A tyrosine-based method for post-modification of polypeptide modules and uses thereof

Abstract

The application belongs to the field of biochemistry, and discloses a tyrosine-based polypeptide modular post-modification method and application thereof. The polypeptide modular post-modification method is as follows: adding a polypeptide, 5-bromo-1,2,3-triazine and an inorganic base in a reaction solvent, stirring and reacting, then adding an electron-rich dienophile, stirring and reacting at a certain temperature, and then concentrating, separating and purifying the mixture to obtain a polypeptide modular post-modification product. The polypeptide modular post-modification method has the advantages of simple raw material synthesis, simple operation, mild reaction conditions, high reaction efficiency, wide substrate application range, and compatibility with liquid phase and solid phase synthesis. In addition, the polypeptide modular post-modification method can be applied to constructing a dual-modular peptide imaging probe, realizing long-acting modification of endogenous polypeptides, and constructing a new type of antibacterial short peptide library, and provides a new and feasible method for the development of polypeptide drugs.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, specifically to a tyrosine-based method for modular modification of peptides. The invention also relates to applications of this method in microarray screening, construction of bimodal peptide imaging probes, long-term modification of endogenous peptides, and construction of novel amphiphilic antimicrobial short peptide libraries. Background Technology

[0002] In recent years, high-throughput screening platforms for peptides have made significant progress. However, most of the seed peptides obtained through biodisplay technology are composed of natural amino acids, which suffer from poor membrane permeability, low oral bioavailability, and poor in vivo stability. This limits their application to some extent. To overcome these limitations, it is particularly important to modify the structure of seed peptides and rapidly derivatize them to obtain non-standard peptides. Non-standard peptides can be obtained by synthesizing peptide-like substances or by post-modification (Drug Discov Today. 2023 May; 28(5):103554.). The former involves selectively integrating non-natural amino acids, such as β-amino acids, D-stereocenters, and N-methyl amino acids, into peptide-like substances. The integration of these special monomers is usually achieved by solid-phase synthesis (SPPS) technology or by recombination methods using engineered protein translation mechanisms. The latter, Ichikawa et al. (Journal of the American Chemical Society, 2023, 145(6):3665–3681.), utilized the classic serine / threonine linker (STL) to chemically synthesize threonine residues into the polymyxin backbone and implemented a "scanning and direct derivatization" strategy of chemically selective acylation. Huang Wei et al. from the Shanghai Institute of Materia Medica first introduced alkyne groups into the vancomycin backbone based on the Click reaction. Heinis et al. utilized the classic reaction between NHS esters and amino groups to construct a combined synthesis and screening method based on sequential cyclization, one-pot peptide acylation, and screening (Nature Chemical Biology, 2023:). These methods all require the introduction of non-natural amino acids in the early stages, which not only makes the reaction steps more complex but also greatly limits the diversity of peptide structures due to the immobility of the linker. Based on this, it is imperative to develop a new strategy that does not require the prior introduction of non-natural amino acids and can achieve modular modification. Summary of the Invention

[0003] Another object of the present invention is to provide the potential of the above-described method in peptide drug development, such as its application in constructing microarray screening, constructing bimodal peptide imaging probes, achieving long-term modification of endogenous peptides, and constructing novel amphiphilic antimicrobial short peptide libraries.

[0004] Based on the above, the purpose of this invention is to provide a tyrosine-based method for post-modification of polypeptide modularization.

[0005] Another object of the present invention is to provide the potential of the above-described method in peptide drug development, such as its application in constructing microarray screening, constructing bimodal peptide imaging probes, achieving long-term modification of endogenous peptides, and constructing novel amphiphilic antimicrobial short peptide libraries.

[0006] To achieve its purpose, the present invention adopts the following technical solution:

[0007] A tyrosine-based method for modular modification of peptides involves the following steps: A base (3) is added to a dry peptide (1) and 5-bromo-1,2,3-triazine (2) in an organic solvent, and the resulting mixture is stirred at a certain temperature for liquid-phase reaction. The corresponding electron-rich diephile (4) is added to the premixed solution, and the resulting mixture is stirred or shaken at a certain temperature for liquid-phase reaction. After the reaction, the mixture is concentrated to remove the solvent, and the residue is purified by silica gel column chromatography or semi-preparative purification to obtain the desired product (5). The reaction formula (I) is as follows:

[0008]

[0009] In the above reaction formula (I), R1 is a dienophile, and the polypeptide (1) can also be attached to the resin for solid-phase reaction.

[0010] As a further preferred embodiment of the technical solution of the present invention, the solid-phase reaction process is as follows:

[0011] Boc-Tyr-OH or Fmoc-Tyr-OH is introduced into the resin via solid-phase peptide synthesis. After the peptide sequence is synthesized, 5-bromotriazine and a base are added sequentially to the peptide synthesis tube, and the reaction is promoted by shaking on a shaker. Subsequently, an electron-rich dienophile is added and reacted on a shaker. After the reaction is completed, the resin is lysed and the peptide is separated and purified by preparative high-performance liquid chromatography to obtain the peptide compound (5). The general synthesis steps are as follows:

[0012]

[0013] Preferably, the molar ratio of the polypeptide (1), 5-bromo-1,2,3-triazine (2), and base (3) is 1:3:6. The molar ratio of the polypeptide (1), electron-rich diene (4), and base (3) is 1:10:6.

[0014] Preferably, the inorganic base is DBU.

[0015] Preferably, the reaction liquid solvent is MeCN and the solid solvent is DMF.

[0016] Preferably, the reaction of 5-bromotriazine with DBU involves a liquid-phase reaction at 37°C and a solid-phase reaction at room temperature. Reactions exceeding 37°C may damage the peptide structure. The liquid-phase reaction time is 30 minutes, and the solid-phase reaction time is 2 hours. Reactions shorter than 30 minutes or shorter than 2 hours may result in residual starting peptides, leading to incomplete reactions.

[0017] Preferably, the subsequent reaction of the dienophile is carried out at a liquid phase temperature of 37°C for 8 hours, and at a solid phase temperature of room temperature for 48 hours.

[0018] Preferably, the liquid-phase reaction and the solid-phase reaction are separated and purified by preparative HPLC.

[0019] Using the above method, peptide compounds with triazine attached to the tyrosine phenolic oxygen site can be obtained, and multiple modules can be efficiently introduced by utilizing the IEDDA reaction characteristics of triazine with "dienophiles".

[0020] Its structural formula is as follows:

[0021]

[0022] R1 and R2 are various diephiles such as ethyl acetoacetate, acetylacetone, butyl acetoacetate, dimethyl acetoacetate, 3-chlorophenylacetonitrile, and benzamide.

[0023] The above-mentioned method based on the tyrosine triazine coupling strategy for post-modification of peptide modularization can be used to achieve solid-phase post-modification of various drug peptides.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. The present invention provides a method for post-modification of peptides based on a tyrosine triazine coupling strategy. The raw materials are inexpensive and commercially available, the synthesis is convenient, the reaction is efficient and simple, and no inert gas protection or organic or metal catalysts are required.

[0026] 2. The substrates of this invention have a wide range of applications, are tolerant and compatible with different active amino acids, and are compatible with various electron-rich dienophile modules.

[0027] 3. Diverse modifications of tyrosine-based bioactive peptides were achieved in the solid phase. This avoids the solubility problems commonly encountered in liquid-phase modifications, while simplifying purification steps and reducing synthesis costs.

[0028] 4. The method constructed in this invention can be applied to multiple key fields, such as the development of dual-mode imaging probes and the construction of novel amphiphilic antimicrobial peptide libraries, bringing new opportunities for peptide drug development. Attached Figure Description

[0029] Figure 1 The results are for the hemolytic activity assay of Z0-Z20.

[0030] Figure 2 The results are the salt stability test results for Z0-Z20.

[0031] Figure 3 The results are the bactericidal kinetics test results for Z0-Z20;

[0032] Figure 4 The results are for serum stability tests of Z0-Z20. Detailed Implementation

[0033] The present invention will be described in detail below with reference to specific embodiments, but these specific embodiments are not intended to limit the actual protection scope of the present invention in any way, nor are they intended to limit the protection scope of the present invention to these embodiments.

[0034] Examples 1-18 Liquid Phase Synthesis

[0035] Example 1

[0036]

[0037] 5-Bromo-1 (stock solution) and DBU (0.1 mmol, 2 eq.) were added to a solution of YPFF (1a, 0.05 mmol, CH3CN stock solution), and the resulting mixture (final peptide concentration 10 mM) was reacted at 37 °C for 0.5 h. Ethyl acetoacetate (3a, 0.5 mmol, 10 eq.) was then added to the premix, and the resulting mixture was incubated at 37 °C in an Eppendorf Thermo Mixer at 800 rpm for 8 h. Semi-preparative purification yielded a white solid 4aa (27 mg, 74%). Mass spectrometry data are shown below.

[0038] HRMS(ESI): m / z calcd for C 41 H 46 N6O7[M+H] +1 735.3506, found 735.3521.

[0039] Example 2

[0040] In this example, substrate 1 was 1b (0.27 mmol scale), and other parameters and reaction conditions were the same as in Example 1. Purification was performed by silica gel column chromatography (PE / EA = 5:1 to 1:1) to obtain a white solid 5ba (110 mg, 62%). The structural formulas and mass spectrometry data of 1b and the product are as follows.

[0041]

[0042] HRMS(ESI): m / z calcd for C 36 H 43 N4O8[M+H] + 659.3075, found 659.3091.

[0043] Example 3

[0044] In this example, substrate 1 was 1c (0.23 mmol scale), and other parameters and reaction conditions were the same as in Example 1. Purification was performed by silica gel column chromatography (PE / EA = 5:1 to 1:1) to obtain a white solid 5ca (105 mg, 67%). The structural formulas and mass spectrometry data of 1c and the product are shown below, and the nuclear magnetic resonance experimental data are shown in Table 1.

[0045]

[0046] HRMS(ESI): m / z calcd for C 35 H 51 N4O 10 [M+H] + 687.3600, found 687.3611.

[0047] Example 4

[0048] In this example, substrate 1 was 1 d (0.28 mmol scale), and other parameters and reaction conditions were the same as in Example 1. Purification was performed by silica gel column chromatography (PE / EA = 4:1 to 1:4) to obtain a white solid 5 da (114 mg, 75%). The structural formulas and mass spectrometry data of 1 d and the product are shown below, and the nuclear magnetic resonance experimental data are shown in Table 1.

[0049]

[0050] HRMS(ESI): m / z calcd for C 27 H 36 N3O9[M+H] + 546.2446, found 546.2453.

[0051] Example 5

[0052] In this example, substrate 1 was 1e (0.23 mmol scale), and other parameters and reaction conditions were the same as in Example 1. Purification was performed by silica gel column chromatography (PE / EA = 5:1 to 1:1) to obtain a white solid 5ea (130 mg, 70%). The structural formulas and mass spectrometry data of 1e and the product are shown below, and the nuclear magnetic resonance experimental data are shown in Table 1.

[0053]

[0054] HRMS(ESI): m / z calcd for C 49 H 52 N5O8[M+H] + 838.3810, found 838.3831.

[0055] Example 6

[0056] In this example, substrate 1 was 1f (0.4 mmol scale), and other parameters and reaction conditions were the same as in Example 1. Purification was performed by silica gel column chromatography (PE / EA = 4:1 to 1:5) to obtain 5fa, a white solid (302 mg, 87%). The structural formulas and mass spectrometry data of 1f and the product are shown below, and the nuclear magnetic resonance experimental data are shown in Table 1.

[0057]

[0058] HRMS(ESI): m / z calcd for C 43 H 59 N6O 11 S[M+H] + 867.3957, found 867.3967.

[0059] Example 7

[0060] In this example, substrate 1 was 1 g (0.2 mmol scale), and other parameters and reaction conditions were the same as in Example 1. It was purified by silica gel column chromatography (PE / EA = 4:1 to 1:1) to obtain 5 g a white solid (126 mg, 76%). The structural formula and mass spectrometry data of 1 g and the product are as follows, and the nuclear magnetic resonance experimental data are shown in Table 1.

[0061]

[0062] HRMS(ESI): m / z calcd for C 46 H 50 N3O8S[M+H] + 804.3313, found 804.3326.

[0063] Example 8

[0064] In this example, substrate 1 was peptide YPFF, substrate 2 was 3b, and other parameters and reaction conditions were the same as in Example 1. After semi-preparative purification, a white solid 4ab (22.1 mg, 63%) was obtained. The structural formula and mass spectrometry data of the product are as follows.

[0065]

[0066] HRMS(ESI): m / z calcd for C 40 H 44 N6O6[M+H] +1 705.3395, found 705.3408.

[0067] Example 9

[0068] In this embodiment, substrate 2 was 3c, and other parameters and reaction conditions were the same as in Example 8. Semi-preparative purification yielded a white solid 4ac (22.7 mg, 59%). The structural formula and mass spectrometry data of the product are as follows.

[0069]

[0070] HRMS(ESI): m / z calcd for C 43 H 44 ClN7O5[M+Na] +1 796.2985, found 796.2970.

[0071] Example 10

[0072] In this example, substrate 2 was 3d, and other parameters and reaction conditions were the same as in Example 8. After semi-preparative purification, a white solid 4ad (18.4 mg, 51%) was obtained. The structural formula and mass spectrometry data of the product are as follows.

[0073]

[0074] HRMS(ESI): m / z calcd for C 42 H 43 N7O5[M+H] +1 726.3398, found 726.3420.

[0075] Example 11

[0076] In this embodiment, substrate 2 was 3e, and other parameters and reaction conditions were the same as in Example 8. After semi-preparative purification, a white solid 4ae (8.3 mg, 24%) was obtained. The structural formula and mass spectrometry data of the product are as follows.

[0077]

[0078] HRMS(ESI): m / z calcd for C 39 H 44 N6O7S[M+H] +1741.3065, found 741.3080.

[0079] Example 12

[0080] In this example, substrate 2 was 3f, and other parameters and reaction conditions were the same as in Example 1. After semi-preparative purification, a white solid 4af (26 mg, 68%) was obtained. The structural formula and mass spectrometry data of the product are as follows.

[0081]

[0082] HRMS(ESI): m / z calcd for C 54 H 56 N6O 13 [M+H] +1 762.3974, found 762.3970.

[0083] Example 13

[0084] Peptide YYY (1h, 30mg, 0.06mmol) was dissolved in 2mL acetonitrile, then 5-bromo-1,2,3-triazine (34.6mg, 0.216mmol, 3.6eq.) was added and mixed thoroughly. DBU (54mg, 0.36mmol, 6eq.) was then added, and the mixture was stirred at 37°C for 30 minutes. Ethyl acetoacetate (3a, 156mg, 1.2mmol, 20eq.) was added to the premixed solution, and the mixture was stirred or shaken at 37°C for 8 hours. After the reaction, the mixture was concentrated to remove the solvent, and the residue was purified semi-preparatively to obtain 5ha (26mg, 43%). The structural formula and mass spectrometry data of the product are as follows.

[0085]

[0086] HRMS(ESI): m / z calcd for C 54 H 56 N6O 13 [M+H] +1 997.3978, found997.3980.

[0087] Example 14

[0088] In this example, substrate 1 was peptide H-RGY-NH2 (0.1 mmol scale), substrate 2 was 3a, and other parameters and reaction conditions were the same as in Example 1. The product was dissolved in 2 mL of DMF and purified semi-preparatively to obtain a white solid 5a (10 mg, 18%). The structural formula and mass spectrometry data of the product are as follows.

[0089]

[0090] HRMS(ESI): m / z calcd for C 35 H 48 N6O 11 [M+H] +1 729.3454, found729.3454.

[0091] Example 15

[0092] In this example, substrate 1 was Ac-KDVY-OH (0.05 mmol scale). Other parameters and reaction conditions were the same as in Example 14. It was dissolved in 2 mL of DMF, and 2.4 equivalents of 5-bromotriazine and 3 equivalents of DBU were added. After semi-preparative purification, a white solid 5ja (6.8 mg, 19%) was obtained. The structural formula and mass spectrometry data of the product are as follows.

[0093]

[0094] HRMS(ESI): m / z calcd for C 35 H 48 N6O 11 [M+H] +1 729.3454, found729.3454.

[0095] Example 16

[0096] In this example, substrate 1 was H-MGYA-NH2 (0.05 mmol scale), and other parameters and reaction conditions were the same as in Example 14. Semi-preparative purification yielded a white solid of 5 kDa (14.3 mg, 48%). The structural formula and mass spectrometry data of the product are as follows.

[0097]

[0098] HRMS(ESI): m / z calcd for C 28 H 38 N6O7S[M+H] +1 603.2595, found 603.2607.

[0099] Example 17

[0100] In this example, substrate 1 was H-RKDVY-OH (0.058 mmol scale), dissolved in 2 mL of DMF, and 2.4 equivalents of 5-bromotriazine and 3 equivalents of DBU were added. Other parameters and reaction conditions were the same as in Example 14. After semi-preparative purification, a white solid 5la (5.6 mg, 12%) was obtained. The structural formula and mass spectrometry data of the product are as follows.

[0101]

[0102] HRMS(ESI): m / z calcd for C 39 H 58 N 10 O 11 [M+H] +1 843.4359, found 843.4345.

[0103] Example 18

[0104] In this example, substrate 1 was H-YGGFL-OH (0.04 mmol scale), and other parameters and reaction conditions were the same as in Example 14. Semi-preparative purification yielded a white solid at 5 mA (22.2 mg, 68%). The structural formula and mass spectrometry data of the product are as follows.

[0105]

[0106] HRMS(ESI): m / z calcd for C 37 H 46 N6O9[M+H] +1 719.3399, found 719.3407.

[0107] Solid-phase synthesis in Examples 19-25

[0108] Example 19

[0109] Accurately weigh 0.465 g (0.2 mmol) of Rink Amide MBHA resin and place it in a 20 mL syringe equipped with a filter cartridge. Then, add 10 mL of anhydrous dichloromethane to swell the resin. After shaking for 20 minutes, remove the dichloromethane and rinse with DMF for 2 minutes each time, repeating this process 3 times to ensure thorough cleaning of the resin.

[0110] Fmoc protecting group removal

[0111] Amino acid condensation: Weigh 3 equivalents of amino acids, HOAT, and HATU, dissolve them in DMF, add 6 equivalents of DIEA, mix well, and then add to a synthesizer with shaking for 1 hour. After completion, drain the liquid and wash the resin with DMF. Confirm the success of condensation by indole assay; if unsuccessful, repeat this step. After successful condensation, repeat steps (3) and (4) according to the peptide chain sequence to perform amino acid condensation until the peptide chain is fully elongated. Special note: When the N-terminus is tyrosine, Boc-Tyr-OH should be used; if the tyrosine is located inside, Fmoc-Tyr-OH should be used. The N-terminal amino acid is Boc-AA-OH. Substrate 1 peptide H-YRAKW-NH2 is obtained.

[0112] Addition and reaction of 5-bromotriazine and DBU: Weigh 3 equivalents of 5-bromotriazine (960 mg) and 6 equivalents of DBU (1.82 mL), and dissolve them separately in 10 mL of DMF. Then, add these solutions sequentially to the peptide synthesis tubes and shake on a shaker for 2 hours to promote the reaction.

[0113] Addition of the dienophile and subsequent reactions: After the previous reaction was completed, the reaction solution was removed, and the peptide synthesis tube was rinsed three times with DMF, shaking for 2 minutes each time. Next, 10 equivalents of the "dienophile" substrate 2,3a were weighed, dissolved in 10 mL of DMF, and added to the peptide synthesis tube. Subsequently, 6 equivalents of DBU (91 mg, 0.6 mmol) were added, and the tube was placed in a shaker at room temperature for 48 hours with shaking.

[0114] Resin Drying: After peptide chain synthesis, the F-moc protecting group of the last amino acid is removed using a deprotecting agent. Subsequently, the resin is washed alternately with DCM and MeOH to ensure complete removal of residual reagents. Finally, the synthesizer is sealed, and the liquid is pumped out until the resin is completely dry to a powder.

[0115] Resin cleavage: A suitable amount of cleavage buffer (TFA:Tis:water = 95%:2.5%:2.5%) was slowly added to the synthesizer and shaken at room temperature for 3 hours. The cleavage buffer was collected into a round-bottom flask, and the TFA was evaporated to dryness to obtain a crude peptide solution. After pre-cooling, cold diethyl ether was added to precipitate the peptide. After centrifugation (4000 rpm, 4℃, 5 minutes), the liquid phase was removed, and the solid was collected, dissolved, and then purified or analyzed by LCMS.

[0116] Purification: Trifluoroacetic acid was removed from the cleavage solution by vacuum concentration, and icy diethyl ether was added to the concentrate, causing the crude peptide product to precipitate as a solid. The crude product was collected and purified by preparative reversed-phase HPLC using a linear gradient. After 13 steps of purification, the product was lyophilized to obtain a white solid 6a (91.5 mg, 52%). The product structure and mass spectrometry data are as follows.

[0117]

[0118] HRMS(ESI): m / z calcd for C 44 H 61 N 12 O8[M+H] + 885.4730, found 885.4731.

[0119] Example 20

[0120] The operation of this embodiment is largely the same as that of Example 19, except that: in this embodiment, substrate 1 is peptide H-YNWNSFGLRF-NH2, and other parameters and reaction conditions are the same as in Example 19. After 23 steps, purification is performed, and lyophilization yields a white solid 6b (28 mg, 28%). The product structure and mass spectrometry experimental data are as follows:

[0121]

[0122] HRMS(ESI): m / z calcd for C 72 H 93 N 18 O 16 [M+H] + 1465.7011, found 1465.7045.

[0123] Example 21

[0124] The operation of this embodiment is largely the same as that of Example 19, except that: in this embodiment, substrate 1 is peptide H-VYIHPF-NH2 (0.2 mmol scale), and other parameters and reaction conditions are the same as in Example 19. After 15 steps, purification is performed, and lyophilization yields a white solid 6c (146 mg, 78%). The product structure and mass spectrometry experimental data are as follows:

[0125]

[0126] HRMS(ESI): m / z calcd for C 49 H 65 N 10 O9[M+H] + 937.4931, found 937.4943.

[0127] Example 22

[0128] The operation of this embodiment is largely the same as that of Example 19, except that: in this embodiment, substrate 1 is H-DRVYIHPFHL-NH2, and other parameters and reaction conditions are the same as in Example 19. After 23 steps, purification is performed, and lyophilization yields a white solid (12.9 mg, 18%) for 6 days. The product structure and mass spectrometry experimental data are as follows:

[0129]

[0130] HRMS(ESI): m / z calcd for C 71 H 100 N 19 O 15 [M+H] + 1458.7641, found 1458.7627.

[0131] Example 23

[0132] The operation of this embodiment is largely the same as that of Example 19, except that: in this embodiment, substrate 1 is peptide H-CYIQNCPLG-NH2 (0.078 mmol scale), substrate 2 is 3g, and other parameters and reaction conditions are the same as in Example 19. After 21 steps, purification is performed, and lyophilization yields a white solid 6e (40.5 mg, 41%). The product structure and mass spectrometry experimental data are as follows:

[0133]

[0134] HRMS(ESI): m / z calcd for C 58 H 90 N 13 O 14 S[M+H] + 1256.6166, found 1256.6186.

[0135] Example 24

[0136] The operation of this embodiment is largely the same as that of Example 19, except that: in this embodiment, substrate 1 is H-YGRKKRRQRRR-NH2 (0.033 mmol scale), substrate 2 is 3h, and other parameters and reaction conditions are the same as in Example 19. After 25 steps, purification is performed, and lyophilization yields a white solid 6f (20 mg, 35%). The product structure and mass spectrometry experimental data are as follows:

[0137]

[0138] HRMS(ESI): m / z calcd for C 75 H 134 N34 O 15 [M+2H] +2 875.5384, found 875.5381.

[0139] Example 25

[0140] The operation of this embodiment is largely the same as that of Example 19, except that: in this embodiment, substrate 1 is peptide H-fPRPGGGGNGDFEEIPEEYL-NH2, and other parameters and reaction conditions are the same as in Example 19. After 43 steps, purification is performed, and lyophilization yields 6g (13.3mg, 11%) of a white solid. The product structure and mass spectrometry experimental data are as follows:

[0141]

[0142] HRMS(ESI): m / z calcd for C 107 H 150 N 26 O 34 [M+2H] +2 1171.5398, found 1171.5441.

[0143] Examples 26-30: Construction of a Dual-Modal Imaging Probe

[0144] Example 26

[0145] The operation of this embodiment is largely the same as that of Example 19, except that: in this embodiment, substrate 1 is peptide NH2-YGRKKRRQRRR-OH (0.02 mmol scale), and other parameters and reaction conditions are the same as in Example 19. After 25 steps, purification is performed, and lyophilization yields a white solid T1 (150 mg, 73%). The product structure and mass spectrometry experimental data are as follows:

[0146]

[0147] HRMS(ESI): m / z calcd for C 91 H 143 N 35 O 19 S[M+2H] +2 1,031.0510,found 1031.5547.

[0148] Example 26

[0149] The operation of this embodiment is largely the same as that of Example 19, except that: in this embodiment, substrate 1 is peptide NH2-YGRKKRRQRRR-OH (0.02 mmol scale), and substrate 2 is [missing information]. Other parameters and reaction conditions are the same as in Example 19. After 25 steps, purification is performed, and lyophilization yields a white solid T2 (15.1 mg, 36%). The product structure and mass spectrometry experimental data are as follows:

[0150]

[0151] HRMS(ESI): m / z calcd for C 95 H 145 N 36 O 20 [M+3H] +3 703.3812, found 703.7189.

[0152] Example 27

[0153] The operation of this embodiment is largely the same as that of Example 19, except that: in this embodiment, substrate 1 is NH2-YGRKKRRQRRR-OH, substrate 2 is [missing information], and other parameters and reaction conditions are the same as in Example 19. After 29 steps, purification is performed, and lyophilization yields a white solid T3 (36.3 mg, 28%). The product structure and mass spectrometry experimental data are as follows:

[0154]

[0155] HRMS(ESI): m / z calcd for C 114 H 175 N 41 O 28 [M+2H] +2 1,283.1764,found 1283.6784.

[0156] Example 29 Flow cytometry experiments at T1, T2, and T3

[0157] First, U87MG cells were evenly seeded in 96-well plates, with approximately 10,000 cells per well, and then the plates were incubated in a cell culture incubator for 24 hours. After incubation, 20 mM of the drug was added to each well, and the plates were incubated for another 2 hours. After incubation, the drug-containing culture medium was gently aspirated, and the cells were then washed three times with DPBS buffer to remove residual culture medium and drug. Next, the cells were digested with trypsin for efficient cell collection. After digestion, the cells were washed three more times with DPBS buffer to ensure the removal of excess trypsin and impurities. After the final wash, the cells were resuspended in DPBS buffer and analyzed by flow cytometry.

[0158] Example 30: Confocal microscopy experiments at T1, T2, and T3

[0159] First, U87MG cells were evenly spread at a density of 100,000 cells per well in glass dishes and incubated in a cell culture incubator for 24 hours to ensure adequate cell adhesion and growth. Then, 20 mM of the drug was added to the culture wells, and incubation continued for 2 hours. After incubation, the drug-containing medium was gently aspirated, and the cells were gently washed three times with DPBS buffer to remove residual drug and medium. Next, the cell nuclei were stained with Hochest 33342 dye for 10 minutes, as instructed in the manufacturer's instructions. After staining, the cells were washed three times again with DPBS buffer to remove excess dye. Then, the cell membranes were stained with Dil dye for 15 minutes. Similarly, the cells were washed three times with DPBS buffer after staining. Finally, an appropriate amount of DMEM medium was added to the culture wells to maintain a normal growth environment for the cells. The cells were observed and photographed using a confocal microscope with different excitation wavelengths. The experiment was repeated three times.

[0160] Tat was selected as the model peptide for further research. Three different conjugates were synthesized: T1, with FITC conjugated at the N-terminus; T2, with FITC conjugated via tyrosine using the YTL strategy; and T3, with DOTA conjugated at the N-terminus and FITC conjugated via tyrosine. These three conjugates were co-incubated with U-87MG cells for 2 hours, and the fluorescence staining status of the cells was observed using confocal laser scanning microscopy. Flow cytometry was used to analyze their permeation. The experimental results showed that T3 had a similar internalization state to T1 and T2, exhibiting good cell penetration ability. This invention provides strong support for constructing novel dual conjugates using the YTL strategy and also provides a novel approach for constructing probes with dual-mode imaging capabilities.

[0161] Examples 34-39: Construction of an amphiphilic antimicrobial short peptide library

[0162] Example 34 Antibacterial activity test of Z0-Z20

[0163] Bacterial strain activation and revival: Add an appropriate amount of bacterial cryopreservation solution to 3 mL of sterile broth medium and incubate overnight at 37°C with shaking at 180 rpm. Use an inoculation loop to take a small amount of bacterial solution and spread it on MH solid medium using the streak plating method. Incubate overnight at 37°C. After single colonies have grown, seal with sealing film and store at 4°C.

[0164] Bacterial amplification: Single colonies were picked using a 200 μL sterile pipette tip and vertically injected into 3 mL of sterile broth. The culture was incubated overnight at 37°C with shaking at 180 rpm until the bacterial culture reached mid-log phase. The bacterial culture was then diluted to 1 x 10⁻⁶ using sterile MH medium. 6 CFU / mL available for use.

[0165] Weigh an appropriate amount of lyophilized polypeptide drug powder, prepare a stock solution of 2048 μg / ml with sterile ultrapure water, and then prepare a drug solution of 128–2 μg / ml by a twofold dilution method.

[0166] Following the concentration gradient, 50 μL of drug solution and 50 μL of corresponding bacterial culture were added sequentially to a 96-well plate, gently shaken to mix, and incubated at 37°C for 16 h.

[0167] The minimum drug concentration (MIC) that completely inhibits the growth of the test bacteria is determined by observing the turbidity of the liquid in the 96-well plate. The experiment should be repeated at least 3 times.

[0168] Twenty amphiphilic short peptides were rapidly constructed using the YTL strategy. As shown in Tables 1 and 2, two antimicrobial short peptides, Z8 and Z10, with excellent antimicrobial activity were successfully screened out by MIC testing.

[0169] Table 1. MICs of antimicrobial peptides against Gram-negative bacteria

[0170]

[0171] Table 2. MICs of antimicrobial peptides against Gram-positive bacteria and fungi.

[0172]

[0173] Example 35: Determination of hemolytic activity of Z0-Z20

[0174] Blood collection from mouse eyeballs: Prepare a 2 mg / mL heparin sodium solution beforehand, add 200 μL to each centrifuge tube, and allow the blood to clot. Pour some ether into a sawdust container, place the mouse in the container, and anesthetize it for 10–15 seconds. Anesthesia is considered complete when the mouse stops struggling violently when lifted. Hold the mouse with your left hand, trim its whiskers with clean scissors, and use tweezers in your right hand to grasp the base of the eyeball and pull it outwards to collect the blood into a centrifuge tube. Gently invert the tube to mix the heparin sodium and plasma. After blood collection, euthanize the mouse by dislocating its cervical spine.

[0175] Centrifuge the plasma at 10,000 rpm for 10 minutes and discard the supernatant. Resuspend the blood cells in 700 μL of physiological saline, centrifuge at 1,000 rpm for 5 minutes, and repeat three times. Prepare an 8% plasma solution using physiological saline.

[0176] Weigh an appropriate amount of lyophilized polypeptide drug powder, prepare a stock solution of 2048 μg / mL with sterile ultrapure water, and then prepare a drug solution of 128–8 μg / mL with physiological saline by a twofold dilution method.

[0177] Following the concentration gradient, 100 μL of drug solution and 100 μL of plasma were added sequentially to a 96-well plate. 2% Triton solution and pure physiological saline were used as positive and negative controls, respectively. The plates were gently shaken to mix and incubated at 37°C for 1 hour.

[0178] Remove the 96-well plate, centrifuge at 800 rpm for 15 min, and aspirate 100 μL of the supernatant into a new 96-well plate.

[0179] Measure the absorbance of the 96-well plate at 490 nm. The experiment should be repeated at least three times. The hemolysis rate is calculated using the following formula: Hemolysis (%) = [OD] 490nm(多肽) –OD 490nm(阴性对照 )] / [OD 490nm(阳性对照) –OD 490nm(阴性对照) ]×100%

[0180] Figure 1 The results showed that Z9 exhibited a relatively balanced characteristic due to its low hemolytic activity and high antibacterial activity, making it a promising candidate compound. While Z8 and Z10 showed better antibacterial effects, they caused greater damage to erythrocytes at high concentrations.

[0181] Salt stability test of Z0-Z20 in Example 36

[0182] Bacterial strain activation and revival: Add an appropriate amount of bacterial cryopreservation solution to 3 mL of sterile broth medium and incubate overnight at 37°C with shaking at 180 rpm. Use an inoculation loop to take a small amount of bacterial solution and spread it on MH solid medium using the streak plating method. Incubate overnight at 37°C. After single colonies have grown, seal with sealing film and store at 4°C.

[0183] Bacterial amplification: Single colonies were picked using a 200 μL sterile pipette tip and vertically injected into 3 mL of sterile broth. The culture was incubated overnight at 37°C with shaking at 180 rpm until the bacterial culture reached mid-log phase. The bacterial culture was then diluted to 1.5 x 10⁻⁶ m² using sterile MH medium. 6 CFU / mL available for use.

[0184] Weigh an appropriate amount of lyophilized polypeptide drug powder, prepare a stock solution of 2048 μg / mL with sterile ultrapure water, and then prepare a drug solution of 96–3 μg / mL by a twofold dilution method.

[0185] Different salt solutions (150mM NaCl, 4.5mM KCl, 2mM CaCl2, 6μM NH4Cl, 8μM ZnCl2, 1mM MgCl2, 4μM FeCl3) were prepared using deionized water and sterilized in an autoclave at 120℃ for 30 minutes before use.

[0186] Following a concentration gradient, add 50 μL of drug solution, 50 μL of corresponding salt solution, and 50 μL of corresponding bacterial culture sequentially to each well of a 96-well plate. Gently shake to mix, and incubate at 37°C for 16 hours. Observe the lowest drug concentration that completely inhibits the growth of the test bacteria. The experiment should be repeated at least three times.

[0187] Figure 2 This indicates that different salt ions have varying effects on the antibacterial activity of Z8, Z9, Z10, and Z16, exhibiting a degree of selectivity and complexity. This invention provides valuable experimental evidence for further optimizing the salt stability of these antimicrobial peptides.

[0188] Example 37: Bactericidal Kinetics Test of Z0-Z20

[0189] Bacterial strain activation and revival: Add an appropriate amount of bacterial cryopreservation solution to 3 mL of sterile broth medium and incubate overnight at 37°C with shaking at 180 rpm. Use an inoculation loop to take a small amount of bacterial solution and spread it on MH solid medium using the streak plating method. Incubate overnight at 37°C. After single colonies have grown, seal with sealing film and store at 4°C.

[0190] Bacterial amplification: Single colonies were picked using a 200 μL sterile pipette tip and vertically injected into 3 mL of sterile broth. The culture was incubated overnight at 37°C with shaking at 180 rpm until the bacterial culture reached mid-log phase. The bacterial culture was then diluted to 1 x 10⁻⁶ using sterile MH medium. 6 CFU / mL available for use.

[0191] Weigh an appropriate amount of lyophilized polypeptide drug powder and prepare a stock solution of 2048 μg / mL with sterile ultrapure water. Then, prepare a drug solution with a concentration of 8×-2×MIC by a twofold dilution method.

[0192] Add an equal volume of diluted bacterial suspension to the drug solution, mix thoroughly by pipetting, and incubate at 37°C. Samples are taken at 0h, 15min, 30min, 1h, 2h, 4h, 6h, and 24h, and plated to estimate bacterial count using the plate count method. Depending on the turbidity of the incubation solution and the incubation time, the collected incubation solution may need to be diluted 100–1,000,000 times. The experiment should be repeated at least three times.

[0193] Figure 3 The results indicate that high concentrations of Z8 exhibit bactericidal effects against all three representative bacterial species, but at low concentrations, it only has an inhibitory effect rather than a bactericidal effect on some species. Z8's bactericidal ability against Gram-positive bacteria is significantly stronger than that against Gram-negative bacteria and fungi.

[0194] Example 38 Cytotoxicity test of Z0-Z20

[0195] HEK-293 cell culture: Add 2700 μL of DMEM medium and 300 μL of Gibco FBS to a 60 mm culture dish and incubate in a cell culture incubator. Once the cells have completely covered the bottom of the dish, begin digestion. Discard the medium in the dish, add 1 mL of PBS to rinse, and discard the PBS. Add 0.5 mL of trypsin and digest for several tens of seconds, then add 0.5 mL of DMEM medium and a few drops of serum. Use a pipette to lift the cells from the bottom of the dish, mix well, and transfer to a 1.5 mL centrifuge tube.

[0196] Cell counting: Centrifuge at 8000r for 15 minutes, discard the supernatant, add 1mL of DMEM medium and mix well by pipetting, then count using a cell counting chamber.

[0197] HEK-293 cells were seeded in 96-well plates (10,000 cells per well) and cultured in a cell culture incubator for 24 hours. Different concentrations of peptide drug solutions (final concentrations ranging from 2 to 128 μg / mL) were added, and the cells were incubated for 48 hours. The culture medium was then discarded, and fresh DMEM medium containing 10% CCK8 reagent was added. The cells were incubated for 40 minutes, and the absorbance at 450 nm and 650 nm was measured.

[0198] The experiment should be repeated at least three times. The formula for calculating cell viability is as follows:

[0199] Cell viability (%) = [(As 450nm –As 650nm )–(Ab 450nm –Ab 650nm )] / [(Ac 450nm –Ac 650nm )–(Ab 450nm –Ab 650nm )]×100%

[0200] As: Absorbance of the experimental group

[0201] Ac: Absorbance of the control group

[0202] Ab: Absorbance of the blank group

[0203] Z9 exhibited relatively low cytotoxicity, a finding consistent with previous hemolytic activity assays.

[0204] Example 39 Serum stability test of Z0-Z20

[0205] The polypeptide solution (200 μM, dissolved in PB buffer) and premium fetal bovine serum were pre-equilibrated to 37°C in separate test tubes for 15 minutes.

[0206] For each replicate experiment, the peptide and serum were mixed in a specific ratio to achieve a final peptide concentration of 100 μM, a serum concentration of 50% (v / v), and a total volume of 1000 μL. (The serum concentration was 50% for the G1 test and 90% for the antimicrobial peptide test).

[0207] The reaction tube was placed in an Eppendorf hot mixer and incubated at 37°C at 300 rpm. At specific time points, 50 μL of the reaction solution was removed and rapidly diluted with four times its volume of cold methanol.

[0208] The diluted sample was allowed to stand at -20°C for 15 minutes to allow serum proteins to precipitate fully. Then, it was centrifuged at 4°C and 14,000g for 10 minutes to separate the supernatant.

[0209] The supernatant was transferred to a new test tube and then analyzed by high-performance liquid chromatography (HPLC). HPLC method F was used, and the changes in peak area of ​​the starting material were recorded and plotted. The experiment was repeated three times.

[0210] Figure 4 This indicates that Z9 has the shortest half-life, approximately 5.5 hours; Z10 has a slightly longer half-life, reaching 8 hours; while Z8 has the longest half-life, approximately 12 hours.

[0211] Table 3 NMR data of the synthesized products in the embodiments of the present invention

[0212]

[0213]

[0214]

Claims

1. A method for post-modification of peptides based on tyrosine, characterized in that, A base (3) was added to a dry polypeptide (1) and 5-bromo-1,2,3-triazine (2) in an organic solvent, and the resulting mixture was stirred at a certain temperature for liquid-phase reaction; the corresponding electron-rich diephile (4) was added to the premixed solution, and the resulting mixture was stirred or shaken at a certain temperature for liquid-phase reaction; after the reaction, the mixture was concentrated to remove the solvent, and the residue was purified by silica gel column chromatography or semi-preparative purification to obtain the desired product (5), the reaction formula of which is as follows: In the above reaction formula, R1 is a dienophile, and the polypeptide (1) can also be attached to the resin for solid-phase reaction.

2. The method for post-modification of peptides based on tyrosine as described in claim 1, characterized in that, The solid-phase reaction process is as follows: Boc-Tyr-OH or Fmoc-Tyr-OH was introduced into the resin via solid-phase peptide synthesis. After the peptide sequence was synthesized, 5-bromotriazine and a base were added sequentially to the peptide synthesis tube, and the reaction was promoted by shaking on a shaker. Subsequently, an electron-rich dienophile was added to the reaction. After the reaction was completed, the resin was lysed and the peptide was separated and purified by preparative high-performance liquid chromatography.

3. A method for post-modification of peptides based on tyrosine as described in claim 1 or 2, characterized in that, The dienophile R 21 The structure shown below, 4. A tyrosine-based peptide modularization post-modification method as described in claim 1 or 2, characterized in that, The molar ratio of the polypeptide (1), 5-bromo-1,2,3-triazine (2), and base (3) is 1:3:6; the molar ratio of the polypeptide (1), electron-rich diene (4), and base (3) is 1:10:6; the inorganic base is DBU; the liquid solvent of the reaction is MeCN, and the solid solvent is DMF.

5. A method for post-modification of peptides based on tyrosine as described in claim 1 or 2, characterized in that, The reaction of 5-bromotriazine with DBU was carried out at a liquid phase temperature of 37°C for 30 minutes and a solid phase reaction at room temperature for 2 hours. The subsequent reaction of the dienophile was carried out at a liquid phase temperature of 37°C for 8 hours and a solid phase reaction at room temperature for 48 hours.

6. A method for post-modification of peptides based on tyrosine as described in claim 1 or 2, characterized in that, The method is used for microarray screening; characterized in that resin is weighed into a 96-well filter plate (1.0 mL), and a 20 μm hydrophobic sieve plate is prepared. The filter plate is fitted with a 96-well plate with a special gasket as a plug. Swelling, deprotection, amino acid condensation, coupling with 5-bromotriazine, dienophilic reaction, and shrinkage acid cleavage are performed in the filter plate. The acid cleavage solution is filtered into a 96-well square hole collection plate (1.6 mL) using a negative pressure device. TFA is dried using a nitrogen blower, precipitated with ice-cold ether, and the crude product is obtained after nitrogen blowing or centrifugation.

7. A method for post-modification of peptides based on tyrosine as described in claim 1 or 2, characterized in that, The method described above is used to construct a dual-mode peptide imaging probe; characterized in that the N-terminus is coupled to an amino group while simultaneously being coupled through a tyrosine modification site to synthesize a novel dual-conjugate; the chelating agent used has the following structure.

8. A method for post-modification of peptides based on tyrosine as described in claim 1 or 2, characterized in that, The method is used to achieve long-term modification of endogenous peptides.

9. A method for post-modification of peptides based on tyrosine as described in claim 1 or 2, characterized in that, The method is used to construct a novel amphiphilic antimicrobial short peptide library to discover novel antimicrobial peptides. The antimicrobial short peptide library uses HYXX-NH2, HXYX-NH2, and HXXY-NH2 as templates, where Y is tyrosine and X is any one of arginine, histidine, ornithine, or lysine, and is post-modified by a dienophile R2.

10. The method for post-modification of peptides based on tyrosine as described in claim 9, characterized in that, A novel amphiphilic antimicrobial short peptide library was constructed to discover novel antimicrobial peptides for antimicrobial activity against Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Candida albicans.

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