CN117925758A

CN117925758A - Ligase-catalyzed polypeptide cyclization method and application thereof in phage display peptide library

Info

Publication number: CN117925758A
Application number: CN202410093169.XA
Authority: CN
Inventors: 方葛敏; 陈颖; 张燕妮; 万晓翠; 孙晓园
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2024-01-23
Filing date: 2024-01-23
Publication date: 2024-04-26

Abstract

The invention provides a ligase-catalyzed polypeptide cyclization method and application thereof in phage display peptide libraries, and relates to the technical field of modification and cyclization of gene-encoded polypeptide libraries. The polypeptide cyclization method realizes that SortaseA enzyme catalysis system is used for preparing side chain to side chain cyclized polypeptides for the first time, and a large-scale gene-encoded cyclic peptide library is obtained by applying the cyclizing method to the cyclization of a phage display polypeptide library and is used for screening cyclic peptide ligands aiming at target proteins. The invention overcomes the defects of the prior art, the enzyme-catalyzed polypeptide cyclization method has mild reaction conditions, high reaction efficiency and high specificity, and the generated phage cyclopeptide library can be used for screening functional cyclopeptide ligands of new structural frameworks, thereby providing a powerful platform technology for the fields of biological medicines and biological materials.

Description

Ligase-catalyzed polypeptide cyclization method and application thereof in phage display peptide library

Technical Field

The invention relates to the technical field of polypeptide library modification and side chain cyclization of gene codes, in particular to a ligase-catalyzed polypeptide cyclization method and application thereof in phage display peptide libraries.

Background

Polypeptide molecules are an important source of drug development and have produced a variety of heavy weight drugs such as semanteme, insulin, teriparatide, and the like. However, short chain linear polypeptides are in a solution generally free of immobilized conformations, have relatively weak binding forces to targets, and are easily degraded by proteolytic enzymes, limiting their use in modern polypeptide drug design and discovery. The polypeptides of the cyclic scaffold have a relatively rigid structure, which has been demonstrated to be significantly superior to linear polypeptides in terms of target protein binding capacity, proteolytic enzyme stability, and cell membrane permeability, as exemplified by cyclosporine and ziconotide. There is a class of cysteine-rich cyclic peptide active molecules in nature that oxidize through the side chains of multiple cysteines to form a disulfide framed cyclic, e.g., conotoxin peptides. However, disulfide bonds are quite unstable in a reducing environment and undergo thiol exchange with free mercapto groups, which in turn leads to erroneous rearrangement of the disulfide bond framework.

The use of small organic molecules to couple two or more amino acid residues in a polypeptide sequence is an important strategy for the construction of stable cyclic peptides. Thioether bonds can overcome the defect of unstable disulfide bonds, and are a common method for cyclizing polypeptides by replacing disulfide bonds. In order to ensure efficient reaction efficiency, thioether linkage-based polypeptide cyclization methods require the use of highly reactive electrophiles, such as bromomethylbenzene or iodomethylbenzene, bromoacetyl or iodoacetyl groups, etc., to couple two or more cysteine residues. Heinis et al (see Nat ChemBiol 2009,5,502-507) constructed thioether-bond-containing bicyclic peptides by reacting tribromomethylbenzene with three cysteine side chains, but the bromomethyl group was able to react with other nucleophilic groups in the polypeptide, such as amino groups, under weakly basic conditions (see SciAdv 2019,5, eaaw 2851).

An important application of polypeptide cyclization is the construction of large-scale libraries of cyclic peptides for the discovery of cyclic peptide ligands for target proteins, in particular for the macrocyclization of libraries of polypeptides encoded by genes, such as phage-displayed polypeptide libraries. When polypeptides cyclize on the surface of phage, tribromomethylbenzene reacts with cysteine of pIII protein, and potential problem of phage infectivity reduction is caused. Low-reactivity electrophiles such as acryl or chloroacetyl can solve the problem of side reactions of bromoacetyl, but their introduction into phage or mRNA-displayed polypeptide libraries typically uses cumbersome codon expansion (see ANGEW CHEM INT ED ENGL 2019,58,15904-15009) or Flexizyme techniques (see Annu Rev Biochem 2022,91,221-243) or structurally complex bifunctional reactive reagents (see JAm Chem Soc 2020,142,5097-5103).

In addition to cyclizing methods based on cysteine side chain polypeptides, cyclizing modification methods based on lysine, tyrosine, tryptophan, or the like have also been developed (see Chem Rev 2020,120,10079-10144). Among these strategies for side chain cyclization of lysine employ succinimide-activated esters, which suffer from instability, susceptibility to hydrolysis and severe side reactions (see JAm Chem Soc2005,127, 14142-14143). In addition, orthogonal unnatural amino acid pairs can be introduced for cyclization of polypeptides, such as azide and alkyne modified amino acids, but the introduction of these unnatural amino acids into phage, E.coli, or mRNA-displayed polypeptide libraries requires cumbersome codon expansion techniques and is limited by patent protection. Furthermore, click chemistry typically requires the use of metal catalysts (see ANGEW CHEM INT ED ENGL 2009,48,6974-6998), which can present toxicity problems for phage or E.coli, and can be prone to metal ion retention problems for cyclic peptides.

With increasing emphasis on cyclic peptide drug discovery, the development of polypeptide cyclization methods compatible with gene coding library technology is an important trend in current basic and application science research. Gene encoding library technology, including phage display, E.coli display, yeast display, and mRNA display technologies, has accelerated the discovery process of cyclopeptide ligands by constructing large-scale random polypeptide libraries for iterative screening of targets. The technology of gene coding polypeptide library is generally carried out in a near neutral aqueous solution, and a polypeptide cyclization method is required to have better biocompatibility. Furthermore, the substrate concentration of the gene-encoded polypeptide library technology is extremely low, and the cyclization reaction is required to have extremely high reaction efficiency and high selectivity. These harsh conditions make the usual chemical cyclization methods either unsuitable for use in gene-encoded polypeptide library technology or less than desired.

SortaseA enzyme is a transpeptidase isolated from gram-positive bacteria capable of inducing covalent attachment of two polypeptides by amide bonds in a sequence-specific manner. Specifically, in the presence of SortaseA enzyme, one polypeptide containing Leu-Pro-Xxx-Thr-Gly (where Xxx is any natural amino acid) will be linked to another polypeptide containing glycine at the N-terminus by a peptide bond (see ANGEW CHEM INT ED ENGL 2011,50,5024-5032). Compared with the polypeptide modified by a common chemical method, the SortaseA enzyme-based reaction has the advantages of milder reaction conditions and higher specificity. However, all of the reported SortaseA enzyme-based modification reactions were used to effect amide linkage of the polypeptide backbone (Commun Chem 2023,6,48), and cyclization of the polypeptide side chains has not been achieved. Although SortaseA enzyme ligation is widely used in the field of protein modification and semisynthesis, it has not been used for the construction of gene-encoded cyclopeptide libraries, such as phage display cyclopeptides libraries. In summary, the development of a novel system for cyclizing polypeptide side chains based on SortaseA enzyme catalysis by deeply exploring SortaseA enzyme catalysis connection systems and applying the novel system to cyclizing a gene-encoded polypeptide library is helpful for the discovery of macrocyclic peptide ligands.

Disclosure of Invention

Aiming at the defects that a cyclopeptide product is unstable under a reduction condition and is easy to cause error rearrangement of disulfide bonds in a polypeptide cyclization method based on disulfide bonds, and meanwhile, aiming at polypeptide libraries such as phage display libraries which have harsh reaction conditions and poor biocompatibility and are not suitable for modifying gene codes in most chemical cyclization methods, the invention provides a SortaseA-catalyzed polypeptide side chain cyclization method with mild reaction conditions, high reaction efficiency and high specificity.

In order to achieve the above object, the technical scheme of the present invention is realized by the following technical scheme:

A method of ligase-catalyzed polypeptide cyclization comprising the steps of:

S1, preparing a polypeptide compound with a side chain containing a SortaseA enzyme recognition function of a low-reactivity group, wherein the compound has the following structural general formula:

wherein X _a is any one of hydrogen, acetyl and oligopeptide group composed of natural or unnatural amino acid except cysteine; x _b is any one of an electrophilic group, an oligopeptidyl group comprising a natural or unnatural amino acid, wherein the electrophilic group comprises a chloroacetyl group, a3, 5-bis [ (2-chloroacetyl) amino ] benzoyl group; x _c is any one of oxygen (oxygen ester bond), nitrogen hydrogen (amide bond) and sulfur (thioester bond), and X _d is any one of amino group and oligopeptide sequence composed of natural or unnatural amino acid except cysteine; n is any number between 1 and 5;

S2, polypeptide connection and polypeptide side chain cyclization are carried out on the polypeptide compound and a polypeptide template with glycine at the N-terminal and cysteine in the sequence in buffer salt solution in the presence of SortaseA enzyme, so as to generate a cyclic peptide molecule;

S3, selecting a polypeptide template with glycine at the N-terminal and cysteine in the sequence in the S2, carrying out gene coding fusion expression on the N-terminal of phage pIII protein, constructing a phage display cyclopeptide library through S2 step operation, and screening cyclopeptide ligand aiming at target protein.

Preferably, the polypeptide templates in S2 are two kinds of:

Template a: g- (X) _m -C; template b: g- (X) _m-C-(X)_y -C;

Wherein, the template a is used for constructing single-ring peptide, the template b is used for constructing double-ring peptide, G represents glycine, X represents any natural L-amino acid, C represents L-cysteine and the position can be changed according to the requirement, and m and y represent the number of amino acids between 3 and 20.

Preferably, the SortaseA enzyme is wild type or mutant thereof.

Preferably, the concentration range of the polypeptide compound in the S2 is 0.1 mu M-10.0 mM, and the concentration range of the Sortase A enzyme is 0.1 mu M-10.0 mM.

Preferably, the buffer salt solution in S2 is a common buffer solution except phosphate, including any one of HEPES (4-hydroxyethyl piperazine ethane sulfonic acid), naOAc (sodium acetate) and Tris (Tris hydroxymethyl amino methane), wherein CaCl ₂ is 0.1-mM mM, TCEP (Tris (2-carboxyethyl) phosphine) is 0.1 mu M-10.0 mM, and the pH range is 6.0-9.0.

Preferably, the time of polypeptide connection and polypeptide side chain cyclization reaction in the S2 is 15 minutes to 6 hours, and the reaction temperature is 20 to 45 ℃.

Preferably, the phage in S3 comprises a phage system consisting of pCANTAB 5E phagemid and helper phage M13KO7 or M13KE phage system.

Preferably, the screening of the cyclopeptide ligand against the target protein in S3 comprises the following steps:

S3-1, constructing a phage display single-ring peptide or double-ring peptide library by utilizing the ligase-catalyzed polypeptide cyclization method;

s3-2, target proteins are biotinylated and fixed on magnetic beads, wherein the single-ring peptide or double-ring peptide library displayed by phage in S3-1 is incubated with the immobilized target proteins, and the phage particles after biopanning are sequenced after 2-4 rounds of biopanning;

s3-3, synthesizing the enriched target cyclopeptide according to a sequencing result, and evaluating the binding force and the biological activity of the target cyclopeptide with the target protein.

The polypeptide cyclization is applied to the construction of a gene coding cyclic peptide library, and comprises the steps of polypeptide connection and side chain cyclization of a phage display polypeptide library, so as to construct phage display single-ring and double-ring peptide libraries.

Preferably, the cyclic peptide ligand obtained by the ligase-catalyzed polypeptide cyclization method is applied to the development of medicines, detection kits or other biomedical and biological materials.

The invention provides a ligase-catalyzed polypeptide cyclization method and application thereof in phage cyclopeptide library, and has the advantages compared with the prior art that:

(1) Compared with the existing SortaseA enzyme-catalyzed polypeptide connection technology, the invention firstly utilizes SortaseA enzyme to mediate and generate side chain-to-side chain cyclic peptide, and polypeptide side chain cyclization is cyclization reaction between electrophilic groups and cysteine residues in an intermediate structure generated by polypeptide connection, thereby constructing cyclic peptide molecules, and the invention is also firstly used for constructing a phage display cyclic peptide library.

(2) Compared with the traditional technology for constructing the cyclopeptide library by a chemical method, the invention does not need an electrophile with high reactivity, does not generate phage toxicity, and is more beneficial to cyclizing modification of a polypeptide library coded by phage.

(3) Compared with the existing cyclic peptide library technology based on codon expansion or Flexizyme, the method does not involve complex codon transformation, does not need to prepare unnatural amino acid modified tRNA, and greatly reduces the construction cost of the cyclic peptide library.

(4) The enzyme-catalyzed polypeptide side chain cyclization method has mild overall reaction condition, high reaction efficiency and high specificity, and the generated phage cyclopeptide library can be used for screening functional cyclopeptide ligands of new structural frameworks, thereby providing a powerful platform technology for the fields of biological medicines and biological materials.

Description of the drawings:

FIG. 1 is a schematic representation of the single cyclization of polypeptide side chains of chloracetyl-containing polypeptide 3 under SortaseA enzyme catalysis to produce a phage single-ring peptide library;

FIG. 2 is a schematic representation of the side chain double cyclization of a3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide 7 under SortaseA enzyme catalysis to produce a phage bicyclic peptide library;

FIG. 3 is a chromatogram and mass spectrum of polypeptides 1 and 2;

FIG. 4 is a chromatogram and a mass spectrum of a chloroacetyl-containing polypeptide 3, a chloroacetyl-containing polypeptide oxygen ester 4, a chloroacetyl-containing polypeptide thioester 5, a chloroacetyl-modified diaminopropionic acid polypeptide 6 prepared separately from examples 1-4;

FIG. 5 is a chromatogram and a mass spectrum of 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide 7, 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide oxy ester 8, 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-modified diaminopropionic acid-containing polypeptide 9, and Biotin-1 of example 10, respectively prepared in examples 5-7;

FIG. 6 is a schematic representation of the synthesis of chloroacetyl-containing polypeptide 3 of example 1;

FIG. 7 is a schematic representation of the synthesis of chlorine-containing acetylpolypeptide oxygen ester 4 of example 2;

FIG. 8 is a schematic representation of the synthesis of chloroacetyl-containing polypeptide thioester 5 of example 3;

FIG. 9 is a schematic representation of the synthesis of polypeptide 6 of example 4, which contains chloroacetyl-modified diaminopropionic acid;

FIG. 10 is a schematic representation of the synthesis of 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide 7 of example 5;

FIG. 11 is a schematic representation of the synthesis of 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide oxygen ester 8 of example 6;

FIG. 12 is a schematic representation of the synthesis of 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl modified diaminopropionic acid-containing polypeptide 9 of example 7;

FIG. 13 is a mass spectrum of SortaseA enzyme-catalyzed side chain monocyclization of polypeptide 1 and chloroacetyl-containing polypeptide 3 and product 10;

FIG. 14 is a mass spectrum of SortaseA enzyme-catalyzed side chain monocyclization of polypeptide 1 and chloroacetyl-containing polypeptide oxygen ester 4 and product 10;

FIG. 15 is a mass spectrum of SortaseA enzyme-catalyzed side chain monocyclization of polypeptide 1 and chloroacetyl-containing polypeptide thioester 5 and product 10;

FIG. 16 is a mass spectrum of SortaseA enzyme-catalyzed side chain monocyclization of polypeptide 1 and of chloroacetyl-modified diaminopropionic acid-containing polypeptide 6 and of the product 11;

FIG. 17 is a mass spectrum of SortaseA enzyme-catalyzed side chain double cyclization of polypeptide 2 and 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide 7 and product 12;

FIG. 18 is a mass spectrum of SortaseA enzyme-catalyzed side chain double cyclization of polypeptide 2 with 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl polypeptide oxo-ester 8 and product 12;

FIG. 19 is a side chain double cyclization and product 13 mass spectrum of SortaseA enzyme catalyzed polypeptide 2 and 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl modified diaminopropionic acid containing polypeptide 9;

FIG. 20 is a bar graph of phage infectivity test results;

FIG. 21 is a graphical representation of the results of a functional monocyclic peptide 14 binding assay to the target protein TEAD 4;

FIG. 22 is a graphical representation of the results of a functional bicyclic peptide 15 binding assay to the target protein TEAD 4.

Detailed Description

In order to more clearly illustrate the invention, it will be further illustrated by the following examples and figures.

The method comprises the following steps:

(1) Two polypeptide backbone templates with glycine at the N-terminus having the following characteristics:

Template a: g- (X) _m -C

Template b: g- (X) _m-C-(X)_y -C

Wherein, the template a is used for constructing single-ring peptide, the template b is used for constructing double-ring peptide, G represents glycine, X represents any one natural L-amino acid, C represents L-cysteine, and m and y represent the number of amino acids between 3 and 20.

According to the template characteristics, two polypeptides with glycine at the N terminal are synthesized as follows:

H-GRYDPANIHPKGWCGGSG-NH ₂ (template polypeptide 1)

H-GRYDPANCIHPKGWCGGSG-NH ₂ (template polypeptide 2)

The amino acids in the polypeptide are all natural L-type amino acids, the N end is amino of glycine, the C end is amide of glycine, and GGSG short-sequence peptide added at the C end is used as a flexible connecting arm to simulate the connection with phage pIII protein. The two polypeptides synthesized are merely illustrative of the design and embodiments of the present technology and should not be considered as being the entire disclosure of this patent.

In order to construct the desired cyclic peptide framework, the position of cysteine in the polypeptide can be changed at will, the number of amino acids in the polypeptide can be increased or decreased according to requirements, and the changed polypeptide is still suitable for the SortaseA enzyme-catalyzed polypeptide side chain cyclization concept disclosed in the patent. Therefore, all changes and modifications in the type of polypeptide backbone made on the basis of this patent are to be considered within the scope of the protection sought by this patent.

(2) A chemically synthesized polypeptide compound having the following characteristics:

Wherein X _a is any one of hydrogen, acetyl and oligopeptide group composed of natural or unnatural amino acid except cysteine; x _b is any one of an electrophilic group, an oligopeptidyl group comprising a natural or unnatural amino acid, wherein the electrophilic group comprises a chloroacetyl group, a 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl group; x _c is any one of oxygen (oxygen ester bond), nitrogen hydrogen (amide bond) and sulfur (thioester bond), and X _d is any one of amino group and oligopeptide sequence composed of natural or unnatural amino acid except cysteine; n is any number between 1 and 5; all simple changes and modifications to the polypeptide compounds made on the basis of this patent are still within the scope of protection of this patent.

(3) Under SortaseA enzyme catalysis, polypeptide connection and polypeptide side chain cyclization are carried out between the chemically synthesized polypeptide compound and the polypeptide template which is glycine at the N-terminal and contains cysteine in the sequence in buffer salt solution;

Wherein SortaseA enzyme is wild type or mutant thereof, sortaseA enzyme concentration is 0.1 mu M-10.0 mM, chemically synthesized polypeptide compound concentration is 0.1 mu M-10.0 mM, buffer salt solution is common buffer solution except phosphate such as HEPES, naOAc and Tris, caCl ₂ (0.1 mM-10.0 mM) and TCEP (tri (2-carboxyethyl) phosphine) (0.1 mu M-10.0 mM), pH of buffer salt solution is 6.0-9.0, polypeptide connection and polypeptide side chain cyclization reaction time is 15 minutes-6 hours, temperature is 20-45 ℃, and all simple changes and modifications of the polypeptide compound made on the basis of the patent are still within the scope of protection of the patent.

(4) Construction and application of phage display cyclic peptide library:

characteristics of the sequences of the library of bicyclic polypeptides displayed on the phage surface:

GXXXXXXCXXXXXXCGGSGGSGG (from N to C terminal, X is any of the natural amino acids, encoded by NNK, a random amino acid mutation is performed at 12 positions in the sequence, wherein GGSGGSGG is a flexible amino acid linker arm between the bicyclic peptide library and the phage surface pIII protein).

Designing and synthesizing a DNA sequence according to the sequence characteristics of the polypeptide:

5'-GCT ggcccagccggcc ATG GCC GGC NNK NNK NNK NNK NNKNNK TGC NNKNNKNNKNNKNNKNNKTGC GGC GGC TCT GGC GGC TCT GGC GGC gcggccgc TAAACTAT-3'(K：G/T,N：A/T/C/G; The recognition sequences for SfiI and NotI endonucleases in the sequences are identified in lowercase letters

The product of the double enzyme digestion of the polypeptide template DNA by SfiI and NotI is mixed with pCANTAB 5E phagemid vector at a ratio of 10:1, then electrotransformed into competent TG1 E.coli, titre-determined library diversity of 2.0X10 ⁸, and packaged with helper phage M13KO7 to form phage displaying the polypeptide pool.

The constructed phage display polypeptide library is incubated with the polypeptide compound containing 3, 5-di [ (2-chloroacetyl) amino ] benzoyl group, a double-ring peptide phage library is generated under the catalysis of SortaseA enzyme, 3-4 rounds of screening are carried out on immobilized TEAD4 target proteins, and phage clones are randomly selected for sequencing.

And (3) obtaining a sequence of the enriched polypeptide according to a sequencing result, synthesizing a bicyclic peptide corresponding to the polypeptide with high enrichment degree, carrying out off-normal fluorescence combined with TEAD4, and determining the actual effect of screening.

(5) The cyclic peptides generated by the cyclization of polypeptide side chains of a series of different polypeptide templates catalyzed by SortaseA enzyme can be applied to the technology of gene-coded polypeptide library to construct a large-scale cyclic peptide library for screening functional macrocyclic peptide ligands. The N end of phage surface display is glycine, and a polypeptide template containing one cysteine in the sequence is incubated with a polypeptide compound containing chloracetyl, and a single-ring peptide phage library is generated under the catalysis of SortaseA enzyme and used for screening functional single-ring peptide ligands. The N end of phage surface display is glycine, and the polypeptide template containing two cysteines in sequence is incubated with 3, 5-di [ (2-chloracetyl) amino ] benzoyl polypeptide compound, and a dicyclic peptide phage library is generated under the catalysis of SortaseA enzyme and used for screening functional dicyclic peptide ligand.

The G- (X) ₁₂ -C polypeptide template is selected to be fused to the surface of phage, and polypeptide connection and side chain cyclization are carried out on the phage and a chloracetyl-containing polypeptide compound under the catalysis of a Sortase A enzyme, so that a phage single-ring peptide library (the storage capacity is 4 multiplied by 10 ⁸, and X is random amino acid) is constructed. Selecting immobilized TEAD4 as a target spot to carry out 3-4 screening, randomly selecting phage clone for sequencing, and finding a highly enriched polypeptide sequence. The single-ring peptide corresponding to the polypeptide with the maximum enrichment degree is chemically synthesized, and the single-ring peptide ligand with the affinity of 2.1 mu M is obtained through a polarized fluorescence experiment and a polarized fluorescence competition experiment of TEAD4, so that the success of the method in constructing a phage single-ring peptide library and screening the functional single-ring peptide ligand is verified.

The G- (X) ₆-C-(X)₆ -C polypeptide template is selected to be fused to the surface of phage, and is catalyzed by SortaseA enzyme to carry out polypeptide connection and side chain cyclization with a polypeptide compound containing 3, 5-di [ (2-chloroacetyl) amino ] benzoyl, so as to construct a phage bicyclo peptide library (the storage capacity is 2 multiplied by 10 ⁸, and X is random amino acid). Selecting fixed TEAD4 as a target spot for 3-4 screening, randomly selecting phage for clone sequencing, and finding a highly enriched polypeptide sequence. The method has the advantages that the bicyclic peptide corresponding to the polypeptide with the maximum enrichment degree is chemically synthesized, and the bicyclic peptide ligand with the affinity of 63.9nM is obtained through a polarized fluorescence experiment and a polarized fluorescence competition experiment of TEAD4, so that the success of the method in constructing a phage bicyclic peptide library and screening the functional bicyclic peptide ligand is verified.

Example 1:

According to the above

Synthesis of chloracetyl polypeptide 3:

200.0. Mu. Mol RINKAMIDE resin (360.0 mg,0.56 mmol/g) was weighed into a 5.0mL solid phase synthesis reactor with filter screen plate, then 3.0mL DMF was added and swollen for 15 minutes at room temperature.

Then 2.0mL of 20% piperidine in DMF was added and the mixture was shaken at room temperature for 6 minutes to remove the Fmoc protecting group of the amino groups on the resin surface, and the Fmoc protecting group removal process was repeated once. The resin was then washed 4 times with DMF, added with an amino acid condensing reagent dissolved in 2.0mL of DMF and containing 4.5 equivalents of Fmoc-Gly-OH (267.0 mg), 4.5 equivalents of oxyma (127.0 mg) and 4.5 equivalents of N, N' -diisopropylcarbodiimide (139.0. Mu.L) and placed in a shaking reactor at 55deg.C for reaction for 40 minutes. The resin was then washed 4 times with DMF and the solid phase polypeptide condensation of Thr, lys, pro and Leu was carried out sequentially according to the same procedure as described above.

The resin was washed 4 times with DMF and 2.0mL of 20% piperidine in DMF was added to remove the Fmoc protecting group of the amino group of Leu. After washing the resin 4 times with DMF, 2.0mL of acetic anhydride blocking reagent was added, wherein the volume ratio of DMF, acetic anhydride and 2, 6-lutidine was 89:5:6. after shaking the resin at normal temperature for 2 minutes, the resin 4 was washed with DMF. After the resin is fully washed by methylene dichloride, the resin is dried at normal temperature, and a trifluoroacetic acid lysate prepared at present is added, wherein the volume ratio of TFA, m-cresol, water and triisopropylsilane is 88:5:5:2. after shaking the resin at normal temperature for 2 hours, the trifluoroacetic acid lysate containing the polypeptide was collected and 9 volumes of pre-ice-cooled diethyl ether were added. Obtaining white powdery crude peptide (sequence is AcNH-Leu-Pro-Lys-Thr-Gly-NH ₂) through centrifugation;

Chloroacetic acid (1.2 mg) and N-hydroxysuccinimide (2.8 mg) were weighed and dissolved in 0.2mL of DMF, N' -diisopropylcarbodiimide (3.7. Mu.L) was added, and the mixture was shaken at room temperature for 60 minutes, followed by addition of 5.5mg of crude peptide (sequence AcNH-Leu-Pro-Lys-Thr-Gly-NH ₂, dissolved in 0.2mL of DMF) and 2.0. Mu.L of N, N-diisopropylethylamine. After shaking for 60 minutes at normal temperature, 3.0mL of water containing 0.1% trifluoroacetic acid was added to quench the reaction system. The target polypeptide product was purified using a reverse-phase high performance liquid chromatograph, and lyophilized to give the target chloroacetyl-containing polypeptide 3 (4.0 mg, sequence AcNH-Leu-Pro- ^ClAcLys-Thr-Gly-NH₂,^ClAc Lys: lys side chain amino modified with chloroacetyl), the preparation scheme is shown in FIG. 6.

And the detection of chloroacetyl-containing polypeptide 3 was: ESI-MS (m/z): calculated for C ₂₇H₄₆ClN₇O₈: 631.3; found 631.1.

The synthesized chloracetyl-containing polypeptide 3 is used for enzymatic side chain cyclization of polypeptides and enzymatic side chain cyclization of phage display polypeptide libraries and screening functional monocyclic peptide ligands.

Example 2:

synthesis of chloroacetyl polypeptide oxygen ester 4:

100.0. Mu. Mol RINKAMIDE resin (180.0 mg,0.56 mmol/g) was weighed into a 5.0mL solid phase synthesis reactor with filter screen plate, then 3.0mL DMF was added and swollen for 15 minutes at room temperature. Then 2.0mL of 20% piperidine in DMF was added and the mixture was shaken at room temperature for 6 minutes to remove the Fmoc protecting group of the amino groups on the resin surface, and the Fmoc protecting group removal process was repeated once. The resin was then washed 4 times with DMF, and glycolic acid condensing agent dissolved in 3.0mL of DMF was added containing 10.0 equivalents of glycolic acid (76.0 mg), 10.0 equivalents oxyma (142.1 mg) and 10.0 equivalents of N, N' -diisopropylcarbodiimide (154.8. Mu.L) and placed in a shaking reactor at 55℃for reaction for 40 minutes. After the resin was washed 4 times with DMF, the resin was treated with 10% hydrazine hydrate in DMF at normal temperature for 30 minutes. After washing the resin with DMF, an amino acid condensing reagent was added to the resin containing Fmoc-Thr (tBu) -OH (0.39 g), HOBt (135.0 mg) and DMAP (2.44 mg) dissolved in 6mL of DMF/DCM solution (1:9, volume ratio), followed by DIC (154.8. Mu.L). After overnight reaction at room temperature, the resin was washed. Then, solid-phase polypeptide condensation of Lys, pro and Leu was performed sequentially. The resin was washed 4 times with DMF and 2.0mL of 20% piperidine in DMF was added to remove the Fmoc protecting group of the amino group of Leu. After washing the resin 4 times with DMF, 2.0mL of acetic anhydride blocking reagent was added, wherein the volume ratio of DMF, acetic anhydride and 2, 6-lutidine was 89:5:6. after shaking the resin at normal temperature for 2 minutes, the resin was washed 4 times with DMF. After the resin is fully washed by methylene dichloride, the resin is dried at normal temperature, and a trifluoroacetic acid lysate prepared at present is added, wherein the volume ratio of TFA, m-cresol, water and triisopropylsilane is 88:5:5:2. after shaking the resin at normal temperature for 2 hours, the trifluoroacetic acid lysate containing the polypeptide was collected and 9 volumes of pre-ice-cooled diethyl ether were added. The white powdery crude peptide was obtained by centrifugation.

Chloroacetic acid (1.2 mg) and N-hydroxysuccinimide (2.8 mg) were weighed and dissolved in 0.2mL of DMF, N' -diisopropylcarbodiimide (3.7. Mu.L) was added, and the mixture was shaken at room temperature for 60 minutes, followed by addition of 6.3mg of the crude oxyester peptide and 2.0. Mu.L of N, N-diisopropylethylamine. After shaking for 60 minutes at normal temperature, 3.0mL of water containing 0.1% trifluoroacetic acid was added to quench the reaction system. Purifying the target polypeptide product by using a reversed-phase high performance liquid chromatograph, and freeze-drying to obtain target chloracetyl polypeptide oxygen ester 4 (4.1 mg, the sequence of which is AcNH-Leu-Pro- ^ClAcLys-Thr-Ogly-NH₂, ogly represents a glycolic acid structure), wherein the synthesis of the chloracetyl polypeptide oxygen ester 4 is shown in figure 7;

And the detection of the chloracetyl polypeptide oxygen ester 4 is as follows: ESI-MS (m/z): calculated for C ₂₇H₄₅ClN₆O₉: 632.2; found 632.7.

The synthesized chloracetyl-containing polypeptide oxygen ester 4 is used for enzyme-catalyzed side chain cyclization of polypeptides, enzyme-catalyzed side chain cyclization of phage display polypeptide libraries and screening of functional monocyclic peptide ligands.

Example 3:

Synthesis of chloracetyl polypeptide thioester 5:

100.0. Mu. Mol of 2-Cl hydrazine resin (180.0 mg,0.56 mmol/g) was weighed into a 5.0mL solid phase synthesis reactor with filter cartridge sieve plates, then 3.0mL DMF was added and the mixture was swelled at room temperature for 15 minutes. Then, the solid-phase polypeptide condensation of Thr, lys, pro and Leu was performed sequentially. The resin was washed 4 times with DMF and 2.0mL of 20% piperidine in DMF was added to remove the Fmoc protecting group of the amino group of Leu. After the resin is washed for 4 to 5 times by DMF, 2.0mL of acetic anhydride blocking reagent is added, wherein the volume ratio of DMF, acetic anhydride and 2, 6-lutidine is 89:5:6. after shaking the resin at normal temperature for 2 minutes, the resin was washed 4 times with DMF. After the resin is fully washed by methylene dichloride, the resin is dried at normal temperature, and a trifluoroacetic acid lysate prepared at present is added, wherein the volume ratio of TFA, m-cresol, water and triisopropylsilane is 88:5:5:2. after shaking the resin at normal temperature for 2 hours, the trifluoroacetic acid lysate containing the polypeptide was collected and 9 volumes of pre-ice-cooled diethyl ether were added. The white powdery crude hydrazide peptide was obtained by centrifugation.

10.0Mg of crude hydrazide peptide was dissolved in 1.0mL of PBS buffer (6.0M guanidine hydrochloride, 0.2M PBS, pH 3). New configuration NaNO ₂ (13.5 mg in 50. Mu.L water) was added to the hydrazide peptide at-15℃in an ice salt bath. After 20 minutes, 175.0. Mu.L of Methyl Thioglycolate (MTG) was added. After 30 minutes of reaction at normal temperature, excess MTG was removed by extraction with diethyl ether, and the white powdered thioester peptide was obtained by HPLC purification and freeze-drying. Chloroacetic acid (1.2 mg) and N-hydroxysuccinimide (2.8 mg) were dissolved in 0.4mL of DMF, and N, N' -diisopropylcarbodiimide (3.8. Mu.L) was added. After 60 minutes of shaking at normal temperature, 5.8mg of the thioetheride was added, and 2.0. Mu.L of N, N-diisopropylethylamine was added. After 60 minutes of reaction at normal temperature, 3.0mL of water containing 0.1% trifluoroacetic acid was added to quench the reaction system. Purifying the target polypeptide product by using a reversed-phase high performance liquid chromatograph, and freeze-drying to obtain target chloracetyl polypeptide thioester 5 (3.2 mg, the sequence is AcNH-Leu-Pro- ^ClAc Lys-Thr-MTG, and the MTG represents a methyl thioglycolate structure); and the preparation flow of the chloracetyl polypeptide thioester 5 is shown in figure 8;

And the detection of chloracetyl polypeptide thioester 5 is: ESI-MS (m/z): calculated for C ₂₈H₄₆ClN₅O₉ S:663.2; found 663.4.

The synthesized chloracetyl-containing polypeptide thioester 5 is used for enzymatic side chain cyclization of polypeptides, enzymatic side chain cyclization of phage display polypeptide libraries and screening of functional monocyclic peptide ligands.

Example 4:

synthesis of polypeptide 6 containing chloracetyl modified diaminopropionic acid:

100.0. Mu. Mol RINKAMIDE resin (180.0 mg,0.56 mmol/g) was weighed into a 5.0mL solid phase synthesis reactor with filter screen plate, then 3.0mL DMF was added and swollen for 15 minutes at room temperature. Then 2.0mL of 20% piperidine in DMF was added and the mixture was shaken at room temperature for 6 minutes to remove the Fmoc protecting group of the amino groups on the resin surface, and the Fmoc protecting group removal process was repeated once. The resin was then washed 4-5 times with DMF, added with an amino acid condensing reagent dissolved in 2.0mL of DMF and containing 4.5 equivalents of Fmoc-Gly-OH (267.0 mg), 4.5 equivalents of oxyma (127.0 mg) and 4.5 equivalents of N, N' -diisopropylcarbodiimide (139.0. Mu.L) and placed in a shaking reactor at 55deg.C for reaction for 40 minutes. The resin was then washed 4 times with DMF and the solid phase polypeptide condensation of Thr, dap ((S) -2, 3-diaminopropionic acid), pro and Leu was performed sequentially according to the same procedure as described above. The resin was washed 4-5 times with DMF and 2.0mL of 20% piperidine in DMF was added to remove the Fmoc protecting group of the amino group of Leu. After washing the resin 4 times with DMF, 2.0mL of acetic anhydride blocking reagent was added, wherein the volume ratio of DMF, acetic anhydride and 2, 6-lutidine was 89:5:6. after shaking the resin at normal temperature for 2 minutes, the resin was washed 4 times with DMF. After the resin is fully washed by methylene dichloride, the resin is dried at normal temperature, and a trifluoroacetic acid lysate prepared at present is added, wherein the volume ratio of TFA, m-cresol, water and triisopropylsilane is 88:5:5:2. after shaking the resin at normal temperature for 2 hours, the trifluoroacetic acid lysate containing the polypeptide was collected and 9 volumes of pre-ice-cooled diethyl ether were added. The crude peptide (sequence AcNH-Leu-Pro-Dap-Thr-Gly-NH ₂) was obtained as a white powder by centrifugation.

Chloroacetic acid (1.2 mg) and N-hydroxysuccinimide (2.8 mg) were weighed and dissolved in 0.4mL of DMF, and N, N' -diisopropylcarbodiimide (3.7. Mu.L) was added thereto, followed by shaking at room temperature for 60 minutes to obtain N-hydroxysuccinimide ester. 5.0mg of crude peptide (sequence AcNH-Leu-Pro-Dap-Thr-Gly-NH ₂) was weighed out in 0.9mL DMF and then 2.0. Mu.L of N, N-diisopropylethylamine was added. After 60 minutes of reaction at normal temperature, the reaction system was quenched in an ice bath. Purifying the target polypeptide product by using a reversed-phase high performance liquid chromatograph, and freeze-drying to obtain target polypeptide 6 containing chloracetyl modified diaminopropionic acid (3.9 mg, the sequence is AcNH-Leu-Pro- ^ClAcDap-Thr-Gly-NH₂,^ClAc Dap: the side chain amino group of the Dap is modified by chloracetyl), wherein the preparation flow of the polypeptide 6 containing chloracetyl modified diaminopropionic acid is shown in figure 9; the detection method comprises the following steps: ESI-MS (m/z): calculated for C ₂₄H₄₀ClN₇O₈: 589.2; found 589.3.

The synthesized polypeptide 6 containing chloracetyl modified diaminopropionic acid is used for enzyme-catalyzed side chain cyclization of polypeptides, enzyme-catalyzed side chain cyclization of phage display polypeptide libraries and screening of functional monocyclic peptide ligands.

Example 5:

Synthesis of 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide 7:

200.0. Mu. Mol RINKAMIDE resin (360.0 mg,0.56 mmol/g) was weighed into a 5.0mL solid phase synthesis reactor with filter screen plate, then 3.0mL DMF was added and swollen for 15 minutes at room temperature. Then 2.0mL of 20% piperidine in DMF was added and the mixture was shaken at room temperature for 6 minutes to remove the Fmoc protecting group of the amino groups on the resin surface, and the Fmoc protecting group removal process was repeated once. The resin was then washed 4 times with DMF, added with an amino acid condensing reagent dissolved in 2.0mL of DMF and containing 4.5 equivalents of Fmoc-Gly-OH (267.0 mg), 4.5 equivalents of oxyma (127.0 mg) and 4.5 equivalents of N, N' -diisopropylcarbodiimide (139.0. Mu.L) and placed in a shaking reactor at 55deg.C for reaction for 40 minutes. The resin was then washed 4 times with DMF and the solid phase polypeptide condensation of Thr, lys, pro and Leu was carried out sequentially according to the same procedure as described above. The resin was washed 4 times with DMF and 2.0mL of 20% piperidine in DMF was added to remove the Fmoc protecting group of the amino group of Leu. After washing the resin 4 times with DMF, 2.0mL of acetic anhydride blocking reagent was added, wherein the volume ratio of DMF, acetic anhydride and 2, 6-lutidine was 89:5:6. after shaking the resin at normal temperature for 2 minutes, the resin was washed 4 times with DMF. After the resin is fully washed by methylene dichloride, the resin is dried at normal temperature, and a trifluoroacetic acid lysate prepared at present is added, wherein the volume ratio of TFA, m-cresol, water and triisopropylsilane is 88:5:5:2. after shaking the resin at normal temperature for 2 hours, the trifluoroacetic acid lysate containing the polypeptide was collected and 9 volumes of pre-ice-cooled diethyl ether were added. The white powdery crude peptide (sequence AcNH-Leu-Pro-Lys-Thr-Gly-NH ₂) was obtained by centrifugation.

3.0G of 3, 5-diaminobenzoic acid was weighed into a 100.0mL round bottom flask equipped with a magneton and dissolved completely in 25.0mL of N-methylpyrrolidone. The round bottom flask was pre-cooled in an ice-salt bath at-15 to-20℃for 30 minutes, then 6.8g of chloroacetyl chloride was added and stirring was continued at this temperature for 30 minutes. The ice-salt bath was removed and the reaction was continued at 0℃for 9 hours, followed by 1 hour at room temperature. The reaction solution was poured into 200.0mL of an aqueous hydrochloric acid solution composed of 10mL of concentrated hydrochloric acid and 190mL of water. The target product is separated out by precipitation, the precipitation is collected by filtration, and the crude product is dried. The crude product was redissolved in 10.0mL of N-methylpyrrolidone and the product was precipitated with 100.0mL of ethanol/water (1:10, volume ratio). The precipitate was collected by filtration and washed thoroughly with water, and dried overnight in a vacuum oven at 70℃to give the desired product, 3, 5-bis [ (2-chloroacetyl) amino ] benzoic acid (4.4 g), as a powder.

3, 5-Bis [ (2-chloroacetyl) amino ] benzoic acid (6.0 mg) and N-hydroxysuccinimide (9.2 mg) were weighed and dissolved in 0.2mL of DMF, N' -diisopropylcarbodiimide (12.6. Mu.L) was added, and shaking was performed at room temperature for 60 minutes, and then 5.5mg of crude peptide (sequence AcNH-Leu-Pro-Lys-Thr-Gly-NH ₂, dissolved in 0.2mL of DMF) was added, followed by 3.0. Mu.L of N, N-diisopropylethylamine. After shaking for 60 minutes at normal temperature, 3.0mL of water containing 0.1% trifluoroacetic acid was added to quench the reaction system. Purifying the target polypeptide product by using a high performance liquid chromatograph, and freeze-drying to obtain target polypeptide 7 containing 3, 5-di [ (2-chloroacetyl) amino ] benzoyl (4.5 mg, the sequence of which is AcNH-Leu-Pro- ^CabLys-Thr-Gly-NH₂,^Cab Lys: lys is modified by 3, 5-di [ (2-chloroacetyl) amino ] benzoyl), and synthesizing polypeptide 7 containing 3, 5-di [ (2-chloroacetyl) amino ] benzoyl, wherein the synthesis of the polypeptide 7 is shown in figure 10, and the detection of the polypeptide is ESI-MS (m/z): calculated for C ₃₆H₅₃Cl₂N₉O₁₀: 841.3; found 841.2.

The synthesized 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl polypeptide 7 is used for enzymatic side chain cyclization of polypeptides, and the phage display of a library of polypeptides, and the functional bicyclic peptide ligands are screened.

Example 6:

synthesis of oxy 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl polypeptide 8:

3, 5-Bis [ (2-chloroacetyl) amino ] benzoic acid (6.0 mg) and N-hydroxysuccinimide (9.2 mg) were weighed and dissolved in 0.2mL of DMF, N' -diisopropylcarbodiimide (12.6. Mu.L) was added, and shaking was performed at room temperature for 60 minutes, then 6.3mg of crude peptide (sequence AcNH-Leu-Pro-Lys-Thr-Ogly-NH ₂, dissolved in 0.2mL of DMF) was added, and 3.0. Mu.L of N, N-diisopropylethylamine was further added. After shaking for 60 minutes at normal temperature, 3.0mL of water containing 0.1% trifluoroacetic acid was added to quench the reaction system. Purifying the target polypeptide product by using a high performance liquid chromatograph, and obtaining target 3, 5-di [ (2-chloroacetyl) amino ] benzoyl polypeptide oxygen ester 8 (5.0 mg, the sequence of which is AcNH-Leu-Pro- ^CabLys-Thr-Ogly-NH₂,^Cab Lys: lys is modified by 3, 5-di [ (2-chloroacetyl) amino ] benzoyl; ogly: glycolic acid) after freeze drying, wherein the preparation process of the 3, 5-di [ (2-chloroacetyl) amino ] benzoyl polypeptide oxygen ester 8 is shown in figure 11, and the detection of the preparation process is ESI-MS (m/z): calculated for C ₃₆H₅₂Cl₂N₈O₁₁: 842.3; found 842.7.

The synthesized polypeptide oxyesters 8 of 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl are useful for enzymatic side chain cyclization of polypeptides and for enzymatic side chain cyclization of phage display polypeptide libraries and screening for functional bicyclic peptide ligands.

Example 7:

synthesis of 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl modified diaminopropionic acid-containing polypeptide 9:

100.0. Mu. Mol RINKAMIDE resin (180.0 mg,0.56 mmol/g) was weighed into a 5.0mL solid phase synthesis reactor with filter screen plate, then 3.0mL DMF was added and swollen for 15 minutes at room temperature. Then 2.0mL of 20% piperidine in DMF was added and the mixture was shaken at room temperature for 6 minutes to remove the Fmoc protecting group of the amino groups on the resin surface, and the Fmoc protecting group removal process was repeated once. The resin was then washed 4 times with DMF, added with an amino acid condensing reagent dissolved in 2.0mL of DMF and containing 4.5 equivalents of Fmoc-Gly-OH (267.0 mg), 4.5 equivalents of oxyma (127.0 mg) and 4.5 equivalents of N, N' -diisopropylcarbodiimide (139.0. Mu.L) and placed in a shaking reactor at 55deg.C for reaction for 40 minutes. The resin was then washed 4 times with DMF and the solid phase polypeptide condensation of Thr, dap, pro and Leu was carried out sequentially according to the same procedure as described above. The resin was washed 4 times with DMF and 2.0mL of 20% piperidine in DMF was added to remove the Fmoc protecting group of the amino group of Leu. After washing the resin 4 times with DMF, 2.0mL of acetic anhydride blocking reagent was added, wherein the volume ratio of DMF, acetic anhydride and 2, 6-lutidine was 89:5:6. after shaking the resin at normal temperature for 2 minutes, the resin was washed 4 times with DMF. After the resin is fully washed by methylene dichloride, the resin is dried at normal temperature, and a trifluoroacetic acid lysate prepared at present is added, wherein the volume ratio of TFA, m-cresol, water and triisopropylsilane is 88:5:5:2. after shaking the resin for 2 hours at normal temperature, collecting the trifluoroacetic acid lysate containing the polypeptide, and adding 8-10 times of pre-ice-cooled diethyl ether. The crude peptide (sequence AcNH-Leu-Pro-Dap-Thr-Gly-NH ₂) was obtained as a white powder by centrifugation.

3, 5-Bis [ (2-chloroacetyl) amino ] benzoic acid (6.0 mg) and N-hydroxysuccinimide (9.2 mg) were weighed and dissolved in 0.2mL of DMF, N' -diisopropylcarbodiimide (12.6. Mu.L) was added, and shaking was performed at room temperature for 60 minutes, and then 5.1mg of crude peptide (sequence AcNH-Leu-Pro-Dap-Thr-Ogly-NH ₂, dissolved in 0.2mL of DMF) was added, followed by 3.0. Mu.L of N, N-diisopropylethylamine. After shaking for 60 minutes at normal temperature, 3.0mL of water containing 0.1% trifluoroacetic acid was added to quench the reaction system. Purifying the target polypeptide product by using a high performance liquid chromatograph, and obtaining the target polypeptide 9 containing 3, 5-di [ (2-chloroacetyl) amino ] benzoyl modified diaminopropionic acid (3.8 mg, the sequence is AcNH-Leu-Pro- ^CabDap-Thr-Gly-NH₂,^Cab Dap: the side chain amino group of the Dap is modified by 3, 5-di [ (2-chloroacetyl) amino ] benzoyl), wherein the synthetic route of the polypeptide 9 containing 3, 5-di [ (2-chloroacetyl) amino ] benzoyl modified diaminopropionic acid is shown in figure 12, and the detection is ESI-MS (m/z): calculated for C ₃₃H₄₇Cl₂N₉O₁₀: 799.2; found 799.2.

The synthesized polypeptide 9 of 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl modified diaminopropionic acid is used for enzyme-catalyzed side chain cyclization of polypeptides, enzyme-catalyzed side chain cyclization of phage display polypeptide libraries and functional bicyclic peptide ligands are screened.

Example 8:

SortaseA enzyme catalyzed preparation of side chain cyclized monocyclic peptides:

SortaseA enzyme Plasmid vector (a SortaseA enzyme mutant with higher enzyme ligation efficiency, from Addgene, plasmid # 75144) was transformed into DH 5. Alpha. Competent cells, and the plates were grown overnight on LB plates containing kanamycin. Several well-grown monoclonal were picked and inoculated in 3.0mL of LB medium containing kanamycin, respectively, for overnight culture. The plasmids were extracted using commercial kits and sequenced to confirm their correctness, and were sub-packaged for storage to-20 ℃ after quantification for later experiments.

The correct SortaseA enzyme plasmid was transformed into BL21 (DE 3) competent cells, plated on LB plates containing kanamycin overnight, and several better-state monoclonal were picked and inoculated into 10.0mL LB medium containing kanamycin, respectively, for overnight culture at 37 ℃. 500.0mL of LB medium was taken, 250. Mu.L of kanamycin mother liquor (100.0 mg/mL) was added, 5.0mL of the overnight cultured strain was added, and the culture was continued at 37℃for 3 hours until the OD600 reached around 0.8, 150.0. Mu.L of IPTG mother liquor (1 mmol/L) was added, and the culture was continued at16℃and 180rpm for 20 hours. The strain is collected by centrifugation, the strain is washed by a lysis buffer (20.0mM HEPES,500.0mM NaCl,5% glycerol, pH 7.5) by centrifugation, 50.0mL of the lysis buffer is added, the strain is broken by ultrasound, the precipitate is removed by centrifugation, a supernatant solution containing the target SortaseA enzyme is obtained, the target protein with the purity of about 90% is obtained by nickel column purification, sortaseA enzyme (9 mg/mL) is obtained by ultrafiltration concentration and replacement into the lysis buffer, split charging is carried out, the split charging is carried out at-80 ℃, each containing 225.0 mug (25.0 mug) of SortaseA enzyme, and the split charging is used for enzyme-catalyzed polypeptide ligation and side chain cyclization reaction.

10.0ML of HEPES buffer (pH 8.0) was prepared containing 50.0mM HEPES, 100.0mM NaCl, 2.0mM CaCl ₂, and 1.0mM TCEP (tris (2-carboxyethyl) phosphine). 1.0mL of HEPES buffer (pH 8.0) was taken, 4.0. Mu.L of polypeptide 1 (sequence H-GRYDPANIHPKGWCGGSG-NH ₂, 94.0. Mu.g, final concentration 50.0. Mu.M) was added, followed by 10.0. Mu.L of SortaseA enzyme (90.0. Mu.g, final concentration 5. Mu.M) and then 4.0. Mu.L of chloroacetyl polypeptide 3 (32.0. Mu.g, final concentration 50.0. Mu.M) was added. The reaction solution was mixed and then subjected to detection by high performance liquid chromatography in a shaking table at 37 ℃. And polypeptide 1 and chloroacetyl-containing polypeptide 3 were pre-dissolved in an aqueous solution containing 1% acetonitrile and 0.1% trifluoroacetic acid.

As shown in FIG. 13, according to the tracking result of high performance liquid chromatography, polypeptide 1 and chloroacetyl-containing polypeptide 3 undergo polypeptide ligation and side chain cyclization reaction in the presence of SortaseA enzyme to produce the target single-ring peptide product 10 (ESI-MS (m/z): calculated for C ₁₀₆H₁₅₇N₃₁O₃₁ S:2393.1; found: 2393.2). In the absence of SortaseA enzyme, polypeptide 1 did not react with chloroacetyl-containing polypeptide 3. These experimental results indicate that the ligation and side chain cyclization reactions of polypeptide 1 and chloroacetyl-containing polypeptide 3 occur enzymatically.

As shown in FIG. 14, according to a similar scheme, polypeptide 1 and chloroacetyl-containing polypeptide oxyesters 4 undergo polypeptide ligation and side chain cyclization to produce the desired monocyclic peptide product 10. As shown in FIG. 15, according to a similar scheme, polypeptide 1 undergoes polypeptide ligation and side chain cyclization with chloroacetyl polypeptide thioester 5 to yield the desired monocyclic peptide product 10. As shown in FIG. 16, according to a similar scheme, polypeptide 1 undergoes polypeptide ligation and side chain cyclization with polypeptide 6 containing chloroacetyl-modified diaminopropionic acid to produce the desired monocyclic peptide product 11 (ESI-MS (m/z): calculated for C ₁₀₃H₁₅₁N₃₁O₃₁ S:2351.1; found: 2351.0).

Example 9:

preparation of side-chain cyclized bicyclic peptides under SortaseA enzyme catalysis

10.0ML of HEPES buffer (pH 7.0) was prepared containing 50.0mM HEPES, 100.0mM NaCl, 2.0mM CaCl ₂, and 1.0mM TCEP (tris (2-carboxyethyl) phosphine). 1.0mL of HEPES buffer (pH 7.0) was added to 4.0. Mu.L of polypeptide 2 (sequence H-GRYDPANCIHPKGWCGGSG-NH ₂, 99.0. Mu.g, final concentration 50.0. Mu.M), followed by 10.0. Mu.L of SortaseA enzyme (90.0. Mu.g, final concentration 5.0. Mu.M), followed by 4.0. Mu.L of 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl polypeptide 7 (42.0. Mu.g, final concentration 50.0. Mu.M). The reaction solution was mixed and then subjected to detection by high performance liquid chromatography in a shaking table at 37 ℃. And peptide 2 and 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide 7 were pre-dissolved in an aqueous solution containing 1% acetonitrile and 0.1% trifluoroacetic acid.

As shown in fig. 17, according to the tracking result of high performance liquid chromatography, polypeptide 2 and 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide 7 undergo polypeptide ligation and side chain cyclization in the presence of SortaseA enzyme, resulting in the desired bicyclic peptide product 12(ESI-MS(m/z)：calculated for C₁₁₈H₁₆₈N₃₄O₃₄S₂:2670.1;found:2670.2)., polypeptide 2 and 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide 7 do not react in the absence of Sortase a enzyme. These experimental results indicate that the peptide ligation and side chain cyclization reactions of polypeptide 2 and 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide 7 occur enzymatically. As shown in FIG. 18, polypeptide 2 undergoes a polypeptide ligation and side chain cyclization reaction with 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl-containing polypeptide oxyester 8 to produce the desired bicyclic peptide product 12 according to a similar scheme. As shown in FIG. 19, according to a similar scheme, polypeptide 2 undergoes polypeptide ligation and side chain cyclization with polypeptide 9 of 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl diaminopropionic acid to produce the desired bicyclic peptide product 13(ESI-MS(m/z)：calculated for C₁₁₅H₁₆₂N₃₄O₃₄S₂:2628.1;found:2628.0).

Example 10:

efficiency of SortaseA enzyme-catalyzed peptide cyclization on phage

1. Construction of M13KE-GX ₁₂ C phage library

A GX ₁₂ C polypeptide library was constructed using an M13KE phage vector (from NEB corporation), where X is the random amino acid encoded by NNK, N is A/T/C/G, and K is G/T. A nucleic acid library was designed based on the cleavage site Kpn I and Eag I cleavage site characteristics of the M13KE vector, and template strands and primer sequences were determined from Bio-Rad (Shanghai) as follows:

Library DNA (template strand 1)：5'-CATGT TTC GGC CGAGCC GCC AGA GCC GCC AGA GCC GCC GCA MNN MNN MNN MNN MNN MNN MNN MNN MNN MNN MNN MNN GCC AGA GTG AGA ATA GAAAGG TAC CCG GGCATG-3'(M A/C);

primer DNA:5'-CATGCCCGGGTACCTTTCTATTCTC-3';

The library DNA and primer DNA were mixed and annealed in equal proportions and extended by Klenow DNA polymerase to produce a double stranded DNA library. The double-stranded DNA library and M13KE vector were treated with Kpn I-HF and Eag I-HF, and recombinant M13KE phage vector with library DNA was constructed, and ER2738 competent cells were electrotransformed. All SOC media containing ER2738 electrotransfer cells were resuscitated for 40 min, inoculated into LB media containing ER2738 in early logarithmic growth phase, and shaken at 37℃for 5 hours to produce phages, with a blue spot titer assay diversity of 10 ⁶.

2. Influence of the reaction conditions based on SortaseA enzyme on phage

Five aliquots of phage (10 ¹² pfu) were taken and dissolved in 1.0mL of five different solutions, solution a: commercial neutral PBS; solution B:50.0mM HEPES,100.0mMNaCl,2.0mM CaCl ₂, 1.0mM TCEP,pH 8.0; solution C:50.0mM HEPES,100.0mM NaCl,2.0mM CaCl ₂, 1.0mM TCEP,5.0 mu M SortaseA enzyme, pH 8.0; solution D:50.0mM HEPES,100.0mM NaCl,2.0mM CaCl ₂, 1.0mM TCEP, 50.0. Mu.M chloroacetyl polypeptide 3 (sequence AcNH-Leu-Pro- ^ClAcLys-Thr-Gly-NH₂), pH 8.0; solution E:50.0mM HEPES,100.0mM NaCl,2.0mM CaCl ₂, 1.0mM TCEP, 5.0. Mu. M SortaseA enzyme, 50.0. Mu.M chloroacetyl-containing polypeptide 3 (sequence AcNH-Leu-Pro- ^ClAcLys-Thr-Gly-NH₂), pH 8.0. After shaking the phage at 37℃for 1 hour, titer detection was performed.

As shown in FIG. 20, the titers of the phages treated by the five solutions are not greatly different (the highest difference and the lowest difference are within 1 time), which indicates that the reaction condition of SortaseA enzyme has little influence on the infectivity of the phages, and the phage cyclization strategy with good biocompatibility is realized.

3. Streptavidin magnetic bead capturing phage

M13KE-GX ₁₂ C phage (2X 10 ¹¹ pfu) was added to 1.0mL of HEPES buffer (50.0mM HEPES,100.0mM NaCl,2.0mM CaCl ₂, 1.0mM TCEP,pH 8.0), 10.0. Mu.L of SortaseA enzyme (final concentration 5.0. Mu.M) and 4.0. Mu.L of Biotin-1 (sequence Biotin-. Beta.Ala-Leu-Pro-ClAcLys-Thr-Gly-NH ₂, final concentration 50.0. Mu.M) and shaken at 37℃for 1 hour. The phage particles were then precipitated with polyethylene glycol (4%PEG8000,0.5M NaCl), resuspended in 1.0mL of TBS buffer, and diluted gradient to a phage solution of predetermined concentration. 20.0. Mu.L phage solution was taken separately in 2 empty 2.0mL centrifuge tubes: a and B. To the pre-washed streptavidin beads were added 50.0. Mu.L of Binding buffer (10.0 mM Tris-Cl, 150.0mM NaCl, 10.0mM MgCl ₂、1.0mM CaCl₂, pH 7.4) and 50.0. Mu. LBlocking buffer (10.0 mM Tris-Cl, 150.0mM NaCl, 10.0mM MgCl ₂、1.0mM CaCl₂, 0.3% Tween-20,3% (w/v) BSA) and incubated by spin at room temperature for 1 hour. 50.0. Mu.L of Binding buffer and 50.0. Mu.L of Blocking buffer were added to phage-dissolved A and B tubes, and incubated for 1 hour at room temperature with rotation. Tube A phage was then added to streptavidin beads, incubated for 30min at room temperature with slow spin, then the beads were captured on a magnetic rack, the supernatant was transferred to a new empty centrifuge tube, and the beads were washed 2 times with 200.0. Mu.L of washing buffer (10.0 mM Tris-Cl, 150.0mM NaCl, 10.0mM MgCl ₂、1.0mM CaCl₂, pH 7.4,0.1% Tween-20) and transferred to the centrifuge tube containing phage supernatant. Titer detection was performed on 620.0. Mu.L of supernatant from tube A captured by magnetic beads and phage from tube B, and the enzyme modification efficiency of phage was: modified phage (%) = [ (titer B-titer a)/titer B ] ×100%. The experiment was repeated 3 times and averaged. For comparison, we performed the same phage capture experiment described above with 4.0. Mu.L of Biotin-1 replaced with 4. Mu.L of HEPES buffer.

Of the phage modified with Biotin-1, 73% of phage particles were captured. In contrast, no apparent change in titer was observed for phage not modified by Biotin-1 before and after bead treatment, indicating that SortaseA enzyme-catalyzed phage surface modification efficiency could reach 73%.

Example 11:

phage monocyclic peptide library construction and functional monocyclic peptide ligand screening

1. Construction of pCANTAB 5E-GX ₁₂ C phage library

A GX ₁₂ C polypeptide library was constructed using pCANTAB 5E phagemid-helper phage (M13 KO 7) (from Sichuan apak Biotechnology Co., ltd.) where X is the random amino acid encoded by NNK, N is A/T/C/G, K is G/T. According to the characteristics of the cleavage sites Sfi I and Not I on the pCANTAB 5E phagemid vector, a nucleic acid library was designed, and the coding strand and primer sequences were customized from general biology (Anhui) Inc. as follows:

library DNA (SfiI and NotI endonuclease recognition sequences in coding strand 1)：5'-GCT ggcccagccggccATG GCC GGC NNK NNK NNK NNK NNK NNK NNK NNK NNK NNK NNK NNK TGC GGC GGC TCT GGC GGC TCT GGC GGC gcggccgc TAAACTAT-3'(K：G/T,N：A/T/C/G; sequences are identified in lowercase);

primer DNA:5'-ATAGTTTAGCGGCCGCGCCGCC-3';

The library DNA and primer DNA were mixed in equal proportions and extended by Klenow DNA polymerase to produce a double stranded DNA library. Double-stranded DNA library and pCANTAB 5E phagemid were treated with Sfi I and Not I to construct recombinant pCANTAB 5E phagemid vector with library DNA, electroporated into TG1 competent cells, plated on 2 XYT (Amp and glucose containing) plates overnight for culture, and titers were determined by dilution to determine library diversity and phage particles were randomly picked to analyze phage quality. The primary large intestine library was collected using glycerol solution and stored at-80 ℃. Phage library diversity was 4×10 ⁸ as determined by phage titer.

2. Biotinylation of target proteins and validation thereof

The biotinylation of proteins can be carried out in two ways. The method comprises the following steps: 600.0 μg of TEAD4 (26.53 kDa, final concentration of 10.0 μM) target protein was dissolved in 2.0mL PBS (pH 7.4) with 20-fold equivalent Biotin-PEG-NHS (commercially available, 1000 g/mol), incubated at room temperature for 1 hour, and excess Biotin reagent was removed using a Zeba ^TM desalting centrifuge plate. The second method is as follows: 200.0. Mu.L of the target protein TEAD4 (26.53 kDa, mother liquor concentration 113.5. Mu.M) was dissolved in 1.8mL of PBS (pH 7.4) together with a 20-fold molar excess of Biotin-PEG-NHS (1000 g/mol) and incubated with slow rotation at room temperature for 1 hour. Ultrafiltration concentration and Lysis buffer (20.0mM HEPES,500.0mM NaCl,5% glycerol, pH 7.5) displacement were used to remove excess Biotin-PEG-NHS.

3. Single-ring peptide ligand screening for immobilized TEAD4

Taking a proper amount of stored split-charging phage glycerinum, melting for a few minutes at room temperature, and inoculating to a 2XYT liquid culture medium (containing glucose and Amp). Reaching 0.3 to 0.5 at 37 ℃ to OD 600. Helper phage M13KO7 was added at a multiplicity of infection (MOI) of 20 (20 times that of TG 1) and infected at 37℃for 1 hour. The supernatant was removed by centrifugation at 4℃and 10000rpm, and the cells were collected and resuspended in 2XYT medium (containing Amp and Kana, without glucose) and cultured at 28℃for 16 hours. Centrifugation was performed at 4℃and 8000rpm for 20 minutes, and the supernatant phage was transferred to a sterile centrifuge bottle, and a pre-chilled 5 XPEG/NaCl solution was added. After ice bath for 1 hour, the supernatant was discarded after centrifugation at 10000rpm at 4℃for 20 minutes. Phage particles were resuspended in 100.0mL TBS, centrifuged at 4℃and 10000rpm for 20 minutes, the supernatant was transferred to a new sterile centrifuge bottle, and the PEG/NaCl precipitation process of phage was repeated once. Phage bacteria liquid was centrifuged at 4℃and 10000rpm for 20 minutes, the supernatant was discarded, resuspended in TBS and filtered with 0.45 μm sterile filter, and stored at 4℃for several days or glycerol for a long period of-20 ℃.

10 ¹¹ Pfu phage were mixed well in 1.0mL HEPES buffer (50.0mM HEPES,100.0mM NaCl,2.0mM CaCl ₂, 1.0mM TCEP,pH 8.0), 10.0. Mu. L SortaseA (9.0 mg/mL) and 4.0. Mu.L of chloroacetyl polypeptide 3 (sequence AcNH-Leu-Pro- ^ClAcLys-Thr-Gly-NH₂, 32.0. Mu.g, final concentration of 50.0. Mu.M) were added and shaken at 37℃for 1 hour at 250 rpm. Then, a pre-chilled 5 XPEG/NaCl solution was added, mixed well, ice-incubated for 30 minutes, centrifuged at 4℃and 10000rpm for 20 minutes, the supernatant was discarded, the pellet was resuspended in 1.5mL binding buffer, 750. Mu.L blocking buffer was added, and the pellet was slowly spun at room temperature for 30 minutes. 40.0. Mu.L of pre-washed streptavidin-coated magnetic beads were resuspended in 376.0. Mu. LPBS, 12.8. Mu.L of Biotin-TEAD4 (5. Mu.g) was added, after 30min of slow rotation at room temperature, the beads were fixed with magnet, the supernatant removed, the beads were washed 3 times with 1.0mL of PBS, and the beads were resuspended in 300.0. Mu. Lbinding buffer and 150.0. Mu. Lblocking buffer and slowly rotated at room temperature for 30 min. Then, phage liquid and bead liquid were mixed, and after 30 minutes of slow rotation at room temperature, the supernatant was discarded on a magnetic rack, and the beads were washed 8 times with 1.0mL of washing buffer, and 2 times with 1.0mL of binding buffer. Then, the beads were resuspended in 100.0. Mu.L of the buffer (pH 2.2), incubated for 5 minutes and placed on a magnetic rack, and the supernatant was transferred to a new centrifuge tube containing 50.0. Mu. L Neutralization buffer (pH 8.0). A small amount of phage was collected for titer determination, and after incubation with TG1 at 37℃for 1 hour, centrifugation was performed for 15 minutes, the supernatant was discarded, the cells were resuspended in 2 XYT, 2 XYT (containing Amp and glucose) plates were plated, inverted overnight at 28℃and scraped off the next day with 2 XYT (containing Amp), and the second, third and fourth rounds of phage preparation and screening were performed according to the procedure described above.

The titer of phage was increased 100-fold over four rounds of screening, indicating enrichment of phage. A third round of phage particle sequencing was randomly selected and a highly enriched polypeptide sequence (sequence GQWPKSFWDFSLGCGGSG) was found. As shown in fig. 21, a fluorescein-modified cyclic peptide 14 (FAM 5 (6) carboxyfluorescein-modified) corresponding to this sequence was synthesized and subjected to polarized fluorescence testing of the target TEAD4, which binds TEAD4 with an affinity of 2.1 μm. These results indicate that the technical scheme in this patent is used for screening the effectiveness of the functional monocyclic peptide.

Example 12:

Phage bicyclic peptide library construction and functional bicyclic peptide ligand screening

1. Construction of pCANTAB 5E-GX ₆CX₆ C phage library

A GX ₆CX₆ C polypeptide library was constructed using pCANTAB 5E phagemid-helper phage (M13 KO 7) (from Sichuan apak Biotechnology Co., ltd.) where X is the random amino acid encoded by NNK, N is A/T/C/G, K is G/T. Nucleic acid libraries were designed based on the characteristics of the cleavage sites Sfi I and Not I on the pCANTAB 5E phagemid vector, and the coding strand and primers were customized from general biosystems (Anhui) Inc. as follows:

library DNA (SfiI and NotI endonuclease recognition sequences in coding strand 2)：5'-GCT ggcccagccggccATG GCC GGC NNK NNK NNK NNK NNK NNK TGC NNK NNK NNK NNK NNK NNK TGC GGC GGC TCT GGC GGC TCT GGC GGC gcggccgc TAAACTAT-3'(K：G/T,N：A/T/C/G; sequences are identified in lowercase);

primer DNA:5'-ATAGTTTAGCGGCCGCGCCGCC-3';

The library DNA and primer DNA were mixed in equal proportions and extended by Klenow DNA polymerase to produce a double stranded DNA library. Double-stranded DNA library and pCANTAB 5E phagemid were treated with Sfi I and Not I to construct recombinant pCANTAB 5E phagemid vector with library DNA, electroporated into TG1 competent cells, plated on 2 XYT (with Amp and glucose) plates for overnight culture, titered to determine library diversity, and phage particles were randomly picked to analyze phage quality. The primary large intestine library was collected from glycerol and stored at-80 ℃. Phage library diversity was 2×10 ⁸ as determined by phage titer.

2. Bicyclic peptide ligand screening for immobilized TEAD4

A suitable amount of stored split-charged phage glycerol bacteria was taken, thawed for several minutes at room temperature and inoculated with 2 XYT liquid medium (containing glucose and Amp). Culturing at 37 deg.c until OD600 reaches 0.3-0.5. Helper phage M13KO7 was added at a multiplicity of infection (MOI) of 20 (20 times that of TG 1) and infected at 37℃for 1 hour. Then, the cells were collected by centrifugation at 10000rpm at 4℃for 15 minutes, the supernatant was discarded, and the cells were resuspended in 2 XYT medium (containing Amp and Kana, without glucose) and cultured with shaking at 28℃for 16 hours. Centrifugation at 4℃and 8000rpm for 20 min, transferring the supernatant phage to a sterile centrifuge bottle, adding a pre-chilled 5 XPEG 8000/NaCl solution, and ice-bathing for 1 hr. Centrifugation at 4℃and 10000rpm for 20 min, discarding the supernatant, re-suspending phage particles in 50-100 mL TBS, centrifugation at 4℃and 10000rpm for 20 min, transferring the supernatant to a new sterile centrifuge bottle, and repeating the PEG/NaCl precipitation process of phage once. Phage bacteria liquid was centrifuged at 4℃and 10000rpm for 20 minutes, the supernatant was discarded, resuspended in TBS and filtered with 0.45 μm sterile filter, and stored at 4℃for several days or glycerol for a long period of-20 ℃.

10 ¹¹ Pfu phage were mixed well in 1.0mL HEPES buffer (50.0mM HEPES,100.0mM NaCl,2.0mM CaCl ₂, 1.0mM TCEP,pH 7.0), 10.0. Mu. L SortaseA (9.0 mg/mL) and 4.0. Mu.L 3, 5-bis [ (2-chloroacetyl) amino ] benzoyl polypeptide 7 (sequence AcNH-Leu-Pro- ^CabLys-Thr-Gly-NH₂, 42.0. Mu.g, final concentration 50.0. Mu.M) were added, and shaking was performed at 37℃for 1 hour at 250 rpm. Then, 0.25mL of a pre-chilled 5 XPEG/NaCl solution was added, mixed well, and then ice-incubated for 30 minutes, centrifuged at 4℃and 10000rpm for 20 minutes, the supernatant was discarded, the pellet was resuspended in 1.5mLbinding buffer, 750. Mu.L of blocking buffer was added, and the pellet was slowly spun at room temperature for 30 minutes. 40. Mu.L of pre-washed streptavidin-coated magnetic beads were resuspended in 376. Mu. LPBS, 12.8. Mu.L of Biotin-TEAD4 (5. Mu.g) was added, after 30 min of slow rotation at room temperature, the beads were fixed with magnet, the supernatant removed, the beads were washed 3 times with 1.0mL PBS, and the beads were resuspended in 300.0. Mu.L of binding buffer and 150.0. Mu.L of blocking buffer and slowly rotated at room temperature for 30 min. Then mixing phage liquid and magnetic bead liquid, slowly rotating for 30 minutes at normal temperature, then discarding the supernatant on a magnetic rack, washing the magnetic beads 8 times with 1.0mL of washing buffer, and washing 2 times with 1.0mLbinding buffer. Then, the beads were resuspended in 100.0. Mu.L of the buffer (pH 2.2), incubated for 5min and placed on a magnetic rack, and the supernatant was transferred to a new centrifuge tube containing 50.0. Mu. LNeutralization buffer (pH 8.0). A small amount of phage was collected for titer determination, and after incubation with TG1 for 1 hour at 37℃and centrifugation at 4℃and 4000rpm for 15 minutes, the supernatant was discarded, the cells were resuspended in 2 XYT, 2 XYT (containing Amp and glucose) plates were applied, inverted overnight at 28℃and scraped off the next day with 2 XYT (containing Amp), and the second, third rounds of phage preparation and screening were performed according to the procedure described above. Note that in the third round of screening we used magnetic beads that did not include the target protein as a control screen.

The titer of phage was increased 10-fold over three rounds of screening, and in particular, it was 135-fold over the third round of control, indicating significant enrichment of phage. A third round of phage particle sequencing was randomly selected and a highly enriched polypeptide sequence (sequence GLTSWVSCHFLRSLCGGSG) was found. As shown in FIG. 22, a fluorescein-modified bicyclic peptide 15 (FITC: fluorescein isothiocyanate modification) corresponding to this sequence was synthesized and subjected to polarized fluorescence testing of the target TEAD4, which bound TEAD4 with an affinity of 63.9nM. These results indicate that the technical scheme in this patent is useful for screening for the effectiveness of functional bicyclic peptides.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method of ligase-catalyzed polypeptide cyclization, the method comprising the steps of:

2. The method of claim 1, wherein the polypeptide templates in S2 are two of:

Template a: g- (X) _m -C; template b: g- (X) _m-C-(X)_y -C;

3. The method of claim 1, wherein the SortaseA enzyme is wild-type or mutant.

4. The method of claim 1, wherein the concentration of the polypeptide compound in S2 is in the range of 0.1 μm to 10.0mM and the concentration of the sortasea enzyme is in the range of 0.1 μm to 10.0mM.

5. The method of claim 1, wherein the buffer salt solution in S2 is a common buffer solution other than phosphate, and comprises any one of HEPES (4-hydroxyethyl piperazine ethane sulfonic acid), naOAc (sodium acetate) and Tris (Tris hydroxymethyl amino methane), wherein CaCl ₂ is 0.1-mM mM, and TCEP (Tris (2-carboxyethyl) phosphine) is 0.1 μm-10.0 mM, and the ph range is 6.0-9.0.

6. The method for cyclizing a polypeptide catalyzed by a ligase according to claim 1, wherein the time of the polypeptide linking and the cyclizing reaction of the polypeptide side chain in the S2 is 15 minutes to 6 hours, and the reaction temperature is 20 ℃ to 45 ℃.

7. The method of claim 1, wherein the phage in S3 comprises a phage system consisting of a pcatab 5E phagemid and a helper phage M13KO7 or an M13KE phage system.

8. The method of claim 1, wherein the step of screening the target protein for a cyclic peptide ligand in S3 comprises the steps of:

9. Use of ligase catalysed polypeptide circularisation according to claims 1 to 8 wherein the polypeptide circularisation is applied in the construction of a gene encoding cyclic peptide library comprising polypeptide ligation and side chain circularisation of a phage displayed polypeptide library to construct phage displayed single and double ring peptide libraries.

10. The use of a ligase catalyzed cyclization of polypeptides according to claim 9 wherein the cyclic peptide ligands obtained from the ligase catalyzed cyclization of polypeptides are useful in the development of pharmaceuticals, test kits or other biomedical and biological materials.