Polypeptide coupling compound as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide coupling compound as well as a preparation method and application thereof. The structure of the polypeptide coupling compound is as follows: # imgabs0 #, L is-CH2CH2-(-CH2-CH2-O-) n-NH-, and n is equal to 2-6; p is a tyrosinase targeting peptide. According to the polypeptide coupling compound provided by the invention, the water solubility of the peptide thimidol is remarkably improved by coupling (-CH2-CH2-O-) n, and the whitening activity of the peptide thimidol is effectively improved by coupling various peptides with a tyrosinase targeting effect, so that the whitening effect is enhanced. In addition, the adopted peptide has good safety and biocompatibility, and a more efficient and safer choice can be provided for skin care.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide technology, and in particular to a polypeptide coupling compound and a preparation method and application thereof. Background Art

[0002] The application of compounds in cosmetics is constantly evolving with technological advances and changes in consumer demand. In recent years, the study of active ingredients has received widespread attention, and many natural extracts and synthetic compounds have been introduced into cosmetics to enhance skin care effects. Although the application of certain compounds in cosmetics has unique activities and diverse functions, there are also some obvious defects, such as poor water solubility. Due to low water solubility, these compounds are often difficult to disperse evenly in the formulation, leading to product stability issues. The bioavailability of poorly soluble compounds is generally low, which limits their effectiveness in skin penetration and absorption. This means that although these compounds have potential skin care effects, in actual applications, consumers may not get the expected results. In addition, poorly soluble compounds may require additional formulation technologies (such as nano-sizing, microemulsification, etc.) to improve their availability in cosmetics, which undoubtedly increases the production cost and complexity of the product.

[0003] In the field of cosmetics, the use of single-functional compounds can provide clear effects in certain specific products, but there are also some significant limitations. First, many traditional cosmetic ingredients have poor water solubility, which leads to limited uniform distribution and skin permeability in the product, affecting the effect of the effect. For example, the cosmetic raw material peptide Anmido (CN105007991A) with whitening effect is limited in its application in cosmetics due to its water insolubility and poor skin permeability. Although peptide Anmido can be dissolved in alcohol solvents, once mixed with water, it will precipitate quickly, affecting its uniform distribution and stability in the product, and also reducing its absorption capacity on the skin. Therefore, improving the water solubility of peptide Anmido becomes the key to improving its application effect. By improving its water solubility, its stability in the formula can be increased, its skin permeability can be improved, and its whitening effect can be enhanced. In addition, single-functional compounds are usually difficult to meet the increasingly diverse needs of modern consumers. Consumers not only expect cosmetics to solve a specific problem (such as anti-wrinkle, whitening or moisturizing), but also hope that the product can provide multiple effects. To this end, improving the water solubility of peptide Anmido can not only enhance its market competitiveness, but also enable it to be better combined with other functional ingredients to meet consumers' demand for all-in-one products. Therefore, seeking technical means to improve the solubility of peptide Anmido while improving bioavailability, skin permeability and how to synergize and enhance synergy has become an important direction of current research. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a polypeptide coupling compound and a preparation method and application thereof, which further expands the function of Peptide Anmido on the basis of solving the water solubility of Peptide Anmido.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] In one aspect, the present invention provides a polypeptide coupling compound, the structure of which is shown in formula (I):

[0007]

[0008] In formula (I), L is -CH2CH2-(-CH2-CH2-O-) n -NH-, n=2-6; P is a tyrosinase targeting peptide.

[0009] As a preferred embodiment, the sequence structure of the tyrosinase targeting peptide is selected from any one of RGDERRRK~ (SEQ ID NO.1), IFENLQNYR~ (SEQ ID NO.2) and MPFRWFKPV~ (SEQ ID NO.3); in SEQ ID NO.3, the third F and W are D-type amino acids;

[0010] In the technical scheme of the present invention, in the polypeptide sequences shown in SEQ ID NO. 1 to 3, the carboxyl group of the C-terminal amino acid is connected to the L group;

[0011] In the technical solution of the present invention, the amino acids in the polypeptide sequences shown in SEQ ID NOs. 1 to 3 are modified or unmodified amino acids.

[0012] In another aspect, the present invention provides a method for preparing the above-mentioned polypeptide coupling compound, the preparation method comprising the following steps:

[0013] (1) Peptide Anmido and Fmoc-NH-(O-CH2-CH2-) n CH2CH2Cl reacts to give Fmoc-NH-(O-CH2-CH2-) n CH2CH2-peptide anmido, and then deprotection to obtain PEGylated peptide anmido intermediate NH2-(O-CH2-CH2-) n CH2CH2-Peptide Anmido;

[0014] (2) performing a coupling reaction between the polypeptide described in any one of SEQ ID NOs. 1 to 3 and the peptide Anmido intermediate obtained in step (1).

[0015] In the technical solution of the present invention, the Fmoc-NH-(O-CH2-CH2-) nThe structural formula of CH2CH2-peptide Anmido is shown in formula (II):

[0016]

[0017] In the technical solution of the present invention, the NH2-(O-CH2-CH2-) n The structural formula of CH2CH2-peptide Anmido is shown in formula (III):

[0018]

[0019] As a preferred embodiment, the polypeptide described in any one of SEQ ID NOs. 1 to 3 is prepared by Fmoc solid phase synthesis method.

[0020] As a preferred implementation, step (1) specifically includes the following steps:

[0021] The peptide Anmido was reacted with Fmoc-NH-(O-CH2-CH2-) n CH2CH2Cl was dissolved in a solvent, NaCl and a catalyst were added, and the reaction was carried out under visible light to obtain Fmoc-NH-(O-CH2-CH2-) n CH2CH2-Peptide Anmido;

[0022] Preferably, the solvent is selected from at least one of dimethyl sulfoxide (DMSO) and dimethylformamide (DMF);

[0023] Preferably, the mass of the NaCl is 0.3 to 5 times the mass of the peptide Anmido;

[0024] Preferably, the wavelength of the visible light source is 400nm to 700nm;

[0025] Preferably, the catalyst is Ag / TiO2;

[0026] Preferably, the reaction temperature is 20°C to 60°C;

[0027] Preferably, the reaction time is 2 to 24 hours;

[0028] Preferably, the reaction is carried out in an inert gas atmosphere;

[0029] Preferably, the peptide Anmido is combined with Fmoc-NH-(O-CH2-CH2-) n The molar ratio of CH2CH2Cl is 1:1-4;

[0030] In certain specific embodiments, the reaction is terminated by adding a cooling solvent; the cooling solvent is ethanol or dichloromethane.

[0031] As a preferred embodiment, in step (2), the coupling reaction is carried out in a solvent;

[0032] Preferably, the solvent is selected from at least one of acetonitrile, dimethyl sulfoxide and dimethylformamide;

[0033] Preferably, the coupling reaction requires the addition of an activation reagent;

[0034] Preferably, the activation reagent is a mixture of 1-hydroxybenzotriazole (HOBt) and N,N'-diisopropylcarbodiimide (DIC);

[0035] Preferably, the molar ratio of the peptide anamido intermediate to 1-hydroxybenzotriazole (HOBt) and N,N'-diisopropylcarbodiimide (DIC) is 2-4:2-6:1-2.

[0036] As a preferred embodiment, the temperature of the coupling reaction is 25-40°C;

[0037] Preferably, the coupling reaction time is 2 to 12 hours.

[0038] In certain specific embodiments, step (2) specifically includes the following steps: dissolving the peptide anamido intermediate and 1-hydroxybenzotriazole (HOBt) in a solvent; then adding N,N'-diisopropylcarbodiimide (DIC) and a polypeptide described in any one of SEQ ID NO.1 to 3, and reacting at 25 to 40°C for 2 to 12 hours; and adding acetic acid to terminate the reaction.

[0039] In the technical solution of the present invention, the structure of the peptide Anmido is as follows:

[0040]

[0041] In another aspect, the present invention provides use of the above-mentioned polypeptide coupling compound in the preparation of whitening cosmetics.

[0042] The present invention has the following advantages:

[0043] (1) The polypeptide coupling compound provided by the present invention is coupled with (-CH2-CH2-O-) n The water solubility of peptide Anmido was significantly improved, solving the problem of its poor solubility in water phase. On this basis, multiple peptides with tyrosinase targeting effects were further coupled to effectively enhance the whitening activity of peptide Anmido, thereby enhancing the whitening effect.

[0044] (2) The polypeptide sequence used in the present invention: The polypeptide shown in SEQ ID NO.1 is an amphiphilic transdermal peptide with tyrosinase targeting effect, which can significantly enhance the solubility, cell penetration and skin permeability of peptide Anmido and promote its effective delivery in the skin; the polypeptide shown in SEQ ID NO.2 is of plant origin and can inhibit the activity of collagenase and prevent collagen degradation through tyrosinase targeting effect, and further enhance the whitening effect of peptide Anmido. After coupling with peptide Anmido, it can simultaneously achieve the dual effects of whitening and anti-aging; the polypeptide shown in SEQ ID NO.3 inhibits the activity of tyrosinase and prevents melanin production by competitively binding with human melanocortical receptors (MC1R) in melanocytes, thereby significantly improving the whitening effect of peptide Anmido. In addition, the polypeptide used in the present invention has good safety and biocompatibility, and can provide a more efficient and safe option for skin care.

[0045] (3) The polypeptide coupling compound provided by the present invention has a simple structure, mild reaction conditions, is easy to prepare, has a high synthesis success rate, and has a high purity of the product. Its application in whitening cosmetics has a good market prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The cytotoxicity test results of the whitening polypeptide coupling compounds synthesized in the examples of the present invention are shown. DETAILED DESCRIPTION

[0047] The following embodiments are only some embodiments of the present invention, rather than all embodiments. Therefore, the detailed description in the embodiments of the present invention provided below is not intended to limit the scope of the present invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the protection scope of the present invention.

[0048] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0049] Example 1 Synthesis of polypeptide sequences

[0050] Take an appropriate amount of 2CTC-Resin with a substitution degree of 0.51 mmol / g and add it to the solid phase reactor. Add N,N-dimethylformamide (DMF) to swell for 30 minutes and wash with DMF three times. NO.1: RGDERRRK, according to the conventional peptide solid phase synthesis method, the amino acid coupling was started from the C-terminus, and Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Boc-Arg(Pbf)-OH were coupled in sequence; after the coupling was completed, the resin was shrunk with methanol for 10 minutes, the methanol was drained, and dried to obtain a dry peptide resin; then the resin was treated with 20% TFE (trifluoroethanol) DMF solution for 1 hour, filtered and dried to obtain a crude product of fully protected peptide I:

[0051] Boc-Arg(Pbf)-Gly-Asp(OtBu)-Glu(OtBu)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Lys(Bo c)-OH.

[0052] Example 2 Synthesis of polypeptide sequences

[0053] Based on the polypeptide sequence SEQ ID NO.2: IFENLQNYR, referring to the method of Example 1, a crude product of fully protected peptide II was obtained:

[0054] Boc-Ile-Phe-Glu(OtBu)-Asn(Trt)-Leu-Gln(Trt)-Asn(Trt)-Tyr(tBu)-Arg(Pbf)-OH.

[0055] Example 3 Synthesis of polypeptide sequences

[0056] Based on the polypeptide sequence SEQ ID NO.3: MPFRWFKPV (wherein the third F and the fifth W are D-phenylalanine and D-tryptophan, respectively), the crude fully protected peptide III was obtained by referring to the method of Example 1:

[0057] Boc-Met-Pro-D-Phe-Arg(Pbf)-D-Trp(Boc)-Phe-Lys(Boc)-Pro-Val-OH.

[0058] Example 4 Preparation of Peptide Anmido-(PEG)6-NH2 Intermediate

[0059] 5mmol of peptide anmido and 5mmol of Fmoc-NH-(O-CH2-CH2-)6CH2CH2Cl were added to the reaction bottle respectively, and 50mL of DMSO was used as the solvent. After fully dissolving, 0.55g of NaCl was added, and then Ag / TiO2 catalyst was added; under a stable visible light source, nitrogen was stirred at 37°C for 24h; after the reaction was completed, the system was added with a cooling solvent of dichloromethane or ethanol to terminate, and solid Fmoc-NH-(O-CH2-CH2-)6CH2CH2-peptide anmido was obtained after drying; the obtained product was then dissolved in a 20% piperidine-dichloromethane (DCM) solution, and after reacting for 30min, the protecting group was removed to obtain NH2-(O-CH2-CH2-)6CH2CH2-peptide anmido intermediate, and its structural formula is shown below:

[0060]

[0061] The preparation process of Fmoc-NH-(O-CH2-CH2-)6CH2CH2Cl is as follows:

[0062] 10 mmol Fmoc-Cl and 10 mmol triethylamine were added to N,N-dimethylformamide (DMF), and then 5 mmol NH2-(O-CH2-CH2-)6CH2CH2Cl was added. The reaction was stirred under nitrogen for 1 h to obtain Fmoc-NH-(O-CH2-CH2-)6CH2CH2Cl.

[0063] Example 5: Preparation of peptide anmido-(PEG)4-NH2 intermediate

[0064] Referring to the synthesis method of Example 4, NH2-(O-CH2-CH2-)4CH2CH2-peptide anmido intermediate was obtained, and its structural formula is shown below:

[0065]

[0066] Example 6: Preparation of peptide anmido-(PEG)2-NH2 intermediate

[0067] Referring to the method of Example 4, NH2-(O-CH2-CH2-)2CH2CH2-peptide anmido intermediate was obtained, and its structural formula is shown below:

[0068]

[0069] Example 7 Preparation of whitening polypeptide coupling compound (I)

[0070] 1 mmol of the intermediate prepared in Example 4, 1 mmol of 1-hydroxybenzotriazole (HOBt), and 10 mL of acetonitrile as solvent were added to a reaction flask; under nitrogen protection, 0.5 mmol of N,N'-diisopropylcarbodiimide (DIC) was added dropwise, and 1.1 mmol of the fully protected peptide I obtained in Example 1 was added, and the reaction was carried out at room temperature for 2 h; 1 mL of acetic acid was added to terminate the reaction; the reaction solution was spin-dried to obtain a light yellow solid;

[0071] Add 30 mL of pre-prepared lysis solution to the above solid, the composition of the lysis solution is: trifluoroacetic acid (TFA): triisopropylsilane (TIS): 1,2-ethanedithiol (EDT): PhOH: H2O in a volume ratio of 90:3:3:2:2; react at room temperature for 1 hour, pour into anhydrous ether to precipitate a white solid, centrifuge, wash the solid with anhydrous ether, and vacuum dry to obtain a white solid coupling compound I; after purification by HPLC, a purified product coupling compound (I) with a purity greater than 99% is obtained, and the structural formula is as follows

[0072]

[0073] Example 8 Preparation of whitening polypeptide coupling compound (II)

[0074] Referring to the coupling method of Example 7, the fully protected peptide I prepared in Example 1 was coupled with the peptide Anmido intermediate prepared in Example 5 to obtain a whitening polypeptide coupling compound (II) with a purity greater than 99%, and the structural formula is as follows:

[0075]

[0076] Example 9 Preparation of whitening polypeptide coupling compound (III)

[0077] Referring to the coupling method of Example 7, the fully protected peptide II prepared in Example 2 was coupled with the peptide Anmido intermediate prepared in Example 4 to obtain a whitening polypeptide coupling compound (III) with a purity greater than 99%, and the structural formula is shown below:

[0078]

[0079] Example 10 Preparation of whitening polypeptide coupling compound (IV)

[0080] Referring to the coupling method of Example 7, the fully protected peptide II prepared in Example 2 was coupled with the peptide Anmido intermediate prepared in Example 6 to obtain a whitening polypeptide coupling compound (IV) with a purity greater than 99%, and the structural formula is shown below:

[0081]

[0082] Example 11 Preparation of whitening polypeptide coupling compound (V)

[0083] Referring to the coupling method of Example 7, the fully protected peptide III prepared in Example 3 was coupled with the peptide Anmido intermediate prepared in Example 4 to obtain a whitening polypeptide coupling compound (V) with a purity greater than 99%, and the structural formula is shown below:

[0084]

[0085] In MPFRWFKPV, the third F and the fifth W are D-phenylalanine and D-tryptophan, respectively.

[0086] Example 12 Preparation of whitening polypeptide coupling compound (VI)

[0087] Referring to the coupling method of Example 7, the fully protected peptide prepared in Example 3 was coupled with the peptide Anmido-(PEG)4-NH2 in Example 5. Referring to the coupling method of Example 7, a whitening polypeptide coupling compound (VI) with a purity greater than 99% was obtained. The structural formula is as follows:

[0088]

[0089] In MPFRWFKPV, the third F and the fifth W are D-phenylalanine and D-tryptophan, respectively.

[0090] Example 13 Compound Toxicity Test

[0091] (1) Cell culture

[0092] Human epidermal melanocytes (HEM) were cultured in DMEM medium containing 1% penicillin / streptomycin and 10% fetal bovine serum, and the cell culture incubator was set at 37°C and 5% CO2.

[0093] (2) Cytotoxicity test

[0094] HEM cells were plated in 96-well plates at 6 × 10 cells per well. 4 Each well was inoculated with cells and cultured for 24 hours until the cells adhered to the wall. The culture medium was aspirated and the culture medium containing different concentrations (100, 200, 400, 600, 800, 1000 mg / L) of the above-mentioned whitening peptide compounds was added to each well. The blank control group was added with culture medium without whitening peptide coupling compounds and cultured at 37°C for 24 hours. The original culture medium was aspirated and 100 μL of 10% CCK-8 solution was added again. After incubation for 1 hour, the absorbance was measured at 450 nm and the HEM cell viability was calculated. The results are shown in Figure 2. Figure 1 As shown in the figure, it can be seen that all concentrations of the whitening polypeptide-coupled compounds have no significant effect on the cell viability of HEM cells, indicating that the compound has no cytotoxicity and has a high safety.

[0095] Example 14 Solubility test of whitening polypeptide coupling compound

[0096] Accurately weigh 1-100 mg of whitening peptide coupling compounds (Ⅰ)~(VI), add the weighed compounds into different ep tubes, add 1 mL of water; use a vortex instrument to mix the solutions for 5 minutes at room temperature, observe the dissolution, and record whether the compounds are completely dissolved. The results are shown in Table 1. As can be seen from Table 1, Peptide Anmido is completely insoluble in water and has a solubility of 0; the solubility of the coupling compounds (Ⅰ)~(VI) of the present invention exceeds 50 g / L, which is significantly improved compared with Peptide Anmido.

[0097] Table 1 Solubility test of peptide coupling compounds

[0098] sample Solubility Whitening peptide conjugated compounds (Ⅰ) 57.2g / L Whitening peptide conjugated compounds (Ⅱ) 54.3g / L Whitening peptide conjugated compounds (Ⅲ) 52.1g / L Whitening peptide conjugated compounds (Ⅳ) 50.4g / L Whitening peptide conjugated compounds (V) 55.7g / L Whitening peptide conjugated compounds (VII) 54.6g / L Peptide Anmito 0

[0099] Example 15 Skin permeability test of whitening polypeptide coupling compounds

[0100] Ten healthy adult male mice were selected and depilated with 10% Na2S. The animals were killed 48 hours later and intact abdominal skin patches were randomly assembled in a skin permeation chamber. 2 200 μL of aqueous solution of whitening peptide coupling compound (I), (III), (V) was dripped onto the skin surface, respectively. The weight of whitening peptide coupling compound (I), (III), (V) was 100 mg, and the coating dosage was 18.51 mg / cm 2 ; 10 ml of redistilled water was injected into the lower chamber as the receiving solution, and the receiving solution was collected every 1, 2, 4, 6, 12, and 24 hours, with 4 parallel samples at each time point. High performance liquid chromatography was used for identification, and the permeability (P), permeation volume (T), and permeation velocity (V) were calculated. The final results are shown in Table 1;

[0101] Permeability T (mg / cm 2 ) = total sample volume in the receiving solution W (mg) / coating area (cm 2 ); Permeability P (%) = total amount of sample in the receiving solution W (mg) / total amount of coating (mg) * 100%.

[0102] The results showed that the whitening peptide coupling compound (I) had better skin permeability than Peptide Anmido. With the increase of time, the permeability and permeation area also continued to increase. The whitening peptide coupling compounds (II) to (III) had no effect on skin permeability.

[0103] Table 2. Permeability test on mouse skin

[0104]

[0105]

[0106] Example 16 In vitro whitening efficacy test of whitening polypeptide coupling compounds

[0107] Using PBS as solvent, the substrate L-DOPA was prepared into a 1 mg / mL solution, and tyrosinase was prepared into an enzyme solution with an activity of 100 U / mL for standby use. The experiment was divided into a reaction group, a reaction control group, a blank group, and a blank control group. The reaction group and the reaction control group were added with 25 μL of 100 ppm of peptide Anmido and whitening peptide coupling compound samples, respectively. At the same time, the blank group and the blank control group were added with an equal volume of PBS solution, and then 25 μL of the above-prepared tyrosinase solution was added to the reaction group and the blank group, and the reaction control group and the blank control group were added with an equal volume of PBS, mixed evenly, and placed in a 37°C incubator for incubation for 5 minutes; all experimental groups added 100 μL of the prepared L-DOPA solution, continued to incubate in a 37°C incubator for 5 minutes, and measured the absorbance (A) value at a wavelength of 475 nm. Repeat the above experiment 3 times, and calculate the tyrosinase inhibition rate according to the following formula:

[0108] Inhibition rate = 1-(reaction group-reaction control group) / (empty group-empty control group)*100%

[0109] The results are shown in Table 3. Due to the coupling of tyrosinase targeting peptide, the whitening peptide-coupled compounds (I) to (VI) all showed higher inhibition rates on tyrosinase than peptide Anmito, among which the coupled compounds (V to VI) had better whitening effects due to the coupling of nonapeptide-1.

[0110] Table 3 Tyrosinase inhibition rate of peptide coupling compounds

[0111]

[0112]

[0113] Example 17 In vitro anti-wrinkle efficacy test of whitening polypeptide coupling compounds

[0114] 200 μL of 0.12 mol Tris-Cl buffer (pH 8.0) was mixed with N-succinyl-tri-L-alanine 4-nitrophenylamine to make the latter reach 1.0 mmol; 30 μL of 100 ppm coupling compound (Formula II), blank control sample and 20 μL of 2.5 U / mL porcine pancreatic elastase were mixed with the solution respectively; after incubation in a 25°C incubator for 20 min, the absorbance at 410 nm was measured with an ELISA reader to obtain the effect of the sample on the activity of elastase, and the calculation formula was as follows:

[0115] Elastase inhibition rate = 1-(experimental group-blank group) / (control-blank)*100%

[0116] Table 4 Inhibition rate of peptide coupling compounds on elastase

[0117] sample Elastase inhibition rate (%) Peptide Anmito 2.4 Whitening peptide conjugated compounds (Ⅰ) 15.7 Whitening peptide conjugated compounds (Ⅱ) 13.1 Whitening peptide conjugated compounds (Ⅲ) 33.6 Whitening peptide conjugated compounds (Ⅳ) 35.6 Whitening peptide conjugated compounds (V) 16.9 Whitening peptide conjugated compounds (VI) 17.3

[0118] The results are shown in Table 4. Compared with peptide Anmido, the whitening polypeptide coupled compounds (III-IV) of the present invention have a significant inhibitory effect on elastase and have good anti-wrinkle and anti-aging effects. This also shows that the whitening polypeptide coupled compounds (III-IV) have both whitening and anti-wrinkle and anti-aging effects.

[0119] Example 18 Application of whitening polypeptide coupling compound in the preparation of essence

[0120] The essence was prepared according to the addition amounts and processes shown in the following table. The percentages in this example are the mass percentages of each component in the final essence.

[0121] Table 5. Whitening Essence Formula

[0122]

[0123] Tables 6-7 show the performance analysis results of the above essences.

[0124] Table 6. Human body efficacy test on freckle removal and whitening (spot area mm 2 )

[0125]

[0126]

[0127] Table 7. Wrinkle removal efficacy test on human body (overall wrinkle area mm 2 )

[0128] Sample(essence) Before use After 28 days of use Whitening peptide conjugated compounds (Ⅰ) 78.44 67.58 Whitening peptide conjugated compounds (Ⅱ) 79.54 67.32 Whitening peptide conjugated compounds (Ⅲ) 81.52 50.23 Whitening peptide conjugated compounds (Ⅳ) 80.64 51.62 Whitening peptide conjugated compounds (V) 76.65 65.21 Whitening peptide conjugated compounds (VI) 75.43 66.37 Peptide Anmito 85.56 78.89

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide coupling compound, characterized in that: The structure is shown in formula (I): In formula (I), L is -CH2CH2-(-CH2-CH2-O-) n -NH-, n=2-6; P is a tyrosinase targeting peptide.

2. The polypeptide-coupled compound according to claim 1, characterized in that: The sequence structure of the tyrosinase targeting peptide is selected from any one of SEQ ID NO.1 to 3: SEQ ID NO.1: RGDERRRK~; SEQ ID NO.2: IFENLQNYR~; SEQ ID NO.3: MPFRWFKPV~; in SEQ ID NO.3, the third F and W are D-type amino acids; The carboxyl group of the C-terminal amino acid in the polypeptide sequence shown in SEQ ID NO. 1 to 3 is connected to the L group; The amino acids in the polypeptide sequences shown in SEQ ID NOs. 1 to 3 are modified or unmodified amino acids.

3. The method for preparing the polypeptide coupling compound according to any one of claims 1 to 2, characterized in that: The steps include: (1) Peptide Anmido and Fmoc-NH-(O-CH2-CH2-) n CH2CH2Cl reacts to give Fmoc-NH-(O-CH2-CH2-) n CH2CH2-peptide anmido, and then deprotection to obtain PEGylated peptide anmido intermediate NH2-(O-CH2-CH2-) n CH2CH2-Peptide Anmido; (2) performing a coupling reaction between the polypeptide described in any one of SEQ ID NOs. 1 to 3 and the peptide Anmido intermediate obtained in step (1).

4. The preparation method according to claim 3, characterized in that: The polypeptide described in any one of SEQ ID NOs. 1 to 3 is prepared by Fmoc solid phase synthesis method.

5. The preparation method according to claim 3, characterized in that: Step (1) specifically includes the following steps: The peptide Anmido was reacted with Fmoc-NH-(O-CH2-CH2-) n CH2CH2Cl was dissolved in a solvent, NaCl and a catalyst were added, and the reaction was carried out under visible light to obtain Fmoc-NH-(O-CH2-CH2-) n CH2CH2-Peptide Anmito.

6. The preparation method according to claim 5, characterized in that: The solvent is selected from at least one of dimethyl sulfoxide and dimethylformamide; Preferably, the mass of the NaCl is 0.3 to 5 times the mass of the peptide Anmido; Preferably, the wavelength of the visible light source is 400nm to 700nm; Preferably, the catalyst is Ag / TiO2.

7. The preparation method according to claim 5, characterized in that: The reaction temperature is 20°C to 60°C; Preferably, the reaction time is 2 to 24 hours; Preferably, the reaction is carried out in an inert gas atmosphere; Preferably, the peptide Anmido is combined with Fmoc-NH-(O-CH2-CH2-) n The molar ratio of CH2CH2Cl is 1:1-4; Preferably, the reaction is terminated by adding a cooling solvent; the cooling solvent is ethanol or dichloromethane.

8. The preparation method according to claim 3, characterized in that: In step (2), the coupling reaction is carried out in a solvent; Preferably, the solvent is selected from at least one of acetonitrile, dimethyl sulfoxide and dimethylformamide; Preferably, the coupling reaction requires the addition of an activation reagent; Preferably, the activation reagent is a mixture of 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide; Preferably, the molar ratio of the peptide anmido intermediate to 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide is 2-4:2-6:1-2.

9. The preparation method according to claim 3, characterized in that: In step (2), the coupling reaction temperature is 25-40°C; Preferably, the coupling reaction time is 2 to 12 hours.

10. The preparation method according to claim 3, characterized in that: Step (2) specifically includes the following steps: dissolving the peptide anmido intermediate and 1-hydroxybenzotriazole in a solvent; then adding N,N'-diisopropylcarbodiimide and any of the polypeptides described in SEQ ID NO.1 to 3, reacting at 25 to 40°C for 2 to 12 hours; and adding acetic acid to terminate the reaction.

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