Solid-phase synthesis method of acetyl hexapeptide-8

Abstract

The invention discloses a solid-phase synthesis method of acetyl hexapeptide-8, which comprises the following steps: by taking RINK AMIDE-AM resin as a carrier, gradually coupling Fmoc-Arg (HCl)-OH, Fmoc-Arg (HCl)-OH, Fmoc-Gln-OH, Fmoc-Met-OH, Fmoc-Glu (OtBu)-OH and Fmoc-Glu (OtBu)-OH amino acids, carrying out deprotection, acetylation and cleavage reaction to prepare crude peptide, and finally purifying to obtain a target product. According to the method, a minimum amino acid protection strategy is adopted, a cracking system is optimized, a modified silica gel chromatographic filler is combined, and a modifier comprises N-(benzo [D] thiazole-2-yl methyl) acrylamide and methyl 2-acrylamido-2-methoxy acetate, so that the column chromatography purification efficiency is improved, the product purity is high, the yield is high, the operation is simple, and the method is suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the field of polypeptide synthesis, and particularly relates to a solid-phase synthesis method of acetyl hexapeptide-8. Background Art

[0002] Acetyl Hexapeptide-8 is a high-quality wrinkle-removing ingredient that can locally block the nerves that transmit muscle contraction signals, affect the nerve conduction of the skin follicle, relax the facial muscles, and smooth out dynamic lines, static lines, and fine lines; it effectively reorganizes collagen elasticity, increases the activity of elastin, relaxes facial lines, smoothes wrinkles, and improves sagging. As an anti-wrinkle ingredient, it is extremely effective with high anti-wrinkle activity and few side effects, and is used in various high-end cosmetics series.

[0003] Currently, the synthesis methods of acetyl hexapeptide-8 include the solid-phase one-by-one coupling method, which uses a method of coupling amino acids with protected side chains one by one to obtain a peptide resin intermediate. The product purity will be low and the raw materials used are expensive; the synthesis method using solid and liquid phase methods is complicated and not suitable for large-scale production; the liquid phase synthesis method uses unprotected arginine and glutamine, has more reaction steps, requires a longer production cycle, and has high requirements for purification technology.

[0004] The present invention addresses the problems of expensive raw materials, complex operations that are unsuitable for large-scale production, numerous reaction steps, and a long production cycle, and proposes a solid-phase synthesis method for acetyl hexapeptide-8. By adopting a minimal amino acid protection strategy and an optimized purification process, the method reduces raw material costs, reduces impurities, simplifies the operation, improves the total yield of acetyl hexapeptide-8, and is suitable for scale-up production. Summary of the Invention

[0005] The object of the present invention is to provide a solid phase synthesis method for acetyl hexapeptide-8 with high yield and purity.

[0006] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are: A solid phase synthesis method for acetyl hexapeptide-8, comprising: The RINK AMIDE-AM resin is coupled with amino acids in sequence, deprotected in a deprotection solution, and acetylated in an acetylation solution to obtain a peptide resin; The peptide resin is placed in a cleavage solution for cleavage, and then placed in a precipitant for precipitation to obtain a crude acetyl hexapeptide-8 peptide; The crude acetyl hexapeptide-8 peptide was purified by column chromatography and lyophilized to obtain the refined acetyl hexapeptide-8; The coupling order of the amino acids is: Fmoc-Arg(HCl)-OH, Fmoc-Arg(HCl)-OH, Fmoc-Gln-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Glu(OtBu)-OH.

[0007] Preferably, the degree of substitution of the RINK AMIDE-AM resin is 0.6-1.2 mmol / g.

[0008] Preferably, the deprotection solution comprises piperidine and N,N-dimethylformamide.

[0009] Preferably, the volume ratio of piperidine to N,N-dimethylformamide is 10-30:80.

[0010] Preferably, the acetylation solution comprises acetic anhydride and pyridine.

[0011] Preferably, the volume ratio of acetic anhydride to pyridine is 15-45:30.

[0012] Preferably, the lysis solution comprises one or more of trifluoroacetic acid, triisopropylsilane, thioanisole and 1,2-ethanedithiol.

[0013] Preferably, the precipitant comprises one or more of methyl tert-butyl ether, diethyl ether and petroleum ether.

[0014] Preferably, the chromatographic column of the column chromatography comprises a C18 chromatographic column, or a modified chromatographic column, and the modified chromatographic column comprises a modified silica gel chromatographic filler.

[0015] Preferably, the modified silica gel chromatographic filler comprises mercapto-functionalized siloxane microspheres grafted with a modifier.

[0016] Preferably, the modifying agent includes N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetate.

[0017] Preferably, the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to mercapto-functionalized silicone microspheres is 0.5-4:20.

[0018] Preferably, the mass ratio of methyl 2-acrylamido-2-methoxyacetate to mercapto-functionalized silicone microspheres is 0.5-4:20.

[0019] N-(Benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetate improve the packing performance in modified silica gel chromatographic packing through a synergistic polymerization reaction, forming a porous network on the silica gel surface, increasing the specific surface area, and enhancing the steric hindrance of the polymer coating; after the two are jointly grafted onto the surface of thiol-functionalized silicone microspheres, a composite coating with balanced hydrophilicity and hydrophobicity and ion exchange capacity is formed. The porous structure increases the adsorption sites and the polarity regulation improves the separation selectivity of acetyl hexapeptide-8 and impurities. At the same time, the packing structure stability is enhanced, thereby significantly improving the purification efficiency and chromatographic column alkali resistance.

[0020] More preferably, the modifier also includes tert-butyl (2-acrylamidoethyl)carbamate, with the mass ratio of (2-acrylamidoethyl)carbamate to thiol-functionalized silicone microspheres being 0.5-4:20. The tert-butyl (2-acrylamidoethyl)carbamate and other modifier monomers together form a three-dimensional network polymer coating on the silica gel surface, optimizing the coating's pore size distribution and specific surface area. This coating reduces direct erosion by alkaline solutions through steric repulsion. Furthermore, its hydrophobicity modifies the polarity distribution of the filler surface, reducing nonspecific adsorption of the target peptide to the filler, thereby achieving efficient impurity separation during purification.

[0021] The present invention also provides a method for preparing a peptide resin, comprising: a. Dissolve Pip in DMF to obtain a deprotection solution.

[0022] Preferably, the volume ratio of Pip to DMF is 10-30:80.

[0023] b. Use DMF as washing solution.

[0024] c. Add RINK AMIDE-AM resin to the reactor, add DMF, swell with nitrogen for 30 minutes, add deprotection solution for 30 minutes, wash with washing solution 5-7 times, and drain for later use.

[0025] Preferably, the degree of substitution of the RINK AMIDE-AM resin is 0.6-1.2 mmol / g, the molar weight of the RINK AMIDE-AM resin is 1 eq, and the mass of the RINK AMIDE-AM resin is W g.

[0026] Preferably, the amount of DMF used is 2-10W mL / g.

[0027] Preferably, the amount of the deprotection solution used is 2-10 W mL / g.

[0028] Preferably, the amount of washing liquid used is 2-10W mL / g.

[0029] d. Dissolve Fmoc-Arg(HCl)-OH and HOBt in DMF, precool to 0-10°C, and activate with DIC for 5-15 minutes. Then, add the mixture to the reactor and react for 2-3 hours. Remove the reaction solution and wash with detergent 2-4 times. Deprotect the mixture with deprotection solution for 25-35 minutes, wash with detergent 5-7 times, drain the solution, and proceed to the next step.

[0030] Preferably, the amount of Fmoc-Arg(HCl)-OH used is 1.5-4 eq.

[0031] Preferably, the amount of HOBt used is 1.5-4 eq.

[0032] Preferably, the amount of DIC used is 1.5-4 eq.

[0033] Preferably, the amount of DMF used is 2-10W mL / g.

[0034] Preferably, the amount of the deprotection solution used is 2-10 W mL / g.

[0035] Preferably, the amount of washing liquid used is 2-10W mL / g.

[0036] e. Dissolve Fmoc-Arg(HCl)-OH and HOBt in DMF, precool to 0-10°C, add DIC for activation for 5-15 minutes, add to the reactor, react for 2-3 hours, remove the reaction solution, wash with detergent 2-4 times, add deprotection solution for 25-35 minutes, wash with detergent 5-7 times, drain the liquid, and proceed to the next step.

[0037] Preferably, the amount of Fmoc-Arg(HCl)-OH used is 1.5-4 eq.

[0038] Preferably, the amount of HOBt used is 1.5-4 eq.

[0039] Preferably, the amount of DIC used is 1.5-4 eq.

[0040] Preferably, the amount of DMF used is 2-10W mL / g.

[0041] Preferably, the amount of the deprotection solution used is 2-10 W mL / g.

[0042] Preferably, the amount of washing liquid used is 2-10W mL / g.

[0043] f. Dissolve Fmoc-Gln-OH and HOBt in DMF, precool to 0-10°C, add DIC for activation for 5-15 minutes, add to the reactor, react for 2-3 hours, remove the reaction solution, wash with detergent 2-4 times, add deprotection solution for 25-35 minutes, wash with detergent 5-7 times, drain the liquid, and proceed to the next step.

[0044] Preferably, the amount of Fmoc-Gln-OH used is 1.5-4 eq.

[0045] Preferably, the amount of HOBt used is 1.5-4 eq.

[0046] Preferably, the amount of DIC used is 1.5-4 eq.

[0047] Preferably, the amount of DMF used is 2-10W mL / g.

[0048] Preferably, the amount of the deprotection solution used is 2-10 W mL / g.

[0049] Preferably, the amount of washing liquid used is 2-10W mL / g.

[0050] g. Dissolve Fmoc-Met-OH and HOBt in DMF, precool to 0-10°C, add DIC for activation for 5-15 minutes, add to the reactor, react for 2-3 hours, remove the reaction solution, wash with detergent 2-4 times, add deprotection solution for 25-35 minutes, wash with detergent 5-7 times, drain the liquid, and proceed to the next step.

[0051] Preferably, the amount of Fmoc-Met-OH used is 1.5-4 eq.

[0052] Preferably, the amount of HOBt used is 1.5-4 eq.

[0053] Preferably, the amount of DIC used is 1.5-4 eq.

[0054] Preferably, the amount of DMF used is 2-10W mL / g.

[0055] Preferably, the amount of the deprotection solution used is 2-10 W mL / g.

[0056] Preferably, the amount of washing liquid used is 2-10W mL / g.

[0057] h. Dissolve Fmoc-Glu(OtBu)-OH and HOBt in DMF, precool to 0-10°C, add DIC for activation for 5-15 minutes, add to the reactor, react for 2-3 hours, remove the reaction solution, wash with detergent 2-4 times, add deprotection solution for 25-35 minutes, wash with detergent 5-7 times, drain the liquid, and proceed to the next step.

[0058] Preferably, the amount of Fmoc-Glu(OtBu)-OH used is 1.5-4 eq.

[0059] Preferably, the amount of HOBt used is 1.5-4 eq.

[0060] Preferably, the amount of DIC used is 1.5-4 eq.

[0061] Preferably, the amount of DMF used is 2-10W mL / g.

[0062] Preferably, the amount of the deprotection solution used is 2-10 W mL / g.

[0063] Preferably, the amount of washing liquid used is 2-10W mL / g.

[0064] i. Dissolve Fmoc-Glu(OtBu)-OH and HOBt in DMF, pre-cool to 0-10°C, add DIC for activation for 5-15 minutes, add to the reactor, react for 2-3 hours, remove the reaction solution, wash with detergent 2-4 times, add deprotection solution for 25-35 minutes, wash with detergent 5-7 times, drain the liquid, and proceed to the next step.

[0065] Preferably, the amount of Fmoc-Glu(OtBu)-OH used is 1.5-4 eq.

[0066] Preferably, the amount of HOBt used is 1.5-4 eq.

[0067] Preferably, the amount of DIC used is 1.5-4 eq.

[0068] Preferably, the amount of DMF used is 2-10W mL / g.

[0069] Preferably, the amount of the deprotection solution used is 2-10 W mL / g.

[0070] Preferably, the amount of washing liquid used is 2-10W mL / g.

[0071] j. Add Ac2O and pyridine to the reactor and acetylate for 0.5-1.5 h. Wash with DMF 3-5 times, wash with DCM 2-4 times, add methanol for shrinkage, and vacuum dry to obtain a peptide resin.

[0072] Preferably, the amount of Ac2O used is 20eq.

[0073] Preferably, the amount of pyridine used is 20eq.

[0074] The present invention also provides a method for preparing crude acetyl hexapeptide-8 peptide, comprising: TFA was dissolved in deionized water and pre-cooled to -15-25°C to obtain a cleavage solution, peptide resin was added, and the reaction was carried out at 20-30°C for 1-3 hours. The reaction was filtered and the filtrate was added to a precipitant pre-cooled to -15-25°C. The precipitated solid was collected and washed three times with a washing solution. The solid was dried at a constant temperature in a vacuum at 15-25°C for 20-25 hours to obtain a crude acetyl hexapeptide-8 peptide.

[0075] Preferably, the volume ratio of TFA to deionized water is 85-105:5.

[0076] More preferably, the lysis solution further comprises TIS.

[0077] Preferably, the volume ratio of TIS to deionized water is 2-10:5.

[0078] Preferably, the mass volume ratio of peptide resin to TFA is 5-15 g:95 mL.

[0079] Preferably, the precipitant comprises one or more of methyl tert-butyl ether or diethyl ether.

[0080] Preferably, the mass volume ratio of the peptide resin to the precipitant is 5-15 g:100 mL.

[0081] Preferably, the washing liquid is one or more of methyl tert-butyl ether or diethyl ether.

[0082] Preferably, the volume-to-mass ratio of the washing liquid to the peptide resin is 100 mL:5-15 g.

[0083] The present invention also provides a method for preparing mercapto-functionalized silicone microspheres, comprising: Preparation of mercapto-functionalized silicone microspheres: Methyltrimethoxysilane is dispersed in deionized water, stirred at 18-20°C and 70-900 rpm for 20-40 minutes, ammonia water is added, stirred for 1-3 minutes, 3-mercaptopropyltrimethoxysilane is added, and the reaction is allowed to stand for 5-6 hours. The mixture is centrifuged at 9000-11000 rpm for 10-20 minutes, the precipitate is washed with ethanol, and vacuum dried at 75-85°C for 10-15 hours to obtain mercapto-functionalized silicone microspheres.

[0084] Preferably, the mass volume ratio of methyltrimethoxysilane to deionized water is 10-30 g:100 mL.

[0085] Preferably, the volume ratio of ammonia water to deionized water is 5-15 μL:100 mL.

[0086] Preferably, the mass ratio of 3-mercaptopropyltrimethoxysilane to methyltrimethoxysilane is 2-10:20.

[0087] The present invention also provides a method for preparing a modified silica gel chromatographic filler, comprising: Preparation of modified silica gel chromatographic filler: Disperse thiol-functionalized siloxane microspheres in toluene, ultrasonicate for 15-25 minutes, add a modifier, add azobisisobutyronitrile, pass nitrogen for 25-35 minutes, react at 65-75°C and 700-900 rpm with stirring for 8-12 hours, cool to room temperature, centrifuge at 9000-11000 rpm for 10-20 minutes, wash the precipitate with ethanol, and dry under vacuum at 45-55°C for 10-15 hours to obtain modified silica gel chromatographic filler.

[0088] Preferably, the mass volume ratio of mercapto-functionalized silicone microspheres to toluene is 10-30 g:300 mL.

[0089] Preferably, the modifier comprises N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetate Preferably, the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to mercapto-functionalized silicone microspheres is 0.5-4:20.

[0090] Preferably, the mass ratio of methyl 2-acrylamido-2-methoxyacetate to mercapto-functionalized silicone microspheres is 0.5-4:20.

[0091] Preferably, the mass ratio of azobisisobutyronitrile to mercapto-functionalized silicone microspheres is 100-300 mg:20 g.

[0092] More preferably, the modifier further comprises tert-butyl (2-acrylamidoethyl)carbamate.

[0093] More preferably, the mass ratio of tert-butyl (2-acrylamidoethyl)carbamate to mercapto-functionalized silicone microspheres is 0.5-4:20.

[0094] The present invention also provides a method for preparing a modified chromatographic column, comprising: Preparation of the modified chromatographic column: Methanol and isopropanol are mixed to obtain a displacement liquid; the modified silica gel chromatographic filler is dispersed in the isopropanol, ultrasonicated for 4-6 minutes, and filled into the chromatographic column tube with the displacement liquid to obtain the modified chromatographic column.

[0095] Preferably, in the displacement fluid, the volume ratio of methanol to isopropanol is 25-75:50.

[0096] Preferably, the mass volume ratio of the modified silica gel chromatographic filler to isopropanol is 2-6 g:60 mL.

[0097] Preferably, the volume of the chromatography column is 2-6 mm×150-300 mm.

[0098] The present invention also provides a method for preparing acetyl hexapeptide-8, comprising: Preparation of acetyl hexapeptide-8: The crude acetyl hexapeptide-8 peptide was purified by RP-HPLC, and acetyl hexapeptide-8 was obtained by elution and collection.

[0099] Preferably, the purification chromatographic column is a C18 chromatographic column, or a modified chromatographic column.

[0100] Preferably, mobile phase A is a 0.05-0.15 wt % TFA aqueous solution, and mobile phase B is a 0.05-0.15 wt % TFA acetonitrile solution.

[0101] Preferably, the volume ratio of mobile phase A to mobile phase B is 70-90:10-30.

[0102] Preferably, the flow rate is 0.8-1.2 mL / min.

[0103] Preferably, the injection volume is 5-15 μL.

[0104] The present invention adopts a minimal amino acid protection strategy and combines it with a modified silica gel chromatography column purification process, thereby having the following beneficial effects: reducing raw material costs and impurity generation, improving coupling and deprotection efficiency, and increasing the purity and yield of acetyl hexapeptide-8, making the preparation process simple and easy to scale up. Therefore, the present invention is a solid-phase synthesis method for acetyl hexapeptide-8 with high yield and purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 Schematic diagram of the solid phase synthesis steps of acetyl hexapeptide-8.

[0106] Figure 2 Schematic diagram of the specific surface area test results of modified silica gel chromatographic packing.

[0107] Figure 3 This is a schematic diagram of the purity test results of acetyl hexapeptide-8.

[0108] Figure 4 Schematic diagram of the yield test results of acetyl hexapeptide-8. DETAILED DESCRIPTION

[0109] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0110] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0111] The Chinese meanings of the abbreviations used in the present invention are shown in Table 1 below.

[0112] Table 1 Chinese meanings of abbreviations

[0113] Example 1: Preparation of peptide resin: a. 20 mL of Pip was dissolved in 80 mL of DMF to obtain a deprotection solution; b. DMF as washing solution; c. 25.01 g of RINK AMIDE-AM resin was added to the reactor, 75 mL of DMF was added, nitrogen was swelled for 30 min, 75 mL of deprotection solution was added for 30 min, and the mixture was washed 6 times with 75 mL of washing solution and dried for later use; the degree of substitution of RINKAMIDE-AM resin was 0.8 mmol / g; d. Dissolve 19.62 g of Fmoc-Arg(HCl)-OH and 5.41 g of HOBt in DMF, precool to 0°C, add 6.25 mL of DIC for activation for 10 minutes, add to the reactor, react for 2.5 hours, remove the reaction solution, wash three times with 75 mL of washing solution, add 75 mL of deprotection solution, deprotect for 30 minutes, wash six times with 75 mL of washing solution, drain the solution, and proceed to the next step; e. Dissolve 19.62 g of Fmoc-Arg(HCl)-OH and 5.41 g of HOBt in DMF, precool to 0°C, add 6.25 mL of DIC for activation for 10 minutes, add to the reactor, react for 2.5 hours, remove the reaction solution, wash three times with 75 mL of washing solution, add 75 mL of deprotection solution for 30 minutes, wash six times with 75 mL of washing solution, drain the liquid, and proceed to the next step; f. Dissolve 15.54 g of Fmoc-Gln-OH and 5.41 g of HOBt in DMF, precool to 0°C, add 6.25 mL of DIC for activation for 10 minutes, add to the reactor, react for 2.5 hours, remove the reaction solution, wash three times with 75 mL of washing solution, add 75 mL of deprotection solution for 30 minutes, wash six times with 75 mL of washing solution, drain the liquid, and proceed to the next step of the reaction; g. Dissolve 16.06g of Fmoc-Met-OH and 5.41g of HOBt in DMF, precool to 0°C, add 6.25mL of DIC for activation for 10 minutes, add to the reactor, react for 2.5 hours, remove the reaction solution, wash three times with 75mL of washing solution, add 75mL of deprotection solution for 30 minutes, wash six times with 75mL of washing solution, drain the liquid, and proceed to the next step; h. Dissolve 18.06 g of Fmoc-Glu(OtBu)-OH and 5.41 g of HOBt in DMF, precool to 0°C, add 6.25 mL of DIC for activation for 10 minutes, add to the reactor, react for 2.5 hours, remove the reaction solution, wash three times with 75 mL of washing solution, add 75 mL of deprotection solution for 30 minutes, wash six times with 75 mL of washing solution, drain the liquid, and proceed to the next step; i. Dissolve 18.06 g of Fmoc-Glu(OtBu)-OH and 5.41 g of HOBt in DMF, precool to 0°C, add 6.25 mL of DIC for activation for 10 min, add to the reactor, react for 2.5 h, remove the reaction solution, wash three times with 75 mL of washing solution, add 75 mL of deprotection solution for 30 min, wash six times with 75 mL of washing solution, drain the liquid, and proceed to the next step of the reaction; j. Add 37.57 mL of Ac2O and 31.19 mL of pyridine to the reactor and acetylate for 1 h. Wash with DMF 4 times and DCM 3 times. Add methanol for shrinkage and vacuum dry to obtain a peptide resin.

[0114] Preparation of crude acetyl hexapeptide-8 peptide: 95 mL of TFA was dissolved in 5 mL of deionized water, precooled to minus 20°C to obtain a lysate, 10 g of peptide resin was added, and the mixture was reacted at 25°C for 2 h. After filtering, the filtrate was added to 100 mL of methyl tert-butyl ether precooled to minus 20°C, the precipitated solid was collected, and the solid was washed three times with 100 mL of methyl tert-butyl ether. The solid was dried at a vacuum constant temperature at 25°C for 24 h to obtain crude acetyl hexapeptide-8 peptide.

[0115] Preparation of acetyl hexapeptide-8: The crude acetyl hexapeptide-8 peptide was purified by RP-HPLC using a 4.6 mm × 250 mm C18 reverse phase chromatographic column. The mobile phase A was a 0.1 wt% TFA aqueous solution, the mobile phase B was a 0.1 wt% TFA acetonitrile solution, the volume ratio of mobile phase A to mobile phase B was 80:20, the flow rate was 1 mL / min, the injection volume was 10 μL, and acetyl hexapeptide-8 was eluted and collected.

[0116] Example 2: This example is different from Example 1 only in that the substitution degree of RINK AMIDE-AM resin in the preparation of the peptide resin is 1.0 mmol / g.

[0117] Example 3: Compared with Example 2, this example differs only in the preparation of crude acetyl hexapeptide-8 peptide.

[0118] Preparation of crude acetyl hexapeptide-8 peptide: 95 mL of TFA and 2.5 mL of TIS were dissolved in 2.5 mL of deionized water, pre-cooled to minus 20°C to obtain a lysate, 10 g of peptide resin was added, and the mixture was reacted at 25°C for 2 h. After filtering, the filtrate was added to 100 mL of methyl tert-butyl ether pre-cooled to minus 20°C, the precipitated solid was collected, and the solid was washed three times with 100 mL of methyl tert-butyl ether, and dried at a vacuum constant temperature at 25°C for 24 h to obtain crude acetyl hexapeptide-8 peptide.

[0119] Example 4: This example is different from Example 3 only in that the substitution degree of RINK AMIDE-AM resin in the preparation of the peptide resin is 1.2 mmol / g.

[0120] Example 5: Compared with Example 3, this example is different only in the preparation of acetyl hexapeptide-8.

[0121] Preparation of mercapto-functionalized siloxane microspheres: Methyltrimethoxysilane was dispersed in deionized water and stirred at 19°C and 800 rpm for 30 minutes. Ammonia was added and stirred for 2 minutes. 3-Mercaptopropyltrimethoxysilane was added and the mixture was allowed to react for 4 hours. The mixture was centrifuged at 10,000 rpm for 15 minutes. The precipitate was washed with ethanol and dried under vacuum at 80°C for 12 hours to obtain mercapto-functionalized siloxane microspheres. The mass-to-volume ratio of methyltrimethoxysilane to deionized water was 20 g:100 mL, the volume ratio of ammonia to deionized water was 10 μL:100 mL, and the mass ratio of 3-Mercaptopropyltrimethoxysilane to methyltrimethoxysilane was 5:20.

[0122] Preparation of modified silica gel chromatographic filler: Disperse thiol-functionalized silicone microspheres in toluene, sonicate for 20 minutes, add N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetate, add azobisisobutyronitrile, pass nitrogen for 30 minutes, react at 70°C and 800 rpm with stirring for 10 hours, cool to room temperature, centrifuge at 10,000 rpm for 15 minutes, wash the precipitate with ethanol, and dry under vacuum at 50°C for 12 hours to obtain modified silica gel chromatographic filler. The mass volume ratio of thiol-functionalized silicone microspheres to toluene is 20 g:300 mL, the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to thiol-functionalized silicone microspheres is 1:20, the mass ratio of methyl 2-acrylamido-2-methoxyacetate to thiol-functionalized silicone microspheres is 1:20, and the mass ratio of azobisisobutyronitrile to thiol-functionalized silicone microspheres is 200 mg:20 g.

[0123] Preparation of the modified chromatographic column: Methanol and isopropanol were mixed to form a displacement fluid; modified silica gel chromatographic filler was dispersed in the isopropanol, sonicated for 5 minutes, and then filled into the chromatographic column tube with the displacement fluid to obtain the modified chromatographic column. The displacement fluid contained a 50:50 volume ratio of methanol to isopropanol, and the mass-to-volume ratio of modified silica gel chromatographic filler to isopropanol was 4 g:60 mL. The chromatographic column had a volume of 4.6 mm x 250 mm.

[0124] Preparation of acetyl hexapeptide-8: The crude acetyl hexapeptide-8 peptide was purified by RP-HPLC using a modified chromatographic column. The mobile phase A was a 0.1 wt% TFA aqueous solution, the mobile phase B was a 0.1 wt% TFA acetonitrile solution, the volume ratio of mobile phase A to mobile phase B was 80:20, the flow rate was 1 mL / min, the injection volume was 10 μL, and acetyl hexapeptide-8 was eluted and collected.

[0125] Example 6: This example differs from Example 5 only in the preparation of the modified silica gel chromatographic filler.

[0126] Preparation of modified silica gel chromatographic filler: Disperse thiol-functionalized silicone microspheres in toluene, sonicate for 20 minutes, add N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetate, add azobisisobutyronitrile, pass nitrogen for 30 minutes, react at 70°C and 800 rpm with stirring for 10 hours, cool to room temperature, centrifuge at 10,000 rpm for 15 minutes, wash the precipitate with ethanol, and dry under vacuum at 50°C for 12 hours to obtain modified silica gel chromatographic filler. The mass volume ratio of thiol-functionalized silicone microspheres to toluene is 20 g:300 mL, the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to thiol-functionalized silicone microspheres is 2:20, the mass ratio of methyl 2-acrylamido-2-methoxyacetate to thiol-functionalized silicone microspheres is 1:20, and the mass ratio of azobisisobutyronitrile to thiol-functionalized silicone microspheres is 200 mg:20 g.

[0127] Example 7: Compared with Example 5, the only difference between this example is the preparation of the modified silica gel chromatographic filler.

[0128] Preparation of modified silica gel chromatographic filler: Disperse thiol-functionalized silicone microspheres in toluene, sonicate for 20 minutes, add N-(benzo[D]thiazol-2-ylmethyl)acrylamide, methyl 2-acrylamido-2-methoxyacetate and tert-butyl (2-acrylamidoethyl)carbamate, add azobisisobutyronitrile, pass nitrogen for 30 minutes, react at 70°C and 800 rpm with stirring for 10 hours, cool to room temperature, centrifuge at 10,000 rpm for 15 minutes, wash the precipitate with ethanol, and dry under vacuum at 50°C for 12 hours to obtain modified silica gel chromatographic filler. The mass volume ratio of thiol-functionalized silicone microspheres to toluene is 20 g:300 mL, the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to thiol-functionalized silicone microspheres is 1:20, the mass ratio of methyl 2-acrylamido-2-methoxyacetate to thiol-functionalized silicone microspheres is 1:20, the mass ratio of (2-acrylamidoethyl)carbamate to thiol-functionalized silicone microspheres is 1:20, and the mass ratio of azobisisobutyronitrile to thiol-functionalized silicone microspheres is 200 mg:20 g.

[0129] Example 8: Compared with Example 5, this example differs only in the preparation of the modified silica gel chromatographic filler.

[0130] Preparation of modified silica gel chromatographic filler: Disperse thiol-functionalized silicone microspheres in toluene, sonicate for 20 minutes, add N-(benzo[D]thiazol-2-ylmethyl)acrylamide, methyl 2-acrylamido-2-methoxyacetate and tert-butyl (2-acrylamidoethyl)carbamate, add azobisisobutyronitrile, pass nitrogen for 30 minutes, react at 70°C and 800 rpm with stirring for 10 hours, cool to room temperature, centrifuge at 10,000 rpm for 15 minutes, wash the precipitate with ethanol, and dry under vacuum at 50°C for 12 hours to obtain modified silica gel chromatographic filler. The mass volume ratio of thiol-functionalized silicone microspheres to toluene is 20 g:300 mL, the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to thiol-functionalized silicone microspheres is 1:20, the mass ratio of methyl 2-acrylamido-2-methoxyacetate to thiol-functionalized silicone microspheres is 1:20, the mass ratio of tert-butyl (2-acrylamidoethyl)carbamate to thiol-functionalized silicone microspheres is 2:20, and the mass ratio of azobisisobutyronitrile to thiol-functionalized silicone microspheres is 200 mg:20 g.

[0131] Comparative Example 1: This comparative example is different from Example 5 only in that N-(benzo[D]thiazol-2-ylmethyl)acrylamide is not used in the preparation of the modified silica gel chromatographic filler.

[0132] Comparative Example 2: This comparative example is different from Example 5 only in that methyl 2-acrylamido-2-methoxyacetate is not used in the preparation of the modified silica gel chromatographic filler.

[0133] Comparative Example 3: This comparative example is different from Example 5 only in that N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetate are not used in the preparation of the modified silica gel chromatographic filler.

[0134] Test Example 1: Specific surface area test of modified silica gel chromatographic packing.

[0135] Test sample: modified silica gel chromatographic filler prepared in Examples 5-8 and Comparative Examples 1-3.

[0136] Test method: The modified silica gel chromatographic packing was degassed at 60°C for 12 hours. An adsorption-desorption experiment was carried out at a liquid nitrogen temperature of -196°C using high-purity nitrogen as the adsorbent. The adsorption-desorption isotherm was plotted based on the nitrogen adsorption amount. The specific surface area of ​​the modified silica gel chromatographic packing was calculated based on the multi-point BET equation.

[0137] The specific surface area test results of the modified silica gel chromatographic filler prepared by the present invention are as follows: Figure 2As shown, Example 5 uses mercapto-functionalized silicone microspheres with N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetate for modification, forming a rich porous structure on the silica gel surface with a high specific surface area; Example 6 increases the amount of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to graft more functional groups to the silica gel surface, forming a denser pore network, and the specific surface area is improved compared with Example 5; Example 7 introduces tert-butyl (2-acrylamidoethyl)carbamate, and the specific surface area is further improved, synergistically acting with other modifiers. , ensuring the richness of pores and optimizing the pore size distribution; Example 8 increases the amount of tert-butyl (2-acrylamidoethyl)carbamate, and the specific surface area reaches the highest value; Comparative Example 1 does not use N-(benzo[D]thiazol-2-ylmethyl)acrylamide, and Comparative Example 2 does not use methyl 2-acrylamido-2-methoxyacetate, and the specific surface area is significantly reduced; Comparative Example 3 lacks both N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetate, and the specific surface area is reduced to the lowest, indicating that these two components play an indispensable synergistic role in improving the specific surface area of ​​the filler.

[0138] Test Example 2: Purity test of acetyl hexapeptide-8.

[0139] Test sample: Acetyl hexapeptide-8 prepared in each example and comparative example.

[0140] Test method: According to the peak area after purification, the purity is calculated as R(%)=S1 / S0×100%, where S1 is the main peak area of ​​acetyl hexapeptide-8 and S0 is the total peak area.

[0141] The purity test results of acetyl hexapeptide-8 prepared by the present invention are as follows Figure 3As shown, Example 1 adopts traditional RP-HPLC purification and uses a C18 reverse phase chromatography column, and the purity of acetyl hexapeptide-8 is relatively low; Example 2 uses a higher resin substitution degree to increase the reaction site density, so that the amino acid coupling is more complete, and the residual unreacted intermediates are reduced, and the purity is improved relative to Example 1; Example 3 adds triisopropylsilane as a cracking aid in the preparation of the crude peptide to enhance the removal efficiency of the protecting group and reduce the generation of impurities, and the purity is improved relative to Example 2; Example 4 further improves the resin substitution degree. The high substitution degree resin causes incomplete release of the target peptide due to its dense structure during the cracking process, accompanied by the generation of more side reaction products, and the purity is reduced compared with Example 3; Example 5 uses a column chromatography modified with mercapto-functionalized siloxane microspheres for purification, and the purity is significantly higher than that of Example 1. The filler of the modified chromatographic column has a large specific surface area and rich surface functional groups, which can adsorb peptides. - The desorption selectivity is stronger, the target product can be separated more efficiently, and the impurity residue is reduced; Example 6 increases the amount of N-(benzo[D]thiazol-2-ylmethyl)acrylamide in the modified filler, and the purity is further improved; Example 7 introduces (2-acrylamidoethyl)carbamic acid tert-butyl ester to modify the filler, and the purity is improved compared with Example 6, which reduces the nonspecific adsorption of the peptide segment and the filler, making the elution of the target product more concentrated; Example 8 increases the amount of (2-acrylamidoethyl)carbamic acid tert-butyl ester, and the purity reaches the highest; Comparative Example 1 does not use N-(benzo[D]thiazol-2-ylmethyl)acrylamide in the modified filler, and Comparative Example 2 does not use methyl 2-acrylamido-2-methoxyacetate, and the purity is significantly reduced compared with Example 5; Comparative Example 3 simultaneously lacks both acrylamide compounds, and the purity is reduced to the lowest, verifying the synergistic necessity of the two components in improving purification efficiency.

[0142] Test Example 3: Yield test of acetyl hexapeptide-8.

[0143] Test sample: Acetyl hexapeptide-8 prepared in each example and comparative example.

[0144] Test method: According to m 纯品 =m1×R to calculate the mass of the pure product, according to S(%)=m 纯品 The yield was calculated as m1 / m0×100%, where m1 is the mass of the purified acetyl hexapeptide-8, R is the purity of the purified acetyl hexapeptide-8, and m0 is the theoretical mass of acetyl hexapeptide-8.

[0145] The yield test results of acetyl hexapeptide-8 prepared by the present invention are as follows Figure 4As shown, in Example 2, the higher degree of resin substitution increases the density of reaction sites, making the amino acid coupling more complete, and the yield is improved relative to Example 1; in Example 3, triisopropylsilane is added as a cleavage aid in the preparation of crude peptides, which enhances the removal efficiency of protective groups and reduces the interference of impurities in purification, and the yield is improved relative to Example 2; in Example 4, the degree of resin substitution is further increased, and the yield is decreased compared with Example 3; in Example 5, column chromatography purification is performed using mercapto-functionalized silicone microspheres modified, the filler has a large specific surface area and strong separation selectivity, and the yield is higher than that of Example 1; in Example 6, the amount of N-(benzo[D]thiazol-2-ylmethyl)acrylamide in the modified filler is increased, and the yield is further improved. Improvement; Example 7 introduces (2-acrylamidoethyl) carbamic acid tert-butyl ester to modify the filler, and the yield is improved compared with Example 6, which reduces the nonspecific adsorption of the peptide segment and the filler, making the elution of the target product more concentrated; Example 8 increases the amount of (2-acrylamidoethyl) carbamic acid tert-butyl ester, and the yield reaches the highest; Comparative Example 1 does not use N-(benzo[D]thiazol-2-ylmethyl)acrylamide in the modified filler, and Comparative Example 2 does not use methyl 2-acrylamido-2-methoxyacetate, and the yield is significantly reduced compared with Example 5; Comparative Example 3 lacks both acrylamide compounds, and the yield is reduced to the lowest, verifying the synergistic necessity of the two components in improving purification efficiency.

[0146] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0147] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A solid phase synthesis method of acetyl hexapeptide-8, comprising: The RINK AMIDE-AM resin is coupled with amino acids in sequence, deprotected in a deprotection solution, and acetylated in an acetylation solution to obtain a peptide resin; The peptide resin is placed in a cleavage solution for cleavage, and then placed in a precipitant for precipitation to obtain a crude acetyl hexapeptide-8 peptide; The crude acetyl hexapeptide-8 peptide was purified by column chromatography and lyophilized to obtain the refined acetyl hexapeptide-8; The coupling order of the amino acids is: Fmoc-Arg(HCl)-OH, Fmoc-Arg(HCl)-OH, Fmoc-Gln-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Glu(OtBu)-OH.

2. The solid phase synthesis method of acetyl hexapeptide-8 according to claim 1, characterized in that: The substitution degree of the RINKAMIDE-AM resin is 0.6-1.2 mmol / g.

3. The solid phase synthesis method of acetyl hexapeptide-8 according to claim 1, characterized in that: The deprotection solution comprises piperidine and N,N-dimethylformamide, and the volume ratio of the piperidine to N,N-dimethylformamide is 10-30:

80.

4. The solid phase synthesis method of acetyl hexapeptide-8 according to claim 1, characterized in that: The acetylation solution includes acetic anhydride and pyridine, and the volume ratio of the acetic anhydride to pyridine is 15-45:

30.

5. The solid phase synthesis method of acetyl hexapeptide-8 according to claim 1, characterized in that: The lysis solution includes one or more of trifluoroacetic acid, triisopropylsilane, thioanisole and 1,2-ethanedithiol.

6. The solid phase synthesis method of acetyl hexapeptide-8 according to claim 1, characterized in that: The precipitant includes one or more of methyl tert-butyl ether, diethyl ether and petroleum ether.

7. The solid phase synthesis method of acetyl hexapeptide-8 according to claim 1, characterized in that: The chromatographic column of the column chromatography comprises a C18 chromatographic column, or a modified chromatographic column, and the modified chromatographic column comprises a modified silica gel chromatographic filler.

8. The solid phase synthesis method of acetyl hexapeptide-8 according to claim 7, characterized in that: The modified silica gel chromatographic filler comprises mercapto-functionalized siloxane microspheres grafted with a modifier, wherein the modifier comprises N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetate.

9. The solid phase synthesis method of acetyl hexapeptide-8 according to claim 8, characterized in that: The mass ratio of the N-(benzo[D]thiazol-2-ylmethyl)acrylamide to the mercapto-functionalized silicone microspheres is 0.5-4:

20.

10. The solid phase synthesis method of acetyl hexapeptide-8 according to claim 8, characterized in that: The mass ratio of the methyl 2-acrylamido-2-methoxyacetate to the mercapto-functionalized silicone microspheres is 0.5-4:20.

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