A polypeptide compound containing a low-reactivity group and application thereof

Abstract

The application provides a polypeptide compound containing a low-reactivity group and an application thereof, and relates to the technical field of polypeptide cyclization modification and cyclic peptide screening. The enzymatic cyclization method uses the polypeptide compound containing the low-reactivity group as a substrate to generate various cyclic peptide skeletons under the catalysis of an enzyme, and is applied to a phage display technology to prepare a large-scale cyclic peptide library and is used for screening of an affinity cyclic peptide ligand of a target protein. The application solves the defects of traditional technologies, the enzymatic cyclization method can realize efficient cyclization modification of the phage peptide library under mild conditions, the phage cyclic peptide library generated by the method is helpful for discovery of cyclic peptides with different characteristic structures, and provides a powerful technical platform for cyclic peptide drug discovery.

Description

Technical Field

[0001] This invention relates to the field of peptide cyclization modification and cyclic peptide screening technology, specifically to a peptide compound containing a low-reactivity group and its applications. Background Technology

[0002] Peptide drugs are a crucial class of drugs, playing a vital role in the treatment of various diseases. Short linear peptides, due to their non-fixed conformation, are easily degraded by proteases in the body, making them unsuitable frameworks for peptide drug development. In contrast, cyclic peptides possess rigid structural features, can withstand protease degradation, exhibit higher binding affinity and targeting specificity to target proteins, and have good tissue permeability, thus serving as the core framework for peptide drug development. Representative examples include cyclosporine and ziconopeptide. Most naturally occurring cyclic peptides are disulfide-bonded framework peptides formed by the oxidation of the side chains of two cysteine ​​residues. Although disulfide-bonded framework cyclic peptides have achieved significant success in peptide drug development, disulfide bonds have inherent defects, such as instability under reducing conditions and susceptibility to intramolecular or intermolecular thiol exchange reactions in the presence of glutathione, potentially leading to physiologically toxic misaligned cyclic peptide molecules.

[0003] Non-disulfide bond cyclic peptide frameworks are an important direction in current cyclic peptide drug development. The most convenient and efficient method is thioether bond cyclic peptides, which typically use highly reactive small organic molecules (such as bromomethylbenzene or bromoacetyl derivatives) to crosslink two or more cysteine ​​residues. A representative method is the bicyclic peptide technique developed by Heinis et al. The bicyclic peptide technique uses tribromomethylbenzene to crosslink three cysteine ​​side chains within a linear peptide to generate bicyclic peptide molecules. Despite its widespread use, this high reactivity has inherent drawbacks; it may undergo side reactions with other nucleophilic side chains in weakly alkaline environments. Of particular concern is that bromomethylbenzene reagents have phage toxicity, which can significantly affect phage infectivity. Therefore, traditional phage-displaying cyclic peptide libraries based on bromomethylbenzene may result in the loss of some highly reactive molecules.

[0004] Enzymatic cyclization is another important method for preparing cyclic peptides, offering greater biocompatibility and widely used in protein modification. Sortase A, a transpeptidase discovered in Gram-positive bacteria, catalyzes the peptide bond linkage between a polypeptide containing the Leu-Pro-Xaa-Thr-Gly motif (where Xaa is any natural L-amino acid) and another polypeptide with an N-terminal Gly. Recently, Fang et al. discovered that the Xaa in the Leu-Pro-Xaa-Thr-Gly recognition sequence of Sortase A is compatible with non-natural amino acids modified with chloroacetyl groups and was used to construct a phage-displayed cyclic peptide system. However, this system has not yet been used with other low-reactivity groups (such as chloromethylbenzene derivatives and fluorobenzene derivatives). Therefore, exploring the Sortase A enzymatic peptide linkage and cyclization system, developing methods for preparing cyclic peptides with different characteristics, and applying them to phage-displayed peptide libraries will be beneficial for promoting the discovery of highly active cyclic peptide molecules. Summary of the Invention

[0005] To address the limitation of Xaa in the Leu-Pro-Xaa-Thr-Gly enzyme recognition motif of Sortase A to non-natural amino acids modified with chloroacetyl groups, this invention provides a polypeptide compound containing a low-reactivity group and its application. This compound is applicable to a novel Sortase A-catalyzed cyclization system for chloromethylbenzene-derived and fluorobenzene-derived peptide substrates, and can generate cyclic peptide backbones with various characteristics.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A polypeptide compound containing a low-reactivity group, the general structural formula of which is as follows: ; Among them, X a 3-(chloromethyl)benzyl: 4-(chloromethyl)benzyl: 2-(chloromethyl)benzyl: 2-(chloromethyl)pyridine methyl derivatives: 4'-(chloromethyl)biphenyl derivatives: , Pentafluorobenzene derivatives: Nonafluorobiphenyl derivatives: And 3,5-di(chloromethyl)benzyl: Any one of them; X b It is any one of hydrogen, acetyl, or oligopeptide group composed of natural or non-natural amino acids other than cysteine; X c It can be any one of O, NH, and S; X dIt is any one of the amino groups or oligopeptide sequences composed of natural or non-natural amino acids other than cysteine.

[0007] The specific operation method of the enzymatic cyclization method using the above-mentioned polypeptide compounds containing low reactive groups as substrates includes the following steps: S1. Under the catalysis of Sortase A enzyme, a polypeptide compound containing a low reactive group is combined with a polypeptide with glycine at the N-terminus and cysteine ​​in the sequence in a non-phosphate buffer to undergo an enzymatic polypeptide linkage and cyclization reaction to generate a cyclic peptide. S2. Select a polypeptide template from S1 with glycine at the N-terminus and containing cysteine ​​in the sequence and fuse it to the N-terminus of the phage pIII protein to prepare a phage cyclic peptide library, and perform in vitro screening of cyclic peptide ligands using target proteins.

[0008] Preferably, the template for the polypeptide in S1 is one of the following two: Template a: G-(X) m -C; Template b: G-(X) m -C-(X) n -C; In this context, template a is used for constructing a monocyclic peptide library, template b is used for constructing a bicyclic peptide library, G is glycine, X is any natural L-amino acid, C is L-cysteine, and m and n are any number of amino acids between 3 and 18.

[0009] Preferably, the concentration range of the polypeptide compound containing low reactive groups in S1 is 0.01µM to 20.0mM, and the concentration range of Sortase A enzyme is 0.01µM to 20.0mM.

[0010] Preferably, the non-phosphate buffer in S1 contains any one of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), imidazole, and tris(hydroxymethyl)aminomethane (Tris), and the non-phosphate buffer contains 0.05mM~50mM CaCl2 and 0.05µM~10.0mM tris(2-carboxyethyl)phosphine (TCEP), with a pH range of 6.0~10.0.

[0011] Preferably, the enzymatic peptide linking and cyclization reaction in S1 takes 15 min to 24 h and the reaction temperature is 15 to 60 °C.

[0012] Preferably, the phage in S2 is either a phage or an M13KE phage generated by packaging pCANTAB 5E phage particles and helper phage M13KO7. Preferably, the in vitro screening method for cyclic peptide ligands using target proteins in S2 includes the following steps: S3-1. Prepare a phage library displaying monocyclic or bicyclic peptides using an enzymatic peptide linking and cyclization method with peptide compounds containing low reactive groups. S3-2. Immobilize the biotin-modified target protein onto magnetic beads and co-incubate it with the phage library from step S3-1. Then perform 1-6 rounds of affinity panning and sequence the enriched phages. S3-3. Analyze the sequencing results, synthesize the enriched cyclic peptides, and evaluate their binding affinity to the target protein.

[0013] The above-mentioned enzymatic cyclization is applied to the construction of cyclic peptide libraries, which include single-ring and double-ring peptide libraries for phage display and mRNA display.

[0014] Preferably, the cyclic peptide ligand obtained by the enzymatic cyclization is used in the development of detection kits, drugs or other biomaterials.

[0015] This invention provides a polypeptide compound containing a low-reactive group and its applications, which have the following advantages compared with the prior art: (1) Compared with the traditional chemical cross-linking cyclic peptide construction method based on bromomethylbenzene, the present invention does not involve highly active electrophiles, does not affect the infectivity of phages, and is conducive to the generation of high-quality phage display cyclized libraries.

[0016] (2) Compared with the reported cyclic peptide library construction method based on Flexizyme, the present invention does not require cumbersome codon modification and does not involve the preparation of non-natural aminoacylated tRNA, thus significantly reducing the construction cost of cyclic peptide libraries.

[0017] (3) Compared with the previously reported SortaseA-catalyzed peptide linking cyclization method, the present invention is applicable to low-reactivity peptide substrates derived from chloromethylbenzene and fluorobenzene, and can be used for cyclic peptide backbones with more structural features, which helps in the discovery of highly active cyclic peptide molecules. Attached Figure Description

[0018] Figure 1 Schematic diagram of the enzymatic cyclization reaction of Pep1 and Pep3 to prepare the monocyclic peptide Pep11. Figure 2 Schematic diagram of the enzymatic cyclization reaction of Pep1 and Pep4 to prepare the monocyclic peptide Pep12. Figure 3 Schematic diagram of the enzymatic cyclization reaction of Pep1 and Pep5 to prepare the monocyclic peptide Pep13. Figure 4 Schematic diagram of the enzymatic cyclization reaction of Pep1 and Pep6 to prepare the monocyclic peptide Pep14. Figure 5 Schematic diagram of the enzymatic cyclization reaction of Pep1 and Pep7 to prepare the monocyclic peptide Pep15. Figure 6Schematic diagram of the enzymatic cyclization reaction of Pep1 and Pep8 to prepare the monocyclic peptide Pep16. Figure 7 Schematic diagram of the enzymatic cyclization reaction of Pep1 and Pep9 to prepare the monocyclic peptide Pep17. Figure 8 Schematic diagram of the enzymatic cyclization reaction of Pep2 and Pep10 to prepare the bicyclic peptide Pep18. Figure 9 A graph showing the titer changes during phage screening; Figure 10 A schematic diagram of surface plasmon resonance between Pep19 and Pep20 and Trop2. Detailed Implementation

[0019] To clearly illustrate the present invention, further explanation is provided below through embodiments and accompanying drawings.

[0020] The present invention includes the following steps: (1) Two polypeptide backbone templates with glycine residues at their N-terminus: Template a: G-(X) m -C; Template b: G-(X) m -C-(X) n -C; In this context, template a is used for constructing a monocyclic peptide library, template b is used for constructing a bicyclic peptide library, G is glycine, X is any natural L-amino acid, C is L-cysteine, and m and n are any number of amino acids between 3 and 18.

[0021] Based on the above template characteristics, two peptides with glycine residues at their N-termini were synthesized according to the following sequence: H-GLYDPANIHPKGWCGGSG-NH2 (template polypeptide Pep1); H-GRYDPANCIHPKGWCGGSG-NH2 (template polypeptide Pep2); Template polypeptide 1 and template polypeptide 2 are both composed of natural L-type amino acids, with an N-terminus of glycine (alfa-amino group) and a C-terminus of glycine (amide group). The C-terminal GGSG motif is a flexible linker arm used to mimic the connection between the polypeptide and the pIII protein of the bacteriophage. These two polypeptides are only used to illustrate the elements and implementation process of the present invention and do not constitute the entirety of this patent.

[0022] To generate the desired cyclic peptide backbone, the position of cysteine ​​residues in the peptide sequence can be arbitrarily changed, and the specific number of amino acids in the sequence can be deleted as needed. The resulting peptide remains applicable to the enzymatic cyclization method for low-reactivity peptide substrates proposed in this patent. Any changes to the peptide backbone type and modification method based on this patent should be within the scope of protection of this patent.

[0023] (2) A low-reactivity polypeptide compound having the following characteristics: X a = ;or ;or ;or ;or ;or ;or ;or ; (3) Under the catalysis of Sortase A, a polypeptide compound containing a low reactive group in S1 is combined with a polypeptide with glycine at the N-terminus and cysteine ​​in the sequence to undergo an enzymatic polypeptide linkage and cyclization reaction in solution to generate a cyclic peptide. The enzyme is either wild-type or a mutant of Sortase A. The concentration range of the low-reactivity polypeptide compound is 0.01 µM to 20.0 mM, and the concentration range of Sortase A enzyme is 0.01 µM to 20.0 mM. The solution is a non-phosphate buffer such as HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), imidazole, and Tris (tris(hydroxymethyl)aminomethane). The solution contains CaCl2 (0.05 mM to 50 mM) and TCEP (tris(2-carboxyethyl)phosphine) (0.05 µM to 10.0 mM). The pH range of the solution is 6.0 to 10.0. The time for the enzymatic polypeptide ligation and cyclization reaction is 15 min to 24 h, and the reaction temperature is 15 to 60 °C. Simple modifications and alterations to this polypeptide compound are still within the scope of protection of this patent.

[0024] (4) Construction and application of phage-displayed cyclic peptide libraries: A bicyclic polypeptide library with the following sequence characteristics was fused to the N-terminus of pIII on the phage surface: GXXXXXCXXXXCGGSGGSGG (N to C terminus, X is any natural L-amino acid, with random amino acid mutations at 9 positions in the sequence, where GGSGGSGG serves as a flexible linker between the bicyclic peptide library and the phage pIII protein).

[0025] Based on the sequence characteristics of the peptide in this library, two DNA sequences with the following values ​​were designed and synthesized: Primer A: 5'-TTGGTCTCGGTGCGCCGGTGCCGTATCCGGATCCGCTG-3'; Primer B: 5'-TTTGGTCTCAGCACCGCCAGAGCCCGCGCAMNNMNNMNNMNNGCAMNNMNNMNNMNNMNNACCGGCCATGGCCGGCTGGGCCGCATAGAAAGG-3' (N stands for A / T / C / G, M stands for C / A, and K stands for G / T).

[0026] Using the pCANTAB 5E mutant vector (without the BsaI restriction site) as a template, primers A and B were used for full-plasmid PCR on KeyPo polymerase (15 PCR cycles, 30 PCR reactions, 20 µL per PCR). The PCR products were directly recovered using a DNA recovery kit (Tiangen) and treated with BsaI and DpnI. The recovered PCR products were ligated overnight using T4 ligase. The recovered plasmid was electroporated into TG1 cells (8 times, 1.0 μg / time), and the number of cell clones obtained was 1.0 × 10⁶. 9 The primary bacterial cells cultured overnight were stored at -80°C with 20% glycerol. The primary glycerol-treated cells were inoculated into 250 mL of 2×YT medium (containing glucose and Amp) to achieve an OD600 of 0.1. After incubation at 37°C until the OD600 reached 0.4, helper phage M13KO7 (MOI 20) was added, and the cells were incubated at 37°C for 1 hour with intermittent shaking. The cells were then collected by centrifugation at 4°C and resuspended in 2×YT medium (containing Kana and Amp) to achieve an OD600 of 0.3. After incubation at 28°C for 16 hours, the liquid was collected by centrifugation at 4°C, and pre-chilled 5×PEG8000 solution was added. After incubation on ice for 1 hour, the phages were collected by centrifugation. 80 mL of TBS and PEG8000 were added sequentially, and the phages were collected by centrifugation at 4°C. The phage particles were resuspended in 2–10 mL of TBS, aliquoted with glycerol, and frozen for storage.

[0027] The phages prepared above were co-incubated with Pep10 and, in the presence of Sortase A, underwent enzymatic peptide linking and cyclization reactions to generate a bicyclic peptide library. Then, four rounds of affinity screening were performed against the immobilized Trop2 target protein. The phage titer collected by magnetic beads in each round was measured, and 40 phages were randomly selected from the fourth round of screening for sequencing.

[0028] Analysis of sequencing results identified the peptide sequences enriched for Trop2. Two corresponding target bicyclic peptides, Pep19 and Pep20, were chemically synthesized, and surface plasmon resonance (SPR) experiments were used to determine their binding energies to Trop2 to be 6.0 µM and 11.7 µM, respectively, thus verifying the actual effectiveness of this invention.

[0029] Example 1: Based on the above content Pep3 synthesis: 200.0 µmol of Rinkamide resin (360.0 mg, 0.56 mmol / g) was transferred to a solid-phase synthesis reactor with a 5.0 mL filter sieve. 3.0 mL of DMF was added to the resin, and the mixture was shaken at room temperature for 15 min. Then, 2.0 mL of a 20% piperidine DMF solution was added to the resin, and the mixture was shaken at room temperature for 6 min, repeating the Fmoc removal process. After washing the resin four times with DMF, 2.0 mL of an amino acid condensation reagent (containing 4.5 equivalents of Fmoc-Arg-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide) was added, and the reaction was carried out at 55 °C for 40 min. The resin was then washed four times with DMF, and solid-phase peptide condensation of Gly, Thr, Cys, Pro, and Leu was performed sequentially according to the peptide synthesis procedure described above. Next, the resin was washed four times with DMF, and 2.0 mL of a 20% piperidine DMF solution was added, repeating the Fmoc removal process. Add 2.0 mL of acetic anhydride blocking reagent (DMF / acetic anhydride / 2,6-dimethylpyridine = 89 / 5 / 6, volume ratio) to the DMF-washed resin. After shaking at room temperature for 2 min, wash the resin thoroughly with DMF and dichloromethane, and air dry the resin at room temperature. Add freshly prepared lysis buffer (containing TFA / m-cresol / water / triisopropylsilane volume ratio = 88 / 5 / 5 / 2) to the resin. After shaking at room temperature for 2 h, collect the lysis buffer, then add ice-cooled diethyl ether (9 times the volume), and centrifuge to obtain linear crude peptide powder with the sequence AcNH-Leu-Pro-Cys-Thr-Gly-Arg-NH2.

[0030] 60.0 mg of the above crude peptide powder was weighed and dissolved in 3.0 mL of DMF. 34.8 mg of 1,3-di(bromomethyl)benzene was added, followed by 45.6 µL of N,N-diisopropylethylamine. After the reaction system was placed on ice for 1 h, 4.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain an intermediate (13.6 mg, 23%). 10.0 mg of this intermediate was dissolved in 0.5 mL of DMF, followed by 4.9 mg of LiCl (dissolved in 0.1 mL H₂O). After reacting at room temperature for 2 h, 3.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain the target product Pep3 (4.3 mg, 43%). Mass spectrometry confirmed the correctness of Pep3 (molecular formula: C). 36 H 57 ClN 10 O8S, [M+H]⁺, theoretical molecular weight: 825.38, observed molecular weight: 825.27 g / mol).

[0031] The structural formula of the Pep3 molecule is as follows: .

[0032] Example 2: Based on the above content Pep4 synthesis: 200.0 µmol of Rinkamide resin (360.0 mg, 0.56 mmol / g) was transferred to a solid-phase synthesis reactor with a 5.0 mL filter sieve. 3.0 mL of DMF was added to the resin, and the mixture was shaken at room temperature for 15 min. Then, 2.0 mL of a 20% piperidine DMF solution was added to the resin, and the mixture was shaken at room temperature for 6 min (Fmoc removal). This removal step was repeated. After washing the resin four times with DMF, 2.0 mL of an amino acid condensation reagent (containing 4.5 equivalents of Fmoc-Arg-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide) was added, and the reaction was carried out at 55 °C for 40 min. The resin was then washed four times with DMF, and solid-phase peptide condensation of Gly, Thr, Cys, Pro, and Leu was performed sequentially according to the peptide synthesis procedure described above. Next, the resin was washed four times with DMF, and 2.0 mL of a 20% piperidine DMF solution was added, repeating the Fmoc removal process. Add 2.0 mL of acetic anhydride blocking reagent (DMF / acetic anhydride / 2,6-dimethylpyridine = 89 / 5 / 6, volume ratio) to the DMF-washed resin. After shaking at room temperature for 2 min, wash the resin thoroughly with DMF and dichloromethane, and air dry the resin at room temperature. Add freshly prepared lysis buffer (containing TFA / m-cresol / water / triisopropylsilane = 88 / 5 / 5 / 2) to the resin. After shaking at room temperature for 2 h, collect the lysis buffer, then add ice-cooled diethyl ether (9 times the volume), and centrifuge to obtain linear crude peptide powder with the sequence AcNH-Leu-Pro-Cys-Thr-Gly-Arg-NH2.

[0033] 60.0 mg of the above crude peptide powder was dissolved in 3.0 mL of DMF, and 34.8 mg of 1,4-di(bromomethyl)benzene was added, followed by 45.6 µL of N,N-diisopropylethylamine. After the reaction system was placed on ice for 1 h, 4.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain an intermediate (14.0 mg, 23%). 10.0 mg of this intermediate was dissolved in 0.5 mL of DMF, and then 4.9 mg of LiCl (dissolved in 0.1 mL H₂O) was added. After reacting at room temperature for 2 h, 3.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain the target product Pep4 (4.3 mg, 43%). Mass spectrometry confirmed the correctness of Pep4 (molecular formula: C). 36 H 57 ClN 10 O8S, [M+H]⁺, theoretical molecular weight: 825.38, observed molecular weight: 825.43 g / mol).

[0034] The structural formula of the Pep4 molecule is as follows: .

[0035] Example 3: Based on the above content Pep5 synthesis: 200.0 µmol of Rinkamide resin (360.0 mg, 0.56 mmol / g) was transferred to a solid-phase synthesis reactor with a 5.0 mL filter sieve. 3.0 mL of DMF was added to the resin, and the mixture was shaken at room temperature for 15 min. Then, 2.0 mL of a 20% piperidine DMF solution was added to the resin, and the mixture was shaken at room temperature for 6 min, repeating the Fmoc removal process. After washing the resin four times with DMF, 2.0 mL of an amino acid condensation reagent (containing 4.5 equivalents of Fmoc-Arg-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide) was added, and the reaction was carried out at 55 °C for 40 min. The resin was then washed four times with DMF, and solid-phase peptide condensation of Gly, Thr, Cys, Pro, and Leu was performed sequentially according to the peptide synthesis procedure described above. Next, the resin was washed four times with DMF, and 2.0 mL of a 20% piperidine DMF solution was added, repeating the Fmoc removal process. Add 2.0 mL of acetic anhydride blocking reagent (DMF / acetic anhydride / 2,6-dimethylpyridine = 89 / 5 / 6, volume ratio) to the DMF-washed resin. After shaking at room temperature for 2 min, wash the resin thoroughly with DMF and dichloromethane, and air dry the resin at room temperature. Add freshly prepared lysis buffer (containing TFA / m-cresol / water / triisopropylsilane = 88 / 5 / 5 / 2) to the resin. After shaking at room temperature for 2 h, collect the lysis buffer, then add ice-cooled diethyl ether (9 times the volume), and centrifuge to obtain linear crude peptide powder with the sequence AcNH-Leu-Pro-Cys-Thr-Gly-Arg-NH2.

[0036] 60.0 mg of the above crude peptide powder was weighed and dissolved in 3.0 mL of DMF. 34.8 mg of 1,2-di(bromomethyl)benzene was added, followed by 45.6 µL of N,N-diisopropylethylamine. After the reaction system was placed on ice for 1 h, 4.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain an intermediate (11.6 mg, 19%). 10.0 mg of this intermediate was dissolved in 0.5 mL of DMF, followed by 4.9 mg of LiCl (dissolved in 0.1 mL H₂O). After reacting at room temperature for 2 h, 3.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain the target product Pep5 (4.5 mg, 45%). Mass spectrometry confirmed the correctness of Pep5 (molecular formula: C). 36 H 57 ClN 10O8S, [M+H]⁺, theoretical molecular weight: 825.38, observed molecular weight: 825.30 g / mol).

[0037] The structural formula of the Pep5 molecule is as follows: .

[0038] Example 4: Based on the above content Synthesis of Pep6: 200.0 µmol of Rinkamide resin (360.0 mg, 0.56 mmol / g) was transferred to a solid-phase synthesis reactor with a 5.0 mL filter sieve. 3.0 mL of DMF was added to the resin, and the mixture was shaken at room temperature for 15 min. Then, 2.0 mL of a 20% piperidine DMF solution was added to the resin, and the mixture was shaken at room temperature for 6 min, repeating the Fmoc removal process. After washing the resin four times with DMF, 2.0 mL of an amino acid condensation reagent (containing 4.5 equivalents of Fmoc-Arg-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide) was added, and the reaction was carried out at 55 °C for 40 min. The resin was then washed four times with DMF, and solid-phase peptide condensation of Gly, Thr, Cys, Pro, and Leu was performed sequentially according to the peptide synthesis procedure described above. Next, the resin was washed four times with DMF, and 2.0 mL of a 20% piperidine DMF solution was added, repeating the Fmoc removal process. Add 2.0 mL of acetic anhydride blocking reagent (DMF / acetic anhydride / 2,6-dimethylpyridine = 89 / 5 / 6, volume ratio) to the DMF-washed resin. After shaking at room temperature for 2 min, wash the resin thoroughly with DMF and dichloromethane, and air dry the resin at room temperature. Add freshly prepared lysis buffer (containing TFA / m-cresol / water / triisopropylsilane = 88 / 5 / 5 / 2) to the resin. After shaking at room temperature for 2 h, collect the lysis buffer, then add ice-cooled diethyl ether (9 times the volume), and centrifuge to obtain linear crude peptide powder with the sequence AcNH-Leu-Pro-Cys-Thr-Gly-Arg-NH2.

[0039] 60.0 mg of the above crude peptide powder was weighed and dissolved in 3.0 mL of DMF. 34.8 mg of 2,6-bis(bromomethyl)pyridine was added, followed by 45.6 µL of N,N-diisopropylethylamine. After the reaction system was placed on ice for 1 h, 4.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain an intermediate (9.6 mg, 16%). 9.6 mg of this intermediate was dissolved in 0.5 mL of DMF, followed by 4.9 mg of LiCl (dissolved in 0.1 mL H₂O). After reacting at room temperature for 2 h, 3.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain the target product Pep6 (4.2 mg, 44%). Mass spectrometry confirmed the correctness of Pep6 (molecular formula: C). 35 H 56 ClN 11 O8S, [M+H]⁺, theoretical molecular weight: 826.37, observed molecular weight: 826.36 g / mol).

[0040] The structural formula of the Pep6 molecule is as follows: .

[0041] Example 5: Based on the above content Pep7 Synthesis: 200.0 µmol of Rinkamide resin (360.0 mg, 0.56 mmol / g) was transferred to a solid-phase synthesis reactor with a 5.0 mL filter sieve. 3.0 mL of DMF was added to the resin, and the mixture was shaken at room temperature for 15 min. Then, 2.0 mL of a 20% piperidine DMF solution was added to the resin, and the mixture was shaken at room temperature for 6 min, repeating the Fmoc removal process. After washing the resin four times with DMF, 2.0 mL of an amino acid condensation reagent (containing 4.5 equivalents of Fmoc-Arg-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide) was added, and the reaction was carried out at 55 °C for 40 min. The resin was then washed four times with DMF, and solid-phase peptide condensation of Gly, Thr, Cys, Pro, and Leu was performed sequentially according to the peptide synthesis procedure described above. Next, the resin was washed four times with DMF, and 2.0 mL of a 20% piperidine DMF solution was added, repeating the Fmoc removal process. Add 2.0 mL of acetic anhydride blocking reagent (DMF / acetic anhydride / 2,6-dimethylpyridine = 89 / 5 / 6, volume ratio) to the DMF-washed resin. After shaking at room temperature for 2 min, wash the resin thoroughly with DMF and dichloromethane, and air dry the resin at room temperature. Add freshly prepared lysis buffer (containing TFA / m-cresol / water / triisopropylsilane = 88 / 5 / 5 / 2) to the resin. After shaking at room temperature for 2 h, collect the lysis buffer, then add ice-cooled diethyl ether (9 times the volume), and centrifuge to obtain linear crude peptide powder with the sequence AcNH-Leu-Pro-Cys-Thr-Gly-Arg-NH2.

[0042] 60.0 mg of the above crude peptide powder was weighed and dissolved in 3.0 mL of DMF. 44.7 mg of 4,4'-di(bromomethyl)biphenyl was added, followed by 45.6 µL of N,N-diisopropylethylamine. After the reaction system was placed on ice for 1 h, 4.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain an intermediate (19.2 mg, 32%). 10.0 mg of this intermediate was dissolved in 0.5 mL of DMF, followed by 4.9 mg of LiCl (dissolved in 0.1 mL H₂O). After reacting at room temperature for 2 h, 3.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain the target product Pep7 (4.8 mg, 48%). Mass spectrometry confirmed the correctness of Pep7 (molecular formula: C). 42 H 61 ClN 10 O8S, [M+H]⁺, theoretical molecular weight: 901.41, observed molecular weight: 901.72 g / mol).

[0043] The structural formula of the Pep7 molecule is as follows: Example 6: Based on the above content Pep8 synthesis: 200.0 µmol of Rinkamide resin (360.0 mg, 0.56 mmol / g) was transferred to a solid-phase synthesis reactor with a 5.0 mL filter sieve. 3.0 mL of DMF was added to the resin, and the mixture was shaken at room temperature for 15 min. Then, 2.0 mL of a 20% piperidine DMF solution was added to the resin, and the mixture was shaken at room temperature for 6 min, repeating the Fmoc removal process. After washing the resin four times with DMF, 2.0 mL of an amino acid condensation reagent (containing 4.5 equivalents of Fmoc-Arg-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide) was added, and the reaction was carried out at 55 °C for 40 min. The resin was then washed four times with DMF, and solid-phase peptide condensation of Gly, Thr, Cys, Pro, and Leu was performed sequentially according to the peptide synthesis procedure described above. Next, the resin was washed four times with DMF, and 2.0 mL of a 20% piperidine DMF solution was added, repeating the Fmoc removal process. Add 2.0 mL of acetic anhydride blocking reagent (DMF / acetic anhydride / 2,6-dimethylpyridine = 89 / 5 / 6, volume ratio) to the DMF-washed resin. After shaking at room temperature for 2 min, wash the resin thoroughly with DMF and dichloromethane, and air dry the resin at room temperature. Add freshly prepared lysis buffer (containing TFA / m-cresol / water / triisopropylsilane = 88 / 5 / 5 / 2) to the resin. After shaking at room temperature for 2 h, collect the lysis buffer, then add ice-cooled diethyl ether (9 times the volume), and centrifuge to obtain linear crude peptide powder with the sequence AcNH-Leu-Pro-Cys-Thr-Gly-Arg-NH2.

[0044] 30.0 mg of the above crude peptide powder was weighed and dissolved in 2.0 mL of DMF. 19.5 μL of hexafluorobenzene was added, followed by 34.8 mg of Tris (dissolved in 0.2 mL of water). The mixture was placed in a vortex reactor and reacted at room temperature for 2 h. Then, 4.0 mL of an acetonitrile-water mixture (3:1 v / v, containing 0.1% TFA) was added. HPLC separation and freeze-drying were then performed to obtain the target product Pep8 (10.8 mg, 36%). Mass spectrometry confirmed the correctness of Pep8 (molecular formula: C). 34 H 49 F5N 10 O8S, [M+H]⁺, theoretical molecular weight: 853.34, observed molecular weight: 853.35 g / mol).

[0045] The structural formula of the Pep8 molecule is as follows: .

[0046] Example 7: Based on the above content Pep9 synthesis: 200.0 µmol of Rinkamide resin (360.0 mg, 0.56 mmol / g) was transferred to a solid-phase synthesis reactor with a 5.0 mL filter sieve. 3.0 mL of DMF was added to the resin, and the mixture was shaken at room temperature for 15 min. Then, 2.0 mL of a 20% piperidine DMF solution was added to the resin, and the mixture was shaken at room temperature for 6 min, repeating the Fmoc removal process. After washing the resin four times with DMF, 2.0 mL of an amino acid condensation reagent (containing 4.5 equivalents of Fmoc-Arg-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide) was added, and the reaction was carried out at 55 °C for 40 min. The resin was then washed four times with DMF, and solid-phase peptide condensation of Gly, Thr, Cys, Pro, and Leu was performed sequentially according to the peptide synthesis procedure described above. Next, the resin was washed four times with DMF, and 2.0 mL of a 20% piperidine DMF solution was added, repeating the Fmoc removal process. Add 2.0 mL of acetic anhydride blocking reagent (DMF / acetic anhydride / 2,6-dimethylpyridine = 89 / 5 / 6, volume ratio) to the DMF-washed resin. After shaking at room temperature for 2 min, wash the resin thoroughly with DMF and dichloromethane, and air dry the resin at room temperature. Add freshly prepared lysis buffer (containing TFA / m-cresol / water / triisopropylsilane = 88 / 5 / 5 / 2) to the resin. After shaking at room temperature for 2 h, collect the lysis buffer, then add ice-cooled diethyl ether (9 times the volume), and centrifuge to obtain linear crude peptide powder with the sequence AcNH-Leu-Pro-Cys-Thr-Gly-Arg-NH2.

[0047] 30.0 mg of the above crude peptide powder was weighed and dissolved in 2.0 mL of DMF. 19.1 mg of decafluorobiphenyl was added, followed by 34.8 mg of Tris (dissolved in 0.2 mL of water). The mixture was placed in a vortex reactor and reacted at room temperature for 2 hours. Then, 4.0 mL of acetonitrile-water mixed solvent (volume ratio 3:1, containing 0.1% TFA) was added. HPLC separation and freeze-drying were then performed to obtain the target product Pep9 (7.2 mg, 24%). Mass spectrometry confirmed the correctness of Pep9 (molecular formula: C). 40 H 49 F9N 10 O8S, [M+H]⁺, theoretical molecular weight: 1001.33, observed molecular weight: 1001.21 g / mol).

[0048] The structural formula of the Pep9 molecule is as follows: .

[0049] Example 8: Based on the above content Synthesis of Pep10: 200.0 µmol of Rinkamide resin (360.0 mg, 0.56 mmol / g) was transferred to a solid-phase synthesis reactor with a 5.0 mL filter sieve. 3.0 mL of DMF was added to the resin, and the mixture was shaken at room temperature for 15 min. Then, 2.0 mL of a 20% piperidine DMF solution was added to the resin, and the mixture was shaken at room temperature for 6 min, repeating the Fmoc removal process. After washing the resin four times with DMF, 2.0 mL of an amino acid condensation reagent (containing 4.5 equivalents of Fmoc-Arg-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide) was added, and the reaction was carried out at 55 °C for 40 min. The resin was then washed four times with DMF, and solid-phase peptide condensation of Gly, Thr, Cys, Pro, and Leu was performed sequentially according to the peptide synthesis procedure described above. Next, the resin was washed four times with DMF, and 2.0 mL of a 20% piperidine DMF solution was added, repeating the Fmoc removal process. Add 2.0 mL of acetic anhydride blocking reagent (DMF / acetic anhydride / 2,6-dimethylpyridine = 89 / 5 / 6, volume ratio) to the DMF-washed resin. After shaking at room temperature for 2 min, wash the resin thoroughly with DMF and dichloromethane, and air dry the resin at room temperature. Add freshly prepared lysis buffer (containing TFA / m-cresol / water / triisopropylsilane = 88 / 5 / 5 / 2) to the resin. After shaking at room temperature for 2 h, collect the lysis buffer, then add ice-cooled diethyl ether (9 times the volume), and centrifuge to obtain linear crude peptide powder with the sequence AcNH-Leu-Pro-Cys-Thr-Gly-Arg-NH2.

[0050] 60.0 mg of the above crude peptide powder was weighed and dissolved in 3.0 mL of DMF. 46.8 mg of 1,3,5-tris(bromomethyl)benzene was added, followed by 45.6 µL of N,N-diisopropylethylamine. After the reaction system was placed on ice for 1 h, 4.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain an intermediate (20.0 mg, 33%). 20.0 mg of this intermediate was dissolved in 0.5 mL of DMF, followed by 8.8 mg of LiCl (dissolved in 0.1 mL H₂O). After reacting at room temperature for 2 h, 3.0 mL of acetonitrile-water mixed solvent (3:1 v / v, containing 0.1% TFA) was added. The mixture was then separated by HPLC and freeze-dried to obtain the target product Pep10 (9.6 mg, 48%). Mass spectrometry confirmed the correctness of Pep10 (molecular formula: C). 37 H 58 Cl2N 10 O8S, [M+H]⁺, theoretical molecular weight: 873.35, observed molecular weight: 873.13 g / mol).

[0051] The structural formula of the Pep10 molecule is as follows: .

[0052] Example 9: Sortase A expression and purification: The Sortase A enzyme plasmid vector (Sortase A mutant, Addgene, Plasmid#75144) was transformed into DH5 competent cells and seeded on LB agar plates (containing kanamycin) for overnight culture. Single colonies were picked and seeded into 3.0 mL of LB medium (containing kanamycin) for overnight culture. The plasmid was extracted and sent to the company for sequencing to confirm its correctness, and then aliquoted and stored in a freezer.

[0053] Sortase A plasmid was transformed into BL21(DE3) competent cells and inoculated onto LB agar plates (containing kanamycin) for overnight culture. Single colonies were picked and inoculated into 10.0 mL of LB medium (containing kanamycin) and cultured overnight at 37°C with a shaker. 500.0 mL of LB medium (containing 250 µL kanamycin, 100.0 mg / mL) was added, along with 5.0 mL of the overnight cultured strain, and cultured at 37°C. After the OD600 of the bacterial culture reached 0.8 (3 h), 150.0 µL of IPTG stock solution (1 mmol / L) was added to the bacterial culture, and the culture was incubated at 16°C (180 rpm). After 20 h, the bacterial culture was centrifuged, the bacterial cells were collected, and washed with lysis buffer (20.0 mM HEPES, 500.0 mM NaCl, 5% glycerol, pH 7.5). 50.0 mL of lysis buffer was added to the bacterial cells, followed by sonication. Cell debris was removed by centrifugation, and the supernatant containing Sortase A was purified by nickel column chromatography to obtain Sortase A with a purity of 90%. The solution was then concentrated by ultrafiltration and replaced with lysis buffer to obtain Sortase A enzyme solution (9 mg / mL). The solution was aliquoted and stored at -80°C, with each aliquot containing 225.0 µg (25.0 µL) of Sortase A enzyme for enzymatic peptide ligation and cyclization.

[0054] Example 10: Enzymatic cyclization of Pep1 and Pep3 to prepare monocyclic peptide Pep11 reaction Prepare 10.0 mL of HEPES buffer (pH 8.0) containing 50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, and 100 µM TCEP (tris(2-carboxyethyl)phosphine). Take 500 µL of HEPES buffer (pH 8.0), add Pep1 (stock concentration 12.5 mM, final concentration 25.0 µM), then add 1.2 µL of Sortase A enzyme (40.0 mg / mL, 48.0 µg, final concentration 5 µM), followed by Pep3 (stock concentration 12.5 mM, final concentration 40.0 µM). After mixing the reaction solutions, detect the reaction using high-performance liquid chromatography (HPLC) at 37°C in a shaker. Pep1 and Pep3 were pre-dissolved in an aqueous solution containing 1% acetonitrile and 0.1% trifluoroacetic acid.

[0055] like Figure 1 As shown, in the presence of Sortase A, Pep1 and Pep3 underwent enzymatic peptide linkage and cyclization to generate the target cyclic peptide Pep11 (ESI-MS (m / z): calculated for C 109 H 156 N 28 O 29 S2 2386.10; Found 2385.94). Experimental results show that the linkage and cyclization reactions of Pep1 and Pep3 are completed after 1 hour of enzyme catalysis.

[0056] Example 11: Enzymatic cyclization of Pep1 and Pep4 to prepare monocyclic peptide Pep12 Prepare 10.0 mL of HEPES buffer (pH 8.0) containing 50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, and 100 µM TCEP (tris(2-carboxyethyl)phosphine). Take 500 µL of HEPES buffer (pH 8.0), add Pep1 (stock concentration 12.5 mM, final concentration 25.0 µM), then add 4.8 µL of Sortase A enzyme (40.0 mg / mL, 192.0 µg, final concentration 20 µM), followed by Pep4 (stock concentration 12.5 mM, final concentration 40.0 µM). After mixing the reaction solutions, incubate at 37°C in a shaker and detect the reaction using high-performance liquid chromatography (HPLC). Pep1 and Pep4 were pre-dissolved in an aqueous solution containing 1% acetonitrile and 0.1% trifluoroacetic acid.

[0057] like Figure 2As shown, in the presence of Sortase A, Pep1 and Pep4 underwent enzymatic peptide linkage and cyclization to generate the target cyclic peptide Pep12 (ESI-MS (m / z): calculated for C 109 H 156 N 28 O 29 S2 2386.10; Found2386.58). Experimental results show that the linkage and cyclization reactions of Pep1 and Pep4 are completed after 1 hour of enzyme catalysis.

[0058] Example 12: Enzymatic cyclization of Pep1 and Pep5 to prepare monocyclic peptide Pep13 Prepare 10.0 mL of HEPES buffer (pH 8.0) containing 50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, and 100 µM TCEP (tris(2-carboxyethyl)phosphine). Take 500 µL of HEPES buffer (pH 8.0), add Pep1 (stock concentration 12.5 mM, final concentration 25.0 µM), then add 1.2 µL of Sortase A enzyme (40.0 mg / mL, 48.0 µg, final concentration 5 µM), followed by Pep5 (stock concentration 12.5 mM, final concentration 40.0 µM). After mixing the reaction solutions, detect the reaction using high-performance liquid chromatography (HPLC) at 37°C in a shaker. Pep1 and Pep5 were pre-dissolved in an aqueous solution containing 1% acetonitrile and 0.1% trifluoroacetic acid.

[0059] like Figure 3 As shown, in the presence of Sortase A, Pep1 and Pep5 underwent enzymatic peptide linkage and cyclization to generate the target cyclic peptide Pep13 (ESI-MS (m / z): calculated for C 109 H 156 N 28 O 29 S2 2386.10; Found 2385.80 g / mol). Experimental results showed that the linkage and cyclization reactions of Pep1 and Pep5 were completed after 1 h of enzyme catalysis.

[0060] Example 13: The enzymatic cyclization of Pep1 and Pep6 to prepare the monocyclic peptide Pep14: Prepare 10.0 mL of HEPES buffer (pH 8.0) containing 50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, and 100 µM TCEP (tris(2-carboxyethyl)phosphine). Take 500 µL of HEPES buffer (pH 8.0), add Pep1 (stock concentration 12.5 mM, final concentration 25.0 µM), then add 1.2 µL of Sortase A enzyme (40.0 mg / mL, 48.0 µg, final concentration 5 µM), followed by Pep6 (stock concentration 12.5 mM, final concentration 40.0 µM). After mixing the reaction solutions, detect the reaction using high-performance liquid chromatography (HPLC) at 37°C in a shaker. The reactive peptides of Pep1 and Pep6 were pre-dissolved in an aqueous solution containing 1% acetonitrile and 0.1% trifluoroacetic acid.

[0061] like Figure 4 As shown, in the presence of Sortase A, Pep1 and Pep6 underwent enzymatic peptide linkage and cyclization to generate the target cyclic peptide Pep14 (ESI-MS (m / z): calculated for C 108 H 155 N 29 O 29 S2 2387.10; Found2387.16). Experimental results show that the linkage and cyclization reactions of Pep1 and Pep6 are completed after 1 hour of enzyme catalysis.

[0062] Example 14: Enzymatic cyclization of Pep1 and Pep7 to prepare monocyclic peptide Pep15 Prepare 10.0 mL of HEPES buffer (pH 8.0) containing 50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, and 100 µM TCEP (tris(2-carboxyethyl)phosphine). Take 500 µL of HEPES buffer (pH 8.0), add Pep1 (stock concentration 12.5 mM, final concentration 25.0 µM), then add 4.8 µL of Sortase A enzyme (40.0 mg / mL, 192.0 µg, final concentration 20 µM), followed by Pep7 (stock concentration 12.5 mM, final concentration 40.0 µM). After mixing the reaction solutions, detect the reaction using high-performance liquid chromatography (HPLC) at 37°C in a shaker. Pep1 and Pep7 were pre-dissolved in an aqueous solution containing 1% acetonitrile and 0.1% trifluoroacetic acid.

[0063] like Figure 5As shown, in the presence of Sortase A, Pep1 and Pep7 underwent enzymatic peptide linkage and cyclization to generate the target cyclic peptide Pep15 (ESI-MS (m / z): calculated for C 115 H 160 N 28 O 29 S2 2462.12; Found2462.44). Experimental results show that the linkage and cyclization reactions of Pep1 and Pep7 are completed after 1 hour of enzyme catalysis.

[0064] Example 15: Enzymatic cyclization of Pep1 and Pep8 to prepare monocyclic peptide Pep16 Prepare 10.0 mL of HEPES buffer (pH 9.0) containing 50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, 100 µM TCEP (tris(2-carboxyethyl)phosphine), and 1 mM reduced glutathione. Take 500 µL of HEPES buffer (pH 9.0), add Pep1 (stock concentration 12.5 mM, final concentration 25.0 µM), then add 14.4 µL of Sortase A enzyme (40.0 mg / mL, 576.0 µg, final concentration 60.0 µM), followed by Pep8 (stock concentration 12.5 mM, final concentration 250.0 µM). After mixing the reaction solutions, detect the reaction using high-performance liquid chromatography (HPLC) in a shaker at 37°C. Pep1 and Pep8 were pre-dissolved in an aqueous solution containing 1% acetonitrile and 0.1% trifluoroacetic acid.

[0065] like Figure 6 As shown, in the presence of Sortase A, Pep1 and Pep8 underwent enzymatic peptide linkage and cyclization to generate the target cyclic peptide Pep16 (ESI-MS (m / z): calculated for C 107 H 148 F4N 28 O 29 S2 2430.03; Found2430.04). Experimental results show that the linkage and cyclization reactions of Pep1 and Pep8 were completed after 6 hours of enzyme catalysis.

[0066] Example 16: Enzymatic cyclization of Pep1 and Pep9 to prepare monocyclic peptide Pep17 reaction Prepare 10.0 mL of HEPES buffer (pH 9.0) containing 50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, 100 µM TCEP (tris(2-carboxyethyl)phosphine), and 1 mM reduced glutathione. Take 500 µL of HEPES buffer (pH 9.0), add Pep1 (stock concentration 12.5 mM, final concentration 25.0 µM), then add 1.2 µL of Sortase A enzyme (40.0 mg / mL, 48.0 µg, final concentration 5 µM), followed by Pep9 (stock concentration 12.5 mM, final concentration 250.0 µM). After mixing the reaction solutions, detect the reaction using high-performance liquid chromatography (HPLC) at 37°C in a shaker. Pep1 and Pep9 were pre-dissolved in an aqueous solution containing 1% acetonitrile and 0.1% trifluoroacetic acid.

[0067] like Figure 7 As shown, in the presence of Sortase A, Pep1 and Pep9 underwent enzymatic peptide linkage and cyclization to generate the target cyclic peptide Pep17 (ESI-MS (m / z): calculated for C 113 H 148 F8N 28 O 29 S2 2578.02; Found2577.86). Experimental results show that the linkage and cyclization reactions of Pep1 and Pep9 were completed after 3 hours of enzyme catalysis.

[0068] Example 17: Enzymatic cyclization of Pep2 and Pep10 to prepare bicyclic peptide Pep18 Prepare 10.0 mL of HEPES buffer (pH 7.0) containing 50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, and 100 µM TCEP (tris(2-carboxyethyl)phosphine). Take 500 µL of HEPES buffer (pH 7.0), add Pep2 (stock concentration 12.5 mM, final concentration 25.0 µM), then add 4.8 µL of Sortase A enzyme (40.0 mg / mL, 192.0 µg, final concentration 20 µM), followed by Pep10 (stock concentration 12.5 mM, final concentration 40.0 µM). After mixing the reaction solutions, detect the reaction using high-performance liquid chromatography (HPLC) at 37°C in a shaker. Pep2 and Pep10 were pre-dissolved in an aqueous solution containing 1% acetonitrile and 0.1% trifluoroacetic acid.

[0069] like Figure 8As shown, in the presence of Sortase A, Pep2 and Pep10 underwent enzymatic peptide linkage and cyclization to generate the target bicyclic peptide Pep18 (ESI-MS (m / z): calculated for C 113 H 132 N 32 O 30 S3 2544.12; Found2544.50). Experimental results showed that the linkage and cyclization reactions of Pep2 and Pep10 were completed after 30 min of enzyme catalysis.

[0070] Example 18: Construction of a phage bicyclic peptide library and screening of functional bicyclic peptide ligands (Trop2) 1. Constructing the pCANTAB 5E-GX5CX4C phage library A phage peptide library displaying GX5CX4C was prepared using pCANTAB 5E phagemid-helper phage (M13KO7) (from Sichuan Apak Biotechnology Co., Ltd.). (X is a random amino acid encoded by MNN, where N is A / T / C / G, M is C / A, and K is G / T). Note that the BsaI restriction site on the pCANTAB 5E phagemid vector was mutated. Two DNA sequences were designed and custom-made from Genscript Biotech (Nanjing) Co., Ltd.: Library DNA (primer B): 5'-TTTGGTCTCAGCACCGCCAGAGCCGCCGCAMNNMNNMNNMNNGCAMNNMNNMNNMNNNNACCGGCCATGGCCGGCTGGGCCGCATAGAAAGG-3' (where N is A / T / C / G, M is C / A, K is G / T, and the underlined bases are BsaI restriction sites); Primer DNA (primer A): 5'-TTGGTCTCGGTGCGCCGGTGCCGTATCCGGATCCGCTG-3'; After mixing the two DNAs, a full-plasmid PCR reaction was performed using the pCANTAB 5E mutant vector (without the BsaI restriction site) as a template under KeyPo enzyme. The recovered DNA was treated with BsaI and DpnI, followed by overnight ligation using T4 ligase. The ligated vector was electroporated into TG1 cells, and the transformed colony count reached 1.0 × 10⁻⁶. 9 After bacterial inoculation and culture, the cells were packaged into a phage peptide library according to standard procedures with the helper phage M13KO7.

[0071] 2. Biotinylation of target proteins 100.0 µg of target protein Trop2 (29.4 kDa, final concentration 10.0 µM) and Biotin-PEG-NHS (20 equivalents, commercially available, 1378 g / mol) were dissolved in 100.0 µL PBS (pH 7.4). After 2 h at room temperature, excess biotin was removed by Zeba™ desalting, and the target protein was obtained by elution.

[0072] 3. Phage selection for Trop2 Cryopreserved glycerol-containing bacteriophages were inoculated into 2×YT liquid medium (containing glucose and Amp) to achieve an OD600 of 0.1. After incubation at 37°C until the OD600 reached 0.3–0.5, helper phage M13KO7 was added at a multiplicity of infection (MOI) of 20, and the culture was incubated at 37°C for 1 hour. The supernatant was removed by centrifugation at 4°C and 10,000 rpm, and the cells were collected and resuspended in 2×YT medium (containing Amp and Kana). After 16 hours (28°C), the cells were centrifuged at 4°C and 8,000 rpm, and the supernatant was collected and pre-chilled 5×PEG8000 / NaCl solution was added. After incubation on ice for 1 hour, the cells were centrifuged at 4°C and 10,000 rpm to collect the phage particles, which were then resuspended in 100 mL TBS. The supernatant was collected by centrifugation at 4°C and 10,000 rpm, and the phages were reprecipitated using PEG8000 / NaCl. Phages collected by centrifugation at 4°C and 10,000 rpm were resuspended in TBS and filtered through a 0.45 µm sterile filter. Phages can be stored at 4°C for several days and in glycerol at -20°C for long-term storage.

[0073] Take 2×10 12PFU phage (diluted to 45 µL with PBS pH 7.2) was added, followed by 5 µL of 10 mM TCEP. The phage was incubated at 37 °C (250 rpm) for 20 min. Then, 450 µL of HEPES buffer (50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, pH 7.0) and Pep10 (stock concentration 12.5 mM, final concentration 50.0 µM) were added, followed by 4.8 µL of Lortase enzyme (final concentration 20.0 µM). The mixture was incubated at 37 °C (250 rpm) for 40 min. Finally, 125.0 µL of 5×PEG8000 / NaCl solution (stored at 4 °C) was added, and the mixture was incubated on ice for 30 min, followed by centrifugation at 4 °C (10000 rpm) for 30 min. Resuspend the phage pellet in 1.5 mL Binding Buffer and 750.0 µL Blocking Buffer, and incubate at 25 °C (slow shaking) for 30 min. Wash 40.0 µL of streptavidin-coated magnetic beads (Dynabeads M-280, 10 mg / mL) three times with 1.0 mL PBS buffer, and then resuspend the streptavidin-coated magnetic beads in 40.0 µL PBS. Add 20.0 µL of the magnetic bead solution to the blocked phage library and incubate at 25 °C (slow shaking) for 30 min. Collect the phage library supernatant, mix it with the remaining 20.0 µL of magnetic bead solution, and incubate at 25 °C (slow shaking) for 30 min.

[0074] Take 20.0 µL of streptavidin-coated magnetic beads, wash three times with 1 mL PBS buffer, resuspend in 200 µL PBS buffer, add 4.3 µL of biotin-labeled TROP2 (5.0 µg), and incubate at 25°C (with slow shaking) for 30 min. Wash the magnetic beads three times with 1.0 mL PBS, then block the magnetic beads with 300.0 µL Binding Buffer and 150.0 µL Blocking Buffer, and incubate at 25°C (with slow shaking) for 30 min. Add the TROP2-coated streptavidin magnetic bead solution to the phage solution treated with streptavidin-coated magnetic beads, and incubate at 4°C (with slow shaking) for 30 min. Wash the magnetic beads eight times with 1.0 mL washing buffer, and then wash twice with 1.0 mL Binding Buffer. To avoid nonspecific binding, change the centrifuge tube three times during the washing process. Then, the magnetic beads were resuspended in 100.0 µL of Elution buffer (pH 2.2), incubated for 5 min, placed on a magnetic rack, and the supernatant was transferred to a new centrifuge tube containing 50.0 µL of Neutralization buffer (pH 8.0).

[0075] A small amount of the collected phages was used for titer determination. The rest were incubated with TG1 at 37°C for 1 hour, centrifuged at 4°C and 4000 rpm for 15 minutes, the supernatant was discarded, and the cells were resuspended in 2×YT. The cells were then plated in 2×YT (containing Amp and glucose) plates and incubated upside down at 28°C overnight. The next day, the cells were scraped off with 2×YT (containing Amp) and the second, third, and fourth rounds of phage preparation and screening were performed according to the above procedure. In the fourth round of screening, magnetic beads uncoated with the target protein were used as a control.

[0076] After four rounds of screening, the phage titer increased by 6250-fold, and its titer was 1000-fold higher than that of the control group in the fourth round, indicating that the phage had undergone significant enrichment. Figure 9 Randomly selected fourth-round phage particle sequencing revealed two highly enriched polypeptide sequences (GWLPLACFYQTCGGK and GWLPMQCFFSSCGGK, respectively). Figure 10 The bicyclic peptides Pep19 and Pep20 corresponding to this sequence were synthesized, and surface plasmon resonance assays were performed on the target Trop2. Their binding affinity to Trop2 was 6.01 µM and 11.70 µM, respectively. These results demonstrate the effectiveness of the technical solution in this patent for screening functional bicyclic peptides.

[0077] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide compound containing a low-reactive group, characterized in that, The general structural formula of the polypeptide compound is as follows: ； Among them, X a 3-(chloromethyl)benzyl: 4-(chloromethyl)benzyl: 2-(chloromethyl)benzyl: 2-(chloromethyl)pyridine methyl derivatives: 4'-(chloromethyl)biphenyl derivatives: , Pentafluorobenzene derivatives: Nonafluorobiphenyl derivatives: And 3,5-di(chloromethyl)benzyl: Any one of them; X b It is any one of hydrogen, acetyl, or oligopeptide group composed of natural or non-natural amino acids other than cysteine; X c It can be any one of O, NH, and S; X d It is any one of the amino groups or oligopeptide sequences composed of natural or non-natural amino acids other than cysteine.

2. A method for enzymatic cyclization using a polypeptide compound containing a low-reactive group as described in claim 1 as a substrate, characterized in that, The specific operation method of the enzyme-catalyzed cyclization reaction includes the following steps: S1. Under the catalysis of Sortase A enzyme, a polypeptide compound containing a low reactive group is combined with a polypeptide with glycine at the N-terminus and cysteine ​​in the sequence in a non-phosphate buffer to undergo an enzymatic polypeptide linkage and cyclization reaction to generate a cyclic peptide. S2. Select a polypeptide template from S1 with glycine at the N-terminus and containing cysteine ​​in the sequence and fuse it to the N-terminus of the phage pIII protein to prepare a phage cyclic peptide library, and perform in vitro screening of cyclic peptide ligands using target proteins.

3. The enzymatic cyclization method according to claim 2, characterized in that: The template for the polypeptide in S1 can be one of the following two types: Template a: G-(X) m -C; Template b: G-(X) m -C-(X) n -C; In this context, template a is used for constructing a monocyclic peptide library, template b is used for constructing a bicyclic peptide library, G is glycine, X is any natural L-amino acid, C is L-cysteine, and m and n are any number of amino acids between 3 and 18.

4. The enzymatic cyclization method according to claim 2, characterized in that: The concentration range of the polypeptide compound containing low reactive groups in S1 is 0.01µM to 20.0mM, and the concentration range of Sortase A enzyme is 0.01µM to 20.0mM.

5. The enzymatic cyclization method according to claim 2, characterized in that: The non-phosphate buffer in S1 contains any one of 4-hydroxyethylpiperazine ethanesulfonic acid, imidazole, and tris(hydroxymethyl)aminomethane, and contains 0.05mM~50mM CaCl2 and 0.05µM~10.0mM tris(2-carboxyethyl)phosphine, with a pH range of 6.0~10.

0.

6. The enzymatic cyclization method according to claim 2, characterized in that: The enzymatic peptide linking and cyclization reaction in S1 takes 15 min to 24 h and is carried out at a temperature of 15 to 60 °C.

7. The enzymatic cyclization method according to claim 2, characterized in that: The phage in S2 is either a phage or an M13KE phage produced by packaging pCANTAB5E phage particles and helper phage M13KO7.

8. The enzymatic cyclization method according to claim 2, characterized in that, The in vitro screening method for cyclic peptide ligands using target proteins in S2 includes the following steps: S3-1. Prepare a phage library displaying monocyclic or bicyclic peptides using an enzymatic peptide linking and cyclization method with peptide compounds containing low reactive groups. S3-2. Immobilize the biotin-modified target protein onto magnetic beads and co-incubate it with the phage library from step S3-1. Then perform 1-6 rounds of affinity panning and sequence the enriched phages. S3-3. Analyze the sequencing results, synthesize the enriched cyclic peptides, and evaluate their binding affinity to the target protein.

9. An application of the enzymatic cyclization method as described in claims 2-8, characterized in that, The enzymatic cyclization is applied to the construction of cyclic peptide libraries, which include single- and double-cyclic peptide libraries for phage display and mRNA display.

10. The application according to claim 9, characterized in that, The cyclic peptide ligands obtained by enzymatic cyclization are used in the development of detection kits, drugs or other biomaterials.

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