A method for solid phase synthesis of acetyl hexapeptide-8

Abstract

The application discloses a solid-phase synthesis method of acetyl hexapeptide-8. RINK AMIDE-AM resin is used as a carrier, 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 are coupled step by step, crude peptides are prepared through deprotection, acetylation and cleavage reaction, and finally the target product is obtained through purification. The method adopts a minimum amino acid protection strategy, optimizes a cleavage system, and combines modified silica gel chromatography packing materials. The modifiers include N-(benzo[D]thiazole-2-ylmethyl) acrylamide and methyl 2-acrylamido-2-methoxyacetate, so that the column chromatography purification efficiency is improved, the product has high purity and high yield, and the operation is simple and suitable for scale-up production.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide synthesis, specifically relating to a solid-phase synthesis method for acetyl hexapeptide-8. Background Technology

[0002] Acetyl hexapeptide-8 is a high-quality anti-wrinkle ingredient that can locally block the transmission of nerve signals to muscle contraction, affecting nerve conduction in the skin follicles, relaxing facial muscles, and smoothing dynamic wrinkles, static wrinkles, and fine lines. It effectively reorganizes collagen and elastin, increases the activity of elastin, relaxes facial lines, smooths wrinkles, and improves sagging. As an anti-wrinkle ingredient, it has excellent effects, high anti-wrinkle activity, and few side effects, and is used in various high-end cosmetic lines.

[0003] Currently, there are several methods for synthesizing acetyl hexapeptide-8, including solid-phase sequential coupling, which involves coupling amino acids with protected side chains one by one to obtain a peptide resin intermediate. However, this method results in lower product purity and requires more expensive raw materials. Solid-liquid phase synthesis is complex and not suitable for large-scale production. Liquid-phase synthesis uses unprotected arginine and glutamine, involves more reaction steps, requires a longer production cycle, and demands high purification technology.

[0004] This invention addresses the problems of expensive raw materials, complex operation unsuitable for large-scale production, numerous reaction steps, and long production cycles by proposing a solid-phase synthesis method for acetyl hexapeptide-8. Through a minimum amino acid protection strategy and optimized purification process, it reduces raw material costs, minimizes impurities, simplifies operation, and improves the overall yield of acetyl hexapeptide-8, making it suitable for scale-up production. Summary of the Invention

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

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0007] A solid-phase synthesis method for acetyl hexapeptide-8, comprising:

[0008] RINK AMIDE-AM resin was sequentially coupled with amino acids, deprotected in a deprotection solution, and acetylated in an acetylation solution to obtain peptide resin.

[0009] The peptide resin was lysed in a lysis buffer and then precipitated in a precipitant to obtain crude acetyl hexapeptide-8 peptide.

[0010] The crude acetyl hexapeptide-8 peptide was purified by column chromatography and lyophilized to obtain high-quality acetyl hexapeptide-8.

[0011] The coupling sequence 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.

[0012] Preferably, the degree of substitution of RINK AMIDE-AM resin is 0.6-1.2 mmol / g.

[0013] Preferably, the deprotection solution comprises piperidine and N,N-dimethylformamide.

[0014] Preferably, the volume ratio of piperidine to N,N-dimethylformamide is 10-30:80.

[0015] Preferably, the acetylation solution comprises acetic anhydride and pyridine.

[0016] Preferably, the volume ratio of acetic anhydride to pyridine is 15-45:30.

[0017] Preferably, the lysis buffer includes one or more of trifluoroacetic acid, triisopropylsilane, benzyl sulfide, and 1,2-ethylenedithiol.

[0018] Preferably, the precipitant includes one or more of methyl tert-butyl ether, diethyl ether, and petroleum ether.

[0019] Preferably, the column used in column chromatography includes a C18 column or a modified column, wherein the modified column includes modified silica gel packing material.

[0020] Preferably, the modified silica gel chromatographic packing material comprises mercapto-functionalized siloxane microspheres grafted with a modifier.

[0021] Preferably, the modifier includes N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl-2-acrylamido-2-methoxyacetic acid ester.

[0022] Preferably, the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to mercaptofunctionalized siloxane microspheres is 0.5-4:20.

[0023] Preferably, the mass ratio of methyl 2-acrylamido-2-methoxyacetic acid ester to mercaptofunctionalized siloxane microspheres is 0.5-4:20.

[0024] N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl-2-acrylamido-2-methoxyacetate improve the performance of modified silica gel chromatographic packing material 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 both are grafted onto the surface of mercaptofunctionalized siloxane microspheres, a composite coating with a balance of hydrophilicity and hydrophobicity and ion exchange capacity is formed. This coating increases adsorption sites through porous structure and improves the separation selectivity of acetyl hexapeptide-8 and impurities through polarity regulation, while also enhancing the structural stability of the packing material, thereby significantly improving purification efficiency and column alkali resistance.

[0025] More preferably, the modifier also includes tert-butyl (2-acrylamidoethyl)carbamate, with a mass ratio of tert-butyl (2-acrylamidoethyl)carbamate to mercapto-functionalized siloxane microspheres of 0.5-4:20. Tert-butyl (2-acrylamidoethyl)carbamate, together with other modifier monomers, forms a three-dimensional network polymer coating on the silica gel surface, optimizing the pore size distribution and specific surface area of ​​the coating. This reduces the direct erosion of the coating by alkaline solutions through steric hindrance, while its hydrophobicity adjusts the polarity distribution of the filler surface, reducing the non-specific adsorption of target peptides to the filler, thereby achieving efficient separation of impurities during purification.

[0026] This invention also provides a method for preparing a peptide resin, comprising:

[0027] a. Dissolve Pip in DMF to obtain a deprotected solution.

[0028] Preferably, the volume ratio of Pip to DMF is 10-30:80.

[0029] b. Use DMF as the washing solution.

[0030] c. Add RINK AMIDE-AM resin to the reactor, add DMF, swell with nitrogen for 30 min, add deprotection solution for 30 min, wash with washing solution 5-7 times, and dry for later use.

[0031] Preferably, the degree of substitution of RINK AMIDE-AM resin is 0.6-1.2 mmol / g, the molar amount of RINK AMIDE-AM resin is 1 eq, and the mass of RINK AMIDE-AM resin is W g.

[0032] Preferably, the amount of DMF used is 2-10 W mL / g.

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

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

[0035] d. Dissolve Fmoc-Arg(HCl)-OH and HOBt in DMF, pre-cool to 0-10℃, add DIC to activate for 5-15 min, then add to the reactor and react for 2-3 h. Remove the reaction liquid, wash 2-4 times with washing solution, add deprotection solution to deprotect for 25-35 min, wash 5-7 times with washing solution, dry the liquid, and proceed to the next step of the reaction.

[0036] Preferably, the amount of Fmoc-Arg(HCl)-OH used is 1.5-4 eq.

[0037] Preferably, the amount of HOBt used is 1.5-4 eq.

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

[0039] Preferably, the amount of DMF used is 2-10 W mL / g.

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

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

[0042] e. Dissolve Fmoc-Arg(HCl)-OH and HOBt in DMF, pre-cool to 0-10℃, add DIC to activate for 5-15 min, then add to the reactor and react for 2-3 h. Remove the reaction liquid, wash 2-4 times with washing solution, add deprotection solution to deprotect for 25-35 min, wash 5-7 times with washing solution, dry the liquid, and proceed to the next step of the reaction.

[0043] Preferably, the amount of Fmoc-Arg(HCl)-OH used is 1.5-4 eq.

[0044] Preferably, the amount of HOBt used is 1.5-4 eq.

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

[0046] Preferably, the amount of DMF used is 2-10 W mL / g.

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

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

[0049] f. Dissolve Fmoc-Gln-OH and HOBt in DMF, pre-cool to 0-10℃, add DIC to activate for 5-15 min, then add to the reactor and react for 2-3 h. Remove the reaction solution, wash 2-4 times with washing solution, add deprotection solution to deprotect for 25-35 min, wash 5-7 times with washing solution, dry the liquid, and proceed to the next step of the reaction.

[0050] Preferably, the amount of Fmoc-Gln-OH used is 1.5-4 eq.

[0051] Preferably, the amount of HOBt used is 1.5-4 eq.

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

[0053] Preferably, the amount of DMF used is 2-10 W mL / g.

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

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

[0056] g. Dissolve Fmoc-Met-OH and HOBt in DMF, pre-cool to 0-10℃, add DIC to activate for 5-15 min, then add to the reactor and react for 2-3 h. Remove the reaction solution, wash 2-4 times with washing solution, add deprotection solution to deprotect for 25-35 min, wash 5-7 times with washing solution, dry the liquid, and proceed to the next step of the reaction.

[0057] Preferably, the amount of Fmoc-Met-OH used is 1.5-4 eq.

[0058] Preferably, the amount of HOBt used is 1.5-4 eq.

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

[0060] Preferably, the amount of DMF used is 2-10 W mL / g.

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

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

[0063] h. Dissolve Fmoc-Glu(OtBu)-OH and HOBt in DMF, pre-cool to 0-10℃, add DIC to activate for 5-15 min, then add to the reactor and react for 2-3 h. Remove the reaction solution, wash 2-4 times with washing solution, add deprotection solution to deprotect for 25-35 min, wash 5-7 times with washing solution, dry the liquid, and proceed to the next step of the reaction.

[0064] Preferably, the amount of Fmoc-Glu(OtBu)-OH is 1.5-4 eq.

[0065] Preferably, the amount of HOBt used is 1.5-4 eq.

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

[0067] Preferably, the amount of DMF used is 2-10 W mL / g.

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

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

[0070] i. Dissolve Fmoc-Glu(OtBu)-OH and HOBt in DMF, pre-cool to 0-10℃, add DIC to activate for 5-15 min, then add to the reactor and react for 2-3 h. Remove the reaction solution, wash 2-4 times with washing solution, add deprotection solution to deprotect for 25-35 min, wash 5-7 times with washing solution, dry the liquid, and proceed to the next step of the reaction.

[0071] Preferably, the amount of Fmoc-Glu(OtBu)-OH is 1.5-4 eq.

[0072] Preferably, the amount of HOBt used is 1.5-4 eq.

[0073] Preferably, the amount of DIC used is 1.5-4 eq.

[0074] Preferably, the amount of DMF used is 2-10 W mL / g.

[0075] Preferably, the amount of deprotection solution used is 2-10 W mL / g.

[0076] Preferably, the amount of washing solution used is 2-10 W mL / g.

[0077] j. Add Ac2O and pyridine to the reactor, 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 peptide resin.

[0078] Preferably, the amount of Ac2O used is 20 eq.

[0079] Preferably, the amount of pyridine used is 20 eq.

[0080] This invention also provides a method for preparing crude acetyl hexapeptide-8 peptide, comprising:

[0081] TFA was dissolved in deionized water and pre-cooled to -15 to -25°C to obtain a lysis buffer. Peptide resin was added and reacted at 20 to 30°C for 1 to 3 hours. The buffer was filtered, and the filtrate was added to a precipitant pre-cooled to -15 to -25°C. The precipitated solid was collected and washed three times with washing liquid. The solid was then dried under vacuum at 15 to 25°C for 20 to 25 hours to obtain crude acetyl hexapeptide-8 peptide.

[0082] Preferably, the volume ratio of TFA to deionized water is 85-105:5.

[0083] More preferably, the lysis buffer also includes TIS.

[0084] Preferably, the volume ratio of TIS to deionized water is 2-10:5.

[0085] Preferably, the mass-to-volume ratio of peptide resin to TFA is 5-15 g: 95 mL.

[0086] Preferably, the precipitant includes one or more of methyl tert-butyl ether or diethyl ether.

[0087] Preferably, the mass-to-volume ratio of peptide resin to precipitant is 5-15 g: 100 mL.

[0088] Preferably, the washing solution is one or more of methyl tert-butyl ether or diethyl ether.

[0089] Preferably, the volume-to-mass ratio of the washing solution to the peptide resin is 100 mL: 5-15 g.

[0090] This invention also provides a method for preparing thiol-functionalized siloxane microspheres, comprising:

[0091] Preparation of thiol-functionalized siloxane microspheres: Methyltrimethoxysilane was dispersed in deionized water and stirred at 18-20℃ and 70-900 rpm for 20-40 min. Ammonia water was added and stirred for 1-3 min. 3-Mercaptopropyltrimethoxysilane was added and allowed to stand for 5-6 h. The mixture was centrifuged at 9000-11000 rpm for 10-20 min, and the precipitate was washed with ethanol. The precipitate was then dried under vacuum at 75-85℃ for 10-15 h to obtain thiol-functionalized siloxane microspheres.

[0092] Preferably, the mass-to-volume ratio of methyltrimethoxysilane to deionized water is 10-30 g: 100 mL.

[0093] Preferably, the volume ratio of ammonia to deionized water is 5-15 μL: 100 mL.

[0094] Preferably, the mass ratio of 3-mercaptopropyltrimethoxysilane to methyltrimethoxysilane is 2-10:20.

[0095] This invention also provides a method for preparing modified silica gel chromatographic packing material, comprising:

[0096] Preparation of modified silica gel chromatographic packing material: Thiol-functionalized siloxane microspheres were dispersed in toluene and sonicated for 15-25 min. Modifiers and azobisisobutyronitrile were added, and nitrogen gas was purged for 25-35 min. The reaction was carried out at 65-75℃ and 700-900 rpm for 8-12 h. After cooling to room temperature, the mixture was centrifuged at 9000-11000 rpm for 10-20 min. The precipitate was washed with ethanol and dried under vacuum at 45-55℃ for 10-15 h to obtain the modified silica gel chromatographic packing material.

[0097] Preferably, the mass-to-volume ratio of mercaptofunctionalized siloxane microspheres to toluene is 10-30 g: 300 mL.

[0098] Preferably, the modifier comprises N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl-2-acrylamido-2-methoxyacetic acid ester.

[0099] Preferably, the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to mercaptofunctionalized siloxane microspheres is 0.5-4:20.

[0100] Preferably, the mass ratio of methyl 2-acrylamido-2-methoxyacetic acid ester to mercaptofunctionalized siloxane microspheres is 0.5-4:20.

[0101] Preferably, the mass ratio of azobisisobutyronitrile to mercaptofunctionalized siloxane microspheres is 100-300 mg: 20 g.

[0102] More preferably, the modifier also includes tert-butyl (2-acrylamidoethyl)carbamate.

[0103] More preferably, the mass ratio of (2-acrylamidoethyl)carbamate tert-butyl ester to mercaptofunctionalized siloxane microspheres is 0.5-4:20.

[0104] This invention also provides a method for preparing a modified chromatographic column, comprising:

[0105] Preparation of modified chromatographic column: Methanol and isopropanol are mixed to obtain a displacement solution; modified silica gel chromatographic packing is dispersed in isopropanol, sonicated for 4-6 min, and then filled into the chromatographic column tube through the displacement solution to obtain the modified chromatographic column.

[0106] Preferably, the volume ratio of methanol to isopropanol in the displacement solution is 25-75:50.

[0107] Preferably, the mass-to-volume ratio of modified silica gel chromatographic packing material to isopropanol is 2-6 g: 60 mL.

[0108] Preferably, the volume of the chromatographic column is 2-6 mm × 150-300 mm.

[0109] This invention also provides a method for preparing acetyl hexapeptide-8, comprising:

[0110] Preparation of acetyl hexapeptide-8: The crude acetyl hexapeptide-8 peptide was purified by RP-HPLC, and the eluted and collected acetyl hexapeptide-8 was obtained.

[0111] Preferably, the purified chromatographic column is a C18 column or a modified chromatographic column.

[0112] 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.

[0113] Preferably, the volume ratio of mobile phase A to mobile phase B is 70-90:10-30.

[0114] Preferably, the flow rate is 0.8-1.2 mL / min.

[0115] Preferably, the injection volume is 5-15 μL.

[0116] This invention, by employing a minimal amino acid protection strategy combined with a modified silica gel column purification process, offers the following advantages: reduced raw material costs and impurity generation; improved coupling and deprotection efficiency; increased purity and yield of acetyl hexapeptide-8; and simplified, easily scaled-up preparation process. Therefore, this invention provides a solid-phase synthesis method for acetyl hexapeptide-8 with high yield and purity. Attached Figure Description

[0117] Figure 1 This is a schematic diagram of the solid-phase synthesis steps of acetyl hexapeptide-8.

[0118] Figure 2 This is a schematic diagram showing the specific surface area test results of the modified silica gel chromatographic packing material.

[0119] Figure 3 This is a schematic diagram showing the purity test results of acetyl hexapeptide-8.

[0120] Figure 4 This is a schematic diagram showing the yield test results of acetyl hexapeptide-8. Detailed Implementation

[0121] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0122] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0123] The Chinese meanings of the abbreviations used in this invention are shown in Table 1 below.

[0124] Table 1. Chinese meanings of the abbreviations

[0125]

[0126] Example 1:

[0127] Preparation of peptide resin:

[0128] a. Dissolve 20 mL of Pip in 80 mL of DMF to obtain a deprotected solution;

[0129] b. Use DMF as a washing solution;

[0130] c. Add 25.01g of RINK AMIDE-AM resin to the reactor, add 75mL of DMF, swell under nitrogen for 30min, add 75mL of deprotection solution for 30min, wash 6 times with 75mL of washing solution, and dry for later use; the degree of substitution of RINKAMIDE-AM resin is 0.8mmol / g.

[0131] d. Dissolve 19.62 g of Fmoc-Arg(HCl)-OH and 5.41 g of HOBt in DMF, pre-cool to 0 °C, add 6.25 mL of DIC to activate for 10 min, then add to the reactor and react for 2.5 h. Remove the reaction solution, wash three times with 75 mL of washing solution, add 75 mL of deprotection solution to deprotect for 30 min, wash six times with 75 mL of washing solution, dry the liquid, and proceed to the next step of the reaction.

[0132] e. Dissolve 19.62 g of Fmoc-Arg(HCl)-OH and 5.41 g of HOBt in DMF, pre-cool to 0°C, add 6.25 mL of DIC to activate for 10 min, then add to the reactor and react for 2.5 h. Remove the reaction solution, wash three times with 75 mL of washing solution, add 75 mL of deprotection solution to deprotect for 30 min, wash six times with 75 mL of washing solution, dry the liquid, and proceed to the next step of the reaction.

[0133] f. Dissolve 15.54 g of Fmoc-Gln-OH and 5.41 g of HOBt in DMF and pre-cool to 0 °C. Add 6.25 mL of LDIC and activate for 10 min. Then add to the reactor and react for 2.5 h. Remove the reaction solution and wash three times with 75 mL of washing solution. Add 75 mL of deprotection solution to deprotect for 30 min and wash six times with 75 mL of washing solution. Drain the liquid and proceed to the next step of the reaction.

[0134] g. Dissolve 16.06 g of Fmoc-Met-OH and 5.41 g of HOBt in DMF, pre-cool to 0°C, add 6.25 mL of DIC to activate for 10 min, then add to the reactor and react for 2.5 h. Remove the reaction solution, wash three times with 75 mL of washing solution, add 75 mL of deprotection solution to deprotect for 30 min, wash six times with 75 mL of washing solution, drain the liquid, and proceed to the next reaction step.

[0135] h. Dissolve 18.06 g of Fmoc-Glu(OtBu)-OH and 5.41 g of HOBt in DMF, pre-cool to 0°C, add 6.25 mL of DIC to activate for 10 min, then add to the reactor and react for 2.5 h. Remove the reaction solution, wash three times with 75 mL of washing solution, add 75 mL of deprotection solution to deprotect for 30 min, wash six times with 75 mL of washing solution, drain the liquid, and proceed to the next reaction step.

[0136] i. Dissolve 18.06 g of Fmoc-Glu(OtBu)-OH and 5.41 g of HOBt in DMF, pre-cool to 0°C, add 6.25 mL of DIC to activate for 10 min, then add to the reactor and react for 2.5 h. Remove the reaction solution, wash 3 times with 75 mL of washing solution, add 75 mL of deprotection solution to deprotect for 30 min, wash 6 times with 75 mL of washing solution, dry the liquid, and proceed to the next step of the reaction.

[0137] j. Add 37.57 mL of Ac2O and 31.19 mL of pyridine to the reactor, acetylate for 1 h, wash 4 times with DMF and 3 times with DCM, add methanol for shrinkage, and dry under vacuum to obtain peptide resin.

[0138] Preparation of crude acetyl hexapeptide-8 peptide: 95 mL of TFA was dissolved in 5 mL of deionized water and pre-cooled to -20 °C to obtain a lysis buffer. 10 g of peptide resin was added and reacted at 25 °C for 2 h. After filtration, the filtrate was added to 100 mL of methyl tert-butyl ether pre-cooled to -20 °C. The precipitated solid was collected and washed three times with 100 mL of methyl tert-butyl ether. The solid was then dried under vacuum at 25 °C for 24 h to obtain crude acetyl hexapeptide-8 peptide.

[0139] Preparation of Acetyl Hexapeptide-8: The crude acetyl hexapeptide-8 peptide was purified by RP-HPLC using a 4.6 mm × 250 mm C18 reversed-phase column. Mobile phase A was 0.1 wt% TFA aqueous solution, and mobile phase B was 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, and the injection volume was 10 μL. Acetyl hexapeptide-8 was obtained by elution and collection.

[0140] Example 2: The only difference between this example and Example 1 is that the degree of substitution of RINK AMIDE-AM resin in the preparation of peptide resin is 1.0 mmol / g.

[0141] Example 3: The only difference between this example and Example 2 is the preparation of the crude acetyl hexapeptide-8 peptide.

[0142] 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 and pre-cooled to -20 °C to obtain a lysis buffer. 10 g of peptide resin was added, and the mixture was reacted at 25 °C for 2 h. After filtration, the filtrate was added to 100 mL of methyl tert-butyl ether pre-cooled to -20 °C. The precipitated solid was collected and washed three times with 100 mL of methyl tert-butyl ether. The solid was then dried under vacuum at 25 °C for 24 h to obtain crude acetyl hexapeptide-8 peptide.

[0143] Example 4: The only difference between this example and Example 3 is that the degree of substitution of RINK AMIDE-AM resin in the preparation of peptide resin is 1.2 mmol / g.

[0144] Example 5: The only difference between this example and Example 3 is the preparation of acetyl hexapeptide-8.

[0145] Preparation of thiol-functionalized siloxane microspheres: Methyltrimethoxysilane was dispersed in deionized water and stirred at 19°C and 800 rpm for 30 min. Ammonia was added, and the mixture was stirred for 2 min. 3-Mercaptopropyltrimethoxysilane was then added, and the mixture was allowed to stand for 4 h. After centrifugation at 10,000 rpm for 15 min, the precipitate was washed with ethanol and dried under vacuum at 80°C for 12 h to obtain thiol-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.

[0146] Preparation of modified silica gel chromatographic packing material: Thiol-functionalized siloxane microspheres were dispersed in toluene and sonicated for 20 min. N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetate were added, followed by azobisisobutyronitrile. Nitrogen gas was purged for 30 min, and the reaction was carried out at 70 °C and 800 rpm for 10 h. After cooling to room temperature, the mixture was centrifuged at 10,000 rpm for 15 min, the precipitate was washed with ethanol, and dried under vacuum at 50 °C for 12 h to obtain the modified silica gel chromatographic packing material. The mass-to-volume ratio of mercaptofunctionalized siloxane microspheres to toluene was 20 g: 300 mL; the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to mercaptofunctionalized siloxane microspheres was 1:20; the mass ratio of methyl 2-acrylamido-2-methoxyacetic acid ester to mercaptofunctionalized siloxane microspheres was 1:20; and the mass ratio of azobisisobutyronitrile to mercaptofunctionalized siloxane microspheres was 200 mg: 20 g.

[0147] Preparation of the modified chromatographic column: Methanol and isopropanol were mixed to obtain a displacement solution; modified silica gel chromatographic packing material was dispersed in isopropanol, sonicated for 5 min, and then filled into the chromatographic column tube through the displacement solution to obtain the modified chromatographic column. In the displacement solution, the volume ratio of methanol to isopropanol was 50:50, the mass-to-volume ratio of modified silica gel chromatographic packing material to isopropanol was 4 g:60 mL, and the column volume was 4.6 mm × 250 mm.

[0148] Preparation of Acetyl Hexapeptide-8: The crude acetyl hexapeptide-8 peptide was purified by RP-HPLC using a modified column. Mobile phase A was 0.1 wt% TFA aqueous solution, and mobile phase B was 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, and the injection volume was 10 μL. Acetyl hexapeptide-8 was obtained by elution and collection.

[0149] Example 6: The only difference between this example and Example 5 is the preparation of the modified silica gel chromatographic packing material.

[0150] Preparation of modified silica gel chromatographic packing material: Thiol-functionalized siloxane microspheres were dispersed in toluene and sonicated for 20 min. N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetate were added, followed by azobisisobutyronitrile. Nitrogen gas was purged for 30 min, and the reaction was carried out at 70 °C and 800 rpm for 10 h. After cooling to room temperature, the mixture was centrifuged at 10,000 rpm for 15 min, the precipitate was washed with ethanol, and dried under vacuum at 50 °C for 12 h to obtain the modified silica gel chromatographic packing material. The mass-to-volume ratio of mercaptofunctionalized siloxane microspheres to toluene was 20 g: 300 mL; the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to mercaptofunctionalized siloxane microspheres was 2:20; the mass ratio of methyl 2-acrylamido-2-methoxyacetic acid ester to mercaptofunctionalized siloxane microspheres was 1:20; and the mass ratio of azobisisobutyronitrile to mercaptofunctionalized siloxane microspheres was 200 mg: 20 g.

[0151] Example 7: The only difference between this example and Example 5 is the preparation of the modified silica gel chromatographic packing material.

[0152] Preparation of modified silica gel chromatographic packing material: Thiol-functionalized siloxane microspheres were dispersed in toluene and sonicated for 20 min. N-(benzo[D]thiazol-2-ylmethyl)acrylamide, methyl 2-acrylamido-2-methoxyacetate and (2-acrylamidoethyl)carbamate tert-butyl ester were added, and azobisisobutyronitrile was added. Nitrogen gas was purged for 30 min, and the reaction was carried out at 70℃ and 800 rpm for 10 h. After cooling to room temperature, the mixture was centrifuged at 10000 rpm for 15 min, the precipitate was washed with ethanol, and dried under vacuum at 50℃ for 12 h to obtain the modified silica gel chromatographic packing material. The mass-to-volume ratio of mercaptofunctionalized siloxane microspheres to toluene was 20 g: 300 mL; the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to mercaptofunctionalized siloxane microspheres was 1:20; the mass ratio of methyl 2-acrylamido-2-methoxyacetic acid ester to mercaptofunctionalized siloxane microspheres was 1:20; the mass ratio of (2-acrylamidoethyl)carbamate tert-butyl ester to mercaptofunctionalized siloxane microspheres was 1:20; and the mass ratio of azobisisobutyronitrile to mercaptofunctionalized siloxane microspheres was 200 mg: 20 g.

[0153] Example 8: The only difference between this example and Example 5 is the preparation of the modified silica gel chromatographic packing material.

[0154] Preparation of modified silica gel chromatographic packing material: Thiol-functionalized siloxane microspheres were dispersed in toluene and sonicated for 20 min. N-(benzo[D]thiazol-2-ylmethyl)acrylamide, methyl 2-acrylamido-2-methoxyacetate and (2-acrylamidoethyl)carbamate tert-butyl ester were added, and azobisisobutyronitrile was added. Nitrogen gas was purged for 30 min, and the reaction was carried out at 70℃ and 800 rpm for 10 h. After cooling to room temperature, the mixture was centrifuged at 10000 rpm for 15 min, the precipitate was washed with ethanol, and dried under vacuum at 50℃ for 12 h to obtain the modified silica gel chromatographic packing material. The mass-to-volume ratio of thiol-functionalized siloxane microspheres to toluene was 20 g: 300 mL; the mass ratio of N-(benzo[D]thiazol-2-ylmethyl)acrylamide to thiol-functionalized siloxane microspheres was 1:20; the mass ratio of methyl 2-acrylamido-2-methoxyacetic acid ester to thiol-functionalized siloxane microspheres was 1:20; the mass ratio of (2-acrylamidoethyl)carbamate tert-butyl ester to thiol-functionalized siloxane microspheres was 2:20; and the mass ratio of azobisisobutyronitrile to thiol-functionalized siloxane microspheres was 200 mg: 20 g.

[0155] Comparative Example 1: The only difference between this comparative example and Example 5 is that N-(benzo[D]thiazol-2-ylmethyl)acrylamide was not used in the preparation of the modified silica gel chromatographic packing.

[0156] Comparative Example 2: This comparative example differs from Example 5 only in that methyl 2-acrylamido-2-methoxyacetic acid ester was not used in the preparation of the modified silica gel chromatographic packing.

[0157] Comparative Example 3: This comparative example differs from Example 5 only in that N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl-2-acrylamido-2-methoxyacetic acid ester were not used in the preparation of the modified silica gel chromatographic packing.

[0158] Experimental Example 1: Specific surface area test of modified silica gel chromatographic packing material.

[0159] Test samples: Modified silica gel chromatographic packing materials prepared in Examples 5-8 and Comparative Examples 1-3.

[0160] Test method: The modified silica gel chromatographic packing was degassed at 60℃ for 12h. Using high-purity nitrogen as the adsorbate, an adsorption-desorption experiment was conducted at liquid nitrogen temperature -196℃. Adsorption-desorption isotherms were plotted based on the amount of nitrogen adsorbed, and the specific surface area of ​​the modified silica gel chromatographic packing was calculated according to the multi-point BET equation.

[0161] The specific surface area test results of the modified silica gel chromatographic packing material prepared in this invention are as follows: Figure 2As shown, Example 5 uses mercapto-functionalized siloxane microspheres modified with N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetic acid ester, 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, allowing more functional groups to be grafted onto the silica gel surface, forming a denser pore network, and the specific surface area is improved compared to Example 5. Example 7 introduces tert-butyl (2-acrylamidoethyl)carbamate, further increasing the specific surface area, and works synergistically with other modifiers. This ensured the abundance of pores and optimized the pore size distribution. In Example 8, the amount of (2-acrylamidoethyl)carbamate tert-butyl ester was increased, resulting in the highest specific surface area. In Comparative Example 1, N-(benzo[D]thiazol-2-ylmethyl)acrylamide was not used, and in Comparative Example 2, methyl 2-acrylamido-2-methoxyacetic acid ester was not used, resulting in a significant decrease in specific surface area. In Comparative Example 3, both N-(benzo[D]thiazol-2-ylmethyl)acrylamide and methyl 2-acrylamido-2-methoxyacetic acid ester were absent, resulting in the lowest specific surface area. This indicates that these two components play an indispensable synergistic role in improving the specific surface area of ​​the filler.

[0162] Experimental Example 2: Purity test of acetyl hexapeptide-8.

[0163] Test samples: Acetyl hexapeptide-8 prepared in each example and comparative example.

[0164] Test method: The purity is calculated based on the purified peak area, R(%) = S1 / S0 × 100%, where S1 is the main peak area of ​​acetyl hexapeptide-8 and S0 is the total peak area.

[0165] The purity test results of the acetyl hexapeptide-8 prepared in this invention are as follows: Figure 3As shown, Example 1 used conventional RP-HPLC purification with a C18 reversed-phase column, resulting in relatively low purity of acetyl hexapeptide-8. Example 2, with its higher resin substitution degree, increased the reaction site density, leading to more complete amino acid coupling and reduced unreacted intermediate residues, resulting in higher purity compared to Example 1. Example 3 added triisopropylsilane as a cleavage aid during crude peptide preparation, enhancing the removal efficiency of protecting groups and reducing impurity generation, thus improving purity compared to Example 2. Example 4 further increased the resin substitution degree; however, the high substitution degree resin, due to its dense structure, resulted in incomplete release of the target peptide during cleavage, accompanied by the generation of more byproducts, leading to a decrease in purity compared to Example 3. Example 5 used column chromatography purification modified with thiol-functionalized siloxane microspheres, achieving significantly higher purity than Example 1. The modified column had a large specific surface area and abundant surface functional groups, enhancing peptide adsorption. - Stronger desorption selectivity, enabling more efficient separation of the target product and reducing impurity residue; Example 6 increased the amount of N-(benzo[D]thiazol-2-ylmethyl)acrylamide in the modified packing material, further improving purity; Example 7 introduced tert-butyl (2-acrylamidoethyl)carbamate modified packing material, resulting in improved purity compared to Example 6, reducing non-specific adsorption of peptides to the packing material, and making the elution of the target product more concentrated; Example 8 increased the amount of tert-butyl (2-acrylamidoethyl)carbamate, achieving the highest purity; Comparative Example 1 did not use N-(benzo[D]thiazol-2-ylmethyl)acrylamide in the modified packing material, and Comparative Example 2 did not use methyl 2-acrylamido-2-methoxyacetate, resulting in a significant decrease in purity compared to Example 5; Comparative Example 3 simultaneously lacked both acrylamide compounds, resulting in the lowest purity, verifying the synergistic necessity of the two components in improving purification efficiency.

[0166] Experimental Example 3: Yield test of acetyl hexapeptide-8.

[0167] Test samples: Acetyl hexapeptide-8 prepared in each example and comparative example.

[0168] Test method: Based on m 纯品 The pure product mass is calculated by m1×R, and according to S(%)=m 纯品 The yield is calculated as / m0×100%. Where m1 is the mass of purified acetyl hexapeptide-8, R is the purity of purified acetyl hexapeptide-8, and m0 is the theoretical mass of acetyl hexapeptide-8.

[0169] The yield test results of acetyl hexapeptide-8 prepared in this invention are as follows: Figure 4As shown, Example 2's higher resin substitution degree increased the reaction site density, resulting in more complete amino acid coupling and a higher yield compared to Example 1. Example 3 added triisopropylsilane as a cleavage aid in the crude peptide preparation, enhancing the removal efficiency of protecting groups and reducing interference from impurities in purification, leading to a higher yield compared to Example 2. Example 4 further increased the resin substitution degree, resulting in a lower yield than Example 3. Example 5 used column chromatography purification modified with mercapto-functionalized siloxane microspheres; the packing material had a large specific surface area and strong separation selectivity, resulting in a higher yield than Example 1. Example 6 increased the amount of N-(benzo[D]thiazol-2-ylmethyl)acrylamide in the modified packing material, further increasing the yield. Improvements were made in the following ways: Example 7 introduced tert-butyl (2-acrylamidoethyl)carbamate modified packing material, resulting in a higher yield than in Example 6. This reduced non-specific adsorption of peptides to the packing material, leading to more concentrated elution of the target product. Example 8 increased the amount of tert-butyl (2-acrylamidoethyl)carbamate, achieving the highest yield. Comparative Example 1 did not use N-(benzo[D]thiazol-2-ylmethyl)acrylamide in its modified packing material, and Comparative Example 2 did not use methyl 2-acrylamido-2-methoxyacetate, resulting in a significantly lower yield than in Example 5. Comparative Example 3, lacking both acrylamide compounds, achieved the lowest yield, verifying the synergistic necessity of the two components in improving purification efficiency.

[0170] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations 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 the technology or embodiments that are substantially the same as the present invention.

[0171] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, 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 situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for solid phase synthesis of acetyl hexapeptide-8, comprising: coupling RINK AMIDE-AM resin with amino acids in sequence, placing in a deprotection solution for deprotection, placing in an acetylation solution for acetylation, to obtain a peptide resin; placing the peptide resin in a cleavage solution for cleavage, placing in a precipitant for precipitation to obtain acetyl hexapeptide-8 crude peptide; purifying the acetyl hexapeptide-8 crude peptide by column chromatography, freeze-drying to obtain fine acetyl hexapeptide-8; the coupling sequence 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; the column chromatography is performed on a modified chromatographic column, and the modified chromatographic column comprises modified silica gel chromatographic filler, and the modifier of the modified silica gel chromatographic filler comprises N-(benzo[D]thiazol-2-ylmethyl) acrylamide and methyl 2-acrylamido-2-methoxyacetate; the preparation method of the modified silica gel chromatographic filler comprises: dispersing mercapto-functionalized siloxane microspheres in toluene, ultrasonicating for 15-25 min, adding a modifier, adding azobisisobutyronitrile, passing nitrogen for 25-35 min, reacting for 8-12 h under the condition of 65-75 DEG C and 700-900 rpm stirring, centrifuging for 10-20 min under the condition of 9000-11000 rpm after cooling to room temperature, washing the precipitate with ethanol, and vacuum constant-temperature drying for 10-15 h under the condition of 45-55 DEG C, to obtain the modified silica gel chromatographic filler; the preparation method of the mercapto-functionalized siloxane microspheres comprises: dispersing methyl trimethoxysilane in deionized water, stirring for 20-40 min under the condition of 18-20 DEG C and 70-900 rpm stirring, adding ammonia water, stirring for 1-3 min, adding 3-mercaptopropyl trimethoxysilane, standing for 5-6 h, centrifuging for 10-20 min under the condition of 9000-11000 rpm, washing the precipitate with ethanol, and vacuum constant-temperature drying for 10-15 h under the condition of 75-85 DEG C, to obtain the mercapto-functionalized siloxane microspheres. The degree of substitution of the RINK AMIDE-AM resin is 0.6-1.2 mmol / g. The deprotection solution comprises piperidine and N,N-dimethylformamide, and the volume ratio of the piperidine and N,N-dimethylformamide is 10-30:

80. The acetylation solution comprises acetic anhydride and pyridine, and the volume ratio of the acetic anhydride and pyridine is 15-45:

30. The cleavage solution comprises one or more of trifluoroacetic acid, triisopropylsilane, benzyl mercaptide and 1,2-ethanedithiol. The precipitant comprises one or more of methyl tert-butyl ether, diethyl ether and petroleum ether. The mass ratio of the N-(benzo[D]thiazol-2-ylmethyl) acrylamide and the mercapto-functionalized siloxane microspheres is 0.5-4:

20. The mass ratio of the methyl 2-acrylamido-2-methoxyacetate and the mercapto-functionalized siloxane microspheres is 0.5-4:

20.

2. The method for solid phase synthesis of acetyl hexapeptide-8 according to claim 1, characterized in that, ​ 3. The method for solid phase synthesis of acetyl hexapeptide-8 according to claim 1, characterized in that, ​ 4. The method for solid phase synthesis of acetyl hexapeptide-8 according to claim 1, characterized in that, ​ 5. The method for solid phase synthesis of acetyl hexapeptide-8 according to claim 1, characterized in that, ​ 6. The method for solid phase synthesis of acetyl hexapeptide-8 according to claim 1, characterized in that, ​ 7. The method of solid phase synthesis of acetyl hexapeptide-8 according to claim 1, characterized in that, ​ 8. The method for solid phase synthesis of acetyl hexapeptide-8 according to claim 1, characterized in that, ​

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