Method for arylating and cyclizing polypeptide

Abstract

The invention provides a method for arylation and cyclization of polypeptide, and the reaction process is as follows: the specific method comprises the following steps: under weakly acidic, neutral or weakly alkaline conditions, carrying out a chemical selective arylation reaction on linear polypeptide II containing two cysteine residues through a dichloro nitrogen-containing heteroaromatic ring reagent I, so as to construct a cyclized stable polypeptide III. The cyclized stable polypeptide III has improved stability and cell transmembrane properties under physiological conditions.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry, and particularly relates to a chemically selective polypeptide arylation and cyclization method based on a dichlorinated nitrogen-containing heteroaromatic ring reagent, and application of the method in preparing highly stable cyclized polypeptides. Background Art

[0002] Peptide drugs have become important drug candidates for the treatment of tumors and metabolic diseases due to their advantages of high targeting and low toxicity. However, their linear structures are susceptible to protease degradation in physiological environments and have low membrane penetration efficiency, which seriously limits their clinical translation. Cyclization modification is a key strategy to improve the stability and bioactivity of peptides. The current mainstream technology relies on disulfide bond cyclization or chemical cross-linkers (such as bismaleimide). However, disulfide bonds are easily broken in the reducing physiological microenvironment, resulting in structural inactivation; traditional cross-linkers rely on strong acidic / alkaline reaction conditions, which are prone to induce non-selective side reactions of peptide side chains (such as random modification of lysine ε-amino groups). Moreover, the resulting products are difficult to achieve a balance between stability and functional activity due to insufficient rigidity.

[0003] In recent years, arylation cyclization technology has been widely used because it can form stable SP 2 Although the cyclization of peptides by polyfluorinated aromatic rings has attracted much attention due to the carbon-sulfur covalent bond, the existing schemes still have significant defects: (1) The mainstream method uses polyfluorinated aromatic ring reagents (such as hexafluorobenzene), whose reaction efficiency is limited by the insufficient activity of the leaving group, and often requires strong alkaline conditions or excessively long reaction times, resulting in the destruction of the peptide conformation or even racemization; (2) The high hydrophobicity of the polyfluorinated benzene group seriously affects the water solubility and in vivo biodistribution of the product; (3) Some methods require the introduction of metal catalysts, which limits their application in biocompatibility scenarios. Therefore, the development of a method for the cyclization of peptide aromatic rings with high selectivity and broad applicability under mild conditions remains a technical challenge that needs to be overcome in this field. Summary of the Invention

[0004] In response to the above-mentioned deficiencies in the prior art, the present invention provides a method for aryl cyclization of polypeptides. The method for aryl cyclization of polypeptides is to chemically and selectively arylize the cysteine ​​residues of the polypeptide, thereby significantly improving the membrane penetration and stability of the product.

[0005] The present invention provides a method for aryl cyclization of a polypeptide, wherein a dichlorinated nitrogen-containing heteroaromatic ring reagent I undergoes an aryl reaction with a linear polypeptide II to generate a cyclized stable polypeptide III, and the general reaction formula is as follows:

[0006]

[0007] Wherein, X, Y, and Z represent any amino acids, LA1 and LA2 represent connecting amino acids; and m, n, a, b, and c are natural numbers.

[0008] Furthermore, the arbitrary amino acid is any one of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, methionine, serine, threonine, proline, histidine or arginine; and the linking amino acid is any one of cysteine, homocysteine ​​or selenocysteine.

[0009] Furthermore, the I is any one of the following structural formulas,

[0010]

[0011] Furthermore, the structural formula of II is

[0012]

[0013] The structural formula of III is

[0014] Furthermore, the structural formula of II is

[0015] FAM-Ahx-Cyclo (CRRRC), the structural formula of I is

[0016]

[0017] Furthermore, when the number of b in the connecting amino acids is different, polypeptides of different ring sizes are formed; when a=0, the cyclization reaction proceeds at the nitrogen end of the polypeptide; when c=0, the cyclization reaction proceeds at the carbon end of the polypeptide; when a and c are both 0, the cyclization reaction proceeds at both ends of the polypeptide.

[0018] Furthermore, when the polypeptide II contains three, four or more ring-closing sites, a bicyclic, tricyclic or polycyclic stable polypeptide is generated.

[0019] Furthermore, in the arylation reaction, the modified atom includes a sulfur atom and other atoms, and the other atom is a selenium atom.

[0020] Furthermore, the dichloro nitrogen-containing heteroaromatic ring reagent I contains at least two chlorine atoms as leaving groups, and the two chlorine atoms are in the para position or the meta position.

[0021] Furthermore, the dichloro nitrogen-containing heteroaromatic ring reagent I is a six-membered nitrogen-containing heteroaromatic ring compound and contains at least one nitrogen atom.

[0022] Furthermore, the cyclization reaction is carried out in a weakly acidic, neutral or weakly alkaline buffer solution, and the pH of the reaction system is 5-8.

[0023] Furthermore, the stabilized polypeptide prepared by the method is used for improving polypeptide stability, drug delivery, or screening protein-protein interaction ligands.

[0024] The present invention uses liquid chromatography (HPLC) and mass spectrometry (MS) technology to confirm the feasibility and universality of the method for the chemical selective arylation reaction of polypeptide cysteine ​​residues.

[0025] The present invention also confirmed through flow cytometry analysis and laser confocal microscopy experiments based on fluorescein-labeled polypeptides that the cell membrane penetration efficiency of cyclized polypeptide III is significantly improved compared with that of linear polypeptide II.

[0026] The present invention also confirms through degradation experiments in buffer and serum environments that the stability of the cyclized polypeptide III is significantly improved compared to the linear polypeptide II.

[0027] Compared with existing technologies, the present invention offers significant technological advancements. Under weakly acidic, neutral, or weakly basic conditions, the present invention uses a dichlorinated nitrogen-containing heteroaromatic ring reagent (I) to chemoselectively arylate a linear polypeptide (II) containing two cysteine ​​residues, thereby constructing a cyclized, stabilized polypeptide (III). The cyclized, stabilized polypeptide (III) exhibits improved stability and cell-penetrating properties under physiological conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the chemical process for the reaction of a series of dichloronitrogen-containing heteroaromatic reagents I with a model peptide II (linear oxytocin) to generate cyclized peptide III.

[0029] Figure 2 The reaction conversion rate of reagent I-1 and polypeptide II (linear oxytocin) under different reaction times and pH gradient (5.5-8.0), as well as the HPLC spectrum comparison and MS spectrum before and after the reaction.

[0030] Figure 3 Flow cytometry was used to analyze the cellular uptake efficiency of cyclized peptides IV-1 and IV-7 in HeLa cells.

[0031] Figure 4 Laser confocal microscopy was used to analyze the cellular uptake behavior of cyclized peptides IV-1 and IV-7 in HeLa cells.

[0032] Figure 5 Stability test data of cyclized peptide IV-1 in buffer (pH 7, GSH or hydrogen peroxide) and mouse serum environment. DETAILED DESCRIPTION

[0033] The following describes the detailed implementation of the method for aryl cyclization of polypeptide cysteine ​​residues using preparation examples, chemical validation, and biological application examples. These examples are provided to illustrate the technical solutions of the present invention and do not constitute limitations on the scope of protection. The specific implementation steps are as follows:

[0034] Example 1: Polypeptide aryl cyclization method

[0035] Dissolve linear peptide II (linear oxytocin, Wuxi Yipeptide) in an acetonitrile / water mixture (25% acetonitrile by volume) and adjust the reaction solution to pH 7.4. Weigh 1.1 molar equivalents of dichloronitrogen-containing heteroaromatic reagent I and dissolve it in N,N-dimethylformamide (DMF) at a solvent volume of 10% of the peptide solution. Add the DMF solution of reagent I to the peptide solution and shake at 37°C for 10 minutes. Monitor the reaction progress by HPLC.

[0036] Figure 1 The sequence of the polypeptide II used and the chemical structure of the reagent I are shown.

[0037] Example 2: Purification and characterization of polypeptides

[0038] The reaction solution obtained in Example 1 was centrifuged (12,000 rpm, 5 min) to remove insoluble matter. After filtration, the product was purified using high-performance liquid chromatography (HPLC, C18 reverse-phase column, acetonitrile / water gradient elution). The conversion rates of the same template polypeptide (linear oxytocin) using different dichloronitrogen-containing heteroaromatic ring reagents (I) were significantly different. The molecular weight of the purified product was confirmed by mass spectrometry. Calculated value [M+H] + =1116.46, [M+Na] + =1138.45; measured value [M+H] + =1116.20, [M+Na] + =1138.35.

[0039] Figure 2 Comparison of HPLC and MS spectra, along with conversion curves, of Reagent I-1 and Peptide II at different reaction times and pH conditions is presented. The results demonstrate that the reaction can be completed within 10 minutes using Reagent I-1, and even at a weakly acidic pH of 5.5, the conversion rate remains above 90%.

[0040] Example 3: Flow cytometry analysis of cellular uptake efficiency of cyclized polypeptides

[0041] HeLa cells were seeded in 24-well plates (5 × 10 4cells / well) and adhered to the wall and grown in a 37°C, 5% CO2 incubator to 80% confluence. 5-Carboxyfluorescein (FAM)-labeled cyclic peptides IV-1, IV-7 and linear peptide IV (control group) were added to the culture medium at a final concentration of 10 μM and incubated at 37°C for 2 hours. The culture medium was discarded, and the cells were washed three times with pre-cooled PBS. After digestion with 0.25% trypsin, the cells were collected by centrifugation (1,000 rpm, 5 min). The cells were resuspended in PBS, filtered through a 40 μm filter, and the FAM fluorescence intensity was detected using a flow cytometer (excitation wavelength 488 nm, emission channel 530 / 30 nm). 10,000 cell events were collected for each sample. Untreated cells were used as blank controls, and the mean fluorescence intensity (MFI) difference was calculated.

[0042] Figure 3 It shows that the MFI of cyclized peptide IV-1 is improved compared with linear peptide II.

[0043] Example 4: Confocal laser microscopy analysis of cellular uptake efficiency of cyclized polypeptides

[0044] HeLa cells were seeded in a confocal microplate (2 × 10 4 Cells were plated (10 μM cells / dish) and treated with FAM-labeled cyclized peptides IV-1 and IV-7 and linear peptide IV (10 μM, incubated at 37°C for 2 hours) according to the procedure in Example 3. The culture medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes, and the nuclei were stained with DAPI (1 μg / mL, 10 minutes). The slides were then mounted with an anti-quenching agent. Z-stack images (0.5 μm slice thickness) were acquired using a laser confocal microscope (excitation wavelength: FITC 488 nm / DAPI 405 nm; emission channels: FITC 525 / 50 nm / DAPI 450 / 50 nm) using a 60x oil immersion lens.

[0045] Figure 4 The results showed that the intracellular fluorescence signal intensity of cyclized peptide IV-1 was significantly enhanced compared with that of linear peptide IV, and its localization was enriched in the cytoplasmic region.

[0046] Example 5: Stability test of cyclized polypeptide

[0047] Cyclic peptide IV-1 and linear peptide IV were dissolved in phosphate buffer (pH 7) or a buffer containing 10 mM GSH or 10 mM hydrogen peroxide to a final concentration of 100 μM and incubated at 37°C with constant shaking (200 rpm). Samples were taken at 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 8 h, 16 h, 24 h, 36 h, and 48 h, filtered through a 0.22 μm filter, and the peak area ratio of the intact peptide was determined by high-performance liquid chromatography (HPLC). The above peptides were added to a buffer containing 30% mouse serum to a final concentration of 100 μM and incubated at 37°C. Samples were taken at 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 8 h, 16 h, and 24 h. The residual rate was calculated with the initial peak area (0 h) as 100%.

[0048] Figure 5 The results showed that even in the presence of GSH, the residual rate of cyclized peptide IV-1 exceeded 80% after 48 hours in buffer, and the residual rate exceeded 60% in the presence of hydrogen peroxide. In serum, IV-1 also showed significantly improved stability compared to linear peptides.

Claims

1. A method for aryl cyclization of a polypeptide, characterized in that: A dichloronitrogen-containing heteroaromatic ring reagent I undergoes an arylation reaction with a linear polypeptide II to generate a cyclized stable polypeptide III. The reaction process is as follows: Wherein, X, Y, and Z represent any amino acids, LA1 and LA2 represent connecting amino acids; and m, n, a, b, and c are natural numbers.

2. The method for aryl cyclization of a polypeptide according to claim 1, characterized in that: The I is any one of the following structural formulas, 3. The method for aryl cyclization of a polypeptide according to claim 1, characterized in that: The structural formula of II is The structural formula of III is 4. The method for aryl cyclization of a polypeptide according to claim 1, characterized in that: The structural formula of II is FAM-Ahx-Cyclo (CRRRC), and the structural formula of I is 5. The method for arylation and cyclization of a polypeptide according to claim 1, characterized in that: The arbitrary amino acid is any one of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, methionine, serine, threonine, proline, histidine or arginine; the linking amino acid is any one of cysteine, homocysteine ​​or selenocysteine.

6. The method for arylation and cyclization of a polypeptide according to claim 1, characterized in that: When the number of b in the middle of the connecting amino acids is different, polypeptides of different ring sizes are formed; when a=0, the cyclization reaction proceeds at the nitrogen end of the polypeptide; when c=0, the cyclization reaction proceeds at the carbon end of the polypeptide; when a and c are both 0, the cyclization reaction proceeds at both ends of the polypeptide.

7. The method for arylation and cyclization of a polypeptide according to claim 1, characterized in that: When the polypeptide II contains three, four or more ring-closing sites, a bicyclic, tricyclic or polycyclic stable polypeptide is generated.

8. The method for arylation and cyclization of a polypeptide according to claim 1, characterized in that: In the arylation reaction, the modified atom includes a sulfur atom and other atoms, and the other atom is a selenium atom.

9. The method for arylation and cyclization of a polypeptide according to claim 1, characterized in that: The dichloro nitrogen-containing heteroaromatic ring reagent I comprises at least two chlorine atoms as leaving groups, and the two chlorine atoms are in the para position or the meta position.

10. The method for arylation and cyclization of a polypeptide according to claim 1, characterized in that: The dichloro nitrogen-containing heteroaromatic ring reagent I is a six-membered nitrogen-containing heteroaromatic ring compound and contains at least one nitrogen atom.

11. The method for arylation and cyclization of a polypeptide according to claim 1, characterized in that: The cyclization reaction is carried out in a weakly acidic, neutral or weakly alkaline buffer solution, and the pH of the reaction system is 5-8.

12. The method for arylation and cyclization of a polypeptide according to claim 1, characterized in that: The stable polypeptide prepared by the method is used for improving polypeptide stability, drug delivery or protein-protein interaction ligand screening.

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