Preparation method of polypeptide

Abstract

The invention relates to the field of bioengineering, in particular to a preparation method of polypeptide. The invention provides a preparation method of polypeptide, which comprises the following steps: coupling initial amino acid with resin, carrying out first acetylation, filtering and washing to obtain coupled resin; performing deprotection on the coupling resin, and sequentially coupling pre-activated amino acids from the C end to the N end to obtain polypeptide resin; the polypeptide resin is subjected to second acetylation, splitting decomposition, purification and concentration, and then the polypeptide is obtained; the starting amino acid comprises: Smoc-Tyr (tBu)-OH; the amino acids comprise Smoc-Val-OH, Smoc-Asp (OtBu)-OH and Smoc-Lys (Boc)-OH, and the amino acids comprise Smoc-Val-OH, Smoc-Asp (OtBu)-OH and Lys (Boc)-OH. According to the method, the resin with special properties and the water-soluble protected amino acid are adopted, and the advantages of solid-phase synthesis and liquid-phase synthesis are combined, so that a very excellent green synthesis method of the acetyl tetrapeptide-2 is formed.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a method for preparing a polypeptide. Background Art

[0002] Acetyl Tetrapeptide-2 is a synthetic peptide that has been shown to fight skin sagging. It helps stimulate the skin's essential proteins, such as collagen and elastin, promoting and improving cohesion between cells and the ECM (extracellular matrix).

[0003] Acetyl Tetrapeptide-2 lifts the skin and maintains facial contours, activating FBLN5 and LOXL1 glycoproteins, essential components for maintaining the normal organization and function of elastin fibers. Together with collagen molecules and elements of the multiprotein complex, they provide firmness and resistance to the skin. Acetyl Tetrapeptide-2 increases collagen and elastin gene expression, helping to protect against the effects of gravity while restructuring elastin and collagen fibers.

[0004] The main problems of the existing technology for the synthesis of acetyl tetrapeptide-2 are: the synthesis process is complicated, and the steps of synthesis through the 2+2 method are cumbersome; a large amount of solvent is used, and the potential for automated synthesis is low; the pure liquid phase method requires a large amount of organic solvents for the synthesis and purification steps, which causes great environmental pollution; at the same time, the use of the classical solid phase synthesis method still requires the use of a large amount of organic solvents, which also limits the size of the batch, which is generally less than 10 kg and is not suitable for large-scale production. Summary of the invention

[0005] In view of this, the present invention provides a method for preparing a polypeptide. The method uses a resin with special properties and a water-soluble protected amino acid, combines the advantages of solid phase synthesis and liquid phase synthesis, and forms a very excellent green synthesis method for acetyl tetrapeptide-2.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a polypeptide, comprising the following steps:

[0008] S1: coupling the starting amino acid with the resin, first acetylation, filtering, and washing to obtain a coupling resin;

[0009] S2: After deprotecting the coupling resin, the preactivated amino acids are sequentially coupled from the C-terminus to the N-terminus to obtain a polypeptide resin;

[0010] S3: After the polypeptide resin is subjected to a second acetylation, cleavage, purification, and concentration, the polypeptide is obtained;

[0011] The starting amino acids include: Smoc-Tyr(tBu)-OH;

[0012] The amino acids include: Smoc-Val-OH, Smoc-Asp(OtBu)-OH and Smoc-Lys(Boc)-OH.

[0013] In some embodiments of the present invention, in the above preparation method, the resin includes: PEG resin.

[0014] In some embodiments of the present invention, in the above preparation method, the molecular weight of the PEG resin is 4000-8000.

[0015] In some embodiments of the present invention, in the above preparation method, the molecular weight of the PEG resin is 5000-7000.

[0016] In some embodiments of the present invention, in the above preparation method, the molecular weight of the PEG resin is 6000.

[0017] In some embodiments of the present invention, in the above preparation method, both the first acetylation and the second acetylation use acetic anhydride and pyridine.

[0018] In some embodiments of the present invention, in the above preparation method, the concentration of the acetic anhydride is 2.5~5 mol; the concentration of the pyridine is 2.5~5 mol.

[0019] In some embodiments of the present invention, in the above preparation method, the deprotection uses a 1M~3M NaOH solution or a 5%~10% (V / V) piperazine solution.

[0020] In some embodiments of the present invention, in the above preparation method, the deprotection adopts 1M NaOH solution or 5% (V / V) piperazine solution.

[0021] In some embodiments of the present invention, in the above preparation method, the coupling in S1 and S2 uses EDC hydrochloride;

[0022] The molar ratio of the resin, the starting amino acid and the EDC hydrochloride in S1 is: 1:2.4:1.2;

[0023] The molar ratio of the resin, the amino acid and the EDC hydrochloride in S2 is 1:2.4:1.2.

[0024] In some embodiments of the present invention, in the above preparation method, the coupling time is 2 to 4 hours.

[0025] In some embodiments of the present invention, in the above preparation method, the coupling time is 3 hours.

[0026] In some embodiments of the present invention, in the above preparation method, the cleavage uses hydrogen chloride / ethyl acetate solution and TIS.

[0027] In some embodiments of the present invention, in the above preparation method, the volume ratio of the hydrogen chloride / ethyl acetate solution to the TIS is 95:5.

[0028] In some embodiments of the present invention, in the above preparation method, the concentration of the hydrogen chloride / ethyl acetate solution is 4~6N.

[0029] In some embodiments of the present invention, in the above preparation method, the concentration of the hydrogen chloride / ethyl acetate solution is 4N.

[0030] In some embodiments of the present invention, in the above preparation method, the concentration of TIS is 5-10% (V / V).

[0031] In some embodiments of the present invention, in the above preparation method, after obtaining the polypeptide, a salt conversion step may be further included to obtain the acetyl tetrapeptide-2 acetate.

[0032] The beneficial effects of the present invention include:

[0033] This method uses a resin with special properties and a water-soluble protected amino acid, combines the advantages of solid phase synthesis and liquid phase synthesis, and forms a very excellent green synthesis method for acetyl tetrapeptide-2.

[0034] 1. The present invention uses water-soluble resin and amino acid, which greatly reduces the pollution to the environment and the use of organic solvents. On the other hand, the water-soluble characteristic of the resin can be dissolved in water during the reaction to form a homogeneous reaction similar to the liquid phase, thereby improving the accessibility of large-scale production and reducing the proportion of reaction raw materials. The insolubility of the resin in n-hexane enables the resin to obtain a simple advantage similar to solid phase synthesis during separation, making the synthesis method more recommended.

[0035] 2. The method of the present invention takes into account the advantages of both the solid phase and the liquid phase, which can not only expand the single batch volume to form a scale advantage, but also simplify the method and retain the potential for automated synthesis; at the same time, compared with the traditional synthesis method, the greenness of the present invention has been greatly improved; BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0037] Figure 1 Shown is the HPLC detection spectrum of acetyl tetrapeptide-2 (PEG6000). DETAILED DESCRIPTION

[0038] The invention discloses a method for preparing a polypeptide.

[0039] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0040] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0041] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0042] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0043] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0044] The synthetic route of the present invention is to sequentially couple each amino acid on the PEG carrier according to the acetyl tetrapeptide-2 peptide sequence, and the coupled amino acids are Smoc-Tyr(tBu)-OH, Smoc-Val-OH, Smoc-Asp(OtBu)-OH, and Smoc-Lys(Boc)-OH. After the coupling is completed, an acetylation reaction is performed. The carrier is removed by hydrochloric acid to obtain the product;

[0045] The present invention comprises the following steps:

[0046] 1) Select a PEG carrier with a suitable molecular weight;

[0047] 2) Sequentially couple acetyl tetrapeptide-2 in a sequential manner from C-terminus to N-terminus, and remove the carrier with hydrochloric acid to obtain acetyl tetrapeptide-2;

[0048] The PEG carrier described in step 1) has a molecular weight of 4000-8000, preferably 5000-7000, and most preferably 6000.

[0049] The Smoc-AA-OH coupled in the order from C-terminus to N-terminus described in step 2) is Smoc-Tyr(tBu)-OH, Smoc-Val-OH, Smoc-Asp(OtBu)-OH, Smoc-Lys (Boc)-OH, and finally N-terminal acetylation is performed.

[0050] The synthesis method comprises:

[0051] 1) Dissolve PEG resin and Smoc-Tyr(tBu)-OH protected amino acid in water, add coupling agent to react, and make the two react in a homogeneous phase until the reaction is terminated by the detection method. After the first amino acid is coupled, it needs to be treated with an acetylation agent to ensure that all possible residual groups on PEG are blocked. After the reaction is completed, add 2 times the volume of n-hexane in the reaction solution to precipitate the PEG resin, filter the reaction solution, and wash 6 times with a mixture of n-hexane and water = 1:1;

[0052] 2) Remove Smoc, then wash the support with solvent and test to ensure complete removal of Smoc;

[0053] 3) Repeat 1) and 2) until all amino acids are coupled. The reagent for removing Smoc is 1M NaOH aqueous solution or 5%-10% piperazine aqueous solution.

[0054] The coupling agent in step 2) of the present invention is EDC hydrochloride, and in the reaction solvent, the ratio of PEG carrier: amino acid: EDC is 1:2.4:1.2

[0055] Step 2) The coupling reaction time of each amino acid is usually 2 to 4 hours, preferably 3 hours; the pressure is preferably normal pressure, and it can also be carried out under appropriately increased or reduced pressure; the temperature is preferably room temperature (i.e., 20±5°C), and it can also be carried out at appropriately increased or reduced temperature.

[0056] In step 2), the N-terminal acetylation reagents are acetic anhydride and pyridine.

[0057] The lysis solution in step 3) is a mixture of hydrogen chloride / ethyl acetate solution and TIS in different proportions, the concentration of hydrogen chloride / ethyl acetate is 4N~6N, and the proportion of TIS is 5%~10%.

[0058] Step 3) After the cleavage reaction, the cleavage solution is evaporated under reduced pressure, and ethanol is added to fully precipitate acetyl tetrapeptide-2, the polyethylene glycol is removed by filtration, and the mixture is dried. Water is added to dissolve and wash acetyl tetrapeptide-2, and the mixture is freeze-dried to obtain GHK solid. Purification and salt conversion are performed to obtain acetyl tetrapeptide-2 acetate;

[0059] In Examples 1 to 9 of the present invention and the verification examples, all the raw materials and reagents used can be purchased from the market.

[0060] The present invention will be further described below in conjunction with embodiments:

[0061] Example 1 Synthesis of Smoc-Tyr(tBu)-PEG(4000)

[0062] Weigh the compound PEG4000 (0.5 mol) and add it to a 5L three-necked flask. Add 1L of water to the reaction flask and add Smoc-Tyr(tBu)-OH (1.2 mol). Stir to dissolve. Add EDC·HCl (0.6 mol) and continue stirring at room temperature for 3 hours. Then add acetic anhydride and pyridine (5 mol / 5 mol) and continue stirring at room temperature for 3 hours. After the reaction is completed, add 2L of n-hexane to the reaction solution and stir for 2 hours. Filter and rinse the filter cake 6 times with n-hexane: water = 1:1 (1000mL×6). Dry the filter cake at 35°C for 8 hours to obtain the compound Smoc-Tyr(tBu)-PEG(4000) (yield 99.9%).

[0063] Example 2 Synthesis of Smoc-Tyr(tBu)-PEG(6000)

[0064] Weigh the compound PEG6000 (0.5 mol) and add it to a 5L three-necked flask. Add 1L of water to the reaction flask, add Smoc-Tyr(tBu)-OH (1.2 mol), and stir to dissolve. Add EDC·HCl (0.6 mol) and continue stirring at room temperature for 3 hours. Then add acetic anhydride and pyridine (5 mol / 5 mol) and continue stirring at room temperature for 3 hours. After the reaction is completed, add 2L of n-hexane to the reaction solution and stir for 2 hours. Filter and rinse the filter cake 6 times with n-hexane: water = 1:1 (1000mL×6). The filter cake was dried at 35°C for 8 hours to obtain the compound Smoc-Tyr(tBu)-PEG (6000) (yield 99.3%).

[0065] Example 3 Synthesis of Smoc-Tyr(tBu)-PEG(8000)

[0066] Weigh the compound PEG8000 (0.5 mol) and add it to a 5L three-necked flask. Add 1L of water to the reaction flask, add Smoc-Tyr(tBu)-OH (1.2 mol), and stir to dissolve. Add EDC·HCl (0.6 mol) and continue stirring at room temperature for 3 hours. Then add acetic anhydride and pyridine (5 mol / 5 mol) and continue stirring at room temperature for 3 hours. After the reaction is completed, add 2L of n-hexane to the reaction solution and stir for 2 hours. Filter and rinse the filter cake with n-hexane: water = 1:1 (1000mL×6) 6 times. The filter cake was dried by air at 35°C for 8 hours to obtain the compound Smoc-Tyr(tBu)-PEG (8000) (yield 98.9%).

[0067] Example 4 Synthesis of Smoc-Val-Tyr(tBu)-PEG(6000)

[0068] Weigh Smoc-Tyr(tBu)-PEG(6000) (0.25 mol) obtained in Example 2 and add it to a 5L three-necked flask. Add 1M NaOH aqueous solution (1L) to the reaction flask, stir to dissolve, and react for 10 minutes. After the reaction is completed, add 2L of n-hexane to the reaction solution, stir and filter, and rinse the filter cake with n-hexane: water = 1: 1 (1000mL×6) 6 times. Repeat the washing once. Take another reaction flask, add Smoc-Val-OH (0.6mol), stir to dissolve, add EDC·HCl (0.3mol) under ice bath conditions, and react in liquid for 10 minutes. Add the reaction solution to the above filter cake and continue to stir and react at room temperature for 3 hours. Monitor the reaction by TLC (DCM: MeOH: HAc = 100: 1: 0.5). Add 2L of n-hexane to the reaction solution, filter, and rinse the filter cake with n-hexane: water = 1: 1 (1000mL×6) 6 times. The filter cake was dried by forced air at 35° C. for 8 hours to obtain the compound Smoc-Val-Tyr(tBu)-PEG(6000) (yield 99.3%).

[0069] Example 5 Synthesis of Smoc-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000)

[0070] Weigh Smoc-Val-Tyr(tBu)-PEG(6000) (0.25 mol) in Example 4 and add it to a 5L three-necked flask. Add 5% piperazine aqueous solution (1L) to the reaction flask, stir to dissolve, and react for 10 minutes. After the reaction is completed, add 2L of n-hexane to the reaction solution, stir and filter, and rinse the filter cake with n-hexane: water = 1: 1 (1000mL×6) 6 times. Repeat the washing once. Take another reaction flask, add Smoc-Asp(OtBu)-OH (0.6 mol), stir to dissolve, add EDC·HCl (0.3 mol) under ice bath conditions, and react in liquid for 10 minutes. Add the reaction solution to the filter cake and continue stirring at room temperature for 4 hours. Monitor the reaction by TLC (DCM: MeOH: HAc = 100: 1: 0.5). Add 2L of n-hexane to the reaction solution, filter, and rinse the filter cake with n-hexane: water = 1: 1 (1000mL×6) 6 times. The filter cake was dried by forced air at 35° C. for 8 hours to obtain the compound Smoc-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000) (yield 98.5%).

[0071] Example 6 Synthesis of Ac-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000)

[0072] Weigh Smoc-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000) (0.25 mol) in Example 5 and add it to a 5L three-necked flask. Add 10% piperazine aqueous solution (1L) to the reaction flask, stir to dissolve, and react for 10 minutes. After the reaction is completed, add 2L of n-hexane to the reaction solution, stir and filter, and rinse the filter cake with n-hexane: water = 1:1 (1000mL×6) 6 times. Repeat the washing once. Take another reaction flask, add Smoc-Lys(Boc)-OH (0.6 mol), stir to dissolve, add EDC·HCl (0.3 mol) under ice bath conditions, and react in liquid for 10 minutes. Add the reaction solution to the filter cake and continue stirring at room temperature for 2 hours. Monitor the reaction by TLC (DCM: MeOH: HAc = 100: 1: 0.5). 2L of n-hexane was added to the reaction solution, filtered, and the filter cake was rinsed 6 times with n-hexane: water = 1:1 (1000mL×6). The filter cake was air-dried at 35°C for 8 hours to obtain the compound Smoc-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000) (yield 98.5%).

[0073] Add the filter cake to a 5L three-necked flask, add 5% piperazine aqueous solution (1L) to the reaction flask, stir to dissolve, react for 10 minutes, add 2L of n-hexane to the reaction solution, stir and filter, and rinse the filter cake with n-hexane: water = 1: 1 (1000mL×6) 6 times. Repeat the washing once. Add acetic anhydride and pyridine (2.5mol / 2.5mol) and continue stirring for 3 hours at room temperature. After the reaction is completed, add 2L of n-hexane to the reaction solution and stir for 2 hours. Filter, rinse the filter cake with n-hexane: water = 1: 1 (1000mL×6) 6 times. The filter cake was dried at 35°C for 8 hours to obtain the compound Ac-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000) (yield 97.8%).

[0074] Example 7 Preparation of Ac-Lys-Asp-Val-Tyr-COOH

[0075] Ac-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000) (about 0.25 mol) obtained in Example 6 was weighed and added to a 5L reaction round-bottom flask, and then 1L of lysis solution (4N hydrogen chloride / ethyl acetate: TIS=95:5) was added to the reaction flask, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to a viscous substance, ethanol (2L) was added, and the mixture was stirred for 2 hours. The mixture was filtered, and the filter cake was rinsed three times with ethanol (500mL×3). The filter cake was dried at 35°C for 8 hours to obtain 146.7g of acetyl tetrapeptide-2, with a detection purity of 93.6% and a yield of 104.3%.

[0076] Example 8 Preparation of Acetyl Tetrapeptide-2 Acetate

[0077] The crude peptide obtained in Example 7 was purified by reverse phase high performance liquid chromatography. Reverse phase octadecylsilane was used as the stationary phase and 0.1% acetic acid aqueous solution / acetonitrile was used as the mobile phase. The target peak fractions were collected, concentrated and freeze-dried to obtain 124.8 g of pure product, with a total yield of 88.3% and a purity of 99.61%. (As Figure 1 shown)

[0078] Example 9

[0079] Ac-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000) (about 0.25 mol) obtained in Example 6 was weighed and added to a 5L reaction round-bottom flask, and then 1L of lysis solution (TFA:TIS=95:5) was added to the reaction flask, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to form a viscous substance, ethanol (2L) was added, and the mixture was stirred for 2 hours. The mixture was filtered, and the filter cake was rinsed three times with ethanol (500mL×3). The filter cake was dried at 35°C for 8 hours to obtain 101.7g of acetyl tetrapeptide-2, with a detection purity of 79.8% and a yield of 71.9%.

[0080] Verification Example

[0081] Ac-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(4000), Ac-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(8000) and Ac-L The preparation method of ys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(12000) is the same as Ac-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000).

[0082] Table 1

[0083]

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a polypeptide, characterized in that: The steps include: S1: coupling the starting amino acid with the resin, first acetylation, filtering, and washing to obtain a coupling resin; S2: After deprotecting the coupling resin, the preactivated amino acids are sequentially coupled from the C-terminus to the N-terminus to obtain a polypeptide resin; S3: After the polypeptide resin is subjected to a second acetylation, cleavage, purification, and concentration, the polypeptide is obtained; The starting amino acids include: Smoc-Tyr(tBu)-OH; The amino acids include: Smoc-Val-OH, Smoc-Asp(OtBu)-OH and Smoc-Lys(Boc)-OH.

2. The preparation method according to claim 1, characterized in that The resin includes: PEG resin.

3. The preparation method according to claim 2, characterized in that: The molecular weight of the PEG resin is 4000-8000.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The first acetylation and the second acetylation both use acetic anhydride and pyridine.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The deprotection adopts 1M~3M NaOH solution or 5%~10% (V / V) piperazine solution.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The coupling described in S1 and S2 used EDC hydrochloride; The molar ratio of the resin, the starting amino acid and the EDC hydrochloride in S1 is: 1:2.4:1.2; The molar ratio of the resin, the amino acid and the EDC hydrochloride in S2 is 1:2.4:1.

2.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The coupling time is 2 to 4 hours.

8. The preparation method according to any one of claims 1 to 7, characterized in that: The cleavage adopts hydrogen chloride / ethyl acetate solution and TIS.

9. The preparation method according to claim 8, characterized in that: The volume ratio of the hydrogen chloride / ethyl acetate solution to the TIS is 95:5.

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