阅读说明：本技术 一种液相合成乙酰基六肽-8的方法 (Method for liquid-phase synthesis of acetyl hexapeptide-8 ) 是由 殷世清 翟涛 刘焕赠 杜传强 于 2021-11-14 设计创作，主要内容包括：本发明涉及多肽合成领域,特别涉及一种液相合成乙酰基六肽-8的方法,本发明首先采用液相合成三个二肽单体：Ac-Glu(O-tBu)-Glu(O-tBu)-OH、H-Met-Gln(Trt)–OH和H-Arg(Pbf)-Arg(Pbf)-NH-(2),然后再顺序合成四肽和六肽的策略合成乙酰基六肽-8。本发明方案扩大了合成规模,减少了有机溶剂的使用,降低了乙酰基六肽-8的生产成本,有利于乙酰基六肽-8在化妆品行业的推广应用。(The invention relates to the field of polypeptide synthesis, in particular to a method for synthesizing acetyl hexapeptide-8 in a liquid phase, which firstly adopts the liquid phase to synthesize three dipeptide monomers: Ac-Glu (O-tBu) -Glu (O-tBu) -OH, H-Met-Gln (Trt) -OH and H-Arg (Pbf) -NH 2 And then acetyl hexapeptide-8 was synthesized following the strategy of sequential synthesis of tetrapeptides and hexapeptides. The scheme of the invention enlarges the synthesis scale, reduces the use of organic solvents, reduces the production cost of the acetyl hexapeptide-8, and is beneficial to the popularization and application of the acetyl hexapeptide-8 in the cosmetic industry.)

1. A method for liquid phase synthesis of acetyl hexapeptide-8 is characterized by comprising the following steps:

(a) Ac-Glu (O-tBu) -OSu and H-Glu (O-tBu) -OH are used as raw materials to synthesize Ac-Glu (O-tBu) -Glu (O-tBu) -OH under alkaline condition, and then the Ac-Glu (O-tBu) -Glu (O-tBu) -OSu is prepared;

(b) adopting Z-Met-OSu and H-Gln (Trt) -OH as raw materials, synthesizing Z-Met-Gln (Trt) -OH under an alkaline condition, and then removing a protecting group Z to obtain H-Met-Gln (Trt) -OH;

(c) using Z-Arg (Pbf) -OH and H-Arg (Pbf) -NH₂Reacting in the presence of a coupling agent to obtain Z-Arg (Pbf) -NH₂Then removing the protecting group Z to obtain H-Arg (Pbf) -NH₂;

(d) Synthesizing Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -OH by adopting Ac-Glu (O-tBu) -Glu (O-tBu) -OSu and H-Met-Gln (Trt) -OH under an alkaline condition;

(e) Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -OH and H-Arg (Pbf) -NH₂Reacting in the presence of a coupling agent to obtain Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -Arg (Pbf) -NH₂；

(f)Ac-Glu(O-tBu)-Glu(O-tBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-NH₂Removing side chain protecting groups, purifying and freeze-drying to obtain the acetyl hexapeptide-8 refined peptide.

2. The method for synthesizing acetyl hexapeptide-8 in liquid phase according to claim 1, wherein the specific operation steps for preparing Ac-Glu (O-tBu) -Glu (O-tBu) -OH in step (a) are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Glu (O-tBu) -OH in a prepared alkali solution at a low temperature bath, wherein the solution is still alkaline after being dissolved; adding an Ac-Glu (O-tBu) -OSu/solvent C solution into the reaction solution at low temperature, heating and continuously stirring for reaction after dropwise adding, and obtaining dipeptide Ac-Glu (O-tBu) -Glu (O-tBu) -OH through concentration, acid regulation, crystallization and recrystallization.

3. The method for synthesizing acetyl hexapeptide-8 in liquid phase according to claim 1, wherein the specific operation steps for preparing Ac-Glu (O-tBu) -Glu (O-tBu) -OSu in step (a) are as follows: dissolving Ac-Glu (O-tBu) -Glu (O-tBu) -OH and HOSu in a solvent C, adding a DCC/solvent C solution under low-temperature stirring, monitoring the end point by TLC, and obtaining Ac-Glu (O-tBu) -Glu (O-tBu) -OSu through filtration, concentration, crystallization and recrystallization.

4. The method for liquid-phase synthesis of acetyl hexapeptide-8 according to claim 1, wherein the specific operation steps for preparing Z-Met-Gln (Trt) -OH in step (b) are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Gln (Trt) -OH in a prepared alkali solution at a low temperature bath, wherein the solution is still alkaline after being dissolved; adding a Z-Met-OSu/solvent C solution into the reaction solution at low temperature, heating after the dropwise addition is finished, continuously stirring for reaction, concentrating, adjusting acid, crystallizing and recrystallizing to obtain the dipeptide Z-Met-Gln (Trt) -OH.

5. The liquid-phase synthesis method of acetyl hexapeptide-8 according to claim 1, wherein in step (c), Z-Arg (Pbf) -NH is prepared₂The specific operation steps are as follows: reacting H-Arg (Pbf) -NH₂Dissolving in a solvent C, adding a mixed solution of Z-Arg (Pbf) -OH/coupling agent/solvent C in a low-temperature bath, heating after the dropwise addition is finished, continuously stirring for reaction, and monitoring the reaction end point by TLC; concentrating, crystallizing and recrystallizing to obtain dipeptide Z-Arg (Pbf) -NH₂。

6. The method for liquid-phase synthesis of acetyl hexapeptide-8 according to claim 1, wherein the specific operation steps for preparing Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -OH in step (d) are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Met-Gln (Trt) -OH in a prepared alkali solution in a low-temperature bath, wherein the solution is still alkaline after being dissolved; adding an Ac-Glu (O-tBu) -Glu (O-tBu) -OSu/solvent C solution into the reaction solution at low temperature, heating after dropwise adding, continuously stirring for reaction, concentrating, adjusting acid, crystallizing and recrystallizing to obtain the tetrapeptide Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -OH.

7. The method for liquid-phase synthesis of acetyl hexapeptide-8 according to claim 1, wherein in step (e), Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -Arg (Pbf) -NH-C₂The specific operation steps are as follows: reacting H-Arg (Pbf) -NH₂Dissolving in solvent C, adding Ac-Glu under low temperature bathAfter the mixed solution of (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -OH/coupling agent/solvent C is added dropwise, the temperature is raised, the reaction is continued to be stirred, and the TLC monitors the end point of the reaction; concentrating, crystallizing and recrystallizing to obtain Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -Arg (Pbf) -NH₂。

8. The method for liquid-phase synthesis of acetyl hexapeptide-8 according to claim 1, wherein in step (f), the reagent for removing the side chain protecting group is a TFA solution added with 1-5% by volume of a scavenger, and the scavenger is one or more of anisole, thioanisole, phenol, water and TIS; the more preferable ratio of the deprotection reagent is as follows: TFA/thioanisole/water/TIS = 87/5/3/5.

9. The method for liquid-phase synthesis of acetyl hexapeptide-8 according to claim 1, wherein the deprotection reagent for protecting group Z in steps (b) and (c) is H₂10% palladium-carbon catalyst or formic acid/10% palladium-carbon catalyst.

10. The liquid phase synthesis method of acetyl hexapeptide-8 according to any one of claims 2-9, wherein the alkali solution prepared by dissolving the alkali A in the solvent B is ammonia, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate aqueous solution or mixed solution of water-miscible organic solvent; or organic solution of organic base such as triethylamine, diethylamine, N-diisopropylethylamine, etc.;

the solvent C is one or more of tetrahydrofuran, dioxane, N-dimethylformamide and acetone;

the coupling agent is one of the following combinations DIC/HOBT, DIC/HOAT, TBTU/HOBT/DIPEA, HBTU/HOBT/DIPEA and HATU/HOAT/DIPEA.

Technical Field

The invention relates to the field of polypeptide synthesis, and particularly relates to a preparation method of acetyl hexapeptide-8.

Technical Field

Acetyl hexapeptide-8, otherwise known as ayorelin, the english name argiline. Acetyl hexapeptide-8 is involved in competing for the site of SNAP-25 at the vacuolar complex, thereby affecting complex formation; when the vesicular complex is slightly unstable, the vesicles cannot effectively release neurotransmitters, thereby causing a decrease in muscle contraction and preventing the formation of wrinkles. Therefore, the composition can inhibit the release of the nerve conduction element acetylcholine and weaken the contraction of muscles, thereby reducing dynamic line expression lines, reducing wrinkle generation, increasing the activity of elastin, maintaining the epidermal renewal capacity to make the skin smooth and compact, challenging the reversal muscle age and showing the youthful luster of the skin.

Acetyl hexapeptide-8 is a biomimetic peptide, an oligopeptide that mimics the N-terminus of SNAP-25 protein and consists of six amino acids. Its polypeptide structure is derived from a fragment of human body SNAP-25 (synaptosome associated protein) end, and some chemical modifications are carried out, and its specific sequence is: Ac-Glu-Glu-Met-Gln-Arg-Arg-NH₂CAS number: 616204-22-9, formula: c₃₄H₆₀N₁₄O₁₂S, molecular weight: 888.91. the structure contains six amino acid residues, and is a short peptide product obtained by chemical synthesis.

Patent CN103694316B discloses a solid-phase preparation method of ayorelin, which adopts amino resin as a solid-phase carrier and utilizes Fmoc chemical strategy to prepare and obtain ayorelin (acetyl hexapeptide-8), the synthesis of the method is limited by the solid-phase synthesis scale, the large-scale production is difficult to realize, and the popularization and application of acetyl hexapeptide-8 in the cosmetic industry are not facilitated.

The acetyl hexapeptide-8 is used as the active ingredient with the efficacy of the anti-wrinkle star with the widest application at present, the market demand is increased year by year, the existing solid phase synthesis process needs to consume a large amount of organic solvent, so that the production cost is high, and greater environmental protection pressure is brought to production enterprises, so that the development of the preparation process suitable for large-scale production of the acetyl hexapeptide-8 is particularly important. The technical scheme of the application adopts a liquid phase method to synthesize the acetyl hexapeptide-8, thereby enlarging the synthesis scale, reducing the use of organic solvents, reducing the production cost of the acetyl hexapeptide-8 and being beneficial to the popularization and application of the acetyl hexapeptide-8 in the cosmetic industry.

Disclosure of Invention

In order to solve the problems in the synthesis process, the invention provides a method for synthesizing acetyl hexapeptide-8 in a liquid phase, and the technical scheme provided by the invention is as follows:

a method for liquid phase synthesis of acetyl hexapeptide-8 is characterized by comprising the following steps:

(a) Ac-Glu (O-tBu) -OSu and H-Glu (O-tBu) -OH are used as raw materials to synthesize Ac-Glu (O-tBu) -Glu (O-tBu) -OH under alkaline condition, and then the Ac-Glu (O-tBu) -Glu (O-tBu) -OSu is prepared;

(b) adopting Z-Met-OSu and H-Gln (Trt) -OH as raw materials, synthesizing Z-Met-Gln (Trt) -OH under an alkaline condition, and then removing a protecting group Z to obtain H-Met-Gln (Trt) -OH;

(c) using Z-Arg (Pbf) -OH and H-Arg (Pbf) -NH₂Reacting in the presence of a coupling agent to obtain Z-Arg (Pbf) -NH₂Then removing the protecting group Z to obtain H-Arg (Pbf) -NH₂;

(d) Synthesizing Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -OH by adopting Ac-Glu (O-tBu) -Glu (O-tBu) -OSu and H-Met-Gln (Trt) -OH under an alkaline condition;

(e) Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -OH and H-Arg (Pbf) -NH₂Reacting in the presence of a coupling agent to obtain Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -Arg (Pbf) -NH₂；

(f)Ac-Glu(O-tBu)-Glu(O-tBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-NH₂Removing side chain protecting groups, purifying and freeze-drying to obtain the acetyl hexapeptide-8 refined peptide.

Preferably, in the step (a), the specific operation steps for preparing Ac-Glu (O-tBu) -Glu (O-tBu) -OH are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Glu (O-tBu) -OH in a prepared alkali solution at a low temperature bath, wherein the solution is still alkaline after being dissolved; adding an Ac-Glu (O-tBu) -OSu/solvent C solution into the reaction solution at low temperature, heating and continuously stirring for reaction after dropwise adding, and obtaining dipeptide Ac-Glu (O-tBu) -Glu (O-tBu) -OH through concentration, acid regulation, crystallization and recrystallization.

Preferably, in the step (a), the specific operation steps for preparing Ac-Glu (O-tBu) -Glu (O-tBu) -OSu are as follows:

dissolving Ac-Glu (O-tBu) -Glu (O-tBu) -OH and HOSu in a solvent C, adding a DCC/solvent C solution under low-temperature stirring, monitoring the end point by TLC, and obtaining Ac-Glu (O-tBu) -Glu (O-tBu) -OSu through filtration, concentration, crystallization and recrystallization.

Preferably, in step (b), the specific operation steps for preparing Z-Met-Gln (Trt) -OH are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Gln (Trt) -OH in a prepared alkali solution at a low temperature bath, wherein the solution is still alkaline after being dissolved; adding a Z-Met-OSu/solvent C solution into the reaction solution at low temperature, heating after the dropwise addition is finished, continuously stirring for reaction, concentrating, adjusting acid, crystallizing and recrystallizing to obtain the dipeptide Z-Met-Gln (Trt) -OH.

Preferably, in step (c), Z-Arg (Pbf) -NH is prepared₂The specific operation steps are as follows: reacting H-Arg (Pbf) -NH₂Dissolving in a solvent C, adding a mixed solution of Z-Arg (Pbf) -OH/coupling agent/solvent C in a low-temperature bath, heating after the dropwise addition is finished, continuously stirring for reaction, and monitoring the reaction end point by TLC; concentrating, crystallizing and recrystallizing to obtain dipeptide Z-Arg (Pbf) -NH₂。

Preferably, in the step (d), the specific operation steps for preparing Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -OH are as follows: dissolving alkali A in a solvent B to prepare an alkaline solution; dissolving H-Met-Gln (Trt) -OH in a prepared alkali solution in a low-temperature bath, wherein the solution is still alkaline after being dissolved; adding an Ac-Glu (O-tBu) -Glu (O-tBu) -OSu/solvent C solution into the reaction solution at low temperature, heating after dropwise adding, continuously stirring for reaction, concentrating, adjusting acid, crystallizing and recrystallizing to obtain the tetrapeptide Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -OH.

Preferably, in step (e), Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -Arg (Pbf) -NH is prepared₂The specific operation steps are as follows: reacting H-Arg (Pbf) -NH₂Dissolving in a solvent C, adding a mixed solution of Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -OH/a coupling agent/a solvent C under low-temperature bath, heating after the dropwise addition is finished, continuously stirring for reaction, and monitoring the reaction end point by TLC; concentrating, crystallizing and recrystallizing to obtain Ac-Glu (O-tBu) -Glu (O-tBu) -Met-Gln (Trt) -Arg (Pbf) -NH₂。

Preferably, in the step (f), the reagent for removing the side chain protecting group is a TFA solution added with 1-5% by volume of a scavenging agent, wherein the scavenging agent is one or more of anisole, dimethyl sulfide, phenol, water and TIS; the more preferable proportion of the cracking reagent is as follows: TFA/thioanisole/water/TIS = 87/5/3/5.

Preferably, in step (b) and step (c), the deprotecting reagent for the protecting group Z is H₂10% palladium-carbon catalyst or formic acid/10% palladium-carbon catalyst.

Preferably, in the above step of the present invention, the alkali a is dissolved in the solvent B to prepare an alkaline solution, which may be an aqueous solution of ammonia, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, or a mixed solution of organic solvents miscible with water; meanwhile, the organic base can also be organic solution of organic base such as triethylamine, diethylamine, N-diisopropylethylamine and the like.

The solvent C can be one or more than one of tetrahydrofuran, dioxane, N-dimethylformamide and acetone; the coupling agent is one of the following combinations DIC/HOBT, DIC/HOAT, TBTU/HOBT/DIPEA, HBTU/HOBT/DIPEA and HATU/HOAT/DIPEA.

Compared with the prior art, the invention has the beneficial effects that: the technical scheme of the application adopts a liquid phase 2+2+2 strategy to synthesize the acetyl hexapeptide-8, so that the synthesis scale is enlarged, the use of organic solvents is reduced, the production cost of the acetyl hexapeptide-8 is reduced, and the popularization and the application of the acetyl hexapeptide-8 in the cosmetic industry are facilitated.

Detailed Description

The present invention will be described in detail with reference to the following specific examples, which are not intended to limit the scope of the present invention; it is within the scope of the present invention to vary the raw material feed ratio, the reaction solvent, the condensing agent, etc. according to the present invention.

Abbreviations used in the specification and claims have the following meanings:

tBu: a tertiary butyl group;

pbf: 2,2,4,6, 7-pentamethylbenzofuran-5-sulfonyl;

trt: a trityl group;

DCM: dichloromethane;

DMF: n, N-dimethylformamide;

DIPEA: n, N-diisopropylethylamine;

DIC: n, N-diisopropylcarbodiimide;

HBTU: benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate;

HATU: 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate;

TBTU: O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate;

HOBT: 1-hydroxybenzotriazole;

HOAT: 1-hydroxy-7-azobenzotriazol;

TFA: trifluoroacetic acid;

and (3) TIS: triisopropylsilane;

su: a succinimide group.

Example 1: synthesis of Ac-Glu (OtBu) -OH

Accurately weighing H-Glu (O-tBu) -OH4.06Kg (20mol) in a 100L reaction kettle, adding 25L of 10% sodium carbonate aqueous solution, and dissolving under stirring; after the solution is dissolved, dropwise adding Ac-Glu (OtBu) -OSu 6.84kg (20mol)/25L tetrahydrofuran solution at low temperature, stirring for reaction, and monitoring the end point by TLC; after decompression concentration, acid adjustment, ethyl acetate extraction, crystallization, recrystallization and drying, dipeptide monomer Ac-Glu (OtBu) -OH7.02kg with the purity of 99.1 percent is obtained, and the yield is 81.6 percent.

Example 2: synthesis of Ac-Glu (OtBu) -OSu

Accurately weighing 7.00kg (16.28mol) of Ac-Glu (OtBu) -OH and 7.00kg (20mol) of HOSu2.30kg (20mol) in a 100L reaction kettle, adding 25L of tetrahydrofuran, and dissolving under stirring; after the solution is dissolved clearly, dropping DCC 4.66kg (20mol)/25L tetrahydrofuran solution at low temperature, stirring for reaction, and monitoring the end point by TLC; filtering, concentrating under reduced pressure, crystallizing, recrystallizing, and dissolving in 25L tetrahydrofuran.

Example 3: synthesis of Z-Met-Gln (Trt) -OH

Accurately weighing H-Gln (Trt) -OH7.77Kg (20mol) in a 100L reaction kettle, adding 25L of 10% sodium carbonate aqueous solution, and dissolving under stirring; after the solution is dissolved clearly, adding 7.61kg (20mol) of Z-Met-OSu/25L tetrahydrofuran solution dropwise at low temperature, stirring for reaction, and monitoring the end point by TLC; vacuum concentrating, adjusting acid, extracting with ethyl acetate, crystallizing, recrystallizing, and drying to obtain dipeptide monomer Z-Met-Gln (Trt) -OH11.05kg with purity of 99.2%, and yield of 82.2%.

Example 4: synthesis of H-Met-Gln (Trt) -OH

Accurately weighing Z-Met-Gln (Trt) -OH11.00Kg (16.4mol) into a 100L reaction kettle, adding 30L of methanol for dissolution, adding 1.10kg of 10% palladium-carbon catalyst/10L of methanol, then adding 6.56kg of formic acid/10L of methanol, stirring for reaction, and monitoring the end point by TLC; filtering, concentrating, crystallizing, recrystallizing, and drying to obtain dipeptide monomer H-Met-Gln (Trt) -OH8.21kg with purity of 99.2%, and yield of 96.1%.

Example 5: Z-Arg (Pbf) -NH₂Synthesis of (2)

Accurately weighing H-Arg (Pbf) -NH₂9.50Kg (20mol) of the mixture was put into a 100L reactor, and 25L of tetrahydrofuran was added and dissolved under stirring; after the mixture is dissolved clearly, adding Z-Arg (Pbf) -OH-CHA13.20kg (20mol), DIC 2.28kg (20mol), HOBT2.70kg (20mol) and 25L tetrahydrofuran solution dropwise at low temperature, stirring for reaction, and monitoring the end point by TLC; vacuum concentrating, adjusting acid, extracting with ethyl acetate, crystallizing, recrystallizing, and drying to obtain dipeptide monomer Z-Arg (Pbf) -NH with purity of 99.2%₂15.71kg, yield 81.2%.

Example 6: H-Arg (Pbf) -NH₂Synthesis of (2)

Accurately weighing Z-Arg (Pbf) -NH₂15.70kg (16.23mol) of the crude product was put into a 100L reactor, dissolved in 30L of methanol, added with 1.60kg of 10% palladium-carbon catalyst/10L of methanol, then added with 6.56kg of formic acid/10L of methanol, stirred for reaction, and monitored by TLC for endpoint; filtering, concentrating, crystallizing, recrystallizing and drying to obtain dipeptide monomer H-Arg (Pbf) -NH with the purity of 99.2 percent₂13.06kg, yield 96.4%.

Example 7: synthesis of Ac-Glu (OtBu) -Met-Gln (Trt) -OH

Accurately weighing H-Met-Gln (Trt) -OH8.20Kg (15.76mol) in a 100L reaction kettle, adding 25L of 10% sodium carbonate aqueous solution, and dissolving under stirring; after the solution is dissolved, dropwise adding the solution of Ac-Glu (OtBu) -OSu tetrahydrofuran obtained in the example 2 at a low temperature, stirring for reaction, and monitoring the end point by TLC; the tetrapeptide Ac-Glu (OtBu) -Met-Gln (Trt) -OH11.78kg with the purity of 98.1 percent is obtained by decompression concentration, acid adjustment, ethyl acetate extraction, crystallization, recrystallization and drying, and the yield is 80.2 percent.

Example 8: Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg(Pbf)-NH₂Synthesis of (2)

Accurately weighing H-Arg (Pbf) -NH₂Adding 13.05Kg (15.64mol) of tetrahydrofuran into a 100L reaction kettle, and dissolving under stirring; after the solution is clear, dropwise adding at low temperature:

（1）Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-OH 11.77kg (12.64mol)，

（2）DIC1.71kg(15mol)，

（3）HOBT2.03kg(15mol)，

(4) 25L of tetrahydrofuran solution, stirring for reaction, and monitoring the end point by TLC; concentrating under reduced pressure, adjusting acid, crystallizing, recrystallizing and drying to obtain hexapeptide Ac-Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -NH with purity of 95.1%₂17.80kg, yield 80.6%.

Example 9: Ac-Glu-Glu-Met-Gln-Arg-Arg-NH₂Preparation of

17.80kg of hexapeptide obtained in example 8:

Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-NH₂adding into frozen 50L lysate (volume ratio is TFA/thioanisole/TIS/H)₂0= 87/5/3/5), the reaction was stirred at room temperature for 4 h; after the reaction is finished, adding 500L of frozen methyl tert-ether into the feed liquid to separate out white precipitate; standing for 30min, filtering, washing with methyl tert-ether for 6 times, and vacuum drying to obtain crude peptide 11.05kg, with a crude peptide yield of 98.6% and a purity of 93.6%.

Example 10: preparation of acetyl hexapeptide-8 refined peptide

The crude peptide obtained in example 9 was dissolved, filtered through a 0.45 μm microfiltration membrane, purified by column preparation using C18 with an inner diameter of 200mm, desalted and lyophilized to obtain 9.15kg of acetyl hexapeptide-8 refined peptide with a purity of 99.91% and a yield of 82.8%.