WO2018126525A1 - Procédé de préparation d'un hexapeptide et produit correspondant - Google Patents

Procédé de préparation d'un hexapeptide et produit correspondant Download PDF

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WO2018126525A1
WO2018126525A1 PCT/CN2017/075125 CN2017075125W WO2018126525A1 WO 2018126525 A1 WO2018126525 A1 WO 2018126525A1 CN 2017075125 W CN2017075125 W CN 2017075125W WO 2018126525 A1 WO2018126525 A1 WO 2018126525A1
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arg
pbf
glu
otbu
fmoc
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PCT/CN2017/075125
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Chinese (zh)
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张忠旗
李乾
王万科
王慧
郭添
苏子梦
韩广
高长波
赵金礼
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陕西慧康生物科技有限责任公司
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Publication of WO2018126525A1 publication Critical patent/WO2018126525A1/fr

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    • 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

Definitions

  • the invention belongs to the technical field of polypeptide synthesis, and in particular relates to a method for synthesizing a hexapeptide by a fragment method.
  • Liusheng peptide is an active peptide containing six amino acids. Its sequence is Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2. It has the biological activity of skin wrinkle removal and is widely used in various high-grade cosmetics. .
  • the synthesis methods of six peptides mainly include solid phase synthesis and liquid phase synthesis.
  • the Fmoc-AM Resin and Fmoc-MBHA Resin used in the solid phase synthesis method are expensive and the degree of resin substitution is small, resulting in high cost of solid phase synthesis of the six peptides.
  • the liquid phase synthesis method greatly reduces the price of raw materials, the liquid phase synthesis method has a complicated synthesis process, a long cycle, low efficiency, and high purification cost.
  • Liusheng peptide has important significance in reducing production cost, improving production efficiency and simplifying the research of synthesis technology.
  • the technical problem solved by the invention is to provide a method of low synthesis cost, high synthesis efficiency and simple synthesis method, which can be used for large-scale synthesis of hexapeptide, which overcomes the high cost of the existing classical solid phase synthesis of hexapeptide resin.
  • the problem of low loading capacity also overcomes the shortcomings of complex process, long synthesis cycle and low efficiency of liquid phase synthesis of hexapeptide.
  • the present invention discloses a method for synthesizing a hexapeptide by synthesizing Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg by 2-Chlorotrityl C hloride Resin (pbf)-O H fully protected fragment peptide, then with the fully protected fragment peptide Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH and hexafluoro Synthesis of Ac-Glu(OtBu)-Glu by benzotriazol-1-yl-oxytripyrrolidinylphosphine, 1-hydroxyphenylpropanetriazole, ammonium chloride and N,N'-diisopropylethylamine (OtBu)-Met-G ln(Trt)-Arg(pbf)-Arg(pbf)-
  • a method for preparing a six-peptide comprising the steps of:
  • step (4) The product obtained in the step (4) is cleaved, separated and purified to obtain a six-peptide product.
  • step (1) is to swell 2-Chlorotrityl Chloride Resin with dichloromethane, and then add dichloromethane, Fmoc-Arg(pbf)-OH, N,N'-diisopropylethylamine to obtain Fmoc.
  • step (2) is: removing Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin obtained in the step (1) twice, and adding N,N-dimethylformamide, Fmoc-Arg(pbf)- OH, 1-hydroxyphenylpropane triazole, benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, N,N'-diisopropylethylamine are reacted to obtain Fmoc- Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin; then Fmoc-Gln(Trt)-OH is sequentially attached to Fmoc-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin according to the method described.
  • step (3) is the removal of the Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin obtained in the step (2).
  • Fmoc- twice adding a mixture of dichloromethane, acetic anhydride, N,N'-diisopropylethylamine to obtain Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg (pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin.
  • the reaction in the step (1), the step (2) and the step (3) is carried out under a nitrogen atmosphere at a normal temperature for 0.5-2 hours; the step (1), the step (2) and the step (3)
  • the intermediate Fmoc is a mixture of piperidine and N,N-dimethylformamide in a volume ratio of 1:4 to remove Fmoc; preferably, step (4) is added with dichloromethane, acetic anhydride, N, N'-di A mixture of isopropylethylamine in a volume ratio of 9:1:0.5.
  • step (4) is to add a cutting solution to Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin for cleavage reaction After separation, purification, and drying to obtain Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH;
  • the step of separating, purifying and drying in the step (4) is to filter the cut product, and the filtrate is washed three times with twice the saturated saline solution, and then an equal volume of saturated saline solution with the cutting liquid is added, and saturated sodium hydrogencarbonate is used.
  • the pH of the solution was adjusted to 7.0, and the organic solvent was evaporated under reduced pressure at 35 ° C to precipitate a precipitate.
  • the mixture was filtered and washed three times with pure water and dried at 40 to 45 ° C to obtain Ac-Glu(OtBu)-Glu(OtBu)-Met- Gln(Trt)-Arg(pbf)-Arg(pbf)-OH.
  • step (5) is to dissolve Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH with N,N-dimethylformamide , adding benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate, 1-hydroxyphenylpropane triazole, ammonium chloride, N,N-diisopropylethylamine to obtain a reaction solution, It is treated, separated, purified and dried to obtain Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH2; then to Ac-Glu(OtBu)- Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH2 was added to the cutting solution for cleavage, and then the crude product was isolated, purified
  • the filtrate is added to cold diethyl ether to precipitate a precipitate, and a crude six-peptide is obtained.
  • the crude six-peptide is purified by reverse phase chromatography and freeze-dried to obtain Ac-Glu-Glu-Met- Gln-Arg-Arg-NH2 product.
  • the cutting liquid in the step (4) is a mixed liquid of trifluoroethanol and acetic acid and dichloromethane in a volume ratio of 2:0.75:7.25; preferably, the cutting liquid in the step (5) is a mass percentage composition of trifluoroethylene.
  • the molar ratio of 2-chlorotrityl chloride resin to Fmoc-Arg(pbf)-OH, N,N'-diisopropylethylamine in the step (1) is 1:1:4.
  • step (2) 2-chlorotrityl chloride resin and Fmoc-Arg(pbf)-OH, The molar ratio of 1-hydroxyphenylpropane triazole, benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, N,N'-diisopropylethylamine 1:2: (2.5 to 3): (2.5 to 3): (2.5 to 3).
  • step (2) Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH and benzotriazole-1-fluorophosphate
  • the invention also provides a six-peptide product prepared by the preparation method, which has a purity higher than 95%.
  • the present invention provides a method for synthesizing a solid phase fragment of a six-peptide, comprising the steps of:
  • a method for synthesizing a six-peptide characterized in that it consists of the following steps:
  • Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin was de-Fmoc- twice with a 1:4 volume ratio of piperidine to N,N-dimethylformamide, and N,N-dimethyl was added.
  • Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH were sequentially attached to Fmoc-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Res in according to the method of Step 2.
  • the above cutting liquid is a mixed liquid of trifluoroethanol and acetic acid and dichloromethane in a volume ratio of 2:0.75:7.25;
  • the above cutting liquid is a mixed liquid having a mass percentage composition of trifluoroacetic acid 83%, phenol 5%, thioanisole 4%, water 3%, and triisopropylsilane 5%.
  • step (1) 2, 2-chlorotrityl chloride resin and Fmoc-Arg(pbf)-OH, 1-hydroxyphenylpropanetriazole, benzotriazole-N, N, N', N
  • the molar ratio of '-tetramethyl urea tetrafluoroboric acid and N,N'-diisopropylethylamine is 1:2: 2.5 to 3: 2.5 to 3: 2.5 to 3.
  • the molar ratio of OH is 1:2, 2-chlorotrityl chloride resin and 1-hydroxyphenylpropane triazole, benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, N,
  • the molar ratio of N'-diisopropylethylamine is 1:2.5 to 3:2.5 to 3:2.5 to 3.
  • the method of obtaining Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH is a cutting liquid Wash 3 times with 2 volumes of saturated saline solution, then add an equal volume of saturated saline solution with the cutting solution, adjust the pH to 7.0 with saturated sodium bicarbonate solution, distill off the organic solvent at 35 ° C by rotary evaporator and precipitate the precipitate. The precipitate was washed 3 times with pure water and dried at 40 to 45 °C.
  • step (2) Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH and benzotriazole hexafluorophosphate
  • the method of obtaining Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH2 is to add 2 to the reaction liquid.
  • a volume of saturated saline solution was precipitated and precipitated, filtered, and the precipitate was washed three times with pure water and dried at 40 to 45 °C.
  • the present invention firstly synthesized a fully protected six-peptide fragment Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)- using a relatively inexpensive 2-Chlorotrityl Chloride Resin- OH, then the fully protected six-peptide fragment carboxy terminal with benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate, 1-hydroxyphenylpropane triazole, ammonium chloride, N, N- Amidation of diisopropylethylamine in N,N-dimethylformamide solution gives Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)- NH2, and then deprotection of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-
  • the invention overcomes the Fmoc-AM Resin and Fmoc used in the traditional six-peptide solid phase synthesis method.
  • - MBHA Resin is expensive and the degree of resin substitution is small, which causes the high cost of solid phase synthesis of Liusheng peptide. It also overcomes the complicated synthesis process of Liusheng peptide liquid phase synthesis method, long cycle, low efficiency and high purification cost. The problem is to provide a synthetic method with simple process, low production cost, easy purification and high efficiency for large-scale production of six peptides.
  • the method of the invention overcomes the problems of high cost and low load of the conventional solid phase synthesis hexapeptide resin, and overcomes the disadvantages of complicated process, long synthesis cycle and low efficiency of liquid phase synthesis of hexapeptide, and has low synthesis cost.
  • the invention has the advantages of high synthesis efficiency and simple synthesis method, and can be used for large-scale synthesis of six peptides.
  • Figure 1 is a mass spectrum of the six-peptide synthesized in Example 1.
  • Figure 2 is a liquid chromatogram of the six-peptide synthesized in Example 1, wherein (a) is a liquid chromatogram and (b) is a treatment channel.
  • Pbf, OtBu, and Trt are all protecting groups, and the names are 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, tert-butoxy, and trityl.
  • the invention provides a method for preparing a six-peptide, characterized in that the method comprises the following steps: (1) synthesizing Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin; (2) synthesizing Fmoc-Glu (OtBu) -Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin;(3) Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt )-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin; (4) Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf) -OH; and (5) the product obtained in the step (4) is cleaved, separated and purified to
  • step (1) is to swell 2-Chlorotrityl Chloride Resin with dichloromethane, then add dichloromethane, Fmoc-Arg(pbf)-OH, N,N'-diisopropyl
  • the ethylamine is reacted to obtain Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin.
  • the synthesis method is the result of the creative work of the inventor, and the synthesis scheme of Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin in step (1) can avoid the use of high cost Rink Amide-AM Resin or Rink Amide-MBHA Resin, etc.
  • the disadvantage of amino resin is effective in reducing the cost; the raw material used is 2-chlorotrityl chloride Resin, which is relatively inexpensive and thus reduces the cost of raw materials.
  • the 2-chlorotrityl chloride Resin which is named 2-chlorotrityl chloride resin, has a degree of substitution of 1.74 mmol/g, but other degrees of substitution of 2-chlorotrityl chloride Resin can also be the same or similar.
  • the technical effects are also within the scope of the present invention.
  • the degree of substitution of 2-chlorotrityl chloride Resin can be all the degree of substitution of the existing commercialization, and the degree of substitution used in the present invention is 1.74 mmol/g, which is the best balance of the yield, purity, and resin utilization ratio of the synthesized fragment. value.
  • step (2) is: removing Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin obtained in the step (1) twice, and adding N,N-dimethylformamide, Fmoc-Arg(pbf)- OH, 1-hydroxyphenylpropane triazole, benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, N,N'-diisopropylethylamine are reacted to obtain Fmoc- Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin; then Fmoc-Gln(Trt)-OH is sequentially attached to Fmoc-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin according to the method described.
  • the step of extending the amino acid chain, wherein the protecting group selected by the side chain protection of each amino acid may be any protecting group having an orthogonal protective effect with Fmoc, as long as the desired technical effect can be achieved, but the invention It is preferred that a person prefers the above substituents.
  • the step (3) is the Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin obtained in the step (2).
  • the amino acid and other reagents should be used in an excessive amount, which is generally 1.8-3 times. If the reaction is difficult, the dosage may be higher. -3 times is an economic increase.
  • step (5) is to dissolve Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH with N,N-dimethylformamide , adding benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate, 1-hydroxyphenylpropane triazole, ammonium chloride, N,N-diisopropylethylamine to obtain a reaction solution, It is treated, separated, purified and dried to obtain Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH2; then to Ac-Glu(OtBu)- Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH2 was added to the cutting solution for cleavage, and then the crude product was isolated, purified
  • the cutting liquid in the step (4) is a mixed liquid of trifluoroethanol and acetic acid and dichloromethane in a volume ratio of 2:0.75:7.25; preferably, the cutting liquid in the step (5) is composed of a mass percentage of three A mixed solution of 83% fluoroacetic acid, 5% phenol, 4% thioanisole, 3% water, and 5% triisopropylsilane.
  • the above various cutting steps are different, and the materials to be cut are different, so the selected cutting liquid is different.
  • step (2) 2-chlorotrityl chloride resin and Fmoc-Arg(pbf)-OH, 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-four
  • the molar ratio of methyl urea tetrafluoroboric acid and N,N'-diisopropylethylamine is 1:2: (2.5 to 3): (2.5 to 3): (2.5 to 3).
  • step (2) Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH and benzotriazole-1-fluorophosphate
  • the ratio of the ingredients between the respective substances is controlled within the above range, which is the result of the inventors' consideration of the preparation method of each step than the consideration of a plurality of factors, and the upper and lower 10% floating within the above protection range.
  • the technical effects described can still be achieved.
  • the preferred technical solution is as above.
  • the invention also provides a six-peptide product prepared by the preparation method, which has a purity higher than 95%.
  • the method for synthesizing a hexapeptide comprises the steps of: (1) synthesizing A c-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg (pbf) )-OH
  • 2-Chlorotrityl Chloride Resin was swelled with dichloromethane for 15 minutes and then filtered with suction to a molar ratio of 2-Chlorotrityl Chloride Resin:Fmoc-Arg(pbf)-OH:N,N'-diisopropylethylamine: 1:4, 2-Chlorotrityl Chloride Resin was added to 10 mL of dichloromethane as a solvent, and stirred under nitrogen for 1.5 hours at room temperature to obtain Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Res in.
  • Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin was de-Fmoc- twice with a 1:4 volume ratio of piperidine to N,N-dimethylformamide, according to 2-Chlorotrityl Chloride Resi n, Fmoc-Arg(pbf)-OH, 1-hydroxyphenylpropanetriazole, benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, N,N'-diisopropyl
  • the molar ratio of ethylamine is 1:2:2.5 ⁇ 3:2.5 ⁇ 3:2.5 ⁇ 3, and each gram of 2-Chlorotr ityl Chloride Resin is added with 10mL of N,N-dimethylformamide as solvent, under the protection of nitrogen, stirring at room temperature 2 In hours, Fmoc-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chlor
  • step (1) 2 to Fmoc-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chlorid
  • Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH are sequentially connected to obtain Fmoc-Glu(OtBu)-Glu(OtBu).
  • the reaction solution was prepared, and 10 mL of the reaction solution was added per gram of 2-Chlorotrityl Chloride Resin, and stirred under nitrogen for 30 minutes at room temperature to obtain Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(p bf )-Arg(pbf)-2-Chlorotrityl Chloride Resin.
  • the cutting solution was prepared according to the volume ratio of trifluoroethanol, acetic acid and dichloromethane of 2:0.75:7.25 (v:v:v), per gram of peptide resin Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt )-Arg(pbf)-Arg(pbf)-2-Chlor otrityl Chloride Resin was added to 10 mL of the cutting solution, stirred at room temperature for 1.5 hours, filtered, and the filtrate was washed 3 times with 2 volumes of saturated saline, and then added to the cutting solution.
  • the degree of substitution of the 2-chlorotrityl chloride resin of the present invention is 1.74 mmol/g, which is commercially available from Tianjin Nankai Hecheng Technology Co., Ltd.
  • the solid phase peptide synthesis reactor is a general-purpose peptide synthesis reactor and can be purchased from CS936X CSBio Peptide Synthesizer of Chengdu Snow Import & Export Co., Ltd.
  • the analytical high performance liquid chromatograph is Hitachi Fully Automatic L2000.
  • the preparative high performance liquid chromatograph is the innovative Hengtong LC3000, the C18 analytical column is 4.6mm ⁇ 250mm of Dalian Institute of Physical Chemistry, and the C18 preparative column is Chengdu Science Biotechnology Co., Ltd. 40.1mm ⁇ 450mm.
  • the LTQ-XL electrospray ionization mass spectrometer is Thermo Finnigan Corporation of the United States.
  • the various organic solvents and organic reagents used in the experiments were domestic AR or CP.
  • the resin was washed twice, 500 mL each time, Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin was removed from Fmoc- twice, and 500 mL of N,N-dimethylformamide and 112.89 g of Fmoc-Arg were added to the synthesizer.
  • the above molar ratios of Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH and 2-chlorotrityl chloride resin are both 2:1, 2 -chlorotrit yl chloride resin with 1-hydroxyphenylpropane triazole, benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboron
  • the molar ratio of acid to N,N'-diisopropylethylamine is 1:2.5 to 3:2.5 to 3:2.5 to 3.
  • reaction was carried out for 20 minutes, suction filtration, and the resin was washed twice with isopropyl alcohol and N,N-dimethylformamide twice, 500 mL each time to complete Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)- Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin was removed from F moc- twice, and 450 mL of dichloromethane, 50 mL of acetic anhydride, 25 mL of N,N'-diisopropylethylamine was added to the synthesizer under nitrogen.
  • the structure of the synthesized product was characterized by mass spectrometer. The results are shown in Fig. 1. It can be seen from the figure that the molecular weight and molecular ion peak of the synthesized product are consistent with the molecular weight and molecular ion peak of the six peptides, indicating that the synthesized product is a six-peptide.
  • the purity of the synthesized six peptides was determined by liquid chromatography. The liquid chromatogram is shown in Fig. 2(a). In combination with Fig. 2(b), the purity of the six peptides is greater than 95%.
  • step (1) of the present embodiment Fmoc-Arg(pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH
  • 2-Chlorotrityl Chloride Resin 2-Chlorotrityl Chloride Resin and 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethylurea IV
  • the molar ratio of fluoroboric acid to N,N'-diisopropylethylamine was 1:2.5:2.5:2.5, and the other steps of this step were the same as in the embodiment (1).
  • step (2) of the present embodiment Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH, benzotriazole hexafluorophosphate-1 - the molar ratio of -oxy-tripyrrolidinylphosphine, 1-hydroxyphenylpropane triazole, ammonium chloride, N,N'-diisopropylethylamine is 1:3:3:3:3, the step of The other steps are the same as in the embodiment (1).
  • Step (3) of the present example was the same as in Example (1), and 62.51 g of hexapeptide was obtained in a yield of 80.71%.
  • the structure of the synthesized product was characterized by mass spectrometry using the same conditions as in Example 1.
  • the molecular weight and molecular ion peak of the synthesized product were consistent with the molecular weight and molecular ion peak of the six peptide.
  • the purity of the synthesized six peptides was determined by liquid chromatography with a purity greater than 95%.
  • the step (1) of the present embodiment is the same as the embodiment (1).
  • step (2) of the present embodiment Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH, benzohexafluorophosphate
  • the molar ratio of triazol-1-yl-oxytripyrrolidinylphosphine, 1-hydroxyphenylpropane triazole, ammonium chloride, N,N'-diisopropylethylamine is 1:3:3:4:4
  • the other steps of this step are the same as in the embodiment (1).
  • Step (3) of the present example was the same as in Example (1) to obtain 62.78 g of hexapeptide, and the yield was 81.06%.
  • the structure of the synthesized product was characterized by mass spectrometry using the same conditions as in Example 1.
  • the molecular weight and molecular ion peak of the synthesized product were consistent with the molecular weight and molecular ion peak of the six peptide.
  • the purity of the synthesized six peptides was determined by liquid chromatography with a purity greater than 95%.

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Abstract

L'invention concerne un procédé de préparation d'un hexapeptide, ledit procédé comprenant les étapes suivantes : (1) synthèse d'une résine de chlorure de Fmoc-Arg(pbf)-2-Chlorotrityle ; (2) synthèse d'une résine de chlorure de Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityle ; (3) synthèse d'une résine de chlorure d'Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityle ; (4) synthèse d'Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH ; et (5) coupe, séparation, amidation et purification du produit obtenu à l'étape (4) pour obtenir un produit hexapeptidique. Le procédé de la présente invention résout les problèmes du coût élevé et de la faible capacité rencontrés avec les techniques classiques existantes de synthèse à l'état solide de résines hexapeptidiques, et corrige également les inconvénients de la synthèse en phase liquide d'hexapeptides ayant un processus compliqué, la période de synthèse étant longue, et l'efficacité étant faible ; l'invention présente les avantages d'une synthèse à faible coût et très efficace qui implique un procédé simple, et peut être utilisée pour synthétiser des hexapeptides à grande échelle.
PCT/CN2017/075125 2017-01-05 2017-02-28 Procédé de préparation d'un hexapeptide et produit correspondant WO2018126525A1 (fr)

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