WO2017114382A1 - Method for synthesizing c-terminal modified peptide - Google Patents

Method for synthesizing c-terminal modified peptide Download PDF

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WO2017114382A1
WO2017114382A1 PCT/CN2016/112317 CN2016112317W WO2017114382A1 WO 2017114382 A1 WO2017114382 A1 WO 2017114382A1 CN 2016112317 W CN2016112317 W CN 2016112317W WO 2017114382 A1 WO2017114382 A1 WO 2017114382A1
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fmoc
resin
mmol
solid phase
modified peptide
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PCT/CN2016/112317
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French (fr)
Chinese (zh)
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伍柯瑾
戴政清
宓鹏程
陶安进
袁建成
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深圳翰宇药业股份有限公司
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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
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • 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
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • 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
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C07K1/062—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for alpha- or omega-carboxy functions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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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  • modified polypeptide drug can significantly reduce immunogenicity, reduce toxic side effects, increase water solubility, prolong the time of action in vivo, change its biodistribution status, etc., and significantly improve the efficacy of the drug.
  • the C-terminal chemical modification of peptides is becoming a research hotspot, and the introduction of some small molecule drugs at the C-end has become a new research direction.
  • the modification of the amino group is generally carried out by amide bond reaction directly with the carboxyl group at the C-terminus of the peptide chain in the liquid phase; for the carboxyl group modification at the C-terminus, it is generally required
  • the introduction of a diamino Linker between the C-terminal carboxyl group of the peptide chain and the modified carboxyl group will undoubtedly increase the chemical synthesis difficulty of such a peptide because it generally performs two amide bond reactions in the liquid phase.
  • the post-treatment difficulty of the synthesis is greatly increased, and the yield of the target peptide is greatly reduced.
  • the main synthetic technique is to first perform the coupling of peptide sequences on 2-Chlorotrityl Chloride Resin using Fmoc solid phase peptide synthesis strategy, in which the N-terminal amino group is protected by Boc and then cleaved. Fully protective peptide, then introduce a diamino Linker (usually ethylenediamine, butanediamine, hexamethylenediamine, octanediamine%) in the liquid phase at the C-terminus of the peptide chain, after post-treatment, again A modified carboxyl group is introduced into the liquid phase.
  • GHWDFRQWWQPSGGGS-hexanediamine-Biotin as an example to illustrate the existing synthetic technical scheme, as shown in Figure 1.
  • the present invention aims to introduce a novel diamino Linker direct coupling resin method.
  • we directly couple the diamino Linker to The solid phase resin is then coupled to the peptide sequence in turn, and then cleaved after coupling to obtain a fully protected peptide.
  • the above fully protected peptide is directly reacted with a carboxyl group-containing modifying group, and the target product is directly obtained in one step.
  • the synthesis of the target product can be accomplished directly on the solid phase, as shown in Figure 2.
  • the method of the present invention accomplishes the coupling of the diamine compound to the solid support and the loading of the first amino acid residue on the solid support by controlling the reaction conditions.
  • the by-product is avoided by a two-step blocking method. Occurs, solving the technical barrier that is difficult to uniformly couple a bifunctional compound to a solid phase carrier in the prior art. This undoubtedly has the advantages of simple operation, energy saving and environmental protection, and improved yield.
  • One aspect of the present invention provides a method for synthesizing a C-terminal modified peptide, which comprises the steps of:
  • the diamino compound is selected from a linear symmetric diamino compound or a branched symmetric diamino compound or a symmetrical spatial diamino compound, preferably ethylenediamine, propylenediamine, butanediamine, pentane Diamine, hexamethylenediamine, heptanediamine, octanediamine,
  • the solid phase synthetic resin is selected from the group consisting of 2-Chlorotrityl Chloride Resin resin, and preferably, the solid phase synthetic resin has a degree of substitution in the range of 0.1 to 1.2 mmol/g, more preferably 0.2 to 0.8 mmol/g, and most preferably 0.3 to 0.5. Mmmol/g.
  • the coupling system used in the Fmoc solid phase peptide synthesis strategy in step 2) is DIC+A or B+A+C, wherein A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA. Or TMP.
  • step 4 a purification step is further included, and the preferred purification step is purification by HPLC method.
  • the solid phase synthetic resin is weighed and placed in a solid phase reaction column, DMF is added, and nitrogen gas is bubbled and swollen; the diamino compound is weighed, dissolved in DMF, and DIPEA is added to the solid.
  • the reaction column after completion of the reaction, methanol and DIPEA were added, and the mixture was sealed for 10 to 60 minutes, washed with DCM, and the resin was shrunk and the resin was drained to obtain a diamino compound-coupled solid phase synthetic resin.
  • the step of coupling the first amino acid to the amino group at the other terminal of the diamino compound in the step 2) is: adding DMF to the diamino compound-coupled solid phase synthetic resin obtained in the step 1), bubbling nitrogen gas Swelling; after activation of the Fmoc-protected amino acid by the coupling system, the activated Fmoc-protected amino acid is added to the reaction column for reaction, then acetic anhydride and pyridine are added, and the mixture is closed to obtain the Fmoc-protected amino acid diamino compound coupled solid phase synthesis. Resin.
  • amino acid sequence on the amino group of the other terminal in which the amino compound is sequentially coupled is selected from a polypeptide comprising 1-20 amino acids, preferably a polypeptide of 5-15 amino acids.
  • carboxyl group-containing modifying group is selected from the group consisting of Biotin, fluorescein, carnitine, formic acid, acetic acid, palmitic acid, stearic acid, cholic acid, a carboxyl group-containing small molecule drug, and a pharmacological functional group.
  • Figure 1 is a flow chart showing the synthesis of a polypeptide C-terminal modified Biotin
  • Figure 2 is a flow chart showing the synthesis of the C-terminal modified Biotin of the polypeptide of the present invention.
  • the structure of the ibirapeptide is: H-Met(O 2 )-Glu-His-Phe-D-Lys-Phe-NH-(CH 2 ) 8 -NH 2 .
  • the reaction was carried out at room temperature for 2.5 hours, the resin was filtered off, the resin was washed with 50 ml of TFA, and the filtrate was combined, and then added to 00 ml of chilled anhydrous diethyl ether to precipitate a white solid.
  • the yield was 99.8%, and the HPLC purity was 91.4%.
  • Example 7 138 g of the peptide resin obtained in Example 7 was placed in a 2000 ml single-necked flask, and 1500 ml of a 0.1% TFA solution of DCM was placed in a flask, and reacted at room temperature for 2.0 hours. The resin was filtered off, and the solvent was evaporated to give a white-protected. The crude peptide was 68 g.
  • Ebolapeptides were prepared by the methods of Examples 1-5 (invention) and Examples 6-11 (prior art) at the same dosage, with a total yield nearly three times different for 7 peptide conjugates.
  • the total yield of sperm peptide can usually reach about 60%, and by the method of the present invention, the inventors unexpectedly found that the effect far exceeds the expected yield, and is as high as 80% or more.
  • Example 12 100 g of NH 2 -(CH 2 ) 2 -NH-2-Chlorotrityl Chloride Resin obtained in Example 12 was weighed into a solid phase reaction column, DMF was added, and nitrogen gas was bubbled and swollen for 60 minutes; Fmoc-Leu-OH 21.2 was weighed. ⁇ (60mmol), HOBt 9.72g (72mmol), dissolved in DMF, added 16.4ml DIC (72mmol) in 0°C ice water bath, activated for 5 minutes, added to the reaction column, after reaction for 2 hours, add 70ml acetic anhydride and 60ml pyridine.
  • the structure of the target compound is prepared: H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH 2 ) 2 -NH-L-Carnitine (L-Carnitine: L-Carnitine: Compounds are used for weight loss, and peptide sequences are linked to increase metabolic stability.
  • Example 14 165 g of the peptide resin obtained in Example 14 was placed in a 2000 ml single-necked flask, and 1800 ml of a 0.1% TFA solution of DCM was placed in a flask, and reacted at room temperature for 2.0 hours. The resin was filtered off, and the solvent was evaporated to give a white-protected. Crude peptide 64g.
  • L-Carnitine 16.1 g (100 mmol) was added to a 500 ml one-necked flask, dissolved in 50 ml of DMF, and 16.3 g (120 mmol) of HOBt, and 15.1 g of DIC (120 mmol) were added.
  • the 64 g of the fully protected crude peptide obtained in Example 15 was dissolved in 200 ml of DCM, and slowly added dropwise to the L-Carnitine solution. After the completion of the dropwise addition, the reaction was continued for 2 hours to complete the reaction.
  • L-Carnitine 16.1 g (100 mmol) was added to a 500 ml one-necked flask, dissolved in 50 ml of DMF, and 16.3 g (120 mmol) of HOBt, and 15.1 g of DIC (120 mmol) were added.
  • the 42 g of the fully protected crude peptide obtained in Example 22 was dissolved in 200 ml of DCM, and slowly added dropwise to the L-Carnitine solution. After the completion of the dropwise addition, the reaction was continued for 2 hours to complete the reaction.
  • Example 24 The crude product obtained in Example 24 was 16.3 g, using a Waters 2454 RP-HPLC system, a wavelength of 220 nm, a column of 100 ⁇ 500 mm reverse phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile solution (v/v); Phase: acetonitrile, gradient: B%: 38% to 68%, flow rate: 6 ml/min, the peak fraction of the target was collected, concentrated by rotary evaporation, and lyophilized to obtain the target sperm peptide 9.7 g, HPLC purity 99.26%, total yield 20.9% .
  • Example 26 100 g of NH 2 -(CH 2 ) 6 -NH-2-Chlorotrityl Chloride Resin obtained in Example 26 was weighed into a solid phase reaction column, DMF was added, and nitrogen gas was bubbled and swollen for 60 minutes; Fmoc-Ser (tBu) was weighed.
  • the structure of the target compound was prepared: GHWDFRQWWQPSGGGS-hexanediamine-Biotin.
  • Biotin 24.4 g (100 mmol) was added to a 1000 ml one-neck flask, dissolved in 100 ml of DMF, and 16.3 g (120 mmol) of HOBt, and 15.1 g DIC (120 mmol) were added.
  • the 169 g of the fully protected crude peptide obtained in Example 29 was dissolved in 500 ml of DCM, and slowly added dropwise to the Biotin solution. After the completion of the dropwise addition, the reaction was continued for 2 hours to terminate the reaction.
  • Example 31 The crude product obtained in Example 31 was obtained from 92.6 g, using a Waters 2454 RP-HPLC system with a wavelength of 220 nm, a column of 100 ⁇ 500 mm reverse phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile solution (v/v); Phase: acetonitrile, gradient: B%: 38% to 68%, flow rate: 6 ml/min, collection of the peak fraction of interest, concentration by rotary evaporation, lyophilization to obtain 40.5 g of the target spermatin, HPLC purity 98.90%, total yield 36.6% .
  • the mixture was shredded with methanol and dried in vacuo to give 268 g of peptide resin.
  • Biotin 24.4 g (100 mmol) was added to a 1000 ml one-neck flask, dissolved in 100 ml of DMF, and 16.3 g (120 mmol) of HOBt, and 15.1 g DIC (120 mmol) were added.
  • the 158 g of the fully protected crude peptide obtained in Example 36 was dissolved in 500 ml of DCM, and slowly added dropwise to the Biotin solution. After the completion of the dropwise addition, the reaction was continued for 2 hours to complete the reaction.
  • Example 38 The crude product obtained in Example 38 was obtained in 45.9 g, using a Waters 2454 RP-HPLC system, a wavelength of 220 nm, a column of 100 ⁇ 500 mm reverse phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile solution (v/v); Phase: acetonitrile, gradient: B%: 38% to 68%, flow rate: 6 ml/min, the peak fraction of the target was collected, concentrated by rotary evaporation, and lyophilized to obtain the target sperm peptide 16.3 g, HPLC purity 99.2%, total yield 14.7% .
  • GHWDFRQWWQPSGGGS-hexanediamine-Biotin was prepared by the method of Examples 26-33 (present invention) and Examples 34-39 (prior art) at the same dosage, with a total yield of more than 2 times, by the method of the present invention, Although only the order of synthesis was changed, the overall yield far exceeded the expected yield.

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Abstract

Provided is a method for synthesizing a C-terminal modified peptide, comprising the following steps: 1) coupling an amine-terminus of a diamino compound to a resin to achieve a solid-phase synthesis; 2) successively coupling an amino acid to the other amine-terminus of the diamino compound using an Fmoc solid-phase peptide synthesis method to obtain a fully-protected polypeptidyl resin; 3) cleaving a fully-protected polypeptide from the resin to obtain the fully-protected polypeptide; 4) removing a protecting group from the fully-protected polypeptide to obtain the target C-terminal modified peptide, or 5) coupling the fully-protected polypeptide to a carboxyl-containing modifying group to obtain the target C-terminal modified peptide.

Description

一种C-端修饰肽的合成方法Method for synthesizing C-terminal modified peptide 技术领域Technical field
一种制备C-端修饰肽的合成方法。A synthetic method for preparing a C-terminal modified peptide.
背景技术Background technique
多肽修饰作为一种改变肽链主链结构和侧链基团的重要手段，在改变肽类化合物的物理化学性质，解决其在体内的有效利用方面发挥越来越重要的作用已有大量文献报道，经过修饰后的多肽药物可以显著降低免疫原性、减少毒副作用、增加水溶性、延长体内作用时间、改变其生物分布状况等等，明显改善了药物的疗效。As an important means to change the main chain structure and side chain groups of peptide chains, peptide modification plays an increasingly important role in changing the physicochemical properties of peptide compounds and solving their effective utilization in vivo. The modified polypeptide drug can significantly reduce immunogenicity, reduce toxic side effects, increase water solubility, prolong the time of action in vivo, change its biodistribution status, etc., and significantly improve the efficacy of the drug.
目前，多肽的C-端化学修饰正成为研究热点，在C-端引入一些小分子药物成为一种新型的研究方向。对于多肽的C-端修饰来说，氨基类基团的修饰，一般在液相中直接与肽链C-端的羧基进行酰胺键反应即可完成；对于C-端进行羧基官能团修饰，一般需要在肽链C-端羧基与修饰的羧基之间引入一个二氨基类的Linker，这样的方法无疑会增加此类肽的化学合成难度，因为其一般在液相中进行两次酰胺键的反应，这样会导致合成的后处理难度大大增加，并且会大大降低目标肽的收率。At present, the C-terminal chemical modification of peptides is becoming a research hotspot, and the introduction of some small molecule drugs at the C-end has become a new research direction. For the C-terminal modification of the polypeptide, the modification of the amino group is generally carried out by amide bond reaction directly with the carboxyl group at the C-terminus of the peptide chain in the liquid phase; for the carboxyl group modification at the C-terminus, it is generally required The introduction of a diamino Linker between the C-terminal carboxyl group of the peptide chain and the modified carboxyl group will undoubtedly increase the chemical synthesis difficulty of such a peptide because it generally performs two amide bond reactions in the liquid phase. The post-treatment difficulty of the synthesis is greatly increased, and the yield of the target peptide is greatly reduced.
目前，针对多肽的C-端羧基修饰，主要的合成技术是先用Fmoc固相肽合成策略在2-Chlorotrityl Chloride Resin上完成肽序的偶联，其中N-端的氨基采用Boc保护，然后裂解得到全保护肽，然后在液相中在肽链的C-端引入一个二氨基类的Linker(一般为乙二胺、丁二胺、己二胺、辛二胺…)，后处理后，再次在液相中引入修饰的羧基基团。以GHWDFRQWWQPSGGGS-己二胺-Biotin为例，说明现有的合成技术方案，具体见附图1。At present, for the C-terminal carboxyl modification of peptides, the main synthetic technique is to first perform the coupling of peptide sequences on 2-Chlorotrityl Chloride Resin using Fmoc solid phase peptide synthesis strategy, in which the N-terminal amino group is protected by Boc and then cleaved. Fully protective peptide, then introduce a diamino Linker (usually ethylenediamine, butanediamine, hexamethylenediamine, octanediamine...) in the liquid phase at the C-terminus of the peptide chain, after post-treatment, again A modified carboxyl group is introduced into the liquid phase. Take GHWDFRQWWQPSGGGS-hexanediamine-Biotin as an example to illustrate the existing synthetic technical scheme, as shown in Figure 1.
发明内容Summary of the invention
为了克服上述C-端修饰肽合成的各种缺点，本发明旨在引入一种新型的二氨基类Linker直接偶联树脂的方法，在本方法中，我们直接把二氨基类的Linker偶联到固相树脂上，然后依次偶联肽序，偶联后裂解得到全保护肽，上述全保护肽直接与带羧基的修饰基团反应，一步反应直接得到目标产物。而对于只要进行C-端进行二氨基修饰的肽(比如依比拉肽)，可以直接在固相上就完成目标产物的合成，具体见附图2。本发明方法通过控制反应条件的方式来完成二胺类化合物与固相载体的偶联以及第一个氨基酸残基在固相载体上的加载量。同时，在偶联二胺类化合物以及第一个氨基酸残基的过程中，通过两步封闭的方法来避免副产物的 发生，解决了现有技术中认为双官能团化合物很难均一偶联到固相载体的技术壁垒。这无疑具有操作简单、节能环保、提高收率等优点。In order to overcome the various shortcomings of the above-mentioned C-terminal modified peptide synthesis, the present invention aims to introduce a novel diamino Linker direct coupling resin method. In this method, we directly couple the diamino Linker to The solid phase resin is then coupled to the peptide sequence in turn, and then cleaved after coupling to obtain a fully protected peptide. The above fully protected peptide is directly reacted with a carboxyl group-containing modifying group, and the target product is directly obtained in one step. For peptides that undergo diamino modification at the C-terminus (such as ibirapeptide), the synthesis of the target product can be accomplished directly on the solid phase, as shown in Figure 2. The method of the present invention accomplishes the coupling of the diamine compound to the solid support and the loading of the first amino acid residue on the solid support by controlling the reaction conditions. At the same time, in the process of coupling the diamine compound and the first amino acid residue, the by-product is avoided by a two-step blocking method. Occurs, solving the technical barrier that is difficult to uniformly couple a bifunctional compound to a solid phase carrier in the prior art. This undoubtedly has the advantages of simple operation, energy saving and environmental protection, and improved yield.
本发明一个方面提供了一种C-端修饰肽的合成方法，其特征在于，包括以下步骤：One aspect of the present invention provides a method for synthesizing a C-terminal modified peptide, which comprises the steps of:
1)将二氨基化合物的一端氨基偶联至固相合成树脂上；1) coupling an amino group at one end of the diamino compound to a solid phase synthetic resin;
2)采用Fmoc固相肽合成策略，将氨基酸依次偶联至二氨基化合物的另一端氨基上，以获得全保护的多肽树脂；2) using an Fmoc solid phase peptide synthesis strategy, the amino acid is sequentially coupled to the amino group at the other end of the diamino compound to obtain a fully protected polypeptide resin;
3)将全保护的多肽从树脂上裂解，以获得全保护的多肽；3) cleavage of the fully protected polypeptide from the resin to obtain a fully protected polypeptide;
4)将全保护的多肽脱除保护基，以获得目标的C-端修饰肽，或者4) removing the protecting group from the fully protected polypeptide to obtain the target C-terminal modified peptide, or
4)将全保护的多肽偶联与含羧基的修饰基团进行偶联，以获得目标的C-端修饰肽。4) Coupling the fully protected polypeptide to the carboxyl-containing modifying group to obtain the target C-terminal modified peptide.
进一步地，所述二氨基化合物选自直链对称二氨基化合物或带支链的对称二氨基化合物或空间结构对称的二氨基类化合物，优选为乙二胺、丙二胺、丁二胺、戊二胺、己二胺、庚二胺、辛二胺、
Figure PCTCN2016112317-appb-000001
Further, the diamino compound is selected from a linear symmetric diamino compound or a branched symmetric diamino compound or a symmetrical spatial diamino compound, preferably ethylenediamine, propylenediamine, butanediamine, pentane Diamine, hexamethylenediamine, heptanediamine, octanediamine,
Figure PCTCN2016112317-appb-000001
进一步地，所述固相合成树脂选自2-Chlorotrityl Chloride Resin树脂，优选地，固相合成树脂替代度范围为0.1～1.2mmol/g，更优选0.2～0.8mmol/g，最优选0.3～0.5mmol/g。Further, the solid phase synthetic resin is selected from the group consisting of 2-Chlorotrityl Chloride Resin resin, and preferably, the solid phase synthetic resin has a degree of substitution in the range of 0.1 to 1.2 mmol/g, more preferably 0.2 to 0.8 mmol/g, and most preferably 0.3 to 0.5. Mmmol/g.
进一步地，步骤2)中的Fmoc固相肽合成策略所用的偶联体系为DIC+A或B+A+C，其中A为HOBt或HOAt，B为HBTU、HATU、TBTU或PyBOP，C为DIPEA或TMP。Further, the coupling system used in the Fmoc solid phase peptide synthesis strategy in step 2) is DIC+A or B+A+C, wherein A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA. Or TMP.
进一步地，步骤3)裂解步骤所用的裂解试剂为TFA、TIS、EDT、H2O的组合物，裂解时间为1.5-3.5小时，优选裂解试剂的体积比为TFA：TIS：EDT：H2O＝85-95:2-5:2-5:1-5，Further, the lysis reagent used in the step 3) is a composition of TFA, TIS, EDT, H2O, and the lysis time is 1.5-3.5 hours, preferably the volume ratio of the lysis reagent is TFA: TIS: EDT: H2O=85-95 :2-5:2-5:1-5,
进一步地，步骤4)之后还包括纯化步骤，优选的纯化步骤为以HPLC方法进行纯化。Further, after step 4), a purification step is further included, and the preferred purification step is purification by HPLC method.
进一步地，其中步骤1)的使用方法为称取固相合成树脂，并置于固相反应柱中，加入DMF，氮气鼓泡溶胀；称取二氨基化合物，用DMF溶解，并将DIPEA加入固相反应柱中，反应完全后，加入甲醇和DIPEA，混合封闭10-60分钟，以DCM洗涤，甲醇收缩后抽干树脂，得到二氨基化合物偶联固相合成树脂。Further, in which the step 1) is used, the solid phase synthetic resin is weighed and placed in a solid phase reaction column, DMF is added, and nitrogen gas is bubbled and swollen; the diamino compound is weighed, dissolved in DMF, and DIPEA is added to the solid. In the reaction column, after completion of the reaction, methanol and DIPEA were added, and the mixture was sealed for 10 to 60 minutes, washed with DCM, and the resin was shrunk and the resin was drained to obtain a diamino compound-coupled solid phase synthetic resin.
进一步地，其中步骤2)中将第一个氨基酸偶联至二氨基化合物的另一端氨基上的步骤为：在步骤1)得到的二氨基化合物偶联固相合成树脂中加入DMF，氮气鼓泡溶胀；以偶联体系活化Fmoc保护的氨基酸后，将活化的Fmoc保护的氨基酸加入反应柱中进行反应，然后加入醋酸酐和吡啶，混合封闭，得到Fmoc保护的氨基酸二氨基化合物偶联固相合成树脂。 Further, the step of coupling the first amino acid to the amino group at the other terminal of the diamino compound in the step 2) is: adding DMF to the diamino compound-coupled solid phase synthetic resin obtained in the step 1), bubbling nitrogen gas Swelling; after activation of the Fmoc-protected amino acid by the coupling system, the activated Fmoc-protected amino acid is added to the reaction column for reaction, then acetic anhydride and pyridine are added, and the mixture is closed to obtain the Fmoc-protected amino acid diamino compound coupled solid phase synthesis. Resin.
进一步地，其中依次偶联至氨基化合物的另一端氨基上氨基酸序列选自包含1-20个氨基酸的多肽，优选为5-15个氨基酸的多肽。Further, the amino acid sequence on the amino group of the other terminal in which the amino compound is sequentially coupled is selected from a polypeptide comprising 1-20 amino acids, preferably a polypeptide of 5-15 amino acids.
进一步地，其中含羧基的修饰基团选自Biotin、荧光素、肉毒碱、甲酸、乙酸、棕榈酸、硬脂酸、胆酸、含羧基的小分子药物及药效官能团。Further, the carboxyl group-containing modifying group is selected from the group consisting of Biotin, fluorescein, carnitine, formic acid, acetic acid, palmitic acid, stearic acid, cholic acid, a carboxyl group-containing small molecule drug, and a pharmacological functional group.
附图说明DRAWINGS
图1为多肽C-端修饰Biotin现有合成流程图；Figure 1 is a flow chart showing the synthesis of a polypeptide C-terminal modified Biotin;
图2为本发明多肽C-端修饰Biotin合成流程图。Figure 2 is a flow chart showing the synthesis of the C-terminal modified Biotin of the polypeptide of the present invention.
具体实施方式detailed description
实施例1 NH2-(CH2)8-NH-2-Chlorotrityl Chloride Resin的制备Example 1 Preparation of NH 2 -(CH 2 ) 8 -NH-2-Chlorotrityl Chloride Resin
称取替代度为0.8mmol/g的2-Chlorotrityl Chloride Resin 250克于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取辛二胺57.7克(400mmol)，用DMF溶解，0℃冰水浴下加入90.0ml DIPEA(500mmol)，加入反应柱，反应1小时后，加入200ml甲醇和200mlDIPEA，混合封闭0.5h，DCM洗涤三次，甲醇收缩后抽干树脂，得到NH2-(CH2)8-NH-2-Chlorotrityl Chloride Resin。250 g of 2-Chlorotrityl Chloride Resin with a degree of substitution of 0.8 mmol/g was weighed into a solid phase reaction column, DMF was added, and nitrogen was bubbled and swelled for 60 minutes; 57.7 g (400 mmol) of octanediamine was weighed and dissolved in DMF, 0 Add 90.0 ml of DIPEA (500 mmol) under ice-cold bath and add to the reaction column. After reacting for 1 hour, add 200 ml of methanol and 200 ml of DIPEA, mix and block for 0.5 h, wash with DCM three times, and shrink the methanol to dry the resin to obtain NH 2 -(CH 2 ) 8 -NH-2-Chlorotrityl Chloride Resin.
实施例2 Fmoc-Phe-NH-(CH2)8-NH-2-Chlorotrityl Chloride Resin的制备Example 2 Preparation of Fmoc-Phe-NH-(CH 2 ) 8 -NH-2-Chlorotrityl Chloride Resin
称取实施例1得到的NH2-(CH2)8-NH-2-Chlorotrityl Chloride Resin 100克于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取Fmoc-Phe-OH 23.22克(60mmol)，HOBt 9.72克(72mmol)，HBTU 33.8克(60mmol)，用DMF溶解，0℃冰水浴下加入15.6ml DIPEA(72mmol)，活化5分钟，加入反应柱，反应2小时后，加入70ml醋酸酐和60ml吡啶，混合封闭24h，DCM洗涤三次，甲醇收缩后抽干树脂，得到Fmoc-Phe-NH-(CH2)8-NH-2-Chlorotrityl Chloride Resin，检测替代度为0.51mmol/g的树脂124g。100 g of NH 2 -(CH 2 ) 8 -NH-2-Chlorotrityl Chloride Resin obtained in Example 1 was weighed into a solid phase reaction column, DMF was added, and nitrogen gas was bubbled and swollen for 60 minutes; Fmoc-Phe-OH 23.22 was weighed.克(60mmol), HOBt 9.72g (72mmol), HBTU 33.8g (60mmol), dissolved in DMF, added 15.6ml DIPEA (72mmol) in 0°C ice water bath, activated for 5 minutes, added to the reaction column, reacted for 2 hours, added 70 ml of acetic anhydride and 60 ml of pyridine were mixed and sealed for 24 h, washed twice with DCM, and the resin was dried and the resin was evaporated to obtain Fmoc-Phe-NH-(CH 2 ) 8 -NH-2-Chlorotrityl Chloride Resin with a detection degree of 0.51 mmol/ g of resin 124g.
实施例3依比拉肽肽树脂的制备Example 3 Preparation of Ibeptidide Peptide Resin
依比拉肽的结构为：H-Met(O2)-Glu-His-Phe-D-Lys-Phe-NH-(CH2)8-NH2。The structure of the ibirapeptide is: H-Met(O 2 )-Glu-His-Phe-D-Lys-Phe-NH-(CH 2 ) 8 -NH 2 .
称取实施例2中替代度为0.51mmol/g的Fmoc-Phe-NH-(CH2)8-NH-2-Chlorotrityl Chloride Resin 98克(50mmol)于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；用DBLK脱除Fmoc保护基团，用DMF洗涤6次。称取Fmoc-D-Lys(Boc)-OH 468克(100mmol)，HOBt 16.3克(120mmol)，用DMF溶解，0℃冰水浴下加入15.1克DIC(120mmol)，活化5分钟，加入反应柱，反应2小时后，DMF洗涤树脂三次，用DBLK脱除Fmoc保护基团，用DMF洗涤6次，DCM洗涤3次。重复上述偶联操作，按照肽序依次偶联Fmoc-Phe-OH，Fmoc-His(Trt)-OH，Fmoc-Glu(OtBu)-OH，Boc-Met(O2)-OH。反应结束后，用甲醇收缩，真空干燥后得到依比拉肽肽树脂142g。Weighed Fmoc-Phe-NH-(CH 2 ) 8 -NH-2-Chlorotrityl Chloride Resin 98 g (50 mmol) in Example 2 with a degree of substitution of 0.51 mmol/g in a solid phase reaction column, and added DMF, nitrogen drum. The bubbles were swelled for 60 minutes; the Fmoc protecting group was removed with DBLK and washed 6 times with DMF. Weighed 468 g (100 mmol) of Fmoc-D-Lys(Boc)-OH, 16.3 g (120 mmol) of HOBt, dissolved in DMF, and added 15.1 g of DIC (120 mmol) in an ice water bath at 0 ° C, activated for 5 minutes, and added to the reaction column. After 2 hours of reaction, the resin was washed three times with DMF, the Fmoc protecting group was removed with DBLK, washed 6 times with DMF and washed 3 times with DCM. The above coupling operation was repeated, and Fmoc-Phe-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Boc-Met(O 2 )-OH was sequentially coupled according to the peptide sequence. After completion of the reaction, the mixture was shredded with methanol, and dried under vacuum to give 142 g of the yopeptide peptide resin.
实施例4依比拉肽粗肽的制备Example 4 Preparation of crude peptide of ibirapeptide
将实施例3得到的142克肽树脂加入到2000ml单口烧瓶中，预先配置裂解液1400ml TFA：TIS：EDT：H2O＝91:3:3:3(体积比)，将裂解液加入到烧瓶中，室温反应2.5小时，滤掉树脂，树脂用50mlTFA洗涤，合并滤液，加入到20000ml的冷冻无水***中，析出白色固体，离心，无水***洗涤固体，真空干燥后得到白色固体49.6克，收率99.8％，HPLC纯度91.4％。142 g of the peptide resin obtained in Example 3 was placed in a 2000 ml single-necked flask, and a lysate of 1400 ml of TFA: TIS:EDT:H 2 O=91:3:3:3 (volume ratio) was placed in advance, and the lysate was added to the flask. The reaction was carried out at room temperature for 2.5 hours, the resin was filtered off, the resin was washed with 50 ml of TFA, and the filtrate was combined, and then added to 00 ml of chilled anhydrous diethyl ether to precipitate a white solid. The yield was 99.8%, and the HPLC purity was 91.4%.
实施例5依比拉肽精肽的制备Example 5 Preparation of Ibeptide Peptide
取实施例4得到的依比拉肽粗肽49.6g，采用Waters 2454 RP-HPLC***，波长220nm，色谱柱为100×500mm反相C18柱，流动相：A相：0.3％TFA/乙腈溶液(v/v)；B相：乙腈，梯度：B％：38％～68％，流速：6毫升/分钟，收集目的峰馏分，旋转蒸发浓缩，冻干得到依比拉肽精肽42.5g，HPLC纯度99.34％，总收率85.4％。49.6 g of the crude peptide of Ibeptide obtained in Example 4 was used, a Waters 2454 RP-HPLC system, a wavelength of 220 nm, a column of 100×500 mm reverse phase C18 column, and a mobile phase: phase A: 0.3% TFA/acetonitrile solution ( v/v); Phase B: acetonitrile, gradient: B%: 38% to 68%, flow rate: 6 ml/min, collection of the peak fraction of interest, concentration by rotary evaporation, lyophilization to obtain 42.5 g of ibirapeptide, HPLC The purity was 99.34%, and the total yield was 85.4%.
实施例6 Fmoc-Phe-2-Chlorotrityl Chloride Resin的制备Example 6 Preparation of Fmoc-Phe-2-Chlorotrityl Chloride Resin
称取替代度为1.0mmol/g的2-Chlorotrityl Chloride Resin 100克于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取Fmoc-Phe-OH 23.22克(60mmol)，HOBt 9.72克(72mmol)，HBTU 33.8克(600mmol)，用DMF溶解，0℃冰水浴下加入15.6ml DIPEA(72mmol)，活化5分钟，加入反应柱，反应2小时后，加入100ml甲醇和100mlDIPEA，混合封闭0.5h，DCM洗涤三次，甲醇收缩后抽干树脂，得到Fmoc-Phe-2-Chlorotrityl Chloride Resin，检测替代度为0.48mmol/g的树脂119g。100 g of 2-Chlorotrityl Chloride Resin with 1.0 mmol/g substitution was weighed into a solid phase reaction column, DMF was added, and nitrogen was bubbled for 60 minutes; Fmoc-Phe-OH 23.22 g (60 mmol) was weighed, and HOBt was 9.72 g. (72mmol), HBTU 33.8g (600mmol), dissolved in DMF, added 15.6ml DIPEA (72mmol) in 0°C ice water bath, activated for 5 minutes, added to the reaction column, after reaction for 2 hours, add 100ml methanol and 100ml DIPEA, mix and close 0.5 h, DCM was washed three times, the methanol was shrunk, and the resin was drained to obtain Fmoc-Phe-2-Chlorotrityl Chloride Resin, and 119 g of a resin having a substitution degree of 0.48 mmol/g was detected.
实施例7 Boc-Met(O2)-Glu(OtBu)-His(Trt)-Phe-D-Lys(Boc)-Phe-2-Chlorotrityl Chloride Resin的制备 Example 7 Preparation of Boc-Met(O 2 )-Glu(OtBu)-His(Trt)-Phe-D-Lys(Boc)-Phe-2-Chlorotrityl Chloride Resin
称取实施例6中替代度为0.48mmol/g的Fmoc-Phe-2-Chlorotrityl Chloride Resin104克(50mmol)于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；用DBLK脱除Fmoc保护基团，用DMF洗涤6次。称取Fmoc-D-Lys(Boc)-OH 468克(100mmol)，HOBt 16.3克(120mmol)，用DMF溶解，0℃冰水浴下加入15.1克DIC(120mmol)，活化5分钟，加入反应柱，反应2小时后，DMF洗涤树脂三次，用DBLK脱除Fmoc保护基团，用DMF洗涤6次，DCM洗涤3次。重复上述偶联操作，按照肽序依次偶联Fmoc-Phe-OH，Fmoc-His(Trt)-OH，Fmoc-Glu(OtBu)-OH，Boc-Met(O2)-OH。反应结束后，用甲醇收缩，真空干燥后得到Boc-Met(O2)-Glu(OtBu)-His(Trt)-Phe-D-Lys(Boc)-Phe-2-Chlorotrityl Chloride Resin 138g。Weighed 104 g (50 mmol) of Fmoc-Phe-2-Chlorotrityl Chloride Resin with a degree of substitution of 0.48 mmol/g in Example 6 in a solid phase reaction column, adding DMF, bubbling with nitrogen for 60 minutes; removing Fmoc protection with DBLK The group was washed 6 times with DMF. Weighed 468 g (100 mmol) of Fmoc-D-Lys(Boc)-OH, 16.3 g (120 mmol) of HOBt, dissolved in DMF, and added 15.1 g of DIC (120 mmol) in an ice water bath at 0 ° C, activated for 5 minutes, and added to the reaction column. After 2 hours of reaction, the resin was washed three times with DMF, the Fmoc protecting group was removed with DBLK, washed 6 times with DMF and washed 3 times with DCM. The above coupling operation was repeated, and Fmoc-Phe-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Boc-Met(O 2 )-OH was sequentially coupled according to the peptide sequence. After completion of the reaction, it was shrunk with methanol and dried in vacuo to give Boc-Met(O 2 )-Glu(OtBu)-His(Trt)-Phe-D-Lys(Boc)-Phe-2-Chlorotrityl Chloride Resin 138 g.
实施例8 Boc-Met(O2)-Glu(OtBu)-His(Trt)-Phe-D-Lys(Boc)-Phe-OH的Example 8 Boc-Met(O 2 )-Glu(OtBu)-His(Trt)-Phe-D-Lys(Boc)-Phe-OH 制备preparation
将实施例7得到的138克肽树脂加入到2000ml单口烧瓶中，预先配置0.1％TFA的DCM溶液1500ml加入到烧瓶中，室温反应2.0小时，滤掉树脂，旋转蒸发掉溶剂，得到白色全保护的粗肽68g。138 g of the peptide resin obtained in Example 7 was placed in a 2000 ml single-necked flask, and 1500 ml of a 0.1% TFA solution of DCM was placed in a flask, and reacted at room temperature for 2.0 hours. The resin was filtered off, and the solvent was evaporated to give a white-protected. The crude peptide was 68 g.
实施例9依比拉肽全保护粗肽的制备Example 9 Preparation of Ibirapeptide Total Protected Crude Peptide
在500ml单口烧瓶中加入辛二胺14.4g(100mmol)，用50mlDMF溶解，加入HOBt 16.3克(120mmol)，和15.1克DIC(120mmol)。将实施例8得到的68克全保护粗肽溶于200ml的DCM中，缓慢滴加到辛二胺溶液中，滴加完毕后，继续反应2h，结束反应。往反应液中加入100ml水，萃取收集有机相(DCM)，旋转蒸发得到白色的全保护的依比拉肽粗肽36g。14.4 g (100 mmol) of octanediamine was added to a 500 ml one-necked flask, dissolved in 50 ml of DMF, and 16.3 g (120 mmol) of HOBt, and 15.1 g of DIC (120 mmol) were added. 68 g of the fully protected crude peptide obtained in Example 8 was dissolved in 200 ml of DCM, and slowly added dropwise to the octanediamine solution. After the completion of the dropwise addition, the reaction was continued for 2 hours to terminate the reaction. 100 ml of water was added to the reaction solution, and the organic phase (DCM) was collected by extraction, and evaporated to give a white, fully-protected crude esomeprin 36 g.
实施例10依比拉肽粗肽的制备Example 10 Preparation of Epirapin Crude Peptide
往实施例9中的全保护依比拉肽粗肽中加入预先配置裂解液TFA：TIS：EDT：H2O＝91:3:3:3(体积比)80ml，室温反应2.5小时，加入到10000ml的冷冻无水***中，析出白色固体，离心，无水***洗涤固体，真空干燥后得到白色固体27.5克，收率55.28％，HPLC纯度84.6％。To the fully protected ippiropin crude peptide in Example 9, a pre-configured lysate TFA: TIS: EDT: H 2 O = 91: 3: 3: 3 (volume ratio) 80 ml was added, and the reaction was carried out at room temperature for 2.5 hours. In 10000 ml of chilled anhydrous diethyl ether, a white solid was precipitated, and the solid was washed with diethyl ether and dried in vacuo to give a white solid, 27.5 g, yield 55.28%, HPLC purity 84.6%.
实施例11依比拉肽精肽的制备Example 11 Preparation of Ibeptide Peptide
取实施例10得到的依比拉肽粗肽27.5g，采用Waters 2454RP-HPLC***，波长220nm，色谱柱为100×500mm反相C18柱，流动相：A相：0.3％TFA/乙腈溶液(v/v)；B相：乙腈，梯度：B％：38％～68％，流速：6毫升/分钟，收集目的峰馏分，旋转蒸发浓缩，冻干得到依比拉肽精肽18.56g，HPLC纯度99.01％，总收率37.3％。27.5 g of the crude epidamide peptide obtained in Example 10, using a Waters 2454 RP-HPLC system, a wavelength of 220 nm, a column of 100 × 500 mm reverse phase C18 column, mobile phase: phase A: 0.3% TFA / acetonitrile solution (v /v); Phase B: acetonitrile, gradient: B%: 38% to 68%, flow rate: 6 ml/min, collection of the peak fraction of interest, concentration by rotary evaporation, lyophilization to obtain 18.56 g of ibirapeptide peptidase, HPLC purity 99.01%, the total yield was 37.3%.
通过实施例1-5(本发明)方法以及实施例6-11(现有技术)方法以相同投料量制备依比拉肽，其总收率相差近3倍，对于7肽缀合物而言，精肽总收率通常能够达到了60％左右，而通过本发明的方法，发明人意外的发现其效果远远超过预期产率，而高达80％以上。Ebolapeptides were prepared by the methods of Examples 1-5 (invention) and Examples 6-11 (prior art) at the same dosage, with a total yield nearly three times different for 7 peptide conjugates. The total yield of sperm peptide can usually reach about 60%, and by the method of the present invention, the inventors unexpectedly found that the effect far exceeds the expected yield, and is as high as 80% or more.
实施例12 NH2-(CH2)2-NH-2-Chlorotrityl Chloride Resin的制备Example 12 Preparation of NH 2 -(CH 2 ) 2 -NH-2-Chlorotrityl Chloride Resin
称取替代度为1.0mmol/g的2-Chlorotrityl Chloride Resin 200克于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取乙二胺24.1克(400mmol)，用DMF溶解，0℃冰水浴下加入90.0ml DIPEA(500mmol)，加入反应柱，反应1小时后，加入200ml甲醇和200mlDIPEA，混合封闭0.5h，DCM洗涤三次，甲醇收缩后抽干树脂，得到NH2-(CH2)2-NH-2-Chlorotrityl Chloride Resin。200 g of 2-Chlorotrityl Chloride Resin with a substitution of 1.0 mmol/g was weighed into a solid phase reaction column, DMF was added, and nitrogen gas was bubbled and swelled for 60 minutes; 24.1 g of ethylenediamine (400 mmol) was weighed and dissolved with DMF, 0 Add 90.0 ml of DIPEA (500 mmol) under ice-cold bath and add to the reaction column. After reacting for 1 hour, add 200 ml of methanol and 200 ml of DIPEA, mix and block for 0.5 h, wash with DCM three times, and shrink the methanol to dry the resin to obtain NH 2 -(CH 2 ) 2 -NH-2-Chlorotrityl Chloride Resin.
实施例13 Fmoc-Leu-NH-(CH2)2-NH-2-Chlorotrityl Chloride Resin的制备Example 13 Preparation of Fmoc-Leu-NH-(CH 2 ) 2 -NH-2-Chlorotrityl Chloride Resin
称取实施例12得到的NH2-(CH2)2-NH-2-Chlorotrityl Chloride Resin 100克于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取Fmoc-Leu-OH 21.2克(60mmol)，HOBt 9.72克(72mmol)，用DMF溶解，0℃冰水浴下加入16.4ml DIC(72mmol)，活化5分钟，加入反应柱，反应2小时后，加入70ml醋酸酐和60ml吡啶，混合封闭24h，DCM洗涤三次，甲醇收缩后抽干树脂，得到Fmoc-Leu-NH-(CH2)2-NH-2-Chlorotrityl Chloride Resin，检测替代度为0.53mmol/g的树脂118g。100 g of NH 2 -(CH 2 ) 2 -NH-2-Chlorotrityl Chloride Resin obtained in Example 12 was weighed into a solid phase reaction column, DMF was added, and nitrogen gas was bubbled and swollen for 60 minutes; Fmoc-Leu-OH 21.2 was weighed.克(60mmol), HOBt 9.72g (72mmol), dissolved in DMF, added 16.4ml DIC (72mmol) in 0°C ice water bath, activated for 5 minutes, added to the reaction column, after reaction for 2 hours, add 70ml acetic anhydride and 60ml pyridine. After mixing and blocking for 24 h, DCM was washed three times, and the methanol was shrunk, and the resin was drained to obtain Fmoc-Leu-NH-(CH 2 ) 2 -NH-2-Chlorotrityl Chloride Resin, and 118 g of a resin having a substitution degree of 0.53 mmol/g was detected.
实施例14 Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH2)2-NHExample 14 Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH 2 ) 2 -NH -2-Chlorotrityl Chloride Resin的制备Preparation of -2-Chlorotrityl Chloride Resin
制备目标化合物的结构为：H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH2)2-NH-L-Carnitine(L-Carnitine：左旋右碱:
Figure PCTCN2016112317-appb-000002
化合物用于减肥，连接肽序提高代谢稳定性。The structure of the target compound is prepared: H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH 2 ) 2 -NH-L-Carnitine (L-Carnitine: L-Carnitine:
Figure PCTCN2016112317-appb-000002
Compounds are used for weight loss, and peptide sequences are linked to increase metabolic stability.
称取实施例13中替代度为0.53mmol/g的Fmoc-Leu-NH-(CH2)2-NH-2-Chlorotrityl Chloride Resin 94克(50mmol)于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；用DBLK脱除Fmoc保护基团，用DMF洗涤6次。称取Fmoc-Cys(Trt)-OH 586克(100mmol)，HOBt 16.3克(120mmol)，HBTU 38克(100mmol)，用DMF溶解，0℃冰水浴下加入16.0克DIPEA(120mmol)，活化5分钟，加入反应柱，反应2小时后，DMF洗涤树脂三次，用DBLK脱除Fmoc保护基团，用DMF洗涤6次，DCM洗涤3次。重复上述偶联操作，按照肽序依次偶联Fmoc-Ser(tBu)-OH，Fmoc-Ala-OH，Fmoc-Gly-OH，Fmoc-Phe-OH，Boc-Phe-OH。反应结束后，用甲醇收缩，真空干燥后得到肽树脂165g。Weigh out Fmoc-Leu-NH-(CH 2 ) 2 -NH-2-Chlorotrityl Chloride Resin 94 g (50 mmol) in Example 13 with a degree of substitution of 0.53 mmol/g in a solid phase reaction column, add DMF, nitrogen drum The bubbles were swelled for 60 minutes; the Fmoc protecting group was removed with DBLK and washed 6 times with DMF. Weigh 586 g (100 mmol) of Fmoc-Cys(Trt)-OH, 16.3 g (120 mmol) of HOBt, 38 g (100 mmol) of HBTU, dissolved in DMF, and added 16.0 g of DIPEA (120 mmol) in an ice water bath at 0 ° C for 5 min. After the reaction was carried out for 2 hours, the resin was washed three times with DMF, and the Fmoc protecting group was removed with DBLK, washed 6 times with DMF and washed 3 times with DCM. The above coupling operation was repeated, and Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Boc-Phe-OH were sequentially coupled according to the peptide sequence. After completion of the reaction, the mixture was shredded with methanol and dried in vacuo to give 165 g of peptide resin.
实施例15 Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH2)2-NH2的制备Example 15 Preparation of Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH 2 ) 2 -NH 2
将实施例14得到的165克肽树脂加入到2000ml单口烧瓶中，预先配置0.1％TFA的DCM溶液1800ml加入到烧瓶中，室温反应2.0小时，滤掉树脂，旋转蒸发掉溶剂，得到白色全保护的粗肽64g。165 g of the peptide resin obtained in Example 14 was placed in a 2000 ml single-necked flask, and 1800 ml of a 0.1% TFA solution of DCM was placed in a flask, and reacted at room temperature for 2.0 hours. The resin was filtered off, and the solvent was evaporated to give a white-protected. Crude peptide 64g.
实施例16 Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH2)2-NH-L-CarnitineExample 16 Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH 2 ) 2 -NH-L-Carnitine 的制备Preparation
在500ml单口烧瓶中加入L-Carnitine 16.1g(100mmol)，用50mlDMF溶解，加入HOBt 16.3克(120mmol)，和15.1克DIC(120mmol)。将实施例15得到的64克全保护粗肽溶于200ml的DCM中，缓慢滴加到L-Carnitine溶液中，滴加完毕后，继续反应2h，结束反应。往反应液中加入100ml水，萃取收集有机相(DCM)，旋转蒸发得到白色的Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH2)2-L-Carnitine粗品41g。L-Carnitine 16.1 g (100 mmol) was added to a 500 ml one-necked flask, dissolved in 50 ml of DMF, and 16.3 g (120 mmol) of HOBt, and 15.1 g of DIC (120 mmol) were added. The 64 g of the fully protected crude peptide obtained in Example 15 was dissolved in 200 ml of DCM, and slowly added dropwise to the L-Carnitine solution. After the completion of the dropwise addition, the reaction was continued for 2 hours to complete the reaction. 100 ml of water was added to the reaction solution, and the organic phase (DCM) was extracted and evaporated to give white Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH 2 ) 2 -L-Carnitine crude 41g.
实施例17 H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH2)2-NH-L-Carnitine粗品的制备Example 17 Preparation of crude H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH 2 ) 2 -NH-L-Carnitine
往实施例16中的粗品中加入预先配置裂解液TFA：TIS：EDT：H2O＝85:5:5:5(体积比)80ml，室温反应2.0小时，加入到10000ml的冷冻无水***中，析出白色固体，离心，无水***洗涤固体，真空干燥后得到白色固体32.4克，收率70.1％，HPLC纯度80.6％。To the crude product of Example 16, a pre-configured lysate TFA: TIS: EDT: H 2 O = 85: 5: 5: 5 (volume ratio) 80 ml was added, and reacted at room temperature for 2.0 hours, and added to 10000 ml of chilled anhydrous diethyl ether. The white solid was precipitated, and the solid was washed with diethyl ether and evaporated to dryness to afford white solid (32.4 g,yield: 70.1%).
实施例18 H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH2)2-NH-L-Carnitine的精制Example 18 Purification of H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH 2 ) 2 -NH-L-Carnitine
取实施例17得到的粗品32.4g，采用Waters 2454RP-HPLC***，波长220nm，色谱柱为100×500mm反相C18柱，流动相：A相：0.3％TFA/乙腈溶液(v/v)；B 相：乙腈，梯度：B％：38％～68％，流速：6毫升/分钟，收集目的峰馏分，旋转蒸发浓缩，冻干得到目标精肽25.2g，HPLC纯度99.70％，总收率54.3％。32.4 g of the crude product obtained in Example 17 was used in a Waters 2454 RP-HPLC system at a wavelength of 220 nm, and the column was a 100×500 mm reverse phase C18 column. Mobile phase: Phase A: 0.3% TFA/acetonitrile solution (v/v); Phase: acetonitrile, gradient: B%: 38% to 68%, flow rate: 6 ml/min, the peak fraction of the target was collected, concentrated by rotary evaporation, and lyophilized to obtain the target sperm peptide 25.2 g, HPLC purity 99.70%, total yield 54.3% .
实施例19 Fmoc-Leu-2-Chlorotrityl Chloride Resin的制备Example 19 Preparation of Fmoc-Leu-2-Chlorotrityl Chloride Resin
称取替代度为1.0mmol/g的2-Chlorotrityl Chloride Resin 100克于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取Fmoc-Leu-OH 21.2克(60mmol)，HOBt 9.72克(72mmol)，HBTU 33.8克(600mmol)，用DMF溶解，0℃冰水浴下加入15.6ml DIPEA(72mmol)，活化5分钟，加入反应柱，反应2小时后，加入100ml甲醇和100mlDIPEA，混合封闭0.5h，DCM洗涤三次，甲醇收缩后抽干树脂，得到Fmoc-Leu-2-Chlorotrityl Chloride Resin，检测替代度为0.46mmol/g的树脂131g。100 g of 2-Chlorotrityl Chloride Resin with 1.0 mmol/g substitution was weighed into a solid phase reaction column, DMF was added, and nitrogen was bubbled for 60 minutes; Fmoc-Leu-OH 21.2 g (60 mmol) was weighed, and HOBt was 9.72 g. (72mmol), HBTU 33.8g (600mmol), dissolved in DMF, added 15.6ml DIPEA (72mmol) in 0°C ice water bath, activated for 5 minutes, added to the reaction column, after reaction for 2 hours, add 100ml methanol and 100ml DIPEA, mix and close 0.5 h, DCM was washed three times, the methanol was shrunk, and the resin was drained to obtain Fmoc-Leu-2-Chlorotrityl Chloride Resin, and 131 g of a resin having a substitution degree of 0.46 mmol/g was detected.
实施例20 Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-2-Chlorotrityl Chloride ResinExample 20 Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-2-Chlorotrityl Chloride Resin 的制备Preparation
称取实施例19中替代度为0.48mmol/g的Fmoc-Leu-2-Chlorotrityl Chloride Resin 109克(50mmol)于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；用DBLK脱除Fmoc保护基团，用DMF洗涤6次。称取Fmoc-Leu-OH 353克(100mmol)，HOBt 16.3克(120mmol)，HBTU 38克(100mmol)，用DMF溶解，0℃冰水浴下加入16.0克DIPEA(120mmol)，活化5分钟，加入反应柱，反应2小时后，DMF洗涤树脂三次，用DBLK脱除Fmoc保护基团，用DMF洗涤6次，DCM洗涤3次。重复上述偶联操作，按照肽序依次偶联Fmoc-Cys(Trt)-OH，Fmoc-Ser(tBu)-OH，Fmoc-Ala-OH，Fmoc-Gly-OH，Fmoc-Phe-OH，Boc-Phe-OH。反应结束后，用甲醇收缩，真空干燥后得到肽树脂178g。Weighed 109 g (50 mmol) of Fmoc-Leu-2-Chlorotrityl Chloride Resin with a degree of substitution of 0.48 mmol/g in Example 19, added DMF, and swelled with nitrogen for 60 minutes; removed Fmoc with DBLK. The protecting group was washed 6 times with DMF. Fmoc-Leu-OH 353 g (100 mmol), HOBt 16.3 g (120 mmol), HBTU 38 g (100 mmol), dissolved in DMF, 16.0 g DIPEA (120 mmol) was added to a 0 ° C ice water bath, activated for 5 minutes, added to the reaction Column, after 2 hours of reaction, the resin was washed three times with DMF, the Fmoc protecting group was removed with DBLK, washed 6 times with DMF and washed 3 times with DCM. The above coupling operation was repeated, and Fmoc-Cys(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Boc- were sequentially coupled according to the peptide sequence. Phe-OH. After completion of the reaction, the mixture was shredded with methanol and dried in vacuo to give 178 g of peptide resin.
实施例21 Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-OH的制备Example 21 Preparation of Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-OH
将实施例20得到的178克肽树脂加入到2000ml单口烧瓶中，预先配置0.1％TFA的DCM溶液1800ml加入到烧瓶中，室温反应2.0小时，滤掉树脂，旋转蒸发掉溶剂，得到白色全保护的粗肽61g。178 g of the peptide resin obtained in Example 20 was placed in a 2000 ml single-necked flask, and 1800 ml of a 0.1% TFA solution of DCM was placed in a flask, and reacted at room temperature for 2.0 hours. The resin was filtered off, and the solvent was evaporated to give a white-protected. Crude peptide 61g.
实施例22 Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH2)2-NH2的制备Example 22 Preparation of Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH 2 ) 2 -NH 2
在500ml单口烧瓶中加入乙二胺6.0g(100mmol)，用50mlDMF溶解，加入HOBt 16.3克(120mmol)，和15.1克DIC(120mmol)。将实施例21得到的61克全保护粗肽溶于200ml的DCM中，缓慢滴加到乙二胺溶液中，滴加完毕后，继续反应2h，结束反应。往反应液中加入100ml水，萃取收集有机相(DCM)，旋转蒸发得到白色的全保护的目标粗肽42g。To a 500 ml one-necked flask was added 6.0 g (100 mmol) of ethylenediamine, dissolved in 50 ml of DMF, and added 16.3 g (120 mmol) of HOBt, and 15.1 g of DIC (120 mmol). 61 g of the fully protected crude peptide obtained in Example 21 was dissolved in 200 ml of DCM, and slowly added dropwise to the ethylenediamine solution. After the completion of the dropwise addition, the reaction was continued for 2 hours to complete the reaction. 100 ml of water was added to the reaction mixture, and the organic phase (DCM) was collected by EtOAc.
实施例23 Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH2)2-NH-L-CarnitineExample 23 Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH 2 ) 2 -NH-L-Carnitine 的制备Preparation
在500ml单口烧瓶中加入L-Carnitine 16.1g(100mmol)，用50mlDMF溶解，加入HOBt 16.3克(120mmol)，和15.1克DIC(120mmol)。将实施例22得到的42克全保护粗肽溶于200ml的DCM中，缓慢滴加到L-Carnitine溶液中，滴加完 毕后，继续反应2h，结束反应。往反应液中加入100ml水，萃取收集有机相(DCM)，旋转蒸发得到白色的Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH2)2-NH-L-Carnitine粗品28g。L-Carnitine 16.1 g (100 mmol) was added to a 500 ml one-necked flask, dissolved in 50 ml of DMF, and 16.3 g (120 mmol) of HOBt, and 15.1 g of DIC (120 mmol) were added. The 42 g of the fully protected crude peptide obtained in Example 22 was dissolved in 200 ml of DCM, and slowly added dropwise to the L-Carnitine solution. After the completion of the dropwise addition, the reaction was continued for 2 hours to complete the reaction. 100 ml of water was added to the reaction solution, and the organic phase (DCM) was extracted and evaporated to give white Boc-Phe-Phe-Gly-Ala-Ser(tBu)-Cys(Trt)-Leu-NH-(CH 2 ) 2 -NH-L-Carnitine crude product 28g.
实施例24 H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH2)2-NH-L-Carnitine粗品的制备Example 24 Preparation of crude H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH 2 ) 2 -NH-L-Carnitine
往实施例23中的粗品中加入预先配置裂解液TFA：TIS：EDT：H2O＝85:5:5:5(体积比)50ml，室温反应2.0小时，加入到5000ml的冷冻无水***中，析出白色固体，离心，无水***洗涤固体，真空干燥后得到白色固体16.3克，收率35.13％，HPLC纯度64.5％。To the crude product of Example 23, a pre-configured lysate TFA: TIS:EDT:H 2 O=85:5:5:5 (volume ratio) 50 ml was added, and reacted at room temperature for 2.0 hours, and added to 5000 ml of chilled anhydrous diethyl ether. The white solid was precipitated, and the solid was washed with diethyl ether and evaporated to dryness, and then evaporated to give a white solid, 16.3 g, yield: 35.
实施例25 H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH2)2-NH-L-Carnitine的精制Example 25 Purification of H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH 2 ) 2 -NH-L-Carnitine
取实施例24得到的粗品16.3g，采用Waters 2454RP-HPLC***，波长220nm，色谱柱为100×500mm反相C18柱，流动相：A相：0.3％TFA/乙腈溶液(v/v)；B相：乙腈，梯度：B％：38％～68％，流速：6毫升/分钟，收集目的峰馏分，旋转蒸发浓缩，冻干得到目标精肽9.7g，HPLC纯度99.26％，总收率20.9％。The crude product obtained in Example 24 was 16.3 g, using a Waters 2454 RP-HPLC system, a wavelength of 220 nm, a column of 100×500 mm reverse phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile solution (v/v); Phase: acetonitrile, gradient: B%: 38% to 68%, flow rate: 6 ml/min, the peak fraction of the target was collected, concentrated by rotary evaporation, and lyophilized to obtain the target sperm peptide 9.7 g, HPLC purity 99.26%, total yield 20.9% .
通过实施例12-18(本发明)方法以及实施例19-24(现有技术)方法以相同投料量制备H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH2)2-NH-L-Carnitine，其总收率相差近3倍，通过本发明的方法，虽然仅改变了合成的顺序，其总收率远远超过预期产率。Preparation of H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH-(CH 2 ) by the same method as in Examples 12-18 (present invention) and Examples 19-24 (prior art) 2- NH-L-Carnitine, the total yield differs by a factor of nearly three, and by the method of the present invention, although only the order of synthesis is changed, the total yield far exceeds the expected yield.
实施例26 NH2-(CH2)6-NH-2-Chlorotrityl Chloride Resin的制备Example 26 Preparation of NH 2 -(CH 2 ) 6 -NH-2-Chlorotrityl Chloride Resin
称取替代度为1.0mmol/g的2-Chlorotrityl Chloride Resin 200克于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取己二胺46.4克(400mmol)，用DMF溶解，0℃冰水浴下加入90.0ml DIPEA(500mmol)，加入反应柱，反应1小时后，加入200ml甲醇和200mlDIPEA，混合封闭0.5h，DCM洗涤三次，甲醇收缩后抽干树脂，得到NH2-(CH2)6-NH-2-Chlorotrityl Chloride Resin。200 g of 2-Chlorotrityl Chloride Resin with a substitution of 1.0 mmol/g was weighed into a solid phase reaction column, DMF was added, and nitrogen gas was bubbled and swollen for 60 minutes; 46.4 g (400 mmol) of hexamethylenediamine was weighed and dissolved with DMF, 0 Add 90.0 ml of DIPEA (500 mmol) under ice-cold bath and add to the reaction column. After reacting for 1 hour, add 200 ml of methanol and 200 ml of DIPEA, mix and block for 0.5 h, wash with DCM three times, and shrink the methanol to dry the resin to obtain NH 2 -(CH 2 6 -NH-2-Chlorotrityl Chloride Resin.
实施例27 Fmoc-Ser(tBu)-NH-(CH2)6-NH-2-Chlorotrityl Chloride Resin的制备Example 27 Preparation of Fmoc-Ser(tBu)-NH-(CH 2 ) 6 -NH-2-Chlorotrityl Chloride Resin
称取实施例26得到的NH2-(CH2)6-NH-2-Chlorotrityl Chloride Resin 100克于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取Fmoc-Ser(tBu)-OH 22.8克(60mmol)，HOBt 9.72克(72mmol)，用DMF溶解，0℃冰水浴下加入16.4ml DIC(72mmol)，活化5分钟，加入反应柱，反应2小时后，加入70ml醋酸酐和60ml吡啶，混合封闭24h，DCM洗涤三次，甲醇收缩后抽干树脂，得到Fmoc-Ser(tBu)-NH-(CH2)6-NH-2-Chlorotrityl Chloride Resin，检测替代度为0.55mmol/g的树脂116g。100 g of NH 2 -(CH 2 ) 6 -NH-2-Chlorotrityl Chloride Resin obtained in Example 26 was weighed into a solid phase reaction column, DMF was added, and nitrogen gas was bubbled and swollen for 60 minutes; Fmoc-Ser (tBu) was weighed. -OH 22.8 g (60 mmol), HOBt 9.72 g (72 mmol), dissolved in DMF, added 16.4 ml DIC (72 mmol) in a 0 ° C ice water bath, activated for 5 minutes, added to the reaction column, after 2 hours of reaction, 70 ml of acetic anhydride and 60ml of pyridine, mixed and blocked for 24h, washed twice with DCM, and the resin was shrunk and the resin was dried to obtain Fmoc-Ser(tBu)-NH-(CH 2 ) 6 -NH-2-Chlorotrityl Chloride Resin with a detection degree of 0.55mmol/g. Resin 116g.
实施例28 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc)Example 28 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc) -Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH2)6-NH-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH 2 ) 6 -NH -2-Chlorotrityl Chloride Resin的制备Preparation of -2-Chlorotrityl Chloride Resin
制备目标化合物的结构为：GHWDFRQWWQPSGGGS-己二胺-Biotin。The structure of the target compound was prepared: GHWDFRQWWQPSGGGS-hexanediamine-Biotin.
称取实施例27中替代度为0.55mmol/g的Fmoc-Ser(tBu)-NH-(CH2)6-NH -2-Chlorotrityl Chloride Resin 91克(50mmol)于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；用DBLK脱除Fmoc保护基团，用DMF洗涤6次。称取Fmoc-Gly-OH 298克(100mmol)，HOBt 16.3克(120mmol)，HBTU 38克(100mmol)，用DMF溶解，0℃冰水浴下加入16.0克DIPEA(120mmol)，活化5分钟，加入反应柱，反应2小时后，DMF洗涤树脂三次，用DBLK脱除Fmoc保护基团，用DMF洗涤6次，DCM洗涤3次。重复上述偶联操作，按照肽序依次偶联Fmoc-Gly-OH，Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Pro-OH，Fmoc-Gln(Trt)-OH，Fmoc-Trp(Boc)-OH，Fmoc-Trp(Boc)-OH，Fmoc-Gln(Trt)-OH，Fmoc-Arg(Pbf)-OH，Fmoc-Phe-OH，Fmoc-Asp(OtBu)-OH，Fmoc-Trp(Boc)-OH，Fmoc-His(Trt)-OH，Boc-Gly-OH。反应结束后，用甲醇收缩，真空干燥后得到肽树脂274g.Fvoc-Ser(tBu)-NH-(CH 2 ) 6 -NH -2-Chlorotrityl Chloride Resin 91 g (50 mmol) with a degree of substitution of 0.55 mmol/g in Example 27 was weighed into a solid phase reaction column, and DMF was added. Nitrogen gas was bubbled for 60 minutes; the Fmoc protecting group was removed with DBLK and washed 6 times with DMF. Fmoc-Gly-OH 298 g (100 mmol), HOBt 16.3 g (120 mmol), HBTU 38 g (100 mmol), dissolved in DMF, 16.0 g DIPEA (120 mmol) was added to a 0 ° C ice water bath, activated for 5 minutes, added to the reaction Column, after 2 hours of reaction, the resin was washed three times with DMF, the Fmoc protecting group was removed with DBLK, washed 6 times with DMF and washed 3 times with DCM. The above coupling operation was repeated, and Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc- were sequentially coupled according to the peptide sequence. Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc -Trp(Boc)-OH, Fmoc-His(Trt)-OH, Boc-Gly-OH. After the reaction was completed, it was shrunk with methanol and dried under vacuum to obtain 274 g of a peptide resin.
实施例29 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc)Example 29 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc) -Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH2)6-NH2的制备-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH 2 ) 6 -NH 2 Preparation
将实施例28得到的274克肽树脂加入到3000ml单口烧瓶中，预先配置0.1％TFA的DCM溶液2800ml加入到烧瓶中，室温反应2.0小时，滤掉树脂，旋转蒸发掉溶剂，得到白色全保护的粗肽169g。274 g of the peptide resin obtained in Example 28 was placed in a 3000 ml single-necked flask, and 2800 ml of a 0.1% TFA solution of DCM was placed in a flask, and reacted at room temperature for 2.0 hours. The resin was filtered off, and the solvent was evaporated to give a white-protected. The crude peptide was 169 g.
实施例30 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc)Example 30 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc) -Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH2)6-NH-Biotin的-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH 2 ) 6 -NH-Biotin 制备preparation
在1000ml单口烧瓶中加入Biotin 24.4g(100mmol)，用100mlDMF溶解，加入HOBt 16.3克(120mmol)，和15.1克DIC(120mmol)。将实施例29得到的169克全保护粗肽溶于500ml的DCM中，缓慢滴加到Biotin溶液中，滴加完毕后，继续反应2h，结束反应。往反应液中加入100ml水，萃取收集有机相(DCM)，旋转蒸发得到白色的Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc)-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH2)6-NH-Biotin粗品161g。Biotin 24.4 g (100 mmol) was added to a 1000 ml one-neck flask, dissolved in 100 ml of DMF, and 16.3 g (120 mmol) of HOBt, and 15.1 g DIC (120 mmol) were added. The 169 g of the fully protected crude peptide obtained in Example 29 was dissolved in 500 ml of DCM, and slowly added dropwise to the Biotin solution. After the completion of the dropwise addition, the reaction was continued for 2 hours to terminate the reaction. 100 ml of water was added to the reaction solution, and the organic phase (DCM) was extracted and evaporated to give white Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt -Trp(Boc)-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH 2 ) 6 -NH-Biotin crude 161 g.
实施例31 H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-Gly-Gly-SerExample 31 H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-Gly-Gly-Ser -NH-(CH2)6-NH-Biotin粗品的制备Preparation of crude -NH-(CH2) 6 -NH-Biotin
往实施例30中的粗品中加入预先配置裂解液TFA：TIS：EDT：H2O＝85:5:5:5(体积比)400ml，室温反应2.0小时，加入到10000ml的冷冻无水***中，析出白色固体，离心，无水***洗涤固体，真空干燥后得到白色固体92.6克，收率83.7％，HPLC纯度56.4％。To the crude product of Example 30, a pre-configured lysate TFA: TIS: EDT: H 2 O = 85: 5: 5: 5 (volume ratio) 400 ml was added, and reacted at room temperature for 2.0 hours, and added to 10000 ml of chilled anhydrous diethyl ether. The white solid was precipitated, and the solid was washed with diethyl ether and evaporated to dryness, and then evaporated to give a white solid (92.6 g, yield: 83.
实施例32 H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-Gly-Gly-SerExample 32 H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-Gly-Gly-Ser -NH-(CH2)6-NH-Biotin的精制-NH-(CH2) 6 -NH-Biotin refining
取实施例31得到的粗品92.6g，采用Waters 2454RP-HPLC***，波长220nm，色谱柱为100×500mm反相C18柱，流动相：A相：0.3％TFA/乙腈溶液(v/v)；B 相：乙腈，梯度：B％：38％～68％，流速：6毫升/分钟，收集目的峰馏分，旋转蒸发浓缩，冻干得到目标精肽40.5g，HPLC纯度98.90％，总收率36.6％。The crude product obtained in Example 31 was obtained from 92.6 g, using a Waters 2454 RP-HPLC system with a wavelength of 220 nm, a column of 100×500 mm reverse phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile solution (v/v); Phase: acetonitrile, gradient: B%: 38% to 68%, flow rate: 6 ml/min, collection of the peak fraction of interest, concentration by rotary evaporation, lyophilization to obtain 40.5 g of the target spermatin, HPLC purity 98.90%, total yield 36.6% .
实施例33 Fmoc-Ser(tBu)-2-Chlorotrityl Chloride Resin的制备Example 33 Preparation of Fmoc-Ser(tBu)-2-Chlorotrityl Chloride Resin
称取替代度为1.0mmol/g的2-Chlorotrityl Chloride Resin 100克于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；称取Fmoc-Ser(tBu)-OH 22.8克(60mmol)，HOBt 9.72克(72mmol)，HBTU 33.8克(600mmol)，用DMF溶解，0℃冰水浴下加入15.6ml DIPEA(72mmol)，活化5分钟，加入反应柱，反应2小时后，加入100ml甲醇和100mlDIPEA，混合封闭0.5h，DCM洗涤三次，甲醇收缩后抽干树脂，得到Fmoc-Leu-2-Chlorotrityl Chloride Resin，检测替代度为0.45mmol/g的树脂139g。100 g of 2-Chlorotrityl Chloride Resin with 1.0 mmol/g substitution was weighed into a solid phase reaction column, DMF was added, and nitrogen was bubbled for 60 minutes; Fmoc-Ser(tBu)-OH 22.8 g (60 mmol) was weighed. HOBt 9.72 g (72 mmol), HBTU 33.8 g (600 mmol), dissolved in DMF, 15.6 ml DIPEA (72 mmol) was added to a 0 ° C ice water bath, activated for 5 minutes, added to the reaction column, after 2 hours of reaction, 100 ml of methanol and 100 ml of DIPEA were added. The mixture was blocked for 0.5 h, washed with DCM three times, and the mixture was evaporated, and then the resin was evaporated to obtain Fmoc-Leu-2-Chlorotrityl Chloride Resin, and 139 g of a resin having a substitution degree of 0.45 mmol/g was detected.
实施例34 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc) -Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-2-Chlorotrityl Chloride Resin的制备 Example 34 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc) -Trp(Boc)-Gln(Trt)-Pro -Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-2-Chlorotrityl Chloride Resin Preparation
称取实施例33中替代度为0.45mmol/g的Fmoc-Ser(tBu)-2-Chlorotrityl Chloride Resin 111克(50mmol)于固相反应柱中，加入DMF，氮气鼓泡溶胀60分钟；用DBLK脱除Fmoc保护基团，用DMF洗涤6次。称取Fmoc-Gly-OH 298克(100mmol)，HOBt 16.3克(120mmol)，HBTU 38克(100mmol)，用DMF溶解，0℃冰水浴下加入16.0克DIPEA(120mmol)，活化5分钟，加入反应柱，反应2小时后，DMF洗涤树脂三次，用DBLK脱除Fmoc保护基团，用DMF洗涤6次，DCM洗涤3次。重复上述偶联操作，按照肽序依次偶联Fmoc-Gly-OH，Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Pro-OH，Fmoc-Gln(Trt)-OH，Fmoc-Trp(Boc)-OH，Fmoc-Trp(Boc)-OH，Fmoc-Gln(Trt)-OH，Fmoc-Arg(Pbf)-OH，Fmoc-Phe-OH，Fmoc-Asp(OtBu)-OH，Fmoc-Trp(Boc)-OH，Fmoc-His(Trt)-OH，Boc-Gly-OH。反应结束后，用甲醇收缩，真空干燥后得到肽树脂268g。Weighed 0.45 mmol/g of Fmoc-Ser(tBu)-2-Chlorotrityl Chloride Resin 111 g (50 mmol) in Example 33 in a solid phase reaction column, added DMF, and swelled with nitrogen for 60 minutes; using DBLK The Fmoc protecting group was removed and washed 6 times with DMF. Fmoc-Gly-OH 298 g (100 mmol), HOBt 16.3 g (120 mmol), HBTU 38 g (100 mmol), dissolved in DMF, 16.0 g DIPEA (120 mmol) was added to a 0 ° C ice water bath, activated for 5 minutes, added to the reaction Column, after 2 hours of reaction, the resin was washed three times with DMF, the Fmoc protecting group was removed with DBLK, washed 6 times with DMF and washed 3 times with DCM. The above coupling operation was repeated, and Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc- were sequentially coupled according to the peptide sequence. Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc -Trp(Boc)-OH, Fmoc-His(Trt)-OH, Boc-Gly-OH. After completion of the reaction, the mixture was shredded with methanol and dried in vacuo to give 268 g of peptide resin.
实施例35 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc)Example 35 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc) -Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-OH的制备Preparation of -Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-OH
将实施例34得到的268克肽树脂加入到3000ml单口烧瓶中，预先配置0.1％TFA的DCM溶液2800ml加入到烧瓶中，室温反应2.0小时，滤掉树脂，旋转蒸发掉溶剂，得到白色全保护的粗肽158g。268 g of the peptide resin obtained in Example 34 was placed in a 3000 ml single-necked flask, and 2800 ml of a 0.1% TFA solution of DCM was placed in a flask, and reacted at room temperature for 2.0 hours. The resin was filtered off, and the solvent was evaporated to give a white-protected. Crude peptide 158g.
实施例36 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc)Example 36 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc) -Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH2)6-NH2的制备-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH 2 ) 6 -NH 2 Preparation
在500ml单口烧瓶中加入己二胺11.6.0g(100mmol)，用50mlDMF溶解，加入HOBt 16.3克(120mmol)，和15.1克DIC(120mmol)。将实施例21得到的61克全保护粗肽溶于200ml的DCM中，缓慢滴加到己二胺胺溶液中，滴加完毕后，继续反应2h，结束反应。往反应液中加入100ml水，萃取收集有机相(DCM)，旋转蒸发得到白色的全保护的目标粗肽158g。 To a 500 ml one-necked flask, 11.6.0 g (100 mmol) of hexamethylenediamine was added, dissolved in 50 ml of DMF, and 16.3 g (120 mmol) of HOBt, and 15.1 g of DIC (120 mmol) were added. 61 g of the fully protected crude peptide obtained in Example 21 was dissolved in 200 ml of DCM, and slowly added dropwise to the hexamethylenediamine solution. After the completion of the dropwise addition, the reaction was continued for 2 hours to complete the reaction. 100 ml of water was added to the reaction mixture, and the organic phase (DCM) was collected by EtOAc.
实施例37 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc)Example 37 Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc) -Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH2)6-NH-Biotin的-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH 2 ) 6 -NH-Biotin 制备preparation
在1000ml单口烧瓶中加入Biotin 24.4g(100mmol)，用100mlDMF溶解，加入HOBt 16.3克(120mmol)，和15.1克DIC(120mmol)。将实施例36得到的158克全保护粗肽溶于500ml的DCM中，缓慢滴加到Biotin溶液中，滴加完毕后，继续反应2h，结束反应。往反应液中加入100ml水，萃取收集有机相(DCM)，旋转蒸发得到白色的Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt)-Trp(Boc)-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH2)6-NH-Biotin粗品126g。Biotin 24.4 g (100 mmol) was added to a 1000 ml one-neck flask, dissolved in 100 ml of DMF, and 16.3 g (120 mmol) of HOBt, and 15.1 g DIC (120 mmol) were added. The 158 g of the fully protected crude peptide obtained in Example 36 was dissolved in 500 ml of DCM, and slowly added dropwise to the Biotin solution. After the completion of the dropwise addition, the reaction was continued for 2 hours to complete the reaction. 100 ml of water was added to the reaction solution, and the organic phase (DCM) was extracted and evaporated to give white Boc-Gly-His(Trt)-Trp(Boc)-Asp(OtBu)-Phe-Arg(Pbf)-Gln(Trt )-Trp(Boc)-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH 2 ) 6 -NH-Biotin crude product 126 g.
实施例38 H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-Gly-Gly-SerExample 38 H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-Gly-Gly-Ser -NH-(CH2)6-NH-Biotin粗品的制备Preparation of crude -NH-(CH2) 6 -NH-Biotin
往实施例37中的粗品中加入预先配置裂解液TFA：TIS：EDT：H2O＝85:5:5:5(体积比)400ml，室温反应2.0小时，加入到10000ml的冷冻无水***中，析出白色固体，离心，无水***洗涤固体，真空干燥后得到白色固体45.9克，收率41.5％，HPLC纯度50.7％。To the crude product of Example 37, a pre-configured lysate TFA: TIS: EDT: H 2 O = 85: 5: 5: 5 (volume ratio) 400 ml was added, and reacted at room temperature for 2.0 hours, and added to 10000 ml of chilled anhydrous diethyl ether. The white solid was precipitated, and the solid was washed with diethyl ether and evaporated to dryness, and then evaporated to give a white solid, 45.9 g, yield 41.
实施例39 H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-Gly-Gly-SerExample 39 H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-Gly-Gly-Ser -NH-(CH2)6-NH-Biotin的精制-NH-(CH2) 6 -NH-Biotin refining
取实施例38得到的粗品45.9g，采用Waters 2454RP-HPLC***，波长220nm，色谱柱为100×500mm反相C18柱，流动相：A相：0.3％TFA/乙腈溶液(v/v)；B相：乙腈，梯度：B％：38％～68％，流速：6毫升/分钟，收集目的峰馏分，旋转蒸发浓缩，冻干得到目标精肽16.3g，HPLC纯度99.2％，总收率14.7％。The crude product obtained in Example 38 was obtained in 45.9 g, using a Waters 2454 RP-HPLC system, a wavelength of 220 nm, a column of 100×500 mm reverse phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile solution (v/v); Phase: acetonitrile, gradient: B%: 38% to 68%, flow rate: 6 ml/min, the peak fraction of the target was collected, concentrated by rotary evaporation, and lyophilized to obtain the target sperm peptide 16.3 g, HPLC purity 99.2%, total yield 14.7% .
通过实施例26-33(本发明)方法以及实施例34-39(现有技术)方法以相同投料量制备GHWDFRQWWQPSGGGS-己二胺-Biotin，其总收率2倍多，通过本发明的方法，虽然仅改变了合成的顺序，其总收率远远超过预期产率。 GHWDFRQWWQPSGGGS-hexanediamine-Biotin was prepared by the method of Examples 26-33 (present invention) and Examples 34-39 (prior art) at the same dosage, with a total yield of more than 2 times, by the method of the present invention, Although only the order of synthesis was changed, the overall yield far exceeded the expected yield.

Claims (10)

  1. 一种C-端修饰肽的合成方法，其特征在于，包括以下步骤：A method for synthesizing a C-terminal modified peptide, comprising the steps of:
    1)将二氨基化合物的一端氨基偶联至固相合成树脂上；1) coupling an amino group at one end of the diamino compound to a solid phase synthetic resin;
    2)采用Fmoc固相肽合成策略，将氨基酸依次偶联至二氨基化合物的另一端氨基上，以获得全保护的多肽树脂；2) using an Fmoc solid phase peptide synthesis strategy, the amino acid is sequentially coupled to the amino group at the other end of the diamino compound to obtain a fully protected polypeptide resin;
    3)将全保护的多肽从树脂上裂解，以获得全保护的多肽；3) cleavage of the fully protected polypeptide from the resin to obtain a fully protected polypeptide;
    4)将全保护的多肽脱除保护基，以获得目标的C-端修饰肽，或者4) removing the protecting group from the fully protected polypeptide to obtain the target C-terminal modified peptide, or
    4)将全保护的多肽偶联与含羧基的修饰基团进行偶联，以获得目标的C-端修饰肽。4) Coupling the fully protected polypeptide to the carboxyl-containing modifying group to obtain the target C-terminal modified peptide.
  2. 根据权利要求1所述的C-端修饰肽的合成方法，其中，所述二氨基化合物选自直链对称二氨基化合物或带支链的对称二氨基化合物或空间结构对称的二氨基类化合物，优选为乙二胺、丙二胺、丁二胺、戊二胺、己二胺、庚二胺、辛二胺、
    Figure PCTCN2016112317-appb-100001
    The method for synthesizing a C-terminal modified peptide according to claim 1, wherein the diamino compound is selected from a linear symmetric diamino compound or a branched symmetric diamino compound or a symmetrical spatial diamino compound. Preferred are ethylenediamine, propylenediamine, butanediamine, pentanediamine, hexamethylenediamine, heptanediamine, octanediamine,
    Figure PCTCN2016112317-appb-100001
  3. 根据权利要求1-2任一项所述的C-端修饰肽的合成方法，其中，所述固相合成树脂选自2-Chlorotrityl Chloride Resin树脂，优选地，固相合成树脂替代度范围为0.1～1.2mmol/g，更优选0.2～0.8mmol/g，最优选0.3～0.5mmol/g。The method for synthesizing a C-terminal modified peptide according to any one of claims 1 to 2, wherein the solid phase synthetic resin is selected from the group consisting of 2-Chlorotrityl Chloride Resin resin, preferably, the solid phase synthetic resin has a degree of substitution of 0.1. ～1.2 mmol/g, more preferably 0.2 to 0.8 mmol/g, most preferably 0.3 to 0.5 mmol/g.
  4. 根据权利要求1-3任一项所述的C-端修饰肽的合成方法，其中，步骤2)中的Fmoc固相肽合成策略所用的偶联体系为DIC+A或B+A+C，其中A为HOBt或HOAt，B为HBTU、HATU、TBTU或PyBOP，C为DIPEA或TMP。The method for synthesizing a C-terminal modified peptide according to any one of claims 1 to 3, wherein the coupling system used in the Fmoc solid phase peptide synthesis strategy in the step 2) is DIC+A or B+A+C. Where A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA or TMP.
  5. 根据权利要求1-4任一项所述的C-端修饰肽的合成方法，其中，步骤3)裂解步骤所用的裂解试剂为TFA、TIS、EDT、H2O的组合物，裂解时间为1.5-3.5小时，优选裂解试剂的体积比为TFA：TIS：EDT：H2O＝85-95:2-5:2-5:1-5，The method for synthesizing a C-terminal modified peptide according to any one of claims 1 to 4, wherein the cleavage reagent used in the step 3) is a composition of TFA, TIS, EDT, H 2 O, and the lysis time is 1.5. -3.5 hours, preferably the volume ratio of the lysis reagent is TFA: TIS: EDT: H 2 O = 85-95: 2-5: 2-5: 1-5,
  6. 根据权利要求1-5任一项所述的C-端修饰肽的合成方法，步骤4)之后还包括纯化步骤，优选的纯化步骤为以HPLC方法进行纯化。The method for synthesizing a C-terminal modified peptide according to any one of claims 1 to 5, further comprising a purification step after the step 4), wherein the preferred purification step is purification by an HPLC method.
  7. 根据权利要求1-6任一项所述的C-端修饰肽的合成方法，其中步骤1)的使用方法为称取固相合成树脂，并置于固相反应柱中，加入DMF，氮气鼓泡溶胀；称取二氨基化合物，用DMF溶解，并将DIPEA加入固相反应柱中，反应完全后，加入甲醇和DIPEA，混合封闭10-60分钟，以DCM洗涤，甲醇收缩后抽干树脂， 得到二氨基化合物偶联固相合成树脂。The method for synthesizing a C-terminal modified peptide according to any one of claims 1 to 6, wherein the method of using the step 1) is to weigh the solid phase synthetic resin, and place it in a solid phase reaction column, and add DMF, a nitrogen drum. Soak the bubble; weigh the diamino compound, dissolve it with DMF, and add DIPEA to the solid phase reaction column. After the reaction is completed, add methanol and DIPEA, mix and seal for 10-60 minutes, wash with DCM, shrink the methanol and drain the resin. A diamino compound-coupled solid phase synthetic resin is obtained.
  8. 根据权利要求1-7任一项所述的C-端修饰肽的合成方法，其中步骤2)中将第一个氨基酸偶联至二氨基化合物的另一端氨基上的步骤为：在步骤1)得到的二氨基化合物偶联固相合成树脂中加入DMF，氮气鼓泡溶胀；以偶联体系活化Fmoc保护的氨基酸后，将活化的Fmoc保护的氨基酸加入反应柱中进行反应，然后加入醋酸酐和吡啶，混合封闭，得到Fmoc保护的氨基酸二氨基化合物偶联固相合成树脂。The method for synthesizing a C-terminal modified peptide according to any one of claims 1 to 7, wherein the step of coupling the first amino acid to the amino group at the other terminal of the diamino compound in the step 2) is: in the step 1) The obtained diamino compound-coupled solid phase synthetic resin is added with DMF, and nitrogen is bubbled and swollen; after the Fmoc-protected amino acid is activated by the coupling system, the activated Fmoc-protected amino acid is added to the reaction column for reaction, and then acetic anhydride is added and Pyridine, mixed and blocked, to obtain an Fmoc-protected amino acid diamino compound coupled solid phase synthetic resin.
  9. 根据权利要求1-8任一项所述的C-端修饰肽的合成方法，其中依次偶联至氨基化合物的另一端氨基上氨基酸序列选自包含1-20个氨基酸的多肽，优选为5-15个氨基酸的多肽。The method for synthesizing a C-terminal modified peptide according to any one of claims 1 to 8, wherein the amino acid sequence of the amino group at the other terminal of the amino compound is sequentially selected from the group consisting of a polypeptide comprising 1-20 amino acids, preferably 5- A 15 amino acid polypeptide.
  10. 根据权利要求1-9任一项所述的C-端修饰肽的合成方法，其中含羧基的修饰基团选自Biotin、荧光素、肉毒碱、甲酸、乙酸、棕榈酸、硬脂酸、胆酸、含羧基的小分子药物及药效官能团。 The method for synthesizing a C-terminal modified peptide according to any one of claims 1 to 9, wherein the carboxyl group-containing modifying group is selected from the group consisting of Biotin, fluorescein, carnitine, formic acid, acetic acid, palmitic acid, stearic acid, Cholic acid, carboxyl-containing small molecule drugs and pharmacodynamic functional groups.
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CN111349147A (en) * 2018-12-21 2020-06-30 国家纳米科学中心 Large-scale preparation method and application of polypeptide
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