Preparation method of palmitoyl tripeptide-8
Through the liquid phase synthesis method, the problems of large amount of amino acids and large amount of solvents in the synthesis of palmitoyl tripeptide-8 were solved, and industrial production with high yields was achieved, which was suitable for large-scale production.
Patent Information
- Application Number
- CN202510630694.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-05
AI Technical Summary
In the prior art, the synthesis method of palmitoyl tripeptide-8 uses a large amount of amino acids and a large amount of solvents, which makes it difficult to produce on a large scale and is difficult to purify.
Palmitoyl tripeptide-8 was prepared by reacting histidine with palmitoyl chloride, then reacting with D-phenylalanine and Boc-arginine, and finally neutralizing and precipitation reaction under specific conditions.
It has achieved small amount of amino acids and small amount of solvents, simple synthesis steps, convenient purification, high yield, and suitable for large-scale industrial production.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of polypeptide synthesis, and in particular to a method for preparing palmitoyl tripeptide-8. Background Art
[0002] Palmitoyl Tripeptide-8 is a signaling peptide that promotes collagen synthesis (types I, II, and IV) and extracellular matrix protein production by increasing matrix cell activity, contributing to a more youthful and elastic appearance. When added to cosmetics, it helps replenish collagen, enhance facial elasticity, and significantly reduce wrinkles. It also boosts cell activity, increases skin hydration, enhances radiance, and improves complexion. By increasing skin hydration and retaining moisture, it leaves skin feeling soft and smooth, brightening the complexion from within and eliminating dullness.
[0003] Palmitoyl tripeptide-8 can inhibit melanin production in melanocytes by inhibiting tyrosinase activity and downregulating the mRNA levels of microphthalmia-associated transcription factor (MITF) and tyrosinase. Chemicalbook suggests that it could be used as a whitening additive in cosmetics. Palmitoyl tripeptide-8 has several benefits for the skin, including moisturizing, antioxidant, anti-inflammatory, damage repair, and collagen production promotion.
[0004] Currently, the synthesis method of palmitoyl tripeptide-8 reported in patents is mainly based on traditional solid-phase peptide synthesis. Due to its short sequence, solid-phase synthesis requires a large amount of amino acids, the use of resin is more expensive, the amount of solvent used is also large, and purification is difficult, making large-scale production impossible. Summary of the Invention
[0005] In view of this, the technical problem to be solved by the present invention is to provide a preparation method of palmitoyl tripeptide-8. The preparation method provided by the present invention has the advantages of small amount of amino acids and solvents, high yield, and can be produced on a large scale.
[0006] The present invention provides a method for preparing palmitoyl tripeptide-8, comprising the following steps:
[0007] Step 1, histidine and palmitoyl chloride react in the presence of diisopropylethylamine to obtain palmitoylhistidine shown in formula (1);
[0008] Step 2: Palmitoyl histidine represented by formula (1) and D-phenylalanine are reacted in the presence of N-hydroxysuccinimide, 1,3-dicyclohexylcarbodiimide, diisopropylethylamine and trimethylsilyl chloride to prepare palmitoyl histidine-D-phenylalanine represented by formula (2);
[0009] Step 3, palmitoylhistidine-D-phenylalanine shown in formula (2) and Boc-arginine shown in formula (3) react in the presence of N-hydroxysuccinimide, 1,3-dicyclohexylcarbodiimide, diisopropylethylamine and trimethylsilyl chloride to prepare palmitoylhistidine-D-phenylalanine-arginine (Boc) shown in formula (4);
[0010] Step 4, palmitoyl histidine-D-phenylalanine-arginine (Boc) shown in formula (4) is reacted in the presence of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, ammonia and acid, and then subjected to neutralization and precipitation reaction to obtain the palmitoyl tripeptide-8;
[0011] Formula (1);
[0012] Formula (2);
[0013] Formula (3);
[0014] Formula (4);
[0015] In some embodiments, step 1 includes:
[0016] Under stirring conditions at 0-5° C., diisopropylethylamine and palmitoyl chloride are sequentially added to the mixture of the histidine and the solvent, reacted, the residue is filtered off, an organic phase is obtained by extraction, and the organic phase is concentrated to obtain palmitoylhistidine represented by formula (1).
[0017] In some embodiments, in step 1,
[0018] The solvent is dichloromethane, the reaction time is 6 to 10 hours, and the extraction reagent is water;
[0019] The mass ratio of the histidine, palmitoyl chloride and diisopropylethylamine is 1: (1-1.5): (2-2.5).
[0020] In some specific embodiments, in step 1,
[0021] The mass ratio of the histidine, palmitoyl chloride and diisopropylethylamine is 50:62.5:106.3.
[0022] In some embodiments, step 2 includes:
[0023] N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide are added to a mixture of palmitoylhistidine represented by formula (1) and a solvent under stirring at 0-5°C, and the mixture is reacted for the first time, and the residue is filtered off to obtain a filtrate 1;
[0024] Add diisopropylethylamine and trimethylsilyl chloride to the mixture of D-phenylalanine and the solvent under stirring at 0-5°C, perform a second reaction, and filter out the residue to obtain filtrate 2;
[0025] Under stirring at 0-5° C., 2 drops of the filtrate are added to the filtrate 1, and the palmitoyl histidine-D-phenylalanine represented by formula (2) is obtained by a third reaction and filtration.
[0026] In some embodiments, in step 2,
[0027] The mass ratio of palmitoyl histidine, N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide shown in formula (1) is (2.5-3):1:(1.8-2.2), and the time of the first reaction is 14-18 hours;
[0028] The mass ratio of D-phenylalanine, diisopropylethylamine and trimethylsilyl chloride is 1: (1-1.5): (0.7-1), and the time of the second reaction is 1-3 hours;
[0029] The time of the third reaction is 5 to 10 hours;
[0030] The solvent is tetrahydrofuran.
[0031] In some specific embodiments, in step 2,
[0032] The mass ratio of palmitoyl histidine, N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide shown in formula (1) is 112.56:39.5:76.7, and the time of the first reaction is 16 hours;
[0033] The mass ratio of D-phenylalanine, diisopropylethylamine and trimethylsilyl chloride is 56.7:73.9:46.6, and the time of the second reaction is 2 hours;
[0034] The third reaction time is 8 hours.
[0035] In some embodiments, step 3 includes:
[0036] N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide are added to a mixture of palmitoylhistidine-D-phenylalanine represented by formula (2) and a solvent under stirring at 0-5°C, and the mixture is reacted for the first time and the residue is filtered off to obtain a filtrate 1;
[0037] Under stirring conditions at 0-5°C, diisopropylethylamine and trimethylsilyl chloride are added to a mixture of Boc-arginine represented by formula (3) and a solvent, and a second reaction is carried out and the residue is filtered off to obtain a filtrate 2;
[0038] Under stirring conditions of 0-5° C., 2 drops of the filtrate are added to the filtrate 1, and the reaction solution is concentrated and extracted to obtain an organic phase, and the organic phase is concentrated to obtain palmitoyl histidine-D-phenylalanine-arginine (Boc) represented by formula (4).
[0039] In some embodiments, in step 3,
[0040] The mass ratio of palmitoyl histidine-D-phenylalanine, N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide shown in formula (2) is (3.5-4.2):1:(1.8-2.2), and the time of the first reaction is 14-18 hours;
[0041] The mass ratio of Boc-arginine, diisopropylethylamine and trimethylsilyl chloride shown in formula (3) is (1.8-2.2): (1.3-1.8): 1, and the time of the second reaction is 1-3 hours;
[0042] The time of the third reaction is 5-10 hours, the temperature of the reaction solution concentration is 40-50°C, the extraction reagent is dichloromethane, and the number of extractions is 2-3 times;
[0043] The solvent is tetrahydrofuran.
[0044] In some specific embodiments, in step 3,
[0045] The mass ratio of palmitoyl histidine-D-phenylalanine, N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide shown in formula (2) is 128.36:32.8:63.7, and the time of the first reaction is 16 hours;
[0046] The mass ratio of Boc-arginine, diisopropylethylamine and trimethylsilyl chloride shown in formula (3) is 78.1:61.4:38.7, and the time of the second reaction is 2 hours;
[0047] The third reaction time is 8 hours, the temperature of the reaction solution concentration is 45°C, and the number of extractions is 2. In some embodiments, step 4 includes:
[0048] Under stirring conditions at 0-5°C, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is added to a mixture of palmitoylhistidine-D-phenylalanine-arginine (Boc) shown in formula (4) and a solvent, and ammonia gas is introduced. After a first reaction, the pH is adjusted to 2-3, and extraction is performed to obtain a palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH aqueous solution;
[0049] 6N hydrochloric acid was added to the palmitoyl histidine-D-phenylalanine-arginine (Boc)-OH aqueous solution, and the mixture was stirred, reacted, neutralized and precipitated, centrifuged, and purified to obtain palmitoyl tripeptide-8.
[0050] In some embodiments, in step 4,
[0051] The mass ratio of palmitoylhistidine-D-phenylalanine-arginine (Boc) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate shown in formula (4) is (1.2-1.5): 1, the time of the first reaction is 14-18 hours, and the solvent is dichloromethane;
[0052] The volume ratio of the palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH aqueous solution to the 6N hydrochloric acid is 1: (0.8-1.2), the stirring reaction time is 14-18 hours, and the stirring reaction temperature is 20-30°C;
[0053] In the neutralization precipitation reaction, sodium hydroxide is used to adjust the pH of the solution after the stirring reaction to 8-9 to precipitate a solid;
[0054] The purification is carried out using methanol.
[0055] In some specific embodiments, in step 4,
[0056] The mass ratio of palmitoylhistidine-D-phenylalanine-arginine (Boc) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate shown in formula (4) is 150.64:107.8, and the time of the first reaction is 16 hours;
[0057] The volume ratio of the palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH aqueous solution and the 6N hydrochloric acid is 1:1, and the stirring reaction time is 16 hours.
[0058] Compared with the prior art, the beneficial effects of the preparation method of palmitoyl tripeptide-8 of the present invention include:
[0059] 1. The liquid phase synthesis method is selected, which has a simple process, few synthesis steps, convenient purification, simple post-processing, and higher yield than the solid phase;
[0060] 2. The raw materials are cheap and easy to obtain, the amount of solvent used is small, it is green and environmentally friendly, the waste liquid can be recycled, and it is suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS
[0061] Figure 1The synthetic route of palmitoyl tripeptide-8 of the present invention is shown, wherein the compound shown in 1 is palmitoyl chloride, the compound shown in 2 is histidine, the compound shown in 3 is palmitoyl histidine, the compound shown in 4 is D-phenylalanine, the compound shown in 5 is palmitoyl histidine-D-phenylalanine, the compound shown in 6 is Boc-arginine, the compound shown in 7 is palmitoyl histidine-D-phenylalanine-arginine (Boc), the compound shown in 8 is palmitoyl histidine-D-phenylalanine-arginine (Boc)-OH, and the compound shown in 9 is palmitoyl tripeptide-8. DETAILED DESCRIPTION
[0062] The present invention provides a method for preparing palmitoyl tripeptide-8. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.
[0063] The present invention provides a method for preparing palmitoyl tripeptide-8, which has mild reaction, simple process and equipment, easy operation and no damage to the environment. The synthetic route is as follows: Figure 1 shown.
[0064] Weigh compound 2 histidine and dichloromethane, add them to a reaction flask and stir evenly, add diisopropylethylamine to the reaction flask, lower the reaction temperature to 0-5°C, weigh compound 1 palmitoyl chloride, maintain the temperature at 0-5°C and add dropwise to the reaction flask, stir at 0-5°C for 6-10 h, filter the reaction solution to remove the residue, extract the reaction solution with water, retain the organic phase, and concentrate to obtain compound 3 palmitoylhistidine.
[0065] Compound 3 and tetrahydrofuran were added to a reaction flask, the temperature was lowered to 0-5°C, N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide were added, the mixture was stirred for 6-10 hours, and filtrated to obtain filtrate 1. Compound 4, D-phenylalanine, and tetrahydrofuran were weighed and added to the reaction flask, the temperature was lowered to 0-5°C, diisopropylethylamine and trimethylsilyl chloride were added, and the mixture was stirred for 2 hours. The reaction mixture was filtered to obtain filtrate 2. Filtrate 2 was added dropwise to filtrate 1, stirred for 6-8 hours, and filtered and dried to obtain compound 5, palmitoylhistidine-D-phenylalanine.
[0066] Compound 5 and tetrahydrofuran were added to a reaction flask, the temperature was lowered to 0-5°C, N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide were added, the mixture was stirred for 6-10 hours, and filtrated to obtain filtrate 1. Compound 6, Boc-arginine, and tetrahydrofuran were weighed and added to the reaction flask, the temperature was lowered to 0-5°C, diisopropylethylamine and trimethylsilyl chloride were added, and the mixture was stirred for 2 hours. The reaction mixture was filtered to obtain filtrate 2. Filtrate 1 was added dropwise to filtrate 2, and the mixture was stirred for 6-8 hours. The reaction mixture was concentrated and extracted with dichloromethane. The organic phase was concentrated to obtain compound 7, palmitoylhistidine-D-phenylalanine-arginine (Boc).
[0067] Compound 7 and dichloromethane were stirred to dissolve, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was added thereto, ammonia gas was introduced thereto at 0-5°C, and the mixture was stirred for 16 h. Water was added to the reaction solution to adjust the pH to 2-3, and the mixture was extracted into the aqueous phase to obtain compound 8 palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH.
[0068] 6N hydrochloric acid was added to compound 8 and stirred at 20-30°C for 16 hours. The pH of the reaction solution was adjusted to 8-9 with sodium hydroxide to precipitate a solid. After centrifugation, the solid was slurried with methanol to obtain compound 9, namely palmitoyl tripeptide-8.
[0069] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention will be further described below with reference to the examples.
[0070] Example 1
[0071] Histidine (50.0 g, 0.32 mol) and 300 mL of dichloromethane were added to a 500 mL reaction flask and stirred evenly. Diisopropylethylamine (62.5 g, 0.48 mol) was added to the reaction flask, and the reaction temperature was lowered to 0-5°C. Palmitoyl chloride (106.3 g, 0.39 mol) was weighed and added dropwise to the reaction flask while maintaining the temperature at 0-5°C. The mixture was stirred at 0-5°C for 6-10 h. The reaction solution was filtered to remove the residue, and the reaction solution was extracted with 300 mL of water. The organic phase was retained and concentrated to obtain 112.56 g of palmitoylhistidine with a yield of 89%.
[0072] Example 2
[0073] In a 1000 mL reaction flask, 112.56 g of palmitoylhistidine and 500 mL of tetrahydrofuran were added, the temperature was lowered to 0-5°C, 39.5 g (0.34 mol) of N-hydroxysuccinimide (HOSU) and 76.7 g (0.37 mol) of 1,3-dicyclohexylcarbodiimide (DCC) were added, and the mixture was stirred for 16 h. The insoluble matter was removed by filtration to obtain filtrate 1. In another 1000 mL reaction flask, 45.47 g of anhydrous sodium carbonate and 200 mL of water were added and stirred to dissolve. 56.7 g (0.34 mol) of D-phenylalanine was added to the reaction flask and stirred evenly. Filtrate 1 was added dropwise to the reaction solution flask at 20-30°C, stirred for 8 h, filtered and dried to obtain 88.9 g of palmitoylhistidine-D-phenylalanine, with a yield of 57.5%.
[0074] Example 3
[0075] Add 112.56 g of palmitoylhistidine and 500 mL of N,N-dimethylformamide to a 1000 mL reaction flask, lower the temperature to 0-5°C, add 39.5 g (0.34 mol) of N-hydroxysuccinimide (HOSU) and 76.7 g (0.37 mol) of 1,3-dicyclohexylcarbodiimide (DCC), stir for 16 h, and filter to remove insoluble matter to obtain filtrate 1. Take another 1000 mL reaction flask and add 56.7 g (0.34 mol) of D-phenylalanine and 300 mL of N,N-dimethylformamide to the reaction flask, lower the temperature to 0-5°C, add 73.9 g (0.57 mol) of diisopropylethylamine and 46.6 g (0.43 mol) of trimethylsilyl chloride, stir for 2 h, and filter to remove insoluble matter to obtain filtrate 2. Filtrate 2 was added dropwise to filtrate 1, stirred for 8 h, filtered and dried to obtain 58.23 g of palmitoyl histidine-D-phenylalanine with a yield of 37.6%.
[0076] Example 4
[0077] Add 112.56 g of palmitoylhistidine and 500 mL of tetrahydrofuran to a 1000 mL reaction flask, lower the temperature to 0-5°C, add 39.5 g (0.34 mol) of N-hydroxysuccinimide (HOSU) and 76.7 g (0.37 mol) of 1,3-dicyclohexylcarbodiimide (DCC), stir for 16 h, and filter to remove insoluble matter to obtain filtrate 1; take another 1000 mL reaction flask, add 56.7 g (0.34 mol) of D-phenylalanine and 300 mL of tetrahydrofuran to the reaction flask, lower the temperature to 0-5°C, add 73.9 g (0.57 mol) of diisopropylethylamine and 46.6 g (0.43 mol) of trimethylsilyl chloride, stir for 2 h, and filter to remove insoluble matter to obtain filtrate 2. Filtrate 2 was added dropwise to filtrate 1, stirred for 8 h, filtered and dried to obtain 128.36 g of palmitoyl histidine-D-phenylalanine, with a yield of 82.9%.
[0078] Example 5
[0079] In a 1000 mL reaction flask, 100 g of palmitoyl histidine-D-phenylalanine and 700 mL of tetrahydrofuran were added, the temperature was lowered to 0-5 °C, 43 g (0.24 mol) of N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) and 45.8 g (0.22 mol) of 1,3-dicyclohexylcarbodiimide (DCC) were added, and the mixture was stirred for 16 h. The insoluble matter was removed by filtration to obtain filtrate 1. In another 1000 mL reaction flask, 300 mL of palmitoyl histidine-D-phenylalanine 55.8 g (0.20 mol) and 47.8 g (0.22 mol) of tetrahydrofuran were added. 1 g (0.37 mol) of NN-diisopropylethylamine (DIEA) was added to the reaction flask, the temperature was lowered to 0-5°C, and 1 drop of the filtrate was added to the reaction flask. The mixture was stirred at 20-30°C for 8 h. The pH of the reaction solution was adjusted to 2-3, and the mixture was extracted twice with 600 mL of dichloromethane. The organic phase was concentrated to obtain 52.47 g of palmitoylhistidine-D-phenylalanine-arginine (Boc), with a yield of 35.6%.
[0080] Example 6
[0081] Add 128.36 g of palmitoylhistidine-D-phenylalanine and 700 mL of tetrahydrofuran to a 1000 mL reaction flask, lower the temperature to 0-5°C, add 32.8 g (0.28 mol) of N-hydroxysuccinimide and 63.7 g (0.31 mol) of 1,3-dicyclohexylcarbodiimide, stir for 16 h, and filter to remove insoluble matter to obtain filtrate 1; take another 1000 mL reaction flask, add 78.1 g (0.28 mol) of Boc-arginine and 300 mL of tetrahydrofuran to the reaction flask, lower the temperature to 0-5°C, add 61.4 g (0.47 mol) of diisopropylethylamine and 38.7 g (0.36 mol) of trimethylsilyl chloride, stir for 2 h, and filter to remove insoluble matter to obtain filtrate 2. Filtrate 2 was added dropwise to filtrate 1, stirred for 8 h, and concentrated at 45°C. The reaction solution was extracted twice with 600 mL of dichloromethane. The organic phase was concentrated to obtain 98.95 g of palmitoylhistidine-D-phenylalanine-arginine (Boc), with a yield of 52.3%.
[0082] Example 7
[0083] In a 1000 mL reaction flask, 95 g of palmitoylhistidine-D-phenylalanine-arginine (Boc) and 500 mL of tetrahydrofuran were added and stirred to dissolve. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (67.98 g, 0.18 mol) was added thereto. 100 mL of ammonia water was added thereto at 0-5°C and stirred for 16 h. 500 mL of water and 500 mL of dichloromethane were added to the reaction solution, the pH was adjusted to 2-3, and the solution was extracted into the aqueous phase to obtain a palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH aqueous solution.
[0084] 500 mL of 6N hydrochloric acid was added to the palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH aqueous solution and stirred at 20-30 ° C for 16 hours. The pH of the reaction solution was adjusted to 8-9 with sodium hydroxide to precipitate a solid. After centrifugation, the solid was slurried with methanol to obtain 35.66 g of palmitoyl tripeptide-8 with a yield of 43%.
[0085] Example 8
[0086] In a 1000 mL reaction flask, 150.64 g of palmitoylhistidine-D-phenylalanine-arginine (Boc) and 500 mL of dichloromethane were added and stirred to dissolve. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (107.8 g, 0.28 mol) was added thereto. Ammonia gas was introduced into the reaction mixture at 0-5°C and stirred for 16 h. 500 mL of water was added to the reaction solution, the pH was adjusted to 2-3, and the mixture was extracted into the aqueous phase to obtain a palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH aqueous solution.
[0087] To a palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH aqueous solution was added 500 mL of 6N hydrochloric acid and stirred at 20-30°C for 16 h. The reaction solution was added to 5 L of acetonitrile to precipitate a solid. After filtration, the product absorbed moisture. The solid after moisture absorption was dissolved in 100 mL of water and the pH was adjusted to 8-9 with sodium hydroxide to precipitate a solid. The solid was filtered to obtain the solid. Purification of the solid with acetonitrile slurry failed, and 20 g of the product was obtained by slurrying with methanol, with a yield of 15.21%.
[0088] Example 9
[0089] In a 1000 mL reaction flask, 150.64 g of palmitoylhistidine-D-phenylalanine-arginine (Boc) and 500 mL of dichloromethane were added and stirred to dissolve. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (107.8 g, 0.28 mol) was added thereto. Ammonia gas was introduced into the reaction mixture at 0-5°C and stirred for 16 h. 500 mL of water was added to the reaction solution, the pH was adjusted to 2-3, and the mixture was extracted into the aqueous phase to obtain a palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH aqueous solution.
[0090] 500 mL of 6N hydrochloric acid was added to the palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH aqueous solution and stirred at 20-30°C for 16 h. The pH of the reaction solution was adjusted to 8-9 with sodium hydroxide to precipitate a solid. After centrifugation, the solid was slurried with methanol to obtain 78.89 g of palmitoyl tripeptide-8, with a yield of 60%.
[0091] The above are only preferred embodiments 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 principles 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 palmitoyl tripeptide-8, characterized in that: The steps include: Step 1, histidine and palmitoyl chloride react in the presence of diisopropylethylamine to obtain palmitoylhistidine shown in formula (1); Step 2: Palmitoyl histidine represented by formula (1) and D-phenylalanine are reacted in the presence of N-hydroxysuccinimide, 1,3-dicyclohexylcarbodiimide, diisopropylethylamine and trimethylsilyl chloride to prepare palmitoyl histidine-D-phenylalanine represented by formula (2); Step 3, palmitoylhistidine-D-phenylalanine shown in formula (2) and Boc-arginine shown in formula (3) react in the presence of N-hydroxysuccinimide, 1,3-dicyclohexylcarbodiimide, diisopropylethylamine and trimethylsilyl chloride to prepare palmitoylhistidine-D-phenylalanine-arginine (Boc) shown in formula (4); Step 4, palmitoyl histidine-D-phenylalanine-arginine (Boc) shown in formula (4) is reacted in the presence of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, ammonia and acid, and then subjected to neutralization and precipitation reaction to obtain the palmitoyl tripeptide-8; Formula (1); Formula (2); Formula (3); Formula (4).
2. The preparation method according to claim 1, characterized in that The step 1 includes: Under stirring conditions at 0-5° C., diisopropylethylamine and palmitoyl chloride are sequentially added to the mixture of the histidine and the solvent, reacted, the residue is filtered off, an organic phase is obtained by extraction, and the organic phase is concentrated to obtain palmitoylhistidine represented by formula (1).
3. The preparation method according to claim 2, characterized in that In the step 1, The solvent is dichloromethane, the reaction time is 6 to 10 hours, and the extraction reagent is water; The mass ratio of the histidine, palmitoyl chloride and diisopropylethylamine is 1: (1-1.5): (2-2.5).
4. The preparation method according to claim 1, characterized in that The step 2 includes: N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide are added to a mixture of palmitoylhistidine represented by formula (1) and a solvent under stirring at 0-5°C, and the mixture is reacted for the first time, and the residue is filtered off to obtain a filtrate 1; Add diisopropylethylamine and trimethylsilyl chloride to the mixture of D-phenylalanine and the solvent under stirring at 0-5°C, perform a second reaction, and filter out the residue to obtain filtrate 2; Under stirring at 0-5° C., 2 drops of the filtrate are added to the filtrate 1, and the palmitoyl histidine-D-phenylalanine represented by formula (2) is obtained by a third reaction and filtration.
5. The preparation method according to claim 4, characterized in that In the step 2, The mass ratio of palmitoyl histidine, N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide shown in formula (1) is (2.5-3):1:(1.8-2.2), and the time of the first reaction is 14-18 hours; The mass ratio of D-phenylalanine, diisopropylethylamine and trimethylsilyl chloride is 1: (1-1.5): (0.7-1), and the time of the second reaction is 1-3 hours; The time of the third reaction is 5 to 10 hours; The solvent is tetrahydrofuran.
6. The preparation method according to claim 1, characterized in that The step 3 includes: N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide are added to a mixture of palmitoylhistidine-D-phenylalanine represented by formula (2) and a solvent under stirring at 0-5°C, and the mixture is reacted for the first time and the residue is filtered off to obtain a filtrate 1; Under stirring conditions at 0-5°C, diisopropylethylamine and trimethylsilyl chloride are added to a mixture of Boc-arginine represented by formula (3) and a solvent, and a second reaction is carried out and the residue is filtered off to obtain a filtrate 2; Under stirring conditions of 0-5° C., 2 drops of the filtrate are added to the filtrate 1, and the reaction solution is concentrated and extracted to obtain an organic phase, and the organic phase is concentrated to obtain palmitoyl histidine-D-phenylalanine-arginine (Boc) represented by formula (4).
7. The preparation method according to claim 6, characterized in that In the step 3, The mass ratio of palmitoyl histidine-D-phenylalanine, N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide shown in formula (2) is (3.5-4.2):1:(1.8-2.2), and the time of the first reaction is 14-18 hours; The mass ratio of Boc-arginine, diisopropylethylamine and trimethylsilyl chloride shown in formula (3) is (1.8-2.2): (1.3-1.8): 1, and the time of the second reaction is 1-3 hours; The time of the third reaction is 5-10 hours, the temperature of the reaction solution concentration is 40-50°C, the extraction reagent is dichloromethane, and the number of extractions is 2-3 times; The solvent is tetrahydrofuran.
8. The preparation method according to claim 1, characterized in that The step 4 includes: Under stirring conditions at 0-5°C, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is added to a mixture of palmitoylhistidine-D-phenylalanine-arginine (Boc) shown in formula (4) and a solvent, and ammonia gas is introduced. After a first reaction, the pH is adjusted to 2-3, and extraction is performed to obtain a palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH aqueous solution; 6N hydrochloric acid was added to the palmitoyl histidine-D-phenylalanine-arginine (Boc)-OH aqueous solution, and the mixture was stirred for reaction, neutralized and precipitated, centrifuged to obtain a precipitate, and the precipitate was purified to obtain palmitoyl tripeptide-8.
9. The preparation method according to claim 8, characterized in that In the step 4, The mass ratio of palmitoylhistidine-D-phenylalanine-arginine (Boc) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate shown in formula (4) is (1.2-1.5):1, the time of the first reaction is 14-18 hours, and the solvent is dichloromethane.
10. The preparation method according to claim 8, characterized in that In the step 4, The volume ratio of the palmitoylhistidine-D-phenylalanine-arginine (Boc)-OH aqueous solution to the 6N hydrochloric acid is 1: (0.8-1.2), the stirring reaction time is 14-18 hours, and the stirring reaction temperature is 20-30°C; In the neutralization precipitation reaction, sodium hydroxide is used to adjust the pH of the solution after the stirring reaction to 8-9 to precipitate a solid; The purification is carried out using methanol.