Dendrimer polypeptide and application thereof

Abstract

The invention discloses a dendritic macromolecular polypeptide, and belongs to the technical field of polypeptides, the dendritic macromolecular polypeptide has a structural formula shown as a formula (a), a formula (b), a formula (c), a formula (d) and a formula (e), and the dendritic macromolecular polypeptide has the beneficial effects that the dendritic macromolecular polypeptide has obvious effects in application of transdermal, anti-inflammatory, whitening and anti-oxidation effects in cosmetics and biological medicine manufacturing; the long-acting anti-wrinkle effect is particularly outstanding.

Description

Technical Field:

[0001] The present invention belongs to the technical field of polypeptides, and relates to a group of dendritic polypeptides, and the application of polypeptides with transdermal, anti-inflammatory, whitening, and antioxidant effects in the manufacture of cosmetics and biopharmaceuticals. Background Art:

[0002] Dendrimers are a class of highly branched macromolecules with specific structures and functions. Dendrimers consist of a central core, an inner layer of branching units, and an outer layer of functional groups. They have a well-defined molecular weight and molecular size, regular structures, and both the molecular volume, shape, and functional groups can be precisely controlled at the molecular level. Dendrimers generally start from the core and continuously branch outwards. When the generation is relatively low, they generally have an open molecular configuration. As the generation increases, the molecule changes from a loose state to a spherical three-dimensional structure with a tight outer and loose inner structure. Common dendrimers include porphyrin-based, aryl ether-based, PAMAM (polyamide-amine), ferrocene-based, etc. Among them, PAMAM is one of the most maturely studied and industrially produced ones.

[0003] Dendrimers have high solubility, a unique relationship between viscosity characteristics and relative molecular mass, and the ability to bind multiple functional groups. Due to their highly branched structure and unique monodispersity, dendrimers have a wide range of applications in the fields of host-guest chemistry, catalysts, metal nanomaterials, nanocomposites, membrane materials, surfactants, medicine, etc. In drug delivery systems, dendrimers show great potential due to their high ability to penetrate cell membranes, non-immunogenicity, and passive targeting ability to tumor tissues.

[0004] In summary, due to their unique structures and multifunctionality, dendrimers show broad application prospects in multiple fields, especially in drug delivery and the biopharmaceutical field. With the in-depth research and technological progress, the applications of dendrimers will be more extensive and in-depth.

[0005] Therapies based on proteins and peptides have high biological activities and specificities, and thus have achieved great success in the medical field. It is reported that seven of the top ten best-selling drugs globally in 2018 were protein drugs. However, these protein drugs are all developed based on extracellular target proteins, and extracellular target proteins only account for a small part of the proteins encoded by the human genome. Therefore, intracellular delivery of proteins or polypeptides will greatly expand the field of protein therapy in the future, and the main obstacle is their membrane impermeability.

[0006] In the past decade or so, many kinds of carriers have been developed to assist in the intracellular delivery of proteins. These carriers show good prospects in the intracellular delivery of proteins, but there are still several problems to be solved. First, it is a challenge to design a robust carrier that can deliver proteins with different isoelectric points. Second, the cargo proteins after endocytosis are trapped in lysosomes, which will lead to a significant reduction in protein activity. Therefore, the most crucial aspects in designing new carriers are strong protein-binding ability and efficient lysosomal escape ability.

[0007] The present invention aims to construct dendritic polypeptides to increase the transdermal permeability of cosmetic polypeptides, thereby prolonging the efficacy of polypeptides. At present, there is no report on new products of dendritic polypeptides.

[0008] Currently, there is a patent reporting the grafting of amino acids on the side chain of amino acids (Patent No. CN106999401B), which uses the side chain of lysine to graft proline or hydroxyproline or pyroglutamic acid. The compound has a certain anti-wrinkle effect, but only one amino acid is grafted, which does not belong to dendritic macromolecules and has no transdermal effect. CN 114903810 B describes a group of compounds with anti-wrinkle effects, but a single product does not have long-term anti-wrinkle effects. Summary of the Invention:

[0009] To solve the above problems, the purpose of the present invention is to provide a group of dendritic polypeptides with long-term anti-wrinkle, transdermal, anti-inflammatory, whitening, and antioxidant effects. Especially after transdermal absorption, the long-term anti-wrinkle effect is particularly prominent. This group of compounds is mainly prepared by condensing amino acids into amide bonds, and their structure is identified and their efficacy is studied. It is found that this group of dendritic polypeptides has very strong skin permeability and has stronger application potential in the fields of beauty skin care, medical aesthetic wrinkle removal, and biopharmaceutical manufacturing.

[0010] The specific technical solution of the present invention to solve the above technical problems is: dendritic polypeptides, the dendritic polypeptides have structural formulas shown in Formula (a), Formula (b), Formula (c), Formula (d), and Formula (e). Formula (a) is Formula (b) is Formula (c) is Formula (d) is Formula (e) is

[0011] Furthermore, the dendritic polypeptides with the structural formulas of Formula (a), Formula (b), Formula (c), Formula (d), and Formula (e) have transdermal, anti-inflammatory, whitening, and antioxidant effects.

[0012] Furthermore, the dendritic polypeptides with the structural formulas of Formula (a), Formula (b), Formula (c), Formula (d), and Formula (e) have long-term anti-wrinkle effects.

[0013] 1) Couple two Boc-lysines to the main chain of carboxyl-protected lysine and the N-terminus of the side chain through amide condensation, and then remove the BOC to obtain the mother nucleus A1, whose structure is as follows:

[0014]

[0015] 2) Couple Boc-lysine to the four N-termini of the A1 mother nucleus through amide condensation and then remove the BOC to obtain a secondary compound; continue to couple Boc-lysine to the eight N-termini of the secondary compound to obtain formula (a).

[0016] 3) Couple Boc-arginine to the four N-termini of the A1 mother nucleus through amide condensation and then remove the BOC to obtain formula (b).

[0017] 4) Couple Boc-lysine to the four N-termini of the A1 mother nucleus through amide condensation and then remove the BOC to obtain a secondary compound; continue to couple Boc-histidine to the eight N-termini of the secondary compound to obtain formula (c).

[0018] 5) Couple the two C-termini of amino-protected glutamic acid to L-glutamic acid di-tert-butyl ester, and then remove the tert-butyl group to obtain a secondary compound; continue to couple L-glutamic acid di-tert-butyl ester to the four C-termini of the secondary compound and then remove the tert-butyl group to obtain the mother nucleus A2, whose structure is as follows:

[0019]

[0020] 6) Couple L-glutamic acid di-tert-butyl ester to the eight C-termini of the A2 mother nucleus through amide condensation and then remove the tert-butyl group to obtain formula (d).

[0021] 7) Couple dipeptide-15 methyl ester to the eight C-termini of the A2 mother nucleus through amide condensation to obtain formula (e).

[0022] 8) The R group of the mother nucleus A1 can be H, C2H5, PhCH2, CH3, tBu.

[0023] 9) The R' group of the mother nucleus A2 can be Cbz, Fmoc, acetyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, hexanoyl, nicotinoyl, biotinoyl, etc.

[0024] The beneficial effects of the present invention are as follows:

[0025] This group of compounds has remarkable effects in the applications of manufacturing cosmetics and biopharmaceuticals with transdermal, anti-inflammatory, whitening, and antioxidant effects;

[0026] The present invention creatively discovers that this group of compounds is a dendrimer, and its application in long-lasting anti-wrinkle effects in cosmetics and biopharmaceuticals is particularly prominent. Description of the Drawings:

[0027] Attached Figure 1 It is the facial fine line diagram processed by VISIA CR in the long - acting anti - wrinkle efficacy test of formula (1) of the present invention;

[0028] Attached Figure 2 It is the facial fine line diagram processed by VISIA CR in the long - acting anti - wrinkle efficacy test of formula (2) of the present invention;

[0029] Attached Figure 3 It is the facial fine line diagram processed by VISIA CR in the long - acting anti - wrinkle efficacy test of formula (3) of the present invention;

[0030] Attached Figure 4 It is the facial fine line diagram processed by VISIA CR in the long - acting anti - wrinkle efficacy test of formula (4) of the present invention;

[0031] Attached Figure 5 It is the facial fine line diagram processed by VISIA CR in the long - acting anti - wrinkle efficacy test of formula (5) of the present invention. Detailed implementation mode:

[0032] In the description of the present invention, specific details are only for fully understanding the embodiments of the present invention. However, those skilled in the art should know that the implementation of the present invention is not limited to these details. In addition, well - known structures and functions are not described or shown in detail to avoid obscuring the key points of the embodiments of the present invention. For those of ordinary skill in the art, the specific meanings of the above - mentioned terms in the present invention can be understood according to specific situations.

[0033] The detailed implementation mode of the present invention:

[0034] Embodiment 1:

[0035] The dendrimer polypeptide with the structural formula of formula (1), the R group of the mother nucleus A1 is methyl (CH3), and the N - terminal of the mother nucleus A1 is coupled with Boc - lysine by amide condensation and then the BOC is removed to obtain the secondary compound; the N - terminal of the secondary compound is prepared by coupling Boc - lysine by amide condensation;

[0036] The structural formula of formula (1) is:

[0037] The specific preparation process is:

[0038] Step 1: Add 10 g of lysine methyl ester dihydrochloride into a 1000 ml three-necked flask, add 32.79 g of N,N'-di-tert-butoxycarbonyl-L-lysine, add 300 ml of dichloromethane, cool the external bath to 0 - 5 °C, add 48.8 g of HBTU, and slowly drip 27.72 g of DIEA. After adding, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 8 h. Wash the liquid material twice with 200 ml of acidic water with pH = 3, then wash it twice with 200 ml of saturated sodium bicarbonate solution, and then wash it twice with 200 ml of saturated brine. Cool the external bath to 0 - 5 °C, add 200 ml of TFA to the organic phase, stir at low temperature for 30 min and then transfer to 25 °C for reaction for 3 h; Concentrate the liquid material under reduced pressure at 40 °C until it basically stops flowing out, drop the liquid material into 150 ml of methyl tert-butyl ether which has been pre-cooled to 0 - 5 °C in advance. A large amount of solid precipitates from the liquid material. Filter by suction, and concentrate the filter cake under reduced pressure at 45 °C to obtain 15 g of off-white solid, with a yield of 83.9%.

[0039] Step 2: Add 15 g of the solid from the previous step into a 2000 ml three-necked flask, add 62.55 g of N,N'-di-tert-butoxycarbonyl-L-lysine, add 400 ml of N,N-dimethylformamide, cool the external bath to 0 - 5 °C, add 68.28 g of HBTU, and slowly drip 37.23 g of DIEA. After adding, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1000 ml of water to the reaction solution, stir and crystallize for 2 h; Filter by suction. Add 200 ml of TFA to the solid, stir at low temperature for 30 min and then transfer to 25 °C for reaction for 1 h; Concentrate the liquid material under reduced pressure at 40 °C until it basically stops flowing out, drop the liquid material into 300 ml of methyl tert-butyl ether which has been pre-cooled to 0 - 5 °C in advance. A large amount of solid precipitates from the liquid material. Filter by suction, and concentrate the filter cake under reduced pressure at 45 °C to obtain 23 g of off-white solid, with a yield of 68.7%.

[0040] Step 3: Add 23 g of the solid from the previous step into a 2000 ml three-necked flask, add 85.33 g of N,N'-di-tert-butoxycarbonyl-L-lysine, add 500 ml of N,N-dimethylformamide, cool the external bath to 0 - 5 °C, add 93.87 g of HBTU, and slowly drip 38.39 g of DIEA. After adding, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1000 ml of acetone to the reaction solution, stir and crystallize for 2 h; Filter by suction. Add 300 ml of TFA to the solid, stir at low temperature for 30 min and then transfer to 25 °C for reaction for 1 h; Concentrate the liquid material under reduced pressure at 40 °C until it basically stops flowing out, drop the liquid material into 400 ml of methyl tert-butyl ether which has been pre-cooled to 0 - 5 °C in advance. A large amount of solid precipitates from the liquid material. Filter by suction, and concentrate the filter cake under reduced pressure at 45 °C to obtain a crude peptide powder; After purification by reverse-phase C18 preparative chromatography and freeze-drying, the compound of formula (1) is obtained.

[0041] C 91 H 184 N30 O 16 , [M+H] + = 1954.45, [M / 2+H] + = 977.72, [M / 3+H] + = 652.15;

[0042] Example 2:

[0043] The dendrimer polypeptide with the structural formula of formula (2), the R group of the mother nucleus A1 is methyl (CH3), and the N-terminus of the mother nucleus A1 is coupled with Boc-arginine by amide condensation and then deprotected to obtain BOC; the structural formula of formula (2) is:

[0044]

[0045] The specific preparation process is as follows:

[0046] Step 1: The same as step 1 of Example 1.

[0047] Step 2: Add 10 g of the above solid to a 2000 ml three-necked flask, add 34.85 g of Boc-arginine, add 400 ml of N,N-dimethylformamide, cool the external bath to 0-5 °C, add 45.52 g of HBTU, and slowly dropwise add 21.72 g of DIEA. After adding, keep the reaction at a constant temperature for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1000 ml of acetone to the reaction solution, stir and crystallize for 2 h; filter by suction. Add 200 ml of TFA to the solid, stir at low temperature for 30 min and then transfer to 25 °C for reaction for 1 h; drop the feed liquid into 500 ml of methyl tert-butyl ether that has been pre-cooled to 0-5 °C, a large amount of solid will precipitate from the feed liquid, filter by suction, and concentrate the filter cake under reduced pressure at 45 °C to obtain a crude peptide powder; after purification by reverse-phase C18 preparative chromatography and freeze-drying, formula (2) is obtained.

[0048] C 43 H 88 O8N 22 , [M+H] + = 1041.72, [M / 2+H] + = 521.36, [M / 3+H] + = 347.90;

[0049] Example 3:

[0050] The dendrimer polypeptide with the structural formula of formula (3), the R group of the mother nucleus A1 is methyl (CH3), and the N-terminus of the A1 mother nucleus is coupled with Boc-lysine by amide condensation and then deprotected to obtain a secondary compound; the eight N-termini of the secondary compound are further coupled with Boc-histidine to obtain, and the structural formula of formula (3) is:

[0051]

[0052] The specific preparation process is as follows:

[0053] Step 1: The same as Step 1 of Example 1.

[0054] Step 2: The same as Step 2 of Example 1.

[0055] Step 3: Add 10 g of the solid obtained in the previous step to a 2000-ml three-necked flask, add 29.2 g of N,N'-di-tert-butoxycarbonyl-L-histidine, add 500 ml of N,N-dimethylformamide, cool the external bath to 0 - 5°C, add 40.81 g of HBTU, and slowly dropwise add 20.86 g of DIEA. After addition, keep the temperature for reaction for 1 h, then raise the temperature to 25°C and react for 12 h. Add 1000 ml of acetone to the reaction solution, stir for crystal cultivation for 2 h; filter by suction. Add 300 ml of TFA to the solid, stir at low temperature for 30 min and then transfer to 25°C for reaction for 1 h; concentrate the material solution under reduced pressure at 40°C until no more material comes out, drop the material solution into 400 ml of methyl tert-butyl ether that has been pre-cooled to 0 - 5°C, a large amount of solid precipitates from the material solution, filter by suction, and concentrate the filter cake under reduced pressure at 45°C to obtain a crude peptide powder; purify it by reverse-phase C18 preparative chromatography and lyophilize to obtain Formula (3).

[0056] C 91 H 144 O 16 N 38 ,[M / 2 + H] + = 1013.58, [M / 3 + H] + = 676.05, [M / 4 + H] + = 507.29;

[0057] Example 4:

[0058] A dendritic polypeptide with the structural formula of Formula (4), where the R' group of the mother nucleus A2 is Cbz, and the C-terminus of the mother nucleus A2 is coupled with di-tert-butyl L-glutamate through an amide condensation method and then the tert-butyl group is removed to prepare it. The structural formula of Formula (4) is:

[0059]

[0060] The specific preparation process is as follows:

[0061] Step 1: Add 10 g of N-carbobenzoxy-L-glutamic acid, 18.44 g of di-tert-butyl L-glutamate, and 300 ml of dichloromethane into a 1000-ml three-necked flask. Cool the external bath to 0 - 5 °C, add 29.66 g of HBTU, and slowly drip 13.79 g of DIEA. After adding, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 8 h. Wash the liquid material twice with 200 ml of acidic water with pH = 3, then wash it twice with 200 ml of saturated sodium bicarbonate solution, and then wash it twice with 200 ml of saturated brine. Cool the external bath to 0 - 5 °C, add 200 ml of TFA to the organic phase, stir at low temperature for 30 min, and then transfer it to 25 °C for reaction for 3 h; Concentrate the liquid material under reduced pressure at 40 °C until it basically stops flowing out, drop the liquid material into 150 ml of methyl tert-butyl ether that has been pre-cooled to 0 - 5 °C in advance. A large amount of solid precipitates from the liquid material. Filter by suction, and concentrate the filter cake under reduced pressure at 45 °C to obtain 16 g of a white solid-like product with a yield of 83.7%.

[0062] Step 2: Add 16 g of the solid obtained in the previous step, 38.6 g of di-tert-butyl L-glutamate, and 400 ml of N,N-dimethylformamide into a 2000-ml three-necked flask. Cool the external bath to 0 - 5 °C, add 56.45 g of HBTU, and slowly drip 26.93 g of DIEA. After adding, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1000 ml of water to the reaction solution and stir for crystal cultivation for 2 h; Filter by suction. Add 200 ml of TFA to the solid, stir at low temperature for 30 min, and then transfer it to 25 °C for reaction for 1 h; Concentrate the liquid material under reduced pressure at 40 °C until it basically stops flowing out, drop the liquid material into 300 ml of methyl tert-butyl ether that has been pre-cooled to 0 - 5 °C in advance. A large amount of solid precipitates from the liquid material. Filter by suction, and concentrate the filter cake under reduced pressure at 45 °C to obtain 22 g of a white solid-like product with a yield of 70.4%.

[0063] Step 3: Add 22 g of the solid obtained in the previous step, 54.34 g of di-tert-butyl L-glutamate, and 600 ml of N,N-dimethylformamide into a 2000-ml three-necked flask. Cool the external bath to 0 - 5 °C, add 79.47 g of HBTU, and slowly drip 32.5 g of DIEA. After adding, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1200 ml of water to the reaction solution and stir for crystal cultivation for 2 h; Filter by suction. Add 200 ml of TFA to the solid, stir at low temperature for 30 min, and then transfer it to 25 °C for reaction for 1 h; Concentrate the liquid material under reduced pressure at 40 °C until it basically stops flowing out, drop the liquid material into 500 ml of methyl tert-butyl ether that has been pre-cooled to 0 - 5 °C in advance. A large amount of solid precipitates from the liquid material. Filter by suction, and concentrate the filter cake under reduced pressure at 45 °C to obtain a crude peptide powder; After purification by reverse-phase C18 preparative chromatography and freeze-drying, the compound of formula (4) is obtained. C 83 H 113 N 15 O 48 ,[M / 3+H] += 697.9, [M / 2 - H] - = 1042.8, [M / 3 - H] - = 694.9;

[0064] Example 5:

[0065] The dendrimer polypeptide with the structural formula of formula (5), the R' group of the mother nucleus A2 is Cbz, and the C-terminal of the mother nucleus A2 is coupled with dipeptide-15 methyl ester by amide condensation to obtain. The structural formula of formula (5) is:

[0066]

[0067] The specific preparation process is as follows:

[0068] Step 1: The same as step 1 of Example 4.

[0069] Step 2: The same as step 2 of Example 4.

[0070] Step 3: Add 20 g of the solid from the previous step to a 2000 ml three-necked flask, add 27.68 g of diglycine methyl ester, add 400 ml of N,N-dimethylformamide, cool the external bath to 0 - 5 °C, add 71.83 g of HBTU, and slowly dropwise add 36.72 g of DIEA. After adding, keep the reaction at a constant temperature for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1200 ml of water to the reaction solution, stir and crystallize for 2 h; filter by suction. The filter cake is dried in vacuo at 45 °C to obtain a crude peptide powder; after purification by reversed-phase C18 preparative chromatography and freeze-drying, the product of formula (5) is obtained.

[0071] C 83 H 121 N 23 O 40 , [M / 2 + H] + = 1040.9, [M / 3 + H] + = 694.27;

[0072] The anti-inflammatory, whitening, antioxidant, and transdermal effects in the above Examples 1 - 5 were detected. Commercially available arginine / lysine polypeptide is one of the components of the composition of Patent CN114903810B, and commercially available oxidized glutathione is the most common compound with amino acid side chain grafted amino acids. Taking these as controls, the detection data are as follows:

[0073] Table 1: Experimental data of the anti-inflammatory effect of the examples of the present invention

[0074]

[0075]

[0076] It can be seen from the data analysis in Table 1 that:

[0077] The dendrimer polypeptide prepared by the present invention can significantly reduce the content of inflammatory factors and has a significant anti-inflammatory effect. Relatively speaking, commercially available polypeptides such as arginine / lysine polypeptide and oxidized glutathione have almost no effect on anti-inflammatory efficacy, indicating that the anti-inflammatory efficacy of this group of compounds is significant.

[0078] Table 2: Experimental data of the examples of the present invention on whitening efficacy

[0079]

[0080] It can be seen from the data analysis in Table 2 that:

[0081] The dendrimer polypeptide prepared by the present invention can significantly reduce the activity of tyrosinase and has a significant whitening effect. Relatively speaking, commercially available polypeptides such as arginine / lysine polypeptide and oxidized glutathione have almost no effect on whitening efficacy, indicating that the whitening efficacy of this group of compounds is significant.

[0082] Table 3: Experimental data of the examples of the present invention on antioxidant efficacy

[0083]

[0084]

[0085] It can be seen from the data analysis in Table 3 that:

[0086] The dendrimer polypeptide prepared by the present invention has an obvious scavenging effect on ROS and has a significant antioxidant effect. Relatively speaking, commercially available polypeptides such as arginine / lysine polypeptide and oxidized glutathione have almost no effect on antioxidant efficacy, indicating that the antioxidant efficacy of this group of compounds is significant.

[0087] Table 4: Experimental data of the examples of the present invention on transdermal efficacy

[0088]

[0089] It can be seen from the data analysis in Table 4 that:

[0090] The dendrimer polypeptide prepared by the present invention has a significant effect on transdermal efficacy. Relatively speaking, commercially available polypeptides such as arginine / lysine polypeptide and oxidized glutathione have general transdermal efficacy, indicating that the transdermal efficacy of this group of compounds is significant.

[0091] In order to more intuitively demonstrate the process advantages of the present invention, a comparison is made by using the replacement method adopted in the present invention.

[0092] Comparative Example 1-1:

[0093] The same mother nucleus A1 as in Example 1 was coupled with H-Ala-Ala-Ala- to obtain Formula (6).

[0094]

[0095] The synthesis method is as follows:

[0096] Step 1: The same as Step 1 in Example 1.

[0097] Step 2: Add 10 g of the solid obtained in the previous step to a 2000 ml three-necked flask, add 22.71 g of BOC-alanyl, add 400 ml of N,N-dimethylformamide, cool the external bath to 0-5 °C, add 45.52 g of HBTU, and slowly add dropwise 24.82 g of DIEA. After adding, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1000 ml of acetone to the reaction solution, stir and crystallize for 2 h; filter by suction. Add 200 ml of TFA to the solid, stir at low temperature for 30 min and then transfer to 25 °C for reaction for 1 h; drop the material solution into 500 ml of methyl tert-butyl ether which has been pre-cooled to 0-5 °C in advance. A large amount of solid precipitates from the material solution. Filter by suction, and dry the filter cake in vacuo at 45 °C for 12 h to obtain 12 g of white powder with a yield of 71.3%.

[0098] Step 3: Add 12 g of the solid obtained in the previous step to a 2000 ml three-necked flask, add 16.2 g of BOC-alanyl, add 400 ml of N,N-dimethylformamide, cool the external bath to 0-5 °C, add 32.47 g of HBTU, and slowly add dropwise 17.70 g of DIEA. After adding, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1000 ml of acetone to the reaction solution, stir and crystallize for 2 h; filter by suction. Add 200 ml of TFA to the solid, stir at low temperature for 30 min and then transfer to 25 °C for reaction for 1 h; drop the material solution into 500 ml of methyl tert-butyl ether which has been pre-cooled to 0-5 °C in advance. A large amount of solid precipitates from the material solution. Filter by suction, and dry the filter cake in vacuo at 45 °C for 12 h to obtain 15 g of white powder with a yield of 88.9%.

[0099] Step 4: Add 15 g of the solid from the previous step to a 2000 ml three-necked flask, add 14.4 g of BOC-alanyl, add 400 ml of N,N-dimethylformamide, cool the external bath to 0 - 5 °C, add 28.87 g of HBTU, and slowly add dropwise 15.74 g of DIEA. After addition, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1000 ml of acetone to the reaction solution, stir and crystallize for 2 h; filter by suction. Add 200 ml of TFA to the solid, stir at low temperature for 30 min and then transfer to 25 °C for reaction for 1 h; drop the material solution into 500 ml of methyl tert-butyl ether that has been pre-cooled to 0 - 5 °C. A large amount of solid precipitates from the material solution, filter by suction, and concentrate the filter cake under reduced pressure at 45 °C to obtain a crude peptide powder; purify it by reverse-phase C18 preparative chromatography and lyophilize to obtain formula (6).

[0100] C 55 H 100 N 18 O 16 ,[M / 2 + H] + = 635.37, [M / 3 + H] + = 423.91;

[0101] Comparative Examples 1 - 2:

[0102] For formula (7), the synthesis method is the same as that in Example 1, except that lysine in the mother nucleus A1 in Example 1 is replaced with 2,5-diaminopentanoic acid, that is, the mother nucleus A3, and the structure is as follows:

[0103] Mother nucleus A3:

[0104]

[0105] C 88 H 178 N 30 O 16 ,[M / 2 + H] + = 956.7, [M / 3 + H] + = 638.1;

[0106] Comparative Example 2 - 1:

[0107] Same as the mother nucleus A1 in Example 2, couple with H-Tyr- to obtain formula (8):

[0108]

[0109] The synthesis method is the same as that in Example 2, except that BOC-arginyl is replaced with BOC-tyrosine.

[0110] C 55 H 76 N 10 O12 , [M / 2 + H] + = 535.28, [M / 3 + H] + = 357.18;

[0111] Comparative Example 2 - 2:

[0112] Equation (9), the synthesis method is the same as that of Example 2, the difference is that lysine in the parent nucleus A1 of Example 2 is replaced with 2,5 - diamino valeric acid, that is, the parent nucleus A3, and the structure is as follows:

[0113]

[0114] C 40 H 82 N 22 O8, [M + H] + = 999.67, [M / 2 + H] + = 500.34;

[0115] Comparative Example 3 - 1:

[0116] Same as the parent nucleus A1 of Example 3, couple H - Ala - Ala - Phe -, to obtain Equation (10):

[0117]

[0118] Step 1: The same as Step 1 of Example 1.

[0119] Step 2: Add 10 g of the above - mentioned solid to a 2000 - ml three - necked flask, add 22.71 g of BOC - alanyl, add 400 ml of N,N - dimethylformamide, cool the external bath to 0 - 5 °C, add 45.52 g of HBTU, and slowly dropwise add 24.82 g of DIEA. After adding, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1000 ml of acetone to the reaction solution, stir and crystallize for 2 h; filter by suction. Add 200 ml of TFA to the solid, stir at low temperature for 30 min and then transfer to 25 °C for reaction for 1 h; drop the material liquid into 500 ml of methyl tert - butyl ether which has been pre - cooled to 0 - 5 °C in advance. A large amount of solid precipitates from the material liquid, filter by suction, and dry the filter cake in vacuum at 45 °C for 12 h to obtain 13 g of white powder, with a yield of 77.2%.

[0120] Step 3: Add 13 g of the solid from the previous step into a 2000 ml three-necked flask, add 17.55 g of BOC-alanyl, add 400 ml of N,N-dimethylformamide, cool the external bath to 0 - 5 °C, add 35.17 g of HBTU, and slowly dropwise add 19.18 g of DIEA. After adding, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1000 ml of acetone to the reaction solution, stir for crystal growth for 2 h; filter by suction. Add 200 ml of TFA to the solid, stir at low temperature for 30 min and then transfer to 25 °C for reaction for 1 h; drop the material solution into 500 ml of methyl tert-butyl ether that has been pre-cooled to 0 - 5 °C in advance. A large amount of solid precipitates from the material solution. Filter by suction, and dry the filter cake under vacuum at 45 °C for 12 h to obtain 16 g of white powder with a yield of 87.5%.

[0121] Step 4: Add 16 g of the solid from the previous step into a 2000 ml three-necked flask, add 21.54 g of BOC-phenylalanyl, add 400 ml of N,N-dimethylformamide, cool the external bath to 0 - 5 °C, add 36.95 g of HBTU, and slowly dropwise add 16.79 g of DIEA. After adding, keep the temperature for reaction for 1 h, then raise the temperature to 25 °C and react for 12 h. Add 1000 ml of acetone to the reaction solution, stir for crystal growth for 2 h; filter by suction. Add 200 ml of TFA to the solid, stir at low temperature for 30 min and then transfer to 25 °C for reaction for 1 h; drop the material solution into 500 ml of methyl tert-butyl ether that has been pre-cooled to 0 - 5 °C in advance. A large amount of solid precipitates from the material solution. Filter by suction, and concentrate the filter cake under reduced pressure at 45 °C to obtain a crude peptide powder; after purification by reverse-phase C18 preparative chromatography and lyophilization, the compound of formula (10) is obtained.

[0122] C 79 H 116 N 18 O 16 ,[M / 2 + H] + = 787.44, [M / 3 + H] + = 525.29;

[0123] Comparative Example 3 - 2:

[0124] For formula (11), the synthesis method is the same as that in Example 3, except that lysine in the mother nucleus A1 of Example 3 is replaced with 2,5-diaminopentanoic acid, that is, the mother nucleus A3, and the structure is as follows:

[0125] C 88 H 178 N 30 O 16 ,[M / 2 + H] + = 956.7, [M / 3 + H] + = 638.1;

[0126] Comparative Example 4 - 1:

[0127] Same as the mother nucleus A2 of Example 4, coupled with tyrosine to obtain Formula (12):

[0128] The synthesis method is the same as that of Example 4, except that di-tert-butyl L-glutamate is replaced with tert-butyl L-tyrosine. C 55 H 76 N 10 O 12 , [M / 3 + H] + = 787.95, [M / 3 - H] - = 785.95;

[0129] Comparative Example 4-2:

[0130] For Formula (13), the synthesis method is the same as that of Example 4, except that glutamate in the mother nucleus A2 of Example 4 is replaced with 2-aminoadipic acid, that is, the mother nucleus A4, and the structure is as follows:

[0131] The structure of Formula (13) is as follows:

[0132] C 90 H 127 N 15 O 48 , [M / 3 + H] + = 729.6, [M / 3 - H] - = 727.6, [M / 4 + H] + = 547.45;

[0133] Comparative Example 5-1:

[0134] Same as the mother nucleus A2 of Example 5, coupled with leucyl leucine methyl ester to obtain Formula (14);

[0135]

[0136] The synthesis method is the same as that of Example 5, except that glycine methyl ester is replaced with leucyl leucine methyl ester. C 148 H 251 N 23 O 40 , [M / 3 + H] + = 998.27, [M / 4 + H] + = 748.95;

[0137] Comparative Example 5-2:

[0138] For Formula (15), the synthesis method is the same as that of Example 5, except that glutamate in the mother nucleus A2 of Example 5 is replaced with 2-aminoadipic acid, that is, the mother nucleus A4, and the structure is as follows:

[0139] C 90 H 135 N 23 O 40 , [M / 3 + H] + = 726.97, [M / 4 + H] + = 545.48;

[0140] Long - term anti - wrinkle efficacy test of polypeptide on human body

[0141] Purpose and principle of the test: In this test, a group of subjects meeting the inclusion and exclusion criteria continuously used the sample for 7 days under the guidance of the testers, then stopped using it, and completed the test content according to the protocol requirements. By comparing the changes in skin test indexes such as the wrinkle index of the tested area before and after the subjects used the sample, the long - term anti - wrinkle efficacy of the tested sample was evaluated.

[0142] Method design principle: Double - blind, self - control before and after;

[0143] Test cycle: A total of 14 days. Visit the laboratory before using the sample (D0), at the last time of using the sample (D1), on the 2nd day after stopping using the sample (D2), on the 3rd day after stopping using the sample (D3), and on the 7th day after stopping using the sample (D7).

[0144] Test environment requirements: Constant temperature and humidity. The indoor temperature is controlled at (21.0 ± 1.0) °C, and the relative humidity is controlled at (50 ± 10) %RH.

[0145] Test process: At the first visit, the volunteers received the test instructions from the testers and the confirmation of eligibility for inclusion in the group. After agreeing to participate in the study, they signed the informed consent form. The subjects used a cleansing product to clean the tested area, dried it, and sat quietly in the constant temperature and humidity room for 30 minutes before conducting the skin test items and the inclusion screening. The test results were used as the baseline value (D0) of the test at the same time. The subjects meeting the inclusion criteria used the sample once under the guidance of the testers; the subjects received the sample and the guidance on using the sample from the testers, and used the sample by themselves according to the sample usage instructions for 6 days.

[0146] The subjects should visit the laboratory at the last time of using the sample (D1), on the 2nd day after stopping using the sample (D2), on the 3rd day after stopping using the sample (D3), and on the 7th day after stopping using the sample (D7). Use a cleansing product to clean the tested area, dry it, and sit quietly under constant temperature and humidity for 30 minutes before conducting the skin test items.

[0147] Skin wrinkle measurement: The eyes were photographed using the multi-functional 3D skin measuring instrument Miravex Antera 3D@, and the ROI area was selected to analyze the fine lines under the eyes. The color images captured by the measuring instrument were transformed into pseudo-color images to make the distribution of the wrinkle directions clearly visible. At the same time, the system could analyze the measurement parameters of fine lines and wrinkles in the ROI selection area. Among them, "length" was the total length of all fine lines / wrinkles in the ROI selection area, unit: mm. The same test area was tracked multiple times to show the most intuitive and subtle changes in the fine lines / wrinkles in the test area.

[0148] Skin surface texture test: The area under the eyes was photographed using the skin surface texture analysis test system Courage﹢Khazaka Visioscan VC20 Plus. It is a dermoscope composed of a uniform annular ultraviolet light (UVA) illumination source and a black-and-white high-resolution CCD camera, which can take images of the skin surface and analyze and process them within the system. In addition, the skin surface gray level distribution was statistically analyzed through the active skin surface analysis software SELS to obtain data on the skin surface texture and roughness in the form of gray level differences. Among them, SEw (Wrinkles) was the wrinkle index, and the larger the value, the more serious the wrinkle condition.

[0149] Facial image capture: The entire face was photographed using the facial image capture and analysis system VISIA CR.

[0150] SPSS Statistic 27.0 was used for data analysis.

[0151] The data were expressed as "mean" and subjected to a normal distribution test. For data that conformed to the normal distribution, a paired t-test was used for comparison before and after itself. For data that did not conform to the normal distribution and ranked data, a non-parametric test was used. A two-tailed test was adopted, and the test level α = 0.05. *P < 0.05 or **P < 0.01 indicated that the difference before and after was statistically significant, and P ≥ 0.05 indicated that the difference before and after was not statistically significant.

[0152] Index change rate or improvement rate (%) = (XT after use - XT before use) / XT before use * 100%

[0153] Test results: The test data are shown in Table 5, Table 6 and the attached drawings;

[0154] Table 5 Statistical results of test data on the length of fine lines under the eyes

[0155]

[0156]

[0157] Table 6 Statistical results of test data on the wrinkle index SEw under the eyes

[0158]

[0159] It can be seen from Table 5 and Table 6 that

[0160] (1) It can be known from the comparison between Examples 1-5 and the comparative examples that

[0161] Based on human tests, by detecting the sub-orbital wrinkle index SEw and the length of sub-orbital fine lines, the long-term anti-wrinkle efficacy of the sample to be tested is evaluated. The change rate of the sub-orbital wrinkle index SEw is greater than 40%, and the change rate of the length of sub-orbital fine lines is greater than 10%. It can be seen that the anti-wrinkle effect of this group of compounds is significant; moreover, after stopping using the sample, from the D3 data analysis, the anti-wrinkle effect of this group of polypeptides decreases slowly, and there is an obvious decrease only from the D7 data. The anti-wrinkle efficacy of Examples 1-5 is retained for a long time;

[0162] For Comparative Example 1 of each example, it is far lower than the change rate of Examples 1-5 and has almost no efficacy in anti-wrinkle; thus, it can be seen that although similar polypeptides can also be prepared after replacing the last conjugated amino acid, the long-term anti-wrinkle efficacy is far lower than that of this group of polypeptides; thus, it can be seen that not all dendrimer polypeptides can have this efficacy; for the comparative examples of each example, although they also have transdermal efficacy, they have almost no effect on anti-wrinkle efficacy. It can be seen that there is no positive correlation between the transdermal performance of polypeptides and long-term anti-wrinkle.

[0163] For Comparative Example 2 of each example, it is far lower than the change rate of Examples 1-5 and has almost no efficacy in anti-wrinkle; thus, it can be seen that although similar dendrimer polypeptides can also be prepared after replacing the parent nucleus, the long-term anti-wrinkle efficacy is far lower than that of this group of polypeptides;

[0164] (2) It can be known from the comparison with commercially available products that

[0165] After stopping using the product with anti-wrinkle efficacy, there is still an anti-wrinkle effect in the short term; as time goes by, the effect almost disappears and skin wrinkles begin to recover; the change rate of the sub-orbital wrinkle index SEw drops to about 20%, and the change rate of the length of sub-orbital fine lines drops to about 5%. It can be seen that the long-term anti-wrinkle effect of this group of polypeptides is significant. Through further research, by increasing the addition amount and frequency of the product with anti-wrinkle efficacy, long-term anti-wrinkle efficacy cannot be obtained from short-term anti-wrinkle effect. Thus, it can be known that the principles of short-term anti-wrinkle effect and long-term anti-wrinkle efficacy are not the same.

[0166] Further investigation was carried out on the replacement of the R group of different parent nuclei A1 with H, C2H5, PhCH2, and tBu, and the transdermal, anti-inflammatory, whitening, antioxidant, and long-acting anti-wrinkle effects of the compounds were detected. After the R' group of different parent nuclei A2 was replaced with acetyl, octanoyl, myristoyl, palmitoyl, hexanoyl, nicotinoyl, and biotinoyl, the transdermal, anti-inflammatory, whitening, antioxidant, and long-acting anti-wrinkle effects of the compounds were detected. The data are shown in Table 7 and Table 8.

[0167] Table 7 Data of the Transdermal, Anti-inflammatory, Whitening, and Antioxidant Effects of Polypeptides

[0168]

[0169]

[0170]

[0171] Table 8 Data of the Long-acting Anti-wrinkle Effect of Polypeptides

[0172]

[0173]

[0174] As can be seen from Tables 7-8:

[0175] After the R group of parent nucleus A1 was replaced with H, C2H5, PhCH2, and tBu, the transdermal, anti-inflammatory, whitening, antioxidant, and long-acting anti-wrinkle effects of the compounds were the same, proving that the effects of the compounds would not change due to different R groups. Further investigation was carried out on the replacement of the R' group of parent nucleus A2 with acetyl, octanoyl, myristoyl, palmitoyl, hexanoyl, nicotinoyl, and biotinoyl, and the transdermal, anti-inflammatory, whitening, antioxidant, and long-acting anti-wrinkle effects of the compounds were the same, proving that the effects of the compounds would not change due to different R' groups.

[0176] To sum up:

[0177] (1) This group of dendritic macromolecule polypeptides has relatively excellent transdermal, anti-inflammatory, whitening, and antioxidant effects;

[0178] (2) This group of compounds is dendritic macromolecule polypeptides, which have significant long-acting anti-wrinkle effects and can reduce the frequency of use.

Claims

1. Dendritic polypeptide, characterized in that The dendritic macromolecule polypeptide has a structural formula shown in formula (a), formula (b), formula (c), formula (d), or formula (e). Formula (a) is Formula (b) is Formula (c) is Formula (d) is; Formula (e) is 2. The dendrimer polypeptide according to claim 1, characterized in that The dendritic macromolecule polypeptide with the structural formula shown in formula (a), formula (b), formula (c), formula (d), or formula (e) has the effects of transdermal, anti-inflammatory, whitening, and antioxidant.

3. The dendrimer polypeptide according to claim 1, characterized in that The dendritic macromolecule polypeptide with the structural formula shown in formula (a), formula (b), formula (c), formula (d), or formula (e) has the effect of long-term anti-wrinkle.

4. The dendrimer polypeptide according to any one of claims 1-3, characterized in that : The dendritic macromolecule polypeptide with the structural formula shown in formula (a) is prepared by coupling Boc-lysine to the N-terminus of the parent nucleus A1 through amide condensation and then removing BOC; the N-terminus of the secondary compound is prepared by coupling Boc-lysine through amide condensation. The dendritic macromolecule polypeptide with the structural formula shown in formula (b) is prepared by coupling Boc-arginine to the N-terminus of the parent nucleus A1 through amide condensation and then removing BOC. The dendritic macromolecule polypeptide with the structural formula shown in formula (c) is prepared by coupling Boc-lysine to the N-terminus of the A1 parent nucleus through amide condensation and then removing BOC to obtain a secondary compound; the eight N-termini of the secondary compound are further coupled with Boc-histidine.

5. The dendrimer polypeptide according to claim 4, wherein The structural formula of the mother nucleus A1 is 6. The dendritic polypeptide according to claim 5, wherein The parent nucleus A1 is prepared by coupling Boc-lysine to the main chain of carboxyl-protected lysine and the N-terminus of the side chain through amide condensation and then removing BOC. The carboxyl protecting group R of the parent nucleus A1 is H, C2H5, PhCH2, CH3, or tBu.

7. The dendrimer polypeptide according to any one of claims 1-3, characterized in that : The dendritic macromolecule polypeptide with the structural formula shown in formula (d) is prepared by coupling di-tert-butyl L-glutamate to the C-terminus of the parent nucleus A2 through amide condensation and then removing the tert-butyl group. The dendritic macromolecule polypeptide with the structural formula shown in formula (e) is prepared by coupling dipeptide-15 methyl ester to the C-terminus of the parent nucleus A2 through amide condensation.

8. The dendrimer polypeptide according to claim 7, wherein The structural formula of the mother nucleus A2 is 9. The dendrimer polypeptide according to claim 8, wherein The parent nucleus A2 is prepared by coupling di-tert-butyl L-glutamate to the two C-termini of amino-protected glutamate and then removing the tert-butyl group to obtain a secondary compound; the four C-termini of the secondary compound are further coupled with di-tert-butyl L-glutamate and then removing the tert-butyl group. The amino protecting group R' of the parent nucleus A2 is Cbz, acetyl, octanoyl, myristoyl, palmitoyl, hexanoyl, nicotinoyl, or biotinoyl.

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