A polypeptide conjugate compound, its preparation method and application

Abstract

This invention discloses a polypeptide coupling compound, its preparation method, and its applications. The structure of the polypeptide coupling compound is shown below: where L is -CH2CH2-(-CH2-CH2-O-). n -NH-, n = 2-6; P is a tyrosinase-targeting peptide. The polypeptide conjugates provided by this invention are coupled with (-CH2-CH2-O-). n This product significantly improves the water solubility of Peptide Anmido and effectively enhances its whitening activity by coupling it with multiple peptides that target tyrosinase, thereby strengthening its whitening effect. Furthermore, the peptides used have good safety and biocompatibility, providing a more efficient and safer option for skin care.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptides, in particular to a polypeptide conjugate compound and its preparation method and application. BACKGROUND

[0002] Compound applications in cosmetics are constantly evolving with technological advancements and changes in consumer demands. In recent years, active ingredient research has received extensive attention, and many natural extracts and synthetic compounds have been introduced into cosmetics to enhance skincare effects. While the use of certain compounds in cosmetics has unique activities and diverse functions, there are also some obvious drawbacks, such as poor water solubility. Due to low water solubility, these compounds often have difficulty in uniform dispersion in formulations, leading to product stability issues. The bioavailability of poorly soluble compounds is generally low, limiting their effectiveness in skin penetration and absorption. This means that, despite the potential skincare efficacy of these compounds, consumers may not achieve the expected results in practical applications. Additionally, poorly soluble compounds may require additional formulation techniques (such as nanonization, microemulsification, etc.) to improve their usability in cosmetics, which undoubtedly increases the production cost and complexity of the product.

[0003] In the field of cosmetics, the use of single-function compounds, while providing clear effects in certain specific products, also has some significant limitations. First, many traditional cosmetic ingredients have limited uniform distribution and skin penetration due to poor water solubility, affecting their effectiveness. For example, the cosmetic raw material peptide anmido (CN105007991A) has whitening efficacy, but its water insolubility and poor skin permeability limit its application in cosmetics. Although peptide anmido can dissolve in alcoholic solvents, it will quickly precipitate once mixed with water, 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 is key to enhancing its application effectiveness. By improving its water solubility, its stability in formulations can be increased, its skin permeability can be improved, and its whitening efficacy can be enhanced. In addition, single-function compounds are often difficult to meet the increasingly diverse needs of modern consumers, who not only expect cosmetics to solve a specific problem (such as anti-aging, whitening, or moisturizing), but also want products to provide multiple effects. Therefore, improving the water solubility of peptide anmido not only enhances its market competitiveness, but also enables it to better combine with other functional ingredients to meet consumer demand for multi-effect products. Therefore, seeking technical means to improve the solubility of peptide anmido while increasing bioavailability, skin permeability, and synergistic effects has become an important direction of current research. SUMMARY

[0004] In order to solve the above technical problems, the present application provides a polypeptide conjugated compound, a preparation method and application thereof, which further expands the function of the peptide amikin on the basis of solving the water-soluble problem of the peptide amikin.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0006] In one aspect, the present application provides a polypeptide conjugated 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 F and W at the third position are D-type amino acids.

[0010] In the technical scheme of the present application, the carboxyl group of the C-terminal amino acid of the polypeptide sequence shown in SEQ ID NO. 1~3 is connected with the L group.

[0011] In the technical scheme of the present application, the amino acids in the polypeptide sequence shown in SEQ ID NO. 1~3 are modified or unmodified amino acids.

[0012] In another aspect, the present application provides a preparation method of the polypeptide conjugated compound, which comprises the following steps:

[0013] (1) reacting the peptide amikin with Fmoc-NH-(O-CH2-CH2-) n CH2CH2Cl to obtain Fmoc-NH-(O-CH2-CH2-) n CH2CH2-peptide amikin, and then deprotecting to obtain the PEGylated peptide amikin intermediate NH2-(O-CH2-CH2-) n CH2CH2-peptide amikin;

[0014] (2) coupling the polypeptide of any one of SEQ ID NO. 1~3 with the peptide amikin intermediate obtained in step (1).

[0015] In the technical scheme of the present application, the Fmoc-NH-(O-CH2-CH2-) nThe structure of CH2CH2-pepamidom is shown in formula (II):

[0016]

[0017] In the technical solution of the present application, the NH2-(O-CH2-CH2-) n The structure of CH2CH2-pepamidom is shown in formula (III):

[0018]

[0019] As a preferred embodiment, the polypeptide according to any one of SEQ ID NO. 1-3 is prepared by Fmoc solid-phase synthesis.

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

[0021] The pepamidom and Fmoc-NH-(O-CH2-CH2-) n CH2CH2Cl are dissolved in a solvent, NaCl and a catalyst are added, and the reaction is carried out under visible light to obtain Fmoc-NH-(O-CH2-CH2-) n CH2CH2-pepamidom;

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

[0023] Preferably, the mass of NaCl is 0.3-5 times the mass of pepamidom;

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

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

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

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

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

[0029] Preferably, the molar ratio of pepamidom to Fmoc-NH-(O-CH2-CH2-) n CH2CH2Cl is 1:1-4;

[0030] In some 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 dimethyl formamide.

[0033] Preferably, the coupling reaction requires the addition of an activating agent.

[0034] Preferably, the activating agent is a mixture of 1-hydroxybenzotriazole (HOBt) and N,N'-diisopropylcarbodiimide (DIC).

[0035] Preferably, the molar ratio of the peptide anmimo intermediate, 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 time of the coupling reaction is 2-12h.

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

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

[0040]

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

[0042] The present application has the following advantages:

[0043] (1) The polypeptide coupling compound provided by the present application improves the water solubility of peptide anmimo by coupling (-CH2-CH2-O-) n significantly improves the water solubility of peptide anmimo, solving the problem of poor solubility in aqueous phase. On this basis, further coupling of multiple peptides with tyrosinase targeting effect effectively enhances the whitening activity of peptide anmimo, thereby enhancing the whitening effect.

[0044] (2) The polypeptide sequence used in the present application: the polypeptide shown in SEQ ID NO. 1 is an amphiphilic transdermal peptide, has tyrosinase targeting effect, can significantly enhance the solubility, cell penetration and skin permeability of peptide anmimo, and promote its effective delivery in the skin; the polypeptide shown in SEQ ID NO. 2 is of plant origin, can inhibit the activity of collagenase through tyrosinase targeting effect, prevent collagen degradation, and further enhance the whitening effect of peptide anmimo, and can realize the dual effects of whitening and anti-aging after coupling with peptide anmimo; the polypeptide shown in SEQ ID NO. 3 inhibits the activity of tyrosinase by competitive binding with human melanocortin receptor (MC1R) in melanocytes, prevents melanin production, and thus significantly improves the whitening effect of peptide anmimo. In addition, the polypeptide used in the present application has good safety and biocompatibility, and can provide more efficient and safe options for skin care.

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

[0046] Figure 1 Cell toxicity test results of the whitening polypeptide coupling compound synthesized in the examples of the present application. DETAILED DESCRIPTION

[0047] The following examples are only a part of the examples of the present application, not all the examples. Therefore, the detailed description of the examples of the present application provided below is not intended to limit the scope of the claimed application, but only represents selected embodiments of the present application. Based on the examples of the present application, all other examples obtained by those skilled in the art without creative work are within the scope of protection of the present application.

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

[0049] Example 1 Synthesis of polypeptide sequence

[0050] A certain amount of 2CTC-Resin with a substitution degree of 0.51 mmol / g was taken into a solid-phase reactor, N,N-dimethylformamide (DMF) was added for swelling for 30 min, and the 2CTC-Resin was washed with DMF for 3 times; for the polypeptide sequence SEQ ID NO. 1: RGDERRRK, according to the conventional polypeptide solid-phase synthesis method, the coupling of amino acids was carried out from the C-terminal, and Fmoc-Lys(Boc)-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 sequentially coupled; after the coupling was completed, the resin was shrunk with methanol for 10 min, the methanol was pumped out, and the resin was dried to obtain a dried peptide resin; then the resin was treated with 20% TFE (trifluoroethanol) in DMF for 1 hour, filtered and dried to obtain a crude full-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 a polypeptide sequence

[0053] Based on the polypeptide sequence SEQ ID NO. 2: IFENLQNYR, according to the method of Example 1, a crude full-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 a polypeptide sequence

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

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

[0058] Example 4 Preparation of a peptide intermediate, aminomethyl-PEG)6-NH2

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

[0060]

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

[0062] In N,N-dimethylformamide (DMF), 10 mmol of Fmoc-Cl and 10 mmol of triethylamine were added, and then 5 mmol of NH2-(O-CH2-CH2-)6CH2CH2Cl was added, and Fmoc-NH-(O-CH2-CH2-)6CH2CH2Cl was obtained after 1 h of reaction under nitrogen stirring.

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

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

[0065]

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

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

[0068]

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

[0070] In a reaction bottle, 1 mmol of the intermediate prepared in Example 4 was added, 1 mmol of 1-hydroxybenzotriazole (HOBt) was added, 10 mL of acetonitrile was used as a solvent; under nitrogen protection, 0.5 mmol of N,N'-diisopropylcarbodiimide (DIC) was added dropwise, 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 rotary evaporated to obtain a light yellow solid;

[0071] To the above solid, 30 mL of a previously prepared cleavage solution was added, and the composition of the cleavage solution was: trifluoroacetic acid (TFA): triisopropylsilane (TIS): 1,2-ethanedithiol (EDT): PhOH: H2O, the volume ratio was 90:3:3:2:2; the reaction was carried out at room temperature for 1 h, and then the white solid was precipitated by pouring into anhydrous ether, centrifuged, and washed with anhydrous ether, and then dried under vacuum to obtain a white solid coupling compound I; after purification by HPLC, the purified product coupling compound (I) with a purity greater than 99% was 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 amido 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 amido intermediate prepared in Example 4 to obtain a whitening polypeptide coupling compound (III) with a purity greater than 99%, and the structural formula is as follows:

[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 amido intermediate prepared in Example 6 to obtain a whitening polypeptide coupling compound (IV) with a purity greater than 99%, and the structural formula is as follows:

[0081]

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

[0083] The full-protected peptide III prepared in Example 3 was coupled with the peptide amido intermediate prepared in Example 4 to obtain the whitening polypeptide conjugate (V) having a purity of more than 99% according to the coupling method of Example 7, and the structure is shown as follows:

[0084]

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

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

[0087] The full-protected peptide prepared in Example 3 was coupled with the peptide amido-(PEG)4-NH2 prepared in Example 5 to obtain the whitening polypeptide conjugate (VI) having a purity of more than 99% according to the coupling method of Example 7, and the structure is shown as follows:

[0088]

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

[0090] Example 13 Toxicity test of compound

[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 conditions were set at 37°C and 5% CO2.

[0093] (2) Cell toxicity test

[0094] HEM cells were inoculated in a 96-well plate at 6x10 4 cells per well, and after 24 h of cell adhesion, the culture medium was removed and replaced with a culture medium containing different concentrations (100, 200, 400, 600, 800, and 1000 mg / L) of the above-mentioned whitening polypeptide compound, and the blank control group was added with a culture medium without the whitening polypeptide conjugate, and incubated at 37°C for 24 h; the original culture medium was removed and replaced with 100 μL of 10% CCK-8 solution, and incubated for 1 h, and then the absorbance value was measured at 450 nm, and the HEM cell viability was calculated, and the results are shown as follows: Figure 1 As can be seen from the figure, all concentrations of the whitening polypeptide conjugate had no significant effect on the cell viability of HEM cells, indicating that the compound had no cytotoxicity and had high safety.

[0095] Example 14 Solubility test of whitening polypeptide conjugated compounds

[0096] Accurately weigh 1-100 mg of whitening polypeptide conjugated compounds (I)-(VI), add the weighed compounds into different ep tubes, and add 1 mL of water; at room temperature, use a vortex to mix the solution for 5 min, observe the dissolution, and record whether the compound is completely dissolved. The results are shown in Table 1. As can be seen from Table 1, the peptide amido is completely insoluble in water, and the solubility is 0; the solubility of the conjugated compounds (I)-(VI) of the application is more than 50 g / L, which is greatly improved compared with the peptide amido.

[0097] Table 1 Solubility test of polypeptide conjugated compounds

[0098] Sample Solubility Whitening polypeptide conjugate (I) 57.2 g / L Whitening polypeptide conjugate (II) 54.3 g / L Whitening polypeptide conjugate (III) 52.1 g / L Whitening polypeptide conjugate (IV) 50.4 g / L Whitening polypeptide conjugate (V) 55.7 g / L Whitening polypeptide conjugate (VI) 54.6 g / L Peptide amyrin 0

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

[0100] Ten healthy adult male mice were depilated with 10% Na2S, and 48 h later, the animals were sacrificed, and their abdominal non-damaged skin pieces were taken and randomly assembled in the skin permeation chamber. 5.4 cm 2 A 200 μL aqueous solution of whitening polypeptide conjugated compounds (I), (III), and (V) was added to the skin surface, wherein the weight of the whitening polypeptide conjugated compounds (I), (III), and (V) was 100 mg, and the coating dose was 18.51 mg / cm 2 ; 10 mL of heavy water was injected into the lower chamber as the receiving liquid, and the receiving liquid was collected every 1, 2, 4, 6, 12, and 24 h, with 4 parallel samples at each time point. High performance liquid chromatography was used for identification, and the penetration rate (P), penetration amount (T), and penetration speed (V) were calculated. The final results are shown in Table 1.

[0101] Penetration amount T (mg / cm 2 ) = total amount of sample in receiving liquid W (mg) / coating area (cm 2 ); penetration rate P (%) = total amount of sample in receiving liquid W (mg) / total amount of coating (mg) * 100%.

[0102] The results show that the whitening polypeptide conjugated compound (I) has better skin permeability than the peptide amido, and with the increase of time, the penetration rate and penetration area also increase, and the whitening polypeptide conjugated compounds (II)-(III) have no effect on skin permeability.

[0103] Table 2. Skin permeability test on mice

[0104]

[0105]

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

[0107] The substrate L-DOPA was prepared into a 1 mg / mL solution with PBS as solvent, and the tyrosinase was prepared into an enzyme solution with activity of 100 U / mL. The experiment was divided into reaction group, reaction control group, blank group and blank control group. 25 μL of 100 ppm peptide anmido, whitening polypeptide conjugate compound sample was added to the reaction group and reaction control group, and the same volume of PBS solution was added to the blank group and blank control group. Then 25 μL of the prepared tyrosinase solution was added to the reaction group and blank group, and the same volume of PBS was added to the reaction control group and blank control group. After mixing, they were incubated in a 37°C incubator for 5 min. 100 μL of the prepared L-DOPA solution was added to all experimental groups, and they were further incubated in a 37°C incubator for 5 min. The absorbance (A) value was measured at a wavelength of 475 nm. The above experiment was repeated three times, and the tyrosinase inhibition rate was calculated according to the following formula:

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

[0109] The results are shown in Table 3. Due to the conjugation of tyrosinase targeting peptide, the whitening polypeptide conjugate compounds (I) - (VI) showed higher inhibition rate than peptide anmido. Among them, conjugate compounds (V-VI) had better whitening effect due to the conjugation of nonapeptide-1.

[0110] Table 3 Inhibition rate of polypeptide conjugate compounds on tyrosinase

[0111]

[0112]

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

[0114] 200 μL of 0.12 mol Tris-Cl buffer (pH 8.0) was mixed with N-succinyl-tri-L-alanine 4-nitrophenylalanine to make the latter reach 1.0 mmol; 30 μL of 100 ppm conjugate compound (Formula II) and blank control sample were mixed with 20 μL of 2.5 U / mL porcine pancreatic elastase solution; after incubation in a 25°C incubator for 20 min, the absorbance at 410 nm was measured by an enzyme marker 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. Elastase inhibition rate of polypeptide conjugate compounds

[0117] Sample Elastase inhibition rate (%) Peptide amyrin 2.4 Whitening polypeptide conjugate (I) 15.7 Whitening polypeptide conjugate (II) 13.1 Whitening polypeptide conjugate (III) 33.6 Whitening polypeptide conjugate (IV) 35.6 Whitening polypeptide conjugate (V) 16.9 Whitening polypeptide conjugate (VI) 17.3

[0118] The results are shown in Table 4. The whitening polypeptide conjugate compounds (III-IV) in the present application have obvious inhibitory effect on elastase compared with the peptide amiloxate, and have good anti-wrinkle and anti-aging effect. This also shows that the whitening polypeptide conjugate compounds (III-IV) have both whitening and anti-wrinkle and anti-aging effects.

[0119] Example 18. Application of whitening polypeptide conjugate compounds in the preparation of serum

[0120] The serum was prepared according to the addition amount and process shown in the following table. The percentage in this embodiment is the mass percentage of each component in the final serum.

[0121] Table 5. Whitening serum formula

[0122]

[0123] The performance analysis results of the above serum are given in Tables 6-7.

[0124] Table 6. Spot-removing and whitening human efficacy test (spot area mm 2 )

[0125]

[0126]

[0127] Table 7. Anti-wrinkle human efficacy test (overall wrinkle area mm 2 )

[0128] Sample (serum) Before use After use for 28 days Whitening polypeptide conjugate (I) 78.44 67.58 Whitening polypeptide conjugate (II) 79.54 67.32 Whitening polypeptide conjugate (III) 81.52 50.23 Whitening polypeptide conjugate (IV) 80.64 51.62 Whitening polypeptide conjugate (V) 76.65 65.21 Whitening polypeptide conjugate (VI) 75.43 66.37 Peptide amyrin 85.56 78.89

[0129] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

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

2. The polypeptide-conjugate 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. 1-3: 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 of the C-terminal amino acid in the polypeptide sequence shown in SEQ ID NO. 1-3 is connected with the L group; The amino acid in the polypeptide sequence shown in SEQ ID NO. 1-3 is a modified or unmodified amino acid.

3. A process for the preparation of a polypeptide conjugate compound according to any one of claims 1-2, characterized in that, The method comprises the following steps: (1) reacting the peptide amantadine with Fmoc-NH-(O-CH2-CH2-) n CH2CH2Cl to obtain Fmoc-NH-(O-CH2-CH2-) n CH2CH2-peptide amantadine, followed by deprotection to obtain the PEGylated peptide amantadine intermediate NH2-(O-CH2-CH2-) n CH2CH2-peptide amantadine; (2) coupling the polypeptide according to any one of SEQ ID NO. 1-3 with the peptide aminoglycoside intermediate obtained in step (1).

4. The production method according to claim 3, characterized by, The polypeptide according to any one of SEQ ID NO. 1-3 is prepared by Fmoc solid-phase synthesis.

5. The preparation method according to claim 3, wherein Step (1) specifically comprises the following steps: Fmoc-NH-(O-CH2-CH2-)peptaminophen is dissolved in a solvent, NaCI and a catalyst are added, and the reaction is carried out under visible light to obtain Fmoc-NH-(O-CH2-CH2-)peptaminophen. n Fmoc-NH-(O-CH2-CH2-)peptaminophen is dissolved in a solvent, NaCI and a catalyst are added, and the reaction is carried out under visible light to obtain Fmoc-NH-(O-CH2-CH2-)peptaminophen. n Fmoc-NH-(O-CH2-CH2-)peptaminophen is dissolved in a solvent, NaCI and a catalyst are added 6. The preparation method according to claim 5, characterized in that, The solvent is selected from at least one of dimethyl sulfoxide and dimethyl formamide.

7. The preparation method according to claim 5, characterized in that, The mass of NaCl is 0.3-5 times the mass of the peptide aminoglycoside.

8. The production method according to claim 5, characterized by, The wavelength of the visible light source is 400-700 nm.

9. The preparation method according to claim 5, characterized in that, The catalyst is Ag / TiO2.

10. The method of claim 5, wherein, The temperature of the reaction is 20-60℃.

11. The method of claim 5, wherein, The reaction time is 2-24 hours.

12. The method of claim 5, wherein, The reaction is carried out in an inert gas atmosphere.

13. The preparation method according to claim 5, characterized in that, The peptide amido with Fmoc-NH-(O-CH2-CH2- n The molar ratio of CH2CH2Cl is 1:1~4.

14. The method of claim 5, wherein, The reaction is terminated by adding a cooling solvent; the cooling solvent is ethanol or dichloromethane.

15. The preparation method according to claim 3, characterized in that, In step (2), the coupling reaction is carried out in a solvent.

16. The method of claim 15, wherein, In step (2), the solvent is selected from at least one of acetonitrile, dimethyl sulfoxide and dimethyl formamide.

17. The preparation method according to claim 3, characterized in that, In step (2), an activating reagent needs to be added.

18. The method of claim 17, wherein, In step (2), the activating reagent is a mixture of 1-hydroxybenzotriazole and N,N'-diisopropyl carbodiimide.

19. The method of claim 18, wherein, In step (2), the molar ratio of the peptide aminoglycoside intermediate to 1-hydroxybenzotriazole, N,N'-diisopropyl carbodiimide is 2-4:2-6:1-2.

20. The method of claim 3, wherein, In step (2), the temperature of the coupling reaction is 25-40℃.

21. The method of claim 3, wherein, In step (2), the coupling reaction time is 2-12 h.

22. The method of claim 3, wherein Step (2) specifically comprises the following steps: dissolving the peptide aminoglycoside intermediate, 1-hydroxybenzotriazole in a solvent; then adding N,N'-diisopropyl carbodiimide and the polypeptide according to any one of SEQ ID NO. 1-3, and reacting at 25-40℃ for 2-12 h; adding acetic acid to terminate the reaction.

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