Phage cyclopeptide library with biphenyl-thiazole linker bond and screening method

Abstract

The invention discloses a phage cyclopeptide library with a biphenyl-thiazole linker bond and a screening method, and relates to a polypeptide cyclization method and a strategy for discovering a protein high-affinity cyclopeptide ligand. The preparation method comprises the following steps: firstly, reacting synthesized new alkene sulfide molecules with linear polypeptide of which the nitrogen terminal and the chain are cysteine to synthesize cyclic peptide; based on the rigid biphenyl structure of the molecule in structure and the high biocompatibility of the reaction, a phage cyclopeptide library of a biphenyl-thiazoline connecting bond is obtained through a post-translational modification reaction, a high-affinity cyclopeptide ligand is screened out for target protein, and more possibilities are provided for the discovery and development of cyclopeptide compounds in drugs.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method for polypeptide cyclization of small molecule compounds, and in particular to a phage library of cyclic peptides with a biphenyl-dihydrothiazole linkage and a screening method. BACKGROUND

[0002] In recent years, with the continuous maturation of polypeptide synthesis technology, and the discovery and application of many high-activity and high-in-vivo-safety polypeptide drugs, the research and development of polypeptide drugs has become one of the hotspots of disease treatment today (Nature reviews Drug discovery 20.4 (2021): 309-325.; Bioorganic & medicinal chemistry, 2018, 26(10): 2700-2707.). The size of polypeptide is between small molecule compounds and biological macromolecular proteins, which has the advantages of high affinity, high biocompatibility, low cost and easy modification, and is an ideal drug for PPIs (Drug Discovery Today, 2021, 26(6): 1521-1531.; Comptes Rendus Chimie, 2016, 19, 19-27.). Therefore, polypeptide drugs are indispensable in new drug development. However, the development and application of natural polypeptides in nature is limited, so the targeted design and development of polypeptides is particularly important (Peptide Science, 2018, 111(1): e24058.; Current Opinion in Chemical Biology, 2017, 38, 52-61.; Journal of the American Chemical Society, 145.3 (2023): 1964-1972.; Nature, 616(7957), pp. 581-589.). Among them, designing and utilizing some small molecule coupling reactions to synthesize cyclic peptides is also an important means to improve the activity and bioavailability of polypeptides (Nature Chemistry, 2011, 3(7): 509-524.; Journal of the American Chemical Society, 141.31 (2019): 12274-12279.; Organic Letters, 2019, 21(12): 4709-4712.; Journal of the American Chemical Society, 1996, 118(42): 10018-10024.; Angewandte Chemie (International Ed in English), 2019, 58(52): 19073-19080.; Bioconjugate Chemistry, 2021, 32(9): 2065-2072.).And get a lot of constraints non-natural macrocyclic peptide or polycyclic peptide not only with the target protein binding entropy consumption is lower (Nature Reviews Drug Discovery, 2008, 7(7): 608-624.), relative to natural disulfide peptide can exist in a reducing environment. These advantages make the polypeptide drug selectivity and in vivo metabolic stability greatly improved (Chemical Reviews, 2010, 110(6): 1-31.), also greatly for the development of polypeptide drugs to expand the space (Journal of Medicinal Chemistry, 2014, 57(2): 278-295.; Current opinion in chemical biology, 38(2017): 24-29.).

[0003] Phage display technology provides a powerful platform for polypeptide drug development (Chemical reviews, 97.2 (1997): 391-410.), and many active polypeptides can be screened using phage display polypeptide library (Nano Letter, 2019, 19: 1467-1478.; Protein Cell, 2019, 10: 787-807.; Angewandte Chemie, 124.8 (2012): 1926-1930.), which also provides more possibilities for the screening of pre-disease drugs of protein-protein interaction mechanism (Chemical Reviews, 2014, 114(9): 4695-748). It is a common strategy to construct phage unnatural cyclic peptide library by chemical modification method and apply it to screening to find active ligands of proteins. Although polypeptides can be constructed into cyclic peptides by amide condensation, click reaction, metal-catalyzed reaction and halogen compound nucleophilic substitution reaction, etc., but in numerous research attempts, the linker molecules with high biocompatibility that can really be constructed into phage cyclic peptide library are few (Nature Chemical Biology, 2009, 5(7): 502-507.; ACS Chemical Biology, 2016, 11(5): 1422-1427.; Chemical Science, 2016, 7(6): 3785-3790.; Chemical Science, 13(28), 8349-8354.; Chemical reviews, 124(9), 6051-6077.). The cyclization molecules with high specific reaction sites are rare. Different structural types of linker molecules often appear in the process of constructing phage cyclic peptide library and screening target proteins, that is, it is difficult to match the target protein with the cyclization effect, or the cyclic peptides with various linkages have great differences in affinity with the target protein. These visible differences are essentially caused by the conformational changes of cyclic peptides with different types of linker molecules, so by introducing effective chemical characteristic groups into the phage library through chemical modification method, the application value of new cyclic peptides with characteristic linkages can be greatly improved, and more possibilities can be provided for the ligand discovery of many disease-related proteins.

[0004] On the other hand, C.L. Wu et al. previously developed an efficient condensation reaction of 1,2-aminothiol with 2-((alkylthio)(aryl)methyl)malononitrile (TAMM) and 1,2-aminoethanethiol, which can generate stable 2-aryl-4,5-dihydrothiazole (ADT) linkage products, and use this highly biocompatible small molecule to construct phage cyclic peptide libraries through post-translational modification (Journal of the American Chemical Society, 142(11), 5097-5103.), which also lays the foundation for the development of protein high-affinity ligands. Based on the reaction advantages of this molecule, modification can obtain cyclic peptide molecules or phage cyclic peptide libraries with characteristic linkages, which provides more possibilities for discovering new cyclic peptide ligands and greatly improves the potential application value of cyclic peptide compounds in disease treatment. SUMMARY

[0005] The first object of the present application is a new rigid long-chain alkenyl sulfide compound.

[0006] The second object of the present application is to provide a method for constructing a phage cyclic peptide library with a novel linkage by post-translational modification using the above-mentioned alkenyl sulfide compound, providing an effective means or strategy for discovering more active cyclic peptide compounds.

[0007] The third object of the present application is to provide a phage cyclic peptide library with a biphenyl-dihydrothiazole linkage constructed using the above-mentioned alkenyl sulfide compound.

[0008] The fourth object of the present application is to provide a method for efficiently synthesizing and screening cyclic peptide ligands or drugs with high affinity to target proteins using the above-mentioned phage cyclic peptide library with a biphenyl-dihydrothiazole linkage, for drug discovery and development. To achieve the above-mentioned objects, the technical solutions of the present application are as follows:

[0009] The present application provides a new rigid long-chain alkenyl sulfide compound, which has the following structure:

[0010]

[0011] The compound has a rigid biphenyl structure, which can improve the stability of the cyclic peptide.

[0012] The synthesis route of the rigid long-chain alkenyl sulfide compound is as follows:

[0013]

[0014] The preparation method of the rigid long-chain alkenyl sulfide compound comprises the following steps:

[0015] 1) using dimethyl diphenyldicarboxylate as raw material, hydrolysis reaction is carried out with sodium hydroxide solution to obtain monomethyl ester biphenyl carboxyl compound;

[0016] 2) monomethyl ester biphenyl carboxyl compound is chlorinated with chlorosulfoxide under the catalysis of N,N-dimethylformamide to form biphenyl chloroform intermediate;

[0017] 3) biphenyl chloroform intermediate is reacted with sodium hydride and malononitrile to form enol compound under the action of sodium hydride;

[0018] 4) enol compound is chlorinated with phosphorus pentachloride to form chloroalkene intermediate;

[0019] 5) chloroalkene intermediate is reacted with sodium bicarbonate and ethanethiol under the action of sodium bicarbonate to form enethioether compound;

[0020] 6) enethioether compound is activated with N-hydroxysuccinimide under the action of carbodiimide hydrochloride to form active ester intermediate;

[0021] 7) active ester intermediate is condensed with chloro amino chain to form ethanethiol substituted enethioether compound;

[0022] 8) ethanethiol substituted enethioether compound is reacted with N-acetyl cysteine under the action of sodium bicarbonate to form the rigid long chain enethioether compound.

[0023] The application provides application of the novel rigid long chain enethioether compound in cyclization polypeptide.

[0024] The application is specifically: cyclization reaction of the novel rigid long chain enethioether compound with linear polypeptide CPARYGWEYEC containing two cysteines under the condition of pH = 7.4; the linear polypeptide is amino at the nitrogen end and is amide at the carbon end; the cyclization polypeptide with biphenyl-dihydrothiazole structure is synthesized by reacting with the linear polypeptide at the nitrogen end and the cysteine in the chain; the reaction condition is mild, the reaction efficiency is high, the product is stable and easy to synthesize.

[0025] The application provides a construction method of a phage cyclization polypeptide library with biphenyl-dihydrothiazole connection, which utilizes the above-mentioned enethioether compound, and the specific steps are as follows:

[0026] Based on high biocompatibility of polypeptide cyclization reaction, the new enethioether molecule is reacted with a polypeptide phage library with multiple skeletons under physiological conditions to construct a phage display macrocyclic polypeptide library.

[0027] 1) constructing CX nC phage polypeptide library, from N-terminal to C-terminal, X represents random amino acids, n random amino acids are introduced by NNK method;

[0028] The CX n C phage polypeptide library skeleton is C(X) n C, the amino acid is an L-type amino acid, X represents a random amino acid, and the subscript n is the number of random amino acids, n = 5-20;

[0029] In a preferred embodiment, n = 10, 11, 12, 13, 14, 15, a total of 6 skeleton groups are selected to form CX 10-15 C phage polypeptide library (Liu Ziyanyan, Construction of chemically modified phage cyclic peptide library and ligand screening[D]. Xiamen: Xiamen University, College of Chemistry and Chemical Engineering, 2022), CX 10-15 The library capacity of the C phage polypeptide library is approximately 2.6*10 8 .

[0030] 2) Use rigid long-chain alkenyl sulfide compounds to modify CX in the cyclization step 1) n C phage polypeptide library, the polypeptide is cyclized to form a macrocyclic polypeptide structure, and the modified and cyclized phage polypeptide library is Library 1, that is, the phage cyclic peptide library with a biphenyl-dihydrothiazole connecting bond.

[0031] The application provides a phage cyclic peptide library with a biphenyl-dihydrothiazole connecting bond.

[0032] The phage cyclic peptide library with a biphenyl-dihydrothiazole connecting bond has the following characteristics:

[0033] A new alkenyl sulfide molecule is synthesized, and a phage polypeptide library with a nitrogen terminal and a cysteine in the chain is obtained through a cyclization reaction; the coupling reaction of the alkenyl sulfide molecule and the phage polypeptide library makes the phage polypeptide library have a structure of a biphenyl-dihydrothiazole connecting bond.

[0034] The application provides a use of the phage cyclic peptide library with a biphenyl-dihydrothiazole connecting bond, and the use is to synthesize and screen a cyclic peptide ligand with high affinity to a target protein.

[0035] 1) The constructed phage cyclic peptide library with a biphenyl-dihydrothiazole connecting bond (Library 1) is used for screening, BCL-X L Protein is used as a target, and three rounds of in-vitro screening are performed;

[0036] 2) After three rounds of screening, high-enrichment phages are obtained, and the enriched monoclonal colonies are picked and sequenced, and the BCL-X LThe linear polypeptides sequenced by protein screening include but are not limited to the following sequences as shown in the sequence table SEQ ID NO. 1-9:

[0037] CRPSWSNTLRPEC (peptide 1)

[0038] CESSVGRATRLYEELC (peptide 2)

[0039] CLENRRETVSQLYNSYC (peptide 3)

[0040] CPSPPTPADLYDEYC (peptide 4)

[0041] CAVSARSLYEIYC (peptide 5)

[0042] CNNEGQRLYEEYC (peptide 6)

[0043] CSETVGDLYQKYC (peptide 7)

[0044] CNATASVYNELFASC (peptide 8)

[0045] CQFDETIPELRNAC (peptide 9)

[0046] Wherein, C is cysteine, R is arginine; P is proline; S is serine; W is tryptophan; N is asparagine; T is threonine; E is glutamic acid; V is valine; G is glycine; A is alanine; L is leucine; Y is tyrosine; I is isoleucine; F is phenylalanine; K is lysine; Q is glutamine; D is aspartic acid; M is methionine; H is histidine.

[0047] 3) The sequence peptide 1 with the highest enrichment degree in step 2) is selected for synthesis, and a rigid long-chain alkenyl sulfide compound is used for cyclization reaction to obtain a cyclic peptide ligand, and the affinity of the cyclic peptide ligand with the target protein BCL-X L is characterized by surface plasmon resonance (SPR).

[0048] The application provides a secondary library Library 2 constructed based on Library 1 for BCL-X L , and the construction method of the Library 2 comprises the following steps:

[0049] 1) The peptide 1 is measured to have an affinity with BCL-X LThe protein has good affinity. Taking peptide 1 as a template, two phage libraries of phage display polypeptide skeletons with conserved sequences are designed, and the library capacity is approximately 2.4*10 8 The two phage display polypeptide skeletons are C(X)6TLRPEC (skeleton 1) and CRPSWS(X)6C (skeleton 2); wherein X represents a random amino acid, and the subscript 6 represents the number of random amino acids.

[0050] 2) The method for synthesizing and screening a cyclic peptide ligand with high affinity to the target protein is used, and the phage library of the two phage display polypeptide skeletons is modified by an alkene sulfide molecule to obtain a phage display cyclic peptide library Library 2.

[0051] The application provides a use of the phage display cyclic peptide library Library 2. The constructed phage macrocyclic polypeptide library Library 2 is used for screening, BCL-X L Protein as a target, two rounds of in-vitro screening are performed, and high enrichment phages are obtained. The enriched single clone colonies are picked and sequenced, and the sequenced linear polypeptide results screened according to BCL-X L Protein include but are not limited to the following sequences, as shown in the sequence table SEQ ID NO. 10-30:

[0052] CTSEWSQTLRPEC (peptide 10)

[0053] CTKEWDETLRPEC (peptide 11)

[0054] CTGEWEATLRPEC (peptide 12)

[0055] CTPEWASTLRPEC (peptide 13)

[0056] CTPEWALTLRPEC (peptide 14)

[0057] CTNEWEGTLRPEC (peptide 15)

[0058] CEHDWEETLRPEC (peptide 16)

[0059] CRDEWVITLRPEC (peptide 17)

[0060] CNAEWSATLRPEC (peptide 18)

[0061] CHVEWASTLRPEC (peptide 19)

[0062] CHPEWASTLRPEC (peptide 20)

[0063] CHPDWDETLRPEC (peptide 21)

[0064] CHEQWEDTLRPEC (peptide 22)

[0065] CLPEWDVTLRPEC (peptide 23)

[0066] CLETWEETLRPEC (peptide 24)

[0067] CLPEWEGTLRPEC (peptide 25)

[0068] CVPEWEDTLRPEC (peptide 26)

[0069] CVPEWAQTLRPEC (peptide 27)

[0070] CRPSWSESLRPEC (peptide 28)

[0071] CRPSWSSTLLPEC (peptide 29)

[0072] CRPSWSCREYLSC (peptide 30)

[0073] The above sequence analysis, select the ranking 4th peptide 13 synthesis, and with alkenyl sulfide molecule cyclization reaction to obtain cyclic peptide ligand (the sequence of the resulting cyclic peptide ligand is CTPEWASTLRPEC, consistent with the original linear polypeptide sequence), and the target protein BCL-X L Ligand affinity was characterized by surface plasmon resonance (SPR).

[0074] The cyclic peptide structure having the same structure as the phage cyclic peptide library is obtained by different libraries such as mRNA display cyclic peptide library, DNA encoded cyclic peptide library and the like.

[0075] The present application utilizes the synthesis of new alkenyl sulfide molecules to construct phage display macrocyclic polypeptide library for protein screening high affinity macrocyclic peptide ligand, the synthesized new alkenyl sulfide compound has a rigid long chain structure and hydrophobic property, and the phage polypeptide library of different polypeptide chain length is modified, so that the small molecule modified multi-chain length phage library can screen more novel structure, biocompatible macrocyclic peptide ligand.

[0076] Compared with the prior art, the present application has the following outstanding advantages and beneficial effects:

[0077] a.The synthetic target olefinsulfide compound described in the application is a new structure, which is applied to cyclization of polypeptides and construction of cyclization phage polypeptide library for the first time, the reaction condition is mild, and the reaction efficiency is high. Modification of phage library with different polypeptide chain lengths greatly improves the structural diversity of phage library, and new cyclic peptide ligand structures can be selected. The reaction product is stable and easy to synthesize. Through the application, more valuable cyclic peptides can be developed.

[0078] b.The Library 1 with BCL-X L Protein as the target screens the active sequence peptide 1, and Library 2 is constructed according to peptide 1 to optimize the higher affinity cyclic peptide ligand. The Library 2 can not only improve the affinity of the cyclic peptide ligand, but also stably screen and find the BCL-X L Ligand series of cyclic peptides. BRIEF DESCRIPTION OF DRAWINGS

[0079] Figure 1 It is a monitoring diagram of the cyclization reaction of the new olefinsulfide molecule and the polypeptide with cysteine at both ends. Wherein, A is the chromatographic detection diagram of the cyclization reaction, and B is the mass spectrum diagram of the cyclic peptide product generated by the cyclization reaction;

[0080] Figure 2 It is a schematic diagram of modification of phage macrocyclic polypeptide library;

[0081] Figure 3 It is a surface plasmon resonance (SPR) curve for characterization of the affinity of the peptide 1 cyclic peptide. Wherein, A is the SPR sensing diagram of BCL-X L Protein and peptide 1 cyclic peptide, B is the affinity fitting curve diagram of BCL-X L Protein and peptide 1 cyclic peptide;

[0082] Figure 4 It is a sequence analysis of the conservative sequence of the Library 2 screening BCL-X L Protein; wherein, A is a sequence analysis of the conservative sequence of the skeleton 1, and B is a sequence analysis of the conservative sequence of the skeleton 2;

[0083] Figure 5 It is a surface plasmon resonance (SPR) curve for characterization of the affinity of the peptide 13 cyclic peptide and a kinetic fitting curve diagram. DETAILED DESCRIPTION

[0084] In order to make the embodiments of the application clearer, the application is further described in detail as follows in combination with the drawings and examples.

[0085] Example 1

[0086] The synthesis of the new thioether compound designed in the embodiments of the present application comprises the following steps:

[0087] Step 1, synthesis of compound 4-[4-(Methoxycarbonyl)-2-Methylphenyl]benzoic acid

[0088]

[0089] Put 2 g (7.4 mmol, 1 eq.) of dimethyl biphenyl dicarboxylate in a round bottom flask, add 100 ml of dichloromethane (DCM) to dissolve, then add 0.33 g (8.1 mmol, 1.1 eq.) of sodium hydroxide solution dissolved in 10 ml of methanol, and stir the system at room temperature overnight. After the reaction is completed, white precipitate is precipitated from the reaction solution, which is centrifuged at high speed, and the supernatant is removed. After the solvent is volatilized, the white solid obtained is added to pure water to disperse the solid uniformly, and concentrated hydrochloric acid is added to adjust the pH to 1-2. After high-speed centrifugation to remove the supernatant, 50 ml of pure water is added to wash the lower solid, and the supernatant is removed after centrifugation. Repeat three times to obtain a solid which is freeze-dried to obtain the product white solid 0.6 g, with a yield of 32%.

[0090] 1 H NMR (500 MHz, DMSO-d6) δ 13.04 (s, 1H), 8.06 (t, J = 8.5 Hz, 4H), 7.89 (dd, J = 11.8, 8.0 Hz, 4H), 3.89 (s, 3H).

[0091] 13 C NMR (126 MHz, DMSO-d6) δ 167.49, 166.42, 143.97, 143.30, 131.10, 130.49, 130.33, 129.63, 127.76, 127.62, 52.68.

[0092] Step 2, synthesis of compound methyl 4'-(chlorocarbonyl)-[1,1'-biphenyl]-4-carboxylate

[0093]

[0094] The white solid product above was weighed 0.6 g (2.3 mmol, 1 eq.) into a round bottom flask, 10 ml of dichloromethane solvent was added, and 2 ml of excess thionyl chloride (SOCl2) chlorinating agent was added, and after adding two drops of N,N-dimethylformamide (DMF), the reaction was stirred at 50°C under nitrogen protection for 24 h. After the reaction was completed, the reaction was cooled to room temperature, and the solvent in the reaction system was removed by distillation under reduced pressure, dichloromethane solvent was added repeatedly, and the residual thionyl chloride in the system was removed by distillation under reduced pressure, dichloromethane (5 x 10 ml) was added to disperse the solid and spin dry, and the yellow-brown acyl chloride product intermediate 0.5 g was obtained with a pungent odor, with a yield of 79%.

[0095] Step 3, synthesis of compound methyl 4'-(2,2-dicyano-1-hydroxyvinyl)-[1,1'-biphenyl]-4-carboxylate

[0096]

[0097] (1) Sodium hydride (0.18 g, 7.2 mmol, 4 eq.) was placed in a round bottom flask, 10 ml of anhydrous tetrahydrofuran (10 ml) was added to disperse, and it was stirred vigorously under nitrogen atmosphere protection at 0°C ice bath. Malononitrile (0.24 g, 3.6 mmol, 2 eq.) was dissolved in 5 mL of anhydrous tetrahydrofuran, and slowly added to the above system. After the addition was completed, the reaction was stirred at 0°C ice bath for 1 h.

[0098] (2) The acyl chloride intermediate (0.5 g, 1.8 mmol, 1 eq.) obtained in step 2) was dissolved in anhydrous tetrahydrofuran (10 mL). While maintaining the 0°C ice bath, the solution was slowly added to the system. After the addition was completed, the reaction system was transferred to room temperature, and it was slowly warmed to room temperature, and the reaction was stirred at room temperature for 1 h.

[0099] (3) The tetrahydrofuran solvent in the system was removed by distillation under reduced pressure. Ice water was added to the residue under magnetic stirring, and after the solid was dispersed, concentrated hydrochloric acid was added to adjust the pH to 1-2. Ethyl acetate (3 x 30 mL) was added for extraction, the organic phase was combined and washed with saturated brine (3 x 10 mL), dried with anhydrous sodium sulfate, and then concentrated to obtain a brown solid 0.42 g with a yield of 76%.

[0100] Step 4, synthesis of compound 4'-(2,2-dicyano-1-hydroxyvinyl)-[1,1'-biphenyl]-4-carboxylic acid

[0101]

[0102] The compound obtained from step 3 reaction (0.2 g, 0.66 mmol, 1 eq.) was dissolved in tetrahydrofuran (10 mL), sodium hydroxide (0.05 g, 1.32 mmol, 2 eq.) dissolved in water (10 mL) was added, the ice bath was removed and the reaction was allowed to warm to room temperature and stirred for 4 h. The tetrahydrofuran solvent in the system was removed by distillation under reduced pressure, 20 mL of water was added, and then washed with ethyl acetate (3 x 10 mL). Hydrochloric acid (1 mol / L) was added to adjust the pH to 1-2, and then extracted with ethyl acetate (3 x 20 mL), the organic phase was combined and washed with saturated brine (3 x 10 mL), dried over anhydrous sodium sulfate, and concentrated to obtain a brown solid (0.16 g) with a yield of 83%.

[0103] ESI-MS (m / z): calcd for C 17 H 10 N2O3[M-H] - : 289.0613; found: 289.20.

[0104] 1 H NMR (500 MHz, DMSO-d6) δ 13.06 (s, 1H), 8.07 (d, J = 8.2 Hz, 2H), 8.00 (d, J = 8.3 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.3 Hz, 2H), 2.81 (q, J = 7.4 Hz, 2H), 1.09 (t, J = 7.4 Hz, 3H).

[0105] Step 5, synthesis of compound 4'-(2,2-dicyano-1-(ethylthio)vinyl)-[1,1'-biphenyl]-4-carboxylic acid

[0106]

[0107] The specific steps are as follows:

[0108] (1) The compound obtained from step 4 reaction (0.1 g, 0.34 mmol, 1 eq.) was dissolved in 20 mL of anhydrous acetonitrile, and phosphorus pentachloride (0.6 g, 3.0 mmol) was added. The reaction was stirred at 50°C under a nitrogen atmosphere for 6 h. After the reaction was completed and cooled to room temperature, the solvent was removed by distillation under reduced pressure. The residue was dissolved in 30 mL of ethyl acetate, washed with water (3 x 10 mL) and saturated brine (2 x 10 mL) in sequence, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a brown solid.

[0109] (2) The brown solid obtained above was dissolved in 10 mL of acetonitrile and water mixture (acetonitrile: water = 1 : 1) and stirred at room temperature for half an hour. Ethane thiol (36 μL, 0.68 mmol, 2 eq.) was added to the solution, which was immediately made alkaline with sodium bicarbonate. The reaction was stirred at room temperature for 2-4 h, and monitored by high performance liquid chromatography. After the reaction was completed, the pH was adjusted to 4-5 with hydrochloric acid (1 mol / L). The acetonitrile solvent was removed by distillation under reduced pressure, and 15 mL of water was added to disperse the residual solid. The crude product was then extracted with ethyl acetate (3 x 20 mL), and the organic phase was washed with saturated brine (3 x 10 mL), dried over anhydrous sodium sulfate, and concentrated by distillation under reduced pressure to obtain 50 mg of brown solid, with a yield of 44%.

[0110] ESI-MS (m / z): calcd for C 19 H 14 N2O2S[M-H] - : 333.0698; found: 333.20.

[0111] Step 6, synthesis of compound 2,5-dioxopyrrolidin-1-yl 4'-(2,2-dicyano-1- (ethylthio)vinyl)-[1,1'-biphenyl]-4-carboxylate

[0112]

[0113] The compound obtained in Step 5 (50 mg, 0.15 mmol, 1 eq.) was dissolved in 10 mL of acetonitrile (ACN), and N-hydroxysuccinimide (34.5 mg, 0.30 mmol, 2 eq.) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (58.0 mg, 0.30 mmol, 2 eq.) were added sequentially. The reaction was stirred at room temperature for 2-4 h, and monitored by high performance liquid chromatography. After the reaction was completed, 30 mL of dichloromethane was added, and the mixture was washed sequentially with water (3 x 10 mL) and saturated brine (3 x 10 mL), dried over anhydrous sodium sulfate, and concentrated by distillation under reduced pressure to obtain 55 mg of brown solid, with a yield of 85%.

[0114] Step 7, synthesis of a new alkenyl sulfide molecule designed in the present application

[0115]

[0116] The compound 55 mg (0.13 mmol, 1 eq.) obtained from step 6 was dissolved in 10 mL acetonitrile, and then the halogenated amide N-(2-aminoethyl)-2-chloroacetamide (35.4 mg, 0.26 mmol, 2 eq.) dissolved in 10 mL phosphate buffer (pH = 7.4, 0.1 M) straight-chain alkane diamine was added, and stirred at room temperature overnight. After monitoring the consumption of the raw material by high performance liquid chromatography, N-acetyl-L-cysteine (NAC, 84.8 mg, 0.52 mmol, 4 eq.) was added to the system, and then the system was adjusted to be alkaline with sodium bicarbonate, and stirred at room temperature for 2 h. The progress of the reaction was monitored by high performance liquid chromatography, and after the reaction was completed, purified by semi-preparative high performance liquid chromatography, and freeze-dried to obtain the final product as a light yellow solid.

[0117] MALDI TOF-MS (m / z): calcd for C 26 H 24 ClN5O5S[M+H] + : 554.1265; found: 554.621.

[0118] 1 H NMR (850 MHz, DMSO-d6) δ 13.10 (s, 1H), 8.64 (s, 1H), 8.41-8.35 (m, 2H), 8.02 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 8.2 Hz, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 8.1 Hz, 2H), 4.37 (td, J = 7.7, 5.3 Hz, 1H), 4.08 (s, 2H), 3.33-3.29 (m, 4H), 3.14-3.11 (m, 2H), 1.87 (s, 3H).

[0119] 13 C NMR (214 MHz, DMSO-d6) δ 181.53, 171.28, 169.88, 166.69, 166.39, 143.13, 141.26, 134.68, 132.04, 130.09, 128.52, 128.07, 127.30, 113.65, 113.26, 79.41, 51.56, 43.15, 39.36, 39.25, 36.33, 22.80.

[0120] Example 2

[0121] Polypeptide cyclization reaction: under the condition of pH = 7.4, the rigid long-chain alkenyl sulfide molecule can be cyclized with the linear polypeptide CPARYGWEYEC containing two cysteines. Since the nitrogen end of the polypeptide is amino and the carbon end is amide, this cyclization reaction is carried out by forming a connecting bond through the alkenyl sulfide molecule. The reaction route is as follows:

[0122]

[0123] Take 50 μL of the stock solution of linear polypeptide CPARYGWEYEC (500 μM) and add it to a centrifuge tube containing 340 μL of phosphate buffer solution (PB, 100 mmol / L, pH = 7.4). Then, add 50 μL of aqueous solution of reducing agent tris (2-carboxyethyl) phosphine salt (TCEP, 1 mM) and 50 μL of aqueous solution of N-acetyl-L-cysteine (NAC, 1 mM) to the system, so that the final concentration of polypeptide is 50 μM (1 eq.), the final concentration of TCEP and NAC is 200 μM (4 eq.), and the final concentration of the rigid long-chain alkenyl sulfide molecule described in the present application is 100 mM (2 eq.). The total system is 500 μL, which is vortexed uniformly and then placed in a 37 °C constant temperature shaker. The reaction is monitored by high performance liquid chromatography (HPLC) (Figure A in Figure 1 ), and after 3 h of reaction, the polypeptide is completely converted to the cyclic peptide product cyclicpeptide, with a polypeptide cyclization rate of 95%. Mass spectrometry detection of the cyclization product is shown in Figure B in Figure 1 , and the structural formula is as follows:

[0124]

[0125] Example 3

[0126] Construction of a phage-displayed macrocyclic polypeptide library Library 1 and screening of BCL-X L Cyclic peptide ligand

[0127] (1) Construction of a phage-displayed macrocyclic polypeptide library Library 1:

[0128] a. Construction of a phage library: insert double-stranded DNA of random 10-15 peptides (C(X) n C) into the sfi I and Not I enzyme digestion sites of the phagemid vector pCantab5E, and electrotransform it into the host bacteria. Calculate the library capacity by determining the number of transformed bacteria.

[0129] b. Modification of phage library: add tri (2 carboxyethyl) phosphine salt solution with final concentration of 1 mM to the phage library described in step a, vortex well, and then place in a 37 °C constant temperature shaker for 30 min. Then add N-acetyl-L-cysteine (NAC) with final concentration of 1.5 mM and alkenyl sulfide molecule solution with final concentration of 0.5 mM to the system, and the reaction system pH = 7.4 is placed in a 37 °C constant temperature shaker for 3 h (see the modification of phage library process in Figure 2 ). After the reaction is completed, add PEG / NaCl to the above reaction solution and place in an ice bath for 30 min, and then high-speed centrifuge for 10 min after white precipitate is precipitated. Discard the supernatant, resuspend the precipitate with phosphate buffer physiological saline (1 × PBS), and then obtain the phage library Library 1, which is stored at 4 °C for screening.

[0130] (2) Library 1 screening against target protein BCL-X L :

[0131] Inoculate E. coli TG1 in 5 ml 2YT medium at 37 °C to the logarithmic growth phase for subsequent titer determination after screening. Add 50 μL of streptavidin-coated magnetic beads (or neutral avidin-coated magnetic beads for the second round of screening) to a 1.5 mL low adsorption centrifuge tube, and then place the centrifuge tube in a magnetic stand, wash twice with 1 mL of binding buffer, and add 100 μL of binding buffer to disperse the magnetic beads. Add 5 μg (2 μg for the second round, and 2 μg for the third round) of biotinylated BCL-X L protein to one of the centrifuge tubes as the experimental group, and add the same volume of 1 × PBS to the other centrifuge tube as the control group. Incubate at room temperature on a 3D shaker for 10 min, then centrifuge and place in a magnetic stand to remove the supernatant, and then wash the magnetic beads three times with 1 mL of binding buffer. Then add 1 mL of blocking buffer to resuspend the magnetic beads, and at the same time, block the phage library Library 1 (titer ≈ 10 12), and incubated on a shaker at room temperature for 2 h for blocking. After the completion of blocking, the phage library Library 1 was divided into two equal parts, which were denoted as the experimental group and the control group. The magnetic beads of the experimental group and the control group were added to the corresponding labeled phage library Library 1, respectively, and mixed well, and then incubated on a shaker at room temperature for 30 min. After the incubation, the supernatant was removed, the magnetic beads were washed with the washing buffer for 9 times, and then washed with the binding buffer for 2 times. During the washing process, the low adsorption centrifuge tube was replaced for 3 times to reduce the non-specific adsorption with the tube wall. Finally, the magnetic beads were resuspended with 200 μL of the elution buffer at room temperature, incubated for 5 min, and then the supernatant was transferred to a new centrifuge tube added with 35 μL of the neutralization buffer for neutralization. After the elution was repeated twice, the eluate was collected for subsequent phage titer determination and amplification. After 3 rounds of screening, the phage monoclonal was randomly selected for sequencing. The sequencing results are shown in Table 1 (in the table, BX-1 to BX-9 correspond to peptide 1 to peptide 9 in the specification, respectively).

[0132] Table 1

[0133]

[0134] Example 4

[0135] Screening and enrichment of sequence loop peptide synthesis and affinity characterization of the target protein of Library 1

[0136] (1) The peptide 1 with the most enriched sequences was selected for synthesis. The linear polypeptide was synthesized by solid-phase polypeptide synthesis, and then was cyclized according to the cyclization method to obtain the loop peptide.

[0137] (2) The affinity of the loop peptide was characterized by surface plasmon resonance SPR (Biacore T200). The biotinylated Bcl-xl protein was diluted to 1 μM, and the protein was immobilized on the streptavidin on the chip surface by manual operation. Different concentrations of polypeptides (loop peptide peptide 1: 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, 800 nM) were used in single cycle / multiple cycle mode to obtain the corresponding sensorgram (FIG. A in L Figure 3 , and the affinity fitting curve (FIG. B in Figure 3 ) obtained the affinity of the loop peptide peptide 1: K D = 71 nM.

[0138] Example 5

[0139] Optimization of template for constructing phage display macrocyclic polypeptide library Library 2 and screening BCL-X L loop peptide ligand ​

[0140] (1) Design two phage display libraries CRPSWS(X)6C and C(X)6TLRPEC containing the screened peptide 1: CRPSWSNTLRPEC as a template

[0141] a. Construction of phage display macrocyclic peptide library: The double-stranded DNA of the random 6-peptide above is inserted into the phagemid vector pCantab5E between the sfi I and Not I enzyme sites, and electroporated into the host bacteria. The library capacity is calculated by determining the number of transformed bacteria.

[0142] b. Modification of the phage library: The modification conditions are the same as those for obtaining Library 1, and the phage library Library 2 is obtained and stored at 4°C for screening.

[0143] (2) Library 2 screening against target protein BCL-X L Screening: The screening operation is referred to the screening process of Library 1. After 2 rounds of screening, high-enrichment phage is obtained, and phage monoclonal is randomly selected for sequencing. The sequencing results are shown in Table 2 (B6X-1 to B6X-21 in the table correspond to peptide 10 to peptide 30 in the specification, respectively).

[0144] Table 2

[0145]

[0146] Example 6

[0147] Synthesis of the sequence-enriched cyclic peptide obtained by screening of Library 2 and characterization of the affinity of the cyclic peptide to the target protein

[0148] (1) Synthesis of the sequence-enriched cyclic peptide obtained by screening of Library 2. After analysis, it is found that the sequencing results (Table 2) are not enriched in one sequence. After sequence conservative amino acid analysis ( Figure 4 ), the 4th ranked peptide 13 with high conservative amino acid is selected. The linear polypeptide is synthesized by solid-phase polypeptide synthesis, and then the cyclic peptide is obtained by the cyclization method.

[0149] (2) The affinity of the cyclic peptide is characterized by surface plasmon resonance SPR (Biacore T200). The biotinylated BCL-X L protein is diluted to 1 μM, and the protein is immobilized on the chip surface by manual operation of the system using streptavidin. Different concentrations of polypeptides (cyclic peptide peptide 1: 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, 800 nM) are used in single cycle / multiple cycle mode to bind BCL-X LProtein binding gives the corresponding curve Figure 5 ), affinity fitting curve gives peptide 13 (named cp-B6X-4) affinity: K D = 12 nM.

[0150] The present application is based on a new synthetic rigid long-chain thio-ene molecule which can cyclize polypeptides and phage libraries. The high biocompatibility reaction modifies the skeleton of polypeptides with different chain lengths, and the obtained phage library Library 1 increases the diversity of small molecules in the construction and screening of modified phage libraries. At the same time, this long-chain rigid thio-ene molecule is relatively hydrophobic, which is conducive to the binding of the hydrophobic pocket of the protein, and Library 2 is screened against BCL-X L Protein to optimize the nearly 6-fold higher affinity cyclic peptide ligand. In summary, the present application synthesizes a new molecule with application value, and provides a new strategy for polypeptide cyclization, construction and screening of phage cyclic peptide library, which has important significance for the development of new functional macrocyclic peptide compounds.

[0151] The above examples are only preferred embodiments of the present application and cannot be considered as limiting the scope of the present application. Any equivalent changes and improvements made within the scope of the present application should still belong to the patent coverage of the present application.

Claims

1. A rigid long-chain alkenyl sulfide compound, characterized by The structural formula is as follows: The compound has a rigid biphenyl structure, which can improve the stability of the cyclic peptide.

2. The method of claim 1, wherein the rigid long-chain alkenyl sulfide compound is prepared by the reaction of a long-chain alkenyl halide compound with a sulfide compound. The synthetic route is as follows: The preparation method of the rigid long-chain alkenyl sulfide compound comprises the following steps: 1) taking dimethyl biphenyl dicarboxylate as a raw material, hydrolysis reaction is carried out with sodium hydroxide solution to obtain monomethyl ester biphenyl carboxyl compound; 2) the monomethyl ester biphenyl carboxyl compound is chlorinated to form biphenyl acyl chloride intermediate under the catalysis of N,N-dimethylformamide and the addition of thionyl chloride; 3) the biphenyl acyl chloride intermediate is reacted with sodium hydride and malononitrile to generate an enol compound under the action of sodium hydride; 4) the enol compound is chlorinated to generate a chloroalkene intermediate by chlorination reaction with phosphorus pentachloride; 5) the chloroalkene intermediate is reacted with sodium bicarbonate and ethyl mercaptan to generate an alkenyl sulfide compound under the action of sodium bicarbonate; 6) the alkenyl sulfide compound is activated by reaction with N-hydroxysuccinimide under the action of carbodiimide hydrochloride to generate an active ester intermediate; 7) the active ester intermediate is condensed with a chloro amino chain to generate an ethyl mercaptan substituted alkenyl sulfide compound; 8) the ethyl mercaptan substituted alkenyl sulfide compound is reacted with N-acetyl cysteine under the action of sodium bicarbonate to generate the rigid long-chain alkenyl sulfide compound.

3. A method for constructing a phage cyclic peptide library having a biphenyl- dihydrothiazole linkage, characterized by The construction method utilizes the alkenyl sulfide compound of claim 1, and specific steps of the method are as follows: 1) Constructing CX n C phage polypeptide library, from N-terminal to C-terminal, X represents random amino acid, n random amino acids are introduced by NNK method; the CX n C phage polypeptide library skeleton is C(X) n C, the amino acid is L-type amino acid, X represents random amino acid, subscript n is the number of random amino acids, n = 5-20; 2) Use of rigid long-chain alkenyl sulfide compounds to modify CX in cyclization step 1) n C phage polypeptide library, the polypeptides are cyclized to form macrocyclic polypeptide structures, the modified and cyclized phage polypeptide library is Library 1, i.e., the phage cyclic peptide library with a biphenyl-dihydrothiazole linkage.

4. A phage cyclic peptide library having a biphenyl-dihydrothiazole linkage, characterized by The phage cyclic peptide library is prepared by the construction method of claim 3 and is named Library 1.

5. The use of the phage cyclic peptide library with biphenyl-dihydrothiazole linkage as described in claim 4, characterized in that... The use is for synthesizing and screening cyclic peptide ligands with high affinity to target proteins, and specific steps are as follows: 1) The constructed phage cyclic peptide library Library 1 with biphenyl- dihydrothiazole linkage was used for screening, with BCL-X L protein as the target, 3 rounds of in vitro screening were performed; 2) After 3 rounds of screening, high enrichment phage was obtained, and the enriched single clone colonies were picked for sequencing, and the BCL-X L Protein screening obtained sequencing linear polypeptides include but are not limited to the following sequences, as shown in the sequence table SEQ ID NO. 1-9: CRPSWSNTLRPEC (peptide 1) CESSVGRATRLYEELC (peptide 2) CLENRRETVSQLYNSYC (peptide 3) CPSPPTPADLYDEYC (peptide 4) CAVSARSLYEIYC (peptide 5) CNNEGQRLYEEYC (peptide 6) CSETVGDLYQKYC (peptide 7) CNATASVYNELFASC (peptide 8) CQFDETIPELRNAC (peptide 9) wherein C is cysteine, R is arginine, P is proline, S is serine, W is tryptophan, N is asparagine, T is threonine, E is glutamic acid, V is valine, G is glycine, A is alanine, L is leucine, Y is tyrosine, I is isoleucine, F is phenylalanine, K is lysine, Q is glutamine, and D is aspartic acid; 3) Select the highest enrichment sequence peptide1 in step 2) for synthesis, cyclize with rigid long-chain alkenyl sulfide compound to obtain cyclic peptide ligand, and use surface plasmon resonance to characterize its affinity with target protein BCL-X L .

6. A method of constructing a phage display cyclic peptide library Library2, characterized in that A phage cyclic peptide library with a biphenyl-dihydrothiazole linkage according to claim 5 is used to target BCL-X L A secondary library, Library2, is constructed by a method comprising the following steps: 1) peptide 1 was found to have good affinity to BCL-X L Protein, two phage libraries with conserved sequence were designed based on peptide 1 as template, and the library capacity was about 2.4 x 10 8 ; the two phage display polypeptide scaffolds were: Scaffold 1: C(X)6TLRPEC and Scaffold 2: CRPSWS(X)6C; wherein X represents a random amino acid, and subscript 6 indicates the number of random amino acids. 2) the alkenyl sulfide molecule is used to modify the phage library of the two phage display polypeptide skeletons to obtain a phage display cyclic peptide library Library 2 by adopting the method of synthesizing and screening cyclic peptide ligands with high affinity to target proteins.

7. A phage display cyclic peptide library, characterized in that The construction method is prepared by claim 6.

8. Use of a phage display cyclic peptide library according to claim 7 for screening of macrocyclic peptide ligands, characterized in that The specific steps of the use are: using the constructed phage macrocyclic polypeptide library Library 2 for screening, two rounds of in vitro screening are carried out with BCL-X L protein as a target, and high enrichment degree phage is obtained, and the enriched single clone colony is picked and sequenced, and the sequenced linear polypeptide result obtained by screening against BCL-X L protein includes but is not limited to the following sequences, as shown in the sequence table SEQ ID NO. 10-30: CTSEWSQTLRPEC (peptide 10) CTKEWDETLRPEC (peptide 11) CTGEWEATLRPEC (peptide 12) CTPEWASTLRPEC (peptide 13) CTPEWALTLRPEC (peptide 14) CTNEWEGTLRPEC (peptide 15) CEHDWEETLRPEC (peptide 16) CRDEWVITLRPEC (peptide 17) CNAEWSATLRPEC (peptide 18) CHVEWASTLRPEC (peptide 19) CHPEWASTLRPEC (peptide 20) CHPDWDETLRPEC (peptide 21) CHEQWEDTLRPEC (peptide 22) CLPEWDVTLRPEC (peptide 23) CLETWEETLRPEC (peptide 24) CLPEWEGTLRPEC (peptide 25) CVPEWEDTLRPEC (peptide 26) CVPEWAQTLRPEC (peptide 27) CRPSWSESLRPEC (peptide 28) CRPSWSSTLLPEC (peptide 29) CRPSWSCREYLSC (peptide 30) wherein C is cysteine, R is arginine; P is proline; S is serine; W is tryptophan; N is asparagine; T is threonine; E is glutamic acid; V is valine; G is glycine; A is alanine; L is leucine; Y is tyrosine; I is isoleucine; F is phenylalanine; K is lysine; Q is glutamine; D is aspartic acid; The peptide 13 ranked 4th in the above sequence analysis was synthesized and cyclized with alkenyl sulfide to obtain the cyclic peptide ligand, which was characterized by surface plasmon resonance for its affinity to the target protein BCL-X L .

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