WO2018126525A1

WO2018126525A1 - Method for preparing hexapeptide, and product thereof

Info

Publication number: WO2018126525A1
Application number: PCT/CN2017/075125
Authority: WO
Inventors: 张忠旗; 李乾; 王万科; 王慧; 郭添; 苏子梦; 韩广; 高长波; 赵金礼
Original assignee: 陕西慧康生物科技有限责任公司
Priority date: 2017-01-05
Filing date: 2017-02-28
Publication date: 2018-07-12
Also published as: CN106749529A; AU2017101705A4

Abstract

Provided is a method for preparing a hexapeptide, said method comprising 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) synthesizing Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin; (4) synthesizing Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH; and (5) cutting, separating, amidating, and purifying the product obtained from step (4) to obtain a hexapeptide product. The method remedies the problems of high cost and low capacity in existing and classic solid-state synthesis of hexapeptide resins, and also resolves the shortcomings of liquid-phase synthesis of hexapeptides having a complicated process, the period of synthesis being long, and efficiency being low; the invention has the advantages of low-cost and highly efficient synthesis that involves a simple method, and may be used for synthesizing hexapeptides on a large scale.

Description

Preparation method of six-peptide and product thereof

Technical field

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.

Background technique

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. .

At present, 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. Although 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. As a raw material for cosmetics, Liusheng peptide has important significance in reducing production cost, improving production efficiency and simplifying the research of synthesis technology.

Therefore, the development of a high-efficiency and low-cost method for synthesizing hexapeptide has become an urgent technical problem to be solved in the art.

Summary of the invention

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.

Specifically, 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)-NH2, then Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-A Rg(pbf)-Arg(pbf)-NH2 removes the protecting group to give a six-peptide.

More specifically, the present invention is achieved by the following technical solutions.

A method for preparing a six-peptide, the method comprising the steps of:

(1) Synthesis of Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin;

(2) Synthesis of 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;

(5) The product obtained in the step (4) is cleaved, separated and purified to obtain a six-peptide product.

Wherein the 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. -Arg(pbf)-2-Chlorotrityl Chloride Resin;

Wherein the 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. , Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, to obtain Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)- Arg(pbf)-2-Chlorotrityl Chloride Resin.

Wherein the 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.

Wherein the 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.

Wherein 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 and dried to obtain a product.

After the step (5) is cut, 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.

Wherein 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. A mixed solution of 83% acetic acid, 5% phenol, 4% thioanisole, 3% water, and 5% triisopropylsilane.

The molar ratio of 2-chlorotrityl chloride resin to Fmoc-Arg(pbf)-OH, N,N'-diisopropylethylamine in the step (1) is 1:1:4.

In the 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).

In the step (2), Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH and benzotriazole-1-fluorophosphate The molar ratio of keto-oxytripyrrolidinylphosphine, 1-hydroxyphenylpropane triazole, ammonium chloride, N,N'-diisopropylethylamine 1:3:3:(3 to 5): (3 ~5).

The invention also provides a six-peptide product prepared by the preparation method, which has a purity higher than 95%.

More specifically, 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:

(1) Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH

1 Synthesis of Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin

After 2-Chlorotrityl Chloride Resin was swelled with dichloromethane for 15 minutes, dichloromethane, Fmoc-Arg(pbf)-OH, N,N'-diisopropylethylamine was added, and the mixture was stirred at room temperature for 1.5 hours under a nitrogen atmosphere. , obtaining Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin;

2 Synthesis of Fmoc-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin

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. Formamide, Fmoc-Arg(pbf)-OH, 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, N,N'-di Isopropylethylamine, under nitrogen atmosphere, stirring at room temperature for 2 hours to obtain Fmoc-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin;

3 Synthesis of Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chl orotrityl Chloride Resin

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. , Fm oc-Glu(OtBu)-OH, to obtain Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Ar g(pbf)-2-Chlorotrityl Chloride Resin;

4 Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chloro trityl Chloride Resin

Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotri tyl Chloride Resin with piperidine and N,N-dimethylformamide The mixture with a volume ratio of 1:4 was de-F moc- twice, and a mixture of dichloromethane, acetic anhydride, N,N'-diisopropylethylamine in a volume ratio of 9:1:0.5 was added under nitrogen protection. , stirring at room temperature for 30 minutes, to obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-2-Chlorotrityl Chloride Resin;

5 cutting treatment

Adding a cutting solution to Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-ChlorotritylChloride Resin, stirring at room temperature for 1.5 hours, filtering, and saturating the filtrate Wash the saline 3 times, then add an equal volume of saturated saline solution with the cutting solution, adjust the pH to 7.0 with a saturated sodium hydrogencarbonate solution, evaporate the organic solvent at 35 ° C by rotary evaporator and precipitate the precipitate, filter, and wash the precipitate with pure water. 3 times, drying at 40 ~ 45 ° C to obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (p bf)-Arg (pbf)-OH;

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;

(2) Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH2

Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH was dissolved in N,N-dimethylformamide, and benzotrifluorophosphate was added. Zin-1-yl-oxytripyrrolidinylphosphine, 1-hydroxyphenylpropane triazole, ammonium chloride, N,N'-diisopropylethylamine, stirred for 3 hours, and added to the reaction solution in 2 volumes Saturated brine and precipitated, filtered, and washed three times with pure water, and dried at 40-45 ° C to obtain Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg (pbf)-NH2;

(3) Synthesis of six peptides Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2

Adding a cutting solution to Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH2 per gram of Ac-Glu(OtBu)-Glu(OtBu)- Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH2 was incubated with 3 to 5 mL of the cutting solution, stirred at room temperature for 1.5 to 2.5 hours, filtered, and the filtrate was added to cold diethyl ether to precipitate a precipitate to obtain a crude six-peptide. The crude six peptides were purified by reverse phase chromatography and lyophilized to obtain Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2;

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%.

Wherein, in the step (1) of the above, 2-chlorotrityl chloride resin and The molar ratio of Fmoc-Arg(pbf)-OH, N,N'-diisopropylethylamine was 1:1:4.

Wherein, in the 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.

Wherein, in the step 3 of the step (1), 2-chlorotrityl chloride resin and Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)- 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.

Wherein, in the step 5 of the step (1), 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.

Wherein, in the step (2), Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH and benzotriazole hexafluorophosphate The molar ratio of -1-yl-oxytripyrrolidinylphosphine, 1-hydroxyphenylpropane triazole, ammonium chloride, N,N'-diisopropylethylamine 1:3:3:3 to 5:3 ~5.

Wherein, in the step (2), 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 beneficial effects of the present invention are as follows:

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)-Arg(pbf)-NH2 to obtain the six-peptide Ac-Glu-Glu-Met-Gln -Arg-Arg-NH ₂ .

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.

DRAWINGS

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.

detailed description

The present invention will be further described in detail below with reference to the drawings and embodiments, but the invention is not limited thereto.

The following short description of the description:

2-chlorotrityl chloride Resin, the name is 2-chlorotrityl chloride resin

Fmoc: 9-fluorene methyleneoxycarbonyl

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 obtain a six-peptide product. Wherein the 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.

Wherein the 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. , Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, to obtain Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)- Arg(pbf)-2-Chlorotrityl Chloride Resin. 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.

Wherein, 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). De-Fmoc- twice, a mixture of dichloromethane, acetic anhydride, N,N'-diisopropylethylamine was added to obtain Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)- Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin; wherein 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 40 minutes - 2 hours; said step (1), step (2) And removing Fmoc in the step (3) by using a mixture of piperidine and N,N-dimethylformamide in a volume ratio of 1:4 to remove Fmoc; preferably adding methylene chloride, acetic anhydride, N in the step (4) a mixture of N'-diisopropylethylamine in a volume ratio of 9:1:0.5; wherein the step (4) is: Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg (pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin is added with a cutting solution for the cleavage reaction. After separation, purification and drying, Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf) is obtained. - Arg(pbf)-OH; wherein 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 saturated saline, and then added to the same volume as the cutting liquid. Saturated brine, adjusted to pH 7.0 with saturated sodium bicarbonate solution, evaporating and removing organic solvent under reduced pressure at 35 ° C, and precipitating the precipitate, filtering, washing the precipitate three times with pure water, drying at 40-45 ° C to obtain Ac-Glu (OtBu) )-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH. The ratio between the respective substances in the preparation process can be appropriately adjusted, and the obtained product can still satisfy the requirements. Therefore, it is also possible to appropriately adjust the ratio between the respective substances in the preparation process and the values of the parameters of the reaction conditions. Within the scope of protection. In the general solid phase peptide synthesis, if the resin is taken as the benchmark, then 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.

Wherein, 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.

Wherein the molar ratio of 2-chlorotrityl chloride resin to Fmoc-Arg(pbf)-OH, N,N'-diisopropylethylamine in the step (1) is 1:1:4.

In the 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). In the step (2), Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH and benzotriazole-1-fluorophosphate The molar ratio of keto-oxytripyrrolidinylphosphine, 1-hydroxyphenylpropane triazole, ammonium chloride, N,N'-diisopropylethylamine 1:3:3:(3 to 5): (3 ~5).

In the preparation step, 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.

In a specific embodiment, 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

1 Synthesis of Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin

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.

2 Synthesis of Fmoc-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin

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 Chloride Resin was obtained.

According to the method in step (1), 2 to Fmoc-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chlorid On the e Resin, 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). -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)-2-Chlor otrityl Chloride Resin

Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotr ityl Chloride Resin with piperidine and N,N-dimethylformamide The mixture with a volume ratio of 1:4 was de-F moc- twice, and the volume ratio of dichloromethane, acetic anhydride, N,N'-diisopropylethylamine was 9:1:0.5 (v:v:v). 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.

5 cutting treatment

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. An equal volume of saturated brine was adjusted to pH 7.0 with saturated sodium bicarbonate solution, and the organic solvent was evaporated on a rotary evaporator at 35 ° C to precipitate a precipitate. The mixture was filtered and washed three times with pure water and dried at 40-45 ° C to obtain Ac- Glu(OtBu)-Glu(Ot Bu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH.

(2) Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH2

Each gram of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH was dissolved in 10 mL of N,N-dimethylformamide according to Ac-Glu ( OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(p bf)-Arg(pbf)-OH, benzotriazol-1-yl-oxytripyrrolidinylphosphonium, 1- The molar ratio of hydroxyphenylpropane triazole, ammonium chloride and N,N'-diisopropylethylamine is 1:3:3:3 to 5:3 to 5, and benzotriazol-1-yl hexafluorophosphate is added. Oxytripyrrolidinylphosphine, 1-hydroxyphenylpropanetriazole, ammonium chloride, N,N'-diisopropylethylamine, stirred for 3 hours, adding 2 volumes of saturated brine to the reaction solution and precipitating The precipitate was precipitated, filtered, and the precipitate was 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)-NH2.

(3) Synthesis of six peptides Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2

According to the ratio of trifluoroacetic acid, phenol, thioanisole, water and triisopropylsilane, 83mL: 5g: 4mL: 3mL: 5mL to prepare the cutting solution, per gram of Ac-Glu(OtBu)-Glu(OtBu)-Met -Gln(Trt)-Arg(p bf)-Arg(pbf)-NH2 was added to 3 to 5 mL of the cutting solution, stirred at room temperature for 1.5 to 2.5 hours, filtered, and the filtrate was added to 5-8 times of cold anhydrous ether to precipitate a precipitate. After filtration, the precipitate was washed three times with anhydrous diethyl ether and dried in vacuo to give a crude crude product of hexapeptide. The crude hexapeptide was purified by reverse phase chromatography and lyophilized to give Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2.

Example

The sources of the respective materials used in the examples are described below. Unless otherwise specified, the materials and instruments used are commercially available, and are conventionally used in the art as long as they meet the experimental needs.

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 various amino acids used and 1-hydroxyphenylpropane triazole, benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, N,N'-diisopropylethylamine were purchased from Shanghai Jill Biochemical Co., Ltd.

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.

Example 1

The preparation method of the six peptides of the present embodiment comprises the following steps:

(1) Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH

1 Synthesis of Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin

50.0 g of 2-Chlorotrityl Chloride Resin was added to the synthesizer, soaked in 500 mL of dichloromethane, swelled for 15 minutes, and then suction filtered, and 500 mL of dichloromethane, 56.45 g of Fmoc-Arg(pbf)-OH, 61.0 mL of N was added to the synthesizer. N'-diisopropylethylamine, 2-Chlorotrityl Chloride Re sin, Fmoc-Arg(pbf)-OH, N,N'-diisopropylethylamine in a molar ratio of 1:1:4, protected by nitrogen The mixture was stirred at room temperature for 1.5 hours, suction filtered, and the resin was washed twice with isopropyl alcohol and N,N-dimethylformamide twice, 500 mL each time to obtain Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin.

2 Synthesis of Fmoc-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin

To the Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin in the synthesizer, a mixture of 500 ml of piperidine and N,N-dimethylformamide in a volume ratio of 1:4 was added for 10 minutes, and filtered by suction. The mixture was mixed with 500 mL of a mixture of piperidine and N,N-dimethylformamide in a volume ratio of 1:4, reacted for 20 minutes, and filtered with suction, using isopropanol and N,N-dimethylformamide. 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. Pbf)-OH, 35.26g 1-hydroxyphenylpropane triazole, 83.81g benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, 45.6mL N,N'-diiso Propylethylamine, under nitrogen atmosphere, stirring at room temperature for 2 hours, suction filtration, washing the resin twice with isopropanol and N,N-dimethylformamide, 500 mL each time, and suction filtration to obtain Fmoc-Arg (p Bf)-Arg(pbf)-2-Chlorotrityl Chloride Resin.

According to the method of 2 in the step (1), 106.26 g of Fmoc-Gln(Trt)-OH and 64.64 g of Fmoc-M were sequentially connected to Fmoc-Arg(pbf)-Arg(pbf)-2-Chlor otrityl Chloride Resin in the synthesizer. Et-OH, 74.04g Fmoc-Glu(OtBu)-OH, 74.04g Fmoc-Glu(OtBu)-OH, other reagents are: 1-hydroxyphenylpropane triazole 35.26g, benzotriazole-N,N, 83.81 g of N',N'-tetramethyluronium tetrafluoroborate and 45.6 mL of N,N'-diisopropylethylamine. The resin was washed twice with isopropyl alcohol and N,N-dimethylformamide. After 500 mL, Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin was obtained.

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.

Add 500 mL of piperidine and N,N-di to Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin in the synthesizer The mixture of methylformamide in a volume ratio of 1:4 was reacted for 10 minutes, and the mixture was removed by suction filtration, and then a mixture of 500 mL of piperidine and N,N-dimethylformamide in a volume ratio of 1:4 was added. The 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. Under the protection, stirring at room temperature for 30 minutes, suction filtration, washing the resin twice with isopropyl alcohol and N,N-dimethylformamide, 500 mL each time, suction filtration, methanol washing the resin 3 times, each time 500 mL, suction filtration, Drying gave Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin.

5 cutting treatment

Add 500 mL of the cutting solution to Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-ChlorotritylChloride Resin. The cutting solution used was trifluoroethanol, acetic acid, A mixture of dichloromethane volume ratio of 2:0.75:7.25 (v:v:v) was stirred at room temperature for 1.5 hours, filtered, and the filtrate was transferred to a 1000 mL separatory funnel, washed three times with 1000 mL of saturated brine, and the filtrate was further filtered. Transfer into a 2000 mL eggplant bottle, add 500 mL of saturated brine, adjust the P H to 7.0 with saturated sodium bicarbonate solution, evaporate the organic solvent at 35 ° C by rotary evaporator and precipitate the precipitate, and filter with a No. 4 sand core funnel. The water was washed 3 times and dried at 40 to 45 ° C to obtain 120.16 g of Ac-Glu(OtBu)-Glu(OtBu)-Me t-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH, yield 78.94%. .

(2) Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH ₂

Add 120.16g (68.68mmol) Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-A rg(pbf)-OH to a 5L beaker and add 1200mL N,N-dimethyl Carboxamide, 107.22g benzotriazol-1-yl-oxytripyrrolidinylphosphonium phosphate, 27.84g 1-hydroxyphenylpropane triazole, 18.37g ammonium chloride, 59.8mL N,N'-diisopropyl Ethylamine, stirring reaction for 3 hours, adding 2400 mL of saturated brine to the reaction solution and precipitating the precipitate, suction filtration with a No. 4 sand core funnel, the precipitate was washed 3 times with pure water, 40-45 After drying at ° C, 113.11 g of Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-N H2 was obtained in a yield of 94.13%.

(3) Synthesis of six peptides Ac-Glu-Glu-Met-Gln-Arg-Arg-NH ₂

-NH ₂ was added to 300mL 113.11gAc-Glu (OtBu) -Glu ( OtBu) -Met-Gln (Trt) -Arg (pbf) -Arg (pbf) cutting fluid cutting fluid used is trifluoroacetic acid, phenol, The ratio of thioanisole, water and triisopropylsilane was 83 mL: 5 g: 4 mL: 3 mL: 5 mL of the mixed solution, stirred at room temperature for 2 hours, suction filtered with a No. 3 sand core funnel, and the filtrate was added to 2400 mL of cold anhydrous diethyl ether. The precipitate was precipitated and filtered with a No. 4 sand core funnel. The precipitate was washed three times with anhydrous diethyl ether and dried in vacuo to give crude hexapeptide. The crude hexapeptide was purified by reverse-phase chromatography and lyophilized to give 63.35 g of hexapeptide. The yield was 81.79%.

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%.

Example 2

In the 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 The molar ratio to 2-Chlorotrityl Chloride Resin is 2:1, 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). In the 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%.

Example 3

The step (1) of the present embodiment is the same as the embodiment (1). In the 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%.

Claims

A method for preparing a six-peptide, characterized in that the method comprises the following steps:

(1) Synthesis of Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin;

(2) Synthesis of 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;

(5) The product obtained in the step (4) is cleaved, separated and purified to obtain a six-peptide product.
The method for preparing a six-peptide according to claim 1, wherein the step (1) is: after swelling 2-Chlorotrityl Chloride Resin with dichloromethane, adding dichloromethane, Fmoc-Arg(pbf)-OH, N, Reaction of N'-diisopropylethylamine gives Fmoc-Arg(pbf)-2-Chlorotrityl Chloride Resin.
The method for preparing a six-peptide according to claim 1 or 2, wherein the 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-hydroxyphenylpropanetriazole, benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, N , N'-diisopropylethylamine is reacted to obtain Fmoc-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin; then Fmoc-Arg(pbf)-Arg(pbf)-2 is used according to the method described. -Chlorotrityl Chloride Resin is sequentially linked to Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH to obtain Fmoc-Glu(OtBu)-Glu ( OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin.
The method for producing a six-peptide according to any one of claims 1 to 3, wherein the step (3) is Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln (Trt) obtained in the step (2). )-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin de-Fmoc- twice, adding dichloromethane, acetic anhydride, N, N'- A mixture of diisopropylethylamine is reacted 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) according to any one of claims 2 to 4 is carried out under a nitrogen atmosphere at a normal temperature for 0.5 to 2 hours; the step (1), In the step (2) and the step (3), the Fmoc is obtained by de-Fmoc using a mixture of piperidine and N,N-dimethylformamide in a volume ratio of 1:4; preferably, dichloromethane is added in the step (4). A mixture of acetic anhydride, N,N'-diisopropylethylamine in a volume ratio of 9:1:0.5.
The method for producing a six-peptide according to any one of claims 1 to 5, wherein the step (4) is: Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf) -Arg(pbf)-2-Chlorotrityl Chloride Resin is added to the cleavage solution for cleavage reaction, separated, purified and dried to obtain Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg (pbf)-OH.
The method for preparing a six-peptide according to claim 6, wherein 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 saturated saline, and then added and cut. An equal volume of saturated saline solution was adjusted to pH 7.0 with a saturated sodium hydrogen carbonate solution, and the organic solvent was evaporated under reduced pressure at 35 ° C. The precipitate was precipitated, filtered, and the precipitate was 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.
A method for producing a six-peptide according to any one of claims 1 to 7, wherein said step (5) is: Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf) -Arg(pbf)-OH is dissolved in N,N-dimethylformamide, benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 1-hydroxyphenylpropane triazole, chlorination The reaction solution is obtained by reacting ammonium, N,N-diisopropylethylamine, and is treated, separated, purified and dried to obtain Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg (pbf) )-Arg(pbf)-NH2; then a cutting solution is added to Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH2 for cleavage, followed by separation The crude product was obtained, purified and dried to give the product.
The method for preparing a six-peptide according to claim 8, wherein the step (5) is followed by cleavage, the filtrate is added to cold diethyl ether, and the precipitate is precipitated to obtain a crude six-peptide, and the crude six-peptide is purified by reverse phase chromatography. And lyophilized to obtain Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 product.
The method for preparing a six-peptide according to any one of claims 6-8, wherein the cutting liquid in the step (4) is a mixture of trifluoroethanol and acetic acid and dichloromethane in a volume ratio of 2:0.75:7.25. Liquid Preferably, the cutting liquid in the step (5) is a mixed liquid having a mass percentage composition of trifluoroacetic acid 83%, phenol 5%, thioanisole 4%, water 3%, and triisopropylsilane 5%.
The method for producing a six-peptide according to any one of claims 2 to 10, wherein in the step (1), 2-chlorotrityl chloride resin and Fmoc-Arg(pbf)-OH, N,N'-diisopropylethylamine The molar ratio is 1:1:4.
The method for synthesizing a hexapeptide according to any one of claims 2 to 11, wherein in the step (2), 2-chlorotrityl chloride resin and Fmoc-Arg(pbf)-OH, 1-hydroxyl The molar ratio of benzotriazole, benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, N,N'-diisopropylethylamine 1:2: (2.5~ 3): (2.5 to 3): (2.5 to 3).
The method for synthesizing a hexapeptide according to claim 1, wherein in the step (2), Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf) -Arg(pbf)-OH with benzotriazol-1-yl-oxytripyrrolidinylphosphonium phosphate, 1-hydroxyphenylpropane triazole, ammonium chloride, N,N'-diisopropyl B The molar ratio of the amine is 1:3:3: (3 to 5): (3 to 5).
The hexapeptide product prepared by the preparation method according to any one of claims 1 to 13, which has a purity higher than 95%.