阅读说明：本技术 一种生长抑素的合成方法 (Synthetic method of somatostatin ) 是由 陈永汉 尹传龙 唐洋明 余品香 于 2020-06-16 设计创作，主要内容包括：本发明公开了一种生长抑素的合成方法,包括以下步骤：1)制备生长抑素肽树脂；2)将生长抑素肽树脂进行裂解,并在裂解的过程中加入氧化剂进行氧化反应得到生长抑素粗肽。该合成方法中采用在粗肽裂解的过程中进行二硫键氧化,采用该氧化方法,首先,缩短了产品的生产时间；其次,避免了液相氧化产生的大量废液；另外,采用反应温和的氧化剂,可以避免相关氧化杂质的形成,提高产品的纯度及收率。本申请的新工艺精肽总收率与现有技术相比要高10％左右。(The invention discloses a synthesis method of somatostatin, which comprises the following steps: 1) preparing somatostatin peptide resin; 2) and (3) cracking the somatostatin peptide resin, and adding an oxidant in the cracking process to perform oxidation reaction to obtain the somatostatin crude peptide. The synthetic method adopts the oxidation of disulfide bonds in the cracking process of crude peptide, and firstly, the production time of the product is shortened by adopting the oxidation method; secondly, a large amount of waste liquid generated by liquid phase oxidation is avoided; in addition, the oxidant with mild reaction is adopted, so that the formation of related oxidation impurities can be avoided, and the purity and the yield of the product are improved. Compared with the prior art, the total yield of the refined peptide of the new process is about 10 percent higher.)

1. A synthesis method of somatostatin is characterized by comprising the following steps:

1) preparing somatostatin peptide resin;

2) and (3) cracking the somatostatin peptide resin, and adding an oxidant in the cracking process to perform oxidation reaction to obtain the somatostatin crude peptide.

2. The method of synthesizing somatostatin according to claim 1, wherein the oxidant in 2) includes 2,2 '-dithiodipyridine, 2' -dithiobis (5-nitropyridine), hydrogen peroxide or dimethyl sulfoxide; preferably, the oxidizing agent is 2,2' -dithiodipyridine, 2' -dithiobis (5-nitropyridine), more preferably, the oxidizing agent is 2,2' -dithiodipyridine.

3. The method for synthesizing somatostatin according to claim 1, wherein the molar ratio of the oxidant to the somatostatin peptide resin in 2) is 1-3: 1, preferably 2: 1.

4. the method for synthesizing somatostatin according to claim 1, wherein the time of the oxidation reaction in 2) is 1-3 hours, preferably, the time of the oxidation reaction is 2 hours.

5. The method for synthesizing somatostatin according to claim 1, wherein the cleavage solution used in the cleavage in 2) is TFA, TIS and H₂Mixture of O, said TFA, TIS and H₂The volume ratio of O is 80-95: 3-10: 2-10, preferably 90:5: 5;

the volume mass ratio of the lysate to the somatostatin peptide resin is 8-12 mL: 1g, preferably 10 mL: 1g of a compound;

the cracking temperature is 10-40 ℃, and preferably 20-30 ℃;

the time for the lysis is 1 to 3 hours, preferably 2 hours.

6. The method for synthesizing somatostatin according to claim 1, wherein the preparation method of the somatostatin peptide resin in 1) is as follows: according to Fmoc solid phase synthesis method, amino acids Fmoc-Cys (Trt) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH and Fmoc-Ala-OH are coupled to a solid phase carrier one by one to obtain a somatostatin peptide resin.

7. The method for synthesizing somatostatin according to claim 6, wherein 1) the solid-phase carrier is 2-CTC resin or Wang resin, preferably the solid-phase carrier is 2-CTC resin;

the substitution degree of the solid phase carrier is 0.6-1.2mmol/g, and preferably, the substitution degree of the solid phase carrier is 1.0 mmol/g.

8. The method for synthesizing somatostatin according to claim 6, wherein the preparation method of the somatostatin peptide resin in 1) specifically comprises: putting the solid phase carrier into a solid phase reaction column, swelling the solid phase carrier by using a solvent, dissolving Fmoc-Cys (Trt) -OH by using the solvent, adding DIPEA for activation, then adding the mixture into the solid phase carrier, and stirring for reacting for a period of time to obtain the Fmoc-Cys (Trt) -solid phase carrier;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating appropriate amount of Fmoc-Ser (tBu) -OH and coupling agent in solvent, and adding into a solid phase reaction column until the reaction termination is detected by detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Thr (tBu) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Phe-OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Thr (tBu) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; after dissolving and activating an appropriate amount of Fmoc-Lys (Boc) -OH and a coupling agent in a solvent, adding the mixture to a solid phase reaction column until the reaction is terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Trp (Boc) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Phe-OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Phe-OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating appropriate amount of Fmoc-Asn (Trt) -OH and coupling agent in solvent, and adding into solid phase reaction column until reaction termination is detected by detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; after dissolving and activating an appropriate amount of Fmoc-Lys (Boc) -OH and a coupling agent in a solvent, adding the mixture to a solid phase reaction column until the reaction is terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Cys (Trt) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating appropriate amount of Fmoc-Gly-OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction termination is detected by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; after the appropriate amount of Fmoc-Ala-OH and coupling reagent are dissolved in solvent and activated, they are added to the solid phase reaction column until the end of the reaction is detected by detection.

9. The method for synthesizing somatostatin according to claim 8, wherein the reagent for removing Fmoc is 20% piperidine/DMF solution;

preferably, the coupling agent is DIPCDI, DIPEA, compound a, a combination of compound B, DIPCDI and compound a or a combination of DIPEA and compound a and compound B, wherein compound a is HOBt or HOAt and compound B is PyBOP, PyAOP, HATU, HBTU or TBTU, preferably the coupling agent is a combination of DIPCDI and HOBt, preferably the molar ratio of DIPCDI to HOBt is 1: 1-2, preferably 1: 1.2.

10. the method for synthesizing somatostatin according to claim 1, wherein the step of synthesizing the crude somatostatin peptide is followed by a step of purifying and salt-converting the crude somatostatin peptide;

preferably, the synthesis of the crude somatostatin peptide further comprises dissolving the crude somatostatin peptide in purified water, purifying the solution by using octadecylsilane chemically bonded silica filler as a stationary phase for two steps, and performing salt conversion by one step to obtain the fine somatostatin peptide.

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to a synthesis method of somatostatin.

Background

Somatostatin is an artificially synthesized cyclic tetradecanoic acid peptide which is identical to natural somatostatin in chemical structure and action mechanism. Physiological somatostatin is mainly present in the hypothalamus and in the gastrointestinal tract. Somatostatin inhibits the secretion of growth hormone, thyroid stimulating hormone, insulin and glucagon and inhibits the secretion of gastric acid by intravenous injection. It also affects the absorption, motility, visceral blood flow and nutritional function of the gastrointestinal tract. Somatostatin can inhibit secretion of gastrin, gastric acid and pepsin, so as to treat upper gastrointestinal hemorrhage, obviously reduce blood flow of internal organs, and does not cause significant change of systemic arterial blood pressure, thus having certain clinical value in treating esophageal variceal hemorrhage. Somatostatin reduces endocrine and exocrine secretion of pancreas and is used for preventing and treating complications after pancreatic surgery. Somatostatin also inhibits glucagon secretion, thereby effectively treating diabetic ketoacidosis.

Somatostatin has the following structural formula:

currently, the following schemes are mainly reported about the synthesis method of somatostatin:

patent CN1552728A discloses a method for synthesizing somatostatin polypeptide, using Wang resin as initial raw material, using Fmoc protected amino acid as monomer, connecting amino acids one by one, using cleavage reagent (TFA/EDT/H)₂O/TIS), adding ether to precipitate crude peptide, introducing air to oxidize at 15-35 ℃ at pH 7.0-10.0, and separating and purifying by using a C18 column, wherein the total yield of the refined peptide is 22.1-22.9%.

Patent CN1923851A discloses a preparation method for synthesizing somatostatin by solid-phase polypeptide. The method comprises the steps of firstly, taking trityl resin, 4-methyl trityl resin or 4-methoxy trityl resin as starting raw materials, sequentially connecting protected amino acids according to a solid-phase synthesis method to obtain protected tetradecapeptide resin, cracking to obtain reduced somatostatin, oxidizing at the pH of 7-11 by using air to obtain a crude somatostatin product, and separating and purifying by using a C18 (or C8) high-pressure column to obtain a refined somatostatin product, wherein the total yield of the refined peptide is 25%.

Patent CN102952175A discloses a method for synthesizing somatostatin by solid-phase polypeptide. The method comprises the steps of taking 2-chlorotrityl alcohol resin as a starting raw material, sequentially connecting amino acids with protecting groups according to a solid-phase synthesis method to obtain protected reduced tetradecapeptide resin, cracking to obtain reduced somatostatin, oxidizing with hydrogen peroxide under the condition that the pH value is 7-9 to obtain a crude somatostatin product, separating and purifying through a C18 high-efficiency liquid-phase column, and freeze-drying to obtain a refined somatostatin product with the total yield of 26.24%.

Patent CN100338090C discloses the oxidation and cyclization of somatostatin linear peptide in hydrogen peroxide or dimethyl sulfoxide aqueous solution to obtain crude somatostatin trifluoroacetate. The crude product is separated and purified by a methanol-water gradient elution method through a C18 bonded silica gel medium-pressure column to obtain a refined product of somatostatin trifluoroacetic acid, and no specific yield data exists.

In the above patents, after peptide resin is synthesized by solid phase, linear crude peptide is obtained by cracking, then the liquid phase is oxidized by air, hydrogen peroxide or dimethyl sulfoxide respectively to obtain crude somatostatin, and then purified to obtain refined somatostatin. In general, 1000kg of oxidizing solution is required to oxidize 1000g of crude peptide.

The disadvantages of liquid phase oxidation are: a large amount of solvent is needed for dissolution and then oxidation reaction is carried out, so that large-scale oxidation equipment is needed; the treatment capacity of the waste liquid after oxidation is large; in addition, strong oxidants such as hydrogen peroxide or dimethyl sulfoxide are adopted to cause oxidation of Trp or Cys in peptide sequences, so that impurities are increased.

In view of the above problems in the prior art, there is a need to develop a new method for synthesizing somatostatin.

Disclosure of Invention

In order to solve the problems of the background art, the present invention aims to provide a method for synthesizing somatostatin. The synthetic method adopts the oxidation of disulfide bonds in the cracking process of crude peptide, and firstly, the production time of the product is shortened by adopting the oxidation method; secondly, a large amount of waste liquid generated by liquid phase oxidation is avoided; in addition, the oxidant with mild reaction is adopted, so that the formation of related oxidation impurities can be avoided, and the purity and the yield of the product are improved. Compared with the prior art, the total yield of the refined peptide of the new process is about 10 percent higher.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a synthesis method of somatostatin, comprising the following steps:

1) preparing somatostatin peptide resin;

2) and (3) cracking the somatostatin peptide resin, and adding an oxidant in the cracking process to perform oxidation reaction to obtain the somatostatin crude peptide.

Further, the oxidizing agent in 2) includes 2,2 '-dithiodipyridine, 2' -dithiobis (5-nitropyridine), hydrogen peroxide or dimethyl sulfoxide; preferably, the oxidizing agent is 2,2' -dithiodipyridine, 2' -dithiobis (5-nitropyridine), more preferably, the oxidizing agent is 2,2' -dithiodipyridine.

Further, the molar ratio of the oxidant to the somatostatin peptide resin in 2) is 1-3: 1, preferably 2: 1.

further, the time of the oxidation reaction in 2) is 1 to 3 hours, and preferably, the time of the oxidation reaction is 2 hours.

Further, the lysis solution used in the cleavage in 2) is TFA, TIS and H₂Mixture of O, said TFA, TIS and H₂The volume ratio of O is 80-95: 3-10: 2-10, preferably 90:5: 5;

the volume mass ratio of the lysate to the somatostatin peptide resin is 8-12 mL: 1g, preferably 10 mL: 1g of a compound;

the cracking temperature is 10-40 ℃, and preferably 20-30 ℃;

the time for the lysis is 1 to 3 hours, preferably 2 hours.

Further, the preparation method of the somatostatin peptide resin in 1) comprises the following steps: according to Fmoc solid phase synthesis method, amino acids Fmoc-Cys (Trt) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH and Fmoc-Ala-OH are coupled to a solid phase carrier one by one to obtain a somatostatin peptide resin.

Further, the solid phase carrier in 1) is 2-CTC resin or Wang resin, preferably, the solid phase carrier is 2-CTC resin; the substitution degree of the solid phase carrier is 0.6-1.2mmol/g, and preferably, the substitution degree of the solid phase carrier is 1.0 mmol/g.

Further, the preparation method of the somatostatin peptide resin in 1) specifically comprises the following steps: putting the solid phase carrier into a solid phase reaction column, swelling the solid phase carrier by using a solvent, dissolving Fmoc-Cys (Trt) -OH by using the solvent, adding DIPEA for activation, then adding the mixture into the solid phase carrier, and stirring for reacting for a period of time to obtain the Fmoc-Cys (Trt) -solid phase carrier;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating appropriate amount of Fmoc-Ser (tBu) -OH and coupling agent in solvent, and adding into a solid phase reaction column until the reaction termination is detected by detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Thr (tBu) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Phe-OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Thr (tBu) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; after dissolving and activating an appropriate amount of Fmoc-Lys (Boc) -OH and a coupling agent in a solvent, adding the mixture to a solid phase reaction column until the reaction is terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Trp (Boc) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Phe-OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Phe-OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating appropriate amount of Fmoc-Asn (Trt) -OH and coupling agent in solvent, and adding into solid phase reaction column until reaction termination is detected by detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; after dissolving and activating an appropriate amount of Fmoc-Lys (Boc) -OH and a coupling agent in a solvent, adding the mixture to a solid phase reaction column until the reaction is terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating an appropriate amount of Fmoc-Cys (Trt) -OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction is detected to be terminated by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; dissolving and activating appropriate amount of Fmoc-Gly-OH and a coupling agent in a solvent, and adding the mixture into a solid-phase reaction column until the reaction termination is detected by a detection method;

removing Fmoc, and washing the resin until the Fmoc is completely removed; after the appropriate amount of Fmoc-Ala-OH and coupling reagent are dissolved in solvent and activated, they are added to the solid phase reaction column until the end of the reaction is detected by detection.

Further, the reagent for removing Fmoc is 20% piperidine/DMF solution;

preferably, the coupling agent is DIPCDI, DIPEA, compound a, a combination of compound B, DIPCDI and compound a or a combination of DIPEA and compound a and compound B, wherein compound a is HOBt or HOAt and compound B is PyBOP, PyAOP, HATU, HBTU or TBTU, preferably the coupling agent is a combination of DIPCDI and HOBt, preferably the molar ratio of DIPCDI to HOBt is 1: 1-2, preferably 1: 1.2.

further, the step of synthesizing the somatostatin crude peptide also comprises the steps of purifying the somatostatin crude peptide and transferring salt;

preferably, the synthesis of the crude somatostatin peptide further comprises dissolving the crude somatostatin peptide in purified water, purifying the solution by using octadecylsilane chemically bonded silica filler as a stationary phase for two steps, and performing salt conversion by one step to obtain the fine somatostatin peptide.

The invention has the beneficial effects that: according to the invention, an oxidant is added in the cracking process to directly oxidize the crude peptide to obtain the crude peptide, and the novel oxidation method can reduce the traditional liquid phase oxidation step, improve the synthesis efficiency and shorten the production time of the product; the new synthesis method can effectively reduce the solvent consumption generated by liquid-phase oxidation, avoid a large amount of waste liquid generated by liquid-phase oxidation and achieve the effect of waste reduction; in addition, the oxidant with mild reaction is adopted, so that the formation of related oxidation impurities can be avoided, and the purity and yield of the product are improved; compared with the prior art, the total yield of the refined peptide of the new process is about 10 percent higher.

Drawings

FIG. 1 is a flow chart of the synthesis of a crude somatostatin peptide according to an embodiment of the invention;

FIG. 2 is an HPLC chromatogram of a somatostatin crude peptide in example 11 of the present invention;

FIG. 3 is an HPLC chromatogram of somatostatin protide in example 12 of the present invention;

FIG. 4 is a mass spectrum of somatostatin protide in example 12 of the present invention.

Detailed Description

For a better understanding of the present invention, the following examples are given to illustrate the present invention, but the present invention is not limited to the following examples.

The meanings of abbreviations used in the specification and claims are listed in the following table:

example 1 Synthesis of peptide resin

The flow is shown in part in figure 1.

40g of 2-CTC Resin with a degree of substitution of 1.0mmol/g were weighed. Adding into a solid phase reaction column, washing with DMF for 2 times, swelling resin with DMF for 30 min, dissolving 46.8g Fmoc-Cys (Trt) -OH with 1000mL DMF, adding 5.0mL DIPEA under ice water bath for activation, adding into the reaction column filled with resin, reacting for 2 hr, adding 15mL anhydrous methanol, and blocking for 0.5 hr. Washing with DMF 3 times, washing with DCM 3 times, shrinking with anhydrous methanol 3 times, draining off solvent, and vacuum drying to obtain Fmoc-Cys (Trt) -2-CTC resin with detection substitution degree of 0.51 mmol/g.

Weighing 39.2g (20mmol) of Fmoc-Cys (Trt) -2-CTC resin with the substitution degree of 0.51mmol/g, adding the resin into a solid-phase reaction column, washing the solid-phase reaction column with DMF for 2 times, swelling the Fmoc-Cys (Trt) -2-CTC resin with DMF for 30 minutes, removing Fmoc protection by DBLK, washing the solid-phase reaction column with DMF for 4 times, washing the solid-phase reaction column with DCM for 2 times, and detecting the color of the resin by an indetrione method, wherein the color of the resin indicates that Fmoc is removed. Dissolving 23.0g of Fmoc-Ser (tBu) -OH (60mmol) and 9.74g of HOBt (72mmol) in a mixed solution of DCM and DMF at a volume ratio of 1: 1, adding 9.84g of DIPCDI (78mmol) in an ice-water bath for activation for 3min, adding the activated product into a solid-phase reaction column, and reacting at room temperature for 2 hours. Detecting and judging the reaction end point by an indetrione method, and if the resin is colorless and transparent, indicating that the reaction is complete; and if the resin is developed, the reaction is not complete, the coupling reaction needs to be carried out for 1 hour, and the judgment standard is suitable for judging the reaction endpoint by using an indetrione method in subsequent contents.

The above steps of removing Fmoc protection and adding the corresponding amino acid for coupling were repeated, and the coupling of Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH, Fmoc-Ala-OH was accomplished in order according to the peptide sequence of somatostatin. The resulting product was washed 3 times with DMF, 5 times with DCM, shrunk with methanol and dried overnight under vacuum to give 80.4g of peptide resin.

Example 2 Synthesis of peptide resin

The flow is shown in part in figure 1.

40g of 2-CTC Resin with a degree of substitution of 1.0mmol/g were weighed. Adding into a solid phase reaction column, washing with DMF for 2 times, swelling resin with DMF for 30 minutes, dissolving 46.8g Fmoc-Cys (Trt) -OH with 1000mL DMF, adding 10.0mL DIPEA under ice water bath for activation, adding into the reaction column filled with resin, reacting for 2 hours, adding 30mL anhydrous methanol, and blocking for 0.5 hour. Washing with DMF 3 times, washing with DCM 3 times, shrinking with anhydrous methanol 3 times, draining off solvent, and vacuum drying to obtain Fmoc-Cys (Trt) -2-CTC resin with detection substitution degree of 0.48 mmol/g.

41.7g (20mmol) of Fmoc-Cys (Trt) -2-CTC resin with the substitution degree of 0.48mmol/g is weighed and added into a solid phase reaction column, the solid phase reaction column is washed with DMF for 2 times, after the Fmoc-Cys (Trt) -2-CTC resin is swelled with DMF for 30 minutes, the Fmoc protection is removed with DBLK, then the solid phase reaction column is washed with DMF for 4 times, DCM is washed for 2 times, and the color of the resin is detected by an indetrione method, and the color of the resin is shown to indicate that the Fmoc is removed. Dissolving 23.0g of Fmoc-Ser (tBu) -OH (60mmol) and 9.74g of HOBt (72mmol) in a mixed solution of DCM and DMF at a volume ratio of 1: 1, adding 9.84g of DIPCDI (78mmol) in an ice-water bath for activation for 3min, adding the activated product into a solid-phase reaction column, and reacting at room temperature for 2 hours. Detecting and judging the reaction end point by an indetrione method, and if the resin is colorless and transparent, indicating that the reaction is complete; and if the resin is developed, the reaction is not complete, the coupling reaction needs to be carried out for 1 hour, and the judgment standard is suitable for judging the reaction endpoint by using an indetrione method in subsequent contents.

The above steps of removing Fmoc protection and adding the corresponding amino acid for coupling were repeated, and the coupling of Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH, Fmoc-Ala-OH was accomplished in order according to the peptide sequence of somatostatin. The resulting product was washed 3 times with DMF, 5 times with DCM, shrunk with methanol and dried overnight under vacuum to give 81.1g of peptide resin.

Example 3 Linear crude peptide cleavage of somatostatin

10g of the peptide resin of example 1 was added to a 500m1 reaction flask, and 100mL of the prepared cleavage reagent (TFA: H)₂TIS ═ 90:5:5) was poured into the above reaction flask, and reacted at room temperature for 2 hours. And after the reaction is finished, filtering the reaction solution, pouring the reaction solution into 1L of glacial ethyl ether, washing out a large amount of white precipitate, centrifuging, washing, drying and weighing to obtain 3.95g of the somatostatin linear crude peptide, wherein the yield is 96.8%, the HPLC purity is 83.7%, and the MS 1639.7 is obtained.

Example 4 Linear crude somatostatin peptide cleavage

10g of the peptide resin of example 2 was added to a 500m1 reaction flask, and 100mL of the formulated cleavage reagent (TFA: H)₂TIS ═ 90:5:5) was poured into the above reaction flask, and reacted at room temperature for 2 hours. And after the reaction is finished, filtering the reaction solution, pouring the reaction solution into 1L of glacial ethyl ether, washing out a large amount of white precipitate, centrifuging, washing, drying and weighing to obtain 3.85g of somatostatin linear crude peptide, wherein the yield is 94.3%, the HPLC purity is 84.2%, and MS 1639.8 is obtained.

Example 5 cleavage of crude somatostatin peptide

The flow is shown in part in figure 1.

10g of the peptide resin of example 1 was added to a 500m1 reaction flask, and 100mL of the prepared cleavage reagent (TFA: H)₂TIS ═ 90:5:5) was poured into the above reaction flask, and reacted at room temperature for 2 hours, then 0.55g of 2,2' -dithiodipyridine was added, and the reaction was continued for 1 hour. And after the reaction is finished, filtering the reaction liquid, pouring the reaction liquid into 1L of glacial ethyl ether, washing out a large amount of white precipitate, centrifuging, washing, drying and weighing to obtain 3.80g of the somatostatin crude peptide, wherein the yield is 93.4%, the HPLC purity is 78.7%, and the MS1637.7 is obtained.

Example 6 cleavage of crude somatostatin peptide

The flow is shown in part in figure 1.

10g of the peptide resin of example 1 was added to a 500m1 reaction flask, and 100mL of the prepared cleavage reagent (TFA: H)₂TIS ═ 90:5:5) was poured into the above reaction flask, and reacted at room temperature for 2 hours, then 1.1g of 2,2' -dithiodipyridine was added, and the reaction was continued for 1 hour. And after the reaction is finished, filtering the reaction liquid, pouring the reaction liquid into 1L of glacial ethyl ether, washing out a large amount of white precipitate, centrifuging, washing, drying and weighing to obtain 3.78g of the somatostatin crude peptide, wherein the yield is 92.9%, the HPLC purity is 81.2%, and the MS 1637.8 is obtained.

Example 7 crude somatostatin peptide cleavage

The flow is shown in part in figure 1.

10g of the peptide resin of example 1 was added to a 500m1 reaction flask, and 100mL of the prepared cleavage reagent (TFA: H)₂TIS ═ 90:5:5) was poured into the above reaction flask, and reacted at room temperature for 2 hours, then 1.1g of 2,2' -dithiodipyridine was added, and the reaction was continued for 2 hours. After the reaction is finished, the reaction solution is filtered and poured into 1L of ethyl glacial ether, a large amount of white precipitate is washed out, centrifuged, washed, dried and weighed, and then 3.86g of the somatostatin crude peptide is obtained, the yield is 94.9%, the HPLC purity is 84.5%, and MS1637.7 is obtained.

Example 8 cleavage of crude somatostatin peptide

The flow is shown in part in figure 1.

10g of the peptide resin of example 1 was added to a 500m1 reaction flask, and 100mL of the prepared cleavage reagent (TFA: H)₂TIS ═ 90:5:5) was poured into the above reaction flask, and reacted at room temperature for 2 hours, then 1.1g of 2,2' -dithiodipyridine was added, and the reaction was continued for 3 hours. And after the reaction is finished, filtering the reaction liquid, pouring the reaction liquid into 1L of glacial ethyl ether, washing out a large amount of white precipitate, centrifuging, washing, drying and weighing to obtain 3.65g of the somatostatin crude peptide, wherein the yield is 89.9%, the HPLC purity is 80.7%, and the MS 1637.8 is obtained.

Example 9 cleavage of crude somatostatin peptide

The flow is shown in part in figure 1.

10g of the peptide resin of example 1 was added to a 500m1 reaction flask100mL of the prepared cleavage reagent (TFA: H)₂TIS ═ 90:5:5) was poured into the above reaction flask, and reacted at room temperature for 2 hours, then 1.65g of 2,2' -dithiodipyridine was added, and the reaction was continued for 2 hours. After the reaction is finished, the reaction solution is filtered and poured into 1L of ethyl glacial ether, a large amount of white precipitate is washed out, centrifuged, washed, dried and weighed, and then 3.82g of crude somatostatin peptide is obtained, the yield is 94.1%, the HPLC purity is 83.7%, and MS 1637.8 is obtained.

Example 10 cleavage of crude somatostatin peptide

The flow is shown in part in figure 1.

10g of the peptide resin of example 1 was added to a 500m1 reaction flask, and 100mL of the prepared cleavage reagent (TFA: H)₂TIS ═ 90:5:5) was poured into the above reaction flask, and reacted at room temperature for 2 hours, followed by addition of 1.54g of 2,2' -dithiobis (5-nitropyridine) and continued reaction for 2 hours. After the reaction is finished, the reaction solution is filtered and poured into 1L of ethyl glacial ether, a large amount of white precipitate is washed out, centrifuged, washed, dried and weighed, and then 3.80g of the somatostatin crude peptide is obtained, the yield is 93.4%, the HPLC purity is 82.3%, and MS1637.7 is obtained.

Example 11 amplification of somatostatin crude peptide cleavage

The flow is shown in part in figure 1.

60g of the peptide resin of example 2 was added to a 1000m1 reaction flask and 600mL of the formulated cleavage reagent (TFA: H)₂TIS ═ 90:5:5) was poured into the above reaction flask, and reacted at room temperature for 2 hours, followed by addition of 6.6g of 2,2' -dithiodipyridine and continued reaction for 2 hours. After the reaction is finished, the reaction solution is filtered and poured into 1L of ethyl glacial ether, a large amount of white precipitate is washed out, centrifuged, washed, dried and weighed to obtain 23.1g of somatostatin crude peptide, the yield is 95.2%, the HPLC purity is 84.7% (see figure 2), and MS1637.7 is obtained.

Example 12 preparation of somatostatin

Sample treatment: dissolving somatostatin synthesized in example 11 in purified water (the dissolving concentration is about 15mg/ml), filtering with a filter membrane with the pore diameter of 0.45um, and collecting the filtrate for later use;

the first step of purification conditions: a chromatographic column: the chromatographic column using octadecylsilane chemically bonded silica filler as a stationary phase has the following diameter and length: 10cm × 25cm, mobile phase: phase A: proportioning a phosphoric acid aqueous solution with the mass percentage concentration of 0.1%, and adjusting the pH value to 2.5 by using triethylamine; phase B: pure acetonitrile. Flow rate: 150-300 ml/min. Detection wavelength: 230 nm. Gradient: the mass percentage concentration of the mobile phase B is as follows: 20-65%, gradient treatment time 40-55 min. The sample injection amount is 12 g;

and (3) purification process: and (3) washing the chromatographic column with acetonitrile with the mass percent concentration of 60%, balancing, and then loading, wherein the loading amount is the sample solution after dissolution and filtration. Carrying out linear gradient elution, collecting a target peak, taking a fraction with the purity of more than or equal to 95 percent as a qualified fraction, and placing the collected peptide solution in a collection bottle for later use; concentrating under reduced pressure to a certain volume except excessive acetonitrile, and preparing for the second purification step;

and the second step of purification conditions: a chromatographic column: the chromatographic column using octadecylsilane chemically bonded silica as a stationary phase has the following diameter and length: 10cm × 25cm, mobile phase: the matching ratio of A contains 0.2 percent of hydrochloric acid aqueous solution by mass percentage; phase B: pure acetonitrile solution. Flow rate: 150-300 ml/min. The detection wavelength is 230 nm. Gradient: mass percentage concentration of mobile phase B: 40-60% and gradient treatment time 45-60 min. The sample amount is sample solution with the purity of more than or equal to 95 percent after the first step of purification and concentration;

and (3) purification process: washing the chromatographic column with acetonitrile with the mass percentage concentration of more than 90%, balancing, and then loading, wherein the loading amount is the sample solution with the purity of more than or equal to 95% after the first-step purification and concentration. Carrying out linear gradient elution, collecting a target peak, taking a fraction with the purity of more than or equal to 98 percent as a qualified fraction, and placing the collected peptide solution in a collection bottle for later use;

the salt conversion condition is as follows: a chromatographic column: the chromatographic column using octadecylsilane chemically bonded silica as a stationary phase has the following diameter and length: 10cm × 25cm, mobile phase: the A matching ratio contains 0.2 percent of acetic acid aqueous solution by mass percentage; phase B: pure acetonitrile solution is washed for 20min by 95% A + 5% B, salts are removed, gradient elution is carried out, phase B gradient change is 10% -50% (40min), products are collected, qualified fractions are concentrated, the concentration temperature does not exceed 40 ℃, and then freeze drying is carried out, so that 8.90g of somatostatin with the purity of more than 99.0% is obtained, the total yield can be 36.7%, the HPLC purity of refined peptide is 99.4% (see attached figure 3), and MS1637.7 (see attached figure 4).

The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.