Preparation method of body protection polypeptide (BPC

CN102229649A - Preparation method of body protection polypeptide (BPC 157 peptide) - Google Patents Preparation method of body protection polypeptide (BPC 157 peptide) Download PDF

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Publication number CN102229649A CN102229649A CN201110114806XA CN201110114806A CN102229649A CN 102229649 A CN102229649 A CN 102229649A CN 201110114806X A CN201110114806X A CN 201110114806XA CN 201110114806 A CN201110114806 A CN 201110114806A CN 102229649 A CN102229649 A CN 102229649A Authority CN China Prior art keywords otbu resin pro gly asp Prior art date 2011-05-05 Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.) Granted Application number CN201110114806XA Other languages Chinese (zh) Other versions CN102229649B (en Inventor 王增禄杨赛张英起 Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.) Medicine Research Institute Of Shaanxi Pharmaceutical Holding Cooperation Original Assignee Fourth Military Medical University FMMU Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.) 2011-05-05 Filing date 2011-05-05 Publication date 2011-11-02 2011-05-05 Application filed by Fourth Military Medical University FMMU filed Critical Fourth Military Medical University FMMU 2011-05-05 Priority to CN201110114806XA priority Critical patent/CN102229649B/en 2011-11-02 Publication of CN102229649A publication Critical patent/CN102229649A/en 2013-06-26 Application granted granted Critical 2013-06-26 Publication of CN102229649B publication Critical patent/CN102229649B/en Status Active legal-status Critical Current 2031-05-05 Anticipated expiration legal-status Critical

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Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS Y02P20/00—Technologies relating to chemical industry Y02P20/50—Improvements relating to the production of bulk chemicals Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

本发明公开了一种合成机体保护多肽(BPC 157肽)的方法,包括以三苯氯甲基树脂、4-甲基-三苯氯甲基树脂、4-甲氧基-三苯氯甲基树脂、2-氯-三苯氯甲基树脂或王树脂中的任何一种为起始原料,用DIC/HOBt或DIC/HOAt或BOP/HOBt或BOP/HOAt或HBTU/HOBt或HBTU/HOAt或HATU/HOBt或HATU/HOAt或HCTU/HOBt中的一种为缩合剂进行接肽反应,获得保护机体保护多肽(BPC 157肽)树脂,其后,同步进行脱侧链保护基团及切肽,获得BPC 157粗品,再经C18(或C8)柱分离纯化,冷冻干燥后,制得机体保护多肽(BPC 157肽)纯品。该方法可用微波辐射以加快反应速度,缩短多肽合成周期,降低生产成本。本发明的方法制备的机体保护多肽(BPC 157肽),具有原辅材料来源方便,工艺稳定,接肽收率高,质量可控,收率高,纯度高,生产成本低,周期短,可规模化生产等诸多优点,完全满足机体保护多肽(BPC 157肽)的应用要求。The invention discloses a method for synthesizing a body-protecting polypeptide (BPC 157 peptide), which comprises using trityl chloromethyl resin, 4-methyl-trityl chloromethyl resin, 4-methoxy-trityl chloromethyl Any one of resin, 2-chloro-trityl chloromethyl resin or Wang resin is a starting material, with DIC/HOBt or DIC/HOAt or BOP/HOBt or BOP/HOAt or HBTU/HOBt or HBTU/HOAt or One of HATU/HOBt or HATU/HOAt or HCTU/HOBt is used as a condensing agent to carry out the peptide-grafting reaction to obtain the resin for protecting the body's protective polypeptide (BPC 157 peptide), and then simultaneously remove the side chain protecting group and cut the peptide. The crude product of BPC 157 is obtained, separated and purified by a C18 (or C8) column, and freeze-dried to obtain a pure body-protecting polypeptide (BPC 157 peptide). In the method, microwave radiation can be used to speed up the reaction speed, shorten the synthesis period of the polypeptide, and reduce the production cost. The body-protecting polypeptide (BPC 157 peptide) prepared by the method of the present invention has the advantages of convenient source of raw and auxiliary materials, stable process, high peptide yield, controllable quality, high yield, high purity, low production cost, short cycle, and can be With many advantages such as large-scale production, it fully meets the application requirements of the body protection polypeptide (BPC 157 peptide).

Description 一种机体保护多肽(BPC 157肽)的制备方法A kind of preparation method of body protection polypeptide (BPC 157 peptide)

技术领域 technical field

本发明属于肽工程领域,具体涉及机体保护多肽(BPC 157肽)的制备方法,尤其适用于放大生产。 The invention belongs to the field of peptide engineering, in particular to a preparation method of a body protection polypeptide (BPC 157 peptide), and is especially suitable for scale-up production. the

背景技术 Background technique

BPC 157(body protection compound)为胃液肽(gastric juice peptide)的片段,由15个氨基酸组成,分子量1419。氨基酸序列为Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val(甘-谷-脯-脯-脯-甘-赖-脯-丙-天冬-天冬-丙-甘-亮-缬)。BPC 157具有细胞保护、抗溃疡和抗炎作用,其作用和细胞保护、NO系统、细胞迁移有关[1-6]。 BPC 157 (body protection compound) is a fragment of gastric juice peptide, consisting of 15 amino acids, with a molecular weight of 1419. The amino acid sequence is Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val (Gly-Gly-Pro-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val) - asparagus - asparagus - acyl - sweet - leuco - valerian). BPC 157 has cytoprotective, anti-ulcer and anti-inflammatory effects, which are related to cytoprotection, NO system and cell migration [1-6] .

发明内容 Contents of the invention

本发明的目的是提供一种固相合成BPC157肽的制备方法,为机体保护多肽(BPC 157肽)的应用提供原料保障。包括手工固相多肽合成法和微波自动固相多肽合成法,以适用于放大生产。 The purpose of the present invention is to provide a preparation method for solid-phase synthesis of BPC157 peptide, which provides raw material guarantee for the application of body protection polypeptide (BPC 157 peptide). Including manual solid-phase peptide synthesis and microwave automatic solid-phase peptide synthesis, suitable for scale-up production. the

本发明提供的技术方案具体为一种BPC157肽的固相制备方法,包括以下步骤: The technical solution provided by the present invention is specifically a solid-phase preparation method of BPC157 peptide, comprising the following steps:

以三苯氯甲基树脂为合成树脂,包括4-甲基-三苯氯甲基树脂、4-甲氧基-三苯氯甲基树脂、2-氯-三苯氯甲基树脂或王氏树脂,以以上树脂中的任何一种为起始原料,按照固相合成的方法依次连接具有Fmoc保护的氨基酸,获的保护的BPC157肽树脂,其间依次脱去Fmoc保护基团,用DIC/HOBt或DIC/HOAt或BOP/HOBt或BOP/HOAt或HBTU/HOBt或HBTU/HOAt或HATU/HOBt或HATU/HOAt或HCTU/HOBt中的一种为缩合剂进行接肽反应,得保护的BPC157肽树脂后,同步进行脱侧链保护基团及切肽,获得BPC157肽粗品,并经C18或C8色谱柱进行分离纯化,获得所需的BPC157肽纯品。 Use trityl chloromethyl resin as synthetic resin, including 4-methyl-trityl chloromethyl resin, 4-methoxy-trityl chloromethyl resin, 2-chloro-trityl chloromethyl resin or Wang's Resin, with any one of the above resins as the starting material, according to the method of solid-phase synthesis, the amino acids with Fmoc protection are sequentially connected to obtain the protected BPC157 peptide resin, during which the Fmoc protection group is sequentially removed, and the DIC/HOBt Or one of DIC/HOAt or BOP/HOBt or BOP/HOAt or HBTU/HOBt or HBTU/HOAt or HATU/HOBt or HATU/HOAt or HCTU/HOBt is a condensing agent for peptide grafting reaction, and the protected BPC157 peptide resin Afterwards, remove the side chain protection group and cut the peptide simultaneously to obtain the crude product of BPC157 peptide, which is separated and purified by C 18 or C 8 chromatographic column to obtain the desired pure product of BPC157 peptide.

更具体地,按照本发明,依次连接具有保护基团的氨基酸,获得保护BPC157肽树脂,其间依次脱去Fmoc保护基团的方法包括以下步骤: More specifically, according to the present invention, the amino acids with protective groups are sequentially connected to obtain the protected BPC157 peptide resin, and the method for sequentially removing the Fmoc protective group includes the following steps:

(1)制备Fmoe-Val-树脂: (1) Preparation of Fmoe-Val-resin:

选用三苯氯甲基树脂、4-甲基-三苯氯甲基树脂、4-甲氧基-三苯氯甲基树脂、2-氯-三苯氯甲基树脂或王氏树脂中任一种树脂,用N,N-二甲基甲酰胺或二氯甲烷浸泡10~60分钟,混合物中,树脂的重量体积浓度为5-20ml/g;然后在上述的树脂中,加入DIEA、DIC或DMAP、Fmoc-Val-OH,10~50°C反应0.5~5小时,再加入封闭试剂甲醇、苯甲酰氯或吡啶10~50°C反应0.2-3小时,树脂用异丙醇、N,N-二甲基甲酰胺洗涤,获得Fmoc-Val-树脂; Choose any of trityl chloromethyl resin, 4-methyl-trityl chloromethyl resin, 4-methoxy-trityl chloromethyl resin, 2-chloro-trityl chloromethyl resin or Wang's resin A kind of resin is soaked with N,N-dimethylformamide or dichloromethane for 10-60 minutes. In the mixture, the weight volume concentration of the resin is 5-20ml/g; then in the above-mentioned resin, add DIEA, DIC or DMAP, Fmoc-Val-OH, react at 10-50°C for 0.5-5 hours, then add blocking reagent methanol, benzoyl chloride or pyridine and react at 10-50°C for 0.2-3 hours, resin with isopropanol, N,N-di Methylformamide washes to obtain Fmoc-Val-resin;

DIEA或DMAP的摩尔数为树脂的2~20倍; The molar number of DIEA or DMAP is 2 to 20 times that of the resin;

Fmoc-Val-OH的摩尔数为树脂的1~10倍: The number of moles of Fmoc-Val-OH is 1 to 10 times that of the resin:

反应溶液中,树脂的浓度为0.1~5ml/g; In the reaction solution, the concentration of resin is 0.1~5ml/g;

(2)制备Fmoc-Leu-Val-树脂: (2) Preparation of Fmoc-Leu-Val-resin:

在步骤(1)的Fmoc-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Leu-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Leu-Val-树脂; In the Fmoc-Val-resin of step (1), add decapping reagent 10~50 °C and react for 5~30 minutes, drain with vacuum pump, add decapping reagent again 10~50 °C and react for 10~60 minutes, decapture, use iso Wash 3 times with propanol, 3 times with DMF, 2 times with distilled DMF, add the mixture of Fmoc-Leu-OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt dissolved in DMF, 10-50 React at °C for 10-60 minutes, dry with a vacuum pump, wash with isopropanol for 3 times, DMF for 3 times, DMF that has been re-distilled for 2 times, and dry to obtain Fmoc-Leu-Val-resin;

所说的脱帽试剂的组分和体积比为:PIP∶DMF=1∶2-5,下同; The component and volume ratio of said decapping reagent are: PIP: DMF=1: 2-5, the same below;

Fmoc-Leu-OH的摩尔数为树脂的1-5倍; The molar number of Fmoc-Leu-OH is 1-5 times that of the resin;

Fmoc-Val-树脂的重量与脱帽试剂的加入量的比例为5~20ml/g; The ratio of the weight of Fmoc-Val-resin to the amount of decapping reagent is 5-20ml/g;

TBTU/HBTU/BOP的摩尔数为树脂的2-5倍; The molar number of TBTU/HBTU/BOP is 2-5 times that of the resin;

HOBT/HOAT摩尔数为树脂的2-5倍; The molar number of HOBT/HOAT is 2-5 times that of the resin;

(3)制备Fmoc-Gly-Leu-Val-树脂: (3) Preparation of Fmoc-Gly-Leu-Val-resin:

在步骤(2)的Fmoc-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3 次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Gly-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Gly-Leu-Val-树脂; In the Fmoc-Leu-Val-resin of step (2), add decapping reagent 10~50 °C and react for 5~30 minutes, drain with a vacuum pump, add decapping reagent 10~50 °C and react for 10~60 minutes, drain, Wash 3 times with isopropanol, DMF 3 times, redistilled DMF 2 times, add the mixture of Fmoc-Gly-OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt dissolved in DMF, 10 React at ~50°C for 10 to 60 minutes, drain with a vacuum pump, wash with isopropanol 3 times, DMF for 3 times, double-distilled DMF for 2 times, and dry to obtain Fmoc-Gly-Leu-Val-resin;

Fmoc-Gly-OH摩尔数为树脂的2~5倍; The molar number of Fmoc-Gly-OH is 2 to 5 times that of the resin;

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(4)制备Fmoc-Ala-Gly-Leu-Val-树脂: (4) Preparation of Fmoc-Ala-Gly-Leu-Val-resin:

在步骤(3)的Fmoc-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Ala-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Ala-Gly-Leu-Val-树脂; In the Fmoc-Gly-Leu-Val-resin of step (3), add decapping reagent 10~50 °C and react for 5~30 minutes, use vacuum pump to dry, add decapping reagent 10~50 °C and react for 10~60 minutes, pump Dry, wash 3 times with isopropanol, 3 times with DMF, 2 times with redistilled DMF, add a mixture of Fmoc-Ala-OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt dissolved in DMF , reacted at 10-50°C for 10-60 minutes, pumped dry with a vacuum pump, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with distilled DMF, and dried to obtain Fmoc-Ala-Gly-Leu- Val-resin;

Fmoc-Ala-OH摩尔数为树脂的2~5倍: The number of moles of Fmoc-Ala-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(5)制备Fmoc-Asp(otBu)-Ala-Gly-Leu-Val-树脂: (5) Preparation of Fmoc-Asp(otBu)-Ala-Gly-Leu-Val-resin:

在步骤(4)的Fmoc-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Asp(otBu)-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Asp(otBu)-Ala-Gly-Leu-Val-树脂; In the Fmoc-Ala-Gly-Leu-Val-resin in step (4), add the decapping reagent at 10-50°C and react for 5-30 minutes, dry it with a vacuum pump, and add the decapping reagent again at 10-50°C for 10-60 minutes , drained, washed 3 times with isopropanol, 3 times with DMF, 2 times with re-distilled DMF, added Fmoc-Asp(otBu)-OH, DIEA, TBTU/HOBt or HBTU/HOBt dissolved in DMF or The mixture of BOP/HOBt was reacted at 10-50°C for 10-60 minutes, dried by vacuum pump, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with re-distilled DMF, and dried to obtain Fmoc-Asp (otBu)-Ala-Gly-Leu-Val-resin;

Fmoc-Asp(otBu)-OH摩尔数为树脂的2~5倍: The number of moles of Fmoc-Asp(otBu)-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(6)制备Fmoc-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂: (6) Preparation of Fmoc-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin:

在步骤(5)的Fmoc-Asp(otBu)-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干, 用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Asp(otBu)-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂; In the Fmoc-Asp(otBu)-Ala-Gly-Leu-Val-resin in step (5), add the decapping reagent at 10~50°C for 5~30 minutes, dry it with a vacuum pump, and add the decapping reagent again at 10~50°C React for 10-60 minutes, drain, wash 3 times with isopropanol, 3 times with DMF, 2 times with distilled DMF, add Fmoc-Asp(otBu)-OH, DIEA, TBTU/HOBt dissolved in DMF Or the mixture of HBTU/HOBt or BOP/HOBt, react at 10-50°C for 10-60 minutes, drain with a vacuum pump, wash with isopropanol for 3 times, wash with DMF for 3 times, wash with re-distilled DMF for 2 times, and drain , obtain Fmoc-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin;

Fmoc-Asp(otBu)-OH摩尔数为树脂的2~5倍: The number of moles of Fmoc-Asp(otBu)-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(7)制备Fmoc-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂: (7) Preparation of Fmoc-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin:

在步骤(6)的Fmoc-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Ala-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂; In the Fmoc-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin in step (6), add the decapping reagent at 10-50°C for 5-30 minutes, dry it with a vacuum pump, and re-add the decapping reagent React the reagent at 10-50°C for 10-60 minutes, drain it, wash it with isopropanol for 3 times, DMF for 3 times, and distilled DMF for 2 times, add Fmoc-Ala-OH, DIEA, TBTU dissolved in DMF /HOBt or HBTU/HOBt or BOP/HOBt mixture, react at 10-50°C for 10-60 minutes, dry with a vacuum pump, wash 3 times with isopropanol, 3 times with DMF, 2 times with re-distilled DMF, Drain, obtain Fmoc-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin;

Fmoc-Ala-OH摩尔数为树脂的2~5倍: The number of moles of Fmoc-Ala-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(8)制备Fmoc-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂: (8) Preparation of Fmoc-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin:

在步骤(7)的Fmoc-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Pro-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂; In the Fmoc-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin of step (7), add decapping reagent 10~50 °C and react for 5~30 minutes, dry with vacuum pump, re- Add capping reagent and react at 10-50°C for 10-60 minutes, pump dry, wash with isopropanol for 3 times, DMF for 3 times, distilled DMF for 2 times, add Fmoc-Pro-OH and DIEA dissolved in DMF , a mixture of TBTU/HOBt or HBTU/HOBt or BOP/HOBt, react at 10-50°C for 10-60 minutes, dry it with a vacuum pump, wash 3 times with isopropanol, wash 3 times with DMF, wash 2 times with re-distilled DMF time, drained to obtain Fmoc-Pro-Ala-Asp (otBu)-Asp (otBu)-Ala-Gly-Leu-Val-resin;

Fmoc-Pro-OH摩尔数为树脂的2~5倍: The number of moles of Fmoc-Pro-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(9)制备Fmoc-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂: (9) Preparation of Fmoc-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin:

在步骤(8)的Fmoc-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用洗涤异丙醇3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Lys(Boc)-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂; In the Fmoc-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin in step (8), add the decapping reagent and react at 10-50°C for 5-30 minutes, and dry it with a vacuum pump , re-add the decapping reagent and react at 10-50°C for 10-60 minutes, drain, wash with isopropanol 3 times, DMF wash 3 times, re-distilled DMF wash 2 times, add Fmoc-Lys (Boc-Lys) dissolved in DMF )-OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt mixture, react at 10-50°C for 10-60 minutes, dry it with a vacuum pump, wash with isopropanol for 3 times, DMF for 3 times, redistill The DMF washed 2 times, drained to obtain Fmoc-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin;

Fmoc-Lys(Boc)-OH摩尔数为树脂的2~5倍: The number of moles of Fmoc-Lys(Boc)-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(10)制备Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂: (10) Preparation of Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin:

在步骤(9)的Fmoc-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Gly-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂树脂; In the Fmoc-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin in step (9), add a decapping reagent and react at 10-50°C for 5-30 minutes , dry it with a vacuum pump, re-add the decapping reagent at 10-50 ° C for 10-60 minutes, drain it, wash it with isopropanol for 3 times, wash it with DMF for 3 times, wash it twice with DMF, add the solution dissolved in DMF Mixture of Fmoc-Gly-OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt, react at 10-50°C for 10-60 minutes, dry it with a vacuum pump, wash with isopropanol for 3 times, DMF for 3 times, The DMF that redistilled is washed 2 times, sucks dry, obtains Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin resin;

Fmoc-Gly-OH摩尔数为树脂的2~5倍: The molar number of Fmoc-Gly-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(11)制备Fmoc-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂: (11) Preparation of Fmoc-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin:

在步骤(10)的Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Pro-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得 Fmoc-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂; In the Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin of step (10), add decapping reagent 10~50 °C and react for 5~ 30 minutes, dry with a vacuum pump, re-add the decapping reagent at 10-50 ° C for 10-60 minutes, dry, wash with isopropanol for 3 times, DMF for 3 times, re-distilled DMF for 2 times, add DMF Dissolved Fmoc-Pro-OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt mixture, reacted at 10-50°C for 10-60 minutes, dried with a vacuum pump, washed 3 times with isopropanol and 3 times with DMF times, the DMF that has been steamed again was washed 2 times, and was drained to obtain Fmoc-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin;

Fmoc-Pro-OH摩尔数为树脂的2~5倍: The number of moles of Fmoc-Pro-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(12)制备Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂: (12) Preparation of Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin:

在步骤(11)的Fmoc-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Pro-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂; In the Fmoc-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin in step (11), add the decapping reagent and react at 10~50°C 5 to 30 minutes, dry with a vacuum pump, re-add the decapping reagent at 10 to 50 ° C for 10 to 60 minutes, dry, wash with isopropanol for 3 times, DMF for 3 times, re-distilled DMF for 2 times, add Dissolve Fmoc-Pro-OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt mixture in DMF, react at 10-50°C for 10-60 minutes, dry with vacuum pump, wash with isopropanol 3 times, DMF Washed 3 times, washed 2 times with re-distilled DMF, and dried to obtain Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu- Val-resin;

Fmoc-Pro-OH摩尔数为树脂的2~5倍: The number of moles of Fmoc-Pro-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(13)制备Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂: (13) Preparation of Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin:

在步骤(12)的Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Pro-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂; In the Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin of step (12), add decapping reagent 10~50 React at °C for 5-30 minutes, drain with a vacuum pump, re-add capping reagent at 10-50°C, react for 10-60 minutes, drain, wash with isopropanol for 3 times, DMF for 3 times, and distilled DMF for 2 times , add the mixture of Fmoc-Pro-OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt dissolved in DMF, react at 10-50°C for 10-60 minutes, dry with a vacuum pump, and wash with isopropanol for 3 times , washed 3 times with DMF, washed 2 times with DMF steamed, and dried to obtain Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala- Gly-Leu-Val-resin;

Fmoc-Pro-OH摩尔数为树脂的2~5倍: The number of moles of Fmoc-Pro-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(14)制备Fmoc-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂: (14) Preparation of Fmoc-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin:

在步骤(13)的Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Glu(otBu)-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂; In the Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin of step (13), add decapping reagent 10 React at ~50°C for 5-30 minutes, drain with a vacuum pump, re-add capping reagent at 10-50°C, react for 10-60 minutes, drain, wash with isopropanol for 3 times, DMF for 3 times, and distilled DMF Twice, add the mixture of Fmoc-Glu(otBu)-OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt dissolved in DMF, react at 10-50°C for 10-60 minutes, dry with a vacuum pump, and use iso Propanol was washed 3 times, DMF was washed 3 times, the DMF that redistilled was washed 2 times, was sucked dry, obtained Fmoc-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp( otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin;

Fmoc-Glu(otBu)-OH摩尔数为树脂的2~5倍: The number of moles of Fmoc-Glu(otBu)-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(15)制备Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂: (15) Preparation of Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin:

在步骤(14)的Fmoc-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,加入用DMF溶解的Fmoc-Gly-OH、DIEA、TBTU/HOBt或HBTU/HOBt或BOP/HOBt的混合物,10~50°C反应10~60分钟,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂; In the Fmoc-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin of step (14) , add decapping reagent at 10-50°C and react for 5-30 minutes, use a vacuum pump to dry up, re-add decapping reagent at 10-50°C and react for 10-60 minutes, drain, wash with isopropanol for 3 times, DMF for 3 times, repeat Wash twice with steamed DMF, add the mixture of Fmoc-Gly-OH, DIEA, TBTU/HOBt or HBTU/HOBt or BOP/HOBt dissolved in DMF, react at 10-50°C for 10-60 minutes, and dry it with a vacuum pump. Washed 3 times with isopropanol, 3 times with DMF, 2 times with re-distilled DMF, and dried to obtain Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro- Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin;

Fmoc-Gly-OH摩尔数为树脂的2~5倍: The molar number of Fmoc-Gly-OH is 2 to 5 times that of the resin:

其余操作以及工艺条件同上; The rest of the operations and process conditions are the same as above;

(16)制备Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂 (16) Preparation of Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin

在步骤(15)的Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂中,加入脱帽试剂10~50°C反应5~30分钟,用真空泵抽干,重新加入脱帽试剂10~50°C反应10~60分钟,抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,甲醇洗涤2次,用甲醇浸泡0.5~3小时,抽干,用氮气吹干树脂成很干的颗粒,所得到的树脂为Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂; In step (15) Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val- Add decapping reagent to the resin, react at 10-50°C for 5-30 minutes, drain with a vacuum pump, re-add capping reagent at 10-50°C, react for 10-60 minutes, drain, wash with isopropanol for 3 times, DMF for 3 times , washed twice with DMF and twice with methanol, soaked in methanol for 0.5 to 3 hours, drained, and dried with nitrogen to form very dry particles. The obtained resin is Gly-Glu(otBu)- Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin;

(17)把吹干的Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-树脂倒入玻璃的切割瓶中,加入预冷至-20~10°C的切肽试剂(TFA/EDT/H2O/Thio=90-95/2-5/2-5/1-5,体积比),10°C-50°C反应1-5小时,过滤除去树脂,加-20~10°C的冰乙醚沉淀,离心收集沉淀物,用乙醚洗涤1~6次,低温冷冻干燥10~24小时,获得BPC157肽粗品; (17) Take the dried Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-resin Pour into a glass cutting bottle, add the peptide cutting reagent (TFA/EDT/H 2 O/Thio=90-95/2-5/2-5/1-5, volume ratio ), reacted at 10°C-50°C for 1-5 hours, filtered to remove the resin, added -20-10°C glacial ether to precipitate, centrifuged to collect the precipitate, washed 1-6 times with ether, and freeze-dried at low temperature for 10-24 hours to obtain Crude product of BPC157 peptide;

切肽试剂中,树脂的浓度为8-12ml/g In the peptide cutting reagent, the concentration of the resin is 8-12ml/g

按照本发明,粗品经C18或C8色谱柱纯化的方法包括如下步骤: According to the present invention, the method for purifying the crude product through C18 or C8 chromatographic column comprises the steps:

流动相有两种:A:0.1%三氟乙酸加99.9%水;B:0.1%三氟乙酸加99.9%乙腈;梯度洗脱;流速为1-150ml/min;检测波长为:215~285nm;用液相色谱仪跟踪收集所需要的流出液,样品峰合并后去盐,冻干,获得成品。 There are two mobile phases: A: 0.1% trifluoroacetic acid plus 99.9% water; B: 0.1% trifluoroacetic acid plus 99.9% acetonitrile; gradient elution; flow rate 1-150ml/min; detection wavelength: 215-285nm; Use a liquid chromatograph to track and collect the required effluent, combine the sample peaks, desalt, and freeze-dry to obtain the finished product. the

在本文中,如未特别指出,“常温”表示室温,即25°C。 Herein, unless otherwise specified, "normal temperature" means room temperature, ie, 25°C. the

尽管理论上有DIC/DMAP或DIC/HOBt或DIC/HOAt或BOP/HOBt或BOP/HOAt或HBTU/HOBt或HBTU/HOAt或HATU/HOBt或HATU/HOAt或HCTU/HOBt或HUTC/HOAt或TBTU/HOBt等试剂可以选择,而且理论上也没有重大区别。但是令人意想不到的是,尽管小试阶段各试剂效果差异不显著,但是进入中试阶段(即,实验放大),我们发现,相对于其他试剂组合,用TBTU/HOBt反应粗品纯度较好,小试试验粗品纯度达到78%以上,而中试批量生产粗品纯度也能在基本不降低产率的条件下达到75%,经过纯化的成品,纯度大于98%,不低于95%的药用纯度要求。 Although theoretically there are DIC/DMAP or DIC/HOBt or DIC/HOAt or BOP/HOBt or BOP/HOAt or HBTU/HOBt or HBTU/HOAt or HATU/HOBt or HATU/HOAt or HCTU/HOBt or HUTC/HOAt or TBTU/ Reagents such as HOBt can be chosen, and theoretically there is no major difference. But what is unexpected is that although the effect of each reagent in the small test stage is not significantly different, but in the pilot test stage (that is, the experimental scale-up), we found that compared with other reagent combinations, the purity of the crude product reacted with TBTU/HOBt is better, and the small The purity of the crude product in the pilot test can reach more than 78%, and the purity of the crude product in the pilot scale production can also reach 75% under the condition of basically not reducing the yield. Require. the

本发明的有益效果是:本发明的BPC157肽固相合成制备方法具有以下特点:原辅材料来源方便,工艺稳定,收率高,质量可控,产品纯度高,生产周期短,成本低,可规模化生产。采用本发明方法制备的BPC157肽极具市场竞争力,其生产规模完全能够满足BPC157肽的应用需求,其质量完全能够达到BPC157肽的应用要求。 The beneficial effects of the present invention are: the preparation method of BPC157 peptide solid-phase synthesis of the present invention has the following characteristics: the source of raw and auxiliary materials is convenient, the process is stable, the yield is high, the quality is controllable, the product purity is high, the production cycle is short, the cost is low, and it can Large-scale production. The BPC157 peptide prepared by the method of the invention is highly competitive in the market, its production scale can fully meet the application requirements of the BPC157 peptide, and its quality can fully meet the application requirements of the BPC157 peptide. the

另外,在本发明的工艺中,采用温和的Fmoc路线,每步脱Fmoc仅用20%的PIP,每步缩合反应时间短,大大缩短了生产周期;采用合理封闭技术,提高粗肽产品的纯度;切割采用TFA,避免了常用的氟化氢,减少了生产中产生的三废,有利于工业化生产。 In addition, in the process of the present invention, a mild Fmoc route is adopted, and only 20% of PIP is used for each step of removing Fmoc, and the condensation reaction time of each step is short, which greatly shortens the production cycle; the rational sealing technology is adopted to improve the purity of the crude peptide product ; Cutting adopts TFA, which avoids the commonly used hydrogen fluoride, reduces the three wastes produced in production, and is beneficial to industrial production. the

前述过程中所采用的原料中英文列表如下: The Chinese and English lists of raw materials used in the aforementioned process are as follows:

具体实施方式 Detailed ways

实施例11g级别王氏树脂(WangResin)的BPC157肽合成过程 The BPC157 peptide synthesis process of embodiment 11g grade Wang's resin (WangResin)

(1)制备Fmoe-Val-WangResin (1) Preparation of Fmoe-Val-WangResin

称取王氏树脂1g(吉尔生化(上海)有限公司;Loadding:1.0mmol/g)倒入反应柱中,加入大约10mlDMF,浸泡30分钟(常温)后用真空泵抽干,加入Fmoc-Val-OH 3.4g,DIC0.7ml,DMAP0.1g,DMF10ml,于常温反应2小时,再加入封闭试剂苯甲酰氯0.6ml,吡啶0.4ml,DCM10ml常温反应0.5小时,树脂用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Val-WangResin; Weigh 1g of Wang's resin (Gill Biochemical (Shanghai) Co., Ltd.; Loading: 1.0mmol/g) and pour it into the reaction column, add about 10ml of DMF, soak for 30 minutes (at room temperature) and dry it with a vacuum pump, add Fmoc-Val-OH 3.4g, DIC0.7ml, DMAP0.1g, DMF10ml, react at room temperature for 2 hours, then add blocking reagent benzoyl chloride 0.6ml, pyridine 0.4ml, DCM10ml, react at room temperature for 0.5 hours, wash the resin with isopropanol 3 times, wash with DMF 3 times, washed 2 times with DMF re-distilled, drained to obtain Fmoc-Val-WangResin;

(2)制备Fmoc-Leu-Val-WangResin (2) Preparation of Fmoc-Leu-Val-WangResin

在步骤(1)的Fmoe-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Leu-OH 0.7g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Leu-Val-WangResin; In the Fmoe-Val-WangResin of step (1), add decapping reagent (DMF solution of 20% piperidine), react at room temperature for 5 minutes, drain, add decapping agent at room temperature and react for 20 minutes, wash 3 times with isopropanol , washed 3 times with DMF, washed 2 times with re-distilled DMF, drained, added 0.7g of Fmoc-Leu-OH, 0.4ml of DIEA, 0.56g of TBTU, 0.2g of HOBT, 10ml of DMF, reacted at room temperature for 1.5 hours, and dried with a vacuum pump. Washed 3 times with isopropanol, 3 times with DMF, 2 times with distilled DMF, and dried to obtain Fmoc-Leu-Val-WangResin;

(3)制备Fmoc-Gly-Leu-Val-WangResin (3) Preparation of Fmoc-Gly-Leu-Val-WangResin

在步骤(2)的Fmoc-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Gly-OH0.7g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Gly-Leu-Val-WangResin; In the Fmoc-Leu-Val-WangResin of step (2), add decapping reagent (DMF solution of 20% piperidine), react at room temperature for 5 minutes, drain, add decapping reagent at room temperature and react for 20 minutes, wash with isopropanol 3 times, washed 3 times with DMF, washed 2 times with re-distilled DMF, drained, added Fmoc-Gly-OH0.7g, DIEA0.4ml, TBTU0.56g, HOBT0.2g, DMF10ml, reacted at room temperature for 1.5 hours, pumped with a vacuum pump Dry, wash 3 times with isopropanol, wash 3 times with DMF, wash 2 times with DMF that has been distilled, and drain to obtain Fmoc-Gly-Leu-Val-WangResin;

(4)制备Fmoc-Ala-Gly-Leu-Val-WangResin (4) Preparation of Fmoc-Ala-Gly-Leu-Val-WangResin

在步骤(3)的Fmoc-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Ala-OH0.7g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Ala-Gly-Leu-Val-WangResin; In the Fmoc-Gly-Leu-Val-WangResin of step (3), add decapping reagent (20% piperidine in DMF solution), react at room temperature for 5 minutes, drain, add decapping reagent at normal temperature and react for 20 minutes, and use isopropyl Wash 3 times with alcohol, 3 times with DMF, 2 times with distilled DMF, drain, add Fmoc-Ala-OH0.7g, DIEA0.4ml, TBTU0.56g, HOBT0.2g, DMF10ml, react at room temperature for 1.5 hours, use Vacuum pump to dry, wash 3 times with isopropanol, DMF wash 3 times, DMF washed twice, dry, obtain Fmoc-Ala-Gly-Leu-Val-WangResin;

(5)制备Fmoc-Asp(otBu)-Ala-Gly-Leu-Val-WangResin (5) Preparation of Fmoc-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(4)的Fmoc-Ala-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Asp(otBu)-OH1.2g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; In the Fmoc-Ala-Gly-Leu-Val-WangResin of step (4), add decapping reagent (DMF solution of 20% piperidine), react at normal temperature for 5 minutes, drain, add decapping agent normal temperature and react for 20 minutes, use Wash 3 times with isopropanol, 3 times with DMF, 2 times with distilled DMF, drain, add Fmoc-Asp(otBu)-OH1.2g, DIEA0.4ml, TBTU0.56g, HOBT0.2g, DMF10ml at room temperature Reacted for 1.5 hours, dried with a vacuum pump, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with re-distilled DMF, and dried to obtain Fmoc-Asp(otBu)-Ala-Gly-Leu-Val- Wang Resin;

(6)制备Fmoc-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin (6) Preparation of Fmoc-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(5)的Fmoc-Asp(otBu)-Ala-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Asp(otBu)-OH1.1g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; In the Fmoc-Asp(otBu)-Ala-Gly-Leu-Val-WangResin in step (5), add decapping reagent (20% piperidine in DMF solution), react at room temperature for 5 minutes, drain, and add decapping reagent at normal temperature React for 20 minutes, wash with isopropanol for 3 times, DMF for 3 times, DMF for redistilled for 2 times, drain, add Fmoc-Asp(otBu)-OH1.1g, DIEA0.4ml, TBTU0.56g, HOBT0 .2g, DMF10ml normal temperature reaction for 1.5 hours, drained with a vacuum pump, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with DMF evaporated, and dried to obtain Fmoc-Asp(otBu)-Asp(otBu )-Ala-Gly-Leu-Val-WangResin;

(7)制备Fmoc-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin (7) Preparation of Fmoc-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(6)的Fmoc-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Ala-OH0.7g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF 10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; In the Fmoc-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin in step (6), add a decapping reagent (20% piperidine in DMF solution), react at room temperature for 5 minutes, and drain, Re-add the capping agent and react at room temperature for 20 minutes, wash with isopropanol for 3 times, DMF for 3 times, re-distilled DMF for 2 times, drain, add Fmoc-Ala-OH0.7g, DIEA0.4ml, TBTU0.56g , HOBT0.2g, DMF 10ml normal temperature reaction 1.5 hours, drained with a vacuum pump, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with DMF evaporated, and dried to obtain Fmoc-Ala-Asp(otBu )-Asp(otBu)-Ala-Gly-Leu-Val-WangResin;

(8)制备Fmoc-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin (8) Preparation of Fmoc-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(7)的Fmoc-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Pro-OH0.8g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; In the Fmoc-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin of step (7), add decapping reagent (DMF solution of 20% piperidine), react at room temperature for 5 minutes, pump Dry, re-add the capping agent and react at room temperature for 20 minutes, wash with isopropanol for 3 times, DMF for 3 times, re-distilled DMF for 2 times, drain, add Fmoc-Pro-OH0.8g, DIEA0.4ml, TBTU0 .56g, HOBT0.2g, DMF10ml, reacted at room temperature for 1.5 hours, dried with a vacuum pump, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with re-distilled DMF, and dried to obtain Fmoc-Pro-Ala- Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin;

(9)制备Fmoc-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin (9) Preparation of Fmoc-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(8)的Fmoc-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Lys(Boc)-OH1.1g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; In the Fmoc-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin of step (8), add a decapping reagent (20% piperidine in DMF solution), and react at room temperature for 5 minutes , drained, re-add capping agent and react at room temperature for 20 minutes, wash 3 times with isopropanol, wash 3 times with DMF, wash 2 times with re-distilled DMF, drain, add Fmoc-Lys(Boc)-OH 1.1g, DIEA0.4ml, TBTU0.56g, HOBT0.2g, DMF10ml reacted at room temperature for 1.5 hours, dried with a vacuum pump, washed 3 times with isopropanol, washed 3 times with DMF, washed twice with distilled DMF, and dried to obtain Fmoc -Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin;

(10)制备Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin (10) Preparation of Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(9)的Fmoc-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Gly-OH0.7g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; In the Fmoc-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin in step (9), add a decapping reagent (20% piperidine in DMF) , reacted at room temperature for 5 minutes, drained, added decapping agent and reacted at room temperature for 20 minutes, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with distilled DMF, drained, added Fmoc-Gly-OH0. 7g, DIEA0.4ml, TBTU0.56g, HOBT0.2g, DMF10ml, react at room temperature for 1.5 hours, use a vacuum pump to dry up, wash with isopropanol for 3 times, DMF for 3 times, distilled DMF for 2 times, and then drain, Obtain Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin;

(11)制备Fmoc-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin (11) Preparation of Fmoc-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(10)的Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Pro-OH0.8g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Pro-Gly-Lys(Boc)-Pro- Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; In step (10) of Fmoc-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin, add decapping reagent (20% piperidine in DMF solution), reacted at room temperature for 5 minutes, drained, added decapping agent and reacted at room temperature for 20 minutes, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with re-distilled DMF, drained, added Fmoc-Pro- 0.8g of OH, 0.4ml of DIEA, 0.56g of TBTU, 0.2g of HOBT, and 10ml of DMF were reacted at room temperature for 1.5 hours, dried with a vacuum pump, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with distilled DMF, pumped Dry, obtain Fmoc-Pro-Gly-Lys (Boc)-Pro-Ala-Asp (otBu)-Asp (otBu)-Ala-Gly-Leu-Val-WangResin;

(12)制备Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin (12) Preparation of Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(11)的Fmoc-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Pro-OH0.8g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; In step (11) of Fmoc-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin, add decapping reagent (20% piperidine DMF solution), reacted at room temperature for 5 minutes, drained, added decapping agent and reacted at room temperature for 20 minutes, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with re-distilled DMF, drained, added Fmoc- Pro-OH0.8g, DIEA0.4ml, TBTU0.56g, HOBT0.2g, DMF10ml, react at room temperature for 1.5 hours, dry with vacuum pump, wash with isopropanol for 3 times, DMF for 3 times, and distilled DMF for 2 times , drained to obtain Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin;

(13)制备Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin (13) Preparation of Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(12)的Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Pro-OH0.8g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; In the Fmoc-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin of step (12), add decapping reagent (20% DMF solution of piperidine), reacted at room temperature for 5 minutes, drained, re-added capping agent and reacted at room temperature for 20 minutes, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with re-distilled DMF, drained, added Fmoc-Pro-OH0.8g, DIEA0.4ml, TBTU0.56g, HOBT0.2g, DMF10ml, react at room temperature for 1.5 hours, dry with a vacuum pump, wash with isopropanol for 3 times, DMF for 3 times, and redestilled DMF for washing 2 times, drained to obtain Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin;

(14)制备 (14) Preparation

Fmoc-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin Fmoc-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(13)的Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Glu(otBu)-OH1.1g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Glu(otBu)- Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; In the Fmoc-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin of step (13), add decapping reagent ( 20% piperidine in DMF), reacted at room temperature for 5 minutes, drained, re-added capping agent and reacted at room temperature for 20 minutes, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with distilled DMF, drained , add Fmoc-Glu(otBu)-OH1.1g, DIEA0.4ml, TBTU0.56g, HOBT0.2g, DMF10ml, react at room temperature for 1.5 hours, dry with a vacuum pump, wash with isopropanol for 3 times, DMF for 3 times, repeat The steamed DMF was washed twice and dried to obtain Fmoc-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly- Leu-Val-Wang Resin;

(15)制备 (15) Preparation

Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(14)的 In step (14) of

Fmoc-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,加入Fmoc-Gly-OH0.8g,DIEA0.4ml,TBTU0.56g,HOBT0.2g,DMF10ml常温反应1.5小时,用真空泵抽干,用异丙醇洗涤3次,DMF洗涤3次,重蒸过的DMF洗涤2次,抽干,获得Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; In Fmoc-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin, add decapping reagent (20 %piperidine in DMF solution), reacted at normal temperature for 5 minutes, drained, added capping agent and reacted at normal temperature for 20 minutes, washed 3 times with isopropanol, washed 3 times with DMF, washed 2 times with DMF evaporated, drained, Add 0.8g of Fmoc-Gly-OH, 0.4ml of DIEA, 0.56g of TBTU, 0.2g of HOBT, and 10ml of DMF to react at room temperature for 1.5 hours, dry it with a vacuum pump, wash 3 times with isopropanol, and wash 3 times with DMF. Wash 2 times and drain to obtain Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu- Val-Wang Resin;

(16)制备Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin (16) Preparation of Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

在步骤(15)的 In step (15) of

Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin Fmoc-Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin

中,加入脱帽试剂(20%哌啶的DMF溶液),常温反应5分钟,抽干,重新加入脱帽剂常温反应20分钟,用DMF洗涤3次,异丙醇洗涤3次,重蒸过的DMF洗涤2次,甲醇洗涤2次,用甲醇浸泡1小时,抽干,用氮气吹干树脂成干燥的颗粒,所得到的树脂为: Add capping reagent (20% piperidine in DMF solution), react at room temperature for 5 minutes, drain, re-add capping agent at room temperature and react for 20 minutes, wash 3 times with DMF, wash 3 times with isopropanol, re-evaporated DMF Wash 2 times, wash 2 times with methanol, soak in methanol for 1 hour, drain, and dry the resin with nitrogen to form dry particles. The obtained resin is:

Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin; Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin;

(17)制备粗品 (17) Preparation of crude product

把吹干的Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin倒入玻璃的切割瓶中,加入预冷的切肽试剂(TFA9.5ml,H2O0.5ml),常温反应1.5小时,过滤除去树脂,加冰乙醚500ml沉淀,3500转3分钟离心收集沉淀物,用乙醚洗涤3次,用纯水溶解后,低温冷冻干燥24小时,获得BPC157肽粗品1.23g,经HPLC测定纯度为78%。 Pour the dried Gly-Glu(otBu)-Pro-Pro-Pro-Gly-Lys(Boc)-Pro-Ala-Asp(otBu)-Asp(otBu)-Ala-Gly-Leu-Val-WangResin into the glass Add pre-cooled peptide cutting reagent (TFA9.5ml, H 2 O0.5ml) to the cutting bottle, react at room temperature for 1.5 hours, filter to remove the resin, add 500ml of glacial ether to precipitate, centrifuge at 3500 for 3 minutes to collect the precipitate, and diethyl ether After washing 3 times, dissolving with pure water, and lyophilizing at low temperature for 24 hours, 1.23 g of crude product of BPC157 peptide was obtained, and the purity was 78% as determined by HPLC.

(18)纯化 (18) Purification

将步骤(17)的BPC157肽粗品1.23g溶于13ml水中,过滤,滤液上样于C18色谱柱(Kromasil-C18,20mm*250mm,汉邦科技色谱公司),流动相为如下A、B液体的混合物:A:0.1%三氟乙酸加99.9%水;B:0.1%三氟乙酸加99.9%乙腈。从A液体占混合物比例100%开始梯度洗脱,期间B液体占混合物比例逐渐升高,直至占100%,流速为10ml/min,检测波长为:215nm。收集目标峰(用相同条件下以实验室级别生产的少量纯品做对照指示目标峰)的流出液,浓缩冻干,获得成品,共获得成品0.69g(MW:1419.6),经HPLC测定纯度大于98%。 Dissolve 1.23 g of the crude BPC157 peptide in step (17) in 13 ml of water, filter, and load the filtrate on a C 18 chromatographic column (Kromasil-C 18 , 20mm*250mm, Hanbang Technology Chromatography Company), the mobile phase is as follows A, B Mixture of liquids: A: 0.1% trifluoroacetic acid plus 99.9% water; B: 0.1% trifluoroacetic acid plus 99.9% acetonitrile. Gradient elution starts from liquid A accounting for 100% of the mixture, during which the proportion of liquid B in the mixture gradually increases until it accounts for 100%, the flow rate is 10ml/min, and the detection wavelength is 215nm. Collect the effluent of the target peak (using a small amount of pure product produced at the laboratory level under the same conditions as a reference to indicate the target peak), concentrate and freeze-dry to obtain the finished product, and obtain a total of 0.69g (MW: 1419.6) of the finished product. The purity measured by HPLC is greater than 98%.

实施例2中试规模的BPC157肽合成过程 The BPC157 peptide synthesis process of the pilot scale of embodiment 2

参照实施例1的具体过程,所不同的是所有试剂直接放大20倍,即称取王氏树脂20g(Loadding:1.0mmol/g)进行反应,也以实施例1的TBTU/HOBt反应,制备树脂-肽接合物,切割制备粗肽,共得粗品24g,HPLC检测纯度为75%。按实施例1中的纯化方法纯化粗肽,色谱柱可用C18色谱柱(Kromasil-C18,30mm*250mm,汉邦科技色谱公司),分多次上样,分别收集目标峰的流出液,最后合并,浓缩冻干,得成品13.6g,HPLC测定纯度大于98%,表明该技术条件可以直接放大生产。 Referring to the specific process of Example 1, the difference is that all reagents are directly amplified 20 times, that is, 20 g of Wang's resin (Loadding: 1.0 mmol/g) is weighed for reaction, and the TBTU/HOBt reaction of Example 1 is also used to prepare the resin - Peptide conjugates, cleaved to prepare crude peptides, a total of 24 g of crude products were obtained, with a purity of 75% as determined by HPLC. Purify the crude peptide according to the purification method in Example 1. The chromatographic column can be a C 18 chromatographic column (Kromasil-C 18 , 30mm*250mm, Hanbang Technology Chromatography Co., Ltd.), and the samples are loaded in multiple times, and the effluents of the target peaks are collected respectively. Finally, they were combined, concentrated and freeze-dried to obtain 13.6 g of the finished product, and the purity determined by HPLC was greater than 98%, indicating that this technical condition can be directly scaled up for production.

参考文献 references

1.薛晓畅,吴勇杰,高明堂,李文广,赵宁,王增禄,颜真,张英起.十五肽BPC157对小型猪皮肤切割伤的保护作用研究.中国新药杂志,2004,13(7):602-605 1. Xue Xiaochang, Wu Yongjie, Gao Mingtang, Li Wenguang, Zhao Ning, Wang Zenglu, Yan Zhen, Zhang Yingqi. Study on the protective effect of pentapeptide BPC157 on cut skin wounds of minipigs. Chinese Journal of New Drugs, 2004, 13(7): 602 -605

2.李雅娟.BPC157可治疗溃疡性结肠炎.国外药讯,2005,12:17-17 2. Li Yajuan . BPC157 can treat ulcerative colitis. Foreign Pharmaceutical News , 2005, 12: 17-17

3.SikiP JeloN.十五肽BPC 157减弱慢性苯丙胺诱导的行为障碍.中国药理学报:英文版,2002,23(5):412-422 3. SikiP JeloN . Pentapeptide BPC 157 Attenuates Chronic Amphetamine-Induced Behavioral Disorders. Chinese Journal of Pharmacology: English Edition , 2002, 23(5): 412-422

4.Hrelec M,Klicek R,Brcic L,Brcic I,Cvjetko I,Seiwerth S, Sikiric P.Abdominal aorta anastomosis in rats and stable gastric pentadecapeptide BPC 157,prophylaxis and therapy.J Physiol Pharmacol.2009,60 Suppl 7:161-5. 4. Hrelec M , Klicek R , Brcic L , Brcic I , Cvjetko I , Seiwerth S , Sikiric P .Abdominal aorta anastomosis in rats and stable gastric pentadecapeptide BPC 157, prophylaxis and therapy. J Physiol Pharmacol. 2009, 60 Suppl 7: 161 -5.

5.Ilic S,Drmic D,Zarkovic K,Kolenc D,Coric M,Brcic L,Klicek R, Radic B,Sever M,Djuzel V,Ivica M,Boban Blagaic A,Zoricic Z,Anic T, Zoricic I,Djidic S,Romic Z,Seiwerth S,Sikiric P.High hepatotoxic dose of paracetamol produces generalized convulsions and brain damage in rats.A counteraction with the stable gastric pentadecapeptide BPC 157(PL 14736).J Physiol Pharmacol.2010,61(2):241-50. 5. Ilic S , Drmic D , Zarkovic K , Kolenc D , Coric M , Brcic L , Klicek R , Radic B , Sever M , Djuzel V , Ivica M , Boban Blagaic A , Zoricic Z , Anic T , Zoricic I , Djidic S , Romic Z , Seiwerth S , Sikiric P. High hepatotoxic dose of paracetamol produces generalized convulsions and brain damage in rats. A counteraction with the stable gastric pentadecapeptide BPC 157(PL 14736). J Physiol Pharmacol .2010, 61(2): 24 -50.

6.Chang CH,Tsai WC,Lin MS,Hsu YH,Pang JH.The promoting effect of pentadecapeptide BPC 157 on tendon healing involves tendon outgrowth,cell survival,and cell migration.J Appl Physiol.2011,110(3):774-80.Epub 2010 Oct 28. 6. Chang CH , Tsai WC , Lin MS , Hsu YH , Pang JH . The promoting effect of pentadecapeptide BPC 157 on tendon healing involves tendon outgrowth, cell survival, and cell migration. J Appl Physiol. 2011, 110(3): 774 -80. Epub 2010 Oct 28.

Claims (7)

1. the method for preparing the BPC157 peptide is characterized in that comprising the steps:

With the triphen chloromethyl resin; 4-methyl-triphen chloromethyl resin; 4-methoxyl group-triphen chloromethyl resin; wherein any of 2-chloro-triphen chloromethyl resin or Wang resin is starting raw material; method according to solid phase synthesis connects the amino acid with the protection of fluorenes methoxy carbonyl acyl group successively; obtain the BPC157 peptide resin of protection; slough fluorenes methoxy carbonyl acyl group blocking group therebetween successively; use N; N-di-isopropyl carbodiimide/1-hydroxyl-benzo-triazole; N; N-di-isopropyl carbodiimide/N-hydroxyl-7-azo benzotriazole; 7-azepine benzothiazole-1-base-oxygen-(three-(dimethylin) phosphines) hexafluorophosphate/1-hydroxyl-benzo-triazole; 7-azepine benzothiazole-1-base-oxygen-(three-(dimethylin) phosphines) hexafluorophosphate/1-hydroxyl-benzo-triazole/N-hydroxyl-7-azo benzotriazole; benzotriazole-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester/1-hydroxyl-benzo-triazole; benzotriazole-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester/N-hydroxyl-7-azo benzotriazole; 2-(7-azo benzotriazole)-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester/1-hydroxyl-benzo-triazole; 2-(7-azo benzotriazole)-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester/N-hydroxyl-7-azo benzotriazole or 6-chlorobenzene and triazole-1; 1; 3; a kind of in 3-tetramethyl-urea phosphofluoric acid ester eight-hydroxyl-benzo-triazole connects reactive polypeptide for condensing agent; behind the BPC157 peptide resin that must protect; take off the side chain protected group synchronously and cut peptide; obtain BPC157 peptide crude product, optional through C 18Or C 8Chromatographic column is carried out separation and purification, obtains said BPC157 peptide.

2. method according to claim 1 uses microwave radiation with fast reaction speed in the preparation process.

3. method according to claim 1 is characterized in that comprising the steps:

(1) preparation Fmoc-Val-resin:

With triphen chloromethyl resin, 4-methyl-triphen chloromethyl resin, 4-methoxyl group-triphen chloromethyl resin, 2-chloro-triphen chloromethyl resin or Wang resin a kind of resin of doing wherein, use N, dinethylformamide or methylene dichloride soaked 10~60 minutes, in the mixture, the bulking value concentration of resin is 5-20ml/g;

Then in above-mentioned resin, add DIEA, DIC or DMAP, Fmoc-Val-OH, 10~50 °C were reacted 0.5~5 hour, adding closed reagent methyl alcohol, Benzoyl chloride or pyridine again reacted 0.2-3 hour for 10~50 °C, resin Virahol, N, the dinethylformamide washing obtains the Fmoc-Val-resin;

The mole number of DIEA or DMAP is 2~20 times of resin;

The mole number of Fmoc-Val-OH is 1~10 times of resin:

In the reaction soln, the concentration of resin is 0.1~5ml/g;

(2) preparation Fmoc-Leu-Val-resin:

In the Fmoc-Val-resin of step (1), add 10~50 °C of reactions of reagent 5~30 minutes of raising one's hat, drain with vacuum pump, again add 10~50 °C of reactions of reagent 10~60 minutes of raising one's hat, drain, with washed with isopropyl alcohol 3 times, DMF washing 3 times, the DMF that heavily steamed washing 2 times, add with DMF dissolved Fmoc-Leu-OH, DIEA, the mixture of TBTU/HOBt or HBTU/HOBt or BOP/HOBt, 10~50 °C were reacted 10~60 minutes, drained with vacuum pump, used washed with isopropyl alcohol 3 times, DMF washing 3 times, the DMF washing of heavily steaming 2 times is drained, and obtains the Fmoc-Leu-Val-resin;

The component of the said reagent of raising one's hat and volume ratio are: PIP: DMF=1: 2-5, down together;

The mole number of Fmoc-Leu-OH is 1-5 a times of resin;

The weight of Fmoc-Val-resin is 5~20ml/g with the ratio of the add-on of the reagent of raising one's hat;

The mole number of TBTU/HBTU/BOP is 2-5 a times of resin;

The HOBT/HOAT mole number is 2-5 a times of resin;

(3) preparation Fmoc-Gly-Leu-Val-resin:

In the Fmoc-Leu-Val-resin of step (2), add 10~50 °C of reactions of reagent 5~30 minutes of raising one's hat, drain with vacuum pump, again add 10~50 °C of reactions of reagent 10~60 minutes of raising one's hat, drain, with washed with isopropyl alcohol 3 times, DMF washing 3 times, the DMF that heavily steamed washing 2 times, add with DMF dissolved Fmoc-Gly-OH, DIEA, the mixture of TBTU/HOBt or HBTU/HOBt or BOP/HOBt, 10~50 °C were reacted 10~60 minutes, drained with vacuum pump, used washed with isopropyl alcohol 3 times, DMF washing 3 times, the DMF washing of heavily steaming 2 times is drained, and obtains the Fmoc-Gly-Leu-Val-resin;

The Fmoc-Gly-OH mole number is 2~5 times of resin;