WO2022141615A1 - Synthesis method for atosiban - Google Patents

Synthesis method for atosiban Download PDF

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Publication number
WO2022141615A1
WO2022141615A1 PCT/CN2021/070062 CN2021070062W WO2022141615A1 WO 2022141615 A1 WO2022141615 A1 WO 2022141615A1 CN 2021070062 W CN2021070062 W CN 2021070062W WO 2022141615 A1 WO2022141615 A1 WO 2022141615A1
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Prior art keywords
resin
fmoc
atosiban
orn
pro
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PCT/CN2021/070062
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French (fr)
Chinese (zh)
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胡鹏
吴峰
刘自成
岳泽乐
钟祥龙
龙镭
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湖北健翔生物制药有限公司
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Priority to CN202180001434.1A priority Critical patent/CN115038711B/en
Priority to PCT/CN2021/070062 priority patent/WO2022141615A1/en
Publication of WO2022141615A1 publication Critical patent/WO2022141615A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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

Definitions

  • the invention relates to the field of polypeptide synthesis, in particular to a method for synthesizing atosiban.
  • Atosiban Acetate Injection was first listed in Austria on March 23, 2000 under the trade name: Atosiban, a new anti-preterm drug developed by Ferring GmbH, is an oxytocin analog, a competitive antagonist of oxytocin receptors in the uterus, decidua, and fetal membranes.
  • the first-line drug recommended by the Medical Association; it can inhibit the binding of oxytocin and oxytocin receptors, thereby directly inhibiting the effect of oxytocin on the uterus and uterine contractions; it can also inhibit the hydrolysis of phosphatidylinositol.
  • Atosiban is a cyclic nonapeptide, its molecular formula is C 43 H 67 N 11 O 12 S 2 ; its molecular weight is 994.19; its CAS registration number is 90779-69-4; its peptide sequence is as follows:
  • the solid-phase synthesis of atosiban uses Rink Amide AM Resin resin solid-phase coupling to obtain Mpa(Trt)-D-Tyr(Et)-Ile-Thr(tBu)- Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-Resin is directly oxidized in solid phase to generate disulfide bonds, and then cleaved to obtain atosiban.
  • the Rink Amide AM Resin resin used in the prior art needs to be cracked in a strongly acidic environment, which is not conducive to product stability and has a greater operational risk; Mpr and Cys both have sulfhydryl groups, and the sulfhydryl groups have the ability to capture tBu to generate double tBu impurities,
  • Mpr and Cys both have sulfhydryl groups, and the sulfhydryl groups have the ability to capture tBu to generate double tBu impurities
  • the Chinese patent with publication number CN105408344B discloses a method for synthesizing atosiban starting from Fmoc-Orn-Gly-NH2, wherein Fmoc-Orn-Gly-NH2 is connected to trityl through the side chain of ornithine On the base resin, impurities can be effectively controlled.
  • the use of dipeptide and trityl-type resin for coupling the resin attached to the Orn side chain of the dipeptide increases the steric hindrance of the subsequent Pro coupling and prolongs the coupling time, which is easy to cause missing peptide impurities.
  • the present invention provides a method for synthesizing atosiban. It includes the following steps:
  • the resin in step 1) is a polymer resin capable of combining with the amino functional group of the side chain of Orn.
  • the polymer resin is selected from trityl type resins.
  • the trityl resin is specifically trityl resin, 2-chloro-trityl resin, 4-methyl-trityl resin, and 4-methoxy-trityl resin.
  • Trityl type resin refers to the resin containing trityl methyl structure, which is easy to react with amino group, can be cracked under mild conditions, is easy to remove, and meets the conditions.
  • the tripeptide can be coupled to the trityl methyl group.
  • the substitution degree of the resin in step 1) is 0.8-1.0 mmol/g, the substitution is too high to increase the low cost, and the substitution is too high to be difficult to couple.
  • step 1) the tripeptide reacts with the resin under the action of DIEA to form a tripeptide resin.
  • DIEA acts as a base to help drive the entire reaction.
  • step 2 ) is specifically: sequentially coupling Fmoc-Cys(Trt)-OH, Fmoc-Asn-OH, Fmoc-Thr-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, Mpa(Trt)-OH give Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys( Trt)-Pro-Orn (trityl-type resin)-Gly- NH2 .
  • the corresponding fragment in the atosiban sequence inserted in step 2) is selected from Mpa(Trt)-D-Tyr(Et)-OH, Fmoc-D-Tyr(Et)- Ile-OH, Fmoc-Asn-Cys(Trt)-OH.
  • the condensing agent in step 2) is selected from any one of HOBt/DIC, HCTU/DIEA, HBTU/DIEA, HATU/HOAt/DIEA, and HBTU/HOBt/DIEA. More preferably, the condensing agent is HOBt/DIC.
  • a low concentration of TFA can separate the peptide from the resin, ensuring the stability of the cyclized peptide chain, and Trt shedding does not require high concentrations of acid.
  • the oxidant in step 3) is selected from iodine and hydrogen peroxide.
  • the resin which can reduce the problem of Pro-deficient peptide impurities due to steric hindrance and prolonged reaction time; the trityl type resin is used as the starting resin , Fmoc-Pro-Orn (trityl resin)-Gly-NH 2 is an intermediate, the active group of Orn in the tripeptide-NH 2 is linked to the resin to reduce a Boc protective group, and Fmoc is used at the same time.
  • the atosiban product obtained by the method has a high quality controllable yield, is beneficial to purification and product quality improvement, is safe and convenient to operate, simple to post-processing, recyclable solvent, reduces production cost, and is suitable for industrial production.
  • Fig. 1 is the HPLC spectrum of atosiban crude peptide prepared in Example 10.
  • FIG. 2 is the HPLC spectrum of the atosiban refined peptide prepared in Example 14.
  • FIG. 2 is the HPLC spectrum of the atosiban refined peptide prepared in Example 14.
  • Fmoc-Pro-OH 134.94 g, 400 mmol
  • N-hydroxysuccinimide 46.00 g, 400 mmol
  • N-hydroxysuccinimide 46.00 g, 400 mmol
  • 1600 ml of tetrahydrofuran 1600 ml
  • the temperature was controlled at about 5°C, and a solution of DCC (90.72g, 440mmol) in tetrahydrofuran (320ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 400 ml of tetrahydrofuran, and H-Gly-NH 2 (29.64 g, 400 mmol) was dissolved in 300 ml of tetrahydrofuran and slowly added dropwise to the above solution, and the reaction was continued at room temperature after dropping, and the monitoring of the raw materials was completed.
  • reaction was filtered, and the filtrate was concentrated under reduced pressure. Dry, add 1000 mL of 5% TFA/DCM solution to the reaction solution, continue to react for 1 h, and concentrate to dryness to obtain a yellow oil, which is recrystallized with isopropanol to obtain 171.56 g of white solid with a yield of 69%.
  • Trityl resin (37.5 g, 30 mmol, substitution degree: 0.80 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1, DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added.
  • 2-CTC Resin resin (30.0 g, 30 mmol, substitution degree: 1.00 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1, DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added.
  • 4-methyl-trityl resin (33.33 g, 30 mmol, substitution degree: 0.90 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1, DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added.
  • 4-Methoxy-trityl resin (30.0 g, 30 mmol, substitution degree: 1.00 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1, DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added.
  • Fmoc-Pro-Orn (trityl resin)-Gly-NH 2 (35.71 g) prepared in Example 2 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod, and the ninhydrin test was positive, indicating that Fmoc had been removed.
  • DBLK solution 20% piperidine/DMF solution, V/V
  • the resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, the DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand.
  • the peptide resin was 48.72g after drying, and the resin weight gain was 89.0%.
  • Fmoc-Pro-Orn(2-CTC Resin)-Gly-NH 2 (30.00 g) prepared in Example 3 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod. The ninhydrin test was positive, indicating that Fmoc had been removed.
  • Fmoc-D-Tyr(Et)-OH (86.30 g, 200 mmol) and N-hydroxysuccinimide (23.00 g, 200 mmol) were weighed into 800 ml of tetrahydrofuran, and stirred at room temperature.
  • the temperature was controlled at about 5°C, and a solution of DCC (45.36g, 220mmol) in tetrahydrofuran (160ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 200 ml of tetrahydrofuran, and H-Ile-OH (26.24 g, 200 mmol) was dissolved in 150 ml of tetrahydrofuran and slowly added dropwise to the above solution. After dropping, the reaction was continued at room temperature. The monitoring of the raw materials was completed. After filtration, the solution was concentrated under reduced pressure.
  • the resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, the DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand.
  • the peptide resin was 42.77g after drying, and the resin weight gain rate was 87.4%.
  • Fmoc-Pro-Orn (4-methyl-trityl resin)-Gly-NH 2 (30.00 g) prepared in Example 4 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod, and the ninhydrin test was positive, indicating that Fmoc had been removed.
  • DBLK solution 20% piperidine/DMF solution, V/V
  • the resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand.
  • the peptide resin was 42.91g after drying, and the resin weight gain rate was 88.3%.
  • Fmoc-Pro-Orn (4-methoxy-trityl resin)-Gly-NH 2 (30.00 g) prepared in Example 5 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod, and the ninhydrin test was positive, indicating that Fmoc had been removed.
  • DBLK solution 20% piperidine/DMF solution, V/V
  • Fmoc-Asn-OH 70.87 g, 200 mmol
  • N-hydroxysuccinimide 23.00 g, 200 mmol
  • the resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, the DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand.
  • the peptide resin was 42.28g after drying, and the resin weight gain was 84.0%.
  • the atosiban crude peptide prepared in Example 10 was dissolved in 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 10.12g , the yield is 64%, the purity is 99%, and the HPLC spectrum of the obtained atosiban refined peptide is shown in Figure 2.
  • the atosiban crude peptide prepared in Example 11 was dissolved in a 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 9.80 g , the yield is 62%, the purity is 99%, and the obtained atosiban peptide HPLC spectrum is similar to Figure 2.
  • the atosiban crude peptide obtained in Example 12 was dissolved in 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 10.28g , the yield is 65%, the purity is 99%, and the HPLC spectrum of the obtained atosiban peptide is similar to that in Figure 2.
  • the atosiban crude peptide obtained in Example 13 was dissolved in 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 10.27g , the yield is 65%, the purity is 99%, and the HPLC spectrum of the obtained atosiban peptide is similar to that in Figure 2.

Abstract

A synthesis method for atosiban, relating to the field of polypeptide synthesis. The method comprises the following steps: synthesizing Fmoc-Pro-Orn-Gly-NH2 tripeptide, and taking a trityl resin as a starting resin to obtain Fmoc-Pro-Orn(trityl resin)-Gly-NH2; using a condensing agent to sequentially introduce corresponding protected amino acids or fragments into an atosiban sequence to obtain an atosiban linear peptide resin; and performing pyrolysis and oxidation to obtain atosiban. The present invention effectively reduces the generation of tBu impurities, can mitigate the problem of lack of peptide impurities in Pro, and provides an atosiban synthesis method that is simple and convenient to operate, easy to control in quality, and beneficial to purification and product quality improvement.

Description

一种阿托西班的合成方法A kind of synthetic method of atosiban 技术领域technical field
本发明涉及多肽合成领域，尤其涉及一种阿托西班的合成方法。The invention relates to the field of polypeptide synthesis, in particular to a method for synthesizing atosiban.
背景技术Background technique
阿托西班注射液(AtosibanAcetate Injection)2000年3月23日首次在奥地利上市，商品名为：
Figure PCTCN2021070062-appb-000001
阿托西班(Atosiban)，一种由Ferring Gmbh研制的新型抗早产药，是一种催产素类似物，是子宫内及蜕膜、胎膜上催产素受体的竞争性拮抗剂，是欧洲医学会推荐的一线用药；它可以抑制催产素和催产素受体结合，从而直接抑制催产素作用于子宫，抑制子宫收缩；也可以抑制磷脂酰肌醇的水解作用。 Atosiban Acetate Injection was first listed in Austria on March 23, 2000 under the trade name:
Figure PCTCN2021070062-appb-000001
Atosiban, a new anti-preterm drug developed by Ferring GmbH, is an oxytocin analog, a competitive antagonist of oxytocin receptors in the uterus, decidua, and fetal membranes. The first-line drug recommended by the Medical Association; it can inhibit the binding of oxytocin and oxytocin receptors, thereby directly inhibiting the effect of oxytocin on the uterus and uterine contractions; it can also inhibit the hydrolysis of phosphatidylinositol.
阿托西班是一个环九肽，其分子式为C 43H 67N 11O 12S 2；分子量为994.19；CAS登记号为90779-69-4；其肽序如下式所示： Atosiban is a cyclic nonapeptide, its molecular formula is C 43 H 67 N 11 O 12 S 2 ; its molecular weight is 994.19; its CAS registration number is 90779-69-4; its peptide sequence is as follows:
Cyclo[Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys]-Pro-Orn-Gly-NH 2 Cyclo[Mpa-D-Tyr(Et)-Ile-Thr-Asn-Cys]-Pro-Orn-Gly-NH 2
公告号为CN101314613B和CN101696236B的中国专利中，阿托西班的固相合成使用Rink Amide AM Resin树脂固相逐步偶联得到Mpa(Trt)-D-Tyr(Et)-Ile-Thr(tBu)-Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-Resin，直接固相氧化生成二硫键，再裂解得到阿托西班。现有技术中采用的Rink Amide AM Resin树脂需要在强酸性环境下才能裂解下来，不利于产品稳定且操作危险性较大；Mpr和Cys都有巯基，巯基有捕获tBu的能力生成双tBu杂质，固相氧化后的肽树脂在裂解脱除保护基和树脂时，由于有tBu或tBu源的Boc保护基，对捕获剂要求高，不利于产品质量控制，降低产品收率。In the Chinese patents with announcement numbers CN101314613B and CN101696236B, the solid-phase synthesis of atosiban uses Rink Amide AM Resin resin solid-phase coupling to obtain Mpa(Trt)-D-Tyr(Et)-Ile-Thr(tBu)- Asn(Trt)-Cys(Trt)-Pro-Orn(Boc)-Gly-Resin is directly oxidized in solid phase to generate disulfide bonds, and then cleaved to obtain atosiban. The Rink Amide AM Resin resin used in the prior art needs to be cracked in a strongly acidic environment, which is not conducive to product stability and has a greater operational risk; Mpr and Cys both have sulfhydryl groups, and the sulfhydryl groups have the ability to capture tBu to generate double tBu impurities, When the peptide resin after solid-phase oxidation is cleaved to remove protecting groups and resins, due to the presence of tBu or tBu-derived Boc protecting groups, high requirements for capture agents are required, which is not conducive to product quality control and reduces product yield.
公告号为CN105408344B的中国专利，公开了一种由Fmoc-Orn-Gly-NH2起始合成阿托西班的方法，其中Fmoc-Orn-Gly-NH2通过鸟氨酸的侧链连接于三苯甲基树脂上，可以有效控制杂质。然而使用二肽与三苯甲基型树脂偶联，二肽Orn侧链上接入的树脂导致后续Pro偶联的空间位阻增大，偶联时间延长，易造成缺失肽杂质。The Chinese patent with publication number CN105408344B discloses a method for synthesizing atosiban starting from Fmoc-Orn-Gly-NH2, wherein Fmoc-Orn-Gly-NH2 is connected to trityl through the side chain of ornithine On the base resin, impurities can be effectively controlled. However, the use of dipeptide and trityl-type resin for coupling, the resin attached to the Orn side chain of the dipeptide increases the steric hindrance of the subsequent Pro coupling and prolongs the coupling time, which is easy to cause missing peptide impurities.
因此，需要寻找一种便于偶联和裂解，有利于产品质量稳定，方便安全操作，产品质量可控，提升产品收率的阿托西班合成方法。Therefore, it is necessary to find a method for synthesizing atosiban which is convenient for coupling and cleavage, is conducive to stable product quality, convenient and safe operation, controllable product quality and improved product yield.
发明内容SUMMARY OF THE INVENTION
针对现有技术中存在异构体、插入肽、tBu阳离子捕获杂质以及位阻和反应时间过长导致Pro缺失肽杂质的问题，本发明提供了一种阿托西班的合成方法，所述方法包括如下步骤：Aiming at the problems in the prior art that isomers, intercalated peptides, tBu cation capture impurities, and Pro deletion peptide impurities caused by steric hindrance and excessive reaction time, the present invention provides a method for synthesizing atosiban. It includes the following steps:
1)合成得到Fmoc-Pro-Orn-Gly-NH 2三肽，之后用树脂与之反应得到Fmoc-Pro-Orn(树脂)-Gly-NH 2肽树脂； 1) Synthesize the Fmoc-Pro-Orn-Gly-NH 2 tripeptide, and then react with the resin to obtain the Fmoc-Pro-Orn (resin)-Gly-NH 2 peptide resin;
2)脱除Fmoc-Pro-Orn(树脂)-Gly-NH 2的Fmoc保护，之后采用缩合剂依次接入阿托西 班序列中相应的保护氨基酸或片段，制得阿托西班线性肽树脂； 2) Remove the Fmoc protection of Fmoc-Pro-Orn (resin)-Gly-NH 2 , then use a condensing agent to sequentially access the corresponding protected amino acids or fragments in the atosiban sequence to obtain atosiban linear peptide resin ;
3)裂解、氧化，得到阿托西班。3) cracking and oxidation to obtain atosiban.
在本发明优选的实施方案中，步骤1)中树脂采用能与Orn的侧链氨基官能团结合的聚合物树脂。进一步优选的，所述聚合物树脂选自三苯甲基型树脂。其中三苯甲基型树脂具体为三苯甲基树脂、2-氯-三苯甲基树脂、4-甲基-三苯甲基树脂、4-甲氧基-三苯甲基树脂。In a preferred embodiment of the present invention, the resin in step 1) is a polymer resin capable of combining with the amino functional group of the side chain of Orn. Further preferably, the polymer resin is selected from trityl type resins. The trityl resin is specifically trityl resin, 2-chloro-trityl resin, 4-methyl-trityl resin, and 4-methoxy-trityl resin.
此工艺既要考虑能将三肽偶联在树脂上，又要考虑将肽较容易的切割下来。三苯甲基型树脂是指含有三苯基甲基结构的树脂，易与氨基反应，使用温和的条件即可裂解，易脱除，符合条件。使用三苯甲基型树脂，三肽可以偶联在三苯基甲基上。In this process, both the tripeptide can be coupled to the resin and the peptide can be easily cleaved. Trityl type resin refers to the resin containing trityl methyl structure, which is easy to react with amino group, can be cracked under mild conditions, is easy to remove, and meets the conditions. Using a trityl type resin, the tripeptide can be coupled to the trityl methyl group.
在本发明优选的实施方案中，步骤1)中树脂的替代度为0.8～1.0mmol/g，替代度过低成本增加，替代度过高偶联困难。In a preferred embodiment of the present invention, the substitution degree of the resin in step 1) is 0.8-1.0 mmol/g, the substitution is too high to increase the low cost, and the substitution is too high to be difficult to couple.
在本发明优选的实施方案中，步骤1)中三肽在DIEA作用下与树脂反应生成三肽树脂。DIEA作为碱有助于推动整个反应的进行。In a preferred embodiment of the present invention, in step 1), the tripeptide reacts with the resin under the action of DIEA to form a tripeptide resin. DIEA acts as a base to help drive the entire reaction.
在本发明优选的实施方案中，步骤2)具体为：在Fmoc-Pro-Orn(树脂)-Gly-NH 2肽树脂上依次偶联Fmoc-Cys(Trt)-OH、Fmoc-Asn-OH、Fmoc-Thr-OH、Fmoc-Ile-OH、Fmoc-D-Tyr(Et)-OH、Mpa(Trt)-OH得到Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Trt)-Pro-Orn(三苯甲基型树脂)-Gly-NH 2。 In a preferred embodiment of the present invention, step 2 ) is specifically: sequentially coupling Fmoc-Cys(Trt)-OH, Fmoc-Asn-OH, Fmoc-Thr-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, Mpa(Trt)-OH give Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys( Trt)-Pro-Orn (trityl-type resin)-Gly- NH2 .
在本发明优选的实施方案中，步骤2)中接入的阿托西班序列中相应的片段选自Mpa(Trt)-D-Tyr(Et)-OH、Fmoc-D-Tyr(Et)-Ile-OH、Fmoc-Asn-Cys(Trt)-OH。In a preferred embodiment of the present invention, the corresponding fragment in the atosiban sequence inserted in step 2) is selected from Mpa(Trt)-D-Tyr(Et)-OH, Fmoc-D-Tyr(Et)- Ile-OH, Fmoc-Asn-Cys(Trt)-OH.
在本发明优选的实施方案中，步骤2)中缩合剂选自HOBt/DIC、HCTU/DIEA、HBTU/DIEA、HATU/HOAt/DIEA、HBTU/HOBt/DIEA中任意一种。更优选的，缩合剂选择HOBt/DIC。In a preferred embodiment of the present invention, the condensing agent in step 2) is selected from any one of HOBt/DIC, HCTU/DIEA, HBTU/DIEA, HATU/HOAt/DIEA, and HBTU/HOBt/DIEA. More preferably, the condensing agent is HOBt/DIC.
在本发明优选的实施方案中，步骤3)裂解试剂选自TFA/DCM，裂解试剂配比进一步优选为TFA：DCM＝1：99～20：80。低浓度的TFA即可将肽和树脂分离，保证已环化肽链的稳定性，同时Trt脱落也不需要高浓度的酸。In a preferred embodiment of the present invention, the cleavage reagent in step 3) is selected from TFA/DCM, and the ratio of the cleavage reagent is more preferably TFA:DCM=1:99～20:80. A low concentration of TFA can separate the peptide from the resin, ensuring the stability of the cyclized peptide chain, and Trt shedding does not require high concentrations of acid.
在本发明优选的实施方案中，步骤3)中氧化剂选自碘、过氧化氢。In a preferred embodiment of the present invention, the oxidant in step 3) is selected from iodine and hydrogen peroxide.
本发明在合成Fmoc-Pro-Orn-Gly-NH 2三肽后与树脂偶联，可以减少因位阻和反应时间延长导致Pro缺失肽杂质的问题；以三苯甲基型树脂作为起始树脂，Fmoc-Pro-Orn(三苯甲基型树脂)-Gly-NH 2为中间体，将三肽中Orn的活性基团-NH 2挂在树脂上偶联减少一个Boc保护基，同时采用Fmoc-Thr-OH，进而减少tBu类杂质的生成，提高主峰纯度，增加收率；使用对酸敏感的树脂，在弱酸性条件下切割肽，仅需裂解一次，保护基即可完全脱除，操作方便安全。使用本发明得到的阿托西班产品质量可控收率高，利于纯化和提升产品质量，同时 操作安全方便，后处理简单，溶剂可回收，降低生产成本，适合工业化生产。 In the present invention, after synthesizing the Fmoc-Pro-Orn-Gly-NH 2 tripeptide, it is coupled with the resin, which can reduce the problem of Pro-deficient peptide impurities due to steric hindrance and prolonged reaction time; the trityl type resin is used as the starting resin , Fmoc-Pro-Orn (trityl resin)-Gly-NH 2 is an intermediate, the active group of Orn in the tripeptide-NH 2 is linked to the resin to reduce a Boc protective group, and Fmoc is used at the same time. -Thr-OH, thereby reducing the generation of tBu impurities, improving the purity of the main peak, and increasing the yield; using acid-sensitive resin to cleave the peptide under weakly acidic conditions, only one cleavage, the protective group can be completely removed, the operation Convenient and safe. The atosiban product obtained by the method has a high quality controllable yield, is beneficial to purification and product quality improvement, is safe and convenient to operate, simple to post-processing, recyclable solvent, reduces production cost, and is suitable for industrial production.
附图说明Description of drawings
图1为采用实施例10制得的阿托西班粗肽HPLC谱图。Fig. 1 is the HPLC spectrum of atosiban crude peptide prepared in Example 10.
图2为采用实施例14制得的阿托西班精肽HPLC谱图。FIG. 2 is the HPLC spectrum of the atosiban refined peptide prepared in Example 14. FIG.
具体实施方式Detailed ways
下面通过实施例对本发明作进一步的详细说明，旨在用于说明本发明而非限定本发明。应当指出，对于本领域技术人员而言，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也同样落入本发明的保护范围之内。The present invention will be further described in detail below through examples, which are intended to illustrate the present invention rather than limit it. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the present invention.
实施例1、Fmoc-Pro-Orn-Gly-NH 2三肽的合成 Example 1. Synthesis of Fmoc-Pro-Orn-Gly-NH 2 tripeptide
称取Fmoc-Pro-OH(134.94g，400mmol)和N-羟基琥珀酰亚胺(46.00g，400mmol)加入到四氢呋喃1600ml中，室温搅拌。控温5℃左右，缓慢地加DCC(90.72g，440mmol)的四氢呋喃(320ml)溶液室温搅拌2.5h，过滤，浓缩后加入到石油醚中重结晶析出固体，洗涤后干燥，将得到的活化酯固体溶于四氢呋喃400ml中，将H-Orn(Boc)-NH 2(92.92g，400mmol)溶于四氢呋喃300ml缓慢的滴加入上述溶液中，滴完室温下继续反应，监控原料反应完全，过滤，滤液减压浓缩干，加入N-羟基琥珀酰亚胺(46.00g，400mmol)和四氢呋喃1600ml溶解，室温搅拌。控温5℃左右，缓慢地加DCC(90.72g，440mmol)的四氢呋喃(320ml)溶液室温搅拌2.5h，过滤，浓缩后加入到石油醚中重结晶析出固体，洗涤后干燥，将得到的活化酯固体溶于四氢呋喃400ml中，将H-Gly-NH 2(29.64g，400mmol)溶于四氢呋喃300ml缓慢的滴加入上述溶液中，滴完室温下继续反应，监控原料反应完全，过滤，滤液减压浓缩干，向反应液中加入1000mL浓度为5％的TFA/DCM溶液，继续反应1h，浓缩干，得黄色油状物，用异丙醇重结晶得到白色固体171.56g，收率69％。 Fmoc-Pro-OH (134.94 g, 400 mmol) and N-hydroxysuccinimide (46.00 g, 400 mmol) were weighed into 1600 ml of tetrahydrofuran, and stirred at room temperature. The temperature was controlled at about 5°C, and a solution of DCC (90.72g, 440mmol) in tetrahydrofuran (320ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 400 ml of tetrahydrofuran, and H-Orn(Boc)-NH 2 (92.92 g, 400 mmol) was dissolved in 300 ml of tetrahydrofuran and slowly added dropwise to the above solution. After dropping, the reaction was continued at room temperature. Concentrate to dryness under reduced pressure, add N-hydroxysuccinimide (46.00 g, 400 mmol) and 1600 ml of tetrahydrofuran to dissolve, and stir at room temperature. The temperature was controlled at about 5°C, and a solution of DCC (90.72g, 440mmol) in tetrahydrofuran (320ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 400 ml of tetrahydrofuran, and H-Gly-NH 2 (29.64 g, 400 mmol) was dissolved in 300 ml of tetrahydrofuran and slowly added dropwise to the above solution, and the reaction was continued at room temperature after dropping, and the monitoring of the raw materials was completed. The reaction was filtered, and the filtrate was concentrated under reduced pressure. Dry, add 1000 mL of 5% TFA/DCM solution to the reaction solution, continue to react for 1 h, and concentrate to dryness to obtain a yellow oil, which is recrystallized with isopropanol to obtain 171.56 g of white solid with a yield of 69%.
实施例2、取代度为0.42mmol/g的Fmoc-Pro-Orn(三苯甲基树脂)-Gly-NH 2肽树脂的合成 Example 2. Synthesis of Fmoc-Pro-Orn (trityl resin)-Gly-NH 2 peptide resin with a degree of substitution of 0.42 mmol/g
称取三苯甲基树脂(37.5g，30mmol，替代度：0.80mmol/g)于固相反应合成柱中。加入400mL干燥的DMF溶胀30min，抽去DMF。用3*400mL干燥的DMF洗涤树脂，抽去DMF。加入实施例1制得的Fmoc-Pro-Orn-Gly-NH 2(37.30g，60mmol)，DIEA(11.63g，90mmol)，加入100mL干燥DMF溶解澄清，加入到树脂中反应2h，加入甲醇(9.61g，300mmol)反应20min，抽干，用3*400mL的CH 2Cl 2洗涤树脂，抽去CH 2Cl 2。取出树脂，25～35℃真空干燥，得到Fmoc-Pro-Orn(三苯甲基树脂)-Gly-NH 2树脂52.14g，测得取代度0.42mmol/g。 Trityl resin (37.5 g, 30 mmol, substitution degree: 0.80 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1, DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added. g, 300 mmol) reacted for 20 min, sucked dry, washed the resin with 3*400 mL of CH 2 Cl 2 , and removed CH 2 Cl 2 . The resin was taken out and dried under vacuum at 25-35° C. to obtain 52.14 g of Fmoc-Pro-Orn (trityl resin)-Gly-NH 2 resin with a measured substitution degree of 0.42 mmol/g.
实施例3、取代度为0.50mmol/g的Fmoc-Pro-Orn(2-CTC Resin)-Gly-NH 2肽树脂的合成 Example 3. Synthesis of Fmoc-Pro-Orn(2-CTC Resin)-Gly-NH 2 peptide resin with a degree of substitution of 0.50 mmol/g
称取2-CTC Resin树脂(30.0g，30mmol，替代度：1.00mmol/g)于固相反应合成柱中。 加入400mL干燥的DMF溶胀30min，抽去DMF。用3*400mL干燥的DMF洗涤树脂，抽去DMF。加入实施例1制得的Fmoc-Pro-Orn-Gly-NH 2(37.30g，60mmol)，DIEA(11.63g，90mmol)，加入100mL干燥DMF溶解澄清，加入到树脂中反应2h，加入甲醇(9.61g，300mmol)反应20min，抽干，用3*400mL的CH 2Cl 2洗涤树脂，抽去CH 2Cl 2。取出树脂，25～35℃真空干燥，得到Fmoc-Pro-Orn(2-CTC Resin)-Gly-NH 2树脂43.80g，测得取代度0.50mmol/g。 2-CTC Resin resin (30.0 g, 30 mmol, substitution degree: 1.00 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1, DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added. g, 300 mmol) reacted for 20 min, sucked dry, washed the resin with 3*400 mL of CH 2 Cl 2 , and removed CH 2 Cl 2 . The resin was taken out and dried under vacuum at 25-35° C. to obtain 43.80 g of Fmoc-Pro-Orn(2-CTC Resin)-Gly-NH 2 resin with a measured substitution degree of 0.50 mmol/g.
实施例4、取代度为0.50mmol/g的Fmoc-Pro-Orn(4-甲基-三苯甲基树脂)-Gly-NH 2肽树脂的合成 Example 4. Synthesis of Fmoc-Pro-Orn (4-methyl-trityl resin)-Gly-NH 2 peptide resin with a degree of substitution of 0.50 mmol/g
称取4-甲基-三苯甲基树脂(33.33g，30mmol，替代度：0.90mmol/g)于固相反应合成柱中。加入400mL干燥的DMF溶胀30min，抽去DMF。用3*400mL干燥的DMF洗涤树脂，抽去DMF。加入实施例1制得的Fmoc-Pro-Orn-Gly-NH 2(37.30g，60mmol)，DIEA(11.63g，90mmol)，加入100mL干燥DMF溶解澄清，加入到树脂中反应2h，加入甲醇(9.61g，300mmol)反应20min，抽干，用3*400mL的CH 2Cl 2洗涤树脂，抽去CH 2Cl 2。取出树脂，25～35℃真空干燥，得到Fmoc-Pro-Orn(4-甲基-三苯甲基树脂)-Gly-NH 2树脂43.89g，测得取代度0.50mmol/g。 4-methyl-trityl resin (33.33 g, 30 mmol, substitution degree: 0.90 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1, DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added. g, 300 mmol) reacted for 20 min, sucked dry, washed the resin with 3*400 mL of CH 2 Cl 2 , and removed CH 2 Cl 2 . The resin was taken out and dried under vacuum at 25-35° C. to obtain 43.89 g of Fmoc-Pro-Orn (4-methyl-trityl resin)-Gly-NH 2 resin with a measured substitution degree of 0.50 mmol/g.
实施例5、取代度为0.50mmol/g的Fmoc-Pro-Orn(4-甲氧基-三苯甲基树脂)-Gly-NH 2肽树脂的合成 Example 5. Synthesis of Fmoc-Pro-Orn (4-methoxy-trityl resin)-Gly-NH 2 peptide resin with a degree of substitution of 0.50 mmol/g
称取4-甲氧基-三苯甲基树脂(30.0g，30mmol，替代度：1.00mmol/g)于固相反应合成柱中。加入400mL干燥的DMF溶胀30min，抽去DMF。用3*400mL干燥的DMF洗涤树脂，抽去DMF。加入实施例1制得的Fmoc-Pro-Orn-Gly-NH 2(37.30g，60mmol)，DIEA(11.63g，90mmol)，加入100mL干燥DMF溶解澄清，加入到树脂中反应2h，加入甲醇(9.61g，300mmol)反应20min，抽干，用3*400mL的CH 2Cl 2洗涤树脂，抽去CH 2Cl 2。取出树脂，25～35℃真空干燥，得到Fmoc-Pro-Orn(4-甲氧基-三苯甲基树脂)-Gly-NH 2树脂43.69g，测得取代度0.50mmol/g。 4-Methoxy-trityl resin (30.0 g, 30 mmol, substitution degree: 1.00 mmol/g) was weighed into a solid-phase reaction synthesis column. 400 mL of dry DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*400 mL of dry DMF, and the DMF was removed. Fmoc-Pro-Orn-Gly-NH 2 (37.30 g, 60 mmol) prepared in Example 1, DIEA (11.63 g, 90 mmol) were added, 100 mL of dry DMF was added to dissolve and clarified, added to the resin to react for 2 h, and methanol (9.61 mmol) was added. g, 300 mmol) reacted for 20 min, sucked dry, washed the resin with 3*400 mL of CH 2 Cl 2 , and removed CH 2 Cl 2 . The resin was taken out and dried under vacuum at 25-35° C. to obtain 43.69 g of Fmoc-Pro-Orn (4-methoxy-trityl resin)-Gly-NH 2 resin with a measured substitution degree of 0.50 mmol/g.
实施例6、阿托西班线性肽树脂的合成1Example 6. Synthesis of Atosiban Linear Peptide Resin 1
称取实施例2制得的Fmoc-Pro-Orn(三苯甲基树脂)-Gly-NH 2(35.71g)于固相反应合成柱中。加入400mL的DMF溶胀30min，抽去DMF。用3*200mL干燥的DMF洗涤树脂，抽去DMF。加入200mL的DBLK溶液(20％哌啶/DMF溶液,V/V)，脱保护两次，第一次5min，第二次15min。脱保护完每次用200mL DMF洗涤树脂，洗涤6遍，第4次洗涤完毕，用玻璃棒取少许树脂，茚三酮检测呈阳性，表明Fmoc已脱。 Fmoc-Pro-Orn (trityl resin)-Gly-NH 2 (35.71 g) prepared in Example 2 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod, and the ninhydrin test was positive, indicating that Fmoc had been removed.
称取17.57g Fmoc-Cys(Trt)-OH和4.86g HOBt，加入100mL DMF溶解，待完全溶解后， 将溶液冷却至5℃以下，然后加入5.68g DIC(预冷至<0℃),于溶液中活化约3～5min，将活化后的溶液控制加入反应柱中，在20～35℃下反应2～3h，茚三酮检测呈阴性，抽掉反应液，加入200mL的DMF洗涤树脂，洗涤6遍。洗毕后，抽除洗涤液，得到Fmoc-Cys(Trt)-Pro-Orn(三苯甲基树脂)-Gly-NH 2。 Weigh 17.57g Fmoc-Cys(Trt)-OH and 4.86g HOBt, add 100mL DMF to dissolve, after complete dissolution, cool the solution to below 5°C, then add 5.68g DIC (pre-cooled to <0°C), Activated in the solution for about 3 to 5 minutes, the activated solution was added to the reaction column under control, and reacted at 20 to 35 °C for 2 to 3 hours. The ninhydrin test was negative. The reaction solution was removed, and 200 mL of DMF was added to wash the resin. 6 times. After washing, the washing liquid was removed to obtain Fmoc-Cys(Trt)-Pro-Orn (trityl resin)-Gly-NH 2 .
重复接肽反应步骤及去除Fmoc保护基的操作，按照阿托西班氨基酸序列，在Fmoc-Cys(Trt)-Pro-Orn(三苯甲基树脂)-Gly-NH 2上依次偶联Fmoc-Asn-OH、Fmoc-Thr-OH、Fmoc-Ile-OH、Fmoc-D-Tyr(Et)-OH、Mpa(Trt)-OH得到Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Trt)-Pro-Orn(三苯甲基树脂)-Gly-NH 2。DMF洗毕后，抽除洗涤液。再每次用200ml DCM洗涤树脂，洗涤4遍，5min/次，抽掉DCM，树脂在室温(20～35℃)下真空抽干至呈流沙状。肽树脂干燥完后为48.72g,树脂增重率89.0％。 Repeat the step of receiving the peptide and removing the Fmoc protective group. According to the amino acid sequence of atosiban, Fmoc-Cys(Trt)-Pro-Orn (trityl resin)-Gly-NH 2 was coupled to Fmoc- Asn-OH, Fmoc-Thr-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, Mpa(Trt)-OH give Mpa(Trt)-D-Tyr(Et)-Ile-Thr- Asn-Cys(Trt)-Pro-Orn (trityl resin)-Gly- NH2 . After washing with DMF, the washing solution was removed. The resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, the DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand. The peptide resin was 48.72g after drying, and the resin weight gain was 89.0%.
实施例7、阿托西班线性肽树脂的合成2Example 7. Synthesis of atosiban linear peptide resin 2
称取实施例3制得的Fmoc-Pro-Orn(2-CTC Resin)-Gly-NH 2(30.00g)于固相反应合成柱中。加入400mL的DMF溶胀30min，抽去DMF。用3*200mL干燥的DMF洗涤树脂，抽去DMF。加入200mL的DBLK溶液(20％哌啶/DMF溶液,V/V)，脱保护两次，第一次5min，第二次15min。脱保护完每次用200mL DMF洗涤树脂，洗涤6遍，第4次洗涤完毕，用玻璃棒取少许树脂，茚三酮检测呈阳性，表明Fmoc已脱。 Fmoc-Pro-Orn(2-CTC Resin)-Gly-NH 2 (30.00 g) prepared in Example 3 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod. The ninhydrin test was positive, indicating that Fmoc had been removed.
称取17.57g Fmoc-Cys(Trt)-OH和13.65g HBTU，加入100mL DMF溶解，待完全溶解后，将溶液冷却至5℃以下，然后加入5.82g DIEA(预冷至<0℃),于溶液中活化约3～5min，将活化后的溶液控制加入反应柱中，在20～35℃下反应2～3h，茚三酮检测呈阴性，抽掉反应液，加入200mL的DMF洗涤树脂，洗涤6遍。洗毕后，抽除洗涤液，得到Fmoc-Cys(Trt)-Pro-Orn(2-CTC Resin)-Gly-NH 2。 Weigh 17.57g Fmoc-Cys(Trt)-OH and 13.65g HBTU, add 100mL DMF to dissolve, after complete dissolution, cool the solution to below 5°C, then add 5.82g DIEA (pre-cooled to <0°C), put it in Activated in the solution for about 3 to 5 minutes, the activated solution was added to the reaction column under control, and reacted at 20 to 35 °C for 2 to 3 hours. The ninhydrin test was negative. The reaction solution was removed, and 200 mL of DMF was added to wash the resin. 6 times. After washing, the washing solution was removed to obtain Fmoc-Cys(Trt)-Pro-Orn(2-CTC Resin)-Gly-NH 2 .
称取Fmoc-D-Tyr(Et)-OH(86.30g，200mmol)和N-羟基琥珀酰亚胺(23.00g，200mmol)加入到四氢呋喃800ml中，室温搅拌。控温5℃左右，缓慢地加DCC(45.36g，220mmol)的四氢呋喃(160ml)溶液室温搅拌2.5h，过滤，浓缩后加入到石油醚中重结晶析出固体，洗涤后干燥，将得到的活化酯固体溶于四氢呋喃200ml中，将H-Ile-OH(26.24g，200mmol)溶于四氢呋喃150ml中缓慢的滴加入上述溶液中，滴完室温下继续反应，监控原料反应完全，过滤后，减压浓缩，将浓缩液加入到石油醚中析出固体，洗涤固体后再干燥，用异丙醇重结晶干燥得到Fmoc-D-Tyr(Et)-Ile-OH 75.60g，收率75％。Fmoc-D-Tyr(Et)-OH (86.30 g, 200 mmol) and N-hydroxysuccinimide (23.00 g, 200 mmol) were weighed into 800 ml of tetrahydrofuran, and stirred at room temperature. The temperature was controlled at about 5°C, and a solution of DCC (45.36g, 220mmol) in tetrahydrofuran (160ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 200 ml of tetrahydrofuran, and H-Ile-OH (26.24 g, 200 mmol) was dissolved in 150 ml of tetrahydrofuran and slowly added dropwise to the above solution. After dropping, the reaction was continued at room temperature. The monitoring of the raw materials was completed. After filtration, the solution was concentrated under reduced pressure. , the concentrated solution was added to petroleum ether to separate out the solid, the solid was washed and then dried, recrystallized and dried with isopropanol to obtain 75.60 g of Fmoc-D-Tyr(Et)-Ile-OH with a yield of 75%.
重复接肽反应步骤及去除Fmoc保护基的操作，按照阿托西班氨基酸序列，在Fmoc-Cys(Trt)-Pro-Orn(2-CTC Resin)-Gly-NH 2上依次偶联Fmoc-Asn-OH、Fmoc-Thr-OH、Fmoc-D-Tyr(Et)-Ile-OH、Mpa(Trt)-OH得到Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Trt)-Pro-Orn(2-CTC  Resin)-Gly-NH 2。DMF洗毕后，抽除洗涤液。再每次用200ml DCM洗涤树脂，洗涤4遍，5min/次，抽掉DCM，树脂在室温(20～35℃)下真空抽干至呈流沙状。肽树脂干燥完后为42.77g,树脂增重率87.4％。 Repeat the step of receiving the peptide and removing the Fmoc protective group. According to the amino acid sequence of atosiban, sequentially couple Fmoc-Asn on Fmoc-Cys(Trt)-Pro-Orn(2-CTC Resin)-Gly-NH 2 -OH, Fmoc-Thr-OH, Fmoc-D-Tyr(Et)-Ile-OH, Mpa(Trt)-OH to give Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Trt )-Pro-Orn(2-CTC Resin)-Gly- NH2 . After washing with DMF, the washing solution was removed. The resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, the DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand. The peptide resin was 42.77g after drying, and the resin weight gain rate was 87.4%.
实施例8、阿托西班线性肽树脂的合成3Example 8. Synthesis of atosiban linear peptide resin 3
称取实施例4制得的Fmoc-Pro-Orn(4-甲基-三苯甲基树脂)-Gly-NH 2(30.00g)于固相反应合成柱中。加入400mL的DMF溶胀30min，抽去DMF。用3*200mL干燥的DMF洗涤树脂，抽去DMF。加入200mL的DBLK溶液(20％哌啶/DMF溶液,V/V)，脱保护两次，第一次5min，第二次15min。脱保护完每次用200mL DMF洗涤树脂，洗涤6遍，第4次洗涤完毕，用玻璃棒取少许树脂，茚三酮检测呈阳性，表明Fmoc已脱。 Fmoc-Pro-Orn (4-methyl-trityl resin)-Gly-NH 2 (30.00 g) prepared in Example 4 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod, and the ninhydrin test was positive, indicating that Fmoc had been removed.
称取17.57g Fmoc-Cys(Trt)-OH、13.65g HBTU和4.05g HOBt，加入100mL DMF溶解，待完全溶解后，将溶液冷却至5℃以下，然后加入5.82g DIEA(预冷至<0℃),于溶液中活化约3～5min，将活化后的溶液控制加入反应柱中，在20～35℃下反应2～3h，茚三酮检测呈阴性，抽掉反应液，加入200mL的DMF洗涤树脂，洗涤6遍。洗毕后，抽除洗涤液，得到Fmoc-Cys(Trt)-Pro-Orn(4-甲基-三苯甲基树脂)-Gly-NH 2。 Weigh 17.57g Fmoc-Cys(Trt)-OH, 13.65g HBTU and 4.05g HOBt, add 100mL DMF to dissolve, after complete dissolution, cool the solution to below 5 ℃, then add 5.82g DIEA (pre-cooled to <0 ℃), activate in the solution for about 3-5min, add the activated solution to the reaction column under control, react at 20-35 ℃ for 2-3h, the ninhydrin test is negative, remove the reaction solution, add 200mL of DMF The resin was washed 6 times. After washing, the washing liquid was removed to obtain Fmoc-Cys(Trt)-Pro-Orn(4-methyl-trityl resin)-Gly-NH 2 .
称取Mpa(Trt)-OH(69.69g，200mmol)和N-羟基琥珀酰亚胺(23.00g，200mmol)加入到四氢呋喃800ml中，室温搅拌。控温5℃左右，缓慢地加DCC(45.36g，220mmol)的四氢呋喃(160ml)溶液室温搅拌2.5h，过滤，浓缩后加入到石油醚中重结晶析出固体，洗涤后干燥，将得到的活化酯固体溶于四氢呋喃200ml中，将H-D-Tyr(Et)-OH(41.85g，200mmol)溶于四氢呋喃150ml中缓慢的滴加入上述溶液中，滴完室温下继续反应，监控原料反应完全，过滤后，减压浓缩，将浓缩液加入到石油醚中析出固体，洗涤固体后再干燥，用异丙醇重结晶干燥得到Mpa(Trt)-D-Tyr(Et)-OH 77.98g，收率72％。Mpa(Trt)-OH (69.69 g, 200 mmol) and N-hydroxysuccinimide (23.00 g, 200 mmol) were weighed into 800 ml of tetrahydrofuran, and stirred at room temperature. The temperature was controlled at about 5°C, and a solution of DCC (45.36g, 220mmol) in tetrahydrofuran (160ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 200 ml of tetrahydrofuran, and H-D-Tyr(Et)-OH (41.85 g, 200 mmol) was dissolved in 150 ml of tetrahydrofuran and slowly added dropwise to the above solution. After dropping, the reaction was continued at room temperature. Concentrate under reduced pressure, add the concentrated solution to petroleum ether to precipitate a solid, wash the solid and then dry, recrystallize and dry with isopropanol to obtain Mpa(Trt)-D-Tyr(Et)-OH 77.98g, yield 72%.
重复接肽反应步骤及去除Fmoc保护基的操作，按照阿托西班氨基酸序列，在Fmoc-Cys(Trt)-Pro-Orn(4-甲基-三苯甲基树脂)-Gly-NH 2上依次偶联Fmoc-Asn-OH、Fmoc-Thr-OH、Fmoc-Ile-OH、Mpa(Trt)-D-Tyr(Et)-OH得到Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Trt)-Pro-Orn(4-甲基-三苯甲基树脂)-Gly-NH 2。DMF洗毕后，抽除洗涤液。再每次用200ml DCM洗涤树脂，洗涤4遍，5min/次，抽掉DCM，树脂在室温(20～35℃)下真空抽干至呈流沙状。肽树脂干燥完后为42.91g,树脂增重率88.3％。 Repeat the step of receiving the peptide and removing the Fmoc protective group, according to the amino acid sequence of atosiban, on Fmoc-Cys(Trt)-Pro-Orn(4-methyl-trityl resin)-Gly-NH 2 Fmoc-Asn-OH, Fmoc-Thr-OH, Fmoc-Ile-OH, Mpa(Trt)-D-Tyr(Et)-OH were sequentially coupled to obtain Mpa(Trt)-D-Tyr(Et)-Ile-Thr -Asn-Cys(Trt)-Pro-Orn(4-methyl-trityl resin)-Gly- NH2 . After washing with DMF, the washing solution was removed. The resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand. The peptide resin was 42.91g after drying, and the resin weight gain rate was 88.3%.
实施例9、阿托西班线性肽树脂的合成4Example 9. Synthesis of atosiban linear peptide resin 4
称取实施例5制得的Fmoc-Pro-Orn(4-甲氧基-三苯甲基树脂)-Gly-NH 2(30.00g)于固相反应合成柱中。加入400mL的DMF溶胀30min，抽去DMF。用3*200mL干燥的DMF洗涤树脂，抽去DMF。加入200mL的DBLK溶液(20％哌啶/DMF溶液,V/V)，脱保护两次， 第一次5min，第二次15min。脱保护完每次用200mL DMF洗涤树脂，洗涤6遍，第4次洗涤完毕，用玻璃棒取少许树脂，茚三酮检测呈阳性，表明Fmoc已脱。 Fmoc-Pro-Orn (4-methoxy-trityl resin)-Gly-NH 2 (30.00 g) prepared in Example 5 was weighed into a solid-phase reaction synthesis column. 400 mL of DMF was added to swell for 30 min, and the DMF was removed. The resin was washed with 3*200 mL of dry DMF, and the DMF was removed. 200 mL of DBLK solution (20% piperidine/DMF solution, V/V) was added and deprotected twice, the first time was 5 min and the second time was 15 min. After deprotection, the resin was washed with 200 mL of DMF each time, and washed 6 times. After the fourth washing, a little resin was taken with a glass rod, and the ninhydrin test was positive, indicating that Fmoc had been removed.
称取Fmoc-Asn-OH(70.87g，200mmol)和N-羟基琥珀酰亚胺(23.00g，200mmol)加入到四氢呋喃800ml中，室温搅拌。控温5℃左右，缓慢地加DCC(45.36g，220mmol)的四氢呋喃(160ml)溶液室温搅拌2.5h，过滤，浓缩后加入到石油醚中重结晶析出固体，洗涤后干燥，将得到的活化酯固体溶于四氢呋喃200ml中，将H-Cys(Trt)-OH(79.96g，200mmol)溶于四氢呋喃150ml中缓慢的滴加入上述溶液中，滴完室温下继续反应，监控原料反应完全，过滤后，减压浓缩，将浓缩液加入到石油醚中析出固体，洗涤固体后再干燥，用异丙醇重结晶干燥得到Fmoc-Asn-Cys(Trt)-OH 102.17g，收率73％。Fmoc-Asn-OH (70.87 g, 200 mmol) and N-hydroxysuccinimide (23.00 g, 200 mmol) were weighed into 800 ml of tetrahydrofuran, and stirred at room temperature. The temperature was controlled at about 5°C, and a solution of DCC (45.36g, 220mmol) in tetrahydrofuran (160ml) was slowly added and stirred at room temperature for 2.5h, filtered, concentrated and added to petroleum ether for recrystallization to precipitate a solid, washed and dried, and the obtained activated ester was The solid was dissolved in 200 ml of tetrahydrofuran, and H-Cys(Trt)-OH (79.96 g, 200 mmol) was dissolved in 150 ml of tetrahydrofuran and slowly added dropwise to the above solution. After dropping, the reaction was continued at room temperature. Concentrate under reduced pressure, add the concentrated solution to petroleum ether to precipitate a solid, wash the solid and then dry, recrystallize and dry with isopropanol to obtain Fmoc-Asn-Cys(Trt)-OH 102.17g, yield 73%.
称取20.99g Fmoc-Asn-Cys(Trt)-OH和13.65g HCTU，加入100mL DMF溶解，待完全溶解后，将溶液冷却至5℃以下，然后加入5.82g DIEA(预冷至<0℃),于溶液中活化约3～5min，将活化后的溶液控制加入反应柱中，在20～35℃下反应2～3h，茚三酮检测呈阴性，抽掉反应液，加入200mL的DMF洗涤树脂，洗涤6遍。洗毕后，抽除洗涤液，得到Fmoc-Asn-Cys(Trt)-Pro-Orn(4-甲氧基-三苯甲基树脂)-Gly-NH 2。 Weigh 20.99g Fmoc-Asn-Cys(Trt)-OH and 13.65g HCTU, add 100mL DMF to dissolve, after complete dissolution, cool the solution to below 5°C, then add 5.82g DIEA (pre-cool to <0°C) , activate in the solution for about 3-5min, add the activated solution to the reaction column, react at 20-35°C for 2-3h, the ninhydrin test is negative, remove the reaction solution, add 200mL of DMF to wash the resin , wash 6 times. After washing, the washing liquid was removed to obtain Fmoc-Asn-Cys(Trt)-Pro-Orn(4-methoxy-trityl resin)-Gly-NH 2 .
重复接肽反应步骤及去除Fmoc保护基的操作，按照阿托西班氨基酸序列，在Fmoc-Asn-Cys(Trt)-Pro-Orn(4-甲氧基-三苯甲基树脂)-Gly-NH 2上依次偶联Fmoc-Thr-OH、Fmoc-Ile-OH、Fmoc-D-Tyr(Et)-OH、Mpa(Trt)-OH得到Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Trt)-Pro-Orn(4-甲氧基-三苯甲基树脂)-Gly-NH 2。DMF洗毕后，抽除洗涤液。再每次用200ml DCM洗涤树脂，洗涤4遍，5min/次，抽掉DCM，树脂在室温(20～35℃)下真空抽干至呈流沙状。肽树脂干燥完后为42.28g,树脂增重率84.0％。 Repeat the step of receiving the peptide and removing the Fmoc protective group. According to the amino acid sequence of atosiban, in Fmoc-Asn-Cys(Trt)-Pro-Orn(4-methoxy-trityl resin)-Gly- Fmoc-Thr-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, Mpa(Trt)-OH were sequentially coupled on NH 2 to obtain Mpa(Trt)-D-Tyr(Et)-Ile- Thr-Asn-Cys(Trt)-Pro-Orn(4-methoxy-trityl resin)-Gly- NH2 . After washing with DMF, the washing solution was removed. The resin was washed with 200 ml of DCM each time, 4 times, 5 min/time, the DCM was removed, and the resin was vacuum-dried at room temperature (20-35° C.) until it was quicksand. The peptide resin was 42.28g after drying, and the resin weight gain was 84.0%.
实施例10、阿托西班粗肽的合成1Example 10. Synthesis of atosiban crude peptide 1
配置TFA/DCM＝2/98(V/V)裂解液487.2ml，冷却到5～10℃，将实施例6制得的48.72g肽树脂加入到裂解液中，室温(20～35℃)下反应5h，过滤，用乙腈洗涤肽树脂2次，50ml/次，合并至滤液中，将滤液旋干，干燥后得到固体，异丙醚洗涤，过滤，在20-35℃减压干燥至恒重得阿托西班线性肽14.77g，将阿托西班线性肽14.30g溶于0.75L冰醋酸溶解，加水6.75L稀释，滴加0.1M/L碘乙醇溶液至溶液变色，室温反应1.0h，即得到阿托西班粗肽，其HPLC谱图见图1。Configure 487.2ml of TFA/DCM=2/98 (V/V) lysis solution, cool to 5-10°C, add 48.72g of peptide resin prepared in Example 6 into the lysis solution, at room temperature (20-35°C) React for 5h, filter, wash the peptide resin twice with acetonitrile, 50ml/time, combine into the filtrate, spin the filtrate to dry, obtain a solid after drying, wash with isopropyl ether, filter, and dry under reduced pressure at 20-35°C to constant weight To obtain 14.77g of atosiban linear peptide, dissolve 14.30g of atosiban linear peptide in 0.75L of glacial acetic acid, add 6.75L of water to dilute, add 0.1M/L iodine ethanol solution dropwise until the solution changes color, react at room temperature for 1.0h, That is, the crude atosiban peptide is obtained, and its HPLC spectrum is shown in Figure 1.
实施例11、阿托西班粗肽的合成2Example 11. Synthesis of atosiban crude peptide 2
配置TFA/DCM＝5/95(V/V)裂解液448.6ml，冷却到5～10℃，将实施例7制得的42.77g肽树脂加入到裂解液中，室温(20～35℃)下反应3h，过滤，用乙腈洗涤肽树脂2次，50ml/次，合并至滤液中，将滤液旋干，干燥后得到固体，异丙醚洗涤，过滤，在20-35℃减压干 燥至恒重得阿托西班线性肽14.21g，将阿托西班线性肽14.21g溶于1.5L冰醋酸溶解，加水6L稀释，滴加0.1M/L碘乙醇溶液至溶液变色，室温反应1.0h，即得到阿托西班粗肽，其HPLC谱图与图1相似。Configure TFA/DCM=5/95 (V/V) lysate 448.6ml, cool to 5～10℃, add 42.77g of peptide resin prepared in Example 7 into the lysate, at room temperature (20～35℃) React for 3h, filter, wash the peptide resin twice with acetonitrile, 50ml/time, combine into the filtrate, spin the filtrate, dry to obtain a solid, wash with isopropyl ether, filter, and dry under reduced pressure at 20-35°C to constant weight To obtain 14.21g of atosiban linear peptide, dissolve 14.21g of atosiban linear peptide in 1.5L of glacial acetic acid, add 6L of water to dilute, add 0.1M/L iodoethanol solution dropwise until the solution changes color, and react at room temperature for 1.0h, that is, The crude atosiban peptide was obtained, and its HPLC chromatogram was similar to that in Figure 1.
实施例12、阿托西班粗肽的合成3Example 12. Synthesis of atosiban crude peptide 3
配置TFA/DCM＝20/80(V/V)裂解液450.5ml，冷却到5～10℃，将实施例8制得的45.05g肽树脂加入到裂解液中，室温(20～35℃)下反应2h，过滤，用乙腈洗涤肽树脂2次，50ml/次，合并至滤液中，将滤液旋干，干燥后得到固体，异丙醚洗涤，过滤，在20-35℃减压干燥至恒重得阿托西班线性肽14.63g，将阿托西班线性肽14.63g溶于1.5L冰醋酸溶解，加水6L稀释，加10％过氧化氢溶液，室温反应1.0h，即得到阿托西班粗肽，其HPLC谱图与图1相似。Configure 450.5ml of TFA/DCM=20/80 (V/V) lysis solution, cool to 5-10°C, add 45.05g of peptide resin prepared in Example 8 into the lysis solution, at room temperature (20-35°C) React for 2h, filter, wash the peptide resin twice with acetonitrile, 50ml/time, combine into the filtrate, spin the filtrate to dry, obtain a solid after drying, wash with isopropyl ether, filter, and dry under reduced pressure at 20-35°C to constant weight To obtain 14.63 g of atosiban linear peptide, dissolve 14.63 g of atosiban linear peptide in 1.5 L of glacial acetic acid, add 6 L of water to dilute, add 10% hydrogen peroxide solution, and react at room temperature for 1.0 h to obtain atosiban Crude peptide, its HPLC chromatogram is similar to Figure 1.
实施例13、阿托西班粗肽的合成4Example 13. Synthesis of atosiban crude peptide 4
配置TFA/DCM＝1/99(V/V)裂解液442.7ml，冷却到5～10℃，将实施例9制得的44.27g肽树脂加入到裂解液中，室温(20～35℃)下反应5h，过滤，用乙腈洗涤肽树脂2次，50ml/次，合并至滤液中，将滤液旋干，干燥后得到固体，异丙醚洗涤，过滤，在20-35℃减压干燥至恒重得阿托西班线性肽14.13g，将阿托西班线性肽14.13g溶于1.5L冰醋酸溶解，加水6L稀释，加30％过氧化氢溶液，室温反应1.0h，即得到阿托西班粗肽，其HPLC谱图与图1相似。Configure TFA/DCM=1/99 (V/V) lysate 442.7ml, cool to 5～10℃, add 44.27g of peptide resin prepared in Example 9 into the lysate, at room temperature (20～35℃) React for 5h, filter, wash the peptide resin twice with acetonitrile, 50ml/time, combine into the filtrate, spin the filtrate to dry, obtain a solid after drying, wash with isopropyl ether, filter, and dry under reduced pressure at 20-35°C to constant weight To obtain 14.13 g of atosiban linear peptide, dissolve 14.13 g of atosiban linear peptide in 1.5 L of glacial acetic acid, add 6 L of water to dilute, add 30% hydrogen peroxide solution, and react at room temperature for 1.0 h to obtain atosiban Crude peptide, its HPLC chromatogram is similar to Figure 1.
实施例14、阿托西班粗肽的纯化1Example 14. Purification of atosiban crude peptide 1
将实施例10制得的阿托西班粗肽用15％乙腈水溶液溶解过滤后，经制备型反相HPLC(C18柱)纯化，转盐，收集大于99％的馏分，浓缩冻干得到10.12g，收率64％，纯度99％，得到的阿托西班精肽HPLC谱图见图2。The atosiban crude peptide prepared in Example 10 was dissolved in 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 10.12g , the yield is 64%, the purity is 99%, and the HPLC spectrum of the obtained atosiban refined peptide is shown in Figure 2.
实施例15、阿托西班粗肽的纯化2Example 15. Purification of atosiban crude peptide 2
将实施例11制得的阿托西班粗肽用15％乙腈水溶液溶解过滤后，经制备型反相HPLC(C18柱)纯化，转盐，收集大于99％的馏分，浓缩冻干得到9.80g，收率62％，纯度99％，得到的阿托西班精肽HPLC谱图与图2相似。The atosiban crude peptide prepared in Example 11 was dissolved in a 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 9.80 g , the yield is 62%, the purity is 99%, and the obtained atosiban peptide HPLC spectrum is similar to Figure 2.
实施例16、阿托西班粗肽的纯化3Example 16. Purification of atosiban crude peptide 3
将实施例12制得的阿托西班粗肽用15％乙腈水溶液溶解过滤后，经制备型反相HPLC(C18柱)纯化，转盐，收集大于99％的馏分，浓缩冻干得到10.28g，收率65％，纯度99％，得到的阿托西班精肽HPLC谱图与图2相似。The atosiban crude peptide obtained in Example 12 was dissolved in 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 10.28g , the yield is 65%, the purity is 99%, and the HPLC spectrum of the obtained atosiban peptide is similar to that in Figure 2.
实施例17、阿托西班粗肽的纯化4Example 17. Purification of atosiban crude peptide 4
将实施例13制得的阿托西班粗肽用15％乙腈水溶液溶解过滤后，经制备型反相HPLC (C18柱)纯化，转盐，收集大于99％的馏分，浓缩冻干得到10.27g，收率65％，纯度99％，得到的阿托西班精肽HPLC谱图与图2相似。The atosiban crude peptide obtained in Example 13 was dissolved in 15% acetonitrile aqueous solution and filtered, purified by preparative reverse-phase HPLC (C18 column), transferred to salt, collected more than 99% of the fraction, concentrated and lyophilized to obtain 10.27g , the yield is 65%, the purity is 99%, and the HPLC spectrum of the obtained atosiban peptide is similar to that in Figure 2.

Claims (10)

  1. 一种固相合成阿托西班的方法，其特征在于，主要包括以下步骤：A method for solid-phase synthesis of atosiban, characterized in that, mainly comprises the following steps:
    1)合成得到Fmoc-Pro-Orn-Gly-NH 2三肽，之后用树脂与之反应得到Fmoc-Pro-Orn(树脂)-Gly-NH 2； 1) Synthesize the Fmoc-Pro-Orn-Gly-NH 2 tripeptide, and then react it with a resin to obtain Fmoc-Pro-Orn (resin)-Gly-NH 2 ;
    2)脱除Fmoc-Pro-Orn(树脂)-Gly-NH 2的Fmoc保护，之后采用缩合剂依次接入阿托西班序列中相应的保护氨基酸或片段，制得阿托西班线性肽树脂； 2) Remove the Fmoc protection of Fmoc-Pro-Orn (resin)-Gly-NH 2 , then use a condensing agent to sequentially access the corresponding protected amino acids or fragments in the atosiban sequence to obtain atosiban linear peptide resin ;
    3)裂解、氧化，得到阿托西班。3) cracking and oxidation to obtain atosiban.
  2. 根据权利要求1所述的方法，其特征在于，所述步骤1)中树脂选自能与Orn的侧链氨基官能团结合的聚合物树脂。The method according to claim 1, wherein the resin in the step 1) is selected from polymer resins that can be combined with the side chain amino functional group of Orn.
  3. 根据权利要求2所述的方法，其特征在于，所述步骤中的聚合物树脂选自三苯甲基型树脂。The method according to claim 2, wherein the polymer resin in the step is selected from trityl type resins.
  4. 根据权利要求3所述的方法，其特征在于，所述树脂的替代度为0.8～1.0mmol/g。The method according to claim 3, wherein the substitution degree of the resin is 0.8-1.0 mmol/g.
  5. 根据权利要求1所述的方法，其特征在于，所述步骤1)中三肽在DIEA作用下与树脂反应生成三肽树脂。The method according to claim 1, wherein in the step 1), the tripeptide reacts with the resin under the action of DIEA to generate the tripeptide resin.
  6. 根据权利要求1所述的方法，其特征在于，所述步骤2)具体为：在Fmoc-Pro-Orn(树脂)-Gly-NH 2肽树脂上依次偶联Fmoc-Cys(Trt)-OH、Fmoc-Asn-OH、Fmoc-Thr-OH、Fmoc-Ile-OH、Fmoc-D-Tyr(Et)-OH、Mpa(Trt)-OH得到Mpa(Trt)-D-Tyr(Et)-Ile-Thr-Asn-Cys(Trt)-Pro-Orn(三苯甲基型树脂)-Gly-NH 2。 The method according to claim 1, wherein the step 2 ) is specifically: sequentially coupling Fmoc-Cys(Trt)-OH, Fmoc-Asn-OH, Fmoc-Thr-OH, Fmoc-Ile-OH, Fmoc-D-Tyr(Et)-OH, Mpa(Trt)-OH give Mpa(Trt)-D-Tyr(Et)-Ile- Thr-Asn-Cys(Trt)-Pro-Orn (trityl type resin)-Gly- NH2 .
  7. 根据权利要求1所述的方法，其特征在于，所述步骤2)中接入的阿托西班序列中相应的片段选自Mpa(Trt)-D-Tyr(Et)-OH、Fmoc-D-Tyr(Et)-Ile-OH、Fmoc-Asn-Cys(Trt)-OH。The method according to claim 1, wherein the corresponding fragment in the atosiban sequence accessed in the step 2) is selected from Mpa(Trt)-D-Tyr(Et)-OH, Fmoc-D -Tyr(Et)-Ile-OH, Fmoc-Asn-Cys(Trt)-OH.
  8. 根据权利要求1所述的方法，其特征在于，所述步骤2)中缩合剂选自HOBt/DIC、HCTU/DIEA、HBTU/DIEA、HATU/HOAt/DIEA、HBTU/HOBt/DIEA中任意一种。The method according to claim 1, wherein in the step 2), the condensing agent is selected from any one of HOBt/DIC, HCTU/DIEA, HBTU/DIEA, HATU/HOAt/DIEA, HBTU/HOBt/DIEA .
  9. 根据权利要求1所述的方法，其特征在于，所述步骤3)裂解试剂选自TFA/DCM，其体积比为TFA：DCM＝1：99～20：80。The method according to claim 1, wherein the step 3) cleavage reagent is selected from TFA/DCM, and the volume ratio thereof is TFA:DCM=1:99～20:80.
  10. 根据权利要求1所述的方法，其特征在于，所述步骤3)中氧化剂选自碘、过氧化氢。The method according to claim 1, wherein the oxidant is selected from iodine and hydrogen peroxide in the step 3).
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