Preparation methods of polypeptides

Abstract

This invention relates to the field of bioengineering, and more particularly to a method for preparing peptides. The invention provides a method for preparing peptides, comprising the following steps: coupling starting amino acids with a resin, performing a first acetylation, filtering, and washing to obtain a coupling resin; deprotecting the coupling resin, and sequentially coupling pre-activated amino acids from the C-terminus to the N-terminus to obtain a peptide resin; performing a second acetylation, cleavage, purification, and concentration of the peptide resin to obtain the peptide; wherein the starting amino acids include: Smoc-Tyr(tBu)-OH; and the amino acids include: Smoc-Val-OH, Smoc-Asp(OtBu)-OH, and Smoc-Lys(Boc)-OH. This method utilizes a resin with special properties and water-soluble protected amino acids, combining the advantages of solid-phase synthesis and liquid-phase synthesis, forming a highly efficient green synthesis method for acetyl tetrapeptide-2.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to a method for preparing polypeptides. Background Technology

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

[0003] Acetyl tetrapeptide-2 lifts the skin and maintains facial contours by activating FBLN5 and LOXL1 glycoproteins, essential components for maintaining the normal tissue structure and function of elastin fibers. Together with collagen molecules and elements of multi-protein complexes, they provide firmness and resilience to the skin. Acetyl tetrapeptide-2 increases collagen and elastin gene expression, helping to resist the effects of gravity while simultaneously remodeling elastin and collagen fibers.

[0004] The main problems with existing methods for synthesizing acetyl tetrapeptide-2 are: the synthesis process is complex, and the steps of the 2+2 synthesis are cumbersome; a large amount of solvent is used, and the potential for automated synthesis is low; pure liquid phase methods require a large amount of organic solvents for synthesis and purification steps, which causes significant environmental pollution; at the same time, the classical solid phase synthesis method still requires a relatively large amount of organic solvents, which also limits the batch size, 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 peptides. This method utilizes resins with special properties and water-soluble protective amino acids, combining the advantages of solid-phase synthesis and liquid-phase synthesis to form a highly efficient green synthesis method for acetyl tetrapeptide-2.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing polypeptides, comprising the following steps:

[0008] S1: The starting amino acid is coupled with the resin, first acetylated, filtered, and washed to obtain the coupling resin;

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

[0010] S3: The polypeptide resin is second-acetylated, cleaved, purified, and concentrated to obtain the polypeptide;

[0011] The starting amino acid includes: 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, the resin in the above preparation method includes PEG resin.

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

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

[0016] In some embodiments of the present invention, the molecular weight of the PEG resin in the above preparation method 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 acetic anhydride is 2.5~5 mol; the concentration of pyridine is 2.5~5 mol.

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

[0021] In some embodiments of the present invention, in the above preparation method, the coupling in S1 and S2 is performed using 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, the coupling time in the above preparation method is 2-4 hours.

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

[0026] In some embodiments of the present invention, the pyrolysis in the above preparation method uses a 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, the concentration of the hydrogen chloride / ethyl acetate solution in the above preparation method is 4~6N.

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

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

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

[0032] The beneficial effects of this invention include:

[0033] This method utilizes resins with special properties and water-soluble protective amino acids, combining the advantages of solid-phase synthesis and liquid-phase synthesis to form an excellent green synthesis method for acetyl tetrapeptide-2.

[0034] 1. This invention uses water-soluble resin and amino acids, which greatly reduces environmental pollution and the use of organic solvents. On the other hand, the water-soluble nature of the resin allows it to dissolve in water during the reaction, forming a homogeneous reaction similar to a liquid phase, which improves the accessibility of large-scale production and reduces the proportion of reaction raw materials used. The insolubility of the resin in hexane allows for the simpler separation of the resin, similar to solid-phase synthesis, making the synthesis method more convenient.

[0035] 2. The method of the present invention combines the advantages of both solid-phase and liquid-phase synthesis, which can both increase the batch size and form economies of scale, and simplify the method while retaining the potential for automated synthesis; at the same time, compared with traditional synthesis methods, the greenness of the present invention is greatly improved. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0037] Figure 1 The HPLC chromatogram of acetyl tetrapeptide-2 (PEG6000) is shown. Detailed Implementation

[0038] This invention discloses a method for preparing polypeptides.

[0039] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

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

[0041] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0042] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0043] Furthermore, 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 precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" 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 this invention involves sequentially coupling amino acids onto a PEG support according to the acetyl tetrapeptide-2 peptide sequence. The coupled amino acids are Smoc-Tyr(tBu)-OH, Smoc-Val-OH, Smoc-Asp(OtBu)-OH, and Smoc-Lys(Boc)-OH. After coupling, an acetylation reaction is performed. The support is removed using hydrochloric acid to obtain the product.

[0045] This invention includes the following steps:

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

[0047] 2) Conjugate acetyl tetrapeptide-2 sequentially from C-terminus to N-terminus, remove the carrier with hydrochloric acid to obtain acetyl tetrapeptide-2.

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

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

[0050] The synthesis method includes:

[0051] 1) PEG resin and Smoc-Tyr(tBu)-OH-protected amino acids were dissolved in water, and a coupling agent was added to react in a homogeneous phase until the reaction was detected as terminated. After the first amino acid was coupled, it was treated with an acetylation reagent to ensure that all possible residual groups on the PEG were blocked. After the reaction was completed, two volumes of hexane were added to precipitate the PEG resin. The reaction solution was filtered and washed six times with a hexane:water mixture of 1:1.

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

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

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

[0055] Step 2) The coupling reaction time for each amino acid is usually 2 to 4 hours, preferably 3 hours; the pressure is preferably atmospheric pressure, but it can also be carried out at an appropriately increased or decreased pressure; the temperature is preferably room temperature (i.e., 20±5℃), but it can also be carried out at an appropriately increased or decreased temperature.

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

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

[0058] After the pyrolysis reaction described in step 3), the pyrolysis solution was rotary evaporated under reduced pressure, and ethanol was added to allow acetyl tetrapeptide-2 to be fully precipitated. The polyethylene glycol was removed by filtration, and the solution was dried. Acetyl tetrapeptide-2 was dissolved and washed with water, then freeze-dried to obtain GHK solid. The solution was purified and converted to salt to obtain acetyl tetrapeptide-2 acetate.

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

[0060] The present invention will be further illustrated below with reference to the embodiments:

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

[0062] Weigh 0.5 mol of compound PEG4000 into a 5 L three-necked flask, add 1 L of water and 1.2 mol of Smoc-Tyr(tBu)-OH. Stir to dissolve. Add 0.6 mol of EDC·HCl 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 complete, add 2 L of n-hexane to the reaction solution and stir for 2 hours. Filter, and wash the filter cake 6 times with n-hexane:water = 1:1 (1000 mL × 6). Dry the filter cake at 35 °C for 8 hours to obtain compound Smoc-Tyr(tBu)-PEG(4000) (yield 99.9%).

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

[0064] Weigh 0.5 mol of compound PEG6000 into a 5 L three-necked flask, add 1 L of water and 1.2 mol of Smoc-Tyr(tBu)-OH, and stir to dissolve. Add 0.6 mol of EDC·HCl 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 complete, add 2 L of n-hexane to the reaction solution and stir for 2 hours. Filter, and wash the filter cake 6 times with n-hexane:water = 1:1 (1000 mL × 6). Dry the filter cake at 35 °C for 8 hours to obtain compound Smoc-Tyr(tBu)-PEG (6000) (yield 99.3%).

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

[0066] Weigh 0.5 mol of compound PEG8000 into a 5 L three-necked flask, add 1 L of water and 1.2 mol of Smoc-Tyr(tBu)-OH, and stir to dissolve. Add 0.6 mol of EDC·HCl 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 complete, add 2 L of n-hexane to the reaction solution and stir for 2 hours. Filter, and wash the filter cake 6 times with n-hexane:water = 1:1 (1000 mL × 6). Dry the filter cake at 35 °C for 8 hours to obtain compound Smoc-Tyr(tBu)-PEG (8000) (yield 98.9%).

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

[0068] Weigh 0.25 mol of Smoc-Tyr(tBu)-PEG(6000) obtained in Example 2 into a 5L three-necked flask. Add 1L of 1M NaOH aqueous solution to the reaction flask, stir to dissolve, and react for 10 min. After the reaction is complete, add 2L of n-hexane to the reaction solution, stir and filter. Wash the filter cake 6 times with n-hexane:water = 1:1 (1000mL × 6). Repeat the washing once. Take another reaction flask, add 0.6 mol of Smoc-Val-OH, stir to dissolve, and add 0.3 mol of EDC·HCl under ice bath conditions. React the liquid for 10 min. Add the reaction solution to the above filter cake and continue stirring 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 wash the filter cake 6 times with n-hexane:water = 1:1 (1000mL × 6). 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 0.25 mol of Smoc-Val-Tyr(tBu)-PEG(6000) from Example 4 into a 5L three-necked flask. Add 1L of 5% piperazine aqueous solution to the reaction flask, stir to dissolve, and react for 10 min. After the reaction is complete, add 2L of n-hexane to the reaction solution, stir and filter. Wash the filter cake 6 times with n-hexane:water = 1:1 (1000mL × 6). Repeat the washing once. Take another reaction flask, add 0.6 mol of Smoc-Asp(OtBu)-OH, stir to dissolve, and add 0.3 mol of EDC·HCl under ice bath conditions. React the liquid for 10 min. 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 wash the filter cake 6 times with n-hexane:water = 1:1 (1000mL × 6). 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 0.25 mol of Smoc-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000) from Example 5 into a 5L three-necked flask. Add 1L of 10% piperazine aqueous solution to the reaction flask, stir to dissolve, and react for 10 min. After the reaction is complete, add 2L of n-hexane to the reaction solution, stir and filter. Wash the filter cake 6 times with n-hexane:water = 1:1 (1000mL × 6). Repeat the washing once. Take another reaction flask, add 0.6 mol of Smoc-Lys(Boc)-OH, stir to dissolve, and add 0.3 mol of EDC·HCl under ice bath conditions. React the liquid for 10 min. 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). 2 L of n-hexane was added to the reaction solution, and the mixture was filtered. The filter cake was washed 6 times with n-hexane:water in a 1:1 ratio (1000 mL × 6). The filter cake was dried in a forced-air condition at 35 °C for 8 hours to obtain the compound Smoc-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000) (yield 98.5%).

[0073] The filter cake was added to a 5L three-necked flask, and 1L of 5% piperazine aqueous solution was added to the reaction flask. The mixture was stirred and dissolved, and the reaction was allowed to proceed for 10 minutes. After the reaction was completed, 2L of n-hexane was added to the reaction solution, and the mixture was stirred and filtered. The filter cake was washed 6 times with n-hexane:water in a 1:1 ratio (1000mL × 6). The washing was repeated once. Acetic anhydride and pyridine (2.5mol / 2.5mol) were added, and the mixture was stirred for 3 hours at room temperature. After the reaction was completed, 2L of n-hexane was added to the reaction solution, and the mixture was stirred for 2 hours. The mixture was filtered, and the filter cake was washed 6 times with n-hexane:water in a 1:1 ratio (1000mL × 6). The filter cake was dried in a forced-air condition at 35℃ for 8 hours to obtain 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] Approximately 0.25 mol of Ac-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000) obtained in Example 6 was weighed and added to a 5 L round-bottom reaction flask. Then, 1 L of lysis buffer (4N hydrogen chloride / ethyl acetate: TIS = 95:5) was added to the flask. 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 consistency. 2 L of ethanol was added, and the mixture was stirred thoroughly for 2 hours. The mixture was then filtered, and the filter cake was washed three times with ethanol (500 mL × 3). The filter cake was dried under forced air at 35 °C for 8 hours to obtain 146.7 g of acetyl tetrapeptide-2, with a 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 reversed-phase high-performance liquid chromatography (RP-HPLC). Using reversed-phase octadecylsilane as the stationary phase and 0.1% acetic acid aqueous solution / acetonitrile as the mobile phase, the target peak fraction was collected, concentrated, and lyophilized to obtain 124.8 g of pure product, with a total yield of 88.3% and a purity of 99.61%. (e.g.) Figure 1 (As shown)

[0078] Example 9

[0079] Approximately 0.25 mol of Ac-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-PEG(6000) obtained in Example 6 was weighed and added to a 5 L round-bottom reaction flask. Then, 1 L of lysis buffer (TFA:TIS = 95:5) was added to the flask. The mixture was reacted at room temperature for 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to a viscous consistency. 2 L of ethanol was added, and the mixture was stirred thoroughly for 2 hours. The mixture was filtered, and the filter cake was washed three times with ethanol (500 mL × 3). The filter cake was dried in a forced-air condition at 35 °C for 8 hours to obtain 101.7 g of acetyl tetrapeptide-2, with a 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 description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing polypeptides, characterized in that, Includes the following steps: S1: The starting amino acid is coupled with the resin, first acetylated, filtered, and washed to obtain the coupling resin; S2: After deprotecting the coupling resin, pre-activated amino acids are sequentially coupled from the C-terminus to the N-terminus to obtain a polypeptide resin; S3: The polypeptide resin is second-acetylated, cleaved, purified, and concentrated to obtain the polypeptide; The starting amino acid is: Smoc-Tyr(tBu)-OH; The amino acids are: Smoc-Val-OH, Smoc-Asp(OtBu)-OH and Smoc-Lys(Boc)-OH; the resin is: PEG resin with a molecular weight of 4000~8000; The pyrolysis was performed using a hydrogen chloride / ethyl acetate solution and TIS; the volume ratio of the hydrogen chloride / ethyl acetate solution to the TIS was 95:

5.

2. The preparation method according to claim 1, characterized in that, Both the first acetylation and the second acetylation use acetic anhydride and pyridine.

3. The preparation method according to claim 1 or 2, characterized in that, The deprotection is performed using a 1M~3M NaOH solution or a 5%~10% V / V piperazine solution.

4. The preparation method according to claim 1 or 2, characterized in that, The coupling described in S1 and S2 uses 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.

5. The preparation method according to claim 1 or 2, characterized in that, The coupling time is 2-4 hours.

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