Cyclic peptide construction method based on sulfydryl-olefin photoclick chemistry and application thereof

Abstract

The invention belongs to the technical field of polypeptide cyclization, and particularly provides a cyclic peptide construction method based on sulfydryl-olefin photoclick chemistry, which comprises the following steps: S1, designing a polypeptide sequence containing a plurality of cysteines; s2, reducing disulfide bonds in the polypeptide through a mercaptan reducing agent; and S3, under the irradiation of ultraviolet light, carrying out sulfydryl-olefin light click chemical reaction on the reduced polypeptide, a cross-linking agent and a photoinitiator to form a cyclic peptide structure. According to the method, the addition reaction of sulfydryl and olefin is excited by ultraviolet light, a stable thioether bond is generated, a cyclic peptide structure is constructed, and the method has high selectivity and high efficiency, is carried out under mild conditions and does not need to use a metal catalyst. The method is applied to construction of a phage display cyclopeptide library, and particularly has wide application potential in construction of a bicyclic peptide library. The stability of the obtained cyclopeptide is remarkably enhanced, and an effective tool is provided for screening high-quality cyclopeptide ligands specifically combined with targets.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide cyclization, and specifically relates to a cyclic peptide construction method based on thiol-olefin photoclick chemistry and an application thereof. Background Art

[0002] In recent years, peptides have been widely used in the fields of chemistry, life sciences, and medicine. As molecules formed by amino acids connected by peptide bonds, peptides have the diversity, high affinity, and high selectivity of biological macromolecules, and the advantages of easy synthesis and modification of small molecules. However, linear peptides usually exhibit flexibility and multi-conformational properties due to the lack of a stable three-dimensional structure, which makes them have weak affinity when binding to targets, easily expose enzyme cleavage sites and be degraded in the body, limiting their biomedical applications. The cyclization method improves the stability, affinity, and selectivity of linear peptides by restricting them to a fixed three-dimensional conformation, and has great potential, especially in drug development.

[0003] At present, common methods for constructing cyclic peptides include disulfide bond cyclization, amidation reaction, and olefin metathesis reaction. Among them, disulfide bond cyclization forms disulfide bonds through the oxidation of the sulfhydryl group of cysteine, but it is prone to mispairing, breakage or aggregation, and the reaction conditions are limited. The amidation reaction of lysine can generate amide bonds, but the commonly used special reagents may trigger side reactions and reduce the reaction efficiency. Although the cyclization method based on olefin metathesis reaction improves the reaction efficiency, it requires the use of highly reactive reagents and metal catalysts, increases the risk of metal contamination and may lead to the formation of by-products. For example, although the cyclization strategy of 1,3,5-tri(bromomethyl)benzene can effectively carry out cyclization, the halogen reactants used are prone to trigger side reactions such as hydrolysis and elimination, affecting the reaction selectivity and efficiency. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems that the existing cyclic peptide construction method is prone to wrong pairing, breakage or aggregation, which may induce side reactions and reduce reaction efficiency.

[0005] To this end, the present invention provides a method for constructing a cyclic peptide based on thiol-olefin photoclick chemistry, comprising the following steps:

[0006] S1. Design a peptide sequence containing multiple cysteines;

[0007] S2, reducing disulfide bonds in the polypeptide using a thiol reducing agent;

[0008] S3. Under ultraviolet light, the reduced polypeptide is subjected to a thiol-olefin photo-click chemical reaction with a cross-linking agent and a photoinitiator to form a cyclic peptide structure.

[0009] Specifically, the common sequence of the above polypeptide is: ACX n-CB or ACX n -CY m -CB; where C represents cysteine, X n represents n random amino acids, Y m represents m random amino acids, A and B represent any number of amino acids at the C-terminus and N-terminus, respectively.

[0010] Specifically, the molar ratio of the thiol reducing agent to the polypeptide is ≥1:1; the molar ratio of the cross-linking agent to the polypeptide is ≥1:1; and the molar ratio of the photoinitiator to the polypeptide is (0.3-1):1.

[0011] Specifically, the thiol reducing agent includes tris(2-carboxyethyl)phosphine salt.

[0012] Specifically, the general formula of the cross-linking agent is R-(CH2CH=CH2)2 or R-(CH2CH=CH2)3, wherein R includes but is not limited to aromatic ring, heterocyclic ring, and alkyl ring.

[0013] Specifically, the photoinitiator includes, but is not limited to, any one of 2,2-dimethoxy-2-phenylacetophenone, bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide and phenyl(2,4,6-trimethylbenzoyl)lithium phosphate.

[0014] Specifically, in the above step S3, the ultraviolet light power includes but is not limited to 24W, and the wavelength includes but is not limited to 365-375nm; the photoclick chemistry reaction temperature is room temperature, and the reaction time is 3-10 minutes.

[0015] The present invention also provides a method for constructing a phage-displayed cyclic peptide library based on thiol-olefin photoclick chemistry, comprising the following steps:

[0016] S1. Displaying a polypeptide sequence on the surface of a phage to obtain a phage-displayed polypeptide library; the polypeptide comprises a plurality of cysteines;

[0017] S2, performing reduction treatment on the phage display peptide library to completely reduce the disulfide bonds in the peptides;

[0018] S3, adding a cross-linking agent and a photoinitiator, and carrying out a thiol-olefin photo-click reaction under ultraviolet light to obtain a cyclized phage-displayed cyclic peptide library.

[0019] The present invention also provides a method for screening cyclic peptide ligands, comprising the following steps:

[0020] S1, immobilize the target protein on the surface of the plate, add the cyclic peptide phage display library for co-incubation;

[0021] S2. After incubation, the specifically bound phages are eluted and recovered to screen out the cyclic peptide ligands with specific binding.

[0022] Specifically, the above-mentioned target protein includes but is not limited to cyclosporine A binding protein.

[0023] Specifically, the amino acid sequences of the affinity peptide ligands for cyclosporine A binding protein are: Ac-CRPYRQCVKGLMC-NH2 and Ac-CLPLRQCLWALIC-NH2.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] The cyclic peptide construction method based on thiol-olefin photoclick chemistry provided by the present invention utilizes ultraviolet light to excite the addition reaction of thiol and olefin to generate a stable thioether bond and construct a cyclic peptide structure, which has high selectivity and high efficiency, and is carried out under mild conditions, without the use of metal catalysts, and does not rely on highly reactive reagents or metal catalysts, is suitable for high-throughput screening, avoids the occurrence of side reactions, and effectively improves the purity and yield of the product. The method can also generate a structure that is more stable than traditional disulfide bonds, and is suitable for application in biological environments.

[0026] This method was applied to the construction of a phage-displayed cyclic peptide library. By introducing a thiol-containing peptide sequence into the phage display system and using thiol-olefin photo-click chemistry to achieve cyclization, a diverse cyclic peptide library was constructed, which expanded the cyclization construction of phage peptide libraries, especially in the construction of bicyclic peptides. The stability of the resulting cyclic peptides was significantly enhanced, and they had good tolerance to acids, bases, exogenous thiols, and oxidants, providing an effective tool for screening high-quality cyclic peptide ligands that specifically bind to targets, overcoming the defects of poor biocompatibility and insufficient stability of traditional methods, and were suitable for the fields of biomedicine and drug development.

[0027] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a structural diagram of the photoinitiator used in Example 1 of the present invention.

[0029] Figure 2 This is a diagram showing the photo-click chemical reaction mechanism between the thiol-containing compound and the olefin in Example 1 of the present invention.

[0030] Figure 3 This is a schematic diagram of the formation of a cyclic peptide by an allyl derivative and a polypeptide containing three cysteines in Example 3 of the present invention.

[0031] Figure 4 This is a HPLC characterization chart of polypeptide cyclization under different buffer solutions in Example 3 of the present invention.

[0032] Figure 5This is the sequencing map of the phage display peptide library constructed in Example 5 of the present invention.

[0033] Figure 6 Schematic diagram of the cyclization of the phage displayed peptide library in Example 5 of the present invention.

[0034] Figure 7 This is a graph of phage infection activity under different reaction conditions in Example 5 of the present invention.

[0035] Figure 8 This is a schematic diagram of the screening of the cyclic peptide phage library constructed in Example 5 of the present invention.

[0036] Fig. 9 It is the polypeptide sequence enriched after screening for the target protein CypA in Example 5 of the present invention.

[0037] Fig.10 HPLC characterization chart of the linear peptide and cyclic peptide of M1 in Example 5 of the present invention.

[0038] Fig.11 This is the SPR sensorgram of the cyclized M1 of the present invention against CypA.

[0039] Fig.12 HPLC characterization chart of the linear peptide and cyclic peptide of M2 in Example 5 of the present invention.

[0040] Fig.13 This is the SPR sensorgram of the cyclized M2 of the present invention against CypA. DETAILED DESCRIPTION

[0041] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be limited by the attached claims and their equivalents.

[0042] The present invention provides a method for constructing a cyclic peptide based on thiol-olefin photoclick chemistry, wherein under the conditions of ultraviolet light irradiation and the presence of a photoinitiator, an allyl derivative is used in a mixed solution of a buffer and an organic solvent to undergo a free radical addition reaction with a polypeptide containing multiple cysteines to form a cyclic peptide. Specifically, the method comprises the following steps:

[0043] S1. Design a peptide sequence containing multiple cysteines.

[0044] The polypeptide sequence preferably includes two or three cysteines (Cys), and the general sequence is: ACX n-CB or ACX n -CY m -CB; where C represents cysteine, X n represents n random amino acids, Y m represents m random amino acids, and the range of n and m is preferably 1 to 10. A and B represent any number of amino acids at the C-terminus and N-terminus, respectively.

[0045] S2. Reduction of disulfide bonds in peptides using a thiol reducing agent.

[0046] Specifically, a polypeptide powder containing cysteine ​​is dissolved in a solvent to obtain a solution 1, and the solution 1 and a thiol reducing agent are added to a buffer solution, wherein the molar ratio of the thiol reducing agent to the polypeptide in the solution 1 is ≥ 1:1, and the disulfide bonds in the polypeptide are completely reduced.

[0047] The thiol reducing agent is preferably tris(2-carboxyethyl)phosphine (TCEP).

[0048] S3. Under ultraviolet light, the reduced polypeptide is subjected to a thiol-olefin photo-click chemical reaction with a cross-linking agent and a photoinitiator to form a cyclic peptide structure.

[0049] Among them, the crosslinking agent is preferably a compound containing an allyl group, with the general formula of R-(CH2CH=CH2)2 or R-(CH2CH=CH2)3, wherein R is a core group, including but not limited to an aromatic ring, a heterocyclic ring, an alkyl group or other small molecular structures. The photoinitiator is preferably one of 2,2-dimethoxy-2-phenylacetophenone, bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide and phenyl(2,4,6-trimethylbenzoyl)lithium phosphate.

[0050] Specifically, the cross-linking agent is dissolved to obtain solution 2, and the photoinitiator is dissolved to obtain solution 3. After the polypeptide is fully reduced, solution 2 and solution 3 are added. The molar ratio of the cross-linking agent in solution 2 to the polypeptide in solution 1 is ≥1:1; the molar ratio of the photoinitiator in solution 3 to the polypeptide in solution 1 is (0.3-1):1. The cyclic peptide product is obtained by reaction under ultraviolet light irradiation, and the product is analyzed and characterized by high performance liquid chromatography and mass spectrometry. The power and wavelength of the ultraviolet light source can be adjusted according to actual conditions. Preferably, a UV lamp with a power of 24W and a wavelength of 365-375nm is used, the reaction temperature is room temperature (generally between 20°C and 25°C), and the time is 3-10 minutes.

[0051] The solvent in the method can be one of water, acetonitrile, methanol or dimethyl sulfoxide; the buffer can be PBS or Tris buffer with a pH of 6-10.

[0052] In a detailed implementation, the allyl derivative powder and the photoinitiator can be dissolved in acetonitrile to obtain a solution 2 or solution 3 with a concentration of 1 mol / L, and then diluted with water to 100 mmol / L or 10 mmol / L for later use.

[0053] Based on the above-mentioned thiol-olefin photo-click chemistry reaction and the highly efficient biocompatible reaction properties of cysteine-containing polypeptides, the present invention also provides a method for constructing a phage-displayed cyclic peptide library based on thiol-olefin photo-click chemistry, wherein a polypeptide library containing cysteine ​​is displayed on a phage, and the polypeptide library is cyclized using an allyl derivative, specifically comprising the following steps:

[0054] S1. Displaying a polypeptide sequence on the surface of a phage to obtain a phage-displayed polypeptide library; the polypeptide comprises a plurality of cysteines.

[0055] Specific, template: ACX n -CB or ACX n -CY m -CB, wherein C represents cysteine, X and Y represent random amino acids, n and m range from 1 to 10, preferably from 3 to 7, and A and B represent any number of amino acids at the C-terminus and N-terminus, respectively.

[0056] Preferably, the polypeptide sequence fused and expressed on the surface of the phage is: CXXXXXXXXXXC or CXXXXXCXXXXXC.

[0057] Wherein, X represents a random amino acid, and random amino acid mutations are introduced at the specified position by the NNK method.

[0058] The corresponding DNA sequence designed according to the above polypeptide sequence is:

[0059] 5'-CTAGGGATCCtgcNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKtgcGGCGGCTCGGCCTCGGGGGC-3' or

[0060] 5'-CTAGGGATCCtgcNNKNNKNNKNNKNNKtgcNNKNNKNNKNNKNNKtgcGGCGGCTCGGCCTCGGGGGC-3'.

[0061] The number of amino acids in the above DNA sequence can be increased or decreased as needed. All changes in polypeptide configuration and other modification methods made in the present invention should be considered within the scope of protection of this patent.

[0062] S2. Perform reduction treatment on the phage display peptide library to completely reduce the disulfide bonds in the peptides.

[0063] The general formula of the cross-linker structure used to construct the phage display cyclic peptide library is:

[0064]

[0065] The general formula is R-(CH2CH=CH2)2 or R-(CH2CH=CH2)3, wherein R is a core group, including but not limited to aromatic rings, heterocyclic rings, alkyl groups or other small molecule structures.

[0066] S3, adding a crosslinking agent and a photoinitiator, and performing a thiol-olefin photo-click reaction under ultraviolet light to obtain a cyclized phage-displayed cyclic peptide library. The specific method can refer to the above-mentioned cyclic peptide construction method.

[0067] The present invention also provides a method for screening cyclic peptide ligands, comprising the following steps:

[0068] S1. Immobilize the target protein on the surface of the plate and add the cyclic peptide phage display library for co-incubation.

[0069] S2. After incubation, unbound phages are washed away, and specifically bound phages are eluted with an acidic solution. The eluted phages are amplified, and multiple rounds of screening are repeated. Monoclonal phages are selected for DNA sequencing, the cyclic peptide sequences are analyzed, and the cyclic peptides with the highest enrichment are synthesized and purified, and affinity tests are performed using surface plasmon resonance (SPR).

[0070] The target protein includes but is not limited to cyclosporine A binding protein (CypA), and the amino acid sequences of the affinity peptide ligands for protein CypA are: Ac-CRPYRQCVKGLMC-NH2 and Ac-CLPLRQCLWALIC-NH2.

[0071] In a detailed embodiment, the cyclic peptide ligand screening method comprises:

[0072] (1) At room temperature, the crosslinking agent is dissolved to obtain solution 2, and the photoinitiator is dissolved to obtain solution 3. 10 TCEP was added to the phage library containing at least two or three cysteines displayed at the nitrogen end at pfu / mL to make the final concentration 1mM to reduce the phage library. Solution 2 was added to make the final concentration 150μM, and solution 3 was added to make the final concentration 75μM. The phage library was irradiated with a UV lamp at a power of 24W and a wavelength of 365nm for 10min. After the reaction, PEG-NaCl solution was added, and the mixture was incubated on ice to precipitate the phage. After precipitation, the mixture was centrifuged, the supernatant was removed, and the precipitate was resuspended in a phosphate buffer solution to obtain a phage cyclopeptide library modified with allyl derivatives.

[0073] The amount of PEG-NaCl added is preferably 1 / 5 of the total volume; the incubation time on ice can be 2 to 6 hours; and the centrifugation condition can be centrifugation at 8000 rpm for 20 minutes.

[0074] (2) Screening the monolithic phage cyclic peptide library: After the target protein is immobilized on a 96-well plate, the cyclic peptide library is added and incubated. After 1 hour, the unbound and low-affinity phages are washed away, and the specifically bound phages are eluted and recovered and amplified for the next round of screening. After multiple rounds of in vitro screening, the phages bound to the target protein are effectively enriched. Single clones of the colonies are picked for DNA sequencing to decode the amino acid sequence bound to the target protein.

[0075] The screening is preferably performed using CypA as the target protein; the in vitro screening can be performed for 3 to 5 rounds.

[0076] After the peptides with higher enrichment are synthesized, they are subjected to cyclization reaction. The affinity between the cyclic peptide ligand and the target protein is verified by biophysical experiments to test the feasibility and effectiveness of the cyclization and screening methods.

[0077] The following specific examples are used to study the effects of the cyclic peptide construction method based on thiol-olefin photoclick chemistry of the present invention and its application.

[0078] Embodiment 1:

[0079] This example explores the effects of different photoinitiators on the thiol-olefin photoclick chemistry reaction, and determines the best photoinitiator by the reaction yield. The reaction formula is as follows:

[0080]

[0081] The specific experimental steps are as follows:

[0082] 1) At room temperature, 0.3 mmol of N-acetyl-L-cysteine ​​methyl ester, 0.1 mmol of triallyl isocyanurate (TAIC) and 0.05 mmol of photoinitiator ( Figure 1 ) was added into a 10 mL test tube, and 1 mL of solvent (acetonitrile: water = 4:1) was added, and irradiated under a UV lamp with a power of 24 W and a wavelength of 365 nm for 10 min. The reaction solution was concentrated under reduced pressure and separated by column chromatography to obtain the product.

[0083] The reaction mechanism is Figure 2 As shown, the reaction yields under different photoinitiators are shown in Table 1, and the best photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

[0084] 1H NMR (600MHz, DMSO-d6) δ8.36(d,J=7.8Hz,1H),4.42(td,J=8.1,5.5Hz,1H),3.80(t,J=7.0Hz,2H),3.63(s,3H),2.85(d d,J=13.7,5.6Hz,1H),2.73(dd,J=13.7,8.2Hz,1H),2.53(t,J=7.4Hz,2H),1.85(s,3H),1.77(dq,J=13.9,7.3Hz,2H).

[0085] 13 C NMR (151MHz, DMSO-d6) δ171.32,169.44,149.10,52.05,51.99,41.47,32.41,28.72,27.10,22.26.

[0086] ESI-HRMS(m / z):calculated for C 30 H 49 N6O 12 S3[M+H] + :781.2571; found:781.2551.

[0087] Table 1 Reaction yields under different photoinitiators

[0088]

[0089] Embodiment 2:

[0090] This example explores the effects of different organic solvents on the thiol-olefin photo-click chemistry reaction, and determines the optimal organic solvent by the reaction yield. The specific experimental steps are as follows:

[0091] 0.3mmol of N-acetyl-L-cysteine ​​methyl ester, 0.1mmol of triallyl isocyanurate (TAIC) and 0.05mmol of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt were added to a 10mL test tube, 1mL of solvent (organic solvent: water = 4:1) was added, and irradiated for 10min under a UV lamp with a power of 24W and a wavelength of 365nm. After the reaction solution was concentrated under reduced pressure, the product was separated by column chromatography. The reaction yield results under different organic solvents are shown in Table 2, and the best organic solvent is acetonitrile.

[0092] Table 2 Reaction yields under different organic solvents

[0093]

[0094] Embodiment 3:

[0095] This example explores the effects of different buffer solutions on polypeptide cyclization, and determines the optimal reaction buffer solution by reaction yield. The reaction formula is as follows:

[0096]

[0097] The specific experimental steps are as follows:

[0098] The cyclization reaction linker triacrylate isocyanurate (TAIC, 100mM, 200μL), the peptide mother solution (100mM, 100μL) and the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (100mM, 50μL) were added to a 10mL reaction tube in sequence, and the system was adjusted with a mixed solvent of buffer and acetonitrile to ensure that the final acetonitrile volume fraction was 20%. The final concentrations of each component in the reaction system were: TAIC 20mM, peptide 10mM, and photoinitiator 5mM. The reaction system was placed at room temperature and irradiated with a UV light source with a power of 24W and a wavelength of 365nm for 10 minutes, followed by separation and purification by preparative reverse high performance liquid chromatography (HPLC), and the target cyclopeptide product was obtained after freeze-drying. Figure 3 The schematic diagram of the formation of cyclic peptide by allyl derivatives and polypeptides containing three cysteines; Figure 4 The HPLC chart of the reaction shows that the retention time of the linear peptide is 14.219 min and the retention time of the cyclized peptide is 15.215 min. Table 3 shows the yield of the cyclization reaction. The best buffer solution is pure water or Tris pH 6.0.

[0099] Table 3 Cyclization reaction yield

[0100]

[0101] ESI-HRMS(m / z):calculated for C 53 H 86 N 17 O 14 S3[M+H] + :1280.5702; found:1280.5731.

[0102] Embodiment 4:

[0103] This example explores the universality of the reaction and uses the reaction to cyclize peptides containing three cysteines and of different lengths. The specific experimental steps are as follows:

[0104] The cyclization reaction linker triacrylate isocyanurate (TAIC, 100mM, 200μL), the peptide mother solution (100mM, 100μL) and the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (100mM, 50μL) were added to a 10mL reaction tube in sequence, and the system was adjusted with a mixed solvent of water and acetonitrile to ensure that the final acetonitrile volume fraction was 20%. The final concentrations of each component in the reaction system were: TAIC 20mM, peptide 10mM, and photoinitiator 5mM. The reaction system was placed at room temperature and irradiated with a UV light source with a power of 24W and a wavelength of 365nm for 10 minutes, and then the reaction was monitored using HPLC. According to HPLC quantification, Table 4 shows the characterization data of the cyclization reaction, and the conversion rate of the peptide is 100%.

[0105] Table 4 Characterization data of cyclization reaction

[0106]

[0107] Embodiment 5:

[0108] CypA (Cyclophilin A) is widely involved in protein folding, signal transduction and other processes, and is closely related to diseases such as viral infection, inflammation and tumors. It is an important drug target. By screening cyclic peptide ligands, it is possible to effectively interfere with CypA-related pathological processes and provide potential candidate molecules for the development of antiviral, anti-inflammatory and anti-tumor drugs.

[0109] This example is based on the thiol-olefin photo-click chemistry modification of the phage library, using CypA as the target protein to screen for cyclic peptide ligands with high affinity.

[0110] (1) The specific steps of the phage display method are as follows:

[0111] a. Construction of phage display cyclic peptide library:

[0112] Template: ACX n -CY m -CB

[0113] Where C is cysteine, X and Y are random amino acids, and A and B represent any number of amino acids at the C-terminus and N-terminus, respectively;

[0114] The polypeptide sequence fused and expressed on the surface of the phage is: CXXXXXCXXXXXC;

[0115] Where X represents a random amino acid, and random amino acid mutations are introduced at the specified position by the NNK method;

[0116] The corresponding DNA sequence designed according to the polypeptide sequence displayed on the surface of the phage is:

[0117] 5'-CTAGGGATCC tgc NNKNNKNNKNNKNNKtgcNNKNNKNNKNNKNNK tgc GGCGGCTCGGCCTCGGGGGC-3′;

[0118] In the phagemid vector pbcomb-3xga, the library fragment was inserted using the BamHI single restriction site. The polypeptide framework in the DNA sequence of the fragment was indicated by lowercase and underline, and the restriction endonuclease cleavage site BamHI was indicated by uppercase and bold;

[0119] The phagemid library was constructed by direct PCR amplification of the pbcomb-3xga plasmid using two primers: MYF: 5'-CTAGGGATCC tgc NNKNNKNNKNNKNNKtgcNNKNNKNNKN NKNNK tgc GGCGGCTCGGCCTCGGGGGC-3' and MYR: 5'-CTAGGGATCCg gccgcctgggccacg-3'; digest the PCR product with restriction endonuclease BamHI, recover the digestion product with 1% agarose gel, connect the digestion product with T4 DNA ligase, and finally remove the phagemid template with DpnI digestion enzyme. The connected plasmid was transformed into competent Escherichia coli TG1 by electroporation to evaluate the connection and transformation efficiency. The transformant was incubated with 1mL LB medium at 37℃220rpm for one hour, 10μL sample was taken for dilution and titer determination, and 20 single clones were selected for DNA sequencing. The sequencing results are shown in Figure 5 ; In the 20 sequencing results, it can be seen that all sites are randomized and unbiased;

[0120] b. Preparation of phage cyclic peptide library:

[0121] The above transformation sample was inoculated into 500 mL of LB medium containing 50 μg / mL carbenicillin, of which 400 mL was cultured at 37°C 220 rpm until OD600 was 0.8-1.0, mixed with 50% glycerol and placed in a -80°C refrigerator for subsequent use; the remaining 100 mL was cultured until OD600 was 0.4-0.6, then helper phage was added for infection for 2 h, 0.5 mM IPTG was added and cultured at 37°C for 12-14 h, centrifuged at 4°C 8000 rpm for 20 min to remove the bacterial precipitate and collect the supernatant, 1 / 5 volume of PEG-NaCl was added to precipitate the supernatant containing phage (it can be placed on ice for 4-6 h or at 4°C overnight), centrifuged at 4°C 8000 rpm for 20 min to collect the precipitate and add 2 mL Resuspend in PBS buffer, add 150 μM TAIC and 75 μM phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, irradiate under ultraviolet light at 24 W and 365 nm for 10 min at room temperature, add 1 / 5 volume of PEG-NaCl to precipitate the phage library, centrifuge at 10000 rpm for 1 min at 4°C to collect the precipitate, and finally resuspend the precipitate with PBS buffer to obtain the phage cyclic peptide library. Figure 6 This is a schematic diagram of constructing a cyclic peptide phage library.

[0122] c. Determination of the titer of phage cyclic peptide library:

[0123] Take 10 μL of the test solution, add it to 990 μL of 2×YT liquid culture medium for dilution, take 10 μL of the dilution solution and add it to 990 μL of 2×YT liquid culture medium for dilution, and then gradually dilute the concentration to 10-8-10-10, take 10 μL of the dilution solution and add it to 150 μL of TG1 in the logarithmic growth phase, place it at 37°C for 30 minutes, then spread the culture evenly on the surface of a solid plate containing 50 μg / mL carbenicillin, invert it in a 37°C constant temperature incubator for 10-12 hours, take it out, count it and calculate its titer.

[0124] d. Coating and sealing:

[0125] The target protein CypA was diluted to 50 μg / mL with 1% NaHCO3 and coated on a 96-well plate at 4°C overnight. At the same time, a group without target protein was set up as a control. The supernatant was removed and 1% NaHCO3 (containing 1% BSA and 1% Tween 20) blocking solution was added to block at 4°C for 2 h, and then washed 6 times with 0.1% PBS-Tween 20.

[0126] e. Combination:

[0127] The phage cyclic peptide library was added and bound at 25°C and 60 rpm for 1 h. After binding, the unbound phage particles were removed and washed 10 times with 0.1% PBS-Tween 20.

[0128] f. Elution:

[0129] After adding 200 μL of Gly-HCl (containing 1% BSA) at pH = 2.2 and eluting at 25°C and 60 rpm for 10 min, 30 μL of Tris-HCl at pH = 9.1 was added for neutralization, the eluate was collected, 10 μL of the eluate was taken and diluted according to the above step b), and then infected with TG1 in the logarithmic growth phase and the titer was determined;

[0130] After repeating the above steps for four rounds of screening of the target protein (the elution concentration in the latter rounds of screening was 0.5% PBS-Tween 20), phage monoclones were randomly selected for sequencing. Figure 7 Schematic diagram of phage display cyclic peptide library and target protein affinity ligand screening. The sequencing results are translated into peptide sequences as shown in Fig. 9 .

[0131] (2) Surface isoelectronic resonance (SPR) analysis:

[0132] SPR analysis was performed using Biacore X100. The protein CypA was immobilized on the chip surface using a standardized amine coupling method. The protein was dissolved in 1x HBS-EP+ buffer at pH = 7.4 to a final protein concentration of 20ug / mL. 1x HBS-EP+ was used as the running buffer. After activating the flow cell Fc2 with a mixture of EDC and NHS, the target protein was coupled to it to achieve a response value of 500RU, and the excess ester was inactivated with ethanolamine. The flow cell Fc1 without fixed protein was used as a reference channel. Serially diluted samples (0, 125, 250, 500, 1000, 2000, 4000 and 8000nM for cyclized peptides) were passed through the flow cells (Fc1 and Fc2) at a flow rate of 30μL / min. For data analysis, all sensorgrams were processed using dual references.

[0133] exist Fig.10 In, select Fig. 9 The peptide sequence with the highest enrichment (numbered M1) was screened and Ac-CRPYRQCVKGLMC-NH2 was synthesized by solid phase synthesis. The obtained linear peptide was purified by preparative reverse HPLC, and its purity and structure were tested and verified by analytical HPLC and high resolution mass spectrometry (ESI-HRMS). Subsequently, the linear peptide was cyclized by thiol-olefin photoclick chemistry reaction, and the obtained cyclic peptide was purified by preparative reverse HPLC, and its purity and structure were further tested and verified by analytical HPLC and ESI-HRMS. Before cyclization: ESI-HRMS (m / z): calculated for C 30 H 49N6O 12 S3[M+H] + :1597.7588; found:1597.7654; after cyclization: ESI-HRMS (m / z): calculated for C 30 H 49 N6O 12 S3[M+H] + :1846.8701; found:1846.8379.

[0134] exist Fig.11 In, yes Fig.10 The cyclized peptide was subjected to surface isoelectronic resonance analysis (SPR). The kinetic data of the binding of the cyclic peptide to the CypA protein were analyzed using a 1:1 binding model and local fitting to obtain the kinetic rate constant (K a and K d ), through K d / K a The dissociation constant was determined, and the reproducibility of the assay was confirmed by repeated measurements.

[0135] exist Fig.12 In, select Fig. 9 The second most abundant peptide sequence (numbered M2) was screened and synthesized by solid phase synthesis. The obtained linear peptide was purified by preparative reverse HPLC, and its purity and structure were verified by analytical HPLC and high resolution mass spectrometry (ESI-HRMS). Subsequently, the linear peptide was cyclized by thiol-olefin photo-click chemistry reaction, and the obtained cyclic peptide was purified by preparative reverse HPLC, and its purity and structure were further verified by analytical HPLC and ESI-HRMS. Before cyclization: ESI-HRMS (m / z): calculated for C 30 H 49 N6O 12 S3[M+H] + :1572.8217; found:1572.8282; after cyclization: ESI-HRMS (m / z): calculated for C 30 H 49 N6O 12 S3[M+H] + :1821.9330; found:1821.9231.

[0136] exist Fig.13 In, yes Fig.12The cyclized peptide was subjected to surface isoelectronic resonance analysis (SPR). The kinetic data of the binding of the cyclic peptide to the CypA protein were analyzed using a 1:1 binding model and local fitting to obtain the kinetic rate constant (K a and K d ), through K d / K a The dissociation constant was determined, and the reproducibility of the assay was confirmed by repeated measurements.

[0137] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

1. A method for constructing a cyclic peptide based on thiol-olefin photoclick chemistry, characterized in that: The following steps are involved: S1. Design a peptide sequence containing multiple cysteines; S2, reducing disulfide bonds in the polypeptide using a thiol reducing agent; S3. Under ultraviolet light, the reduced polypeptide is subjected to a thiol-olefin photo-click chemical reaction with a cross-linking agent and a photoinitiator to form a cyclic peptide structure.

2. The method for constructing a cyclic peptide based on thiol-olefin photoclick chemistry according to claim 1, characterized in that: The general sequence of the polypeptide is: ACX n -CB or ACX n -CY m -CB; where C represents cysteine, X n represents n random amino acids, Y m represents m random amino acids, A and B represent any number of amino acids at the C-terminus and N-terminus, respectively.

3. The method for constructing a cyclic peptide based on thiol-olefin photoclick chemistry according to claim 1, characterized in that: The thiol reducing agent includes tris(2-carboxyethyl)phosphine salt.

4. The method for constructing a cyclic peptide based on thiol-olefin photoclick chemistry according to claim 1, characterized in that: The general formula of the cross-linking agent is R-(CH2CH=CH2)2 or R-(CH2CH=CH2)3, wherein R includes but is not limited to aromatic ring, heterocyclic ring, and alkyl ring.

5. The method for constructing a cyclic peptide based on thiol-olefin photoclick chemistry according to claim 1, characterized in that: The photoinitiator includes, but is not limited to, 2,2-dimethoxy-2-phenylacetophenone, bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide, and phenyl(2,4,6-trimethylbenzoyl)phosphate lithium salt.

6. The method for constructing a cyclic peptide based on thiol-olefin photoclick chemistry according to claim 1, characterized in that: In step S3, the ultraviolet light power is 24W, and the wavelength is 365-375nm; the photo-click chemistry reaction temperature is room temperature, and the reaction time is 3-10 minutes.

7. A method for constructing a phage-displayed cyclic peptide library based on thiol-olefin photoclick chemistry, characterized in that: The following steps are involved: S1. Displaying a polypeptide sequence on the surface of a phage to obtain a phage-displayed polypeptide library; the polypeptide comprises a plurality of cysteines; S2, performing reduction treatment on the phage display peptide library to completely reduce the disulfide bonds in the peptides; S3, adding a cross-linking agent and a photoinitiator, and carrying out a thiol-olefin photo-click reaction under ultraviolet light to obtain a cyclized phage-displayed cyclic peptide library.

8. A method for screening cyclic peptide ligands, characterized in that: The following steps are involved: S1, immobilize the target protein on the surface of the plate, add the cyclic peptide phage display library for co-incubation; S2. After incubation, the specifically bound phages are eluted and recovered to screen out the cyclic peptide ligands with specific binding.

9. The method for screening cyclic peptide ligands according to claim 8, characterized in that: The target protein includes but is not limited to cyclosporine A binding protein.

10. The method for screening cyclic peptide ligands according to claim 9, characterized in that: The amino acid sequences of affinity peptide ligands for cyclosporine A binding protein are: Ac-CRPYRQCVKGLMC-NH2 and Ac-CLPLRQCLWALIC-NH2.

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