Oligopeptidic cyclic peptides and uses thereof

Abstract

This invention provides an oligopeptide cyclic peptide and its applications. The cyclic peptide is obtained by truncating the linear Hst1 sequence to the shortest possible sequence that retains Hst1 activity. Then, a triazole ring-containing oligopeptide is prepared by a cycloaddition reaction under specific conditions targeting this shortest sequence. Its molecular weight is only 1 / 3 that of linear histamine, and its biological activity is 15 times that of linear histamine. To achieve the same activity, the cost of the oligopeptide cyclic peptide is only 1 / 100 that of linear histamine, and the yield is significantly higher than that of cyclic peptides directly cyclized with amide bonds. It also exhibits significantly improved stability and resistance to biodegradation. The entire cyclization process of this oligopeptide cyclic peptide is mild, simple, and efficient, and can produce high-purity oligopeptide cyclic peptides. It can be used to prepare anti-aging and repairing skincare products, health products, or pharmaceuticals, and has excellent prospects for large-scale industrialization.

Description

[0001] This application claims priority to the earlier Chinese application, application number 202110851741.0, filed on July 27, 2021; all its contents are part of this invention. Technical Field

[0002] This invention relates to the fields of biomedical technology and personal care, and more specifically, to an oligopeptide cyclic peptide and its applications. Background Technology

[0003] Linear peptides exhibit excellent bioactivity and stability in vitro, but their activity rapidly diminishes after in vivo. This is due to the complex in vivo environment, which contains a wide variety of enzymes. Linear peptides are easily degraded and metabolized by these enzymes, thus losing their activity. To obtain peptides with good bioactivity, long half-life, and higher receptor selectivity, many peptide modification methods have been reported in the literature, including converting linear peptides into cyclic peptides. These macrocyclic molecules have a well-defined and fixed conformation, enabling them to better bind to receptors and significantly reducing their sensitivity to aminopeptidase and carboxypeptidase. Therefore, cyclic peptides have much higher metabolic stability and bioavailability than linear peptides.

[0004] Cyclic peptides are classified according to their cyclization mechanism into head-to-tail cyclic peptides, sidechain-to-sidechain cyclic peptides, sidechain-to-end cyclic peptides, disulfide-bridged cyclic peptides, stapled peptides, and cyclic peptides with other bridging structures. From a synthetic perspective, head-to-tail cyclic peptides are the most difficult to synthesize. This is because the peptide bonds in linear peptides have a strong p-bond characteristic, and the molecules prefer to form a trans conformation, exhibiting an extended state. This results in the carboxyl and amino groups at the terminal groups, which are the reaction center, being spatially distant, which is unfavorable for intramolecular condensation reactions and instead favors intermolecular condensation.

[0005] Cyclic peptides with connected ends are usually linear peptides with free N-terminus and C-terminus in dilute solutions (10). -3 ~10 -4 M) is synthesized by forming an amide bond between a carboxyl group and an amino group. The type and number of amino acids in the linear precursor play a crucial role in the ease of cyclization and the yield of the cyclic peptide.

[0006] The wide variety of amino acid numbers and types contained in linear peptides, the precursors of cyclic peptides, has led to a diverse range of methods for synthesizing cyclic peptides. Reagents and methods that exhibit efficient and rapid condensation for one type of linear peptide may become inefficient or ineffective for another. Therefore, finding the corresponding cyclic peptide synthesis method based on the sequence of the target cyclic peptide requires long-term, meticulous exploration and arduous effort.

[0007] Cyclic peptides are an important method for improving the rigidity and stability of peptide structures. Exopeptidases, such as aminopeptidases or carboxypeptidases, can recognize and hydrolyze the N- or C-terminal groups of linear peptides. Therefore, cyclic peptides are more resistant to enzymatic hydrolysis than linear peptides. The cyclic structure pre-restricts the conformation of cyclic peptides, blocking the exposed amino groups at both ends, resulting in poorer enzymatic cleavage and thus reducing the entropy cost during receptor binding. This feature increases their binding affinity and specificity to receptors and protein targets. Therefore, they are suitable for probing and regulating protein-protein interactions. With proper design, cyclic peptides can mimic protein secondary structures (such as α-helices and β-hairpins) that are key modules in receptor recognition. All these favorable pharmacological features make cyclic peptides potential drug candidates.

[0008] Peptide synthesis methods mainly include liquid-phase synthesis and solid-phase synthesis. Liquid-phase synthesis uses fully protected linear precursors (except for the two cyclized ends) for direct coupling in the presence of a coupling agent. However, this strategy has several drawbacks, leading to low synthesis efficiency. First, cyclization must be carried out in highly diluted solutions to prevent / reduce oligomerization caused by intermolecular reactions, as peptide cyclization is an entropy-reducing process, resulting in significant solvent waste and cumbersome operation and post-processing. Second, activated C-terminal carboxylic acids are prone to epimerization, producing a pair of cyclic isomers. These epimers can also be generated under alkaline conditions through α-protonation deprotonation, enolizing the activated C-terminal carboxylic acid. Therefore, liquid-phase synthesis requires very high purification standards, leading to reduced synthesis yield. Finally, some fully protected linear peptide precursors have poor solubility in organic solvents, which can prevent cyclization. Therefore, solid-phase peptide synthesis (SPPS) is more competitive.

[0009] Currently available methods for cyclization mainly include: on-resin cyclization, chemical ligation, azide-alkyne cycloaddition, ring-closing metathesis, and electrostatically-controlled macro-cyclizations.

[0010] There are existing reports on the preparation of cyclized Hst1 using the transpeptidase Sortase A method (Sortase A as a tool for high-yield histatin cyclization, Jan GMBolscher, The FASEB Journal. Research Communication), which synthesizes highly active Hst1 cyclic peptides. However, the Sortase A method is cumbersome, the resulting cyclic peptides have poor solubility, and Sortase A is expensive and, being a bacterial enzyme, is prone to bacterial product residues, leading to process safety issues such as immunogens and pathogenic microorganisms. This makes it difficult to prepare high-purity Hst1 cyclic peptides for large-scale industrial production. In addition, this method requires large recognition units and introduces redundant amino acid sequences at both ends, unnecessarily increasing the length and sequence complexity of the original peptide, making it particularly unsuitable for the cyclization of small oligopeptides. In addition, CN103249426A discloses a histone-rich cyclic analog prepared by disulfide bond method. However, the disulfide bond method usually produces cyclic peptides linked to side chains, making it difficult to obtain stable head-to-tail linked cyclic peptides, and it is not suitable for polypeptide sequences that do not contain cysteine ​​residues.

[0011] The linear Hst1 sequence can be truncated to obtain the shortest sequence Hst1-MAD that retains Hst1 activity (see Structure-activity analysis of histatin, a potent wound healing peptide from human saliva: cyclization of histatin potentiates molar activity 1000-fold, The FASEB Journal. Research Communication). Because the molecular weight of the truncated sequence is significantly reduced, the cost required to achieve the same activity will also be significantly lower. Further preparation of the first and last cyclic peptides of Hst1-MAD to improve its activity and stability will further reduce the production cost per unit of activity. Therefore, there is an urgent need for a simpler, more efficient, less reactive, highly soluble, safe, sterile, and scalable method for preparing the shortest Hst1-MAD sequence that retains Hst1 activity, thereby obtaining oligopeptides with higher activity, better stability, and lower cost. Summary of the Invention

[0012] To address the problems existing in the prior art, this invention provides an oligopeptide cyclic peptide and its applications. This cyclic peptide is obtained by truncating the linear Hst1 (histamine 1) sequence to obtain the shortest sequence that retains Hst1 activity. Then, a triazole ring-containing oligopeptide is prepared by a cycloaddition reaction of this shortest sequence under specific conditions. Its molecular weight is only 1 / 3 that of linear histamine. When the concentration is only 1 / 10 that of linear histamine, its biological activity is increased by 1.5 times compared to linear histamine. To achieve the same activity, the cost of the oligopeptide cyclic peptide is only 1 / 100 that of linear histamine, and the yield is significantly higher than that of cyclic peptides directly cyclized with amide bonds. It also possesses significantly improved stability and resistance to biodegradation. The entire cyclization process of this oligopeptide cyclic peptide is mild, simple, and efficient, and can produce high-purity oligopeptide cyclic peptides. It can be used to prepare anti-aging and repairing skincare products, health products, or pharmaceuticals, and has excellent prospects for large-scale industrialization.

[0013] On the one hand, the present invention provides an oligopeptide cyclic peptide, the structural formula of which is shown in Formula 1 or Formula 2:

[0014]

[0015] Wherein, Xaa1, Xaa2, Xaa3, and Xaa4 are any one amino acid; the Hst1-MAD has an amino acid sequence as shown in SEQ ID NO.1 in the sequence listing; or a sequence and fragment thereof having more than 80% homology with SEQ ID NO.1, or a derivative modified peptide of SEQ ID NO.1.

[0016] In some embodiments, Xaa1, Xaa2, Xaa3, and Xaa4 may be the same or different amino acids.

[0017] Hst1-MAD is obtained by truncating the complete Hst1 (histamine 1) sequence. The shortest sequence that can maintain Hst1 activity is Hst1 (20-32), which is the sequence of amino acids 20 to 32 in the histamine 1 sequence.

[0018] The construction of head-to-tail linked cyclic peptides of Hst1-MAD is difficult, requiring stringent reaction conditions and is highly susceptible to side reactions. This invention successfully synthesizes Hst1-MAD oligopeptides containing triazole rings via an azide-alkynyl cycloaddition reaction, after extensive screening of reagents and optimization of process conditions. The resulting peptide exhibits activity approximately 15 times that of linear histamine.

[0019] The Hst1-MAD oligopeptide cyclic peptide containing a triazole ring provided by this invention can improve the biological activity and clinical efficacy of histamine and significantly reduce costs. The molecular weight of the Hst1-MAD oligopeptide cyclic peptide is only 1 / 3 that of linear histamine. When the concentration is only 1 / 10 that of linear histamine, the biological activity is increased by 1.5 times compared with linear histamine. It can be seen that its activity can reach about 15 times that of linear histamine, while the mass concentration is reduced by 4 / 5. When the same activity is achieved, the cost of the oligopeptide cyclic peptide is only 1 / 100 of that of linear histamine.

[0020] Furthermore, cyclization can lock the spatial conformation of peptide molecules, improving their stability and thus enhancing efficacy and in vivo half-life. By introducing azido and alkynyl groups into the head and tail of Hst1-MAD and simultaneously binding it with resin, two types of cyclic peptides, as shown in Formulas 1 and 2, can be prepared via an azido-alkynyl cycloaddition reaction.

[0021] Due to the construction of the triazole ring, the oligopeptide cyclic peptide provided by this invention can resist biological metabolic degradation, thus possessing excellent metabolic stability. It can form hydrogen bonds with biomolecular targets, mimic the trans structure of natural polypeptide peptide bonds or secondary structures such as β-sheets and turns, improve solubility, resist enzyme degradation, and exhibit excellent stability against hydrolysis and oxidation reactions.

[0022] Furthermore, the derivative modification includes one or more of the following: alkylation, acylation, esterification, phosphorylation, sulfonation, glycosylation, PEGylation, biotin labeling, fluorescent labeling, isotope labeling, D-type, linker, carrier protein coupling, or specific amino acid.

[0023] In some embodiments, the specific amino acids include derived amino acids from the 20 basic amino acids that make up natural proteins, and specific amino acids that make up certain special proteins, such as 4-hydroxyproline, 5-hydroxylysine, N-methyllysine, N-formylmethionine, γ-carboxyglutamic acid, selenocysteine, pyrrolidone, desmosin, isodesmosin, and β, γ, δ-amino acids, etc.

[0024] In some embodiments, the derivative modified peptide of SEQ ID NO.1 includes, but is not limited to, methylation, alkylation, acylation, esterification, phosphorylation, sulfonation, glycosylation, PEGylation, biotin labeling, fluorescent labeling or isotope labeling, D-type, linking to various linkers, carrier protein coupling or specific amino acids, etc.

[0025] In some embodiments, the derived modified peptide of SEQ ID NO.1 includes peptides obtained by adding / removing several amino acids from the beginning and end of SEQ ID NO.1, changing L-type amino acids to D-type amino acids, or by phosphorylation, acylation, glycosylation, methylation, hydroxylation, ubiquitination, lipidation, aliphatic hydrocarbon group, alicyclic hydrocarbon group, aromatic hydrocarbon group, heterocyclic group, or halogenation of amino acid groups.

[0026] In some embodiments, the derived modified peptide of SEQ ID NO.1 includes N-terminal modifications of SEQ ID NO.1 (such as Ac acetylation); N-terminal fatty acid modifications (Myr myristic acid, Pal palmitic acid, Ste stearic acid, lauric acid, decanoic acid, caprylic acid, etc.); and C-terminal modifications (-NH2 amidation, -PNA, -AMC, -OME, -OET, etc.).

[0027] In some embodiments, the derived modified peptide of SEQ ID NO.1 includes fluorescent labeling modification of SEQ ID NO.1, such as Cy3, Cy5, Cy5.5, Cy7, FAM, FITC, Rhodamine B, TAMRA, etc.

[0028] In some embodiments, the derived modified peptide of SEQ ID NO.1 includes biotinylation (such as BIOTIN), PEGylation (modification sites at the N-terminus and C-terminus of the peptide, the Lys side chain and the thiol group of Cys, etc.), phosphorylation (such as p-Ser, p-Thr, p-Tyr, etc.), and methylation (such as Lys(Me), Arg(Me), etc.).

[0029] Furthermore, its structural formula is shown in Equation 3 or Equation 4:

[0030]

[0031] Where n = 1 to 6, m = 0 to 3, x = 0 or 1, a = 1 to 6, b = 0 to 2, and R group is the R group of any amino acid.

[0032] The R group can be the R group of any amino acid, including natural and non-natural amino acids, essential and non-essential amino acids, L-type amino acids, special amino acids (D-type amino acids, beta amino acids, homo amino acids), and other various side-chain modified amino acids.

[0033] In some embodiments, the R group includes, but is not limited to, H, aliphatic hydrocarbon group, alicyclic hydrocarbon group, aromatic hydrocarbon group, heterocyclic group, halogenated group, etc.

[0034] In some embodiments, the R group is the fourth group attached to a C atom in one of 20 natural amino acids. The R group varies depending on the amino acid and mainly includes: -H, -CH3, -CH2OH, -CH2SH, -CH(OH)CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2CH2SCH3, -C7H7, -CH2-Benzene ring-OH, -CH2COOH, -CH2CONH2, -CH2CH2COOH, -CH2CH2CONH2, -CH2CH2CH2CH2NH2, -CH2CH2NHCNH2, wait.

[0035] Furthermore, the R group includes (substituted) aliphatic hydrocarbon groups, alicyclic hydrocarbon groups, aromatic hydrocarbon groups, heterocyclic groups, or halogenated groups, etc.

[0036] Furthermore, the R group includes -H, or any one of methyl, ethyl, and phenyl.

[0037] Furthermore, the amino and / or carboxyl groups in Formula 3 or Formula 4 can be derivatized to obtain histamine cyclic peptides derivatized with -NH2 and / or -COOH.

[0038] Further, the derivatization of the amino group in Formula 3 or Formula 4 includes any one or more of acylation, alkylation, PEGylation, biotin labeling, or fluorescent labeling; the derivatization of the carboxyl group in Formula 3 or Formula 4 includes any one or more of amidation, esterification, glycosylation, or PEGylation.

[0039] The amino-derived modifications include, but are not limited to, acylation, alkylation, PEGylation, biotin labeling, and fluorescent labeling; the carboxyl-derived modifications include, but are not limited to, amidation, esterification, glycosylation, and PEGylation.

[0040] The modified derivatives obtained by amino-NH2 derivatization such as amidation; the modified derivatives obtained by carboxyl-COOH derivatization such as esterification, palmitoylation, myristoylation, acetylation, etc.

[0041] Furthermore, the R base is -H.

[0042] Furthermore, the R group of the oligopeptide cyclic peptide is H, and the structural formula of the oligopeptide cyclic peptide is shown in Formula 5 or Formula 6:

[0043]

[0044] Where n = 1 to 6, m = 0 to 3, x = 0 or 1, a = 1 to 6, b = 0 to 3.

[0045] Furthermore, its structural formula is shown in Equation 7 or Equation 8:

[0046]

[0047] Where n = 1 to 3, a = 4, and x = 0 or 1.

[0048] Furthermore, the carbon chains of Xaa1, Xaa2, Xaa3, and Xaa4 connected to the triazole ring may contain any one or more of benzene rings, alicyclic rings, aromatic rings, heterocyclic rings, or halogenated rings.

[0049] Furthermore, the oligopeptide cyclic peptide is obtained by attaching any amino acid containing an alkynyl group to one end of Hst1-MAD and any amino acid containing an azide group to the other end, and binding resin to either end, wherein the alkynyl group and the azide group undergo cyclization addition, and the resin is removed.

[0050] On the other hand, the present invention provides the use of an oligopeptide cyclic peptide in promoting wound healing or skin care, wherein the structural formula of the oligopeptide cyclic peptide is shown in Formula 7 or Formula 8:

[0051]

[0052] Where n = 1 to 3, a = 4, x = 0. Or 1

[0053] In another aspect, the present invention provides a method for preparing an oligopeptide cyclic peptide, which involves attaching an arbitrary amino acid containing an alkynyl group to one end of a derivative modified product of Hst1-MAD and its fragment, attaching an arbitrary amino acid containing an azido group to the other end, and binding a resin to either end. The alkynyl group and the azido group undergo cyclization addition, and the resin is removed to obtain the oligopeptide cyclic peptide. The Hst1-MAD has the amino acid sequence shown in SEQ ID NO.1 in the sequence listing; or a sequence and its fragment having more than 80% homology with SEQ ID NO.1, or a derivative modified peptide of SEQ ID NO.1.

[0054] Furthermore, by linking an alkynyl group (Xaa1) to the C-terminus of Hst1-MAD and its fragment derivatives, and an azide group (Xaa2) to the N-terminus, with resin bound to Xaa2, the alkynyl group attached to Xaa1 and the azide group attached to Xaa2 undergo cyclization addition, followed by resin removal, a histamine cyclic peptide is obtained, as shown in Formula 9:

[0055]

[0056] Alternatively, by linking an azide-containing Xaa3 to the C-terminus of a derivative of Hst1-MAD and its fragments, and an alkynyl-containing Xaa4 to the N-terminus, with resin bound to Xaa4, the azide group of Xaa3 and the alkynyl group of Xaa4 undergo cyclization addition, followed by resin removal, an oligopeptide cyclic peptide is obtained, as shown in Formula 10:

[0057]

[0058] Among them, Xaa1, Xaa2, Xaa3, and Xaa4 are any one of the amino acids.

[0059] The derivative modifications include, but are not limited to, alkylation, acylation, esterification, phosphorylation, glycosylation, PEGylation, biotin labeling, fluorescent labeling, isotope labeling, or specific amino acids.

[0060] Furthermore, the structural formula of the alkynyl-containing Xaa1 or Xaa4 is shown in Formula 11, or the amino and / or carboxyl-modified derivatives shown in Formula 11:

[0061]

[0062] The structural formula of Xaa2 or Xaa3 containing an azide group is shown in Formula 12, or the amino and / or carboxyl modified derivatives shown in Formula 12:

[0063]

[0064] Where n = 1 to 6, m = 0 to 3, R group is any amino acid R group, and x = 0 or 1.

[0065] In some embodiments, the R group includes, but is not limited to, H, aliphatic hydrocarbon group, alicyclic hydrocarbon group, aromatic hydrocarbon group, heterocyclic group, and halogenated group.

[0066] Furthermore, the amino and / or carboxyl groups in Formula 11 or Formula 12 can be further derivatized to obtain histamine cyclic peptides derivatized with -NH2 and / or -COOH.

[0067] The derivatization of the amino and / or carboxyl groups includes, but is not limited to, acylation, alkylation, PEGylation, biotin labeling, and fluorescent labeling of amino groups, and, but is not limited to, amidation, esterification, glycosylation, and PEGylation of carboxyl groups.

[0068] In some methods, the amino-NH2 derivatization, such as amidation, yields modified derivatives; the carboxyl-COOH derivatization, such as esterification, palmitoylation, myristoylation, acetylation, yields modified derivatives.

[0069] Furthermore, the R base is -H.

[0070] In some methods, the derivatization includes, but is not limited to, acylation, alkylation, PEGylation, biotin labeling, fluorescent labeling, etc.

[0071] Furthermore, before the reaction, a protecting group Fmoc needs to be attached to the -NH2 of Xaa1, Xaa3, Xaa3 or Xaa4. After the reaction is completed, the protecting group Fmoc is removed to become naked -NH2, or the naked -NH2 is further derivatized.

[0072] In some methods, the derivatization includes, but is not limited to, acylation, alkylation, PEGylation, biotin labeling, fluorescent labeling, etc.

[0073] Furthermore, before the reaction, a protecting group Fmoc needs to be attached to the -NH2 of Xaa1 or Xaa3. After the reaction is completed, the protecting group Fmoc is removed to become the exposed -NH2, or the exposed -NH2 is further derivatized.

[0074] In some methods, the derivatization includes, but is not limited to, acylation, alkylation, PEGylation, biotin labeling, fluorescent labeling, etc.

[0075] The modified derivatives obtained by amino-NH2 derivatization, such as amidation.

[0076] Furthermore, the structural formula of the alkynyl-containing Xaa1 is shown in Formula 13:

[0077]

[0078]

[0079] The structural formula of Xaa2 containing the azide group is shown in Formula 14:

[0080]

[0081] The structural formula of the Xaa3 containing the azide group is shown in Formula 15:

[0082]

[0083] The structural formula of the alkynyl group-containing Xaa4 is shown in Formula 16:

[0084]

[0085] Where n = 1 to 4, a = 4, and x = 0 or 1.

[0086] Furthermore, the carbon chains of Xaa1 and / or Xaa4 connected to the alkynyl group may contain any one or more of benzene rings, alicyclic rings, aromatic rings, or heterocyclic rings; the carbon chains of Xaa2 and / or Xaa3 connected to the azide group may contain any one or more of benzene rings, alicyclic rings, aromatic rings, heterocyclic rings, or halogenated groups.

[0087] In some embodiments, the reaction formula of the preparation method is shown in Formula 17.

[0088]

[0089] Furthermore, the method shown includes the following steps:

[0090] 1) The linear peptide prepared by solid-phase polypeptide synthesis process is placed in a container and a solvent is added; the linear peptide is a linear histamine with an alkynyl group connected to the C-terminus of Xaa1 and an azide group connected to the N-terminus of Xaa2, wherein the Xaa2 is bound to resin; or a linear histamine with an azide group connected to the C-terminus of Xaa3 and an alkynyl group connected to the N-terminus of Xaa4, wherein the Xaa4 is bound to resin.

[0091] 2) Add a catalyst;

[0092] 3) Add ligands;

[0093] 4) Use nitrogen gas to blow bubbles;

[0094] 5) Tightly cover and seal the container, then let it stand and stir.

[0095] 6) Wash with disodium ethylenediaminetetraacetate to remove the washing solution;

[0096] 7) Rinse with water or an organic solvent to remove the detergent solution;

[0097] 8) Vacuum drying;

[0098] 9) Add the cleavage mixture to cleave the peptides from the resin;

[0099] 10) Purify the polypeptide.

[0100] In some embodiments, the solvent may be a protic solvent such as methanol, ethanol, tert-butanol, polyethylene glycol (PEG), trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), or water; a dipolar aprotic solvent such as acetonitrile (MeCN), dimethyl sulfoxide (DMSO), acetone, N,N-dimethylformamide (DMF), N,N-diisopropylethylamine (DIPEA), N-methylpyrrolidone (NMP), pyridine, or piperidine; or a nonpolar solvent such as dichloromethane (DCM), chloroform, tetrahydrofuran (THF), ethyl acetate, diethyl ether, benzene, toluene, carbon tetrachloride, dioxane, n-hexane, or cyclohexane. It may be a mixture of two or three of these solvents in a certain proportion, such as tert-butanol / water, methanol / dichloromethane, or dichloromethane / Acetonitrile, dichloromethane / water, acetonitrile / dimethyl sulfoxide / water, acetone / dimethyl sulfoxide, ethanol / water, N-methylpyrrolidone / acetonitrile, N-methylpyrrolidone / dichloromethane / water, n-hexane / dioxane, N,N-dimethylformamide / trifluoroethanol, N,N-dimethylformamide / hexafluoroisopropanol, etc.

[0101] The steric hindrance, electrical properties, polarity, protonicity, hydrogen bonding ability, and proportion of the solvent affect the reaction rate, side reactions, byproducts such as dimers, trimers, polymers, and decomposition products.

[0102] Furthermore, the present invention uses a mixed solution of acetonitrile and dimethyl sulfoxide as a solvent.

[0103] Further, the solvent in step 1) is a solution prepared by mixing acetonitrile and dimethyl sulfoxide (MeCN:DMSO) in a 2:1 ratio.

[0104] Studies have found that the concentration of Hst1-MAD oligopeptide in solution affects the reaction rate, leading to an increase in the proportion of byproducts such as dimers, trimers, and polymers. However, in general, SPPS solid-phase synthesis is more conducive to intramolecular cyclization than intermolecular cyclization and other side reactions compared to liquid-phase synthesis methods. This is one of the reasons why SPPS was chosen in this invention.

[0105] In some embodiments, the present invention preferably uses 750 mg of Hst1-MAD oligopeptide, added to 20 ml of a solvent of acetonitrile and dimethyl sulfoxide (MeCN:DMSO = 4:1).

[0106] Furthermore, the resin described in this invention is selected from any one of Wang Resin, Rink Amide AM Resin, Rink Amide MBHA Resin, 2-Chlorotrityl Resin, PEGylated Rink-modified TentaGel (TGR)resin, Aminomethyl Resin, Sieber Amide Resin, PAM Resin, etc.

[0107] Different resins can affect the reaction rate and lead to an increase in side reactions and byproducts.

[0108] In some embodiments, the resin used is Rink Amide MBHA Resin, which can accelerate the reaction rate and significantly reduce the occurrence of side reactions and byproducts.

[0109] Furthermore, the catalyst described in this invention can be a copper or rhodium reagent.

[0110] In some embodiments, the catalyst of the present invention is a copper reagent, which may be selected from monovalent copper salts and their complexes such as cuprous iodide CuI, cuprous chloride CuCl, cuprous iodide / triethyl phosphite CuI·P(OEt)3, cuprous bromide, cuprous bromide dimethyl sulfide, tris(triphenylphosphine)cuprous bromide [CuBr(PPh3)3], tris(triphenylphosphine)cuprous fluoride [CuF(PPh3)3], and tetraethyl copper tetrafluoroborate [Cu(MeCN)4]B. F4, copper hexafluorophosphate tetraethyl cyanophosphate [Cu(MeCN)4]PF6, copper trifluoromethanesulfonate tetraethyl cyanophosphate [Cu(MeCN)4]OTf, various Cu(I)-carbene complexes; or combinations of divalent copper salts and their complexes such as copper sulfate CuSO4, copper chloride CuCl2, copper acetate Cu(OAc)2, copper nitrate Cu(NO3)2, copper acetylacetone Cu(acac)2) and reducing agents such as sodium ascorbate, or nano copper and its combinations.

[0111] In addition, the loading of copper / rhodium reagents also affects the degree of reaction completion to some extent, ranging from 0.05 eq to 4 eq. Non-copper / rhodium reagents such as HBTU / HOBt can also be used, but the effect varies significantly.

[0112] Furthermore, the copper catalyst described in this invention is cuprous iodide.

[0113] Furthermore, the amount of cuprous iodide added is 20 mM.

[0114] Furthermore, the ligands described in this invention are generally nitrogen-containing ligands, selectable from 2,6-dimethylpyridine (2,6-lutidine), diethylamine, triethylamine, n-propylamine, diisopropylamine, tributylamine, diisopropylethylamine DIPEA, dimethylaminopyridine DMAP, 1,8-diazabicyclo[5.4.0]undec-7-ene DBU, pentamethyldiethylenetriamine PMDETA, hexamethyltriethylenetetramine HMTETA, tris(2-dimethylaminoethyl)amine Me6TREN, piperidine, pyridine, bispyridine, 2,2′:6′,2″-terpyridine tpy, tris(2-pyridylmethyl)amine TPMA, bipyridine, tert-butyltrichloroacetylimine ester TBTA, tris(3-hydroxypropyltriazolylmethyl)amine THPTA, TTTA, BTTAA, TABTA, BTTE, The ligand can be any one of BTTP; it can also be a phosphorus-containing ligand, selected from any one of triphenylphosphine, tricyclohexylphosphine PCy3, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine)phosphine BINAP, 1,2-bis(diphenylphosphine)ethane DPPE, 1,3-bis(diphenylphosphine)propane DPPP, 1,4-bis(diphenylphosphine)butane DPPB, 1,5-bis(diphenylphosphine)pentane DPPPe; it can also be a sulfur-containing ligand, selected from any one of 4,4'-thiobis(6-tert-butyl-3-methylphenol) BPS, BTTES, BTTPS.

[0115] When the catalyst described in this invention is a copper reagent, the basicity, donor properties, and steric hindrance of the ligand can stabilize monovalent copper ions, preventing them from being oxidized or undergoing disproportionation reactions. At the same time, it promotes the formation of cuprous acetylene complex, reduces the loading of copper reagent, and improves the cyclization reaction efficiency. The ratio of ligand to copper varies from 0.5 / 1 to 4 / 1 and even in excess, depending on the relative activity, polarity, and donor capacity of the ligand, copper reagent, and solvent.

[0116] Furthermore, the ligand described in this invention is 2,6-dimethylpyridine (2,6-lutidine).

[0117] Furthermore, the amount of 2,6-dimethylpyridine added in this invention is 30 μL.

[0118] Furthermore, the reaction conditions for the azide-alkynyl cycloaddition reaction described in this invention are also strictly limited.

[0119] In some embodiments, the reaction system of the present invention can tolerate a wide range of acidities from 4 to 12.

[0120] Furthermore, the reaction process of this invention must involve deoxygenation;

[0121] Oxygen can oxidize copper reagents, causing the cyclization process to fail. Therefore, deoxygenation is important, and humidity also affects reaction efficiency.

[0122] Furthermore, the reaction process of this invention requires nitrogen bubbling for 2 to 5 minutes. Nitrogen bubbling for 2 to 5 minutes can be used to ensure deoxygenation during the reaction process.

[0123] Furthermore, it is necessary to isolate the air: cover tightly and seal it to isolate oxygen.

[0124] Furthermore, the reaction temperature of this invention is from room temperature to 100°C. The reaction temperature affects the reaction rate and the relative proportions of products / byproducts and decomposition products, as well as their stability.

[0125] Furthermore, the reaction time of the present invention is from 10 min to 100 h.

[0126] In some embodiments, the reaction of the present invention can also be carried out under microwave conditions (room temperature to 100°C), which can accelerate the reaction; however, our research has shown that microwave conditions readily produce byproducts and decomposition products.

[0127] Furthermore, after the reaction is complete, the mixture needs to be washed with a saturated solution of disodium ethylenediaminetetraacetate (EDTA), with the chelating agent EDTA used to remove copper ions.

[0128] Furthermore, the sample was washed twice with EDTA by shaking, 10 minutes each time.

[0129] Furthermore, it is necessary to rinse once with water, once with acetonitrile, once with dichloromethane, and once with ether to remove the washing solution.

[0130] Furthermore, after washing, the floor needs to be vacuum dried for 1 hour.

[0131] Further, after drying, lysis was performed: following the standard procedure: 20 ml of a mixture (95.5% trifluoroacetic acid, 2% phenol aqueous solution, 2% anisole sulfide, 0.5% triisopropylsilane), shaken for 3 hours to shear the peptides from the resin. The peptides were then filtered through cold ether, washed three times, and dried.

[0132] Further purification is required by high-performance liquid chromatography-mass spectrometry (HPLC), using an aqueous solution of acetonitrile containing 0.1% (v / v) trifluoroacetic acid as the mobile phase for gradient elution, and a detection wavelength of 210 nm.

[0133] Furthermore, the preparation method includes the following steps:

[0134] 1) A linear peptide prepared by a 750mg solid-phase polypeptide synthesis process is placed in a container, and 20ml of a mixed solution of acetonitrile and dimethyl sulfoxide in a ratio of 1:1 to 10:1 is added; the linear peptide is a linear histamine with an alkynyl group connected to its C-terminus Xaa1 and an azide group connected to its N-terminus Xaa2, wherein resin is bound to Xaa2; or a linear histamine with an azide group connected to its C-terminus Xaa3 and an alkynyl group connected to its N-terminus Xaa4, wherein resin is bound to Xaa4.

[0135] 2) Add 750mg of resin;

[0136] 3) Add 20mM cuprous iodide;

[0137] 4) Add 30 μL of 2,6-dimethylpyridine;

[0138] 5) Blow nitrogen gas for 2-5 minutes;

[0139] 6) Tightly cover and seal the container;

[0140] 7) Stir and react at room temperature for 40–80 minutes;

[0141] 8) Wash twice with a saturated disodium ethylenediaminetetraacetate solution, 10 minutes each time, and remove the washing solution;

[0142] 9) Rinse once with water, once with acetonitrile, once with dichloromethane, and once with ether to remove the washing solution;

[0143] 10) Vacuum dry for 1 hour;

[0144] 11) Cutting: Prepare a cutting mixture comprising trifluoroacetic acid:phenol: anisole:triisopropylsilane = 95.5:2:2:0.5 (v / v). Add 20 ml of the cutting mixture to the sample obtained in step 10), shake for 3 hours, and cut the peptide from the resin. Filter the peptide with cold ether, wash 3 times, and dry.

[0145] 12) High performance liquid chromatography-mass spectrometry purification.

[0146] Studies have shown that the cyclization method provided by this invention can be used to cyclize long peptide chains head-to-tail, and can successfully cyclize any histamine Hst1 to Hst12, thereby obtaining histamine cyclic peptides containing triazole rings. The preparation process of cyclic peptides is simple, has high purity, high activity, and is easy to scale up for industrial production.

[0147] This invention provides a copper-catalyzed azido-acetylene cycloaddition reaction to prepare a histamine cyclic peptide containing a triazole ring. This reaction yields only 1,4-disubstituted [1,2,3]triazole products, which share many similarities with natural peptide bonds, such as the ability to form hydrogen bonds, planarity, distance between the 1 and 4 substituents, and conformational constraints of the peptide backbone. Therefore, replacing the peptide bond with a triazole in this modified peptide can produce a secondary structure similar to that of the natural polypeptide.

[0148] The beneficial effects of this invention are as follows:

[0149] 1. A series of Hst1-MAD oligopeptides with triazole rings were creatively prepared by copper-catalyzed azide-alkyne cycloaddition reaction;

[0150] 2. The molecular weight of the prepared Hst1-MAD oligopeptide cyclic peptide is only 1 / 3 that of linear histamine, and its biological activity is 15 times that of linear histamine.

[0151] 3. The prepared Hst1-MAD oligopeptide has excellent stability and resistance to biodegradation, and can improve solubility. It has a unique conformation and significantly enhances the bioactivity and clinical efficacy of histamine.

[0152] 4. To achieve the same activity, the cost of oligopeptide proximal and cyclic peptides is only 1 / 100 of that of linear histamine.

[0153] 5. The cyclization process used in this invention is mild, simple, and efficient, and the resulting oligopeptides have higher purity at both the first and last cyclic peptides, showing very good prospects for large-scale and industrial applications. Attached Figure Description

[0154] Figure 1 The mass spectrometry (MS) spectrum of the cyclization reaction product in Example 1;

[0155] Figure 2 The HPLC chromatogram of the cyclization reaction product in Example 1 is shown.

[0156] Figure 3 The infrared (IR) spectrum of the cyclization reaction product in Example 1;

[0157] Figure 4 These are photographs of the wound healing status of four groups of mice in Example 10 at 0, 3, and 5 days.

[0158] Figure 5 This is a schematic diagram comparing the wound healing rates of the four groups of mice in Example 10 at 3, 5, and 10 days.

[0159] Figure 6 These are photographs of the epidermal regeneration of the four groups of wound tissues in Example 10, taken on day 10 after HE staining.

[0160] Figure 7 This is a comparative schematic diagram of the thickness of new epidermal tissue regeneration in four groups of wounds in Example 10;

[0161] Figure 8 These are photographs of collagen regeneration in four groups of wound tissues from Example 10, taken on days 5 and 10 after Masson staining.

[0162] Figure 9 This is a comparative diagram of collagen regeneration in the four groups of wound tissues on days 5 and 10 in Example 10.

[0163] Figure 10 This is a schematic diagram showing the number of newly formed CD31-positive blood vessels in the four groups of wound tissues on days 5 and 10 in Example 10;

[0164] Figure 11 This is a schematic diagram comparing the quantitative analysis of the number of newly formed CD31 positive blood vessels in the four groups of wound tissues on days 5 and 10 in Example 10;

[0165] Figure 12 This is a schematic diagram showing the VEGF expression levels of the four groups of wound tissues in Example 10 on days 5 and 10.

[0166] Figure 13 This is a schematic diagram showing the quantitative analysis and comparison of VEGF expression levels in four groups of wound tissues on days 5 and 10 in Example 10.

[0167] Figure 14 These are photographs of the dendritic cells (MHCⅡ+ and CD11c+) in four groups of wound tissues on days 3 and 5, as detected by immunofluorescence double staining in Example 10.

[0168] Figure 15 This is a comparative analysis of the effects of the four groups of drugs in Example 10 on the regulation of dendritic cells (MHCⅡ+ and CD11c+) in the wound.

[0169] Figure 16 These are photographs of the M1 macrophages (CD68+ and CD80+) in four groups of wound tissues on days 3 and 5, as detected by immunofluorescence double staining in Example 10.

[0170] Figure 17 This is a comparative analysis of the effects of the four groups of drugs in Example 10 on the regulation of M1 macrophages (CD68+ and CD80+) in the wound.

[0171] Figure 18 These are photographs of the M2 macrophages (CD68+ and CD206+) in four groups of wound tissues on days 3 and 5, as detected by immunofluorescence double staining in Example 10.

[0172] Figure 19 This is a comparative analysis of the effects of the four groups of drugs in Example 10 on the regulation of M2 macrophages (CD68+ and CD206+) in the wound.

[0173] Figure 20 This is a graph showing the M1 / M2 macrophage results analysis in Example 10;

[0174] Figure 21 These are photographs of the expression of Claudin1, a wound junction protein, in four groups as detected by immunofluorescence double staining in Example 10.

[0175] Figure 22 This is a comparative analysis of the effects of the four groups of drugs in Example 10 on the regulation of wound connexin (Claudin1);

[0176] Figure 23 These are photographs of the expression of Claudin2, a wound healing protein, detected by immunofluorescence double staining in four groups in Example 10.

[0177] Figure 24 This is a comparative analysis of the effects of the four groups of drugs in Example 10 on the regulation of wound connexin (Claudin2);

[0178] Figure 25 The images show the expression of the Nrf2 / HO-1 / NQO1 signaling pathway in four groups of wounds as detected by Western blotting in Example 10.

[0179] Figure 26 This is a comparative analysis of the expression of the Nrf2 / HO-1 / NQO1 signaling pathway in the wound on days 3 and 5 of the four drug groups in Example 10, reflecting the effects of oxidative stress.

[0180] Figure 27 The images show the expression of inflammatory factors IL-6, TNF-α, iNOS, and MIP-1β in four groups of wounds as detected by Western blotting in Example 10.

[0181] Figure 28 This is a comparative chart showing the effects of four drug groups in Example 10 on the expression of inflammatory factors IL-6, TNF-α, iNOS, and MIP-1β on the wound on days 3 and 5. Detailed Implementation

[0182] The present invention will be further described in detail below with reference to embodiments. It should be noted that the embodiments described below are intended to facilitate understanding of the present invention and are not intended to limit it in any way. All reagents used in the present invention are commercially available reagents.

[0183] Example 1: Preparation of propynolyl glycine-Hst1-MAD cyclic peptide

[0184] The preparation method in this embodiment is as follows:

[0185] 1. An alkynyl group and a protecting group Fmoc are introduced onto glycine, and an azido group and a protecting group Fmoc are introduced onto lysine to obtain Fmoc-propynylglycine (Formula 18) for C-terminal attachment (Fmoc-propynylglycine purchased from Merck KGaA in this embodiment) and Fmoc-azidolysine (Formula 19) for N-terminal attachment (Fmoc-azidolysine purchased from Merck KGaA in this embodiment), with the following structural formulas:

[0186]

[0187] 2. Based on Fmoc-azidolysine, from right to left, straight-chain amino acids of Hst1-MAD (SEQ ID NO.1) were synthesized step by step using solid-phase peptide synthesis (SPPS), and Fmoc-propynylglycine was attached to the C-terminus of Hst1-MAD (SEQ ID NO.1);

[0188] 3. Using the linear Hst1-MAD polypeptide prepared in step 2, a cyclic peptide of Hst1-MAD (SEQ ID NO.1) (Click-Hst1-MAD) is prepared via a copper-catalyzed azide-alkyne cycloaddition reaction. The preparation method includes the following steps:

[0189] 1) Place 750 mg of the domain of Hst1 linear polypeptide in a container and add 20 ml of a mixed solution of acetonitrile and dimethyl sulfoxide in a 4:1 ratio;

[0190] 2) Add 750mg of Rink Amide MBHA Resin (purchased from Merck KGaA);

[0191] 3) Add 20mM cuprous iodide;

[0192] 4) Add 30 μl of 2,6-rutidine (dimethylpyridine);

[0193] 5) Blow nitrogen gas for 5 minutes;

[0194] 6) Tightly cover and seal the container;

[0195] 7) Stir and react at room temperature for 40 minutes;

[0196] 8) Wash twice with a saturated disodium ethylenediaminetetraacetate solution, 10 minutes each time, and remove the washing solution;

[0197] 9) Rinse once with water, once with acetonitrile, once with dichloromethane, and once with ether to remove the washing solution;

[0198] 10) Vacuum dry for 1 hour;

[0199] 11) Shearing: Prepare a shearing mixture comprising 95.5% trifluoroacetic acid, 2% phenol aqueous solution, 2% anisole sulfide, and 0.5% triisopropylsilane. Add 20 ml of the shearing mixture to the sample obtained in step 10), shake for 3 hours, and shear the peptide from the template. Filter the peptide with cold ether, wash 3 times, and dry.

[0200] 12) High-performance liquid chromatography-mass spectrometry (HPLC) purification was performed using an aqueous solution of acetonitrile containing 0.1% (v / v) trifluoroacetic acid as the mobile phase for gradient elution, with a detection wavelength of 210 nm.

[0201] 707 mg of propynoglycine-Hst1-MAD cyclic peptide was obtained.

[0202] The structural formula of the propynoglycine-Hst1-MAD cyclic peptide is shown in Formula 20:

[0203]

[0204] Mass spectrometry analysis showed the following chromatogram: Figure 1 As shown, its purity is above 95%, and its recovery rate is 94%. HPLC analysis yielded the following chromatogram: Figure 2 As shown; the detection spectrum obtained by infrared (IR) detection is as follows. Figure 3 As shown, it exhibits the characteristic peak of triazole (3100 cm⁻¹). -1 1400cm -1 1100cm -1 700cm -1 ), without azide (2100cm) -1 ) and acetylene (2200cm) -1 Characteristic peaks of ).

[0205] Example 2: Preparation of high-propynylglycine-Hst1-MAD cyclic peptide

[0206] The preparation method in this embodiment is as follows:

[0207] 1. An alkynyl group and a protecting group Fmoc are introduced onto glycine, and an azido group and a protecting group Fmoc are introduced onto lysine to prepare Fmoc-propynylglycine (Formula 21) for C-terminal attachment (Fmoc-propynylglycine purchased from Merck KGaA in this embodiment) and Fmoc-azidolysine (Formula 19) for N-terminal attachment (Fmoc-azidolysine purchased from Merck KGaA in this embodiment), with the following structural formulas:

[0208]

[0209] 2. Based on Fmoc-azidolysine, from right to left, straight-chain amino acids of Hst1-MAD (SEQ ID NO.1) were synthesized step by step using solid-phase peptide synthesis (SPPS), and Fmoc-propynylglycine was linked to the C-terminus of Hst1-MAD.

[0210] 3. Using the Hst1-MAD linear polypeptide prepared in step 2, a copper-catalyzed azido-alkyne cycloaddition reaction is used to prepare the Hst1-MAD cyclic peptide, wherein the preparation method is in accordance with the method provided in Example 1.

[0211] 698 mg of propynoglycine-Hst1-MAD cyclic peptide was obtained.

[0212] The structural formula of the propynoglycine-Hst1-MAD cyclic peptide is shown in Formula 22:

[0213]

[0214] Mass spectrometry analysis showed a purity of over 95% and a recovery rate of 93%. Infrared spectroscopy (IR) analysis revealed the presence of a triazole characteristic peak (3100 cm⁻¹). -1 1400cm -1 1100cm -1 700cm -1 ), without azide (2100cm) -1 ) and acetylene (2200cm) -1 Characteristic peaks of ).

[0215] Example 3: Preparation of dual-high-propynylglycine-Hst1-MAD cyclic peptide

[0216] The preparation method in this embodiment is as follows:

[0217] 1. An alkynyl group and a protecting group Fmoc are introduced onto glycine, and an azido group and a protecting group Fmoc are introduced onto lysine to prepare Fmoc-propynylglycine (Formula 23) for C-terminal attachment (Fmoc-propynylglycine purchased from Merck KGaA in this embodiment) and Fmoc-azidolysine (Formula 19) for N-terminal attachment (Fmoc-azidolysine purchased from Merck KGaA in this embodiment), with the following structural formulas:

[0218]

[0219] 2. Based on Fmoc-azidolysine, from right to left, straight-chain amino acids of Hst1-MAD (SEQ ID NO.1) were synthesized step by step using solid-phase peptide synthesis (SPPS), and Fmoc-propynylglycine was linked to the C-terminus of Hst1-MAD.

[0220] 3. Using the Hst1-MAD linear polypeptide prepared in step 2, a copper-catalyzed azido-alkyne cycloaddition reaction is used to prepare the Hst1-MAD cyclic peptide, wherein the preparation method is in accordance with the method provided in Example 1.

[0221] 699 mg of propynoglycine-Hst1-MAD cyclic peptide was obtained.

[0222] The structural formula of the propynoglycine-Hst1 cyclic peptide is shown in Formula 24:

[0223]

[0224] Mass spectrometry analysis showed a purity of over 95% and a recovery rate of 93%. Infrared spectroscopy (IR) analysis revealed the presence of a triazole characteristic peak (3100 cm⁻¹). -1 1400cm -1 1100cm -1 700cm -1 ), without azide (2100cm) -1 ) and acetylene (2200cm) -1 Characteristic peaks of ).

[0225] Example 4: Effect of linear oligopeptide concentration on cyclization reaction

[0226] In this embodiment, propyne glycine-Hst1-MAD cyclic peptides were prepared according to the method provided in Example 1. 250, 500, 750, and 1000 mg of the linear oligopeptide were added to 20 ml of a mixed solution of acetonitrile and dimethyl sulfoxide in a 4:1 ratio, and the reaction was carried out. The required reaction time was recorded, and the yield of the reaction product was detected by HPLC. The proportion of side reactions occurring in the reaction product (including diploid, triploid, and tetraploid byproducts of intermolecular condensation, which were confirmed by molecular weight analysis using mass spectrometry, thus calculating the proportion of byproducts to the first and last cyclic peptides of the oligopeptide Hst1-MAD) was investigated. The effect of different concentrations of linear Hst1-MAD oligopeptide on the cyclization reaction was examined, and the results are shown in Table 1.

[0227] Table 1 Effect of different linear Hst1-MAD oligopeptides on the cyclization reaction

[0228] Increased dosage of linear oligopeptides (mg) Reaction time (min) Yield (%) Percentage of side reactions occurring (%) 250 40 82 15 500 40 91 7 750 40 94 3 1000 60 80 16

[0229] As shown in Table 1, different amounts of linear Hst1-MAD oligopeptide have a significant impact on the cyclization reaction. As the amount of linear oligopeptide increases, the reaction rate slows down, and the yield and the proportion of side reactions also change accordingly. When the amount of linear oligopeptide is 750 mg, the reaction rate is still relatively fast, the yield reaches 94%, and the proportion of side reactions is very low. Therefore, the optimal amount of linear oligopeptide to add in 20 ml of solvent is 750 mg.

[0230] Example 5: Effect of different solvents on cyclization reaction

[0231] In this embodiment, propyne glycine-Hst1-MAD cyclic peptide was prepared according to the method provided in Example 1. Different solvents as shown in Table 2 were used for the reaction, the required reaction time was recorded, and the yield of the reaction product was detected by HPLC. The proportion of side reactions in the reaction product was calculated to investigate the effect of different solvents on the cyclization reaction. The results are shown in Table 2.

[0232] Table 2 Effect of different solvents on cyclization reaction

[0233] solvent Reaction time (min) Yield (%) Percentage of side reactions occurring (%) methanol 120 44 51 tert-Butanol 90 54 41 dimethyl sulfoxide 60 67 29 Acetonitrile 90 57 40 Acetonitrile: Dimethyl sulfoxide (1:1) 60 84 11 Acetonitrile: Dimethyl sulfoxide (2:1) 40 94 3 Acetonitrile: Dimethyl sulfoxide (4:1) 40 91 7 Acetonitrile: Dimethyl sulfoxide (8:1) 60 89 9

[0234] As shown in Table 2, different solvents have a significant impact on the cyclization reaction. This is mainly because different solvents produce different steric hindrances, electrical properties, polarity, protonation, hydrogen bonding strength, and ratios, which affect the reaction rate, side reactions, byproducts such as dimers, trimers, polymers, and decomposition products. Therefore, acetonitrile:dimethyl sulfoxide (2:1) is preferred as the solvent.

[0235] Example 6: Effect of different resins on the cyclization reaction

[0236] In this embodiment, propyne glycine-Hst1-MAD cyclic peptide was prepared according to the method provided in Example 1. Eight different resins were used for the reaction: WangResin, Rink Amide AM Resin, Rink Amide MBHA Resin, 2-Chlorotrityl Resin, PEGylated Rink-modified TentaGel (TGR)resin, Aminomethyl Resin, Sieber Amide Resin, and PAM Resin. The required reaction time was recorded, and the yield of the reaction product was detected by HPLC. The proportion of side reactions in the reaction product was calculated to investigate the effect of different resins on the cyclization reaction. The results are shown in Table 3.

[0237] Table 3 Effect of different resins on cyclization reaction

[0238]

[0239] As shown in Table 3, the selection of different resins has a significant impact on the cyclization reaction, with significant changes in reaction rate, yield, and the proportion of side reactions. The optimal resin is Rink Amide MBHA Resin, which can achieve a product yield of over 94% and reduce the proportion of side reactions to 3%. This shows that combining with Rink Amide MBHA Resin can prevent the occurrence of side reactions and improve the efficiency of the cyclization reaction.

[0240] Example 7: Effect of different catalysts on cyclization reaction

[0241] In this embodiment, propyne glycine-Hst1-MAD cyclic peptide was prepared according to the method provided in Example 1. Copper iodide CuI, cuprous chloride CuCl, cuprous iodide / triethyl phosphite CuI·P(OEt)3, cuprous bromide, cuprous dimethyl sulfide bromide, tris(triphenylphosphine) fluoride [CuF(PPh3)3], tetraethyl copper tetrafluoroborate [Cu(MeCN)4]BF4, and tetraethyl copper hexafluorophosphate [Cu(MeCN)4]PF6 were used as catalysts for the reaction. The required reaction time was recorded, and the yield of the reaction product was detected by HPLC. The proportion of side reactions occurring in the reaction product was calculated to investigate the effect of different catalysts on the cyclization reaction. The results are shown in Table 4.

[0242] Table 4. Effects of different catalysts on the cyclization reaction

[0243] catalyst Reaction time (min) Yield (%) Percentage of side reactions occurring (%) Cuprous iodide 40 94 3 Cuprous chloride 60 89 9 Cuprous iodide / triethyl phosphite 40 56 41 Cuprous bromide 70 77 20 Cuprous dimethyl sulfide 60 69 25 Tri(triphenylphosphine)cuprous fluoride 80 70 27 Copper tetrafluoroborate 60 61 36 Copper hexafluorophosphate tetraethyl cyanophosphate 60 55 43

[0244] As shown in Table 4, different catalysts have a significant impact on the cyclization reaction, with obvious changes in reaction rate, yield, and the proportion of side reactions. Only when cuprous iodide is used as the catalyst can the product yield reach 94% and the proportion of side reactions be minimized. Therefore, cuprous iodide is the optimal catalyst.

[0245] Example 8: Effect of different ligands on cyclization reaction

[0246] In this embodiment, propyne glycine-Hst1-MAD cyclic peptide was prepared according to the method provided in Example 1. 2,6-dimethylpyridine (2,6-lutidine), diethylamine, triethylamine, n-propylamine, diisopropylamine, tributylamine, diisopropylethylamine (DIPEA), and dimethylaminopyridine (DMAP) were used as ligands for the reaction. The reaction time was recorded, and the yield of the reaction product was detected by HPLC. The proportion of side reactions occurring in the reaction product was calculated to investigate the effect of different ligands on the cyclization reaction. The results are shown in Table 5.

[0247] Table 5. Effects of different ligands on cyclization reaction

[0248] ligands Reaction time (min) Yield (%) Percentage of side reactions occurring (%) 2,6-Dimethylpyridine 40 94 3 Diethylamine 60 87 11 Triethylamine 50 66 31 n-Propylamine 70 78 20 diisopropylamine 60 70 26 Tributylamine 80 64 30 Diisopropylethylamine (DIPEA) 60 79 20 Dimethylaminopyridine DMAP 60 81 15

[0249] As shown in Table 5, the selection of different ligands has a significant impact on the cyclization reaction, with obvious changes in reaction rate, yield, and the proportion of side reactions. Therefore, 2,6-dimethylpyridine is the optimal ligand.

[0250] Example 9: Effect of reaction conditions on cyclization reaction

[0251] In this embodiment, propyne glycine-Hst1-MAD cyclic peptide was prepared according to the method provided in Example 1. The reaction was carried out under different reaction conditions as shown in Table 6. The reaction time was recorded, and the yield of the reaction product was detected by HPLC. The proportion of side reactions in the reaction product was calculated to investigate the effect of different reaction conditions on the cyclization reaction. The results are shown in Table 6.

[0252] Table 6. Effects of different reaction conditions on the cyclization reaction

[0253] ligands Reaction time (min) Yield (%) Percentage of side reactions occurring (%) Nitrogen gas bubble for 5 minutes at 25°C 40 94 3 Nitrogen gas bubble for 5 minutes at 50°C 60 87 10 Nitrogen gas bubble for 5 minutes at 100℃ 50 54 44 Nitrogen bubble for 5 minutes, then microwave. 60 77 20 Nitrogen-free bubble blowing, 25℃ 40 11 84

[0254] As shown in Table 6, different reaction conditions have a significant impact on the cyclization reaction, with significant changes in reaction rate, yield, and the proportion of side reactions. Therefore, the optimal reaction conditions are nitrogen bubbling for 5 minutes at 25°C.

[0255] Example 10: The effect of Hst1-MAD cyclic peptide on wound repair

[0256] In this embodiment, animal experiments were conducted using the propynoglycine-Hst1-MAD cyclic peptide (Click-Hst1-MAD), linear Hst1-MAD, and linear peptide Hst1 (linear histamine 1) provided in Example 1. A control group was also set up to observe the effect of Click-Hst1-MAD on the wound healing rate in mice. The concentrations of linear peptide Hst1, linear Hst1-MAD, and Click-Hst1-MAD were all 10 μM.

[0257] 1. Materials and Methods

[0258] This study was approved by the Medical Animal Center and Ethics Committee of the Southern Theater General Hospital of the Chinese People's Liberation Army.

[0259] 1.1 Animals and sources of major reagents and instruments

[0260] Thirty-six healthy male C57 BL / 6 mice, aged 6-8 weeks and weighing 22-28g, were purchased from the Guangdong Provincial Laboratory Animal Center. Diaminobenzidine (DAB) was purchased from Wuhan Boster Biological Engineering Co., Ltd. Rabbit anti-CD31 antibody, mouse anti-VEGF antibody, goat anti-rabbit secondary antibody, and goat anti-mouse secondary antibody were purchased from Servicebio, USA. Ethylenediaminetetraacetic acid (EDTA) antigen buffer, immunohistochemical pen, bovine serum albumin, collagenase A, and hematoxylin were all purchased from Wuhan Google Biotechnology Co., Ltd. Inverted fluorescence microscope was purchased from Nikon Corporation, Japan. A dehydrator was purchased from Wuhan Junjie Electronics Co., Ltd., and a paraffin microtome was purchased from Leica Instruments, Shanghai.

[0261] 1.2 Animal grouping and treatment

[0262] Thirty-six healthy male specific pathogen-free C57 BL / 6 mice (purchased from Guangdong Provincial Experimental Animal Center), aged 6-8 weeks and weighing 22-28g, were anesthetized by intraperitoneal injection of 5ml / kg 1% sodium pentobarbital. After the mice lost their corneal reflex, the hair on their backs was carefully removed and the wounds were disinfected with alcohol. Two 1cm × 1cm full-thickness skin defects were prepared on the backs of each mouse using a circular skin punch. The 36 mice were randomly divided into three groups using a random number table: a control group (n=9), a 10μM linear peptide Hst1 (Histatin1) group (n=9), a 1μM Hst1-MAD linear peptide group (n=9), and a 1μM propargyl glycine-Hst1-MAD cyclic peptide group (Click-Hst1-MAD) group (n=9). Each wound was treated with 0.5 ml of one of the four drugs daily until the wounds in the experimental group healed. Photos were taken on days 0, 3, 5, and 10 post-surgery, and tissue samples were taken from the wounds on days 3, 5, and 10 for subsequent pathological staining.

[0263] 1.3 Observation Indicators

[0264] 1.3.1 Wound healing rate

[0265] The mice were observed for wound healing at 0, 3, 5, and 10 days post-injury, and photographs were taken using a digital camera (see [link to article]). Figure 4 And calculate the wound healing rate (see...) Figure 5 Wound healing rate = (immediate post-injury wound area – unhealed wound area at each time point) ÷ immediate post-injury wound area × 100%.

[0266] 1.3.2 Tissue Morphology Examination

[0267] Wound specimens were fixed in 4% paraformaldehyde for 48 hours, rinsed with tap water for 10 minutes, dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and sectioned to a thickness of 4 μm. Selected sections were subjected to hematoxylin and eosin (HE) staining to assess epidermal thickness and Masson staining to assess collagen regeneration. The effects of the four drug groups on epidermal regeneration on day 10 were evaluated using hematoxylin-eosin (HE) staining. Figure 6 and Figure 7 The effects of four drug groups on collagen regeneration in the wound were assessed on days 5 and 10 using Masson staining. Figure 8 and Figure 9 Five slices were taken from each mouse, and five fields of view were selected from each slice. Data analysis was performed using Image-pro Plus software.

[0268] 1.3.3 Immunohistochemical staining

[0269] Paraffin sections prepared in section 1.3.2, 10 days post-injury, were dewaxed and rehydrated with xylene, then subjected to antigen heat retrieval for 20 minutes in 0.1 mol / L citrate buffer (pH 6.0), followed by inactivation of endogenous peroxidase with 10% hydrogen peroxide for 10 minutes, and blocking with 50 g / L bovine serum albumin for 2 hours. Rabbit anti-mouse CD31 primary antibody (dilution 1:300) and mouse anti-VEGF (dilution 1:100) were added, and the sections were incubated overnight at 4°C, followed by washing with PBS. The sections were then warmed for 1 hour, washed with PBS, and incubated for 2 hours with ready-to-use biotinylated goat anti-mouse and goat anti-rabbit IgG secondary antibodies, followed by washing with PBS. DAB staining, hematoxylin counterstaining, graded ethanol dehydration, xylene clearing, and resin mounting were performed. Five sections were taken from each mouse, and five fields of view were selected from each section. The sections were observed under a 400x inverted fluorescence microscope, and the number of CD31-positive vessels was analyzed using Image-pro Plus software (see [link to image]). Figure 10 ) and VEGF expression levels (see Figure 11 ).

[0270] 1.3.4 Immunofluorescence double staining detection of dendritic cells, M1 macrophages, Claudin1 and Claudin2

[0271] Immunofluorescence double staining detection of dendritic cells (MHCⅡ+ and CD11c+): The effect of 1 μM Hst1-MAD on the regulation of dendritic cells (MHCⅡ+ and CD11c+) in the wound was evaluated by immunofluorescence double staining on days 3 and 5.

[0272] Immunofluorescence double staining method for detecting M1 macrophages (CD68+ and CD80+) and M2 macrophages (CD68+ and CD206+): The effect of 1 μM Hst1-MAD on the regulation of wound macrophages on days 3 and 5 was evaluated by immunofluorescence double staining.

[0273] Immunofluorescence double staining method for detecting Claudin1: The effect of 1 μM Hst1-MAD on wound junction protein (Claudin1) on day 10 was evaluated by immunofluorescence double staining.

[0274] Immunofluorescence double staining method for detecting Claudin2: The effect of 1 μM Hst1-MAD on wound connexin (Claudin2) on day 10 was evaluated by immunofluorescence double staining.

[0275] Specific steps: The preceding steps are the same as in 1.3.3. Antibodies MHC II (orb101661; 1:500; Biobyt Biotechnology Co., Ltd., Wuhan, CN), CD11c (97585; 1:200; Cell Signaling Technology Inc., Boston, MA), CD68 (GB11067; 1:300; Servicebio Inc.), CD80 (GB11034; 1:300; Servicebio Inc.), CD206 (GB11062; 1:500; Servicebio Inc.), claudin1 (37-4900; 1:100; Thermo Fisher Scientific Co., Ltd., Shanghai, CN), and claudin2 (32-5600; 1:200; Thermo Fisher Scientific Co., Ltd.) were incubated overnight at 4°C. Fluorescently labeled secondary antibody was incubated at room temperature for 1 hour, followed by inoculation with 4',6-diamidino-2'-phenylindole (DAPI) (G1012; Google Biotechnology Co., Ltd., Wuhan, CN). Stained sections were photographed using a fluorescence microscope (Nikon Eclipse TI-SR, Tokyo, Japan). Positive cells were automatically counted and analyzed using Image-Pro Plus software. The expression levels of claudin1 and claudin2 were quantified using MOD values.

[0276] 1.3.5 Western Blotting

[0277] Western blotting method for detecting the Nrf2 / HO-1 / NQO1 signaling pathway: The effects of 1 μM Hst1-MAD and Click-Hst1-MAD on day 3 and day 5 against oxidative stress on the Nrf2 / HO-1 / NQO1 signaling pathway were evaluated by WB experiments.

[0278] Western blotting method for detecting inflammatory factor expression: The effects of 1 μM Hst1-MAD and Click-Hst1-MAD on the regulation of inflammatory factor expression in wounds on days 3 and 5 were evaluated by WB experiments.

[0279] Specific steps: To detect the expression of inflammatory factors and the regulation of oxidative stress, wound tissue was collected for Western blot analysis. The wound tissue was washed with PBS, lysis buffer was added, and the mixture was homogenized and centrifuged at 12000g for 10 min. The supernatant was collected. The bicinchoninic acid (BCA) kit (p0010; Beyotime Biotechnology Co., Ltd., Shanghai, CN) was mixed with reduced sodium dodecyl sulfate (SDS) sample buffer, and then boiled for 5 min. The sample was loaded onto an SDS-PAGE gel, followed by electrophoresis at 80 volts for 30 min and then at 120 volts for 1 h. After being transferred into a polyvinylidene fluoride (PVDF) membrane, the sample was incubated with 5% skim milk powder for 1 hour, and the primary antibodies Nrf2 (ab137550; 1:1000; Abcam Trade Co., Ltd.), NQO1 (ab28947; 1:1000; Abcam Trade Co., Ltd.), TNF-α (ab6671; 1:1000; Abcam Trade Co., Ltd.), IL-6 (ab9324; 1:1000; Abcam Trade Co., Ltd.), and macrophage inflammatory protein-1β (MIP-1β) (C04131; 1:1000; Signalway Antibody Co., Ltd., Nanjing, CN) were incubated overnight at 4°C. Subsequently, the samples were incubated at room temperature for 1 hour with enzyme-labeled goat anti-rabbit secondary antibody (SA00001-2; 1:3000; Proteintech Group Inc., Wuhan, CN) and HRP-labeled goat anti-mouse secondary antibody (GB23301; 1:3000; Proteintech Group Inc.), followed by chemiluminescence visualization. Western blot analysis of the above proteins was performed using ImageJ software to detect their expression.

[0280] 1.4 Statistical Analysis

[0281] Quantitative data are expressed as mean ± SD. SPSS 20.0 software was used to perform one-way ANOVA and independent samples t-tests to detect statistical significance among groups. Bonferroni tests were used for pairwise comparisons. *p<0.05, **p<0.01, ***p<0.001 were considered statistically significant, while p>0.05 was not statistically significant.

[0282] 2 Results

[0283] 2.1 Gross observation and wound healing rate

[0284] Postoperative days 3, 5, and 10 showed that the wound areas in all four groups gradually decreased over time. The 1μM propargyl glycine-Hst1-MAD cyclic peptide group (Click-Hst1-MAD) had smaller wound areas at days 3 and 5 compared to the other three groups. Figure 4 ).Depend on Figure 5 As can be seen, on days 3 and 5, the wound healing rate of the 1μM Click-Hst1-MAD group was significantly higher than that of the Control, Histatin1, and Hst1-MAD linear peptide groups (P < 0.05). By day 10, the mouse wounds had basically been repaired, so the results were not relevant. Specifically, the 1μM Click-Hst1-MAD, 1μM Hst1-MAD, and 10μM Hst groups showed significant differences compared to the Control group (P < 0.05). This indicates that the 1μM Click-Hst1-MAD group can better promote wound healing, with effects not only better than the 1μM Hst1-MAD linear peptide group but also better than the 10μM Histatin group. Furthermore, the effects of the 1μM Hst1-MAD linear peptide group are comparable to those of the 10μM Histatin linear peptide group.

[0285] 2.2 Observation of tissue morphology

[0286] To assess the effects of the four groups on epidermal thickness and dermal collagen expression, wound tissue was stained with hematoxylin and eosin (HE). Figure 6 On day 10, the wound epidermis was thickest in the 1μM Click-Hst1-MAD group, followed by the 10μM Histatin1 group, while the epidermis was thinnest in the Control group and the 1μM Hst1-MAD group (P<0.05). Figure 7 ).

[0287] Masson staining results for collagen regeneration expression are shown below. Figure 8 On days 5 and 10, the collagen regeneration expression level in the 1 μM Click-Hst1-MAD group was significantly higher than that in the other three groups. Figure 9At day 5, the dermal collagen expression levels in the 1 μM Click-Hst1-MAD group and the 10 μM Histatin1 group were comparable. Specifically, there were significant differences between the 1 μM Click-Hst1-MAD group, the 1 μM Hst1-MAD group, the 10 μM Histatin1 group, and the Control group (P < 0.05). This indicates that the 1 μM Click-Hst1-MAD group can better promote collagen regeneration.

[0288] 2.3 Immunohistochemical staining

[0289] CD31 and VEGF are angiogenesis markers. Immunohistochemical staining was used to assess the effects of 1 μM Click-Hst1-MAD group, 1 μM Hst1-MAD group, 10 μM Histatin1 and Control group on wound angiogenesis.

[0290] Immunohistochemical staining assessment of the number of newly formed CD31-positive vessels in the wounds of the four groups showed the following results: Figure 10 and 11 As shown. By Figure 10 As can be seen, at 5 and 10 days post-operation, the number of CD31-positive vessels per field of view in the 1 μM Click-Hst1-MAD group, 1 μM Hst1-MAD group, and 10 μM Histatin1 group was significantly higher than that in the Control (arrow indicated) scale bar = 50 μm; Figure 11 As can be seen from quantitative analysis of CD31-positive vessel count, the 1μM Click-Hst1-MAD group had a higher number of newly formed vessels than the other three groups. §: There were significant differences between the 1μM Click-Hst1-MAD group, the 10μM Histatin1 group, the 1μM Hst1-MAD group, and the Control group (P<0.05).

[0291] Immunohistochemical staining assessment of VEGF expression levels in four wound groups showed the following results: Figure 12 and 13 As shown. By Figure 12 Ten days post-surgery, the VEGF-positive expression levels were significantly higher in the 1 μM Click-Hst1-MAD group and the 10 μM Histatin1 group (arrows indicated). Scale bar = 50 μm. Figure 13 Among them, (1) is the result of VEGF integral density analysis, and (2) is the result of VEGF expression area analysis. Figure 13Quantitative analysis of VEGF in (1) and (2) showed that at 5 days, the VEGF expression levels in the 1 μM Click-Hst1-MAD group, the 10 μM Histatin1 group, and the 1 μM Hst1-MAD group were comparable; at 10 days, the VEGF expression level in the 1 μM Click-Hst1-MAD group was significantly higher than that in the other three groups. §There were significant differences between the 1 μM Click-Hst1-MAD group, the 10 μM Histatin1 group, the 1 μM Hst1-MAD group, and the Control group (P < 0.05).

[0292] 2.4 Immunofluorescence double staining

[0293] Images of dendritic cells (MHC II+ and CD11c+) detected by immunofluorescence double staining are shown below. Figure 14 The effects of four drugs on the regulation of dendritic cells (MHC II+ and CD11c+) in wounds were analyzed as follows: Figure 15 .Depend on Figure 15 As can be seen from the assessment on days 3 and 5, on day 3 post-surgery, the number of dendritic cells in the 10μM Hst1 group and the 1μM Click-Hst1-MAD group were 1.75 times and 1.95 times that of the control group, respectively. The number of dendritic cells in the 1μM Hst1-MAD group was slightly lower than that in the 10μM Histatin1 group, but both were significantly higher than those in the Control group. The number of dendritic cells in the Click-Hst1-MAD group was significantly higher than that in the other three groups. It can be seen that the 1μM Click-Hst1-MAD group (1μM propargyl glycine-Hst1-MAD cyclic peptide) can significantly promote the growth of dendritic cells (MHCⅡ+ and CD11c+) compared with the linear Hst1-MAD peptide.

[0294] Images of M1 macrophages (CD68+ and CD80+) detected by immunofluorescence double staining are shown below. Figure 16 The effects of four drugs on the regulation of M1 macrophages (CD68+ and CD80+) in the wound were analyzed as follows: Figure 17 .Depend on Figure 17The results were assessed on days 3 and 5. The number of M1 macrophages (CD68+ and CD80+) in the 1μM Hst1-MAD group was slightly higher than that in the 10μM Histatin1 group, but both were lower than those in the Control group. This indicates that both the Hst1-MAD and Histatin1 groups can reduce the number of M1 macrophages (CD68+ and CD80+) in the wound. On day 3, the number of M1 macrophages (CD68+ and CD80+) in the Click-Hst1-MAD group was comparable to that in the Control group. However, on day 5, the number of M1 macrophages (CD68+ and CD80+) in the Click-Hst1-MAD group was significantly lower than that in the other three groups. This shows that 1μM propargylglycine-Hst1-MAD cyclic peptide significantly reduced the number of M1 macrophages (CD68+ and CD80+) after day 5, indicating that 1μM propargylglycine-Hst1-MAD cyclic peptide can better alleviate the inflammatory response in the later stages of inflammation.

[0295] Images of M2 macrophages (CD68+ and CD206+) detected by immunofluorescence double staining are shown below. Figure 18 The effects of four drugs on the regulation of M2 macrophages (CD68+ and CD206+) in the wound were analyzed as follows: Figure 19 .Depend on Figure 19 On day 3, the number of M2 macrophages (CD68+ and CD206+) in the Click-Hst1-MAD group was significantly higher than that in the other three groups. The number of M2 macrophages in the 1μM Click-Hst1-MAD group (16.40±5.86) and the 10μM Hst1 group (12.67±7.34) was higher than that in the control group (12.17±4.44). On day 5, the number of M2 macrophages (CD68+ and CD206+) in the Click-Hst1-MAD group was also slightly higher than that in the other three groups. M2 cells secrete inflammatory factors that can promote wound healing, indicating that 1μM propargyl glycine-Hst1-MAD cyclic peptide can more effectively promote wound healing.

[0296] As can be seen, the M2 / M1 macrophage ratio in the Click-Hst1-MAD group was significantly higher than that in the control group (2.61 times and 2.17 times, respectively), the 10μM Histatin1 group, and the 1μM Hst1-MAD group. This indicates that the Click-Hst1-MAD group can better promote the conversion of M1 pro-inflammatory macrophages to M2 pro-healing macrophages. Figure 20 ).

[0297] Images of Claudin1, a wound connexin detected by immunofluorescence double staining, are shown below. Figure 21The effects of four drugs on the regulation of wound connexin 1 (Claudin1) were analyzed as follows: Figure 22 .Depend on Figure 22 On day 10, the Claudin1 expression intensity in the Click-Hst1-MAD group was significantly greater than that in the other three groups. The 10μM Histatin1 group and the 1μM Hst1-MAD group promoted Claudin1 expression to a similar degree, while the 1μM Click-Hst1-MAD group could further and effectively promote Claudin1 expression.

[0298] Images of Claudin2, a wound connexin detected by immunofluorescence double staining, are shown below. Figure 23 The effects of four drugs on the regulation of wound connexin 2 (Claudin2) were analyzed as follows: Figure 24 .Depend on Figure 24 On day 10, the Claudin2 expression intensity in the Click-Hst1-MAD group was significantly higher than that in the other three groups, followed by the 1μM Hst1-MAD group. The 10μM Histatin1 group showed no significant effect in promoting Claudin2 expression, and the difference was not significant compared to the Control group. This indicates that compared to the 10μM Histatin1 linear peptide, the Hst1-MAD linear peptide and the Click-Hst1-MAD cyclic peptide are more effective in promoting Claudin2 expression, especially the Click-Hst1-MAD cyclic peptide (1μM propargylglycine-Hst1-MAD cyclic peptide).

[0299] 2.5 Western Blotting

[0300] The experimental results of Western blotting detection of the Nrf2 / HO-1 / NQO1 signaling pathway are shown in the image below. Figure 25 As shown, the analysis of the effects of the four drugs on the Nrf2 / HO-1 / NQO1 signaling pathway under oxidative stress on days 3 and 5 is as follows. Figure 26 As shown. By Figure 25 and 26It can be seen that on the 3rd day after surgery, the expression levels of Nrf2 and HO-1 in the 10μM Histatin1 group were 1.27 and 1.80 times that of the control group, respectively. The expression level of NQO1 in the 10μM Hst1 group was significantly higher than that in the control group (2.08 times). The expression levels of Nrf2 and HO-1 in the Click-Hst1-MAD group and the Hst1-MAD group were also higher than those in the control group and comparable to those in the Histatin1 group. On the 5th day after surgery, the expression levels of Nrf2, HO-1 and NQO1 signaling pathways in the 1μM Click-Hst1-MAD group were significantly higher than those in the control group and the Histatin1 group.

[0301] The experimental results of detecting the expression levels of inflammatory factors using Western blotting are shown in the image. Figure 27 As shown, the analysis of the effects of the four drug groups on the regulation of wound inflammatory factor expression on days 3 and 5 is as follows. Figure 28 As shown. By Figure 27 and 28 It can be seen that, compared with the control group, all three drugs can effectively inhibit the expression levels of inflammatory factors IL-6, TNF-α, iNOS, and MIP-1β. Among them, for the inflammatory factor TNF-α, the inhibitory effect of the 1μM Click-Hst1-MAD group was significantly better than the other three groups, both on days 3 and 5 postoperatively. For the inflammatory factor iNOS, the inhibitory effect of the 1μM Click-Hst1-MAD group was significantly better than the other three groups on day 3 postoperatively. On day 5 postoperatively, the inhibitory effect of the 1μM Click-Hst1-MAD group was similar to that of the 1μM Hst1-MAD group, and significantly better than the control group and the 10μM Histatin1 group. For the inflammatory factors IL-6 and MIP-1β, the inhibitory effects of the 1μM Click-Hst1-MAD group and the 1μM Hst1-MAD group were also significantly better than those of the control group and the 10μM Histatin1 group.

[0302] Example 11: Comparison of the activities of cyclic peptides

[0303] This embodiment uses propargylglycine-Hst1-MAD cyclic peptide, high propargylglycine-Hst1-MAD cyclic peptide, and double high propargylglycine-Hst1-MAD cyclic peptide prepared in Examples 1, 2, and 3, respectively, and Hst1-MAD head-and tail cyclic peptide prepared using the amide bond cyclization method provided in the literature "Sortase A as a tool for high-yield histatin cyclization, Jan GMBolscher, The FASEB Journal. Research Communication". Reaction times were recorded, and the yield of the reaction products was detected by HPLC. The proportion of side reactions in the reaction products was calculated, and activity comparisons were performed. The activity comparison method was to calculate the dose required to achieve the same efficacy through a scratch assay. Specifically, fibroblasts (NIH / 3T3; GNM 6; Chinese Academy of Sciences, China) were simultaneously subjected to a 2×10⁻⁶... 6 The cells were seeded into 6-well plates and cultured to 70-80% confluence. Cells were starved for 8-12 hours before the experiment. Mitomycin C (Sigma Aldrich, USA) was added to the culture medium to a concentration of 15 μg / ml, and the medium was changed after 3 hours. Using a 200 μl pipette tip, the cells were scratched perpendicularly to the bottom of the plate with the same force. After scratching, the cells were washed twice with PBS. An appropriate amount of DEME medium (10% FBS, 1% penicillin-ethyl) was added to each well. Except for the blank control group, 10 μM of linear Hst1, 1 μM of propargyl glycine-Hst1-MAD cyclic peptide, 1 μM of propargyl glycine-Hst1-MAD cyclic peptide, 1 μM of propargyl glycine-Hst1-MAD cyclic peptide, 1 μM of linear Hst1-MAD, and 1 μM of Hst1-MAD cyclic peptide prepared by amide bond cyclization were added to each well, forming the experimental groups. Cells were then cultured at 37°C with 5% CO2. The scratch area was calculated using imageJ (Rawak Software, Inc., Germany) software at 12 and 24 hours post-culture. The scratch area at 0 hours was considered the initial scratch area. The scratch healing rate was calculated as follows: W% (scratch healing rate) = (W0 - Wt) / W0 × 100%, where W0 = initial scratch area and Wt = remaining scratch area. The results are shown in Table 7.

[0304] Table 7 Comparison of Activities

[0305]

[0306]

[0307] As shown in Table 7, the Hst1-MAD oligopeptide cyclic peptide prepared by the method provided in this invention has more than 15 times higher activity than the linear peptide Hst1, and the yield is also higher with very few side reactions. In contrast, Hst1-MAD prepared by direct cyclization using conventional amide bonds has lower activity, even lower than the linear peptide Hst1-MAD, with a yield of only 10% and a side reaction rate as high as 89%. The operation process is cumbersome, the reaction time is long, and it is difficult to prepare Hst1-MAD cyclic peptide.

[0308] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for preparing oligopeptides and cyclic peptides, characterized in that, The structural formula of the oligopeptide cyclic peptide is shown below: The amino acid sequence of the Hst1-MAD is shown in SEQ ID NO.1 of the sequence listing; The method for preparing the oligopeptide cyclic peptide is as follows: 1) The Hst1-MAD linear peptide prepared by solid-phase peptide synthesis was placed in a container, and a mixed solution of acetonitrile and dimethyl sulfoxide in a 4:1 ratio was added; the Hst1-MAD linear peptide is Hst1-MAD with Fmoc-propynylglycine linked to the C-terminus and Fmoc-azidolysine linked to the N-terminus, and the Fmoc-azidolysine is bound to a resin; the resin is Rink AmideMBHA Resin; 2) Add a catalyst; the catalyst is cuprous iodide; 3) Add a ligand, wherein the ligand is 2,6-dimethylpyridine; 4) Use nitrogen gas to blow bubbles; 5) Tightly cover and seal the container, then let it stand and stir. 6) Wash with disodium ethylenediaminetetraacetate to remove the washing solution; 7) Rinse with water or an organic solvent to remove the detergent solution; 8) Vacuum drying; 9) Add the cutting mixture to cut the oligopeptide cyclic peptides off the resin; 10) Purify oligopeptides and cyclic peptides.

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