detecting the wavelength to be 220nm, tracking the collected solution by using HPLC, and freeze-drying by using a conventional method to obtain the thymalfasin refined peptide, wherein the mass is 36.3g, the purity is 99.61%, and the total yield is 39.0%.

The synthetic route of the invention is shown in figure 1, and the HPLC (high performance liquid chromatography) map of the invention is shown in figure 2, wherein the purity is 99.61%, the maximum single impurity is 0.13%, and the total impurity is 0.39%.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.