Palmitoyl tripeptide-38 as well as preparation method and application thereof

Abstract

The invention relates to palmitoyl tripeptide-38 and a preparation method and application thereof.According to the palmitoyl tripeptide-38, low-toxicity and biodegradable NFM and anisole are compounded according to the volume ratio of 1: 1, and the palmitoyl tripeptide-38 has strong polarity and moderate hydrophobicity and can completely replace DMF. According to the mixed solvent, the solubility of Fmoc amino acid monomers, activating reagents and resin is improved, the risk of reproductive toxicity is avoided, and the volatility is reduced compared with that of DMF (Dimethyl Formamide); the process is synergistically enhanced, namely, the mass transfer limitation of the traditional intermittent reaction is overcome through the synergism of the laminar shear effect and the ultrasonic cavitation effect of a continuous flow reaction device, so that the coupling reaction time of the palmitoyl tripeptide-38 is shortened, the occurrence rate of side reaction is reduced, and the universality verifies that the solvent system has excellent performance in synthesis of the palmitoyl tripeptide-38, and the solvent system is suitable for industrial production. And the coupling efficiency is stable.

Description

Technical Field

[0001] This invention relates to the field of solid-phase polypeptide preparation technology, specifically to a palmitoyl tripeptide-38, its preparation method, and its applications. Background Technology

[0002] In traditional solid-phase peptide synthesis (SPPS) processes, N,N-dimethylformamide (DMF) is widely used in the dissolution and coupling reactions of amino acids due to its excellent solubility. However, DMF has been clearly classified as a reproductive toxicant and has environmental accumulation properties, posing a potential threat not only to the health of operators but also to ecological and environmental safety.

[0003] Although various solvents have been developed to replace DMF in existing technologies, such as N-methyl-2-pyrrolidone (NMP), there are still many limitations, mainly including: 1. Imbalance between toxicity and economy: Although NMP is less toxic than DMF, it is still considered to have reproductive hazards and is more expensive; while solvents with lower toxicity, such as anisole, are not good at dissolving Fmoc-protected amino acids when used alone, which can lead to a decrease in coupling efficiency and limit their application in SPPS. 2. Low reaction efficiency: Existing SPPS mostly adopts batch operation, and the reaction process relies on diffusion mass transfer mechanism, which leads to long time consumption for key steps such as coupling and deprotection. Especially when preparing long-chain or complex peptides, the overall cycle can be extended to several days, which significantly affects production efficiency. 3. Poor adaptability of solvent systems: Current alternative solvents are insufficient in terms of swelling capacity of peptide resins, making it difficult to meet the reaction space required for the preparation of long-chain peptides. At the same time, they have poor compatibility with advanced preparation processes such as continuous flow, which limits the integration and automation of the process. 4. High purification difficulty: Byproducts are easily generated during the reaction process, and some solvents are difficult to remove, resulting in limited purity of the target product. Multiple purification steps are required, which not only increases the complexity of the operation, but also increases the overall cost and environmental burden.

[0004] To address the shortcomings of existing solvent systems, this invention, through systematic screening (see Tables 1-4), discovered that the NFM / An (N-formylmorpholine / anisole = 1:1, v / v) mixed solvent possesses both strong polarity and moderate hydrophobicity. Its solubility and swelling properties for Fmoc amino acid monomers (such as Fmoc-Phe-OH), activating agents (such as OxymaPure / DIC), and resins are superior to traditional DMF and other green solvents (NOP / An, NCP / An). Specifically: Solubility: It has better compatibility with the poorly soluble reagent HATU (Table 1); Swelling properties: The swelling degree of CTC resin reaches 5.0 mL·g -1 (Table 2) To avoid the risk of excessive swelling; Reaction efficiency: Coupling rate under ultrasound assistance (k~obs~=0.556 min) -1 It is 4.5 times better than DMF (Table 3-4); Side reaction control: the epimerization rate was reduced to 2.5% (Table 5).

[0005] Based on the above advantages, the following process will specifically demonstrate the synergistic application of NFM / An in the synthesis of palmitoyl tripeptide-38. Summary of the Invention

[0006] Therefore, it is necessary to provide a palmitoyl tripeptide-38, its preparation method, and its application to address the deficiencies in the existing technology.

[0007] To achieve the above objectives, the present invention provides a technical solution: A method for preparing palmitoyl tripeptide-38, wherein the sequence of palmitoyl tripeptide-38 is H-Pal-Lys-Met(O2)-Lys-OH, includes the following steps: S100. The resin is loaded into a continuous flow reaction system and pretreated using a green solvent. The green solvent is a mixed solution of N-formylmorpholine and anisole in a volume ratio of 1:1.

[0008] The specific steps are as follows: The resin was loaded into a PTFE tubular reactor, rinsed with a green solvent, and swollen with ultrasonic assistance.

[0009] In some embodiments, the resin includes at least one of CTC resin (2-chlorotriphenylmethyl chloride resin) and ClT resin (chlorotriphenylmethyl resin), with a substitution value of 0.4-3.0 mmol / g.

[0010] In some embodiments, preferably, the resin is CTC resin.

[0011] In some embodiments, the continuous flow reaction has a flow rate of 1-3 mL / min, a reactor temperature of 25°C-40°C, an ultrasonic power of 100 W-400 W, and a residence time of 30 min-60 min.

[0012] S200. Through a continuous flow reaction system, the coupling and deprotection steps of three Fmoc-protected amino acids are completed sequentially to obtain a polypeptide chain; The amino acids protected by Fmoc are, in order: Fmoc-Lys(Boc)-OH, Fmoc-Met(O2)-OH, and Fmoc-Lys(Boc)-OH.

[0013] The specific steps are as follows: S210. The coupling and deprotection steps of Fmoc-Lys(Boc)-OH are completed through a continuous flow reaction system.

[0014] The coupling steps of S211.Fmoc-Lys(Boc)-OH are as follows: Fmoc-Lys(Boc)-OH and a coupling agent are dissolved in a green solvent and injected into a syringe pump to react with the swollen CTC resin from step S100, thereby loading amino acids onto the resin. The coupling agent includes N,N'-diisopropylcarbodiimide.

[0015] The deprotection steps of S212.Fmoc-Lys(Boc)-OH are as follows: The deprotecting agent / green solvent solution is reacted with the product from step S211, and ultrasonic-assisted treatment is performed. The waste liquid is directly introduced into a waste liquid tank. The deprotecting agent is DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene), and the volume fraction of DBU in the deprotecting agent / green solvent mixed solution is 5%.

[0016] The product was washed with methanol and then with green solvent in sequence by switching solvent pumps.

[0017] S220. The coupling and deprotection steps of Fmoc-Met(O2)-OH are completed through a continuous flow reaction system.

[0018] The coupling steps of S221. Fmoc-Met(O2)-OH are as follows: Fmoc-Met(O2)-OH and the coupling agent are dissolved in a green solvent, pre-activated by an online mixing module, and then injected into the product of step S212. The coupling agent includes at least one of 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione and N,N'-diisopropylcarbodiimide.

[0019] Reaction parameters: The continuous flow reaction rate was 1 mL / min - 3 mL / min, the reactor temperature was 25℃ - 40℃, the ultrasonic power was 100 W - 140 W, and the coupling reaction residence time was 10 min - 30 min. Coupling completion was confirmed by online ninhydrin detection, and the resin was washed using a solvent pump.

[0020] The deprotection steps of S222. Fmoc-Met(O2)-OH are as follows: The mixed solution of deprotecting agent / green solvent is pumped into the reactor and reacted with the product of step S221. Ultrasonic treatment is performed, and the waste liquid is directly discharged into a waste liquid tank. The deprotecting agent is DBU, and the volume fraction of DBU in the mixed solution of deprotecting agent / green solvent is 5%.

[0021] The reaction products were washed sequentially with methanol and green solvent by switching solvent pumps.

[0022] S230. The coupling and deprotection steps of Fmoc-Lys(Boc)-OH are completed through a continuous flow reaction system.

[0023] The coupling steps of S231. Fmoc-Lys(Boc)-OH are as follows: Fmoc-Lys(Boc)-OH and the coupling agent are dissolved in a green solvent, pre-activated by an online mixing module, and then injected into the product of step S222. The coupling agent includes at least one of 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione and N,N'-diisopropylcarbodiimide.

[0024] Reaction parameters: The continuous flow reaction rate was 1 mL / min - 3 mL / min, the reactor temperature was 25℃ - 40℃, the ultrasonic power was 100 W - 140 W, and the coupling reaction residence time was 10 min - 30 min. Coupling completion was confirmed by online ninhydrin detection, and the reaction products were washed using a solvent pump.

[0025] The deprotection steps of S232. Fmoc-Lys(Boc)-OH are as follows: The mixed solution of deprotectant / green solvent is pumped into the reactor and injected into the product of step S231. Ultrasonic treatment is then performed to obtain the polypeptide chain, and the waste liquid is directly discharged into a waste liquid tank. The deprotectant is DBU, and the volume fraction of DBU in the mixed solution of deprotectant / green solvent is 5%.

[0026] The reaction products were washed sequentially with methanol and green solvent by switching solvent pumps.

[0027] S300. The polypeptide chain and palmitic acid are coupled using a continuous flow reaction system to obtain the final polypeptide chain. In some embodiments, in step S300, the molar ratio of the polypeptide chain to palmitic acid is 1:(1-3).

[0028] The specific steps are as follows: Palmitic acid and a coupling agent are dissolved in a green solvent, mixed and activated online, and then added to the polypeptide chain of the product from step S232. The coupling agent includes at least one of 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione and N,N'-diisopropylcarbodiimide.

[0029] Reaction parameters: The flow rate of the continuous flow reaction is 1 mL / min - 3 mL / min, the reactor temperature is 25℃ - 40℃, the ultrasonic power is 100 W - 140 W, and the residence time of the coupling reaction is 10 min - 30 min.

[0030] S400. The final peptide chain is cleaved to obtain the palmitoyl tripeptide-38.

[0031] The specific steps are as follows: S410, peptide chain cleavage: Add the cutting fluid to the product of step S300, shake the reaction, filter and collect the pyrolysis liquid, combine the filtrates, and remove the organic solvent by rotary evaporation under reduced pressure. The cutting fluid is a mixture of hexafluoroisopropanol (HFIP) and dichloromethane (DCM), wherein the volume fraction of HFIP in the mixture is 20%.

[0032] S420, Purification: Add cold benzyl tert-butyl ether to the product of step S410 to precipitate the crude peptide. After centrifugation and freeze-drying, purify by reversed-phase high-performance liquid chromatography to obtain palmitoyl tripeptide-38H-Pal-Lys(Boc)-Met(O2)-Lys(Boc)-OH with a protecting group. Then treat it with a mixture of trifluoroacetic acid / dichloromethane, in which the volume fraction of trifluoroacetic acid is 30%. Remove the organic solvent by rotary evaporation under reduced pressure to obtain palmitoyl tripeptide-38H-Pal-Lys-Met(O2)-Lys-OH.

[0033] In some embodiments, the chromatographic conditions for the liquid chromatography purification are as follows: Column: Yuexu LP-C18; Mobile phase: water and acetonitrile; Gradient: 1-10 min, acetonitrile from 10%-90%, 10-15 min, acetonitrile from 90%-10%; Flow rate: 1 ml / min; Injection volume: 5 μL; Detection wavelength: 215nm.

[0034] The present invention also provides a palmitoyl tripeptide-38, wherein the purity of the palmitoyl tripeptide-38 is ≥98%.

[0035] This invention also provides the application of palmitoyl tripeptide-38 in the preparation of anti-aging cosmetics.

[0036] The beneficial effects of this invention are: 1. Precise formulation with green solvent: A 1:1 volume ratio of low-toxicity, biodegradable NMF and anisole is used, combining strong polarity with moderate hydrophobicity, making it a complete replacement for DMF. This mixed solvent improves the solubility of Fmoc amino acid monomers, activating agents, and resins, poses no reproductive toxicity risk, and has lower volatility than DMF. 2. Synergistic enhancement of process: By combining the laminar shear effect and ultrasonic cavitation of the continuous flow reactor, the mass transfer limitations of traditional batch reaction are overcome, which shortens the coupling reaction time of palmitoyl tripeptide-38 and reduces the incidence of side reactions. 3. Universality verification: The solvent system performed well in the synthesis of palmitoyl tripeptide-38, with a coupling efficiency of over 95% for each time. Attached Figure Description

[0037] Figure 1 The NMR spectrum of palmitoyl tripeptide-38, a product from one embodiment, is shown below. Figure 2 NMR spectrum of palmitoyl tripeptide-38; Figure 3 Screenshot of the effects of palmitoyl tripeptide-38 in the Meili Xiuxing app; Figure 4 Screenshot of the effects of palmitoyl tripeptide-38 in the Meili Xiuxing app. Detailed Implementation

[0038] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0039] In the embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0040] To replace the traditional toxic solvent DMF, this invention systematically screens combinations of low-toxicity, biodegradable, and green solvents. By comparing four core indicators—solubility, resin swelling, coupling efficiency, and side reaction rate—the optimal solvent system was determined (test results are summarized in Tables 1-5).

[0041] Example 0 (Performance verification of green solvent NFM / An): To verify the solvent's universality, model reactions were used to test the performance of NFM / An: 1. Solubility test of green solvent; The solubility of the green solvent (NFM / An) for amino acids, coupling reagents, and reaction byproducts was tested.

[0042] Known poorly soluble amino acids Fmoc-Phe-OH, the classic coupling reagent combination OxymaPure / DIC (ethyl cyanohydroxyimino ester / N,N'-diisopropylcarbodiimide), OxymaB / DIC (5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione / N,N'-diisopropylcarbodiimide), HATU / DIPEA (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate / N,N-diisopropylethylamine), PyAOP / DIPEA ((7-azabenzotriazol-1-oxy)tripyrrolidinephosphine hexafluorophosphate / N,N-diisopropylethylamine), and the coupling reaction byproduct DIU (diisopropylurea) were selected as references for preliminary screening at a concentration of 0.5%. The solubility of these materials in DMF and NFM / An (1:1, v:v) was tested under M conditions, and the results are shown in Table 1.

[0043] Table 1. Solubility of amino acids, coupling reagents, and DIU in green mixed solvents. In the table, unless the concentration is specified as 0.125 M, the solubility is monitored at a concentration of 0.5 M and the mixture is irradiated with ultrasound at 30 °C for 10 minutes.

[0044] As shown in Table 1, the solubility of NFM / An in Fmoc-Phe-OH and OxymaPure / DIC is comparable to that of DMF.

[0045] The solubility of NFM / An for DIU is 125 M, consistent with that of DMF. DIU can be prevented from precipitating by subsequent dilution.

[0046] 2. Resin swelling test; The swelling degree of the CTC resin (substitution value 0.4 mmol / g-3.0 mmol / g) used in this patent was measured under ultrasound at 30°C.

[0047] In solid-phase peptide synthesis, resin swelling is a key characteristic of the solvent.

[0048] Resin swelling energy: (1) Increase the resin volume and internal pores to enhance the mass transfer of reactants to the resin; (2) Improve the diffusion of reactants inside the resin and their contact rate with the reaction sites; (3) Improve the elution efficiency of by-products and excess reagents; (4) Reduce collisions between resin microspheres to prevent breakage.

[0049] Therefore, the swelling degree of the solvent on the resin needs to be tested before it is used in the reaction, and the results are shown in Table 2.

[0050] Table 2. Swelling degree of resin in solvent (ml g) -1 ) a test In the table, NFM / An was added to CTC resin (i.e., P-2CTC-Cl resin, 1.0 g) to make its final volume 10 mL. After swelling under ultrasonic conditions at 30 °C for 30 min, the resin volume was measured. The average value was taken from three measurements.

[0051] 4 mL . g -1 Insufficient swelling; 4-7 mL . g -1 For sufficient swelling; >7 mL . g -1 This is due to excessive expansion.

[0052] As shown in Table 2, the swelling degree of CTC resin in NFM / An is 5.0 mL·g. -1 Within the ideal range (4 mL·g) -1 -7 mL·g -1 ).

[0053] 3. Coupling reaction rate test: Solid-phase peptide synthesis (SPPS) involves multiple steps, and solvents and process enhancement techniques (ultrasonic / vibration conditions) can both affect its performance.

[0054] Given the advantages of ultrasound-assisted technology, such as high energy utilization, low amplification difficulty, and low equipment cost, and the fact that it is currently a blank in green solvent SPPS, this test combines a novel green solvent (NFM / An) with ultrasound-assisted technology to test the performance of this scheme in the multi-step SPPS process.

[0055] Coupling is a key step in SPPS, and the solvent has a significant impact on the coupling rate, thus it can be used as an important indicator of solvent performance.

[0056] This test uses the standard method of the Pedersen team: Fmoc-Gly-OH reacts with CTC resin loaded with tripeptide H-Pro-Phe-Ala-2-CTC-PS, and samples are taken at regular intervals to analyze the conversion rate and determine the rate.

[0057] Based on this method, the performance of the ultrasound-assisted green solvent NFM / An in the coupling step was tested. Specifically, the kinetic conversion rate of Fmoc-Gly-OH coupled to PFA-CTC (i.e., Pro-Phe-Ala-CTC peptide-resin, that is, the tripeptide Pro-Phe-Ala linked to CTC resin) was measured. After removing Fmoc, washing the resin, and cleaving the peptide, the conversion of the tripeptide to the tetrapeptide was quantified by HPLC analysis. The results are shown in Table 3.

[0058] Table 3. Coupling kinetic conversion data under different conditions In the table: NFM / An a This refers to the molecular ratio of 1.3 eq. DIC / OxymaB / AA being 1:1:1 under ultrasonic conditions, where AA refers to amino acids.

[0059] NFM / An b This refers to the molecular ratio of 1.3 eq. DIC / OxymaB / AA being 1:1:1 under vibration conditions.

[0060] NFM / An c This refers to the molecular ratio of 1.3 eq. DIC / OxymaB / AA being 1:0.2:1 under ultrasonic conditions.

[0061] The coupling reaction rate on the resin is negligible due to the concentration of activated amino acids, and can be approximated as a pseudo-first-order reaction of the NH2 group of the resin peptide: ; In the formula k obs - Apparent reaction rate constant (min) -1 ); a0 - The conversion rate of raw materials when the reaction is complete (%); t - reaction time (min); y - Raw material conversion rate (%) at the corresponding time point.

[0062] Table 4. Fitting results of the dynamic model As shown in Table 4, the pseudo-first-order reaction model fits well in all cases (R0). 2 >0.95). NFM / An exhibits the highest apparent rate constant (k... obs = 0.5563 min -1 The final conversion rate (98.3%) was comparable to that of DMF.

[0063] Ultrasonic conditions significantly enhanced the coupling rate (compared to vibrational conditions) and promoted reaction kinetics. Even under the catalytic concentration of OxymaB (k obs = 0.2546 min -1 The rate is still higher than that of DMF or vibration conditions.

[0064] In summary, NFM / An is an excellent green solvent, with a coupling rate significantly higher than that of the control group (DMF), and ultrasound assistance effectively enhances the reaction rate in the green solvent.

[0065] 4. Side reaction control test; Epimerization is a common side reaction in solid-phase peptide synthesis, which can reduce yield and increase purification difficulty.

[0066] The side reaction rate was tested using the epimerization model peptide Fmoc-Phg-Pro-NH2. This reaction utilizes the active and easily isomerized α-hydrogen in Phg and the steric hindrance of Phg / Pro to compare the degree of isomerization under different conditions. The results are shown in Table 5.

[0067] Table 5 Epimerization rate of Fmoc-Phg-Pro-NH2 coupling under green solvent conditions a In the table, all reactions are carried out in the solution phase.

[0068] DL (%) is (Fmoc-D-Phg-Pro-NH2) / (Fmoc-D-Phg-Pro-NH2+ Fmoc-L-Phg-Pro-NH2)×100.

[0069] After synthesizing Fmoc-D-Phg-Pro-NH2 using Fmoc-D-Phg-OH as the starting material, its position in HLPC was determined.

[0070] OxymaB is 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione, and OxymaPure is ethyl (hydroxyimino)cyanocyanate.

[0071] The DIC / 1.3eq.OxymaB combination exhibits a diastereomeric rate of 2.5% in NFM / An, which is lower than that of DMF or pure NFM.

[0072] The DIC / 0.6eq.OxymaB combination had the lowest epimerization rate in NFM / An (1.2%), while other combinations had higher rates (>3%) in NFM / An.

[0073] The NFM / An green solvent system has been validated in four aspects: solubility, swelling, reaction efficiency, and control of side reactions. It is particularly suitable for the synthesis of palmitoyl tripeptide-38 containing thioether bonds (Met) and long-chain fatty acid modification (Pal), providing a basis for process design in subsequent examples.

[0074] Example 1 A method for preparing palmitoyl tripeptide-38, wherein the sequence number of palmitoyl tripeptide-38 is: H-Pal-Lys-Met-Lys-OH, and the C-terminus to N-terminus sequence is: Lys(Boc)→Met(O2)→Lys(Boc)→Pal; the synthetic route is as follows. Figure 1 As shown.

[0075] The green solvent used in this embodiment refers to N-formylmorpholine: anisole (1:1, volume ratio). equipment: Continuous flow reaction system: including an injection pump (flow rate accuracy ±1%), a PTFE tubular reactor (inner diameter 2 mm, volume 30 mL), and an online mixing module; Ultrasonic reaction tank (40 kHz, power adjustable up to 200 W); Vacuum filtration device; HPLC monitoring system.

[0076] Preparation steps S100. The resin is loaded into a continuous flow reaction system and pretreated using a green solvent.

[0077] The specific steps are as follows: 1.0 g of CTC (substitution value 0.8 mmol / g) resin was loaded into a PTFE tubular reactor, rinsed with green solvent (flow rate 5 mL / min) for 5 min, and ultrasonically assisted swelling for 30 min at a power of 100 W.

[0078] S200. Through a continuous flow reaction system, the coupling and deprotection steps of three Fmoc-protected amino acids are completed sequentially to obtain a polypeptide chain; The amino acids protected by Fmoc are, in order: Fmoc-Lys(Boc)-OH, Fmoc-Met(O2)-OH, and Fmoc-Lys(Boc)-OH.

[0079] The specific steps are as follows: S210. The coupling and deprotection steps of Fmoc-Lys(Boc)-OH are completed through a continuous flow reaction system.

[0080] The coupling steps of S211.Fmoc-Lys(Boc)-OH are as follows (amino acid anchoring): Fmoc-Lys(Boc)-OH (2.4 mmol, 3.0 equiv.) and N,N-diisopropylethylamine (4.8 mmol, 6.0 equiv.) were dissolved in a green solvent (15 mL) and injected into a syringe pump to react with the swollen CTC resin from step S100.

[0081] Continuous flow parameters: flow rate 2 mL / min, reactor temperature 25℃, ultrasonic power 100 W, residence time 60 min, solvent pump was switched to wash 3 times each with methanol and green solvent (flow rate 5 mL / min), methanol (3×10 mL) and green solvent (3×10 mL).

[0082] The deprotection steps of S212.Fmoc-Lys(Boc)-OH are as follows: A mixed solution of 5 v / v% DBU / green solvent was pumped into the reactor at a flow rate of 3 mL / min to react with the product in step S211. The mixture was then treated with ultrasound assistance (120 W) for 2 min, and the waste liquid was directly introduced into the waste liquid tank.

[0083] Switch the solvent pump and wash with methanol (3×10 mL) and green solvent (3×10 mL) in sequence (flow rate 5 mL / min).

[0084] S220. The coupling and deprotection steps of Fmoc-Met(O2)-OH are completed through a continuous flow reaction system.

[0085] The coupling steps of S221. Fmoc-Met(O2)-OH are as follows: Fmoc-Met(O2)-OH (2.4 mmol), 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (0.48 mmol), and N,N'-diisopropylcarbodiimide (3.2 mmol) were dissolved in a green solvent (10 mL), pre-activated for 30 s in an online mixing module, and then injected into the product of step S212.

[0086] Reaction parameters: flow rate 3 mL / min, ultrasonic power 120 W, residence time 12 min. Coupling was confirmed to be complete by online ninhydrin detection. The resin was then washed by switching the solvent pump.

[0087] The deprotection steps of S222. Fmoc-Met(O2)-OH are as follows: A 5% v / v DBU / green solvent mixture was pumped into the reactor at a flow rate of 3 mL / min and injected into the product of step S221. The mixture was then treated with ultrasound assistance (120 W) for 2 min, and the waste liquid was directly introduced into the waste liquid tank.

[0088] The solvent pump was switched to wash with methanol and green solvent in sequence (flow rate 5 mL / min, 3 times each).

[0089] S230. The coupling and deprotection steps of Fmoc-Lys(Boc)-OH are completed through a continuous flow reaction system.

[0090] The coupling steps of S231. Fmoc-Lys(Boc)-OH are as follows: Fmoc-Lys(Boc)-OH (2.4 mmol), 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (0.48 mmol), and N,N'-diisopropylcarbodiimide (3.2 mmol) were dissolved in a green solvent (10 mL), pre-activated for 30 s in an online mixing module, and then injected into the product of step S222.

[0091] Reaction parameters: flow rate 3 mL / min, ultrasonic power 120 W, residence time 12 min. Coupling was confirmed to be complete by online ninhydrin detection. The resin was then washed by switching the solvent pump.

[0092] The deprotection steps of S232. Fmoc-Lys(Boc)-OH are as follows: A mixed solution of 5 v / v% DBU / green solvent was pumped into the reactor at a flow rate of 3 mL / min and reacted with the product of step S231. The mixture was then treated with ultrasound assistance (120 W) for 2 min to obtain the polypeptide chain. The waste liquid was directly introduced into the waste liquid tank.

[0093] The solvent pump was switched to wash with methanol and green solvent in sequence (flow rate 5 mL / min, 3 times each).

[0094] S300. The polypeptide chain and palmitic acid are coupled using a continuous flow reaction system to obtain the final polypeptide chain.

[0095] The specific steps are as follows: Palmitic acid (2.4 mmol), 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (0.96 mmol), and N,N'-diisopropylcarbodiimide (3.2 mmol) were dissolved in a green solvent (10 mL), mixed and activated online for 30 s, and then added to the polypeptide chain of the product from step S232.

[0096] Reaction parameters: flow rate 2 mL / min, ultrasonic power 120 W, residence time 12 min, reaction temperature 30℃.

[0097] S400. The final peptide chain is cleaved to obtain the palmitoyl tripeptide-38.

[0098] The specific steps are as follows: S410, peptide chain cleavage: Add 15 mL of a 20% hexafluoroisopropanol (HFIP) / dichloromethane (DCM) mixture to the product of step S300, shake at room temperature for 1.5 hours, filter and collect the lysate, combine the filtrates, and remove the organic solvent by rotary evaporation under reduced pressure.

[0099] S420, Purification: Add 40 mL of cold benzyl tert-butyl ether to the product from step S410 to precipitate the crude peptide. After centrifugation and lyophilization, purify by reversed-phase high-performance liquid chromatography to obtain palmitoyl tripeptide-38 H-Pal-Lys(Boc)-Met(O2)-Lys(Boc)-OH with a protecting group. Then treat it with a mixed solution of 30 v / v% trifluoroacetic acid / dichloromethane for 1 h, and remove the organic solvent by rotary evaporation under reduced pressure to obtain palmitoyl tripeptide-38 (serial number H-Pal-Lys-Met(O2)-Lys-OH). The product yield is 72.80% and the purity is 95.03%. The NMR chromatogram of the product palmitoyl tripeptide-38 is shown below. Figure 2 As shown.

[0100] The chromatographic conditions are as follows: Column: Yuexu LP-C18; Mobile phase: water and acetonitrile; Gradient: 1-10 min, acetonitrile from 10%-90%, 10-15 min, acetonitrile from 90%-10%; Flow rate: 1 ml / min; Injection volume: 5 μL; Detection wavelength: 215nm.

[0101] Test Example 1 The other steps are the same as in Example 1, except that different processes are used for coupling and deprotection. The yields of the obtained palmitoyl tripeptide-38H-Pal-Lys-Met(O2)-Lys-OH are shown in Table 6.

[0102] Table 6. Yield of palmitoyl tripeptide-38H-Pal-Lys-Met(O2)-Lys-OH Application Example 1 The palmitoyl tripeptide-38 prepared in Example 1 was applied to anti-aging cosmetics, such as... Figure 3 and Figure 4 As shown, a total of 526 products containing palmitoyl tripeptide-38 were found in the Meili Xiuxing APP, which have multiple uses, such as facial care, lip care, makeup, eye care, and neck care.

[0103] It should be noted that the specific parameters or reagents in the above embodiments are specific or preferred embodiments under the concept of the present invention, and not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of the present invention.

Claims

1. A method for preparing palmitoyl tripeptide-38, characterized by, The sequence of the palmitoyl tripeptide-38 is H-Pal-Lys-Met(O2)-Lys-OH, comprising the following steps: S100. The resin is loaded into a continuous flow reaction system, and a green solvent is used for pretreatment; S200. Through the continuous flow reaction system, the coupling and deprotection steps of three Fmoc-protected amino acids are sequentially completed to obtain a polypeptide chain; The Fmoc-protected amino acids are Fmoc-Lys(Boc)-OH, Fmoc-Met(O2)-OH, and Fmoc-Lys(Boc)-OH in sequence; S300. The polypeptide chain and palmitic acid are coupled by the continuous flow reaction system to obtain a final peptide chain; S400. The final peptide chain is cut to obtain the palmitoyl tripeptide-38. The green solvent is a mixed solution of N-formylmorpholine and anisole.

2. The method of preparing palmitoyl tripeptide-38 according to claim 1, characterized in that, In step S100, the resin includes at least one of CTC resin and ClT resin, and the substitution value is 0.4 mmol / g-3.0 mmol / g.

3. The method of preparing palmitoyl tripeptide-38 according to claim 1, characterized in that, In step S100, the flow rate of the continuous flow reaction is 1-3 mL / min, the reactor temperature is 25℃-40℃, the ultrasonic power is 100 W-400 W, and the residence time is 30 min-60 min.

4. The method of preparing palmitoyl tripeptide-38 according to claim 1, characterized in that, In step S200, the flow rate of the continuous flow reaction is 1 mL / min -3 mL / min, the reactor temperature is 25℃-40℃, the ultrasonic power is 100 W -140W, and the coupling reaction residence time is 10 min -30min.

5. The method of preparing palmitoyl tripeptide-38 according to claim 1, characterized in that, In step S200, in the coupling step, the coupling reagent includes at least one of 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione and N,N'-diisopropyl carbodiimide.

6. The method of preparing palmitoyl tripeptide-38 according to claim 1, characterized in that, In step S300, the molar ratio of the polypeptide chain to palmitic acid is 1:(1-3), the coupling temperature is 25-40℃, and the residence time is 10 min -30min.

7. The method for preparing palmitoyl tripeptide-38 according to claim 1, characterized in that, In step S400, after the final peptide chain is cut, the palmitoyl tripeptide-38 is obtained after purification by reverse phase high performance liquid chromatography.

8. The method for preparing palmitoyl tripeptide-38 according to claim 7, characterized in that, The chromatographic conditions of the liquid chromatography purification are: Chromatographic column: Lux RP-C18; Mobile phase: water and acetonitrile; Gradient: 1-10 min, acetonitrile from 10%-90%; 10-15 min: acetonitrile from 90%-10%; Flow rate: 1 ml / min; Injection volume: 5uL; Detection wavelength: 215nm.

9. Palmitoyl tripeptide-38 prepared by the method according to any one of claims 1 to 8, characterized by The purity of the palmitoyl tripeptide-38 is ≥95%.

10. The use of the palmitoyl tripeptide-38 prepared by the method of any one of claims 1-8 or the palmitoyl tripeptide-38 of claim 9 in the preparation of anti-aging cosmetics.

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