Preparation method and application of bionic collagen cyclic hexapeptide

Abstract

The invention provides a preparation method and application of bionic collagen cyclic hexapeptide, and belongs to the technical field of biological medicine. According to the method, a liquid phase synthesis strategy is adopted, and the bionic collagen cyclohexapeptide shown in the formula (I) is efficiently and highly selectively constructed through an optimized reaction route and a protecting group strategy. The method overcomes the defects that a traditional solid-phase synthesis process is complex and high in cost, and collagen hydrolysate is complex in component, non-uniform in quality and the like, and large-scale preparation of the high-purity bionic collagen cyclohexapeptide which is clear in structure and single in molecular weight is achieved. The prepared bionic collagen cyclic hexapeptide has the advantages of biological activity of collagen peptide and stability of cyclic peptide, and has a good application prospect in the fields of skin aging resistance, tissue repair and biological materials. Formula (I)

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing and using a biomimetic collagen cyclic hexapeptide. Background Technology

[0002] Collagen is the most abundant structural protein in animals, and its molecular structure typically presents as a (Gly-XY)n repeating sequence. The X site is primarily composed of proline (Pro), and the Y site is primarily composed of hydroxyproline (Hyp). Therefore, Gly-Pro-Hyp is the most representative repeating unit in collagen. Studies have shown that intact large collagen molecules are difficult to absorb transdermally due to their high molecular weight, while low-molecular-weight collagen peptides have excellent transdermal properties and exhibit multiple anti-aging effects, including repairing skin damage, increasing skin hydration, enhancing skin elasticity, and reducing wrinkles. Collagen peptides containing hydroxyproline are considered key components for these biological activities (Seong SH et al., J Cosmet Dermatol, 2024; Wang L et al., Curr Opin Food Sci, 2023). In addition, collagen peptides have been shown to promote cell proliferation, support angiogenesis and tissue repair (Ohara H et al., J Dermatol, 2010; Zhang Z et al., J Sci FoodAgric, 2011), and have important application value in cell culture, medical repair and skin care.

[0003] Current research and applications mainly focus on linear collagen peptides. However, linear peptides are prone to oxidation and hydrolysis during storage and use, and their membrane permeability and structural stability still need to be improved, which limits their bioavailability and long-lasting effects.

[0004] Compared to linear peptides, cyclic peptides have attracted significant attention in the development of active ingredients for pharmaceuticals and cosmetics due to their conformational constraints, high enzymatic stability, excellent target binding efficiency, and good biocompatibility. Cyclic peptides can penetrate the skin barrier more effectively, binding to cell surface receptors by mimicking endogenous signaling molecules, thereby regulating collagen synthesis, inhibiting melanin production, and neutralizing free radicals, thus exhibiting potential advantages in anti-aging, whitening, and antioxidant effects. Early cyclic peptides were mainly derived from plant extracts; however, natural sources have low cyclic peptide content, complex extraction processes, high costs, and resource and environmental limitations, making it difficult to meet large-scale demand.

[0005] Current synthesis of cyclic peptides mainly relies on solid-phase synthesis strategies: first, linear precursors are gradually coupled on resin, followed by cleavage, purification, and solution-phase cyclization. While this route is relatively mature, the overall process is cumbersome, requiring expensive protective amino acid monomers, often involving excessive feedstock input to ensure coupling efficiency, and involving multiple purification and processing steps, resulting in high production costs and complex processes, thus hindering its large-scale preparation and application. Furthermore, microbial fermentation methods for preparing cyclic peptides make it difficult to introduce non-natural amino acids such as hydroxyproline, and product quality consistency is difficult to control.

[0006] Another common route to obtain collagen peptides is the hydrolysis of natural collagen. However, the product obtained by this method is a mixture of peptides with different chain lengths and structures, with complex composition, wide molecular weight distribution, and potential risk of pathogen residues, making it difficult to obtain single cyclic collagen peptides with well-defined structures and high purity.

[0007] Therefore, developing a simple, cost-effective, and scalable synthetic method for accurately constructing cyclic peptides containing hydroxyproline units has become a pressing technical challenge in this field. In particular, achieving efficient and selective cyclization in a liquid-phase system while overcoming potential side reactions caused by the hydroxyl groups on the hydroxyproline side chain is one of the key challenges in synthesizing biomimetic collagen cyclic hexapeptides. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing and using biomimetic collagen cyclic hexapeptide, aiming to overcome the shortcomings of existing technologies such as high cost, complex process, and difficulty in large-scale synthesis of biomimetic cyclic peptides containing hydroxyproline units.

[0009] The specific technical solution of this invention is as follows: This invention provides a method for preparing a biomimetic collagen cyclic hexapeptide, wherein the biomimetic collagen cyclic hexapeptide has the structure shown in formula (I), and the synthesis method is a liquid-phase synthesis method, comprising the following steps: Formula (I) (1) Cyclization reaction: the linear hexapeptide precursor is cyclized. Deprotection of the amino and carboxyl ends, followed by intramolecular cyclization, forms a biomimetic collagen cyclic hexapeptide with protected side chain hydroxyl groups. R1 is a hydroxyl protecting group; (2) Removal of protecting groups: The biomimetic collagen cyclic hexapeptide described in step (1) is deprotected under alkaline conditions to obtain the biomimetic collagen cyclic hexapeptide shown in formula (I).

[0010] Further, in step (1), the intramolecular cyclization reaction is carried out in any of the following ways: (I) After activating the carboxyl terminus of the linear hexapeptide precursor to an activated ester and removing the amino terminus protecting group, cyclization is carried out in an organic solvent; or, (II) After removing the amino and carboxyl protecting groups from the linear hexapeptide precursor to obtain the free linear hexapeptide acid, it is directly cyclized with a condensing agent and a base. In step (2), the removal of hydroxyl protection includes the following steps: reacting the biomimetic collagen cyclic hexapeptide with an organic solvent and an inorganic base to obtain the biomimetic collagen cyclic hexapeptide shown in formula (I).

[0011] Furthermore, step (II) includes the following sub-steps: ① The linear hexapeptide precursor, organic solvent, catalyst, and reducing agent are reacted to obtain intermediate product A. R3 is an amino protecting group; ② React intermediate product A, activating ester reagent, activator, and organic solvent to obtain intermediate product B. ;R 4 To activate ester groups; ③ React intermediate product B, organic solvent, and acid to obtain intermediate product C. X is an organic or inorganic acid; ④ The intermediate product C, an organic solvent, and an organic base are reacted to obtain a biomimetic collagen cyclic hexapeptide with protected side chain hydroxyl groups. .

[0012] Further, each of the organic solvents is independently selected from N,N-dimethylformamide, dichloromethane, or methanol; each of the activator and condensing agent is independently selected from the following reagents: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, O-benzotriazole-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and ethyl 2-oximenitrile; each of the organic bases is independently selected from N,N-di... Isopropyl ethylamine, triethylamine, or N-methylmorpholine; each of the inorganic bases is independently selected from sodium carbonate, sodium hydroxide, potassium hydroxide, or potassium carbonate; each of the acids is independently selected from trifluoroacetic acid or 1,4-dioxane hydrochloric acid solution; each of the catalysts is independently selected from palladium on carbon or palladium alumina; each of the reducing agents is independently selected from hydrogen; and the activating ester reagent is selected from pentafluorophenol, N-hydroxysuccinimide, N-(benzyloxycarbonyloxy)succinimide, or 9-fluorenemethoxycarbonylsuccinimide.

[0013] Furthermore, the preparation method of the linear hexapeptide precursor includes the following steps: (A) Synthesizing tripeptide units; (B) Synthesis of linear hexapeptide precursor; The structure of the tripeptide unit is as follows: R2 is a carboxyl protecting group.

[0014] Further, in step (A), the synthesis of the tripeptide unit includes the following sub-steps: (a) Hydroxyproline with a protecting group and a basic catalyst were dissolved in an organic solvent, and glycine with a protecting group was added for a coupling reaction. Then, a protecting agent, an imidazole compound, and a catalyst were added for an upprotection reaction. After purification, a dipeptide product with a protecting group was obtained. ; (b) The dipeptide product protected by the protecting group and the deprotecting agent are dissolved in an organic solvent to carry out the deprotection reaction, and the deprotected dipeptide product is obtained after purification. ; (c) The protected proline was dissolved in an organic solvent, the deprotected dipeptide product was added, and a coupling reaction was carried out under an alkaline catalyst. After purification, the tripeptide unit was obtained. ; In step (B), the synthesis of the linear hexapeptide precursor includes the following sub-steps: (i) Using a tripeptide protected by a protecting group as a starting material, a deprotection reaction was carried out with a metal catalyst in an organic solvent, and the intermediate product 1 was obtained after purification. ; (ii) The tripeptide protected by the protecting group and the deprotecting agent were dissolved in an organic solvent to carry out the deprotection reaction, and the intermediate product 2 was obtained after purification. ; (iii) Dissolve intermediate 1 in an organic solvent, add intermediate 2, and carry out a coupling reaction under a basic catalyst. After purification, a linear hexapeptide precursor is obtained. .

[0015] Further, in step (A) and / or step (B), the coupling reaction is carried out in the presence of a carboxyl-activated system; the carboxyl-activated system is selected from one or a mixture of two or more of N,N'-diisopropylcarbodiimide, O-benzotriazole-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and ethyl 2-oximenitrile.

[0016] Further, in step (a), the hydroxyproline protected by the protecting group is Boc-L-hydroxyproline, the carboxyl activation system of the coupling reaction is benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; the basic catalyst is N,N-diisopropylethylamine, the glycine protected by the protecting group is glycine benzyl ester hydrochloride, the organic solvent is N,N-dimethylformamide, and the hydroxyproline protected by the protecting group is benzotriazole-N,N,N' The equivalence ratio of N'-tetramethylurea hexafluorophosphate:N,N-diisopropylethylamine:glycine protected by the protecting group is 1:0.5-1:2-5:1-1.2, and the condensation reaction time is 1-5 hours; the protecting agent is acetic anhydride, the imidazole compound is imidazole, the catalyst is 4-dimethylaminopyridine, and the equivalence ratio of acetic anhydride:imidazole:4-dimethylaminopyridine is 1:1-3:1-3, and the upper protection reaction time is 1-3 hours; In step (b), the deprotecting agent is 1,4-epoxyhexacyclohexane or its salt, the organic solvent is dichloromethane, the equivalence ratio of the dipeptide product protected by the protecting group to the deprotecting agent is 1:9-11, the mass-volume ratio of the dipeptide product protected by the protecting group to the organic solvent is 1:0.5-2, and the deprotection reaction time is 0.5-2 hours. In step (c), the proline protected by the protecting group is Boc-L-proline; the organic solvent is dichloromethane; the basic catalyst is triethylamine; the equivalence ratio of the proline protected by the protecting group, the deprotected dipeptide product, the carboxyl activation system, and the basic catalyst is 1:1-3:2-4:1-2; the mass-volume ratio of the proline protected by the protecting group to the organic solvent is 1:9-11; and the reaction time is 2-6 hours. Preferably, in step (a), the equivalence ratio of the protecting group-protected hydroxyproline: benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate: N,N-diisopropylethylamine: protecting group-protected glycine is 1:0.95:3:1.05, and the condensation reaction time is 4 hours; the equivalence ratio of the protecting group-protected acetic anhydride:imidazolium:4-dimethylaminopyridine is 1:2:2; and the upper protection reaction time is 2 hours. In step (b), the deprotecting agent is 1,4-epoxyhexacyclohydrochloride, the equivalent ratio of the dipeptide product protected by the protecting group to the deprotecting agent is 1:10, the mass-volume ratio of the dipeptide product protected by the protecting group to the organic solvent is 1:1, and the deprotection reaction time is 1 hour. In step (c), the carboxyl activation system for the coupling reaction is ethyl 2-oxime cyanoacetate and N,N'-diisopropylcarbodiimide. The equivalent ratio of the protected proline, the deprotected dipeptide product, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide, and the basic catalyst is 1:2:2:1.1:1.1. The mass-to-volume ratio of the protected proline to the organic solvent is 1:10. The reaction time is 4 hours.

[0017] In step (i), the metal catalyst is carbon-supported palladium; the equivalent ratio of the tripeptide protected by the protecting group to the metal catalyst is 1:0.005-0.02; the organic solvent is methanol; and the reaction time is 3-8 hours.

[0018] Preferably, in step (i), the equivalent ratio of the tripeptide protected by the protecting group to the metal catalyst is 1:0.01; and the reaction time is 6 hours.

[0019] Further, the hydroxyl protecting group is acetyl, tert-butoxycarbonyl, tert-butyldiphenylsilyl or methoxymethyl, the carboxyl protecting group is benzyl, acetyl, tert-butoxycarbonyl, tert-butyldiphenylsilyl or methoxymethyl, and the amino protecting group is tert-butoxycarbonyl, acetyl, tert-butyldiphenylsilyl or methoxymethyl. Preferably, the hydroxyl protecting group is an acetyl group, the carboxyl protecting group is a benzyl group, and the amino protecting group is a tert-butoxycarbonyl group.

[0020] Furthermore, after each reaction step is completed, one or more of the following purification steps are also included: extraction, precipitation, concentration, and chromatographic purification.

[0021] Further, the purification method described in step (a) is as follows: after the reaction is completed, water is added to the reaction solution, the product is extracted by ethyl acetate in the forward direction, then back-extracted with water and saturated brine, and then water is absorbed by anhydrous sodium sulfate and concentrated under reduced pressure to obtain the product. The purification method described in step (b) is as follows: after the reaction is complete, remove the solvent and precipitate with ice-cold diethyl ether to obtain the product; The purification method described in step (c) is as follows: After the reaction is completed, water is added to the reaction solution. First, the product is extracted by forward extraction with ethyl acetate, then back-extracted with water and saturated saline solution, and then water is absorbed by anhydrous sodium sulfate and concentrated under reduced pressure to obtain the final product.

[0022] This invention also provides the use of the biomimetic collagen cyclic hexapeptide of formula (I) in the preparation of a drug that promotes fibroblast proliferation: Formula (I).

[0023] The present invention has achieved the following beneficial effects: (1) Achieved efficient and low-cost large-scale preparation: Compared with the traditional solid-phase synthesis method, the liquid-phase synthesis strategy adopted in this invention simplifies the process steps, avoids cumbersome operations such as the use of solid-phase carriers, repeated coupling and pyrolysis, significantly reduces raw material consumption and production costs, and provides a feasible technical path for the large-scale production of biomimetic collagen cyclic hexapeptide.

[0024] (2) Achieved precise and controllable synthesis of biomimetic collagen cyclic hexapeptides: The method of this invention can accurately synthesize biomimetic collagen short peptides with well-defined sequences, single molecular weights, and high purity in a liquid-phase system. By precisely controlling the coupling sequence of amino acids, the composition, topological structure (especially cyclic configuration), and stereochemistry (such as hydroxyproline configuration) of the polypeptide sequence can be precisely controlled, thereby obtaining ideal products with clear structural definitions and uniform quality, overcoming the shortcomings of complex composition and non-uniform quality of collagen hydrolysis products.

[0025] (3) Effectively solves the side reaction problem of key amino acids: In response to the technical problem that the hydroxyl side chain hydroxyl group is prone to side reactions during cyclization, this invention effectively shields the interference of hydroxyl group by optimizing the synthesis route and reaction conditions (such as the preferred protection strategy of acetyl group), which significantly improves the efficiency and specificity of cyclization reaction and provides a reliable guarantee for the efficient and highly selective synthesis of hydroxyproline-containing biomimetic collagen cyclic hexapeptide.

[0026] (4) A high-performance biomimetic collagen cyclic hexapeptide product was obtained: The prepared biomimetic collagen cyclic hexapeptide has both the bioactivity of collagen characteristic sequence (Gly-Pro-Hyp) and the unique advantages of cyclic molecules. It is expected to have higher enzyme stability, better membrane permeability and stronger ability to bind to biological targets, showing broad application potential in the fields of skin care (such as anti-aging, moisturizing and repair), tissue engineering, and biomedical materials.

[0027] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0028] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0029] Figure 1 Boc-O OAc HPLC chromatogram and mass spectrometry of G-OBn.

[0030] Figure 2HCl·NH2-O OAc HPLC chromatogram of G-OBn.

[0031] Figure 3 For Boc-PO OAc HPLC chromatogram and mass spectrometry of G-OBn.

[0032] Figure 4 HCl·NH2-PO OAc G-OBn and Boc-PO OAc HPLC chromatogram of G-OH.

[0033] Figure 5 For Boc-(PO OAc HPLC chromatogram and mass spectrometry of G)2-OBn.

[0034] Figure 6 For Boc-(PO OAc HPLC chromatogram and mass spectrometry of G)2-OH.

[0035] Figure 7 For Boc-(PO OAc G)2-PFP and TFA·NH2-(PO OAc HPLC chromatogram of G)2-PFP.

[0036] Figure 8 For c(PO) OAc HPLC chromatogram and mass spectrometry of G)2.

[0037] Figure 9 The HPLC and mass spectra of c(POG)2 are shown.

[0038] Figure 10 To screen the HPLC chromatograms of Boc-OG-OBn synthesized by different methods.

[0039] Figure 11 To obtain the HPLC chromatogram of Boc on Boc-OG-OBn using trifluoroacetic acid (TFA).

[0040] Figure 12 To screen the HPLC chromatograms of HCl H2N-OG-OBn synthesized by different methods.

[0041] Figure 13 To screen the HPLC chromatograms of Boc-POG-OBn synthesized by different methods.

[0042] Figure 14 To screen the HPLC chromatograms of c(POG)2 synthesized by different methods.

[0043] Figure 15To screen the HPLC chromatograms of Boc-(POG)2-PFP and TFA NH2-(POG)2-PFP synthesized by different methods.

[0044] Figure 16 The results show the effects of peptide samples on the proliferation of human fibroblasts (HSF).

[0045] Figure 17 The results show the effects of peptide samples on the proliferation of mouse embryonic fibroblasts (NIH-3T3). Detailed Implementation

[0046] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0047] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further explained and described below in conjunction with the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0048] In this invention, "room temperature" means 25±5℃.

[0049] Example 1: Preparation of biomimetic collagen cyclic hexapeptide The structure of the biomimetic collagen cyclic hexapeptide in this embodiment of the invention is as follows: (1) Dipeptide Boc-O OAc G-OBn synthesis: Boc-L-hydroxyproline (Boc-O OH The mixture of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIEA) was dissolved in 100 mL of N,N-dimethylformamide (DMF), and glycine benzyl ester hydrochloride (HCl·NH2-G-OBn) was added, wherein the equivalent ratio was: Boc-O OH -OH: HBTU: DIEA: HCl·NH2-G-OBn = 1: 0.95: 3: 1.05, react for 4 hours. After the reaction is complete, acetic anhydride liquid and imidazole are slowly added dropwise, followed by 4-dimethylaminopyridine solid (DMAP), with an equivalent ratio of imidazole:DMAP:acetic anhydride = 2: 2: 1, and the reaction is continued for 2 hours.

[0050] After the reaction was complete, an equal volume of water and two-thirds volume of ethyl acetate (EA) were added to the reaction solution for forward extraction to obtain the product. The product was then back-extracted three times with pure water and saturated brine to remove excess DMF from the EA. The EA layer was dehydrated using anhydrous sodium sulfate and then filtered to obtain an EA solution containing the product. Silica gel was then added to the EA solution, and the solution was evaporated to dryness. The product was then purified by column chromatography, with the eluent being a petroleum ether (PE) solution containing 45%–60% EA, yielding Boc-O.OAc G-OBn. Purity and molecular weight were confirmed by HPLC and LC-MS. Figure 1 The yield is higher than 92%, and the purity is higher than 97%.

[0051] (2) HCl·NH2-O OAc Synthesis of G-OBn: The Boc-O obtained in step (1) OAc G-OBn was dissolved in dichloromethane (DCM), and 1,4-epoxyhexane hydrochloride was added, with an equivalence ratio of Boc-O OAc G-OBn: 1,4-epoxyhexane hydrochloride = 1:10, Boc-O OAc The mass-to-volume ratio of G-OBn to DCM was 1:1 g / mL. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was removed, and the product was precipitated using icy diethyl ether. The filter cake was dried to obtain HCl·NH2-O. OAc G-OBn. HCl·NH2-O OAc The HPLC chromatogram of G-OBn is as follows: Figure 2 As shown. The yield is higher than 91%, and the purity is higher than 95%.

[0052] (3) Tripeptide Boc-PO OAc Synthesis of G-OBn: Boc-L-proline (Boc-P-OH) was mixed with ethyl 2-oxime cyanoacetate (Oxyma) and N,N'-diisopropylcarbodiimide (EDCI) and dissolved in DCM. HCl·NH2-O obtained in step (2) was then added. OAc G, reacted at room temperature for 4 hours under alkaline conditions (pH=8) provided by triethylamine. The equivalence ratio was Boc-P-OH : Oxyma : EDCI : HCl·NH2-O. OAc G: Triethylamine = 1: 2: 2: 1.1: 1.1, and the mass-to-volume ratio of Boc-P-OH to DCM is 1:10 g / mL.

[0053] After the reaction was complete, an equal volume of water was added to the reaction solution. First, the product was extracted using ethyl acetate (EA) in the forward direction. Then, it was back-extracted three times with pure water and saturated brine to remove any remaining DMF from the EA. Finally, the solution was dehydrated with anhydrous sodium sulfate and concentrated under reduced pressure to an oily liquid, yielding Boc-PO4. OAc G-OBn. Purity and molecular weight were confirmed by HPLC and LC-MS. Figure 3 The yield is higher than 82%, and the purity is higher than 97%.

[0054] (4) Boc-PO OAc Synthesis of G-OH: The Boc-PO obtained in step (3) OAcG-OBn is dissolved in methanol (MeOH), and a catalyst supported on carbon palladium (Pd / C) is added, with an equivalence ratio of Boc-PO. OAc G-OBn: Pd / C = 1: 0.01, Boc-PO OAc The mass-to-volume ratio of G-OBn to MeOH was 1:10 g / mL. Hydrogen gas was introduced, and the reaction was carried out for 5 hours. After the reaction was complete, the mixture was filtered under reduced pressure, and the solvent was evaporated to obtain Boc-PO. OAc G-OH. Boc-PO OAc The HPLC chromatogram of G-OH is as follows: Figure 4 As shown. The yield is higher than 92%, and the purity is higher than 96%.

[0055] (5) HCl·HN2-PO OAc Synthesis of G-OBn: The Boc-PO obtained in step (3) OAc G-OBn was dissolved in DCM, and 1,4-epoxyhexane hydrochloride was added, with an equivalent ratio of Boc-PO. OAc G-OBn: 1,4-epoxyhexane hydrochloride = 1:10, Boc-PO OAc The mass-to-volume ratio of G-OBn to DCM was 1:1 g / mL. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was removed, and the product was precipitated using ice-cold diethyl ether. The filter cake was dried to obtain HCl·HN2-PO4. OAc G-OBn. HCl·NH2-PO OAc The HPLC chromatogram of G-OBn is as follows: Figure 4 As shown. The yield is higher than 92%, and the purity is higher than 96%.

[0056] (6) Boc-(PO) OAc Synthesis of G)2-OBn: The Boc-PO obtained in step (4) OAc G-OH is dissolved in DMF with Oxyma and EDCI, and HCl·HN-PO4 obtained in step (5) is added. OAc G-OBn was reacted for 4 hours under alkaline conditions (pH=8) provided by triethylamine. The equivalence ratio was Boc-PO. OAc G-OH: Oxyma: EDCI: HCl·HN-PO OAc G-OBn:triethylamine = 1:2:2:1.1:1.1, Boc-PO OAc The mass-to-volume ratio of G-OH to DMF is 1:10 g / mL.

[0057] After the reaction was completed, the reaction solution was concentrated under reduced pressure, and extracted three times with pure water and DCM in a 1:1 ratio. The DCM layer was collected, extracted twice with saturated Na₂CO₃ solution, and washed once with saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to obtain a pale yellow oily liquid. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 60 / 40) to obtain Boc-(PO₄)₂. OAc G)2-OBn. Purity and molecular weight were confirmed by HPLC and LC-MS. Figure 5 The yield is higher than 86%, and the purity is higher than 97%.

[0058] (7) Boc-(PO) OAc Synthesis of Boc-(PO)2-OH obtained in step (6): OAc When G)2-OBn is mixed with Pd / C, the equivalence ratio of Boc-(PO) OAc G)2-OBn: Pd / C = 1:0.005, anhydrous ethanol was added, and Bn was removed under hydrogen atmosphere. After the reaction was complete, the mixture was filtered through a membrane, and the filtrate was rotary evaporated under reduced pressure to obtain Boc-(PO)2-OBn: Pd / C = 1:0.005. OAc G)2-OH. Boc-(PO OAc The HPLC and mass spectra of G)2-OH are as follows: Figure 6 As shown. The yield is higher than 91%, and the purity is higher than 98%.

[0059] (8) Boc-(PO) OAc Synthesis of Boc-(PO)2-PFP: The Boc-(PO)2-PFP obtained in step (7) is synthesized by... OAc G)2-OH was dissolved in DCM, and PFP and DIC were added sequentially, followed by stirring and activation. The molar ratio was Boc-(PO)2-OH. OAc G)2-OH: PFP: DIC: DCM = 1: 1.07:1.1: 9. The reaction was carried out for 1.25 hours. After activation, the reaction solution was washed and extracted with an equal volume of water. The DCM layer containing the product was collected and dehydrated with anhydrous sodium sulfate. The solvent was then removed by rotary evaporation to obtain Boc-(PO)2-OH: PFP: DIC: DCM = 1: 1.07:1.1: 9. OAc G)2-PFP. Boc-(PO) OAc The HPLC chromatogram of G)2-PFP is as follows: Figure 7 As shown. The yield is higher than 89%, and the purity is higher than 96%.

[0060] (9) TFA·NH2-(PO OAc Synthesis of Boc-(POG)2-PFP: The Boc-(POG)2-PFP obtained in step (8) was dissolved in twice the mass of DCM, and then trifluoroacetic acid was added and stirred at room temperature for 1-2 h; wherein the equivalence ratio of Boc-(POG)2-PFP was as follows:OAc G)2-PFP: TFA = 1:15. After the reaction is complete, methyl tert-butyl ether is added and stirred to precipitate. After the product is completely precipitated, it is filtered and the residue is dried. The obtained product can be used directly without further purification. TFA·NH2-(PO OAc The HPLC chromatogram of G)2-PFP is as follows: Figure 7 As shown. The yield is higher than 92%, and the purity is higher than 98%.

[0061] (10) c(PO) OAc Synthesis of G)2: The TFA·NH2-(PO obtained in step (9) is used to synthesize the TFA·NH2-(PO)2. OAc G)2-PFP is dissolved in DMF, and DIEA is added, wherein the molar ratio is TFA·NH2-(PO OAc G)2-PFP: DIEA: DMF = 1: 2: 80, stirred for 2 hours; after the reaction, methyl tert-butyl ether was added to precipitate, followed by filtration, collection and drying of the precipitate, purification by high performance liquid chromatography (HPLC), and collection of the pure fraction, which was then freeze-dried to obtain c(PO) OAc G)2. Purity and molecular weight were confirmed by HPLC and LC-MS. Figure 8 The yield is higher than 86%, and the purity is higher than 98%.

[0062] (11) Removal of Ac protecting group: The c(PO) obtained in step (10) is removed OAc G)2 was dissolved in methanol, and anhydrous potassium carbonate was added and stirred for 4 hours, wherein the equivalence ratio c(PO) OAc G)2: Anhydrous potassium carbonate = 1: 3. After the reaction, the filtrate was collected by filtration, methanol was removed by rotary evaporation, and the resulting product was purified by high performance liquid chromatography (HPLC). The pure fraction was collected and lyophilized to obtain c(POG)2. The purity and molecular weight were confirmed by HPLC and LC-MS. Figure 9 The yield is higher than 89%, and the purity is higher than 98%.

[0063] The following experimental examples demonstrate the beneficial effects of the present invention.

[0064] Experimental Example 1: Screening of Preparation Process 1. Boc-PO OAC G-OBn Synthesis Screening In previous studies, we attempted peptide synthesis without protecting the hydroxyproline hydroxyl group. Experimental results showed that when the reaction proceeded to the synthesis of the tripeptide Boc-POG-OBn, further synthesis was difficult to advance.

[0065] To address the aforementioned problems, this invention employs the following synthetic method: Boc-L-hydroxyproline (Boc-O-OH) is reacted with O-benzotriazole-tetramethylurea hexafluorophosphate, glycine benzyl ester hydrochloride (HCl·NH2-G-OBn), and N,N-diisopropylethylamine (DIEA) in N,N-dimethylformamide (DMF) for 2 hours. The molar ratio of Boc-O-OH, HBTU, HCl·NH2-G-OBn, DIEA, and DMF is 1:0.95:1.05:3:10. After the reaction is complete, the product is first extracted using ethyl acetate (EA) in a forward direction. The EA layer is then extracted three times with a saturated sodium bicarbonate aqueous solution to remove HOBt; subsequently, the EA layer is extracted 2-3 times with a 0.5M potassium bisulfate aqueous solution to remove excess alkali. After dehydration of the EA layer using anhydrous sodium sulfate, it is filtered to obtain an EA solution containing Boc-OG-OBn. EA was then removed by rotary evaporation to obtain Boc-OG-OBn (yield > 86%, purity > 91%). HPLC analysis of Boc-OG-OBn is as follows. Figure 10 As shown.

[0066] Boc-OG-OBn was dissolved in dichloromethane (DCM), and trifluoroacetic acid (TFA) was added to remove Boc. The mixture was stirred at room temperature for 1–4 hours. After the reaction was complete, DCM and TFA were removed by rotary evaporation. The resulting product was impure, and HPLC analysis showed two peak shapes, as shown below. Figure 11 As shown, no further reaction can be carried out.

[0067] After replacing the Boc removal reagent for Boc-OG-OBn with 1,4-dioxane hydrochloric acid solution, the purity of the obtained HCl H2N-OG-OBn (higher than 91%) was sufficient for the next experimental step. Analysis of HCl H2N-OG-OBn is as follows... Figure 12 As shown.

[0068] HCl H₂N-OG-OBn, Et₃N, ethyl 2-oxime cyanoacetate (oxyma), and N,N'-diisopropylcarbodiimide (DIC) were dissolved in dimethyl methacrylate (DCM), followed by the addition of Boc-P-OH. The molar ratio of Boc-P-OH, HCl H₂N-OG-OBn, oxyma, Et₃N, DIC, and DCM was 1:1.1:2:1.1:2:10. After the reaction was complete, the mixture was extracted with one-third of the volume of dilute hydrochloric acid solution, followed by extraction with one-third of the volume of saturated sodium bicarbonate solution. The DCM layer containing the product was collected, and the DCM layer solution was dehydrated using anhydrous sodium sulfate. The resulting DCM solution was then filtered and concentrated. The concentrated DCM solution was precipitated with methyl tert-butyl ether to obtain the Boc-POG-OBn tripeptide. HPLC analysis of Boc-POG-OBn is as follows. Figure 13Its purity is insufficient for the next reaction.

[0069] Therefore, the Boc-O-OH raw material was replaced with Boc-4 acetyl-L-hydroxyproline (Boc-O) which is protected by an acetyl group. OAc High-purity Boc-PO4 can be obtained by following the method described in Example 1 (-OH). OAC G-OBn.

[0070] The above results indicate that in the synthesis of Boc-PO OAC When performing G-OBn, a strategy for protecting the hydroxyl group of hydroxyproline needs to be controlled. Specifically, using an acetyl (Ac) group to protect the hydroxyl group of hydroxyproline (i.e., using Boc-OOAc-OH instead of Boc-O-OH), and using a 1,4-dioxane hydrochloric acid solution instead of trifluoroacetic acid (TFA) to remove the Boc protecting group, can avoid the formation of byproducts and ensure the purity of the intermediate and the smooth progress of the reaction.

[0071] 2. c(POG)2 synthesis and screening Synthesis of the fully protected hexapeptide Boc-(PO) OAc After G)2-OBn, the benzyl group is removed by hydrogenation on palladium on carbon to obtain Boc-(PO) OAc G)2-OH, then TFA is used to remove Boc to obtain TFA·NH2-(PO OAc G)2-OH. TFA·NH2-(PO OAc G)2-OH is dissolved in DMF, and condensing agent HBTU and organic base DIEA are added to obtain c(PO)2-OH. OAc G)2-cyclic peptide. TFA·NH2-(PO OAc The molar ratio of G)2-OH, HBTU, DIEA, and DMF is 1:2:2:80. After the cyclization reaction, there are many impurities, including a significant number of peptide-related impurities, making purification difficult and resulting in a yield of less than 60%. This is in contrast to the reaction using TFA NH2-(PO) OAc The synthesis of cyclic peptides using G)2-PFP differs significantly. HPLC analysis of the cyclic peptides obtained by the two methods is as follows: Figure 14 As shown.

[0072] Synthesis of the fully protected hexapeptide Boc-(PO) OAcAfter removing the Ac protecting group from Boc-(POG)2-OBn, methanol and anhydrous potassium carbonate were used to obtain Boc-(POG)2-OBn. Then, the benzyl group was removed under palladium on carbon hydrogenation to obtain Boc-(POG)2-OH. Boc-(POG)2-OH, PFP, and EDC were used similarly to obtain Boc-(POG)2-PFP according to the method described in Example 1, followed by Boc removal for final cyclization. The product generated after Boc removal from Boc-(POG)2-PFP showed non-uniqueness on HPLC, with significant impurities, and could not be separated by column chromatography, failing to meet the conditions for final cyclization. However, the acetyl-protected Boc-(POG)2-OBn... OAc After the removal of Boc, G)2-PFP exhibits a singlet peak. Therefore, the cyclization reaction is only easily carried out when the hydroxyl group of hydroxyproline is protected by Ac, as described in the previous patent. HPLC analysis of Boc-(POG)2-PFP and TFA NH2-(POG)2-PFP is as follows... Figure 15 As shown.

[0073] The above results indicate that the activation mode of the intermediate before cyclization and the order of removal of the protecting group need to be controlled during the synthesis of c(POG)2. Specifically, under the condition that the hydroxyproline hydroxyl group is protected by acetyl (Ac), the activated ester (PFP ​​ester) of the fully protected hexapeptide is prepared first, and then the cyclization reaction is carried out. If the Ac protecting group is removed in advance or the carboxylic acid and condensing agent (such as HBTU) are used directly for cyclization, it will lead to an increase in product impurities and a significant decrease in yield. By using the activated ester method and maintaining Ac protection until the cyclization step, high-purity cyclic peptides can be obtained.

[0074] Experimental Example 2: The effect of biomimetic collagen cyclic hexapeptide on promoting fibroblast proliferation 1. Experimental Methods Cell proliferation was tested using CCK-8 assay: Cells cultured the previous day were digested, centrifuged, and counted. 100 μL of cell suspension (5000 cells / well) was seeded into 96-well plates, with 5 replicates per group. 200 μL of PBS was added around the perimeter of each well to prevent excessive evaporation of the culture medium. Blank and control experimental groups were set up (blank: culture medium only; control: cells plus culture medium). Cells were cultured at 37°C, 5% CO2, and 90% humidity for 24 hours.

[0075] Prepare cyclic peptide c (POG) at a concentration of 0.1 mg / mL. 2、 Culture medium for the linear peptide H-POG2-OH.

[0076] Aspirate the culture medium and replace it with peptide-containing medium, adding 100 μL to each well of a 96-well plate. (Incubation times are 24 h, 48 h, and 72 h.) CCK8 assay: In the dark, prepare 10% CCK8 medium (100 μL / well), remove the original medium, add directly, and incubate for 2-4 hours. Measure the absorbance at 450 nm using a microplate reader. Calculate cell viability.

[0077] 2. Experimental Results Results of peptide samples promoting human fibroblast (HSF) proliferation ( Figure 16 Results of the proliferation of mouse embryonic fibroblasts (NIH-3T3) and peptide samples ( Figure 17 The results showed that biomimetic collagen hexapeptide has the effect of promoting fibroblast proliferation, and biomimetic collagen cyclic hexapeptide has a better proliferative effect.

[0078] In summary, this invention provides a method for preparing biomimetic collagen cyclic hexapeptides and their applications. The method employs a liquid-phase synthesis strategy, utilizing optimized reaction routes and protecting group strategies to efficiently and selectively construct biomimetic collagen cyclic hexapeptides. This method overcomes the drawbacks of traditional solid-phase synthesis processes, such as complexity, high cost, and the complex composition and inconsistent quality of collagen hydrolysis products. It achieves the large-scale preparation of high-purity biomimetic collagen cyclic hexapeptides with well-defined structures and uniform molecular weights. The resulting biomimetic collagen cyclic hexapeptide combines the bioactivity of collagen peptides with the stability of cyclic peptides, showing promising applications in skin anti-aging, tissue repair, and biomaterials.

Claims

1. A method for preparing a biomimetic collagen cyclic hexapeptide, characterized in that, The biomimetic collagen cyclic hexapeptide has the structure shown in formula (I), and the synthesis method is a liquid-phase synthesis method, including the following steps: Formula (I) (1) Cyclization reaction: the linear hexapeptide precursor is cyclized. Deprotection of the amino and carboxyl ends, followed by intramolecular cyclization, forms a biomimetic collagen cyclic hexapeptide with protected side chain hydroxyl groups. R1 is a hydroxyl protecting group; (2) Removal of protecting groups: The biomimetic collagen cyclic hexapeptide described in step (1) is deprotected under alkaline conditions to obtain the biomimetic collagen cyclic hexapeptide shown in formula (I).

2. The synthesis method according to claim 1, characterized in that, In step (1), the intramolecular cyclization reaction is carried out in any of the following ways: (I) After activating the carboxyl terminus of the linear hexapeptide precursor to an activated ester and removing the amino terminus protecting group, cyclization is carried out in an organic solvent; or, (II) After removing the amino and carboxyl protecting groups from the linear hexapeptide precursor to obtain the free linear hexapeptide acid, it is directly cyclized with a condensing agent and a base. In step (2), the removal of hydroxyl protection includes the following steps: reacting the biomimetic collagen cyclic hexapeptide with an organic solvent and an inorganic base to obtain the biomimetic collagen cyclic hexapeptide shown in formula (I).

3. The synthesis method according to claim 2, characterized in that, Step (II) includes the following sub-steps: ① The linear hexapeptide precursor, organic solvent, catalyst, and reducing agent are reacted to obtain intermediate product A. R3 is an amino protecting group; ② React intermediate product A, activating ester reagent, activator, and organic solvent to obtain intermediate product B. ;R 4 To activate ester groups; ③ React intermediate product B, organic solvent, and acid to obtain intermediate product C. X is an organic or inorganic acid; ④ The intermediate product C, an organic solvent, and an organic base are reacted to obtain a biomimetic collagen cyclic hexapeptide with protected side chain hydroxyl groups. .

4. The synthesis method according to any one of claims 1 to 3, characterized in that, Each of the organic solvents is independently selected from N,N-dimethylformamide, dichloromethane, or methanol; each of the activator and condensing agent is independently selected from the following reagents: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, O-benzotriazole-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and ethyl 2-oximenitrile; each of the organic bases is independently selected from N,N-diisopropyl... Ethylamine, triethylamine, or N-methylmorpholine; each of the inorganic bases is independently selected from sodium carbonate, sodium hydroxide, potassium hydroxide, or potassium carbonate; each of the acids is independently selected from trifluoroacetic acid or 1,4-dioxane hydrochloric acid solution; each of the catalysts is independently selected from palladium on carbon or palladium alumina; each of the reducing agents is independently selected from hydrogen; and the activating ester reagent is selected from pentafluorophenol, N-hydroxysuccinimide, N-(benzyloxycarbonyloxy)succinimide, or 9-fluorenemethoxycarbonylsuccinimide.

5. The synthesis method according to claim 1, characterized in that, The preparation method of the linear hexapeptide precursor includes the following steps: (A) Synthesizing tripeptide units; (B) Synthesis of linear hexapeptide precursor; The structure of the tripeptide unit is as follows: R2 is a carboxyl protecting group.

6. The synthesis method according to claim 5, characterized in that, In step (A), the synthesis of the tripeptide unit includes the following sub-steps: (a) Hydroxyproline with a protecting group and a basic catalyst were dissolved in an organic solvent, and glycine with a protecting group was added for a coupling reaction. Then, a protecting agent, an imidazole compound, and a catalyst were added for an upprotection reaction. After purification, a dipeptide product with a protecting group was obtained. ; (b) The dipeptide product protected by the protecting group and the deprotecting agent are dissolved in an organic solvent to carry out the deprotection reaction, and the deprotected dipeptide product is obtained after purification. ; (c) The protected proline was dissolved in an organic solvent, the deprotected dipeptide product was added, and a coupling reaction was carried out under an alkaline catalyst. After purification, the tripeptide unit was obtained. ; In step (B), the synthesis of the linear hexapeptide precursor includes the following sub-steps: (i) Using a tripeptide protected by a protecting group as a starting material, a deprotection reaction was carried out with a metal catalyst in an organic solvent, and the intermediate product 1 was obtained after purification. ; (ii) The tripeptide protected by the protecting group and the deprotecting agent were dissolved in an organic solvent to carry out the deprotection reaction, and the intermediate product 2 was obtained after purification. ; (iii) Dissolve intermediate 1 in an organic solvent, add intermediate 2, and carry out a coupling reaction under a basic catalyst. After purification, a linear hexapeptide precursor is obtained. .

7. The synthesis method according to claim 5, characterized in that, In step (A) and / or step (B), the coupling reaction is carried out in the presence of a carboxyl-activated system; the carboxyl-activated system is selected from one or a mixture of two or more of N,N'-diisopropylcarbodiimide, O-benzotriazole-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and ethyl 2-oximenitrile.

8. The synthesis method according to claim 5, characterized in that, In step (a), the hydroxyproline protected by the protecting group is Boc-L-hydroxyproline, the carboxyl activation system of the coupling reaction is benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; the basic catalyst is N,N-diisopropylethylamine, the glycine protected by the protecting group is glycine benzyl ester hydrochloride, the organic solvent is N,N-dimethylformamide, and the hydroxyproline protected by the protecting group is benzotriazole-N,N,N',N The equivalence ratio of '-tetramethylurea hexafluorophosphate:N,N-diisopropylethylamine:glycine protected by the protecting group is 1:0.5-1:2-5:1-1.2, and the condensation reaction time is 1-5 hours; the protecting agent is acetic anhydride, the imidazole compound is imidazole, the catalyst is 4-dimethylaminopyridine, and the equivalence ratio of acetic anhydride:imidazole:4-dimethylaminopyridine is 1:1-3:1-3, and the upper protection reaction time is 1-3 hours; In step (b), the deprotecting agent is 1,4-epoxyhexacyclohexane or its salt, the organic solvent is dichloromethane, the equivalence ratio of the dipeptide product protected by the protecting group to the deprotecting agent is 1:9-11, the mass-volume ratio of the dipeptide product protected by the protecting group to the organic solvent is 1:0.5-2, and the deprotection reaction time is 0.5-2 hours. In step (c), the proline protected by the protecting group is Boc-L-proline; the organic solvent is dichloromethane; the basic catalyst is triethylamine; the equivalence ratio of the proline protected by the protecting group, the deprotected dipeptide product, the carboxyl activation system, and the basic catalyst is 1:1-3:2-4:1-2; the mass-volume ratio of the proline protected by the protecting group to the organic solvent is 1:9-11; and the reaction time is 2-6 hours. In step (i), the metal catalyst is carbon-supported palladium; the equivalent ratio of the tripeptide protected by the protecting group to the metal catalyst is 1:0.005-0.02; the organic solvent is methanol; and the reaction time is 3-8 hours.

9. The synthesis method according to claim 5, characterized in that, The hydroxyl protecting group is acetyl, tert-butoxycarbonyl, tert-butyldiphenylsilyl or methoxymethyl; the carboxyl protecting group is benzyl, acetyl, tert-butoxycarbonyl, tert-butyldiphenylsilyl or methoxymethyl; and the amino protecting group is tert-butoxycarbonyl, acetyl, tert-butyldiphenylsilyl or methoxymethyl. Preferably, the hydroxyl protecting group is an acetyl group, the carboxyl protecting group is a benzyl group, and the amino protecting group is a tert-butoxycarbonyl group.

10. Use of the biomimetic collagen cyclic hexapeptide shown in formula (I) in the preparation of drugs that promote fibroblast proliferation: Formula (I).

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