Palmitoyl pentapeptide-3 as well as preparation method and application thereof

Abstract

The invention relates to palmitoyl pentapeptide-3 and a preparation method and application thereof.According to the palmitoyl pentapeptide-3, low-toxicity and biodegradable NFM and anisole are compounded according to the volume ratio of 1: 1, and the palmitoyl pentapeptide-3 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 batch-type 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 palmitoyl pentapeptide-3 is shortened, the occurrence rate of side reaction is reduced, and the universality verifies that the solvent system has excellent performance in synthesis of palmitoyl pentapeptide-3, and has a wide application prospect in the synthesis of palmitoyl pentapeptide-3. And the coupling efficiency is stable.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of solid-phase polypeptide preparation, in particular to a palmitoyl pentapeptide-3 and a preparation method and application thereof. BACKGROUND

[0002] In the traditional solid-phase polypeptide preparation (SPPS) process, N,N-dimethylformamide (DMF) is widely used in the dissolution and coupling reaction of amino acids due to its excellent solubility. However, DMF has been explicitly listed as a substance with reproductive toxicity and environmental accumulation, which not only poses a potential threat to the health of operators, but also has an adverse impact on the safety of the ecological environment.

[0003] Although various solvents have been developed to replace DMF in the prior art, such as N-methyl-2-pyrrolidone (NMP), there are still many limitations, mainly including: 1. Imbalance between toxicity and economy: NMP is less toxic than DMF, but it is still considered to have reproductive hazards and is more expensive; solvents such as anisole, which have lower toxicity, have insufficient solubility for Fmoc-protected amino acids when used alone, which easily leads to a decrease in coupling efficiency, limiting their application in SPPS; 2. Low reaction efficiency: current SPPS mostly uses batch operation, and the reaction process relies on the diffusion mass transfer mechanism, resulting in a long time consumption in key steps such as coupling and deprotection, especially when preparing long-chain or complex polypeptides, the overall cycle can be extended to several days, which significantly affects the production efficiency; 3. Poor adaptability of solvent system: the current replacement solvents have insufficient swelling capacity for polypeptide resins, which is difficult to meet the reaction space required for the preparation of long-chain polypeptides, and have poor compatibility with advanced preparation processes such as continuous flow, which limits the integration and automation development of the process; 4. Difficulty in purification: by-products are easily generated during the reaction process, and some solvents are difficult to remove, which limits the purity of the target product, and multiple purification steps are required, which not only increases the complexity of the operation, but also increases the overall cost and environmental burden. SUMMARY

[0004] Therefore, it is necessary to provide a palmitoyl pentapeptide-3 and a preparation method and application thereof to solve the defects in the prior art.

[0005] To achieve the above-mentioned purpose, the present application provides a technical solution: A preparation method of a palmitoyl pentapeptide-3, the sequence of the palmitoyl pentapeptide-3 being H-Pal-Lys-Thr-Thr-Lys-Ser-OH, comprising the following steps: S100. The resin is loaded into a continuous flow reaction system, and pretreatment is performed using a green solvent; S200. The coupling and deprotection steps of five Fmoc-protected amino acids are sequentially completed by the continuous flow reaction system to obtain a polypeptide chain; The Fmoc-protected amino acids are Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-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 pentapeptide-3. The green solvent is a mixed solution of N-formylmorpholine and anisole in a volume ratio of 1:1.

[0006] Preferably, 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.

[0007] Preferably, 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.

[0008] Preferably, 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 coupling reaction residence time is 10 min-30 min.

[0009] Preferably, 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.

[0010] Preferably, 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-30 min.

[0011] Preferably, in step S400, after the final peptide chain is cut, the palmitoyl pentapeptide-3 is obtained after purification by reverse phase high performance liquid chromatography.

[0012] Preferably, the chromatographic conditions of the liquid chromatography purification are as follows: Chromatographic column: YMC 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 uL; Detection wavelength: 215 nm.

[0013] The application also provides a palmitoyl pentapeptide-3, the purity of which is greater than or equal to 93%.

[0014] The application also provides a palmitoyl pentapeptide-3 for use in medicine, cosmetics and diagnostic reagents.

[0015] The application has the following advantages: 1. Green solvent precise compounding: NFM and anisole are compounded at a volume ratio of 1:1, which has strong polarity and moderate hydrophobicity, and can completely replace DMF. The mixed solvent improves the solubility of Fmoc amino acid monomers, activating reagents and resins, and has no reproductive toxicity risk and reduced volatility compared with DMF; 2. Process synergy enhancement: through the synergy of laminar shear effect and ultrasonic cavitation effect of the continuous flow reaction device, the mass transfer limitation of traditional batch reaction is overcome, the coupling reaction time of palmitoyl pentapeptide-3 is shortened, and the occurrence rate of side reactions is reduced; 3. Universality verification: the solvent system performs excellently in the synthesis of palmitoyl pentapeptide-3, and the coupling efficiency is stable. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a synthesis route map in an embodiment; Figure 2 is an NMR chart of the product palmitoyl pentapeptide-3 in an embodiment; Figure 3 is a product in which palmitoyl pentapeptide-3 is applied to anti-aging cosmetics. DETAILED DESCRIPTION

[0017] In order to better illustrate the purpose, technical scheme and advantages of the application, the application will be further described below in combination with specific embodiments.

[0018] In the embodiments, the test methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0019] To replace the traditional toxic solvent DMF, the system screens a combination of low-toxicity, biodegradable green solvents. By comparing the solubility, resin swelling, coupling efficiency and side reaction rate of four core indicators, the optimal solvent system is determined (see Tables 1-5 for test results).

[0020] Example 0 (Performance verification of green solvent NFM / An): To verify the universality of the solvent, the performance of NFM / An was tested using a model reaction: 1. Solubility test of green solvent; Test the solubility of green solvent (NFM / An) for amino acids, coupling reagents and reaction by-products.

[0021] The known poorly soluble amino acid Fmoc-Phe-OH, the classic coupling reagent combination OxymaPure / DIC (ethyl cyanohydroxyimine acid ester / N,N'-diisopropyl carbodiimide), OxymaB / DIC (5- (hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione / N,N'-diisopropyl carbodiimide), HATU / DIPEA (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate / N,N- diisopropylethylamine), PyAOP / DIPEA (hexafluorophosphate (7-azabenzotriazol-1-oxyl) tripyrrolidine phosphorus / N,N-diisopropylethylamine) and coupling reaction by-product DIU (diisopropyl urea) were selected as reference for preliminary screening, and their solubility in DMF, NFM / An (1:1, v:v) was tested at a concentration of 0.5 M, and the test results are shown in Table 1.

[0022] Table 1 Solubility of amino acids, coupling reagents and DIU in green mixed solvent In the table, unless the specified concentration is 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.

[0023] As can be seen from Table 1, NFM / An has comparable solubility to DMF for Fmoc-Phe-OH and OxymaPure / DIC.

[0024] The solubility of NFM / An for DIU is 125 M, which is consistent with DMF, and DIU can be avoided by dilution in the future.

[0025] 2. Resin swelling test; The swelling degree of CTC resin (substitution value 0.4 mmol / g-3.0 mmol / g) used in the present patent was measured under ultrasonic condition at 30°C.

[0026] In solid-phase polypeptide synthesis, resin swelling is a key property of solvents.

[0027] Resin swelling can: (1) increase the volume and internal porosity of the resin, enhancing 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 the collision between resin microspheres and prevent breakage.

[0028] Therefore, the solvent used before the reaction needs to be tested for its swelling degree on the resin, and the results are shown in Table 2.

[0029] Table 2 Swelling degree of resin in solvents (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 the final volume 10 mL, and the resin volume was measured after swelling for 30 min under ultrasonic condition at 30°C, and the average value was taken from three measurements.

[0030] 4 mL . g -1 under-swelling; 4-7 mL . g -1 swelling sufficient; >7 mL . g -1 over-swelling.

[0031] From the results in Table 2, the swelling degree of CTC resin in NFM / An is 5.0 mL·g -1 , which is within the ideal range (4 mL·g -1 -7 mL·g -1 ).

[0032] 3. Coupling reaction rate test: Solid-phase polypeptide synthesis (SPPS) involves multiple steps, and solvents and process intensification techniques (ultrasonic condition / shaking condition) can affect its performance.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] NFM / An b This refers to the molecular ratio of 1.3 eq. DIC / OxymaB / AA being 1:1:1 under vibration conditions.

[0039] NFM / An c This refers to the molecular ratio of 1.3 eq. DIC / OxymaB / AA being 1:0.2:1 under ultrasonic conditions.

[0040] 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 - raw material conversion rate (%) at the completion of the reaction; t - reaction time (min); y - raw material conversion rate (%) at the corresponding time point.

[0041] Table 4 Kinetic model fitting results As can be seen from Table 4, the pseudo-first-order reaction model is well fitted (R 2 > 0.95) in all cases. NFM / An shows the highest apparent rate constant (k obs = 0.5563 min -1 ) and the final conversion rate (98.3%) is comparable to DMF.

[0042] Ultrasonic conditions significantly improve the coupling rate (compared to vibration conditions) and promote the reaction kinetics. Even under catalytic amount of OxymaB (k obs = 0.2546 min -1 ), the rate is still higher than DMF or vibration conditions.

[0043] In summary, NFM / An is an excellent green solvent, and its coupling rate is significantly better than the control group (DMF), and ultrasonic assistance effectively improves the reaction rate in green solvents.

[0044] 4. Side reaction control test; Epimerization is a common side reaction in solid-phase peptide synthesis, which reduces the yield and increases the difficulty of purification.

[0045] The epimerization rate was tested using the model peptide Fmoc-Phg-Pro-NH2, which is prone to epimerization. This reaction takes advantage of the active α-hydrogen in Phg and the steric hindrance characteristics of Phg / Pro to compare the degree of epimerization under different conditions. The results are shown in Table 5.

[0046] Table 5 Epimerization rate of Fmoc-Phg-Pro-NH2 coupling under green solvent conditions a In the table, all reactions were carried out in solution phase.

[0047] DL (%) = (Fmoc-D-Phg-Pro-NH2) / (Fmoc-D-Phg-Pro-NH2 + Fmoc-L-Phg-Pro-NH2) x 100.

[0048] After synthesizing Fmoc-D-Phg-Pro-NH2 using Fmoc-D-Phg-OH as the starting material, its position in HPLC was determined.

[0049] OxymaB is 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione, and OxymaPure is (hydroxyimino)cyanoacetate ethyl ester.

[0050] The epimerization rate of DIC / 1.3 eq. OxymaB combination in NFM / An is 2.5%, which is lower than that in DMF or pure NFM.

[0051] The epimerization rate of DIC / 0.6 eq. OxymaB combination in NFM / An is the lowest (1.2%), and the epimerization rate of other combinations in NFM / An is higher than 3%.

[0052] The NFM / An green solvent system is particularly suitable for the synthesis of palmitoyl pentapeptide-3 by the above-mentioned fourfold verification of solubility, swelling, reaction efficiency and side reaction control, and provides a process design basis for subsequent examples.

[0053] Example 1 A preparation method of palmitoyl pentapeptide-3, the sequence of which is: H-Pal-Lys-Thr-Thr-Lys-Ser-OH; the synthesis route is as shown in Figure 1 .

[0054] C-terminal to N-terminal: Ser(tBu)→Lys(Boc)→Thr(tBu)→Thr(tBu)→Lys(Boc)→Pal; The green solvent in this embodiment refers to N-formylmorpholine: anisole (1:1, by volume); Materials and equipment Resin: 2-chlorotrityl chloride resin (CTC resin, substitution value 0.8 mmol / g), amount 1.0 g; Deprotection solution: a mixed solution of DBU / green solvent, wherein the volume fraction of DBU in the mixed solution is 5%.

[0055] Cutting solution: a mixed solution of HFIP / DCM, wherein the volume fraction of HFIP in the mixed solution is 20%.

[0056] Equipment: Continuous flow reaction system: Injection pump (flow rate range 0.1-10 mL / min, flow rate accuracy ±1%); PTFE tubular reactor (inner diameter 2 mm, volume 30 mL, filled with CTC resin bed); Online mixing module (pre-activation reagent); Ultrasonic reaction tank: 40 kHz, power adjustable (50-200 W); Real-time detection unit (Kaiser / indole triketone test paper combination); vacuum filtration device, HPLC monitoring system; Preparation steps: S100. Load the resin into the continuous flow reaction system and pretreat it using green solvent.

[0057] The specific steps are as follows: Load 1.0 g of CTC resin (substitution value 0.8 mmol / g) into a PTFE tubular reactor, flush with green solvent (flow rate 5 mL / min) for 10 min, and ultrasonic-assisted swelling for 30 min (150 W).

[0058] S200. Complete the coupling and deprotection steps of five Fmoc-protected amino acids in sequence through the continuous flow reaction system to obtain a polypeptide chain; The Fmoc-protected amino acids are in sequence: Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH.

[0059] The specific steps are as follows: S210. Complete the coupling and deprotection steps of Fmoc-Ser(tBu)-OH through the continuous flow reaction system.

[0060] S211. The coupling step of Fmoc-Ser(tBu)-OH is as follows: Dissolve Fmoc-Ser(tBu)-OH (2.4 mmol, 3.0 equiv.) and N,N-diisopropylethylamine (4.8 mmol, 6.0 equiv.) in green solvent (15 mL), inject into the syringe pump, and react with the product in step S200.

[0061] Continuous flow parameters: flow rate 2 mL / min, reactor temperature 25℃, ultrasonic power 100 W, residence time 60 min.

[0062] After confirming the disappearance of free amino groups through online Kaiser detection (reactor outlet), switch the solvent pump to wash with methanol and green solvent each 3 times (flow rate 5 mL / min), methanol (3x10 mL) and green solvent (3x10 mL).

[0063] S212. The deprotection step of Fmoc-Ser(tBu)-OH is as follows: Pump 5 v / v% DBU / green solvent solution into the reactor at a flow rate of 3 mL / min, react with the product in step S211, and treat with ultrasound assistance (120 W) for 2 min x 2 times, and the waste liquid is directly introduced into the waste liquid tank.

[0064] Switch the solvent pump to wash with methanol (3 x 10 mL) and green solvent (3 x 10 mL) in turn (flow rate 5 mL / min, 3 times each).

[0065] S220. Complete the coupling and deprotection steps of Fmoc-Thr(tBu)-OH through a continuous flow reaction system.

[0066] S221. The coupling step of Fmoc-Lys(Boc)-OH is as follows (amino acid anchoring): Dissolve Fmoc-Lys(Boc)-OH (2.4 mmol, 3.0 equiv.) with 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (4.8 mmol, 6.0 equiv.) and N,N'-diisopropylcarbodiimide (3.2 mmol) in green solvent (15 mL), inject into the syringe pump, and react with the product in step S210.

[0067] Continuous flow parameters: flow rate 2 mL / min, reactor temperature 25°C, ultrasonic power 100 W, residence time 30 min.

[0068] Switch the solvent pump to wash with methanol and green solvent for 3 times each (flow rate 5 mL / min), methanol (3 x 10 mL) and green solvent (3 x 10 mL).

[0069] S222. The deprotection step of Fmoc-Lys(Boc)-OH is as follows: Pump 5 v / v% DBU / green solvent solution into the reactor at a flow rate of 3 mL / min, react with the product in step S221, and treat with ultrasound assistance (120 W) for 2 min x 2 times, and the waste liquid is directly introduced into the waste liquid tank.

[0070] Switch the solvent pump to wash with methanol (3 x 10 mL) and green solvent (3 x 10 mL) in turn (flow rate 5 mL / min, 3 times each).

[0071] S230. Complete the coupling and deprotection steps of Fmoc-Thr(tBu)-OH through a continuous flow reaction system.

[0072] S231. The coupling step of Fmoc-Thr(tBu)-OH is as follows: Fmoc-Thr(tBu)-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 green solvent (10 mL), pre-activated for 30 s via an online mixing module and injected into the product of step S222.

[0073] Reaction parameters: flow rate 3 mL / min, ultrasonic power 120 W, residence time 12 min, completion of coupling confirmed by online ninhydrin detection, switching of solvent pumps to wash the resin.

[0074] S232. Deprotection of Fmoc-Thr(tBu)-OH was performed as follows: A 5 v / v% DBU / green solvent solution was pumped into the reactor at a flow rate of 3 mL / min and reacted with the product of step S221, treated with ultrasonic assistance (120 W) for 2 min and the waste was directly introduced into the waste tank.

[0075] The solvent pumps were switched to wash the resin with methanol, green solvent in turn (flow rate 5 mL / min, 3 times each).

[0076] S240. Coupling and deprotection of Fmoc-Thr(tBu)-OH were performed by the continuous flow reaction system, online HPLC detection was used to ensure that the coupling efficiency was > 97%, and the byproduct would increase when the coupling efficiency was < 97%.

[0077] S241. Coupling of Fmoc-Thr(tBu)-OH was performed as follows Fmoc-Thr(tBu)-OH (2.4 mmol), 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (0.48 mmol), N,N'-diisopropylcarbodiimide (3.2 mmol) were dissolved in green solvent (10 mL), pre-activated for 30 s via an online mixing module and injected into the product of step S222.

[0078] Reaction parameters: flow rate 3 mL / min, ultrasonic power 120 W, residence time 12 min, completion of coupling confirmed by online ninhydrin detection, switching of solvent pumps to wash the resin.

[0079] S242. Deprotection of Fmoc-Thr(tBu)-OH was performed as follows: Pump 5% DBU in green solvent solution into the reactor at a flow rate of 3 mL / min, ultrasonic-assisted (120 W) for 2 min, inject into the product of step S241, to obtain the polypeptide chain, the waste liquid is directly introduced into the waste liquid tank.

[0080] Switch the solvent pump to wash with methanol, green solvent in turn (flow rate 5 mL / min, 3 times respectively).

[0081] S250. The coupling and deprotection steps of Fmoc-Lys(Boc)-OH are completed by a continuous flow reaction system, and the online HPLC detection is used to ensure that the coupling efficiency is ≥97%.

[0082] S251. The coupling step of Fmoc-Lys(Boc)-OH is as follows: Fmoc-Lys(Boc)-OH (2.4 mmol), ethyl cyanohydroxy imidic acid (0.48 mmol), DIC (3.2 mmol) are dissolved in green solvent (10 mL), pre-activated for 30 s through the online mixing module, and injected into the product of step S242.

[0083] Reaction parameters: flow rate 3 mL / min, ultrasonic power 120 W, residence time 12 min, coupling completion is confirmed by online ninhydrin detection, and the resin is washed by switching the solvent pump.

[0084] S252. The deprotection step of Fmoc-Lys(Boc)-OH is as follows: Pump 5% DBU in green solvent solution into the reactor at a flow rate of 4 mL / min, ultrasonic-assisted (150 W) for 2 min, inject into the product of step S251, to obtain the polypeptide chain, the waste liquid is directly introduced into the waste liquid tank.

[0085] Switch the solvent pump to wash with methanol, green solvent in turn (flow rate 5 mL / min, 3 times respectively).

[0086] S300. The polypeptide chain and palmitic acid are coupled by a continuous flow reaction system to obtain the final peptide chain.

[0087] 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), N,N'-diisopropyl carbodiimide (3.2 mmol) are dissolved in green solvent (10 mL), online mixing activation for 30 s, and added to the product polypeptide chain of step S232.

[0088] Reaction parameters: flow rate 4 mL / min, ultrasonic power 150 W, residence time 12 min, reaction temperature 30°C.

[0089] S400. cleaving the terminal peptide chain to obtain the palmitoyl pentapeptide-3.

[0090] The specific steps are as follows: S410, cleaving the peptide chain: To the product of step S300, 20% hexafluoroisopropanol (HFIP) / dichloromethane (DCM) mixture (15 mL) was added, and the reaction was oscillated at room temperature for 1.5 hours. The cleavage solution was collected by filtration, and the filtrate was combined. The organic solvent was removed by rotary evaporation under reduced pressure.

[0091] S420, purification: To the product of step S410, 40 mL of cold benzyl tert-butyl ether was added to precipitate the crude peptide. After centrifugal freeze-drying, the protected palmitoyl pentapeptide-3 H-Pal-Lys (Boc)-Thr (tBu)-Thr (tBu)-Lys (Boc)-Ser (tBu)-OH was obtained by reverse phase high performance liquid chromatography (C18 column, acetonitrile / water gradient elution). Then it was treated with 30% trifluoroacetic acid / dichloromethane for 1 h, and the organic solvent was removed by rotary evaporation under reduced pressure. The palmitoyl pentapeptide-3 (sequence number: H-Pal-Lys-Thr-Thr-Lys-Ser-OH) was obtained, with a purity of 93.91%, and the yield of the product palmitoyl pentapeptide-3 was 65.02%. The NMR chart is shown in Figure 2 .

[0092] The chromatographic conditions for liquid chromatography purification are as follows: Chromatographic column: YMC 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 uL; Detection wavelength: 215 nm.

[0093] Test Example 1 The other steps are the same as in Example 1, except that different processes are used for coupling and deprotection. The yield table of the obtained palmitoyl pentapeptide-3 H-Pal-Lys-Thr-Thr-Lys-Ser-OH is shown in Table 6.

[0094] Table 6 Yield table Application Example 1 The palmitoyl pentapeptide-3 prepared in Example 1 was applied to anti-aging cosmetics, such as Figure 3 As shown in Table 1, the palmitoyl pentapeptide-3 has the effect of wrinkle mark improvement.

[0095] It should be noted that the specific parameters or some reagents in the above examples are specific examples or preferred examples under the concept of the present application, but not a limitation; those skilled in the art can make adaptive adjustments within the concept and protection scope of the present application.

Claims

1. A method for preparing palmitoyl pentapeptide-3, characterized in that, The palmitoyl pentapeptide-3 has the sequence H-Pal-Lys-Thr-Thr-Lys-Ser-OH and includes the following steps: S100. The resin is loaded into a continuous flow reaction system and pretreated with a green solvent; S200. The coupling and deprotection steps of five Fmoc-protected amino acids are completed sequentially through a continuous flow reaction system to obtain a polypeptide chain; The amino acids protected by Fmoc are, in order: Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Lys(Boc)-OH; S300. The polypeptide chain and palmitic acid are coupled using a continuous flow reaction system to obtain the final polypeptide chain; S400. The final peptide chain is cleaved to obtain palmitoyl pentapeptide-3; The green solvent is a mixed solution of N-formylmorpholine and anisole in a volume ratio of 1:

1.

2. The method for preparing palmitoyl pentapeptide-3 according to claim 1, characterized in that, In step S100, the resin includes at least one of CTC resin and ClT resin, with a substitution value of 0.4 mmol / g to 3.0 mmol / g.

3. The method for preparing palmitoyl pentapeptide-3 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 for preparing palmitoyl pentapeptide-3 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 - 140 W, and the residence time of the coupling reaction is 10 min - 30 min.

5. The method for preparing palmitoyl pentapeptide-3 according to claim 1, characterized in that, In step S200, during the coupling step, the coupling agent includes at least one of 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione and N,N'-diisopropylcarbodiimide.

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

7. The method for preparing palmitoyl pentapeptide-3 according to claim 1, characterized in that, In step S400, after the final peptide chain is cleaved, the palmitoyl pentapeptide-3 is obtained by purification using reversed-phase high-performance liquid chromatography.

8. The method for preparing palmitoyl pentapeptide-3 according to claim 7, characterized in that, 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% to 90%; 10-15 min: acetonitrile from 90% to 10%; Flow rate: 1 ml / min; Injection volume: 5 μL; Detection wavelength: 215nm.

9. A palmitoyl pentapeptide-3 prepared by the method according to any one of claims 1 to 8, characterized in that, The purity of the palmitoyl pentapeptide-3 is ≥93%.

10. The use of palmitoyl pentapeptide-3 prepared by any one of claims 1 to 8 or the palmitoyl pentapeptide-3 of claim 9 in pharmaceuticals, cosmetics and diagnostic reagents.

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