阅读说明：本技术 一种替度鲁肽的制备方法 (Preparation method of teduglutide ) 是由 刘宏 邬莉娜 周琳 赵春林 胡沙 李�浩 于 2021-08-04 设计创作，主要内容包括：本发明涉及药物合成领域,特别涉及一种替度鲁肽的制备方法。该方法包括：制备Phe-Ile-Asn(trt)-Trp(Boc)-Leu五肽片段、Asp(tBu)-Asn(trt)-Leu三肽片段(Ⅰ)、His(trt)-Gly-Asp(tBu)三肽片段(Ⅱ)、Ile-Gln(trt)-Thr(tBu)-Lys(Boc)-Ile-Thr(tBu)-Asp(tBu)第一肽树脂；在第一肽树脂上依次偶联氨基酸和多肽片段,将所得替度鲁肽树脂裂解纯化。该方法成功的突破了替度鲁肽～(1)His消旋杂质的控制难点、规避了～(24)Asn发生分子内自主成环并提高了～(16)Asn的偶联效率。(The invention relates to the field of drug synthesis, and particularly relates to a preparation method of teduglutide. The method comprises the following steps: preparing a first peptide resin of Phe-Ile-Asn (trt) -Trp (Boc) -Leu pentapeptide fragment, Asp (tBu) -Asn (trt) -Leu tripeptide fragment (I), His (trt) -Gly-Asp (tBu) tripeptide fragment (II), Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu); sequentially coupling amino acid and polypeptide fragment on the first peptide resin, and cracking and purifying the obtained teduglutide resin. The method successfully breaks through the teduglutide 1 The control difficulty of His racemic impurities is avoided 24 Asn occurs intramolecularly self-formingIs surrounded by and improves 16 Coupling efficiency of Asn.)

1. A preparation method of teduglutide is characterized by comprising the following steps:

step (1): preparing Phe-Ile-Asn (trt) -Trp (Boc) -Leu-OH to obtain a pentapeptide fragment;

asp (tBu) -Asn (trt) -Leu-OH is prepared to obtain tripeptide fragment (I);

preparing His (trt) -Gly-Asp (tBu) -OH to obtain tripeptide fragment (II);

preparing Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin to obtain a first peptide resin;

step (2): coupling the pentapeptide fragment with a first peptide resin to obtain a second peptide resin;

and (3): sequentially coupling Asp (tBu), Arg (pbf), Ala, tripeptide fragment (I), Leu, Ile, Thr (tBu), Asn (trt), Met, Glu (tBu), Asp (tBu), Ser (tBu), Phe, Ser (tBu), Gly and tripeptide fragment (II) on the second peptide resin to obtain the teduo peptide resin;

and (4): cracking the teduglutide resin to obtain a crude teduglutide product;

and (5): and purifying the crude teduglutide.

2. The method according to claim 1, wherein the step (1) is:

preparing Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-OH to obtain a pentapeptide fragment;

preparing Fmoc-Asp (tBu) -Asn (trt) -Leu-OH to obtain tripeptide fragment (I);

preparing Boc-His (trt) -Gly-Asp (tBu) -OH to obtain tripeptide fragment (II);

preparation of Fmoc-Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin to obtain a first peptide resin.

3. The method according to claim 1, wherein the step (3) is: Fmoc-Asp (tBu) -OH, Fmoc-Arg (pbf) -OH, Fmoc-Ala-OH, tripeptide fragment (I), Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (trt) -OH, Fmoc-Met-OH, Fmoc-Glu (tBu) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH and tripeptide fragment (II) are coupled in this order on the second peptide resin to obtain the tetrodol peptide resin.

4. The method of claim 1, wherein the resin is a 2-ClCTC resin or a Wang resin.

5. The method of claim 1, wherein the coupling agent is one of HATU/HOBt/DIEA, HBTU/HOBt/DIEA, PyBOP/HOBt/DIEA, DIC/HOBt, DIC/HOAt, and DIC/Cl-HOBt.

6. The method of claim 1, wherein the cleavage is performed with a cleavage agent system selected from the group consisting of TFA/Tis/EDT, TFA/Tis/H₂O, TFA/water, TFA/phenol/thiobenzol/Tis/EDT/water.

7. The method of claim 1, wherein the purification is performed by Pre-HPLC.

8. The method of claim 1, wherein the Pre-HPLC conditions are: c₁₈A reversed phase column; and (3) taking a 15-25 mM ammonium acetate water solution as a mobile phase A and acetonitrile as a mobile phase B, and performing gradient elution.

9. The method according to claim 8, wherein the gradient of the mobile phase B is 20 to 60% in 60 min.

10. The method of any one of claims 1 to 9, wherein the purification further comprises a lyophilization step.

Technical Field

The invention relates to the field of drug synthesis, and particularly relates to a preparation method of teduglutide.

Background

Teduglutide (Teduglutide) is a glucagon-like peptide 2(GLP-2) analog that reduces gastric emptying and secretion, and regulates the growth, proliferation and repair of intestinal membrane cells, thereby increasing intestinal absorption and reducing diarrhea. Developed by NPS Pharmaceuticals, approved by the european union and FDA in 2012 to be marketed under the trade name Gattex/Revestive, is an orphan drug for the treatment of Short Bowel Syndrome (Short Bowel Syndrome). The structural formula and the peptide sequence are shown in figure 1.

The most effective chemical synthesis method of the polypeptide drug at present is the Fmoc method of solid phase synthesis, namely, starting from-COOH of polypeptide, connecting the first amino acid to resin, removing Fmoc protecting group at N terminal of the amino acid, coupling the next amino acid in sequence, removing N terminal protection, recoupling, and then deprotecting until the last N terminal amino acid is coupled, then cleaving the polypeptide from the resin and removing all protecting groups simultaneously to obtain a complete polypeptide, and the method has obvious advantages in the synthesis of polypeptides with sequence length of 10-25.

Chemical synthesis methods of teduglutide have been reported in the literature, but the reported preparation methods have different degrees of defects and disadvantages. For example, in CN201310102159, a solid-phase stepwise synthesis method is adopted to prepare the teduglutide molecule, and a large amount of missing peptide impurities are generated due to incomplete reaction of individual amino acids in the teduglutide sequence, and the nature similarity of the missing peptide impurities is high, which causes difficulty in separation and purification and low yield; in CN111018962A, a coupling mode of large fragment peptide (more than 10 amino acids) is adopted to prepare a target molecule, the coupling condensation efficiency between large polypeptide fragments is low, the content of the missing peptide impurities is high, and the total yield is low; CN11217506A, CN106749614A and CN109456404A adopt a large number of short peptide fragments (2-5 amino acids) as intermediates to prepare target molecules, and the procedures are complicated and are not suitable for the development of production processes; CN201310102450, CN201310369843 and CN201510274924 adopt solid-phase fragment condensation method to prepare target molecules, and are all around control¹His racemization was studied, but solid phase fragment selection was rationalizedInsufficient results in low crude product purity and poor total yield.

In the development and research of a synthesis method of the teduglutide, the inventor finds that the amino acids of the teduglutide are sequentially coupled according to a stepwise synthesis method, so that a target molecule cannot be effectively obtained due to the reason that the synthesis is completed to²⁴Asn and¹⁶asn is the point where the subsequent amino acid of teduglutide cannot be coupled further. Complete the process¹A large amount of racemization impurities of the amino acid appear when His exists, and the impurities are difficult to separate and purify, so that the total yield is low. The preparation method of the present invention was developed based on the above-mentioned disadvantages and experimental results of the reported literature methods.

Disclosure of Invention

In view of the above, the present invention provides a preparation method of teduglutide. The method can effectively avoid incomplete amino acid coupling, ring closing phenomenon in molecules and amino acid racemization phenomenon in the process of synthesizing the teduolutide, reduce a large amount of impurities with small structural difference, improve the purity of the crude teduolutide product, simplify the purification method of the crude teduolutide product, improve the yield of the product, and ensure the product quality and industrialization realization basis of the crude medicine of the teduolutide.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of teduglutide, which comprises the following steps:

step (1): preparing Phe-Ile-Asn (trt) -Trp (Boc) -Leu-OH to obtain a pentapeptide fragment;

asp (tBu) -Asn (trt) -Leu-OH is prepared to obtain tripeptide fragment (I);

preparing His (trt) -Gly-Asp (tBu) -OH to obtain tripeptide fragment (II);

preparing Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin to obtain a first peptide resin;

step (2): coupling the pentapeptide fragment with a first peptide resin to obtain a second peptide resin;

and (3): sequentially coupling Asp (tBu), Arg (pbf), Ala, tripeptide fragment (I), Leu, Ile, Thr (tBu), Asn (trt), Met, Glu (tBu), Asp (tBu), Ser (tBu), Phe, Ser (tBu), Gly and tripeptide fragment (II) on the second peptide resin to obtain the teduo peptide resin;

and (4): cracking the teduglutide resin to obtain a crude teduglutide product;

and (5): and purifying the crude teduglutide.

Preferably, step (1) is:

preparing Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-OH to obtain a pentapeptide fragment;

preparing Fmoc-Asp (tBu) -Asn (trt) -Leu-OH to obtain tripeptide fragment (I);

preparing Boc-His (trt) -Gly-Asp (tBu) -OH to obtain tripeptide fragment (II);

preparation of Fmoc-Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin to obtain a first peptide resin.

Preferably, the first peptide resin is prepared by the following method: after activation of the protected amino acid, Fmoc-Asp (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Lys (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Gln (trt) -OH and Fmoc-Ile-OH are coupled stepwise onto the resin to form Fmoc-Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin.

Preferably, the method for preparing the pentapeptide fragment comprises the following steps: after protective amino acid is taken for activation, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (trt) -OH, Fmoc-Ile-OH and Fmoc-Phe-OH are coupled step by step and are cracked to obtain Fmoc-Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-OH.

Preferably, the tripeptide fragment (I) is prepared by the following method: after protective amino acid is taken for activation, Fmoc-Leu-OH, Fmoc-Asn (trt) -OH and Fmoc-Asp (tBu) -OH are coupled step by step and are cracked to obtain Fmoc-Asp (tBu) -Asn (Trt) -Leu-OH.

Preferably, the tripeptide fragment (II) is prepared by the following method: activating protected amino acid, gradually coupling Fmoc-Asp (tBu) -OH, Fmoc-Gly-OH and Boc-His (trt) -OH, and cracking to obtain Boc-His (Trt) -Gly-Asp (tBu) -OH.

Intermediate fragment resin cleavage agent is TFA/DCM-10/9, 5/95, 2/98; or TFE/DCM-1/4, 1/2, 1/1, wherein TFE/DCM-1/1 is preferred.

Preferably, step (3) is: Fmoc-Asp (tBu) -OH, Fmoc-Arg (pbf) -OH, Fmoc-Ala-OH, tripeptide fragment (I), Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (trt) -OH, Fmoc-Met-OH, Fmoc-Glu (tBu) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH and tripeptide fragment (II) are coupled in this order on the second peptide resin to obtain the tetrodol peptide resin.

Preferably, the resin is a 2-Cl CTC resin or Wang resin.

Preferably, the resin is a 2-Cl CTC resin.

Preferably, the coupling agent used for coupling is one of HATU/HOBt/DIEA, HBTU/HOBt/DIEA, PyBOP/HOBt/DIEA, DIC/HOBt, DIC/HOAt or DIC/Cl-HOBt.

Preferably, the coupling reagent used for the coupling is DIC/HOBt.

Preferably, the cleavage is performed with a cleavage system of TFA/Tis/EDT, TFA/Tis/H₂O, TFA/water, TFA/phenol/thiobenzol/Tis/EDT/water.

Preferably, the cleavage is performed with a cleavage system of TFA/Tis/H₂O。

Preferably, the purification is carried out by Pre-HPLC.

Preferably, the conditions of Pre-HPLC are: c₁₈A reversed phase column; and (3) taking a 15-25 mM ammonium acetate water solution as a mobile phase A and acetonitrile as a mobile phase B, and performing gradient elution.

Preferably, the gradient of mobile phase B is 20% to 60% in 60 min.

Preferably, the gradient of mobile phase B is 25% to 55% over 60 min.

Preferably, the loading is 0.5% to 1% (mass ratio of sample to filler).

Preferably, the amount is 0.8%.

Preferably, the flow rate is 100 to 200 mL/min.

Preferably, the flow rate is 180 mL/min.

Preferably, the salt conversion conditions are as follows: 0.05% acetic acid water solution, acetonitrile gradient 35%, 40min, 40% -50%, 30 min.

Preferably, C is₁₈The parameters of the reversed phase column are: 10 μm, aperture of 100-120A.

Preferably, the purification step further comprises a lyophilization step.

The invention provides a preparation method of teduglutide. The preparation method comprises the following steps: preparing Phe-Ile-Asn (trt) -Trp (Boc) -Leu-OH to obtain a pentapeptide fragment; asp (tBu) -Asn (trt) -Leu-OH is prepared to obtain tripeptide fragment (I); preparing His (trt) -Gly-Asp (tBu) -OH to obtain tripeptide fragment (II); preparing Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin to obtain a first peptide resin; coupling the pentapeptide fragment with a first peptide resin to obtain a second peptide resin; sequentially coupling Asp (tBu), Arg (pbf), Ala, tripeptide fragment (I), Leu, Ile, Thr (tBu), Asn (trt), Met, Glu (tBu), Asp (tBu), Ser (tBu), Phe, Ser (tBu), Gly and tripeptide fragment (II) on the second peptide resin to obtain the teduo peptide resin; and (3) cracking and purifying the teduglutide resin. The invention has the technical effects that:

by introducing the fragment method, the method successfully breaks through the teduglutide¹The control difficulty of His racemic impurities is avoided²⁴Asn generates intramolecular autonomous ring formation and is improved¹⁶The coupling efficiency of Asn ensures the high-efficiency coupling of the whole sequence of the tedulpit, reduces the purification difficulty of the crude tedulpit, improves the yield of the product, greatly reduces the material cost and the purification cost, ensures the quality of the product, improves the single-batch production scale, and can realize the mass production of the tedulpit.

Drawings

FIG. 1 is the chemical structure of teduglutide;

FIG. 2 is a flow chart of the present invention for the synthesis of teduglutide;

FIG. 3 is a chromatogram of a purified teduglutide prepared in example 1 of the present invention;

FIG. 4 is a chromatogram of a purified teduglutide prepared in example 2 of the present invention;

FIG. 5 is a chromatogram of a purified teduglutide prepared in example 3 of the present invention.

Detailed Description

The invention discloses a preparation method of teduglutide, which can be realized by a person skilled in the art by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Some abbreviations used in the present invention have the following meanings:

the invention provides a preparation method of teduglutide, which comprises the following steps:

Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-OH (pentapeptide fragment), Fmoc-Asp (tBu) -Asn (trt) -Leu-OH [ tripeptide fragment (I) ], Boc-His (trt) -Gly-Asp (tBu) -OH [ tripeptide fragment (II) ] and Fmoc-Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin (first peptide resin) were prepared in advance;

then Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-OH (pentapeptide fragment) is coupled with Fmoc-Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin (first peptide resin) to obtain Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-Ile-Gln (trt) -Thr (tBu) -Lys (Lys) (Ile-Thr) (tBu) -Asp (tBu) -resin (second peptide resin);

Fmoc-Asp (tBu) -OH, Fmoc-Arg (pbf) -OH, Fmoc-Ala-OH, Fmoc-Asp (tBu) -Asn (trt) -Leu-OH [ tripeptide fragment (I) ], Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (trt) -OH, Fmoc-Met-OH, Fmoc-Glu (tBu) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-trOH and Boc-His (t) -Gly-Asp (tBu) -OH [ fragment (II ], obtaining Boc-His (trt) -Gly-Asp (tBu) -Gly-Ser (tBu) -Phe-Ser (tBu) -Asp (tBu) -Glu (tBu) -Met-Asn (trt) -Thr (tBu) -Ile-Leu-Asp (tBu) -Asn (trt) -Leu-Ala-Arg (pbf) -Asp (tBu) -Phe-Ile-Asn (trt) -Trp (Boc) -Leu-Ile-Gln (trt) -Thr (tBu) -Lys Boc-Ile-Thr (tBu) -Asp (tBu) -resin (tedu peptide resin);

and cracking the obtained teduglutide resin according to a polypeptide solid phase synthesis cracking method to obtain a crude product of the teduglutide.

And finally, purifying the crude product of the teduglutide by Pre-HPLC and freeze-drying to obtain a finished product of the teduglutide.

In a specific embodiment of the present invention, a specific method for the synthesis of teduglutide comprises the following steps:

(1) stepwise coupling Fmoc-Asp (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Lys (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Gln (trt) -OH, Fmoc-Ile-OH to form Fmoc-Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin (first peptide resin) after activation of the protected amino acid based on the amino acid sequence of the tedulpit peptide;

(2) after protective amino acid is taken and activated according to the amino acid sequence of the teduolutide, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (trt) -OH, Fmoc-Ile-OH and Fmoc-Phe-OH are coupled step by step and are cracked to obtain Fmoc-Phe-Ile-Asn (Trt) -Trp (Boc) -Leu-OH (pentapeptide fragment);

(3) after protective amino acid is taken and activated according to the amino acid sequence of the teduolutide, Fmoc-Leu-OH, Fmoc-Asn (trt) -OH and Fmoc-Asp (tBu) -OH are coupled step by step and are cracked to obtain Fmoc-Asp (tBu) -Asn (trt) -Leu-OH tripeptide fragment (I);

(4) according to the amino acid sequence of the teduglutide, after protective amino acid is activated, Fmoc-Asp (tBu) -OH, Fmoc-Gly-OH and Boc-His (trt) -OH are coupled step by step and are cracked to obtain Boc-His (Trt) -Gly-Asp (tBu) -OH tripeptide fragment (II);

(5) taking a first peptide resin, removing the N-terminal Fmoc protection, and coupling a pentapeptide fragment to obtain Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin (second peptide resin);

(6) taking a second peptide resin according to the amino acid sequence of the teduo peptide, after removing the Fmoc protection at the N-terminal, coupling Fmoc-Asp (tBu) -OH, Fmoc-Arg (pbf) -OH, Fmoc-Ala-OH, Fmoc-Asp (tBu) -Asn (trt) -Leu-OH [ tripeptide fragment (I) ], Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (trt) -OH, Fmoc-Met-OH, Fmoc-Glu (tBu) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH and Boc-His trt (t) -Asp (tBu) -OH [ tripeptide fragment (II) ], obtaining teduglutide resin;

(7) cracking the teduglutide resin to obtain a crude teduglutide product;

(8) and purifying the crude product of the teduglutide by Pre-HPLC and freeze-drying to obtain a finished product of the teduglutide.

In the step (7), the cleavage agent system of the teduglutide resin is TFA/Tis/EDT, TFA/Tis/H₂O, TFA/water or TFA/phenol/thioanisole/Tis/EDT/water, wherein TFA/Tis/H is preferred₂And (4) combining.

The resin in the steps (1), (2), (3) and (4) is 2-Cl CTC resin or Wang resin, wherein the 2-Cl CTC resin is preferred.

In the steps (1) to (6), the coupling agent is any one combination of HATU/HOBt/DIEA, HBTU/HOBt/DIEA, PyBOP/HOBt/DIEA, DIC/HOBt, DIC/HOAt or DIC/Cl-HOBt, wherein DIC/HOBt is preferred.

In the step (8), the Pre-HPLC purification method comprises the following steps: 10 μm, pore size of 100-120A, C₁₈The reverse phase column is characterized in that the purification condition is 15-25 mM (preferably 20mM) ammonium acetate aqueous solution, the acetonitrile gradient is 20-60%, preferably 25-55%, and 60min, the sample loading amount is 0.5-1% (mass ratio of sample to filler), preferably 0.8%, the salt conversion condition is 0.05% acetic acid aqueous solution, and the acetonitrile gradient is 35%, 40min, 40-50%, and 30 min.

The reagents or apparatus used in the present invention are commercially available.

The invention is further illustrated by the following examples:

example 1

A method for synthesizing teduglutide, as shown in fig. 2, comprising the following steps:

1. synthesis of first peptide resin

Weighing 2-Cl CTC resin (1.500g,1.5mmol) with the substitution degree of 1.013mmol/g, adding into a solid phase reaction vessel, adding DCM (15mL) for swelling for 10min, washing the resin with DCM for 3 times (15mL each time) after swelling is finished, and decompressing and draining the resin for later use.

Fmoc-Asp (tBu) — OH (0.191g, 0.45mmol) and DIEA (0.4mL, 2.25mmol) are dissolved in DCM (15mL), and after the solution is clear, the solution is added into the 2-Cl CTC resin for starting reaction, the reaction temperature is controlled between 25 ℃ and 30 ℃, and the reaction lasts for 2.5 hours. After the reaction was complete, the resin was washed 3 times with 15mL of DCM. The resin was charged with methanol/DIEA/DCM (volume ratio) (15mL) 1/2/7 and capped at 25-30 ℃ for 15min, after the reaction was complete the resin was drained and the capping operation repeated again 1 time. After blocking, the resin was washed 8 times with 15mL portions of DCM to obtain Fmoc-Asp- (tBu) -resin, which was sampled and tested for substitution by UV-spectrophotometer to find 0.281 mmol/g.

Fmoc-Asp (tBu) -resin is added into a solid phase reaction vessel, 20% piperidine-DMF solution (15mL) is added to remove the Fmoc protecting group, and after 15min of reaction, a sample of ninhydrin is taken for detection, so that the resin presents uniform blue color, which shows that the deprotection reaction is complete. The resin was drained and washed with DMF 8 times, 15mL each time, and the resin was drained under reduced pressure for use. Fmoc-Thr (tBu) -OH (0.630g,1.5mmol), HOBt (0.210g,1.5mmol) and DIC (0.194g,1.5mmol) are added into DMF (15mL) and stirred for 20min at room temperature, after the stirring is finished, the solution is added into the resin, the reaction is carried out at 25-30 ℃, after 1h, the resin is sampled and detected by ninhydrin, the resin is in a colorless transparent state, the reaction is shown to be finished, the resin is drained and washed with DMF for 8 times, 15mL each time, and Fmoc-Thr (tBu) -Asp (tBu) -resin is obtained.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially coupling Fmoc-Asp (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Lys (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Gln (trt) -OH and Fmoc-Ile-OH according to the sequence of teduolutide to obtain a first peptide resin, wherein the sequence is Fmoc-Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin.

2. Synthesis of pentapeptide fragments

Weighing 2-Cl CTC resin (3.000g,3.00mmol) with the substitution degree of 1.013mmol/g, adding into a solid phase reaction vessel, adding DCM (30mL) for swelling for 10min, washing the resin with DCM for 3 times (30mL each time) after swelling is finished, and draining the resin under reduced pressure for standby.

Dissolving Fmoc-Leu-OH (2.184g,6.00mmol) and DIEA (5.5mL, 30.00mmol) in DCM (30mL), adding the mixture into the 2-Cl CTC resin for starting reaction after the mixture is dissolved and clarified, controlling the reaction temperature to be between 25 and 30 ℃, and reacting for 3 hours. After the reaction was complete, the resin was washed 8 times with 30mL portions of DCM. The resin was charged with 1/2/7 (vol/vol) (30mL) methanol/DIEA/DCM and blocked at 25-30 ℃ for 15 min. And (4) repeating the end capping for 1 time, draining the resin after the reaction is finished, and repeating the end capping operation for 1 time again. After the end-capping, the resin was washed 8 times with 30mL portions of DCM. After the end-capping was completed, the resin was washed 8 times with DCM, 30mL each time, and the resin was drained for use to obtain Fmoc-Leu-resin.

Adding Fmoc-Leu-resin into a solid phase reaction container, adding 20% piperidine-DMF solution (30mL), removing Fmoc protecting groups for 15min, after the reaction is finished, sampling the resin, detecting with ninhydrin, wherein the resin presents uniform blue color, completely deprotecting, washing with DMF for 8 times after the resin is dried, each time, keeping the volume at 30mL, and drying the resin under reduced pressure for later use. Fmoc-Trp (Boc) -OH (3.196g,6.00mmol), HOBt (0.824g,6.00mmol) and DIC (1mL,6.00mmol) are added into DMF (30mL) and stirred for 20min at room temperature, after the stirring is finished, the solution is added into resin, reaction is carried out at 25-30 ℃, after 1h of reaction, the resin is sampled and detected by ninhydrin, the resin is colorless and transparent, the reaction is shown to be finished, after the resin is drained, the resin is washed by DMF for 8 times, 30mL is carried out every time, the resin is drained under reduced pressure for standby, and Fmoc-Trp (Boc) -Leu-resin is obtained.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially completing the coupling of Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (trt) -OH, Fmoc-Ile-OH and Fmoc-Phe-OH according to the sequence of teduglutide to obtain the pentapeptide fragment resin with the sequence of Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-resin.

Adding the pentapeptide fragment resin into TFE/DCM (1/4) (30mL), cracking for 1h at 25-30 ℃, collecting lysate, washing the resin with DCM for 2 times, and combining the lysate and the washing solution. The above lysis step was repeated 1 time, the pools were combined and concentrated to dryness under reduced pressure (-0.90 MPa, 35 ℃). The residue was added to MTBE (400mL), stirred for 30min and filtered, the filter cake was collected and dried under reduced pressure (35 ℃ C., -100Pa) to obtain Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-OH (pentapeptide fragment).

3. Synthesis of tripeptide fragment (I)

Weighing 2-Cl CTC resin (5.000g,5.00mmol) with the substitution degree of 1.013mmol/g, adding the 2-Cl CTC resin into a solid phase reaction vessel, adding DCM (50mL) to swell for 10min, washing the resin with DCM for 3 times after swelling, wherein 50mL of the resin is used for each time, and performing reduced pressure draining on the resin for standby.

Dissolving Fmoc-Leu-OH (3.541g, 10.00mmol) and DIEA (9.0mL, 50.00mmol) in DCM (50mL), adding the mixture into the 2-Cl CTC resin for starting reaction after the mixture is dissolved and clarified, controlling the reaction temperature to be between 25 and 30 ℃, and reacting for 2 hours. After the reaction was complete, the resin was washed 8 times with 50mL of DCM and the resin was drained under reduced pressure for further use. The resin was added with 1/2/7 (vol/vol) (50mL) methanol/DIEA/DCM, capped at 25-30 ℃ for 15min and repeated 1 time. After the end-capping was completed, the resin was washed 8 times with 50mL of DCM and the resin was drained under reduced pressure to obtain Fmoc-Leu-resin.

Adding Fmoc-Leu-resin into a solid phase reaction container, adding 20% piperidine-DMF solution (50mL), removing Fmoc protecting groups for 15min, after the reaction is finished, sampling ninhydrin from the resin for detection, detecting the resin to show uniform blue color, completely deprotecting, washing the resin with DMF for 8 times after draining, 50mL each time, and decompressing and draining the resin for later use. Adding Fmoc-Asn (trt) -OH (6.029g,10.00mmol), HOBt (1.362g,10.00mmol) and DIC (1.5mL,10.00mmol) into DMF (50mL) and stirring at room temperature for 15min, adding the solution into resin after stirring, reacting at 25-30 ℃, detecting the resin sample with ninhydrin after 1h reaction, displaying that the reaction is complete, washing the resin with DMF for 8 times after draining, and draining the resin at reduced pressure for 50mL each time for standby to obtain Fmoc-Asn (trt) -Leu-resin.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially completing the coupling of Fmoc-Leu-OH, Fmoc-Asn (trt) -OH and Fmoc-Asp (tBu) -OH according to the sequence of the teduo peptide to obtain a third peptide fragment (I) resin, wherein the sequence of the third peptide fragment (I) resin is Fmoc-Asp (tBu) -Asn (trt) -Leu-resin.

Adding the tripeptide fragment resin (I) into TFE/DCM (1/4) (50mL), cracking for 1h at 25-30 ℃, collecting lysate, washing the resin with DCM for 2 times, and combining the lysate and the washing solution. The above lysis step was repeated 1 time, the pools were combined and concentrated to dryness under reduced pressure (-0.90 MPa, 35 ℃). The residue was added to MTBE (500mL), stirred for 30min and filtered, and the filter cake was collected and dried under reduced pressure (35 ℃ C., -100Pa) to obtain Fmoc-Asp (tBu) -Asn (trt) -Leu-OH [ tripeptide fragment (I) ].

4. Synthesis of tripeptide fragment (II)

Weighing 2-Cl CTC resin (5.00g,5.00mmol) with the substitution degree of 1.013mmol/g, adding into a solid phase reaction vessel, adding DCM (50mL) for swelling for 10min, washing the resin with DCM for 3 times after swelling, each time 50mL, and draining the resin under reduced pressure for standby.

Dissolving Fmoc-Asp (tBu) -OH (4.128g, 10.00mmol) and DIEA (6.773g, 50.00mmol) in DCM (50mL), adding the mixture into the 2-Cl CTC resin for starting reaction after the mixture is dissolved and clarified, controlling the reaction temperature to be between 25 and 30 ℃, and reacting for 2.5 hours. After the reaction, the resin was washed with DCM for 8 times (50mL each time) and the resin was drained for further use. To the resin was added 1/2/7 (vol/vol) (50mL) methanol/DIEA/DCM, and the blocking was repeated 1 time at 25-30 ℃ for 15 min. After the end capping was completed, the resin was washed 8 times with DCM, 50mL each time, and the resin was dried under reduced pressure to obtain Fmoc-Asp (tBu) -resin.

Adding Fmoc-Asp (tBu) -resin into a solid-phase reaction vessel, adding 20% piperidine-DMF solution (50mL) to remove Fmoc protecting groups for 15min, after the reaction is finished, sampling the resin, detecting with ninhydrin, wherein the resin presents uniform blue color, completely deprotecting is shown, after the resin is decompressed and drained, washing with DMF for 8 times, 50mL each time, and draining the resin for standby. Adding Fmoc-Gly-OH (2.982g, 10.00mmol), HOBt (1.401g, 10.00mmol) and DIC (1.285g, 10.00mmol) into DMF (50mL), stirring for 15min, adding the solution into resin after stirring, reacting at 25-30 ℃, detecting the resin sample by ninhydrin after reacting for 1 hour, displaying that the resin is colorless and transparent, sucking the resin under reduced pressure, washing 8 times with DMF, 50mL each time, sucking the resin under reduced pressure for standby, and obtaining Fmoc-Gly-Asp (tBu) -resin.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially coupling Fmoc-Asp (tBu) -OH, Fmoc-Gly-OH and Boc-His (trt) -OH according to the sequence of the teduo peptide to obtain tripeptide fragment (II) resin with the sequence of Boc-His (trt) -Gly-Asp (tBu) -resin.

Adding the tripeptide fragment (II) resin into TFE/DCM 1/4(50mL), cracking for 1h at 25-30 ℃, collecting lysate, washing the resin with DCM for 2 times, and combining the lysate and the washing solution. The above lysis step was repeated 1 time, the pools were combined and concentrated to dryness under reduced pressure (-0.90 MPa, 35 ℃). The residue was added to MTBE (500mL), stirred for 30min and filtered, the filter cake was collected and dried under reduced pressure (35 ℃ C., -100Pa) to obtain Boc-His (trt) -Gly-Asp (tBu) -OH [ tripeptide fragment (II) ].

5. Synthesis of second peptide resin

And taking the first peptide resin and the pentapeptide fragment, and obtaining the second peptide resin according to the steps of removing the Fmoc protecting group and coupling amino acid.

6. Synthesis of crude teduglutide

Taking the second peptide resin, repeating the steps of removing Fmoc protecting group and coupling amino acid, and sequentially completing Fmoc-Asp (tBu) -OH, Fmoc-Arg (pbf) -OH, Fmoc-Ala-OH, Fmoc-Asp (tBu) -Asn (trt) -Leu-OH [ third peptide fragment (I) ], Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (trt) -OH, Fmoc-Met-OH, Fmoc-Glu (tBu) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH and Boc-trt-Gly (t) -Asp (tBu) -OH [ tripeptide fragment (II) ], and obtaining the teduglutide resin.

Mixing TFA, Tis and H₂Preparing a lysate (15mL) from O according to a volume ratio of 95: 5, adding the lysate into the teduglutide resin, cracking for 2 hours at room temperature (25 +/-5 ℃), filtering and collecting the lysate, washing the resin with the lysate (1.5mL), collecting filtrate, combining the filtrates, and concentrating to dryness (25-30 ℃ and-100 Pa). The residue was added to MTBE (150mL) and stirred for 0.5h, and the white solid was collected by filtration and dried under reduced pressure (25-30 ℃, -100Pa) to obtain 1.246g of crude teduglutide with a purity of 95.33% and a yield of 73.82%.

7. Purification of crude teduglutide

Sample pretreatment: the crude teduglutide (0.600g) was added to 0.1% TFA water (15mL) to dissolve clear and filter the filtrate until use.

Purification chromatographic conditions: detection wavelengths of 220nm and 254 nm; the sample loading amount is 30 mL; DAC80 nanometer/micrometer 10-100C of chromatographic column₁₈(ii) a The mobile phase A is 20mM ammonium acetate water solution, and the phase B is acetonitrile; the elution gradient is that the phase B is gradually increased from 20 percent to 60 percent within 60 min; the flow rate is 180 mL/min; samples were collected in stages and tested by HPLC, and pools containing no more than 0.1% of a single impurity were pooled and diluted with 2Bv volumes of purified water.

And (3) transferring salt chromatographic conditions: DAC80 nanometer 10-100C of chromatographic column₁₈(ii) a The mobile phase A is 0.05% acetic acid water solution, and the phase B is acetonitrile; the elution gradient is that after the B phase is kept at 25% within 40min, the concentration of the B phase is increased from 35% to 60% within 41-70 min; the flow rate is 180 mL/min; collecting samples by stages, detecting by HPLC, combining the collected liquid with single impurity not more than 0.1%, concentrating (30 degrees) until the volume is reduced to 1/3, and freeze-drying in a freeze dryer.

Purifying and lyophilizing to obtain 0.736g of teduglutide product with purity of 99.527%, as shown in FIG. 3; the purification yield was 59.00%; the total yield was 43.55%.

Example 2

A method for synthesizing teduglutide comprises the following steps:

1. synthesis of first peptide resin

Weighing 2-Cl CTC resin (3.000g,3.00mmol) with the substitution degree of 1.013mmol/g, adding into a solid phase reaction vessel, adding DCM (30mL) for swelling for 10min, washing the resin with DCM for 3 times (30mL each time) after swelling is finished, and draining the resin under reduced pressure for standby.

Dissolving Fmoc-Asp (tBu) — OH (0.381g, 0.90mmol) and DIEA (0.8mL, 2.50mmol) in DCM (30mL), adding the mixture into the 2-Cl CTC resin for starting reaction after the mixture is dissolved and clarified, and reacting for 2.5 hours at the reaction temperature of 25-30 ℃. After the reaction was complete, the resin was washed 3 times with 30mL portions of DCM. Adding methanol/DIEA/DCM (volume ratio) 1/2/7 (30mL) into the resin, blocking the resin at 25-30 ℃ for 15min, draining the resin after the reaction is finished, and repeating the blocking operation for 1 time again. After the end-capping, the resin was washed 8 times with 30mL of DCM to obtain Fmoc-Asp- (tBu) -resin, which was sampled and tested for substitution by UV-spectrophotometry, found to be 0.288 mmol/g.

Fmoc-Asp (tBu) -resin was added to the solid phase reaction vessel, and the Fmoc protecting group was removed by adding 20% piperidine-DMF solution (30mL), and after 15min of reaction, ninhydrin detection was sampled and the resin appeared uniformly blue, indicating complete deprotection. The resin was drained and washed with DMF 8 times, 30mL each time, and the resin was drained under reduced pressure for use. Fmoc-Thr (tBu) -OH (1.260g, 3.00mmol), HOBt (0.424g, 3.00mmol) and DIC (0.393g, 3.00mmol) are added into DMF (30mL) and stirred for 20min at room temperature, after the stirring is finished, the solution is added into the resin, the reaction is carried out at 25-30 ℃, after 1h, the resin is sampled and detected by ninhydrin, the resin is in a colorless transparent state, the reaction is shown to be finished, the resin is drained and washed with DMF for 8 times, 30mL each time, and Fmoc-Thr (tBu) -Asp (tBu) -resin is obtained.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially coupling Fmoc-Asp (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Lys (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Gln (trt) -OH and Fmoc-Ile-OH according to the sequence of teduolutide to obtain a first peptide resin, wherein the sequence is Fmoc-Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin.

2. Synthesis of pentapeptide fragments

Weighing 2-Cl CTC resin (3.000g,3.00mmol) with the substitution degree of 1.013mmol/g, adding into a solid phase reaction vessel, adding DCM (30mL) for swelling for 10min, washing the resin with DCM for 3 times (30mL each time) after swelling is finished, and draining the resin under reduced pressure for standby.

Dissolving Fmoc-Leu-OH (2.188g, 6.00mmol) and DIEA (5.5mL, 30.00mmol) in DCM (30mL), adding the mixture into the 2-Cl CTC resin for starting reaction after the mixture is dissolved and clarified, controlling the reaction temperature to be between 25 and 30 ℃, and reacting for 3 hours. After the reaction was complete, the resin was washed 8 times with 30mL portions of DCM. The resin was charged with 1/2/7 (vol/vol) (30mL) methanol/DIEA/DCM and blocked at 25-30 ℃ for 15 min. And (4) repeating the end capping for 1 time, draining the resin after the reaction is finished, and repeating the end capping operation for 1 time again. After the end-capping, the resin was washed 8 times with 30mL portions of DCM. After the end-capping was completed, the resin was washed 8 times with DCM, 30mL each time, and the resin was drained for use to obtain Fmoc-Leu-resin.

Adding Fmoc-Leu-resin into a solid phase reaction container, adding 20% piperidine-DMF solution (30mL), removing Fmoc protecting groups for 15min, after the reaction is finished, sampling the resin, detecting with ninhydrin, wherein the resin presents uniform blue color, completely deprotecting, washing with DMF for 8 times after the resin is dried, each time, keeping the volume at 30mL, and drying the resin under reduced pressure for later use. Fmoc-Trp (Boc) -OH (3.201g, 6.00mmol), HOBt (0.833g, 6.00mmol) and DIC (1.0mL, 6.00mmol) are added into DMF (30mL) and stirred for 20min at room temperature, after the stirring is finished, the solution is added into resin, reaction is carried out at 25-30 ℃, after 1h of reaction, the resin is sampled and detected by ninhydrin, the resin is colorless and transparent, the reaction is shown to be finished, after the resin is drained, the resin is washed by DMF for 8 times, 30mL is carried out each time, the resin is drained under reduced pressure for standby, and Fmoc-Trp (Boc) -Leu-resin is obtained.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially completing the coupling of Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (trt) -OH, Fmoc-Ile-OH and Fmoc-Phe-OH according to the sequence of teduglutide to obtain the pentapeptide fragment resin with the sequence of Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-resin.

Adding the pentapeptide fragment resin into TFE/DCM (1/4) (30mL), cracking for 1h at 25-30 ℃, collecting lysate, washing the resin with DCM for 2 times, and combining the lysate and the washing solution. The above lysis step was repeated 1 time, the pools were combined and concentrated to dryness under reduced pressure (-0.90 MPa, 35 ℃). The residue was added to MTBE (400mL), stirred for 30min and filtered, the filter cake was collected and dried under reduced pressure (35 ℃ C., -100Pa) to obtain Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-OH (pentapeptide fragment).

3. Synthesis of tripeptide fragment (I)

Weighing 2-Cl CTC resin (5.000g,5.00mmol) with the substitution degree of 1.013mmol/g, adding the 2-Cl CTC resin into a solid phase reaction vessel, adding DCM (50mL) to swell for 10min, washing the resin with DCM for 3 times after swelling, wherein 50mL of the resin is used for each time, and performing reduced pressure draining on the resin for standby.

Dissolving Fmoc-Leu-OH (3.544g, 10.00mmol) and DIEA (9.0mL, 50.00mmol) in DCM (50mL), adding the mixture into the 2-Cl CTC resin for starting reaction after the mixture is dissolved and clarified, controlling the reaction temperature to be between 25 and 30 ℃, and reacting for 2 hours. After the reaction was complete, the resin was washed 8 times with 50mL of DCM and the resin was drained under reduced pressure for further use. The resin was added with 1/2/7 (vol/vol) (50mL) methanol/DIEA/DCM, capped at 25-30 ℃ for 15min and repeated 1 time. After the end-capping was completed, the resin was washed 8 times with 50mL of DCM and the resin was drained under reduced pressure to obtain Fmoc-Leu-resin.

Adding Fmoc-Leu-resin into a solid phase reaction container, adding 20% piperidine-DMF solution (50mL), removing Fmoc protecting groups for 15min, after the reaction is finished, sampling ninhydrin from the resin for detection, detecting the resin to show uniform blue color, completely deprotecting, washing the resin with DMF for 8 times after draining, 50mL each time, and decompressing and draining the resin for later use. Adding Fmoc-Asn (trt) -OH (6.033g, 10.00mmol), HOBt (1.360g, 10.00mmol) and DIC (1.5mL,10.00mmol) into DMF (50mL) and stirring at room temperature for 15min, adding the solution into resin after stirring, reacting at 25-30 ℃, detecting the resin sample with ninhydrin after 1h reaction, displaying that the reaction is complete, washing the resin with DMF for 8 times after draining, and draining the resin at reduced pressure for 50mL each time for standby to obtain Fmoc-Asn (trt) -Leu-resin.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially completing the coupling of Fmoc-Leu-OH, Fmoc-Asn (trt) -OH and Fmoc-Asp (tBu) -OH according to the sequence of the teduo peptide to obtain a third peptide fragment (I) resin, wherein the sequence of the third peptide fragment (I) resin is Fmoc-Asp (tBu) -Asn (trt) -Leu-resin.

Adding the tripeptide fragment resin (I) into TFE/DCM (1/4) (50mL), cracking for 1h at 25-30 ℃, collecting lysate, washing the resin with DCM for 2 times, and combining the lysate and the washing solution. The above lysis step was repeated 1 time, the pools were combined and concentrated to dryness under reduced pressure (-0.90 MPa, 35 ℃). The residue was added to MTBE (500mL), stirred for 30min and filtered, and the filter cake was collected and dried under reduced pressure (35 ℃ C., -100Pa) to obtain Fmoc-Asp (tBu) -Asn (trt) -Leu-OH [ tripeptide fragment (I) ].

4. Synthesis of tripeptide fragment (II)

Weighing 2-Cl CTC resin (5.00g,5.00mmol) with the substitution degree of 1.013mmol/g, adding into a solid phase reaction vessel, adding DCM (50mL) for swelling for 10min, washing the resin with DCM for 3 times after swelling, each time 50mL, and draining the resin under reduced pressure for standby.

Dissolving Fmoc-Asp (tBu) -OH (4.131g, 10.00mmol) and DIEA (6.782g, 50.00mmol) in DCM (50mL), adding the mixture into the 2-Cl CTC resin for starting reaction after the mixture is dissolved and clarified, controlling the reaction temperature to be between 25 and 30 ℃, and reacting for 2.5 hours. After the reaction, the resin was washed with DCM for 8 times (50mL each time) and the resin was drained for further use. To the resin was added 1/2/7 (vol/vol) (50mL) methanol/DIEA/DCM, and the blocking was repeated 1 time at 25-30 ℃ for 15 min. After the end capping was completed, the resin was washed 8 times with DCM, 50mL each time, and the resin was dried under reduced pressure to obtain Fmoc-Asp (tBu) -resin.

Adding Fmoc-Asp (tBu) -resin into a solid-phase reaction vessel, adding 20% piperidine-DMF solution (50mL) to remove Fmoc protecting groups for 15min, after the reaction is finished, sampling the resin, detecting with ninhydrin, wherein the resin presents uniform blue color, completely deprotecting is shown, after the resin is decompressed and drained, washing with DMF for 8 times, 50mL each time, and draining the resin for standby. Fmoc-Gly-OH (2.990g, 10.00mmol), HOAt (1.411g, 10.00mmol) and DIC (1.280g, 10.00mmol) are added into DMF (50mL) and stirred for 15min, after stirring, the solution is added into resin and reacted at 25-30 ℃, after 1 hour of reaction, the resin is sampled and detected by ninhydrin, the resin is colorless and transparent, the reaction is shown to be completed, the resin is decompressed, drained and washed by DMF for 8 times, 50mL each time, and the resin is decompressed and drained for standby, thus obtaining Fmoc-Gly-Asp (tBu) -resin.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially coupling Fmoc-Asp (tBu) -OH, Fmoc-Gly-OH and Boc-His (trt) -OH according to the sequence of the teduo peptide to obtain tripeptide fragment (II) resin with the sequence of Boc-His (trt) -Gly-Asp (tBu) -resin.

Adding the tripeptide fragment (II) resin into TFE/DCM 1/4(50mL), cracking for 1h at 25-30 ℃, collecting lysate, washing the resin with DCM for 2 times, and combining the lysate and the washing solution. The above lysis step was repeated 1 time, the pools were combined and concentrated to dryness under reduced pressure (-0.90 MPa, 35 ℃). The residue was added to MTBE (500mL), stirred for 30min and filtered, the filter cake was collected and dried under reduced pressure (35 ℃ C., -100Pa) to obtain Boc-His (trt) -Gly-Asp (tBu) -OH [ tripeptide fragment (II) ].

5. Synthesis of second peptide resin

And taking the first peptide resin and the pentapeptide fragment, and obtaining the second peptide resin according to the steps of removing the Fmoc protecting group and coupling amino acid.

6. Synthesis of crude teduglutide

Taking the second peptide resin, repeating the steps of removing Fmoc protecting group and coupling amino acid, and sequentially completing Fmoc-Asp (tBu) -OH, Fmoc-Arg (pbf) -OH, Fmoc-Ala-OH, Fmoc-Asp (tBu) -Asn (trt) -Leu-OH [ third peptide fragment (I) ], Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (trt) -OH, Fmoc-Met-OH, Fmoc-Glu (tBu) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH and Boc-trt-Gly (t) -Asp (tBu) -OH [ tripeptide fragment (II) ], and obtaining the teduglutide resin.

Mixing TFA, Tis and H₂Preparing a lysate (15mL) from O according to a volume ratio of 95: 5, adding the lysate into the teduglutide resin, cracking for 2 hours at room temperature (25 +/-5 ℃), filtering and collecting the lysate, washing the resin with the lysate (1.5mL), collecting filtrate, combining the filtrates, and concentrating to dryness (25-30 ℃ and-100 Pa). The residue was added to MTBE (150mL) and stirred for 0.5h, the white solid was collected by filtration and dried under reduced pressure (25-30 ℃, -100Pa) to obtain 2.739g of crude teduglutide with a purity of 92.38% and a crude yield: 81.11 percent.

7. Purification of crude teduglutide

Sample pretreatment: the crude teduglutide (1.800g) was added to 0.1% TFA water (45mL) to dissolve clear and filter the filtrate until use.

Purification chromatographic conditions: detection wavelengths of 220nm and 254 nm; the sample loading amount is 30 mL; DAC80 nanometer/micrometer 10-100C of chromatographic column₁₈(ii) a The mobile phase A is 20mM ammonium acetate water solution, and the phase B is acetonitrile; the elution gradient is that the phase B is gradually increased from 20 percent to 60 percent within 60 min; the flow rate is 180 mL/min; samples were collected in stages and tested by HPLC, and pools containing no more than 0.1% of a single impurity were pooled and diluted with 2Bv volumes of purified water.

And (3) transferring salt chromatographic conditions: DAC80 nanometer 10-100C of chromatographic column₁₈(ii) a The mobile phase A is 0.05% acetic acid water solution, and the phase B is acetonitrile; the elution gradient is that after the B phase is kept at 25% within 40min, the concentration of the B phase is increased from 35% to 60% within 41-70 min; the flow rate is 180 mL/min; collecting samples by stages, detecting by HPLC, combining the collected liquid with single impurity not more than 0.1%, concentrating (30 degrees) until the volume is reduced to 1/3, and freeze-drying in a freeze dryer.

Purifying and freeze-drying to obtain 1.729g of the finished product of the teduglutide with the purity of 99.761 percent, as shown in figure 4; the purification yield was 61.31%; the total yield was 56.64%.

Example 3

A method for synthesizing teduglutide comprises the following steps:

1. synthesis of first peptide resin

Weighing 2-Cl CTC resin (30.000g,30.00mmol) with the substitution degree of 1.013mmol/g, adding into a solid phase reaction vessel, adding DCM (300mL) for swelling for 20min, washing the resin with DCM for 3 times (300mL each time) after swelling is finished, and draining the resin under reduced pressure for standby.

Dissolving Fmoc-Asp (tBu) -OH (3.734g, 9.00mmol) and DIEA (8mL, 45.00mmol) in DCM (300mL), adding the mixture into the 2-Cl CTC resin for starting reaction after the mixture is dissolved and clarified, controlling the reaction temperature to be between 25 and 30 ℃, and reacting for 3 hours. After the reaction was complete, the resin was washed 3 times with 300mL portions of DCM. Adding methanol/DIEA/DCM (volume ratio) 1/2/7 (300mL) into the resin, blocking the mixture at 25-30 ℃ for 30min, draining the resin after the reaction is finished, and repeating the blocking operation for 1 time again. After the end-capping, the resin was washed 8 times with DCM, 300mL each, giving Fmoc-Asp (tBu) -resin, and the resin was sampled and tested for substitution by UV-Spectrophotometry, found to be 0.285 mmol/g.

Fmoc-Asp (tBu) -resin was added to the solid phase reaction vessel, and the Fmoc protecting group was removed by adding 20% piperidine-DMF solution (300mL), and after 15min of reaction, ninhydrin detection was sampled and the resin appeared uniformly blue, indicating complete deprotection. The resin was drained and washed with DMF 8 times, 300mL each time, and the resin was drained under reduced pressure for use. Adding Fmoc-Thr (tBu) -OH (11.996g,30.00mmol), HOBt (4.203g,30.00mmol) and DIC (5mL,30.00mmol) into DMF (300mL) and stirring at room temperature for 20min, adding the solution into resin after stirring, reacting at 25-30 ℃, detecting the resin sample with ninhydrin after 1h reaction, displaying that the reaction is finished, draining the resin and washing the resin with DMF for 8 times, 300mL each time, and obtaining Fmoc-Thr (tBu) -Asp (tBu) -resin.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially coupling Fmoc-Asp (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Lys (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Gln (trt) -OH and Fmoc-Ile-OH according to the sequence of teduolutide to obtain a first peptide resin, wherein the sequence is Fmoc-Ile-Gln (trt) -Thr (tBu) -Lys (Boc) -Ile-Thr (tBu) -Asp (tBu) -resin.

2. Synthesis of pentapeptide fragments

Weighing 2-Cl CTC resin (50.000g,50.00mmol) with the substitution degree of 1.013mmol/g, adding into a solid phase reaction vessel, adding DCM (300mL) for swelling for 10min, washing the resin with DCM for 3 times (300mL each time) after swelling is finished, and draining the resin under reduced pressure for standby.

Dissolving Fmoc-Leu-OH (17.805g,50.00mmol) and DIEA (45mL, 250.00mmol) in DCM (300mL), adding the mixture into the 2-Cl CTC resin for starting reaction after the mixture is dissolved and clarified, and reacting for 3 hours at the reaction temperature controlled between 25 and 30 ℃. After the reaction was complete, the resin was washed 8 times with 300mL portions of DCM. The resin was charged with 1/2/7 (vol/vol) (300mL) methanol/DIEA/DCM and blocked at 25-30 ℃ for 15 min. And (4) repeating the end capping for 1 time, draining the resin after the reaction is finished, and repeating the end capping operation for 1 time again. After the end-capping, the resin was washed 8 times with 300mL portions of DCM. After the end capping was completed, the resin was washed 8 times with DCM, 300mL each, and the resin was drained to dryness to obtain Fmoc-Leu-resin.

Adding Fmoc-Leu-resin into a solid phase reaction container, adding 20% piperidine-DMF solution (300mL), removing Fmoc protecting groups for 15min, after the reaction is finished, sampling the resin, detecting with ninhydrin, wherein the resin presents uniform blue color, showing complete deprotection, washing with DMF for 8 times after the resin is drained, each time, 300mL, and decompressing and draining the resin for later use. Fmoc-Trp (Boc) -OH (52.713g,100.00mmol), HOBt (13.817g,100.00mmol) and DIC (16mL,100.00mmol) are added into DMF (500mL) and stirred for 20min at room temperature, after stirring, the solution is added into resin for reaction at 25-30 ℃, after 1h of reaction, the resin is sampled and detected by ninhydrin, the resin is colorless and transparent, the reaction is shown to be completed, after the resin is drained, the resin is washed by DMF for 8 times and 500mL each time, the resin is drained under reduced pressure for standby, and Fmoc-Trp (Boc) -Leu-resin is obtained.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially completing the coupling of Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (trt) -OH, Fmoc-Ile-OH and Fmoc-Phe-OH according to the sequence of teduglutide to obtain the pentapeptide fragment resin with the sequence of Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-resin.

Adding the pentapeptide fragment resin into TFE/DCM (1/4) (500mL), cracking for 1h at 25-30 ℃, collecting lysate, washing the resin with DCM for 2 times, and combining the lysate and the washing solution. The above lysis step was repeated 1 time, the pools were combined and concentrated to dryness under reduced pressure (-0.90 MPa, 35 ℃). The residue was added to MTBE (1000mL), stirred for 30min and filtered, the filter cake was collected and dried under reduced pressure (35 ℃ C., -100Pa) to obtain Fmoc-Phe-Ile-Asn (trt) -Trp (Boc) -Leu-OH (pentapeptide fragment).

3. Synthesis of tripeptide fragment (I)

Weighing 2-Cl CTC resin (40.000g,40.00mmol) with the substitution degree of 1.013mmol/g, adding the 2-Cl CTC resin into a solid phase reaction vessel, adding DCM (400mL) to swell for 10min, washing the resin with DCM for 3 times after swelling, each time 400mL, and draining the resin under reduced pressure for standby.

Dissolving Fmoc-Leu-OH (28.465g, 80.00mmol) and DIEA (70.0mL, 400.00mmol) in DCM (400mL), adding the mixture into the 2-Cl CTC resin for starting reaction after the mixture is dissolved and clarified, and reacting for 2 hours at the reaction temperature of 25-30 ℃. After the reaction was complete, the resin was washed 8 times with 50mL of DCM and the resin was drained under reduced pressure for further use. The resin was added with 1/2/7 (vol/vol) (400mL) methanol/DIEA/DCM and capped at 25-30 ℃ for 15min, and capping was repeated 1 time. After the end-capping was completed, the resin was washed 8 times with DCM, 400mL each time, and the resin was drained under reduced pressure for use to obtain Fmoc-Leu-resin.

Adding Fmoc-Leu-resin into a solid phase reaction container, adding 20% piperidine-DMF solution (400mL), removing Fmoc protecting groups for 15min, after the reaction is finished, sampling ninhydrin from the resin for detection, detecting the resin to show uniform blue color, completely deprotecting, washing the resin with DMF for 8 times after draining, each time 50mL, and decompressing and draining the resin for later use. Adding Fmoc-Asn (trt) -OH (47.775g,80.00mmol), HOBt (11.028g,80.00mmol) and DIC (13mL,80.00mmol) into DMF (400mL) and stirring for 15min at room temperature, adding the solution into resin after stirring, reacting at 25-30 ℃, sampling the resin after 1h of reaction and detecting with ninhydrin, displaying that the resin is in a colorless transparent state, completely reacting, washing the resin with DMF for 8 times after draining, and draining the resin at 400mL each time under reduced pressure for standby to obtain Fmoc-Asn (trt) -Leu-resin.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially completing the coupling of Fmoc-Leu-OH, Fmoc-Asn (trt) -OH and Fmoc-Asp (tBu) -OH according to the sequence of the teduo peptide to obtain a third peptide fragment (I) resin, wherein the sequence of the third peptide fragment (I) resin is Fmoc-Asp (tBu) -Asn (trt) -Leu-resin.

Adding the tripeptide fragment resin (I) into TFE/DCM (1/4) (400mL), cracking for 1h at 25-30 ℃, collecting lysate, washing the resin with DCM for 2 times, and combining the lysate and the washing solution. The above lysis step was repeated 1 time, the pools were combined and concentrated to dryness under reduced pressure (-0.90 MPa, 35 ℃). The residue was added to MTBE (2000mL), stirred for 30min and filtered, and the filter cake was collected and dried under reduced pressure (35 ℃ C., -100Pa) to obtain Fmoc-Asp (tBu) -Asn (trt) -Leu-OH [ tripeptide fragment (I) ].

4. Synthesis of tripeptide fragment (II)

Weighing 2-Cl CTC resin (50.00g,5.00mmol) with the substitution degree of 1.013mmol/g, adding into a solid phase reaction vessel, adding DCM (500mL) for swelling for 10min, washing the resin with DCM for 3 times after swelling, each time for 500mL, and draining the resin under reduced pressure for standby.

Fmoc-Asp (tBu) -OH (20.694g, 50.00mmol) and DIEA (44mL, 250.00mmol) are dissolved in DCM (500mL), and after the solution is clear, the solution is added into the 2-Cl CTC resin for starting reaction, the reaction temperature is controlled between 25 ℃ and 30 ℃, and the reaction lasts for 2.5 hours. After the reaction, the resin was washed with DCM 8 times (500mL each time) and the resin was drained for use. To the resin was added 1/2/7 (vol/vol) (50mL) methanol/DIEA/DCM, and the blocking was repeated 1 time at 25-30 ℃ for 15 min. After the end capping was completed, the resin was washed 8 times with DCM (500mL each) and the resin was dried under reduced pressure to obtain Fmoc-Asp (tBu) -resin.

Adding Fmoc-Asp (tBu) -resin into a solid-phase reaction vessel, adding 20% piperidine-DMF solution (500mL) to remove Fmoc protecting groups for 15min, after the reaction is finished, sampling the resin, detecting with ninhydrin, wherein the resin presents uniform blue color, completely deprotecting is shown, after the resin is decompressed and drained, washing with DMF for 8 times, 500mL each time, and draining the resin for standby. Adding Fmoc-Gly-OH (30.050g, 100.00mmol), HOBt (13.705g, 100.00mmol) and DIC (16mL,100.00mmol) into DMF (500mL), stirring for 15min, adding the solution into resin after stirring, reacting at 25-30 ℃, detecting the resin sample by ninhydrin after reacting for 1 hour, and displaying that the resin is colorless and transparent, and draining the resin under reduced pressure and washing the resin with DMF for 8 times, 500mL each time, and draining the resin under reduced pressure for standby use to obtain Fmoc-Gly-Asp (tBu) -resin.

Repeating the steps of removing the Fmoc protecting group and coupling the amino acid, and sequentially coupling Fmoc-Asp (tBu) -OH, Fmoc-Gly-OH and Boc-His (trt) -OH according to the sequence of the teduo peptide to obtain tripeptide fragment (II) resin with the sequence of Boc-His (trt) -Gly-Asp (tBu) -resin.

Adding the tripeptide fragment (II) resin into TFE/DCM (1/4) (500mL), cracking for 1h at 25-30 ℃, collecting lysate, washing the resin with DCM for 2 times, and combining the lysate and the washing solution. The above lysis step was repeated 1 time, the pools were combined and concentrated to dryness under reduced pressure (-0.90 MPa, 35 ℃). The residue was added to MTBE (500mL), stirred for 30min and filtered, the filter cake was collected and dried under reduced pressure (35 ℃ C., -100Pa) to obtain Boc-His (trt) -Gly-Asp (tBu) -OH [ tripeptide fragment (II) ].

5. Synthesis of second peptide resin

And taking the first peptide resin and the pentapeptide fragment, and obtaining the second peptide resin according to the steps of removing the Fmoc protecting group and coupling amino acid.

6. Synthesis of crude teduglutide

Taking the second peptide resin, repeating the steps of removing Fmoc protecting group and coupling amino acid, and sequentially completing Fmoc-Asp (tBu) -OH, Fmoc-Arg (pbf) -OH, Fmoc-Ala-OH, Fmoc-Asp (tBu) -Asn (trt) -Leu-OH [ third peptide fragment (I) ], Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (trt) -OH, Fmoc-Met-OH, Fmoc-Glu (tBu) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH and Boc-trt-Gly (t) -Asp (tBu) -OH [ tripeptide fragment (II) ], and obtaining the teduglutide resin.

Mixing TFA, Tis and H₂Preparing a lysate (300mL) from O according to the volume ratio of 95: 5, adding the lysate into the teduglutide resin, cracking for 2 hours at room temperature (25 +/-5 ℃), filtering and collecting the lysate, washing the resin with the lysate (300mL), collecting filtrate, combining the filtrates, and concentrating to dryness (25-30 ℃ and-100 Pa). The residue was added to MTBE (1500mL) and stirred for 0.5h, the white solid was collected by filtration and dried under reduced pressure (25-30 ℃, -100Pa) to obtain 27.021g of crude teduuide with a purity of 93.12% and a crude yield: 80.02 percent.

7. Purification of crude teduglutide

Sample pretreatment: the crude teduglutide (0.600g) was added to 0.1% TFA water (15mL) to dissolve clear and filter the filtrate until use.

Purification chromatographic conditions: detection wavelengths of 220nm and 254 nm; the sample loading amount is 30 mL; DAC80 nanometer/micrometer 10-100C of chromatographic column₁₈(ii) a The mobile phase A is 20mM ammonium acetate water solution, and the phase B is acetonitrile; the elution gradient is that the phase B is gradually increased from 20 percent to 60 percent within 60 min; the flow rate is 180 mL/min; samples were collected in stages and tested by HPLC, and pools containing no more than 0.1% of a single impurity were pooled and diluted with 2Bv volumes of purified water.

And (3) transferring salt chromatographic conditions: DAC80 nanometer 10-100C of chromatographic column₁₈(ii) a The mobile phase A is 0.05% acetic acid water solution, and the phase B is acetonitrile; the elution gradient is that after the B phase is kept at 25% within 40min, the concentration of the B phase is increased from 35% to 60% within 41-70 min; the flow rate is 180 mL/min; collecting samples by stages, detecting by HPLC, combining the collected liquid with single impurity not more than 0.1%, concentrating (30 degrees) until the volume is reduced to 1/3, and freeze-drying in a freeze dryer.

Purifying and freeze-drying to obtain 17.064g of the finished product of the teduglutide with the purity of 99.580 percent, as shown in figure 5; the purification yield is 63.15%; the total yield was 50.53%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.