Glycosylated polypeptide-riboflavin composite material capable of repairing human tissue aged skin, synthetic method and application

Abstract

The invention belongs to the technical field of pharmaceutical chemistry synthesis, and discloses a synthesis method of a deglycosylated polypeptide-riboflavin composite material capable of repairing aged skin of human tissue, which comprises the following steps: dissolving riboflavin in N, N-dimethylformamide, adding a maleimide compound and a catalyst, culturing in a constant-temperature dark environment, filtering, washing, and drying to obtain the polypeptide-riboflavin composite material capable of repairing aged skin of human tissue. Adding a catalyst to obtain a riboflavin-maleimide intermediate; and putting the intermediate into N, N-dimethylformamide, adding polypeptide and triethylamine, culturing in a constant-temperature dark environment, dialyzing, and freeze-drying to obtain the polypeptide-riboflavin composite material. The polypeptide and riboflavin are combined to obtain a novel composite material, riboflavin small molecules are introduced while the polypeptide has a targeting function, and are converted into FMN < + > through FMN in a human body, so that the polypeptide has an obvious deglycosylation effect, has a good deglycosylation effect, and has a great application prospect in health care products, cosmetics and medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemical synthesis, and in particular to a deglycosylated polypeptide-riboflavin composite material capable of repairing aged skin of human tissue, a synthesis method and application thereof. Background Art

[0002] Advanced glycation end products (AGEs) are a general term for a series of complex compounds generated by non-enzymatic reactions of reducing sugars, proteins, amino acids, etc. These products can be naturally generated in the human body or ingested through food. Numerous studies have confirmed that the increase and accumulation of AGEs in the human body are related to many chronic diseases of the elderly, such as atherosclerosis and diabetes. At the same time, the accumulation of AGEs is also considered to be one of the important factors of skin aging. Therefore, establishing a safe and efficient method for removing glycation is crucial to maintaining human health interests.

[0003] At present, the removal of AGEs is mainly divided into physical-assisted (pressure cycle technology, microwave-assisted technology), chemical (hydrazinolysis, periodate oxidation-β elimination, trifluoroacetic acid or trifluoromethanesulfonic acid) and biological (enzyme degradation) methods. Neither physical nor chemical methods are suitable for the removal of glycosylation in the human body, and there are various problems such as secondary damage, low efficiency and high cost, which greatly limits their practical application for human deglycosylation. In contrast, biological methods have the advantages of strong specificity, mild action and no harm to the human body, and have gradually become a research hotspot. Current studies have found that fructosamine oxidase is an oxidoreductase that can catalyze the cleavage of glycosylation products, and its cofactor is FMN (flavin mononucleotide). The product is hydrolyzed to produce glucose ketoaldehyde, amino acids and hydrogen peroxide. However, the purification and separation methods of natural fructosamine oxidase are complex, the production cost is high, and the reaction conditions are strict, which seriously limits its research and further application. Therefore, it is of great significance to develop a mimetic enzyme with natural fructosamine oxidase activity.

[0004] This material is an artificial enzyme synthesized based on the structural characteristics of natural enzymes and the working mechanism of natural enzymes, which provides more possibilities for biocatalysis. So far, many types of simulated enzymes have been constructed, such as simulated enzymes of dendrimer polymers, supramolecular simulated enzymes, nanomaterial simulated enzymes and peptide simulated enzymes. Peptides are ideal materials for constructing simulated enzymes because of their low toxicity, biocompatibility, structural adjustability and biodegradability. Riboflavin is a type of B vitamin. Its function is mainly related to the active conjugated double bonds existing at the 1,5 positions of N on the isoalloxazine in the molecule. It can be used as both a hydrogen donor and a hydrogen transmitter. In the human body, it participates in redox reactions in the form of flavin mononucleotide (FMN), plays the role of hydrogen delivery, and is a cofactor for some important redox enzymes in the body.

[0005] As we age, proteins in the skin, especially collagen, are prone to glycation reactions, forming advanced glycation end products (AGEs). The accumulation of these AGEs can cause aging phenomena such as loss of skin elasticity, wrinkles and sagging. Decomposing the already formed AGEs through the action of enzymes is a potential treatment method for repairing aging skin in human tissue. Peptides are a safe and effective way to repair aging skin in human tissue. These molecules can penetrate deep into the skin layer, exert various biological activities, and promote skin health and rejuvenation. They can be used through topical application (such as essences, creams) or oral supplementation (such as drinks). Therefore, the invention of a deglycosylated composite material using peptides and riboflavin has broad application prospects. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a deglycosylated polypeptide-riboflavin composite material capable of repairing aged skin of human tissue, a synthesis method and application.

[0007] The technical solution adopted by the present invention to solve its technical problem is:

[0008] A method for synthesizing a deglycosylated polypeptide-riboflavin composite material capable of repairing aged skin of human tissue comprises the following steps:

[0009] (1) dissolving riboflavin in N,N-dimethylformamide (DMF), adding a maleimide compound and a catalyst, and culturing the mixture under a constant temperature and dark environment to obtain a riboflavin-maleimide intermediate;

[0010] (2) placing the intermediate in N,N-dimethylformamide (DMF), adding the polypeptide and triethylamine, culturing in a constant temperature dark environment, dialyzing, and freeze-drying to obtain a polypeptide-riboflavin composite material, which is a deglycosylated polypeptide-riboflavin composite material that can repair aged skin of human tissue.

[0011] Furthermore, the polypeptide in step (2) is a mixture of one or more polypeptides of the following amino acid sequences:

[0012] (1) the amino acid sequence as shown in SEDID NO.5, SEDID NO.5: CVPGAKRPGHE;

[0013] (2) SEDIDNO.5 introduces D-amino acids or unnatural amino acids;

[0014] (3) The amino acid sequence shown in SEDIDNO.5 is modified by acetylation, amidation, or phosphorylation;

[0015] (4) an amino acid sequence formed by adding one or more amino acids before or after the amino acid sequence shown in SEDIDNO.5;

[0016] (5) an amino acid sequence in which the functional amino acid in the amino acid sequence shown in SEDID NO.5 is replaced by an amino acid residue of the same type;

[0017] (6) a degenerate sequence, an extended sequence, or a position-interchange sequence of the sequence shown in SEDIDNO.5;

[0018] (7) The polypeptides also include: SEDIDNO.1 to SEDIDNO.20; molecular docking technology was used to predict the binding energy between the polypeptides and the glycosylated substrates. After 100 cycles of docking, these 20 polypeptides all had certain binding energies with the substrate fructosamine, proving that these polypeptides all have certain potential for removing glycosylation. See Table 1 for details.

[0019] Table 1

[0020] SEDIDNO Peptide sequence Binding energy (kcal / mol) SEDIDNO: 1 QCG -2.75 SEDIDNO: 2 CVPGAHPG -3.29 SEDIDNO: 3 CVPGAHAPGV -3.43 SEDIDNO: 4 CVPGAHEPGKR -3.02 SEDIDNO: 5 CVPGAKRPGHE -4.04 SEDIDNO: 6 CVPGAEKEHAPGV -2.96 SEDIDNO:7 CVPGAEREHAPGV -2.45 SEDIDNO:8 CHHHVPGAHHH -2.8 SEDIDNO:9 CHHVPGAHHHVPGA -3.0 SEDIDNO:10 CVPGA -2.85 SEDIDNO:11 CHVPGAHH -2.97 SEDIDNO:12 CVPGAHHH -2.6 SEDIDNO:13 CAVPGAVH -2.75 SEDIDNO:14 CAVPGAVHHH -3.12 SEDIDNO:15 CGVPGAH -2.87 SEDIDNO:16 CGVPGAHHH -3.11 SEDIDNO:17 CVPGAHVPGA -2.97 SEDIDNO:18 CVPGAHHHVPGA -3.13 SEDIDNO:19 CAVPGAVHHHVPGA -3.26 SEDIDNO:20 CAVPGAVHVPGA -2.52

[0021] Alternatively, the maleimide compound in step (1) is trans-4-(maleimidomethyl)cyclohexanecarboxylic acid or other maleimide compounds;

[0022] Alternatively, the catalyst in step (1) is a compound that can catalyze the esterification reaction of hydroxyl-carboxyl groups, which is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI and 4-(dimethylamino)pyridine DMAP;

[0023] Furthermore, the amino acid sequence of the SEDIDNO.5 polypeptide comprises a hairpin structure functional region, a cysteine ​​connection functional region, and a catalytic activity functional region;

[0024] Alternatively, the molar mass ratio of riboflavin: maleimide compound: EDCI: DMAP is 0-3: 1: 0-3: 0.1. Preferably, the molar mass ratio of riboflavin: maleimide compound: EDCI: DMAP is 1.5: 1: 1.5: 0.1.

[0025] Furthermore, the backbone amino acids in the hairpin functional region are D-proline and / or glycine and / or valine and / or alanine, and the amino acid repeating unit in the hairpin structure functional region is greater than or equal to 1; the backbone amino acids in the cysteine ​​linking functional region are cysteine, and the amino acid repeating unit in the linking functional region is greater than or equal to 1; the backbone amino acids in the catalytic activity functional region are histidine and / or glutamic acid and / or lysine and / or arginine, and the amino acid repeating unit in the catalytic activity functional region is greater than or equal to 1.

[0026] Furthermore, the dark culture time in step (1) and step (2) is more than 24 hours;

[0027] Alternatively, the constant temperature environment in step (1) and step (2) is 20-50° C., and the culture time is 12-48 hours;

[0028] Alternatively, in step (1), the molar ratio of riboflavin: maleimide compound: catalyst is 1.5:1:0.1, and the mass ratio of riboflavin to N,N-dimethylformamide in step (1) is 1:5 in mg:ml; in step (2), the mass ratio of riboflavin-maleimide intermediate: polypeptide is 10:0-5, and the amount of triethylamine added in step (2) is one tenth of the mass of the polypeptide; preferably, the mass ratio of intermediate: polypeptide is 10:2.5, and the addition ratio of riboflavin-maleimide intermediate to N,N-dimethylformamide in step (2) is 1:1 in mg:ml.

[0029] The deglycosylated polypeptide-riboflavin composite material prepared by the method described above can repair aged skin of human tissue.

[0030] Furthermore, the molecular structural formula of the polypeptide-riboflavin composite material is one or more of the following:

[0031]

[0032]

[0033] Use of the polypeptide-riboflavin composite material as described above in the preparation of fructosamine and / or fructosyl lysine and / or glycopeptide and / or glycated bovine serum albumin and / or glycated hemoglobin and / or glycated collagen and / or glycated cells and / or glycated skin.

[0034] Use of the polypeptide-riboflavin composite material as described above in the removal of sugar from glycosylated substances in vitro and / or in vivo.

[0035] A method for removing glycosylation using the polypeptide-riboflavin composite material as described above, the method comprising the following steps:

[0036] The reaction temperature for deglycosylation is 30-60° C., and more preferably, the reaction temperature is 40° C., the deglycosylation is carried out in an environment of pH 8, and the reaction time for deglycosylation is 24 hours.

[0037] The advantages and positive effects achieved by the present invention are:

[0038] 1. The present invention combines polypeptide with riboflavin to obtain a new composite material. While the polypeptide itself has a targeting function, riboflavin small molecules are introduced and converted into FMN+ through FMN in the human body, so that it has an obvious deglycosylation effect and has a good deglycosylation effect. It has great application prospects in health products, cosmetics, and medicines.

[0039] 2. The present invention combines polypeptide with riboflavin, and the functional polypeptide carries riboflavin to the glycosylation site to exert its effect. This changes the disadvantage that small molecule riboflavin cannot reach glycosylation, and greatly improves the functional characteristics of polypeptide deglycosylation.

[0040] 3. The polypeptide-riboflavin composite material of the present invention is non-toxic and has little harm to the human body.

[0041] 4. The present invention prepares a mimetic enzyme by changing the amino acid sequence of a polypeptide, thereby achieving an efficient and stable deglycosylation effect, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the MALDI-TOF mass spectrum of the deglycosylated polypeptide (SED ID NO.5)-riboflavin composite material in the present invention;

[0043] Figure 2 This is a transmission electron microscopy image of the deglycosylated polypeptide (SED ID NO.5)-riboflavin composite material of the present invention;

[0044] Figure 3 This is a graph showing the results of product content analysis of the deglycosylated polypeptide (SED ID NO.1-6)-riboflavin composite material in the present invention in removing glycosylated substances from glycosylated bovine serum albumin;

[0045] Figure 4 This is a result diagram of the molecular weight of substrate glycoprotein after the deglycosylated polypeptide (SED ID NO.1-6)-riboflavin composite material of the present invention removes glycosylated substances from glycosylated bovine serum albumin;

[0046] Figure 5 This is a toxicity analysis diagram of the deglycosylated polypeptide (SED ID NO.5)-riboflavin composite material of the present invention. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0048] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0049] A method for synthesizing a deglycosylated polypeptide-riboflavin composite material capable of repairing aged skin of human tissue comprises the following steps:

[0050] (1) dissolving riboflavin in N,N-dimethylformamide (DMF), adding a maleimide compound and a catalyst, and culturing the mixture under a constant temperature and dark environment to obtain a riboflavin-maleimide intermediate;

[0051] (2) placing the intermediate in N,N-dimethylformamide (DMF), adding the polypeptide and triethylamine, culturing in a constant temperature dark environment, dialyzing, and freeze-drying to obtain a polypeptide-riboflavin composite material, which is a deglycosylated polypeptide-riboflavin composite material that can repair aged skin of human tissue.

[0052] Preferably, the polypeptide in step (2) is a mixture of one or more polypeptides of the following amino acid sequences:

[0053] (1) The amino acid sequence shown in SED ID NO.5, SED ID NO.5: CVPGAKRPGHE;

[0054] (2) SED ID NO.5 introduces D-amino acids or unnatural amino acids;

[0055] (3) The amino acid sequence shown in SED ID NO.5 is modified by acetylation, amidation, or phosphorylation;

[0056] (4) an amino acid sequence formed by adding one or more amino acids before or after the amino acid sequence shown in SED ID NO.5;

[0057] (5) an amino acid sequence in which the functional amino acid in the amino acid sequence shown in SED ID NO.5 is replaced by an amino acid residue of the same type;

[0058] (6) a degenerate sequence, an extended sequence, or a position-interchange sequence of the sequence shown in SED ID NO.5;

[0059] (7) The polypeptides also include: SED ID NO.1 to SED ID NO.20; molecular docking technology was used to predict the binding energy between the polypeptides and the glycosylated substrates. After 100 cycles of docking, these 20 polypeptides all had certain binding energies with the substrate fructosamine, proving that these polypeptides all have certain potential for removing glycosylation. See Table 1 for details.

[0060] Table 1

[0061]

[0062]

[0063] Alternatively, the maleimide compound in step (1) is trans-4-(maleimidomethyl)cyclohexanecarboxylic acid or other maleimide compounds;

[0064] Alternatively, the catalyst in step (1) is a compound that can catalyze the esterification reaction of hydroxyl-carboxyl groups, which is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI and 4-(dimethylamino)pyridine DMAP;

[0065] Preferably, the amino acid sequence of the SEDIDNO.5 polypeptide comprises a hairpin structure functional region, a cysteine ​​connection functional region, and a catalytic activity functional region;

[0066] Alternatively, the molar mass ratio of riboflavin: maleimide compound: EDCI: DMAP is 0-3: 1: 0-3: 0.1. Preferably, the molar mass ratio of riboflavin: maleimide compound: EDCI: DMAP is 1.5: 1: 1.5: 0.1.

[0067] Preferably, the backbone amino acids in the hairpin functional region are D-proline and / or glycine and / or valine and / or alanine, and the amino acid repeating unit in the hairpin functional region is greater than or equal to 1; the backbone amino acids in the cysteine ​​linking functional region are cysteine, and the amino acid repeating unit in the linking functional region is greater than or equal to 1; the backbone amino acids in the catalytically active functional region are histidine and / or glutamic acid and / or lysine and / or arginine, and the amino acid repeating unit in the catalytically active functional region is greater than or equal to 1.

[0068] Preferably, the dark culture time in step (1) and step (2) is more than 24 hours;

[0069] Alternatively, the constant temperature environment in step (1) and step (2) is 20-50° C., and the culture time is 12-48 hours;

[0070] Alternatively, in step (1), the molar ratio of riboflavin: maleimide compound: catalyst is 1.5:1:0.1, and the mass ratio of riboflavin to N,N-dimethylformamide in step (1) is 1:5 in mg:ml; in step (2), the mass ratio of riboflavin-maleimide intermediate: polypeptide is 10:0-5, and the amount of triethylamine added in step (2) is one tenth of the mass of the polypeptide; preferably, the mass ratio of intermediate: polypeptide is 10:2.5, and the addition ratio of riboflavin-maleimide intermediate to N,N-dimethylformamide in step (2) is 1:1 in mg:ml.

[0071] The deglycosylated polypeptide-riboflavin composite material prepared by the method described above can repair aged skin of human tissue.

[0072] Furthermore, the molecular structural formula of the polypeptide-riboflavin composite material is one or more of the following:

[0073]

[0074]

[0075]

[0076] Use of the polypeptide-riboflavin composite material as described above in the preparation of fructosamine and / or fructosyl lysine and / or glycopeptide and / or glycated bovine serum albumin and / or glycated hemoglobin and / or glycated collagen and / or glycated cells and / or glycated skin.

[0077] Use of the polypeptide-riboflavin composite material as described above in the removal of sugar from glycosylated substances in vitro and / or in vivo.

[0078] A method for removing glycosylation using the polypeptide-riboflavin composite material as described above, the method comprising the following steps: the reaction temperature for removing glycosylation is 30-60°C, more preferably, the reaction temperature is 40°C, the removal of glycosylation is carried out in an environment of pH 8, and the reaction time for removing glycosylation is 24 hours.

[0079] Specifically, the relevant preparation and detection are as follows:

[0080] Example 1

[0081] A method for synthesizing a deglycosylated polypeptide-riboflavin composite material capable of repairing aged skin of human tissue comprises the following steps:

[0082] The riboflavin is dissolved in N,N-dimethylformamide (DMF), a maleimide compound and a catalyst are added, and the mixture is cultured in a constant temperature dark environment to obtain a riboflavin-maleimide intermediate; the mass ratio of riboflavin to N,N-dimethylformamide is 1:5 in mg:ml;

[0083] The intermediate is placed in N,N-dimethylformamide (DMF), and a polypeptide and triethylamine are added. The mixture is cultured in a constant temperature dark environment, dialyzed, and freeze-dried to obtain a polypeptide-riboflavin composite material; the addition ratio of riboflavin-maleimide intermediate to N,N-dimethylformamide is 1:1 in mg:ml.

[0084] Among them, the molar mass ratio of riboflavin: maleimide compound: 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride EDCI: 4-(dimethylamino)pyridine DMAP is 1.5:1:1.5:0.1; the added molar ratio of riboflavin-maleimide intermediate: polypeptide: triethylamine is 4:1:0.1.

[0085] The polypeptide sequences are SED ID NO.1: QCG, SED ID NO.2: CVPGAHPG, SED ID NO.3: CVPGAHAPGV, SED ID NO.4: CVPGAHEPGKR, SED ID NO.5: CVPGAKRPGHE, SED ID NO.6: CVPGAEKEHAPGV.

[0086] As the same inventive concept as the present invention, the same type of amino acid residue substitution occurs on the basis of SEQ ID NO. 5, and the activity of the polypeptide does not change substantially, so the polypeptide can still have the function of removing glycosylation. Here, the replacement of the same amino acid residue may occur at any position of the polypeptide.

[0087] As the same inventive concept as the present invention, there are modifications of amino acid residues based on SEQ ID NO.5, including acetylation, amidation, phosphorylation modification, etc. The modification of the polypeptide usually does not change the amino acid sequence structure, so the polypeptide can still have the function of removing glycosylation.

[0088] Therefore, in summary, homologous substitutions of amino acid sequences related to the polypeptides provided by the present invention, modifications of amino acid residues, etc. should all be included in the scope of the present invention.

[0089] The following structural formula is the molecular structure of the deglycosylated polypeptide (SED ID NO.1-6)-riboflavin composite material in the present invention;

[0090]

[0091]

[0092] The molecular weight of the deglycosylated polypeptide (SEDIDNO.5)-riboflavin composite material was determined in the reverse positive ion mode using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with α-cyano-4-hydroxycinnamic acid as the matrix. Figure 1 As shown, it can be clearly observed that the peak of the mass spectrum of the material appears at 1788 [M+H] +, and the molecular weight shown by this value is consistent with the theoretical molecular weight, which indicates that the deglycosylated polypeptide (SEDIDNO.5)-riboflavin composite material was successfully synthesized by chemical synthesis.

[0093] The morphology of the deglycosylated polypeptide (SEDIDNO.5)-riboflavin composite material was observed using a transmission electron microscope (TEM). First, a 2wt% (mass concentration) phosphotungstic acid solution was prepared for subsequent staining, and it was prepared on demand. Then, the 5mM composite material solution at the end of incubation was diluted to 0.1mM with the corresponding buffer solution (1mM pH7 phosphate buffer solution). 10μL of the solution was aspirated and dropped onto the surface of the TEM-specific copper grid. After standing and adsorbing for about 0.5h, excess solution on the surface was removed with filter paper. Finally, 10μL of phosphotungstic acid was used for staining for about 120s, and the solution was repeatedly washed with deionized water for several times to remove excess dye on the surface. The solution was then naturally dried at room temperature before measurement. The results are shown in the figure. Figure 2 As shown, linear fibers are clearly observed, which indicates that the material is stable.

[0094] Example 2: Application of different polypeptide-riboflavin composite materials in saccharification substances

[0095] The application of different polypeptide-riboflavin composite materials in saccharification materials in this embodiment is prepared by the method in Example 1. The present invention detects the deglycosylation effect of different polypeptide-riboflavin composite materials, and the specific implementation steps are as follows:

[0096] Application of different glycosylated peptide-riboflavin composite materials in in vitro glycosylation of bovine serum albumin

[0097] The amino acid sequences of the polypeptides are SEDID NO.1: QCG, SEDID NO.2: CVPGAHPG, SEDID NO.3: CVPGAHAPGV, SEDID NO.4: CVPGAHEPGKR, SEDID NO.5: CVPGAKRPGHE, SEDID NO.6: CVPGAEKEHAPGV.

[0098] The concentration of glycosylated bovine serum albumin was 300mM, and 1mM of the polypeptide-riboflavin composite material was added. The system without the composite material was used as a control, and the reaction system was repeated 3 times. The reaction was carried out at 37°C and pH 7.4 for 24 hours, and then deionized water was added in an amount three times the volume of the original solution to terminate the reaction. The effect of the polypeptide-riboflavin composite material was analyzed by the microplate reader method. The parameters of the microplate reader were: plate type: 96-well plate; detection wavelength: 320nm; temperature: 25°C.

[0099] The results are as follows Figure 3 As shown in the figure, this figure shows that various polypeptide-riboflavin composite materials have the effect of removing glycosylation in glycated bovine serum albumin. However, the hydrolysis activity is different. Among materials 1-6, the activity of P1 polypeptide-riboflavin material is the lowest, which is 0.04mM, and the P5 polypeptide-riboflavin composite material has the highest ability to remove glycosylation and the highest activity, which is 0.43mM.

[0100] The concentration of glycated bovine serum albumin was 300mM, and 1mM of the polypeptide-riboflavin composite material was added. The system without the composite material was used as a control, and the reaction system was repeated 3 times. The reaction was carried out at 37°C and pH7.4 for 24 hours, and then deionized water was added in an amount three times the volume of the original solution to terminate the reaction. The molecular weight of the substance after glycated bovine serum albumin was deglycosylated was analyzed by ultra-high performance liquid chromatography-mass spectrometry, which further illustrates the hydrolytic activity of the polypeptide-riboflavin composite material in deglycosylation. Chromatographic conditions: C18 column (Agilent, 3.5um, 2.1x100mm); mobile phase A was 0.1% formaldehyde aqueous solution, mobile phase B was acetonitrile, elution program: 0.01min, 1% B; 1.5min, 1% B; 15min, 99% B; 20min, 99% B; 21min, 1% B; 30min, stop. Flow rate: 0.3ml / min, column temperature: 30°C; injection volume: 1ul. Mass spectrometry conditions: positive ion mode; temperature: 550°C; scanning range m / z: 800-3000. The experimental results were analyzed using PeakView software.

[0101] The molecular weight of glycated bovine serum albumin is concentrated around 69,000. The molecular weight of glycated bovine serum albumin after the glycosylation substances are removed is as follows Figure 4 As shown, the product of material 1 contains substances with molecular weights of 69000 (80%), 68000 (40%), 67000 (20%), and 66000 (20%); the product of material 2 contains substances with molecular weights of 69000 (60%), 68000 (80%), 67000 (80%), and 66000 (60%); the product of material 3 contains substances with molecular weights of 69000 (10%), 68000 (40%), 67000 (90%), and 66000 (40%). The product of material 4 contained substances with molecular weights of 69000 (40%), 68000 (50%), 67000 (60%), and 66000 (80%); the product of material 5 contained substances with molecular weights of 69000 (0%), 68000 (70%), 67000 (90%), and 66000 (40%); the product of material 6 contained substances with molecular weights of 69000 (80%), 68000 (50%), 67000 (70%), and 66000 (80%). According to the above results, it can be seen that different composite materials have different hydrolysis activities on glycated bovine serum albumin, among which the substances with molecular weights of 69000 and 68000 in material 3 and material 5 are relatively small, so it can be considered that the removal ability of material 3 and material 5 is higher than that of the other four materials. In addition, it can be further shown that a variety of polypeptide-riboflavin composite materials have removal effects on glycated bovine serum albumin.

[0102] Example 3: Biological toxicity evaluation of deglycosylated polypeptide-riboflavin composite materials

[0103] This example is a toxicity evaluation of a polypeptide-riboflavin composite material with deglycosylation, and the material is prepared in Example 1. The present invention detects the toxicity of the polypeptide-riboflavin composite material, and the specific implementation steps are as follows:

[0104] The biological toxicity of the simulated enzyme was evaluated by measuring the effect of the mimetic enzyme on the survival rate of Caco-2 cells by the MTT method. The cells were co-cultured with the polypeptide-riboflavin composite material (concentrations of 0, 0.1, 0.25, 0.5, and 1 mM) for 24 hours, and the absorbance (OD value) was measured at 490 nm after the end. The blank group was a well without cells and without the addition of the polypeptide-riboflavin composite material, the control group was a well containing only cells, and the experimental group was a well containing cells and with the addition of the polypeptide-riboflavin composite material. The cell viability calculation formula is as follows

[0105]

[0106] The results are as follows Figure 5 The results showed that the polypeptide-riboflavin composite material had no significant effect on cell viability and did not cause cell damage, and the cell survival rate remained above 90%, indicating that the composite material had low toxicity and excellent biocompatibility, and could be further studied and applied to the degradation of glycosylation in various fields.

[0107] After analysis, the principle of glycosylation removal by the polypeptide-riboflavin composite material is as follows: the polypeptide-riboflavin composite material promotes the conversion of the glycosylation CN bond of the substrate into C=N, and then the Schiff base is degraded into glucosone and free amine when it comes into contact with water.

[0108]

[0109] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for synthesizing a deglycosylated polypeptide-riboflavin composite material capable of repairing aged skin of human tissue, characterized in that: The steps include: (1) dissolving riboflavin in N,N-dimethylformamide, adding a maleimide compound and a catalyst, and culturing in a constant temperature dark environment to obtain a riboflavin-maleimide intermediate; (2) placing the intermediate in N,N-dimethylformamide (DMF), adding the polypeptide and triethylamine, culturing in a constant temperature dark environment, dialyzing, and freeze-drying to obtain a polypeptide-riboflavin composite material, which is a deglycosylated polypeptide-riboflavin composite material that can repair aged skin of human tissue.

2. The synthesis method according to claim 1, characterized in that: The polypeptide in step (2) is a mixture of one or more polypeptides of the following amino acid sequences: (1) The amino acid sequence shown in SED ID NO.5, SED ID NO.5: CVPGAKRPGHE; (2) SED ID NO.5 introduces D-amino acids or unnatural amino acids; (3) The amino acid sequence shown in SED ID NO.5 is modified by acetylation, amidation, or phosphorylation; (4) an amino acid sequence formed by adding one or more amino acids before or after the amino acid sequence shown in SED ID NO.5; (5) an amino acid sequence in which the functional amino acid in the amino acid sequence shown in SED ID NO.5 is replaced by an amino acid residue of the same type; (6) a degenerate sequence, an extended sequence, or a position-interchange sequence of the sequence shown in SED ID NO.5; (7) The polypeptide further comprises: SED ID NO.1 to SED ID NO.20; or, the maleimide compound in step (1) is trans-4-(maleimidomethyl)cyclohexanecarboxylic acid; Alternatively, the catalyst in step (1) is a compound that can catalyze the esterification reaction of hydroxyl-carboxyl groups, such as 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI and 4-(dimethylamino)pyridine DMAP.

3. The synthesis method according to claim 2, characterized in that: The amino acid sequence of the SED ID NO.5 polypeptide comprises a hairpin structure functional region, a cysteine ​​connection functional region, and a catalytic activity functional region; Alternatively, the molar mass ratio between riboflavin:maleimide compound:EDCI:DMAP is 0-3:1:0-3:0.

1.

4. The synthesis method according to claim 3, characterized in that: The backbone amino acids of the hairpin functional region are D-proline and / or glycine and / or valine and / or alanine, and the amino acid repeating unit of the hairpin structure functional region is greater than or equal to 1; the backbone amino acids of the cysteine ​​connecting functional region are cysteine, and the amino acid repeating unit of the connecting functional region is greater than or equal to 1; the backbone amino acids of the catalytic activity functional region are histidine and / or glutamic acid and / or lysine and / or arginine, and the amino acid repeating unit of the catalytic activity functional region is greater than or equal to 1.

5. The synthesis method according to claim 1, characterized in that: The dark culture time in step (1) and step (2) is more than 24 hours; Alternatively, the constant temperature environment in step (1) and step (2) is 20-50° C., and the culture time is 12-48 hours; Alternatively, in step (1), the molar ratio of riboflavin: maleimide compound: catalyst is 1.5:1:0.1, and the mass ratio of riboflavin to N,N-dimethylformamide in step (1) is 1:5 in mg:ml; in step (2), the mass ratio of riboflavin-maleimide intermediate: polypeptide is 10:0-5, and the amount of triethylamine added in step (2) is one tenth of the mass of the polypeptide; preferably, the mass ratio of intermediate: polypeptide is 10:2.5, and the addition ratio of riboflavin-maleimide intermediate to N,N-dimethylformamide in step (2) is 1:1 in mg:ml.

6. The deglycosylated polypeptide-riboflavin composite material for repairing aged skin of human tissue obtained by the method according to any one of claims 1 to 5.

7. The polypeptide-riboflavin composite material according to claim 6, characterized in that: The molecular structural formula of the polypeptide-riboflavin composite material is one or more of the following:

8. Use of the polypeptide-riboflavin composite material as claimed in claim 6 or 7 in the preparation of fructosamine and / or fructosyl lysine and / or glycopeptide and / or glycated bovine serum albumin and / or glycated hemoglobin and / or glycated collagen and / or glycated cells and / or glycated skin.

9. Use of the polypeptide-riboflavin composite material according to claim 6 or 7 in removing sugar from glycosylated substances in vitro and / or in vivo.

10. A method for removing glycosylation using the polypeptide-riboflavin composite material according to claim 6 or 7, characterized in that: The method comprises the following steps: The reaction temperature of deglycosylation is 30-60° C., the deglycosylation is carried out in an environment of pH 8, and the reaction time of deglycosylation is 24 hours.

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