Method for preparing Pal-GQPR from LPPS
The low purification efficiency and isomerization problems in the preparation of palmitoyl tetrapeptide-7 were solved by liquid phase peptide synthesis (LPPS). Using inexpensive raw materials and environmentally friendly solvents, high yield and high purity of palmitoyl tetrapeptide-7 were achieved, which is suitable for industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- FLAMMA HONKAI (DALIAN) PHARM CO LTD
- Filing Date
- 2026-01-22
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies for preparing palmitoyl tetrapeptide-7 suffer from problems such as low purification efficiency, difficulty in improving purity, and limited scalability. Furthermore, liquid-phase peptide synthesis is prone to intramolecular or intermolecular condensation and epimerization, which affect the product yield and optical purity.
The liquid-phase peptide synthesis (LPPS) method was adopted. Through reasonable route design, the racemization and intramolecular condensation of Gln residues were avoided. Inexpensive and readily available raw materials and environmentally friendly solvents were used. Condensing agents such as HBTU and DSC and DIPEA were used as bases. The reaction conditions were optimized, unnecessary purification steps were reduced, and a compact synthetic route was designed.
This method achieves high yield and high purity of palmitoyl tetrapeptide-7, reduces production costs, improves the environmental compatibility and operational safety of the process, is suitable for industrial-scale production, and produces products with good quality stability and consistency.
Smart Images
Figure CN122011092A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of polypeptide compound synthesis, specifically relating to a method for preparing palmitoyl tetrapeptide Pal-Gly-Gln-Pro-Arg-OH that avoids self-condensation and epimerization. Background Technology
[0002] Palmitoyl Tetrapeptide-7 (Pal-GQPR, structure shown in the image below) is a lipopeptide developed by Sederma to mimic the effects of DHEA and restore cytokine balance in mature skin. With age, DHEA levels decline, leading to an increase in IL-6, an inflammatory mediator that accelerates extracellular matrix degradation, ultimately causing wrinkles, sagging skin, and decreased elasticity. Studies have shown that palmitoyl tetrapeptide-7 can effectively reduce IL-6 production by 40% and exhibits significant anti-inflammatory effects in UV-induced inflammation. Comparable to the anti-inflammatory capabilities of DHEA, it helps reduce roughness, fine lines, and wrinkles, improves uneven skin tone, and aids in skin repair and regeneration. This ingredient can be used alone or in combination with palmitoyl tripeptide-1 to promote collagen production, accelerate skin repair, and improve firmness and smoothness. Therefore, palmitoyl tetrapeptide-7, as a potent anti-inflammatory and anti-aging ingredient, is widely used in anti-aging skincare products, significantly improving skin appearance and texture.
[0003]
[0004] In existing technologies, its preparation largely relies on solid-phase synthesis (SPPS) process. This technology is based on a solid support and uses repeated steps of "amino acid fixation-deprotection-carboxyl activation-peptide bond formation" to gradually extend the peptide chain from the C-terminus. Finally, the peptide is deprotected and the support linkage is cut to obtain a crude peptide. The crude peptide then needs to be purified by reversed-phase chromatography to meet the purity requirements. Early SPPS technology developed by Li Qian, Zhang Zhongqi, and others laid the foundation for the preparation of palmitoyl tetrapeptide-7. However, this peptide has extremely poor water solubility due to the presence of hydrophobic palmitoyl groups and multiple hydrophobic amino acids. During reverse-phase purification, it is prone to precipitation and low loading concentration, resulting in low purification efficiency, difficulty in improving purity, and limited scalability. Furthermore, traditional C18 packing material is expensive and has a short lifespan, increasing production costs. CN109748948B uses polystyrene divinylphenyl packing material (such as PS10-300) to replace C18, simultaneously achieving purification and salt conversion. Although this increases purity to over 98%, yield to around 70%, and reduces packing material costs, it does not solve the core water solubility problem, limiting loading to only 10 g levels and limiting scalability potential. CN112110984A improves water solubility by temporarily coupling 3-6 lysine residues and a hydrophilic fragment composed of p-hydroxybenzoic acid, achieving a purity of 99.66%, but still relies on RinkAmide. Specialized solid-phase carriers such as Resin require additional fragment coupling and removal processes. Furthermore, the pyrolysis and resin removal process requires a complex mixture of TFA: m-cresol: benzyl sulfide: water (92.5:2.5:2.5:2.5), resulting in significant process complexity, impurity risks, and environmental pressures.
[0005] Liquid-phase peptide synthesis (LPPS) methods are prone to intramolecular or intermolecular condensation (such as dipeptide-dipeptide cyclization to generate DKP impurities) during the synthesis of Pal-Gly-Gln-OH, leading to decreased yield. Furthermore, configuration inversion can occur during condensation, resulting in epimerization (racemization), which severely affects the optical purity of the product. For example, CN112830956A discloses a method for preparing palmitoyl tetrapeptide-7 (Pal-GQPR). Its core is the preparation of the target product through seven key synthetic steps (including the synthesis of Boc-Pro-OH and Pal-Gly-Gln-ONp) and a purification process. However, this method requires the prior synthesis of Pal-Gly-Gln-ONp before condensation with H-Pro-Arg-OH. Since Gln residues directly participate in amide bond formation reactions, the α-position configuration is prone to inversion under activation and basic conditions, which can lead to racemization. Therefore, the risk of racemization during the condensation stage of Gln residues cannot be avoided.
[0006] Therefore, there is an urgent need for a new synthetic route that can simultaneously suppress the self-condensation of the Pal-Gly-Gln-OH stage and the racemization of the Gln site, and is industrially feasible. Summary of the Invention
[0007] To address the aforementioned problems, the present invention aims to provide an economical, environmentally friendly, and commercially viable method for synthesizing Pal-GQPR. In this application, through a rational route design, the first amino acid preceding the carboxyl group in the condensation step either lacks a racemic form or is difficult to racemate, thereby significantly reducing the risk of epimerization (racemization).
[0008] Based on this, this application proposes a method for preparing palmitoyl tetrapeptide-7 and its salts via liquid-phase peptide synthesis (LPPS), wherein the method includes: Step 1: Palmitoyl chloride (Pal-Cl) is obtained from palmitic acid (Pal) via an acyl chloride reaction, and then palmitoyl glycine (Pal-Gly-OH) is generated by a substitution reaction with glycine (Gly). Step 1': N-tert-butoxycarbonyl-glutamine (Boc-Gln-Pro-OBzl) is synthesized from N-tert-butoxycarbonyl-glutamine (Boc-Gln-OH) and benzyl proline hydrochloride (H-Pro-OBzl·HCl) via dipeptide condensation reaction, and glycyl-glutamine-proline benzyl hydrochloride (H-Gln-Pro-OBzl.HCl) is obtained by deprotection. Step 2: Palmitoyl-glycyl-glutamine-proline benzyl ester (Pal-Gly-Gln-Pro-OBzl) is obtained by tripeptide condensation reaction of Pal-Gly-OH and H-Gln-Pro-OBzl.HCl. Step 3: Remove the protecting group via hydrolysis to prepare palmitoyl-gly-glutamine-proline (Pal-Gly-Gln-Pro-OH); and Step 4: Pal-Gly-Gln-Pro-OH is prepared into an active ester, palmitoyl-glycyl-glutamine-proline succinimide ester (Pal-Gly-Gln-Pro-OSu), via ester condensation reaction. Then, palmitoyl tetrapeptide-7 (Pal-Gly-Gln-Pro-Arg-OH) is obtained by reacting Pal-Gly-Gln-Pro-OSu with arginine (Arg) via tetrapeptide condensation reaction.
[0009] Therefore, in the synthesis method of this application, the carboxyl groups of Pal-Gly-OH and Pal-Gly-Gln-Pro-OH are connected to the α-carbon of glycine and proline, respectively. Since glycine has no chiral center and proline has a secondary amine structure, it is not easy for them to racemize at the α-position, which reduces the possibility of racemization from the perspective of molecular design.
[0010] In some embodiments, the acyl chloride reaction is carried out in a first solvent and at a first temperature in the presence of an acylating agent; wherein the first solvent is selected from one or more of toluene, n-heptane, and methyl tert-butyl ether.
[0011] In some embodiments, the acyl chloride reaction specifically includes using n-heptane as a solvent to react Pal-OH with the acylating agent for 2-3 hours; after the reaction is complete, the solution is concentrated to obtain an oily substance, Pal-Cl.
[0012] In some embodiments, the acylation agent is selected from one or a combination of thionyl chloride, oxalyl chloride, phosphorus oxychloride, and phosphorus pentachloride. In some specific embodiments, the acylation agent is thionyl chloride.
[0013] In some embodiments, the molar ratio of Pal-OH to thionyl chloride in the acyl chloride reaction is 1:(1.5~3), preferably 1:2.5.
[0014] In some embodiments, the acyl chloride reaction is carried out at a temperature of 20°C to 60°C. In some specific embodiments, the acyl chloride reaction is carried out at a temperature of 40°C to 50°C.
[0015] In some embodiments, the reaction temperature in the substitution reaction is 0-30 degrees Celsius, preferably 0-10 degrees Celsius.
[0016] In some embodiments, the molar ratio of Pal-Cl to glycine in the substitution reaction is 1:(1.0~2.0), preferably 1:1.5.
[0017] In some embodiments, the substitution reaction is carried out at a pH of 9 to 12. In some specific embodiments, the substitution reaction is carried out at a pH of 10 to 11.
[0018] In some embodiments, the substitution reaction specifically involves reacting Pal-Cl and glycine in a solvent of 2-methyltetrahydrofuran and water for 3-5 hours. After the reaction is complete, the pH is adjusted to 2-3, and then the mixture is filtered and dried to obtain Pal-Gly-OH.
[0019] In some specific embodiments, the volume ratio of 2-methyltetrahydrofuran to water in the substitution reaction is 1:(1.0~4.0). In some more specific embodiments, the volume ratio of 2-methyltetrahydrofuran to water is 1:4.0.
[0020] In some embodiments, the dipeptide condensation reaction is carried out in the presence of a first condensation reagent. In some embodiments, the dipeptide condensation reaction is carried out in the presence of both a first condensation reagent and a first base.
[0021] In some embodiments, the first condensing agent is selected from one or more of TBTU, HBTU, DCC, HOBt, HOSu, and DSC. In some specific embodiments, the first condensing agent is HBTU.
[0022] In some embodiments, the first base is selected from one or more of DIPEA (N,N-diisopropylethylamine), TEA (triethylamine), and NMM (N-methylmorpholine). In some specific embodiments, the first base is DIPEA.
[0023] In some embodiments, the dipeptide condensation reaction comprises reacting H-Pro-OBzl.HCl, Boc-Gln-OH, a first condensing agent (such as HBTU), and a first base (such as DIPEA) in 2-methyltetrahydrofuran as a solvent at room temperature for 2-5 hours. After the reaction is complete, the mixture is concentrated, water is added, and the mixture is stirred for 2-3 hours. The mixture is then filtered and dried to obtain Boc-Gln-Pro-OBzl.
[0024] In some embodiments, the molar ratio of H-Pro-OBzl.HCl:Boc-Gln-OH:HBTU:DIPEA is 1:(1.0~1.3):(1~1.5):(2~4.0). In some specific embodiments, the molar ratio of H-Pro-OBzl.HCl:Boc-Gln-OH:HBTU:DIPEA is 1:1.15:1.2:3.0.
[0025] In some embodiments, deprotection is carried out in the presence of trifluoroacetic acid, an aqueous solution of hydrogen chloride, an acetonitrile solution of hydrogen chloride, or an ethyl acetate solution of hydrogen chloride. In some specific embodiments, deprotection is carried out in the presence of an acetonitrile solution of hydrogen chloride.
[0026] In some embodiments, deprotection involves reacting Boc-Gln-Pro-OBn and the BOC-deprotecting agent at room temperature for 2-3 hours using acetonitrile as a solvent. After the reaction is complete, the mixture is cooled to 5-10 degrees Celsius, stirred for 30 minutes, filtered, and dried to obtain the target compound.
[0027] In some embodiments, the tripeptide condensation reaction is carried out in the presence of a second condensing agent. In some embodiments, the tripeptide condensation reaction is carried out in the presence of a second condensing agent and a second base.
[0028] In some embodiments, the second base is selected from one or more of DIPEA (N,N-diisopropylethylamine), TEA (triethylamine), and NMM (N-methylmorpholine). In some specific embodiments, the second base is DIPEA.
[0029] In some embodiments, the second condensing agent is selected from one or more of TBTU, HBTU, DCC, HOBt, HOSu, and DSC. In some specific embodiments, the second condensing agent is HBTU.
[0030] In some embodiments, the tripeptide condensation reaction comprises reacting Pal-Gly-OH, H-Gln-Pro-OBzl.HCl, a second condensing agent (such as HBTU), and a second base (such as DIPEA) in 2-methyltetrahydrofuran as a solvent at room temperature for 2-3 hours. After the reaction is complete, the mixture is concentrated, water is added, and the mixture is stirred for 2-3 hours. The mixture is then filtered and dried to obtain Pal-Gly-Gln-Pro-OBzl.
[0031] In some embodiments, the molar ratio of H-Gln-Pro-OBzl.HCl:Pal-Gly-OH:HBTU:DIPEA in the tripeptide condensation reaction is 1:(1.0~1.3):(1~1.5):(2~4.0). In some specific embodiments, the molar ratio of H-Gln-Pro-OBzl.HCl:Pal-Gly-OH:HBTU:DIPEA is 1:1.15:1.2:3.0.
[0032] In some embodiments, the hydrolysis reaction is carried out in the presence of one or more of lithium hydroxide, sodium hydroxide, and potassium hydroxide. In some specific embodiments, the hydrolysis reaction is carried out in the presence of lithium hydroxide.
[0033] In some embodiments, the hydrolysis reaction specifically includes adding the product Pal-Gly-Gln-Pro-OBzl from the previous step and the reagent for the first hydrolysis of benzyl ester to isopropanol as a solvent, and reacting at room temperature for 2-3 hours. After the reaction is complete, the mixture is concentrated, acetonitrile is added, and the mixture is stirred for 2-3 hours. The mixture is then filtered and dried to obtain Pal-Gly-Gln-Pro-OH.
[0034] In some embodiments, the ester condensation reaction is carried out in the presence of a third condensing agent, and the third condensing agent is selected from one or more of TBTU, HBTU, DCC, HOBt, HOSu, and DSC. In some specific embodiments, the third condensing agent is DSC.
[0035] In some embodiments, the ester condensation reaction includes adding the product from the previous step, Pal-Gly-Gln-Pro-OH, a third condensing agent (such as DSC), and a third base (such as DIPEA) to acetonitrile as a solvent, and reacting at room temperature for 2-3 hours. After the reaction is complete, a Pal-Gly-Gln-Pro-OSu solution is obtained.
[0036] In some embodiments, the molar ratio of Pal-Gly-Gln-Pro-OH:DSC:DMAP in the ester condensation reaction is 1:(1.0~1.3):(0.05~0.5). In some specific embodiments, the molar ratio of Pal-Gly-Gln-Pro-OH:DSC:DMAP is 1:1.2:0.2.
[0037] In some embodiments, the tetrapeptide condensation reaction is carried out in a second solvent and in the presence of a base.
[0038] In some embodiments, the second solvent is selected from one or more of 2-methyltetrahydrofuran, tetrahydrofuran, and acetonitrile. In some specific embodiments, the second solvent is acetonitrile.
[0039] In some embodiments, the molar ratio of Pal-Gly-Gln-Pro-OSu to base is 1:(1.0~3.0). In some embodiments, the molar ratio of Pal-Gly-Gln-Pro-OSu to base is 1:3.0.
[0040] In some embodiments, the fourth condensation reaction comprises adding H-Arg-OH and DIPEA, along with a solution of the previous product Pal-Gly-Gln-Pro-OSu, to acetonitrile as a solvent and reacting at room temperature for 1-2 hours. After the reaction is complete, the mixture is filtered and dried to obtain Pal-GQPR.
[0041] In some embodiments, the molar ratio of H-Arg-OH to DIPEA in the tetrapeptide condensation reaction is 1:(1.0~3.0). In some specific embodiments, the molar ratio of H-Arg-OH to DIPEA is 1:3.0.
[0042] In one specific embodiment, the method for preparing palmitoyl tetrapeptide-7 according to this application is carried out as follows:
[0043]
[0044] S1: Starting with palmitic acid, palmitoyl chloride is produced by acyl chloride reaction via thionyl chloride. S2: Palmitoyl chloride undergoes a substitution reaction with glycine (H-Gly-OH) to give palmitoylglycine (Pal-Gly-OH). S3: Boc-Gln-OH and H-Pro-OBzl were subjected to a dipeptide condensation reaction under the conditions of HBTU as condensing agent and DIPEA as base to prepare Boc-Gln-Pro-OBzl. S4: Remove the Boc protecting group under acidic conditions to obtain H-Gln-Pro-OBzl.HCl; S5: Pal-Gly-OH and H-Gln-Pro-OBzl.HCl were subjected to tripeptide condensation reaction in the presence of HBTU and DIPEA to obtain Pal-Gly-Gln-Pro-OBzl. S6: The benzyl ester protecting group is hydrolyzed under alkaline conditions to generate Pal-Gly-Gln-Pro-OH; S7: Under the condition of DSC as a condensing agent, Pal-Gly-Gln-Pro-OH is prepared into the active ester Pal-Gly-Gln-Pro-OSu; S8: Pal-Gly-Gln-Pro-OSu undergoes a condensation reaction with H-Arg-OH to finally obtain the target compound Pal-Gly-Gln-Pro-Arg-OH.
[0045] Therefore, this invention provides a liquid-phase peptide synthesis (LPPS) method using readily available and inexpensive palmitic acid, glycine, Boc-Gln-OH, H-Pro-OBzl.HCl, and arginine as main raw materials, and using condensing agents such as HBTU and DSC, and DIPEA as a base. Pal-Gly-OH, Boc-Gln-Pro-OBzl, H-Gln-Pro-OBzl.HCl, Pal-Gly-Gln-Pro-OBzl, Pal-Gly-Gln-Pro-OH, and Pal-Gly-Gln-Pro-OSu are prepared sequentially according to the synthesis steps, and finally condensed with arginine to obtain the target compound Pal-Gly-Gln-Pro-Arg-OH.
[0046] From a sustainability perspective, this method uses environmentally friendly solvents such as 2-methyltetrahydrofuran and n-heptane, and the solvent system is singular in each step, making it easy to recover and recycle, thus reducing the overall consumption of organic solvents. The reaction conditions are mild, mainly carried out at room temperature or in the range of 0–50°C, reducing energy consumption. Furthermore, the synthetic route avoids the use of perfluoroalkyl substances and reagents related to carcinogenicity, mutagenicity, and reproductive toxicity, improving the environmental compatibility of the process and the safety of operators.
[0047] From a production perspective, this method features a compact step design, reducing unnecessary purification steps by optimizing the reaction sequence and intermediate handling. The reaction conditions at each step are easy to control, and the intermediates exhibit good stability, facilitating storage and transportation. The process is relatively simple to operate, suitable for scale-up production, and helps improve production efficiency and reduce costs. Currently, this process has been scaled up to a 100 kg scale, with an overall yield of 50-60% and a purity of no less than 97.0%.
[0048] From a product quality perspective, this method effectively avoids intramolecular / intermolecular self-condensation of the Pal-Gly-Gln-OH intermediate through ingenious molecular design and protection strategies. By selecting appropriate condensing agents (such as HBTU and DSC) and reaction conditions, the risk of epimerization is significantly reduced. Simultaneously, mild reaction conditions and precise pH control minimize side reactions, contributing to the acquisition of a high-purity final product. The well-designed separation and purification steps for the intermediate and product further ensure the stability and consistency of product quality.
[0049] In summary, this method features a rationally designed process route, mild reaction conditions, low organic solvent consumption with easy recovery, and simple operation. It effectively controls key issues such as self-condensation and epimerization, resulting in high product yield and purity. It is an economical and environmentally friendly method for the commercial synthesis of high-quality palmitoyl tetrapeptide-7, with promising industrial application prospects. Attached Figure Description
[0050] Figure 1 The synthetic route diagram of this application is shown.
[0051] Figure 2 The HPLC spectrum of Pal-Gly-OH obtained according to embodiment S2 of this application is shown.
[0052] Figure 3 The HPLC chromatogram of Boc-Gln-Pro-OBzl obtained in S3 according to an embodiment of this application is shown.
[0053] Figure 4 The HPLC spectrum of H-Gln-Pro-OBzl.HCl obtained in S4 according to an embodiment of this application is shown.
[0054] Figure 5 The HPLC chromatogram of Pal-Gly-Gln-Pro-OBzl obtained in S5 according to an embodiment of this application is shown.
[0055] Figure 6 The HPLC chromatogram of Pal-Gly-Gln-Pro-OH obtained in S6 according to an embodiment of this application is shown.
[0056] Figure 7 The HPLC spectrum of Pal-GQPR-OH obtained according to an embodiment of this application is shown.
[0057] Figure 8 The positive ion mode mass spectrum of Pal-GQPR-OH obtained according to an embodiment of this application is shown.
[0058] Figure 9The secondary mass spectrum of Pal-GQPR-OH obtained according to an embodiment of this application is shown.
[0059] Figure 10 The Pal-GQPR-OH obtained according to an embodiment of this application is shown. 1 H NMR spectrum. Detailed Implementation
[0060] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on this application. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0061] This invention provides a method for preparing palmitoyl tetrapeptide-7, which uses simple and inexpensive starting materials and natural amino acids to produce high-quality palmitoyl tetrapeptide-7 in a relatively mild reaction condition with a high yield. The post-processing method is simple and conducive to industrial-scale production.
[0062] As can be seen from the specific embodiments below, the method of the present invention has stable yield and high product purity in kilogram-scale experiments.
[0063] The present invention will now be further described using a particularly preferred synthetic route. It will be understood by those skilled in the art that the present invention is not limited to the specific embodiments described below. The reactants, catalysts, solvents, etc., used in the following preparation examples are commercially available.
[0064] The abbreviations and their corresponding English translations used in this invention are shown in the table below:
[0065] In the reaction, HPLC is used to detect the reaction and product purity. The specific method of HPLC is as follows: Analysis Method 1, used for S1 and S2 Column: Kromasil C18 250 × 4.6 mm 5 μm Column temperature: 30 degrees Celsius Flow rate: 1.0 mL / min Detector: CAD Mobile phase: A. Chromatographic grade acetonitrile containing 0.05% trifluoroacetic acid; B. Chromatographic grade water containing 0.05% trifluoroacetic acid Gradient: 0 minutes 80%A and 20%B 10 minutes 100% A and 0% B 20 minutes 100%A and 0%B Pal-Gly-OH has a shelf life of 10.2 minutes. Pal-OH retention time: 14.6 minutes Analysis Method 2 is used for S3, S4, S5, S6, S7, and S8. Column: Kromasil C18 250 × 4.6 mm 5 μm Column temperature: 25 degrees Celsius Flow rate: 1.0 mL / min Detector: UV (210 nm) Mobile phase: A. Chromatographic grade water containing 0.1% trifluoroacetic acid; B-grade acetonitrile Gradient: 0 minutes 70%A and 30%B 10 minutes 30%A and 70%B Boc-Gln-Pro-OBn retention time: 9.1 minutes H-Gln-Pro-OBn.HCl retention time: 4.3 minutes Pal-Gly-Gln-Pro-OBn retention time: 24.0 minutes Pal-Gly-Gln-Pro-OH, shelf life: 18.7 minutes Pal-Gly-Gln-Pro-OSu retention time: 20.5 minutes Pal-Gly-Gln-Pro-Arg-OH, retention time: 13.1 minutes. Example 1 S1: At 20-25℃, add 178 g of palmitic acid and 700 mL of n-heptane to a reactor equipped with an acidic gas absorption device. Raise the temperature to 45-50℃ and stir for 15 minutes. Then, add 200 g of thionyl chloride dropwise over at least 15 minutes, maintaining the temperature at 45-50℃. After the addition is complete, continue the reaction for 2 hours. Concentrate under reduced pressure until no fraction remains, add 360 mL of n-heptane, and concentrate again to obtain 200 g of a colorless, transparent oily substance, Pal-Cl, which can be used directly in the next step.
[0066] S2: At 20-25℃, add 800 mL of water, 78.2 g of H-Gly-OH, and 200 mL of 2-methyltetrahydrofuran to a 3 L reaction flask, and stir until the solid is completely dissolved. Adjust the pH to 10.0-10.8 by adding 30% NaOH aqueous solution dropwise, and cool to 0-5℃. At 0-5℃, add a mixed solution of 200 g Pal-Cl and 200 mL of 2-methyltetrahydrofuran dropwise, while simultaneously adding 30% NaOH aqueous solution dropwise to maintain the pH at 10-11. The dropwise addition time should be controlled at more than 3 hours, and stirring should continue for 2.5-3 hours at 0-5℃ and pH 10-11. Adjust the pH to 2.5-3.0 by adding concentrated hydrochloric acid aqueous solution dropwise, stir for 0.5-1 hours, filter, and collect the filter cake. Dry at 40-45℃ to obtain 175.5 g of white solid Pal-Gly-OH, with an overall yield of 82.0% for steps 1 and 2.
[0067] S3: At 20-25℃, add 103 g Boc-Gln-OH, 87.9 g H-Pro-OBzl.HCl, and 620 mL 2-methyltetrahydrofuran to a 2 L reaction flask, add 141 g DIPEA dropwise, stir for 5-10 minutes, then add 165.5 g HBTU in portions, stir at room temperature for 2-3 hours until clear; concentrate at T<40℃ to 610 g of oil, add 650 mL EA, wash successively with water, saturated NaHCO3 aqueous solution, and 10% citric acid aqueous solution, add 200 mL of water and adjust pH to 6.5-7.0 with saturated NaHCO3 aqueous solution; concentrate at T<40℃ to 300 g of solid-liquid mixture, add 800 mL of water, stir at room temperature for 2-3 hours, filter, and dry at 45℃ to obtain 116 g of solid, yield 73%.
[0068] S4: Add 200 g of Boc-Gln-Pro-OBzl and 2 L of acetonitrile to a 3 L reaction flask. Cool to 5-10 °C and, under nitrogen protection, add 255 g of 19.8% HCl acetonitrile solution dropwise over approximately 45 minutes. After the addition is complete, gradually raise the temperature to 20-25 °C and stir for 1-2 hours. Continue stirring at room temperature for at least 3 hours. Cool to 5-10 °C and stir for 0.5 hours, then filter and dry at 45-50 °C to obtain 150.2 g of white solid, yield 88%.
[0069] S5: Add 12.5 g Pal-Gly-OH, 125 mL 2-methyltetrahydrofuran, and 5.7 g DIPEA to a 500 mL reaction flask. Stir at 20-25 °C for 5 minutes until clear. Add 16.7 g HBTU and continue stirring for 5 minutes. Add 16.3 g H-Gln-Pro-OBzl.HCl in portions. Then, add a mixed solution of 8 g DIPEA and 12.5 mL Me-THF dropwise over 30 minutes. Stir at room temperature for 3 hours. Add 100 mL EA and 100 mL water to the reaction mixture. Stir for 10-20 minutes and let stand for 30 minutes. Collect the organic phase by separation. Wash once with 100 mL of 10% citric acid aqueous solution, 50 mL of water, and 100 mL of saturated NaHCO3 aqueous solution. Collect the organic phase and concentrate under reduced pressure at 40-45 °C to an oily substance.
[0070] S6: Add 30.0 g of oily Pal-Gly-Gln-Pro-OBzl, 100 mL of IPA, and 50 mL of water to a 250 mL flask, and stir at 20-25 °C for 5 minutes until clear; add a mixture of 1.85 g of LiOH·H2O and 12.5 mL of water to adjust the pH to 12.0-12.5, and stir at room temperature for 2 hours; adjust the pH to 6.5-7.5 with HCl solution, concentrate under reduced pressure at 40-45 °C to remove IPA, cool to room temperature, add 100 mL of water, and adjust the pH to 2.0-2.5 with HCl solution; add 100 mL of EA, stir, and separate the liquids; wash the organic phase twice with 50 mL of water, and concentrate under reduced pressure at 40-45 °C; add 180 mL of acetonitrile, stir at room temperature for 1 hour, and dry at 45-50 °C to obtain 17.0 g of white solid. The overall yield of steps 5 and 6 is 78.9%.
[0071] S7: Add 5.0 g Pal-Gly-Gln-Pro-OH, 50 mL ACN, 2.9 g DSC and 0.1 g DMAP to a 250 mL reaction flask A, and stir at 30~35℃ for 0.5-1.5 hours; S8: Take another 250 mL reaction flask B, add 1.6 g H-Arg-OH and 12 mL water and stir until clear. After stirring at room temperature for 15 minutes, add 3.6 g DIPEA. Add the mixture from flask A in step 7 dropwise to flask B at 20-25℃ over 1 hour. After stirring at room temperature for 0.5-1 hour, add 60 mL acetonitrile and filter. Add 50 mL methanol and 1.2 g acetic acid to the filter cake and stir until clear. Add 50 mL water and 50 mL acetonitrile and cool to room temperature. Stir for 3-4 hours and filter. Dry at 45℃ to obtain 5.6 g of white solid. The total yield of steps 7 and 8 is 80%.
[0072] Example 2 S1: At 20-25°C, add 3.6 kg of palmitic acid and 14 L of n-heptane to a 30 L reactor. Raise the temperature to 45-50°C and stir for 15 minutes. Then, add 4 kg of thionyl chloride dropwise over at least 15 minutes, maintaining the temperature at 45-50°C. After the addition is complete, continue the reaction for 2 hours. Concentrate under reduced pressure until no fraction remains. Add 7.2 L of n-heptane and concentrate again to obtain 4 kg of a colorless, transparent oily substance, Pal-Cl. Use 2 kg of Pal-Cl directly for the next step.
[0073] S2: At 20-25℃, add 8 L of water, 0.78 kg of H-Gly-OH, and 2 L of 2-methyltetrahydrofuran to a 30 L reaction flask, and stir until the solid is completely dissolved. Adjust the pH to 10-11 by adding 30% NaOH aqueous solution dropwise, and cool to 0-5℃. At 0-5℃, add a mixed solution of 2 kg Pal-Cl and 2 L of 2-methyltetrahydrofuran dropwise, while simultaneously adding 30% NaOH aqueous solution dropwise to maintain the pH at 10-11. The dropwise addition time should be controlled to be more than 3 hours, and stirring should continue for 2.5-3 hours at 0-5℃ and pH 10-11. Adjust the pH to 2.5-3.0 by adding concentrated hydrochloric acid aqueous solution dropwise, stir for 0.5-1 hours, filter, collect the filter cake, and the product has a chemical purity of 99.4% (the obtained spectrum is shown in...). Figure 2 (In the middle). Drying at 40-45℃, 1.8 kg of white solid Pal-Gly-OH was obtained, with an overall yield of 83.2% for steps 1 and 2.
[0074] S3: At 20-25℃, add 2 kg Boc-Gln-OH, 1.75 kg H-Pro-OBzl.HCl, and 12 L 2-methyltetrahydrofuran to a 30 L reaction flask. Add 2.8 kg DIPEA dropwise, stir for 5-10 minutes, then add 3.3 kg HBTU in portions. Stir at room temperature for 2-3 hours until clear. Concentrate to 1.2 kg of oil at T<40℃, add 13 L EA, and wash successively with water, saturated NaHCO3 aqueous solution, and 10% citric acid aqueous solution. Add 4 L of water and adjust the pH to 6.5-7.0 with saturated NaHCO3 aqueous solution. Concentrate to 6 kg of solid-liquid mixture at T<40℃, add 16 L of water, stir at room temperature for 2-3 hours, filter, and dry at 45℃ to obtain 2.4 kg of solid with a chemical purity of 99.2% (the obtained spectrum is shown in...). Figure 3 (In the middle), the yield was 75.5%.
[0075] S4: Add 2 kg of Boc-Gln-Pro-OBzl and 20 L of acetonitrile to a 30 L reaction flask. Cool to 5-10 °C and, under nitrogen protection, add 2.5 kg of 19.8% HCl acetonitrile solution dropwise over approximately 45 minutes. After the addition is complete, gradually raise the temperature to 20-25 °C and stir for 1-2 hours. Continue stirring at room temperature for at least 3 hours. Cool to 5-10 °C and stir for 0.5 hours, then filter. Dry at 45-50 °C to obtain 1.54 kg of white solid with a chemical purity of 99.5% (the resulting spectrum is shown in...). Figure 4 (Medium), yield 90%.
[0076] S5: Add 750 g Pal-Gly-OH, 7.5 L 2-methyltetrahydrofuran, and 342 g DIPEA to a 30 L reaction flask. Stir at 20-25°C for 5 minutes until clear. Add 1 kg HBTU and continue stirring for 5 minutes. Add 9.8 kg H-Gln-Pro-OBzl.HCl in portions, followed by dropwise addition of a mixed solution of 480 g DIPEA and 750 mL Me-THF over 30 minutes. Stir at room temperature for 3 hours. Add 6 L EA and 6 L water to the reaction mixture, stir for 10-20 minutes, and let stand for 30 minutes. Collect the organic phase separately and wash it once each with 6 L of 10% citric acid aqueous solution, 3 L of water, and 6 L of saturated NaHCO3 aqueous solution. Collect the organic phase and concentrate it under reduced pressure at 40-45°C to an oily substance for direct use in the next step. The chemical purity is 98.1% (the resulting spectrum is shown in...). Figure 5 middle).
[0077] S6: Add 3.6 kg of the oily compound Pal-Gly-Gln-Pro-OBzl, 12 L of IPA, and 6 L of water to a 30 L flask, and stir at 20-25°C for 5 minutes until clear; adjust the pH to 12.0-12.5 by adding a mixture of 222 g LiOH·H2O and 1.5 L of water, and stir at room temperature for 2 hours; perform HPLC analysis (the resulting chromatogram is shown in...). Figure 6 After the reaction was complete, the pH was adjusted to 6.5-7.5 with HCl solution, and the mixture was concentrated under reduced pressure at 40-45℃ to remove IPA. After cooling to room temperature, 12 L of water was added, and the pH was adjusted to 2.0-2.5 with HCl solution. 12 L of EA was added, and the mixture was stirred and separated. The organic phase was washed twice with 6 L of water and concentrated under reduced pressure at 40-45℃. 21.6 L of acetonitrile was added and stirred at room temperature for 1 hour. The mixture was dried at 45-50℃ to obtain 2 kg of white solid. The overall yield of steps 5 and 6 was 77.4%.
[0078] S7: Add 600 g Pal-Gly-Gln-Pro-OH, 6 L ACN, 348 g DSC and 12 g DMAP to a 30 L reaction flask A, and stir at 30~35℃ for 1-2 hours; S8: In a separate 30 L reaction flask B, add 192 g H-Arg-OH and 1.4 L water and stir until clear. After stirring at room temperature for 15 minutes, add 432 g DIPEA. Add the mixture from flask A in step 7 dropwise to flask B at 20-25°C over 1 hour. After stirring at room temperature for 0.5-1 hour, add 7.2 L acetonitrile and filter. Add 6 L methanol and 144 g acetic acid to the filter cake and stir until clear. Add 6 L water and 6 L acetonitrile and cool to room temperature. Stir for 3-4 hours and filter. Dry at 45°C to obtain 706 g of white solid with a chemical purity of 97.4% (the obtained spectrum is shown in...). Figure 7 (In the middle), the overall yield of steps 7 and 8 was 84%. (Positive ion mode mass spectrum of Pal-GQPR-OH (the obtained spectrum is shown in...) Figure 8 (middle); secondary mass spectrum (the resulting spectrum is shown in...) Figure 9 middle) The obtained product Pal-GQPR was subjected to NMR analysis, and the results were obtained... 1 H NMR (the resulting spectrum is shown in) Figure 10 The test results are as follows (in Chinese). The peaks at chemical shifts of 3.71 and 6.10 originate from the internal standard 1,3,5-trimethoxybenzene. : 1 H NMR (400 MHz, DMSO) δ 8.38 (d, 1H), 8.24 (d, 1H), 8.10 (d, 1H), 8.00 (s, 1H), 7.93 (d, 1H), 7.57 (s, 1H), 7.41 (d, 1H), 7.10 (d, 5H), 4.67 (d, 1H), 4.50 (d, 1H), 4.39 (d, 1H), 4.21 (d, 1H), 3.12 (d, 2H), 2.33 – 2.00 (m, 5H), 1.99 – 1.72 (m, 8H), 1.72 – 1.40 (m, 6H), 1.24 (s, 24H), 0.86 (t, J = 6.2 Hz, 3H).
[0079] The above description is merely an embodiment of this application and is not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, and improvements made within the spirit and scope of this application are included within the scope of protection of this application.
Claims
1. A method for preparing palmitoyl tetrapeptide-7 and its salts via liquid-phase peptide synthesis (LPPS), wherein, The method includes: Step 1: Palmitoyl chloride (Pal-Cl) is obtained from palmitic acid (Pal) via an acyl chloride reaction, and then palmitoyl glycine (Pal-Gly-OH) is generated by a substitution reaction with glycine (Gly). Step 1': N-tert-butoxycarbonyl-glutamine (Boc-Gln-Pro-OBzl) is synthesized from N-tert-butoxycarbonyl-glutamine (Boc-Gln-OH) and benzyl proline hydrochloride (H-Pro-OBzl·HCl) via dipeptide condensation reaction, and then deprotected to obtain benzyl glutamine hydrochloride (H-Gln-Pro-OBzl.HCl). Step 2: Palmitoyl-glycyl-glutamine-proline benzyl ester (Pal-Gly-Gln-Pro-OBzl) is obtained by tripeptide condensation reaction of Pal-Gly-OH and H-Gln-Pro-OBzl.HCl. Step 3: Remove the protecting group via hydrolysis to prepare palmitoyl-gly-glutamine-proline (Pal-Gly-Gln-Pro-OH); and Step 4: Pal-Gly-Gln-Pro-OH is prepared into an active ester, palmitoyl-glycyl-glutamine-proline succinimide ester (Pal-Gly-Gln-Pro-OSu), via ester condensation reaction. Then, palmitoyl tetrapeptide-7 (Pal-Gly-Gln-Pro-Arg-OH) is obtained by reacting Pal-Gly-Gln-Pro-OSu with arginine (Arg) via tetrapeptide condensation reaction.
2. The method according to claim 1, wherein, The acyl chloride reaction is carried out in the presence of an acylating agent in a first solvent and at a first temperature, wherein the acylating agent is selected from one or more of thionyl chloride, oxalyl chloride, phosphorus oxychloride and phosphorus pentachloride, preferably thionyl chloride; the first temperature is 20°C to 60°C, preferably 40°C to 50°C; the first solvent is selected from one or more of toluene, n-heptane and methyl tert-butyl ether, preferably n-heptane.
3. The method according to any one of claims 1 to 2, wherein, The substitution reaction is carried out at a pH of 9 to 12, preferably 10 to 11, at a temperature of 0 to 30°C, preferably 0 to 10°C; and the molar ratio of palmitoyl chloride (Pal-Cl) to glycine (Gly) is 1:(1.0~2), preferably 1:1.
5.
4. The method according to any one of claims 1 to 3, wherein, The dipeptide condensation reaction is carried out in the presence of a first condensation reagent, which is selected from one or more of TBTU, HBTU, DCC, HOBt, HOSu, and DSC, with HBTU being preferred.
5. The method according to any one of claims 1 to 4, wherein, The deprotection is carried out in the presence of trifluoroacetic acid, an aqueous solution of hydrogen chloride, an acetonitrile solution of hydrogen chloride, and an ethyl acetate solution of hydrogen chloride; preferably, it is carried out in the presence of an acetonitrile solution of hydrogen chloride.
6. The method according to any one of claims 1 to 5, wherein, The tripeptide condensation reaction is carried out in the presence of a second condensation reagent, which is selected from one or more of TBTU, HBTU, DCC, HOBt, HOSu, and DSC; preferably HBTU.
7. The method according to any one of claims 1 to 6, wherein, The hydrolysis reaction is carried out in the presence of one or more of lithium hydroxide, sodium hydroxide, and potassium hydroxide; preferably, the hydrolysis reaction is carried out in the presence of lithium hydroxide.
8. The method according to any one of claims 1 to 7, wherein, The ester condensation reaction is carried out in the presence of a third condensing agent, which is selected from one or more of TBTU, HBTU, DCC, HOBt, HOSu, and DSC; preferably DSC.
9. The method according to any one of claims 1 to 8, wherein, The tetrapeptide condensation reaction is carried out in a second solvent and in the presence of a base, wherein the second solvent is selected from one or more of 2-methyltetrahydrofuran, tetrahydrofuran and acetonitrile, preferably acetonitrile; the molar ratio of H-Arg-OH to base is 1:(1.0-3.0), preferably 1:3.0.