A method for constructing cyclic peptides based on thiol-ene photo-click chemistry and application thereof

Abstract

The application belongs to the technical field of polypeptide cyclization, and specifically provides a cyclic peptide construction method based on thiol-alkene photo-click chemistry, comprising the following steps: S1, designing a polypeptide sequence containing multiple cysteines; S2, reducing disulfide bonds in the polypeptide through a thiol reducing agent; S3, under ultraviolet light irradiation, performing thiol-alkene photo-click chemistry reaction on the reduced polypeptide, a crosslinking agent and a photoinitiator to form a cyclic peptide structure. The method uses ultraviolet light to excite the addition reaction of thiol and alkene, generates stable thioether bonds, constructs a cyclic peptide structure, has high selectivity and high efficiency, and is performed under mild conditions without using a metal catalyst. The method is applied to the construction of a phage display cyclic peptide library, and has wide application potential in the construction of a bicyclic peptide library. The obtained cyclic peptide has significantly enhanced stability, and provides an effective tool for screening high-quality cyclic peptide ligands that specifically bind to a target.

Description

Technical Field

[0001] This invention belongs to the field of peptide cyclization technology, specifically relating to a method for constructing cyclic peptides based on mercapto-olefin photoclick chemistry and its application. Background Technology

[0002] In recent years, peptides have been widely used in chemistry, life sciences, and medicine. As molecules formed by amino acids linked by peptide bonds, peptides possess both the diversity, high affinity, and high selectivity of biomacromolecules, and the advantages of being small molecules that are easy to synthesize and modify. However, linear peptides, lacking a stable three-dimensional structure, typically exhibit flexible and multi-conformal characteristics, resulting in weak affinity when binding to targets, easy exposure of enzyme cleavage sites, and degradation in vivo, thus limiting their biomedical applications. Cycloning methods, by restricting linear peptides to a fixed three-dimensional conformation, improve their stability, affinity, and selectivity, and hold great potential, particularly in drug development.

[0003] Currently, common methods for constructing cyclic peptides include disulfide cyclization, amidation, and olefin metathesis. Disulfide cyclization involves the oxidation of the thiol group of cysteine ​​to form a disulfide bond; however, it is prone to mispairing, breakage, or aggregation, and the reaction conditions are limited. Lysine amidation can generate amide bonds, but commonly used special reagents may trigger side reactions, reducing reaction efficiency. Cyclolysis methods based on olefin metathesis improve reaction efficiency, but require highly reactive reagents and metal catalysts, increasing the risk of metal contamination and potentially leading to byproduct formation. For example, while the cyclization strategy for 1,3,5-tris(bromomethyl)benzene is effective, the halogen reactants used are prone to hydrolysis and elimination side reactions, affecting reaction selectivity and efficiency. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of existing cyclic peptide construction methods, such as easy mispairing, breakage or aggregation, which may lead to side reactions and reduce reaction efficiency.

[0005] Therefore, this invention provides a method for constructing cyclic peptides based on mercapto-olefin photoclick chemistry, comprising the following steps:

[0006] S1. Design a polypeptide sequence containing multiple cysteine ​​residues;

[0007] S2. Reduce disulfide bonds in polypeptides using thiol reducing agents;

[0008] S3. Under ultraviolet light irradiation, the reduced peptide is reacted with a cross-linking agent and a photoinitiator to undergo a thiol-olefin photoclick chemical reaction to form a cyclic peptide structure.

[0009] Specifically, the universal sequence of the above-mentioned polypeptide is: ACX n-CB or ACX n -CY m -CB; where C represents cysteine, X n Y represents n random amino acids m Let A represent m random amino acids, and let 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 crosslinking 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 aforementioned thiol reducing agents include tris(2-carboxyethyl)phosphine salts.

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

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

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

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

[0016] S1. Display the polypeptide sequence on the surface of a phage to obtain a phage display polypeptide library; the polypeptide contains multiple cysteine ​​residues.

[0017] S2. Reduce the phage-displayed peptide library to completely reduce the disulfide bonds in the peptides.

[0018] S3. Add crosslinking agent and photoinitiator, and carry out thiol-olefin photoclick reaction under ultraviolet light irradiation to obtain cyclized phage display cyclic peptide library.

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

[0020] S1. Immobilize the target protein on the plate surface and add the cyclic peptide phage display library for co-incubation;

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

[0022] Specifically, the target proteins mentioned above include, but are not limited to, cyclosporine A-binding proteins.

[0023] Specifically, the amino acid sequences of the affinity peptide ligands for the 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] This invention provides a cyclic peptide construction method based on thiol-olefin photoclick chemistry. It utilizes ultraviolet light to excite the addition reaction between thiol groups and olefins, generating stable thioether bonds to construct cyclic peptide structures. This method exhibits high selectivity and efficiency, operates under mild conditions, requires no metal catalysts, and is independent of highly reactive reagents or metal catalysts. It is suitable for high-throughput screening, avoids side reactions, and effectively improves product purity and yield. Furthermore, this method can generate structures more stable than traditional disulfide bonds, making it suitable for applications in biological environments.

[0026] This method was applied to the construction of phage-displayed cyclic peptide libraries. By introducing thiol-containing peptide sequences into the phage display system and achieving cyclization using thiol-olefin photoclick chemistry, a diverse range of cyclic peptide libraries were constructed, expanding the application potential of phage peptide library cyclization, particularly in the construction of bicyclic peptides. The resulting cyclic peptides exhibited significantly enhanced stability, demonstrating good tolerance to acids, alkalis, exogenous thiol groups, and oxidants. This provides an effective tool for screening high-quality cyclic peptide ligands that specifically bind to targets, overcoming the shortcomings of poor biocompatibility and insufficient stability of traditional methods, and is applicable to the fields of biomedical and drug development.

[0027] The present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

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

[0029] Figure 2 This is a diagram illustrating the photoclick 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 cysteine ​​residues in Example 3 of the present invention.

[0031] Figure 4 The images show the HPLC characterization of peptide cyclization under different buffer solutions in Example 3 of this invention.

[0032] Figure 5This is a sequencing diagram of the phage display peptide library construction in Example 5 of the present invention.

[0033] Figure 6 This is a schematic diagram of phage display peptide library cyclization in Example 5 of the present invention.

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

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

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

[0037] Figure 10 The images show the HPLC characterization of linear and cyclic peptides of M1 in Example 5 of this invention.

[0038] Figure 11 This is the SPR sensing image of M1 after ringing according to the present invention for CypA.

[0039] Figure 12 The images show the HPLC characterization of linear and cyclic peptides of M2 in Example 5 of this invention.

[0040] Figure 13 This is the SPR sensing image of M2 after circumduction for CypA according to the present invention. Detailed Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand 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 defined by the appended claims and their equivalents.

[0042] This invention provides a method for constructing cyclic peptides based on mercapto-olefin photoclick chemistry. Under ultraviolet light irradiation and in the presence of a photoinitiator, an allyl derivative undergoes a free radical addition reaction with a polypeptide containing multiple cysteine ​​residues in a mixed solution of buffer and organic solvent to form a cyclic peptide. Specifically, the method includes the following steps:

[0043] S1. Design a polypeptide sequence containing multiple cysteine ​​residues.

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

[0045] S2, reduce disulfide bonds in polypeptides using thiol reducing agents.

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

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

[0048] S3. Under ultraviolet light irradiation, the reduced peptide is reacted with a cross-linking agent and a photoinitiator to undergo a thiol-olefin photoclick chemical reaction to form a cyclic peptide structure.

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

[0050] Specifically, the cross-linking agent is dissolved to obtain solution 2, and the photoinitiator is dissolved to obtain solution 3. After the peptide is fully reduced, solutions 2 and 3 are added. The molar ratio of the cross-linking agent to the peptide in solution 1 in solution 2 is ≥1:1; the molar ratio of the photoinitiator to the peptide in solution 1 in solution 3 is (0.3 - 1):1. The reaction is carried out under ultraviolet light irradiation to obtain a cyclic peptide product, which 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 the actual situation. A 24W ultraviolet lamp with a wavelength of 365-375 nm is preferred. The reaction temperature is room temperature (generally between 20℃ and 25℃), and the reaction time is 3-10 minutes.

[0051] The solvent used in this 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 more detailed embodiment, 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, which can then be diluted with water to 100 mmol / L and 10 mmol / L for later use.

[0053] Based on the highly biocompatible reaction properties of thiol-olefin photoclick chemistry and cysteine-containing peptides, this invention also provides a method for constructing a phage-display cyclic peptide library based on thiol-olefin photoclick chemistry. This method displays a cysteine-containing peptide library on a phage and cyclizes the peptide library using an allyl derivative. The specific steps include:

[0054] S1. Display the polypeptide sequence on the surface of the phage to obtain a phage display polypeptide library; the polypeptide contains multiple cysteine ​​residues.

[0055] Specifically, the template is: ACX. n -CB or ACX n -CY m -CB, where 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 expressed on the surface of the phage is: CXXXXXXXXXXC or CXXXXXCXXXXXC.

[0057] Here, X represents a random amino acid, which is introduced at a specified position using the NNK method.

[0058] Based on the above polypeptide sequence, the corresponding DNA sequence is designed as follows:

[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 conformation and other modifications made in this invention shall be considered within the scope of protection of this patent.

[0062] S2. The phage-displayed peptide library is reduced to completely reduce the disulfide bonds in the peptides.

[0063] The general formula for the crosslinker structure used to construct the phage display cyclic peptide library is:

[0064]

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

[0066] S3. Add a cross-linking agent and a photoinitiator, and perform a thiol-olefin photoclick reaction under ultraviolet light irradiation to obtain a cyclized phage display cyclic peptide library. The specific method can be found in the cyclic peptide construction method described above.

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

[0068] S1. The target protein is immobilized on the plate surface and co-incubated with a cyclic peptide phage display library.

[0069] S2. After incubation, unbound phages are washed away, and specifically bound phages are recovered by elution with an acidic solution. The eluted phages are amplified and screened repeatedly in multiple rounds. Single phage clones are selected for DNA sequencing to resolve cyclic peptide sequences. The cyclic peptides with the highest enrichment levels are synthesized and purified, and affinity is tested using surface plasmon resonance (SPR).

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

[0071] In a refined embodiment, the cyclic peptide ligand screening method includes:

[0072] (1) At room temperature, the crosslinking agent was dissolved to obtain solution 2, and the photoinitiator was dissolved to obtain solution 3. For titers greater than 10... 10 TCEP was added to a phage library containing at least two or three cysteine ​​residues at the N-terminus to reduce the phage library to 1 mM (pfu / mL). Solution 2 was added to reduce the final concentration to 150 μM, and solution 3 was added to reduce the final concentration to 75 μM. The mixture was irradiated with a 24 W UV lamp at 365 nm for 10 min. After the reaction, PEG-NaCl solution was added, and the mixture was incubated on ice to precipitate the phages. After precipitation, the mixture was centrifuged, the supernatant was removed, and the precipitate was resuspended in phosphate buffer to obtain the allyl derivative-modified phage cyclic peptide library.

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

[0074] (2) Screening of the mononucleotide phage cyclic peptide library: After immobilizing the target protein on a 96-well plate, the cyclic peptide library was added and co-incubated. After 1 h, unbound and low-affinity phages were washed away, and the specifically bound phages were washed and recovered and amplified for the next round of screening. After multiple rounds of in vitro screening, the phages bound to the target protein were effectively enriched. Single colony clones were picked and DNA sequencing was performed to decode the amino acid sequence bound to the target protein.

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

[0076] After synthesizing peptides with high enrichment, a cyclization reaction was carried out. The affinity between the cyclic peptide ligand and the target protein was verified by biophysical experiments to test the feasibility and effectiveness of the cyclization and screening methods.

[0077] The following specific examples illustrate the effects of the cyclic peptide construction method based on mercapto-olefin photoclick chemistry of the present invention on its application.

[0078] Example 1:

[0079] This embodiment investigates the effects of different photoinitiators on the photoclick chemical reaction of mercapto-olefins, and determines the optimal photoinitiator based on the reaction yield. The reaction formula is shown below:

[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 Add the solution to a 10 mL test tube, add 1 mL of solvent (acetonitrile: water = 4:1), irradiate under a 24 W, 365 nm UV lamp for 10 min, concentrate the reaction solution under reduced pressure, and then separate the product by column chromatography.

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

[0084] 1H NMR (600 MHz, DMSO-d6) δ 8.36 (d, J = 7.8 Hz, 1H), 4.42 (td, J =8.1, 5.5 Hz, 1H), 3.80 (t, J = 7.0 Hz, 2H), 3.63 (s, 3H), 2.85 (dd, J = 13.7,5.6 Hz, 1H), 2.73 (dd, J = 13.7, 8.2 Hz, 1H), 2.53 (t, J = 7.4 Hz, 2H), 1.85(s, 3H), 1.77 (dq, J = 13.9, 7.3 Hz, 2H).

[0085] 13 C NMR (151 MHz, DMSO-d6) δ 171.32, 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] Example 2:

[0090] This embodiment investigated the effects of different organic solvents on the photoclick chemistry reaction of mercapto-olefins, and determined the optimal organic solvent based on the reaction yield. The specific experimental steps are shown below:

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

[0092] Table 2. Reaction yields under different organic solvents

[0093]

[0094] Example 3:

[0095] This embodiment investigated the effect of different buffer solutions on peptide cyclization, and determined the optimal reaction buffer solution based on the reaction yield. The reaction formula is shown below:

[0096]

[0097]

[0098] The specific experimental steps are as follows:

[0099] The cyclization linker, triallyl isocyanurate (TAIC, 100 mM, 200 μL), the peptide stock solution (100 mM, 100 μL), and the photoinitiator, lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (100 mM, 50 μL), were sequentially added to a 10 mL reaction tube. The system was adjusted using a mixture of buffer and acetonitrile to ensure a final acetonitrile volume fraction of 20%. The final concentrations of each component in the reaction system were: TAIC 20 mM, peptide 10 mM, and photoinitiator 5 mM. The reaction system was placed at room temperature and irradiated with a 24 W, 365 nm UV light source for 10 min. Subsequently, preparative reversed-phase high-performance liquid chromatography (HPLC) was used for separation and purification, followed by lyophilization to obtain the target cyclic peptide product. Figure 3 A schematic diagram of the formation of a cyclic peptide by an allyl derivative and a polypeptide containing three cysteine ​​residues; Figure 4 The HPLC chromatogram shows the linear peptide retention time at 14.219 min and the cyclized peptide retention time at 15.215 min. Table 3 shows the yield of the cyclization reaction. The optimal buffer solution is pure water or Tris at pH 6.0.

[0100] Table 3. Cyclization reaction yield

[0101]

[0102] ESI-HRMS (m / z): calculated for C 53 H 86 N 17 O 14 S3 [M + H] + : 1280.5702;found: 1280.5731.

[0103] Example 4:

[0104] This embodiment explores the universality of the reaction by cyclizing it with polypeptides containing three cysteine ​​residues of varying lengths. The specific experimental steps are shown below:

[0105] The cyclization linker, triallyl isocyanurate (TAIC, 100 mM, 200 μL), the peptide stock solution (100 mM, 100 μL), and the photoinitiator, lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (100 mM, 50 μL), were sequentially added to a 10 mL reaction tube. The system was adjusted using a mixed solvent of water and acetonitrile to ensure a final acetonitrile volume fraction of 20%. The final concentrations of each component in the reaction system were: TAIC 20 mM, peptide 10 mM, and photoinitiator 5 mM. The reaction system was placed at room temperature and irradiated with a 24 W, 365 nm UV light source for 10 minutes. The reaction was then monitored using HPLC. Table 4 shows the characterization data of the cyclization reaction based on HPLC quantification; the peptide conversion rate was 100%.

[0106] Table 4 Characterization data of cyclization reaction

[0107]

[0108] Example 5:

[0109] CypA (Cyclophilin A) is widely involved in protein folding and signal transduction processes, and is closely related to diseases such as viral infections, inflammation, and tumors, making it an important drug target. By screening cyclic peptide ligands, it is possible to effectively interfere with CypA-related pathological processes, providing potential candidate molecules for the development of antiviral, anti-inflammatory, and anti-tumor drugs.

[0110] This embodiment is based on a thiol-olefin photoclick chemically modified phage library, with CypA as the target protein, to screen for cyclic peptide ligands with high affinity.

[0111] (1) The specific steps of the phage display method are as follows:

[0112] a. Construction of a phage-displaying cyclic peptide library:

[0113] Template: ACX n -CY m -CB

[0114] 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.

[0115] The polypeptide sequence expressed on the surface of the fusion phage is: CXXXXXCXXXXXC;

[0116] Where X represents a random amino acid, and random mutations of amino acids are introduced at a specified position using the NNK method;

[0117] The corresponding DNA sequence was designed based on the polypeptide sequence displayed on the surface of the bacteriophage:

[0118] 5'-CTAGGGATCC tgc NNKNNKNNKNNKNNKtgcNNKNNKNNKNNKNNK tgc GGCGGCTCGGCCTCGGGGGC-3';

[0119] In the phage vector pbcomb-3xga, the library fragment was inserted using the BamHI single restriction enzyme site. In the DNA sequence of this fragment, the polypeptide frame is represented by lowercase and underline, and the restriction endonuclease cleavage site BamHI is represented by uppercase and bold.

[0120] Phagemid libraries were constructed by directly amplifying the pbcomb-3xga plasmid using PCR, using two primers: MYF: 5'-CTAGGGATCC. tgc NNKNNKNNKNNKNNKtgcNNKNNKNNKNNKNNK tgc The PCR products GGCGGCTCGGCCTCGGGGGC-3' and MYR:5'-CTAGGGATCCggccgcctgggccacg-3' were digested with the restriction endonuclease BamHI. The digested products were recovered using a 1% agarose gel, ligated with T4 DNA ligase, and finally, the phage template was removed using DpnI digestion enzyme. The ligated plasmids were transformed into competent *E. coli* TG1 cells by electroporation to evaluate ligation and transformation efficiency. Transformants were incubated in 1 mL of LB medium at 37°C and 220 rpm for one hour. 10 μL of the sample was diluted to determine the titer, and 20 single cloning sites were picked for DNA sequencing. The sequencing results are shown below. Figure 5 The 20 sequencing results show that all sites are randomized and unbiased.

[0121] b. Preparation of phage cyclic peptide libraries:

[0122] The transformed samples were inoculated into 500 mL of LB medium containing 50 μg / mL carbenicillin. 400 mL was incubated at 37°C and 220 rpm until an OD600 of 0.8–1.0 was reached, then mixed with 50% glycerol and stored at -80°C for later use. The remaining 100 mL was incubated until an OD600 of 0.4–0.6 was reached, then helper phages were added for infection for 2 h. 0.5 mM IPTG was added, and the mixture was incubated at 37°C for 12–14 h. The culture was then centrifuged at 4°C and 8000 rpm for 20 min to remove the bacterial pellet and collect the supernatant. 1 / 5 volume of PEG-NaCl was added to precipitate the supernatant containing the phages (this can be allowed to settle on ice for 4–6 h or overnight at 4°C). The pellet was then centrifuged at 4°C and 8000 rpm for 20 min, collected, and resuspended in 2 mL of PBS buffer. 150 μM TAIC and 75 μM lithium phenyl (2,4,6-trimethylbenzoyl)phosphate were added, and the mixture was incubated at room temperature with a power of 24... Irradiate the bacteriophage library under a 365 nm UV lamp for 10 min, 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.

[0123] c. Determination of phage cyclic peptide library titers:

[0124] Take 10 μL of the test solution and dilute it with 990 μL of 2 × YT liquid medium. Take 10 μL of the diluted solution and dilute it with 990 μL of 2 × YT liquid medium. Then, dilute the concentration stepwise to 10⁻⁸-10⁻¹⁰. Take 10 μL of the diluted solution and add it to 150 μL of logarithmic growth phase TG1 medium. After standing at 37 ℃ for 30 min, spread the culture evenly on the surface of a solid plate containing 50 μg / mL carbenicillin. Invert the plate and incubate it in a constant temperature incubator at 37 ℃ for 10-12 h. Take the plate out and count the titers.

[0125] d. Encapsulation and sealing:

[0126] The target protein CypA was diluted to 50 μg / mL with 1% NaHCO3 and coated onto a 96-well plate overnight at 4°C. A control group without target protein was also set up. After removing the supernatant, 1% NaHCO3 (containing 1% BSA and 1% Tween 20) blocking buffer was added and the plate was blocked at 4°C for 2 h. The plate was then washed 6 times with 0.1% PBS-Tween 20.

[0127] e. Combination:

[0128] Phage cyclic peptide libraries were added and bound at 25°C and 60 rpm for 1 h. After binding, unbound phage particles were removed, and the phage was washed 10 times with 0.1% PBS-Tween 20.

[0129] f. Washing:

[0130] After adding 200 μL of Gly-HCl (containing 1% BSA) at pH = 2.2 and eluting at 25℃ and 60 rpm for 10 min, add 30 μL of Tris-HCl at pH = 9.1 for neutralization, collect the eluent, take 10 μL of the eluent and dilute it according to step b) above, and then infect the TG1 in the logarithmic growth phase and determine the titer;

[0131] After repeating the above steps to screen the target protein four times (using 0.5% PBS-Tween 20 as the elution concentration in the later screening rounds), phage single clones were randomly selected for sequencing. Figure 7 This diagram illustrates the phage display of a cyclic peptide library and target protein affinity ligand selection. Sequencing results translated into polypeptide sequences are shown below. Figure 9 .

[0132] (2) Surface isoelectronic resonance (SPR) analysis:

[0133] SPR analysis was performed using a Biacore X100. The CypA protein was immobilized on the chip surface using a standardized amine conjugation method. The protein was dissolved in 1 x HBS-EP+ buffer at pH 7.4 to a final concentration of 20 μg / mL. 1 x HBS-EP+ was used as the run buffer. After activating flow cell Fc2 with a mixture of EDC and NHS, the target protein was conjugated to achieve a response of 500 RU, and excess esters were inactivated with ethanolamine. The unimmobilized flow cell Fc1 was used as the reference channel. Serially diluted samples (cyclized peptides at 0, 125, 250, 500, 1000, 2000, 4000, and 8000 nM) were passed through the flow cells (Fc1 and Fc2) at a flow rate of 30 μL / min. For data analysis, all sensor maps were processed using dual references.

[0134] exist Figure 10 From, select Figure 9The peptide sequence with the highest enrichment (numbered M1) was obtained through screening. Ac-CRPYRQCVKGLMC-NH2 was synthesized using solid-phase synthesis. The resulting linear peptide was purified by preparative reversed-phase high-performance liquid chromatography (HPLC), and its purity and structure were verified by analytical HPLC and high-resolution mass spectrometry (ESI-HRMS). Subsequently, the linear peptide was cyclized using a mercapto-olefin photoclick chemistry reaction. The resulting cyclic peptide was then purified by preparative reversed-phase 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] + : 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.

[0135] exist Figure 11 In China, Figure 10 The cyclic peptide was subjected to surface isoelectronic resonance (SPR) analysis. The kinetic data of the binding of this cyclic peptide to CypA protein were analyzed using a 1:1 binding model and local fitting to obtain the kinetic rate constant (K0). a and K d ), through K d / K a The dissociation constant was determined, and the repeatability of the measurement was confirmed through repeated measurements.

[0136] exist Figure 12 From, select Figure 9 The peptide sequence with the second highest enrichment (numbered M2) was obtained through screening. Ac-CLPLRQCLWALIC-NH2 was synthesized using solid-phase synthesis. The resulting linear peptide was purified by preparative reversed-phase high-performance liquid chromatography (HPLC), and its purity and structure were verified by analytical HPLC and high-resolution mass spectrometry (ESI-HRMS). Subsequently, the linear peptide was cyclized using a mercapto-olefin photoclick chemistry reaction. The resulting cyclic peptide was then purified by preparative reversed-phase 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 N6O12 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.

[0137] exist Figure 13 In China, Figure 12 The cyclic peptide was subjected to surface isoelectronic resonance (SPR) analysis. The kinetic data of the binding of this cyclic peptide to CypA protein were analyzed using a 1:1 binding model and local fitting to obtain the kinetic rate constant (K0). a and K d ), through K d / K a The dissociation constant was determined, and the repeatability of the measurement was confirmed through repeated measurements.

[0138] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A method for the construction of cyclic peptides based on thiol-ene photo-click chemistry, characterized in that, Includes the following steps: S1. Design a polypeptide sequence containing multiple cysteine ​​residues; the universal sequence of the polypeptide is AC-Xn-C-Ym-CB; where C represents cysteine, Xn represents n random amino acids, Ym represents m random amino acids, and A and B represent any number of amino acids at the C-terminus and N-terminus, respectively; n and m range from 1 to 10. S2. Reduce disulfide bonds in polypeptides using thiol reducing agents; S3. Under ultraviolet light irradiation, the reduced peptide is subjected to a mercapto-olefin photoclick chemical reaction with a crosslinking agent and a photoinitiator to form a cyclic peptide structure; the crosslinking agent is triallyl isocyanurate (TAIC).

2. The thiol-ene photo-click chemistry based cyclic peptide construction method according to claim 1, wherein: The thiol reducing agent includes tris(2-carboxyethyl)phosphine salt.

3. The thiol-ene photoclick chemistry based cyclic peptide construction method according to claim 1, wherein: The photoinitiator includes any one of 2,2-dimethoxy-2-phenylacetophenone, bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide, and lithium phenyl(2,4,6-trimethylbenzoyl)phosphate.

4. The method of thiol-ene photo-click chemistry based cyclic peptide construction according to claim 1, wherein: In step S3, the ultraviolet light power is 24 W and the wavelength is 365-375 nm; the photoclick chemical reaction temperature is room temperature and the reaction time is 3-10 minutes.

5. A method for phage display cyclic peptide library construction based on thiol-alkene photoclick chemistry, characterized in that, Includes the following steps: S1. Display the polypeptide sequence on the surface of a phage to obtain a phage display polypeptide library; the polypeptide is the polypeptide containing multiple cysteine ​​residues as described in claim 1; S2. Cyclic peptide construction method according to any one of claims 1-4 is used to construct cyclic peptides from peptides in the phage display peptide library to obtain a cyclic phage display peptide library.

6. A method of screening cyclic peptide ligands, characterized by, Includes the following steps: S1. A phage display cyclic peptide library is constructed using the phage display cyclic peptide library construction method described in claim 5. The target protein is immobilized on the surface of a plate, and the phage display cyclic peptide library is added for co-incubation. S2. After incubation, the specifically bound phages are washed away and screened for cyclic peptide ligands with specific binding.

7. The method of screening cyclic peptide ligands according to claim 6, wherein: The target protein includes cyclosporine A-binding protein.

8. The cyclic peptide ligand screening method as described in claim 7, characterized in that, The amino acid sequences of the affinity peptide ligands for cyclosporine A binding protein are: Ac-CRPYRQCVKGLMC-NH2 and Ac-CLPLRQCLWALIC-NH2.

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