Tetrapeptide-30 as well as preparation method and application thereof

Abstract

The invention relates to tetrapeptide-30 and a preparation method and application thereof.According to the tetrapeptide-30, low-toxicity and biodegradable NFM and anisole are compounded according to the volume ratio of 1: 1, and the tetrapeptide-30 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 method has the advantages that the process is synergistically strengthened, 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, the coupling reaction time of the tetrapeptide-30 is shortened, the side reaction occurrence rate is reduced, and the universality verifies that the solvent system has excellent performance and stable coupling efficiency in the synthesis of the tetrapeptide-30.

Description

Technical Field

[0001] This invention relates to the field of solid-phase polypeptide preparation technology, specifically to a tetrapeptide-30, its preparation method, and its application. 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. Summary of the Invention

[0004] Therefore, it is necessary to provide a tetrapeptide-30, its preparation method, and its application to overcome the deficiencies in the existing technology.

[0005] To achieve the above objectives, the present invention provides a technical solution: A method for preparing tetrapeptide-30, wherein the sequence of tetrapeptide-30 is as follows: H-Pro-Lys-Glu-Lys-OH 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 four Fmoc-protected amino acids are completed sequentially through a continuous flow reaction system to obtain the final peptide chain; The amino acids protected by Fmoc are, in order: Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Pro-OH; S300. The final peptide chain is cleaved to obtain the tetrapeptide-30; 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, with a substitution value of 0.4 mmol / g to 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, the coupling agent in the coupling step includes at least one of 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione and N,N'-diisopropylcarbodiimide.

[0010] Preferably, in step S300, after the final peptide chain is cleaved, the tetrapeptide-30 is obtained by purification using reversed-phase high-performance liquid chromatography.

[0011] Column: Yuexu LP-C18; Mobile phase: water; Gradient: 100% water, 1-15 min; Flow rate: 1 ml / min; Injection volume: 5 μL; Detection wavelength: 215nm.

[0012] The present invention also provides a tetrapeptide-30, wherein the purity of the tetrapeptide-30 is ≥98%.

[0013] On the other hand, the present invention also provides the application of tetrapeptide-30 in pharmaceuticals, cosmetics and diagnostic reagents.

[0014] 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 reactions are overcome, shortening the coupling reaction time of tetrapeptide-30 and reducing the incidence of side reactions. 3. Verification of universality: The solvent system performed well in the synthesis of tetrapeptide-30, with a coupling efficiency of over 95% for each coupling. Attached Figure Description

[0015] Figure 1 This is a synthesis roadmap under implementation; Figure 2 The NMR spectrum of tetrapeptide-30, a product from one embodiment, is shown below. Figure 3 Screenshot showing the effects of tetrapeptide-30 in the Meili Xiuxing app. Detailed Implementation

[0016] 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.

[0017] 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.

[0018] 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).

[0019] 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.

[0020] 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.

[0021] 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.

[0022] As shown in Table 1, the solubility of NFM / An in Fmoc-Phe-OH and OxymaPure / DIC is comparable to that of DMF.

[0023] The solubility of NFM / An for DIU is 125 M, consistent with that of DMF. DIU can be prevented from precipitating by subsequent dilution.

[0024] 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.

[0025] In solid-phase peptide synthesis, resin swelling is a key characteristic of the solvent.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 4 mL . g -1 Insufficient swelling; 4-7 mL . g -1 For sufficient swelling; >7 mL . g -1 This is due to excessive expansion.

[0030] 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 ).

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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 to determine the rate.

[0035] 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.

[0036] 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.

[0037] NFM / An b This refers to the molecular ratio of 1.3 eq. DIC / OxymaB / AA being 1:1:1 under vibration conditions.

[0038] NFM / An c This refers to the molecular ratio of 1.3 eq. DIC / OxymaB / AA being 1:0.2:1 under ultrasonic conditions.

[0039] 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.

[0040] 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.

[0041] Ultrasonic enhancement significantly increased 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.

[0042] 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.

[0043] 4. Side reaction control test; Epimerization is a common side reaction in solid-phase peptide synthesis, which can reduce yield and increase purification difficulty.

[0044] 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.

[0045] 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.

[0046] DL (%) is (Fmoc-D-Phg-Pro-NH2) / (Fmoc-D-Phg-Pro-NH2+ Fmoc-L-Phg-Pro-NH2)×100.

[0047] After synthesizing Fmoc-D-Phg-Pro-NH2 using Fmoc-D-Phg-OH as the starting material, its position in HLPC was determined.

[0048] OxymaB is 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione, and OxymaPure is ethyl (hydroxyimino)cyanocyanate.

[0049] 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.

[0050] 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.

[0051] 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 tetrapeptide-30, providing a basis for process design in subsequent examples.

[0052] Example 1 A method for preparing tetrapeptide-30, with the sequence number H-Pro-Lys-Glu-Lys-OH; the synthetic route is as follows: Figure 2 As shown From C-end to N-end: Lys(Boc)→Glu(OtBu)→Lys(Boc)→Pro; In this embodiment, the green solvent refers to N-formylmorpholine: anisole (1:1, volume ratio). Materials and Equipment: Resin: 2-chlorotriphenylmethyl chloride resin (CTC resin, substitution value 0.8 mmol / g), dosage 1.0 g Reagents and amino acids: Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH.

[0053] Green solvent, N,N-diisopropylethylamine, 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione, N,N'-diisopropylcarbodiimide (DIC).

[0054] Deprotection solution: 5% DBU / green solvent solution.

[0055] Cutting fluid: 20% HFIP / DCM.

[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-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. The resin is loaded into a continuous flow reaction system and pretreated using a green solvent.

[0057] The specific steps are as follows: 1.0 g of CTC resin (substitution value 0.8 mmol / g) was loaded into a PTFE tubular reactor, rinsed with green solvent (flow rate 5 mL / min) for 10 min, and then ultrasonically assisted to swell for 30 min (150 W).

[0058] S200. The coupling and deprotection steps of four Fmoc-protected amino acids are completed sequentially through a continuous flow reaction system to obtain the final peptide chain; The amino acids protected by Fmoc are, in order: Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Pro-OH.

[0059] 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.

[0060] 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 product from step S200.

[0061] Continuous flow parameters: flow rate 2 mL / min, reactor temperature 25℃, ultrasonic power 100 W, residence time 30 min.

[0062] The solvent pump was switched to wash three times each with methanol and green solvent (flow rate 5 mL / min), and the solvent was used to wash three times with methanol (3 × 10 mL) and green solvent (3 × 10 mL).

[0063] The deprotection steps of S212.Fmoc-Lys(Boc)-OH are 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 in step S211. The reaction was performed with ultrasonic assistance (120 W) for 2 min × 2 times. The waste liquid was directly introduced into the waste liquid tank.

[0064] The solvent pump was switched and the solution was washed with methanol (3 × 10 mL) and green solvent (3 × 10 mL) in sequence (flow rate 5 mL / min, 3 times each).

[0065] S220. The coupling and deprotection steps of Fmoc-Glu(OtBu)-OH are completed through a continuous flow reaction system.

[0066] The coupling steps of S221.Fmoc-Glu(OtBu)-OH are as follows: Fmoc-Glu(OtBu)-OH (2.4 mmol), 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (0.48 mmol), and DIC (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.

[0067] Reaction parameters: flow rate 3 mL / min, ultrasonic power 120 W, residence time 12 min. Coupling was confirmed by online ninhydrin detection. The resin was then washed using a solvent pump.

[0068] The deprotection steps of S222. Fmoc-Glu(OtBu)-OH are 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. The mixture was then treated with ultrasonic assistance (120 W) for 2 min, and the waste liquid was directly introduced into the waste liquid tank.

[0069] The solvent pump was switched to wash with methanol and green solvent in sequence (flow rate 5 mL / min, 3 times each).

[0070] S230. The coupling and deprotection steps of Fmoc-Lys(Boc)-OH are completed through a continuous flow reaction system, with a coupling efficiency of ≥98%.

[0071] 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 DIC (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.

[0072] Reaction parameters: flow rate 3 mL / min, ultrasonic power 120 W, residence time 12 min. Coupling was confirmed by online ninhydrin detection. The resin was then washed using a solvent pump.

[0073] The deprotection steps of S232. Fmoc-Lys(Boc)-OH are as follows: A 5% DBU / green solvent solution was pumped into the reactor at a flow rate of 3 mL / min, treated with ultrasound assistance (120 W) for 2 min, and then injected into the product of step S231 to obtain the final peptide chain. The waste liquid was directly introduced into the waste liquid tank.

[0074] The solvent pump was switched to wash with methanol and green solvent in sequence (flow rate 5 mL / min, 3 times each).

[0075] S240. The coupling and deprotection steps of Fmoc-Pro-OH are completed through a continuous flow reaction system, with a coupling efficiency of ≥98%.

[0076] The coupling steps of S241.Fmoc-Pro-OH are as follows: Fmoc-Pro-OH (2.4 mmol), 5-(hydroxyimino)-1,3-dimethyl-1,3-thiazine-2,4,6-trione (0.48 mmol), and DIC (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 S232.

[0077] Reaction parameters: flow rate 3 mL / min, ultrasonic power 120 W, residence time 12 min. Coupling was confirmed by online ninhydrin detection. The resin was then washed using a solvent pump.

[0078] The deprotection steps for S242.Fmoc-Pro-OH are as follows: A 5% DBU / green solvent solution was pumped into the reactor at a flow rate of 4 mL / min, treated with ultrasound assistance (150 W) for 2 min, and then injected into the product of step S241 to react and obtain the final peptide chain. The waste liquid was directly introduced into the waste liquid tank.

[0079] The solvent pump was switched to wash with methanol and green solvent in sequence (flow rate 5 mL / min, 3 times each).

[0080] S300. The final peptide chain is cleaved to obtain the tetrapeptide-30.

[0081] The specific steps are as follows: S310, 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.

[0082] S320, 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 (C18 column, acetonitrile / water gradient elution) to obtain a tetrapeptide-30H-Pro-Lys-Glu-Lys-OH with a protecting group. Then treat it with 30% trifluoroacetic acid / dichloromethane for 1 h, and remove the organic solvent by rotary evaporation under reduced pressure to obtain tetrapeptide-30 (serial number: H-Pro-Lys-Glu-Lys-OH). The NMR chromatogram of the product tetrapeptide-30 is shown below. Figure 2 As shown, the purity is 98.03%.

[0083] The chromatographic conditions for liquid chromatography purification are as follows: Column: Yuexu LP-C18; Mobile phase: water; Gradient: 100% water, 1-15 min; Flow rate: 1 ml / min; Injection volume: 5 μL; Detection wavelength: 215nm.

[0084] 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 resulting tetrapeptide-30H-Pro-Lys(Boc)-Glu(OtBu)-Lys(Boc)-OH are shown in Table 6.

[0085] Table 6 Yield Results Application Example 1 The tetrapeptide-30 prepared in Example 1 is applied in pharmaceuticals, cosmetics, and diagnostic reagents, such as... Figure 3 As shown in the Meili Xiuxing APP, tetrapeptide-30 has the effect of removing freckles and whitening skin.

[0086] 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 process for the preparation of tetrapeptide-30, characterized in that, The sequence of the tetrapeptide-30 is H-Pro-Lys-Glu-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 four Fmoc-protected amino acids are sequentially completed to obtain a final peptide chain; The Fmoc-protected amino acids are Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Pro-OH in sequence; S300. The final peptide chain is cut to obtain the tetrapeptide-30; The green solvent is a mixed solution of N-formylmorpholine and anisole in a volume ratio of 1:

1.

2. The method of claim 1, wherein the tetrapeptide-30 is prepared by, 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 for preparing tetrapeptide-30 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 30min-60min.

4. The method of claim 1, wherein the tetrapeptide-30 is prepared by, 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 -30min.

5. The method for preparing tetrapeptide-30 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 claim 1, wherein the tetrapeptide-30 is prepared by, In step S300, after the final peptide chain is cut, the tetrapeptide-30 is obtained after purification by reverse phase high performance liquid chromatography.

7. The method of claim 6, wherein the tetrapeptide-30 is prepared by, The chromatographic conditions of the liquid chromatography purification are as follows: Chromatographic column: Lux LP-C18; Mobile phase: water; Gradient: 100% water, 1-15 min; Flow rate: 1 ml / min; Injection volume: 5uL; Detection wavelength: 215nm.

8. Tetrapeptide-30 prepared according to the method of any one of claims 1 to 7, characterized by The purity of the tetrapeptide-30 is ≥98%.

9. The tetrapeptide-30 prepared by the method of any one of claims 1-7 or the tetrapeptide-30 of claim 8 for use in medicine, cosmetics, and diagnostic reagents.

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