Method for synthesizing acetyl tetrapeptide-9

Abstract

The invention provides a method for synthesizing acetyl tetrapeptide-9, which comprises the following steps: taking a plurality of solid-phase synthetic resins, and sequentially carrying out deprotection and coupling reaction on the plurality of solid-phase synthetic resins to obtain a peptide chain, the solid-phase synthetic resins being amino acid derivatives pre-loaded with 9-fluorenylmethoxycarbonyl protection; cracking and separating the peptide chain, and drying to obtain a crude target peptide; loading the crude target peptide to a first liquid chromatographic column, and carrying out first gradient elution on the crude target peptide by using a flowing phase of an eluent composed of a phase A and a phase B to obtain an eluent fraction; the eluent fraction is loaded to a second liquid chromatographic column, an eluent composed of an A1 phase, an A2 phase and a B phase is used for second gradient salt conversion elution, purified target peptide is obtained after concentration and freeze-drying, the target peptide is acetyl tetrapeptide-9, deep graded removal of impurities can be achieved, the separation efficiency and the resolution ratio can be improved, and the purity of the target peptide is improved. And the product purity and yield are improved.

Description

Technical Field

[0001] This invention relates to the field of peptide synthesis technology, and in particular to a method for synthesizing acetyl tetrapeptide-9. Background Technology

[0002] Acetyl tetrapeptide-9, an active ingredient with anti-wrinkle and firming effects, has broad application prospects in the high-end cosmetics field. However, its traditional chemical synthesis and purification processes have significant bottlenecks, restricting its industrialization. While traditional liquid-phase peptide synthesis methods can be used for fragment condensation, the steps are cumbersome and the overall yield is low, making it difficult to meet industrial needs. Furthermore, single-pass chromatographic separation cannot simultaneously achieve high resolution and high recovery rates. Summary of the Invention

[0003] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention provides a method for synthesizing acetyl tetrapeptide-9, which improves separation efficiency, resolution, product purity, and yield.

[0004] To achieve the above objectives, this application provides a method for synthesizing acetyl tetrapeptide-9, comprising: Multiple solid-phase synthetic resins were taken, and the multiple solid-phase synthetic resins were subjected to deprotection and coupling reactions in sequence to obtain peptide chains. The solid-phase synthetic resins were amino acid derivatives pre-loaded with 9-fluorenemethyloxycarbonyl groups. The peptide chain was cleaved, separated, and dried to obtain the crude target peptide. The crude target peptide is loaded onto a first liquid chromatography column, and the crude target peptide is eluted using a first gradient elution with a flow of eluent composed of phase A and phase B to obtain an eluent fraction. The eluent fraction was loaded onto a second liquid chromatography column, and a second gradient salt elution was performed using an eluent consisting of phases A1, A2, and B. After concentration and lyophilization, the purified target peptide was obtained, which is acetyl tetrapeptide-9.

[0005] In one possible embodiment, phase B of the mobile phase is acetonitrile, methanol, or ethanol; and phase A is an aqueous solution of trifluoroacetic acid. The first gradient elution is a linear gradient, the proportion of phase B increases from 0% to 12%, the proportion of phase A decreases from 100% to 88%, and the gradient run time is 0 to 65 minutes.

[0006] In one possible embodiment, the second gradient elution includes salt conversion elution and equilibrium elution, wherein the A1 phase is used for the salt conversion elution and the A2 phase is used for the equilibrium elution; the B phase of the mobile phase is acetonitrile, methanol, or ethanol; the A1 phase is an aqueous solution of ammonium acetate and the A2 phase is an aqueous solution of acetic acid.

[0007] In one possible embodiment, during the salt-elution process, the proportion of phase B is 0, the proportion of phase A1 is 100%, and the gradient run time is 0 to 14 minutes. During the equilibrium elution process, the proportion of phase B increases from 0% to 20%, and the proportion of phase A2 decreases from 100% to 80%, with a gradient run time of 0 to 95 minutes.

[0008] In one possible embodiment, the liquid chromatography column comprises a 300 DAC dynamic column; a wavelength of 230 nm; a Daiso-C18-8-100 packing material; and a flow rate of 1600 ml / min.

[0009] In one possible embodiment, the lysis and separation process includes: adding the peptide chain and lysis buffer to a lysis vessel for lysis, allowing it to stand to obtain a product to be separated, performing a separation operation on the product to be separated to obtain a separated product, and verifying the pH of the separated product until the pH meets a preset pH value, including pH=4.5; the lysis buffer includes a lysis system composed of isopropyl ether, trifluoroacetic acid and water.

[0010] In one possible embodiment, the pyrolysis includes primary pyrolysis and secondary pyrolysis. The primary pyrolysis is performed at a temperature of 15°C for 10 minutes. The secondary pyrolysis is performed at a temperature of 23°C to 28°C for 120 minutes. The time between the primary pyrolysis and the secondary pyrolysis does not exceed 20 minutes.

[0011] In one possible embodiment, the separation product comprises a filter and an isopropyl ether washing solution, wherein the filter is the undried crude target peptide, and the separation operation comprises: centrifuging and / or filtering the product to be separated to obtain a crude peptide; adding isopropyl ether to the crude peptide for washing and filtration to obtain the filter and the isopropyl ether washing solution.

[0012] In one possible embodiment, the plurality of solid-phase synthetic resins include Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH and 2-CTC resin.

[0013] In one possible embodiment, the step of sequentially deprotecting and coupling the plurality of solid-phase synthetic resins to obtain peptide chains includes: Add the solid-phase synthetic resin to the swelling agent, and add the condensing agent and activator to dissolve and cool. The cooling temperature is -10℃ and the cooling time is 20-30 minutes. The condensing agent is added to the cooled solid-phase synthetic resin and the system of the condensing agent and activator to dissolve, coupled for 2-5.5 hours and washed to obtain the peptide chain.

[0014] By implementing the embodiments of this application, various solid-phase synthetic resins are taken, and the various solid-phase synthetic resins are sequentially subjected to deprotection and coupling reactions to obtain peptide chains. The solid-phase synthetic resins are amino acid derivatives pre-loaded with 9-fluorenemethyloxycarbonyl groups for protection. The peptide chains are cleaved, separated, and dried to obtain crude target peptides. The crude target peptides are loaded onto a first liquid chromatography column, and the crude target peptides are eluted using a first gradient elution with a mobile phase consisting of phases A and B to obtain an eluent fraction. The eluent fractions are loaded onto a second liquid chromatography column, and a second gradient salt-transfer elution is performed using an eluent consisting of phases A1, A2, and B. After concentration and lyophilization, the purified target peptide is obtained. The target peptide is acetyl tetrapeptide-9, which can achieve deep fractional removal of impurities, improve separation efficiency and resolution, and increase product purity and yield.

[0015] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the background art, the accompanying drawings used in the embodiments of the present invention or the background art will be described below.

[0017] Figure 1 This is a schematic flowchart of a method for synthesizing acetyl tetrapeptide-9 provided in an embodiment of this application; Figure 2 This is a schematic diagram of the synthesis and purification process of acetyl tetrapeptide-9 provided in the embodiments of this application. Detailed Implementation

[0018] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0019] The terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish different objects, not to describe a specific order. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or electronic device that includes a series of steps or units is not limited to the listed steps or units, but in an alternative example also includes steps or units not listed, or in an alternative example also includes other steps or units inherent to these processes, methods, products, or electronic devices.

[0020] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0021] Acetyl tetrapeptide-9 is a bioactive peptide commonly used in cosmetics. Its sequence is typically Ac-Gln-Asp-Val-His or a similar structure, and it possesses effects such as promoting collagen synthesis, improving skin barrier function, and anti-wrinkle properties. With its increasingly widespread application in skincare products, efficient, economical, and environmentally friendly synthesis and purification methods have become a research focus in related fields. While traditional liquid-phase peptide synthesis methods can be used for fragment condensation, the procedures are cumbersome and the overall yield is low, making it difficult to meet industrial-scale demands.

[0022] To address the aforementioned problems, this application provides a method for synthesizing acetyl tetrapeptide-9, which enables deep fractional removal of impurities, improves separation efficiency and resolution, and increases product purity and yield. The following is a detailed description of this application.

[0023] Please see Figure 1 , Figure 1 This is a schematic flowchart of a method for synthesizing acetyl tetrapeptide-9 provided in an embodiment of this application, as shown below. Figure 1 As shown, the method includes: S110, take a variety of solid-phase synthetic resins, and perform deprotection and coupling reactions on the various solid-phase synthetic resins in sequence to obtain peptide chains, wherein the solid-phase synthetic resins are amino acid derivatives pre-loaded with 9-fluorenemethyloxycarbonyl groups.

[0024] In this embodiment, the 9-fluorenemethyloxycarbonyl group is Fmoc. The various solid-phase synthetic resins used specifically refer to a group of 2-chlorotriphenylmethyl chloride resins that have been covalently linked to specific Fmoc-protected amino acids. Specifically, these include: Fmoc-His(Trt)-OH, whose C-terminus is fixed to the resin; this C-terminus is a carboxyl group, the amino group is protected by Fmoc, and the imidazole group of histidine's side chain is protected by triphenylmethyl (Trt) to prevent side reactions during coupling. Fmoc-Val-OH, the α-amino group of valine is protected by Fmoc, and its side chain is an inert alkyl group, requiring no additional protection. Fmoc-Asp(OtBu)-OH, the carboxyl group of aspartic acid's side chain is protected by tert-butyl ester (OtBu) to prevent incorrect linkage with the growing peptide chain. Fmoc-Gln(Trt)-OH, the amide group of glutamine's side chain is protected by Trt to avoid the formation of byproducts under strong acid conditions. Using 2-CTC resin as a universal carrier, its advantage lies in its ability to achieve efficient peptide chain cleavage under mild acidic conditions, and the resulting target peptide has a free carboxylic acid at its C-terminus, which helps to form the target sequence, which is Ac-Gln-Asp-Val-His, with a free carboxylic acid at the C-terminus, namely the sequence of acetyl tetrapeptide-9.

[0025] In this synthesis, the four resins loaded with different starting amino acids were placed in multiple independent reaction columns or reactors for parallel operation. In each synthesis cycle, each resin was first treated with a 20% piperidine N,N-dimethylformamide solution to remove the outermost Fmoc protecting group at the N-terminus of the peptide chain, exposing the free α-amino group and preparing for the next coupling step. After each deprotection and coupling reaction, resin samples were rapidly tested using the ninhydrin test or bromophenol blue indicator to verify the completeness of the reaction in real time. Through these cycles, Val, Asp, and Gln were sequentially linked to the selected main-chain resin to form a fully side-chain protected peptide resin. In this embodiment, the main-chain resin was Fmoc-His(Trt)-OH resin. This improved the synthetic flexibility and resource utilization.

[0026] In one possible embodiment, the step of sequentially deprotecting and coupling the various solid-phase synthetic resins to obtain peptide chains includes: adding a swelling agent to the solid-phase synthetic resin, and adding a condensing agent and an activator to dissolve and cool the resin at a temperature of -10°C for 20-30 minutes; adding the condensing agent to the cooled solid-phase synthetic resin and the system of the condensing agent and activator to dissolve the resin, coupling for 2-5.5 hours and washing to obtain the peptide chains.

[0027] In each coupling round, the solid-phase synthetic resin loaded with Fmoc-protected amino acids used in that round is placed in a solid-phase synthesis reactor, and a sufficient amount of swelling agent is added to fully swell the resin. The swelling agent can be dichloromethane or N,N-dimethylformamide to expose the internal reaction sites, ensuring that subsequent reagents can fully contact all functional groups. A condensing agent and an activator are added to the system and dissolved. The activator can be hydroxybenzotriazole (HOBt), and in some cases, 1-hydroxy-7-azabenzotriazole (HOAt) can also be selected. Before adding the condensing agent, the entire system containing the resin and activator is cooled to -10°C and maintained at this temperature for 20 to 30 minutes. After the system has fully cooled and stabilized, the condensing agent is added to the low-temperature system and dissolved. The condensing agent can include diisopropylcarbodiimide (DIC), etc. The condensing agent and activator work synergistically to undergo a coupling reaction with the terminal amino groups of the resin that have been deprotected from the Fmoc group, under conditions maintained at low temperature throughout the process. The coupling time was set to 2 to 5.5 hours. After the reaction, the resin was thoroughly washed with solvents such as DMF, DCM, and methanol to completely remove unreacted reagents, byproducts, and solvents, yielding a peptide chain-resin complex with the next amino acid linked to it. This process maximizes the suppression of side reactions, improves coupling efficiency, and ensures the accuracy of the final peptide chain sequence.

[0028] After the coupling reaction, end-capping is performed, similar to the coupling reaction process. Finally, acetic anhydride is added for deprotection, washing, and verification of complete reaction.

[0029] In one possible embodiment, the amount of material fed in step S110 can be referenced as shown in Table 1 below: Table 1

[0030] S120, the peptide chain is cleaved, separated, and dried to obtain crude target peptide.

[0031] Among them, the cleavage separation achieves efficient and selective dissociation of peptide chains from resin carriers through precisely controlled cleavage reaction system and conditions, while removing all side chain protecting groups, and obtaining crude solid products suitable for purification through subsequent processing.

[0032] In the separation process, the capped peptide chain is added to a lysis buffer for lysis. The lysis buffer can be a mixed solution with trifluoroacetic acid as the main component and a specific scavenging agent. For example, a mixed solution with a volume ratio of trifluoroacetic acid:water:triisopropylsilane:1,2-ethanedithiol = 92.5:2.5:2.5:2.5 can be used. Trifluoroacetic acid can cleave the ester bond between the peptide chain and 2-chlorotriphenylmethyl chloropolymer, generating a peptide with a free carboxylic acid at the C-terminus, and completely remove all acid-labile side-chain protecting groups, such as His's Trt and Asp's OtBu. 1,2-ethanedithiol is a thiol scavenger that can remove the triphenylmethyl cation generated by the cleavage of the Trt protecting group; triisopropylsilane can rapidly capture the triphenylmethyl and other carbocations released by the trifluoroacetic acid, preventing them from attacking the side chains of sensitive amino acids in the peptide chain and avoiding side reactions such as alkylation.

[0033] The cleavage reaction process includes: transferring the synthesized peptide resin complex, after thorough washing and drying, into a dedicated cleavage reactor. A pre-prepared cleavage solution, cooled to a low temperature, is added, followed by stirring at room temperature. The reaction time is typically controlled to 2-3 hours. This mild temperature and sufficient time window ensure complete cleavage and deprotection reactions while minimizing peptide chain degradation or side reactions that may be caused by prolonged exposure to strong acid.

[0034] After lysis, trifluoroacetic acid can be used for washing and separation to obtain crude peptide products.

[0035] In one possible embodiment, the lysis and separation process includes: adding the peptide chain and lysis buffer to a lysis vessel for lysis, allowing it to stand to obtain a product to be separated, performing a separation operation on the product to be separated to obtain a separated product, and verifying the pH of the separated product until the pH meets a preset pH value, including pH=4.5; the lysis buffer includes a lysis system composed of isopropyl ether (Tis), trifluoroacetic acid (TFA), and water (H2O).

[0036] Please refer to Table 2 below for the preparation ratio of the lysis buffer: Table 2

[0037] The separation process can be sedimentation followed by filtration. The filtration can be performed using a membrane or a centrifuge, and there are no specific limitations on this.

[0038] The process of verifying the pH of the separated product until it meets the preset pH value includes: adding an appropriate amount of room temperature isopropyl ether to the separated product, stirring and breaking it into a suspension, and repeating centrifugation or filtration until the pH of the product isopropyl ether washing solution is 4-5.

[0039] In one possible embodiment, the pyrolysis includes primary pyrolysis and secondary pyrolysis. The primary pyrolysis is performed at a temperature of 15°C for 10 minutes. The secondary pyrolysis is performed at a temperature of 23°C to 28°C for 120 minutes. The time between the primary pyrolysis and the secondary pyrolysis does not exceed 20 minutes.

[0040] The first pyrolysis involves a first pyrolysis at a low temperature of 15°C for 10 minutes, followed by a second pyrolysis at a temperature range of 23°C to 28°C for 120 minutes.

[0041] For example, a possible specific embodiment of the lysis separation process is provided: Pre-frozen lysis buffer is added to a lysis vessel, and peptide resin is slowly added while stirring, maintaining the temperature below 10°C. After addition, the temperature of the lysis vessel is maintained below 15°C for the first 10 minutes, then increased to 23-28°C within 20 minutes, and maintained at 23-28°C for 120 minutes. After lysis, the lysis buffer is filtered, and the resin is washed twice with 1.6L TFA, each time for 3 minutes. The lysis buffers are combined and concentrated to 1 / 3 volume. During concentration, the water bath temperature is 30-35°C, and then 8 times the volume of pre-frozen isopropyl ether precipitate is added while stirring. The precipitate is allowed to stand for at least 20 minutes. If obvious stratification occurs in the mixture, the supernatant is removed. The lower layer is shaken and mixed, then filtered, centrifuged, or both simultaneously. Regardless of centrifugation or filtration, the crude peptide is washed until the pH is between 4 and 5. The filter membrane is made of PP material, and the centrifuge speed is 3500 rpm for 3 minutes. The above times can be adjusted according to the specific equipment. During washing, whether by centrifugation or filtration, the following procedure should be followed: Add an appropriate amount of room-temperature isopropyl ether to the sample product obtained after filtration or centrifugation, stir to break it into a suspension, and repeat centrifugation or filtration until the pH of the isopropyl ether washing solution is 4-5. Place the obtained crude peptide in a drying tray, control the vacuum to below -0.08 MPa, maintain the desiccator temperature at 15-30℃, dry for >1 hour, ensure the vacuum gauge reading is ≤-0.1 and the sample thickness is <3 cm, and obtain the crude product by vacuum drying. Sample the crude peptide for testing to determine its purity, estimate the net content of the crude peptide, label it, and store it at -20℃.

[0042] In one possible embodiment, phase B of the mobile phase is acetonitrile, methanol, or ethanol; phase A is an aqueous solution of trifluoroacetic acid; the first gradient elution is a linear gradient, the proportion of phase B increases from 0% to 12%, the proportion of phase A decreases from 100% to 88%, and the gradient run time is 0 to 65 minutes.

[0043] For the gradient elution gradients used in the purification analysis, please refer to Table 3 below: Table 3

[0044] The solution can be prepared by accurately weighing an appropriate amount of acetyl tetrapeptide-9 reference standard, dissolving it in water, quantifying it, and diluting it to prepare a solution with a concentration of approximately 1 mg / mL. The injection volume is 10 μL (peak height approximately 900).

[0045] For the test solution, inject 2 μL directly before and after the peak of the distillate, inject 1 μL at the peak top, and inject 5-10 μL after diluting the sample 1 50 times. The peak height and peak area after injection should not differ much from those of the reference sample.

[0046] The parameters of the C18 chromatographic column are: inner diameter 2.1~4.6 mm, column length 100~250 mm, and packing particle size 3~5 μm. In this embodiment, the preferred parameters are an inner diameter of 4.6 mm, a column length of 250 mm, and a packing particle size of 5 μm.

[0047] Thus, the reversed-phase C18 column and the mobile phase system containing trifluoroacetic acid are adapted to the hydrophobicity and ionization characteristics of acetyl tetrapeptide, which can effectively separate the target product from the synthesis byproducts; gradient elution not only ensures baseline separation of acetyl tetrapeptide from adjacent impurities, but also shortens the analysis cycle.

[0048] S130, the crude target peptide is loaded onto the first liquid chromatography column, and the crude target peptide is eluted using a first gradient elution with a flow of eluent composed of phase A and phase B to obtain the eluent fraction.

[0049] The crude target peptide was dissolved in a small amount of phase A and filtered before being loaded onto the first liquid chromatography column. The first liquid chromatography column could be a 300 DAC dynamic column, packed with Daiso-C18-8-100 material, with a wavelength of 230 nm and a flow rate of 1600 ml / min. High-throughput, high-capacity preliminary separation was achieved through gradient elution on the first liquid chromatography column, rapidly removing most impurities whose physicochemical properties differed significantly from the target peptide.

[0050] Before loading the sample, a pre-loading equilibration is performed. Specifically, a gradient equilibration is conducted between the aqueous phase and the acetonitrile phase. The core purpose is to ensure that the stationary phase on the surface of the chromatographic column packing reaches a stable hydrophobic state, eliminating interference from residual mobile phase or impurities from the previous experiment, and guaranteeing the consistency of target analyte retention time and the stability of resolution. As a preferred example, see Table 4 below, which shows the elution time and gradient during the pre-loading equilibration process: Table 4

[0051] During sample loading, the amount of arginine peptides in the crude product is first determined; a certain amount of crude product corresponding to the amount of arginine peptides is taken, dissolved in water by ultrasonication, filtered through a 0.45μm polypropylene membrane, and then loaded onto the sample.

[0052] In one possible embodiment, phase B of the mobile phase is acetonitrile, methanol, or ethanol; phase A is an aqueous solution of trifluoroacetic acid; the first gradient elution is a linear gradient, the proportion of phase B increases from 0% to 12%, the proportion of phase A decreases from 100% to 88%, and the gradient run time is 0 to 65 minutes.

[0053] For example, please refer to Table 5, which shows an example of the elution gradient and elution time in this embodiment: Table 5

[0054] Based on the test results, the purity of the main peak of different samples is determined and corresponding sample processing is performed. For sample 1, if the purity of the main peak is >98.5%, it will proceed to the two-step salt conversion process; for sample 2, if the purity of the main peak is >85% and <98.5%, it will be treated as tailings and recycled in one step.

[0055] After the first gradient elution, column washing is performed with the following parameters: water (H2O) and acetonitrile (ACN) are used as washing solvents, and the solvent volume ratio is kept constant throughout the washing process, with water accounting for 20% and acetonitrile for 80%. The column is continuously washed for 15 minutes at this ratio. This ensures that the solvent ratio in this washing step is close to that of mobile phase B in the purification process, guaranteeing both elution efficiency and consistency with the overall purification process solvent system. This avoids introducing new reagents and meets the requirements of process continuity and economy.

[0056] S140, the eluent fraction is loaded onto the second liquid chromatography column, and a second gradient salt conversion elution is performed using an eluent consisting of phases A1, A2 and B. After concentration and lyophilization, the purified target peptide is obtained, which is acetyl tetrapeptide-9.

[0057] The second liquid chromatography column used a 300 DAC dynamic column with a wavelength of 230 nm; the packing material was Daiso-C18-8-100; and the flow rate was 1600 ml / min.

[0058] Optionally, phase B is acetonitrile, methanol, or ethanol. Preferably, in this embodiment, phase A1 is an aqueous solution of ammonium acetate (NH4Ac), and phase A2 is an aqueous solution of acetic acid (HAc); specifically, phase A1 is an 8 g / L aqueous solution of ammonium acetate, phase A2 is a 0.03% aqueous solution of acetic acid, and phase B is acetonitrile (ACN).

[0059] Before loading the sample, a pre-loading equilibration is performed. Specifically, a gradient equilibration is conducted between the aqueous phase and the acetonitrile phase. The core purpose is to ensure that the stationary phase on the surface of the chromatographic column packing reaches a stable hydrophobic state, eliminating interference from residual mobile phase or impurities from the previous experiment, and guaranteeing the consistency of target analyte retention time and the stability of resolution. As a preferred example, see Table 6 below, which shows the elution time and gradient during the pre-loading equilibration process: Table 6

[0060] After the sample is equilibrated, the sample 1 obtained in step S130 is subjected to nanofiltration, preferably nanofiltration to a concentration of about 30 g / L.

[0061] After sample loading, a salt conversion process is initiated, using phases A1 and B as the solvent system. The solvent ratio is maintained constant from 0 to 15 minutes throughout the conversion process, with phase A1 comprising 100% and phase B 0%. This ratio is maintained for 15 minutes. Following salt conversion, equilibrium elution is performed using phases A2 and B as the solvent system. The solvent ratio is maintained constant from 0 to 15 minutes throughout the conversion process, with phase A2 comprising 100% and phase B 0%. From 15 to 95 minutes, the initial ratio is linearly adjusted to 80% for phase A2 and 20% for phase B, and this ratio is maintained for the subsequent elution process. After elution, based on the obtained main peak purity, the sample consolidation criteria and corresponding subsequent processing requirements are determined: For sample consolidation 3, if the main peak purity is >99%, the consolidated solution is sent to the concentration and freeze-drying process; for sample consolidation 4, if the main peak purity is >90%, the consolidated solution is treated as waste material for recycling. After completing the above elution and sample merging operations, column washing is performed using water (H2O) and acetonitrile (ACN) as washing solvents. The solvent volume ratio is kept constant from 0 to 15 minutes throughout the entire column washing process, with water accounting for 50% and acetonitrile accounting for 50%. The column is continuously washed for 15 minutes in this ratio.

[0062] For example, the concentration process includes nanofiltration concentration, specifically including: loading sample 3 into the 120L tank of a nanofiltration machine for nanofiltration, using a nanofiltration membrane or reverse osmosis membrane with a specification model within 200, controlling the refrigerant temperature at -5-0℃, the frequency at 20Hz, the pressure at 0.5-1Mpa, the flow rate at 1200L / h, nanofiltration to a sample concentration of approximately 100g / L, releasing it in one go and labeling the content; Take 20L of the last nanofiltration permeate and add it to the feed tank, then add 20L of water. Circulate the nanofiltration solution until it reaches 18-20L, then release it a second time and label the content. Then rinse the nanofiltration machine with 20L of water, circulate it again, and release it a third time. Combine the first and second release solutions. If the content of the third release solution is 2% of the total, it can be mixed into the combined solution; otherwise, concentrate and freeze-dry it separately. Filter the combined solution under pressure using a 0.22μm water film. After filtration, label the content and freeze-dry it. The freeze-drying concentration is 50±15g / L.

[0063] For example, the freeze-drying process includes: sterilizing all necessary instruments and equipment at high temperature before freeze-drying; evenly loading the filtered sample into freeze-drying trays, with each tray containing approximately 1-1.2 L of sample; please refer to Table 7, which shows the freeze-drying curve data for the freeze-drying process: Table 7

[0064] Thus, by combining first-gradient elution and second-gradient elution, the overall purification yield is maximized, and by converting to a milder, less volatile acetate form, the storage stability, safety, and application performance of the target peptide are significantly improved.

[0065] In summary, please refer to Figure 2 , Figure 2 This is a schematic diagram of the synthesis and purification process of acetyl tetrapeptide-9 provided in the embodiments of this application, as shown below. Figure 2 As shown, the peptide resin synthesis involves sequential coupling of amino acids to generate the peptide resin; crude product acquisition involves cleaving the peptide resin to obtain the crude product; crude product quality inspection and processing involves first testing the crude product, followed by preparation operations to obtain a qualified sample product; concentration and quality inspection involves concentrating the qualified sample product to obtain a concentrated solution, which is then tested; freeze-drying and finished product processing involves freeze-drying and mixing the concentrated solution to obtain a freeze-dried powder, which is finally tested to form the finished product, namely the target peptide acetyl tetrapeptide-9.

[0066] As can be seen, by implementing the embodiments of this application, multiple solid-phase synthetic resins are taken, and the multiple solid-phase synthetic resins are sequentially subjected to deprotection and coupling reactions to obtain peptide chains. The solid-phase synthetic resins are pre-loaded with amino acid derivatives protected by Fmoc. The peptide chains are cleaved, separated, and dried to obtain crude target peptides. The crude target peptides are loaded onto a first liquid chromatography column, and the crude target peptides are eluted using a first gradient elution with a mobile phase consisting of phases A and B to obtain an eluent fraction. The eluent fractions are loaded onto a second liquid chromatography column, and the crude target peptides are eluted using a second gradient salt conversion elution with a mobile phase consisting of phases A1, A2, and B. After concentration and lyophilization, the purified target peptide is obtained. The target peptide is acetyl tetrapeptide-9, which can achieve deep fractional removal of impurities, improve separation efficiency and resolution, and increase product purity and yield.

[0067] It should be noted that, based on the steps of the above embodiments, it is possible to achieve a purity ≥99%, moisture ≤8%, acetic acid ≤5%, TFA ≤1.0%, peptide content ≥85%, and pH 4.0-6.0 when synthesizing 2kg of acetyl tetrapeptide-9 product.

[0068] It should be noted that, for the sake of simplicity, the foregoing method embodiments are all described as a series of actions. However, those skilled in the art should understand that this application is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to this application. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to this application.

[0069] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0070] The embodiments of this application have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A method for synthesizing acetyl tetrapeptide-9, characterized in that the method... The application relates to a method for preparing a target peptide. The method comprises the following steps: taking a plurality of solid-phase synthesis resins, and sequentially performing deprotection and coupling reaction on the plurality of solid-phase synthesis resins to obtain a peptide chain, wherein the solid-phase synthesis resins are pre-loaded with 9-fluorenylmethyloxycarbonyl-protected amino acid derivatives; performing cleavage separation and drying on the peptide chain to obtain a crude target peptide; loading the crude target peptide into a first liquid chromatography column, and performing first gradient elution on the crude target peptide by using a mobile phase formed by an eluent of A phase and B phase to obtain an eluent fraction; loading the eluent fraction into a second liquid chromatography column, and performing second gradient salt conversion elution by using an eluent formed by A1 phase, A2 phase and B phase, and then performing concentration and freeze-drying to obtain a purified target peptide, wherein the target peptide is acetyl tetrapeptide-9.

2. The method of claim 1, wherein, The B phase of the mobile phase is acetonitrile, methanol or ethanol; and the A phase is trifluoroacetic acid aqueous solution. The first gradient elution is linear gradient, the proportion of the B phase is increased from 0% to 12%, the proportion of the A phase is decreased from 100% to 88%, and the gradient operation time is 0-65 minutes.

3. The method of claim 1, wherein, The second gradient elution comprises salt conversion elution and balance elution, the A1 phase is used for the salt conversion elution, and the A2 phase is used for the balance elution; the B phase of the mobile phase is acetonitrile, methanol or ethanol; the A1 phase is ammonium acetate aqueous solution, and the A2 phase is acetic acid aqueous solution.

4. The method of claim 3, wherein, During the salt conversion elution, the proportion of the B phase is 0, the proportion of the A1 phase is 100%, and the gradient operation time is 0-14 minutes. During the balance elution, the proportion of the B phase is increased from 0% to 20%, the proportion of the A2 phase is decreased from 100% to 80%, and the gradient operation time is 0-95 minutes.

5. The method according to any one of claims 1 to 4, characterized in that, The liquid chromatography column comprises a 300 DAC dynamic column, a wavelength of 230 nm, Daiso-C18-8-100 filler and a flow rate of 1600 ml / min.

6. The method of claim 1, wherein, The cleavage separation process comprises the following steps: adding the peptide chain and a cleavage solution into a cleavage kettle for cleavage, standing to obtain a to-be-separated product, performing separation operation on the to-be-separated product to obtain a separated product, and performing solution pH value verification on the separated product until the pH value meets a preset pH value, wherein the pH value is 4.5; and the cleavage solution comprises a cleavage system composed of isopropyl ether, trifluoroacetic acid and water.

7. The method according to claim 1 or 6, characterized in that, The cleavage comprises primary cleavage and secondary cleavage, the primary cleavage is performed at a cleavage temperature of 15 DEG C for 10 minutes, the secondary cleavage is performed at a temperature of 23 DEG C to 28 DEG C for 120 minutes, and the time between the primary cleavage and the secondary cleavage is not more than 20 minutes.

8. The method of claim 1 or 6, wherein, The separated product comprises a filtrate and an isopropyl ether washing solution, the filtrate is the crude target peptide without drying, and the separation operation comprises the following steps: performing centrifugal operation and / or filtration operation on the to-be-separated product to obtain a crude peptide; and adding isopropyl ether into the crude peptide for washing and filtration to obtain the filtrate and the isopropyl ether washing solution.

9. The method of claim 1, wherein, The plurality of solid-phase synthesis resins include Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH and 2-CTC resin.

10. The method according to claim 1 or 9, characterized in that, The plurality of solid-phase synthesis resins are sequentially subjected to deprotection and coupling reactions to obtain a peptide chain. The solid-phase synthesis resin is added with a swelling agent, and a condensing agent and an activating agent are dissolved and cooled, with a cooling temperature of -10°C and a cooling time of 20-30 minutes. The condensing agent is added to the cooled solid-phase synthesis resin and the condensing agent and activating agent system to dissolve, and coupling is performed for 2-5.5 hours and washing is performed to obtain the peptide chain.

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