Cartamyl pentapeptide-17 as well as preparation method and application thereof

Abstract

The invention relates to myristoyl pentapeptide-17 and a preparation method and application thereof.According to the myristoyl pentapeptide-17, low-toxicity and biodegradable NFM and anisole are compounded according to the volume ratio of 1: 1, and the myristoyl pentapeptide-17 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 the myristoyl pentapeptide-17 is shortened, the occurrence rate of side reaction is reduced, and the universality verifies that the solvent system has excellent performance in the synthesis of the myristoyl pentapeptide-17, and has a wide application prospect in the synthesis of the myristoyl pentapeptide-17. 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 myristoyl pentapeptide-17 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 has 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: the toxicity of NMP is lower than that of DMF, but it is still considered to have reproductive hazards, and the price is relatively high; and solvents such as anisole have lower toxicity, but when used alone, the solubility of Fmoc-protected amino acids is insufficient, which easily leads to a decrease in coupling efficiency, limiting its application in SPPS; 2. Low reaction efficiency: the existing SPPS mostly uses intermittent 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 in the preparation of long-chain or complex polypeptides, the overall cycle can be prolonged to several days, which significantly affects the production efficiency; 3. Poor adaptability of solvent system: the current alternative 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 at the same time, they 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 operation complexity, but also increases the overall cost and environmental burden. SUMMARY

[0004] Therefore, it is necessary to provide a myristoyl pentapeptide-17 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: The present application provides a preparation method of a myristoyl pentapeptide-17, the sequence of the myristoyl pentapeptide-17 being H-Myr-Lys-Leu-Ala-Lys-Lys-NH2, comprising the following steps: S100. loading resin into a continuous flow reaction system, pre-treatment is performed using a green solvent; S200. sequentially completing the coupling and deprotection steps of five Fmoc-protected amino acids by a continuous flow reaction system to obtain a polypeptide chain; The Fmoc-protected amino acids are Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, and Fmoc-Lys(Boc)-OH in sequence. S300. coupling and treating the polypeptide chain and myristic acid by a continuous flow reaction system to obtain a final peptide chain; S400. cleaving the final peptide chain to obtain the myristoyl pentapeptide-17. The green solvent is a mixed solution of N-formyl morpholine and anisole in a volume ratio of 1:1.

[0006] Preferably, in step S100, the resin includes at least one of RINK AMIDE resin and Sieber Amide 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, 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.

[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 myristic 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 cleaving the final peptide chain, the myristoyl pentapeptide-17 is obtained after purification by reverse phase high performance liquid chromatography.

[0012] Preferably, the chromatographic conditions of the liquid chromatography purification are: Chromatographic column: Lux 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 myristoyl pentapeptide-17 with a purity of ≥ 87%.

[0014] The application also provides a use of the myristoyl pentapeptide-17 in medicine, cosmetics and diagnostic reagents.

[0015] The application has the following advantages: 1. Toxicity and economic imbalance: NMP has slightly lower toxicity than DMF, but still has potential reproductive hazards, and the cost is high; the solubility of Fmoc-protected amino acids is insufficient when using low-toxicity solvents such as anisole alone, resulting in a decrease in coupling efficiency; 2. Low reaction efficiency: traditional batch reactions rely on diffusion mass transfer, and the coupling and deprotection steps take a long time, and the synthesis of complex polypeptides can take several days; 3. Poor solvent compatibility: existing alternative solvent systems have insufficient swelling ability for long-chain polypeptides, and are difficult to adapt to the mass transfer needs of continuous flow processes; 4. Difficulty in purification: byproduct and solvent residues limit the purity of the final product, which requires multiple purification steps, increasing process complexity. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a synthesis route map in an embodiment; Figure 2 is an NMR chart of the product myristoyl pentapeptide-17 in an embodiment; Figure 3 is a product applying myristoyl pentapeptide-17 to cosmetics; Figure 4 is a product applying myristoyl pentapeptide-17 to cosmetics and its efficacy. DETAILED DESCRIPTION

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

[0018] In the embodiments, the test methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0019] To replace the traditional toxic solvent DMF, the present system screens a low-toxic, biodegradable green solvent combination. 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) to 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 / 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'-tetramethyluronium hexafluorophosphate / N,N- diisopropylethylamine), PyAOP / DIPEA (hexafluorophosphate (7-azabenzotriazol-1-oxyl) tripyrrolidine phosphorus / N,N-diisopropylethylamine) and the coupling reaction by-product DIU (diisopropyl urea) were selected as reference materials 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

[0023] 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.

[0024] As can be seen from Table 1, the solubility of NFM / An to Fmoc-Phe-OH, OxymaPure / DIC is comparable to DMF.

[0025] The solubility of NFM / An to DIU is 125 M, which is consistent with DMF, and DIU can be avoided by dilution in the future.

[0026] 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.

[0027] In solid-phase polypeptide synthesis, resin swelling is a key property of solvents.

[0028] 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 reaction sites; (3) improve the elution efficiency of by-products and excess reagents; (4) reduce the collision between resin microspheres and prevent breakage.

[0029] 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.

[0030] Table 2 Swelling degree of resin in solvents (ml g -1 ) a Test

[0031] In Table 2, NFM / An was added to CTC resin (i.e. P-2CTC-Cl resin, 1.0 g) to a final volume of 10 mL, and the resin volume was measured after swelling for 30 min under ultrasonic condition at 30°C, with three measurements taken and averaged.

[0032] 4 mL·g -1 is insufficient swelling; 4-7 mL·g -1 is sufficient swelling; >7 mL·g -1 is over-swelling.

[0033] 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 ).

[0034] 3. Coupling reaction rate test: Solid-phase polypeptide synthesis (SPPS) involves multiple steps, and both solvents and process intensification techniques (ultrasonic condition / vibration condition) can affect its performance.

[0035] Given the advantages of ultrasonic-assisted technology, such as high energy utilization rate, low difficulty in scaling up, and low cost of equipment, and the fact that it is still a blank in green solvent SPPS, the present test combines a new green solvent (NFM / An) with ultrasonic-assisted technology to test the performance of this scheme in multiple steps of SPPS.

[0036] Coupling is a key step in SPPS, and the solvent has a significant impact on the coupling rate, so it can be an important indicator of solvent performance.

[0037] The test uses the standard method of Pedersen's team: Fmoc-Gly-OH is reacted 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 to determine the rate.

[0038] Based on this method, the performance of the ultrasonic-assisted green solvent NFM / An in the coupling step was tested, specifically the kinetic conversion rate of Fmoc-Gly-OH coupling to PFA-CTC (i.e. Pro-Phe-Ala-CTC peptide-resin, which is a tripeptide Pro-Phe-Ala connected to CTC resin). After removing Fmoc, resin washing and peptide cleavage, the conversion of tripeptide to tetrapeptide was quantified by HPLC analysis, and the results are shown in Table 3.

[0039] Table 3 Kinetic conversion rate data of coupling under different conditions

[0040] In the table: NFM / An a refers to the molecular ratio of 1:1:1 of 1.3 eq. DIC / OxymaB / AA under ultrasonic conditions, and AA refers to amino acids.

[0041] NFM / An b refers to the molecular ratio of 1:1:1 of 1.3 eq. DIC / OxymaB / AA under vibration conditions.

[0042] NFM / An c refers to the molecular ratio of 1:0.2:1 of 1.3 eq. DIC / OxymaB / AA under ultrasonic conditions.

[0043] The coupling reaction rate on the resin can be approximated as a pseudo-first-order reaction of the resin peptide NH2 group, ignoring the activated amino acid concentration: ; where k obs is the apparent reaction rate constant (min -1 ); a0is the raw material conversion rate (%) at the end of the reaction; t is the reaction time (min); y is the raw material conversion rate (%) at the corresponding time point.

[0044] Table 4 Kinetic model fitting results

[0045] From Table 4, the pseudo-first-order reaction model fits well (R 2 > 0.95) in all cases. NFM / An shows the highest apparent rate constant (k obs = 0.5563 min -1 ) and comparable final conversion (98.3%) to DMF.

[0046] Ultrasonic conditions significantly improve the coupling rate (compared to shaking conditions) and promote the reaction kinetics. Even with catalytic amounts of OxymaB (k obs = 0.2546 min -1 ), the rate is still higher than DMF or shaking conditions.

[0047] In summary, NFM / An is an excellent green solvent with significantly better coupling rates than the control (DMF), and ultrasonic assistance effectively improves the reaction rate in green solvents.

[0048] 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.

[0049] The model peptide Fmoc-Phg-Pro-NH2, which is prone to epimerization, was used to test the side reaction rate. 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.

[0050] Table 5 Epimerization rate of Fmoc-Phg-Pro-NH2 coupling under green solvent conditions a

[0051] In Table 5, all reactions were carried out in solution phase.

[0052] DL (%) = (Fmoc-D-Phg-Pro-NH2) / (Fmoc-D-Phg-Pro-NH2 + Fmoc-L-Phg-Pro-NH2) x 100.

[0053] After synthesizing Fmoc-D-Phg-Pro-NH2 using Fmoc-D-Phg-OH as the starting material, its position in HPLC was determined.

[0054] OxymaB is 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione, and OxymaPure is ethyl (hydroxyimino)cyanocyanate.

[0055] The DIC / 1.3eq.OxymaB combination exhibited a diastereomeric rate of 2.5% in NFM / An, which is lower than that of DMF or pure NFM.

[0056] 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.

[0057] The NFM / An green solvent system has passed the quadruple verification of solubility, swelling, reaction efficiency, and side reaction control, and is particularly suitable for the synthesis of myristoyl pentapeptide-17, providing a basis for process design in subsequent examples.

[0058] Example 1 A method for preparing myristoyl pentapeptide-17, H-Myr-Lys-Leu-Ala-Lys-Lys-NH2, C-terminus to N-terminus: NH2→Lys→Lys→Ala→Leu→Lys→Myr); the synthetic route is as follows: Figure 1 As shown.

[0059] In this embodiment, the green solvent refers to N-formylmorpholine: anisole (1:1, volume ratio). Materials and Equipment Resin: RINK AMIDE resin (substitution value 0.6 mmol / g), dosage 1.0 g; Deprotection solution: A mixed solution of DBU and green solvent, wherein the volume fraction of DBU in the mixed solution is 5%.

[0060] Cutting fluid: A mixture of HFIP and DCM, with HFIP comprising 20% ​​by volume.

[0061] 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 RINK AMIDE resin bed); Online mixing module (pre-activated reagent); Ultrasonic reaction tank: 40 kHz, power adjustable (50-200 W); Real-time detection unit (Kaiser / ninhydrin test strips); vacuum filtration device; HPLC monitoring system; Preparation steps: S100. Load the resin into the continuous flow reaction system, pre-treat with green solvent.

[0062] The specific steps are as follows: Load 1.0 g RINK AMIDE resin (substitution value 0.6 mmol / g) into a PTFE tubular reactor, rinse with green solvent (flow rate 5 mL / min) for 10 min, ultrasonic-assisted swelling for 30 min (150 W).

[0063] Pump 5 v / v% DBU / green solvent solution into the reactor at a flow rate of 3 mL / min, react with the RINK AMIDE resin, deprotect the RINK AMIDE resin, ultrasonic-assisted (120 W) treatment for 2 min x 2 times, and the waste liquid is directly introduced into the waste liquid tank.

[0064] S200. Complete the coupling and deprotection steps of five Fmoc-protected amino acids in turn through the continuous flow reaction system to obtain a polypeptide chain; The Fmoc-protected amino acids are Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, and Fmoc-Lys(Boc)-OH in turn. The specific steps are as follows: S210. Complete the coupling and deprotection steps of Fmoc-Lys(Boc)-OH through the continuous flow reaction system.

[0065] S211. The coupling step of Fmoc-Lys(Boc)-OH is as follows: Dissolve Fmoc-Lys(Boc)-OH (2.4 mmol, 3.0 equiv.) with 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (0.48 mmol), N,N'-diisopropylcarbodiimide (2.4 mmol) (4.8 mmol, 6.0 equiv.) in green solvent (15 mL), inject into the syringe pump, and react with the product in step S200.

[0066] Continuous flow parameters: flow rate 2 mL / min, reactor temperature 25℃, ultrasonic power 100 W, residence time 60 min.

[0067] After confirming the disappearance of free amino groups by online Kaiser detection (reactor outlet), switch the solvent pump to wash with methanol and green solvent for 3 times (flow rate 5 mL / min), methanol (3x10 mL) and green solvent (3x10 mL).

[0068] S212. 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 S211, ultrasonic assisted (120 W) treatment 2 min x 2 times, the waste liquid is directly introduced into the waste tank.

[0069] Switch the solvent pump to wash with methanol (3 x 10 mL), green solvent (3 x 10 mL) in turn (flow rate 5 mL / min, 3 times each).

[0070] S213. The coupling step of Fmoc-Lys(Boc)-OH is as follows: Dissolve Fmoc-Lys(Boc)-OH (2.4 mmol, 3.0 equiv.) with 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (0.48 mmol), N,N'-diisopropylcarbodiimide (2.4 mmol) (4.8 mmol, 6.0 equiv.) in green solvent (15 mL), inject into the syringe pump, and react with the product in step S200.

[0071] Continuous flow parameters: flow rate 2 mL / min, reactor temperature 25°C, ultrasonic power 100 W, residence time 60 min.

[0072] After confirming the disappearance of free amino groups by online Kaiser detection (reactor outlet), 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).

[0073] S214. 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 S211, ultrasonic assisted (120 W) treatment 2 min x 2 times, the waste liquid is directly introduced into the waste tank.

[0074] S220. The coupling and deprotection steps of Fmoc-Ala-OH are completed by a continuous flow reaction system.

[0075] S221. The coupling step of Fmoc-Ala-OH is as follows: Fmoc-Lys(Boc)-OH (2.4 mmol, 3.0 equiv.) was dissolved in green solvent (15 mL) with 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (0.48 mmol), N,N'-diisopropylcarbodiimide (2.4 mmol) and injected into the product from step S210 using a syringe pump.

[0076] Continuous flow parameters: flow rate 2 mL / min, reactor temperature 25 °C, ultrasonic power 100 W, residence time 30 min.

[0077] The solvent pump was switched to wash the resin with methanol and green solvent each 3 times (flow rate 5 mL / min), methanol (3 x 10 mL) and green solvent (3 x 10 mL).

[0078] The deprotection step for Fmoc-Leu-OH was 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 from step S221 with ultrasonic assistance (120 W) for 2 min x 2 times, and the waste was directly introduced into the waste tank.

[0079] The solvent pump was switched to wash the resin with methanol (3 x 10 mL), green solvent (3 x 10 mL) each 3 times (flow rate 5 mL / min) to remove residual basic reagents.

[0080] S230. The coupling and deprotection steps for Fmoc-Lys(Boc)-OH were completed by the continuous flow reaction system.

[0081] S231. The coupling step for Fmoc-Lys(Boc)-OH was as follows: Fmoc-Lys(Boc)-OH (2.4 mmol), 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (0.48 mmol), N,N'-diisopropylcarbodiimide (2.4 mmol) were dissolved in green solvent (10 mL) and pre-activated for 30 s using an online mixing module and injected into the product from step S222. The online HPLC detection was used to ensure that the coupling efficiency was >95%.

[0082] Reaction parameters: flow rate 3 mL / min, ultrasonic power 120 W, residence time 12 min, the completion of the coupling was confirmed by online ninhydrin detection, and the solvent pump was switched to wash the resin.

[0083] S232. The deprotection step for Fmoc-Lys(Boc)-OH was as follows: A 5 v / v% DBU / green solvent solution was pumped into the reactor at a flow rate of 3 mL / min and injected into the product of step S221 for reaction, ultrasonic-assisted (120 W) treatment for 2 min, and the waste liquid was directly introduced into the waste liquid tank.

[0084] The solvent pump was switched to sequentially wash with methanol and green solvent (flow rate of 5 mL / min, 3 times for each).

[0085] S300. The polypeptide chain and myristic acid were coupled by a continuous flow reaction system to obtain a final peptide chain.

[0086] The specific steps are as follows: Myristic acid (Myr 1.8 mmol), 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (0.48 mmol), and N,N'-diisopropylcarbodiimide (2.4 mmol) were dissolved in green solvent (10 mL), mixed online for activation for 30 s, and added to the product polypeptide chain of step S232.

[0087] Reaction parameters: flow rate of 4 mL / min, ultrasonic power of 150 W, residence time of 12 min, and reaction temperature of 30°C.

[0088] S400. The final peptide chain was cleaved to obtain the myristoyl pentapeptide-17.

[0089] The specific steps are as follows: S410, peptide chain cleavage: 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 liquid was collected by filtration, and the filtrate was combined and the organic solvent was removed by rotary evaporation under reduced pressure.

[0090] S420, purification: To the product of step S410, cold benzyl tert-butyl ether (40 mL) was added to precipitate the crude peptide. After centrifugal freeze-drying, the protected myristoyl pentapeptide-17 H-Pal-Lys(Boc)-Met(O2)-Lys(Boc)-OH was obtained by reverse phase high performance liquid chromatography (C18 column, acetonitrile / water gradient elution) purification, and then 95% TFA / 2.5% H2O / 2.5% TIS (20 ml) was used to remove the organic solvent by rotary evaporation under reduced pressure to obtain the myristoyl pentapeptide-17 (sequence number: H-Myr-Lys(Boc)-Leu-Ala-Lys(Boc)-Lys(Boc)-NH2). The NMR chart of the product myristoyl pentapeptide-17 is shown in Figure 2 , and the purity was 87.32%.

[0091] Chromatographic conditions for liquid chromatography purification were as follows: Chromatographic column: YMC 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 uL; Detection wavelength: 215 nm.

[0092] Test Example 1 Other steps were the same as in Example 1, except that different processes were used for coupling and deprotection. The obtained myristoyl pentapeptide-17 The yield table of H-Myr-Lys-Leu-Ala-Lys-Lys-NH2 is shown in Table 6.

[0093] Table 6 Yield table

[0094] Application Example 1 As shown in Figure 3 and Figure 4 , the myristoyl pentapeptide-17 prepared in Example 1 was applied in anti-aging cosmetics.

[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, and are not limited thereto; those skilled in the art can make adaptive adjustments within the scope of the concept and protection of the present application.

Claims

1. A process for the preparation of myristoyl pentapeptide- 17, characterized by, The sequence of the myristoyl pentapeptide-17 is H-Myr-Lys-Leu-Ala-Lys-Lys-NH2, comprising the following steps: S100. The resin is loaded into a continuous flow reaction system, and a green solvent is used for pretreatment; 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-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, and Fmoc-Lys(Boc)-OH in sequence; S300. The polypeptide chain and myristic 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 myristoyl pentapeptide-17. The green solvent is a mixed solution of N-formylmorpholine and anisole in a volume ratio of 1:

1.

2. The process for preparing myristoyl pentapeptide-17 according to claim 1, characterized in that, In step S100, the resin includes at least one of RINK AMIDE resin and Sieber Amide resin, and the substitution value is 0.4 mmol / g-3.0 mmol / g.

3. The process for preparing the myristoyl pentapeptide-17 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 process for preparing myristoyl pentapeptide-17 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 coupling reaction residence time is 10 min-30 min.

5. The process for the preparation of the myristoyl pentapeptide-17 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 process for the preparation of a pentapeptide-17 according to claim 1, characterized in that, In step S300, the molar ratio of the polypeptide chain to myristic acid is 1:(1-3), the coupling temperature is 25-40℃, and the residence time is 10 min-30 min.

7. The process for preparing the myristoyl pentapeptide-17 according to claim 1, characterized in that, In step S400, after the final peptide chain is cut, the myristoyl pentapeptide-17 is obtained after purification by reverse phase high performance liquid chromatography.

8. The process for preparing myristoyl pentapeptide-17 according to claim 7, characterized in that, The chromatographic conditions of the liquid chromatography purification are as follows: 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: 5 uL; Detection wavelength: 215 nm.

9. A myristoyl pentapeptide-17 prepared by the method according to any one of claims 1 to 8, characterized in that, The purity of the myristoyl pentapeptide-17 is ≥87%.

10. The myristoyl pentapeptide-17 prepared by the method of any one of claims 1-8 or the myristoyl pentapeptide-17 of claim 9 is used in medicine, cosmetics, and diagnostic reagents.

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