Polypeptide compound, method for preparing cyclopeptide oxygen ester from polypeptide compound and application of polypeptide compound

Abstract

The invention provides a polypeptide compound as well as a method for preparing cyclopeptide oxygen ester and application thereof, and relates to the field of chemoenzymatic modification of a gene encoding peptide library. According to the preparation method of the cyclopeptide oxygen ester, the preparation of the cyclopeptide molecule connected with the side chain to the side chain oxygen ester by a Sortase A enzyme system is realized for the first time, and the cyclopeptide oxygen ester is applied to the oxygen ester bond cyclization modification of a phage display polypeptide library to generate a gene coding cyclopeptide oxygen ester library and is used for finding the cyclopeptide oxygen ester molecule combined with a target protein. The defects in the aspect of preparation of the cyclopeptide oxygen ester library in the prior art are overcome, the preparation method of the cyclopeptide oxygen ester has mild reaction conditions and high reaction efficiency, and the generated gene coding cyclopeptide oxygen ester library can be used for screening functional cyclopeptide oxygen ester. And a powerful platform technology is provided for the fields of drug discovery and biological materials.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical enzymatic modification of gene-encoded peptide library, and particularly relates to a polypeptide compound and a method for preparing a cyclic peptide ester and application thereof. BACKGROUND

[0002] Cyclic peptide esters are a unique type of cyclic peptides containing ester bonds, which exhibit a variety of biological activities, including anti-tumor, antibacterial and immunosuppressive functions. Compared with amide bonds, ester bonds lack a hydrogen bond donor, which can endow cyclic peptides with unique structural features and biochemical properties. A large number of bioactive cyclic peptide esters have been discovered from microorganisms, some of which, such as romidepsin and daptomycin, have been approved by FDA as clinical drugs. The discovery and structural analysis of cyclic peptide esters from microorganisms rely on tedious in vitro purification, spectroscopic analysis and bioinformatics techniques. In addition, the limited sequence and structural diversity of cyclic peptide esters extracted from nature may lead to some highly bioactive cyclic peptide esters being difficult to be discovered. Therefore, developing an efficient method for preparing cyclic peptide esters and applying it to generate a cyclic peptide ester molecule library from a gene-encoded peptide library can promote the exploration of this unique chemical space and facilitate the discovery of cyclic peptide esters with ideal biological activities.

[0003] Cyclic peptide esters mainly have three different forms. The first type is main-chain ester bond cyclic peptide, which refers to the replacement of amide in the polypeptide main chain with ester bond, mainly used to study the influence of local hydrogen bond on protein structure and function. The second type is side chain-tail cyclic peptide ester, which specifically refers to the connection between the side chain hydroxyl of Thr or Ser in the polypeptide sequence and the C-terminal carboxyl with an ester bond. Based on the principle of combinatorial chemistry, Inoue et al. constructed a side chain-tail cyclic peptide ester library, but the diversity of the library was limited and involved tedious and high-intensity chemical synthesis. Recently, Suga et al. used ribosome to synthesize side chain-tail cyclic peptide ester through intramolecular S-to-O acyl transfer reaction. It should be pointed out that side chain-tail cyclic peptide ester is difficult to be displayed by phage or mRNA, because the C-terminal carboxyl of the peptide library needs to be covalently connected with the pIII protein of phage or the puromycin of mRNA by amide bond. The third type is side chain-side chain cyclic peptide ester, which specifically refers to the connection between the side chain hydroxyl of Thr or Ser in the polypeptide sequence and the side chain carboxyl of Asp or Glu in the sequence with an ester bond. Recently, people have discovered a variety of bioactive side chain-side chain cyclic peptide esters from microorganisms, but there is currently a lack of efficient method for constructing a gene-encoded side chain-side chain cyclic peptide ester molecule library.

[0004] Sortase A, a transpeptidase isolated from Gram-positive bacteria, mediates peptide bond linkage between a polypeptide containing the Leu-Pro-Xxx-Thr-Gly motif (where Xxx represents any natural amino acid) and another polypeptide with an N-terminal glycine residue. Compared to traditional peptide chemical modification methods, the Sortase A-based enzymatic method offers milder reaction conditions and higher specificity. Sortase A ligation reactions are widely used in protein semi-synthesis and peptide modification. However, this system has not yet been used to prepare cyclic peptide oxyesters. Therefore, further exploration of the Sortase A-catalyzed ligation system, development of novel methods for preparing cyclic peptide oxyesters, and application to gene-encoded peptide libraries will contribute to the discovery of cyclic peptide oxyester ligand molecules. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a polypeptide compound, a method for preparing cyclic peptide oxyesters, and its applications, enabling the preparation of cyclic peptide oxyesters under mild reaction conditions and with high reaction efficiency catalyzed by Sortase A.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A polypeptide compound having the following general structural formula: Among them, X a It is a serine derivative: Threonine derivatives: or proline derivatives: Any one of them; R is any one of the electrophilic group and oligopeptide groups composed of natural or non-natural amino acids containing an electrophilic group. The electrophilic group includes any one of chloroacetyl, 3,5-di[(2-chloroacetyl)amino]benzoyl, 4-chloroacetamylbenzoyl, 3,5-di(chloromethyl)benzoyl, 2-(chloromethyl)benzoyl, 3-(chloromethyl)benzoyl, 4-(chloromethyl)benzoyl, and 4-(chloromethyl)bibenzoyl. 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 (oxyester bond), NH (amide bond), or S (thioester bond); X d It is any one of the amino groups or oligopeptide sequences composed of natural or non-natural amino acids other than cysteine.

[0007] Method for preparing cyclic peptide oxyesters using the above-mentioned polypeptide compounds: S1, polypeptide compound is connected with N-terminal glycine and polypeptide template containing cysteine in the sequence in the presence of Sortase A enzyme in the buffer salt solution, polypeptide connection and intramolecular cyclization reaction occur, and a cyclic peptide ester molecule is generated; S2, the N-terminal glycine and polypeptide template containing cysteine in the sequence in S1 are selected, and the phage display cyclic peptide ester library is constructed by gene coding fusion expression at the N-terminal of phage pIII protein, and the cyclic peptide ligand is screened for the target protein through the operation of S2.

[0008] The polypeptide template is as follows: Template a: G-(X)m-C; template b: G-(X)m-C-(X)n-C; Wherein, template a is used to construct a single cyclic peptide ester, template b is used to construct a double cyclic peptide ester, G represents glycine, X represents any kind of natural L-amino acid, C represents L-cysteine and the position can be changed according to requirements, m and n represent the number of amino acids between 3 and 20.

[0009] Preferably, the concentration of the polypeptide compound in S1 is 0.1 µM~100.0 mM, and the concentration of Sortase A enzyme is 0.1 µM~10.0 mM.

[0010] Preferably, the buffer salt solution in step S1 is a non-phosphate buffer solution, including any one of HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), NaOAc (sodium acetate), MOPS (3-(N-morpholine) propanesulfonic acid), MES (2-morpholine ethanesulfonic acid), imidazole and Tris (trihydroxymethyl aminomethane), wherein CaCl2 is 0.1 mM~100 mM, and TCEP (tris (2-carboxyethyl) phosphine) is 0.1 µM~100.0 mM, and the pH range is 6.0~9.0.

[0011] Preferably, the polypeptide connection and intramolecular cyclization reaction in S1 is 15 min~24 h, and the reaction temperature is 0~60℃.

[0012] Preferably, the phage in S2 is a phage system composed of pCANTAB 5E phagemid and helper phage M13KO7 or M13KE phage system.

[0013] Preferably, the screening of the cyclic peptide ligand for the target protein in S2 includes the following steps: S2-1, constructing a phage display single cyclic peptide ester or double cyclic peptide ester library by using the cyclic peptide ester preparation method; S2-2, the target protein is biotinylated and immobilized on magnetic beads, the phage-displayed monocyclic or bicyclic peptide library in S2-1 is co-incubated with the immobilized target protein, and after 2-4 rounds of biopanning, the phage particles after biopanning are sequenced; Preferably, S2-3, the enriched target cyclic peptide is synthesized according to the sequencing results, and the binding force and biological activity with the target protein are evaluated.

[0014] The polypeptide compound is used for preparing a cyclic peptide library, and the polypeptide library displayed by phage or mRNA is connected and intramolecularly cyclized to generate a monocyclic or bicyclic peptide library.

[0015] Preferably, the cyclic peptide prepared by the polypeptide compound is used for developing drugs, detection kits or other biomedical and biomaterials.

[0016] The present application provides a polypeptide compound, a method for preparing a cyclic peptide and application thereof, and has the following advantages compared with the prior art: (1) Compared with the reported Sortase A enzyme catalyzed polypeptide modification technology, the side chain-side chain cyclic peptide is generated by Sortase A enzyme mediation for the first time, so as to construct the cyclic peptide molecule, and it is also the first time to be used for constructing a gene coded peptide library to generate a large-scale cyclic peptide library.

[0017] (2) Compared with the reported traditional combinatorial chemical library, the present application does not involve a complicated chemical synthesis process, and can reduce the construction cost of the cyclic peptide library.

[0018] (3) Compared with the Flexizyme system, the present application does not need to prepare natural aminoacyl tRNA, and can more conveniently obtain the cyclic peptide library. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Reaction diagram for preparing monocyclic peptide Pep9 by Sortase A enzyme catalysis of Pep1 and Pep3; Figure 2 Reaction diagram for preparing monocyclic peptide Pep10 by Sortase A enzyme catalysis of Pep1 and Pep4; Figure 3 Reaction diagram for preparing monocyclic peptide Pep11 by Sortase A enzyme catalysis of Pep1 and Pep5; Figure 4 Reaction diagram for preparing monocyclic peptide Pep12 by Sortase A enzyme catalysis of Pep2 and Pep6; Figure 5 Reaction diagram for preparing monocyclic peptide Pep13 by Sortase A enzyme catalysis of Pep2 and Pep7; Figure 6 Reaction diagram for Sortase A catalyzed reaction of Pep2 with Pep8 to produce mono-epoxide ester Pep14; Figure 7 Titer change diagram for phage selection; Figure 8 Polarization fluorescence diagram for bis-cyclic peptide epoxide and TEAD4; Figure 9 Schematic diagram for Sortase A catalyzed system to construct gene encoded cyclic peptide epoxide library. DETAILED DESCRIPTION

[0020] In order to make the present application clearer, the following will be further explained by means of embodiments and drawings.

[0021] The present application comprises the following steps: (1) Two polypeptide backbone templates with N-terminal glycine having the following characteristics: Template a: G-(X)m-C Template b: G-(X)m-C-(X)n-C Wherein, template a is used to construct mono-cyclic peptide epoxide, template b is used to construct bis-cyclic peptide epoxide, G represents glycine, X represents any one of natural L-amino acids, C represents L-cysteine and the position can be changed according to requirements, and m and n represent the number of amino acids between 3 and 20.

[0022] According to the above template characteristics, two polypeptides with N-terminal glycine are synthesized as follows: H-GLYDPANIHPKGWCGGSG-NH2 (template polypeptide Pep1) H-GLYDPANCIHPKGWCGGSG-NH2 (template polypeptide Pep2) The polypeptide is composed of natural L-type amino acids, the N-terminal is the amino group of glycine, the C-terminal is the amide of glycine, and the short sequence peptide GGSG at the C-terminal is a flexible linker arm simulating the phage pIII protein. The two polypeptides (Pep1 and Pep2) here are only to illustrate the design principle and implementation process of the present application, and should not be considered as the entire content of the present patent. The N-terminal glycine polypeptide produced by protease treatment should also fall within the scope of protection of the present patent.

[0023] In order to construct cyclic peptide frameworks with different characteristics, the position of cysteine residues in the polypeptide can be changed arbitrarily, and the number of amino acid residues in the polypeptide can be increased or decreased according to requirements, and the changed polypeptide is still applicable to the concept of Sortase A enzyme catalyzed preparation of cyclic peptide epoxide proposed in the present patent. Therefore, all changes and modifications of the cyclic peptide backbone type based on the present patent still fall within the scope of protection of the present patent.

[0024] (2) A chemically synthesized polypeptide compound with the following characteristics: The polypeptide compounds Pep3, Pep4 and Pep5 are used to react with Pep1 to produce monocyclic peptide ester molecules; the polypeptide compounds Pep6, Pep7 and Pep8 are used to react with Pep2 to produce bicyclic peptide ester molecules. The six polypeptide compounds here are only to illustrate the design principle and implementation process of the technology, and should not be considered as the whole content of the patent. All simple changes and modifications of the polypeptide compounds made on the basis of the patent are still within the scope of protection of the patent.

[0025] (3) Under the catalysis of Sortase A enzyme, the above-mentioned chemically synthesized polypeptide compound reacts with Pep1 or Pep2 in a buffer salt solution to produce a polypeptide linkage and intramolecular cyclization reaction, producing a cyclic peptide ester molecule; wherein the Sortase A enzyme is its wild type or mutant, the concentration of the Sortase A enzyme is 0.1 µM~10.0 mM, the concentration of the chemically synthesized polypeptide compound is 0.1 µM~100.0 mM, the buffer salt solution is any one of HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), NaOAc (sodium acetate), MOPS (3-(N-morpholine) propanesulfonic acid), MES (2-morpholine ethanesulfonic acid), imidazole and Tris (trihydroxymethyl aminomethane), the buffer salt solution contains CaCl2 (0.1 mM~100.0 mM) and TCEP (tris (2-carboxyethyl) phosphine) (0.1 µM~100.0 mM), the pH of the buffer salt solution is 6.0~9.0, the polypeptide linkage and intramolecular cyclization reaction time is 15 minutes~24 hours, and the temperature is 0~60℃.

[0026] (4) Construction and application of a phage-displayed cyclic peptide library: Sequence characteristics of the phage-displayed bicyclic polypeptide library: GX3CX6CGGSGG, GX4CX5CGGSGG, GX5CX4CGGSGG and GX6CX3CGGSGG (from N to C terminal, X is any one of natural amino acids, encoded by NNK, random amino acid mutation at 9 positions in the sequence, and GGSGG is a flexible amino acid linker between the bicyclic peptide library and the pIII protein on the phage surface).

[0027] The pCANTAB 5E phagemid vectors of the above-mentioned four libraries are constructed, and are mixed in equal proportions and electrotransformed into competent TG1 E. coli, and the titer determination library diversity is 7.0×10 8and then packaged into phage by helper phage M13KO7.

[0028] The phage constructed above was co-incubated with Pep6, and a bicyclic peptide phage library was generated under the catalysis of Sortase A enzyme, followed by 3 rounds of screening with magnetic beads immobilized with TEAD4 target protein, and the phage titer was determined in each round, and phage clones after the third round of screening were randomly selected for sequencing.

[0029] According to the sequencing results, a highly enriched polypeptide sequence was determined, and the target bicyclic peptide ester Pep15 was synthesized, and the ability to bind TEAD4 was evaluated by polarized fluorescence as 2.2 μM, which verified the success of the method of the present patent in constructing a gene-encoded cyclic peptide ester molecule library.

[0030] Example 1: According to the above content Synthesis of Pep3: 100.0 μmol of Rinkamide resin was weighed and transferred to a solid-phase synthesis reactor with a filter screen plate, and DMF was added for swelling at room temperature for 15 minutes. After the resin was treated with 2.0 mL of 20% piperidine in DMF, the resin was washed 4 times with DMF. Fmoc-Arg(Pbf)-OH, oxyma, and N,N'-diisopropylcarbodiimide were dissolved in 1.0 mL of DMF to form a condensation reagent containing 4.5 equivalents of Fmoc-Arg(Pbf)-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide, and the mixture was reacted in a 55°C shaker for 40 minutes.

[0031] Subsequently, the resin was washed 4 times with DMF, and solid-phase polypeptide condensation of Gly, Gly, Thr, Ser, Pro, and Leu was performed in the order mentioned above (wherein the condensation amino acid for modifying the Ser residue of chloroacetic acid was Fmoc-Ser-OH without side chain protection).

[0032] After the Fmoc protecting group of N-terminal Leu was removed, the resin was washed 4 times with DMF, and then 1.0 mL of DMF blocking reagent containing 2.0 equivalents of acetic acid-N-succinimidyl ester and 3.0 equivalents of N,N-diisopropylethylamine was added. After shaking at room temperature for 40 minutes, the resin was washed 4 times with DMF, and 4.0 mL of DMF / DCM (1 / 4, by volume) condensation reagent containing 5.0 equivalents of chloroacetic acid, 8.2 equivalents of HOBt, 8.2 equivalents of EDCI, and 10.0 equivalents of N-methylmorpholine was added. After 3 hours of reaction at 18°C, the resin was washed with DMF and DCM, respectively. After the resin was air-dried, trifluoroacetic acid cleavage solution was added, with a composition of TFA / m-cresol / water / triisopropylsilane (volume ratio of 88 / 5 / 5 / 2).

[0033] After 2 hours at room temperature, the trifluoroacetic acid cleavage solution was collected and 9 volumes of ice-cold diethyl ether was added. The white crude peptide was obtained by centrifugation. The target polypeptide was purified by liquid chromatography and obtained as Pep3 (15.4 mg) after freeze-drying. The mass spectrum confirmed the correctness of Pep3 (molecular formula: C 32 H 54 ClN 11 O 11 , theoretical molecular weight: 803.37, observed molecular weight: 803.44.

[0034] The structural formula of Pep3 molecule is as follows: Example 2: Synthesis of Pep4: 100.0 pmol of Rinkamide resin was weighed and transferred to a solid-phase synthesis reactor with filter screen, and DMF was added for swelling at room temperature for 15 minutes. After the resin was treated with 2.0 mL of 20% piperidine in DMF, the resin was washed 4 times with DMF. The resin was added with amino acid condensation reagent dissolved in 1.0 mL of DMF, containing 4.5 equivalents of Fmoc-Arg(Pbf)-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide, and reacted in a 55°C shaker for 40 minutes.

[0035] Subsequently, the resin was washed 4 times with DMF, and solid-phase polypeptide condensation of Gly, Gly, Thr, Thr, Pro, and Leu was sequentially performed according to the similar procedure described above (wherein the condensation amino acid for modifying the Thr residue of chloroacetic acid was side chain-unprotected Fmoc-Thr-OH).

[0036] After the Fmoc protecting group of the N-terminal Leu was removed, the resin was washed 4 times with DMF, and then 1.0 mL of DMF blocking reagent containing 2.0 equivalents of acetic acid-N-succinimidyl ester and 3.0 equivalents of N,N-diisopropylethylamine was added. After shaking at room temperature for 40 minutes, the resin was washed 4 times with DMF, and 4.0 mL of DMF / DCM (1 / 4, by volume) condensation reagent containing 5.0 equivalents of chloroacetic acid, 8.2 equivalents of HOBt, 8.2 equivalents of EDCI, and 10.0 equivalents of N-methylmorpholine was added. After 3 hours of reaction at 18°C, the resin was washed with DMF and DCM, respectively. After the resin was air-dried, trifluoroacetic acid cleavage solution was added, with a composition of TFA / m-cresol / water / triisopropylsilane (volume ratio of 88 / 5 / 5 / 2).

[0037] After 2 hours at room temperature, the trifluoroacetic acid cleavage solution was collected and 9 volumes of ice-cold diethyl ether was added. The white crude peptide was obtained by centrifugation. The target polypeptide was purified by liquid chromatography and was obtained as Pep4 (14.7 mg) after freeze-drying. The mass spectrum confirmed the correctness of Pep4 (molecular formula: C 33 H 56 ClN 11 O 11 , theoretical molecular weight: 817.38, observed molecular weight: 817.42.

[0038] The structural formula of Pep4 molecule is as follows: Example 3: Synthesis of Pep5: 100.0 pmol of Rinkamide resin was weighed and transferred to a solid-phase synthesis reactor with filter screen, and DMF was added for swelling at room temperature for 15 minutes. After the resin was treated with 2.0 mL of 20% piperidine in DMF, the resin was washed 4 times with DMF. The resin was added with amino acid condensation reagent dissolved in 1.0 mL of DMF, which contained 4.5 equivalents of Fmoc-Arg(Pbf)-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide, and reacted in a 55°C shaker for 40 minutes.

[0039] Subsequently, the resin was washed 4 times with DMF, and solid-phase polypeptide condensation of Gly, Gly, Thr, Hyp (hydroxyproline), Pro, and Leu was sequentially performed according to the above similar process (wherein the condensation amino acid for modifying the Hyp residue of chloroacetic acid was Fmoc-Hyp-OH without side chain protection).

[0040] After the Fmoc protecting group of the N-terminal Leu was removed, the resin was washed 4 times with DMF, and then 1.0 mL of DMF blocking reagent containing 2.0 equivalents of acetic acid-N-succinimidyl ester and 3.0 equivalents of N,N-diisopropylethylamine was added. After shaking at room temperature for 40 minutes, the resin was washed 4 times with DMF, and 4.0 mL of DMF / DCM (1 / 4, by volume) condensation reagent containing 5.0 equivalents of chloroacetic acid, 8.2 equivalents of HOBt, 8.2 equivalents of EDCI, and 10.0 equivalents of N-methylmorpholine was added. After 3 hours of reaction at 18°C, the resin was washed with DMF and DCM, respectively. After the resin was air-dried, trifluoroacetic acid cleavage solution was added, with a composition of TFA / m-cresol / water / triisopropylsilane (volume ratio of 88 / 5 / 5 / 2).

[0041] After 2 hours at room temperature, the trifluoroacetic acid cleavage solution was collected and 9 volumes of ice-cold diethyl ether was added. The white crude peptide was obtained by centrifugation. The target polypeptide was purified by liquid chromatography and Pep5 (9.1 mg) was obtained after freeze-drying. The mass spectrum confirmed the correctness of Pep5 (molecular formula: C 34 H 56 ClN 11 O 11 , theoretical molecular weight: 829.38, observed molecular weight: 829.59.

[0042] The structural formula of Pep5 molecule is as follows: Example 4: Synthesis of Pep6: 100.0 μmol of Rinkamide resin was weighed and transferred to a solid-phase synthesis reactor with filter screen, and DMF was added for swelling at room temperature for 15 minutes. After the resin was treated with 2.0 mL of 20% piperidine in DMF, the resin was washed with DMF 4 times. The resin was added with amino acid condensation reagent dissolved in 1.0 mL of DMF, which contained 4.5 equivalents of Fmoc-Arg(Pbf)-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide, and reacted in a 55°C shaker for 40 minutes.

[0043] Subsequently, the resin was washed with DMF 4 times, and solid-phase polypeptide condensation of Gly, Gly, Thr, Ser, Pro, and Leu was sequentially performed according to the similar procedure described above (wherein the condensation amino acid for modifying the chloroacetyl group of the Ser residue was Fmoc-Ser-OH without side chain protection).

[0044] After the Fmoc protecting group of the N-terminal Leu was removed, the resin was washed with DMF 4 times, and then 1.0 mL of DMF blocking reagent containing 2.0 equivalents of acetic acid-N-succinimidyl ester and 3.0 equivalents of N,N-diisopropylethylamine was added. After shaking at room temperature for 40 minutes, the resin was washed with DMF 4 times, and 4.0 mL of DMF / DCM (1 / 4, by volume) condensation reagent containing 5.0 equivalents of 3,5-bis[(2-chloroacetyl)amino]benzoic acid, 8.2 equivalents of HOBt, 8.2 equivalents of EDCI, and 10.0 equivalents of N-methylmorpholine was added. After 3 hours of reaction at 18°C, the resin was washed with DMF and DCM, respectively. After the resin was air-dried, trifluoroacetic acid cleavage solution was added, with a composition of TFA / m-cresol / water / triisopropylsilane (volume ratio of 88 / 5 / 5 / 2).

[0045] After 2 hours at room temperature, the trifluoroacetic acid lysis buffer was collected, and 9 volumes of ice-cooled diethyl ether were added. Centrifugation yielded a white crude peptide. The target peptide was purified by liquid chromatography, lyophilized, and Pep6 (13.8 mg) was obtained. Mass spectrometry confirmed the correctness of Pep6 (molecular formula: C). 41 H 61 Cl2N 13 O 13 Theoretical molecular weight: 1013.39, observed molecular weight: 1013.43.

[0046] The structural formula of the Pep6 molecule is as follows: Example 5: Pep7 Synthesis: In the reactor, DMF was added and allowed to swell at room temperature for 15 minutes. The resin was treated with 2.0 mL of 20% piperidine DMF solution, followed by washing the resin four times with DMF. An amino acid condensation reagent dissolved in 1.0 mL of DMF, containing 4.5 equivalents of Fmoc-Arg(Pbf)-OH, 4.5 equivalents of oxyma, and 4.5 equivalents of N,N'-diisopropylcarbodiimide, was added to the resin, and the reaction was carried out in a shaker at 55°C for 40 minutes. Subsequently, the resin was washed four times with DMF, and solid-phase peptide condensation of Gly, Gly, Thr, Thr, Pro, and Leu was performed sequentially following a similar procedure. Note that the condensed amino acid used to modify the Thr residue of the chloroacetyl group is an unprotected Fmoc-Thr-OH. After removing the Fmoc protecting group of the N-terminal Leu, the resin was washed four times with DMF. Then, 1.0 mL of DMF blocking reagent was added, containing 2.0 equivalents of N-succinimide acetate and 3.0 equivalents of N,N-diisopropylethylamine. After shaking at room temperature for 40 minutes, the resin was washed four times with DMF. Then, 4.0 mL of DMF / DCM (1 / 4, volume ratio) condensation reagent was added, containing 5.0 equivalents of 3,5-bis[(2-chloroacetyl)amino]benzoic acid, 8.2 equivalents of HOBt, 8.2 equivalents of EDCI, and 10.0 equivalents of N-methylmorpholine. After reacting at 18°C ​​for 3 hours, the resin was washed with DMF and DCM, respectively. After drying the resin, trifluoroacetic acid lysis buffer was added, with the composition TFA / m-cresol / water / triisopropylsilane (volume ratio 88 / 5 / 5 / 2). After 2 hours at room temperature, the trifluoroacetic acid lysis buffer was collected, and 9 volumes of ice-cooled diethyl ether were added. Centrifugation yielded a white crude peptide. The target peptide was purified by liquid chromatography, lyophilized, and Pep7 (7.6 mg) was obtained. Mass spectrometry confirmed the correctness of Pep7 (molecular formula: C). 42 H 63 Cl2N 13 O 13 Theoretical molecular weight: 1027.40, observed molecular weight: 1027.83.

[0047] The structure of Pep7 is as follows: Example 6: Synthesis of Pep8: In the reactor, DMF was added and swelled for 15 minutes. After the resin was treated with 2.0 mL of 20% piperidine in DMF, the resin was washed with DMF four times. The resin was added with amino acid condensation reagent dissolved in 1.0 mL of DMF, which contained 4.5 equivalents of Fmoc-Arg(Pbf)-OH, 4.5 equivalents of oxyma and 4.5 equivalents of N,N'-diisopropylcarbodiimide, and reacted in a 55°C shaker for 40 minutes.

[0048] Subsequently, the resin was washed with DMF four times, and solid-phase polypeptide condensation of Gly, Gly, Thr, Hyp (hydroxyproline), Pro and Leu was sequentially performed according to the similar procedure described above (wherein the condensation amino acid for modifying the chloroacetyl group of the Hyp residue was Fmoc-Hyp-OH without side chain protection).

[0049] After the Fmoc protecting group of the N-terminal Leu was removed, the resin was washed with DMF four times, and then 1.0 mL of DMF blocking reagent containing 2.0 equivalents of acetic acid-N-succinimidyl ester and 3.0 equivalents of N,N-diisopropylethylamine was added. After shaking at room temperature for 40 minutes, the resin was washed with DMF four times, and 4.0 mL of DMF / DCM (1 / 4, by volume) condensation reagent containing 5.0 equivalents of 3,5-bis[(2-chloroacetyl)amino]benzoic acid, 8.2 equivalents of HOBt, 8.2 equivalents of EDCI and 10.0 equivalents of N-methylmorpholine was added. After reacting at 18°C for 3 hours, the resin was washed with DMF and DCM, respectively. After the resin was air-dried, trifluoroacetic acid cleavage solution was added, which was composed of TFA / m-cresol / water / triisopropylsilane (volume ratio of 88 / 5 / 5 / 2).

[0050] After 2 hours at room temperature, the trifluoroacetic acid cleavage solution was collected, 9 times the volume of ice-cooled ether was added, and a white crude peptide was obtained by centrifugation. The target polypeptide was purified by liquid chromatography, and Pep8 (5.7 mg) was obtained after freeze-drying. Mass spectrometry confirmed the correctness of Pep8 (molecular formula: C 43 H 63 Cl2N 13 O 13 , theoretical molecular weight: 1039.40, observed molecular weight: 1039.11.

[0051] The structure of Pep8 is as follows: Example 7: Sortase A enzyme catalyzed preparation of monocyclic peptidyl ester: Sortase A was expressed and purified according to the same method reported in the paper (Chemical Science, 2024, 15, 9649-9656) and stored in aliquots at -80 °C. 1.5 μL Sortase A (85.0 mg / mL, 127.5 μg, final concentration 15.0 μM) was taken from the -80 °C freezer and added to 0.5 mL of HEPES buffer (pH 7.0, 50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, 1.0 mM TCEP (tris(2-carboxyethyl)phosphine). Then, 20.0 μg of Pep3 (final concentration 50.0 μM) and 50.0 μg of Pep1 (final concentration 55.0 μM) were added to the buffer in turn. After incubation of the reaction system at 37 °C for 1 hour, the reaction solution was analyzed by HPLC, As Figure 1 shown, according to the data of HPLC analysis and mass spectrometry, under the catalysis of Sortase A enzyme, Pep1 and Pep3 occurred polypeptide ligation and intramolecular cyclization to form the target monocyclic peptidyl ester Pep9 (molecular formula: C 103 H 150 N 28 O 31 S theoretical molecular weight: 2307.07, observed molecular weight: 2307.18).

[0052] According to a similar procedure, the enzyme ligation cyclization reaction of Pep4 and Pep1 under the catalysis of Sortase A was tested. As expected, as Figure 2 shown, under the catalysis of Sortase A enzyme, Pep1 and Pep4 occurred polypeptide ligation and intramolecular cyclization to form the target monocyclic peptidyl ester Pep10 (molecular formula: C 104 H 152 N 28 O 31 S theoretical molecular weight: 2321.09, observed molecular weight: 2320.94).

[0053] According to a similar procedure, the enzyme ligation cyclization reaction of Pep5 and Pep1 under the catalysis of Sortase A was tested. As expected, as Figure 3 shown, under the catalysis of Sortase A enzyme, Pep1 and Pep5 occurred polypeptide ligation and intramolecular cyclization to form the target monocyclic peptidyl ester Pep11 (molecular formula: C 105 H 152 N 28 O 31S (Theoretical molecular weight: 2333.02, observed molecular weight: 2333.09).

[0054] Example 8: Preparation of bicyclic peptide oxyesters catalyzed by Sortase A enzyme: Following the same method described in the previously reported paper (Chemical Science, 2024, 15, 9649-9656), Sortase A was expressed and purified, and aliquoted and stored at -80°C. 1.5 μL of Sortase A (85.0 mg / mL, 127.5 μg, final concentration 15.0 μM) was taken from the -80°C freezer and added to 0.5 mL of HEPES buffer (pH 7.0, 50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, 1.0 mM TCEP (tris(2-carboxyethyl)phosphine)). Then, 30.0 μg of Pep6 (final concentration 60.0 μM) and 60.0 μg of Pep2 (final concentration 60.0 μM) were added sequentially to the buffer. After incubating the reaction system at 37°C for 1 hour, the reaction solution was analyzed by HPLC. like Figure 4 As shown, based on HPLC analysis and mass spectrometry data, under the catalysis of Sortase A enzyme, Pep2 and Pep6 underwent peptide linkage and intramolecular cyclization to form the target bicyclic peptide oxyester Pep12 (molecular formula: C). 115 H 162 N 34 O 34 S2 theoretical molecular weight: 2627.14, observed molecular weight: 2627.48).

[0055] Following a similar procedure, the enzymatic cyclization reaction of Pep7 and Pep2 catalyzed by Sortase A was tested. As expected, ... Figure 5 As shown, under the catalysis of Sortase A, Pep2 and Pep7 underwent peptide linkage and intramolecular cyclization to form the target bicyclic peptide oxyester Pep13 (molecular formula: C). 116 H 164 N 34 O 34 S2 theoretical molecular weight: 2641.16, observed molecular weight: 2641.06).

[0056] Following a similar procedure, the enzymatic cyclization reaction of Pep8 and Pep2 catalyzed by Sortase A was tested. As expected, ... Figure 6As shown, under the catalysis of Sortase A, Pep2 and Pep8 underwent peptide linkage and intramolecular cyclization to form the target bicyclic peptide oxyester Pep14 (molecular formula: C 117 H 164 N 34 O 34 S2 theoretical molecular weight: 2653.16, observed molecular weight: 2653.28).

[0057] Example 9: Phage display-based screening of bicyclic peptide oxyester ligands 1. Constructing the pCANTAB 5E phage library Both the pCANTAB 5E phage particle and the helper phage (M13KO7) were derived from Sichuan Apak Biotechnology Co., Ltd. Using the pCANTAB 5E vector as a template, the BsaI recognition site of the vector was point-mutated via homologous recombination to obtain the pCANTAB 5E' vector (5'-GAGCGTGGGTCTCGCGGTATCATTGCAGCAC-3' mutated to 5'-GAGCGTGGGTCGCGCGGTATCATTGCAGCAC-3'). The specific homologous recombination procedure was strictly followed according to the previously reported paper Organic Letters, 2024, 26, 2601-2605. Five primers (M is C or A; N is A, C, T, or G) were custom-made from General Biotechnology (Anhui) Co., Ltd., with the following sequences: Primer 1: 5'-TTTGGTCTCAGCACCGCCAGAGCCGCCGCAMNNMNNMNNMNNMNNMNNGCAMNNMNNMNNACCGGCCATGGCCGGCTGGGCCGCATAGAAAGG-3'; Primer 2: 5'-TTTGGTCTCAGCACCGCCAGAGCCGCCGCAMNNMNNMNNMNNMNNGCAMNNMNNMNNMNNACCGGCCATGGCCGGCTGGGCCGCATAGAAAGG-3'; Primer 3: 5'-TTTGGTCTCAGCACCGCCAGAGCCGCCGCAMNNMNNMNNMNNGCAMNNMNNMNNMNNMNNACCGGCCATGGCCGGCTGGGCCGCATAGAAAGG-3'; Primer 4: 5'-TTTGGTCTCAGCACCGCCAGAGCCGCCGCAMNNMNNMNNGCAMNNMNNMNNMNNMNNMNNACCGGCCATGGCCGGCTGGGCCGCATAGAAAGG-3'; Primer 5: 5'-TTGGTCTCGGTGCGCCGGTGCCGTATCCGGATCCGCTG-3'; PCR was performed with pCANTAB 5E' as template, primer 1 / primer 5, primer 2 / primer 5, primer 3 / primer 5 and primer 4 / primer 5 respectively, 15 cycles, 25 μL volume for each PCR, KeyPo DNA polymerase (from Monarch Biosciences). PCR products were recovered by DNA recovery kit (TIANGEN) and treated with Bsal and Dpnl. Four kinds of equal amount of mixed products were connected by T4 ligase overnight, then electrotransformed into TG1 cells (5 times, 1.0 μg / time), and the diversity was determined by titer as 7.0x10 8 The primary bacteria were collected and stored in -80℃ refrigerator with glycerol. The primary glycerol bacteria were inoculated in 2xYT medium (containing glucose and Amp), incubated at 37℃ until OD 600 reached 0.4, and helper phage M13KO7 (MOI 20) was added. After 1 hour of infection at 37℃, the bacteria were collected by centrifugation at 4℃ (10000 rpm), and resuspended in 2xYT medium (containing Kana and Amp, without glucose). After 16 hours of culture at 28℃, the supernatant was collected by centrifugation at 4℃ (8000 rpm, 20 minutes), and 1 / 5 volume of pre-cooled 5xPEG8000 was added. After 1 hour of ice bath, the phage was collected by centrifugation at 4℃ (10000 rpm, 20 minutes), resuspended in 80 mL TBS, and centrifuged at low temperature, then PEG8000 precipitation. The phage was dissolved in TBS, filtered by 0.45 μm filter, and stored in -20℃ after adding appropriate amount of glycerol. After the above process, phage containing four kinds of mixed peptide library was obtained, and the four peptide libraries were GX3CX6CGGSGG, GX4CX5CGGSG, GX5CX4CGGSG and GX6CX3CGGSGG (from N to C terminal, X is any one of natural amino acids, encoded by NNK, random amino acid mutation at 9 positions in the sequence, and GGSGG as a double-ring peptide library and flexible amino acid linker between phage surface pIII protein).

[0058] To 500.0 μΐ^ of HEPES buffer (50.0 mM HEPES, 100.0 mM NaCl, 2.0 mM CaCl2, 1.0 mM TCEP, pH 7.0) was added 1 x 10 12 pfu phage and 3.0 μΐ^ of Pep6 (12.5 mM stock, final concentration 60.0 μΜ). Then 3.0 μΐ^ of Sortase A (final concentration 30.0 μΜ) was added and incubated at 37 °C for 60 min (250 rpm). 125.0 μΐ^ of 5x PEG8000 / NaCl solution was added and centrifuged at 4 °C (10,000 rpm, 30 min), 1.5 mL of binding buffer and 750.0 μΐ^ of blocking buffer were added and incubated at 25 °C for 30 min. Meanwhile, 100.0 μΐ^ of streptavidin-coated magnetic beads (Dynabeads M-280) were washed with 1.0 mL of PBS buffer for three times. 50.0 μΐ^ of magnetic beads solution was added to the phage library and incubated at room temperature for 30 min. The supernatant was collected and mixed with the remaining magnetic beads solution (50.0 μΐ^). After incubation at room temperature for another 30 min, the phage supernatant was collected. For the first background subtraction, 10.0 μΐ^ of streptavidin magnetic beads were washed with 500 μΐ^ of PBS buffer, 170.0 μΐ^ of PBS buffer and 3.0 μΐ^ of biotinylated TEAD4 (5.0 μg) were added and incubated at 25 °C for 30 min. The beads were washed with 1.0 mL of PBS buffer for three times, followed by 300.0 μΐ^ of binding buffer and 150.0 μΐ^ of blocking buffer and incubated at 25 °C for 30 min. The magnetic beads solution of TEAD4 was mixed with the phage solution at 4 °C for 30 min. The beads were washed with 1.0 mL of washing buffer for eight times and 1.0 mL of binding buffer for two times. 100.0 μΐ^ of pH 2.2 elution buffer was added (5 min, room temperature). The eluate was mixed with 50.0 μΐ^ of pH 8.0 neutralization buffer. 10.0 μΐ^ was taken for titer determination. The remaining phage solution was mixed with TG1 cells (20.0 mL, OD600= ~0.4) and amplified according to the general procedure for the following round of selection. The cells were mixed with equal volume of glycerol (40% glycerol water, v / v, sterilized) and stored at -80 °C for long-term preservation. The collected TG1 cells were used for the second / third round of amplification. (The phage added in the second round of selection was the phage particles collected after the first round of selection, the input was 10.0 μΐ^ (1.0 x 10 12 pfu phage and 3.0 μΐ^ of Pep6 (12.5 mM stock, final concentration 60.0 μΜ). Then 3.0 μΐ^ of Sortase A (final concentration 30.0 μΜ) was added and incubated at 37 °C for 60 min (250 rpm). 125.0 μΐ^ of 5x PEG8000 / NaCl solution was added and centrifuged at 4 °C (10,000 rpm, 30 min), 1.5 mL of binding buffer and 750.0 μΐ^ of blocking buffer were added and incubated at 25 °C for 30 min. Meanwhile, 100.0 μΐ^ of streptavidin-coated magnetic beads (Dynabeads M-280) were washed with 1.0 mL of PBS buffer for three times. 50.0 μΐ^ of magnetic beads solution was added to the phage library and incubated at room temperature for 30 min. The supernatant was collected and mixed with the remaining magnetic beads solution (50.0 μΐ^). After incubation at room temperature for another 30 min, the phage supernatant was collected. For the first background subtraction, 10.0 μΐ^ of streptavidin magnetic beads were washed with 500 μΐ^ of PBS buffer, 170.0 μΐ^ of PBS buffer and 3.0 μΐ^ of biotinylated TEAD4 (5.0 μg) were added and incubated at 25 °C for 30 min. The beads were washed with 1.0 mL of PBS buffer for three times, followed by 300.0 μΐ^ of binding buffer and 150.0 μΐ^ of blocking buffer and incubated at 25 °C for 30 min. The magnetic beads solution of TEAD4 was mixed with the phage solution at 4 °C for 30 min. The beads were washed with 1.0 mL of washing buffer for eight times and 1.0 mL of binding buffer for two times. 100.0 μΐ^ of pH 2.2 elution buffer was added (5 min, room temperature). The eluate was mixed with 50.0 μΐ^ of pH 8.0 neutralization buffer. 10.0 μΐ^ was taken for titer determination. The remaining phage solution was mixed with TG1 cells (20.0 mL, OD600= ~0.4) and amplified according to the general procedure for the following round of selection. The cells were mixed with equal volume of glycerol (40% glycerol water, v / v, sterilized) and stored at -80 °C for long-term preservation. The collected TG1 cells were used for the second / third round of amplification. (The phage added in the second round of selection was the phage particles collected after the first round of selection, the input was 10.0 μΐ^ (1.0 x 10Figure 7 As shown, after three rounds of screening, the phage titer was increased by 23000 times compared with the first round of screening, and was 103 times higher than that of empty magnetic beads. A highly enriched polypeptide was obtained by sequencing a single clone selected at random, and the corresponding bicyclic peptide ester Pep15 was synthesized in vitro, which contained a fluorescein isothiocyanate (FITC) modification. The affinity with TEAD4 was 2.2 μM, as shown in the figure. These results demonstrate the effectiveness of the present application. Figure 8 As shown, after three rounds of screening, the phage titer was increased by 23000 times compared with the first round of screening, and was 103 times higher than that of empty magnetic beads. A highly enriched polypeptide was obtained by sequencing a single clone selected at random, and the corresponding bicyclic peptide ester Pep15 was synthesized in vitro, which contained a fluorescein isothiocyanate (FITC) modification. The affinity with TEAD4 was 2.2 μM, as shown in the figure. These results demonstrate the effectiveness of the present application.

[0059] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A polypeptide compound, characterized in that, The polypeptide compounds have the following general structural formula: Among them, X a It is a serine derivative: Threonine derivatives: or proline derivatives: Any one of them; R is any one of the electrophilic group and oligopeptide groups composed of natural or non-natural amino acids containing an electrophilic group. The electrophilic group includes any one of chloroacetyl, 3,5-di[(2-chloroacetyl)amino]benzoyl, 4-chloroacetamylbenzoyl, 3,5-di(chloromethyl)benzoyl, 2-(chloromethyl)benzoyl, 3-(chloromethyl)benzoyl, 4-(chloromethyl)benzoyl, and 4-(chloromethyl)bibenzoyl. 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 preparing cyclic peptide oxyesters using polypeptide compounds as described in claim 1 above: S1. In the presence of Sortase A, polypeptide compounds are linked to a polypeptide template with glycine at the N-terminus and cysteine ​​in the sequence in a buffer salt solution to undergo polypeptide linkage and intramolecular cyclization reaction, producing cyclic peptide oxyester molecules. S2. Select a polypeptide template from S1 with glycine at the N-terminus and containing cysteine ​​in the sequence. Encode and express it at the N-terminus of the phage pIII protein through gene encoding. Then, construct a phage-displayed cyclic peptide oxyester library through the S2 step and screen cyclic peptide oxyester ligands for the target protein.

3. The method for preparing cyclic peptide oxyesters from polypeptide compounds according to claim 2, characterized in that, The polypeptide template is one of the following two types: Template a: G-(X)mC; Template b: G-(X)mC-(X)nC; Template a is used to construct monocyclic peptide oxyesters, template b is used to construct bicyclic peptide oxyesters, G represents glycine, X represents any natural L-amino acid, C represents L-cysteine ​​and its position can be changed as required, and m and n represent the number of amino acids between 3 and 20.

4. The method for preparing cyclic peptide oxyesters from polypeptide compounds according to claim 2, characterized in that: The concentration range of the polypeptide compound in S1 is 0.1 µM to 100.0 mM, and the concentration range of Sortase A enzyme is 0.1 µM to 10.0 mM.

5. The method for preparing cyclic peptide oxyesters from polypeptide compounds according to claim 2, characterized in that: The buffer salt solution in step S1 is a non-phosphate buffer solution, comprising any one of 4-hydroxyethylpiperazine ethanesulfonic acid, sodium acetate, 3-(N-morpholino)propanesulfonic acid, 2-morpholinoethanesulfonic acid, imidazole, and tris(hydroxymethyl)aminomethane, wherein it contains 0.1 mM to 100 mM CaCl2 and 0.1 µM to 100.0 mM tris(2-carboxyethyl)phosphine, and the pH range is 6.0 to 9.

0.

6. The method for preparing cyclic peptide oxyesters from polypeptide compounds according to claim 2, characterized in that: The time for peptide linking and intramolecular cyclization reactions in S1 is 15 min to 24 h, and the reaction temperature is 0 to 60 °C.

7. The method for preparing cyclic peptide oxyesters from polypeptide compounds according to claim 2, characterized in that: The phage in S2 is a phage system composed of pCANTAB 5E phage particles and helper phage M13KO7 or an M13KE phage system.

8. The method for preparing cyclic peptide oxyesters from polypeptide compounds according to claim 2, characterized in that, The S2 step of screening cyclic peptide ligands for the target protein includes the following steps: S2-1. Construct a phage-displaying monocyclic or bicyclic peptide oxygen ester library using the method described above. S2-2, The target protein is biotinylated and immobilized on magnetic beads. The monocyclic peptide oxyester or bicyclic peptide oxyester library displayed by the phage in S2-1 is co-incubated with the immobilized target protein. After 2-4 rounds of biopanning, the biopanned phage particles are sequenced. S2-3. Based on the sequencing results, synthesize the enriched target cyclic peptide oxyesters and evaluate their binding affinity and biological activity to the target protein.

9. An application of the polypeptide compound as described in claims 2-8 in the preparation of cyclic peptide oxyesters, characterized in that: The polypeptide compounds are used to prepare cyclic peptide oxyesters for the construction of gene-encoded cyclic peptide libraries, including peptide linking and intramolecular cyclization of phage-displayed and mRNA-displayed polypeptide libraries to generate monocyclic peptide oxyester and bicyclic peptide oxyester libraries.

10. The application according to claim 9, characterized in that, The cyclic peptide oxyesters prepared from the polypeptide compounds are used in the development of drugs, diagnostic kits, or other biomedical and biomaterials.

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